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Coronavirus written by Awal Syed (M.Phil. Biological Science Quaid-i-Azam University Islamabad)

The contents copied from standard Microbiology and Virology books (1. Simian Virolgy Alexander F. Voevodin, MD, PhD, DSC, FRCPath, 2. Principle
and practice of clinical virology J. Zukerman , 3. Principles of Molecular Virology 4th Edition Alan J. Cann University of Leicester, UK, 4. VIROLOGY
PRINCIPLES AND APPLICATIONS John B. Carter and Venetia A. Saunders School of Biomolecular Sciences, Liverpool John Moores University, UK, 5.
Bacteria and Viruses / edited by Kara Rogers 6. Color Atlas of Medical Microbiology (Kayser, Thieme 2005) 7. desk encyclopedia of human and medical
virology editors-in-chief dr brian w j mahy and dr march v van regenmortel 8. Lippincott’s Illustrated Reviews: Microbiology Third Edition

Coronavirus

1. What is corona virus?

Coronaviruses are known to cause disease in humans, other mammals, and birds. They cause
major economic loss, sometimes associated with high mortality, in neonates of some domestic
species (e.g., chickens, pigs). In humans, they are responsible for respiratory and enteric
diseases. As a group, coronaviruses are not limited to particular organs; target tissues include
the nervous system, immune system, kidney, and reproductive tract in addition to many parts
of the respiratory and enteric systems.
2. Why it is called corona?

The name (corona, as in wreath or crown) refers to the appearance of the viruses.

3. What does mean COVID19?

CO: Corona

VI: Virus

D: Disease

19: 2019

4. Taxonomy and Classification of corona virus

the genus Coronavirus together with the genus Torovirus form the family Coronaviridae;
members of these two genera are similar morphologically. The Coronaviridae, Arteriviridae,
and Roniviridae are within the order Nidovirales. Members of this order have a similar genome
organization and produce a nested set of subgenomic mRNAs (nidus, Latin for nest). To date,
coronaviruses have been placed into one of three groups (Table 1). Initially, this was on the
basis of serological relationships which subsequently have been supported by gene sequencing.
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Coronavirus written by Awal Syed (M.Phil. Biological Science Quaid-i-Azam University Islamabad)

There are two serotypes of Corona virus called 229E and OC43. The genome sequence of the
coronavirus that caused the SARS (CoV-SARS) outbreak is different from the existing human
strains. The Coronaviridae family includes several viral species that can infect vertebrates
such as dogs, cats, cattle, pigs, rodents, and poultry.
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5. Structure and composition of corona virus

Coronaviruses have a non-segmented, single-stranded, positive-polarity RNA genome (Figure
a). They are enveloped viruses with a helical nucleocapsid. In the electron microscope,
prominent club-shaped spikes in the form of a "corona" (halo) can be seen. All coronaviruses
have four structural proteins in common (Figure a): a large surface glycoprotein (S; 1150–1450
amino acids); a small envelope protein (E; 100 amino acids, present in very small amounts in
virions); integral membrane glycoprotein (M; 250 amino acids); and a phosphorylated
nucleocapsid protein (N; 500 amino acids), have an additional structural glycoprotein, the
hemagglutinin-esterase protein (HE; 425 amino acids). This is not essential for replication in
vitro and may affect tropism in vivo.

