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Outline
BASIC VIROLOGY 1. Definition of virus

IN MEDICINE 2.
3.
Virus morphology and structure
Virus infection and replication
4. Virus taxonomy and classification
5. Diagnostic tools in virus infection
Priyo Budi Purwono
Department of Microbiology 6. Antiviral drugs
Faculty of Medicine, Universitas Airlangga 7. Vaccine against viruses
October 2023

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VIRUS (Definition and PROFILE) Virus structure

Virion: the intact virus particle
1. Viruses are Small in Size Capsid: the protein coat
The smallest of viruses 20 nm in diameter, Influenza
virus 100 nm in diameter. Average human cells are  Capsomeres: the protein structural units of capsid
10–30 μm (microns)
2. Viruses are obligate intracellular parasites  Nucleic acid genome: either DNA or RNA
Completely dependent upon the internal  Envelope: some virus particles are surrounded by a lipoprotein
environment of the cell to create new infectious
virus particles, or virions envelope; containing viral antigens
3. The genetic material of viruses can be composed
of DNA or RNA. Virus genomes
All living cells, whether human, animal, plant, or o DNA or RNA
bacterial, have dsDNA as their genetic material. o Single or double stranded
Viruses, on the other hand, composed of DNA or
RNA (but not both). o intact or segmented
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STRUCTURE (ENVELOPE and NON-ENVELOPE) Shapes of viruses

• Bullet shaped with
helical nucleocapsid
• Long helical tubes (up to
1000 nm)
• Icosahedral shaped

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Nomenclature (International Committee on

Taxonomy of viruses/ICTV)
• The ICTV has adopted a formal nomenclature for viruses, specifying sufixes for the
various taxa, and rules for written descriptions of viruses.
• Orders = -virales; families –viridae; subfamilies= -virinae, genera= -virus  all be single
words
• Species names may contain more than one word and have no specific ending
• In written usage, the formal virus taxonomic names are capitalized and written in italics,
and preceded by the name of the taxon, which is neither capitalized nor italicized
• As an example: order Mononegavirales, family Paramyxoviridae, subfamily
Pneumovirinae, genus Pneumovirus, species Human respiratory syncytial virus
• The criteria applied for such groupings typically include nature of the viral genome (DNA
or RNA), strandedness of the viral genome (single stranded or double stranded), polarity
of the genome (positive sense, negative sense, or ambisense), and reverse transcription
 Baltimore Classification : seven categories of viruses

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BALTIMORE Virus names

CLASSIFICATION
1. Viruses named after the clinical conditions they cause
• The Baltimore HIV, Hepatitis virus, HPV, Rabies (madness)
classification system,
named after its creator 2. Viruses named after their location of discovery
David Baltimore, is a Coxsackievirus, Ebola virus, Nipah virus, West Nile virus
widely used scheme
based on the nature of 3. Viruses named after their properties
the genome packaged
in virions and the Coronavirus (Latin corona, meaning crown), Herpesviruses (Greek
pathway of nucleic acid herpein, “to creep,”), Influenza virus (“influence of the stars”),
synthesis that each Poliovirus (Greek polios, meaning “gray,” referring to the gray
group takes to matter/cell bodies in the spinal cord)
accomplish messenger
RNA (mRNA) synthesis 4. Viruses named after people (historically assigned)
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Epstein–Barr virus, JC Virus PBP@2023 10

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Virus replication cycle Attachment to cell receptor • Viruses make initial contact with cells at
the plasma membrane.
• the process of replication to create new, • The binding of a virion is a specific process
infectious virions that are able to infect that involves the virus attachment protein
binding to a cell surface receptor
other cells of the body or subsequent
hosts • Determines the tropism of the virus 
specific cells, tissue, host
• After gaining entry into the body, a virus • Some viruses require co-receptors for
makes physical contact with and crosses entry
the plasma membrane of a target cell.
• Virus attachment proteins will be located
• Inside, it releases and replicates its on the outermost surface of the virion,
genome while facilitating the whether that is the envelope or capsid (for
1. Attachement non-enveloped viruses)
2. Penetration manufacture of its proteins by host
3. Uncoating ribosomes • Eg. HIV-1 requires CD4 as a receptor and
4. Biosynthesis (transcription, translation and chemokine receptors CCR5 or CXCR4 as
genome replication) • Virus particles are assembled from these coreceptors. Influenza viruses bind to
5. Assembly newly synthesized biological molecules terminal sialic acid
6. Release and become infectious virions. Finally,
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PENETRATION Uncoating • the breakdown of the viral capsid,

