MLS 3A MODULE 1: ❏ The term ‘virus’ derives from the Latin for slimy liquid or

BASIC CONCEPTS IN VIROLOGY poison and was gradually introduced during this period to
INTRODUCTION replace the term ‘filterable agents’.
❏ Until the latter half of the nineteenth century, it was believed
that certain submicroscopic infectious agents of mammalian TOBACCO MOSAIC VIRUS (TMV)
hosts were merely small forms or bacteria. Scientists in ❏ The first virus to be visualized by x-ray crystallography and
these earlier times rationalized that their inability to cultivate electron microscopy which was reported in 1939 and 1941,
these small bacteria only implied they were fastidious in their respectively. These advances introduced the notion that
nutritional requirements. After the turn of the century, viruses were structurally composed of repeating subunits
scientists proposed that these submicroscopic forms of life
be called viruses. In 1915, even bacteria were discovered to
be capable of being infected by viruses. The bacterial
invaders were called bacteriophages or phages.
❏ Not until the discovery of the electron microscope and other
technological advances were scientists able to discard fact
from fiction regarding viruses.
❏ A virus is now defined as a subcellular agent consisting of a
core of nucleic acid surrounded by a protein coat that must
use the metabolic machinery of a living host to replicate and
produce more viral particles.

BACKGROUND AND DISCOVERY

❏ The concept behind modern virology can be traced back to
Adolf Mayer, Dimitri Ivanofsky, and Martinus Beijerinck
who, independently in the late 1880’s, discovered what was
.
later to be called tobacco mosaic virus (TMV), first virus
❏ Frederick Twort and Felix d’Herelle, working
discovered.
independently, are credited with the discovery of viruses
❏ Their discoveries led to the descriptions of filterable agents,
which could infect and lyse bacteria in 1915. They called this
too small to be seen with the light microscope, that could be
“bacteriophage”, a virus that infects bacteria. This discovery
grown in living cells and cause disease. They called this
of this agent gave birth to Modern Virology.
agent “filterable virus”.
❏ D’Herelle introduced the term ‘bacteriophages’ for these
❏ Foot and Mouth Disease Virus
agents and also described the concepts of virus adsorption
➔ the first filterable agent from animals
to its target, cell lysis and release of infectious particles.
➔ was described by Loeffler and Frosch in 1898
Over the next 35-40 years, work with phages led to
❏ Yellow Fever Virus
numerous discoveries including how the introduction of DNA
➔ the first human filterable agent discovered was
into a target cell could reproduce itself and the regulation of
➔ discovered by Walter Reed in 1901
cellular macromolecular synthesis directed by viruses. In

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 essence, the field of molecular biology was opened up ➔ Non-enveloped viruses or naked viruses are
during this period. those that have capsid but do not have
❏ Advances in animal virology were noted throughout the 20th envelopes.
century but the major breakthrough came through the 2. Nucleocapsid.
development of tissue culture systems that led, for example, ➔ The complete protein-nucleic acid complex.
to the isolation of poliovirus by Enders et al. in 1949. This ➔ Composed of nucleic acid core and capsid.
markedly facilitated detailed study of this agent and, most
importantly, the development of poliovirus vaccines. The B. SATELLITE OR DEFECTIVE VIRUSES
ensuing 60 years have seen diagnostic virology mature as a ❏ Viruses which require a second virus (helper virus) for
field with the discovery of new agents and diseases and the replication.
parallel determination of the importance of viruses in our ❏ Hepatitis Delta Virus is the major human pathogen
understanding of molecular biology and cancer. example. It requires the presence of hepatitis B virus to
complete its replication cycle.
DEFINITIONS
VIRUS C. VIROIDS
❏ The smallest replication intracellular microorganism that ❏ Viroids are the smallest known autonomously replicating
comprises sets of genes either DNA (DNA viruses) or RNA molecules.
(RNA viruses). ❏ They consist of single-stranded, circular RNA molecules that
❏ viruses are parasites at the genetic level, replicating only in lack protein shells, 240-375 residues in length and are plant
living cells and are inert in the extracellular environment. pathogens.
❏ A single virus is called a Virus particle or virion
D. PRIONS
A. VIRUS PARTICLE / VIRION ❏ These are not viruses but are often discussed within this
❏ An infectious agent composed of nucleic acid (RNA or DNA), microbiologic category. They are not genetic materials
a protein shell (capsid) and, in some cases, a lipid envelope. (neither RNA or DNA).
❏ Virions have full capacity for replication when a susceptible ❏ Misfolded or defective proteins that cause normal proteins in
target cell is encountered. the brain to misfold because they have the ability to transmit
❏ The virion is the entire infectious unit of the virus. Its basic their misfolded shape onto normal variants of the same
unit is the nucleic acid (RNA OR DNA) encased in capsid protein.
with or without envelope. ❏ The pathogenic prion protein, PrPSc, is formed from a
1. Capsid and capsomeres normal human protein, PrPC, through post-translational
➔ The protein coat that surrounds the viral processing.
nucleic acid. This is composed of repeating ❏ Infectious protein molecules that contain no definable nucleic
protein MID 29 subunits called capsomeres. acid and are responsible for the transmissible and familial
Generally, capsids have either helical or spongiform encephalopathies:
icosahedral symmetry. ➔ Kuru
➔ Capsid protects the nucleic acid, the capsid ➔ Creutzfeldt Jakob disease
may also be surrounded by a lipid-containing ➔ Gerstmann-Straussler- Sheinker syndrome
membrane (envelope). ➔ Fatal Familial Insomnia

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 ➔ Scrapie in sheep
➔ Bovine Spongiform Encephalopathy (BSE) in cattle
(“mad cow disease”)

CLASSIFICATION
❏ Viral classification has been confusing and oft-changing over
the years.
❏ In the past, viruses were often classified by host, target
organ or vector and these are still used vernacularly (e.g.,
the hepatitis viruses and arboviruses). Modern
classification is based on the following four characteristics:
1. Type of viral nucleic acid (RNA or DNA,
single-stranded or double-stranded) and its
replication strategy.
2. Capsid symmetry (icosahedral or helical).
3. Presence or absence of lipid envelope. Figure 1.2: Viral Capsid Shape
4. Structure

Figure 1.1: Structure of Virions

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 ADDITIONAL NOTE: (Sir Robert)
Basis of classification of viruses:
1. Morphology of the Virion
2. Genome properties of the virus
3. Physicochemical properties of the virion
4. Virus protection properties
5. Genome organization and replication
6. Antigenic properties
7. Biologic properties

A. GENOME STRUCTURE
❏ The genome of the viruses can either be RNA or DNA, but
not both.
❏ DNA or RNA genomes may be single or double stranded
❏ May be linear or circular
❏ Some genomes are segmented, every segment can encode
a protein
❏ RNA genomes could be:
➔ Positive (+) sense - can be translated directly into a
viral protein, also called “Translated ready genome”.
These are infectious genetic material.
➔ Negative (-) sense - can’t be translated directly to
viral protein, it needs to be transformed to +ssRNA
by RNA dependent RNA polymerase (RdRP). These
are non-infectious genetic material.
➔ Ambisense (+/-) sense

DAVID BALTIMORE - classified viruses using their nucleic acid

bases

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 B. CAPSID STRUCTURE 3. COMPLEX SYMMETRY
❏ Made up of capsomeres (virus-encoded specific proteins) ❏ Some virus particles do not exhibit simple cubic or
that protect the genome and confer shapes to viruses. helical symmetry but are more complicated in
❏ Present in all viruses and very specific for each type structure
❏ Functions: ➔ Ex. Pox Virus and Bacteriophage
➔ It protects the nucleic acid core from the external NOTE: All DNA viruses (except poxviruses) and most of the RNA
environment viruses have icosahedral symmetry
➔ In non-enveloped viruses, it initiates the first step of
viral replication by attaching to specific receptors on
the host cells thus facilitating the entry of the virus
➔ It is antigenic and specific from each virus
NOTE: Purpose of capsid is to bind to specific receptors that would
initiate replication.

TYPE OF SYMMETRY
1. HELICAL SYMMETRY
❏ Simplest structure for capsid
❏ Protein subunits are bound in a periodic way to the
viral nucleic acid, winding it into a helix
❏ The filamentous viral nucleic acid-protein complex C. ENVELOPE STRUCTURE
(nucleocapsid) is then coiled inside a lipid containing ❏ Many human viruses have an outer lipid bilayer membrane
envelope. that is derived from cellular membranes, mainly the plasma
❏ It is not possible for “empty” helical particles to form membrane, but also, in some cases, cytoplasmic or nuclear
because it needs genetic material to form a membranes
nucleocapsid. Most common example: Tobacco ❏ The viral envelope lipid layer membrane contains
mosaic virus (TMV) virus-encoded glycoproteins called “spikes” or “peplomers”
or “viral envelope proteins”
2. CUBIC SYMMETRY ❏ The envelope spikes bind to the receptor on the host cells,
❏ Aka spherical or icosahedral symmetry help the virus envelope membrane fuse with the cellular
❏ Composed of a number of repeated protein subunits membrane of the host cells and act as principal antigens
(polypeptides) called capsomeres against which the host mounts immune response for the
❏ All cubic symmetry observed with animal viruses is of recognition of the virus
the icosahedral pattern, the most efficient ❏ Envelope virus are only infectious if they acquire envelop
arrangement for subunits in a closed shell. ➔ Example: SARS-CoV 2
❏ Most viruses that have icosahedral symmetry do not
have an icosahedral shape-rather, the physical NOTE:
appearance of the particle is spherical ❏ Enveloped viruses are more sensitive to detergents,
solvents, ethanol, ether, and heat compared with non

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 enveloped (naked capsid) viruses whose outer coat is capsid RNA VIRUSES
protein
❏ Main purpose of spikes is to initiate replication or attach to
receptor present in human cells. Spikes can be found on
envelopes if viruses have envelopes. Viruses with no
evelope, it can be found on capsid.

VIRUSES AND THEIR STRUCTURE

DNA VIRUSES

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 3. TRANSMISSION FROM ANIMAL TO ANIMAL, WITH
HUMANS AS AN ACCIDENTAL HOST
Spread may be:
➔ By bite - (e.g. rabies)
➔ By droplets or aerosol infection from
rodent-contaminated quarters (e.g. hantaviruses,
arenaviruses)

4. TRANSMISSION BY MEANS OF ARTHROPOD VECTOR

➔ (E.g. dengue virus)

NOTE:
❏ All Icosahedral RNA viruses are naked except
Togaviridae
❏ All Helical and Complex RNA viruses are enveloped

MODE OF TRANSMISSION
Viruses may be transmitted in the following ways:
1. DIRECT TRANSMISSION FROM PERSON TO PERSON
BY CONTACT VIRAL PHYSIOLOGY
The major means of transmission include: VIRAL REPLICATION
➔ Droplet or aerosol (Influenza, Rhinovirus, Measles ❏ Viruses can only multiply inside living cells and require a
and Smallpox) host cell to survive. The host cell must contain the required
➔ By sexual contact (Papilloma virus, Hepatitis B, machinery to synthesize the viral proteins and nucleic acids.
HSV 2 and HIV)
➔ Hand-mouth, hand-eye, or mouth-mouth contact Viral replication occurs in several stages, namely;
(HSV1-found in mouth and HSV2- found in genitals)
➔ By exchange of contaminated blood (Hepatitis B, 1. ATTACHMENT – The virus becomes attached to the cell by
Hepatitis C and HIV) specific cellular receptors which can be glycoproteins,
phospholipids or glycolipids.
2. INDIRECT TRANSMISSION 2. ENTRY– Following attachment, the virus can enter the cell,
➔ By the fecal-oral route - improper sanitation (e.g most commonly via receptor mediated endocytosis
Rotavirus, enteroviruses, Hepa A virus) (sometimes through fission of envelope). This is the same
➔ By fomites - non-living things that may harbor
process by which many hormones enter the cell.
pathogens (e.g. Norwalk virus, Rhinovirus)

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3. UNCOATING– Once inside the host cell, the viral capsid
must be uncoated to release the viral nucleic acid. Uncoating
may be achieved by host or viral enzymes that will degrade
the capsid.
4. REPLICATION– Once uncoated, viruses (DNA or RNA)
replicate by switching the host machinery from cellular
protein synthesis to viral synthesis and viral proteins are
produced.
5. ASSEMBLY– Newly synthesised viral proteins are
post-transcriptionally modified and packaged into virions that
can be released from the infected host cell to infect other
cells.
6. RELEASE – Virions are released from the cell either by lysis
or budding. In lysis, the infected cell dies and the virions are
released. In budding, the virion takes some of the host cell’s
membrane with it as it leaves – this normally does not kill
the infected cell.
SPECIMEN COLLECTION, PROCESSING & HANDLING
❏ Laboratory diagnosis of viral infections requires an
understanding of the pathogenesis of the suspected agent,
stage of infection, as well as age and immunocompetence of
the infected individual.
❏ It is important to select the appropriate specimens, collect
the specimen carefully to optimize recovery of the infectious
agent, and transport the specimens as directed so as to
maintain viability and minimize overgrowth with
contaminating organisms.

SELECTION OF SPECIMENS
❏ The specimen should be collected from the target organ
most closely associated with clinical symptoms to identify
etiologic agents responsible for the patient's disease.

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NOTE: animal or arthropod contacts or bites, recent travel to areas
❏ Selection of the appropriate type of specimen is one of the of endemic infections, and recent vaccines.
keys to a correct test result
❏ Specimen for the detection of virus should be collected as GUIDELINES FOR THE CHOICE AND HANDLING OF
early as possible after the onset of symptomatic disease SPECIMENS
- Virus may no longer be present as early as 2 days Table 1.1 Specimen for viral isolation and their preparation
after the appearance of symptoms (since the virus
Specimen Preparation
does not stay at the site of infection after 2 days)
❏ Calcium Alginate Swabs interfere with PCR, the recovery
The patient gargles with 10 mL of sterile
of some enveloped viruses and fluorescent-antibody tests balanced salt solution (Hank's or Earle's
and therefore should not be used. Throat wash
BSS) or trypticase soy broth (TSB);
expelled into a sterile container.
SPECIMEN COLLECTION GUIDELINES
❏ Collect specimens as soon as possible after the onset of Random clean-catch sample collected in a
Urine
symptoms. The chance of virus recovery is best during the sterile container
first 3 days after onset and is greatly reduced beyond 5
days with many viruses. Autopsy samples need to be Collect 7-10 mL of blood in a Yellow ACD
collected as soon as possible after death before tissues start tube (Solution A or B). Blood more than 72
Blood
hrs old from time of collection to the time of
decomposing.
processing will be rejected.
❏ Refer to the specimen collection guide table for viral culture.
In general, place swabs into a tube containing a small Insert a flexible fine-shafted swab through
volume of VTM, and scrapings and small pieces of tissue Naso-Pharyngeal the nostril into the nasopharynx and rotate
into a tube containing a small volume of VTM or saline. Swab the swab gently a few times. Place into M4
Place fluid and bulk specimens (e.g., tissue) into a sterile viral transport media.
leak proof container; add a volume of VTM sufficient to
Withdraw vesicle fluid using a tuberculin
prevent drying of tissue.
syringe or swab the vesicle and place it in
❏ Collect specimens as aseptically as possible to avoid an M4 Culture Transport Tube or 1 ml of
introduction of contaminating organisms that can take over Vesicle fluids
BSS or TSB; crusted, dry lesions are not
the culture later and make accurate diagnosis difficult. vesicles and are not appropriate for virus
❏ Place each specimen into a separate container labeled with isolation.
the patient's name and identification number, the collection
site, the date of collection, and the time of the collection.
❏ Obtain a complete patient history, including the date of
onset of symptoms, clinical findings, recent exposure history,

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 ➔ It should also not be allowed to stay at higher
Collect aseptically and keep moist by temperatures, since enveloped viruses are sensitive
placing in a sterile container with 2 mL of
Tissue to heat, alcohol, ether and detergents.
BSS (Hanks Phosphate Buffered Saline),
TSB, saline or viral transport media M4. ❏ Specimen should be kept cool (4OC) and immediately
transported to the laboratory
Stool Approximately 5 gm of stool placed in a ➔ For storage up to 5 days: 4OC
tightly-capped sterile container without ➔ Storage for 6 days or longer: -20OC or preferable
preservatives. A swab covered with stool is -70OC
acceptable for culture but not preferred.
Swabs are UNACCEPTABLE for
MATERIALS
Clostridium difficile toxin.
A. REAGENTS
VIRAL TRANSPORT MEDIUM (VTM)
NOTE: ❏ prevents specimen drying
❏ All specimens except blood should be tightly sealed, kept ❏ maintains viral viability
cold, and sent on wet ice or a "cool pack" (not frozen) to the ❏ retards the growth of microbial contaminants (All
laboratory as soon as possible, preferably on the day of contaminants are fast growers and may deplete the nutrients
collection. present in the medium needed by the desired pathogens to
❏ Specimens for cytomegalovirus (CMV) isolation should be grow)
sent as soon as possible to our laboratory since this virus is ❏ VTM contains gelatin and antimicrobial agents in a buffered
very heat labile and the probability of isolating it decreases salt solution.
rapidly with time. ❏ Some specimes for virus culture are collected in tube with
❏ If absolutely necessary, all specimens except blood and VTM
those for CMV, RSV and VZ can be frozen at -50°C or ❏ Laboratories have different guidelines in different specimen
below (do not use -20°C) and sent to the laboratory on dry for viral culture
ice.
B. SUPPLIES
SPECIMEN TRANSPORT AND STORAGE ❏ Sterile, leakproof, screw-cap containers including urine cups,
❏ Ideally all specimens collected for detection of virus should disposable centrifuge tubes (15 and 50 mL), and smaller
be processed immediately tubes (e.g., 4-mL Nunc vials, 13x100 tubes), suitable for
❏ Specimen for viral isolation should not be allowed to sit at holding 1-2 mL of VTM.
room or higher temperature ❏ Sterile cotton-, Dacron-, or rayon-tipped swabs with plastic or
➔ If viruses stay at RT, it loses its structure and aluminum shafts small-tip flexible (fine- shafted aluminum)
infectivity and must be preserved through swabs are used for certain samples such as urethral swabs.
refrigeration.

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 Do not use calcium alginate swabs or wooden-shafted ❏ A serological diagnosis can be made by the detection of
swabs. rising titers of antibody between acute and convalescent
❏ Tuberculin syringe with 26- or 27-gauge needle for aspirating stages of infection, or the detection of IgM.
vesicular fluid ❏ In general, the majority of common viral infections can be
❏ Blood collection tubes containing anticoagulant (ACD-yellow diagnosed by serology. The specimen used for direction
top). For serologic testing, use a red-topped tube. detection and virus isolation is very important. A positive
result from the site of disease would be of much greater
C. EQUIPMENT diagnostic significance than those from other sites. For
❏ Refrigerator (2-8OC) example, in the case of herpes simplex encephalitis, a
positive result from the CSF or the brain would be much
LABORATORY TESTS FOR IDENTIFICATION OF VIRUSES greater significance than a positive result from an oral ulcer,
OVERVIEW OF DIAGNOSTIC METHODS since reactivation of oral herpes is common during times of
❏ In general, diagnostic tests can be grouped into 3 categories: stress.

A. DIRECT DETECTION / EXAMINATION DIAGNOSTIC METHODS:

❏ In direct examination, the clinical specimen is examined A. DIRECT EXAMINATION OF SPECIMEN
directly for the presence of virus particles, virus antigen or ❏ Electron Microscopy morphology / immune electron
viral nucleic acids. microscopy
❏ Cytological and Histological Appearance through Light ❏ Light microscopy histological appearance - e.g. inclusion
microscope bodies
❏ Electron Microscope ❏ Antigen detection immunofluorescence, ELISA etc.
❏ Immunodiagnosis (Antigen Detection) ❏ Molecular techniques for the direct detection of viral
❏ PCR genomes

B. INDIRECT EXAMINATION (VIRUS ISOLATION) NOTES FROM SIR ROBERT

❏ In indirect examination, the specimen into cell culture, eggs UNDER DIRECT EXAMINATION:
or animals in an attempt to grow the virus: this is called CYTOLOGICAL AND HISTOLOGICAL APPEARANCE
virus isolation. ❏ Uses light microscope
❏ Usually performed for the detection of viral inclusions
C. SEROLOGY / SEROLOGIC EXAMINATION ❏ Viral inclusions are intracellular structures formed by
❏ Serology actually constitutes by far the bulk of the work of aggregates of virus or viral components in an infected cell of
any virology laboratory. abnormal accumulations of cellular materials resulting from
virus-induced metabolic disruption.
❏ Inclusions occur in single or syncytial cells

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 ❏ Syncytial cells are aggregates of cells fused to form one
large cell with multiple nuclei allowing it to be visible under
the light microscope

Adenovirus - naked and may not be destroyed using alcohol

NOTE: Familiarize the Inclusion bodies
2. IMMUNODIAGNOSIS
❏ Direct and Indirect Immunofluorescent method
➔ Both usually use FITC (Fluorescein Isothiocyanate)
as the labelled antibody
➔ Direct Method: best suited in dealing with high
concentration of viruses
E.g. Intracytoplasmic Virus ➔ Indirect Method: suitable for low concentration of
viruses
1. ELECTRON MICROSCOPY ❏ Enzyme Immunoassay
❏ Most helpful for detecting viruses that do not grow readily in - E.g ELISA
cell culture ❏ Latex Agglutination
➔ But labor intensive and relative insensitive - Easy method but lacks sensitivity
❏ Works best if the titer of the virus is at least 10^6 to 10^7 ❏ Immunoperoxidase Test
particles per mL - Stains histologic section
❏ Immune Electron Microscopy (IEM) allows visualization of
virus particles present in numbers too small for easy direct 3. POLYMERASE CHAIN REACTION
detection (Modified electron microscopy) ❏ Target amplification
➔ The addition of specific antiserum to the ❏ Divided into three: denaturation, annealing, and synthesizing
suspension causes the virus particles to form ❏ Purpose: to increase the target and to visualize the genetic
antibody-bound aggregates which are more easily material of viruses through amplification or increasing the
detected than are single virus particles concentration.
➔ Antiserum contains antibodies that will bind with the
virus to form aggregates.

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❏ Replication is performed to increase the concentration of
viruses.
❏ Denaturation @ 95OC: Parent DNA is separated into two
strands
❏ Annealing @ 55OC: Addition of DNA Primer to the template
strand or the INitial replication.
❏ Synthesizing @ 72OC

B. INDIRECT EXAMINATION
❏ Cell Culture - cytopathic effect, haemadsorption,
confirmation by neutralization, interference,
immunofluorescence etc.
➔ Viruses are strict intracellular parasites,
requiring a living cell for multiplication and
reproduction
➔ There are three basic types of conventional
cell culture: Primary, Secondary, and Tertiary
❏ Eggs pocks on CAM - haemagglutination, inclusion
bodies
❏ Animals disease or death confirmation by
neutralization

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NOTE: DIRECT EXAMINATION
PRIMARY CELL CULTURE ❏ Direct examination methods are often also called rapid
❏ widely acknowledged as the best cell culture systems diagnostic methods because they can usually give a result
available since they support the widest range of viruses. either within the same or the next day.
❏ However, they are very expensive and it is often difficult to ❏ This is extremely useful in cases when the clinical
obtain a reliable supply. Continuous cells are the easiest to management of the patient depends greatly on the rapid
handle but the range of viruses supported is often limited. availability of laboratory results e.g. diagnosis of RSV
TERTIARY CELL CULTURE infection in neonates, or severe CMV infections in
❏ For Tertiary Culture, it cannot be used for vaccine immunocompromised patients. However, it is important to
production since it is derived from cancer cells and may realize that not all direct examination methods are rapid, and
replicate indefinitely conversely, virus isolation and serological methods may
❏ Supply is unlimited coming from cancer cells sometimes give a rapid result. With the advent of effective
antiviral chemotherapy, rapid diagnostic methods are
C. SEROLOGY / SEROLOGIC EXAMINATION expected to play an increasingly important role in the
➔ Detection of rising titers of antibody between acute and diagnosis of viral infections.
convalescent stages of infection, or the detection of IgM in
primary infection. The serological methods involved in the A. ANTIGEN DETECTION
identification are listed in Table 1.2. ❏ Examples of antigen detection include:
➔ Usually, a method is used to detect antibody concentration in ➔ Immunofluorescence testing of nasopharyngeal
the plasma or serum of a patient. aspirates for respiratory viruses e.g.. RSV, flu A, flu
B, and adenoviruses
Table 1.2 Serologic methods in the identification of viral antibodies ➔ Detection of rotavirus antigen in faeces
Classical Techniques Newer Techniques
➔ pp65 CMV antigenemia test
➔ Detection of HSV and VZV in skin scrapings, and
❏ Complement fixation tests ❏ Radioimmunoassay (RIA) ➔ Detection of HBsAg in serum.
(CFT) ❏ Enzyme linked ❏ (However, the latter is usually considered as a serological
❏ Hemagglutination immunosorbent assay
inhibition tests (EIA) test). The main advantage of these assays is that they are
❏ Immunofluorescence ❏ Particle agglutination rapid to perform with the result being available within a few
techniques (IF) ❏ Western Blot (WB) hours. However, the technique is often tedious and time
❏ Neutralization tests ❏ Recombinant immunoblot
consuming, the result difficult to read and interpret, and the
❏ Single Radial Hemolysis assay (RIBA)
❏ Line Immunoassay sensitivity and specificity poor.
❏ The quality of the specimen obtained is of utmost importance
in order for the test to work properly.

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 Figure 1.6 Electron micrographs of viruses commonly found in stool
specimens from patients suffering from gastroenteritis. From left to
right: rotavirus, adenovirus, astroviruses, Norwalk-like viruses.
B. ELECTRON MICROSCOPY (EM)
C. LIGHT MICROSCOPY
❏ Virus particles are detected and identified on the basis of
❏ Replicating viruses often produce histological changes in
morphology. A magnification of around 50,000 is normally
infected cells. These changes may be characteristic or
used. EM is now mainly used for the diagnosis of viral
non-specific. Viral inclusion bodies are basically collections
gastroenteritis by detecting viruses in feces e.g. rotavirus,
of replicating virus particles either in the nucleus or
adenovirus, astrovirus, calicivirus and Norwalk-like
cytoplasm.
viruses. Occasionally it may be used for the detection of
❏ Examples of inclusion bodies include the negri bodies and
viruses in vesicles and other skin lesions, such as
cytomegalic inclusion bodies found in rabies and CMV
herpesviruses and papillomaviruses.
infections, respectively. Although not sensitive or specific,
❏ The sensitivity and specificity of EM may be enhanced by
histology nevertheless serves as a useful adjunct in the
immune electron microscopy, whereby virus specific
diagnosis of certain viral infections.
antibody is used to agglutinate virus particles together and
thus making them easier to recognize, or to capture virus
particles onto the EM grid. The main problem with EM is the
expense involved in purchasing and maintaining the facility.
In addition, the sensitivity of EM is often poor, with at least
10^5 to 10^6 virus particles per ml in the sample required for
visualization. Therefore, the observer must be highly skilled.
❏ With the availability of reliable antigen detection and
Figure 1.7 Inclusion bodies. Left: Negri bodies pointed by the arrow;
molecular methods for the detection of viruses associated
Right CMV inclusion body pointed by the arrow.
with viral gastroenteritis, EM is becoming less and less
widely used.

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D. VIRAL GENOME DETECTION to interpret as it does not necessarily indicate the presence
❏ Methods based on the detection of viral genome are also of disease.
commonly known as molecular methods. It is often said that ❏ This problem is particularly great in the case of latent viruses
molecular methods are the future direction of viral diagnosis. such as CMV, since latent CMV genomes may be amplified
However, in practice, although the use of these methods is from the blood of healthy individuals. Despite all this, PCR is
indeed increasing, the role played by molecular methods in a being increasingly used for the rapid diagnosis of rapid. This
routine diagnostic virus laboratory is still small compared to is especially as the cost of the assay comes down and the
conventional methods. availability of closed automated systems that could also
❏ It is certain though that the role of molecular methods will perform quantification (Quantitative PCR) e.g. real-time PCR
increase rapidly in the near future. Classical molecular and Cobas Amplicor.systems. Other amplification techniques
techniques such as dot-blot and Southern- blot depend on such as LCR and NASBA are just as susceptible to
the use of specific DNA/RNA probes for hybridization. contamination as PCR but that is ameliorated to a great
❏ The specificity of the reaction depends on the conditions extent by the use of proprietary closed systems.
used for hybridization. These techniques may allow for the ❏ It is unlikely though that other amplification techniques will
quantification of DNA/RNA present in the specimen. challenge the dominance of PCR since it is much easier to
However, it is often found that the sensitivity of these set up a house PCR assay than other assays. One
techniques is not better than conventional viral diagnostic advantage of PCR assays is that the PCR product can be
methods. sequenced and thus used for epidemiological investigation
❏ Newer molecular techniques such as the polymerase chain and drug susceptibility testing.
reaction (PCR), ligase chain reaction (LCR), nucleic
acid-based amplification (NASBA), and branched DNA VIRUS ISOLATION
(bDNA) depend on some form of amplification, either the ❏ Cell cultures, eggs, and animals may be used for isolation.
target nucleic acid, or the signal itself. However, eggs and animals are difficult to handle and most
❏ bDNA is essentially a conventional hybridization technique viral diagnostic laboratories depend on cell culture only.
with increased sensitivity. However, it is not as sensitive as ❏ There are 3 types of cell cultures:
PCR and other amplification techniques.
❏ PCR is the only amplification technique which is in common TYPES OF CELL CULTURES
use. PCR is an extremely sensitive technique: it is possible A. Primary cells - e.g. Monkey Kidney. These are essentially
to achieve a sensitivity of down to 1 DNA molecule in a normal cells obtained from freshly killed adult animals. These
clinical specimen. However, PCR has many problems, the cells can only be passaged once or twice.
chief among which is contamination, since only a minute
amount of contamination is needed to give a false positive B. Semi-continuous cells - e.g. Human embryonic kidney and
result. In addition, because PCR is so sensitive compared to skin fibroblasts. These are cells taken from embryonic tissue,
other techniques, a positive PCR result is often very difficult and may be passaged up to 50 times.

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 C. Continuous cells - e.g. HeLa, Vero, Hep2, LLC-MK2, BGM.
These are immortalized cells i.e. tumour cell lines and may
be passaged indefinitely.
➔ Primary cell culture is widely acknowledged as the
best cell culture systems available since they support
the widest range of viruses. However, they are very
expensive and it is often difficult to obtain a reliable
supply. Continuous cells are the easiest to handle but Figure 1.8 Left to Right: Cytopathic effect of HSV, enterovirus 71,
the range of viruses supported is often limited. and RSV in cell culture. Note the ballooning of cells in the cases of
HSV and enterovirus 71. Note syncytia formation in the case of
NOTE: Passage - number of times you allowed a culture to be RSV.
multiplied.
PROBLEMS WITH CELL CULTURE
IDENTIFICATION OF GROWING VIRUS ❏ The main problem with cell culture is the long period (up to 4
❏ The presence of growing virus is usually detected by: weeks) required for a result to be available. Also, the
➔ Cytopathic Effect (CPE) - may be specific or sensitivity is often poor and depends on many factors, such
non-specific e.g. HSV and CMV produce a specific as the condition of the specimen, and the condition of the
CPE, whereas enteroviruses do not. cell sheet. Cell cultures are also very susceptible to bacterial
➔ Haemadsorption - cells acquire the ability to stick to contamination and toxic substances in the specimen. Lastly,
mammalian red blood cells. Hemadsorption is mainly many viruses will not grow in cell culture at all e.g. Hepatitis
used for the detection of influenza and parainfluenza B and C, Diarrheal viruses, parvovirus etc.
viruses.
❏ Confirmation of the identity of the virus may be carried out RAPID CULTURE TECHNIQUES
using neutralization, hemadsorption-inhibition, ❏ Rapid culture techniques are available whereby viral
immunofluorescence, or molecular tests. antigens are detected 2 to 4 days after inoculation.
Examples of rapid culture techniques include shell vial
cultures and the CMV DEAFF test. In the CMV DEAFF test,
the cell sheet is grown on individual cover slips in a plastic
bottle. After inoculation, the bottle is then spun at a low
speed for one hour (to speed up the adsorption of the virus)
and then incubated for 2 to 4 days. The cover slip is then
taken out and examined for the presence of CMV early
antigens by immunofluorescence.

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 ➔ Take note of the suffix:
- Subgenus & Genus: Virus
- Subfamily: Virinae
- Family: Viridae

PREVENTION AND CONTROL

PREVENTION AND THERAPY
Figure 1.9 Left: Haemadsorption of red blood cells onto the surface ❏ Immunizations are available for some viruses capable of
of a cell sheet infected by mumps virus. Also note the presence of causing human disease.
syncytia which is indistinguishable from that of RSV. ❏ For viruses for which there are no available vaccines, the
most effective means of preventing viral infection involves:
❏ The role of cell culture (both conventional and rapid ➔ Regular, thorough handwashing (easiest way to
techniques) in the diagnosis of viral infections is being break chain of infection)
increasingly challenged by rapid diagnostic methods i.e. ➔ Avoiding contact with others during episodes of
antigen detection and molecular methods. Therefore, the evident signs and symptoms
role of cell culture is expected to decline in future and is ● Fever
likely to be restricted to large central laboratories. ● Cough
● Diarrhea
ADDITIONAL NOTE: NOMENCLATURE ● Respiratory Infections

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 MODULE 2: PATHOGENESIS OF VIRAL DISEASE CHAIN OF INFECTION

OUTLINE OF THE LESSON

I. INTRODUCTION
A. Chain of infection
II. VIRAL PATHOGENESIS
A. Specific steps involved in viral pathogenesis
III. PATHOGENESIS OF VIRAL DISEASE
A. Key elements of the virus-host interaction
IV. PATHOGENIC STEPS IN HUMAN INFECTION
A. Viruses and their specific receptor
VIRAL PATHOGENESIS
V. VIRAL REPLICATION PROCESS
VI. VIRAL INTERACTION ❏ process by which viruses cause disease in the host
A. Ways of viral interaction ➔ From the ​entry to the excretion of the virus from the
VII. TERMINOLOGIES host
A. Terms describing​ infections of an organism ➔ Viruses cause disease when they breach the host’s
primary physical and natural protective barriers;
B. Terms describing ​virus transmission
VIII. IMMUNE RESPONSE TO VIRAL INFECTIONS evade local, and immune defenses; spread in the
A. Non-specific / innate immunity body; and destroy cells either directly or via
B. Specific immunity / adaptive bystander immune and inflammatory responses.
C. Intense immunologic reaction ➔ Signs and symptoms of a disease usually appear
once the host’s cells are destroyed and the virus has
undergone replication.
INTRODUCTION
❏ Once the virus is introduced into a host, the virus infects SPECIFIC STEPS INVOLVED IN VIRAL PATHOGENESIS​:
susceptible cells, frequently in the upper respiratory tract. 1. Viral entry into the host and primary viral replication (​viral
❏ Viral infections may produce one of three characteristic replication can occur in in the ​nucleus​ or the ​cytoplasm​)
clinical presentation: 2. Viral spread and tropism
➔ Acute viral infection 3. Cell injury and clinical illness
➔ Latent infection 4. Recovery from infection
➔ Chronic infection 5. Virus shedding
❏ In this module, we will discuss the mechanism of viral
disease and know the sequence of events involved in viral
replication.

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 PATHOGENESIS OF VIRAL DISEASE THE NET RESULT OF THIS INTERACTION MAY BE:
❏ As with other infectious agents which cause human disease, 1. No infection
the ​outcome of the interaction of a particular virus with the 2. Abortive infection with limited viral replication
human host is dependent ​on both ​pathogen ​and ​host ➔ Cells that have been infected with a virus but did not
factors​. produce any progeny virus, infection was stopped.
❏ Viral strains within a genus may have differential cell 3. Asymptomatic infection
tropisms, replication capacities, and cytopathogenic effects. 4. Symptomatic infection
❏ Example: ​Strains of HIV​ may preferentially: 5. Depending upon the agent and the immune status of the
➔ target ​monocyte/macrophages or T-lymphocytes​, host,
may use ​5 different co- receptors (e.g., the ➔ Persistent/latent or
chemokine receptors​, ​CCR5 or ​CXCR4)​ on the cell ➔ Self limited infection
surface
➔ may r​eplicate to different levels and may ​induce PATHOGENETIC STEPS IN HUMAN INFECTION
different degrees of cell killing​. ❏ A generalized schema of viral infection leading to disease in
These traits have direct clinical correlates for HIV the human host is as follows:
infected persons with respect to the rates of CD4 cell (SIR ROBERT)
decline and progression to clinical AIDS. 1. VIRAL ENTRY​ AND R ​ EPLICATION
❏ On the ​host side​, the ​nature of the exposure and the ​host ❏ Most viral infections are initiated when viruses attach
immune status are probably the ​two most important and enter cells of one of the body surfaces
determinants of outcome​. ➔ skin, respiratory tract, gastrointestinal tract,
urogenital tract, or the conjunctiva.
KEY ELEMENTS OF THE VIRUS-HOST INTERACTION ❏ The majority of viruses enter their hosts through the
1. Viral Strain mucosa of the respiratory or gastrointestinal tract
➔ i.e New strains are usually more virulent/ pathogenic (these surfaces have a ​direct contact with the outside
than older strains. environment
2. Inoculum Size ❏ Depending upon the agent, the ​virus enters​ through:
➔ i.e ID50 and LD50 ➔ Skin,
3. Route of exposure ➔ Mucous membranes,
4. Susceptibility of host ➔ Respiratory tract,
➔ i.e Is there pre-existent from past exposure or ➔ Gastrointestinal tract,
vaccination?) ➔ Conjunctiva
5. Immune Status ​and ​Age of Host ➔ Urogenital tract
➔ i.e babies and pregnant women ➔ via a transfusion or transplanted organ
➔ via maternal-fetal transmission.

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 ❏ After entry, the viral nucleic acid and ➔ Peripheral nerve routes to the central
virion-associated proteins interact with cellular nervous system ​(e.g.,​ ​rabies virus​).
macromolecules to ultimately produce new virions ➔ For other neurotrophic agents, the central
that are released from the host cells by shedding or nervous system is seeded following ​viremia​.
cell lysis. ❏ For many agents, there is ​replication in regional
❏ There is ​local replication at the site of the lymph nodes ​with subsequent viremia and spread to
inoculation. Certain agents exhibit pathology at the target organs. Some viruses travel:
skin or mucous membrane​ surface ➔ Bloodstream free in plasma (e.g.,
➔ e.g., ​herpes simplex virus​, human picornaviruses​)
papillomavirus​. ➔ Cell associated ​(e.g., ​cytomegalovirus​).
NOTE: the first step in viral pathogenesis is the viral entry and ➔ Replication in target organs may lead to
replication inside the host cell local damage and further rounds of viremia.