6. Replication and Transcription of CoV RNA

the replication and transcription of CoV RNA takes place in the cytoplasm of infected cells
(Figure 3). The CoV virion attaches to the host cell receptor via the spike glycoprotein and,
depending on the virus strain, the spike mediates fusion directly with the plasma membrane or
the virus undergoes receptor-mediated endocytosis and spike-mediated fusion with endosomal
membranes to release the viral gRNA into the cytoplasm (figure 3). Once the positive-strand
RNA genome is released, it acts as a messenger RNA is translated by ribosomes to generate
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the viral RNA-dependent RNA polymerase polyprotein, termed the viral replicase. The
replicase polyprotein is processed by replicase-encoded proteases (papain-like proteases and a
poliovirus 3C-like protease) to generate 16 mature replicase products. These viral replicase
proteins sequester host cell membranes to generate distinctive double membrane vesicles
(DMVs) that have been shown to be the site of CoV RNA synthesis. The replicase complex on
the DMVs then mediates the replication of the positive-strand RNA genome to generate full-
length and subgenomic negative-strand RNAs, and the subsequent production of positive-
strand gRNAs and sgmRNAs. The sgmRNAs are translated to generate viral structural and
accessory proteins, and virus particles assemble with positive-strand gRNA in the endoplasmic
reticulum Golgi intermediate compartment (ERGIC) and bud into vesicles, with subsequent
release from the cell. Depending on the virus strain, this replication can be robust and result in
destruction of the host cell or a low-level, persistent infection that can be maintained in cultured
cells or infected animals. A hallmark of CoV transcription is the generation of a nested set of
mRNAs, with each mRNA having the identical ‘leader’ sequence of approximately 65–90 nt
at the 5 end (Figure 4). The leader sequence is encoded only once at the 5 end of the gRNA.
Each subgenomic mRNA (sgmRNA) has the identical leader sequence fused to the 5 end of
the body sequence. How are the leader containing mRNAs generated during CoV
transcription? Current evidence supports a model of discontinuous transcription, whereby the
replicase complex switches templates during the synthesis of negative-strand RNA (Figure 3).
The key sequence element in this process is the transcriptional regulatory sequence (TRS). The
TRS is a sequence of approximately 6–9 nt (50-ACGAAC-30 for SARS-CoV) which is found
at the end of the leader sequence and at each intergenic region Site-directed mutagenesis and
deletion analysis has revealed the critical role of the TRS in mediating transcription of
sgmRNAs. Deletion of any intergenic TRS results in loss of production of the corresponding
sgmRNA. In addition, the CoV leader TRS and the intergenic TRS sequences must be identical
for optimal production of the sgmRNAs. A three-step working model for template switching
during negative strand RNA synthesis has been proposed to describe the process for the
generation of CoV leader-containing sgmRNAs. In this process, the 5 end and 3 end of the
gRNA form a complex with host cell factors and the viral replication complex. The 3 end of
the positive strand is used as the template for the initiation of transcription of negative-strand
RNA. Negative-strand RNA synthesis continues up to the point of the TRS. At each TRS, the
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viral replicase may either read through the sequence to generate a longer template, or switch
templates to copy the leader sequence. The template switch allows the generation of a leader-
containing sgmRNA. In this model, alignment of the leader TRS, the newly synthesized
negative-strand RNA, and the genomic TRS is critical for the template switching to occur.
Disruption of the complex, or loss of base-pairing within the complex, will result in the loss of
production of that sgmRNA. Further studies of the CoV replication complex may yield new
insights into the role of the viral helicase and endoribonuclease in the generation of the leader-
containing CoV RNAs. Another hallmark of CoV replication is high-frequency RNA
recombination. RNA recombination occurs when a partially synthesized viral RNA dissociates
from one template and hybridizes to similar sequences present in a second template. Viral RNA
synthesis continues and generates a progeny virus with sequences from two different parental
genomes. This RNA recombination event is termed copy-choice recombination. Copy-choice
RNA recombination can be demonstrated experimentally when two closely related CoV
strains.
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7. Epidemiology (occurrence, distribution)