releasing the genome into the cell
• viruses cross the plasma membrane
• Enveloped viruses: by fusion of virus
• Viruses take advantage of normal cellular envelope with cell membrane.
processes to gain entry into the cytoplasm.
• Cell enzymes strip off the virus protein
• One common method involves endocytosis. coat
This can occur through endocytosis of
caveolin- or clathrin-coated pits, bulk-phase • Many viruses achieve uncoating by
endocytosis, or phagocytosis escaping from the endosome that they
used to enter the cell.
• Some enveloped viruses use fusion to enter
the cell. This process is mediated by viral • Other viruses do not completely uncoat
fusion proteins and merges the viral and use the remaining capsid as a
envelope with the cell membrane home base for replication processes.
• Fusion: HIV, Influenza, Coronavirus, • Some viruses uncoat at the nuclear
Denguevirus, Herpes simplex virus, Ebola envelope immediately before
virus transporting the genome into the
nucleus.
• Clathrin-mediated endocytosis:
Coronavirus, Dengue virus, HCV, adenovirus • A few viruses are small enough to pass
through the nuclear pores and uncoat
• Cavolin-mediated endocytosis: HPV, HBV in the nucleus.

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BIOSYNTHESIS (REPLICATION) ASSEMBLY AND RELEASE

• Seven classes of viruses in the Baltimore classification system,
• based upon the type of nucleic acid and replication strategy of viruses: • Assembly : the packaging of the
copied viral genome with newly
1. dsDNA manufactured viral proteins to
create a virion.
2. ssDNA • Maturation : the final changes
3. dsRNA that must occur within the
virion to create an infectious
4. +ssRNA virion rather than an inert
particle  the modification of
5. −ssRNA cell surface receptors, the
cleavage of viral polyproteins, or
6. RNA viruses changes to the viral capsid
reverse transcribe • Release : the exit of the virion
from the cell. This most often
7. DNA viruses occurs through the budding of
enveloped viruses or via cell
reverse transcribe lysis.
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Five types of virus 1. Productive infection : the virus infection that leads
to the production of progeny virus Transmission route of viruses
infections • lytic infection produces a progeny virus via cell lysis
(eg, adenovirus and influenza virus)
• persistent infection continues to produce a progeny
virus for a long period either without cell death 1. Conjunctiva: Adenovirus
[eg, hepatitis B virus and hepatitis C virus (HCV)] or
with cell death but leaving long-lasting reservoir 2. Respiratory : Coronavirus, Influenza virus,
cells (eg, HIV) rhinovirus
2. Nonproductive infection: the virus infections that 3. Gastrointestinal : Rotavirus
do not lead to the production of a progeny virus
• Latent infection (eg, herpesvirus and HIV) maintains 4. Urogenital: Herpes simplex virus, HPV
the viral genomes stably in the infected cell without 5. Trans-placental : Hepatitis B virus
producing a progeny virus
• Transforming infection (eg, human papillomavirus) 6. Skin lesion : Varicella zoster virus
harbors the viral genome as a chromosomally 7. Blood/body fluid : Human immunodeficiency
integrated form without producing a progeny virus
 the infected cells are transformed to cancerous virus, Hepatitis B and C virus
cells
8. Arthropod : Dengue virus
• Abortive: the viral genome replication may not
occur after entry to target cells due to strong host 9. Bite of vertebra : Rabies virus
immune response or host restriction factors

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Stages of viral Common Lab methods for virus diagnosis

infection in general
Methods Advantage Disadvantage
• The incubation period is the time
between when the virus initially infects 1. Virus isolation/culture Produces further material for study of agent Selection of cell type, etc., may be critical
the host and when symptoms appear. - Pathogenesis study (Plaque assay, can be difficult and expensive
immunopathogenesis)
• The prodromal period occurs after the - Drug and Vaccine studies
incubation period and is when symptoms
first appear. Nonspecific, mild : malaise, 2. Direct observation by Detects viruses that cannot be grown in Limited to a few infections
muscle aches, or a low-grade fever electron microscopy culture

