understanding genomic diversity, pan-genome ...understanding genomic diversity,...

Understanding genomic diversity,pan-genome, and evolution of SARS-CoV-2Arohi Parlikar*, Kishan Kalia*, Shruti Sinha, Sucheta Patnaik,Neeraj Sharma, Sai Gayatri Vemuri and Gaurav Sharma

Institute of Bioinformatics and Applied Biotechnology (IBAB), Bengaluru, Karnataka, India* These authors contributed equally to this work.

ABSTRACTCoronovirus disease 2019 (COVID-19) infection, which originated from Wuhan,China, has seized the whole world in its grasp and created a huge pandemic situationbefore humanity. Since December 2019, genomes of numerous isolates have beensequenced and analyzed for testing confirmation, epidemiology, and evolutionarystudies. In the first half of this article, we provide a detailed review of the history andorigin of COVID-19, followed by the taxonomy, nomenclature and genomeorganization of its causative agent Severe Acute Respiratory Syndrome-relatedCoronavirus-2 (SARS-CoV-2). In the latter half, we analyze subgenus Sarbecovirus(167 SARS-CoV-2, 312 SARS-CoV, and 5 Pangolin CoV) genomes to understandtheir diversity, origin, and evolution, along with pan-genome analysis of genusBetacoronavirus members. Whole-genome sequence-based phylogeny of subgenusSarbecovirus genomes reasserted the fact that SARS-CoV-2 strains evolved from theircommon ancestors putatively residing in bat or pangolin hosts. We predicted a fewcountry-specific patterns of relatedness and identified mutational hotspots with high,medium and low probability based on genome alignment of 167 SARS-CoV-2strains. A total of 100-nucleotide segment-based homology studies revealed that themajority of the SARS-CoV-2 genome segments are close to Bat CoV, followed bysome to Pangolin CoV, and some are unique ones. Open pan-genome of genusBetacoronavirus members indicates the diversity contributed by the novel virusesemerging in this group. Overall, the exploration of the diversity of these isolates,mutational hotspots and pan-genome will shed light on the evolution andpathogenicity of SARS-CoV-2 and help in developing putative methods of diagnosisand treatment.

Subjects Bioinformatics, Evolutionary Studies, Genomics, Microbiology, VirologyKeywords COVID-19, SARS, Coronavirus, Pandemic, Viral disease, Genome, Bioinformatics,Genomics, Mutations

INTRODUCTIONThe emergence of coronavirus disease 2019 (COVID-19) has sent people from across theworld into a state of high alert, and they are trying to survive complete or partial lockdowns(Lescure et al., 2020). The causative agent of this pandemic is a novel Severe AcuteRespiratory Syndrome (SARS) Coronavirus, Severe Acute Respiratory Syndrome-relatedCoronavirus-2 (SARS-CoV-2). Since 2000, the world has witnessed two major coronavirusoutbreaks: the first, of SARS, caused by SARS-CoV, in 2002 in China (Zhong et al., 2003),

How to cite this article Parlikar A, Kalia K, Sinha S, Patnaik S, Sharma N, Vemuri SG, Sharma G. 2020. Understanding genomic diversity,pan-genome, and evolution of SARS-CoV-2. PeerJ 8:e9576 DOI 10.7717/peerj.9576

Submitted 29 April 2020Accepted 29 June 2020Published 17 July 2020

Corresponding authorGaurav Sharma,[email protected]

Academic editorAbhishek Kumar

Additional Information andDeclarations can be found onpage 24

DOI 10.7717/peerj.9576

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and the second, of Middle East Respiratory Syndrome (MERS) in 2012 in Saudi Arabia(Zaki et al., 2012). Amongst coronaviruses, SARS-CoV and MERS are known as highlypathogenic viruses that cause pneumonia and respiratory system infections (Coleman &Frieman, 2014). Besides these, several other low pathogenic strains are also known thatcause mild respiratory diseases and infect majorly the upper respiratory tract (Han et al.,2020). After the diagnosis of the first reported case in Wuhan, China, and genomesequencing of the Wuhan Hu-1 strain (SARS-CoV-2 reference genome), scientists acrossthe world are striving to develop vaccines and diagnostic kits and understand itsevolution and epidemiology. Several potential drug molecules to combat COVID-19 areundergoing clinical trials. As no vaccine or drugs are available at present, the only waysknown to contain transmission are social distancing, regular washing of hands, andcovering mouth and nose while coughing or sneezing, thereby, preventing direct or indirectphysical contact and containing the transfer of infected respiratory droplets.

This study comprises a detailed overview of the history and origin of COVID-19along with the taxonomy, nomenclature and genome structure of its causative agentSARS-CoV-2. Following that, we analyze 167 SARS-CoV-2, 312 SARS-CoV and fivePangolin CoV genomes to study their genomic variability, evolution and mutationhotspots. We also characterize the pan-genome of the genus Betacoronavirus (includingthe recently sequenced Pangolin CoV) to classify their proteins-coding regions into core,accessory, and unique categories within each genome group.

TAXONOMY AND NOMENCLATURECoronaviruses are members of family Coronaviridae that include enveloped positive sensessRNA containing viruses, taxonomically classified amongst order Nidovirales in the realmRiboviria. Like other viruses, they thrive in the gray area of living and non-living asthey do not have the machinery to survive outside a living host cell. Therefore, they cannotreplicate outside the host. The Riboviruses or realm Riboviria members replicate byutilizing RNA-dependent RNA-polymerases (RdRps). Their genetic material that is, RNAserves as messenger RNA (mRNA), directly translating into proteins and undergoesgenetic recombination in the presence of another viral genome in the host cell(Barr & Fearns, 2010).

Members of order Nidovirales have a positive sense linear (capped and polyadenylated)RNA molecule, known for causing severe infections. The word “Nido” stands for “nest”implying that all Nidoviruses express subgenomic mRNAs (sgRNA) in a nested form. Theyare known to infect hosts within three important classes in Vertebrates, namely mammals(Mammalia), birds (Aves) and fishes (Pisces). Coronaviruses (family Coronaviridae;subfamily Orthocoronavirinae) commonly infect both mammals and birds; Torovirus(family Tobaniviridae; subfamily Torovirinae) infect specifically mammals and Bafinivirus(family Tobaniviridae; subfamily Piscanivirinae) infect fishes. Coronavirus virions arespherical, Torovirus are crescent-shaped, and Bafinivirus are rod-like; however, allmembers of this family are adorned with a crown or club-shaped surface proteinscalled the Spike proteins. Because of their crown-like morphology observed in electronmicrographs, they are named Coronaviruses. Family Coronaviridae has a single

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subfamily Orthocoronavirinae and its members form a monophyletic clade. They arefurther classified into four defined genera based on phylogenetic studies of conservedgenome regions and their serological cross-reactivity: Alphacoronavirus, Betacoronavirus,Gammacoronavirus and Deltacoronavirus. Under genus Betacoronavirus, five lineagesdiverge: Lineage A (subgenus: Embecovirus) includes HCoV-OC43 and HCoV-HKU1,Lineage B (subgenus: Sarbecovirus) includes SARS-CoV and SARS-CoV-2, Lineage C(subgenus: Merbecovirus) contains Bat coronavirus BtCoV-HKU4 and BtCoV-HKU5,Lineage D (subgenus: Nobecovirus) includes Rousettus Bat coronavirus BtCoV-HKU9and lineage E (subgenus: Hibecovirus) includes Bat Hp-betacoronavirus/Zhejiang2013.The subgenus Sarbecovirus members tend to undergo deep recombination events leadingto the formation of new alleles (Boni et al., 2020) and zoonotic transfer.

ORIGIN AND HISTORY OF THE CORONAVIRUSINFECTIONSThe first Coronavirus associated disease was described in 1931 (Peiris, 2012). The viruseswere first isolated from humans in the UK and USA around the same time. The firstisolated specimen was B814, taken from a boy exhibiting symptoms of the common cold in1960 and cultured in human embryonic tracheal organ culture (Tyrrell & Bynoe, 1966).The specimen was deemed distinct from Adeno-, Entero- or Rhinoviruses, being etherlabile and propagated only in organ cultures (Kendall, Bynoe & Tyrrell, 1962; Tyrrell &Bynoe, 1966). Later, the gradual discoveries of 229E in 1966 (Hamre & Procknow, 1966),OC43 in 1967 (McIntosh, Becker & Chanock, 1967), SARS-CoV in 2002, Bovine CoV in2006, MERS-CoV in 2012 and SARS-CoV-2 in 2019 (Zhu et al., 2020) were majorsignificant steps in coronavirus history.