Generally HCV epidemics occur during the winter and early spring but the peak period may
vary by several months. However, other periods of high infection rates have been observed
and it appears that major peaks may occur at any time of the year. The incidence of OC43 and
229E types (Strains of CORONA virus) of HCV (Human corona virus) has been studied in
depth among approximately 1000 people in Tecumseh, USA, over a period of 4 years. Serology
revealed that 17% of individuals had a cold caused by OC43 in any one year (Monto and Lim,
1974). Infections by 229E in the same community averaged 8% each year, while double this
rate was detected elsewhere in another study. In some years the incidence of disease was much
greater than the average. In a study in the Rhone Alpes region of France, virus was isolated
from approximately one-third of nasal swabs, 18% of these yielding HCV (Lina et al., 1996).
7.a. From where this virus arise (origin)? Is it old or just arise in 2019?
One coronavirus species (human coronavirus, HuCV) is known since some time to be a human
pathogen. It has at least two serotypes (as discussed in start) and probably a number of
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serological variants. In November 2002, a new coronavirus emerged in China and, after
originally being mistaken as a new influenza recombinant, was identified as the causative agent
of a typical pneumonia, later termed SARS, occurred in the southern Chinese city of
Guangzhou (Guangdong Province). Where 300 people became ill and at least five died. Then
in February of 2003, the world was alerted about the lung disease, shortly before it escaped
China, when one of the doctors who had been treating patients travelled to Hong Kong, where
he became ill and died and transmitted it to other 29 countries by summer 2003 including
Singapore, Vietnam, Canada and the US, taking the infectious agent with them. The causative
agent was found to be a new coronavirus. Coronaviruses with 99% sequence similarity to the
surface spike protein of human SARS isolates have been isolated in Guangdong, China, from
apparently healthy masked palm civets, a cat-like mammal closely related to the mongoose.
The unlucky palm civet is regarded as a delicacy in Guangdong and it is believed that humans
became infected as they raised and slaughtered the animals rather than by consumption of
infected meat. The outbreak of corona resulted in 8,098 persons with possible SARS, including
744 deaths reported by the World Health Organization. There were 373 possible SARS cases
in the United States; however, SARS-CoV identification has been confirmed in only 8 of them.
Seven of the eight cases were likely due to exposure during international travel, and the eighth
case was probably due to exposure to one of the other seven. The genome sequence of SARS-
CoV strains provides an explanation for the apparent disappearance of the disease (see
portion: Why this disease disappear after 2003).

7.b. Who is more prone/ vulnerable and exposed to COVID?

1. All age groups are infected with HCVs and infection rates have been shown to be relatively
uniform for all age groups (Monto and Lim, 1974). This is different from the situation with some
other respiratory viruses, such as respiratory syncytial virus, where there is a distinct decrease in
infection rates with increase in age. 2. it’s mostly lethal for immunocompromised patient (having
weakened immune system) i.e. Diabetes, heart disease. 3. Medical staff, veterinary staff and other
personnel dealing with humans and animals infected with high risk viruses.

7.c. Transmission

Transmission of the virus is by droplets, produced by coughing and sneezing through close contact
(“household transmission”) but other routes of infection may also be involved, such as fecal
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contamination. The SARS virus is transmitted aerogenically with an incubation time of 2 to 10

days with a mean of 5 days.