• The illness period occurs when specific 3. Identification of virus or Rapid and sensitive Interpretation may be difficult
symptoms of the disease occur. At this antigen, for example, EIA Readily available, often as diagnostic kits Not as sensitive as PCR
point, the virus is multiplying to high 4. Detection of viral Rapid, very sensitive Risk of DNA contamination
levels and the immune system has been genomes by PCR Good quantitation of load Needs good quality control
activated Potentially applicable to all viruses Cannot distinguish infectious and non-infectious
virus particles
• In immunocompetent hosts with
functioning immune systems, infected 5. Antibody seroconversion Useful if appropriate samples for direct Slow, late (retrospective)
cells will eventually be eliminated and the (acute and convalescent detection cannot be obtained, or to exclude Interpretation may be difficult
amount of virus within the host will sera) a particular infection retrospectively
decline
6. IgM serology Rapid False positives may occur
• the symptoms of the disease subside as
the host begins feeling better, having 7. Gene sequencing To identify virus gene mutation Very expensive
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entered into the convalescent period Able to detect unknown virus Common in Research purpose

Specimens for virus laboratory diagnosis Virus culture for isolation

Can be in : 1. Cells (primary or cell line) 2. Embryonated egg 3. animal

The recognition of a virus infection in cell culture: cytopathic effect and

hemadsorption
1. Cytopathic effect (CPE) comprises two different phenomena:
(a) Morphological : changes induced in individual cells or groups of cells by virus
infection that are easily recognizable under a light microscope
(b) inclusion bodies, which are more subtle alterations to the intracellular
architecture of individual cells. For example, Negri bodies formed during a
rabies virus infection
Cytopathic effect is the simplest and most widely used criterion for infection;
however, not all viruses cause a cytopathic effect

2. Hemadsorption : the ability of red blood cells to attach specifically to virus-

infected cells.
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Virus culture and cytopathic effect Hemaglutination test with RBC

Cytopathic effect of viruses in cell cultures • Useful in influenza virus
(A) Confluent monolayer of uninfected diagnosis/study
Vero cells. • Seven different samples of
(B) Vero cells infected with the SARS influenza virus mixed with
coronavirus (SARS-CoV) 24 hours post- chicken RBCs, and incubated
infection. Massive cytopathic effect (CPE), on ice for 1 to 2 hours.
with cells rounded up and detaching from
the substrate. • Erythrocytes that are not
(C) Confluent monolayer of uninfected agglutinated are free to roll to
Vero cells. the bottom of the well
(D) Vero cells infected with herpes simplex • Erythrocytes that are
virus 1 (HSV-1) at 24 hours post-infection. agglutinated coat the bottom
A single large syncytium of at least 50 cells
(massed nuclei) which is unstable and surface of the well to form a
later progresses to a necrotic/apoptotic shield.
CPE.
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Embryonated chicken egg Observation of embryonated egg culture

• Specimens are inoculated

into pathogen-free fertilized
eggs of 10-11 days
• incubated for 2-9 days
before harvesting the viruses
• Growth and multiplication of
the viruses are indicated by
the death of the embryo, or
by the formation of typical
pocks or lesions on the egg
membranes
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Biological Assays in virus quantification Plaque assay to quantify the number of

THE PLAQUE ASSAY
infectious virus particle
• The plaque assay is the most elegant, the most quantitative, and the most useful biological assay for viruses.
• The plaque assay is based simply on the ability of a single infectious virus particle to give rise to a macroscopic area of
cytopathology of cultured cells • the African green monkey
kidney cell line BSC40
THE FOCUS ASSAY
were infected with 10-fold
serial dilutions of wild-
• Some tumor viruses, most notably retroviruses, normally transform cells rather than killing them but can nevertheless type vaccinia virus
be quantified by taking advantage of the transformation cytopathology
• quantified on treatment of an infected monolayer with an appropriate stain • incubated at 31°C or 40°C
for 7 days
THE ENDPOINT METHOD • in the presence of
• to calculate the dilution of virus that results in infection in 50% of replicate inoculations= the infectious dose 50 (ID50).
isatinthiosemicarbazone
(IBT) or in the absence of
• cytopathic effect in cultured cells= tissue culture infective dose 50 (TCID50) drug
• cytopathology or embryonic death in inoculated embryonated chicken eggs= egg infective dose 50 (EID50); • Plaque = infectious virus
• death of an experimental laboratory animal, yielding lethal dose 50 (LD50)