2. VIRAL SPREAD​ AND ​CELL TROPISM VIRUSES AND THEIR SPECIFIC RECEPTOR
❏ Mechanisms of viral spread vary, but the most
VIRUS RECEPTOR
common route is via the​ bloodstream​ or​ lymphatics​.
❏ The presence of virus in the blood is called​ ​viremia Adenovirus Integrins
❏ Virus tend to exhibit organ and cell-type specificities,
or​ Viral tropism Arenavirus a-dystroglycan
❏ Tropism ​is the capacity of viruses to infect a specific Cytomegalovirus Heparan sulfate
cell type within a tissue or organ
❏ Tropism is determined by the specific interactions Coronavirus Aminopeptidase N
between the viral surface proteins and cellular
receptors. Sulfated glycosaminoglycans
➔ Example: HIV contains ​glycoproteins ​that Lectin
allows specific ​binding to the CD4+ cell
receptor​. Once the virus binds to the receptor, Epstein barr virus CR2 (CD21)
it then replicates itself and spreads to other
Filovirus(ebola and marburg) TIM-1
CD4+ cells.
❏ For some ​neurotropic viruses there may be spread Hantavirus Integrins
along:
➔ Peripheral nerve routes to ​ganglia Hepatitis A virus HAVCR1/TIM1
(e.g., ​herpes simplex virus​)
Herpes simplex Heparan sulfate

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 ➔ Clinical illness from viral infections is the
Human herpes 7 CD4
result of complex serie of events
HIV CD4, CXCR4, CCRS ● The destruction of cells and the
reaction of our immune system
Influenza A Sialic acid ➔ Many of the factors that determine the degree
Measles CD46 of illness are still unknown
➔ Non-specific and specific host immune
Papillomavirus Alpha-6-beta-4 integrin responses come into play to try to ​control
and downregulate the viral replicative
Rabies Acetylcholine receptor process​.
Reovirus Sialic acid
EGF receptor 4. VIRAL SHEDDING
❏ The last stage in viral pathogenesis
Rhinoviruses ICAM-1 NOTE: ​It is important for a virus to shed in order to ​maintain its
pathogenicity
Alpha1-beta1 and alpha2-
beta2 Integrins ❏ This is a necessary step to maintain a viral infection
in populations of hosts
vaccinia EGF receptor ❏ Shedding usually occurs from the body surfaces
involved in viral entry
NOTE: ❏ Shedding occurs at different stages of disease
❏ People that do not have the coreceptors CXCR4 and CCRS depending on the particular virus involved
receptor are resistant to HIV. ❏ Shedding represents the time at which an infected
individual is infectious. In some viral infections, such
3. CELL INJURY AND CLINICAL ILLNESS as rabies, human represent dead-end infections, and
❏ Destruction of virus infected cells in the target tissue shedding does not occur
➔ Presence of virus does not necessarily cause
illness but the destruction
➔ Physiologic alterations produced in the host
by tissue injury responsible for the
development of the disease
➔ Some physiologic effects may result from
nonlethal impairment of specialized function
of cells

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EXAMPLE:
STAGES OF POLIOVIRUS PATHOGENESIS

STEP 1 ➔ Poliovirus is ingested

➔ The virus will enter the oropharynx

STEP 2 ➔ After ingestion the virus will migrate to the gut

associated lymphoid tissue
➔ Tonsils, peyer’s patches

STEP 3 ➔ Will then spread via the lymphatic vessels

➔ Regional lymph nodes
➔ Virus replicates

STEP 4 ➔ Distributed via the bloodstream

STEP 5 ➔ Crosses the blood-brain barrier

● Virus crossed the endothelium
➔ Spinal cord
● Virus replicates in anterior horn cells
● Affects motor functions
● Results in destruction of spinal cord Figure 2.1​ Schematic representation of viral infection from entry to
● Paralysis the signs of the disease

STEP 6 ➔ Sheds through the gut VIRAL REPLICATION PROCESS

➔ Excreted in the feces GENERAL STEPS
❏ Viruses do not undergo binary fission unlike bacteria, but
undergo a complex way of replication
❏ Replication of viruses through six sequential steps

1. ATTACHMENT
❏ The attachment is the first step in viral infection, interaction
of virion with a specific receptor on the host cell

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 ❏ Receptor molecules are generally glycoproteins
❏ Each susceptible cell may contain upto 100,000 receptors for
a given virus,

2.PENETRATION / ENTRY
❏ Also known as ​engulfment
❏ Virus particle is taken up inside the cell
❏ Sometimes accomplished through ​receptor-mediated
endocytosis with ​uptake of the ingested virus particles
within an ​endosome
➔ Virus that exhibit cell tropism
❏ Sometimes accompanied through ​direct penetration of
virus ​particles across the ​plasma membrane
❏ Sometimes accomplished by ​fusion ​of the ​virion envelope 4. EXPRESSION OF VIRAL GENOME
with the plasma membrane cell ❏ Occurs after uncoating of the viral genome
➔ Fusion is possible because the virion envelope is ❏ Various classes of viruses use different pathways to
also from the host cell synthesize mRNAs depending on the structure of the viral
nucleic acid
3. U ​ NCOATING ➔ Specific mRNA are transcribed from the viral nucleic
❏ Occurs with penetration or shortly after penetration acid for successful expression and duplication of viral
❏ Physical separation of viral nucleic acid from the ​outer genome
structural components of the virion TERMS:
❏ Release of genetic material from a nucleocapsid. Genome VIRAL REPLICATION
may be released as a​ free nucleic acid ​or as ​nucleocapsid ❏ duplicates the genetic material of the virus
❏ Nucleocapsid usually contains polymerase VIRAL TRANSLATION
➔ Polymerase ​is responsible for replication inside the ❏ produces a capsomere that will form a capsid that will house
nucleocapsid the replicated genome
NOTE: ​Once nucleic acid is released into the cell, the virus CAPSID
opportunistically replicates using the host’s organelles ❏ is the protein shell of a virus, enclosing its genetic material.
❏ It may require an ​acidic pH in an endosome. ​The infectivity CAPSOMERE
of the parental virus is lost at the uncoating stage ❏ is a subunit of the capsid, an outer covering of protein that
protects the genetic material of a virus. Capsomeres
self-assemble to form the capsid

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 NOTE:
5. SYNTHESIS OF VIRAL COMPONENTS ❏ Budding ​enables viruses to exit the host cell and is mostly
used by enveloped viruses which must acquire a
host-derived membrane enriched in viral proteins to form
their external envelope.
❏ Enveloped virus are not infectious unless the acquire their
envelopes

Enveloped virus (Budding)

6. MORPHOGENESIS
❏ Newly synthesized viral genomes and polypeptides
assemble together to from progeny viruses
❏ Capsids of ​icosahedral viruses ​can condense in the
absence of nucleic acid
❏ Capsids of ​helical viruses​ cannot form without RN

7. RELEASE
❏ Naked viruses accumulate in infected cells and the cells
eventually lyse and release the virus
❏ Enveloped viruses​ are released through​ budding

Naked virus

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 VIRAL INTERACTION NOTE:
❏ When two or more viruses infect the same host cell, they ❏ In complementation the normal cell will provide the defective
may interact in a number of ways: cell with the necessary structure in order to produce a
➔ Recombination functioning virus
➔ Complementation
➔ Phenotypic mixing 3. PHENOTYPIC MIXING
➔ Interferences ❏ A special case of complementation
❏ This occurs when the genome of one virus becomes
WAYS OF VIRAL INTERACTION: randomly incorporated within capsid proteins specified by a
1. RECOMBINATION different virus or a capsid consisting of components of both
❏ Results in the formation of progeny virus (recombinant) that viruses
carries traits not found in either parent ❏ It usually occurs between different members o the same
➔ The resulting progeny virus is different from the virus family;the intermixed capsid proteins must be able to
parent virus interact correctly to form a structurally intact capsid
❏ The classic mechanism is that the nucleic acid strands ❏ There is no genetic change only the phenotype
break, and a part of the genome of one parent is joined to ❏ Mixing of the capsid proteins
part of the genome of the second parent ❏ Can result to resistance to antibody neutralization
❏ The recombinant virus is genetically stable, yielding progeny
similar to itself upon replication 4. INTERFERENCES
NOTE: ❏ Infection with 2 viruses often leads to an ​inhibition of
❏ Recombination hinders vaccine studies due to the constant multiplication of one of the viruses
production of new strains that are different from their parent ❏ Several mechanisms have been elucidated as cause of
viirus interference:
1. One virus may inhibit the ability of the second to
2. COMPLEMENTATION adsorb to the cell, either by blocking its
❏ This refers to the interaction of viral gene products in cells receptors(retroviruses, enteroviruses) or by
infected with two viruses, one or both of which may be destroying its receptors (orthomyxovirus)
defective 2. One virus may compete with the second for
❏ It results in the replication of one or both under conditions in components of the replication apparatus(polymerase,
which replication would not ordinarily occur translation initiation factor).
❏ The basis for complementation is that one virus provides a NOTE:
gene product in which the second is defective, allowing the ❏ The first virus may cause the infected cell to produce an
second virus to grow. The genotype of the two viruses inhibitor that prevent replication of the second virus
remain unchanged

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 TERMINOLOGIES ACUTE INFECTION
❏ It is a ​rapid onset of disease symptoms resulting in severe
TERMS DESCRIBING​ INFECTIONS OF AN ORGANISM illness or death of the infected animal (influenza, viral
hemorrhagic fever).
CHRONIC INFECTION
❏ It is a ​prolonged infection in which the organism is not
immediately killed and may carry the virus for long periods of
time (hepatitis, HIV).

TERMS DESCRIBING ​VIRUS TRANSMISSION

HORIZONTAL TRANSMISSION
❏ is defined as the transmission of virus or other pathogen to
host​ at any age after birth​.

VERTICAL TRANSMISSION
❏ is the passage of a virus from ​mother to the new born child​.

ZOONOSIS
LYTIC INFECTION
❏ is defined as the disease which is naturally transmitted
❏ When a virus enters the cell and hijacks its cellular
between animals and man (Rabies, H1N1 influenza virus,
machinery to rapidly multiply and in the process ​kills the cell
Rift valley fever virus).
is termed as lytic infection (many influenza viruses).
❏ Sometimes, the virus can be transmitted through an ​insect
vector (arboviruses). Viruses present in the saliva of the
LYSOGENIC INFECTION
infected insect are transmitted during feeding of blood meal
❏ It is the process characterized by the ​incorporation of viral
to the susceptible host.
DNA to the cellular DNA. Once incorporated, the viral DNA
replicates along with the host DNA. The incorporated viral
PERSISTENT INFECTION
DNA permits the host cell to undergo normal cell cycle.
❏ is a condition where the ​virus remains associated with the
(Does not cause damage to cell & transforms the cell into
cell without actively multiplying or killing it​. This often occurs
oncogenic cell)
when the viral genome gets integrated into the host genome
(retroviruses) and sometimes without integration
(Herpesvirus).

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 ❏ CATEGORIES:
1. Virus genome persists ​within the cell without actual
release of the virus​, eg. Some​ retroviruses​.
2. Virus released ​sporadically but remains in a state of
"​latent​" for most of the time (​herpes simplex​).
3. Virus released ​continuously without lysis of the host
cell​, eg. ​hepatitis B virus​. (lysogenic cycle, budding)

MULTIPLICITY OF INFECTION (M.O.I.)

Figure 2.2​ Schematic diagrams showing the patterns of viral
❏ This is the ​ratio of total virus infected to the number of target
infections
cells in an infection condition​. This is usually used to
describe the infection of a cell type grown invitro in a culture
IMMUNE RESPONSE TO VIRAL INFECTIONS
system.(ex.​ 5:10​, 5 virus and 10 target cells) A. NON-SPECIFIC / INNATE IMMUNITY
❏ refers to those elements of the immune system that
“INFECTIOUS DOSE 50” (ID50) can clear virus or virus infected cells immediately
❏ Median Infectious Dose upon or shortly after viral exposure and which are ​not
❏ The dose required to​ infect 50%​ of the inoculated animals. dependent upon immunologic memory​. Non-specific
immunity may include:
“LETHAL DOSE 50” (LD50) ➔ Phagocytic cells ​(neutrophils and monocytes
❏ The dose required to ​kill 50%​ of the inoculated animals. / macrophages).
➔ Cytokines ​(e.g., interferons) and
INCUBATION PERIOD chemokines​.
❏ The ​time between the initial infection to the actual onset of ➔ Natural killer cells​ -​ kills infected cells
disease symptoms​. This period can range from a few days ➔ Poorly defined antiviral factors ​that may
(cold viruses) to years (HIV). exist in blood or body fluids.

B. SPECIFIC IMMUNITY / ADAPTIVE

❏ refers to ​antigen specific B and T cell ​responses that
lead to the development of antibodies, cytotoxic T
cells and antibody dependent cellular cytotoxicity.

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C. INTENSE IMMUNOLOGIC REACTION
❏ In some instances, an ​intense immunologic
reaction to a viral agent can result in
immunopathology and a serious clinical syndrome. A
prime example is ​dengue hemorrhagic fever which
is likely due to antibody dependent enhancement and
T cell activation on re-exposure to dengue virus.

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 MODULE 3: CHARACTERISTICS, LABORATORY TEST, EPIDEMIOLOGY, Although the incidence varies inversely with age (ie, greater
PREVENTION AND CONTROL OF RESPIRATORY AND among younger children than healthy young adults), the
CHILDHOOD FEVER VIRUS morbidity is significantly higher in elderly population.
Seasonality is also a feature; ​incidence is lowest in the
OUTLINE OF THE LESSON summer months and highest in the winter​.
❏ In addition to the ability to cause a variety of Acute
I. INTRODUCTION Respiratory Disease syndromes, this group of viruses
II. RESPIRATORY AND CHILDHOOD FEVER VIRUSES
discussed in this module shares a ​relatively short incubation
A. Parvoviridae
B. Adenoviridae period (1-4 days) and a ​person-to-person mode of spread​.
C. Poxviridae Transmission is direct, by infectious droplet nuclei, or
D. Orthomyxoviridae indirect, by hand transfer of contaminated secretions to nasal
➔ Influenza A Virus or conjunctival epithelium. These respiratory viral agents are
E. Paramyxoviridae associated with an increased risk of bacterial superinfection
➔ Parainfluenza of the damaged tissue of the respiratory tract, and all have a
➔ Respiratory Syncytial Virus (RSV)
worldwide distribution.
➔ Mumps (Rubula) Virus
➔ Measles (Rubeola) Virus
➔ German Measles (Rubella) Virus (SIR ROBERT)
F. Coronaviridae ❏ Viral infections account for most of acute morbidity
➔ SARS-CoV associated with respiratory diseases
➔ SARS-CoV 2 ❏ Respiratory viruses are represented from different virus
➔ MERS-CoV families:
HIGHLIGHTING OF TOPICS BULLET PLACEMENT ➔ Parvoviridae
➔ Adenoviridae
AAAAAA​ - Family of virus ❏ (Main definition) ➔ Poxviridae
AAAAAA​ ​- Main topic ➔ (Sub-definition/Enumerate) ➔ Orthomyxoviridae
AAAAAA​ - Subtopics ● (Super Sub-detail) ➔ Paramyxoviridae and Rubella
➔ Coronaviridae
INTRODUCTION ❏ Transmission of respiratory viruses by ​droplet, nuclei, ​or
❏ Respiratory disease accounts for an estimated 75% to 80% hand transfer of contaminated secretions
of all acute morbidity and most of these illnesses
(approximately 80%) are viral infections. Although a majority
of the episodes may not require medical attention, the ​overall
average is three to four illnesses per year per person​.

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 RESPIRATORY AND CHILDHOOD FEVER VIRUSES
❏ Biopsy
PARVOVIRIDAE Portal of Entry Pharynx of the respiratory route

Mode of ❏ Droplets
GENERAL CHARACTERISTICS OF ​PARVOVIRIDAE
Transmission ❏ Blood
Family Parvoviridae ❏ Transplacental

Common Name Parvovirus Diseases ❏ Erythema infectiosum (fifth disease)

❏ Aplastic crisis in patients with chronic
Virus Parvovirus B-19 hemolytic anemia
❏ Fetal infection and stillbirth
Characteristics ❏ Single-stranded DNA virus
❏ Icosahedral capsid, no envelope Treatment Supportive
❏ Parvovirus B-19 is the ​only known human
pathogen Prevention Avoid contact
❏ Icosahedral
❏ 18-26 nm in diameter GENERAL CHARACTERISTICS
❏ 32 capsomeres (made up of proteins: ❏ Parvovirus came from a Latin word “​parvus​”, meaning
VP1 ​and ​VP2​)
small​. Parvoviruses are the ​smallest DNA animal viruses​.
Genome ❏ Single-stranded DNA It is widely distributed among warm blooded animals.
❏ Linear ❏ Parvovirus B19 represents the ​one human pathogen in the
family. Its replication in human cells is restricted to ​erythroid
Proteins ❏ VP1 (minor protein) progenitor ​cells, making ​adult bone marrow and fetal
❏ VP 2 (major protein)
liver​, the ​site of erythropoiesis during fetal development​, the
Envelope None major sites of viral replication​.
❏ Parvovirus B19 is pathogenic for humans and has tropism
Replication Nucleus for erythroid progenitor cells
➔ Globoside or ​Blood Group P Antigen (antigen on
Outstanding ❏ Environmentally stable
erythroid progenitor cells in which the Parvovirus B19
Characteristics ❏ Parvovirus has tropism for RBC
progenitor binds to)

Detection ❏ Serology/Antigen Detection

❏ PCR

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 STRUCTURE OF PARVOVIRUSES PARVOVIRUSES REPLICATION
- Parvovirus B19 encodes ​two capsid proteins (​VP1 and ​VP2​) ❏ Parvovirus B19 has a tropism for cells with group P antigen
that encapsulate a single stranded DNA molecule into (Globoside) receptors
icosahedral symmetry. ❏ Globoside ​is expressed on
- VP2 is the ​major capsid protein that comprises almost 90% ➔ Mature erythrocyte
of the virion capsid. ➔ Erythroid progenitors
➔ Megakaryocytes
➔ Endothelial cells
➔ Placenta
➔ Fetal liver
➔ Heart
❏ Replication occurs in the nucleus (because the ​RNA
polymerase of the host cell is in the nucleus so in order to
replicate the virus must be inside the nucleus)
❏ All DNA viruses replicate in the nucleus ​(except
poxviruses) because they need to be converted into mRNA
Figure 3.1​ Structure of Parvovirus (left) and Slapped Cheek Rash first.
caused by Parvovirus B19 ❏ All RNA viruses replicate in the cytoplasm (except
orthomyxoviruses & retroviruses) because it is where
ribosomes are found. The ribosomes are the ones
responsible for the translation of RNA into viral proteins.

PATHOGENESIS
GENERAL PATHOGENESIS:
❏ The virus is spread through the respiratory route. ​Pharynx
is the site of viral shedding for parvovirus B19 .
❏ The virus can be transmitted parenterally by blood
transfusions or by infected blood products (coagulation
PARVOVIRUSES CLASSIFICATION factors and immunoglobulin concentrates) and vertically from
There are two subfamilies of Parvoviridae: mother to fetus.
A. Parvovirinae ​- ​infect vertebrates; ​Human parvovirus B19 ​is ❏ Parvovirus B19 can survive in the coagulation factors and
the most common member of this subfamily immunoglobulin concentrates because it is naked, which
B. Densovirinae -​ i​nfect insects makes it resistant to harsh environments.

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 ❏ Occur throughout the year in all age groups and many B. ARTHRALGIA-ARTHRITIS
infections are subclinical. ❏ Joint involvement is prominent in adult cases. There
❏ Parvovirus causes a ​biphasic illness​ in humans. will be formation of antigen-antibody complexes.
a. FIRST PHASE - comprises fever, malaise, myalgia, These complexes will be deposited in the joints
and chills​, which corresponds to peak levels of virus causing inflammation and pain. Joint symptoms
destruction or erythroblasts. This phase when mild, mimic rheumatoid arthritis.
may be overlooked or considered ​nonspecific viral
disease. C. TRANSIENT APLASTIC CRISIS
b. SECOND PHASE - ​includes ​rash and arthralgia ❏ There will be transient aplastic crisis in ​patients with
occurring after the virus has disappeared but at a Hemolytic disorders which might lead to severe acute
time when parvovirus- specific antibody can be anemia. Remember that the receptor for parvovirus
detected, consistent with the conclusion that the rash B19 is present on the surface of RBCs.
is ​caused by immune complexes in the capillaries of
the skin. D. PURE RED CELL APLASIA
❏ Manifests in ​immunodeficient hosts which will lead to
DISEASES CAUSED: chronic anemia.
A. ERYTHEMA INFECTIOSUM (FIFTH DISEASE)
❏ Most common manifestation of human parvovirus E. HYDROPS FETALIS
B19 infection ❏ Happens when a pregnant woman is infected with
❏ Fifth disease because there are six other diseases parvovirus B19. The virus will cross the placenta and
that cause exanthems and Erythema infectiosum is the baby will develop fetal anemia.
the fifth.
❏ Most common manifestation of human parvovirus LABORATORY DIAGNOSIS
B19 infection. It is a characteristic cutaneous rash. ❏ Laboratory diagnosis is accomplished by using
❏ Most common in children of early school age and parvovirus-specific IgM or ​virus-specific IgG antibody
occasionally affects adults. Fever and mild rash testing with acute and convalescent sera​. Parvovirus
“​slapped-cheek​” appearance manifest in children. cannot be cultivated in usual cells available in clinical
❏ The appearance of ​erythematous facial rash and virology laboratories. PCR, serologic assays, and
lacelike rash on the limbs and trunks will ​appear 2 immunohistochemistry can be used.
weeks after the start of incubation​. ➔ PCR ​is the most sensitive among the three.
➔ IgM ​or ​cold antibody ​is seen in recent/acute
infection. It is the first antibody produced during
infection.

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 ➔ IgG ​or warm antibody indicates past infection and
❏ Fiber projects from each vertex
immunity.
➔ Immunohistochemistry ​can also be used to detect Composition 13% ​- DNA
parvovirus B19 antigens in fetal tissues and bone 87% ​- Protein
marrow.
Genome ❏ Double-stranded DNA
❏ Linear
TREATMENT, PREVENTION, AND CONTROL ❏ Protein bound to terminal portion
❏ Erythema Infectiosum (Fifth disease) and transient aplastic
crisis are treated symptomatically. There is no antiviral drug Proteins Important antigens (240 hexon, 12 penton
therapy parvovirus B19. base, fibers) are associated with the major
❏ Commercial immunoglobulin preparations contain outer capsid proteins
neutralizing antibodies to human parvovirus used for Envelope None
treatment and prevention of B19 infection.
❏ There is no vaccine against human parvovirus but there are Replication Nucleus
effective vaccines for use in cats, dogs and pigs.
❏ Good hygienic practices for prevention Outstanding Excellent models for molecular studies of
Characteristics eukaryotic cell processes

ADENOVIRIDAE Site of Latency Replication in oropharynx

Disease ❏ Pharyngitis
GENERAL CHARACTERISTICS OF ​ADENOVIRIDAE ❏ Pharyngoconjunctival fever
❏ Keratoconjunctivitis (pink eye)
Family Adenoviridae
❏ Pneumonia
Common Adenovirus ❏ Hemorrhagic cystitis
Name ❏ Disseminated disease
❏ Gastroenteritis in children
Virus Adenovirus
Diagnosis ❏ Cell culture (Hep-2 and other continuous
Characteristics ❏ Double-stranded DNA genome human epithelial lines)
❏ Icosahedral capsid ❏ EIA for gastroenteritis serotypes 40-41
❏ No envelope
❏ Approximately 50 human serotypes Treatment Supportive
❏ 70-90 nm in diameter
Prevention Vaccine (adenovirus serotypes 4 and 7)
❏ 252 capsomeres

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 KNOB DOMAIN
❏ Found at the end of a fiber
❏ detects the receptor of the adenovirus (integrins) and
bind to the host cell or the surface of epithelial cells

COXSACKIE-ADENOVIRUS RECEPTOR (CAR)

❏ Integrin - ​receptor where adenovirus binds
❏ found in epithelial cells
❏ provides and maintain tight junction integrity in epithelial
cells
Figure 3.2​ Virion structure of Adenovirus

GENERAL CHARACTERISTICS
❏ Adenovirus is a member of family Adenoviridae and the
genus Mastadenovirus. The virus has a linear
double-stranded DNA​, ​icosahedral symmetry​, and ​a size of
70-90 nm​.
❏ Adenoviruses were first isolated ​from human adenoid
tissues​. At present, ​approximately 50 serotypes of human
adenoviruses have been described; however, most disease
is associated with only one third of these types.
Adenoviruses cause less than 5% of all acute respiratory ADENOVIRIDAE ​CLASSIFICATION
disease in the general population. In addition, adenovirus ❏ Adenoviruses have been recovered from a wide variety of
serotypes 40 and 41 cause ​gastroenteritis in infants and species and grouped into​ five genera​.
young children. Other diseases occur but are less common. ❏ All of the human adenoviruses are classified in the
Mastadenovirus ​genus​.
PROPERTIES OF ​ADENOVIRIDAE ❏ Human adenoviruses are divided into ​seven ​groups (​A-G​)
❏ Adenovirus is a ​non-enveloped ​virus on the basis of their ​genetic​, ​physical​, ​chemical​, and ​biologic
❏ 252 capsomeres = 240 hexon and 12 penton base properties.
➔ Each penton base has a projected fiber
➔ The importance of the fiber is for the attachment of
the virus

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 ADENOVIRIDAE ​REPLICATION ❏ The name “​adenovirus​” reflects the ​recovery of the initial
❏ Adenoviruses can replicate and produce disease in the isolate from explanations of human adenoids​.
respiratory, gastrointestinal, and urinary tracts and in the ❏ Adenoviruses are responsible for about 5% of acute
eye. respiratory disease in young children, but they account for
❏ Adenovirus only replicates well in cells of epithelial origin and much less in adults.
inside the nucleus.
❏ Adenoviruses infect and replicate in epithelial cells of the
respiratory tract, eye, gastrointestinal tract, and urinary tract.
❏ They usually do not spread beyond the regional lymph
nodes.

PATHOGENESIS
❏ Adenoviruses are spread by:
➔ Direct Contact
➔ Fecal-oral route
➔ Respiratory droplets (replicates in the epithelial cells) NOTE:
➔ Contaminated fomites ❏ Bold numbers - serotypes that causes outbreak in the
❏ About ​one-third ​of the known human serotypes are specific syndromes
commonly associated with ​human illness. ➔ Childhood febrile illness - 3, 7a
❏ It should be noted that a single serotype may cause different ➔ Pneumonia - 3, 5, 7a, 7b, 14a
clinical diseases and, conversely, that more than one type ➔ Pertussis-like - 3, 19
may cause the same clinical illness. ➔ Keratoconjunctivitis - 3, 19
❏ Most infections are mild and self-limited. ❏ Pharyngoconjunctival fever - swimming pool conjunctivitis
❏ The viruses occasionally cause disease in other organs, ❏ Conjunctivitis/ Keratoconjunctivitis - looks like sore eyes;
particularly the eye and the gastrointestinal tract. highly contagious
❏ Many adenovirus infections are subclinical, and viruses may
LABORATORY DIAGNOSIS
persist in the host for months.
❏ Specimens to be collected include ​throat swabs, nasal
❏ Group C viruses persist as latent infections for years in
washings, conjunctival swabs or scrapings, or feces​.
adenoids and tonsils and are shed in the feces for many
Laboratory diagnosis is accomplished by conventional cell
months after the initial infection.
culture using ​HEp-2 cells​ and​ serologic methods​.
(Adenoids - secondary lymphoid organs present behind
nasal cavity)

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A. DETECTION, ISOLATION, AND IDENTIFICATION OF ❏ Adenovirus (​naked​ ​capsid​) is relatively ​resistant to
VIRUS disinfectants​ and more stable than other enveloped
❏ Adenovirus may be recovered from stool, urine, respiratory viruses
throat, conjunctiva or rectum:
➔ Virus isolation in cell cultures TREATMENT, PREVENTION AND CONTROL
➔ Immunofluorescence ❏ There is no specific treatment for adenoviruses infection
➔ Hemagglutination-inhibition and Neutralization ➔ Adenovirus is self-limiting therefore the immune
tests system of an immunocompetent individual can
➔ Cell staining handle the infection)
➔ PCR (most sensitive) - for DNA virus ❏ Ways to prevent Adenovirus infections:
➔ RT-PCR - for mRNA virus ➔ Careful handwashing
➔ Electron microscopy ➔ DIsinfection of environmental surfaces
➔ ELISA ➔ Chlorination of swimming pools
➔ Latex-Agglutination Test ➔ Adequate sterilization of ophthalmic equipment
(adenoviruses can infect the eyes)
B. SEROLOGICAL TESTS ❏ Attempts to control adenovirus infections in the military have
❏ Complement fixing antibodies (least specific, least focused on vaccines.
sensitive) ➔ Live adenovirus vaccine containing types 4 and 7,
❏ Hemagglutination-inhibition tests (more specific, encased in gelatin-coated capsules and ​given orally,
more sensitive) was introduced in 1971.
❏ Neutralization Tests (most specific, most sensitivity) ➔ Virus first replicates in the respiratory tract. The
vaccine is given orally to bypass the replication of
EPIDEMIOLOGY virus and for it to be killed immediately by the
❏ Adenoviruses exist in all parts of the world immune system)
❏ They are present year-round and usually do not cause ❏ The vaccine proved highly effective but was discontinued in
community outbreaks of disease 1999, and was reapproved in 2011 for US military personnel
❏ Infections with Types 1,2,5 and 6 occur chiefly during the only.
first years of life
❏ Infections with Types 3 and 7 are contracted during school
years (take note of this)
❏ Infections with types 4, 8, and 19 are encountered during
adulthood

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 POXVIRIDAE
❏ Virus encoded proteins to help evade host's
immune defense system
GENERAL CHARACTERISTICS OF ​POXVIRUSES ❏ Smallpox was the first viral disease in the
world
Family Poxviridae
Transmission Respiratory droplets (smallpox); direct contact
Common Name Poxvirus (molluscum and orf)

Virus ❏ Small, molluscum contagiosum, and orf Disease All disease of the skin; smallpox is a
virus generalized infection with pustular rash
❏ Complex structure (10-25% fatal); molluscum manifests as benign
❏ Oval or brick shaped nodules of skin; orf manifests as localized
❏ 300-400 nm in length papules/vesicles of the skin
❏ 230 nm in diameter
Detection Electron Microscopy - skin lesion material
Characteristics ❏ Largest and most complex of all viruses
❏ Brick-shaped virion with nonconforming Epidemiology: Smallpox eradicated from world in 1977;
symmetry reffered to as complex smallpox and molluscum and limited to
❏ Double-stranded DNA genome humans; orf is zoonotic

Composition 3%​ - DNA Treatment: Supportive

90%​ - Proteins
5%​ - Lipids Prevention: Vaccine for smallpox.
Avoid contact for others.
Genome ❏ Double-stranded DNA
❏ Linear

Proteins ❏ Contains >100 polypeptides

❏ Many enzymes are present in core,
including transcriptional system

Envelope Formation of multiple membranes

Replication Cytoplasmic factories

Outstanding ❏ Large and complex viruses

characteristics ❏ Very resistant to inactivation

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 GENERAL CHARACTERISTICS ❏ Pox virus replicates in the cytoplasm because it can make its
❏ The ​Poxviruses are the largest and most complex of all the own DNA and RNA polymerase. DNA viruses only enter the
viruses, with a ​size of 225 x 300 nm​. These are nucleus because RNA polymerase is only present in the
DNA-containing viruses that are ​enveloped with complex nucleus of the host’s cell.
coats with brick shape​ and ​complex morphology​.
❏ The Family includes ​variola​, the ​smallpox virus​, and STRUCTURE OF THE ​POXVIRUSES
vaccinia virus​, as well as the ​agents of cowpox,
monkeypox and canarypox​.

POXVIRUS REPLICATION

NOTE:
❏ Poxviridae that infects vertebrates are classified into 8
genera
❏ Among these 8 genus, only 4 genus can cause diseases to
humans.

PROPERTIES OF ​POXVIRUSES
❏ All DNA viruses replicate in the nucleus except for Pox
viruses
❏ It is unique since it contains an enzyme in its core which can
produce its own RNA

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 PATHOGENESIS ❏ The virus spreads through the capillaries to the skin followed
❏ Diseases caused by poxviruses include ​smallpox​, ; again by a viral replication and evolution of rash
molluscum​ ​contagiosum​,; and ​orf​, . ❏ By the virus further spread cell-to-cell or through the mid and
A. VARIOLA VIRUS (SMALLPOX) basal layers of the skin causing necrosis and pustules
❏ a devastating and frequently fatal disease of (pantal of lesions)
historical importance (eliminated from the world in ❏ Later, pustules breakdown and will form crusts and
1980) discharge the virus into the environment of the smallpox
❏ Deadly; Highly contagious and can survive well in the patient
extracellular environment.
❏ In 1067 the WHO) launched an ambitious program to
eradicate smallpox because of two main reasons:
➔ Humans are the only reservoir of smallpox.
➔ Asymptomatic carriage apparently did not
occur
❏ Global eradication of Smallpox was confirmed in
1979 and accepted by WHO in May 1980. Since
then, the virus has been solely secured in two
WHO-restricted laboratories: ​USA​ and ​Russia
❏ 1st virus to be eradicated

MECHANISM OF VARIOLA VIRUS

❏ Infections with most pox viruses are characterized by a rash,
although lesions induced by some members of the family are
markedly proliferative.
❏ The virus enters the mucous membranes of the upper
respiratory tract through inhalation followed by viral
replication at the site of entry and infection of mononuclear
phagocytic cells in regional lymph nodes.
❏ Viremia allows the virus be transported to the liver, spleen
and other tissues
❏ At the end of the incubation period (10 to 14 days)
inflammatory mediators are released causing fever and other
symptoms.

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 B. MOLLUSCUM CONTAGIOSUM ❏ Antigenic cross - reactivity was so much that ​Variola was
❏ an ​exclusively human disease of the skin that is able to eradicate variola globally
self-limited but may recur during periods of severe ❏ Vaccinia virus
immunosuppression ❏ Pathogenic to animals
❏ Non-pathogenic to humans
C. ORF ❏ Used for smallpox vaccine
❏ a localized infection of the skin caused by viruses
responsible for ​dermatitis in sheep, goats, and B. COWPOX VACCINE
related animals​ (zoonotic) ❏ Discovered by Edward Jener (the father of
Vaccination) was in use before vaccinia was
LABORATORY DIAGNOSIS OF VARIOLA VIRUS available
❏ Direct examination of material from skin lesions NOTE:
❏ Cell culture can be used for virus isolation ❏ Cowpox vaccine was replaced with live vaccinia vaccine
❏ Viral DNA detection via PCR because the vaccinia vaccine had less severe effects and
❏ Identification of antigen from lesions was more effective.
❏ Demonstration of antibodies in the blood
C. VARIOLATION
VACCINATION ❏ Was the first attempt of providing artificial immunity
A. LIVE VACCINIA VACCINE against Smallpox it was in use even before the
❏ Was​ highly effective cowpox vaccine was available
❏ Used live Vaccinia Virus
❏ it was given as single dose between one and two ADDITIONAL INFO:
years of age ❏ Since the Variola virus (pox viruses) is stable in the outside
❏ as an un-attenuated live virus was used adverse environment, they have ​high potential for bioterrorism
reactions were common; such as mild vaccinia
induced rashes
❏ Eradicated smallpox
NOTE:
❏ The mechanism behind the vaccine was Cross-immunity
because Vaccinia virus is serologically related to smallpox,
although its exact origin is unknown
❏ Vaccinia cross-reacts with variola and the antibodies
produced against vaccinia are protective against variola.

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 ORTHOMYXOVIRIDAE
❏ Influenza causes worldwide epidemics

GENERAL CHARACTERISTICS OF ​ORTHOMYXOVIRIDAE INFLUENZA A INFLUENZA B

Family Orthomyxoviridae
Transmission Contact with respiratory secretions
Common Name Orthomyxovirus
Disease ❏ Influenza (malaise, Similar to mild
Characteristics ❏ Segmented (eight separate molecules) headache, myalgia, influenza
❏ Single-stranded RNA genome cough)
❏ Spherical (Because of the presence of the ❏ primary Influenza
envelope) pneumonia
❏ Helical nucleocapsid, 9nm ❏ in children, croup,
❏ Three major antigenic types: Influenza A, B, bronchiolitis, and
and C. otitis media
➔ Type A and B cause nearly all human
disease. Detection Cell culture (PMK), EIA, FA stain
Composition ❏ RNA (1%) Epidemiology Viral subtypes based Antigenic drift only​,
❏ Protein (73%) on ​hemagglutinin and resulting in local
❏ Lipid (20%) neuraminidase outbreaks every 1-3
❏ Carbohydrate(6%) glycoproteins years
abbreviated “H” and
Genome ❏ Single stranded RNA “N”, respectively (e.g.,
❏ Segmented (eight molecules) H1N1 or H3N2); infects
❏ Negative-sense humans and other
animals;
Proteins ❏ Nine structural proteins, ❏ Antigenic drift​,
❏ One non-structural
resulting in minor
Envelope Contains viral ​hemagglutinin and antigenic change,
neuraminidase​ proteins causes local
outbreaks of
Replication Nuclear transcription influenza every 1-3
years;
Outstanding ❏ Genetic reassortment common among ❏ Antigenic shift​,
Characteristics members of the same genus resulting in major
antigenic change,

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 GENERAL CHARACTERISTICS
causes periodic
worldwide ❏ Causative agents of Flu (trangkaso)
outbreaks ❏ Influenza viruses (​A, B, ​and ​C​)
❏ The orthomyxoviridae (influenza viruses) are a major
Treatment ❏ Supportive ❏ Supportive determinant of morbidity and mortality caused by respiratory
❏ Antivirals ❏ Zanamivir and disease and outbreaks of infection sometimes occur in
❏ Amantadine and Oseltamivir
worldwide epidemics
rimantadine
(Influenza A only) ❏ The viruses contain ​positive sense, single-stranded RNA and
❏ Zanamivir and helical symmetry and are ​enveloped​. Influenza viruses range
Oseltamivir for in size from: 80-120 nm.
influenza A and B ❏ Their structures transform that is why yearly you can be
infected with the flu
❏ Influenza has been responsible for millions of deaths
Prevention Influenza vaccine or antiviral prophylaxis
worldwide
NOTE:
“​H or HA​” = Hemagglutinin STRUCTURE OF THE ​ORTHOMYXOVIRIDAE
“​N​” = Neuraminidase ❏ Because of the segmented nature of the viral genome (​eight
segments​), when a cell is co-infected of two strains of
Orthomyxoviruses, there is a possibility of recombination
❏ Influenza viruses are divided into three types: A, B, C based
on ​antigenic differences in their ​ribonucleoprotein (NP) and
matrix (M) protein antigens
❏ Influenza A virus has the greatest virulence and
predominance in epidemic spreads, because of their ability
to undergo genetic changes and existence in several species

Table 3.4​ Virion Structure of Orthomyxoviruses

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FEATURE INFLUENZA A INFLUENZA B INFLUENZA C RNA

Gene 8 8 7 6 NA ​- Neuraminidase Virus release from

segments infected cells

Unique M2 NB HEF 7 M1​,​M2 - matrix ​proteins Matrix, ion channel

proteins
8 NS1​,​NS2​ Nonstructural NS1 in interferon
Host range Humans,swine, Humans, seals Humans, swine proteins antagonist
avians,
equines,marine
mammals, bats NOTE:
❏ Segment 6​ of RNA is not present in INFLUENZA B
Disease Often severe Occasionally Usually mild ❏ RNA 1,2,3​ produces ​RNA Polymerase
severity severe
IMPORTANCE OF HEMAGGLUTININ (HA)
Epidemic Extensive, Outbreaks, Limited
potential epidemics and occasionally outbreaks ❏ Hemagglutinin attach to ​N-Acetylneuraminic (sialic acid)
pandemics epidemics (antigenic drift acid-only containing glycoprotein or glycolipid receptor
(antigenic drifts (antigenic drift only) sites ​on human respiratory cell surfaces, which is critical first
and shifts) only) step in initiating infection of the cell
❏ HA also promotes the fusion of the viral envelope to the host
cell membrane and can hemagglutinate human, chicken, and
RNA PROTEINS FUNCTION guinea pig red blood cells. HA also elicits the neutralizing
SEGMENT
antibody response.
1 PB2 ​- Polymerase RNA synthesis, virulence ❏ Influenza viruses display cell tropism towards respiratory
component cells, because respiratory cells contain sialic acid
(N-Acetylneuraminic acid)
2 PB1- ​polymerase RNA synthesis
component
IMPORTANCE OF NEURAMINIDASE
3 PA ​- Polymerase RNA synthesis ❏ Neuraminidase promotes a smooth passage for the virus in
component respiratory tract by inactivating mucoprotein receptors in
respiratory secretions
4 HA ​- Hemagglutinin Viral attachment ❏ Neuraminidase destroys viral receptors, thus preventing
5 NP ​- Nucleocapsid RNA synthesis, binds to aggregation and superinfection in infected cells

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 ❏ NA permits the virus ​entry into the host cel​l and also ​cleaves Example : A/Goose/Guangdong/1/1997 (H5N1)
the sialic acid on the viral particle (uncoating), which helps to ❏ If the infected is human, no need to put the description of the
release the virus from the host cells. infected animal.
NOTE: ❏ H5N1​- Bird Flu
❏ Neuraminidase will destroy the sialic acid to allow the
release of the influenza virus outside the host cell. PROPERTIES OF ​ORTHOMYXOVIRIDAE
❏ The ​Orthomyxoviridae family includes the ​influenza (A, B
ORTHOMYXOVIRUS REPLICATION and C) viruses​. Important characteristics of the family
❏ Orthomyxoviridae are RNA viruses, but they replicate inside includes the presence of ​hemagglutinin antigen (HA) and
the nucleus because they first need to steal the ​Cap neuraminidase antigen (NA)​.
sequences of the host’s mRNA and transfer it to their own ❏ Mutability and high frequency of genetic reassortment and
mRNA for replication of viral mRNA. resultant antigenic changes in the viral surface glycoproteins
❏ Once the virus obtains the cap sequence, it then goes to the make influenza viruses from edible challenges for control
cytoplasm to replicate. efforts

ORTHOMYXOVIRIDAE NOMENCLATURE ANTIGENIC DRIFT

IN ORDER: ❏ Caused by mutation during genome replication, results in
1. Antigenic type minor antigenic change and relatively mild influenza
2. Infected animal outbreaks every 1-3 years.
3. Geographic site of isolation ❏ Gene recombination occurs when influenza viruses
4. strain # re-assort, accumulation of a series of ​minor genetic
5. Year of isolation mutations​, HA antigen changes change.
6. (Antigenic description H-Hemagglutinin and
N-Neuraminidase)

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ANTIGENIC SHIFT
❏ caused by reassortment or mixing of the segmented viral
genome during co-infection in nonhuman animals, results in
major antigenic change and periodic worldwide outbreaks
(pandemics). Co-infection occurs when a human virus infects
a cell at the same time as an animal influenza virus.
❏ Genetic change that enables a flu strain to jump from one
animal virus to another which allows gene recombination
making a​ new strain (yearly) ​.