Coronaviruses have been associated with mild respiratory infections and cold inhumans and animals (specifically poultry and livestock). These viruses were notconsidered treacherous until the SARS epidemic that emerged in China in November 2002.The associated SARS-CoV species were first classified as a separate clade underBetacoronavirus after this outbreak, post which new species including those of humancoronaviruses (hCoVs) have been identified. The 2002 SARS epidemic ultimately infected8,096 people, with 774 deaths. Later, MERS-CoV emerged in Saudi Arabia in September2012, causing 2,494 confirmed cases of infection and 858 deaths across 27 countries(https://www.who.int/emergencies/diseases/en/).

The current pandemic, COVID-19, is caused by the virus, initially designated “2019novel coronavirus” or “2019-nCoV”, later re-named SARS-CoV-2, segregating it as a novelSARS species (Huang et al., 2020). Now, it forms a new clade in the subgenus Sarbecovirusand is established as the 7th member of the family which infects humans (Simmonds et al.,2017; Gorbalenya et al., 2020; Zhu et al., 2020). The first patients of COVID-19 wereidentified in late December 2019 when several local health centers reported clusters ofpatients with pneumonia caused by an unknown pathogen, epidemiologically linked toliving animal and seafood wholesale market in Wuhan. The Chinese Center for DiseaseControl and Prevention called a rapid action team for the epidemiological investigation.Bronchoalveolar-lavage fluids were propagated on human respiratory tract epithelial cell


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cultures for 4–6 weeks. Extracted nucleic acid was identified as viral RNA by real-timereverse transcription PCR targeting the RdRp region of pan-BetaCoV. The electronmicrographs revealed the presence of distinctive spikes of length 9–12 nm, establishingmorphological resemblance with the Coronaviridae family (Zhu et al., 2020). Followingthis, SARS-CoV-2 infection has been spreading worldwide. As of 29 April 2020(submission date), 213 countries and territories around the world are under surveillance,with more than 3.15 million confirmed cases and over 218,490 reported fatalities, and thesenumbers continue to exponentially increase day by day.

Since December 2019, numerous SARS-CoV-2 genomes have been sequenced, whichallows researchers to address many critical questions regarding its origin, transmission,epidemiology, and most importantly to design vaccines and viral detection kits. Viralgenome sequencing and multiple sequence alignment of SARS-CoV-2 genomes haverevealed its identity with Bat CoV RaTG13 (MN996532) and Pangolin-CoV (Cui, Li &Shi, 2019; Boni et al., 2020; Rehman et al., 2020). A comparative phylogenetic studyrevealed that the Pangolin-CoV genes shared a high level of sequence identity withSARS-CoV-2 genes, specifically orf1b, the spike (S), orf7a and orf10 genes (Zhang, Wu &Zhang, 2020). Higher identity of S protein sequence implies the functional similaritybetween Pangolin-CoV and SARS-CoV-2 as compared to the RaTg1 strain (Zhang, Wu &Zhang, 2020) further suggesting Pangolin as an intermediate host for infection and naturalreservoir of SARS-CoV-2-like strains.

CORONAVIRUS GENOME ORGANIZATIONCoronaviruses belong to family Coronaviridae and comprise enveloped viruses thatreplicate in the cytoplasm of animal host cells; distinguished by the presence of asingle-stranded positive-sense RNA genome (about 30 kb in length) (Marra et al., 2003).Coronaviruses possess the largest genomes among all known RNA viruses (Fehr &Perlman, 2015), ranging from 25.32 kb in Porcine Deltacoronavirus PD-CoV to 31.775 kbin Beluga whale coronavirus SW1 (information available on NCBI Virus webpagehttps://www.ncbi.nlm.nih.gov/labs/virus/). SARS-CoV-2 is a spherical or pleomorphicenveloped particle, containing single-stranded, positive-sense RNA associated with anucleoprotein, lying inside a capsid composed of matrix protein (Lai et al., 2020).

The SARS-CoV-2 reference genome (NC_045512; Wuhan Hu-1 strain) is 29,903nucleotides long, which consists of A (8,954 nt, 29.94%), G (5,863 nt, 19.61%), C (5,492 nt,18.37%), T (9,594 nt, 32.08%). Compared to SARS-CoV-2, the SARS-CoV referencegenome (NC_004718; Tor1 strain) is a little larger, that is, 29,751 nucleotides and itsnucleotide composition is marginally different: A (8,481 nt, 28.50), G (6,187 nt, 20.80%),C (5,940 nt, 19.97%), T (9,143 nt, 30.73%). We identified that the GC content (averageof all genomes under this study) of SARS-CoV, SARS-CoV-2, and Pangolin CoV is40.81%, 38% and 38.52% respectively. Like other Betacoronavirus members, this genomecontains two flanking untranslated regions (UTRs), 5′-UTR (265 nt) and 3′-UTR (229 nt)sequences.

A typical CoV genome contains at least six ORFs (Graham et al., 2008). The genomes ofall coronaviruses usually encode four well-conserved and characterized structural proteins:


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S (spike), E (envelope), M (membrane) and N (nucleocapsid) (Graham et al., 2008;Fuk-Woo Chan et al., 2020), encoded by sgRNA 2, 4, 5 and 9a respectively present at 3′endand sharing 1/3 of the genome in SARS-CoV (Graham et al., 2008).

SARS-CoV-2 Proteins: The SARS-CoV-2 genome has 12 protein-coding regions, whichencode two categories of proteins: first, Structural proteins, which give characteristicstructure to the virus and are involved in viral entry, and second, Non-structural (NS)or accessory proteins, which help in viral replication (Marra et al., 2003; Graham et al.,2008; Hu et al., 2018; Fuk-Woo Chan et al., 2020). Most of the information available so farabout these proteins is based on SARS-CoV and other members of the genusBetacoronavirus.

STRUCTURAL PROTEINSSpike proteinThe S protein is a 1,273 aa trimeric, cell-surface glycoprotein consisting of two subunits(S1 and S2), encoded by the gene S. The S1 subunit is responsible for receptor binding(Hu et al., 2018). This is also important for mediating the fusion of viral and hostmembranes. Both these processes are critical for virus entry into host cells (Tan, Lim &Hong, 2005). SARS-CoV-2 has a polybasic cleavage site RRAR, at the junction of S1 and S2subunits, which enables effective cleavage by Furin and other proteases (Andersen et al.,2020; Zhang, Wu & Zhang, 2020). Furthermore, one proline residue is also inserted atthe leading cleavage site of SARS-CoV-2, making “PRRA”, the final inserted sequence.Comparison of this site within different Betacoronavirus members revealed that theinsertion of a Furin cleavage site at the S1–S2 junction of SARS-CoV-2 enhances cell-cellfusion (Follis, York & Nunberg, 2006;De Haan et al., 2008; Andersen et al., 2020). Similarly,an effective cleavage of the polybasic cleavage site in Hemagglutinin esterase proteinfacilitated the inter-species transmission of MERS-like coronaviruses from bats to humans(Andersen et al., 2020). Majorly, variations in the S protein are responsible for twoattributes, tissue tropism and host ranges of different CoVs (Hu et al., 2018). It wasobserved that S protein underwent several drastic changes during the viral infection.The S protein of SARS-CoV-2 is more vulnerable to mutations, especially in the aminoacids associated with the spike protein-cell receptor interface. Interestingly, the amino acidsequence represented ~19% changes with four major insertions and ~81% sequencesimilarity in contrast to SARS-CoV (Ahmed, Quadeer & McKay, 2020; Rehman et al.,2020).

Nucleocapsid proteinN proteins are 419 aa long phosphoproteins weighing ~46kDa. These have helix bindingproperties. Coronavirus N proteins possess three easily distinguishable and highlyconserved domains; the N-terminal domain (NTD) (N1b), the C-terminal domain (CTD)(N2b) and the N3 region (Grossoehme et al., 2009; Cong et al., 2019). The N proteinplays a vital role in virion structure formation as it is localized in both the replication-transcription region and the ERGIC (Endoplasmic reticulum-Golgi apparatusIntermediate Compartment), the site of virion assembly (Tok & Tatar, 2017).



