Evolution of the SARS-CoV-2 pandemic in India.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a bat-derived betacoronavirus, that emerged around December 2019. In spite of the lesser genomic diversity of CoVs in general, a steady accumulation of mutations spread over its genome have been noted, resulting in the emergence of several clades and lineages. Majority of these mutations are random and non-functional changes; however a few variants of concern (VOC) and variants of interest (VOI) designated by the WHO since late 2020 have implications to diagnostics, pathogenicity and immune escape. This review discusses the various nomenclatures depicting the SARS-CoV-2 evolution, the designated VOCs and VOIs and the mutations characterizing these variants. The evolution of SARS-CoV-2 in India and the implications to vaccine efficacy and breakthrough infections is also addressed.

Perspectives for repurposing drugs for the coronavirus disease 2019.

The newly emerged 2019 novel coronavirus (CoV), named as severe acute respiratory syndrome CoV-2 (SARS-CoV-2), like SARS-CoV (now, SARS-CoV-1) and Middle East respiratory syndrome CoV (MERS-CoV), has been associated with high infection rates with over 36,405 deaths. In the absence of approved marketed drugs against coronaviruses, the treatment and management of this novel CoV disease (COVID-19) worldwide is a challenge. Drug repurposing that has emerged as an effective drug discovery approach from earlier approved drugs could reduce the time and cost compared to de novo drug discovery. Direct virus-targeted antiviral agents target specific nucleic acid or proteins of the virus while host-based antivirals target either the host innate immune responses or the cellular machineries that are crucial for viral infection. Both the approaches necessarily interfere with viral pathogenesis. Here we summarize the present status of both virus-based and host-based drug repurposing perspectives for coronaviruses in general and the SARS-CoV-2 in particular.

Full-genome sequences of the first two SARS-CoV-2 viruses from India.

Background & objectives: Since December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has globally affected 195 countries. In India, suspected cases were screened for SARS-CoV-2 as per the advisory of the Ministry of Health and Family Welfare. The objective of this study was to characterize SARS-CoV-2 sequences from three identified positive cases as on February 29, 2020. Methods: Throat swab/nasal swab specimens for a total of 881 suspected cases were screened by E gene and confirmed by RdRp (1), RdRp (2) and N gene real-time reverse transcription-polymerase chain reactions and next-generation sequencing. Phylogenetic analysis, molecular characterization and prediction of B- and T-cell epitopes for Indian SARS-CoV-2 sequences were undertaken. Results: Three cases with a travel history from Wuhan, China, were confirmed positive for SARS-CoV-2. Almost complete (29,851 nucleotides) genomes of case 1, case 3 and a fragmented genome for case 2 were obtained. The sequences of Indian SARS-CoV-2 though not identical showed high (~99.98%) identity with Wuhan seafood market pneumonia virus (accession number: NC 045512). Phylogenetic analysis showed that the Indian sequences belonged to different clusters. Predicted linear B-cell epitopes were found to be concentrated in the S1 domain of spike protein, and a conformational epitope was identified in the receptor-binding domain. The predicted T-cell epitopes showed broad human leucocyte antigen allele coverage of A and B supertypes predominant in the Indian population. Interpretation & conclusions: The two SARS-CoV-2 sequences obtained from India represent two different introductions into the country. The genetic heterogeneity is as noted globally. The identified B- and T-cell epitopes may be considered suitable for future experiments towards the design of vaccines and diagnostics. Continuous monitoring and analysis of the sequences of new cases from India and the other affected countries would be vital to understand the genetic evolution and rates of substitution of the SARS-CoV-2.

Whole-genome-based characterization of three human Rotavirus C strains isolated from gastroenteritis outbreaks in Western India and a provisional intra-genotypic lineage classification system.

