SARS Corona-virus 2: Genome Sequencing And Its Application

SARS Coronavirus 2: Genome Sequencing
And Its Application
Prepared By:-
Adyasha Nayak
Sarbajit Ray
Badri Prasad Sarangi
Sugata Lahiri

Introduction
• Coronaviruses (CoVs) belong to the family of Coronaviridae and were first isolated from humans in
1962.
• The CoVs were thought to cause only mild respiratory and gastrointestinal infections in human and
animals.
• The outbreaks of Severe Acute Respiratory Syndrome-CoronaVirus 1 (SARS-CoV-1) in 2002–2003 in
Guangdong province, China.
• Middle East Respiratory Syndrome CoronaVirus (MERS-CoV) in the Middle Eastern countries,
particularly Saudi Arabia in 2012.
• CoVs can be divided in four genera, Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and,
Deltacoronavirus . Alpha and Beta coronaviruses more commonly cause infections in humans and
mammal and in particular, Beta coronaviruses include severe acute respiratory syndrome
coronavirus (SARS-CoV).
• Both the viruses originated in bats and their chain of transmission established between animal to
human and human to human.
• A distinguishing feature of SARS-CoV-2 is its incorporation of a polybasic site cleaved by furin,
which appears to be an important element enhancing its virulence.

● There are two scenarios that can explain the origin of SARS-CoV-2:
(i)natural selection in an animal host before zoonotic transfer; and (ii)
natural selection in humans following zoonotic transfer. Also possibility of
an inadvertent laboratory release of SARS-CoV-2 cannot be denied.
● Phylogenetic analysis also indicates that a virus from Rhinolophus affinis,
designated RaTG13, has a 96.1% resemblance to SARS-CoV-2. This
sequence was the closest known to SARS-CoV-2 at the time of its
identification, but it is not its direct ancestor.
● Bats are considered the most likely natural reservoir of SARS-CoV-2.
Differences between the bat coronavirus and SARS-CoV-2 suggest that
humans may have been infected via an intermediate host, although the
source of introduction into humans remains unknown.
● Although the role of Pangolins as an intermediate host was initially
posited but pangolin virus samples are too distant to SARS-CoV-2 i.e.,
only 92% identical in sequence to the SARS-CoV-2 genome. In addition,
despite similarities in a few critical amino acids, pangolin virus samples
exhibit poor binding to the human ACE2 receptor.
● Human to human transmission was initially assumed to occur by
respiratory droplets but indirect transmission was confirmed latter. One
meta-analysis found that 17% of infections are asymptomatic, and
asymptomatic individuals were 42% less likely to transmit the virus

● The International Committee on Taxonomy of Viruses (ICTV) has named the novel
Coronavirus as Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
● Disease caused by this virus has been officially named as COVID-19 by WHO.
● The COVID-19 has been regarded as Public Health Emergency of International Concern
(PHEIC) on January 30, 2020 by WHO.
● SARS-CoV-2 shares 96% genome similarity with a bat Coronavirus.
● Several notable variants of SARS-CoV-2 emerged in late 2020 :
○ Alpha: Lineage B.1.1.7 emerged in the UK in Sept 2020, with evidence of increased
transmissibility and virulence. Notable mutations include N501Y, P681H & E484K
○ Beta: Lineage B.1.351 emerged in South Africa in May 2020, with evidence of increased
transmissibility and changes to antigenicity. Notable mutations include K417N, E484K
and N501Y.
○ Gamma: Lineage P.1 emerged in Brazil in November 2020, also with evidence of
increased transmissibility and virulence, alongside changes to antigenicity.Notable
mutations also include K417N, E484K and N501Y.
○ Delta: Lineage B.1.617.2 emerged in India in October 2020. There is also evidence of
increased transmissibility and changes to antigenicity.
○ Omicron: Lineage B.1.1.529 emerged in Botswana in November 2021.
● There is uncertainty about reinfection and long-term immunity. It is not known how
common reinfection is, but reports have indicated that it is occurring with variable severity.