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The N protein of SARS-CoV is primarily expressed during the early stages of SARS-CoVinfection (Surjit & Lal, 2008; Grossoehme et al., 2009; Cong et al., 2019). It is importantas a diagnostic marker as it induces a strong immune response (Hu et al., 2018).Evolutionary analysis has shown 89–91% sequence homology between the N proteins inthe SARS-CoV and Bat SL-CoV (Hu et al., 2018; Ahmed, Quadeer & McKay, 2020).The N protein can bind to Nsp3 protein to help bind the genome to replication/transcription complex (RTC) (Cong et al., 2019) and package the encapsulated genomeinto virions (Chen, Liu & Guo, 2020). IBV, SARS CoV and MHV N protein undergophosphorylation and allow discrimination of non-viral mRNA binding from viralmRNA binding (Cong et al., 2019). N protein is also an antagonist of interferon (IFN)(Kopecky-Bromberg et al., 2007) and virus-encoded repressor of RNA interference (RNAi),therefore, it appears to benefit the viral replication (Chen, Liu & Guo, 2020).

Membrane proteinThe most abundant structural protein in the genome is the membrane (M) glycoprotein,which spans across the membrane bilayer three times. Thus, M glycoproteins have threetransmembrane regions, leaving a short NH2-terminal domain outside the virus and along COOH terminus (cytoplasmic domain) inside the virion (Tok & Tatar, 2017;Mousavizadeh & Ghasemi, 2020). The M protein plays a key role in regenerating virions inthe cell. M proteins undergo glycosylation in the Golgi apparatus which is crucial forthe virion to fuze into the cell and to make antigenic proteins. As mentioned before, Nprotein forms a complex by binding to genomic RNA andM protein triggers the formationof interacting virions in the endoplasm (Tok & Tatar, 2017).

Envelope glycoproteinE glycoproteins are composed of approximately 76–109 amino acids in other coronavirusspecies. E protein plays a crucial role in the assembly and morphogenesis of virionswithin the host. About 30 amino acids in the N-terminus of the E protein enablebinding to the viral membranes. Co-expression of E and M proteins with mammalianexpression vectors enable the formation of virus-like structures within the cell (Tok &Tatar, 2017).

NON-STRUCTURAL (ACCESSORY) PROTEINSORF1ab and ORF1aThe SARS 5′ proximal gene 1 (~22 kb) comprises two long overlapping open readingframes, orf1a and orf1ab, encoding for polyproteins 1a and 1ab. These polyproteins arecleaved by viral proteases that is, PLpro (papain-like protease) and 3CLpro (chymotrypsin-like protease) into 16 non-structural proteins, Nsp1–Nsp16. Expression of orf1abinvolves a (−1) ribosomal frameshift upstream of orf1a stop codon, thus forming thefull-length ORF1ab. ORF1a polyprotein of ~500 kDa encodes for Nsp 1–11, while ORF1abpolyprotein of ~800 kDa encodes for all Nsp1–16. The ORF1a and ORF1ab polyproteinsundergo post-translational modifications to form mature proteins and hence are not



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detected during infection (Graham et al., 2008; Lei, Kusov & Hilgenfeld, 2018). A briefdescription of these proteins is provided below:

i. Nsp1: SARS-CoV Nsp1 is an N-terminal protein coded by the first gene of ORF1ab,which plays an important role in SARS-CoV pathogenesis by inhibiting the hostgene expression via binding to the small subunit of the ribosome and then truncatingthe translation activity. Nsp1 induces endonucleolytic RNA cleavage of a cappedhost mRNA, making it translationally incompetent (Tanaka et al., 2012). Nsp1inhibits the type-1 IFN expression and other antiviral signaling pathways, thussuppressing the innate immune system. The viral mRNA is resistant to Nsp1-mediated RNA cleavage in the infected host cells, however, the mechanism throughwhich it achieves this is unknown (Tanaka et al., 2012).

ii. Nsp2: the function of Nsp2 in SARS-CoV is unknown. The experimental results showthat the deletion of the nsp2 gene from MHV and SARS-CoV was tolerated withmodest growth and some RNA defect. Also, there was no observable effect on thepolyprotein processing in the mutants. Another evidence shows that neither theNsp2 protein nor the nsp2 gene is involved in pathogenesis (Graham et al., 2005).

iii. Nsp3: the multi-domain SARS-CoV Nsp3 is a 215 kDa glycosylated transmembranemultidomain protein involved in viral replication and transcription. It may serveas a scaffolding protein for numerous other proteins (Angelini et al., 2013).The organization of various Nsp3 domains differs amongst the Coronavirusgenomes. However, eight domains are common to all CoVs: ubiquitin-like domain 1(Ubl1), the glutamate-rich acidic domain also known as “hypervariable region”, an Xmacrodomain, ubiquitin-like domain 2 (Ubl2), PL2pro (papain-like protease 2),ectodomain 3Ecto, also known as “zinc-finger domain”, and domains Y1 and CoV-Y(unknown functions). Nsp3 releases itself, Nsp1 and Nsp2 from the polyproteins.It alters the post-translational modification of host proteins to antagonize theinnate immune response by de-MARylating, de-PARylating, de-ubiquitinating,or de-ISGylating the host proteins. Meanwhile, Nsp3 modifies itself by theN-glycosylation of the ectodomain and can also interact with host proteins (such asRCHY1) to enhance viral survival (Lei, Kusov & Hilgenfeld, 2018).

iv. Nsp4: it is known that Coronaviruses induce double-membrane vesicles (DMVs).Any alteration in the DMVs’ morphology impairs the RNA synthesis andtherefore growth of Nsp4 mutants (Gadlage et al., 2010).

v. Nsp5: the Nsp5 protease also known as 3CLpro or Mpro, cleaves the Nsp peptides at11 cleavage sites (Stobart et al., 2013). Nsp5 of Porcine Deltacoronavirus (PDCoV)is a type I IFN antagonist. It disrupts the IFN signaling pathway by cleaving theNF-κB essential modulator (NEMO), thus impairing the host’s ability to activate theIFN response (Zhu et al., 2017).

vi. Nsp6: Nsp6, along with Nsp3 and Nsp4, has membrane proliferation ability. Thus, itcan induce perinuclear vesicles localized around the microtubule-organizingcenter and DMV formation (Angelini et al., 2013). The coronavirus Nsp6 protein



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restricts the autophagosome expansion either directly or indirectly throughstarvation or chemical inhibition of MTOR signaling (Cottam, Whelband &Wileman, 2014).

vii. Nsp7: the Nsp12 RNA-dependent RNA polymerase cannot act on its own anddepends upon stimulation by a complex of Nsp7 and Nsp8. Thus, Nsp7 acts asone of the cofactors along with Nsp8 to stimulate Nsp12. It is hypothesized that Nsp7and Nsp8 heterodimers stabilize the Nsp12 regions involved in RNA binding; also,Nsp8 acts as a minor RdRp (Kirchdoerfer & Ward, 2019).

viii. Nsp8: Nsp8 is a 22 kDa non-canonical polymerase shown to be capable of de novoRNA synthesis with low fidelity on single-stranded RNA templates. The ‘main’ RdRpin Coronaviruses is the Nsp12 which employs a primer-dependent initiationmechanism. These observations led to a hypothesis that Nsp8 would act as an RNAprimase and synthesize a short primer that will be extended by Nsp12 (Te Velthuis,Van den Worm & Snijder, 2012).

ix. Nsp9: Nsp9 is an RNA-binding subunit in the replication complex in allcoronaviruses (Zeng et al., 2018).

x. Nsp10: SARS-CoV Nsp10 binds to and stimulates the exoribonucleases, Nsp14, andNsp16, thus playing an important role in the replication-transcription complex(RTC) formation (Bouvet et al., 2014). Nsp10 acts as an essential co-factor intriggering Nsp16 2′O-MTase activity, which suggests its involvement in theregulation of capping of viral RNA (Lugari et al., 2010).

xi. Nsp11: the exact function of Nsp11 in Coronaviridae is unknown. Arterivirus Nsp11was identified as NendoU (Nidoviral uridylate-specific endoribonucleases) that playsa role in the viral life cycle. Nsp11 could be essential to produce helicase (Woo et al.,2005).

xii. Nsp12: Nsp12 is a 102 kDa RNA-dependent RNA polymerase (RdRp) featuringall conserved motifs of the known RdRps. It is the most conserved protein incoronaviruses and assumes a center stage in the viral RTC. The Nsp7/Nsp8 complexenhances the binding of Nsp12 to RNA. Nsp8 is the second, non-canonical RdRp incoronaviruses (Subissi et al., 2014).

xiii. Nsp13: Nsp13 is a 66.5 kDa multi-functional protein. The N terminal has azinc-binding domain while the C-terminal harbors a helicase domain-containingconserved motif of superfamily-1 helicases (Subissi et al., 2014).

xiv. Nsp14: Nsp14 is a 60 kDa bifunctional enzyme. Its N-terminal is a 3′–5′exoribonuclease involved in the mismatch repair system, improving the fidelityof virus replication via RNA proofreading. Yeast trans-complementationstudies have shown that the C-terminal domain of SARS-CoV Nsp14 is anS-adenosylmethionine-dependent guanine-N7-methyltransferase (MTase) (Subissiet al., 2014; Zeng et al., 2016) with no RNA sequence specificity (Subissi et al., 2014).

xv. Nsp15: SARS-CoV Nsp15 is a uridine specific ribonuclease that cleaves the 3′ ofuridylates, producing 2′–3′ cyclic phosphate ends (Subissi et al., 2014).