INFLUENZA A VIRUS
INFLUENZA A VIRUS
❏ Influenza A virus genome has eight negative sense RNA
segments each encoding at least one protein
❏ A unique aspect of this virus is their ability to ​develop a wide
variety of subtypes through the process of ​mutation and
Whole-gene swapping​ between strains called​ reassortment
❏ Mutation ​(​antigenic drift​) and ​reassortment ​(​antigenic
shift​) which produces antigenic changes in the virus
➔ That is why yearly, you can be infected by influenza
virus. Because no two viruses are the same
❏ 18 recognized subtypes of Hemagglutinin (HA) and 11
Neuraminidase (N) subtypes are known to exist among
influenza A viruses
❏ Three subtypes of H (H1,H2, and H3) and two subtypes of
N(N1 and N2) exist in humans
➔ This is why we are not infected by other influenza
❏ These subtypes are designated according to the H and N
antigens on their surface (eg, H1N1 , H3N2)

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 ❏ Subtle changes known as Antigenic drift (mutation) occurs in LABORATORY DIAGNOSIS OF INFLUENZA A VIRUS
all strains, whereas drastic changes as antigenic shift ❏ Specimens collected for identification of influenza virus
(reassortment) occurs when two closely related strains of include throat swabs and nasal aspirates or nasopharyngeal
influenza A infect the same cell swabs. Influenza virus disease is detected in the laboratory
❏ Antigenic drift occurs every year to few years with influenza by ​cell culture, fluorescent antibody (FA) staining or
A viruses, whereas antigenic shift occurs abruptly and other antigen detection methods, serologic testing and
unpredictably PCR​.
❏ Nasopharyngeal swabs and nasal aspirate or lavage fluid
PANDEMICS CAUSED BY INFLUENZA A are the best specimens for diagnostic testing and should be
A. SWINE INFLUENZA obtained within 3 days after the onset of symptoms.
❏ An infectious viral disease of the swine ➔ RT-PCR
❏ Swine viruses such as A(H1N1) and A(H3N2) have ➔ Isolation and Identification of Influenza virus using
caused serious infections in humans cell cultures and inoculation in embryonated egg
➔ Serological method for detection of influenza virus
B. AVIAN INFLUENZA protein antibodies
❏ Commonly called “​Bird​ ​Flu​”
❏ An infectious viral disease of birds EPIDEMIOLOGY OF INFLUENZA A VIRUS
❏ Most avian influenza viruses do not infect humans ❏ Humans are the major hosts of the influenza viruss, and
❏ A(H5N1) is a highly pathogenic avian influenza A severe respiratory disease is the primary manifestation of
virus infection.
❏ Influenza occur worldwide and cause annual outbreaks
INFLUENZA A TARGETS especially during cold months
❏ H1N1 (human) and H5N1 (avian) target different regions of ❏ Every 10-40 years, a new subtype of influenza A appears to
the ​respiratory tract cause pandemics.
❏ Receptors for ​H1N1​ ​(human)​ are dominant in the ​upper​ part
of the respiratory tract
❏ H5N1​ ​(avian)​ receptors are found in the​ lower ​portion of the
lung in humans
❏ H1N1 (swine)​ interacts with both receptors in the​ upper and
lower​ respiratory tract

NOTE: ​H5N1 (avian)​ is rare due to the position of receptors (​lower

portion of the lungs)

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 ❏ Lipid (20%)
TREATMENT ❏ Carbohydrates (6%)

Proteins 6-8 structural proteins

Envelope ❏ Contains​ viral glycoprotein G​ (RSV),

❏ H ​(measles) or ​HN ​(paramyxovirus & mumps)
➔ for viral attachment which sometimes carries
hemagglutinin or neuraminidase activity)
NOTE: ​Amantadine ​and ​Rimantadine ​are ​not currently ❏ Fusion (f) glycoproteins
recommended for use due to ​resistance ➔ causes syncytia / multinucleated cell
➔ very fragile

PREVENTION AND CONTROL Replication Cytoplasm; particles bud from plasma membrane
❏ The best available control method of controlling influenza NOTE:​ All RNA viruses replicates in the cytoplasm
infections is to ​annually vaccinate all people aged 6 months except Orthomyxoviridae and Reoviridae
and older. Outstanding ❏ Antigenically stable (not capable of antigenic shift &
❏ Studies shown that handwashing with soap and water of the Characteristics drift)
use of alcohol-based hand rubs is highly effective at ❏ Particles are labile yet highly infectious
reducing the amount of virus in hands
Respiratory
Measles Virus Mumps Parainfluenza Syncytial
PARAMYXOVIRIDAE Virus

Transmission
GENERAL CHARACTERISTICS OF ​PARAMYXOVIRIDAE
Contact with Person-to-person Contact with Person-to-pers
Family Paramyxoviridae respiratory contact, respiratory on by hand and
secretions; presumably secretions respiratory
Common Name Paramyxovirus extremely respiratory contact
contagious droplets
Characteristics ❏ Single stranded
❏ RNA genome Disease
❏ Linear
❏ Helical capsid with envelope Measles, atypical Mumps Adults: upper Primarily in
❏ No segmented genomes like orthomyxoviruses measles (occurs respiratory, infants and
❏ Negative sense in those with rarely children.
waning pneumonia Infants:
Composition ❏ RNA (1%) “vaccine”, Children: bronchiolitis,
❏ Protein (73%) respiratory pneumonia and

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 and subacute including croup, croup. transmission
sclerosing bronchiolitis, Children: upper with isolation
panencephalitis and pneumonia respiratory and cohorting.

Detection
GENERAL CHARACTERISTICS
Cell culture Cell Culture Cell culture Cell culture ❏ The ​Paramyxoviruses include the genera ​Paramyxovirus,
(PMK) and (PMK) and (PMK), shell (Hep-2 cells),
Morbillivirus​, and ​Pneumovirus​. The family possesses
serology serology viral culture, EIA, and FA
and FA stain stain negative sense​, ​single stranded RNA​, ​helical symmetry​, an
Four serotypes, envelope​, and an average size of 150 – 300 nm.
disease occurs Disease occurs ❏ Paramyxoviruses do not have segmented genomes, and
year-round annually late
therefore do not undergo antigenic shift like the
fall through
early spring; orthomyxoviruses. The ​Morbillivirus includes ​rubeola which
nosocomial causes measles. The Genus ​Pneumovirus includes the
transmission respiratory syncytial virus (RSV​).
can occur
readily
❏ The paramyxoviruses include the most important agents of
respiratory infections of ​infants and young children (RSV &
Treatment Parainfluenza Virus) as well as the causative agents of two
of the most common contagious diseases of childhood
Supportive; Supportive Supportive Supportive;
immunocompromi treat severe (Mumps & Measles).
sed patients can disease in ❏ All members of the Paramyxoviridae family initiate infection
be treated with compromised via the respiratory tract. Whereas replication of the
immune serum infants with
respiratory pathogen is limited to the ​respiratory epithelia​,
globulin ribavirin
measles and mumps become disseminated throughout the
Prevention body and produce generalized disease.
❏ Laboratory detection is performed using ​cell culture with
Prevention: Mumps Vaccine Avoid contact Avoid contact
Measles vaccine with virus with viruses.
hemadsorption, FA staining​, or​ enzyme immunoassay​.
Immune
globulin for PARAMYXOVIRIDAE CLASSIFICATION
infants with ❏ The Paramyxoviridae family is divided into two subfamilies
underlying lung
disease; and seven genera, six of which contain human pathogens
prevent ❏ Most of the members are monotypic
nosocomial

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CLASSIFICATION: PARAINFLUENZA
❏ Parainfluenza viruses are ubiquitous and cause common
PARAMYXOVIRINAE
respiratory illness in persons of all ages.
Respirovirus Rubulavirus Morbillivirus Henipavirus ❏ They are major pathogens of severe respiratory tract disease
in ​infants and young children​ (​parainfluenza 1 & 3​)
Parainfluenza Mumps Measles Hendra ❏ Reinfections with parainfluenza viruses are common (due to
1 and 3
many serotype)
Parainfluenza Nipah
2, 4a,and 4b
PATHOGENESIS
❏ Parainfluenza virus replication in the immunocompetent host
PNUEMOVIRINAE appears to be limited to respiratory epithelia
❏ The infection may involve only the nose and throat, resulting
Pneumovirus Metapneumovirus in a “common cold” syndrome
Respiratory Syncytial Human Metapneumovirus ❏ The production of ​virus-specific IgE antibodies during
Virus (RSV) primary infections has been associated with ​disease
severity
❏ The mechanism may involve release of mediators of
REPLICATION OF PARAMYXOVIRIDAE
inflammation that alter airway function

TYPES OF PARAINFLUENZA
A. Type 1 & 2
❏ May involve the ​larynx and the upper trachea​,
resulting in croup (​laryngotracheobronchitis​)
❏ Croup ​is characterized by ​respiratory obstruction
caused by ​swelling of the larynx and ​related
structures
❏ Croup is more likely to occur in older children
between 6-18 months of age

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 EPIDEMIOLOGY
❏ Parainfluenza viruses are widely distributed geographically
❏ Parainfluenza viruses are a major cause of lower respiratory
tract disease in young children
❏ Parainfluenza viruses are spread by:
➔ Direct person to person contact
➔ Large droplet aerosols

TREATMENT , PREVENTION, AND CONTROL

❏ Contact isolation precaution
❏ Proper hand hygiene
B. Type 3 ❏ Ribavirin has been used with some benefit in treatment of
❏ May spread deeper to the lower trachea and the immunocompromised patients with lower respiratory tract
ronchi to become pneumonia or bronchiolitis disease
❏ No vaccine available
C. Type 4
❏ Does not cause serious disease even on the first RESPIRATORY SYNCYTIAL VIRUS (PNEUMOVIRUS)
infection ❏ RSV is the single most important agent of ​bronchiolitis and
pneumonia​ in infants younger than 1 year of age
LABORATORY DIAGNOSIS ❏ RSV replication occurs initially in epithelial cell of the
❏ Nucleic acid amplification tests RT-PCR are the preferred nasopharynx
diagnostic methods because of their sensitivity and ❏ Virus may spread into lower respiratory tract and cause
specificity​, their ability to ​detect a broad range of viruses​, bronchiolitis and pneumonia
and the ​rapidity of results​. ❏ Viral antigen can be detected in the upper respiratory tract
❏ Antigen detection methods are also useful for rapid and in shed epithelial cells
diagnosis.
❏ A ​continuous monkey kidney cell line​, ​LLC-MK2​, is NOTE:
suitable for isolation of parainfluenza viruses (multinucleated ❏ Respiratory syncytial virus (RSV) ​contains a surface
giant cell appearance in cultured cells). protein called ​F (fusion) protein​.
❏ Antibody responses can be measured using neutralization, ❏ F PROTEIN
hemagglutination-inhibition (HI), or enzyme-linked ➔ mediates host cell fusion into ​syncytial cells​, which
immunosorbent assay (ELISA) tests​. are the hallmark of RSV infections. RSV disease in

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 newborns with underlying medical conditions, TREATMENT, PREVENTION, AND CONTROL
especially premature children with developing lung ❏ The antiviral drug ​ribavirin ​is approved for treatment of
functions, can be prophylaxed with RSV immune lower respiratory tract disease caused by RSV
serum that prevents severe RSV bronchiolitis during ❏ No vaccine available
susceptible early months of life. Except for RSV, the ❏ Contact isolation precaution
paramyxoviruses hemasorb guinea pig red blood ❏ Proper hand hygiene
cells. ❏ Supportive care

LABORATORY DIAGNOSIS MUMPS (RUBULA) VIRUS

❏ RSV differs from other Paramyxoviruses as ​RSC does not ❏ An acute contagious disease characterized ​by
have hemagglutinin nonsuppurative enlargement of one or both salivary glands
➔ Diagnostic methods for RSV infections cannot use ❏ Mostly causes a mild childhood disease, but in adults
hemagglutinin or hemadsorption complications incuding erningit6ius andf orchtitis are fairly
➔ Antigen detection common
➔ Isolation and identification of RSV from nasal ❏ Causes parotitis, a painful infection of the ​parotid glands
secretions on human HeLa and Hep-2 cell lines characterized by swelling behind the ears and difficulty of
➔ Detection of viral RNA using RT-PCR swallowing. Mumps virus possesses both HA ​and NA
antigens and a hemolysin Secondary target is gonads but it
EPIDEMIOLOGY can cause sterility.
❏ Distributed worldwide and is recognized as the major ❏ Humans are the only natural hosts for mumps virus. Primary
pediatric respiratory tract pathogen replication occurs in nasal or upper respiratory tract epithelial
❏ About 70% infants are infected by age 1 and almost all by cells
AGE 2 years ❏ Viremia then disseminates the virus to the salivary glands
❏ The virus can be isolated from most infants younger than and other major organ systems
age 6 months with bronchiolitis, but it is almost never
isolated from healthy infants
❏ Subgroup A infections appear to cause more severe illness
than subgroup B infections
❏ Most common cause of viral pneumonia in children younger
that age 5 years but may also cause pneumonia in elderly
adults or in immunocompromised persons

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 IMMUNITY
❏ Immunity is ​permanent​ after single infection
❏ There is only one antigenic type of mumps virus and it does
not exhibit significant antigens variation
❏ Antibodies to the HN glycoprotein, the F glycoprotein, and
the nucleocapsid protein (NP) develop in serum after natural
infection
❏ Antibodies to the NP proteins appear earliest (3-7 days after
the onset of clinical symptoms) but are transient and are
usually gone within 6 months
❏ Antibodies to HN antigen develop more slowly (~4 weeks
after onset) but persist for years

MUMPS VIRUS COMPLICATION WITHOUT PAROTITIS LABORATORY DIAGNOSIS

❏ The diagnosis of typical cases usually can be made on the
Meningitis Approximately 10% of all infected patients basis of clinical findings
developed meningitis. It is usually mild, but ➔ RT-PCR is a very sensitive method that can detect
can be confused with bacterial meningitis mumps genome sequence in clinical samples
Encephalitis Encephalitis is occasionally severe ➔ Isolation and identification of Mumps virus from
saliva, CSF, and urine using monkey kidney cell
Pancreatitis Suggested by upper abdominal pain, nausea cultures
and vomiting ➔ Serology

Orchitis Orchitis (inflammation of the testes) is

estimated to occur in 10% to 20 % of infected EPIDEMIOLOGY
men, which could be unilateral or bilateral in ❏ High frequency of mumps in 5 to 15 years age group
post-pubertal men. Although subsequent ❏ Person-to-person transmission via respiratory route
sterility is a concern,it appears that this ❏ High infectivity is resent 7 days before and 9 days after onset
outcome is rare. of illness
❏ Replicate in the upper respiratory tract epithelium and local
Oophoritis Oophoritis (inflammation of ovaries) is an
lymph nodes
unusual, usually benign, inflammation of the
ovarian glands

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 MEASLES (RUBEOLA) VIRUS ❏ Isolation and identification of Measles virus from
❏ An acute, highly infectious disease characterized by fever nasopharyngeal and conjunctiva swabs, bloods, respiratory
respiratory symptoms and a maculopapular rash secretions,and urine collected during febrile phase on
❏ possesses hemagglutinin antigen (HA) and a hemolysin but monkey , human, or B95
does not possess neuraminidase antigen​.
❏ Humans are the only natural hosts for measles virus, IMMUNITY
although numerous other species, including monkeys, dogs ❏ Only one antigenic type of measles virus
and mice can be experimentally infected. ❏ Infection confers lifelong immunity
❏ Causative agent of first disease
EPIDEMIOLOGY
PATHOGENESIS ❏ The key epidemiologic features of measle are as follow:
❏ The virus gains access to the human body via the respiratory ➔ The virus is highly contagious
tract, where it multiplies locally; the infection then spread to ➔ There is a single serotype
the regional lymphoid tissue, where further multiplication ➔ There is no animal reservoir
occurs ➔ Inapparent infections are rare
❏ Primary viremia disseminates the virus, which then replicates ➔ Infection confers lifelong immunity
in the reticuloendothelial system ❏ Transmission occurs predominantly via the respiratory route
❏ Finally, a secondary viremia seeds the epithelial surface of (by inhalation of large droplets of infected secretions)
the body, including the skin respiratory tract , and the
conjunctiva , where focal replication occurs. TREATMENT, PREVENTION, AND CONTROL
❏ Live attenuated rubella vaccine (​MR​)
❏ Live attenuated rubella and mumps vaccines (​MMR​)
❏ Live attenuated varicella vaccine (​MMRV​)

GERMAN MEASLES (RUBELLA) VIRUS

❏ Rubella (German measles; 3-day measles) is an acute
febrile illness characterized by a rash and lymphadenopathy
that affects children and young adults
LABORATORY DIAGNOSIS ❏ It is the mildest of common viral exanthems
❏ Detection of Measle antigen in epithelial cells from ❏ However infection during early pregnancy may result in
respiratory secretions, the nasopharynx , conjunctiva , and serious abnormalities of the fetus, including congenital
urine in Serology malformations and mental retardation
❏ Detection of antibodies to Measles nucleoproteins ❏ Causative agent for ​third disease

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NOTE:
VIRAL EXANTHEMS
➔ 1st disease : ​Measles
➔ 2nd disease: ​Scarlet Fever (​Streptococcus pyogenes​)
➔ 3rd disease: ​German measles (Rubella)
➔ 4th disease: ​Staphylococcus aureus
➔ 5th disease:​ Parvovirus B-19
➔ 6th disease : ​Herpes NOTE: ​ ​E1​ - initiation of replication

CLASSIFICATION OF RUBELLA VIRUS RUBELLA VIRUS REPLICATION

❏ A member of the ​Togaviridae family​, is the sole member of
the genus Rubivirus
❏ It is simple, icosahedral, enveloped virus, and contains a
single-stranded , positive-sense RNA genome
❏ Although its morphologic features and physicochemical
properties place it in the togavirus group, rubella is not
transmitted by arthropods.

STRUCTURE OF RUBELLA VIRUS

❏ There is a single species of capsid protein, and the lipid
bilayer envelope contains two glycoproteins -- E1 and E2 NOTE:
❏ E1 ​interacts with the receptor on the host cell and comprises ➔ Replicates in cytoplasms
the principal antigenic determinants or epitopes involved in ➔ Envelope of RNA virus is from plasma membrane
virus neutralization and hemagglutination.
❏ E2 interacts with capsid and E1 to reach the Golgi apparatus CLINICAL INFECTION
for viral assembly. ❏ German measles infection are classified as either:
1. Postnatal Rubella
2. Congenital Rubella Syndrome (CRS)
3. Progressive Rubella Panencephalitis (20 yrs old
above)

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PATHOGENESIS LABORATORY DIAGNOSIS:
A. Postnatal Rubella ➔ RT-PCR
❏ The virus enters the host through the upper ➔ ELISA
respiratory tract, replicartes, and then spreads by the
bloodstream to distant sites, including lymphoid EPIDEMIOLOGY
tissue , skin, and organs ❏ Rubella Virus has ​high infectivity but low virulence
❏ A major focus of concern is susceptible women of
childbearing age, who carry a risk of exposure during
pregnancy and transmitting the virus to their babies
(congenital infection )
❏ Congenital Rubella Syndrome ( CRS) ​is the highest in
Africa and Southeast Asia where the vaccination is the
lowest. The disease is preventable by vaccination​.

IMMUNITY
❏ Natural infection al;so results in the procuring of specific
secretory IgA antibodies in the respiratory tractImmunity to
disease is nearly always lifelong; however m, re-exposure
B. CRS can lead to transient respiratory tract infection, with an
❏ Congenital infection occurs as a result of maternal anamnestic rise in IgG and secretory IgA antibodies, but
viremia that leads to placental infection and then without resultant viremia or illness.
transplacental spread to the fetus
❏ CRS Classic Triad TREATMENT, PREVENTION, AND CONTROL
❏ Rubella is mild, self-limiting which requires no treatment
❏ Attenuated live Rubella vaccines may be monovalent ot
given in combination with Measles and Mumps
❏ Primary purpose of your Rubella vaccination is to ​prevent
congenital rubella infections

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 Characteristics ❏ Single-stranded
❏ RNA genome
❏ Helical capsid with envelope
❏ Spherical, 120-160 nm in diameter

Genome ❏ Single-stranded RNA

❏ Linear
❏ Non-segmented
❏ Positive sense

Proteins ❏ Two glycoproteins and one

phosphoprotein
❏ Some viruses contain a third
glycoprotein (hemagglutinin esterase)

Envelope Contain large, widely spaced,

club-or-petal-shaped spikes

Replication Cytoplasm; particles mature by budding

into endoplasmic reticulum and Golgi.

Outstanding ❏ Cause colds, SARS, and MERS

Characteristics ❏ Display high frequency of recombination
❏ Difficult to grow in a cell culture

Figure 3.5​ Virion structure of Paramyxoviruses Transmission Unknown, probably direct contact or
aerosol
CORONAVIRIDAE
Disease Common cold; possibly gastroenteritis,
especially in children
GENERAL CHARACTERISTICS OF ​CORONAVIRIDAE
Detection Electron Microscopic
Family Coronaviridae
Treatment Supportive
Common Name Coronaviruses
Prevention Avoid contact with virus
Virus Coronavirus

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 GENERAL CHARACTERISTICS ❏ Mechanism of replication is in the ​cytoplasm
❏ Coronaviruses are large, enveloped RNA viruses ❏ Its ​envelope i​ s formed from the ​endoplasmic reticulum
❏ The human coronaviruses cause common colds, may cause
lower respiratory tract infections, and have been implicated
in gastroenteritis in infants.
❏ Novel coronaviruses have been identified as the cause of
severe acute respiratory syndrome (SARS) and Middle East
respiratory syndrome (MERS). Many coronaviruses of
humans and other animals exist.
❏ The prefix corona- is used because of the crown- like surface
projections that are seen when the virus is examined by Figure 3.6​ Virion Structure of Coronaviruses
electron microscopy. Human respiratory coronaviruses
cause colds, and occasionally, pneumonia in adults. PATHOGENESIS
❏ Coronaviruses are thought to cause diarrhea in infants ❏ Coronavirus infections in humans usually, but not always,
based on the presence (using electron microscopy) of remain limited to the respiratory tract.
coronavirus-like particles in stool of symptomatic patients.
SEVERE ACUTE RESPIRATORY SYNDROME - CORONAVIRUS
❏ Characterized by serious respiratory illness, including
pneumonia and progressive respiratory failure.
❏ The SARS virus probably originated in a nonhuman host,
most likely bats.
❏ ACE 2​- receptor of SARS-CoV

SEVERE ACUTE RESPIRATORY SYNDROME - CORONAVIRUS 2

❏ Just like SARS-CoV and MERS-CoV, SARS-CoV 2
originates from ​bats​.
❏ SARS-CoV 2 uses the ​angiotensin-converting enzyme 2
(ACE 2) receptor​ to facilitate viral entry into the target cells.
NOTE: CORONAVIRUS DISEASE (COVID-19) / (SARS-Cov-2​)
❏ Peplomers - ​appearance is like ​“crown of thorns” ​or ❏ is an infectious disease caused by a newly discovered
“solar corona; ​spike glycoproteins of the virus that will coronavirus. Most people infected with the COVID-19 virus
detect the receptors will experience ​mild to moderate respiratory illness and
❏ Coronavirus ​is an ​RNA ​virus recover without requiring special treatment.

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 ❏ Older people, and those with underlying medical problems been reported. Some laboratory-confirmed cases of
like cardiovascular disease, diabetes, chronic respiratory MERS-CoV infection are reported as ​asymptomatic​,
disease, and cancer are more likely to develop serious meaning that they do not have any clinical symptoms, yet
illness. they are positive for MERS-CoV infection following a
❏ The COVID-19 virus spreads primarily through ​droplets of laboratory test.
saliva or ​discharge from the nose when an infected person ❏ Most of these asymptomatic cases have been detected
coughs or sneezes, so it’s important that you also practice following aggressive contact tracing of a
respiratory etiquette (for example, by coughing into a flexed laboratory-confirmed case. Approximately ​35% of reported
elbow). patients with MERS-CoV infection have died.
❏ The new strain lineage ​B.1.1.7 is characterized by ​17 ❏ Middle East respiratory syndrome (MERS)​, although most
mutations that cause amino acid changes, 8 of which occur human cases of MERS-CoV infections have been attributed
in the gene for the spike (S) protein​. There are mutations in to human-to-human infections in health care settings, in
other regions of the SARS-CoV-2 genome that provide a current scientific evidence suggests that dromedary
genomic signature for this lineage. camels are a major reservoir host for MERS-CoV and an
❏ Another highly transmissible strain lineage, designated as animal source of MERS infection in humans. However, the
B.1.351 (also known as ​variant 501Y.V2​), was recently exact role of dromedaries in transmission of the virus and the
identified and found to have multiple mutations in the ​S exact route(s) of transmission are unknown.
gene​.
LABORATORY DIAGNOSIS
MIDDLE EAST RESPIRATORY SYNDROME - CORONAVIRUS ❏ Coronavirus antigens in cells in respiratory secretions may
❏ characterized by pneumonia and respiratory failure, though be detected using the ELISA test if a high-quality antiserum
most patients who died had medical comorbidities. is available.
❏ MERSCoV likely originated in ​bats ​and became widespread ❏ Polymerase chain reaction (PCR) assays are the preferred
in ​camels​. methods to detect coronavirus nucleic acid in respiratory
❏ A viral respiratory disease caused by a novel coronavirus secretions and stools.
(​Middle East respiratory syndrome coronavirus​, or ❏ Isolation of viruses using Vero monkey kidney cells
MERS-CoV​) that was first identified in Saudi Arabia in 2012. ❏ ELISA, indirect immunofluorescent antibody assays, and
Typical MERS symptoms include ​fever, cough and shortness hemagglutination tests may be used.
of breath​.
❏ CD26 / Dipeptidyl Peptidase-4 (DPP4) ​- receptor of MERS- EPIDEMIOLOGY
CoV ❏ Coronaviruses are distributed worldwide and are a major
❏ Pneumonia is common, but not always present. cause of respiratory illness in adults.
Gastrointestinal symptoms, including ​diarrhea​, have also

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 ❏ Coronaviruses are transmitted by contact with respiratory
droplets, contaminated surfaces and fomites (contaminated
inanimate objects).

TREATMENT, CONTROL, AND PREVENTION

❏ There is no proven treatment for coronavirus infections and
no vaccine.
❏ SARS and MERS vaccines are under development
❏ Control measures that were effective in stopping the spread
of SARS included isolation of patients, quarantine of those
who had been exposed, and travel restrictions, as well as the
“​Di ka nag-iisa sa laban na to.”
use of gloves, gowns, goggles and respirators by health care
workers.

Figure 3.7​ SARS-CoV-2 Origin and Transmission

ADDITIONAL NOTE:
❏ Alpha & Beta strain - ​ zoonotic infection, more virulent
❏ Delta & Gamma strain - ​not common
❏ Spike protein mutations ​may be due to​ post translational
factors
❏ RNA viruses are faster to mutate than DNA viruses.

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 MODULE 4: HERPESVIRUSES ➔ Cytomegalovirus (CMV) can also cause mononucleosis
symptoms in adults, and is a leading cause of congenital
OUTLINE OF THE LESSON blindness;
➔ HHV types 6 and 7 (HHV-6 and HHV-7)​, which cause
I. INTRODUCTION roseola in infants; and
II. PROPERTIES OF HERPES VIRUSES ➔ Kaposi Sarcoma (KS)-associated herpesvirus (KSHV)​,
III. CLASSIFICATION OF HERPES VIRUSES also known as HHV-8.
IV. HERPES VIRUS REPLICATION
V. CYTOPATHIC EFFECT OF HERPES VIRUSES REPLICATION NOTE: ​They can all undergo latency. Once you get infected by herpes, you
VI. HERPES INFECTION IN HUMANS will forever have herpes. They can reactivate if the immune system is
A. HSV 1&2 weakened and can cause infection.
B. Varicella-Zoster Virus (HHV3)
C. Epstein-Barr Virus (HHV4) ❏ In addition, the simian herpesvirus, herpes B virus, has
D. Cytomegalovirus (HHV5) occasionally caused lethal human disease in primate center
E. Human Herpesvirus 6 workers.
F. Human Herpesvirus 7 ❏ All herpesviruses establish lifelong latent infections in their hosts
G. Human Herpesvirus 8 with periodic reactivation events.
❏ Herpes viruses are widely disseminated among animal species.
HIGHLIGHTING OF TOPICS BULLET PLACEMENT However, the zoonotic forms of herpes do not infect humans,
except for herpes B virus from non-human primates but is not
AAAAAA​ - Family of virus ❏ (Main definition) counted among the eight human herpes viruses.
AAAAAA​ ​- Main topic ➔ (Sub-definition/Enumerate)
AAAAAA​ - Subtopics ● (Super Sub-detail) PROPERTIES OF HERPES VIRUSES

INTRODUCTION PROPERTY DESCRIPTION

❏ The ​Herpesviridae is composed of ​large, enveloped,
double-stranded DNA viruses​. There are eight known human Virion ❏ Spherical
herpesviruses (HHVs) and a very large number of animal ❏ 150–200 nm in diameter
herpesviruses. ❏ Icosahedral symmetry
❏ The word “​herpes​” is derived from the Greek word meaning “​to
creep​” (hide from the immune cells) and was historically used to Genome ❏ Double-stranded DNA
describe the spreading, ulcerative skin lesions typically seen in ❏ Linear reiterated (or repeated) genes
HSV (Herpes simplex virus) infection.
❏ Eight ​human herpes group viruses have been described: Proteins More than 35 proteins in virion
➔ Herpes simplex virus-1 (HSV-1) and HSV-2​, which
cause facial and genital lesions; Envelope ❏ Contains viral glycoproteins
➔ Varicella-zoster virus (VZV)​, which causes chickenpox ❏ Fc receptors
and later in life can reactivate to cause shingles;
➔ Epstein-Barr virus (EBV)​, an infectious cause of Replication ❏ Nucleus
mononucleosis as well as Burkitt lymphoma (BL) among ❏ Bud from nuclear membrane
other B-cell lymphomas;
Outstanding ❏ Encode many enzymes

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 CLASSIFICATION OF HERPES VIRUSES
❏ Establish ​latent infections
❏ Persist indefinitely​ in infected hosts
❏ Frequently reactivated in immunosuppressed hosts
❏ Some cause cancer
Table 4.1​ Properties of Herpes Virus

❏ The capsid is surrounded by the ​tegument​, a relatively amorphous

protein-filled region unique to herpesviruses. The tegument
contains viral proteins and enzymes that play a structural role and
many are required immediately for viral replication upon initial
infection​.
❏ Surrounding the tegument is a lipoprotein envelope originally
derived from the nuclear membrane of the infected host cell. The
envelope contains multiple viral glycoproteins that act as viral
binding, fusion, and entry proteins.

Table 4.2 ​Classification of Human Herpesviruses

1. Alphaherpesviruses are ​fast-growing​, ​cytolytic (lyse cells causing

lesions) viruses that tend to ​establish latent infections in neurons​;
➔ HSV 1​ and​ 2​ (genus Simplex Virus)
➔ VZVs ​(genus Varicellovirus) are members.
2. Betaherpesviruses are ​slow growing and may be cytomegalic
(massive enlargements of infected cells) and become latent in
secretory glands​ and ​kidneys​;
➔ CMV is classified in the Cytomegalovirus genus. They are
latent in ​glands ​and in ​kidneys​.
➔ HHV-6 and ​HHV-7​, classified under the genus
Roseolovirus. They are latent in ​lymphoid tissue​. By
Figure 4.1 ​Diagram of Herpes complex virus particle structure biologic criteria, they are similar to gammaherpesviruses
because they infect lymphocytes (T lymphotropic), but
molecular analyses of their genomes reveal that they are
NOTE:
What should you remember about Herpes structure? more closely related to the betaherpesviruses.
3. Gammaherpesviruses​, exemplified by
❏ Double-stranded DNA
➔ EBV (genus Lymphocryptovirus), infect and become latent
❏ Presence of envelope
❏ Icosahedral in lymphoid cells
➔ HHV-8/KSHV ​is classified in the Rhadinovirus genus.
❏ Presence of tegument

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 HERPES VIRUSES REPLICATION 2. Viral nucleocapsid is transported to the nuclear pore
❏ Replication of herpesviruses takes place in the host cell ​nucleus​, 3. Uncoating releases linear DNA into the nucleus
and is similar to replication of any other dsDNA virus. The only 4. Linear DNA become circular DNA
difference is that the linear dsDNA of herpesviruses becomes 5. Circular DNA is transcribed to form immediate-early mRNA
circular inside the host cell and then replicates by rolling circle 6. Immediate-early mRNA is transported to cytoplasm
mechanism. 7. Immediate-early mRNA in the cytoplasm is translated to α-proteins

EARLY PHASE
1. α-proteins are transported back to the nucleus and is the start of
the early phase
2. α-proteins transcribe circular DNA in nucleus to form Early mRNA
3. Early mRNA is transported to cytoplasm
4. Early mRNA in the cytoplasm is translated to β-proteins

LATE PHASE
1. β-proteins are transported back to the nucleus
2. β-proteins transform circular DNA in nucleus to concatemeric DNA
➔ Repeated sequences of nucleotides
3. Concatemeric DNA is replicated and transcribed to form late
mRNA
4. Late mRNA is transported to the cytoplasm
5. Replicated concatemeric DNA is cleaved to form viral DNA
6. Some late mRNAs in the cytoplasm are translated to structural
У-proteins
7. Structural У-proteins are transported to the nucleus
8. Some late mRNAs in the cytoplasm are translated to viral
glycoproteins
9. Viral glycoproteins will attach to the nuclear membrane
10. Viral nucleocapsids bud off from nuclear membrane with viral
glycoprotein to form enveloped progeny viruses
Figure 4.2​ Replication cycle of herpes simplex virus 11. Enveloped progeny viruses pass through ER and Golgi apparatus
12. Enveloped progeny viruses are released from Golgi apparatus to
❏ The replication of the herpesviruses is divided into: outside the cell by exocytosis.
A. Immediate-early phase
B. Early phase CYTOPATHIC EFFECTS OF HERPESVIRUSES REPLICATION
C. Late phase ❏ HSV​ in Hep-2 cells cause swollen, rounded cells
❏ VZV in human kidney cells cause multinucleated giant cells with
IMMEDIATE-EARLY PHASE acidophilic intranuclear inclusion
1. Viral envelope glycoproteins fuse with the cell surface ❏ CMV shows multinucleated giant cells with acidophilic intranuclear
glycosaminoglycans (GAGs) especially Heparan Sulfate and cytoplasmic inclusions
➔ Cellular receptor of herpesviruses ➔ Different from adenovirus because CMV has 2 nuclei
➔ Has a similar structure with heparin which is a natural
anticoagulant but the heparin is more sulfated

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 Figure 4.3​ Multinucleated giant cells (Owl’s eye inclusion)

HERPES INFECTION IN HUMANS

1. Herpes Simplex Virus 1
2. Herpes Simplex Virus 2
3. Varicella-Zoster Virus
4. Epstein-Barr Virus
5. Cytomegalovirus
6. Human Herpesvirus 6
7. Human Herpesvirus 7
8. Human Herpesvirus 8

HERPES SIMPLEX VIRUSES (HSV)

INTRODUCTION
❏ Herpes simplex viruses belong to the α-subfamily of Herpesviridae.
They are extremely widespread and exhibit a broad host range;
can infect many types of cells and different animals.
❏ However, the human herpesviruses infect exclusively men. They
replicate fast (8 - 16 hours cycle), spread fast and are cytolytic.
❏ They can cause a spectrum of diseases, involving skin, mucosa
and various organs.
❏ They undergo ​latency in nerve cells​; ​reactivate later causing
recurrent lesions​. Herpes simplex viruses (HSV) are of two distinct
types: ​HSV-1 and ​HSV-2​. They differ from each other in many
aspects

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 HSV ACID FAST STAIN
❏ Multinucleated giant cells and intranuclear inclusion bodies. The
pink areas within the epithelial cell nuclei are intranuclear inclusion
bodies.

A. PRIMARY / ACUTE INFECTION

❏ Both HSV-1 and HSV-2 initially infect and replicate in the
muco-epithelial cells and ​initiate lytic or productive
infection at the site of contact (​lesions are produced).
❏ Transmission occurs through abraded skin or mucosa from
any site, but more commonly by: HSV-1: Oropharyngeal
contact with infected saliva or direct skin contact for
HSV-1, and Sexual contact or rarely vertical mode (from
PATHOGENESIS AND PATHOLOGY mother 10 fetus) for HSV-2. HSV-1 is more often
❏ Characteristic histopathologic changes include ballooning of associated with disease “above the waist” or facial herpes,
infected cells, production of ​Cowdry type A intranuclear whereas HSV-2 is most often associated with genital
inclusion bodies, margination of chromatin, and formation of infections or “below the waist” infections.
multinucleated giant cells. ❏ Virus then invades the local nerve ending and is
transported by retrograde axonal flow to the dorsal root
ganglia, where it replicates further, and then undergoes
latency.
❏ Primary HSV infections are usually mild; in fact, most are
asymptomatic. However, in immunocompromised hosts,
viremia occurs that leads to widespread organ involvement
and systemic manifestations.

B. LATENT INFECTION
❏ In humans, latent infection by
➔ HSV-1 has been demonstrated in ​trigeminal​,
superior cervical​, and ​vagal nerve ganglia​, and
Figure 4.4​ Multinucleated and Balloon cells occasionally in the S2-S3 dorsal sensory nerve
root ganglia.
➔ HSV-2 infection has been demonstrated in the
sacral​ (S2-S3) region​.

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NOTE: ​Herpes virus hides in these regions, since these regions are
nearest to the site of entry​.

❏ HSV does not replicate in latent stage except for a small RNA,
called micro-RNA (encoded by a latency- associated viral gene) CLINICAL FINDINGS
which maintains the latent infection and prevents cell death. ❏ Infection with herpes simplex virus may take several clinical forms.
The infection is most often not apparent. The usual clinical
C. RECURRENT INFECTION (REACTIVATION) manifestation is a vesicular eruption of the skin or mucous
❏ Reactivation of the latent virus can occur following various membranes. Infection is sometimes seen as severe keratitis,
provocative stimuli, such as ​fever​, ​axonal injury ​(release of meningoencephalitis and a disseminated illness of the newborn.
virus), ​physical or emotional stress​, and ​exposure to
ultraviolet light​.
❏ Via the axonal spread, the virus goes back to the
peripheral site and further replicates in skin or mucosa
producing secondary lesions. Recurrent infections are less
extensive and less severe because of the presence of
pre-existing host immunity that limits the local viral
replication.
❏ The mechanisms by which latent infection is reactivated
are unknown.

Table 4.4​ HSV-1 and HSV-2 Clinical Association

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NOTE: ​How to prevent neonatal herpes?
❏ Perform cesarean section since there will be no exposure to
genitals of mother

DISEASES CAUSED BY HSV 1 & HSV 2

A. HERPES LABIALIS
❏ Caused by HSV 1
❏ Also known as cold sores or ​herpes febrilis
❏ The most common recurrent disease produced by type 1.
❏ Clusters of localized vesicles occur, usually at the
mucocutaneous junction of the lips.
❏ The vesicle ruptures, leaving a painful ulcer that heals
without scarring. C. GENITAL HERPES
❏ The lesions may recur, repeatedly and at various intervals ❏ Cause by HSV 2
of time, in the same location. ❏ Characterized by vesiculoulcerative lesions of the penis of
❏ The permanent site of latent herpes simplex virus is the male or the cervix, vulva, vagina and perineum of the
trigeminal-ganglion​. female.
❏ The lesions are more severe during primary infection and
may be associated with fever, malaise, and inguinal
lymphadenopathy.
❏ Remains latent in lumbar and sacral ganglia

HSV 1 lesions outside the mouth

Herpes labialis or cold sores

B. KERATOCONJUNCTIVITIS HSV 2 lesions in the genitals

❏ Cause by HSV 1 Herpes progenitalis or genital herpes
❏ Initial infection may be in the eye, producing severe
keratoconjunctivitis. D. NEONATAL HERPES
❏ Recurrent infection of the eye appears as dendritic keratitis ❏ Caused by HSV 2
or corneal ulcers or as vesicles on the eyelids. ❏ transmitted to the newborn during birth by contact with
❏ With recurrent keratitis, there may be progressive herpetic lesions in the birth canal.
involvement of the corneal stroma, with permanent ❏ Spectrum of illness may vary from subclinical of local to
opacification and blindness. severe generalized disease with a fatal outcome.
❏ Severely affected infants who survive may have
permanent brain damage.

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 LABORATORY DIAGNOSIS
POLYMERASE CHAIN REACTION (PCR) ASSAYS
❏ can be used to detect viruses in vesicle swabs, blood, CSF, and
tissue and are sensitive and specific. PCR amplification of viral
DNA from cerebrospinal fluid is the most sensitive means of
detection and is recommended for diagnosis of herpes
meningitis/encephalitis.

VIRUS CULTURE
❏ is commonly used, particularly for diagnosis of mucocutaneous
disease. Inoculation of tissue cultures is used for viral isolation.
HSV is relatively easy to cultivate, with ​cytopathic effects typically
occurring in 2–3 days ​(18-36 hours)​. The agent is then identified
by neutralization test or immunofluorescence staining with specific
antiserum. HV, Immunofluorescence test
NOTE: ​Cytopathic effect - infected cells develop intranuclear acidophilic
inclusion and then undergo necrosis EPIDEMIOLOGY
❏ HSV are ​worldwide in distributio​n. No animal reservoirs or vectors
are involved with the human viruses.
➔ Humans are only reservoirs
❏ Transmission is by ​contact with infected secretions​. The
epidemiology of HSV-1 and HSV-2 differs.
➔ HSV-1 is ​more constantly present in humans than any
other virus.
➔ HSV-2​ is usually acquired as a ​STD​.

TREATMENT, PREVENTION, CONTROL

❏ Several antiviral drugs have proved effective against HSV
RAPID CYTOLOGIC METHOD infections, including ​acyclovir​, ​valacyclovir​, and ​vidarabine​.
❏ is to stain scrapings obtained from the base of a vesicle (eg, with Newborns and persons with eczema should be protected from
Giemsa’s stain); the presence of ​multinucleated giant cells exposure to persons with active herpetic lesions. Patients with
indicates that herpesvirus (HSV-1, HSV-2, or varicella-zoster) is genital herpes should be counseled that asymptomatic shedding is
present. frequent and that the risk of transmission can be reduced by
antiviral therapy and condom usage. Experimental vaccines of
SEROLOGY various types are being developed.
❏ Antibodies appear in 4–7 days after infection and reach a peak in
2–4 weeks. They persist with minor fluctuations for the life of the ACTION OF ANTIVIRAL DRUGS
host. Detection methods available include ​neutralization​, ➔ Suppress clinical manifestation
immunofluorescence​, and e​nzyme-linked immunosorbent assay​. ➔ Shorten time of acute infection
➔ Speed up healing but can’t remove virus in neurons

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 VARICELLA- ZOSTER VIRUS ➔ lesions are only located where the varicella-zoster virus
was latent
Other name: HUMAN HERPES VIRUS 3 / HHV-3 ❏ Both diseases are caused by the same virus. Whereas varicella is
the acute disease that follows primary contact with the virus, zoster
INTRODUCTION is the response of the partially immune host to reactivation of
❏ Varicella (chickenpox) is a mild, highly contagious disease, chiefly varicella virus present in latent form in neurons in sensory ganglia.
of children, characterized clinically by a generalized vesicular ➔ reactivated form of chickenpox after undergoing latency
eruption of the skin and mucous membranes. The disease may be ❏ Varicella-zoster virus is morphologically identical to HSV. It has no
severe in adults and in immunocompromised individuals. animal reservoir. The virus propagates in cultures of human
embryonic tissue and produces typical intranuclear inclusion
Reasons why it is called chickenpox: bodies
1. The lesions looked liked chicken peas
2. The rash that is produced looked like peck marks of chicken PATHOGENESIS AND PATHOLOGY
VARICELLA​:
❏ Route of infection is probably the mucosa of the upper respiratory
CHICKENPOX SMALLPOX
tract.
❏ Virus then circulates in the blood and localizes in the skin where
Causative agent Varicella-zoster virus Variola virus
there will be swelling of the epithelial cells, ballooning,
degeneration, and the accumulation of tissue fluids result in vesicle
Lesions Vary in appearance undergo stages of
formation.
and stages. development and are
❏ In nuclei of infected cells, particularly in early stages, eosinophilic
overall the same
inclusion bodies are found.