Genomic organization of SARS-CoV-2
• The SARS-CoV-2 is a single stranded positive sense RNA virus of ~29.9 kB in
size.
• The SARS-CoV-2 is a non-segmented enveloped virus with a diameter of 50–
200 nm.
• The SARS-CoV-2 has 14 open reading frames (ORFs), which encodes for 27
different proteins.
• The SARS-CoV-2 genome shares 82 percent sequence commonality with
SARS-CoV and MERS-CoV, with key enzymes and structural components
sharing >90 percent sequence identity. Because of the good degree of the
sequence, a common pathogenic mechanism was discovered, allowing for
therapeutic targeting.
• It has 5′ untranslated region (UTR), replication complex (ORF1a and ORF1b),
Spike (S) gene, Envelope (E) gene, Membrane (M) gene, Nucleocapsid (N)
gene, 3′ UTR, several unidentified non-structural ORFs and a poly (A) tail.

• The viral genome having a Receptor Binding Domain (RBD) for the
interaction with host cell receptors is covered by the Spike
glycoprotein.
• The M glycoprotein is responsible for the assembly of viral particles
has three domains, the cytoplasmic domain, the transmembrane
domain, and the N hydrophilic domain
• The Envelope protein is reported to play role in pathogenesis.
• The nucleocapsid protein packs the viral genome into a
ribonucleoprotein complex.
• The nucleocapsid, a phosphoprotein plays role in viral genome
replication and the cell signaling pathway.
• The SARS-CoV-2 genome is unstable at elevated temperature
because of highly enriched A+U content (62%) and reduced G+C
content (38%).

FIG-
The 5′ UTR and 3′ UTR and coding region of COVID-19, SARS-CoV, and MERS-CoV.
The numbers of base pairs among betacoronaviruses are shown. This figure is modified from the
sequence comparison and genomic organization of 2019-nCoV, 2020.
The differences in the arrangement of the envelope (E), membrane (M), and nucleoprotein (N)
among COVID-19, SARS-CoV, and MERS-CoV are shown at 3′ end.

• The full-length RNA genome comprises of ~29,903 nucleotides and has a
replicase complex (comprised of ORF1a and ORF1b) at the 5′UTR.
• The ORF1a gene codes for the polyprotein pp1a, which has ten nsps (nsp1–
nsp10). The ORF1b gene, which is found near ORF1a, produces the
polyprotein pp1ab, which has 16 nsps(nsp1–nsp16).
• SARS-CoV-2 has 13–15 (12 functional) open reading frames (ORFs) with a
total of 30,000 nucleotides in its genetic profile. The genome has 11
protein-coding genes, with 12 expressed proteins.
• Four genes that encode for the Structural proteins are Spike gene,
Envelope gene, Membrane gene, Nucleocapsid gene and a poly (A) tail at
the 3′UTR.
• 16 nonstructural, 4 structural, and 6 accessory proteins are encoded by the
SARS-CoV-2 genome.
• The accessory genes are distributed in between the structural genes.

Schematic presentation of the SARS-CoV-2 genome Structure

Features of the spike protein in human SARS-CoV-2 and
related coronaviruses

Technical aspects of genomic sequencing and
analysis of SARS-CoV-2

The acquisition of sufficient, high quality SARS-CoV-2 RNA helps to
maximize sequencing yield and the ultimate quality of genome
sequence data. The quantity and quality of an RNA sample are affected
by: choice of clinical sample; handling of clinical sample; method of
viral RNA isolation; and the technical proficiency of personnel.
Where several different sample types are available, it is beneficial to
select one that has a high viral load and low levels of human or
bacterial genetic material contaminants. Such samples can be
sequenced using both metagenomic and SARS-CoV-2 targeted
techniques.

Whole genome sequencing of COVID-19
5
4
3
2
1

Metagenomics studies
• Metagenomic protocols permit untargeted sequencing of nucleic
acid in a sample, including viral genomic material if present.
• These protocols offer a hypothesis-free approach to pathogen
discovery, as they require little prior knowledge of the pathogen of
interest

Sequencing
Capture-based approaches
are used to enrich the viral
genome. It mainly involves
hybridization of viral cDNA to
DNA or RNA baits which are
made complimentary to
SARS-CoV-2 genome.
Sample collection
Nasopharyngeal swabs and
similar samples containing
high viral load needs to be
isolated like oral samples,
tissue samples(autopsy) and
blood samples.
Pretreatment
Since metagenomics refers to
the study of a variety of
DNA/RNA together, there
need to be pretreatment
procedures like centrifugation
to extract viral RNA