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xvi. Nsp16: Nsp16 is a 2′-O-Methyl Transferase (Zeng et al., 2016) and requires Nsp10 asa stimulatory factor to exhibit its MTase activity (Chen et al., 2011). EukaryoticmRNA has a unique 5′ cap; to evade the host machinery, the viral RNA must bemade indistinguishable from the host mRNA by capping the viral RNA (Menachery,Debbink & Baric, 2014). Both Nsp14 and Nsp16 are involved in the modificationof viral RNA cap structure (Zeng et al., 2016) to maintain the viral RNA stability,ensure protein translation and immune escape (Lugari et al., 2010; Subissi et al.,2014). The SARS-CoV genome encodes two SAM-dependent methyltransferases(Zeng et al., 2016); Nsp14 N7 methyltransferase, and Nsp16 2′-O-methyl transferase,that methylates the RNA at N7 of guanosine and ribose 2′O sites, respectively(Chen et al., 2011). This process also involves Nsp10 for stabilizing the SAM bindingregion and RNA binding of Nsp16 (Subissi et al., 2014).

ORF3a proteinThe orf3a gene lies between S and E genes and encodes for a transmembrane protein(McBride & Fielding, 2012). SARS-CoV-2 ORF3a protein shows 97.82% homology to NS3 ofBat coronavirus RaTG13 and 72% homology to SARS-CoV ORF3a protein (Issa et al., 2020).It is localized to the cell membrane and partly to the ER and the Golgi perinuclearspace in the host cell. ORF3a is known to activate PERK (PKR-like ER kinase) signalingpathway through which the viral particles escape ER-associated degradation and theresulting ER stress also induces apoptosis. ORF3a of both SARS-CoV and SARS-CoV-2 havean APA3_viroporin (a pro-apoptosis protein) conserved domain (McBride & Fielding, 2012;Issa et al., 2020). ORF3a has been shown to activate NF-κB and JNK. This leads to theupregulation of the chemokine named RANTES (Regulated upon Activation, Normal T CellExpressed and Secreted) along with IL-8 in A549 and HEK293T cells (Liu et al., 2014).The extracellular N-terminus of protein can evoke a humoral immune response. Bindingof ORF3a protein to caveolin-1 may be required for viral uptake and release (Liu et al., 2014).ORF3a can augment the activation of the p38 MAPK pathway and induce the mitochondriato leak inducing apoptosis (Liu et al., 2014).

ORF6 proteinSARS-CoV ORF6/Protein 6 is a 63-aa polypeptide. It has an amphipathic 1–40 aaN-terminal portion and a highly polar C-terminal. The amino acid residues 2–32 in theN-terminal form an a-helix which is embedded in the cell membrane. There are twosignal sequences in the C-terminal; aa 49–52 sequence YSEL targets proteins forincorporating into endosomes and the acidic tail which signals ER export. This proteinis localized in the ER and Golgi apparatus. ORF6 is incorporated into VLPs, whenco-expressed with SARS-CoV S, M and E structural proteins. It enhances viral replication,thus serving an important role in pathogenesis during SARS-CoV infection (Liu et al.,2014). ORF6 is also well known as a β-interferon antagonist; its overexpression inhibitsnuclear import of STAT1 in IFN-β treated cells. Along with ORF3b and N protein, theSARS-CoV ORF6 inhibits activation of IRF-3 via phosphorylation and binding of IRF-3 to



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a promoter with IRF-3 binding sites. IRF-3 is an important protein for the expression ofIFN. ORF6 may disturb the ER/Golgi transport necessary for the interferon response(Kopecky-Bromberg et al., 2007). During SARS-CoV infection, the C-terminal domain ofORF6 modulates the host protein nuclear transport and type-I interferon signaling, thusplaying an important role in immune evasion (Liu et al., 2014).

ORF7a proteinSARS-CoV ORF7a/ Protein 7a is a 122 aa type-I transmembrane protein (Liu et al., 2014).It consists of 15 aa N terminal signal peptide sequence, an 81 aa ectodomain, 21 aa Cterminal transmembrane domain, and a cytoplasmic tail of 5 aa residues. The ectodomainof ORF7a binds to human LFA-1 (lymphocyte function-related antigen 1) on Jurkat cellsvia the alpha integrin-I domain of LFA-1. This suggests that probably LFA-1 is abinding factor or receptor for SARS-CoV on human leukocytes (McBride & Fielding,2012). SARS-CoV ORF7a is localized in the ER-Golgi Intermediate Compartment(ERGIC), the assembly point of coronaviruses. It interacts with M and E structuralproteins, indicating a possible role in viral assembly during SARS-CoV replication.In mammalian cells infected with SARS-CoV, ORF7a has been known to inducecaspase-dependent apoptosis by cleaving PARP (poly(ADP-ribose) polymerase), anapoptotic marker. Its pro-apoptotic nature is a result of the interaction between itstransmembrane domain with Bcl-XL, an anti-apoptotic protein of the Bcl-2 family(Liu et al., 2014). Like ORF3a, overexpression of ORF7a activates NF-κB and c-Jun N-terminal kinase (JNK), augmenting the production of pro-inflammatory cytokines such asinterleukin 8 (IL-8) and RANTES (Liu et al., 2014). ORF7a overexpression inducedapoptosis can occur via a caspase-3 dependent pathway as well as p38 MAPK pathway(McBride & Fielding, 2012).

ORF7b proteinSARS-CoV ORF7b/Protein 7b is a 44 aa long, highly hydrophobic, integraltransmembrane protein with a luminal N-terminal and cytoplasmic C-terminal.The expression of ORF7a is reduced significantly when the orf7a start codon (upstreamof 7b) is mutated to a strong Kozak sequence or when an additional start codon AUG isinserted upstream of the orf7b start codon. Thus, ORF7b may be expressed by “leakyscanning” of the ribosome (Liu et al., 2014). The Golgi-restricted localization of ORF7bwas attributed to the transmembrane domain (Liu et al., 2014). However, the role ofORF7a and ORF7b during the replication of SARS-CoV remains uncertain (McBride &Fielding, 2012).

ORF8 proteinHuman SARS-CoV-2 isolated from early patients, Civet SARS-CoV and other batSARSr-CoV contains the full-length ORF8 (Fuk-Woo Chan et al., 2020). Two genomes ofgenus Betacoronavirus isolated from horseshoe bat, SARS-Rf-BatCoV YNLF_31C andYNLF_34C are 93% identical to the human and civet SARS-CoV genome. However, allHuman SARS-CoV isolates from mid and late-phase patients contain a signature 29



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nucleotide deletion in the orf8 gene, splitting it into orf8a and orf8b. This suggests thatORF8 may play a role in interspecies transmission. The generation of civet SARSr-CoVcould be a result of a potential recombination event between SARSr-Rf-BatCoVs andSARSr-Rs-BatCoVs identified around ORF8 (Lau et al., 2015). Interestingly, the newSARS-CoV-2 ORF8 shares very less homology with the conserved ORF8 or ORF8bisolated from Human SARS-CoV or its related viruses, Civet SARS-CoV, Bat-CoVYNLF_31C andYNLF_34C. The ORF8 lacks any known functional domain or motif.SARS-CoV ORF8b has an aggregation motif VLVVL (75–79aa) which is known to activateNLRP3 inflammasomes and trigger intracellular stress pathways (Fuk-Woo Chan et al.,2020), but this is not conserved in SARS-CoV-2. Notably, both ORF3 and ORF8 inSARS-CoV-2 are highly divergent from the interferon antagonist ORF3a andinflammasome activator ORF8b in SARS-CoV (Yuen et al., 2020).

