SARS- COV-2 Virus

BIO 254

Genetics Laboratory

March 31, 2021

 SARS coronavirus infections in human
• Severe acute respiratory syndrome (SARS)
• is the first emerging infectious disease of the 21st century
• highly transmissible and fatal
• caused by a previously unknown coronavirus (SARS-CoV).
• The SARS epidemic in 2003 resulted in more than 8400 SARS cases and
approximately 800 deaths.
• the emergence of the Middle East Respiratory Syndrome-CoV (MERS-
CoV) outbreak in 2012.
• Currently, 2019 novel CoV (SARS-CoV-2) that was initially identified in
the city of Wuhan, China in December 2019.
• The 2019 novel CoV virus (2019-nCoV) was named as SARS-CoV-2 by
the World Health Organization (WHO).
• The disease is named as COVID-19.
 SARS coronavirus infections in human
By 13 January 2021, globally, there have been 90,054,813 confirmed cases
of COVID-19, including 1,945,610 deaths, reported to WHO.
By 31 March 2021, WHO reports …throughout the world.
• Confirmed cases  127.349.248
• Confirmed deaths  2.787.593

• In Turkey by January 12 2021, 2.346.285 people are infected and 23.152

people died as reported by Ministry of Health.
By 31 March 2021, Ministry of Health reports …throughout Turkey.
• Confirmed cases  3.277.880
• Confirmed deaths  31.385

 The death rate of SARS-CoV-2 is currently lower than SARS-CoV but it is

highly contagious based on the number of infected cases to date.
 Comparison between SARS-CoV, MERS-CoV, and
the SARS-CoV-2
Intermediate Incubation Number of Number of
Virus Receptor Primary Host Fatality R0
Host Period Cases Deaths

Typically
Civets and between 2 and
SARS-CoV ACE2 Bats 8098 774 ≈ 10% 2–5
raccoon dogs 10 days, and
up to 14 days

Typically
MERS-CoV DPP4 (CD26) Bats Camels between 2 and 2506 862 ≈ 35% 2–5
14 days

Current
estimates Over 92,000 as Over 3000 as
Most probably Most probably 3.4% as of 3
SARS-CoV-2 Not identified between 2 and of 3 March of 3 March 2–3.5
ACE2 Bats March 2020
10 days, and 2020 2020
up to 14 days

***basic reproduction number (R0)

Origin and evolution of
(A) SARS-CoV,
(B) MERS-CoV, and
(C) SARS-CoV-2 in different hosts.