Applications of genomics to
SARS-CoV-2

Applications of genomics to SARS-CoV-2
Understanding the emergence of SARS-CoV-2
Understanding the biology of SARS-CoV-2
Improving diagnostics and therapeutics
Investigating virus transmission and spread
Inferring epidemiological parameters

Identifying the
causative agent of
COVID-19
Determining times of
origin and early
diversification
Identifying the zoonotic
origin
1
2
3

1. SARS-CoV-2 was independently identified and sequenced in early 2020. Several
different metagenomic next-generation sequencing (mNGS) approaches were used
to identify the causative pathogen of COVID-19. Metagenomic sequencing permits
non targeted sequencing of nucleic acid in a sample, and can therefore identify viral
RNA or DNA if present at high enough copy numbers relative to DNA or RNA from
other sources.
1. It was particularly important to determine when SARS-CoV-2 first emerged in
humans, since this could provide an indication of whether there was a long period of
undetected transmission before the first clinical cases were seen.
1. SARS-CoV-2 genome sequences and related virus genomes from other animals have
been analysed phylogenetically in an attempt to determine the zoonotic reservoir
from which SARS-CoV-2 emerged. To date, there has been relatively limited
sampling with the aim of identifying the animals involved in the genesis of SARS-
CoV-2 and determining when, where and how the virus emerged in humans.

Host receptor usage
SARS-CoV-2 evolution:
identifying candidate
genomic sites that may
confer phenotypic
changes
1
2

1. Since viruses can replicate only inside the living cells of a host organism,
determining the host cellular receptor used by SARS-CoV-2 is essential to
understanding its basic biology. Receptor binding is mediated by the S protein of the
virus. Genetic similarities in the S protein receptor-binding motif between SARS-
CoV-2 and other, previously investigated coronaviruses have helped to identify the
cellular receptor to which SARS-CoV-2 binds, and hence the cell types that it might
infect. Most amino acid residues that are known to be essential for ACE2
binding by SARS-CoV are conserved in SARS-CoV-2.
1. All viruses acquire genetic changes as they evolve, and most acquired genetic
changes do not substantially affect virulence or transmissibility. Variants between
virus genomes sampled from different locations cannot be assumed to cause
observed epidemiological differences in COVID-19 between those locations and
are instead likely to be stochastic. Despite this, it is possible that a genetic change
may occur that causes a corresponding phenotypic change in SARS-CoV-2 of public
health importance.

Improving molecular
diagnostics
Supporting the design
and sensitivity
monitoring of
serological assays Supporting vaccine
design
1
2
3
Supporting design of
antiviral therapy
Identifying antiviral
resistance or vaccine
escape mutations
4
5

1. The development of rapid, inexpensive and sensitive nucleic acid amplification tests (NAATs)
for routine molecular detection of SARS-CoV-2 was prioritized as metagenomic sequencing is too
time-consuming and costly. As SARS-CoV-2 continues to acquire genetic changes over time
during this pandemic, continued generation and sharing of virus genomes will be vital for
monitoring the expected sensitivity of the various diagnostic assays in different locations.
2. Virus genomic sequence data can be important in helping to identify virus proteins that are likely
to be strongly antigenic, and to indicate how these antigens can be produced for serological assays.
3. SARS-CoV-2 genome sequences have been used in the design of candidate vaccines that rely on
inoculation with antigens or mRNA/DNA to stimulate, directly or indirectly, antibody production
and cellular responses.
4. Genetic and structural information can reveal similarities in proteolytic and replication
pathways between SARS-CoV-2 and other viruses for which antiviral therapy is already
available, and therefore help to determine which existing antivirals might be repurposed.
5. In-depth genomic sequencing may be useful in exploring the impact of intra-host diversity on
antiviral resistance and vaccine escape (if these occur) or pathogenesis. Genetic sequencing of
specific regions of interest, such as the spike gene, may be sufficient to assess the prevalence of
specific known variants in pre-identified regions.