8. Pathogenesis, Immunity Host Range

Human coronaviruses are known to be associated with enteric and respiratory diseases (e.g.,
diarrhea), in addition to respiratory disease. SARS-CoV was also associated with diarrhea in
humans, in addition to serious lung disease. Coronavirus infection is typically limited to the
mucosal cells of the respiratory tract. Approximately 50% of infections are asymptomatic, and
it is unclear what role they play in the spread of infection. Immunity following infection
appears to be brief and reinfection can occur. Pneumonia caused by SARS coronavirus is
characterized by diffuse edema resulting in hypoxia. The binding of the virus to angiotensin-
converting enzyme-2 (Recently, the receptor involved in the entry of the SARS virus into the
cell was reported to be the angiotensin-converting enzyme 2 (ACE2) on the surface of
respiratory tract epithelium may contribute to the dysregulation of fluid balance that causes the
edema in the alveolar space.
Avian IBV (bird’s corona virus) not only causes respiratory disease but can also damage
gonads in both females and males, and causes serious kidney disease (dependent on the strain
of virus, and to some extent on the breed of chicken). Some coronaviruses have their most
profound effect in the alimentary tract (e.g., porcine TGEV causes 90% mortality in neonatal
pigs). Coronavirus replication and disease are not necessarily restricted to a single host species.
Canine enteric CoV and feline CoV can replicate and cause disease in pigs; these two viruses
have proteins with very high amino acid identity to those of porcine TGEV. Canine respiratory
CoV has proteins, including the S protein (which is the attachment protein and a determinant
of host range), with very high amino acid identity (95%) to other group 2 viruses Hu CoV-
OC43 and BCoV (bovine corona virus). This raises the possibility of co-infection in these
hosts. Bovine CoV causes enteritis in turkeys following experimental oral infection. There is
evidence that pheasant CoV can infect chickens, and IBV infect teal (a duck), though without
causing disease. The most dramatic demonstration that coronaviruses can have a wide host
range was provided by SARSCoV. This may have had its origin in bats, was transferred to
various other species (e.g., civet cat) that were captured for trade, and then caused lethal disease
in humans. Following infection of very young chickens, IBV is re excreted when hens start to
lay eggs. The trigger for release is probably the stress of coming into lay. The S protein is a
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determinant of both tissue tropism within a host and host range. This has been elegantly
demonstrated by genetic manipulation of the genome of MHV (murine hepatitis virus), which
is unable to attach to feline cells. Replacement of the MHV S protein gene with that of CoV
from feline coronavirus resulted in a recombinant virus that was able to attach, and
subsequently replicate in, feline cells. However, other proteins can also affect pathogenicity.
Currently, the most widely used prophylactics for control of IBV in chickens include killed
vaccines and live vaccines attenuated by passage in embryonated eggs. However, disease
control is complicated by extensive variation in the S1 protein which is the inducer of
protective immunity
9. Sign and Symptoms (Clinical Findings)
SARS (Severe acute respiratory syndrome) is a type of viral pneumonia, resemble those of
influenza and include fever or febrile (100.4°F or 38°C), aching muscles, coryza (rhinorrhea,
runny nose), sore throat, headaches, cough, dyspnea, and hypoxia, Chills, rigors, malaise, and
shortness of breath. Leukopenia and thrombocytopenia are also seen. About 90 percent of
patients recovered, but for the remainder the infection proved to be lethal. On the face of it
SARS is a respiratory tract disease, but in many patients the infection spread to other parts of
the body. SARS patients may develop a dry cough after a few days, and most will develop
pneumonia. About 10 to 20% of patients have diarrhea. If not detected early, this disease can
be fatal even with supportive care. Death may result from progressive respiratory failure due
to lung damage.
10. Mortality Rate/ death ratio
The only preventive measure to date is exposure prevention. Under therapy with ribavirin and
intensive care, mortality of SARS is around 10%.
11. Diagnosis:
Serology or electron microscopy for common cold strains; PCR or isolation for SARS &
Pathogen. The common-cold coronavirus can be grown in organ cultures of human tracheal
tissue or in human diploid cells. Isolating the viruses for diagnostic purposes is not routine.
Serodiagnosis (complement-binding reaction, immunofluorescence or enzyme immunoassay)
and electron microscopy are feasible methods. The SARS virus can be identified by PCR or
isolated in the Vero cell line.
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12. Treatment/Vaccines and Antiviral Drug Development