The number of whole-genome sequences of human rotavirus C (RVC) strains available in public databases is recently increasing. Thus far from India only a single whole genome of human RVC of a sporadic case was available. In this study, nearly full-length genome sequencing and phylogenetic analyses of three RVC strains isolated from three different gastroenteritis outbreaks during 2010-2014 in Western India was carried out. Further, an intra-genotypic lineage classification system for human RVCs based on the nucleotide divergence cut-off values was proposed by using the algorithm of the Rotavirus Classification Working Group. Two lineages could be defined for all the genes except the VP7 gene and the M3 VP3 genotype. Provisional classification of the lineages indicated the absence of reassortment events in the genomic constellation of Indian strains, contrary to earlier reports. The comparatively higher variability of the NSP1, NSP3, NSP5 and M2 VP3 genotype, emphasizes their utility in lineage classification.

Computational analysis of the effect of polymerase acidic (PA) gene mutation F35L in the 2009 pandemic influenza A (H1N1) virus on binding aspects of mononucleotides in the endonuclease domain.

An F35L mutation in the N-terminal domain of the polymerase acidic protein (PA-Nter), which contains the active site of the endonuclease, has been reported to result in higher polymerase activity in mouse-adapted strains of the 2009 pandemic influenza A H1N1 virus. We modeled wild and mutant complexes of uridine 5'-monophosphate (UMP) as the endonuclease substrate and performed molecular dynamics simulations. The results demonstrated that the F35L mutation could result in a changed orientation of a helix containing active site residues and improve the ligand affinity in the mutant strain. This study suggests a molecular mechanism of enhanced polymerase activity.

Genetic characterization of chikungunya viruses isolated during the 2015-2017 outbreaks in different states of India, based on their E1 and E2 genes.

During 2015-2017, chikungunya virus (CHIKV) showed a resurgence in several parts of India with Karnataka, Maharashtra and New Delhi accounting for a majority of the cases. E2-E1 gene based characterization revealed Indian subcontinent sublineage strains possessing Aedes aegypti mosquito-adaptive mutations E1: K211E and E2:V264A, with the 211 site positively selected. Novel mutational sites E1: K16E/Q, E1: K132Q/T, E1: S355T, E2: C19R and E2:S185Y could be associated with epitopes or virulence determining domains. The study examines the role of host, vector and viral factors and fills gaps in our molecular epidemiology data for these regions which are known to possess a dynamic population.

A molecular modelling approach to understand the effect of co-evolutionary mutations (V344M, I354L) identified in the PB2 subunit of influenza A 2009 pandemic H1N1 virus on m7GTP ligand binding.

The cap binding domain of the polymerase basic 2 (PB2) subunit of influenza polymerases plays a critical role in mediating the 'cap-snatching' mechanism by binding the 5' cap of host pre-mRNAs during viral mRNA transcription. Monitoring variations in the PB2 protein is thus vital for evaluating the pathogenic potential of the virus. Based on selection pressure analysis of PB2 gene sequences of the pandemic H1N1 (pH1N1) viruses of the period 2009-2014, we identified a site, 344V/M, in the vicinity of the cap binding pocket showing evidence of adaptive evolution and another co-evolving residue, 354I/L, in close vicinity. Modelling of the three-dimensional structure of the pH1N1 PB2 cap binding domain, docking of the pre-mRNA cap analogue m7GTP and molecular dynamics simulation studies of the docked complexes performed for four PB2 variants observed showed that the complex possessing V344M with I354L possessed better ligand binding affinity due to additional hydrogen bond contacts between m7GTP and the key residues His432 and Arg355 that was attributed to a displacement of the 424 loop and a flip of the side chain of Arg355, respectively. The co-evolutionary mutations identified (V344M, I354L) were found to be established in the PB2 gene of the pH1N1 viral population over the period 2010-2014. The study demonstrates the molecular basis for the enhanced m7GTP ligand binding affinity with the 344M-354L synergistic combination in PB2. Furthermore, the insight gained into understanding the molecular mechanism of cap binding in pH1N1 viruses may be useful for designing novel drugs targeting the PB2 cap binding domain.

