Evolution of Delta variant by non-Spike signature co-appearing mutations: trailblazer of COVID-19 disease outcome (preprint)

The high transmissibility and infectivity of a SARS-CoV-2 variant is usually ascribed to the Spike mutations, while emerging non-spike mutations might be a serious threat to the current Spike-recombinant vaccines. In addition to mutations in structural Spike glycoprotein, rapid accumulation of mutations across non-structural genes is leading to continuous virus evolution, altering its pathogenicity. We performed whole genome sequencing of SARS-CoV-2 positive samples collected from different clinical groups from eastern India, during the second pandemic wave (April-May, 2021). In addition to the several common spike mutations in Delta variant, two mutually explicit signature constellations of non-spike co-appearing mutations were identified, driving symptomatic and asymptomatic infections. We attempted to correlate these unique signatures of non-Spike co-appearing mutations to COVID-19 disease outcome. Results revealed that the Delta strains harboring a unique constellation of 9 non-spike co-appearing mutations could be the wheeler and dealer of symptomatic infection, even post vaccination. The strains predominantly driving asymptomatic infection possessed 7 non-spike co-appearing mutations, which were mutually exclusive in contrast to the set of mutations causing symptomatic disease. Phylodynamic analysis depicted high probability of emergence of these unique sub-clusters within India, with subsequent spread worldwide. Interestingly, some mutations of this signature were selected in Omicron and IHU variants, which suggest that gradual accumulation of such co-existing mutations may lead to emergence of more vaccine- evading variants in future. Hence, unfaltering genome sequencing and tracking of non-Spike mutations might be significant in formulation of any future vaccines against emerging SARS- CoV-2 variants that might evade the current vaccine-induced immunity.

S glycoprotein diversity of the Omicron variant (preprint)

On the backdrop of ongoing Delta variant infection and vaccine-induced immunity, the emergence of the new Variant of Concern, the Omicron, has again fuelled the fears of COVID-19 around the world. Currently, very little information is available about the S glycoprotein mutations, transmissibility, severity, and immune evasion behaviour of the Omicron variant. In the present study, we have performed a comprehensive analysis of the S glycoprotein mutations of 309 strains of the Omicron variant and also discussed the probable effects of observed mutations on several aspects of virus biology based on known available knowledge of mutational effects on S glycoprotein structure, function, and immune evasion characteristics.

SARS-CoV-2 Delta derivatives impact on neutralization of Covishield recipient sera (preprint)

The emergence of SARS-CoV-2 Delta variant and its derivatives has created grave public health problem worldwide. The high transmissibility associated with this variant has led to daily increase in the number of SARS-CoV-2 infections. Delta variant has slowly dominated the other variants of concern. Subsequently, Delta has further mutated to Delta AY.1 to Delta AY.126. Of these, Delta AY.1 has been reported from several countries including India and considered to be highly infectious and probable escape mutant. Considering the possible immune escape, we had already evaluated the efficacy of the BBV152 against Delta and Delta AY.1 variants. Here, we have evaluated the neutralizing potential of sera of COVID-19 naive vaccinees (CNV) immunized with two doses of vaccine, COVID-19 recovered cases immunized with two doses of vaccine (CRV) and breakthrough infections (BTI) post immunization with two doses of vaccine against Delta, Delta AY.1 and B.1.617.3 using 50% plaque reduction neutralization test (PRNT50). Our study observed low NAb titer in CNV group against all the variants compared to CRV and BTI groups. Delta variant has shown highest reduction of 27.3-fold in NAb titer among CNV group compared to other groups and variants. Anti-S1-RBD IgG immune response among all the groups was also substantiated with NAb response. Compromised neutralization was observed against Delta and Delta AY.1 compared B.1 in all three groups. However, it provided protection against severity of the disease and fatality.

Clinical characterization and Genomic analysis of COVID-19 breakthrough infections during second wave in different states of India (preprint)