there is no antiviral therapy or vaccine available. A combination of ribavirin and steroids has
been tried in the treatment of life-threatening cases of SARS, but their efficacy is uncertain.
Because of the economic importance of CoV infection to livestock and domestic animals, a
variety of live-attenuated and killed CoV vaccines have been tested in animals. Vaccines have
been developed against IBV, TGEV, CCoV, and FIPV (TABLE 1). However, these vaccines
do not seem to provide complete protection from wild-type virus infection. Killed virus
vaccines may also be problematic for some CoV infections. Vaccination of cats with a killed
FIPV vaccine has been shown to exacerbate disease when cats are challenged with wild-type
virus. A variety of approaches are currently under investigation for developing a SARS-CoV
vaccine, including analysis of killed virus vaccines, live attenuated virus vaccines, DNA
immunization, and viral vector vaccines (such as modified vaccine virus Ankara, canarypox,
alphavirus, and adenovirus vectors). The development of improved animal models for SARS
will be essential for evaluating SARS-CoV candidate vaccines. Transgenic mice expressing
human ACE-2 (angiotensin-converting enzyme 2) may be an appropriate small animal model.
13. Prevention:
The SARS outbreak was brought under control by quarantine measures, but only after there
had been over 8000 cases with nearly 800 deaths. The virus was isolated in February 2003 and
three months later its genome had been sequenced. The following year a paper was published
reporting compounds that inhibit replication of the virus, while other papers reported the cell
receptor of the virus and the structure of its replicase protein. Diagnostic laboratory methods
to detect evidence of SARS coronavirus in samples from patients rapidly became available;
tests based on immunofluorescence and RT-PCR were developed. The health of international
travelers was monitored and those found to be infected with the virus were nursed in isolation.
These measures brought the SARS outbreak under control. When there is an outbreak of a
highly infectious virus, such as the SARS coronavirus or Ebola virus, infected patients and
their contacts are quarantined. Control measures for some outbreaks include slaughter of
animals that are infected and those that have been in contact with infected animals. Medical
staff, veterinary staff and other personnel dealing with humans and animals infected with high
risk viruses must take precautions to protect themselves and to avoid spreading infection.
Precautions include wearing protective clothing and breathing filtered air. Must use ether and
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chloroform as a disinfector as the envelop of virus are destroyed by this.

14. from where corona come? Natural reservoir for corona
Researchers suspected that SARS-CoV had “jumped” from its animal host to humans, so
samples of animals at open markets in Guangdong were taken for nucleotide sequencing. These
studies revealed that catlike animals called masked palm civets (Paguma larvata) harbored
variants of the SARS-CoV. Although thousands of civets were then slaughtered, further studies
failed to find widespread infection of domestic or wild civets. In addition, experimental
infection of civets with human SARS-CoV strains made these animals ill, making the civet an
unlikely candidate for the reservoir species. Such a species would be expected to harbor SARS-
CoV without symptoms so that it could efficiently spread the virus. Bats are hosts of several
viruses spread from animals to people (zoonotic viruses), including the emerging Hendra and
Nipah viruses that have been found in Australia and East Asia, respectively. Thus it was
perhaps not too surprising when in 2005 two groups of international scientists independently
demonstrated that Chinese horseshoe bats (genus Rhinolophus) are the natural reservoir of a
SARS-like coronavirus. When the genomes of the human and bat viruses are aligned, 92% of
the nucleotides are identical. More revealing is alignment of the translated amino acid
sequences of the proteins encoded by each virus. The amino acid sequences are 96 to 100%
identical for all proteins except the receptor-binding spike proteins, which are only 64%
identical. The SARS-CoV spike protein mediates both host cell surface attachment and
membrane fusion. Thus a mutation of the spike protein allowed the virus to “jump” from bat
host cells to those of another species. It is not clear if the SARS-CoV was transmitted directly
to humans (bats are eaten as a delicacy and bat feces are a traditional Asian cure for asthma)
or if transmission to humans occurred through infected civets.
So we concluded that the horseshoe bat appears to be the natural reservoir for CoV-SARS,
with the civet cat serving as an intermediate host.
15. Why this disease disappear after 2003? Evolutionary reason
The relationship between the viruses found in civets and humans has also been studied in
detail and offers insight into why there have been no additional cases of SARS since 2004
(at least as this book went to press). The region of the SARS-CoV spike protein that binds
to the surface membrane: Evolution of a Virus host receptor, angiotensin-converting
enzyme-2 (ACE2), forms a shallow pocket into which ACE2 rests. The region of the spike
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protein that makes this pocket is called the receptor-binding domain (RBD). Of the
approximately 220 amino acids within the RBD, only four differ between civet and human.
Two of these amino acids appear to be critical. As shown in below box figure, compared
to the spike RBD in the SARS-CoV that caused the 2002–2003 epidemic, the civet spike
has a serine (S) substituted for a threonine (T) at position 487 (T487S) and a lysine (K) at
position 479 instead of asparagine (N), N479K. This causes a 1,000-fold decrease in the
capacity of the virus to bind to human ACE2. Furthermore, the spike found in SARS-CoV
isolated from patients in 2003 and 2004 also has a serine at position 487 as well as a proline
(P) for leucine (L) substitution at position 472 (L472P). These amino acid substitutions
could be responsible for the reduced virulence of the virus found in these more recent
infections. In other words, these mutations could be the reason the SARS virus appears to
have “died out.” Meanwhile a SARS vaccine based on the virulent 2002–2003 strain is
being tested. This raises additional questions. Does the original virulent SARS-CoV strain
still exist? Will the most recently identified SARS-CoV continue to evolve into less
virulent forms? If not, will this vaccine be effective in preventing another highly infective
SARS outbreak? Unfortunately, these questions cannot be easily answered.
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16. Why Corona more dangerous and why not curable by vaccine?
Reason 1: Virions (entire virus particle including capsid and nucleic acid) are 120 nm in
diameter, although they can be up to twice that size, and the ring of S protein spikes is
approximately 20 nm deep. In some species, the S protein is cleaved into two subunits (fig),
the N-terminal S1 fragment being slightly smaller than the C-terminal S2 sequence. The S
protein is anchored in the envelope by a transmembrane region near the C-terminus of S2.
The functional S protein is highly glycosylated and exists as a trimer. The bulbous outer
part of the mature S protein is formed largely by S1 while the stalk is formed largely by
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S2, having a coiled-coil structure. S1 is the most variable part of the S protein; some
serotypes of IBV or Avian infectious bronchitis virus differ from one another by 40% of
S1 amino acids. S1 is the major inducer of protective immune responses. Variation in the
S1 protein enables one strain of virus to avoid immunity induced by another strain of the
same species.