Genomic characterization of coxsackievirus type B3 strains associated with acute flaccid paralysis in south-western India.

Acute flaccid paralysis (AFP) associated with coxsackievirus type B3 (CV-B3) of the species Enterovirus B is an emerging concern worldwide. Although CV-B3-associated AFP in India has been demonstrated previously, the genomic characterization of these strains is unreported. Here, CV-B3 strains detected on the basis of the partial VP1 gene in 10 AFP cases and five asymptomatic contacts identified from different regions of south-western India during 2009-2010 through the Polio Surveillance Project were considered for complete genome sequencing and characterization. Phylogenetic analysis of complete VP1 gene sequences of global CV-B3 strains classified Indian CV-B3 strains into genogroup GVI, along with strains from Uzbekistan and Bangladesh, and into a new genogroup, GVII. Genomic divergence between genogroups of the study strains was 14.4 % with significantly lower divergence (1.8 %) within GVI (n = 12) than that within GVII (8.5 %) (n = 3). The strains from both AFP cases and asymptomatic contacts, identified mainly in coastal Karnataka and Kerala, belonged to the dominant genogroup GVI, while the GVII strains were recovered from AFP cases in north interior Karnataka. All study strains carried inter-genotypic recombination with the structural region similar to reference CV-B3 strains, and 5' non-coding regions and non-structural regions closer to other enterovirus B types. Domain II structures of 5' non-coding regions, described to modulate virus replication, were predicted to have varied structural folds in the two genogroups and were attributed to differing recombination patterns. The results indicate two distinct genomic compositions of CV-B3 strains circulating in India and suggest the need for concurrent analysis of viral and host factors to further understand the varied manifestations of their infections.

Phylogeographic analysis of Japanese encephalitis virus in India (1956-2012).

Japanese encephalitis virus (JEV) isolates from India phylogenetically belong to two genotypes, III and I. We used envelope gene sequences from GenBank, representing different states of India and other countries, to study the spatiotemporal transmission histories of these two JEV genotypes separately. Genotype III was found to have been successively introduced in the 1930s, 1950s and 1960s, followed by genotype I twice around 2003-2006. Changes in JEV disease patterns in India over the last five decades could thus be attributed to multiple introductions of JEV strains from neighboring Asian countries along with increased transmission potential due to altered ecological settings.

Evolution, dispersal and replacement of American genotype dengue type 2 viruses in India (1956-2005): selection pressure and molecular clock analyses.

This study reports the phylogeny, selection pressure, genotype replacement and molecular clock analyses of many previously unstudied dengue type 2 virus (DENV-2) strains, isolated in India over a time span of almost 50 years (1956-2005). Analysis of complete envelope (E) gene sequences of 37 strains of DENV-2 from India, together with globally representative strains, revealed that the American genotype, which circulated predominantly in India during the pre-1971 period, was then replaced by the Cosmopolitan genotype. Two previously unreported amino acid residues, one in the American (402I) and one in the Cosmopolitan (126K) genotypes, known to be involved functionally in the cellular tropism of the virus, were shown to be under positive selection pressure. The rate of nucleotide substitution estimated for DENV-2 was 6.5x10(-4) substitutions per site year(-1), which is comparable with earlier estimates. The time to the most recent common ancestor of the pre-1971 Indian strains and the American genotype was estimated to be between 73 and 100 years (1905-1932), which correlates with the historical record of traffic between India and South America and suggests transportation of the virus from the Americas. Post-1971 Indian isolates formed a separate subclade within the Cosmopolitan genotype. The estimated time to the most recent common ancestor of the Indian Cosmopolitan strains was about 47 years, with further estimates indicating the migration of DENV-2 from India to countries across the Indian ocean between 1955 and 1966. Overall, the present study increases our understanding of the events leading to the establishment and dispersal of the two genotypes in India.

Longer duration of viremia and unique amino acid substitutions in a hepatitis A virus strain [corrected] associated with Guillain-Barré syndrome (GBS).