ZOSTER:
❏ In addition to the skin lesions, histopathologically identical with
those of varicella, there is an inflammatory reaction of the dorsal
nerve roots and sensory ganglia.
❏ Often only a single ganglion may be involved. As a rule, ​the
distribution of the lesions in the skin corresponds closely to the
areas of innervation from an individual dorsal root ganglia​.
❏ There is cellular infiltration, necrosis of nerve cells, and
inflammation of the ganglion sheath.

❏ Herpes zoster (shingles) is a sporadic, incapacitating disease of

elderly or immunocompromised individuals that is characterized by
pain and vesicular rash limited in distribution to the skin innervated
by a single sensory ganglion. The lesions are similar to those of
varicella/chickenpox.

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 NOTE:
➔ #5 from figure 4.5: ​Once infected by the varicella zoster virus it
will undergo latency in the nerve cells. The immune system would
only destroy those viruses that do not undergo latency.

NOTE: ​If a person is exposed to someone who has shingles, you will still
get chickenpox because shingles can only be acquired after chickenpox​.

CLINICAL FINDINGS
VARICELLA (ACUTE INFECTION)
❏ Also known as​ chickenpox
❏ Mild, highly infectious disease, mainly by children, characterized by
vesicular eruption of the skin and mucous membranes.
❏ Subclinical varicella is unusual. The incubation period of typical
disease is 10–21 days.
❏ Malaise and fever are the earliest symptoms, soon followed by the
rash, first on the trunk and then on the face, the limbs, and the
buccal and pharyngeal mucosa in the mouth.
❏ Successive fresh vesicles appear in crops, so that all stages of
macules, papules, vesicles, and crusts may be seen at one time.
❏ The rash lasts about 5 days, and most children develop several
hundred skin lesions.
Figure 4.5​ The pathogenesis of primary infection with varicella-zoster virus ❏ Immunocompromised patients are at increased risk of
complications of varicella, including those with malignancies, organ
transplants, or HIV infection and those receiving high doses of
corticosteroids. Disseminated intravascular coagulation may occur

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 that is rapidly fatal. Children with leukemia are especially prone to
developing severe, disseminated VZV disease.

Figure 4.6​ Multiple stages or “crops” of varicella skin lesions

HERPES ZOSTER
❏ Also known as​ Shingles or Zona (REACTIVATION) Figure 4.7​ Shingles
❏ Usually occurs in persons immunocompromised as a result of
disease, therapy, or aging, but it occasionally develops in healthy LABORATORY DIAGNOSIS
young adults. ​CYTOPATHOLOGY
➔ It is a reactivation of varicella virus present in the sensory ❏ Giemsa staining of the scrapings from the ulcer base (Tzanck
ganglia. smear) reveals cytopathological changes similar to that of HSV
❏ It usually starts with severe pain in the area of skin or mucosa infection, such as formation of multinucleated giant cells.
supplied by one or more groups of sensory nerves and ganglia and ➔ Its cell culture and cytopathic effects are the same
is often unilateral. with ​HSV-1​ and ​HSV-2​.
❏ Within a few days after onset, a crop of vesicles appears over the VIRUS ISOLATION
skin supplied by the affected nerves. ❏ Virus isolation in various cell lines can also produce HSV-like
❏ The trunk, head, and neck are most commonly affected. cytopathic effects such as diffuse rounding and ballooning of
❏ The most common complication of zoster in elderly adults is infected cells.
postherpetic neuralgia—protracted pain that may continue for
months. VZV-SPECIFIC METHODS
❏ Sporadic, incapacitating disease of adults (rare in children) that is ❏ Specific antigen detection by direct immunofluorescence staining
characterized by an ​inflammatory reaction of the posterior nerve and ​PCR​ detecting VZV-specific genes.
roots and ganglia​, accompanied by crops of vesicles (like those of ➔ Direct Immunofluorescence: detect antigens
varicella) over the skin supplied by the affected sensory nerve. produced
➔ PCR:​ detect varicella zoster genome

EPIDEMIOLOGY
❏ Varicella and herpes zoster occur worldwide. Varicella
(chickenpox) is highly communicable and is a common epidemic
disease of childhood (most cases occur in children < 10 years of
age). Adult cases do occur. It is much more common in winter and

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 spring than in summer in temperate climates. Herpes zoster occurs
sporadically, chiefly in adults and without seasonal prevalence.
❏ Contact infection is less common in zoster

TREATMENT, PREVENTION, CONTROL

❏ Varicella in normal children is a mild disease and requires no
treatment.
➔ Self-limiting
❏ Neonates and immunocompromised patients with severe infections
should be treated.
➔ Since their system can’t produce antibody themselves
❏ High risk patients exposed to Varicella can be protected by
immunoglobulin before onset of varicella. Several antiviral
compounds provide effective therapy for varicella, including Figure 4.8​ Attachment of EBV to B cells
acyclovir, valacyclovir, famciclovir, and foscarnet.
❏ Use of live attenuated Varicella vaccine to prevent chickenpox in PATHOGENESIS
children and the use of a more potent vaccine to prevent shingles A. PRIMARY INFECTION
in adults are also widely used to prevent acquiring the disease. ❏ EBV is transmitted by oropharyngeal contact through
➔ Chickenpox can still be developed even after vaccination infected salivary secretions.
but milder ❏ EBV binds to specific receptors present on ​B cells (CD21
or CR2) which are also receptors for the C3b component of
EPSTEIN-BARR VIRUS complement.
❏ Such receptors are also present on pharyngeal epithelial
Other name: HUMAN HERPESVIRUS 4 / HHV-4 cells.
➔ Primary infection occurs in the oropharynx.
INTRODUCTION ❏ EBV replicates in epithelial cells or surface B lymphocytes
❏ EBV is a ubiquitous herpesvirus that is the causative agent of of the pharynx and salivary glands.
acute infectious mononucleosis and is associated with ❏ Following entry into the B cells, EBV directly enters into
nasopharyngeal carcinoma, Burkitt lymphoma, Hodgkin and latent phase without completing the viral replication.
non-Hodgkin lymphomas, other lymphoproliferative disorders in ❏ Though the majority of the infected cells are eliminated, a
immunodeficient individuals, and gastric carcinoma. small number of infected cells may persist for lifetime.
❏ The major target cell for EBV is the B lymphocyte. ❏ Virus spreads from the oropharynx to other sites of the
➔ r​eceptor of virus is located in B lymphocytes (CR2/ CD21) body and is capable of undergoing reactivation later. Viral
❏ When human B lymphocytes are infected with EBV, continuous shedding continues in oropharyngeal secretions at low
cell lines can be established, indicating that cells have been levels for weeks to months and serves as a source of
immortalized by the virus. infection.
➔ EBV is a herpes virus and needs to undergo latency in the ❏ In children, most primary infections are subclinical, but
B lymphocytes young adults often develop a condition called acute
➔ Very few of the immortalized cells produce infectious infectious mononucleosis.
viruses. EBV initiates infection of B cells by binding to the NOTE: ​Mononucleosis ​is a polyclonal stimulation of lymphocytes.
viral receptor, which is the receptor for the C3d component EBV-infected B cells synthesize immunoglobulin. Autoantibodies are
of complement ​(CR2 or CD21)​.

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typical of the disease, with heterophile antibodies that react with antigens
on sheep erythrocytes detectable in acute cases.

REMEMBER:
❏ In Children – Subclinical
❏ In Adults – Infectious Mononucleosis

B. REACTIVATION FROM LATENCY

❏ Reactivation is due to immunosuppression
❏ Reactivation of EBV latent infections can occur or be
observed when there is:
➔ Increased levels of virus in saliva
➔ Increased levels of DNA in blood cells

CLINICAL FINDINGS
A. INFECTIOUS MONONUCLEOSIS
❏ Primary Infection of Epstein-Barr Virus
❏ Aka Kissing Disease
➔ Disease can be get through kissing because of
increased levels of virus in saliva
➔ Virus can be isolated in the saliva of an infected
person B. CANCERS ASSOCIATED WITH EBV
❏ After an incubation period of 30–50 days, symptoms of ❏ Nasopharyngeal carcinoma
headache, fever, malaise, fatigue, and sore throat occur. ❏ Burkitt Lymphoma (Cancer of the B lymphocyte)
Enlarged lymph nodes and spleen are characteristic. ➔ Respond to chemotherapy (few weeks/ month)
Some patients develop signs of hepatitis. ❏ Hodgkin Lymphoma
❏ The typical illness is self-limited and lasts for 2–4 weeks. ❏ Non-hodgkin lymphoma
➔ Disease is self-limiting as long as your immune ❏ Gastric Carcinoma
system is competent ❏ Oral Hairy Leukoplakia
❏ During the disease, there is an increase in the number of
circulating white blood cells, with a predominance of ONCOGENIC PROPERTIES
lymphocytes. ❏ Herpesviruses have been linked with malignant disease in
❏ Rely on serologic test for diagnosis humans:
❏ Many of these are large, atypical T lymphocytes. ➔ Herpes simplex virus type 2 and vulvar carcinoma
➔ Larger with more cytoplasm than the nucleus and ➔ EB virus with Burkilt’s lymphoma of African children and
have nucleoli with nasopharyngeal carcinoma.
➔ “Dutch skirt" appearance because of RBC that
sticks in the cytoplasm and forms indentions
❏ Low-grade fever and malaise may persist for weeks to
months after acute illness.

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 LABORATORY DIAGNOSIS CYTOMEGALOVIRUS
MOLECULAR TEST
❏ detection of the ​genome ​of the virus Other name: HUMAN HERPESVIRUS 5
❏ Nucleic acid hybridization is the most sensitive means of detecting
EBV in patient materials. It can be isolated from saliva, peripheral INTRODUCTION
blood or lymphoid tissue
❏ PCR ❏ CMV is a ubiquitous herpesvirus that is a common cause of human
disease. CMV is the ​most common cause of congenital infection,
SEROLOGIC TEST which can lead to severe abnormalities​. Inapparent infection is
❏ detection of the ​antigen ​and ​antibody common during childhood and adolescence. Severe CMV
❏ Common serological procedures for detecting EBV antibodies infections are frequently found in adults who are
include: immunosuppressed.
➔ ELISA ➔ It can be found in the cervix of up to 10% of healthy
➔ Immunoblot assays women.
➔ Indirect immunofluorescence ➔ It affects pregnant women at an increased severity since it
can affect the fetus.
EPIDEMIOLOGY ❏ CMV is named for the cytopathic effect it produces in cell culture.
❏ EBV is common in all parts of the world, with more than 90% of In addition to nuclear inclusions (​“​owl’s eye cells​” or “owl’s eye
adults being seropositive. It is transmitted primarily ​by contact with inclusion”​, CMV produces perinuclear cytoplasmic inclusions and
oropharyngeal secretions​. enlargement of the cell (cytomegaly). CMV possesses the largest
❏ In developing areas, infections occur early in life; more than 90% genome of the HHVs.
of children are infected by age 6 years. ❏ Inapparent infection is common during childhood and adolescence.
❏ In almost half of cases, the infection is manifested by infectious Severe CMV infections are frequently found in adults receiving
mononucleosis. immunosuppressive therapy.
❏ Note: Infectious Mononucleosis - Acute infection of epstein-barr ❏ It affects the vascular endothelial cells and wbcs
virus and characteristic is the presence of atypical lymphocytes.
❏ Transmitted by intimate contact

TREATMENT, PREVENTION AND CONTROL

❏ There is ​no EBV vaccine available.
❏ Acyclovir reduces EBV shedding from the oropharynx during the
period of drug administration, but it does not affect the number of
EBV-immortalized B cells.
➔ This is used to minimize the transmission of the virus to
others. Since it is a herpesvirus, there is latency infection
occurring inside the monocyte. However, it does not
remove this infection. Also, this is a self-limiting virus in 1-2
months.

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 PATHOGENESIS B. CMV INFECTION IN IMMUNOCOMPROMISED HOSTS
❏ Cytomegalovirus can cause persistent infection in various tissues, ❏ CMV may involve the lungs, GIT, liver retina
including those of the salivary glands, breasts, kidneys, ❏ CMV produces markedly severe infection in
endocervix, seminal vesicles and peripheral leukocytes. immunosuppressed individuals; most of which are due to
❏ This persistent infection leads to chronic viral excretion by the reactivation of their own latent viruses.
involved organ. ❏ ln AIDS patients with CD4 T cell count <50/uL, CMV may
❏ Transmission of virus through contact with infected secretions cause chorioretinitis, gastroenteritis, dementia and other
❏ Kidneys of organ donors can be a source of CMV for the recipient disseminated CMV infection.
and that peripheral blood leukocytes. ❏ CMV is probably the most common viral infection that
❏ The average incubation period is 4-6 weeks. occurs usually between 1- and 4-months following
transplantation and presents in various forms such as:
A. CMV INFECTION IN NORMAL HOSTS ➔ Bilateral interstitial pneumonia is the most
❏ In healthy adults, CMV produces an infection following common form, seen in 15-20% of bone marrow
blood transfusion called ​mononucleosis-like syndrome​. transplant recipients.
This condition is similar to infectious mononucleosis ➔ Febrile leukopenia is seen among solid organ
caused by EBV transplant recipients
❏ Mononucleosis syndrome (fever, malaise, atypical ➔ Obliterative bronchiolitis​ in lung transplants.
lymphocytosis, pharyngitis and rarely, cervical adenopathy ➔ Graft atherosclerosis​ in heart transplants
or hepatitis. ➔ Rejection of renal allografts
❏ The third clinical entity is cytomegalovirus infection in
severely immunocompromised patients C. CONGENITAL CMV INFECTION
❏ CMV is probably the most common intrauterine infection
associated with congenital defects. About 1 % of infants
born are infected with CMV. Cytomegalic inclusion disease
develops in about 5% of the infected fetus, the remaining
are although asymptomatic at birth, 5-25% of them may
develop significant psychomotor, hearing, ocular, or dental
defects within 2 years. Mortality rate is very high (20%).
❏ Hepatosplenomegaly, retinitis, petechial/purpuric skin rash,
and involvement of the CNS.

D. PERINATAL CMV INFECTION

❏ Transmission to the newborn occurs either during:
➔ Delivery ​- through infected birth canal or
➔ Postnatal ​- through infected breast milk /
Table 4.5​ Comparison of infectious and mononucleosis-like syndrome secretions from mother.
❏ Most of the infected infants remain asymptomatic, but
NOTE: shed virus in urine from 8- 12 weeks of age, up to several
➔ Both EBV and CMS contain atypical lymphocytes. years. Few infants, especially premature babies develop
➔ They have specific antibodies (CMV and EBV) interstitial pneumonitis.
➔ Heterophile antibodies is elevated in EBV

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 LABORATORY DIAGNOSIS
presence of CMV infection, but does not provide
❏ POLYMERASE CHAIN REACTION
proof that CMV is the cause of disease unless
❏ SEROLOGIC TESTS
other pathogens are excluded. Seroconversion is
➔ CMV IgG Antibodies: Indicative of Past Infection
diagnostic but rarely occurs, especially in patients
● IgG ​is only produced after an infection that’s why it
with AIDS, because more than 95% of these
indicates a past infection
patients are seropositive for CMV before infection
➔ CMV IgM Antibodies: Indicative of Current Infection
with human immunodeficiency virus (HIV).
● IgM ​is first produced by the body that’s why it
indicates a current infection
CMV Infection in Seroconversion and presence of IgM antibody
❏ Humans are the only known host for CMV
Normal Hosts specific for CMV are best indicators of primary
❏ Transmission requires close person-to-person contact
infection. Urine culture positivity supports the
❏ Virus may be shed in urine, saliva, semen, breast milk, and
diagnosis of CMV infection, but may reflect remote
cervical secretions and is carried in circulating white blood cells.
infection because positivity may continue for
➔ CMV is carried by WBCs
months to years. A positive blood assay for CMV
➔ CMV is a sexually-transmitted virus
antigen or DNA, however, is diagnostic in this
❏ Oral and respiratory spread are probably the dominant routes of
patient population.
CMV transmission
➔ Most common MOT Table 4.6​ Recommended procedure to facilitate the diagnosis of CMV
❏ CMV can be transmitted by blood transfusion, though the risk is infection
low with leuko-reduced blood products
➔ Since CMV is carried by wbcs, blood products must be EPIDEMIOLOGY
converted into leuko-reduced products to reduce the risk. ❏ CMV is endemic in all parts of the world; epidemics are unknown.
WBC concentration must be reduced in the blood. It is present throughout the year, with no seasonal variation seen in
infection rates.
❏ Humans are the only known host for CMV. Transmission requires
Clinical Laboratory Diagnosis
close person-to-person contact. Virus may be shed in urine, saliva,
Condition
semen, breast milk, and cervical secretions and is carried in
circulating white blood cells. Oral and respiratory spread are
Congenital CMV Virus culture or viral DNA assay positive at birth or
probably the dominant routes of CMV transmission. CMV can be
Infection within 1 to 2 weeks (to distinguish from natally or
transmitted by blood transfusion, though the risk is low with
perinatally infected infants, who will not begin to
leuko-reduced blood products.
excrete virus until 3-4 weeks after delivery)
TREATMENT, PREVENTION AND CONTROL
Perinatal CMV Culture-negative specimens at birth but positive
❏ Drug treatments of CMV infections have shown some encouraging
Infection specimens at 4 weeks or more after birth suggest
results. ​Ganciclovir has been shown to be successful in treating
natal or early postnatal acquisition. Seronegative
serious life-threatening CMV infections.
infants may acquire CMV from exogenous
➔ Other drugs used to treat CMV infections are ​Foscarnet,
sources, such as from blood transfusion.
Acyclovir and Valacycovir
❏ These drugs are used for immunocompromised individuals since
CMV Infection in Demonstration of virus by viral antigen or DNA in
CMV is also a self-limiting virus.
Immunocompro blood documents viremia. Demonstration of
mised hosts inclusions or viral antigen in diseased tissue (e.g, ❏ Vaccine is under development.
lung, esophagus, or colon) establishes the

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 HUMAN HERPES VIRUS 6 susceptible to acyclovir, because the virus has no thymidine
INTRODUCTION kinase.
❏ In 1986, a herpesvirus, now called human herpesvirus type 6 ❏ No specific treatment and it is self-limiting.
(HHV-6), was identified in cultures of peripheral blood lymphocytes
from patients with lymphoproliferative diseases. HHV-6 is a
β-herpesvirus subfamily. The virus morphologically similar to other HUMAN HERPES VIRUS 7
herpesviruses with similar replication patterns of other ❏ Isolation of human herpesvirus 7 (HHV-7) was first reported in
herpesviruses. HHV-6 replicates in lymphoid tissue, especially 1990. The virus was isolated from activated CD4+ T lymphocytes
CD4+ T lymphocytes​, and has two distinct variants, A and B, that of a healthy individual.
are genetically disparate enough that some consider them different ❏ The ​CD4 molecule ​appears to be a receptor for virus attachment.
species. ❏ HHV-7 is closely related to HHV-6 and is in the β-herpesvirus
genus.
CLINICAL FINDINGS ❏ Seroepidemiologic studies indicate that this virus usually does not
❏ HHV-6 type B is the ​main etiologic agent of ​exanthem subitum infect children until after infancy, but that nearly 90% of children
(roseola) or 6th disease​, and both types A and B can cause are antibody positive by 3 years of age.
acute febrile illnesses with or without seizures or rashes. ❏ As with HHV-6, this virus is frequently isolated from saliva, and
Exanthem subitum generally ​occurs in infants aged 6 months to 1 close personal contact is the probable means of transmission.
year. In the first 6 months, infants are generally protected by the ❏ There is little disease associated with HHV-7, however, it may also
mother’s IgG. Exanthem subitum is characterized by fever (usually be a cause of exanthem subitum, but the association has only
about 39°C) for 3 days, followed by a faint maculopapular rash been found in rare cases.
spreading from the trunk to the extremities, which begins during ❏ The diagnosis of acute infection can be made by the
defervescence. Exanthem subitum is one of the six classic demonstration of seroconversion. No treatment has been
childhood exanthems. identified.
NOTE:​ ​HHV-6 and HHV-7 are the etiologic agents of exanthem subitum. NOTE:​ ​No specific treatment

EPIDEMIOLOGY (SIR TUBOLA)

❏ Of the herpesviruses, HHV-6 is the most rapidly spread and is Both HHV-6 & HHV-7
shed in the throats of 10% of babies by age 5 months, 70% by 12 A. PROPERTIES OF HHV-6 AND 7
months, and 30% of adults. Greater than 90% of the population ❏ Belong to the betaherpesvirus subfamily of herpesvirus
has antibodies to this virus by the age of 5 years. ❏ HHV-6 and HHV-7 share limited nucleotide homology and
antigenic cross-reactivity.
LABORATORY DIAGNOSIS
❏ Primary virus infection can be documented by seroconversion. B. EPIDEMIOLOGY AND PATHOGENESIS
Active virus infection can be documented by culture, antigenemia, ❏ HHV-6 and 7 are ubiquitous and are found worldwide
or DNA detection in the blood (by PCR). Because asymptomatic ❏ They are transmitted mainly through contact with saliva
viremic reactivation is common, it is very difficult to use these tools and through breast feeding
to identify HHV-6 as the cause of febrile or other miscellaneous ❏ HHV-6 and HHV-7 infection are acquired rapidly after the
syndromes. age of 4 months when the effect of maternal antibody
wears off
TREATMENT, PREVENTION AND CONTROL ❏ By the time of adulthood 90-99 % of the population had
❏ Definitive therapy has not been established, but like the better been infected by both viruses.
characterized β-herpesvirus, CMV, HHV- 6 appears to be
susceptible in vitro to ganciclovir and foscarnet. It is less

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 ❏ Like other herpesviruses, HHV-6 and HHV-7 remains ❏ Therefore very few virology laboratories offer a diagnostic
latent in the body after primary infection and reactivation service for HHV-6 or HHv-7 infection.
from time to time. ❏ The technique for virus isolation is complicated and thus
not practicable as a routine diagnostic procedure
C. CLINICAL MANIFESTATIONS ❏ Therefore serology is the mainstay diagnosis where
❏ Primary HHV-6 infection is associated with Roseola specific IgG and IgM are detected
Infantum, which is a classical disease of childhood ❏ There is no specific antiviral treatment for HHV-6 infection
❏ Most cases occur in infants between the ages of 4 months
and two years HUMAN HERPES VIRUS 8
❏ A spiking fever develops over a period of 2 days followed INTRODUCTION
by a mild rash. The fever is high enough to cause febrile ❏ During the AIDS epidemic in the 1980, in the United States,
convulsions. Kaposi Sarcoma (KS) occurred in 20% to 30% of gay or bisexual
❏ There are reports that the disease may be complicated bt males with AIDS but in only around 1% of hemophiliacs with AIDS.
encephalitis This led to the proposal that there was another infectious agent
❏ If primary infection is delayed until adulthood, there is a associated with KS.
small chance that an infectious mononucleosis-like ❏ In 1994, unique viral DNA sequences were identified in KS tumors
disease may develop in a similar manner to EBV and CMV using subtractive hybridization analysis. The sequences bore
❏ There is no firm evidence linking HHV-6 to lymphomas or homology to γ-herpesviruses and were used to clone the entire
lymphoproliferative diseases. 165 kbp genome of the eighth HHV, commonly known as
❏ There is no firm disease associations with HHV-7 at KS-associated herpesvirus (KSHV) or HHV-8. KSHV is ​found in
present 100% of KS tumors.
❏ Although both viruses may be reactivated in ❏ KSHV is cancer associated in the mouth
immunocompromised patients, it is yet uncertain whether ❏ Belong to gammaherpesviruses subfamily of herpesviruses
they cause significant disease since CMV is almost ❏ Originally isolated from cells of Kaposi’s sarcoma (KS)
invariably present. ❏ Now appears to be firmly associated with Kaposi’s sarcoma as well
as lesser known malignancies such as Castleman’s disease and
primary effusion lymphomas
❏ HHV-8 DNA is found in almost 100% of cases of Kaposi’s sarcoma
❏ Most patients with KS have antibodies against HHV-8
❏ The seroprevalence of HHV-8 is low among the general population
but is high in groups of individuals susceptible to KS, such as
homosexuals
❏ Unlike other herpes viruses, HHV-8 does not have a ubiquitous
distribution

Roseola Infantum

D. DIAGNOSIS AND MANAGEMENT

❏ Roseola Infantum has a very characteristic presentation
and a diagnosis can usually be made on clinical grounds
alone.

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 PATHOGENESIS
❏ KSHV infects the ​oral epithelium and can be ​shed into saliva for
Note: ​HIV must be treated first for Epidemic Kaposi
transmission​. In B cells, KSHV is predominantly in the latent state
sarcoma to be treated.
although lytic antigens can be found in a low percentage of the
cells. In KS tumors, KSHV is found in the main tumor cell, the Table 4.7 ​The four main forms of Kaposi Sarcoma
spindle cell, a cell of endothelial origin. KSHV is found in all spindle
cells in later stage tumors. Again, the virus is found predominantly
in the latent state, though 1% to 5% of the spindle cells support
lytic antigens and likely replication and virus production. In culture,
KSHV can infect many cell types where it establishes latency in
most of the cells. Similar to the KS tumor, a low percentage of
endothelial cells infected in culture also express lytic antigens.

CLINICAL FINDINGS

Kaposi
Sarcoma Description
KAPOSI’S SARCOMA
Forms ❏ Human herpes virus 8 or Kaposi sarcoma associated herpes virus
(KSHV) is also associated with:
Classic Originally described in the 1800s by Moriz Kaposi, it is
➔ Primary Effusion Lymphoma (PEL))
Kaposi a rare, fairly indolent ​tumor mainly found on the lower
➔ Multicentric Castleman disease (MCD)
Sarcoma extremities. It is mostly seen in elderly men of
Mediterranean origin and was also described in
EPIDEMIOLOGY
Ashkenazi jews.
❏ KSHV is the least widespread HHV. As noted earlier, KS was
common in the gay and bisexual AIDS community where the
Endemic In the middle of the 20th century, KS became common
seroprevalence rates perfectly match the KS rates at around 25%,
Kaposi in central Africa, where in countries like Uganda it is the
whereas in hemophiliacs with AIDS the seroprevalence rates were
Sarcoma most common tumor reported in hospitals. It is more
similar to healthy blood donors.
aggressive than classic KS and tumors can be seen
❏ The virus is ​not found in sexual secretions but is shed in saliva.
higher on the extremities and in the ​oral cavity and the
Because the virus is ​not ubiquitous like the other saliva-transmitted
torso.
herpesvirus​, it is not likely to be easily transmitted by kissing and
may require more prolonged intimate contact.
Iatrogenic KS also arises in ​posttransplant patients​, but generally
NOTE:
Kaposi regresses upon the removal of immunosuppression.
❏ Immunocompromised individuals are only infected
Sarcoma
❏ HSV8 is spread through sexual intercourse, blood transfusion, or
Note: Immune system must be weakened to protect the
vertical transmission or from mother to fetus.
transplanted organs.
LABORATORY DIAGNOSIS
Epidemic or This is the ​most aggressive form of KS, with the tumors
❏ Diagnosis for KSHV infection is currently imperfect.
AIDS-associ often ​appearing first in the mouth, on the torso, and
Immunofluorescence with sera from infected patients is a standard
ated Kaposi face, and can also be found on internal organs​. Without
technique but has a sensitivity of only 70% to 90%.
Sarcoma treatment for HIV, it can lead to death.
❏ PCR

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 ❏ SEROLOGIC TESTS:​ to detect antigen produced by HHV-8
➔ Indirect Immunofluorescence
➔ Western blot
➔ ELISA

TREATMENT, PREVENTION AND CONTROL

❏ A number of anti herpesvirus drugs inhibit lytic replication of KSHV
with foscarnet being the most active followed by ganciclovir. There
is evidence that treatment with ganciclovir is positively indicated for
MCD because it is a more lytic disease.
❏ No treatment for latently infected cells or vaccines is available.
❏ Antiviral drugs:
➔ Foscarnet
➔ Ganciclovir
➔ Cidofovir

TAKEAWAY NOTES:
❏ HHSV undergoes latency and there is a possibility of reactivation.
It is only reactivated once the immune system is severely
compromised or due to old age resulting in a weakened immune
system. Once infected, it will stay in the host’s body forever.

Figure 4.11​ Kaposi Sarcoma of the Oral Cavity

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 MODULE 5: HEPATITIS VIRUSES possible. The severity and course of the disease depend on the
particular virus and the state of the host.
OUTLINE OF THE LESSON ❏ Hepatitis is diagnosed serologically through the identification of
specific antigens or antibodies. Also, liver enzymes, including
I. INTRODUCTION Alanine Aminotransferase (ALT), Aspartate Aminotransferase
II. PROPERTIES OF HEPATITIS VIRUS (AST), Lactate dehydrogenase (LD), and Alkaline Phosphatase
III. HEPATITIS VIRUSES (ALP) may be increased up to 10 times the normal value in
A. Hepatitis A Virus (HAV) hepatitis. The serum bilirubin level also is increased in
B. Hepatitis B Virus (HBV) symptomatic patients.
C. Hepatitis C Virus (HCV)
D. Hepatitis D Virus (HDV) PROPERTIES OF HEPATITIS VIRUS
E. Hepatitis E Virus (HEV)

HIGHLIGHTING OF TOPICS BULLET PLACEMENT

AAAAAA - Family of virus ❏ (Main definition)

AAAAAA - Main topic ➔ (Sub-definition/Enumerate)
AAAAAA - Subtopics ● (Super Sub-detail)

INTRODUCTION
❏ Viral hepatitis is a systemic disease primarily involving the liver.
Most cases of acute viral hepatitis in children and adults are
caused by one of the following five agents: hepatitis A virus
(HAV), hepatitis B virus (HBV), hepatitis C virus (HCV),
hepatitis D (HDV), or hepatitis E virus (HEV).
❏ Hepatitis viruses produce acute inflammation of the liver,
resulting in a clinical illness characterized by fever, gastrointestinal
symptoms such as nausea and vomiting, and jaundice. Hepatitis
viruses cause similar appearing histopathologic lesions in the liver
during acute disease.

Currently, there are five recognized hepatitis viruses:

1. Hepatitis A Virus (HAV)
2. Hepatitis B Virus (HBV)
3. Hepatitis C Virus (HCV) – previously known as non-A, non-B
(NANB)
4. Hepatitis D Virus (HDV)
5. Hepatitis E Virus (HEV)

❏ Viral hepatitis, or infectious disease from hepatitis viruses, may

range from mild and self-limiting disease to acute fulminating
cirrhosis; chronic disease and asymptomatic carriage also are
Table 5.1 Characteristics of Hepatitis Virus

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NOTE: Take note of the family, genus, envelope, genome, and NOTE: Take note of the definitions in the table.
transmission. ➔ Core antigen is found in the nucleocapsid.
❏ All of them have vaccines except Hepatitis C ➔ In HBV, HBsAg, HBeAg, HBcAg are the antigens being detected
❏ Most common in the world: Hepatitis A and B in the laboratory.
❏ Classified according to host cell and diseases caused, but they are ➔ Immune globulins (IG) or IGIV (Intravenous) is a type of passive
not genetically related. immunity made for immunocompromised persons. Antibodies are
❏ Parenteral: other routes aside from the enteral or GIT route. directly injected into the body.
❏ Fulminant disease: end-stage disease, frequent in Hepatitis D
infection IMPORTANT INFO:
❏ Oncogenic disease: can cause cancer Hepatitis viruses do not cause cross-reactivity since they don’t have similar
antigenic properties. They are just named because they affect the liver and
the letters signify the sequence of discovery.

HEPATITIS A VIRUS (HAV)

INTRODUCTION
❏ HAV is a distinct member of the picornavirus family. HAV is a
spherical particle with cubic symmetry containing a linear
single-stranded RNA genome. It is assigned to the picornavirus
genus, Hepatovirus. There is no antigenic cross-reactivity with the
other hepatitis viruses. It is also known as “Infectious Hepatitis
Virus”.
❏ Alpha2-macroglobulin in the hepatocytes is the receptor of
Hepatitis A.
❏ It has only one serotype or monotypic, once you get infected and
recovered, you will acquire life-long immunity.
❏ There is no antigenic cross-reactivity with the other hepatitis virus
since they are from different families.
❏ HAV is stable (because it is naked) to treatment with 20% ether,
acid (pH 1.0 for 2 hours), and heat (60°C for 1 hour), and its
infectivity can be preserved for at least 1 month after being dried
and stored at 25°C (air conditioned room) or for years at −20°C
(freezer).
❏ The virus is destroyed by:
➔ Autoclaving (121°C for 20 minutes)
➔ Boiling in water for 5 minutes
➔ Dry heat (oven) (180°C for 1 hour)
➔ Ultraviolet irradiation (1 minute at 1.1 watts
➔ Treatment with formalin (1:4000 for 3 days at 37°C)
➔ Treatment with chlorine (10–15 ppm for 30 minutes).
❏ Heating food to above 85°C (185°F) for 1 minute and disinfecting
surfaces with sodium hypochlorite (1:100 dilution of chlorine
Table 5.2 Nomenclature and Definitions of Hepatitis Viruses, Antigens and bleach) are necessary to inactivate HAV. The relative resistance of
Antibodies

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 HAV to disinfection procedures emphasizes the need for extra
Assay Result Interpretation
precautions in dealing with hepatitis patients and their products.
❏ HAV initially was identified in stool and liver preparations by using
Anti- HAV IgM Positive Acute Infection with HAV
immune electron microscopy. Sensitive serologic assays and
polymerase chain reaction (PCR) methods have made it possible
Anti-HAV IgG Positive Past Infection with HAV
to detect HAV in stools and other samples and to measure specific
antibodies in serum. Various primate cell lines will support growth Table 5.3 Interpretation of HAV Serologic MArkers in Patients with
of HAV, although fresh isolates of virus are difficult to adapt and Hepatitis
grow. Usually, no cytopathic effects are apparent. Mutations in the
viral genome are selected during adaptation to tissue culture.

LABORATORY TESTS
❏ Virus appears early in the disease and disappears within 2 weeks
following the onset of jaundice. HAV can be detected in the liver,
stool, bile, and blood of naturally infected humans and
experimentally infected non-human primates by immunoassays,
nucleic acid hybridization assays, or PCR. HAV is detected in the
stool from about 2 weeks prior to the onset of jaundice up to 2
weeks after.
❏ RT-PCR is the method of choice for mRNA viruses.
❏ The Anti-HAV is an antibody usually used to detect the infection
caused by the Hepatitis A Virus. Anti- HAV appears in the IgM
fraction during the acute phase, peaking about 2 weeks after
elevation of liver enzymes. Anti-HAV IgM appears soon after the
onset of disease and persists for decades. Thus, detection of
IgM-specific anti-HAV in the blood of an acutely infected patient
confirms the diagnosis of hepatitis
❏ ELISA is the method of choice for measuring HAV antibodies.

Figure 5-2. Immunologic and biologic events associated with human

infection with hepatitis A virus. IgG, immunoglobulin G; IgM,
immunoglobulin M.

❏ Virus in the blood (viremia) appears 2 weeks after exposure. By

the time signs & symptoms appear, the virus in the blood is already
undetectable. This is the reason why blood could not be used as a
diagnostic specimen for HAV. During the appearance of signs &
symptoms, HAV RNA appears in the feces (virus shedding
occurs).

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 ❏ Jaundice / symptoms appear 4 weeks after exposure. Liver ➔ Persons with clotting factor disorders
enzymes will also start to be elevated. ➔ Persons working with non-human primates.
❏ HAV undergoes replication inside the hepatocytes and they will be
released through lysis. Aminotransferases elevate before the ❏ Individuals with chronic liver disease are at increased risk for
appearance of jaundice, this means that there is inflammation in fulminant hepatitis if a hepatitis A infection occurs. These groups
the liver (hepatitis). should be vaccinated.
❏ Since there is inflammation in the liver, bilirubin could not be
converted into a soluble substance for excretion, there will be TREATMENT
excess bilirubin resulting in jaundice. ❏ Treatment of patients with hepatitis A is supportive and directed at
❏ IgM appears approximately 4 weeks after exposure, during the allowing hepatocellular damage to resolve and repair itself.
acute infection. ❏ Food supplements and vitamins like Liver Aid
❏ IgG will gradually rise and will confer life-long immunity against
HAV. PREVENTION AND CONTROL
❏ FORMALIN-INACTIVATED HAV VACCINES
EPIDEMIOLOGY ➔ made from cell culture adapted virus are safe, effective,
❏ HAV is widespread throughout the world. Outbreaks of type A and recommended for use in persons more than 1 year of
hepatitis are common in families and institutions, summer camps, age. Routine vaccination of all children is now
day care centers, neonatal intensive care units, and among military recommended, as is vaccination of persons at increased
troops. risk, including international travelers, men who have sex
❏ The most likely mode of transmission under these conditions is by with men, and drug users.
the fecal–oral route through close personal contact. Stool
specimens may be infectious for up to 2 weeks before to 2 weeks ❏ Until all susceptible at-risk groups are immunized, prevention and
after onset of jaundice. Under crowded conditions and poor control of hepatitis A still must emphasize interrupting the chain of
sanitation, HAV infections occur at an early age; most children in transmission and using passive immunization. The appearance of
such circumstances become immune by age 10 years. hepatitis in camps or institutions is often an indication of poor
❏ Clinical illness is uncommon in infants and children; disease is sanitation and poor personal hygiene.
most often manifest in children and adolescents, with the highest ❏ Control measures are directed toward the prevention of fecal
rates in those between 5 and 14 years of age. contamination of food, water, or other sources by the individual.
❏ HAV is seldom transmitted by the use of contaminated needles ➔ Reasonable hygiene—such as handwashing,
and syringes or through the administration of blood. ➔ The use of disposable plates and eating utensils, and
➔ the use of 0.5% sodium hypochlorite (eg, 1:10 dilution of
Transfusion-associated hepatitis A is rare because: chlorine bleach) as a disinfectant—is essential in
➔ The viremic stage of infection occurs during the preventing the spread of HAV during the acute phase of
prodromal phase and is of short duration, the illness.
➔ The titer of virus in the blood is low,
➔ And there is no carrier state. There is little evidence for ❏ IMMUNE (γ) GLOBULIN (Ig)
HAV transmission by exposure to urine or nasopharyngeal ➔ passive immunity
secretions of infected patients. ➔ is prepared from large pools of normal adult plasma and
confers passive protection in about 90% of those
Groups that are at increased risk of acquiring hepatitis A are: exposed when given within 1–2 weeks after exposure to
➔ Travelers to developing countries from developed countries hepatitis A.
➔ Men who have sex with men, users of injection and non-injection ➔ Its prophylactic value decreases with time, and its
drugs administration more than 2 weeks after exposure or after

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 onset of clinical symptoms is not indicated. In the doses
generally prescribed, IG does not prevent infection but
rather makes the infection mild or subclinical and permits
active immunity to develop. HAV vaccine produces a more
enduring immunity and should replace the use of IG.
NOTE:
❏ The purpose of the Immune (y) globulin (IG) is for prophylaxis, it
gives the body (active immunity) enough time to produce
antibodies against the virus.

HEPATITIS B VIRUS
INTRODUCTION
❏ HBV is classified as a Hepadnavirus. HBV establishes chronic
infections, especially in those infected as infants; it is a major
factor in the eventual development of liver disease and
hepatocellular carcinoma in those individuals.
❏ The Hepa B virus targets the liver because the receptor of the
Hepatitis B virus are the NTCP (Sodium taurocholate
Co-transporting polypeptides) which is found on the hepatocytes
❏ Electron microscopy of hepatitis B surface antigen (HBsAg)- FORMS DESCRIPTION
positive serum reveals three morphologic forms.
Pleomorphic / ● Most numerous form and contain only HBsAg
THREE MORPHOLOGICAL FORMS OF HEPATITIS B VIRUS: Spherical ● Not a complete virion
1. SPHERICAL / PLEOMORPHIC Particles ● Non-infectious
➔ Most numerous; these small particles are made up
exclusively of HBsAg—as are tubular or filamentous Filamentous / ● Contains only HBsAg but are longer
forms—and result from overproduction of HBsAg. Elongated ● Not a complete virion
2. ELONGATED / FILAMENTOUS particles which have the same Form ● Non-infectious
components as the spherical ones.
3. SPHERICAL VIRIONS Spherical ● The complete virion
➔ Larger than the two Virion (Dane ● Infectious
➔ originally referred to as Dane particles Particle)
➔ are less frequently observed. The outer surface, or Table 5.4 Morphologic Features of HBV
envelope, contains HBsAg and surrounds an inner
nucleocapsid core that contains hepatitis B core antigen NOTE: Increased surface antigen production, little capsid and genome
(HBcAg). The viral genome (Figure 5-3A) consists of production.
partially double-stranded circular DNA.
❏ The virus is stable at 37°C for 60 minutes and remains viable after
being dried and stored at 25°C for at least 1 week.
❏ HBV (but not HBsAg) is sensitive to higher temperatures (100°C
for 1 minute) or to longer incubation periods (60°C for 10 hours).