ORF10 proteinThe SARS-CoV-2 ORF10 protein has no homologs in SARS-CoV and any other membersof genus Betacoronavirus (Xu et al., 2020). Viruses are known to sabotage ubiquitinationpathways for replication and pathogenesis. The ORF10 interacts with specifically theCUL2ZYG11B complex and the rest of the members of the Cullin-2 (CUL2) RING E3 ligasecomplex. ZYG11B degrades substrates with exposed N-terminal glycine residues.By studying the ORF10 interactome, it was observed that it interacts the most with theCULZYG11B complex (Gordon et al., 2020). This evidence shows that ORF10 might bind tothis complex and hijack its ubiquitination of restriction factors (Gordon et al., 2020).

METHODOLOGY (GENOMES, DATABASES, AND TOOLSUSED IN THIS STUDY)Complete genomes, belonging to SARS-CoV-2 (167 genomes), SARS-CoV (312 genomes)and Pangolin CoV (five genomes) were downloaded from NCBI Virus webpage(https://www.ncbi.nlm.nih.gov/labs/virus/) as on 29 March 2020. Similarly, RefSeqassemblies of 56 suborder Cornidovirineae genomes (including 18 from Betacoronavirusgenus) along with the genome of Paguronivirus-1 (as an outgroup) were downloadedas on 1 April 2020. The latest version of the Virus database was downloaded from theNCBI Virus page as on 6 April 2020.

For alignments of the studied genomes, as required for phylogeny and mutationalstudies, we have used MUSCLE v3.8.1551 and MEGAX (Edgar, 2004; Kumar et al., 2018)tools as per their default settings. To generate maximum likelihood phylogenies ofgenome-based alignments, RAxML v8.2.12 (Stamatakis, 2014) was used to performrapid bootstrap analysis and search for bestscoring ML tree in one program run (“-f a”parameter) using GTRGAMMA as nucleotide substitution model (-m) with 100 bootstrapvalues. After obtaining the newik trees from RAxML, an online iTOL platform (Letunic &Bork, 2019) was used to visualize phylogenetic trees as shown in this study. For eachexperiment in respective result sections, we have provided comprehensive details about thegenomes under study, research methodology, and used parameters.


https://www.ncbi.nlm.nih.gov/labs/virus/


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For the pangenome study, Proteinortho v6.0.12 (Lechner et al., 2011) was used with anE value cutoff 1E−05 for each identified hit along with other default settings. It utilizesdiamond v0.8.36.98 and BLAST 2.8.1+ (Christiam et al., 2009; Buchfink, Xie & Huson,2014) to identify ortholog proteins using reciprocal blast strategy. For the identificationof pairwise average nucleotide identity (ANI) values within all genomes under study,PYANI v0.1.2 (Pritchard et al., 2016) program was used. Throughout this study, theWuhan Hu-1 strain has been used as a reference for SARS-CoV-2 genomes.

RESULTS AND DISCUSSIONHuman SARS-CoV-2 genome is quite disparate as compared to otherBetacoronavirus genomesThe SARS outbreak in 2003 and MERS in 2012 had already set a precedent for widespreadgenome analysis, therefore, with the recent emergence of COVID-19 in November2019, extensive sequencing and genome analysis of SARS-CoV-2 isolates are beingperformed. Since February 2020, several Bat and Pangolin coronaviruses have also beensequenced to understand the probable origin of SARS-CoV-2. In this study, we haveanalyzed 312 SARS-CoV, 167 SARS-CoV-2 and 5 Pangolin CoV genomes to understandtheir genomic conservation, unique genes, mutational hotspots and respective evolutionand origin.

Phylogeny relationship at suborder Cornidovirineae levelSARS-CoV-2 is a member of suborder Cornidovirineae, family Coronaviridae, genusBetacoronavirus, and subgenus Sarbecovirus. In this study, we have tried to understand thestrain diversity and evolutionary relationships at different taxonomy levels in a top-bottomclassification scheme. For this analysis, the complete whole-genome sequences of 56suborder Cornidovirineae reference genomes were analyzed, which included membersfrom five genera i.e. 23 Alphacoronavirus, 20 Betacoronavirus, 10 Deltacoronavirus andthree Gammacoronavirus and the genome of Paguronivirus-1 was included as an outgroupfor this study. Their genome length varied from 25,425-31,686 nucleotides. ClustalWalignment (using MEGA-X) generated an alignment of 35,601 nucleotides off which17,284 conserved sites were stripped and used to generate an ML-based phylogeny usingRAxML. We noted that this phylogeny is unambiguously able to demarcate all the generainto their respective monophyletic clades (Fig. S1). The significant variations amongstbranch length distances indicate the relative diversity within each genus.

Phylogeny relationship at genus Betacoronavirus levelWe generated the phylogeny of 18 Betacoronavirus reference strains (along with fivePangolin CoV strains) using their complete genomes ranging within 29,114–31,526nucleotides. ClustalW based whole genome alignment using MEGAX generated analignment of length 33,346 from which 24,555 conserved nucleotide sites were strippedand used to generate ML phylogeny (Fig. S2). Three distinct clades were seen in thephylogeny: the first clade includes strains from subgenus Sarbecovirus, Nobecovirus andHibecovirus, second with Embecovirus members, and the third clade of members of


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subgenus Merbecovirus. Within the first clade, both members of subgenus Sarbecovirus(SARS-CoV and SARS-CoV-2) are grouped with Pangolin CoV strains, which is supportedby their percentage identity. All Pangolin CoV genomes are >99.8% identical to each otherwhereas their closest relatives are the SARS-CoV-2 reference strain (~85% identity),followed by the SARS-CoV reference strain (~79% identity). These three groups are closest(~57% identity) to their sister branch occupied by subgenus Hibecovirus strain: BatHp-betacoronavirus Zhejiang 2013. Two reference strains of another subgenusNobecovirus (~67% identity with each other) form a separate clade that is closest (~52%identity with other group members) to the above-mentioned clade comprising subgenusSarbecovirus and Hibecovirus. The second clade only has representatives of subgenusEmbecovirus including Murine hepatitis virus, Bovine CoV, Rabbit CoV, Rat CoV, etc.Some of these strains form respective subclades in the phylogeny based on their closeness.Strains belonging to subgenus Merbecovirus that include the MERS, form the thirdclade. MERS genomes (England 1 and MERS Co.) are 99.7% identical to each other andtherefore form a separate sub-clade. As observed from their phylogenetic branch lengthsand percentage identity matrix, all subgenera are quite distinct from each other.As expected, members of each subgenus are closer to each other, therefore formingrespective monophyletic clades.

Phylogeny relationship at subgenus Sarbecovirus levelTo understand the similarities and differences among the strains of subgenus Sarbecovirus,we analyzed the complete genomes of 103 SARS-CoV-2 and 312 SARS-CoV isolatesincluding the reference strains of Wuhan Hu-1 (SARS-CoV-2) and Tor2 (SARS-CoV).Since Pangolin CoV belongs to subgenus Sarbecovirus, we also included 5 Pangolin CoVgenome sequences in our dataset and aligned them using MUSCLE. The genome size ofSARS-CoV, Pangolin CoV and SARS-CoV-2 strains were in the range of 29,013–30,311nucleotides, 29,795–29,806 nucleotides and 29,325–29,945 nucleotides, respectively.The alignment of length 31,718 nucleotides was stripped to a conserved region of 26,762nucleotides (sites with gaps in between them were excluded) which was used to generate amaximum likelihood phylogeny using RAxML (Fig. 1; Fig. S3). We noted from thephylogeny that SARS-CoV-2 strains form a single monophyletic clade as compared to theSARS-CoV. SARS-CoV strain RaTG13 procured from the bat in 2013 from Yunnan,China is closest to SARS-CoV-2 strains, suggesting that they might have diverged from acommon ancestor. Average Nucleotide Identity (ANI) studies also indicated that theSARS-CoV-2 reference genome and the Bat RaTG13 branch share 96.11% identity at thegenome level, a finding supported by earlier reports (Lv et al., 2020; Zhou et al., 2020).The next closest strains in the phylogeny were those of Pangolin CoV, all forming asubclade. Other close relatives are the SARS-CoV strains Bat CoVZC45 and BatCoVZXC21, procured from Zhoushan, eastern China in 2018. The phylogeny suggeststhat Pangolin CoVs are closer to SARS-CoV-2 strains as compared to CoVZC45 andCoVZXC21, however, genome-wide percentage identity suggests the opposite—thegenomes of both CoVZC45 and CoVZXC21 are ~89% identical to SARS-CoV-2, asopposed to its 86% identity with Pangolin CoVs.