All viruses initially existed in bats as CoV-related viruses (SARSr-CoV, MERSr-

CoV, and SARSr-CoV-2) before acquiring mutations and adapting to
intermediate hosts and ultimately humans.
 SARS-CoV-2 –R0 value
• the basic reproduction number (R0)
• the most important number for public health experts.
• This number measures the disease’s potential.
• It represents the average number of people who will catch the disease
from one infected person in a population that has never had the disease
in the past.
• R0 for the SARS-CoV-2  around 2.35 (1 infected person spread the
disease to at least two individuals).
• Any R0 above 1 should be taken seriously.
• The goal will be to reduce the R0 to a value that is below 1.
 SARS-CoV-2 –R0 value
**R value calculation assumes that every infected person and every
contact they make follows the same pattern..
R0 estimates can be higher than the “true” R0 values because of two main
reasons:
1. infected people who did not show symptoms and infected people who
did not report their symptoms.
2. R0 estimates are highest at the beginning of an outbreak and then
subside gradually once countries become aware of the outbreak and
manage to put in effective control measures to prevent the spread of the
virus.
 SARS-CoV-2 and K value
• Doctors have designed a new model to help them monitor the spread of
coronavirus.
• Now, the K number takes into account anomalies such as super
spreaders, which have become more common across the globe.
• Some people might have high viral loads or might simply cough more
and hence spread the virus more effectively.
• Different pathogens will have different ways in which they spread.
• Statisticians use K, the so-called dispersion parameter, to describe how
variable the infection can be.
• For some diseases, the variation will not be large, as shown below.
• A low K value suggests that a small number of infected people are
responsible for large amounts of disease transmission.
Ex: For the 1918 influenza, the number K is thought to be around 1, and
perhaps 40% of infected people might not pass on the virus to anybody
else.
Ex: For diseases like Sars, Mers and COVID-19 with K as low as 0.1, this
proportion rises to 70%. Large outbreaks will be initiated by only few
super-spreaders, as shown below.
 SARS-CoV-2
• To date, the fatality of SARS-CoV-2 (3.5%) appears to be less than that
observed in SARS-CoV (10%) and MERS-CoV (35%) infections.
• However, the fatality of this virus may keep changing and will not be
accurately calculated until after the end of this outbreak.
• The virus appears to be more fatal in elderly patients or patients with
comorbidities.
• There could be cases that went undetected, which makes it hard to
accurately calculate the fatality of this new virus.
• The practice of eating raw meat and the close contact between humans
and animals are both risk factors for the initiation of a new human CoV
outbreak.
• This is due to the constant exposure of humans in these cultures to the
ever-changing mutant CoVs.
 SARS-CoV-2
• The current outbreak represents the sixth time WHO has declared
a global public emergency since it gained the power to declare an
international emergency in 2005.
• The previous five times were the
• 2009 H1N1 swine flu,
• 2013 Ebola outbreak in West Africa,
• 2014 polio outbreak,
• 2016 Zika outbreak,
• 2019 Ebola outbreak in the Democratic Republic of Congo.
• None of these previous emergencies led a worldwide pandemic.
• For the first time WHO declared a pandemic at 12 March 2020.
Classification of different types of coronaviruses;
family  Coronaviridae,
subfamily  Orthocoronavirinae,
genera alpha-, beta-, gamma-, and deltacoronaviruses

The SARS-CoV-2 is classified as a betacoronavirus.

 Characteristic features of Nidovirales

They are as follows: they

(1) contain very large genomes for RNA viruses,
(2) are highly replicative due to conserved genomic organization,
(3) exhibit several unique enzymatic activities and
(4) have extensive ribosomal frameshifting due to the expression of
numerous non-structural genes.
 Host range
• CoVs are typically harbored in mammals and birds and are common in
camels, cattle, cats, bats, and other animals.
• Alpha and betacoronaviruses circulate in mammals, including bats
• Gammacoronaviruses mostly infect avian species and a few mammalian
species,
• Deltacoronaviruses infect birds and mammals.
• Animal CoVs are known to cause important diseases in animals.
• Responsible for economic losses in domestic animals or birds.
• avian infectious bronchitis virus (IBV),
• transmissible gastroenteritis virus (TGEV),
• porcine epidemic diarrhea virus (PEDV),
• swine acute diarrhea syndrome-CoV (SADS-CoV).
• Although rare, animal CoVs have the ability to infect humans and could
further spread through human-to-human transmission.
 Royal crowns…
• CoVs, including the newly discovered SARS-CoV-2, are spherical positive
single-stranded RNA viruses that are characterized by spike proteins
projecting from the virion surface.
• The spherical morphology of the viral particle together with the spike
projections led to the name coronavirus from the Latin word corona
that means the crown. The virion looks like a royal crown in electron
micrographs.
 SARS-CoV genome
• The SARS-CoV has approximately 30 thousand nucleotides,
• positive-sense (directly translated in the host cell), single-stranded RNA
genome,
• containing 14 functional open reading frames (ORFs),
• 2 large ORFs constitute the replicase gene which encodes proteins
required for viral RNA syntheses,
• The remaining 12 ORFs encode the 4 structural and eight accessory
proteins.
• The structural proteins are:
spike, membrane, nucleocapsid and envelope;

Ashour H. M. et al., Pathogens, March 2020

 SARS-CoV-2 Envelope Proteins
The S, M, and E proteins are all
embedded in the viral envelope;
• CoVs are enveloped viruses,
• envelope is a lipid bilayer derived from
the host cell membrane
• the viral structure formed primarily of
structural proteins such as
• spike (S),
• membrane (M),
• envelope (E),
• nucleocapsid (N) proteins,
• hemagglutinin-esterase proteins in some
betacoronaviruses.
 Structure of CoVs
N protein interacts with the viral RNA and is located in the core of the viral
particle, forming the nucleocapsid.
The S protein is a heavily glycosylated protein that forms homotrimeric
spikes on the surface of the viral particle and mediates viral entry into host
cells.
 Proteins of CoVs