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Lateral Flowstrip test (Rapid test) ELISA/EIA (Enzyme immune assay)

• the principle of lateral flow
strip presented with
sandwich assay.
• Sample solution is added to
sample pad. Antigen
attaches to antibodies in
conjugate pad and the
complex formed attach to
test line antibodies.
• The excess labeled • Can be qualitative or
Quantitative (titre)
antibodies bind with • Read the plate on
antibodies in control line spectrophotometer in
particular wavelength
• Qualitative method
• Absorbance value
• Eg. HBsAg, anti-HBs titer
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Immunostaining for Diagnosis of PCR (Polimerase Chain Reaction)

rabies
• (A) Direct immunofluorescence on • The method relies on thermal cycling, that is, cycles of
impression smear of fox brain repeated heating and cooling to melt and re-anneal DNA
(possible human contact), showing along with enzymatic replication of the targeted melted
prominent brilliant apple-green strands of DNA.
masses of viral antigen (Negri • Primers : short DNA fragments containing sequences
bodies of light microscopy) and complementary to each end of the target region of the DNA
punctate “dust,” against a dark of interest  must be specific
background. FITC-labeled anti-rabies • Basic steps:
globulin, UV microscopy. 1. Denaturation: the two strands of the DNA double helix are
• (B) Histopathology on human physically separated at a high temperature (>90c)
postmortem brain section. Finding 2. Annealing: the temperature is lowered and the two DNA
Negri bodies (shown here in the strands become templates for DNA synthesis (50-60c)
cytoplasm of a Purkinje cell in the 3. Elongation/Extention: In the presence of heat-resistant
cerebellum) often requires careful DNA polymerase and deoxynucleotide triphosphates, two
searching. H&E staining new copies of the desired region of the DNA are produced
(70-75c)
• (C) Immunohistochemistry used in • the DNA template is exponentially amplified
rabies research and in special
diagnostic situations, for example, • After the first few cycles, virtually all the templates consist of
for postmortem diagnosis in just the short region chosen for amplification by the choice
patients infected via organ of primers. After 30-40 cycles, this region has been amplified
many million fold
transplants. Here viral antigen fills
the axoplasm of peripheral nerves • Detection of Amplicon: Gel electrophoresis
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Reverse Transcript PCR = RNA DNA

Modifikasi metode molekuler Real Time PCR (qPCR) = deteksi realtime

1. PCR konvensional  kualitatif (positif/negatif)

2. Reverse transcriptase (RT) PCR : deteksi RNA --> one step vs two step
3. qPCR : kuantitatif  viral load (copies/ml)
4. qRT-PCR
a. SYBR GREEN
b. LUX (hairpin loop primer)
c. TaqMAN KEUNGGULAN qRT-PCR
• Sangat Sensitif and Reliable
d. droplet digital PCR (ddPCR) • Deteksi sinyal fluorescence secara Real Time
• Dapat mendeteksi DNA/RNA lebih awal daripada PCR
konvensional
• Data Kualitatif dan Kuantitatif
• Ct value = cycle threshold value, the lower value= the
earlier detection= the higher number of virus gene
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Antivirus drugs Vaccine to prevent virus infection

• Antiviral drugs block various

stages of viral replication.
• A generic replication cycle of
viruses in cells showing the
stages of infection, which
different drug classes block.
• These targets include :
attachment and entry,
uncoating, gene expression,
genome replication,
assembly and maturation,
releasese
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Highlights
1. What is viruses?
2. How the viruses classified ?
• the First vaccine used in humans—live
3. How the viruses infect the cells? How many stages ?
cowpox virus
• In 1796, Edward Jenner inoculated 8 year
4. What is virus tropism ?
old James Phipps with purulent material
taken from a cowpox pustule on the hand 5. What is the impact of virus to the cells ?
of milkmaid Sarah Nelmes and introduced
it into an incision on the boy’s arm. 6. How to diagnose the virus infection ?
• the boy was protected from an inoculation
of material from a smallpox lesion. 7. What is the strategies to eliminate virus infection ?
• Jenner published the work in 1798
• the word vaccine from the Latin vacca for
cow, designating the process vaccination.

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