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 HBsAg is stable at a pH of 2.4 for up to 6 hours, but HBV 3. HBeAg
infectivity is lost. ❏ Hepatitis B e antigen
➔ Sodium hypochlorite, 0.5% (eg, 1:10 chlorine bleach), ❏ Secreted from nucleocapsid core of unknown function
destroys antigenicity within 3 minutes at low protein ❏ Used as an indication of active HBV replication
concentrations, but undiluted serum specimens require ❏ Marker of potential infectivity
higher concentrations (5%). ❏ If a test is (+) for HBeAg this indicates active Hepatitis B
❏ HBsAg is not destroyed by ultraviolet irradiation of plasma or other infection
blood products, and viral infectivity may also resist such treatment.
HBV REPLICATION
❏ Replication occurs in the nucleus of hepatocytes

Figure 5.4 The complete HBV Virion

Figure 5-5. HBV Replication Cycle
THE COMPLETE HBV VIRION
1. HBsAg ❏ The infectious virion attaches to cells and becomes uncoated. In
❏ Hepatitis B surface antigen the nucleus, the partially double-stranded viral genome is
❏ Found on the HBV envelope converted to covalently closed circular double-stranded DNA
❏ Always in excess (cccDNA).
❏ If (+) in a test, it Indicates acute infection of Hepatitis B ➔ The cccDNA serves as a template for all viral transcripts,
including a 3.5-kb pre-genome RNA.
2. HBcAg ❏ The pre-genome RNA becomes encapsulated with newly
❏ Hepatitis B core antigen synthesized HBcAg.
❏ Found on the HBV nucleocapsid core\ ❏ Within the cores, the viral polymerase synthesizes by reverse
❏ Cannot be found in serum transcription a negative-strand DNA copy. The polymerase starts
❏ Can be found in the nucleus of the hepatocytes where the to synthesize the positive DNA strand, but the process is not
replication of the virus occurs completed.

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 ❏ Cores bud from the pre-Golgi membranes, acquiring HBsAg ❏ Recovery from the hepatitis B virus, the HbsAg and HbeAg will
containing envelopes, and may exit the cell. Alternatively, cores decrease because the antibodies will increase
may be reimported into the nucleus and initiate another round of ❏ Window period or equivalence zone - small period characterized
replication in the same cell. by the disappearance of HBsAg and before the appearance of
Anti-HBsAg
LABORATORY TESTS ➔ Might give false negative results if samples are collected
❏ PCR - method of choice for detection of viral DNA during this period.
❏ ELISA - method of choice for detecting viral antigen and antibody ➔ Anti-HBc is detected in this period
❏ Once Anti HBsAg develops, it indicates immunity against Hepatitis
B virus

RISE OF SEROLOGIC MARKERS (during Hepatitis B Virus infection):

1. HBsAG - first to increase
2. HBSeAG - after 1-2 months
3. HBcAG - more or less 2 months
4. Anti HBcAG
5. Anti HBeAg
6. Anti HBsAg- Indicates immunity against Hepatitis B

NOTE: During vaccination, HBsAg is given.

❏ HBcAG is only present during Hepatitis B virus infection that is why
during vaccination there is no HBcAg and Anti HBcAg detected,
only Anti HBsAG
❏ Common on screening tests are HBsAg and Anti HBs, but if there
is an issue Anti HBc is used.
❏ Level of detection - Minimum level of detection of antigen
❏ (+/-) - would mean it's either above or below the line (refer to the
table)
Figure 5-6. Clinical and serologic events occurring in a patient with acute
hepatitis B virus infection.
NOTE: (SIR ROBERT)
❏ During the acute episode of the disease, if there is active
replication, there will be large amounts of HBSAg and HBeAG in
the serum.
➔ The increase in HBSAg indicates an ongoing
infection(replication)of Hepatitis B virus
➔ There is no detection of HBcAG in the serum because it is
only found in hepatocytes
➔ During the start of an acute infection the antibody against
HBcAg will quickly rise resulting in the removal of the
antigen in the serum

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 EPIDEMIOLOGY TREATMENT
❏ HBV is worldwide in distribution. Most individuals infected as ❏ Treatment of HBV infection is recommended for patients with
infants develop chronic infections. As adults, they are subject to chronic active hepatitis to prevent progression of liver fibrosis and
liver disease and are at high risk of developing hepatocellular development of hepatocellular carcinoma.
carcinoma. The major modes of HBV transmission during infancy
are from an infected mother to her newborn during delivery and THERAPIES FOR HEPATITIS B
from an infected household contact to an infant. 1. First-line therapies for Hepatitis B
❏ There is no seasonal trend for HBV infection and no high ➔ Pegylated interferon alfa-2a
predilection for any age group, although there are definite high-risk ➔ Entecavir
groups, such as: ➔ Tenofovir
➔ parenteral drug abusers, NOTE: Medication is given as Peglylated interferon alfa-2a with either
➔ institutionalized persons, Entecavir or Tenofovir
➔ health care personnel, 2. Second-line therapy for Hepatitis B
➔ multiple transfused patients, ➔ Telbivudine
➔ organ transplant patients, 3. Third-line therapy for Hepatitis B
➔ hemodialysis patients and staff, ➔ Lamivudine, also known as 3TC
➔ highly promiscuous persons, ➔ Adefovir
➔ newborn infants born to mothers with hepatitis B.
❏ Mandatory screening of blood donors for markers of HBV infection PREVENTION AND CONTROL
(HBsAg, HBc Ab, and HBV DNA) has substantially reduced the ❏ Hepatitis B Virus infection can be prevented by currently available
number of cases of transfusion-associated hepatitis. People have vaccine
been infected by improperly sterilized syringes, needles, or
scalpels and even by tattooing or ear piercing. HEPATITIS C VIRUS (HCV)
❏ HBsAg can be detected in: INTRODUCTION
➔ Saliva ❏ Clinical and epidemiologic studies and cross- challenge
➔ Nasopharyngeal washings experiments in chimpanzees in the past had suggested that there
➔ Semen were several non-A, non-B (NANB) hepatitis agents that, based
➔ Menstrual fluid on serologic tests, were not related to HAV or HBV. The major
➔ Vaginal secretions agent was identified as HCV.
➔ Blood ❏ Most cases of post-transfusion NANB hepatitis were caused by
❏ Transmision from carriers to close contacts by the oral route or by HCV. Heparan sulfate proteoglycans are the receptors for
sexual or other intimate exposure occurs. Hepatitis C which is found in the hepatocytes
❏ There is strong evidence of transmission from persons with
subclinical cases and carriers of HBsAg to homosexual and
heterosexual long-term partners. Transmission by the fecal–oral
route has not been documented.
❏ Recalling that there may be more than 1 billion virions/mL of blood
from an HBeAg positive carrier and that the virus is resistant to
drying, it should be assumed that all bodily fluids from
HBV-infected patients may be infectious. Subclinical infections are
common, and these unrecognized infections represent the
principal hazard to hospital personnel.
Figure 5.7 HCV Structure

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 ❏ Anti-HCV antibodies can be detected in 50–70% of patients at the
❏ HCV is a positive-stranded RNA virus, classified as family onset of symptoms, but in others, antibody appearance is delayed
Flaviviridae, genus Hepacivirus. Most cases of post-transfusion 3–6 weeks.
NANB hepatitis were caused by HCV. Most new infections with ❏ Antibodies are directed against core, envelope, and NS3 and NS4
HCV are subclinical. proteins and tend to be relatively low in titer. Nucleic acid-based
❏ The majority (70–90%) of HCV patients develop chronic hepatitis, assays (eg, reverse transcription PCR) detect the presence of
and many are at risk of progressing to chronic active hepatitis and circulating HCV RNA and are useful for diagnosis of acute infection
cirrhosis (10–20%). In 1–5% of infected individuals, HCV leads to soon after exposure and for monitoring patients on antiviral
hepatocellular carcinoma, which is the fifth most common cause therapy.
of cancer worldwide.
ASSAY RESULT INTERPRETATION
LABORATORY TESTS
Anti-HCV Positive Current or Past infection with HCV

EPIDEMIOLOGY
❏ Infections by HCV are extensive throughout the world. The World
Health Organization estimates that about 1% of the world
population has been infected, with population subgroups in Africa
having prevalence rates as high as 10%. Other high-prevalence
areas are found in South America and Asia.
❏ HCV is transmitted primarily through direct percutaneous
exposures to blood, although in 10–50% of cases, the source of
HCV cannot be identified. In roughly decreasing order of
prevalence of infection are:
➔ Injecting drug users (∼80%)
➔ Individuals with hemophilia treated with clotting factor
products before 1987
➔ Recipients of transfusions from HCV-positive donors,
chronic hemodialysis patients (10%)
➔ Persons who engage in high-risk sexual practices
➔ Health care workers (1%)
Figure 5.8 Clinical and serologic events associated with hepatitis C virus ❏ The virus can be transmitted from mother to infant, although not as
(HCV) frequently as for HBV. Estimates of mother-to-child vertical
INFECTION: transmission vary from 3% to 10%. Mothers with higher HCV viral
❏ RT PCR - confirmation of ELISA loads or coinfection with HIV more frequently transmit HCV. No risk
❏ ELISA of transmission has been associated with breastfeeding. HCV was
NOTE: ALT increases since HCV affects the liver. found in saliva from more than one-third of patients with HCV and
HIV co infections.
❏ Serologic assays are available for diagnosis of HCV infection. ❏ HCV infection has been associated with tattooing and, in some
Enzyme immunoassays detect antibodies to HCV but do not countries, with folk medicine practices.
distinguish among acute, chronic, or resolved infection ❏ HCV can be transmitted to an organ transplant recipient from an
because anti-HCV antigens persist for life HCV-positive donor.

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❏ The average incubation period for HCV is 6–7 weeks. The
average time from exposure to seroconversion is 8–9 weeks,
and about 90% of patients are anti-HCV positive within 5 months.

TREATMENT
❏ Orthotopic liver transplantation is a treatment for chronic
hepatitis B and C end-stage liver damage.
❏ Pegylated interferon combined with ribavirin has been the
standard treatment for chronic hepatitis C.
❏ First-generation protease inhibitor drugs
➔ Telaprevir
➔ Boceprevir
● They are given in combination with interferon and
ribavirin.
❏ Second-generation protease inhibitor drugs
➔ Sofosbuvir Figure 5.9 Structural representation of hepatitis B and delta viruses.
● These drugs have less toxicity than
first-generation antivirals, and greater efficacy. LABORATORY TESTS
❏ RT-PCR
PREVENTION AND CONTROL ❏ ELISA
❏ There is currently no vaccine for Hepatitis C

HEPATITIS D VIRUS (HDV)

INTRODUCTION
❏ An antigen–antibody system termed the delta antigen (deltaAg)
and antibody (anti-delta) is detected in some HBV infections. The
antigen is found within certain HBsAg particles. In blood, HDV
(delta agent) contains delta-Ag (HDAg) surrounded by an HBsAg
envelope.
❏ HDV is a defective virus that requires the HBsAg coat for
transmission that is why they are only found in individuals with
acute or chronic Hepatitis B infection. No Hep B, no Hep D
❏ The genome of HDV consists of single-stranded, circular,
negative-sense RNA.
❏ It is the smallest of known human pathogens and resembles
subviral plant pathogens (ie, viroids. HDAg is the only protein
coded for by HDV RNA and is distinct from the antigenic
determinants of HBV. HDV is a defective virus that requires the Figure 5.10
HBsAg coat for transmission.
❏ It is often associated with the most severe forms of hepatitis in ❏ Because HDV depends on a coexistent HBV infection, acute type
HBsAg-positive patients. It is classified in the Deltavirus genus, D infection occurs either as a simultaneous infection (coinfection)
which is not assigned to any virus family. with HBV or as a superinfection of a person chronically infected
with HBV.

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 ❏ In the coinfection pattern, antibody to HDAg develops late in the transfusions, intravenous drug abusers, and their close contacts
acute phase of infection and may be of low titer. Assays for HDAg are at high risk.
or HDV RNA in the serum or for IgM-specific anti-HDV are ❏ The primary routes of transmission are believed to be similar to
preferable. All markers of HDV replication disappear during those of HBV, although HDV does not appear to be a sexually
convalescence; even the HDV antibodies may disappear within transmitted disease.
months to years. ❏ Infection depends on HBV replication because HBV provides
❏ Superinfection by HDV usually results in persistent HDV infection an HBsAg envelope for HDV. The incubation period varies from 2
(>70% of cases). High levels of both IgM and IgG anti-HD persist, to 12 weeks, being shorter in HBV carriers who are superinfected
as do levels of HDV RNA and HDAg. HDV superinfections may be with the agent than in susceptible persons who are simultaneously
associated with fulminant hepatitis. infected with both HBV and HDV. HDV has been transmitted
perinatally, but fortunately, it is not prevalent in regions of the world
(e.g, Asia) where perinatal transmission of HBV occurs frequently.
ASSAY RESULT INTERPRETATION
TREATMENT
Anti-HDV (+), HBsAg(+) Infection with HDV
❏ Treatment of patients with hepatitis D is supportive (treatment of
symptoms only) and directed at allowing hepatocellular damage
Anti-HDV (+), Anti-HBc IgM (+) Coinfection with HDV and HBV
to resolve and repair itself.
❏ Vaccination for Hepatitis B can prevent Hepatitis D since HBV is
Anti-HDV (+), Anti-HBc IgM (-) Superinfection of Chronic HBV
needed by HDV.
infection with HDV
PREVENTION AND CONTROL
SIR ROBERT ❏ Delta hepatitis can be prevented by vaccinating HBV susceptible
persons with hepatitis B vaccine.
Anti HDV Anti HBc IgM Total Anti HBc
❏ However, vaccination does not protect hepatitis B carriers from
superinfection by HDV.
+ + + coinfection
HEPATITIS E VIRUS (HEV)
+ - + superinfection
INTRODUCTION
❏ HEV is transmitted enterically and occurs in epidemic form in
NOTE: developing countries, where water or food supplies are sometimes
❏ IgG cannot be detected on its own, that is why we test for the total fecally contaminated. (can cause epidemic)
Anti HBc. ❏ The viral genome has been cloned and is a positive-sense,
❏ If Anti HBc IgM is negative but positive for total HBc this means single-stranded RNA. The virus is classified in the virus family,
that the one responsible for making the total anti HBc positive was Hepeviridae, in the genus Hepevirus. HEV resembles, but is
anti HBc IgG. IgG is present in chronic Hepatitis B distinct from, caliciviruses.
❏ Animal strains of HEV are common throughout the world. There is
EPIDEMIOLOGY evidence of HEV or HEV-like infections in rodents, pigs, sheep,
❏ HDV is found throughout the world but with a nonuniform and cattle in the United States, with occasional transmission to
distribution. Its highest prevalence has been reported in Italy, the humans. (zoonotic disease)
Middle East, central Asia, West Africa, and South America. HDV
infects all age groups. Persons who have received multiple EPIDEMIOLOGY
❏ Hepatitis E is found worldwide, but the prevalence is highest in
East and South Asia

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 TREATMENT
❏ Treatment of patients with hepatitis E is supportive and directed at
allowing hepatocellular damage to resolve and repair itself.
❏ Self limiting
❏ A vaccine to prevent hepatitis E virus infection has been developed
and is licensed in China, but is not yet available elsewhere.
❏ Immunization only provides short-term protection and
there is no specific therapy available

PREVENTION AND CONTROL

❏ A vaccine to prevent hepatitis E virus infection has been developed
and is licensed in China, but is not yet available elsewhere.
Immunization only provides short-term protection and there is no
specific therapy available
NOTE: Hepatitis transmission:
❏ Fecal oral : A and E
❏ Parenteral: B,C,D

NEVER SHALL WE FAIL!

#GetThatRMTBro!

MLS 13A: MYCOLOGY & VIROLOGY | RMT 2022 | Page 12

 MODULE 6
Characteristics, Laboratory Tests,
Epidemiology, Prevention, and
Control of Picornaviruses

Prepared by: John Robert D. Fundal, RMT

INTRODUCTION
Picornaviruses

• Picornaviruses represent a very large virus family with respect to the

number of members but one of the smallest in terms of virion size and
genetic complexity.

• They include two major groups of human pathogens:

1. Enteroviruses

2. Rhinoviruses
Properties of Picornaviruses
Property Description
Virion ✓ Icosahedral
✓ 28–30 nm in diameter
✓ Contains 60 subunits
Composition ✓ RNA (30%)
✓ Protein (70%)
Genome ✓ Single-stranded RNA
✓ Linear
✓ Positive sense
✓ 7.2–8.4 kb in size
✓ Molecular weight 2.5 millio
✓ Infectious
✓ Contains genome-linked protein (VPg)
Proteins ✓ Four major polypeptides cleaved from a large precursor polyprotein.
✓ Surface capsid proteins VP1 and VP3 are major antibody-binding sites.
✓ VP4 is an internal protein.
Envelope ✓ None
Replication ✓ Cytoplasm
Outstanding Characteristics Family is made up of many enterovirus and rhinovirus types that infect humans and lower
animals, causing various illnesses ranging from poliomyelitis to aseptic meningitis to the
common cold.
Structure of Picornaviruses
Classification of Picornaviruses
Replication of Picornaviruses
Serotypes of some Picornaviruses

Class Serotypes

Poliovirus 3

Coxsackievirus
Group A 23

Group B 6
Echovirus 32

Enterovirus 4

Parechovirus 8
GENUS ENTEROVIRUS
Enteroviruses
• Species and subtypes to consider:

1. Poliovirus

2. Coxsackievirus

3. ECHO Viruses and other Enterovirus

4. Enterovirus in the Environment

1. Poliovirus

• Poliomyelitis is an acute infectious disease that in its serious form affects

the central nervous system (CNS).
✓ The destruction of motor neurons in the spinal cord results in
flaccid paralysis.

• Most poliovirus infections are subclinical.

• Poliovirus has served as a model enterovirus in many laboratory studies

of the molecular biology of picornavirus replication.
Pathogenesis of Poliomyelitis
Pathogenesis of Poliomyelitis
Clinical Findings
Disease Description
Mild Disease ✓ Most common form of poliovirus infection
✓ Symptoms: Fever, Malaise, Drowsiness, Headache, Nausea,
Vomiting, Constipation and Sore Throat
✓ Complete recovery occurs in few days
Non-Paralytic Poliomyelitis (Aseptic ✓ Same symptoms with Mild Disease with added features of
Meningitis) stiffness and pain in back and neck
✓ Disease lasts for 2-10 days with rapid and complete recovery
Paralytic Poliomyelitis ✓ The destruction of lower motor neurons in the spinal cord
results in flaccid paralysis
✓ Invasion of Poliovirus in the brainstem may lead to
incoordination and painful muscle spasms
✓ Maximal recovery in six months
Progressive Post Poliomyelitis Muscle ✓ Infected individuals may develop paralysis and muscle
Atrophy wasting years after poliovirus infection
Laboratory Diagnosis
• RT-PCR

• Human or monkey cell culture

• Sample of choice:

✓ Throat swabs

✓ Rectal swabs

✓ Stool samples
Epidemiology
• Before global eradication efforts began, poliomyelitis occurred
worldwide—year-round in the tropics and during summer and fall in the
temperate zones.

• There are three wild types of poliovirus (WPV)

• The disease occurs in all age groups, but children are usually more
susceptible than adults because of the acquired immunity of the adult
population.

• Humans are the only known reservoir of infection.

Prevention and Control
• There are no antiviral drugs for treatment of Poliovirus infection. The
WHO launched a campaign for global eradication of Poliovirus in 1988
through Vaccination:

1. Formalin Inactivated Poliovirus vaccine (Salk) – Injection

2. Live Attenuated Polio vaccine (Sabin) – Oral Administration

PEOPLE NEED TO BE PROTECTED AGAINST ALL THREE

TYPES IN ORDER TO PREVENT POLIO DISEASE
2. Coxsackieviruses

• Coxsackieviruses are named for Coxsackie, New York, where the viruses
were first isolated.

• Coxsackieviruses, a large subgroup of the enteroviruses, were divided into

two groups:

1. Coxsackievirus A

2. Coxsackievirus B
Clinical Findings
Clinical Findings
 NOTE

Hand-foot-and-Mouth disease is not to be

confused with foot-and-mouth disease of cattle,
which is caused by an unrelated picornavirus that
does not normally infect humans.
Laboratory Diagnosis
• Sample of Choice:
✓ Throat swab and washings
✓ Nasal Secretions
✓ CSF
✓ Stool

• Molecular Diagnostic Test

✓ RT-PCR

• Serologic Test
✓ Immunofluorescence
Epidemiology
• Encountered around the globe.

• Isolations have been made mainly from human feces, pharyngeal swabs,
and sewage.

• The most frequent types of coxsackieviruses recovered worldwide over an

8-year period (1967–1974) were types A9 and B2–B5.

• Familial exposure is important in the acquisition of infections with

coxsackieviruses
Prevention and Control
• There are no vaccines or antiviral drugs currently available for prevention
or treatment of diseases caused by coxsackieviruses

• Supportive treatment
3. ECHO viruses and other Enterovirus
A. ECHO virus (Enteric Cytopathogenic Human Orphan Viruses)

• Grouped together because they infect the human enteric tract

and because they can be recovered from humans only by
inoculation of certain tissue cultures.

• More than 30 serotypes are known but not all have been
associated with human illness.

• Many echoviruses have been associated with aseptic meningitis.

3. ECHO viruses and other Enterovirus
Enterovirus 68 Enterovirus 70 Enterovirus 71
Shares several characteristics with The chief cause of acute The most common Enterovirus
rhinoviruses hemorrhagic conjunctivitis. recovered from fecal samples of
patients with acute flaccid paralysis .
Previously classified as rhinovirus 87

Acute Hemorrhagic Conjunctivitis

Laboratory Diagnosis
• RT-PCR

• Serologic tests
✓ Impractical because of the many different viral types

• Specimen of choice:
✓ Throat swabs
✓ Stool
✓ Rectal swabs
✓ CSF
Epidemiology
• ECHO Virus and other Enteroviruses occur in all parts of the globe and are
usually found in younger individuals.

• In the temperate zone, infections occur chiefly in the summer and

autumn and are about five times more prevalent in children of lower
income families
Treatment, Prevention and Control
• Avoidance of contact with patients exhibiting acute febrile illness is
advisable for very young children.

• There are no antivirals or vaccines (other than polio vaccines) available for
the treatment or prevention of any enterovirus diseases.
4. Enterovirus in the Environment
• Fecal contamination (hands, utensils, food, water) is the usual avenue of
virus spread.

• Enteroviruses are present in variable amounts in sewage.

• This may serve as a source of contamination of water supplies used for

drinking, bathing, irrigation, or recreation.

• Enteroviruses survive exposure to the sewage treatments and

chlorination in common practice, and human wastes in much of the world
are discharged into natural waters with little or no treatment.
Transmission
5. Parechovirus
• This genus was defined in the 1990s and contains 19 types, of which
types 1 and 2 were originally classified as echoviruses 22 and 23

• The viruses replicate in the respiratory and gastrointestinal tracts.

• Human parechovirus cannot be detected by enterovirus-specific nucleic

acid typing assays commonly used, so it may be underreported
GENUS RHINOVIRUS
Rhinovirus

• Rhinoviruses are the common cold viruses.

• They are the most commonly recovered agents from people with mild
upper respiratory illnesses.

• They are usually isolated from nasopharyngeal secretions but may also be
found in throat and oral secretions.
Rhinovirus

• Human rhinoviruses can be divided into major and minor receptor

groups:

1. Major group of Rhinovirus - use intercellular adhesion molecule-1

(ICAM-1) as receptor

2. Minor group of Rhinovirus - bind members of the low-density

lipoprotein receptor (LDLR) family.
Clinical Findings

• The incubation period is brief—from 2 to 4 days—and the acute illness

usually lasts for 7 days, although a nonproductive cough may persist for
2–3 weeks.

• The average adult has one or two attacks each year

Epidemiology

• The disease occurs throughout the world.

• The virus is believed to be transmitted through close contact by means of

virus-contaminated respiratory secretions.

• The fingers of a person with a cold are usually contaminated, and

transmission to susceptible persons then occurs by hand-to-hand, hand-
to-eye, or hand-to-object-to-hand (e.g. doorknob) contamination.
Treatment, Prevention and Control

• No specific prevention method or treatment is available.

GENUS APHTHOVIRUS
Aphthovirus
• Causes Foot-and-Mouth disease.

• This highly infectious disease of cloven-hoofed animals such as cattle,

sheep, pigs, and goats.

• It may be transmitted to humans by contact or ingestion.

Foot and Mouth Disease
Prevention and Control
• Slaughtering all exposed animals and destroying their carcasses.

• Vaccines could be given to animals.

• Strict quarantine can also help control the spread of virus:

✓ Area is not presumed to be safe until susceptible animals fail to

develop symptoms within 30 days
✓ Quarantine the herd and vaccinate all unaffected animals
✓ Forbid the importation of potentially infective materials such as
meat.
END
 MODULE 7
Characteristics, Laboratory Tests,
Epidemiology, Prevention, and
Control of Gastrointestinal Viruses

Prepared by: John Robert D. Fundal, RMT

INTRODUCTION
Introduction
• Viral gastroenteritis is caused by rotaviruses, caliciviruses, astroviruses,
and some serotypes of adenoviruses, which results in vomiting and/or
diarrhea.

Family Reoviridae Family Caliciviridae Family Astroviridae

• Genus Orthoreovirus • Genus Norovirus (Norwalk • Genus Mamastrovirus
• Genus Coltivirus viruses)
• Genus Rotavirus • Genus Sapovirus
• Genus Orbivirus • Genus Nebovirus
• Genus Reovirus • Genus Lagovirus
• Genus Vesivirus
Etiologic agents of Gastroenteritis
Etiologic agents of Gastroenteritis
Special Feature Rotavirus Calicivirus Astrovirus Adenovirus
Biologic
Nucleic acid ds RNA ss (+) RNA ss (+) RNA ds DNA
Shape Naked, icosahedral Naked, icosahedral Naked, star- Naked,
shaped icosahedral
Pathogenic
Site of Infection Duodenum, Jejunum Small Intestine Small Intestine
Jejunum
Epidemiologic
Epidemicity Epidemic or Sporadic Family and Sporadic Sporadic
Community
Outbreaks
Seasonality Usually winter Unknown Unknown Unknown
Ages primarily Infants, Children Older children and Infants, Infants,
affected aged <2 years old adults Children Children
Method of Fecal-oral Fecal-oral Fecal-oral Fecal-oral
Transmission
Major diagnostic RT-PCR, EIA, EM RT-PCR, EM, IEM RT-PCR, EM PCR, EIA, EM
Tests
FAMILY REOVIRIDAE
Reoviridae

Subfamily Sedoreovirinae
Subfamily Spinareovirinae • Genus Rotavirus
• Genus Orthoreovirus - Has 8 Species (A-H)
- Only A, B and C can infect Humans
• Genus Coltivirus
• Genus Orbivirus
Properties of Reoviridae
Property Description
Virion ✓ Icosahedral
✓ 60–80 nm in diameter
✓ Double capsid shell
Composition ✓ RNA (15%)
✓ Protein (85%)
Genome ✓ Double-stranded RNA
✓ Linear
✓ Segmented (10–12 segments)
✓ Total genome size 16–27 kbp
Proteins ✓ Nine structural proteins
✓ Core contains several enzymes
Envelope ✓ None (transient pseudoenvelope is present during
rotavirus particle morphogenesis)
Replication ✓ Cytoplasm; virions not completely uncoated
Outstanding Characteristics ✓ Genetic reassortment occurs readily
✓ Rotaviruses are the major cause of infantile diarrhea
✓ Reoviruses are good models for molecular studies of
viral pathogenesis
Replication of Reoviridae
1. Genus Rotavirus
Pathogenesis of Rotavirus
Clinical Findings

Rotaviruses cause the

major portion of diarrheal
illness in infants and
children worldwide but
not in adults.
Laboratory Diagnosis

• Sample of Choice:
✓ Stool

• RT-PCR

• Serologic test:
✓ EIA
Epidemiology

• Rotaviruses are the single most important worldwide cause of

gastroenteritis in young children.

• Rotavirus is transmitted by fecal–oral route, and the virus particle is

partially digested in the gastrointestinal tract
Treatment

• Treatment of gastroenteritis is supportive to correct the loss of water and

electrolytes that may lead to dehydration, acidosis, shock, and death.

• Rotavirus vaccine
2. Reovirus

• The viruses of this genus, which have been studied most thoroughly by
molecular biologists, are not known to cause human disease.

• Reoviruses contain a hemagglutinin for human group O or bovine

erythrocytes.
3. Genus Orbivirus

• They commonly infect insects, and many are transmitted by insects to

vertebrates.

• None of these viruses cause serious clinical disease in humans, but they
may cause mild fevers.

• Serious animal pathogens include:

✓ Bluetongue virus of sheep
✓ African horse sickness virus.
4. Genus Coltivirus

• Colorado Tick Fever Virus

✓ Transmitted by ticks
✓ Able to infect humans
✓ Clinical Findings:
▪ Fever, chills, headache, photophobia
▪ Myalgia, arthralgia, lethargy
▪ Rash
▪ systemic symptoms in infected patients
✓ Colorado Tick Fever Virus infection can lead to encephalitis or
meningitis
FAMILY CALICIVIRIDAE
Properties of Caliciviruses
Property Description
Virion ✓ Icosahedral
✓ 27–40 nm in diameter
✓ Cup-like depressions on capsid surface
Genome ✓ Single-stranded RNA
✓ Linear
✓ Positive-sense
✓ Non-segmented
✓ 7.4–8.3 kb in size
✓ Contains genome-linked protein (VPg)
Proteins ✓ Polypeptides cleaved from a precursor polyprotein;
capsid is composed of a single protein
Envelope ✓ None
Replication ✓ Cytoplasm
Outstanding Characteristics ✓ Noroviruses are major cause of nonbacterial
epidemic gastroenteritis
✓ Human viruses are non-cultivable
1. Genus Norovirus

• Noroviruses or Norwalk viruses

✓ the most important cause

of epidemic viral
gastroenteritis in adults
Laboratory Diagnosis
• Specimens/Samples:
✓ Feces
✓ Vomitus
✓ Food
✓ Water

• RT-PCR

• Electron microscope
✓ Immune Electron Microscopy

• ELISA
Epidemiology

• The viruses are most often associated with epidemic outbreaks of

waterborne, foodborne, and shellfish-associated gastroenteritis.

• Fecal–oral spread is the primary means of transmission.

• Characteristics of norovirus include a low infectious dose (as few as 10

virus particles), relative stability in the environment, and multiple modes
of transmission.
Treatment, Prevention and Control

• Symptomatic Treatment

• There is currently no vaccine

available
FAMILY ASTROVIRIDAE
Astroviruses
• Exhibit a distinctive starlike morphology
in the electron microscope.

• They contain single-stranded, positive-

sense RNA

• Contains two genera; all human viruses

are classified in the Mamastrovirus
genus
Epidemiology

• Cause diarrheal illness and may be shed in extraordinarily large quantities

in feces.

• The viruses are transmitted by the fecal–oral route through contaminated

food or water, person-to-person contact, or contaminated surfaces.
Laboratory Diagnosis
• RT-PCR

• Electron microscopy
ENTERIC ADENOVIRUS AND
“CANDIDATE VIRUSES”
1. Enteric Adenovirus
• Adenoviruses that can cause gastroenteritis:
✓ Adenovirus serotype 38
✓ Adenovirus serotype 40
✓ Adenovirus serotype 41

• They are transmitted by fecal–oral route and the incubation period is 8

to 10 days and the symptoms of gastroenteritis last for 5 to 12 days.
2. Candidate Viruses
• Some other candidate viruses such as coronavirus-like agents, toroviruses
may cause diarrhea

• Group A coxsackieviruses may cause gastroenteritis in severely

immunocompromised patients
END
 University of San Agustin
ILOILO CITY, PHILIPPINES
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS
MEDICAL LABORATORY SCIENCE PROGRAM

MODULE PACKETS IN MLS 13A:

MYCOLOGY AND
VIROLOGY
M8

Matthew I. Tubola, RMT, MSMT

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John Robert D. Fundal, RMT

MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
© 2021. Printed in Calibri.
Cover Design: J.R.D. Fundal, RMT
All rights reserved. Copyright applied for. No part of this module may be reproduced, stored in retrieval
system or transmitted in any forms or by any means, electronic, mechanical, photocopying or otherwise,
without written permission from the authors.

The authors have done everything possible to make this module accurate and in accordance with accepted
standards. The authors are not responsible for errors or omissions or for consequences (loss, damage, or
disruption) from application of the module, and make no warranty, expressed or implied in regard to the
contents of the module. Any practice described in this module should be applied by the reader in accordance
with accepted standards used in regard to unique circumstances that may apply in each situation.

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LEARNER’S HONOR CODE STATEMENT FOR THIS MODULE PACKET

“I affirm that I will not give or receive any unauthorized help on this MODULE, and that all
work will be my own.”
“I affirm that I have not given or received any unauthorized help on this assignment, and that
this work is my own.”

_________________________
Signature over printed name

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 ABOUT THE AUTHORS

MATTHEW I. TUBOLA, RMT, MSMT

Hello Augustinians!
Welcome to MLS-13A (Mycology and Virology Lecture), I will be your lecturer for this subject. I am a
graduate of BSMT of this University and also finished my Master’s degree in Medical Technology in this
University. I have been teaching in this University for 12 years teaching Medical Technology Professional
Subjects.

JOHN ROBERT D. FUNDAL, RMT

Hello Augustinians!
Welcome to MLS-13A (Mycology and Virology Lecture), I will also be your lecturer for this subject. I
obtained my Bachelor’s Degree in Medical Laboratory Science from University of San Agustin. This is
currently my third semester in teaching MLS subjects.

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 TABLE OF CONTENTS
Disclaimer 1
Honor Code 2
About the Authors 3
Content

Arboviruses
Family Bunyaviridae 9
Family Flaviviridae 10
Family Reoviridae 14
Family Togaviridae 15
Non-Arboviruses
Family Arenaviridae 16
Family Bunyaviridae 19
Family Filoviridae 19
Family Rhabdoviridae 21
References 24

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 UNIT MAP
Characteristics, Laboratory Tests,
Epidemiology, Prevention, and
Control of Zoonotic Viruses

ARBOVIRUSES NON-ARBOVIRUSES

Family Bunyaviridae Family Arenaviridae

Family Flaviviridae Family Bunyaviridae

Family Reoviridae Family Filoviridae

Family Togaviridae Family Rhabdoviridae

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 PART 1: OVERVIEW OF DISCUSSION

The zoonotic viruses comprise of more than 400 viral agents, one or more of which occur in most parts
of the world. Members of the group have their ultimate reservoirs in insects or lower vertebrates.
The arthropod-borne viruses (arboviruses) and rodent-borne viruses represent ecologic groupings of
viruses with complex transmission cycles involving arthropods or rodents. These viruses have diverse physical
and chemical properties and are classified in several virus families.

PART 2: LEARNING OBJECTIVES

At the end of the lecture, the student shall be able to:

• Know the properties of Arthropod-borne viruses and Zoonotic viruses

• Describe the clinical findings and laboratory diagnosis of human infections of Arthropod-borne viruses
and Zoonotic viruses
• Describe the epidemiology, methods of treatment, prevention, and control of Arthropod-borne viruses
and Zoonotic viruses

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 PART 3: DISCUSSION

A. INTRODUCTION

The zoonotic viruses discussed here are divided into two groups: Arthropod-borne (arboviruses) and non-
arthropod-borne zoonotic viruses.
Arthropod-borne Viruses (Arbovirus) Non-Arthropod-borne Viruses
• Transmitted to humans by infected blood- • Transmitted by inhalation of infected animal excretions,
sucking insects, such as mosquitoes, ticks, by the conjunctival route, or occasionally by direct
and Phlebotomus flies (sandflies). contact with infected animal (e.g., animal bite)

In most cases, the zoonotic viruses were first named after the place or region of initial isolation or reported
infection (e.g., St. Louis encephalitis virus, West Nile virus, Zika virus) or after the disease produced (e.g., yellow fever).
More recent studies have assigned the majority to families and genera on the basis of properties including morphologic
and genetic features, geographic distribution, and disease spectrum.

B. CLASSIFICATION OF ARBOVIRUSES

Table 8.1 Arboviruses of Major Importance to Humans

Genus and Members Primary Arthropod Vector Usual Disease Expression
FAMILY BUNYAVIRIDAE
Bunyavirus
• California Virus Mosquito Encephalitis
• Bunyamwera virus Mosquito Febrile illness

Phlebovirus
• Rift valley fever virus Mosquito Febrile illness
• Sandfly fever virus Phlebotomus Febrile illness
• Heartland virus Tick Febrile illness

Nairovirus
• Crimean-Congio hemorrhagic Tick Febrile illness
fever virus
FAMILY FLAVIVIRIDAE
Flavivirus
• St. Louis encephalitis virus Mosquito Encephalitis
• Japanese B encephalitis virus Mosquito Encephalitis
• Dengue virus Mosquito Febrile illness or hemorrhagic fever
• Yellow fever virus Mosquito Hepatic necrosis, hemorrhage
• West Nile virus Mosquito Febrile illness or Encephalitis
• Zika virus Mosquito Febrile illness, birth defects
• Murray Valley encephalitis virus Mosquito Encephalitis
• Powassan virus Tick Encephalitis
FAMILY REOVIRIDAE
Reovirus
• Coltivirus
• Colorado tick fever virus Tick Febrile illness
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 Genus and Members Primary Arthropod Vector Usual Disease Expression
FAMILY TOGAVIRIDAE
Alphavirus
• Western equine encephalitis Mosquito Encephalitis
virus
• Eastern equine encephalitis Mosquito Encephalitis
virus
• Venezuelan equine encephalitis Mosquito Encephalitis
virus
• Chikungunya virus Mosquito Febrile illness
• Ross River virus Mosquito Febrile illness

C. CLASSIFICATION OF NON-ARBOVIRUSES

Table 8.2 Non-Arbovirus of Major Importance to Humans

Genus and Members Primary Vector Usual Disease Expression
FAMILY ARENAVIRIDAE
Junin virus Calomys musculinus (Drylands Vesper Mouse) Argentinean hemorrhagic fever

Lassa virus Mastomys natalensis (Natal Multimammate Lassa fever

Mouse)

Machupo virus Larger vasper mouse Bolivian hemorrhagic fever

Whitewater Arroyo virus Woodrat Hemorrhagic fever

Chapare virus Rodent Hemorrhagic fever

Lugo virus Rodent Hemorrhagic fever

Lymphocytic choriomeningitis House mouse, Hamsters CNS infections

virus (LCMV)
FAMILY BUNYAVIRIDAE
Hantavirus
• Hantavirus Peromyscus maniculatus Deer mouse Hantavirus pulmonary syndrome

• Hantaan virus Apodemus species (rodent) Hemorrhagic fever with renal syndrome

• Puumala virus Clethrionomys (bank vole) Hemorrhagic fever with renal syndrome

• Dobrava virus Apodemus species Hemorrhagic fever with renal syndrome

• Seoul virus Rattus (brown rat) Hemorrhagic fever with renal syndrome

• Saaremaa virus Apodemus species Hemorrhagic fever with renal syndrome

FAMILY FILOVIRIDAE
Marburg virus African monkeys Hemorrhagic fever

Ebola virus Fruit bats, apes, monkeys and Duikers Hemorrhagic fever

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 ARBOVIRUS INFECTION IN HUMANS
A. FAMILY BUNYAVIRIDAE

Table 8.3 Properties of Bunyaviruses

Property Description
Virion • Spherical
• 80-120 nm
Genome • Single-stranded
• Negative-sense or ambisense RNA
• Triple segmented
Envelope • Present with 2 glycoproteins
Replication • Cytoplasm

Figure 8.1 Virion structure of Bunyaviruses. The virions of bunyaviruses contain single-stranded, negativesense RNA
viruses that are spherical and enveloped with an external diameter of 80 to 120 nm. The envelope contains two
glycoproteins, G1 and G2, and encloses three helical nucleocapsids containing RNA, namely, large (L), medium (M), and
small (S), associated with an RNA-dependent RNA polymerase (L) and nonstructural proteins (N).

There are four genera of Bunyaviridae family:

1. Bunyavirus – Negative sense RNA

2. Phlebovirus – Negative sense RNA
3. Nairovirus - Ambisense RNA
4. Hantavirus – Negative sense RNA

All bunyaviruses are arboviruses, except Hantavirus, which is a non-arthropod zoonotic virus and discussed later.

A.1. La Crosse virus

La Crosse virus is a significant virus under the California encephalitis virus complex, and is a major cause of
encephalitis and aseptic meningitis in children in the upper Midwest part of the world.

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 The onset of California encephalitis viral infection is abrupt, typically with severe headache, fever, and in some
cases vomiting and convulsions. About half of patients develop seizures, and the case-fatality rate is about 1%. Less
frequently, patients have only aseptic meningitis. The illness lasts from 10 to 14 days, although convalescence may be
prolonged. Neurologic sequelae are rare. There are many infections for every case of encephalitis. Serologic confirmation
by Hemagglutination Inhibition, ELISA, or neutralization tests is done on acute and convalescent specimens

A.2. Sandfly Fever Virus

Sandfly fever is a mild, insect-borne disease. Sandfly fever (also called Phlebotomus fever) is caused by a
bunyavirus in the Phlebovirus genus. The disease is transmitted by the female sandfly, Phlebotomus papatasi.