Supporting or rejecting
evidence for transmission
routes or clusters
Identifying and quantifying
periods of transmission
1
2
Identifying importation
events and local
circulation
Discerning involvement of
other species
Discerning transmission
chains between patients
using intra-host viral
diversity
3
4
5

1. The placement of sequences within a phylogenetic tree can be used to investigate hypotheses of
transmission routes. For example, Considerable phylogenetic separation of virus sequences from
two patients would indicate that the two patients had acquired infections from different sources.
2. Once there is sufficient genetic diversity within a virus lineage, the rate of evolutionary change
(substitution rate) can be estimated. If the substitution rate can be estimated, genetic diversity
between two sampled viruses with known sampling dates can be used to estimate the Time to Most
Recent Common Ancestor (TMRCA). The TMRCA of a group of viruses provides a lower-limit
estimate of the duration of its circulation within the sampled population.
3. If metadata on sampling location are available, sequencing of SARS-CoV-2 genomes can help to
determine whether infections have resulted from local transmission or have been imported.
Such transmission dynamics may be interpreted cautiously and informally via sequence positioning
within a phylogeny.
4. A number of non-human animal species can become naturally infected with SARS-CoV-2. Where
multiple animals are infected, phylogenetic investigations of clustering can be used to demonstrate
that the animals became infected through different routes or a common route. (Eg. In Netherlands,
SARS-CoV-2 genome sequence sampled from a human was consistent with sample collected a
Mink. Hence, humans becoming infected from animals.)

Reproduction number
Scale of outbreak over
time and infection-to-
case reporting ratio
1
2

1. R0 is the basic reproduction number is an epidemiological metric used to measure
the transmissibility of infectious agents. R0 can be estimated using population
genetic modelling.
2. The final size of an epidemic can be defined informally as the total number of
people experiencing infection during the outbreak. Estimating absolute epidemic
size from genetic data has been attempted only recently and is an active area of
phylodynamic methodological development. A variety of experimental methods
have been applied in the current COVID-19 epidemic. In general, any method for
reconstructing epidemic size should account for the major factors that influence
genetic diversity within the sampling frame, including: geographical structure,
variance in transmission rates, exponential growth and nonlinear population
dynamics, and the generation time distribution.

References
1. World Health Organization. (2021). Genomic sequencing of SARS-CoV-2: a guide to implementation for
maximum impact on public health, 8 January 2021. https://www.who.int/publications/i/item/9789240018440
2. Wang, F., Huang, S., Gao, R., Zhou, Y., Lai, C., Li, Z., ... & Liu, L. (2020). Initial whole-genome sequencing
and analysis of the host genetic contribution to COVID-19 severity and susceptibility. Cell discovery, 6(1), 1-
16. https://www.nature.com/articles/s41421-020-00231-4
3. Umair, M., Ikram, A., Salman, M., Khurshid, A., Alam, M., Badar, N., ... & Klena, J. (2021). Whole-genome
sequencing of SARS-CoV-2 reveals the detection of G614 variant in Pakistan. Plos one, 16(3), e0248371.
https://doi.org/10.1371/journal.pone.0248371
4. Aggarwal, D., Myers, R., Hamilton, W. L., Bharucha, T., Tumelty, N. M., Brown, C. S., ... & Page, A. J.
(2021). The role of viral genomics in understanding COVID-19 outbreaks in long-term care facilities. The
Lancet Microbe.
5. Yadav, P. D., Potdar, V. A., Choudhary, M. L., Nyayanit, D. A., Agrawal, M., Jadhav, S. M., ... & Cherian, S.
S. (2020). Full-genome sequences of the first two SARS-CoV-2 viruses from India. The Indian journal of
medical research, 151(2-3), 200.
6. Naqvi, A.A.T., Fatima, K., Mohammad, T., Fatima, U., Singh, I.K., Singh, A., Atif, S.M., Hariprasad, G.,
Hasan, G.M. and Hassan, M.I., 2020. Insights into SARS-CoV-2 genome, structure, evolution, pathogenesis
and therapies: Structural genomics approach. Biochimica et Biophysica Acta (BBA)-Molecular Basis of
Disease, 1866(10), p.165878.
7. Rastogi, M., Pandey, N., Shukla, A. and Singh, S.K., 2020. SARS coronavirus 2: from genome to infectome.
Respiratory Research, 21(1), pp.1-15.
8. Brant, A.C., Tian, W., Majerciak, V., Yang, W. and Zheng, Z.M., 2021. SARS-CoV-2: from its discovery to
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