The molecular characteristics of hepatitis A virus (HAV) have been studied widely though there is a paucity of data on the correlation with virological and serological findings. In the present study, the whole genome of an Indian HAV strain associated with Guillain-Barré syndrome (GBS) was characterized vis-à-vis two other Indian HAV genotype IIIA strains, associated with a self-limiting disease. The percentage nucleotide divergence displayed by the Indian strains (CP-IND, PN-IND, and GBS-IND) varied from 3 to 6, whereas the percentage amino acid divergence varied from 0.1 to 0.7 as compared to the other HAV IIIA strains (n = 5) available in the GenBank. The GBS-IND strain showed an increased rate of nonsynonymous substitutions as well as a larger number of unique and heterologous amino acid substitutions compared to the HAV IIIA GenBank strains. These amino acid substitutions in the GBS-IND strain were detected in a nonstructural protein (2C-251F) and the B-cell epitope regions of structural proteins (VP1-29E, VP1-91S, VP3-50Y, and VP4-5S). In a comparative analysis of HAV strains, homology-based models of the capsid proteins indicated a localized alteration in the surface charge distribution on the VP1 protein of GBS-IND strain and involvement of its unique amino acid substitutions in the predicted antigenic determinants. Overall, the study suggests that the unique amino acid substitutions in the GBS-IND strain may have contributed to neutralization escape of the virus leading to a longer duration of viremia.

Chikungunya phylogeography reveals persistent global transmissions of the Indian Ocean Lineage from India in association with mutational fitness.

Since the resurgence of chikungunya virus (CHIKV) in India in 2005, the Indian subcontinent sublineage of the Indian Ocean lineage (IOL) has continued transmission in India and also radiation from India causing additional outbreaks in surrounding countries. This study was undertaken for an in-depth understanding of the evolutionary dynamics of the IOL, the global transmission routes in the Indian context and possible association with mutational fitness. The whole genome sequencing of Indian isolates representing CHIKV outbreaks (2014-2018) from selected States of India was carried out, followed by phylogeography analysis of the IOL using the Bayesian Markov chain Monte Carlo method and selection pressure analysis. Phylogeography analysis of IOL strains revealed indigenous evolution in India at least at three time points, with specific mutations that conferred viral fitness in the Aedes vector species. Further dispersal of the strains from India was noted to neighbouring and distant countries with multiple exportations to Sri Lanka, Bangladesh and China. The study reveals India as an endemic reservoir for CHIKV and persistent global transmissions from India. Though natural selection does not appear to play a major role in establishment of the IOL, sustainable efforts towards vector control can help address the issues.

Full genome based sequence and structural characterization of an unusual group A rotavirus G12P[11] isolated from neonates in Pune, western India.

Studies conducted at neonatal intensive care units in Pune, western India, suggested early exposure to rotaviruses and predominance of unusual human-bovine-like G12P[11] strains. The whole genome sequencing and phylogenetic analyses of a naturally attenuated, culture adapted neonatal strain, (NIV-1740121) revealed multiple-gene reassortment events, containing ROTAVAC® vaccine strain, 116E-like VP4, VP6, NSP3, NSP5 genes, VP7 gene of G12 origin and VP3 gene of porcine ancestry in a human Wa-like backbone. Analysis of 3D structure modeling of the VP7 and VP4 proteins with respect to 116E suggested amino acid variations in the major neutralizing epitopes of VP7, contributed to a modified charge density. Visualization of receptor-glycan interaction structures of NIV-1740121 and 116E VP8* showed type I glycan binds with a similar conformation at the same active site as represented in the available crystal structure of G10P[11] VP8*. The study adds to the knowledge of age restricted tropism of P[11] strains in neonates.

Phylogeography of Kyasanur Forest Disease virus in India (1957-2017) reveals evolution and spread in the Western Ghats region.