The disease begins abruptly after an incubation period of 3–6 days. The virus is found in the blood briefly near the
time of onset of symptoms. Clinical features consist of headache, malaise, nausea, fever, photophobia, stiffness of the
neck and back, abdominal pain, and leukopenia. All patients recover and there is no specific treatment.

Prevention of disease in endemic areas relies on use of insect repellents during the night and residual insecticides
around living quarters.

A.3. Rift Valley Fever Virus

A mosquito-borne zoonotic virus pathogenic primarily for domestic livestock. Humans are secondarily infected
during the course of epizootics in domesticated animals.

Epizootics occur periodically after heavy rains that allow hatches of the primary vector and reservoir (Aedes
species mosquitoes). Viremia in animals leads to infection of other vectors with collateral transmission to humans.
Transmission to humans is primarily by contact with infected animal blood and body fluids and mosquito bites.

Disease in humans is usually a mild febrile illness that is short lived, and recovery almost always is complete.
Complications include retinitis, encephalitis, and hemorrhagic fever. Permanent loss of vision may occur (1–10% of cases
with retinitis). About 1% of infected patients die.

A.4. Heartland Virus

Patients infected with this virus presented fever, fatigue, anorexia, nausea, or diarrhea and had leukopenia,
thrombocytopenia, and elevated liver enzymes. It is thought that Lone Star ticks transmit the virus. Another related
Phlebovirus, Lone Star virus, has been recovered from Lone Star ticks and can infect human cell lines, but no human cases
have been reported.

B. FAMILY FLAVIVIRIDAE

Table 8.4 Properties of Flaviviruses

Property Description
Virion • Icosahedral capsid
• 40-60 nm
Genome • Single-stranded
• Positive sense RNA
Envelope • Present with 2 glycoproteins
Replication • Cytoplasm
Outstanding Characteristics • All flaviviruses are antigenically related
• Hepatitis C virus, classified in a separate genus in the
Flaviviridae family, has no arthropod vector and is not an
arbovirus

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Figure 8.2 Virion Structure of Flaviviridae Two types of virions, intracellular and extracellular virions, are shown. The
positive-sense, single stranded RNA genome is packaged into an icosahedral capsid wrapped into a lipid bilayer envelope
containing membrane (M) protein and spike glycoprotein (E). The prM is the precursor to M protein. The size of flavivirus
virion ranges from 40 to 60 nm in diameter. There are two major differences between intracellular and extracellular
virions; intracellular virions have only prM and E as monomer, whereas extracellular virions have prM and M and E as
dimer.

A. Pathogenesis of Flaviviruses
In susceptible vertebrate hosts, primary viral multiplication occurs either in myeloid and lymphoid cells or in
vascular endothelium. Multiplication in the central nervous system depends on the ability of the virus to pass the blood–
brain barrier and to infect nerve cells. In natural infection of birds and mammals, an inapparent infection is usual. For
several days there is viremia, and arthropod vectors acquire the virus by sucking blood during this period—the first step
in its dissemination to other hosts.

In the vast majority of infections, the virus is controlled before neuro-invasion occurs. Invasion depends on many
factors, including the level of viremia, the genetic background of the host, the host innate and adaptive immune responses,
and the virulence of the virus strain. Humans show an age-dependent susceptibility to central nervous system infections,
with infants and elderly adults being most susceptible

B. Laboratory Diagnosis for most Arboviruses

Samples Direct Detection Serology
• Blood • Common Cell lines • Hemagglutination Inhibition
• CSF • Mosquito Cell lines Test
• Intracerebral inoculation of • ELISA
suckling mice or hamsters
• RT-PCR

B.1. St. Louis Encephalitis Virus

St. Louis encephalitis virus is the most important cause of epidemic encephalitis of humans in North America. The
presence of infected mosquitoes is required before human infections can occur, although socioeconomic and cultural
factors (air conditioning, screens, mosquito control) affect the degree of exposure of the population to these virus-carrying
vectors.
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 B.2 Japanese B Encephalitis Virus
Japanese B encephalitis is the leading cause of viral encephalitis in Asia. The mortality rate can exceed 30%. A high
percentage of survivors (up to 50%) are left with neurologic and psychiatric sequelae. Infections during the first and second
trimesters of pregnancy have reportedly led to fetal death.

. There is no treatment. Several effective Japanese encephalitis vaccines are available in Asia. An inactivated Vero
cell culture-derived vaccine was licensed in the United States in 2009.

B.3. West Nile Fever Virus

It is now the leading cause of arboviral encephalitis in the United States. It is estimated that about 80% of West
Nile infections are asymptomatic, with about 20% causing West Nile fever and less than 1% causing neuro-invasive disease
(meningitis, encephalitis, or acute flaccid paralysis).

West Nile virus produces viremia and an acute, mild febrile disease with lymphadenopathy and rash. Transitory
meningeal involvement may occur during the acute stage. Only one antigenic type of virus exists, and immunity is
presumably permanent.

A West Nile vaccine for horses became available in 2003. There is no human vaccine. Prevention of West Nile virus
disease depends on mosquito control and protection against mosquito bites.

B.4. Zika Virus

Zika virus can be found in blood, urine, and other body fluids including semen, leading to potential sexual
transmission. Most infected individuals are asymptomatic, others can develop rash, arthralgia, conjunctivitis, and fever.
Severe cases are rare; however, the virus can pass through the placenta during pregnancy and infect fetal neuronal tissue,
leading to microcephaly and neurologic abnormalities.

Treatment is generally supportive, and screening is available for pregnant women potentially exposed to infection.
Prevention of mosquito exposure in endemic regions is important to reduce infection rates.

B.5. Yellow Fever Virus

Introduction
Yellow fever virus is the prototype member of the Flaviviridae family. It causes yellow fever, an acute, febrile,
mosquito-borne illness that occurs in the tropics and subtropics of Africa and South America. Severe cases are
characterized by liver and renal dysfunction and hemorrhage, with a high mortality rate. There is a single serotype.

Pathogenesis
The virus is introduced by a mosquito through the skin, where it multiplies. It spreads to the local lymph nodes,
liver, spleen, kidney, bone marrow, and myocardium, where it may persist for days. It is present in the blood early during
infection.
The lesions of yellow fever are caused by the localization and propagation of the virus in a particular organ.
Infections may result in necrotic lesions in the liver and kidney. Degenerative changes also occur in the spleen, lymph
nodes, and heart. Serious disease is characterized by hemorrhage and circulatory collapse. Virus injury to the myocardium
may contribute to shock.

Laboratory Diagnosis
Samples Direct Detection Serology
• Blood • RT-PCR • ELISA – detection of IgM
• Postmortem tissue • Hemagglutination Inhibition
Test
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Epidemiology
There are two major epidemiologic cycles of transmission of yellow fever
Urban Yellow fever Sylvatic or Jungle yellow fever
Involves person-to-person transmission by domestic Primarily a disease of monkeys. Transmitted from
Aedes mosquitoes. monkey to monkey by arboreal mosquitoes. Persons
involved in forest-clearing activities come in contact
with these mosquitoes in the forest and become
infected.

Treatment, Prevention and Control

There is no antiviral drug therapy. The 17D strain of yellow fever virus is an excellent attenuated live-virus
vaccine.

B.6. Dengue Fever Virus

Introduction
DF Fever virus has four serotypes (DEN-1 to DEN-4). Recently, the fifth serotype (DEN-5) was discovered in 2013
from Bangkok. It causes Breakbone fever that is characterized by fever, severe headache, muscle and joint pain, nausea
and vomiting, eye pain, and rash. Severe forms of the disease, dengue hemorrhagic fever and dengue shock syndrome,
principally affect children. Aedes aegypti is the principal vector followed by Aedes albopictus. They bite during die day
time.

Pathogenesis
Primary dengue infection occurs when a person is infected with dengue virus for the first lime with any one
serotype. Months to years later, a more severe form of dengue illness may appear (called secondary dengue infection)
due to infection with another second serotype which is different from the first serotype causing primary infection. The
reason why secondary dengue infection is more severe is because of the Antibody Dependent enhancement.

Clinical Classifications
There are two classifications of Dengue infection

1. The traditional (1997) WHO Classifications

2. 2009 WHO Classifications
Traditional (1997) WHO Classification 2009 WHO
Classifications
1. Dengue Fever (DF) 2. Dengue hemorrhagic fever 3. Dengue shock 1. Dengue with or
(DHF) syndrome (DSS) without warning
• Abrupt onset of high fever • High grade continuous All the criteria of DHF are signs
(also called biphasic fever, fever present, and in addition
break bone fever or saddle • Hepatomegaly manifestations of shock 2. Severe dengue
back fever) • Thrombocytopenia are present, such as:
• Maculopapular rashes over • Raised hematocrit by 20%
the chest and upper limbs • Evidence of hemorrhages • Rapid and weak pulse
• Severe frontal headache which can be detected by: • Narrow pulse pressure
• Muscle and Joint pains (< 20 mm Hg) or
• Lymphadenopathy 1. Positive tourniquet test hypotension
• Retro orbital pain (>20 petechial spots per square • Presence of cold and
• Loss of appetite, nausea inch area in cubital fossa. clam my skin
and vomiting • Restlessness
2. Spontaneous bleeding from
skin, nose, mouth and gums.
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 Figure 8.3 2009 WHO Classification of Dengue Infection

Laboratory Diagnosis
Direct Detection Serology
• RT-PCR • ELISA – detection of IgG and IgM
• Hemagglutination Inhibition Test

Epidemiology
There are more than 100 countries where dengue has become endemic. These viral agents are widespread
throughout the world, particularly Africa, the Americas, the Eastern Mediterranean, South Asia, South-east Asia and the
Western Pacific, the Middle East, Africa, the Far East, and the Caribbean Islands.

Globally, it is estimated that about 100 million people are infected by dengue virus, 500 000 dengue hemorrhagic
fever cases, and 22 000 deaths mostly in children every year.

Treatment, Prevention and Control

There is no antiviral drug therapy. Dengue hemorrhagic fever can be treated by fluid replacement therapy. There
is no vaccine, but candidate vaccines are under development. Vaccine development is difficult because a vaccine must
provide protection against all four serotypes of virus. Therapeutic antibodies able to neutralize multiple genotypes of
dengue are also under development.

Control depends on anti-mosquito measures, including elimination of breeding places and the use of insecticides.
Screened windows and doors can reduce exposure to the vectors.

C. FAMILY REOVIRIDAE
Reoviruses are spherical, naked capsid icosahedral, double-stranded segmented RNA viruses. The reoviruses
described here are arboviruses that are transmitted through insect (tick) bites. The most important North American
arbovirus of this family, which is a member of the genus Coltivirus, causes Colorado tick fever in humans. The other
arboviruses from the Reoviridae family are Orbivirus which includes African horse sickness and bluetongue viruses, mainly
causing disease in animals.
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 D. FAMILY TOGAVIRIDAE
Alphavirus genus includes arboviruses within this family that infect humans.

Table 8.5 Properties of Alphaviruses

Property Description
Virion • Capsid is icosahedral
• Approximately 70mm
Genome • Positive-sense
• Single-stranded, linear RNA
Envelope • Present with 2 glycoproteins
Replication • Cytoplasm
Outstanding Characteristics • Alphaviruses have the ability to hemagglutinate via fusion of
E1 glycoprotein to lipids in erythrocyte membrane and E2
also participates in this process.

Figure 8.3 Virion structure of alphavirus. The single-stranded, positive-sense RNA genome is encapsidated into an
icosahedral capsid (C protein) wrapped by a lipid bilayer envelope (viral membrane) containing viral-encoded
glycoproteins (spikes), E1 and E2 with an external diameter of 70 nm. E1 has the ability to hemagglutinate via fusion to
lipids on erythrocyte membrane and E2 also participates in this process.

Pathogenesis

Same as Flaviviruses’

Laboratory Diagnosis

Same as Flaviviruses’

D.1. Western Equine Encephalitis

The virus is transmitted through mosquito (Culex tarsalis) bites. Horses and humans represent blind-end hosts;
both are susceptible to infection and illness, commonly manifested as encephalitis. Although human infection in endemic
areas is commonplace, overall, only 1 of 1000 infections causes clinical symptoms. However, in young infants, 1 of every
25 infections may produce severe illness. The attack rates are therefore far higher in young infants than in other groups.
The disease spectrum may range from mild, nonspecific febrile illness to aseptic meningitis or severe, overwhelming
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encephalitis. Mortality rate is estimated at 5% for cases of encephalitis. It is a very serious disease in infants less than 1
year of age; as many as 60% of survivors have permanent neurologic impairment.

D.2. Eastern Equine Encephalitis

The mosquito vector (principally Culiseta melanura) generally restricts its feeding to horses and birds, although
occasional outbreaks among humans have occurred. Increasing numbers of human infections have been observed in 2005
and 2012, which is a cause of concern. The virus can cause severe encephalitis in horses and also in wild birds. The mortality
rate for eastern equine encephalitis among humans is estimated at 33% for individuals of all ages, and the incidence of
severe sequelae among survivors is high.

D.3. Chikungunya Virus

Chikungunya (a native term for “that which bends up”) is an Alphavirus (Togaviruses) transmitted by mosquitoes
(A aegypti and some other species), particularly in urban areas of Asia, Africa, and most recently in limited areas of
Southern Europe and the Caribbean.

The incubation period is between 2 and 12 (average 3-7) days and a majority of infected people develop some
symptoms. Illness is characterized by an abrupt onset of fever, accompanied by excruciating myalgia and polyarthritis.
Infected people may experience additional symptoms such as headache, myalgia, arthritis, conjunctivitis, nausea,
vomiting, or maculopapular rash. Symptoms usually last 1 week, but the musculoskeletal complaints can sometimes
persist for weeks to months. The disease is usually not fatal.

Diagnosis is done by detecting IgM or RNA by RT-PCR. There is no specific treatment or vaccine.

NON-ARBOVIRUS INFECTION IN HUMANS

The non-arthropod-borne zoonotic viruses are those that are not transmitted through arthropod vectors but
transmitted through small mammals and rodents. These viruses include arenaviruses, hantaviruses (bunyavirus), and
filoviruses.

A. FAMILY ARENAVIRIDAE
Table 8.6 Properties of Arenaviridae
Property Description
Virion • Pleomorphic
• 50–300 nm
Genome • Bisegmented
• One large negative sense RNA
• One small ambisense RNA
Envelope • Present with large, club-shaped peplomers
Replication • Cytoplasm
Outstanding Characteristics • The virion contains host cell ribosomes in their interior.
• These ribosomes confer a granular appearance to the viruses;
hence their name (from the Latin arenosus for “sandy”).

The most significant arenavirus infections in humans are the hemorrhagic fevers caused by Lassa virus in West
Africa. In addition, the South American hemorrhagic fevers are caused by arenaviruses, including Junin virus, Machupo
virus, Guanarito virus, and Sabia virus. LCMV is occasionally transmitted to humans from infected mice and other rodents,
and associated with CNS infection that may persist for several months.

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Figure 8.4 Virion Structure of Arenaviridae. Arenaviruses are enveloped containing two surface glycoproteins, G1 and G2,
and the RNA genome comprises large (L) single-stranded, negative-sense (–) and a small (S) ambisense (–/+) RNA that
form L and S nucleocapsids. The size of virions ranges from 50 to 300 nm in diameter. The virion contains host cell
ribosomes inside the virus particle. These ribosomes confer a granular or sandy appearance to the virions; hence their
name (from the Latin arenosus for “sandy”).

Arenaviruses associated with Hemorrhagic Fevers Arenaviruses Associated With CNS Infections
• Lassa Virus • Lymphocytic Choriomeningitis Virus
• Lujo Virus
• Junin Virus
• Machupo Virus
• Sabia Virus
• Guanarito Virus
• Chapare Virus

A.1 Lassa Virus

Lassa virus is highly virulent—the mortality rate is about 15% for patients hospitalized with Lassa fever. Overall,
about 1% of Lassa virus infections are fatal.

The incubation period for Lassa fever is 1–3 weeks from time of exposure. The disease can involve many organ
systems, although symptoms may vary in the individual patient. Onset is gradual, with fever, vomiting, and back and chest
pain. The disease is characterized by very high fever, mouth ulcers, severe muscle aches, skin rash with hemorrhages,
pneumonia, and heart and kidney damage. Deafness is a common complication, affecting about 25% of patients during
recovery; hearing loss is often permanent

Lassa virus infections cause fetal death in more than 75% of pregnant women. During the third trimester,
maternal mortality is increased (30%), and fetal mortality is very high (>90%)
Laboratory Diagnosis
Direct Detection Serology
• RT-PCR • ELISA – detection of IgG and IgM
• Immunohistochemistry

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Epidemiology

A house rat (Mastomys natalensis) is the principal rodent reservoir of Lassa virus. The virus can be transmitted by
human-to-human contact. When the virus spreads within a hospital, human contact is the mode of transmission.
Meticulous barrier nursing procedures and standard precautions to avoid contact with virus-contaminated blood and body
fluids can prevent transmission to hospital personnel.

Treatment, Prevention and Control

Rodent control measures are one way to minimize virus spread but are often impractical in endemic areas. The
antiviral drug ribavirin is the drug of choice for Lassa fever and is most effective if given early in the disease process. No
vaccine exists, although a vaccinia virus recombinant that expresses the glycoprotein gene of Lassa virus is able to induce
protective immunity both in guinea pigs and in monkeys.

A.2 Lujo Virus

Lujo virus was identified in 2008 as a cause of hemorrhagic fever in South Africa. The source of infection is
unknown. Rodents are thought to be the primary host, similar to other arenaviruses.

A.3 Junin Virus

Agent of Junin hemorrhagic fever (Argentine hemorrhagic fever), a major public health problem in certain
agricultural areas of Argentina.

The infection occurs almost exclusively among workers in maize and wheat fields who are exposed to the reservoir
rodent, Calomys musculinus.

Junin virus produces both humoral and cell-mediated immunodepression; deaths caused by Junin hemorrhagic
fever may be related to an inability to initiate a cell-mediated immune response.

An effective live attenuated Junin virus vaccine is used to vaccinate high-risk individuals in South America.

A.4 Machupo Virus

Agent of Machupo hemorrhagic fever (Bolivian hemorrhagic fever) that was identified in Bolivia in 1962. An
effective rodent control program directed against infected Calomys callosus, the host of Machupo virus, was undertaken
in Bolivia and has greatly reduced the number of cases of Machupo hemorrhagic fever.

A.5 Guanarito virus

The agent of Venezuelan hemorrhagic fever, and was identified in 1990; it has a mortality rate of about 33%. Its
emergence was tied to clearance of forest land for small farm use.

A.6 Sabia virus

Was isolated in 1990 from a fatal case of hemorrhagic fever in Brazil.

A.7 Chapare virus

Was isolated in Bolivia and is the causative agent of Chapare (Bolivia) hemorrhagic fever

A.8 Lymphocytic Choriomeningitis Virus

Lymphocytic choriomeningitis (LCM) virus was discovered in 1933 and is widespread in Europe and in the United
States. Its natural vector is the wild house mouse, Mus musculus.

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 The illness usually consists of fever, headache, and myalgia, although meningitis or meningoencephalitis also
occurs occasionally. Such CNS infections may persist as long as 3 months. There is also evidence that transplacental
infection can occur in humans, resulting in fetal death, hydrocephalus, or chorioretinitis. No person-to-person
transmission of infection has been documented.

The diagnosis of lymphocytic choriomeningitis is suggested by a history of rodent contact. The virus may be
isolated in the early stages of disease by cell culture or intracerebral inoculation of blood or CSF into weanling mice or
young guinea pigs. Serologic testing of acute and convalescent sera is usually performed by indirect immunofluorescence.
RT-PCR to detect viral RNA is also available.

B. FAMILY BUNYAVIRIDAE
Hantaviruses are classified in the Hantavirus genus of the Bunyaviridae family. The viruses are found worldwide
and cause two serious and often fatal human diseases:
1. Hemorrhagic fever with renal syndrome
2. Hantavirus Pulmonary Syndrome
Hemorrhagic fever with renal syndrome Hantavirus Pulmonary Syndrome
An acute viral infection that causes an interstitial nephritis A severe, sometimes fatal, respiratory disease in humans
that can lead to acute renal insufficiency and renal failure
in severe forms of the disease.
Hantavirus associated with these infections and their rodent vectors
• Hantaan virus Apodemus species (rodent) • Sin Nombre virus Deer mouse (Peromyscus
• Dobrava virus Apodemus species maniculatus)
• Saaremaa virus Apodemus species
• Seoul virus Rattus (brown rat) • New York hantavirus White footed mouse
• Puumala virus Clethrionomys (bank vole) • Black Creek hantavirus Cotton rat
Transmission
Inhalation of excreta of the rodents by the conjunctival Inhalation of infectious rodent excreta, by the conjunctival
route or by direct contact with skin breaks route, or by direct contact with skin breaks.
Treatment, Prevention and Control
• No vaccine • No vaccine
• Supportive treatment • Supportive treatment

C. FAMILY FILOVIRIDAE
Table 8.7 Properties of Filoviridae
Property Description
Virion • Filamentous and highly pleomorphic
• Helical capsid
Genome • Single-stranded
• Negative sense RNA
Envelope • Present with 10 nm peplomers or spikes
Replication • Cytoplasm
Outstanding Characteristics • Two members of filoviruses: Marburg (0 subtypes) and Ebola
(4 subtypes) viruses that cause hemorrhagic fevers

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 Figure 8.5 Virion Structure of Filoviruses. Filoviruses are enveloped, single-stranded, negative-
sense RNA viruses with filamentous and highly pleomorphic virions, averaging 80 nm in diameter
and 300 to 14 000 nm in length. The nucleocapsid (Np) has a helical symmetry and the envelope
is derived from plasma membrane containing 10 nm peplomers or spikes, the (GP) glycoprotein,
which mediate virus entry into susceptible cells.

Filoviruses are highly virulent and require maximum containment facilities (Biosafety
Level 4) for laboratory work. Filovirus infectivity is destroyed by heating for 30 minutes at 60°C,
by ultraviolet and γ-irradiation, by lipid solvents, and by bleach and phenolic disinfectants. The
natural hosts and vectors are suspected to be African fruit bats.

Filoviruses have a tropism for cells of the macrophage system, dendritic cells, interstitial
fibroblasts, and endothelial cells. Very high titers of virus are present in many tissues, including
the liver, spleen, lungs, and kidneys, and in blood and other fluids. These viruses have the highest
mortality rates (25–90%) of all the viral hemorrhagic fevers.

Epidemiology
It is probable that Marburg and Ebola viruses have a reservoir host, most likely the fruit bat, and become
transmitted to humans only accidentally. Monkeys are not considered to be reservoir hosts because most infected animals
die too rapidly to sustain virus survival.

Human infections are highly communicable to human contacts, generally by direct contact with blood, body fluids,
or recently deceased victims.

Laboratory Diagnosis

Direct Detection Serology

• RT-PCR • ELISA – detection of viral antibodies and
• Fresh virus isolates can be cultured in cell lines antigen
such as Vero and MA-104 monkey cell lines

Treatment, Prevention and Control

There are no specific antiviral therapies available. Treatment is directed at maintaining renal function and
electrolyte balance and combating hemorrhage and shock. An experimental vaccine is now available and is being tested
for effectiveness as a targeted outbreak control measure.

Because the natural reservoirs of Marburg and Ebola viruses are still unknown, no control activities can be
organized. The use of isolation facilities in hospital settings remains the most effective means of controlling Ebola disease
outbreaks

C.1 Marburg Virus

Marburg virus disease was recognized in 1967 among laboratory workers exposed to tissues of African green
monkeys (Cercopithecus aethiops) imported into Germany and Yugoslavia.

Causes acute diseases characterized by fever, headache, sore throat, and muscle pain followed by abdominal pain,
vomiting, diarrhea, and rash, with both internal and external bleeding, often leading to shock and death.
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 Transmission from patients to medical personnel occurred, with high mortality rates. Antibody surveys have
indicated that the virus is present in East Africa and causes infection in monkeys and humans. Recorded cases of the
disease are rare, but outbreaks have been documented in Kenya, South Africa, Democratic Republic of the Congo, and
Angola. Marburg virus can infect guinea pigs, mice, hamsters, monkeys, and various cell culture systems.

C.2 Ebola Virus

In 1976, severe outbreaks of hemorrhagic fever occurred in northern Zaire and southern Sudan, with case fatality
rates of 90% and 50%, respectively. The illnesses were similar to those described for Marburg virus, but were later shown
to be caused by an antigenically different agent known as Ebola virus. Ebola virus has four subtypes (Zaire, Sudan, Reston,
Ivory Coast).

Causes acute diseases characterized by fever, headache, sore throat, and muscle pain followed by abdominal
pain, vomiting, diarrhea, and rash, with both internal and external bleeding, often leading to shock and death, similar to
Marburg virus.

The largest known Ebola outbreak occurred in western Africa in 2014–2016, with over 28,000 cases and 11,000
deaths in Guinea, Liberia, and Sierra Leone. Intense international emergency response and quarantine measures followed,
eventually containing the outbreak in June 2016.

D. FAMILY RHABDOVIRIDAE

Table 8.8 Properties of Rhabdoviruses

Property Description
Virion • Bullet-shaped
• 75 nm in diameter × 180 nm in length
• Nucleocapsid is Helical
Genome • Single-stranded, Linear
• Negative-sense RNA
Envelope • Present with knob-like glycoproteins
Replication • Cytoplasm

Figure 8.6 Virion structure of Rhabdoviruses.

A. Electron micrograph of bullet-shaped particle typical of the

rhabdovirus family

B. Schematic model of rabies virus showing the surface

glycoprotein spikes extending from the lipid envelope that
surrounds the internal nucleocapsid and the matrix protein
lining the envelope

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Classification
Rabies viruses belong to the Lyssavirus, which is a genus under the family Rhabdoviridae. The rhabdoviruses are
very widely distributed in nature, infecting vertebrates, invertebrates, and plants. Rabies is the major medically important
rhabdovirus. Many of the animal rhabdoviruses infect insects, but rabies virus does not.

Antigenic Properties
There is a single serotype of rabies virus. However, there are strain differences among viruses isolated from
different species (raccoons, foxes, skunks, canines, bats) in different geographic areas.

Pathogenesis
Rabies virus multiplies in muscle or connective tissue at the site of inoculation and then enters peripheral nerves
at neuromuscular junctions and spreads up the nerves to the central nervous system. However, it is also possible for rabies
virus to enter the nervous system directly without local replication. It multiplies in the central nervous system and
progressive encephalitis develops. The virus then spreads through peripheral nerves to the salivary glands and other
tissues. The organ with the highest titers of virus is the submaxillary salivary gland. Other organs where rabies virus has
been found include pancreas, kidney, heart, retina, and cornea. Rabies virus has not been isolated from the blood of
infected persons.

Pathology
Rabies virus produces a specific eosinophilic cytoplasmic inclusion, the Negri body, in infected nerve cells. Negri
bodies are filled with viral nucleocapsids. The presence of such inclusions is pathognomonic of rabies but is not observed
in at least 20% of cases. Therefore, the absence of Negri bodies does not rule out rabies as a diagnosis.

Figure 8.6 Negri bodies.

Clinical Findings
Rabies is primarily a disease of lower animals and is spread to humans by bites of rabid animals or by contact with
saliva from rabid animals. It presents as an acute, fulminant, fatal encephalitis; human survivors have been reported only
occasionally. The clinical stages of rabies infection are summarized in Table 8.9 below.

Table 8.9 Clinical Stages of Rabies Virus Infection

Stages of Infection Time Frame Symptoms Site of Virus Replication
Incubation Period 10-365 days None Site of bite, Muscle cells
(Average: 20-90 days)
Prodromal Stage 2-10 days Nonspecific symptoms, malaise, Virus replication in the
headache, fever, nausea, vomiting, CNS
upper respiratory distress, subtle
mental changes (insomnia), pain,
itching, tingling at the site of bite
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 Stages of Infection Time Frame Symptoms Site of Virus Replication
Acute neurologic stage 2-7 days Furious or dumb presentation Virus replication in brain
and transported to other
sites (salivary glands and
other organs)
Coma 0-14 days Patient in coma; respiratory Virus replication in brain
paralysis, cardiac arrest, drop in and transported to other
blood pressure, secondary organs
infections
Death Extreme rare survival

Epidemiology
Rabies is enzootic in both wild and domestic animals. Domestic animal bites are very important sources of rabies
in developing countries because of lack of enforcement of animal immunization. Infection in domestic animals usually
represents a spillover from infection in wildlife reservoirs. Human infection tends to occur where animal rabies is common
and where there is a large population of unimmunized domestic animals.

Human-to-human rabies infection is very rare. The only documented cases involve rabies transmitted by corneal
and organ transplants.

Laboratory Diagnosis

Samples Direct Detection Serology

• Saliva • RT-PCR • Immunofluorescence tests
• Neck biopsy • Negri bodies in the brain or the
• Serum spinal cord
• CSF in human ante
mortem

Treatment, Prevention and Control

There is no specific treatment available, thus Prevention is the mainstay of controlling rabies in humans
immediately after exposure by starting the rabies vaccination process. The prevention of rabies is divided into preexposure
prophylaxis (PreEP) and postexposure prophylaxis (PEP).

All vaccines for human use contain only inactivated rabies virus.

1. Human diploid cell vaccine (HDCV)

2. Purified chick embryo cell vaccine (PCEC)

Pre-exposure Prophylaxis Post-exposure Prophylaxis

Recommended for individuals with high risk of The decision to administer rabies antibody or rabies vaccine—or
contact with rabies virus, such as both—depends on several factors:
veterinarians, spelunkers, laboratory workers,
and animal handlers. 1. The nature of the biting animal (species, state of health, domestic,
or wild) and its vaccination status
2. The availability of the animal for laboratory examination (all bites
by wild animals and bats require rabies immune globulin and vaccine)
3. The existence of rabies in the area
4. The manner of attack (provoked or unprovoked)
5. The severity of the bite and contamination by saliva of the animal
6. Advice from local public health officials
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 PART 4: LIST OF REFERENCES

• Books, G, et.al. Jawetz, Melnick & Adelberg’s Medical Microbiology, 27th Ed. McGraw-Hill. Copyright 2015.

• Bulmer, Glenn. Fungus Diseases in the Orient. Rex Bookstore. Manila. 1991.

• Mahon and Manusells (ed.) Textbook of Diagnostic Microbiology. Elsevier (Singapore) Pte Ltd. 2014

• Forbes, B., Sahm, D. & WEissfeld, A. Bailey & Scott’s Diagnostic Microbiology, 13th ed. Elsevier Science
(Singapore) Pte Ltd. Copyright 2014

• Davey, F., et.al (ed). John Bernard Henry, Clinical Diagnosis and Management by Laboratory Methods. 22nd ed.
W.B. Saunders Co. Philadelphia. 2007

• Black, Jacquelyn G. Microbiology, Principles and Explorations, 5th edition. USA: McGraw-Hill Companies Inc.
2005

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 University of San Agustin
ILOILO CITY, PHILIPPINES
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS
MEDICAL LABORATORY SCIENCE PROGRAM

MODULE PACKETS IN MLS 13A:

MYCOLOGY AND
VIROLOGY
M9

Matthew I. Tubola, RMT, MSMT

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John Robert D. Fundal, RMT

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© 2021. Printed in Calibri.
Cover Design: J.R.D. Fundal, RMT
All rights reserved. Copyright applied for. No part of this module may be reproduced, stored in retrieval
system or transmitted in any forms or by any means, electronic, mechanical, photocopying or otherwise,
without written permission from the authors.

The authors have done everything possible to make this module accurate and in accordance with accepted
standards. The authors are not responsible for errors or omissions or for consequences (loss, damage, or
disruption) from application of the module, and make no warranty, expressed or implied in regard to the
contents of the module. Any practice described in this module should be applied by the reader in accordance
with accepted standards used in regard to unique circumstances that may apply in each situation.

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LEARNER’S HONOR CODE STATEMENT FOR THIS MODULE PACKET

“I affirm that I will not give or receive any unauthorized help on this MODULE, and that all
work will be my own.”
“I affirm that I have not given or received any unauthorized help on this assignment, and that
this work is my own.”

_________________________
Signature over printed name

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 ABOUT THE AUTHORS

MATTHEW I. TUBOLA, RMT, MSMT

Hello Augustinians!
Welcome to MLS-13A (Mycology and Virology Lecture), I will be your lecturer for this subject. I am a
graduate of BSMT of this University and also finished my Master’s degree in Medical Technology in this
University. I have been teaching in this University for 12 years teaching Medical Technology Professional
Subjects.

JOHN ROBERT D. FUNDAL, RMT

Hello Augustinians!
Welcome to MLS-13A (Mycology and Virology Lecture), I will also be your lecturer for this subject. I
obtained my Bachelor’s Degree in Medical Laboratory Science from University of San Agustin. This is
currently my third semester in teaching MLS subjects.

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 TABLE OF CONTENTS
Disclaimer 1
Honor Code 2
About the Authors 3
Content

Human Immunodeficiency Virus 7

Papillomavirus 14
References 17

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 UNIT MAP
Characteristics, Laboratory Tests,
Epidemiology, Prevention, and
Control of Sexually Transmitted
Viruses

HIV Papillomavirus

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 PART 1: OVERVIEW OF DISCUSSION

Sexually transmitted diseases (STDs) — or sexually transmitted infections (STIs) — are generally
acquired by sexual contact. The organisms (bacteria, viruses or parasites) that cause sexually transmitted
diseases may pass from person to person in blood, semen, or vaginal and other bodily fluids. Sometimes these
infections can be transmitted non-sexually, such as from mother to infant during pregnancy or childbirth, or
through blood transfusions or shared needles.

PART 2: LEARNING OBJECTIVES

At the end of the lecture, the student shall be able to:

• Know the properties of Papillomaviruses and Retroviruses

• Describe the clinical findings and laboratory diagnosis of human infections of Papillomaviruses and
Retroviruses
• Describe the epidemiology, methods of treatment, prevention, and control of Papillomaviruses and
Retroviruses

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 PART 3: DISCUSSION

Viruses to consider in this module:

1. Family Retroviridae

• Genus Lentivirus
✓ Human Immunodeficiency Virus 1 and 2 (HIV 1and 2)

2. Family Papovaviridae

• Papilloma viruses

HUMAN IMMUNODEFICIENCY VIRUSES (HIVs)

INTRODUCTION

Human immunodeficiency virus (HIV) types, derived from primate lentiviruses, are the etiologic agents of
Acquired Immune Deficiency Syndrome (AIDS). The illness was first described in 1981, and HIV-1 was isolated by the end
of 1983. Initial reports were based on an unusual increase in the incidence of Kaposi sarcoma (KS) and Pneumocystis
pneumonia (PCP), diseases that were considered at that time to occur rarely.

There are two types: HIV-1 and HIV-2, which cause AIDS. A devastating disease worldwide, for which there is no
permanent cure or preventive vaccine for protection, AIDS has spurred unprecedented research efforts to determine the
nature and immunopathogenic mechanisms of the virus in the hope of finding more and new effective drugs and a
preventive AIDS vaccine. Most of our present knowledge of HIV is derived from studies on HIV-1, which is the major cause
of AIDS worldwide.

The development of highly active antiretroviral therapy (HAART) for chronic suppression of HIV replication and
prevention of AIDS has been a major achievement in HIV medicine.

Table 9.1 Properties of HIV

Property Description
Virion ✓ Spherical
✓ 80–100 nm in diameter
✓ Cylindrical core
Genome ✓ Single-stranded RNA
✓ Linear
✓ Positive-sense
✓ Diploid
✓ Contains up to six additional replication genes
Proteins ✓ Envelope glycoprotein undergoes antigenic variation
✓ Reverse transcriptase enzyme contained inside virions
✓ Protease required for production of infectious virus
Envelope ✓ Present with glycoprotein spikes
Replication ✓ Nucleus
Outstanding Characteristics ✓ Members are non-oncogenic and may be cytocidal
✓ Infect cells of the immune system

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Figure 9.1 General Structure of Human Immunodeficiency Virus (HIV) - 1

Figure 10.1

Structural Proteins:

• The HIV matrix proteins - Consisting of the p17 protein, lie between the envelope and core.

• Gag proteins - The gag gene gives rise to the 55 kilodalton Gag precursor protein, also called p55, which is
expressed from the unspliced viral mRNA.

• Envelope proteins - Env exists as a multimer, most likely a trimer, on the surface of the cell of the virion.
Interactions between HIV and the virion receptor, CD4, are mediated through specific domains of gp120.

Non- Structural Proteins & Enzymes:

• HIV-1 protease - The HIV-1 protease is an aspartyl protease that acts as a dimer. Protease activity is required
precursors during virion maturation.

• Reverse transcriptase - During the process of reverse transcription, the polymerase makes a double stranded DNA
copy of the dimer of single stranded genomic RNA present in the virion.

• Integrase - The integrase protein mediates the insertion of the HIV proviral DNA into the genomic DNA of an
infected cell.

• Regulator proteins:
o Tat - Tat is a transcriptional transactivator that is essential for HIV-1 replication.
o Rev - Rev is a 13-kD sequence-specific RNA binding protein. Rev acts to induce the transition from the
early to the late phase of HIV gene expression.

• Accessory proteins:
o Vpr - Vpr can block cell division.
o Vpu - Vpu also increases the release of HIV from the surface of an infected cell.

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Table 9.2 Major Retroviral Genes and Proteins
Genes Products encoded Function
gag • p17 Structural
• p24 Structural
• p7 Structural
• Protease Gag-Pol protein processing
pol • Protease Gag-Pol protein processing
• Reverse Transcriptase DNA synthesis
• Integrase
Integration
env • gp120 Adsorption
• gp41 Fusion of envelope with plasma
membrane

REPLICATION CYCLE OF HIV

1. HIV entry - CD4 as a primary receptor for HIV embedded in the outer membrane of the HIV virion bind to receptors on
the surface of target cells. T-cells (white blood cells) have CD4 and CCR5 receptors to which HIV can bind. Binding of the
HIV envelope protein to CD4 and CCR5 allows the HIV-1 outer membrane to fuse with the cell’s outer membrane and the
contents of the virus particle to enter the cell.

2. Fusion and Release – The HIV envelope and the CD4 cell membrane fuse (join together), which allows HIV to enter the
cell.

3. Reverse transcription - The genetic material of the virus is in the form of RNA, or ribonucleic acid. There are two strands
of RNA in each HIV-1 virus particle. An enzyme known as reverse transcriptase initiates the formation of one double-
stranded molecule of viral DNA (deoxyribonucleic acid) by copying the sequence of the RNA strands contained in the virus
particle.

4. Integration of the viral DNA into cellular genomic DNA - The viral DNA enters the nucleus of the host and becomes
integrated into the host’s DNA. An enzyme called integrase is key in this process. Once the viral DNA has integrated into
the cell’s DNA, the cell is infected for the remainder of its life. The integrated viral DNA is now referred to as a provirus.

5. Replication:

a. Transcription - The provirus DNA serves as a template for the creation of new viral RNA via a process
known as transcription. The host cell’s own machinery that is normally used for the transcription of human
genes is used by the virus to create new viral RNA molecules. The newly formed viral RNA moves out of
the infected cell’s nucleus.

b. Translation - The viral RNA carries code for the synthesis of viral proteins and enzymes. The code is
translated into long chains of amino-acids, known as a polypeptide chains, which fold to form the protein
and enzyme components of new virus particles.

6. Assembly - Components that are required to build new virus particles, namely viral proteins, enzymes and genetic
material (viral RNA) move to the cell’s outer membrane where they accumulate and assemble in the form of a bud.

7. Release and Maturation - Host-cell proteins cut the virus bud from the cell’s outer membrane, thereby releasing a new
virus particle. During and after assembly and release, a viral enzyme called protease cuts the HIV polypeptide chains at
several positions, in a process called maturation, to make the finished components of the new, infectious, virus particle.
A single infected cell can release many new HIV particles which move on to infect other cells in various parts of the body,
where the viral life cycle is repeated. The infected cells are eventually destroyed.

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Figure 9.2 Replication Cycle of HIV

A. Viral entry and post entry (reverse transcription, DNA synthesis, and integration) events; B. Viral gene expression
(transcription and protein synthesis); C. Virus assembly and release.

CLINICAL MANIFESTATION

In 1993, the CDC definition of AIDS stated that all patients who are HIV antibody positive and have CD4+ T-
lymphocyte counts lower than 200/mm3 or less than 14% of total T-lymphocytes have the disease. HIV-1 infection is
characterized as a four-stage process.

STAGE 1
Patients who are asymptomatic or have persistent generalized lymphadenopathy (lymphadenopathy of at least two
sites [not including inguinal] for longer than 6 months) are categorized as being in stage 1, where they may remain for
several years.

Figure 9.3 Generalized Lymphadenopathy

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 STAGE 2
Even in early HIV infection, patients may demonstrate several clinical manifestations. Clinical findings included in stage
2 (mildly symptomatic stage) are unexplained weight loss of less than 10 percent of total body weight and recurrent
respiratory infections (such as sinusitis, bronchitis, otitis media, and pharyngitis), as well as a range of dermatological
conditions including herpes zoster flares, angular cheilitis, recurrent oral ulcerations, papular pruritic eruptions,
seborrhoeic dermatitis, and fungal nail infections.

STAGE 3
As disease progresses, additional clinical manifestations may appear. Those encompassed by the WHO clinical stage 3
(the moderately symptomatic stage) category are weight loss of greater than 10 percent of total body weight,
prolonged (more than 1 month) unexplained diarrhea, pulmonary tuberculosis, and severe systemic bacterial
infections including pneumonia, pyelonephritis, empyema, pyomyositis, meningitis, bone and joint infections, and
bacteremia.

Mucocutaneous conditions, including recurrent oral candidiasis, oral hairy leukoplakia, and acute necrotizing ulcerative
stomatitis, gingivitis, or periodontitis, may also occur at this stage.

