Molecular and Structural Evolution of Porcine Epidemic Diarrhea Virus.

To analyze the evolutionary characteristics of the highly contagious porcine epidemic diarrhea virus (PEDV) at the molecular and structural levels, we analyzed the complete genomes of 647 strains retrieved from the GenBank database. The results showed that the spike (S) gene exhibited larger dS (synonymous substitutions per synonymous site) values than other PEDV genes. In the selective pressure analysis, eight amino acid (aa) sites of the S protein showed strong signals of positive selection, and seven of them were located on the surface of the S protein (S1 domain), suggesting a high selection pressure of S protein. Topologically, the S gene is more representative of the evolutionary relationship at the genome-wide level than are other genes. Structurally, the evolutionary pattern is highly S1 domain-related. The haplotype networks of the S gene showed that the strains are obviously clustered geographically in the lineages corresponding to genotypes GI and GII. The alignment analysis on representative strains of the main haplotypes revealed three distinguishable nucleic acid sites among those strains, suggesting a putative evolutionary mechanism in PEDV. These findings provide several new fundamental insights into the evolution of PEDV and guidance for developing effective prevention countermeasures against PEDV.

Adaptive Evolution of the Spike Protein in Coronaviruses.

Coronaviruses are single-stranded, positive-sense RNA viruses that can infect many mammal and avian species. The Spike (S) protein of coronaviruses binds to a receptor on the host cell surface to promote viral entry. The interactions between the S proteins of coronaviruses and receptors of host cells are extraordinarily complex, with coronaviruses from different genera being able to recognize the same receptor and coronaviruses from the same genus able to bind distinct receptors. As the coronavirus disease 2019 pandemic has developed, many changes in the S protein have been under positive selection by altering the receptor-binding affinity, reducing antibody neutralization activities, or affecting T-cell responses. It is intriguing to determine whether the selection pressure on the S gene differs between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other coronaviruses due to the host shift from nonhuman animals to humans. Here, we show that the S gene, particularly the S1 region, has experienced positive selection in both SARS-CoV-2 and other coronaviruses. Although the S1 N-terminal domain exhibits signals of positive selection in the pairwise comparisons in all four coronavirus genera, positive selection is primarily detected in the S1 C-terminal domain (the receptor-binding domain) in the ongoing evolution of SARS-CoV-2, possibly owing to the change in host settings and the widespread natural infection and SARS-CoV-2 vaccination in humans.

Natural selection differences detected in key protein domains between non-pathogenic and pathogenic feline coronavirus phenotypes.

Feline coronaviruses (FCoVs) commonly cause mild enteric infections in felines worldwide (termed feline enteric coronavirus [FECV]), with around 12 per cent developing into deadly feline infectious peritonitis (FIP; feline infectious peritonitis virus [FIPV]). Genomic differences between FECV and FIPV have been reported, yet the putative genotypic basis of the highly pathogenic phenotype remains unclear. Here, we used state-of-the-art molecular evolutionary genetic statistical techniques to identify and compare differences in natural selection pressure between FECV and FIPV sequences, as well as to identify FIPV- and FECV-specific signals of positive selection. We analyzed full-length FCoV protein coding genes thought to contain mutations associated with FIPV (Spike, ORF3abc, and ORF7ab). We identified two sites exhibiting differences in natural selection pressure between FECV and FIPV: one within the S1/S2 furin cleavage site (FCS) and the other within the fusion domain of Spike. We also found fifteen sites subject to positive selection associated with FIPV within Spike, eleven of which have not previously been suggested as possibly relevant to FIP development. These sites fall within Spike protein subdomains that participate in host cell receptor interaction, immune evasion, tropism shifts, host cellular entry, and viral escape. There were fourteen sites (twelve novel sites) within Spike under positive selection associated with the FECV phenotype, almost exclusively within the S1/S2 FCS and adjacent to C domain, along with a signal of relaxed selection in FIPV relative to FECV, suggesting that furin cleavage functionality may not be needed for FIPV. Positive selection inferred in ORF7b was associated with the FECV phenotype and included twenty-four positively selected sites, while ORF7b had signals of relaxed selection in FIPV. We found evidence of positive selection in ORF3c in FCoV-wide analyses, but no specific association with the FIPV or FECV phenotype. We hypothesize that some combination of mutations in FECV may contribute to FIP development, and that it is unlikely to be one singular 'switch' mutational event. This work expands our understanding of the complexities of FIP development and provides insights into how evolutionary forces may alter pathogenesis in coronavirus genomes.