• M protein gives the virus its shape and is critical together with E protein
in orchestrating the assembly of the virus and in forming mature viral
envelopes.
• The E protein also functions in release of viral particles from host cells,
in addition to other functions.
• N protein binds the viral RNA and is required for packaging of viral RNA
into the viral particle during viral assembly.
• HE is a hemagglutinin similar to influenza virus hemagglutinin and binds
sialic acid on host cell. HE enhance entry and pathogenesis of
coronaviruses that contain such proteins in their viral structure.
 SARS-CoV-2 and its S protein
• S protein is the viral protein that mediates the entry of CoVs into host
cells.
• The tissue tropism of CoVs is determined by the S protein interaction
with the receptors on host cells.
• Receptor-binding domain (RBD) within the S1 domain mediates binding
to the cognate host cell receptor;
• the S2 domain mediates the fusion events, between viral membrane
and host cell membrane, that are required for entry of CoVs into host
cells.
• The RBD is located at N-terminus of the S1 subunit as in mouse hepatitis
virus (MHV) or at the C- terminus as in SARS-CoV and MERS-CoV.
Genome Organization
• The RNA genome of CoVs is the
second largest of all RNA
viruses, ranging from 26 to 32
kilobases (kb) in size.
• The largest genome of all RNA
viruses is that of the recently
described planarian secretory
cell nidovirus, PSCNV (41.1 kb
genome size).
• Viral RNA codes for structural
and nonstructural proteins.
• The structural proteins together
with a few nonstructural
proteins, with different
functions, are coded within the
3’end of the viral genome.
• However, the 5’ two-thirds of
the genome codes for
nonstructural proteins that are
important in viral replication,
including the RNA-dependent
RNA polymerase (RdRP).
SARS-CoV-2 virus and the S potein
 Coronavirus receptors
• Several cellular receptors were described as receptors for CoVs.
• Aminopeptidase N (APN) as the receptor for several alphacoronaviruses.
• Angiotensin-converting enzyme 2 (ACE2) as the receptor for SARS-CoV,
HCoV- NL63 and possibly for the newly discovered SARS-CoV-2,
• CEACAM1 as the receptor for MHV,
• dipeptidyl-peptidase 4 as the receptor for MERS-CoV.
 Infection by SARS-CoV-2
• Once the viral genome is inside the host cell cytoplasm translation of
the 5’ end of viral RNA produces the RdRP.
• RdRP uses viral RNA as a template to generate virus-specific mRNAs
(subgenomic mRNAs) from subgenomic negative strand intermediates.
• Subgenomic mRNAs share the same 3’ ends and the same leader
sequence of 70–90 nucleotides at their 5’ ends.
• Translation of subgenomic mRNAs leads to production of structural and
nonstructural viral proteins.
• Once sufficient structural proteins and genomic viral RNA are formed,
viral RNA is then assembled with viral structural proteins into virions.
• Viral assembly and budding occur in smooth-walled vesicles in the
endoplasmic reticulum–Golgi intermediate compartment (ERGIC).
Infection by SARS-CoV-2
 SARS-CoV-2 genome

• two notable features of the SARS-CoV-2 genome:

• (i) SARS-CoV-2 appears to be optimized for binding to the human ACE2
receptor;
• (ii) the highly variable spike (S) protein of SARS-CoV-2 has a polybasic
(furin) cleavage site at the S1 and S2 boundary via the insertion of
twelve nucleotides.
• The receptor binding domain (RBD) in the spike protein of SARS-CoV and
SARS-related coronaviruses is the most variable part of the virus
genome.
• Six residues in the RBD appear to be critical for binding to the human
ACE2 receptor and determining host range.
• Five of these six residues are mutated in SARS-CoV-2 compared to its
most closely related virus, RaTG13 sampled from a Rhinolophus
affinis bat, to which it is ~96% identical.
 SARS-CoV-2/ hACE2 binding