STAGE 4
The WHO clinical stage 4 (the severely symptomatic stage) designation includes all of the AIDS-defining illnesses. Clinical
manifestations for stage 4 disease that allow presumptive diagnosis of AIDS to be made based on clinical findings alone
are HIV wasting syndrome, Pneumocystis pneumonia (PCP), recurrent severe or radiological bacterial pneumonia,
extrapulmonary tuberculosis, HIV encephalopathy, CNS toxoplasmosis, chronic (more than 1 month) or orolabial
herpes simplex infection, esophageal candidiasis, and Kaposi’s sarcoma.

Other conditions that should arouse suspicion that a patient is in clinical stage include cytomegaloviral (CMV) infections
(CMV retinitis or infection of organs other than the liver, spleen or lymph nodes), extrapulmonary cryptococcosis,
disseminated endemic mycoses (e.g., coccidiomycosis, penicilliosis, histoplasmosis), cryptosporidiosis, isosporiasis,
disseminated non-tuberculous mycobacteria infection, tracheal, bronchial or pulmonary candida infection, visceral

Figure 9.4 HIV/AIDS Disease Progression

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EPIDEMIOLOGY OF HIV

AIDS was first recognized in the United States in 1981, when it became apparent that an unusual number of rare
skin cancers (Kaposi sarcoma) and opportunistic infections were occurring among male homosexuals. These patients were
found to have a marked reduction in CD4+ T-lymphocytes and were subject to a wide range of opportunistic infections
normally controlled by an intact immune system. The disease was found to progress relentlessly to a fatal outcome and
was first identified in male homosexuals, hemophiliacs, who were receiving blood-derived coagulation factors, and
injection drug users.

Figure 9.3 Adults and children estimated to be

living with HIV/AIDS, by continent or region,
as of December 2009, totaling 33.3 million. It
is estimated that about 1.8 million people
worldwide died of HIV/AIDS in 2009. (Data
from the Joint United Nations Program on
HIV/AIDS.)

Figure 9.4 HIV data and statistics

TRANSMISSION OF HIV

HIV is transmitted through:

• Unprotected sexual contact with an infected partner

• Exposure of broken skin or wound to infected blood or body fluids.
• Transfusion with HIV-infected blood
• Injection with contaminated objects
• Mother to child during pregnancy, birth, or breastfeeding

LABORATORY DIAGNOSIS

1. Serologic Method

Antibodies to HIV can be measured by a variety of techniques. None of these detect HIV itself, but rather detect
an immune response to the virus, and therefore take some time to develop and become reactive (or positive) after HIV
infection has been acquired. Antibodies to HIV-1 and HIV-2 are detected by EIA, also known as enzyme-linked
immunosorbent assay (ELISA), simple/rapid test devices, and western blot (WB) tests.

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 Figure 9.5 Serologic Profile of HIV-1 Infection

2. Nucleic Acid Detection

1. Polymerase chain reaction (PCR): it exponentially amplifies the DNA target by many orders of magnitude by
cycling the temperature of the reaction several times. DNA primers define the region of the target sequence to
be amplified by a DNA polymerase that duplicates the number of sequences in each cycle of the reaction. In the
case of RNA specimens such as HIV, the RNA must be converted to DNA by a reverse transcriptase enzyme in an
isothermal reaction before initiating the PCR. This reaction is referred to as RT-PCR.
2. Nucleic acid sequence-based amplification (NASBA): it selectively amplifies the target RNA through isothermal
production of an intermediate DNA by a reverse transcriptase. This DNA serves as a template for the cyclical
amplification of RNA using an RNA polymerase. Fluorescent-labelled probes hybridize to newly synthesized RNA
and quantification is determined by comparing the fluorescence of the target with internal standards.
3. Branched-chain DNA (b-DNA): it is based on amplifying the signal rather than the target RNA.
4. Transcription-mediated amplification (TMA): this is a qualitative method that can detect either RNA or DNA. It
uses the same principle as the NASBA methodology.

TREATMENT
A growing number of antiviral drugs are approved for treatment of HIV infections. Therapy with combinations of
antiretroviral drugs, referred to as HAART (Highly active antiretroviral therapy), became available in 1996. It often can
suppress viral replication to below limits of detection in plasma, decrease viral loads in lymphoid tissues, allow the
recovery of immune responses to opportunistic pathogens, and prolong patient survival.
There is currently no cure for HIV, but the availability of HAART means that HIV is manageable through lifelong
treatment. When HAART is discontinued or there is treatment failure, virus production rebounds.

Table 9.3 Antiretroviral Drugs

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 HUMAN PAPILLOMA VIRUS

INTRODUCTION

HPV is a very common virus that can be spread from one person to another person through anal, vaginal, or oral
sex, or through other close skin-to-skin touching during sexual activity. There were about 43 million HPV infections in
2018, many among people in their late teens and early 20s. Nearly all sexually active people who do not get the HPV
vaccine get infected with HPV at some point in their lives.

Table 9.4 Properties of Papillomaviruses

Property Description
Virion ✓ Icosahedral
✓ 55 nm in diameter
Genome ✓ Double-stranded DNA
✓ Circular
Proteins ✓ Two structural proteins
✓ Cellular histones condense DNA in virion
Envelope ✓ None
Replication ✓ Nucleus
Outstanding Characteristics ✓ Stimulate cell DNA synthesis
✓ Restricted host range and tissue tropism
✓ Significant cause of human cancer, especially cervical cancer
✓ Viral oncoproteins interact with cellular tumor suppressor proteins

Figure 9.6 Structure of Papillomavirus

PATHOGENESIS

HPV targets stratified squamous epithelium through the damaged area of the epithelium and infects the basal
cells. Although the incidence of infection is high, most infections resolve spontaneously. A small proportion of infected
persons become persistently infected; persistent infection is the most important risk factor for the development of cervical
cancer precursor lesions. The most common clinically significant manifestation of persistent genital HPV infection is
cervical intraepithelial neoplasia, or CIN. Within a few years of infection, low-grade CIN—called CIN 1—may develop,
which may spontaneously resolve and the infection clear.

Persistent HPV infection, however, may progress directly to high-grade CIN, called CIN2 or CIN3. High-grade
abnormalities are at risk of progression to cancer and so are considered cancer precursors. A small proportion of high-
grade abnormalities spontaneously regress.

If left undetected and untreated, years or decades later CIN2 or 3 can progress to cervical cancer. Infection with
one type of HPV does not prevent infection with another type. Of persons infected with mucosal HPV, 5% to 30% are
infected with multiple types of the virus.

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CLINICAL FEATURES

Most HPV infections are asymptomatic and result in no clinical disease. Clinical manifestations of HPV infection
include anogenital warts, recurrent respiratory papillomatosis, cervical cancer precursors (cervical intraepithelial
neoplasia), and cancers, including cervical, anal, vaginal, vulvar, penile, and some head and neck cancer.

Table 9.5 Examples of Association of Human Papillomaviruses with Clinical Lesions

HPV Type Clinical Lesion Suspected Oncogenic Potential
1 Plantar warts Benign
2, 4, 27, 57 Common skin warts Benign
3, 10, 28, 49, 60, 76, 78 Cutaneous lesions Low
5, 8, 9, 12, 17, 20, 36, 47 Epidermodysplasia verruciformis Mostly benign, but some progress to
malignancy
6, 11, 40, 42–44, 54, 61, Anogenital condylomas; laryngeal papillomas; Low
70, 72, 81 dysplasias and intraepithelial neoplasias (mucosal
sites)
7 Hand warts of butchers Low
16, 18 Can progress to high-grade dysplasias and High correlation with genital and oral
carcinomas of genital mucosa; laryngeal and carcinomas, especially cervical cancer
esophageal carcinomas
30, 31, 33, 35, 39, 45, Can progress to high-grade dysplasias and Moderate correlation with genital
51–53, 56, carcinomas of genital mucosa; laryngeal and and oral carcinomas, especially
58, 59, 66, 68, 73, 82 esophageal carcinomas. Moderate correlation with cervical cancer, considered high-risk
genital and oral carcinomas, especially cervical HPV types
cancer, considered high-risk HPV types

LABORATORY DIAGNOSIS

HPV has not been isolated in culture. Infection is identified by detection of HPV DNA from clinical samples.

1. Nucleic Acid Detection

Epidemiologic and basic research studies of HPV generally use nucleic acid amplification methods that generate
type specific results. The PCR assays used most commonly in epidemiologic studies target genetically conserved regions
in the L1 gene.

Currently, only the Digene Hybrid Capture 2 ® (hc2) High Risk HPV DNA Test is approved by the Food and Drug
Administration for clinical use. The hc2 uses liquid nucleic acid hybridization and detects 13 high-risk types (HPV 16, 18,
31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68). Results are reported as positive or negative and are not type-specific. The hc2
test is approved for triage of women with equivocal Papanicolaou (Pap) test results (ASC-US, atypical cells of undetermined
significance) and in combination with the Pap test for cervical cancer screening in women 30 years of age and older. The
test is not clinically indicated nor approved for use in men.

2. Serologic Method

The most frequently used HPV serologic assays are VLP-based enzyme immunoassays. However, laboratory
reagents used for these assays are not standardized and there are no standards for setting a threshold for a positive result.

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3. Cytologic Method

Most cases and deaths from cervical cancer can be prevented through detection of precancerous changes within
the cervix by cervical cytology using the Pap test. Currently available Pap test screening can be done by a conventional
Pap or a liquid-based cytology. CDC does not issue recommendations for cervical cancer screening, but various
professional groups have published recommendations. The American College of Obstetricians and Gynecologists (ACOG),
the American Cancer Society (ACS), and the U.S. Preventive Services Task Force (USPSTF) guidelines recommend that all
women should have a Pap test for cervical cancer screening within 3 years of beginning sexual activity or by age 21,
whichever occurs first. While the USPSTF recommends a conventional Pap test at least every 3 years regardless of age,
ACS and ACOG recommend annual or biennial screening of women younger than age 30, depending on use of conventional
or liquid–based cytology. According to these organizations, women over age 30 with three normal consecutive Pap tests
should be screened every 2 to 3 years. The use of HPV vaccine does not eliminate the need for continued Pap test
screening, since 30% of cervical cancers are caused by HPV types not included in the vaccine.

EPIDEMIOLOGY

HPV infection occurs throughout the world. Humans are the only natural reservoir of HPV. It is transmitted by
direct contact, usually sexual, with an infected person. Transmission occurs most frequently with sexual intercourse but
can occur following nonpenetrative sexual activity. Studies of newly acquired HPV infection demonstrate that infection
occurs soon after onset of sexual activity. Genital HPV infection also may be transmitted by nonsexual routes, but this
appears to be uncommon. Non-sexual routes of genital HPV transmission include transmission from a woman to a
newborn infant at the time of birth.

HPV is presumably communicable during the acute infection and during persistent infection. This issue is difficult
to study because of the inability to culture the virus. Communicability can presumed to be high because of the large
number of new infections estimated to occur each year.

TREATMENT, PREVENTION AND CONTROL

There is no specific treatment for HPV infection. Medical management depends on treatment of the specific
clinical manifestation of the infection (such as genital warts or abnormal cervical cell cytology).

HPV transmission can be reduced but not eliminated with the use of physical barriers such as condoms. Recent
studies demonstrated a significant reduction in HPV infection among young women after initiation of sexual activity when
their partners used condoms consistently and correctly. Abstaining from sexual activity (i.e., refraining from any genital
contact with another individual) is the surest way to prevent genital HPV infection. For those who choose to be sexually
active, a monogamous relationship with an uninfected partner is the strategy most likely to prevent future genital HPV
infections.

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 PART 4: LIST OF REFERENCES

• Books, G, et.al. Jawetz, Melnick & Adelberg’s Medical Microbiology, 27th Ed. McGraw-Hill. Copyright 2015.

• Bulmer, Glenn. Fungus Diseases in the Orient. Rex Bookstore. Manila. 1991.

• Mahon and Manusells (ed.) Textbook of Diagnostic Microbiology. Elsevier (Singapore) Pte Ltd. 2014

• Forbes, B., Sahm, D. & WEissfeld, A. Bailey & Scott’s Diagnostic Microbiology, 13th ed. Elsevier Science
(Singapore) Pte Ltd. Copyright 2014

• Davey, F., et.al (ed). John Bernard Henry, Clinical Diagnosis and Management by Laboratory Methods. 22nd ed.
W.B. Saunders Co. Philadelphia. 2007

• Black, Jacquelyn G. Microbiology, Principles and Explorations, 5th edition. USA: McGraw-Hill Companies Inc.
2005

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A.Y. 2020 - 2021
 University of San Agustin
ILOILO CITY, PHILIPPINES
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS
MEDICAL LABORATORY SCIENCE PROGRAM

MODULE PACKETS IN MLS 13A:

MYCOLOGY AND
VIROLOGY
M10

Matthew I. Tubola, RMT, MSMT

0|Page

John Robert D. Fundal, RMT

MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
© 2021. Printed in Calibri.
Cover Design: J.R.D. Fundal, RMT
All rights reserved. Copyright applied for. No part of this module may be reproduced, stored in retrieval
system or transmitted in any forms or by any means, electronic, mechanical, photocopying or otherwise,
without written permission from the authors.

The authors have done everything possible to make this module accurate and in accordance with accepted
standards. The authors are not responsible for errors or omissions or for consequences (loss, damage, or
disruption) from application of the module, and make no warranty, expressed or implied in regard to the
contents of the module. Any practice described in this module should be applied by the reader in accordance
with accepted standards used in regard to unique circumstances that may apply in each situation.

1|Page
MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
LEARNER’S HONOR CODE STATEMENT FOR THIS MODULE PACKET

“I affirm that I will not give or receive any unauthorized help on this MODULE, and that all
work will be my own.”
“I affirm that I have not given or received any unauthorized help on this assignment, and that
this work is my own.”

_________________________
Signature over printed name

2|Page
MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
 ABOUT THE AUTHORS

MATTHEW I. TUBOLA, RMT, MSMT

Hello Augustinians!
Welcome to MLS-13A (Mycology and Virology Lecture), I will be your lecturer for this subject. I am a
graduate of BSMT of this University and also finished my Master’s degree in Medical Technology in this
University. I have been teaching in this University for 12 years teaching Medical Technology Professional
Subjects.

JOHN ROBERT D. FUNDAL, RMT

Hello Augustinians!
Welcome to MLS-13A (Mycology and Virology Lecture), I will also be your lecturer for this subject. I
obtained my Bachelor’s Degree in Medical Laboratory Science from University of San Agustin. This is
currently my third semester in teaching MLS subjects.

3|Page
MYCOLOGY AND VIROLOGY
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 TABLE OF CONTENTS
Disclaimer 1
Honor Code 2
About the Authors 3
Content

Prions 7
Prion Diseases 7
References 10

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 UNIT MAP
General Characteristics and
Mechanisms of Pathogenesis of
Bovine Spongiform Encephalopathy
Agents and other Prions

Classic Creutzfeldt-Jakob
Disease (CJD)

Variant Creutzfeldt-Jakob
Disease (vCJD)

Kuru

Scrapie

Bovine Spongiform
Encephalitis

Chronic wasting disease

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 PART 1: OVERVIEW OF DISCUSSION

Prion diseases or transmissible spongiform encephalopathies (TSEs) are a family of rare progressive
neurodegenerative disorders that affect both humans and animals. They are distinguished by long incubation
periods, characteristic spongiform changes associated with neuronal loss, and a failure to induce inflammatory
response.
The causative agents of TSEs are believed to be prions. The term “prions” refers to abnormal,
pathogenic agents that are transmissible and are able to induce abnormal folding of specific normal cellular
proteins called prion proteins that are found most abundantly in the brain. The functions of these normal
prion proteins are still not completely understood. The abnormal folding of the prion proteins leads to brain
damage and the characteristic signs and symptoms of the disease. Prion diseases are usually rapidly
progressive and always fatal.

PART 2: LEARNING OBJECTIVES

At the end of the lecture, the student shall be able to:

• Know the general characteristics of Bovine Spongiform Encephalopathy Agents and other Prions
• Know the mechanisms of pathogenesis of Bovine Spongiform Encephalopathy Agents and other Prions

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 PART 3: DISCUSSION

PRIONS

Prion diseases or transmissible spongiform encephalopathies (TSEs) are a family of rare progressive
neurodegenerative disorders that affect both humans and animals. They are distinguished by long incubation periods,
characteristic spongiform changes associated with neuronal loss, and a failure to induce inflammatory response.

The causative agents of TSEs are believed to be prions. The term “prions” refers to abnormal, pathogenic agents
that are transmissible and can induce abnormal folding of specific normal cellular proteins called prion proteins that are
found most abundantly in the brain. The functions of these normal prion proteins are still not completely understood. The
abnormal folding of the prion proteins leads to brain damage and the characteristic signs and symptoms of the disease.
Prion diseases are usually rapidly progressive and always fatal.

PRNP is a gene in your Prion protein or PrP is a Prion is an infectious

DNA which encodes for protein on the surface particle made up of
prion protein. of your cells. misfolded prion proteins.

PRION PROTEINS

Prion protein (PrP) appears to be the major, and possibly exclusive component of prions. PrPc (cellular) is the protein
product that is thought to be the target for prion disease. In the wild type, it is a normal host glycoprotein encoded by a
single exon of a single copy gene (PRNP on chromosome 20). It assumes an alpha helical structure and is located on the
cell surface with a glycoinositol phospholipid anchor. Treatment with proteases results in complete digestion.

In infected individuals, the PrPc protein is deranged to become the PrPsc (scrapie) protein. This glycoprotein assembles
into beta-sheets and is located in cytoplasmic vesicles. The insoluble PrPsc accumulates inside cells instead of being
located on the cell surface. It is only incompletely digested by proteases and this insolubility is thought to contribute to
storage problems and aggregation.

PRION DISEASES

There are several distinguishing hallmarks of these prion diseases. Although the etiologic agent may be
recoverable from other organs, the diseases are confined to the nervous system. The basic features are
neurodegeneration and spongiform changes. Amyloid plaques may be present. Long incubation periods (months to
decades) precede the onset of clinical illness and are followed by chronic progressive disease (weeks to years). The
diseases are always fatal, with no known cases of remission or recovery.

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 The host shows no inflammatory response and no immune response (the agents do not appear to be antigenic);
no production of interferon is elicited; and there is no effect on host B-cell or T-cell function. Immunosuppression of the
host has no effect on pathogenesis; however, chronic inflammation induced by other factors (viruses, bacteria,
autoimmunity) may affect prion pathogenesis. It has been observed that prions accumulate in organs with chronic
lymphocytic inflammation. When coincident with nephritis, prions are excreted in urine.

HUMAN PRION DISEASES ANIMAL PRION DISEASES

• Creutzfeldt-Jakob Disease (CJD), Classic • Bovine Spongiform Encephalopathy (BSE)
• Variant Creutzfeldt-Jakob Disease (vCJD) • Chronic Wasting Disease (CWD)
• Gerstmann-Straussler-Scheinker Syndrome • Scrapie
• Fatal Familial Insomnia • Transmissible mink encephalopathy
• Kuru • Feline spongiform encephalopathy
• Parkinson’s disease • Ungulate spongiform encephalopathy
• Alzheimer’s Disease
• Huntington’s Disease
• Lou Gehrig's disease

1. Classic Creutzfeldt-Jakob Disease (CJD)

Creutzfeldt-Jakob disease (CJD) is a rapidly progressive, invariably fatal neurodegenerative disorder believed to
be caused by an abnormal isoform of a cellular glycoprotein known as the prion protein. CJD occurs worldwide and the
estimated annual incidence in many countries has been reported to be about one case per million population.

Treatment of prion diseases remains supportive; no specific therapy has been shown to stop the progression of
these diseases.

Figure 10.1 Pathology of Normal brain (left) and pathology of the brain of a patient with CJD (right). Note the spongiform
pathology of the left picture.

2. Variant Creutzfeldt-Jakob Disease (vCJD)

Variant Creutzfeldt-Jakob disease (vCJD) is a prion disease that was first described in 1996 in the United Kingdom.
There is now strong scientific evidence that the agent responsible for the outbreak of prion disease in cows, bovine
spongiform encephalopathy (BSE or ‘mad cow’ disease), is the same agent responsible for the outbreak of vCJD in humans.

Variant CJD (vCJD) is not the same disease as classic CJD (often simply called CJD). It has different clinical and
pathologic characteristics from classic CJD. Each disease also has a particular genetic profile of the prion protein gene.

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Both disorders are invariably fatal brain diseases with unusually long incubation periods measured in years, and are caused
by an unconventional transmissible agent called a prion.

Treatment of prion diseases remains supportive; no specific therapy has been shown to stop the progression of
these diseases.

Table 10.1 Comparison of Classic CJD and Variant CJD

3. Kuru

Kuru occurred only in the eastern highlands of New Guinea and was spread by customs surrounding ritual
cannibalism of dead relatives. Since the practice has ceased, the disease has disappeared.

In the Fore language (spoken in Papua New Guinea), the term “kuru” refers to body tremors, which are a
characteristic feature of the disease. Symptoms progress from unsteady gait and tremors, to loss of muscle coordination,
severe ataxia, and death, over the course of 3 months to 2 years.

4. Scrapie

Scrapie, also called rida or tremblante du mouton, is a fatal neurodegenerative disease of sheep and goats. Scrapie
has a long incubation time, typically between about 18 months and five years following transmission. The first signs to
arise are usually behavioral changes such as general apprehensiveness and nervousness. As the disease progresses, the
animal loses weight and weakens, develops head and neck tremors, loses muscular coordination, and begins to rub or
scrape its body against objects, wearing away its fleece or hair—hence the name “scrapie”. The disease inevitably causes
death within one to six months. No treatment or palliative measures are known.

5. Bovine Spongiform Encephalopathy

BSE (bovine spongiform encephalopathy) is a progressive neurological disorder of cattle that results from infection
by an unusual transmissible agent called a prion. BSE possibly originated as a result of feeding cattle meat-and-bone meal
that contained BSE-infected products from a spontaneously occurring case of BSE or scrapie-infected sheep products.

If a cow is infected, it has trouble walking and getting up. It may also act very nervous or violent, which is why BSE
is often called “mad cow disease.”

6. Chronic Wasting Disease

A scrapie-like disease, designated chronic wasting disease, is found in mule deer and elk in the United States and
Canada. It is laterally transmitted with high efficiency, but there is no evidence that it has been transmitted to humans.
Infectivity has been detected in feces of deer before they become ill; the agent is retained in the soil, where it can then
be ingested by other deer and elk.

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MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
 PART 4: LIST OF REFERENCES

• Books, G, et.al. Jawetz, Melnick & Adelberg’s Medical Microbiology, 27th Ed. McGraw-Hill. Copyright 2015.

• Bulmer, Glenn. Fungus Diseases in the Orient. Rex Bookstore. Manila. 1991.

• Mahon and Manusells (ed.) Textbook of Diagnostic Microbiology. Elsevier (Singapore) Pte Ltd. 2014

• Forbes, B., Sahm, D. & WEissfeld, A. Bailey & Scott’s Diagnostic Microbiology, 13th ed. Elsevier Science
(Singapore) Pte Ltd. Copyright 2014

• Davey, F., et.al (ed). John Bernard Henry, Clinical Diagnosis and Management by Laboratory Methods. 22nd ed.
W.B. Saunders Co. Philadelphia. 2007

• Black, Jacquelyn G. Microbiology, Principles and Explorations, 5th edition. USA: McGraw-Hill Companies Inc.
2005

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MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
 University of San Agustin
ILOILO CITY, PHILIPPINES
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS
MEDICAL LABORATORY SCIENCE PROGRAM

MODULE PACKETS IN MLS 13A:

MYCOLOGY AND
VIROLOGY
M11

Matthew I. Tubola, RMT, MSMT

0|Page

John Robert D. Fundal, RMT

MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
© 2021. Printed in Calibri.
Cover Design: J.R.D. Fundal, RMT
All rights reserved. Copyright applied for. No part of this module may be reproduced, stored in retrieval
system or transmitted in any forms or by any means, electronic, mechanical, photocopying or otherwise,
without written permission from the authors.

The authors have done everything possible to make this module accurate and in accordance with accepted
standards. The authors are not responsible for errors or omissions or for consequences (loss, damage, or
disruption) from application of the module, and make no warranty, expressed or implied in regard to the
contents of the module. Any practice described in this module should be applied by the reader in accordance
with accepted standards used in regard to unique circumstances that may apply in each situation.

1|Page
MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
LEARNER’S HONOR CODE STATEMENT FOR THIS MODULE PACKET

“I affirm that I will not give or receive any unauthorized help on this MODULE, and that all
work will be my own.”
“I affirm that I have not given or received any unauthorized help on this assignment, and that
this work is my own.”

_________________________
Signature over printed name

2|Page
MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
 ABOUT THE AUTHORS

MATTHEW I. TUBOLA, RMT, MSMT

Hello Augustinians!
Welcome to MLS-13A (Mycology and Virology Lecture), I will be your lecturer for this subject. I am a
graduate of BSMT of this University and also finished my Master’s degree in Medical Technology in this
University. I have been teaching in this University for 12 years teaching Medical Technology Professional
Subjects.

JOHN ROBERT D. FUNDAL, RMT

Hello Augustinians!
Welcome to MLS-13A (Mycology and Virology Lecture), I will also be your lecturer for this subject. I
obtained my Bachelor’s Degree in Medical Laboratory Science from University of San Agustin. This is
currently my third semester in teaching MLS subjects.

3|Page
MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
 TABLE OF CONTENTS
Disclaimer 1
Honor Code 2
About the Authors 3
Content

Introduction 7
General Features and Characteristics 7
Specimen Collection, Transport and Processing 10
Laboratory Diagnosis 11
References 14

4|Page
MYCOLOGY AND VIROLOGY
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 UNIT MAP

Basic Concepts of Mycology

Introduction

General features and

characteristics

Specimen Collection, Transport

and Processing

Laboratory Diagnosis

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 PART 1: OVERVIEW OF DISCUSSION

The fungal kingdom encompasses a diverse and rich group of organisms ranging from microscopic yeasts
to mushrooms. Most fungi are free-living in nature where they function as decomposers in the energy cycle. Of
the more than 90 000 known fungal species, fewer than 200 have been reported to produce disease in humans.
Once considered clinical rarities, human fungal infections are becoming increasingly common, especially among
immunocompromised patients. Therefore, it is important to understand the unique clinical and microbiological
features of these diseases.

PART 2: LEARNING OBJECTIVES

At the end of the lecture, the student shall be able to:

• To know the general features and characteristics of fungi

• To identify ways on how fungi is transmitted
• To learn about the methods of collecting, transporting, and handling of specimen for fungal tests
• To enumerate the laboratory tests for identification of fungal infections
• To state ways on how fungal infections can be prevented and controlled

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 PART 3: DISCUSSION

INTRODUCTION

Mycology is the study of fungi, which are eukaryotic organisms that evolved in tandem with the animal kingdom.
However, unlike animals, most fungi are nonmotile and possess a rigid cell wall. Unlike plants, fungi are non-
photosynthetic.

Like all eukaryotes, each fungal cell has at least one nucleus with a nuclear membrane, endoplasmic reticulum,
mitochondria, and secretory apparatus. Most fungi are obligate or facultative aerobes. They are chemotrophic, secreting
enzymes that degrade a wide variety of organic substrates into soluble nutrients that are then passively absorbed or taken
into the cell by active transport.

CELLULAR STRUCTURE

Figure 11.1 A yeast cell showing the cell wall and internal structures of the fungal eukaryotic cell plan.

Fungal cells have a rigid cell wall external to the cytoplasmic membrane, which differs in its chemical
composition from the cell walls of bacteria and plants. In addition to the cell wall, another important difference from
mammalian cells is the sterol makeup of the cytoplasmic membrane. In mammalian cells, the dominant membrane
sterol is cholesterol; in fungi, it is ergosterol. Fungi are usually haploid in their DNA content, although diploid nuclei are
formed through nuclear fusion in the process of sexual reproduction.

The chemical structure of the cell wall in fungi is markedly different from that of bacterial cells in that it does not
contain peptidoglycan, glycerol, teichoic acids, or lipopolysaccharide. In their place are complex polysaccharides such as
mannans, glucans, and chitins in close association with each other and with structural proteins.

GENERAL FEATURES AND CHARACTERISTICS

Fungi that cause human infections can be broadly divided based on their morphological forms.

1. Yeast - single cells, usually spherical to ellipsoid in shape and varying in diameter from 3 to 15 µm.
2. Molds - fungi that primarily grow as filamentous, tube-like structures called hyphae that vary in
diameter from 2 to 10 µm.
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Table 11.1 Yeast vs. Molds
Point of Distinction Yeast Molds
Structure Mostly unicellular and existing individually Multicellular with tubular, filamentous
or with buds growing on them hyphae (branches)
Appearance Round or oval-shaped Threadlike
Method of Reproduction Budding or binary fission Production of sexual or asexual spores

Figure 11.2 Yeast (left) and mold (right) forms of fungal growth

TYPES OF HYPHAE

Hyphae can be divided into three based on their appearance on culture media.

1. Vegetative Hyphae – penetrate the media and absorb food

2. Aerial Hyphae – directed above the surface of the media
3. Reproductive Hyphae – aerial hyphae that carry reproductive spores

Figure 11.3 Types of Hyphal Growth

Although it is useful to consider this basic distinction based on cell shape, it is important to remember that some
fungi can transition between yeast-like and hyphal morphologies. Often, this plasticity of shape is directly related to
pathogenesis since different forms may be better suited for different microenvironments.

Table 11.2 Plasticity of Shape of Fungi

Fungal Forms Growth Phase
Monomorphic Fungi Has one growth phase (Yeast or Mold)
Dimorphic Fungi Mold at Room Temperature
Yeast at 37°C and in tissues
Diphasic Fungi Mold at Room temperature and at 37°C
Yeast in tissues

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METHODS OF REPRODUCTION
Fungi may reproduce by either asexual or sexual process. The asexual form is called the anamorph, and its
reproductive elements are termed conidia. The sexual form is called the teleomorph, and its reproductive structures are
called spores (eg, ascospores, zygospores, basidiospores).
Asexual reproduction involves mitotic division of the haploid nucleus and is associated with production by
budding, spore-like conidia or, alternatively by the separation of hyphal elements.
In sexual reproduction, the haploid nuclei of donor and recipient cells fuse to form a diploid nucleus, which then
divides by classic meiosis.

Table 11.3 Spores Involved in Sexual Reproduction

Spores Description
1. Ascospores Spores are contained in a saclike ascus.

2. Zygospores Involve the fusion of two identical cells arising from

the same hyphae.

3. Oospores Involve the fusion of cells from two separate, non-

identical hyphae.

4. Basidiospores Spores are contained in a club-shaped basidium.

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Table 11.4 Spores Involved in Asexual Reproduction
Spores Description
1. Conidia Spores produced singly or multiply in long
chains or clusters by specialized vegetative
haphae known as conidiophores.

Macroconidia – large, multicellular

Microconidia – small, unicellular
2. Blastoconidia Develops as daughter cell buds off the
(Blastospores) mother cell and is pinched off

3. Chlamydoconidia Thick walled, resistant, resting spores

(Chlamydospores) produced by rounding up and enlargement of
hyphal part.

Terminal – end of hypha

Intercalary – within hypha
Sessile – side of hypha
4. Arthroconidia Involve the simple fragmentation of the
(Arthrospores) mycelium. It appears “jointed”,
rectangular/barrel-shaped spores

5. Sporangiospores Spores contained in a sporangia or sacs that

are produced terminally on sporangiophore
or aseptate hyphae

MODE OF TRANSMISSION

Fungal infections are most often acquired from the external environment. One common mechanism of infection
is by the inhalation of infectious conidia generated from environmental molds. Some of these molds are ubiquitous,
whereas others are restricted to specific endemic areas and geographic regions whose climate favors their growth. Many
fungi produce disease only after they are accidentally injected past the skin/mucosal barrier, especially in
immunocompromised patients. Other pathogenic fungi have more sophisticated means of tissue penetration and
invasion.

SPECIMEN COLLECTION

Skin specimens should be cleaned with 70% alcohol to remove dirt, oil and surface saprophytes. Same procedure
must be done if the specimen is a nail, but it should be clipped and needs to be finely minced before inoculating to media.
Hair can be obtained by plucking, brushing, or with sticky tape. Normal sterile procedure must be done if the specimen is
a body fluid.
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 SPECIMEN TRANSPORT AND HANDLING

Hair & nails sent in a dry envelope, inside a proper container. Other specimens are usually sent frozen or on dry
ice. Specimen must be inside a packaging with biohazard regulations.

Any growing cultures must be on tube media, not in plates. Aluminum screw-capped inner with outer cardboard
mailing tube is usually the container of fungal specimen.

Inside labeling information must contain Patient ID, specimen source, suspected organism. Outside labeling
information must state, WARNING: POTENTIAL PATHOGEN. This labeling format still depends on the protocol of every
laboratory.

SPECIMEN PROCESSING

Table 11.5 Specimen processing to recover fungi.

Specimen Processing
Skin, nails, & hair Direct exam following KOH preparation
CSF Centrifuged; examine sediment microscopically, inoculate
media
Pleural fluid, sputum, Specimen must be fresh as saprophytes would overgrow
and bronchial pathogens such as H. capsulatum.
aspiration
Specimens may be refrigerated up to 2 hours
Gastric washings Same as for pleural fluids
Genito-urinary First morning specimen preferred
specimens
Blood/bone marrow Generally inoculated directly to BHI broth and BHI slant.
Wound abscess or Should be cultured anaerobically, especially if actinomycosis
drainage is suspected
Tissue specimens Examine for pus, caseous material or granules; mince
aseptically, can use small amount of sterile saline and the
supernatant inoculated

LABORATORY DIAGNOSIS

DIRECT EXAMINATION OF SPECIMENS

Fungi can often be identified by directly observing their distinctive morphologic features on direct microscopic
examination of infected pus, fluids, or tissues. Direct microscopic exam is required on any material sent to lab for fungus
culture. Medical technologists usually look for spores, hyphae, mycelial elements, budding yeast, mycotic granules.

1. Wet mount preparation

Good for yeast because examination is done in natural environment, so loss of fragile structure is minimized.

2. 10% KOH Mount

Done on skin scrapings, hail, nails, sputum, vaginal specimens, etc. The KOH clears the specimen’s tissue cells,
mucous, etc., without destroying the cell wall so fungal elements can be seen.

3. Fungal Stains

Direct examinations can be aided by the use fungal stain that can enhance the visualization of fungal structures
under the microscope.
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Table 11.6 Selected Fungal Stains
Stains Use
Lactophenol Cotton Blue (LPCB) Very popular for quick evaluation of fungal structures; will
stain the chitin in cell walls of fungi
Periodic Acid - Schiff Stain (PAS) Stains certain polysaccharide in the cell walls of fungi. Fungi
stain purplish-red with blue nuclei
Gomori Methenamine Silver Stain Outlines fungi in black due to the silver precipitating on the
fungi cell wall. Internal structures are deep rose to black;
background is light green
Gridley Stain Hyphae and yeast stain dark blue or rose. Tissues stain deep
blue and background is yellow
Mayer Mucicarmine Stain Will stain capsules of Cryptococcus neoformans deep rose
Fluorescent Antibody Stain Simple, sensitive, and specific. Applications for many
different fungal organisms
Papanicolaou Stain Good for initial differentiation of dimorphic fungi. Works
well on sputum smears
Gram Stain Most fungi are gram-positive
Giemsa Stain Used on blood and bone marrow specimens.
India Ink Demonstrates the capsule of Cryptococcus neoformans in
CSF specimens

FUNGAL CULTURE

In most cases, the culture is more sensitive than the direct examination, and a portion of the material collected
for microscopy should be cultured. Culture must be held for 21 days at room temperature, 25-30°C. Yeasts grow better at
37°C and molds at 30°C.

Table 11.7 Selected Fungal Culture Media

Stains Use
Birdseed Agar Isolation and preliminary identification of C. neoformans.
Appear as black colonies
Cornmeal Agar Cultivation of chlamydospore-bearing C. albicans
Cornmeal Agar with 1% Dextrose Improves the pigment production to differentiate T. rubrum
and T. mentagrophytes.

T. rubrum – produces red pigment

T. mentagrophytes – does not produce red pigment
Czapek’s agar Used to isolate Aspergillus spp.
Dermatophytes Test medium Used to isolate dermatophytes from cutaneous specimens
Mycosel/Mycobiotic Agar Selective media used to isolate pathogenic fungi,
dermatophytes, and systemic fungi.

Contains cycloheximide, which suppresses the growth of

saprophytic fungi, and chloramphenicol, which inhibits
bacterial contaminants
Sabouraud dextrose agar (SDA) Contains glucose and modified peptone (pH 7.0), supports
the growth of fungi, and restricts the growth of bacteria.

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MISCELLANEOUS TESTS

1. Germ Tube

• Yeasts are incubated with sterile serum at 37°C for up to 3 hours.

• Test for C. albicans

2. L-DOPA Ferric Citrate Test

• For phenol oxidase of C. neoformans

• Positive color is BLACK

3. Urease Test (+) Control

• C. neoformans is positive for this test

4. Hair Baiting test

• V-shaped penetration of hair shaft

• T. mentagrophytes – positive
• T. rubrum – negative

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 PART 4: LIST OF REFERENCES

• Books, G, et.al. Jawetz, Melnick & Adelberg’s Medical Microbiology, 27th Ed. McGraw-Hill. Copyright 2015.

• Bulmer, Glenn. Fungus Diseases in the Orient. Rex Bookstore. Manila. 1991.

• Mahon and Manusells (ed.) Textbook of Diagnostic Microbiology. Elsevier (Singapore) Pte Ltd. 2014

• Forbes, B., Sahm, D. & WEissfeld, A. Bailey & Scott’s Diagnostic Microbiology, 13th ed. Elsevier Science
(Singapore) Pte Ltd. Copyright 2014

• Davey, F., et.al (ed). John Bernard Henry, Clinical Diagnosis and Management by Laboratory Methods. 22nd ed.
W.B. Saunders Co. Philadelphia. 2007

• Black, Jacquelyn G. Microbiology, Principles and Explorations, 5th edition. USA: McGraw-Hill Companies Inc.
2005

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 University of San Agustin
ILOILO CITY, PHILIPPINES
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS
MEDICAL LABORATORY SCIENCE PROGRAM

MODULE PACKETS IN MLS 13A:

MYCOLOGY AND
VIROLOGY
M12

Matthew I. Tubola, RMT, MSMT

0|Page

John Robert D. Fundal, RMT

MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
© 2021. Printed in Calibri.
Cover Design: J.R.D. Fundal, RMT
All rights reserved. Copyright applied for. No part of this module may be reproduced, stored in retrieval
system or transmitted in any forms or by any means, electronic, mechanical, photocopying or otherwise,
without written permission from the authors.

The authors have done everything possible to make this module accurate and in accordance with accepted
standards. The authors are not responsible for errors or omissions or for consequences (loss, damage, or
disruption) from application of the module, and make no warranty, expressed or implied in regard to the
contents of the module. Any practice described in this module should be applied by the reader in accordance
with accepted standards used in regard to unique circumstances that may apply in each situation.

1|Page
MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
LEARNER’S HONOR CODE STATEMENT FOR THIS MODULE PACKET

“I affirm that I will not give or receive any unauthorized help on this MODULE, and that all
work will be my own.”
“I affirm that I have not given or received any unauthorized help on this assignment, and that
this work is my own.”

_________________________
Signature over printed name

2|Page
MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
 ABOUT THE AUTHORS

MATTHEW I. TUBOLA, RMT, MSMT

Hello Augustinians!
Welcome to MLS-13A (Mycology and Virology Lecture), I will be your lecturer for this subject. I am a
graduate of BSMT of this University and also finished my Master’s degree in Medical Technology in this
University. I have been teaching in this University for 12 years teaching Medical Technology Professional
Subjects.

JOHN ROBERT D. FUNDAL, RMT

Hello Augustinians!
Welcome to MLS-13A (Mycology and Virology Lecture), I will also be your lecturer for this subject. I
obtained my Bachelor’s Degree in Medical Laboratory Science from University of San Agustin. This is
currently my third semester in teaching MLS subjects.

3|Page
MYCOLOGY AND VIROLOGY
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 TABLE OF CONTENTS
Disclaimer 1
Honor Code 2
About the Authors 3
Content

Introduction 7
Superficial Mycoses 7
Cutaneous Mycoses 8
References 12

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 UNIT MAP
Morphology, Identification
Techniques, Pathology, Prevention,
and Control of Superficial and
Cutaneous Dermatophytes

Introduction

Superficial Mycoses

Cutaneous Mycoses

Laboratory Diagnosis

Treatment

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 PART 1: OVERVIEW OF DISCUSSION

The least invasive of the pathogenic fungi are the dermatophytes and other superficial fungi that are adapted to
the keratinized outer layers of the skin. Dermatophytoses are slowly progressive eruptions of the skin and its appendages.
Although often unsightly, they are not typically painful or life-threatening. The manifestations vary depending on the site
of infection and vigor of the host response, but they often involve erythema, induration, itching, and scaling. The most
familiar name is “ringworm,” describing the annular shape of the advancing edge of this cutaneous infection.

PART 2: LEARNING OBJECTIVES

At the end of the lecture, the student shall be able to:

• Describe the morphology of Superficial and Cutaneous Dermatophytes

• Enumerate identification techniques of Superficial and Cutaneous Dermatophytes
• Know the pathology of Superficial and Cutaneous Dermatophytes
• State ways on how to prevent and control Superficial and Cutaneous Dermatophytes

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 PART 3: DISCUSSION

INTRODUCTION

The least invasive of the pathogenic fungi are the dermatophytes and other superficial fungi that are adapted to
the keratinized outer layers of the skin.