During March to June 2021 India has experienced a deadly second wave of COVID19 with an increased number of post vaccination breakthrough infections reported across the country. To understand the possible reason of these breakthroughs we collected 677 clinical samples (throat swab/ nasal swabs) of individuals who had received two doses (n=592) and one dose (n=85) of vaccines (Covishield and Covaxin,) and tested positive for COVID19, from 17 states/Union Territories of country. These cases were telephonically interviewed and clinical data was analyzed. A total of 511 SARS-CoV-2 genomes were recovered with genome coverage of higher than 98% from both the cases. Analysis of both the cases determined that 86.69% (n=443) of them belonged to the Delta variant along with Alpha, Kappa, Delta AY.1 and Delta AY.2. The Delta variant clustered into 4 distinct sub-lineages. Sub-lineage I had mutations: ORF1ab, A1306S, P2046L, P2287S, V2930L, T3255I, T3446A, G5063S, P5401L, A6319V and N G215C; Sub lineage II : ORF1ab P309, A3209V, V3718A, G5063S, P5401L and ORF7a L116F; Sub lineage III : ORF1ab A3209V, V3718A, T3750I, G5063S, P5401L and Spike A222V; Sub-lineage IV ORF1ab P309L, D2980N, F3138S and spike K77T. This study indicated that majority of the clinical cases in the breakthrough were infected with the Delta variant and only 9.8% cases required hospitalization while fatality was observed in only 0.4% cases. This clearly suggests that the vaccination does provide reduction in hospital admission and mortality.

Emergence of a new SARS-CoV-2 variant from GR clade with a novel S glycoprotein mutation V1230L in West Bengal, India (preprint)

India is currently facing the devastating second wave of COVID-19 pandemic resulting in approximately 4000 deaths per day. To control this pandemic continuous mutational surveillance and genomic epidemiology of circulating strains is very important. In this study, we performed mutational analysis of the protein coding genes of SARS-CoV-2 strains (n=2000) collected during January 2021 to March 2021. Our data revealed the emergence of a new variant in West Bengal, India, which is characterized by the presence of 11 co-existing mutations including D614G, P681H and V1230L in S-glycoprotein. This new variant was identified in 70 out of 412 sequences submitted from West Bengal. Interestingly, among these 70 sequences, 16 sequences also harbored E484K in the S glycoprotein. Phylogenetic analysis revealed strains of this new variant emerged from GR clade (B.1.1) and formed a new cluster. We propose to name this variant as GRL or lineage B.1.1/S:V1230L due to the presence of V1230L in S glycoprotein along with GR clade specific mutations. Co-occurrence of P681H, previously observed in UK variant, and E484K, previously observed in South African variant and California variant, demonstrates the convergent evolution of SARS-CoV-2 mutation. V1230L, present within the transmembrane domain of S2 subunit of S glycoprotein, has not yet been reported from any country. Substitution of valine with more hydrophobic amino acid leucine at position 1230 of the transmembrane domain, having role in S protein binding to the viral envelope, could strengthen the interaction of S protein with the viral envelope and also increase the deposition of S protein to the viral envelope, and thus positively regulate virus infection. P618H and E484K mutation have already been demonstrated in favor of increased infectivity and immune invasion respectively. Therefore, the new variant having G614G, P618H, P1230L and E484K is expected to have better infectivity, transmissibility and immune invasion characteristics, which may pose additional threat along with B.1.617 in the ongoing COVID-19 pandemic in India.

Detecting Pathogen-Associated RNA via Piecewise Isothermal Testing achieving Sample-to-Result Integration (preprint)

We report a novel piece-wise isothermal nucleic acid test (PINAT) for diagnosing pathogen-associated RNA that embeds an exclusive DNA-mediated specific probing reaction with the backbone of an isothermal reverse-transcription cum amplification protocol as a unified single-step procedure. This single step sample-to-result test method has been seamlessly integrated in an inexpensive, scalable, pre-programmable and portable instrument, resulting in a generic platform technology for detecting nucleic acid from a wide variety of pathogens. The test exhibited high sensitivity and specificity of detection of SARS-CoV-2 infection when assessed using 200 double-blind patient samples, conducted by the Indian Council of Medical Research (ICMR), reporting a positive and negative percent agreement of 94.6% and 98% respectively. We also established its efficacy in detecting Influenza-A virus infection, performing the diagnosis at the point of collection with uncompromised detection rigor. The envisaged trade-off between advanced laboratory-based procedures with the elegance of common rapid tests renders the innovation to be ideal for deployment in resource-limited settings towards catering the needs of the underserved.