• They determined the crystal structure of the SARS-CoV-2 receptor-

binding domain (RBD) in complex with hACE2.
• Compared with the SARS-CoV RBD, a hACE2-binding ridge in SARS-CoV-2
RBD takes a more compact conformation.
• moreover, several residue changes in SARS-CoV-2 RBD stabilize
two virus-binding hotspots at the RBD/hACE2 interface.
• These structural features of SARS-CoV-2 RBD enhance its hACE2-binding
affinity.

Shang et al., Nature. 2020 Mar 30. doi: 10.1038/s41586-020-2179-y

 S protein and ACE2 interaction
• A recent study published in Science showed that SARS-CoV-2 S protein
has higher affinity to ACE2 than SARS-CoV S protein.
• It is demonstrated that SARS-CoV-2 uses the SARS-CoV receptor ACE2
for entry and the serine protease TMPRSS2 for S protein priming.
• The spike (S) protein of coronaviruses facilitates viral entry into target
cells.
• Entry depends on binding of the surface unit, S1, of the S protein to a
cellular receptor, which facilitates viral attachment to the surface of
target cells.
• In addition, entry requires S protein priming by cellular proteases, which
entails S protein cleavage at the S1/S2 and the S2’ site.
• This allows fusion of viral and cellular membranes, a process driven by
the S2 subunit.
• SARS-S engages angiotensin-converting enzyme 2 (ACE2) as the entry
receptor and employs the cellular serine protease TMPRSS2 for S
protein priming.
 Inhibition of Coronavirus Entry
• Antiviral small molecules that inhibited cathepsin L were able to inhibit SARS-
CoV infections in vitro.
• It also inhibits other viruses that depend on cathepsin L for entry, such as Ebola,
Hendra, and Nipah viruses.
• The presence of a serine protease TMPRSS2 cleavage site in SARS-CoV-2 S
protein (S1/S2 site) allowed used of its inhibitor in inhibiting SARS-CoV-2
infections.
• Antibodies against RBD and S2 domain of SARS-CoV and MERS-CoV S proteins
have been found effective in neutralizing infections of permissive cell lines in
vitro.
• In addition, neutralizing antibodies were capable of treating infections in
experimental animals and in infected patients during these major outbreaks.
• In one study, several SARS-CoV RBD-specific monoclonal antibodies did not bind
to SARS-CoV-2 S protein.
• Another study showed that the SARS-CoV-specific monoclonal antibody,
CR3022, bound with high affinity to SARS-CoV-2 RBD.
• The previous studies suggest that there are differences between the two RBDs
which impact the cross reactivity of many neutralizing antibodies.
• However, neutralizing antibodies against SARS-CoV-2, once developed, could
be promising in controlling the current SARS-CoV-2 outbreak.
 SARS-CoV-2

• It is not well understood why outbreaks of CoV infections are mostly

occurring in China.
• Those viruses may be predominantly circulating in animals in China
rather than other animals in different parts of the world.
• One of the reasons for these sudden outbreaks could be the close
interactions with live and wild animals that are consumed as food in
wholesale food markets in China.
 SARS-CoV-2

• In a very recent study, the genomes of CoVs isolated from nine patients
having viral pneumonia in Wuhan were analyzed.
• The study showed that the genomes of these viruses differed by less
than 0.1 percent (more than 99.98% of sequence identity), which
indicates that the virus has only recently emerged in humans and has
been detected rapidly after its emergence.
• As the virus keeps spreading to more individuals, more mutations arise
which can potentially make the virus more virulent and thus constant
surveillance will be necessary.
 Mutations in SARS-CoV-2