Superficial Mycoses Agents Cutaneous Mycoses Agents

• Malassezia spp. • Microsporum species
• Hortaea werneckii • Trichophyton species
• Trichosporon spp. • Epidermophyton floccosum
• Piedraia hortae
SUPERFICIAL MYCOSES

MALASSEZIA SPP.
Pityriasis (tinea) versicolor is a very common superficial fungal infection of the skin. It is characterized by discrete
patches of either hypopigmentation or hyperpigmentation, especially on the skin of the torso and upper arms.
There are 14 currently recognized species of Malassezia, but the vast majority of cases of pityriasis versicolor are
caused by Malassezia globosa, Malassezia furfur, or Malassezia sympodialis.

Figure 12.1 Tinea versicolor

HORTAEA WERNECKII
Tinea nigra (or tinea nigra palmaris) is a superficial chronic and asymptomatic infection of the stratum corneum
caused by the dematiaceous fungus Hortaea (Exophiala) werneckii. The lesions appear as a dark (brown to black)
discoloration, often on the palm.

Figure 12.2 Tinea nigra

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PIEDRAIA HORTAE
Causative agent of Black piedra which is a nodular infection of the hair shaft.

TRICHOSPORON CUTANEUM
Causative agent of White Piedra. This superficial mycosis presents as larger, softer, yellowish nodules on the hairs

Figure 12.3 Piedra is an infection of the hair characterized by black (left) or white (right) nodules attached to the
hair shaft.

CUTANEOUS MYCOSES

Cutaneous mycoses are caused by fungi that infect only the keratinized tissue (skin, hair, and nails). The most
important of these are the dermatophytes, a group of about 40 related fungi that belong to three genera: Microsporum,
Trichophyton, and Epidermophyton.

PATHOGENESIS

Dermatophytoses begin when the infecting fungus comes in contact with skin, especially if there are minor breaks
in the skin integrity. Detached hair and skin scales containing dermatophytes can remain infectious for months in the
environment. Once the stratum corneum is penetrated, the organism can proliferate in the keratinized layers of the skin,
with a variety of proteinases helping to establish infection. Most dermatophyte infections are self-limited, spontaneously
resolving with time.

CLINICAL MANIFESTATIONS

Dermatophyte infections range from inapparent colonization to chronic progressive eruptions that last months or
years, causing considerable discomfort and disfiguration. Dermatologists often give each infection its own “disease” name
based on the Latin name for the anatomic site at which the infection is found. For example, these names include tinea
capitis (scalp), tinea pedis (feet, athlete’s foot), tinea manuum (hands), tinea cruris (groin), tinea barbae (beard, hair), and
tinea unguium (nail beds). Skin infections otherwise not included in this anatomic list are called tinea corporis (body).
There are certain clinical, etiologic, and epidemiologic differences among these syndromes, but they are basically the same
disease in different locations.

1. Tinea Pedis (Athlete’s Foot)

Tinea pedis is the most prevalent of all dermatophytoses. It usually occurs as a chronic infection of the toe webs.
Initially, there is itching between the toes and the development of small vesicles that rupture and discharge a thin fluid.
The skin of the toe webs becomes macerated and peels, whereupon cracks appear that are prone to develop secondary

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bacterial infection. When the fungal infection becomes chronic, peeling and cracking of the skin are the principal
manifestations, accompanied by pain and pruritus.

Figure 12.4 Tinea Pedis

2. Tinea Unguium (Onychomycosis)

Nail infection may follow prolonged tinea pedis. With hyphal invasion, the nails become yellow, brittle,
thickened, and crumbly. One or more nails of the feet or hands may be involved.

Figure 12.5 Tinea unguium

3. Tinea Corporis

Superficial fungal infection of the skin that can affect any part of the body, excluding the hands and feet, scalp,
face and beard, groin, and nails. It is commonly called 'ringworm' as it presents with characteristic ring-shaped lesions.

Figure 12.6 Tinea Corporis (Ringworm of the Body)

4. Tinea Cruris

Ringworm of the groin

Figure 12.7 Tinea Cruris

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5. Tinea manuum

Ringworm of hands, palms, and between fingers

Figure 12.8 Tinea manuum

6. Tinea capitis

Infection of hair and scalp begins with an erythematous papule around the hair shaft, which progresses to
scaling of the scalp, and discoloration/fracture of the shaft. Spread to adjacent hair follicles progresses in a ring-like
fashion, leaving behind broken, discolored hairs, and sometimes black dots where the hair is absent but the infection
has invaded the follicle.

Figure 12.9 Tinea capitis

7. Tinea barbae

Ringworm of the beard and mustache

Figure 12.10 Tinea barbae

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LABORATORY DIAGNOSIS

• Lactophenol Cotton Blue Stains

Figure 12.11 Microscopic appearance of Microscporum (Left), Trichophyton (Middle) and Epidermophyton (Right)

• Hair baiting technique

✓ V-shaped penetration of hair shaft
✓ T. mentagrophytes – positive
✓ T. rubrum – negative

• Wood’s lamp
✓ Some species of dermatophyte fluoresce when exposed to ultraviolet light

Figure 12.13 Wood’s lamp test of the scalp

TREATMENT

Many local skin infections resolve spontaneously without therapy. Those that do not resolve may be treated with
topical terbinafine or azoles (miconazole, ketoconazole). More extensive skin infections, especially those involving the
scalp, often require systemic therapy with griseofulvin, itraconazole, or oral terbinafine, often combined with topical
therapy. Nail infections are especially difficult to cure, likely due to the slow turnover of the infected nail and poor
penetration of antifungal agents. Therapy for nail infections must be continued over weeks to months, and relapses may
occur. Keratolytic agents (Whitfield’s ointment) may be useful for reducing the size of hyperkeratotic lesions.
Dermatophyte infections can usually be prevented simply by observing general hygiene measures. No specific preventive
measures such as vaccines exist.
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 PART 4: LIST OF REFERENCES

• Books, G, et.al. Jawetz, Melnick & Adelberg’s Medical Microbiology, 27th Ed. McGraw-Hill. Copyright 2015.

• Bulmer, Glenn. Fungus Diseases in the Orient. Rex Bookstore. Manila. 1991.

• Mahon and Manusells (ed.) Textbook of Diagnostic Microbiology. Elsevier (Singapore) Pte Ltd. 2014

• Forbes, B., Sahm, D. & WEissfeld, A. Bailey & Scott’s Diagnostic Microbiology, 13th ed. Elsevier Science
(Singapore) Pte Ltd. Copyright 2014

• Davey, F., et.al (ed). John Bernard Henry, Clinical Diagnosis and Management by Laboratory Methods. 22nd ed.
W.B. Saunders Co. Philadelphia. 2007

• Black, Jacquelyn G. Microbiology, Principles and Explorations, 5th edition. USA: McGraw-Hill Companies Inc.
2005

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MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
 University of San Agustin
ILOILO CITY, PHILIPPINES
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS
MEDICAL LABORATORY SCIENCE PROGRAM

MODULE PACKETS IN MLS 13A:

MYCOLOGY AND
VIROLOGY
M13

Matthew I. Tubola, RMT, MSMT

0|Page

John Robert D. Fundal, RMT

MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
© 2021. Printed in Calibri.
Cover Design: J.R.D. Fundal, RMT
All rights reserved. Copyright applied for. No part of this module may be reproduced, stored in retrieval
system or transmitted in any forms or by any means, electronic, mechanical, photocopying or otherwise,
without written permission from the authors.

The authors have done everything possible to make this module accurate and in accordance with accepted
standards. The authors are not responsible for errors or omissions or for consequences (loss, damage, or
disruption) from application of the module, and make no warranty, expressed or implied in regard to the
contents of the module. Any practice described in this module should be applied by the reader in accordance
with accepted standards used in regard to unique circumstances that may apply in each situation.

1|Page
MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
LEARNER’S HONOR CODE STATEMENT FOR THIS MODULE PACKET

“I affirm that I will not give or receive any unauthorized help on this MODULE, and that all
work will be my own.”
“I affirm that I have not given or received any unauthorized help on this assignment, and that
this work is my own.”

_________________________
Signature over printed name

2|Page
MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
 ABOUT THE AUTHORS

MATTHEW I. TUBOLA, RMT, MSMT

Hello Augustinians!
Welcome to MLS-13A (Mycology and Virology Lecture), I will be your lecturer for this subject. I am a
graduate of BSMT of this University and also finished my Master’s degree in Medical Technology in this
University. I have been teaching in this University for 12 years teaching Medical Technology Professional
Subjects.

JOHN ROBERT D. FUNDAL, RMT

Hello Augustinians!
Welcome to MLS-13A (Mycology and Virology Lecture), I will also be your lecturer for this subject. I
obtained my Bachelor’s Degree in Medical Laboratory Science from University of San Agustin. This is
currently my third semester in teaching MLS subjects.

3|Page
MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
 TABLE OF CONTENTS
Disclaimer 1
Honor Code 2
About the Authors 3
Content

Introduction 7
Sporotrichosis 7
Chromoblastomycosis 8
Phaeohyphomycosis 9
Mycetoma 9
References 10

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MYCOLOGY AND VIROLOGY
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 UNIT MAP
Morphology, Identification
Techniques, Pathology, Prevention,
and Control of Subcutaneous
Mycosis

Introduction

Sporotrichosis

Chromoblastomycosis

Phaeohyphomycosis

Mycetoma

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MYCOLOGY AND VIROLOGY
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 PART 1: OVERVIEW OF DISCUSSION

Many fungal pathogens can produce subcutaneous lesions as part of their disease spectrum. Those considered
here are introduced traumatically through the skin, with infection typically limited to subcutaneous tissues, lymphatic
vessels, and contiguous tissues. These fungi rarely spread to distant organs. The diseases they cause include
Sporotrichosis, Chromoblastomycosis, Phaeohyphomycosis and Mycetoma.

PART 2: LEARNING OBJECTIVES

At the end of the lecture, the student shall be able to:

• Describe the morphology of Subcutaneous fungi

• Enumerate identification techniques of Subcutaneous fungi
• Know the pathology of Subcutaneous fungi
• State ways on how to prevent and control Subcutaneous fungi

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 PART 3: DISCUSSION

INTRODUCTION

The fungi that cause subcutaneous mycoses normally reside in soil or on vegetation. They enter the skin or
subcutaneous tissue by traumatic inoculation with contaminated material. For example, a superficial cut or abrasion may
introduce an environmental mold with the ability to infect the exposed dermis. In general, the lesions become
granulomatous and expand slowly from the area of implantation. Extension via the lymphatics draining the lesion is slow
except in sporotrichosis. These mycoses are usually confined to the subcutaneous tissues, but in rare cases they become
systemic and produce life-threatening disease.

Subcutaneous mycoses to consider:

1. Sporotrichosis
2. Chromoblastomycosis
3. Phaeohyphomycosis
4. Mycetoma

SPOROTRICHOSIS

S. schenckii is a thermally dimorphic fungus that lives on vegetation. It is associated with a variety of plants—
grasses, trees, sphagnum moss, rose bushes, and other horticultural plants. At ambient temperatures, it grows as a mold,
producing branching, septate hyphae and conidia, and in tissue or in vitro at 35–37°C as a small budding yeast. Following
traumatic introduction into the skin, S. schenckii causes sporotrichosis, a chronic granulomatous infection. The initial
episode is typically followed by secondary spread with involvement of the draining lymphatics and lymph nodes.

MORPHOLOGY OF S. schenckii

Sporothrix schenckii is a dimorphic fungus that grows as a cigar-shaped, 3- to 5-mm yeast in tissues and in culture
at 37°C. The mold, which grows in cultures incubated at 25°C, is presumably the infectious form in nature.

Figure 13.1 Yeast (left) and Mold (Right) form of S. schenckii

DIAGNOSTIC LABORATORY TESTS

Specimens, like biopsy material or exudate from granulomatous or ulcerative lesions, are examined directly with
KOH or calcofluor white stain, the yeasts are rarely found. The use of specials stains like Gomori methenamine silver
(stains the cell walls black), Periodic Acid-Schiff stain (imparts a red color to the cell walls), and fluorescent antibody
staining are used to enhance the sensitivity of the test.

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 Another structure termed an asteroid body is often seen in tissues of individuals infected with S. schenckii. In
hematoxylin and eosin-stained tissue, the asteroid body consists of a central basophilic yeast cell surrounded by radiating
extensions of eosinophilic material, which are depositions of antigen–antibody complexes and complement.

Figure 13.2 Asteroid Body

The most reliable method of diagnosis is culture. Specimens are streaked on Inhibitory Mold Agar or Sabouraud
Dextrose Agar containing antibacterial antibiotics and incubated at 25–30°C. The identification is confirmed by growth at
35°C and conversion to the yeast form.

TREATMENT, PREVENTION AND CONTROL

In some cases, the infection is self-limited. Although the oral administration of saturated solution of potassium
iodide in milk is quite effective, it is difficult for many patients to tolerate. The treatment of choice is oral itraconazole or
another azole. For systemic disease, amphotericin B is given.

Prevention includes measures to minimize accidental inoculation and the use of fungicides, where appropriate, to
treat wood.

CHROMOBLASTOMYCOSIS (CHROMOMYCOSIS)

Chromoblastomycosis (chromomycosis) is a subcutaneous mycotic infection that is usually caused by traumatic

inoculation of any of the recognized fungal agents, which reside in soil and vegetation. All are dematiaceous fungi, having
melanized cell walls: Phialophora verrucosa, Fonsecaea pedrosoi, Fonsecaea compacta, Rhinocladiella aquaspersa, and
Cladophialophora carrionii. The infection is chronic and characterized by the slow development of progressive
granulomatous lesions that in time induce hyperplasia of the epidermal tissue.

MORPHOLOGY OF DEMATIACEOUS FUNGI

The dematiaceous fungi are similar in their pigmentation, antigenic structure, morphology, and physiologic
properties. The colonies are compact, deep brown to black, and develop a velvety, often wrinkled surface. The agents of
chromoblastomycosis are identified by their modes of conidiation. In tissue they appear the same, producing spherical
brown cells termed muriform or sclerotic bodies that divide by transverse septation. Septation in different planes with
delayed separation may give rise to a cluster of four to eight cells.

Figure 13. 3 Sclerotic bodies

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DIAGNOSTIC LABORATORY TEST

Specimens of scrapings or biopsies from lesions are examined microscopically in KOH for dark, spherical cells.
Specimens should be cultured on IMA or SDA with antibiotics. The dematiaceous species is identified by its characteristic
conidial structures. There are many similar saprophytic dematiaceous molds, but they differ from the pathogenic species
in being unable to grow at 37°C and being able to digest gelatin.

TREATMENT, PREVENTION AND CONTROL

Surgical excision with wide margins is the therapy of choice for small lesions. Chemotherapy with flucytosine or
itraconazole may be efficacious for larger lesions. The application of local heat is also beneficial. Relapse is common.

The disease occurs chiefly on the legs of barefoot agrarian workers following traumatic introduction of the fungus.
Chromoblastomycosis is not communicable. Wearing shoes and protecting the legs probably would prevent infection

PHAEOHYPHOMYCOSIS

Phaeohyphomycosis is a term applied to infections characterized by the presence of darkly pigmented septate
hyphae in tissue. Both cutaneous and systemic infections have been described.

Table 13.1 Causative agents of Phaeohyphomycosis

Subcutaneous Systemic
Phaeohyphomycosis Phaeohyphomycosis
• Exophiala jeanselmei • Exophiala jeanselmei
• Phialophora richardsiae • Phialophora richardsiae
• Bipolaris spicifera • Bipolaris spicifera
• Wangiella dermatitidis • Wangiella dermatitidis
• Exserohilum rostratum
• Alternaria species
• Curvularia species
In tissue, the hyphae are large (5–10 µm in diameter), often distorted and may be accompanied by yeast cells, but
these structures can be differentiated from other fungi by the melanin in their cell walls. Specimens are cultured on routine
fungal media to identify the etiologic agent. In general, itraconazole or flucytosine is the drug of choice for subcutaneous
phaeohyphomycosis. Brain abscesses are usually fatal, but when recognized, they are managed with amphotericin B and
surgery. The leading cause of cerebral phaeohyphomycosis is C. bantiana.

MYCETOMA

Mycetoma is a chronic subcutaneous infection induced by traumatic inoculation with any of several saprophytic
species of fungi or actinomycetous bacteria that are normally found in soil. The infection is characterized by local swelling
of the infected tissue and interconnecting, often draining, sinuses or fistulae that contain granules, which are
microcolonies of the agent embedded in tissue material.

An actinomycetoma is a mycetoma caused by an actinomycete; a eumycetoma (maduromycosis, Madura foot) is

a mycetoma caused by a fungus. The fungal agents of mycetoma include, among others, Pseudallescheria boydii,
Madurella mycetomatis, Madurella grisea, Exophiala jeanselmei. E. jeanselmei and the Madurella species are
dematiaceous molds. These molds are identified primarily by their mode of conidiation.

In tissue, the mycetoma granules may range up to 2 mm in size. The color of the granule may provide information
about the agent. For example, the granules of mycetoma caused by P. boydii and A. falciforme are white; those of M.
grisea and E. jeanselmei are black; and M. mycetomatis produces a dark red to black granule. These granules are hard and
contain intertwined, septate hyphae (3–5 µm in width). The hyphae are typically distorted and enlarged at the periphery
of the granule
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 Figure 13.4 Madura Foot

DIAGNOSTIC LABORATORY TEST

Granules can be dissected out from the pus or biopsy material for examination and culture on appropriate media.
The granule color, texture, and size and the presence of hyaline or pigmented hyphae (or bacteria) are helpful in
determining the causative agent.

TREATMENT, PREVENTION AND CONTROL

The management of eumycetoma is difficult, involving surgical debridement or excision and chemotherapy. P.
boydii is treated with topical nystatin or miconazole. Itraconazole, ketoconazole, and even amphotericin B can be
recommended for Madurella infections and flucytosine for E. jeanselmei. Chemotherapeutic agents must be given for long
periods to adequately penetrate these lesions.

The organisms producing mycetoma occur in soil and on vegetation. Barefoot farm laborers are therefore
commonly exposed. Properly cleaning wounds and wearing shoes are reasonable control measures.

PART 4: LIST OF REFERENCES

• Books, G, et.al. Jawetz, Melnick & Adelberg’s Medical Microbiology, 27th Ed. McGraw-Hill. Copyright 2015.

• Bulmer, Glenn. Fungus Diseases in the Orient. Rex Bookstore. Manila. 1991.

• Mahon and Manusells (ed.) Textbook of Diagnostic Microbiology. Elsevier (Singapore) Pte Ltd. 2014

• Forbes, B., Sahm, D. & WEissfeld, A. Bailey & Scott’s Diagnostic Microbiology, 13th ed. Elsevier Science
(Singapore) Pte Ltd. Copyright 2014

• Davey, F., et.al (ed). John Bernard Henry, Clinical Diagnosis and Management by Laboratory Methods. 22nd ed.
W.B. Saunders Co. Philadelphia. 2007

• Black, Jacquelyn G. Microbiology, Principles and Explorations, 5th edition. USA: McGraw-Hill Companies Inc.
2005

10 | P a g e
MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
 University of San Agustin
ILOILO CITY, PHILIPPINES
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS
MEDICAL LABORATORY SCIENCE PROGRAM

MODULE PACKETS IN MLS 13A:

MYCOLOGY AND
VIROLOGY
M14

Matthew I. Tubola, RMT, MSMT

0|Page

John Robert D. Fundal, RMT

MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
© 2021. Printed in Calibri.
Cover Design: J.R.D. Fundal, RMT
All rights reserved. Copyright applied for. No part of this module may be reproduced, stored in retrieval
system or transmitted in any forms or by any means, electronic, mechanical, photocopying or otherwise,
without written permission from the authors.

The authors have done everything possible to make this module accurate and in accordance with accepted
standards. The authors are not responsible for errors or omissions or for consequences (loss, damage, or
disruption) from application of the module, and make no warranty, expressed or implied in regard to the
contents of the module. Any practice described in this module should be applied by the reader in accordance
with accepted standards used in regard to unique circumstances that may apply in each situation.

1|Page
MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
LEARNER’S HONOR CODE STATEMENT FOR THIS MODULE PACKET

“I affirm that I will not give or receive any unauthorized help on this MODULE, and that all
work will be my own.”
“I affirm that I have not given or received any unauthorized help on this assignment, and that
this work is my own.”

_________________________
Signature over printed name

2|Page
MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
 ABOUT THE AUTHORS

MATTHEW I. TUBOLA, RMT, MSMT

Hello Augustinians!
Welcome to MLS-13A (Mycology and Virology Lecture), I will be your lecturer for this subject. I am a
graduate of BSMT of this University and also finished my Master’s degree in Medical Technology in this
University. I have been teaching in this University for 12 years teaching Medical Technology Professional
Subjects.

JOHN ROBERT D. FUNDAL, RMT

Hello Augustinians!
Welcome to MLS-13A (Mycology and Virology Lecture), I will also be your lecturer for this subject. I
obtained my Bachelor’s Degree in Medical Laboratory Science from University of San Agustin. This is
currently my third semester in teaching MLS subjects.

3|Page
MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
 TABLE OF CONTENTS
Disclaimer 1
Honor Code 2
About the Authors 3
Content

Introduction 7
Histoplasmosis 7
Coccidioidomycosis 8
Blastomycosis 10
Paracoccidioidomycosis 11
References 12

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 UNIT MAP
Morphology, Identification
Techniques, Pathology, Prevention,
and Control of Endemic Mycoses

Introduction

Histoplasmosis

Coccidioidomycosis

Blastomycosis

Paracoccidioidomycosis

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 PART 1: OVERVIEW OF DISCUSSION

The fungi discussed in this module cause a variety of infections, each ranging in severity from subclinical to
progressive, debilitating disease. Some of these species are dimorphic, growing in the infectious mold form in the
environment but switching to a round, yeast-like form in infected tissues. They differ from the opportunistic fungi in their
ability to cause disease in previously healthy persons. However, the most serious infections still occur in patients with
compromised immune systems. Each of these fungi is restricted to geographic niches corresponding to the environmental
habitats of the mold form of the species (Endemic). None of these infections is transmitted from human to human.

PART 2: LEARNING OBJECTIVES

At the end of the lecture, the student shall be able to:

• Describe the morphology of Systemic Mycoses

• Enumerate identification techniques of Systemic Mycoses
• Know the pathology of Systemic Mycoses
• State ways on how to prevent and control Systemic Mycoses

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 PART 3: DISCUSSION

INTRODUCTION

Coccidioidomycosis, Histoplasmosis, Blastomycosis, and Paracoccidioidomycosis are systemic mycoses that are
geographically restricted to specific areas of endemicity.

Table 14.1 Summary of Endemic Mycoses

Mycoses Agents Habitat Tissue form
Histoplasmosis Histoplasma capsulatum Avian and bat habitats Oval yeasts, 2 × 4 μm;
(guano) mixed with Intracellular in macrophages
alkaline soil
Coccidioidomycosis Coccidioides posadasii Dry soil Spherules, 10–80 μm,
Coccidioides immitis containing endospores, 2–4 μm
Blastomycosis Blastomyces dermatitidis Unknown (riverbanks) Thick-walled yeasts with broad-
based, usually single, buds,
8–15 μm
Paracoccidioidomycosis Paracoccidioides brasiliensis Unknown (soil) Large, multiply budding yeasts,
15–30 μm
HISTOPLASMOSIS

H. capsulatum is a dimorphic soil saprophyte that causes histoplasmosis, the most prevalent pulmonary fungal
infection in humans and animals. In nature, H. capsulatum grows as a mold in association with soil and avian habitats,
being enriched by alkaline nitrogenous substrates in guano.

MORPHOLOGY OF H. capsulatum

At temperatures below 37°C, primary isolates of H. capsulatum often develop brown mold colonies, but the
appearance varies. In tissue or in vitro on rich medium at 37°C, the hyphae and conidia convert to small, oval yeast cells
(2 × 4 µm).

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LABORATORY DIAGNOSIS

Table 14.2
SPECIMENS MICROSCOPIC CULTURE
• Sputum • Use of fungal wall stains to visualize • Glucose-cysteine-blood agar at
• Urine the small ovoid cells. 37°C
• Scrapings from superficial
lesions 1. GMS • SDA or IMA at 25–30°C.
• Bone marrow aspirates 2. PAS
• Buffy coat blood cells. 3. Giemsa

TREATMENT, PREVENTION AND CONTROL

Acute pulmonary histoplasmosis is managed with supportive therapy and rest. Itraconazole is the treatment for
mild to moderate infection. In disseminated disease, systemic treatment with amphotericin B is often curative, though
patients may need prolonged treatment and monitoring for relapses. Patients with AIDS typically relapse despite therapy
that would be curative in other patients. Therefore, AIDS patients require maintenance therapy with itraconazole.

COCCIDIOIDOMYCOSIS

Coccidioidomycosis is caused C. posadasii or C. immitis. They are phenotypically indistinguishable, cause similar
clinical manifestations, and are not differentiated in the clinical laboratory.

Acute primary infection with C. immitis is most often asymptomatic, but it can manifest as a complex of symptoms
called “Valley Fever” by residents of the endemic areas. Valley Fever includes fever, malaise, dry cough, joint pains, and
sometimes a rash.

Figure 14.1 Erythema multiforme due to coccidioidomycosis .

MORPHOLOGY OF C. posadasii and C. immitis

These agents produce a white to tan cottony colony. The hyphae form chains of arthroconidia (arthrospores),
which often develop in alternate cells of a hypha. These chains fragment into individual arthroconidia, which are readily
airborne and highly resistant to adverse environmental conditions. These small arthroconidia (3 × 5 µm) remain viable for
years and are highly infectious. Following their inhalation, the arthroconidia become spherical and enlarge, forming
spherules that contain endospores

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 Figure 14.2 The saprobic cycle is found in the environment and produces infectious arthroconidia. They may become
airborne and be inhaled by the host or may return to the environment to continue the saprobic life cycle.

DIAGNOSTIC LABORATORY TESTS

Table 14.3
SPECIMENS MICROSCOPIC CULTURE
• Sputum • KOH • IMA
• Exudate from cutaneous • Calcofluor white stain • Brain–heart infusion blood agar
lesions slant
• Spinal fluid Histologic Stains
• Blood NOTE: Incubate at room temperature
• Urine • H&E or at 37°C
• Tissue biopsies • GMS
• PAS

TREATMENT, PREVENTION AND CONTROL

In most persons, symptomatic primary infection is self-limited and requires only supportive treatment, although
itraconazole may reduce the symptoms. However, patients who have severe disease require treatment with amphotericin
B, which is administered intravenously.

Cases of coccidioidal meningitis have been treated with oral fluconazole, which has good penetration of the
central nervous system; however, long-term therapy is required, and relapses have occurred.

The disease is not communicable from person to person, and there is no evidence that infected rodents contribute
to its spread. Some measure of control can be achieved by reducing dust, paving roads and airfields, planting grass or
crops, and using oil sprays.

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 BLASTOMYCOSIS

B. dermatitidis causes blastomycosis, a chronic infection with granulomatous and suppurative lesions that is
initiated in the lungs, whence dissemination may occur to any organ but preferentially to the skin and bones. The disease
has been called North American blastomycosis because it is endemic, and most cases occur in the United States and in
Canada.

B. dermatitidis is a thermally dimorphic fungus that grows as a mold in culture, producing hyaline, and branching
septate hyphae and conidia. At 37°C or in the host, it converts to a large, singly budding yeast cell

Figure 14.2 Extraplumonary Blastomycosis

MORPHOLOGY OF B. dermatitidis

When B. dermatitidis is grown on SDA at room temperature, a white or brownish colony develops, with branching
hyphae bearing spherical, ovoid, or piriform conidia (3–5 µm in diameter) on slender terminal or lateral conidiophores. In
tissue or culture at 37°C, B. dermatitidis grows as a thick-walled, multinucleated, spherical yeast (8–15 µm) that usually
produces single buds.

Figure 14.3 Mold form (L) and Yeast form (R) of B. dermatitidis

LABORATORY DIAGNOSIS

Table 14.4
SPECIMENS MICROSCOPIC CULTURE
• Sputum • Wet mounts of specimens may show • Colonies usually develop within 2
• Pus broadly attached buds on thick-walled weeks on Sabouraud’s or enriched
• Exudates yeast cells. These may also be apparent blood agar at 30°C
• Urine in histologic section
• Biopsies from lesions

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TREATMENT, PREVENTION AND CONTROL

Severe cases of blastomycosis are treated with amphotericin B. In patients with confined lesions, a 6-month course
of itraconazole is very effective.

Blastomycosis is a relatively common infection of dogs (and rarely other animals) in endemic areas. Blastomycosis
cannot be transmitted by animals or humans. Unlike C. immitis and H. capsulatum, B. dermatitidis has only rarely (and not
reproducibly) been isolated from the environment, so its natural habitat is unknown. However, the occurrence of several
small outbreaks has linked B. dermatitidis to rural river banks.

PARACOCCIDIOIDOMYCOSIS

Paracoccidioidomycosis is progressive mycosis of the lungs, skin, mucous membranes, lymph nodes, and internal
organs caused by Paracoccidioides brasiliensis.

P. brasiliensis is the thermally dimorphic fungal agent of paracoccidioidomycosis (South American blastomycosis),
which is confined to endemic regions of Central and South America.

P. brasiliensis is inhaled, and initial lesions occur in the lung. After a period of dormancy that may last for decades,
the pulmonary granulomas may become active, leading to chronic, progressive pulmonary disease or dissemination.

Figure 14.4 Paracoccidioidomycosis lesion

MORPHOLOGY OF P. brasiliensis

Cultures of the mold form of P. brasiliensis grow very slowly and produce chlamydospores and conidia. The features are
not distinctive. At 36°C, on rich medium, it forms large, multiply budding yeast cells (up to 30 µm). The yeasts are larger
and have thinner walls than those of B. dermatitidis. The buds are attached by a narrow connection

Figure 14.5 Mold (left) and Yeast (right) form of P. brasiliensis

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LABORATORY DIAGNOSIS

Table 14.5
MICROSCOPIC CULTURE
• Yeasts are often apparent on direct microscopic • Cultures on Sabouraud’s or yeast extract agar are
examination with KOH or calcofluor white incubated at room temperature and confirmed by
conversion to the yeast form by in vitro growth at
36°C.

TREATMENT, PREVENTION AND CONTROL

Itraconazole appears to be most effective against paracoccidioidomycosis, but ketoconazole and trimethoprim–
sulfamethoxazole are also efficacious. Severe disease can be treated with amphotericin B.

Since P. brasiliensis has only rarely been isolated from nature, its natural habitat has not been definitively
determined. As with the other endemic mycoses, paracoccidioidomycosis is not communicable.

PART 4: LIST OF REFERENCES

• Books, G, et.al. Jawetz, Melnick & Adelberg’s Medical Microbiology, 27th Ed. McGraw-Hill. Copyright 2015.

• Bulmer, Glenn. Fungus Diseases in the Orient. Rex Bookstore. Manila. 1991.

• Mahon and Manusells (ed.) Textbook of Diagnostic Microbiology. Elsevier (Singapore) Pte Ltd. 2014

• Forbes, B., Sahm, D. & WEissfeld, A. Bailey & Scott’s Diagnostic Microbiology, 13th ed. Elsevier Science
(Singapore) Pte Ltd. Copyright 2014

• Davey, F., et.al (ed). John Bernard Henry, Clinical Diagnosis and Management by Laboratory Methods. 22nd ed.
W.B. Saunders Co. Philadelphia. 2007

• Black, Jacquelyn G. Microbiology, Principles and Explorations, 5th edition. USA: McGraw-Hill Companies Inc.
2005

12 | P a g e
MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
 University of San Agustin
ILOILO CITY, PHILIPPINES
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS
MEDICAL LABORATORY SCIENCE PROGRAM

MODULE PACKETS IN MLS 13A:

MYCOLOGY AND
VIROLOGY
M15

Matthew I. Tubola, RMT, MSMT

0|Page

John Robert D. Fundal, RMT

MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
© 2021. Printed in Calibri.
Cover Design: J.R.D. Fundal, RMT
All rights reserved. Copyright applied for. No part of this module may be reproduced, stored in retrieval
system or transmitted in any forms or by any means, electronic, mechanical, photocopying or otherwise,
without written permission from the authors.

The authors have done everything possible to make this module accurate and in accordance with accepted
standards. The authors are not responsible for errors or omissions or for consequences (loss, damage, or
disruption) from application of the module, and make no warranty, expressed or implied in regard to the
contents of the module. Any practice described in this module should be applied by the reader in accordance
with accepted standards used in regard to unique circumstances that may apply in each situation.

1|Page
MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
LEARNER’S HONOR CODE STATEMENT FOR THIS MODULE PACKET

“I affirm that I will not give or receive any unauthorized help on this MODULE, and that all
work will be my own.”
“I affirm that I have not given or received any unauthorized help on this assignment, and that
this work is my own.”

_________________________
Signature over printed name

2|Page
MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
 ABOUT THE AUTHORS

MATTHEW I. TUBOLA, RMT, MSMT

Hello Augustinians!
Welcome to MLS-13A (Mycology and Virology Lecture), I will be your lecturer for this subject. I am a
graduate of BSMT of this University and also finished my Master’s degree in Medical Technology in this
University. I have been teaching in this University for 12 years teaching Medical Technology Professional
Subjects.

JOHN ROBERT D. FUNDAL, RMT

Hello Augustinians!
Welcome to MLS-13A (Mycology and Virology Lecture), I will also be your lecturer for this subject. I
obtained my Bachelor’s Degree in Medical Laboratory Science from University of San Agustin. This is
currently my third semester in teaching MLS subjects.

3|Page
MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
 TABLE OF CONTENTS
Disclaimer 1
Honor Code 2
About the Authors 3
Content

Introduction 7
Candidiasis 7
Aspergillosis 8
Mucormycosis 9
References 11

4|Page
MYCOLOGY AND VIROLOGY
A.Y. 2020 - 2021
 UNIT MAP

Morphology, Identification
Techniques, Pathology, Prevention,
and Control Opportunistic Mycoses

Introduction

Candidiasis

Aspergillosis

Mucormycosis

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 PART 1: OVERVIEW OF DISCUSSION

The “opportunistic fungi” are usually found as members of the resident human microbiota or as saprophytes in
the environment. With the breakdown of host defenses, they can cause infections ranging from skin/mucous membrane
involvement to life-threatening, systemic disease.

PART 2: LEARNING OBJECTIVES

At the end of the lecture, the student shall be able to:

• Describe the morphology of Opportunistic Mycoses

• Enumerate identification techniques of Opportunistic Mycoses
• Know the pathology of Systemic Mycoses
• State ways on how to prevent and control Opportunistic Mycoses

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 PART 3: DISCUSSION

INTRODUCTION

The “opportunistic fungi” are usually found as members of the resident human microbiota or as saprophytes in
the environment. With the breakdown of host defenses, they can cause infections ranging from skin/mucous membrane
involvement to life-threatening, systemic disease.

Candida and related yeasts are endogenous opportunists. Other opportunistic mycoses are caused by exogenous
fungi that are globally present in soil, water, and air.

The coverage here will focus on the more common pathogens and the diseases they cause—candidiasis,
aspergillosis and mucormycosis.

CANDIDIASIS

Candidiasis is the most prevalent systemic mycosis, and the most common agents are C. albicans, Candida
parapsilosis, Candida glabrata, Candida tropicalis, Candida guilliermondii, and Candida dubliniensis.

Candidiasis occurs in localized and disseminated forms. Localized disease is seen as erythema and white plaques
in moist skinfolds (diaper rash) or on mucosal surfaces (oral thrush). It may also cause the itching and thick white discharge
of vulvovaginitis. Deep tissue and disseminated infections are limited almost exclusively to the immunocompromised.

MORPHOLOGY

Candida albicans, the most common cause of human invasive fungal infections, is also able to form hyphae
triggered by changes in conditions such as temperature, pH, and available nutrients. When observed in their initial stages
of germination from the yeast cell, these nascent hyphae resemble sprouts and are called “germ tubes”. Other elongated
forms with restrictions at regular intervals are called pseudohyphae because they lack the parallel walls and septation of
true hyphae.

Figure 15.1 Three forms of Candida albicans

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LABORATORY DIAGNOSIS

Table 15.1
SPECIMENS MICROSCOPIC CULTURE
• Swabs and scrapings from • Tissue biopsies, centrifuged spinal • All specimens are cultured on fungal or
superficial lesions fluid, and other specimens may be bacteriologic media at room
• Blood examined in Gram-stained smears temperature or at 37°C.
• Spinal fluid or histopathologic slides for
• Tissue biopsies pseudohyphae and budding cells • Yeast colonies are examined for the
• Urine presence of pseudohyphae.
• Exudates • As with dermatophytosis, skin or
• Material from removed nail scrapings are first placed in a • C. albicans is identified by the production
intravenous catheters. drop of KOH and calcofluor white of germ tubes or chlamydospores.

• CHROMagar® is a useful commercial

medium for the rapid identification of
several Candida species based on fungal
enzymatic action on chromogenic
substrates in the medium.

TREATMENT, PREVENTION AND CONTROL

Thrush and other mucocutaneous forms of candidiasis are usually treated with topical nystatin or oral
ketoconazole or fluconazole. Systemic candidiasis is treated with amphotericin B, sometimes in conjunction with oral
flucytosine, fluconazole, or caspofungin.

The most important preventive measure is to avoid disturbing the normal balance of microbiota and intact host
defenses. Candidiasis is not communicable, since virtually all persons normally harbor the organism.

ASPERGILLOSIS

Aspergillosis is a spectrum of diseases that may be caused by a number of Aspergillus species. Aspergillus species
are ubiquitous saprobes in nature, and aspergillosis occurs worldwide. A. fumigatus is the most common human
pathogen, but many others, including Aspergillus flavus, Aspergillus niger, Aspergillus terreus, and Aspergillus lentulus,
may cause disease. This mold produces abundant small conidia that are easily aerosolized. Following inhalation of these
conidia, atopic individuals often develop severe allergic reactions to the conidial antigens. In immunocompromised
patients— especially those with leukemia, stem cell transplant patients, and individuals taking corticosteroids—the
conidia may germinate to produce hyphae that invade the lungs and other tissues.

Aspergillus infections typically present in one of four major ways, with the clinical findings completely dependent
on the immune status of the host. These clinical presentations include:

1. Aspergillus pneumonia
2. Disseminated aspergillosis
3. Allergic respiratory disease
4. Aspergilloma (fungus ball)

MORPHOLOGY
Aspergillus species grow rapidly, producing aerial hyphae that bear characteristic conidial structures: long
conidiophores with terminal vesicles on which phialides produce basipetal chains of conidia (see figure 15.2). The species
are identified according to morphologic differences in these structures, including the size, shape, texture, and color of the
conidia.
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 Figure 15.2 Aspergillus

LABORATORY DIAGNOSIS

Table 15.2
SPECIMENS MICROSCOPIC CULTURE
• Sputum • KOH • Aspergillus species grow within a few
• Other respiratory tract • Calcofluor white days on most media at room
specimens • Histologic stains temperature
• Lung biopsy tissue

TREATMENT, PREVENTION AND CONTROL

Voriconazole, are the preferred treatments for invasive aspergillosis. Caspofungin and amphotericin B are
alternatives. No regimen is considered highly effective because the mortality rate of invasive disease is high. Surgical
removal of localized lesion is sometimes helpful, even in the brain. Construction of rooms with filtered air has been
effective in reducing exposure to environmental conidia.

ZYGOMYCOSIS (MUCORMYCOSIS)

Zygomycosis (mucormycosis) is the term applied to infection with any of a group of zygomycetes, the most
common of which are Absidia, Rhizopus, and Mucor. These fungi are ubiquitous saprophytes in soil and are commonly
found on bread and many other food-stuff. They occasionally cause disease in persons with diabetes mellitus and in
immunosuppressed patients receiving corticosteroid therapy. Diabetic ketoacidosis has a particularly strong association
with zygomycosis.

Pulmonary or rhinocerebral disease is acquired by inhalation of conidia.

• The pulmonary form has clinical findings similar to those of other fungal pneumonias.
• The rhinocerebral form produces a dramatic clinical syndrome in which agents of zygomycosis show
striking invasive capacity. They penetrate the mucosa of the nose, paranasal sinuses, or palate, often
resulting in ulcerative lesions. Once beyond the mucosa, they progress through tissue, nerves, blood
vessels, fascial

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 Figure 15.3 Rhinocerebral disease

LABORATORY DIAGNOSIS

Direct examination or culture of nasal discharge, tissue, or sputum will reveal broad hyphae (10–15 µm) with
uneven thickness, irregular branching, and sparse septations. These fungi grow rapidly on laboratory media, producing
abundant cottony colonies. Identification is based on the sporangial structures.

Figure 15.3 Broad, ribbon-like, non-septate (aseptate) hyphae

TREATMENT

Treatment consists of aggressive surgical debridement, rapid administration of amphotericin B, and control of the
underlying disease. Many patients survive, but there may be residual effects such as partial facial paralysis or loss of an
eye.

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 PART 4: LIST OF REFERENCES

• Books, G, et.al. Jawetz, Melnick & Adelberg’s Medical Microbiology, 27th Ed. McGraw-Hill. Copyright 2015.

• Bulmer, Glenn. Fungus Diseases in the Orient. Rex Bookstore. Manila. 1991.

• Mahon and Manusells (ed.) Textbook of Diagnostic Microbiology. Elsevier (Singapore) Pte Ltd. 2014

• Forbes, B., Sahm, D. & WEissfeld, A. Bailey & Scott’s Diagnostic Microbiology, 13th ed. Elsevier Science
(Singapore) Pte Ltd. Copyright 2014

• Davey, F., et.al (ed). John Bernard Henry, Clinical Diagnosis and Management by Laboratory Methods. 22nd ed.
W.B. Saunders Co. Philadelphia. 2007

• Black, Jacquelyn G. Microbiology, Principles and Explorations, 5th edition. USA: McGraw-Hill Companies Inc.
2005

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A.Y. 2020 - 2021