Molecular evolution and structural analyses of the spike glycoprotein from Brazilian SARS-CoV-2 genomes: the impact of selected mutations.

The COVID-19 pandemic caused by SARS-CoV-2 has reached by February 2022 more than 380 million cases and 5.5 million deaths worldwide since its beginning in late 2019, leading to enhanced concern in the scientific community and the general population. One of the most important pieces of this host-pathogen interaction is the spike protein, which binds to the hACE2 cell receptor, mediates the membrane fusion and is the major target of neutralizing antibodies against SARS-CoV-2. The multiple amino acid substitutions observed in this region, specially in RBD have enhanced the hACE2 binding affinity and led to several modifications in the mechanisms of SARS-CoV-2 pathogenesis, improving the viral fitness and/or promoting immune evasion, with potential impact in the vaccine development. In this work, we identified 48 sites under selective pressures, 17 of them with the strongest evidence by the HyPhy tests, including VOC related mutation sites 138, 142, 222, 262, 484, 681, and 845, among others. The coevolutionary analysis identified 28 sites found not to be conditionally independent, such as E484K-N501Y. The molecular dynamics and free energy estimates showed the structural stabilizing effect and the higher impact of E484K for enhanced binding affinity between the spike RBD and hACE2 in P.1 and P.2 lineages (specially with L452V). Structural changes were also identified in the hACE molecule when interacting with B.1.1.7 RDB. Despite some destabilizing substitutions, a stabilizing effect was identified for the majority of the positively selected mutations.Communicated by Ramaswamy H. Sarma.

The Gift of Ancient Times:a Breakthrough in The Tracing ofModern Human Genes br

The2022Nobel Prize in Physiology or Medicine was awarded to Swedish biologist Svante Paabo forhis decisive contribution to paleoanthropogenomics and human origins.There are various theories about theorigin of human beings,and the current mainstream view is:out of the African doctrine.In other words,ancienthumans had about three times of migrations.The first time wasHomo erectus,the second was Neanderthals andDenisovans,and the third was the ancestors of modern humans.All migrated from Africa to Eurasia.Whilepioneering a new discipline,paleoanthropogenomics,Svante Paabo has been refining the"Out of Africa Theory".With the help of various biological techniques,he delved into the origin of human beings from the perspective ofgenomics and found that some genetic imprints from ancient humans were retained in our bodies.For example,the STAT2gene and TLR gene associated with immunity,the EPAS1gene that contributes to hypoxic respirationand the six genes of chromosome3are highly positively correlated with the incidence of COVID-19.Thisresearch means that we can go back to the root of certain diseases,rather than limiting our eyes to the genesthemselves,and exploring where a gene comes from will be a new way of studying diseases.We summarized hisinnovations in related biotechnology in the process of research,his exploration of ancient humans based onmitochondrial and nuclear genes and related results,and introduced some genes derived from ancient humans andtheir related information

Vaccinia Virus-Based Reverse Genetics for Feline Coronaviruses

For decades, the genetic modification of coronavirus genomes and the generation of recombinant coronaviruses have been hampered mostly due to the extraordinary large size of the coronaviral genome. The very first reverse genetic system for feline coronaviruses (FCoVs) was established in the early 2000s;the respective approach exclusively enabled the manipulation of the 3'-third of the viral genome. Later on, vaccinia virus-and bacterial artificial chromosome (BAC)-based systems have been developed. Both systems have the advantage that the entire FCoV genome is amenable for mutagenesis. The main focus of this chapter is the vaccinia virus-based reverse genetic system for FCoVs. Here we present protocols for (1) the generation of a full-length cDNA clone, (2) the manipulation of the FCoV genome, and (3) the rescue of recombinant FCoVs. Copyright © Springer Science+Business Media New York 2016.