• A variant of SARS-CoV-2 with a D614G substitution in the gene encoding

the spike protein emerged in late January or early February 2020.
• In several months, the D614G mutation replaced the initial SARS-CoV-2
strain identified in China and by June 2020 became the dominant form
of the virus circulating globally.
• Studies demonstrated that compared to the initial virus strain, the strain
with the D614G substitution has increased infectivity and transmission.
• It does not cause more severe illness or alter the effectiveness of
existing laboratory diagnostics, therapeutics, vaccines, or public health
preventive measures.
 Mutations in SARS-CoV-2
• A SARS-CoV-2 variant was reported in North Jutland, Denmark in August
2020.
• It was spread to human from farmed mink
• The variant, referred to as the “Cluster 5” variant by Danish authorities,
has a combination of mutations that were not previously observed.
• To date, Danish authorities have identified only 12 human infected with
the Cluster 5 variant.
• It does not appear to have spread widely.
 Mutations in SARS-CoV-2
• On 14 December 2020, authorities of the United Kingdom reported to
WHO a variant referred to by the United Kingdom as SARS-CoV-2 (VOC
202012/01).
• This variant contains 23 nucleotide substitutions.
• How and where SARS-CoV-2 VOC 202012/01 originated is unclear.
• It appeared in South East England but within a few weeks began to
replace other virus lineages.
• It has increased transmissibility.
• There is no change in disease severity.
 Mutations in SARS-CoV-2