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Taxonomy

SARS-CoV-2

SARS-CoV

Pangolin CoV

Host

Human

Bat

Pangolin

Monkey

Civets

Mouse

Pig

Unknown

Figure 1 Maximum Likelihood (ML) phylogeny representing relationship amongst 312 SARS-CoV, 103 SARS-CoV-2, and five Pangolin CoVstrains. The whole-genome sequences of 420 isolates were aligned using MUSCLE and stripped to include the highly conserved alignments across allstrains. The final alignment was subjected to RAxML to generate the ML phylogeny utilizing the GTRGAMMA model of nucleotide substitutionwith 100 bootstrap replicates. The phylogeny is depicted with branch length consideration. The inner-circle represents the taxonomy of all strains(depicting SARS-CoV, SARS-CoV-2 and Pangolin CoV). The outermost circle represents the respective host of each strain. Inner Blue and Grayalternative dashed lines represent an internal tree scale with a branch length increment of 0.04 from inside to outside.

Full-size DOI: 10.7717/peerj.9576/fig-1


http://dx.doi.org/10.7717/peerj.9576/fig-1


https://peerj.com/

We also examined the ANI scores of the above-identified closest strains of SARS-CoV-2with all SARS-CoV, SARS-CoV-2 and Pangolin CoV genomic sequences used in this study(Table S2). We found that the Bat RaTG13 strain is 95.97–96.12% identical whencompared to all SARS-CoV-2 genomes under study. Amongst the SARS-CoV strains, BatCoVZC45 and Bat CoVZXC21 are its closest neighbors with 89% genome identity,indicating a common ancestor in bat coronaviruses, whereas, with Pangolin CoV, theyshare lowest (~86%) nucleotide identity. Bat CoVZC45 and Bat CoVZXC21 are ~97%identical to each other, whereas their identity with SARS-CoV-2 (~89%) is higher thanthat with SARS-CoV (86–89%) and lowest with Pangolin CoV (~85%). Similarly,Pangolin CoVs share maximum identity with Bat RaTG13 SARS-CoV (86.50%) followedby SARS-CoV-2 (86.30–86.38%) and lowest with other SARS-CoV. Therefore, we canargue that as SARS-CoV-2, Bat RaTG13 and Pangolin CoV have considerable genomesimilarity, they possibly diverged from a common ancestor and SARS-CoV-2 was latertransmitted to humans through recombination and transformation events via an unknownintermediate host.

Phylogeny relationship at SARS-CoV-2 levelSARS-CoV-2 strains, like other viruses, have a high mutation rate for better adaptabilityand survival. Therefore, one of our aims was to understand the diversity amongSARS-CoV-2 isolates. Whole-genome sequences of 167 SARS-CoV-2 were aligned usingMUSCLE (alignment length 29,950 nucleotides) and the conserved sites of length 29,725were used to generate a maximum-likelihood phylogeny using RAxML (Fig. 2; Fig. S4).Irrespective of high similarity amongst all (>99.90% identity), several subclades can beidentified from the phylogeny based on their distance from each other, depicting theirsubtypes. Our data has 97 isolates from the USA, 46 from China, five from Japan, fourfrom Spain, three from Taiwan, two from Vietnam and India, one isolate each fromSweden, South Korea, Pakistan, Nepal, Italy, Finland, Brazil and Australia. From thegenome sequence data of 15 distinct geographical locations, we were able to identify a fewphylogenetic clusters depicting country-specific patterns. We identified multiple clustersper country suggesting the existence of multiple subtypes. The KMS1 isolate fromChina was found to be the most distinct one, followed by WA-UW230, WA-UW194,WA-UW218 isolates from the USA. The Wuhan Hu-1 isolate shares a sister cladewith the USA Cruise samples, indicative of high similarity. Isolates sampled at theUniversity of Washington (WA, USA) form two separate subclades within two differentclades in the phylogeny, suggesting that even amongst people residing in a limitedarea in the state of Washington, multiple strains exist and are causing COVID-19.Furthermore, both the WA subclades have Valencia-Spain isolates as the closestbranch/subclade, suggesting that at least two individuals from these two locationsindependently encountered each other and transmitted different subtypes of the virus.This study also included two distinct genomes sampled from India which weredistributed by the phylogeny into two separate clusters signifying the existence ofdifferent subtypes.





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As strains from diverse locations continue to be sequenced and analyzed, more reliablecountry-specific patterns showing relatedness can be obtained. Similar to several cuttingedge studies (Woo et al., 2010; Fuk-Woo Chan et al., 2020; Zhang, Wu & Zhang, 2020;Rehman et al., 2020), this study also provides an example of how phylogenetic analysiscan help generate clusters of identical strains, further enabling the identification ofsignature mutations amongst those diverse groups.

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Geo Location:

China

USA

India

Spain

Vietnam

Pakistan

Sweden

Japan

Italy

Brazil

Figure 2 ML phylogeny representing relationship amongst 167 SARS-CoV-2 strains. The whole-genome sequences of 167 isolates were alignedusing MUSCLE, stripped to include only conserved alignments, and subjected to RAxML to generate the ML phylogeny utilizing the GTRGAMMAmodel of nucleotide substitution with 100 bootstrap replicates. The phylogeny is depicted with branch length consideration. The inner circlerepresents the respective geo-location of each strain. Inner Blue and Gray alternative dashed lines represent an internal tree scale with a branchlength increment of 0.00005 from inside to outside. Full-size DOI: 10.7717/peerj.9576/fig-2




https://peerj.com/

Mutations are continuously diversifying the Human SARS-CoV-2strainsLike the previous section, the whole genome sequence alignment of 167 SARS-CoV-2isolates were used for this study. As anticipated, ~100 nucleotides at both N andC-terminals were highly diverse and, therefore, changes in these sites were not consideredin this study. For the rest of the alignment, the percentage of nucleotide occurrence at eachsite (along with gap and other non-standard nucleotides) was calculated using theSARS-CoV-2 Wuhan Hu-1 genome as the reference (Fig. 3). The mutational probabilitywas classified into four categories based on random distribution patterns: high (>19%variation), medium (4–19% variation), low (2–3% variation) and very low (<2% variation).Overall, 415 mutation sites were identified, among which non-standard nucleotideswere present in the genome at 125 sites, which have been ignored in this study.We ultimately selected 290 sites with a confirmed variation. Amongst these, we found43 sites with high, medium and low mutation probabilities within 9 out of 12protein-coding regions in SARS-CoV-2 genomes, which also included the genes (S, M andN), identified as core proteins across genus Betacoronavirus. Amongst all mutation sites,we found 21 sites within the gene encoding Spike protein, 18 sites in the gene encodingNucleocapsid protein and three sites in gene encoding M protein, suggesting that eventhese highly conserved protein-coding regions (core proteins as later identified viapan-genome analysis) are capable of mutating. Also, we recognized 142 mutational sites inorf1a, 50 in orf1b, 6 in orf3a, 2 in the envelope protein-coding gene, 4 in orf6, and orf8, 3 inorf7b and 1 in orf10. We also identified 36 sites within non-coding regions.