Accelerated LD-based selective sweep detection using GPUs and FPGAs

Selective sweep detection carries theoretical significance and has several practical implications, from explaining the adaptive evolution of a species in an environment to understanding the emergence of viruses from animals, such as SARS-CoV-2, and their transmission from human to human. The plethora of available genomic data for population genetic analyses, however, poses various computational challenges to existing methods and tools, leading to prohibitively long analysis times. In this work, we accelerate LD (Linkage Disequilibrium) - based selective sweep detection using GPUs and FPGAs on personal computers and datacenter infrastructures. LD has been previously efficiently accelerated with both GPUs and FPGAs. However, LD alone cannot serve as an indicator of selective sweeps. Here, we complement previous research with dedicated accelerators for the ω statistic, which is a direct indicator of a selective sweep. We evaluate performance of our accelerator solutions for computing the w statistic and for a complete sweep detection method, as implemented by the open-source software OmegaPlus. In comparison with a single CPU core, the FPGA accelerator delivers up to 57.1× and 61.7× faster computation of the ω statistic and the complete sweep detection analysis, respectively. The respective attained speedups by the GPU-accelerated version of OmegaPlus are 2.9× and 12.9×. The GPU-accelerated implementation is available for download here: https://github.com/MrKzn/omegaplus.git. © 2022 IEEE.

Contrasting Patterns in the Early Stage of SARS-CoV-2 Evolution between Humans and Minks.

One of the unique features of SARS-CoV-2 is its apparent neutral evolution during the early pandemic (before February 2020). This contrasts with the preceding SARS-CoV epidemics, where viruses evolved adaptively. SARS-CoV-2 may exhibit a unique or adaptive feature which deviates from other coronaviruses. Alternatively, the virus may have been cryptically circulating in humans for a sufficient time to have acquired adaptive changes before the onset of the current pandemic. To test the scenarios above, we analyzed the SARS-CoV-2 sequences from minks (Neovision vision) and parental humans. In the early phase of the mink epidemic (April to May 2020), nonsynonymous to synonymous mutation ratio per site in the spike protein is 2.93, indicating a selection process favoring adaptive amino acid changes. Mutations in the spike protein were concentrated within its receptor-binding domain and receptor-binding motif. An excess of high-frequency derived variants produced by genetic hitchhiking was found during the middle (June to July 2020) and late phase I (August to September 2020) of the mink epidemic. In contrast, the site frequency spectra of early SARS-CoV-2 in humans only show an excess of low-frequency mutations, consistent with the recent outbreak of the virus. Strong positive selection in the mink SARS-CoV-2 implies that the virus may not be preadapted to a wide range of hosts and illustrates how a virus evolves to establish a continuous infection in a new host. Therefore, the lack of positive selection signal during the early pandemic in humans deserves further investigation.

Distinct evolutionary trajectories of SARS-CoV-2-interacting proteins in bats and primates identify important host determinants of COVID-19.

The coronavirus disease 19 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a coronavirus that spilled over from the bat reservoir. Despite numerous clinical trials and vaccines, the burden remains immense, and the host determinants of SARS-CoV-2 susceptibility and COVID-19 severity remain largely unknown. Signatures of positive selection detected by comparative functional genetic analyses in primate and bat genomes can uncover important and specific adaptations that occurred at virus-host interfaces. We performed high-throughput evolutionary analyses of 334 SARS-CoV-2-interacting proteins to identify SARS-CoV adaptive loci and uncover functional differences between modern humans, primates, and bats. Using DGINN (Detection of Genetic INNovation), we identified 38 bat and 81 primate proteins with marks of positive selection. Seventeen genes, including the ACE2 receptor, present adaptive marks in both mammalian orders, suggesting common virus-host interfaces and past epidemics of coronaviruses shaping their genomes. Yet, 84 genes presented distinct adaptations in bats and primates. Notably, residues involved in ubiquitination and phosphorylation of the inflammatory RIPK1 have rapidly evolved in bats but not primates, suggesting different inflammation regulation versus humans. Furthermore, we discovered residues with typical virus-host arms race marks in primates, such as in the entry factor TMPRSS2 or the autophagy adaptor FYCO1, pointing to host-specific in vivo interfaces that may be drug targets. Finally, we found that FYCO1 sites under adaptation in primates are those associated with severe COVID-19, supporting their importance in pathogenesis and replication. Overall, we identified adaptations involved in SARS-CoV-2 infection in bats and primates, enlightening modern genetic determinants of virus susceptibility and severity.