• On 18 December, national authorities in South Africa announced the

detection of a new variant of SARS-CoV-2 that is rapidly spreading.
• South Africa has named this variant 501Y.V2, because of a N501Y
mutation.
• While SARS-CoV-2 from the UK also has the N501Y mutation,
phylogenetic analysis has shown that 501Y.V2 from South Africa are
different virus variants.
• This new SARS-CoV-2 variant has largely replaced other SARS-CoV-2
viruses circulating in South Africa.
• Preliminary studies suggest the variant is associated with a higher viral
load, which may suggest potential for increased transmissibility.
• As of 30 December, the 501Y.V2 variant from South Africa has been
reported from four other countries to date.
Structurally characterized non-structural proteins of SARS-CoV-2.
Highlighted in pink are mutations found when aligning the proteins against their homologs
from the closest related coronaviruses: human SARS-CoV, bat coronavirus BtCoV, and another
bat betacoronavirus BtRf-BetaCoV.
The structurally resolved part of wNsp7 shares 100% sequence identity to its homolog.
Structurally characterized structural proteins and an ORF of SARS-CoV-2.
Highlighted in pink are mutations found when aligning the proteins against their homologs from the closest
related coronaviruses: human SARS-CoV, bat coronavirus BtCoV, and another bat betacoronavirus BtRf-
BetaCoV.
Highlighted in yellow are novel protein inserts found in wS.
 CoVs infecting human
• The first discovered CoVs were IBV that causes respiratory disease in
chickens and
• the human CoVs, human CoV-229E (HCoV-229E) and human CoV-OC43
(HCoV-OC43), which cause the common cold in humans.
• Then several other HCoVs were discovered, such as Severe Acute
Respiratory Syndrome-CoV (SARS-CoV) in 2002,
• HCoV-NL63 in 2004,
• HCoV-HKU1 in 2005, Middle East Respiratory Syndrome-CoV (MERS-
CoV) in 2012.
• Starting December 2019, there were reports of patients presenting with
severe viral pneumonia in the city of Wuhan, China.
• Sequencing of the virus from these patients has identified a novel CoV
as the causative agent of this respiratory disease.
 SARS-CoV-2
• Genetic analyses of viral samples from patients with SARS-CoV-2
infections revealed that the SARS-CoV-2 is a betacoronavirus.
• It has 88% sequence identity to two bat SARSr-CoV:
• 79% identity to SARS-CoV
• 50% identity to MERS-CoV.
• The previous findings suggest that SARS-CoV-2 is a new virus that is
distinct from SARS-CoV and MERS-CoV but most probably originated in
bats, similar to SARS-CoV and MERS-CoV.
• Another recent study confirmed that SARS-CoV-2 significantly clustered
with a sequence from the bat SARS-like CoV that was isolated in 2015.
• However, the existence of an intermediate host for SARS-CoV-2 is still
not verified.
 Protective Measures to Prevent Spread of
the Disease
• air travel should be restricted.
• Respiratory viruses spread through respiratory droplets that are
produced when an infected person coughs or sneezes.
• It is a highly contagious virus
• The ways of transmission is still not clear.
• The most obvious measure is to avoid contact with people who are sick.
• If someone is sick, they should stay home.
• Once they recover, they may consider using disposable face masks
(while frequently changing them) and avoiding close contact with
coworkers.
• Small droplets can travel farther than large droplets and can be inhaled
around the sides of the masks.
 Protective Measures to Prevent Spread of
the Disease
• Being an enveloped virus, washing hands with water and soap for at
least 30 s would be beneficial in deactivating the SARS-CoV-2.
• Hand sanitizers can also be used if water and soap are not readily
available, while touching eyes, nose, and mouth should be prevented.
• Disinfection of different environmental surfaces, tools, and objects is
crucial in limiting the spread of the virus.
• Even after success with the pandemic the patients who are cured and
declared virus-free should be followed.
• In Ebola outbreak, in which some patients who walked out from the
hospital “virus-free” were found later to harbor the Ebola virus that was
carefully hiding itself in other parts of the body, such as the immune-
privileged eye.
• Although this hidden Ebola virus was no longer transmissible to other
humans, it rendered the label “virus-free” incorrect with these
individuals.
 Protective Measures to Prevent Spread of
the Disease
• The current diagnostic test for the virus is qRT-PCR.
• This test typically takes 48 h, but new tests have been developed that
takes about 40 minutes.
 MERS CoV
• MERS-CoV was first described in 2012 as a new CoV that causes a severe
respiratory disease in Saudi Arabia.
• Similar to SARS-CoV, MERS-CoV infects unciliated bronchial epithelial
cells and type II pneumocytes and causes severe illness of the
respiratory tract.
• It is characterized by fever, cough, shortness of breath, and severe
complications such as pneumonia and kidney failure.
• The incubation period of MERS-CoV is quite similar to SARS-CoV and
ranges from 2 to 14 day.
• As of January 2020 and since 2012, 862 of 2506 infected cases in 27
countries have died (≈35% fatality), which is more than three times the
fatality seen in SARS-CoV infections.
• However, unlike SARS-CoV, human-to-human transmission of MERS-CoV
is not easy and has not been confirmed except in cases of very close
contact with infected patients in health care settings.
 MERS CoV
• MERS-related CoVs (MERSr-CoVs) were detected in bats, suggesting a
potential bat origin.
• MERS-CoV was transmitted to humans from dromedary camels.
• Studies have also shown that camel MERS-CoV strains are almost
identical to human MERS-CoV strains.
• It was postulated that MERS-CoV existed in camels at least 30 years ago
since antibodies to MERS-CoV were detected in samples that were
collected from camels in 1983.
• Sequence analyses have shown that MERSr-CoVs’ RBDs share only 60–
70% sequence identity with that of human and camel MERS-CoVs.
• Similar to the adaptation of SARS-CoV to human host, MERSr-CoVs that
are circulating in bats had to undergo several amino acid changes in RBD
of S protein to become capable of infecting camels and humans.
• We believe that the amino acid changes in MERSr-CoVs’ RBD led to the
emergence of MERS-CoV strains that are capable of binding to human
DPP4 with high affinity, infecting humans, and causing the 2012
outbreak.
 SARS-CoV

• Studies in bats from different regions of China have identified several

SARSr-CoVs.
• The previous finding indicates that SARS-CoV has been circulating in
bats for a long time before genetically changing and jumping to humans.
• Since ACE2 was identified as the receptor for SARS-CoV, it is not
surprising that SARS-CoV has adapted itself to bind human ACE2 and
efficiently infect human cells.
• That sort of adaptation required a set of amino acid changes in the RBD
of S protein of SARS viruses that were circulating in bats.
• Therefore, we conclude that the human-to-human transmission that
was seen during the SARS-CoV outbreak is attributed to the ability of
SARS-CoV to adapt its S protein (particularly RBD) to efficiently bind to
human ACE2 and infect airway epithelia.