Amongst sites with high mutation probability, C8,782T in orf1a showed ~35% variationfrom C to T, suggesting a transition mutation. Likewise, T28,144C in orf8 also has~35% transition mutation variations. We found three hotspot mutational sites withinthe orf1b region of orf1ab, which has three transitions, two C to T (C17,747T andC18,060T; ~20% variation) and one A to G (A17,858G; 19% variation). In the spikeprotein-coding region, we were able to identify one transition site (A23,403G nucleotide)with medium (11%) mutational probability. Overall, we were able to identify nine hotspotswith more than two consecutive probable mutational nucleotides in vicinity (197–210(non-coding), 508–522 (orf1a), 686–694 (orf1a), 4,879–4,881 (orf1a), 20,298–20,300(orf1b), 21,385–21,389 (orf1b), 21,991–21,993 (S), 28,878–28,883 (N), 29,750–29,759(non-coding)). Most of these hotspot sites showed ~1–2% mutational probabilities andnone of these have high or medium mutational probability as discussed in Fig. 3.We believe these genomic locations are the probable sites of evolutionary divergence, andtherefore govern the evolutionary capabilities of these viruses.

We also analyzed transition and transversion possibilities within all these mutationalcombinations. Out of the 290 identified mutational sites, 244 had a nucleotide substitution,distributed into 158 transitions and 86 transversions. Transition events are known to bemore frequent and less likely to cause a change at the protein level as compared totransversions (Sanjuán et al., 2010). Most transition events are silent mutations, whiletransversion events may cause change at the protein level and therefore impact the



https://peerj.com/

pathogenesis of the disease (Lyons & Lauring, 2017). Therefore, we suggest that amongstSARS-CoV-2 genomes, transversion events, that are occurring in significant numbers,would provide more evolutionary diversity and possibly lead to divergence, as compared totransition events.

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Figure 3 Mutation sites and hotspots distribution amongst 167 SARS-CoV-2 genomes as compared to Wuhan Hu-1. MUSCLE-basedalignment was used to analyze the distribution of all nucleotides (including gap and other nucleotides) at each alignment site and the relativepercentage of each nucleotide is represented here in a circular manner respectively for each nucleotide. The innermost circle represents the respectiveorf and non-coding DNA according to the location as mentioned in the third circle from inside. The second circle represents the mutationalprobability score of each locus distributed in high, medium, low and very low categories. Full-size DOI: 10.7717/peerj.9576/fig-3




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Evolutionary relationship amongst SARS-CoV-2, Bat CoV andPangolin CoVTo re-examine the closest hosts of these viruses, we performed homology studies on smallgenome fragments (100 nucleotides) to identify their close relatives. We segmented each ofthe SARS-CoV-2, Pangolin CoV, SARS-CoV and MERS genome (reference genome ofeach category) into 100 nucleotide fragments, identified their closest homologs in thevirus database, filtered the strains belonging to its taxonomy, selected top 50 hits persegment, calculated the host CoV distribution and represented it as a heatmap for eachsegment/strain along with the closest (topmost) hit per segment/strain (Fig. 4).MERS-CoV is already known to originate from the bat, however, the infection spread viacamels either directly/indirectly (Azhar et al., 2014). On excluding Camel CoVs fromthe data, we found several segments showing maximum similarity representation in BatCoVs, followed by Hedgehog and Human CoV strains, however, most of the parts wereuniquely present in MERS. As expected, when Camel CoV strains were included, they werethe closest best hit as indicated in the “MERS Closest” category (Fig. 4).

When we further investigated the same patterns in the SARS-CoV genome, weidentified the Bat CoV as the closest strain for most of the reference segments, followedby SARS-CoV-2 and Pangolin CoV. In the closest category, we found Bat CoV as theclosest suggesting their evolutionary origin. However, many segments do not showany closeness with any strain, indicating their uniqueness in the reference genome.We performed the same analysis on the Pangolin CoV strain GX-P5E and found thatseveral segments have close homology-distribution within SARS-CoV and SARS-CoV-2genomes. Interestingly, as visible in the “Pangolin CoV Closest” category, several hits showmaximum similarity with Bat SARS-CoV, followed by SARS-CoV-2 and a few HumanCoVs.

Likewise, SARS-CoV-2 genome fragments show maximum homology representation inBat CoV followed by Pangolin CoV. In the “closest category”, Bat CoV indisputably standsout as the closest host, which is corroborated by whole-genome phylogeny and thepercentage identity matrix studies. However, the presence of several Pangolin CoVhomologous segments in this analysis indicates that certain specific segments in theSARS-CoV-2 genome share a closer relationship with Pangolin CoV strains as comparedto Bat CoVs.

Overall, our analysis indicates that the SARS-CoV-2 genome has a close relationshipwith Bat CoV and Pangolin CoV, along with the presence of a few unique segments.This reasserts that SARS-CoV-2 strains might have evolved from a common ancestorof Bat SARS-CoV and Pangolin CoV strains and during evolution, several segments in theSARS-CoV-2 genome might have diverged as compared to Bat SARS-CoV and PangolinCoV. The recognized unique genomic segments in this study may be used as potentialtargets for diagnostic kits.

Pan-genome (Core, accessory and unique gene) analysisRegardless of their approximately similar genome size, the members of Betacoronavirusharbor huge diversity (5–14 CDS) in the number of protein-coding regions, as per the



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available NCBI genome annotations. SARS-CoV and SARS-CoV-2, belonging to subgenusSarbecovirus, have 14 and 12 protein-coding regions respectively, revealing the diversitywithin the same subgenus. Similarly, Pangolin CoV (studied strain: GX-P5E), which alsobelongs to subgenus Sarbecovirus, has only nine protein-coding regions. To identify the

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Figure 4 Host distribution of each 100-nucleotide segment in the reference genomes of MERS, SARS-CoV, Pangolin CoV, and SARS-CoV-2.Each 100-nucleotide segment per strain was subjected to BLASTn against the virus database. Self-category hits were removed from the analysis, hostinformation of top 50 hits was identified, and the relative percentage is represented here as a heatmap for each segment in each genome. In the closestcategory, the host information of best hit (after removing self-hit) is represented in the form of a multi-color stripe. The innermost circle representsthe location of each segment to start from 1 to 100 nucleotides to the total genome length of the genome.

Full-size DOI: 10.7717/peerj.9576/fig-4




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presence of (co-)orthologous proteins present across all these genomes, a bidirectional(reciprocal) best BLAST hit-based homology detection strategy was followed using the toolProteinortho (Lechner et al., 2011) (Fig. 5; Fig. S5). A total of 44 protein-coding regionclusters represent the pan-genome of nineteen studied genus Betacoronavirus membersamongst which, 3 protein-coding regions (S, M and N proteins) were found to beconserved across all genomes, representing the core genome. A similar analysis hasbeen performed on all Betacoronavirus species; however, it does not include PangolinCoV (Alam et al., 2020). Our study also recognized that the pan-genome of genusBetacoronavirus is open, which means that with the addition of a new species, thepan-genome is continuously increasing (Fig. S6). The increase in the pan-genome at eachstep is approximately proportional to the increase in unique genes. Since each newlyidentified strain has additional unique gene sequences, it is effectively impossible to predicttheir complete pan-genome. As expected, the members of each subgenus, which areclosest to each other, have only a few variations, however, with the addition of a newsubgenus member(s), the pan-genome expands immediately.

orf1ab, the largest coding region, codes for two polyproteins ORF1a and ORF1ab(266–13,468,13,468–21,555) because of the “leaky mechanism” of the ribosome, a (-1)frameshift just upstream of the orf1a translation termination codon. All CoVs, except forstrain MHV A59 of subgenus Embecovirus, translate the full-length orf1ab. MHV A59,

Figure 5 Pan-genome (Core, accessory, and unique) analysis amongst 19 Betacoronavirus referencegenomes (including one Pangolin CoV). The X-axis represents the strain combinations as black-filledcircles, whereas Y-axis represents the distribution of genes in the respective combinations and inside eachvertical bar, those gene names are written. For each strain, their respective subgenus category has beenpointed out on the left side as the colored scale in the corner. In the left-down corner, the number ofgenes encoded by each genome is depicted as a horizontal bar. In the lowermost panel, pan-genomecategories (core, accessory and unique genes) are shown. Full-size DOI: 10.7717/peerj.9576/fig-5