Emergence of SARS-CoV-2 stains harbouring the signature mutations of both A2a and A3 clade (preprint)

SARS-CoV-2 strains with both high transmissibility and potential to cause asymptomatic infection is expected to gain selective advantage over other circulating strains having either high transmissibility or ability to trigger asymptomatic infection. The D614G mutation in spike glycoprotein, the characteristic mutation A2a clade, has been associated with high transmissibility, whereas the A3 clade specific mutation L37F in NSP6 protein has been linked with asymptomatic infection. In this study, we performed a comprehensive mutational analysis of 3,77,129 SARS-CoV-2 genomes collected during January, 2020 to December, 2020 from all across the world for the presence of D614G and L37F mutations. Out of 3,77,129 SARS-CoV-2 strains analysed, 14, 598 (3.87%) were found to harbour both the D614G and L37F mutations. Majority of these double mutant SARS-CoV-2 strains were identified in Europe (11097) followed by North America (1915), Asia (980), Oceania (242), Africa (219), and South America (145). Geographical root surveillance revealed their first emergence during February-March in all the six continents. Temporal prevalence analysis from February, 2020 to December, 2020 showed a gradual upsurge in their frequencies worldwide, which strongly demonstrated the adaptive selection of these double mutants. Evolutionary analysis depicted that these double mutants emerged as a new clade in the dendrogram (named as A2a/3), and were sub-divided into four distinct clusters (Cluster I, II, III and IV) according to different sets of coexisting mutations. The frequency distribution pattern showed the global predominance of cluster III (41.42%), followed by cluster IV (23.31%), cluster II (21.02%) and cluster I (14.25%). Overall, our study highlighted the emergence of a unique phylogenetic clade encompassing the double-mutant SARS-CoV-2 strains which may provide a fitness advantage during course of virus evolution.

Comprehensive analysis of genomic diversity of SARS-CoV-2 in different geographic regions of India: An endeavour to classify Indian SARS-CoV-2 strains on the basis of co-existing mutations (preprint)

Accumulation of mutations within the genome is the primary driving force for viral evolution within an endemic setting. This inherent feature often leads to altered virulence, infectivity and transmissibility as well as antigenic shift to escape host immunity, which might compromise the efficacy of vaccines and antiviral drugs. Therefore, we aimed at genome-wide analyses of circulating SARS-CoV-2 viruses for the emergence of novel co-existing mutations and trace their spatial distribution within India. Comprehensive analysis of whole genome sequences of 441 Indian SARS-CoV-2 strains revealed the occurrence of 33 different mutations, 21 being distinctive to India. Emergence of novel mutations were observed in S glycoprotein (7/33), NSP3 (6/33), RdRp/NSP12 (4/33), NSP2 (2/33) and N (2/33). Non-synonymous mutations were found to be 3.4 times more prevalent than synonymous mutations. We classified the Indian isolates into 22 groups based on the co-existing mutations. Phylogenetic analyses revealed that representative strain of each group divided themselves into various sub-clades within their respective clades, based on the presence of unique co-existing mutations. India was dominated by A2a clade (55.60%) followed by A3 (37.38%) and B (7%), but exhibited heterogeneous distribution among various geographical regions. The A2a clade mostly predominated in East India, Western India and Central India, whereas A3 clade prevailed in South and North India. In conclusion, this study highlights the divergent evolution of SARS-CoV-2 strains and co-circulation of multiple clades in India. Monitoring of the emerging mutations would pave ways for vaccine formulation and designing of antiviral drugs.

The novel Coronavirus enigma: Phylogeny and mutation analyses of SARS-CoV-2 viruses circulating in India during early 2020. (preprint)

BackgroundThis is a comprehensive analysis of 46 Indian SARS-CoV-2 genome sequences available from the NCBI and GISAID repository during early 2020. Evolutionary dynamics, gene-specific phylogeny and emergence of the novel co-evolving mutations in nine structural and non-structural genes among circulating SARS-CoV-2 strains in ten states of India have been assessed. Materials and methods46 SARS-CoV-2 nucleotide sequences submitted from India were downloaded from the GISAID (39/46) or from NCBI (7/46) database. Phylogenetic study and analyses of mutation were based on the nine structural and non-structural genes of SARS-CoV-2 strains. Secondary structure of RdRP/NSP12 protein was predicted with respect to the novel A97V mutation. ResultsPhylogenetic analyses revealed the evolution of "genome-type clusters" and adaptive selection of "L" type SARS-CoV-2 strains with genetic closeness to the bat SARS-like coronaviruses than pangolin or MERS-CoVs. With regards to the novel co-evolving mutations, 2 groups are seen to circulate in India at present: the "major group" (52.2%) and the "minor group" (30.4%), harboring four and five co-existing mutations, respectively. The "major group" mutations fall in the A2a clade. All the minor group mutations, except 11083G>T (L37F, NSP6) were unique to the Indian isolates. ConclusionThe study highlights rapidly evolving SARS-CoV-2 virus and co-circulation of multiple clades and sub-clades, driving this pandemic worldwide. This comprehensive study is a potential resource for monitoring the novel mutations in the viral genome, changes in viral pathogenesis, for designing vaccines and other therapeutics.