The assembled and annotated genome of the masked palm civet (Paguma larvata).

BACKGROUND: The masked palm civet (Paguma larvata) acts as an intermediate host of severe acute respiratory syndrome coronavirus (SARS-CoV), which caused SARS, and transfered this virus from bats to humans. Additionally, P. larvata has the potential to carry a variety of zoonotic viruses that may threaten human health. However, genome resources for P. larvata have not been reported to date. FINDINGS: A chromosome-level genome assembly of P. larvata was generated using PacBio sequencing, Illumina sequencing, and Hi-C technology. The genome assembly was 2.44 Gb in size, of which 95.32% could be grouped into 22 pseudochromosomes, with contig N50 and scaffold N50 values of 12.97 Mb and 111.81 Mb, respectively. A total of 21,582 protein-coding genes were predicted, and 95.20% of the predicted genes were functionally annotated. Phylogenetic analysis of 19 animal species confirmed the close genetic relationship between P. larvata and species belonging to the Felidae family. Gene family clustering revealed 119 unique, 243 significantly expanded, and 58 significantly contracted genes in the P. larvata genome. We identified 971 positively selected genes in P. larvata, and one known human viral receptor gene PDGFRA is positively selected in P. larvata, which is required for human cytomegalovirus infection. CONCLUSIONS: This high-quality genome assembly provides a valuable genomic resource for exploring virus-host interactions. It will also provide a reliable reference for studying the genetic bases of the morphologic characteristics, adaptive evolution, and evolutionary history of this species.

The early SARS-CoV-2 epidemic in Senegal was driven by the local emergence of B.1.416 and the introduction of B.1.1.420 from Europe.

Molecular surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is growing in west Africa, especially in the Republic of Senegal. Here, we present a molecular epidemiology study of the early waves of SARS-CoV-2 infections in this country based on Bayesian phylogeographic approaches. Whereas the first wave in mid-2020 was characterized by a significant diversification of lineages and predominance of B.1.416, the second wave in late 2020 was composed primarily of B.1.1.420. Our results indicate that B.1.416 originated in Senegal and was exported mainly to Europe. In contrast, B.1.1.420 was introduced from Italy, gained fitness in Senegal, and then spread worldwide. Since both B.1.416 and B.1.1.420 lineages carry several positive selected mutations in the spike and nucleocapsid genes, each of which may explain their local dominance, their mutation profiles should be carefully monitored. As the pandemic continues to evolve, molecular surveillance in all regions of Africa will play a key role in stemming its spread.

Self-mediated positive selection of T cells sets an obstacle to the recognition of nonself.

Adaptive immune recognition is mediated by the binding of peptide-human leukocyte antigen complexes by T cells. Positive selection of T cells in the thymus is a fundamental step in the generation of a responding T cell repertoire: only those T cells survive that recognize human peptides presented on the surface of cortical thymic epithelial cells. We propose that while this step is essential for optimal immune function, the process results in a defective T cell repertoire because it is mediated by self-peptides. To test our hypothesis, we focused on amino acid motifs of peptides in contact with T cell receptors. We found that motifs rarely or not found in the human proteome are unlikely to be recognized by the immune system just like the ones that are not expressed in cortical thymic epithelial cells or not presented on their surface. Peptides carrying such motifs were especially dissimilar to human proteins. Importantly, we present our main findings on two independent T cell activation datasets and directly demonstrate the absence of naïve T cells in the repertoire of healthy individuals. We also show that T cell cross-reactivity is unable to compensate for the absence of positively selected T cells. Additionally, we show that the proposed mechanism could influence the risk for different infectious diseases. In sum, our results suggest a side effect of T cell positive selection, which could explain the nonresponsiveness to many nonself peptides and could improve the understanding of adaptive immune recognition.