The Kyasanur Forest Disease (KFD) has become a major public health problem in the State of Karnataka, India where the disease was first identified and in Tamil Nadu, Maharashtra, Kerala, and Goa covering the Western Ghats region of India. The incidence of positive cases and distribution of the Kyasanur Forest Disease virus (KFDV) in different geographical regions raises the need to understand the evolution and spatiotemporal transmission dynamics. Phylogeography analysis based on 48 whole genomes (46 from this study) and additionally 28 E-gene sequences of KFDV isolated from different regions spanning the period 1957-2017 was thus undertaken. The mean evolutionary rates based the E-gene was marginally higher than that based on the whole genomes. A subgroup of KFDV strains (2006-2017) differing from the early Karnataka strains (1957-1972) by ~2.76% in their whole genomes and representing spread to different geographical areas diverged around 1980. Dispersal from Karnataka to Goa and Maharashtra was indicated. Maharashtra represented a new source for transmission of KFDV since ~2013. Significant evidence of adaptive evolution at site 123 A/T located in the vicinity of the envelope protein dimer interface may have functional implications. The findings indicate the need to curtail the spread of KFDV by surveillance measures and improved vaccination strategies.

Evolutionary rates and timescale comparison of Chikungunya viruses inferred from the whole genome/E1 gene with special reference to the 2005-07 outbreak in the Indian subcontinent.

Chikungunya (CHIK) virus reemerged during 2005-07 as an important pathogen causing massive disease outbreaks affecting India and several countries of the Indian Ocean. Knowledge of the evolutionary rates and divergence times of the CHIK virus may help to better understand the disease epidemiology. Considering the limited availability of such information, we estimated the substitution rates and the ancestral times for all the CHIK genotypes and also the time to the most recent common ancestor (tMRCA) of the 2005-07 isolates. Using whole genomes and partial E1 gene datasets, we applied the Bayesian Markov Chain Monte Carlo (MCMC) framework that explicitly accounts for lineage-specific evolutionary rates through the use of 'relaxed' molecular clock models. Under a constant population relaxed clock model, the evolutionary timescale of CHIK viruses in this study was estimated to be in the last 300 years. The progenitor of the 2005-07 viruses was found to have existed around 9 years ago, and to have originated from Central Africa. The presence of a strain in India in 2000 that bears 99% identity with a Ugandan strain of 1982, which correlates with the tMRCA of the Indian and Indian Ocean isolates, confirms our earlier report that the progenitor of the 2005-07 isolates originates from Uganda's neighbourhood. The 'A226V' mutation that existed in the Indian Ocean isolates since late 2005 was found to occur only in the 2007 isolate from India. The study confirms the epidemiological data, specifically with regard to the re-emergence of CHIKV and throws light on the evolutionary dynamics of CHIK viruses.

A unique influenza A (H5N1) virus causing a focal poultry outbreak in 2007 in Manipur, India.

BACKGROUND: A focal H5N1 outbreak in poultry was reported from Manipur, a north-eastern state, of India, in 2007. The aim of this study was to genetically characterize the Manipur isolate to understand the relationship with other H5N1 isolates and to trace the possible source of introduction of the virus into the country. RESULTS: Characterization of the complete genome revealed that the virus belonged to clade 2.2. It was distinctly different from viruses of the three EMA sublineages of clade 2.2 but related to isolates from wild migratory waterfowl from Russia, China and Mongolia. The HA gene, had the cleavage site GERRRRKR, earlier reported in whooper swan isolates from Mongolia in 2005. A stop codon at position 29 in the PB1-F2 protein could have implications on the replication efficiency. The acquisition of polymorphisms as seen in recent isolates of 2005-07 from distinct geographical regions suggests the possibility of transportation of H5N1 viruses through migratory birds. CONCLUSION: Considering that all eight genes of the earlier Indian isolates belonged to the EMA3 sublineage and similar strains have not been reported from neighbouring countries of the subcontinent, it appears that the virus may have been introduced independently.