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however, has separate polyproteins from orf1a and orf1b. Due to the absence of ribosomalslippage reported in several coronaviruses, ORF1a is encoded by only 9 out of 18genomes, making it an accessory protein cluster of the pan-genome. ORF1b polyprotein isencoded as a part of orf1ab in most of the species belonging to genus Betacoronavirus,again except for strain MHV A59, where it is encoded separately as RdRp, forming aunique gene. Envelope protein (76–82 aa across Betacoronavirus) is another accessoryprotein encoded by 15 out of 18 genomes. In another cluster, we identified the presence ofsmall envelope protein in two out of those three genomes where E protein is absent(both in subgenus Nobecovirus). No E protein-encoding region is found in the MHV A59genome annotations. The gene encoding ORF3 is distributed into two subunits (orf3aand orf3b) in SARS-CoV (via an internal ribosomal entry mechanism), whereas onlythe first subunit (orf3a; 276 aa) is present in SARS-CoV-2. The gene for ORF3a isconserved only amongst subgenus Sarbecovirus organisms. This protein activates thepro-transcription of gene IL-1β and helps its maturation; both signals are prerequisitesfor NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasomeactivation during infection (Siu et al., 2019). Conservation of ORF3a across SARS-CoV-2,SARS-CoV and Pangolin CoVs provides a clue about its unique infection strategy. ORF3b(155 aa) is only present in SARS-CoV and absent in all other coronaviruses. It hasbeen suggested that ORF3b supports SARS-CoV infection by inhibiting the type-1Interferon (IFN) synthesis and therefore, IFN signaling (McBride & Fielding, 2012).ORF6 is also a subgenus Sarbecovirus specific protein (conserved across SARS-CoV,SARS-CoV-2 and Pangolin CoV) localized in the Endoplasmic Reticulum or the Golgimembrane. During infection, it interacts with and interrupts the nuclear import complexformation. During infection-caused interferon signaling, this interruption inhibits STAT1transportation to the nucleus and blocks the expression of genes involved, thereforemimicking an antiviral state (Frieman et al., 2007).

Another accessory protein of the Betacoronavirus pan-genome is ORF7a, which is aunique protein amongst subgenus Sarbecovirus members. In SARS-CoV, it is knownthat bone marrow stromal antigen-2 (BST-2 or tetherin or CD317) can restrict the virusrelease from inside the cell causing partial inhibition of infection. However, ORF7a caninhibit the action of this protein, thus promoting the infection (Taylor et al., 2015). ORF7bis present in both SARS-CoV-2 and SARS-CoV but absent in Pangolin CoV NCBIannotations. Like orf7b in SARS-CoV, its initiation codon overlaps with orf7a viaribosome leaky scanning and it works as a structural component (Schaecher, Mackenzie &Pekosz, 2007). orf8 gene (365 nucleotides) is uniquely present in SARS-CoV-2 andPangolin CoV but absent in SARS-CoV. Distinctively, orf8 in SARS-CoV is separatedinto two proteins ORF8a (119 nucleotides) and ORF8b (254 nucleotides). ORF8 inSARS-CoV-2 is highly divergent from the inflammasome activator ORF8b in SARS-CoV(Yuen et al., 2020). The function of this protein is not clear yet, but it has been suggestedthat overall ORF8ab or ORF8a and ORF8b together modulate viral replication andpathogenesis in unknown fashion (McBride & Fielding, 2012). ORF10 is a unique proteinin SARS-CoV-2 strains. We also performed the homology analysis of ORF10 protein



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against the NCBI Non-redundant database and found no hits, which suggests that thisprotein is specific to SARS-CoV-2.

This analysis also revealed that some proteins are specific to a subgenus. SubgenusEmbecovirus has a unique combination of proteins, namely HE, ORF4, internal protein,NS2a and NS3a, that are present in most genomes of this subgenus. These proteins mightbe considered unique to subgenus Embecovirus as compared to the rest of the genusBetacoronavirus. Hemagglutinin Esterase glycoprotein (HE) is a nonessential proteinwith sialic acid-binding and acetyl esterase activity that create tinier spikes (secondattachment factor) on the viral envelope in several MHV strains. orf4 (also annotated asorf5a, ns12.9) encodes a nonstructural accessory protein that is, known to function as typeI interferon antagonist. The internal gene is encoded within the N gene in MHV CoV;however, its function is not well-known. Similarly, subgenus Merbecovirus members alsohave some unique proteins (NS3a, NS3b, NS4b, ORF5), that are absent in other subgenera.We also identified unique proteins in each organism, which were absent in otherBetacoronavirus. Overall, it can be argued that although genus Betacoronavirus membershave ~10 proteins coding regions per genome, they have conserved similarity in only~10% (3–4 proteins) of the genes and their diversity (~40% accessory genes and ~50%unique genes) spans the rest of the genome, leading to their different pathogenesis acrossdiverse hosts and distinct evolution. We identified that even among subgenus Sarbecovirusmembers (SARS-CoV, SARS-CoV-2 and Pangolin CoV), orf3b, orf7b, orf8, orf9b andorf10 are uniquely distributed within at least one genome, therefore depicting widediversity within the same subgenus.

CONCLUSIONSHumanity has already encountered coronavirus infections twice in the past two decades,in the form of the SARS and MERS epidemics. The latest coronavirus infection,COVID-19, caused by the highly pathogenic and transmissible SARS-CoV-2, turned into apandemic in sheer three months. This work provides a brief review of the taxonomy,history, origin, and genome organization of the virus, followed by comparative genomicsresearch focused on understanding the evolutionary relationship amongst 167SARS-CoV-2, 312 SARS-CoV and 5 Pangolin CoV genome sequences as available on29 March 2020. We identified that SARS-CoV-2 strains form a monophyletic clade distinctfrom SARS-CoV and Pangolin CoV. This study reaffirmed that they are closest to theBat CoV RaTG13 strain followed by Pangolin CoV, suggesting that SARS-CoV-2 evolvedfrom a common ancestor putatively residing in bat or pangolin hosts. We identified severalmutation sites and hotspots within SARS-CoV genomes with high, medium and lowprobabilities. Homology studies based on 100 nucleotide segments in the SARS-CoV-2reference genome pointed out their close relationships with Bat and Pangolin CoVs, alongwith the unique nature of a few segments. We recognized that 44 protein-coding regionsconstitute the pan-genome for nineteen genus Betacoronavirus strains. Moreover, theirpan-genome is open, highlighting the wide diversity provided by newly identified novelstrains. Even members of subgenus Sarbecovirus are diverse relative to each other due to



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the relative presence of unique protein-coding regions orf3b, orf7b, orf8 and orf9b andorf10. Overall, this review and research highlight the diversity within the availableSARS-CoV-2 genomes, their potential mutational sites hotspots, and probableevolutionary relationship with other coronaviruses, which might further assist in ourunderstanding of their evolution, epidemiology and pathogenicity.

ACKNOWLEDGEMENTSWe acknowledge Tanvi Kale, IBAB MSc (2018–20) student for graciously offering herartwork to use as a cover image for this manuscript.

ADDITIONAL INFORMATION AND DECLARATIONS

FundingDepartment of Science and Technology (DST), Government of India and IBAB, Bengaluruprovided financial and infrastructure support to Gaurav Sharma. The funders had no rolein study design, data collection and analysis, decision to publish, or preparation of themanuscript.

Grant DisclosuresThe following grant information was disclosed by the authors:DST-INSPIRE Faculty Award from Department of Science and Technology (DST),Government of India to GS.

Competing InterestsThe authors declare that they have no competing interests.

Author Contributions� Arohi Parlikar performed the experiments, analyzed the data, authored or revieweddrafts of the paper, and approved the final draft.

� Kishan Kalia performed the experiments, analyzed the data, prepared figures and/ortables, authored or reviewed drafts of the paper, and approved the final draft.

� Shruti Sinha performed the experiments, analyzed the data, authored or reviewed draftsof the paper, and approved the final draft.

� Sucheta Patnaik performed the experiments, analyzed the data, authored or revieweddrafts of the paper, and approved the final draft.

� Neeraj Sharma performed the experiments, analyzed the data, prepared figures and/ortables, authored or reviewed drafts of the paper, and approved the final draft.

� Sai Gayatri Vemuri performed the experiments, analyzed the data, authored or revieweddrafts of the paper, and approved the final draft.

� Gaurav Sharma conceived and designed the experiments, performed the experiments,analyzed the data, prepared figures and/or tables, authored or reviewed drafts of thepaper, and approved the final draft.



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Data AvailabilityThe following information was supplied regarding data availability:

The strains and genomes used in this research were retrieved from NCBI Virus(https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/) on 29 March 2020. This information isprovided in Table S1.

Supplemental InformationSupplemental information for this article can be found online at http://dx.doi.org/10.7717/peerj.9576#supplemental-information.

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understanding genomic diversity, pan-genome ...understanding genomic diversity,...

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