Selection Analysis Identifies Clusters of Unusual Mutational Changes in Omicron Lineage BA.1 That Likely Impact Spike Function.

Among the 30 nonsynonymous nucleotide substitutions in the Omicron S-gene are 13 that have only rarely been seen in other SARS-CoV-2 sequences. These mutations cluster within three functionally important regions of the S-gene at sites that will likely impact (1) interactions between subunits of the Spike trimer and the predisposition of subunits to shift from down to up configurations, (2) interactions of Spike with ACE2 receptors, and (3) the priming of Spike for membrane fusion. We show here that, based on both the rarity of these 13 mutations in intrapatient sequencing reads and patterns of selection at the codon sites where the mutations occur in SARS-CoV-2 and related sarbecoviruses, prior to the emergence of Omicron the mutations would have been predicted to decrease the fitness of any virus within which they occurred. We further propose that the mutations in each of the three clusters therefore cooperatively interact to both mitigate their individual fitness costs, and, in combination with other mutations, adaptively alter the function of Spike. Given the evident epidemic growth advantages of Omicron overall previously known SARS-CoV-2 lineages, it is crucial to determine both how such complex and highly adaptive mutation constellations were assembled within the Omicron S-gene, and why, despite unprecedented global genomic surveillance efforts, the early stages of this assembly process went completely undetected.

Computational Inference of Selection Underlying the Evolution of the Novel Coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2.

The novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that recently emerged in China is thought to have a bat origin, as its closest known relative (BatCoV RaTG13) was described previously in horseshoe bats. We analyzed the selective events that accompanied the divergence of SARS-CoV-2 from BatCoV RaTG13. To this end, we applied a population genetics-phylogenetics approach, which leverages within-population variation and divergence from an outgroup. Results indicated that most sites in the viral open reading frames (ORFs) evolved under conditions of strong to moderate purifying selection. The most highly constrained sequences corresponded to some nonstructural proteins (nsps) and to the M protein. Conversely, nsp1 and accessory ORFs, particularly ORF8, had a nonnegligible proportion of codons evolving under conditions of very weak purifying selection or close to selective neutrality. Overall, limited evidence of positive selection was detected. The 6 bona fide positively selected sites were located in the N protein, in ORF8, and in nsp1. A signal of positive selection was also detected in the receptor-binding motif (RBM) of the spike protein but most likely resulted from a recombination event that involved the BatCoV RaTG13 sequence. In line with previous data, we suggest that the common ancestor of SARS-CoV-2 and BatCoV RaTG13 encoded/encodes an RBM similar to that observed in SARS-CoV-2 itself and in some pangolin viruses. It is presently unknown whether the common ancestor still exists and, if so, which animals it infects. Our data, however, indicate that divergence of SARS-CoV-2 from BatCoV RaTG13 was accompanied by limited episodes of positive selection, suggesting that the common ancestor of the two viruses was poised for human infection.IMPORTANCE Coronaviruses are dangerous zoonotic pathogens; in the last 2 decades, three coronaviruses have crossed the species barrier and caused human epidemics. One of these is the recently emerged SARS-CoV-2. We investigated how, since its divergence from a closely related bat virus, natural selection shaped the genome of SARS-CoV-2. We found that distinct coding regions in the SARS-CoV-2 genome evolved under conditions of different degrees of constraint and are consequently more or less prone to tolerate amino acid substitutions. In practical terms, the level of constraint provides indications about which proteins/protein regions are better suited as possible targets for the development of antivirals or vaccines. We also detected limited signals of positive selection in three viral ORFs. However, we warn that, in the absence of knowledge about the chain of events that determined the human spillover, these signals should not be necessarily interpreted as evidence of an adaptation to our species.