Reason 2: the wild-type CoV (corona virus) rapidly evolves to escape neutralization by
vaccine-induced antibodies. In studies of vaccinated chickens, a live-attenuated IBV
(Avian infectious bronchitis virus) vaccine has been shown to undergo RNA recombination
with wildtype virus to generate vaccine escape mutants.
Reason 3: The immunity conferred by infection, apparently IgA-dependent, is short-lived.
Reinfections are therefore frequent, whereby the antigenic variability of the virus may be
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a contributing factor. Various enteral coronaviruses with morphologies similar to the

respiratory types have also been described in humans.

It would seem that the SARS coronavirus evolved from a rare virus that had this ability.

17. Virus Isolation

Virus isolation can be done from nasal and throat swabs, and nasopharyngeal aspirates
taken from infected individuals. 229E and related strains can be isolated in roller culture
monolayers of human embryonic lung fibroblasts, such as W138 and MRCS cells. In virus-
positive cultures a gradual cytopathic effect consisting of small, granular, round cells
appears through-out the monolayers, although especially around the periphery of the
monolayers. However, cell sheets are rarely destroyed completely on initial isolation.
Isolates are generally confirmed as being 229E-related by standard serum neutralization
tests.
18. Detection of HCV Proteins
HCV particles can be detected in epithelial cells shed from the nasopharynx of individuals
with coronavirus infections. Indirect immunofluorescence assays have shown that
coronavirus fluorescence can be visualized in nasopharyngeal cells from volunteers
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inoculated with HCVs. An antigen-capture ELISA has been used to detect HCV antigens
in nasal and throat swabs, and nasopharyngeal aspirates, taken from children suffering from
acute respiratory infections. Nasal swabs were the best specimens for obtaining suitable
quantities of antigen for the assay.