Two-step fitness selection for intra-host variations in SARS-CoV-2.

Spontaneous mutations introduce uncertainty into coronavirus disease 2019 (COVID-19) control procedures and vaccine development. Here, we perform a spatiotemporal analysis on intra-host single-nucleotide variants (iSNVs) in 402 clinical samples from 170 affected individuals, which reveals an increase in genetic diversity over time after symptom onset in individuals. Nonsynonymous mutations are overrepresented in the pool of iSNVs but underrepresented at the single-nucleotide polymorphism (SNP) level, suggesting a two-step fitness selection process: a large number of nonsynonymous substitutions are generated in the host (positive selection), and these substitutions tend to be unfixed as SNPs in the population (negative selection). Dynamic iSNV changes in subpopulations with different gender, age, illness severity, and viral shedding time displayed a varied fitness selection process among populations. Our study highlights that iSNVs provide a mutational pool shaping the rapid global evolution of the virus.

Detection of Neanderthal Adaptively Introgressed Genetic Variants That Modulate Reporter Gene Expression in Human Immune Cells.

Although some variation introgressed from Neanderthals has undergone selective sweeps, little is known about its functional significance. We used a Massively Parallel Reporter Assay (MPRA) to assay 5,353 high-frequency introgressed variants for their ability to modulate the gene expression within 170 bp of endogenous sequence. We identified 2,548 variants in active putative cis-regulatory elements (CREs) and 292 expression-modulating variants (emVars). These emVars are predicted to alter the binding motifs of important immune transcription factors, are enriched for associations with neutrophil and white blood cell count, and are associated with the expression of genes that function in innate immune pathways including inflammatory response and antiviral defense. We combined the MPRA data with other data sets to identify strong candidates to be driver variants of positive selection including an emVar that may contribute to protection against severe COVID-19 response. We endogenously deleted two CREs containing expression-modulation variants linked to immune function, rs11624425 and rs80317430, identifying their primary genic targets as ELMSAN1, and PAN2 and STAT2, respectively, three genes differentially expressed during influenza infection. Overall, we present the first database of experimentally identified expression-modulating Neanderthal-introgressed alleles contributing to potential immune response in modern humans.

Adaptation of the endemic coronaviruses HCoV-OC43 and HCoV-229E to the human host.

Four coronaviruses (HCoV-OC43, HCoV-HKU1, HCoV-NL63, and HCoV-229E) are endemic in human populations. All these viruses are seasonal and generate short-term immunity. Like the highly pathogenic coronaviruses, the endemic coronaviruses have zoonotic origins. Thus, understanding the evolutionary dynamics of these human viruses might provide insight into the future trajectories of SARS-CoV-2 evolution. Because the zoonotic sources of HCoV-OC43 and HCoV-229E are known, we applied a population genetics-phylogenetic approach to investigate which selective events accompanied the divergence of these viruses from the animal ones. Results indicated that positive selection drove the evolution of some accessory proteins, as well as of the membrane proteins. However, the spike proteins of both viruses and the hemagglutinin-esterase (HE) of HCoV-OC43 represented the major selection targets. Specifically, for both viruses, most positively selected sites map to the receptor-binding domains (RBDs) and are polymorphic. Molecular dating for the HCoV-229E spike protein indicated that RBD Classes I, II, III, and IV emerged 3-9 years apart. However, since the appearance of Class V (with much higher binding affinity), around 25 years ago, limited genetic diversity accumulated in the RBD. These different time intervals are not fully consistent with the hypothesis that HCoV-229E spike evolution was driven by antigenic drift. An alternative, not mutually exclusive possibility is that strains with higher affinity for the cellular receptor have out-competed strains with lower affinity. The evolution of the HCoV-OC43 spike protein was also suggested to undergo antigenic drift. However, we also found abundant signals of positive selection in HE. Whereas such signals might result from antigenic drift, as well, previous data showing co-evolution of the spike protein with HE suggest that optimization for human cell infection also drove the evolution of this virus. These data provide insight into the possible trajectories of SARS-CoV-2 evolution, especially in case the virus should become endemic.

The emergence and ongoing convergent evolution of the SARS-CoV-2 N501Y lineages.

The independent emergence late in 2020 of the B.1.1.7, B.1.351, and P.1 lineages of SARS-CoV-2 prompted renewed concerns about the evolutionary capacity of this virus to overcome public health interventions and rising population immunity. Here, by examining patterns of synonymous and non-synonymous mutations that have accumulated in SARS-CoV-2 genomes since the pandemic began, we find that the emergence of these three "501Y lineages" coincided with a major global shift in the selective forces acting on various SARS-CoV-2 genes. Following their emergence, the adaptive evolution of 501Y lineage viruses has involved repeated selectively favored convergent mutations at 35 genome sites, mutations we refer to as the 501Y meta-signature. The ongoing convergence of viruses in many other lineages on this meta-signature suggests that it includes multiple mutation combinations capable of promoting the persistence of diverse SARS-CoV-2 lineages in the face of mounting host immune recognition.

A computational analysis of molecular evolution for virulence genes of zoonotic novel coronavirus (COVID-19).

Zoonotic Novel coronavirus disease 2019 (COVID-19) is highly pathogenic and transmissible considered as emerging pandemic disease. The virus belongs from a large virus Coronaviridae family affect respiratory tract of animal and human likely originated from bat and homology to SARA-CoV and MERS-CoV. The virus consists of single-stranded positive genomic RNA coated by nucleocapsid protein. The rate of mutation in any virulence gene may influence the phenomenon of host radiation. We have studied the molecular evolution of selected virulence genes (HA, N, RdRP and S) of novel COVID-19. We used a site-specific comparison of synonymous (silent) and non-synonymous (amino acid altering) nucleotide substitutions. Maximum Likelihood genealogies based on differential gamma distribution rates were used for the analysis of null and alternate hypothesis. The null hypothesis was found more suitable for the analysis using Likelihood Ratio Test (LRT) method, confirming higher rate of substitution. The analysis revealed that RdRP gene had the fastest rate evolution followed by HA gene. We have also reported the new motifs for different virulence genes, which are further useful to design new detection and diagnosis kit for COVID -19.

Positive selection as a key player for SARS-CoV-2 pathogenicity: Insights into ORF1ab, S and E genes.

The human ß-coronavirus SARS-CoV-2 epidemic started in late December 2019 in Wuhan, China. It causes Covid-19 disease which has become pandemic. Each of the five-known human ß-coronaviruses has four major structural proteins (E, M, N and S) and 16 non-structural proteins encoded by ORF1a and ORF1b together (ORF1ab) that are involved in virus pathogenicity and infectivity. Here, we performed detailed positive selection analyses for those six genes among the four previously known human ß-coronaviruses and within 38 SARS-CoV-2 genomes to assess signatures of adaptive evolution using maximum likelihood approaches. Our results suggest that three genes (E, S and ORF1ab genes) are under strong signatures of positive selection among human ß-coronavirus, influencing codons that are located in functional important protein domains. The E protein-coding gene showed signatures of positive selection in two sites, Asp 66 and Ser 68, located inside a putative transmembrane α-helical domain C-terminal part, which is preferentially composed by hydrophilic residues. Such Asp and Ser sites substitutions (hydrophilic residues) increase the stability of the transmembrane domain in SARS-CoV-2. Moreover, substitutions in the spike (S) protein S1 N-terminal domain have been found, all of them were located on the S protein surface, suggesting their importance in viral transmissibility and survival. Furthermore, evidence of strong positive selection was detected in three of the SARS-CoV-2 nonstructural proteins (NSP1, NSP3, NSP16), which are encoded by ORF1ab and play vital roles in suppressing host translation machinery, viral replication and transcription and inhibiting the host immune response. These results are insightful to assess the role of positive selection in the SARS-CoV-2 encoded proteins, which will allow to better understand the virulent pathogenicity of the virus and potentially identifying targets for drug or vaccine strategy design.