The SARS-CoV-2 B.1.617.2 (Delta) variant was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha) 1 . In vitro, B.1.617.2 is 6-fold less sensitive to serum neutralising antibodies from recovered individuals, and 8-fold less sensitive to vaccine-elicited antibodies as compared to wild type (WT) Wuhan-1 bearing D614G. Serum neutralising titres against B.1.617.2 were lower in ChAdOx-1 versus BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies against the receptor binding domain (RBD) and N-terminal domain (NTD). B.1.617.2 demonstrated higher replication efficiency in both airway organoid and human airway epithelial systems compared to B.1.1.7, associated with B.1.617.2 spike in a predominantly cleaved state compared to B.1.1.7. The B.1.617.2 spike protein was able to mediate highly efficient syncytium formation that was less sensitive to inhibition by neutralising antibody as compared to WT spike. Additionally we observed that B.1.617.2 had higher replication and spike mediated entry as compared to B.1.617.1, potentially explaining B.1.617.2 dominance. In an analysis of over 130 SARS-CoV-2 infected healthcare workers across three centres in India during a period of mixed lineage circulation, we observed reduced ChAdOx-1 vaccine effectiveness against B.1.617.2 relative to non-B.1.617.2, with the caveat of possible residual confounding. Compromised vaccine efficacy against the highly fit and immune evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era. India's first wave of SARS-CoV-2 infections in mid-2020 was relatively mild and was controlled by a nationwide lockdown. Since easing of restrictions, India has seen expansion in cases of COVID-19 since March

After escaping relatively unscathed during the first wave of the COVID-19 pandemic, India witnessed a ferocious second COVID-19 wave, starting in March 2021 and accounting for about half of global cases by the first week of May. SARS-CoV-2 had spread widely throughout India in the first wave, with the third national serosurvey in January 2021 finding that 21.4% of adults and 25.3% of 10-to 17-year-old adolescents were seropositive (1). Delhi, the national capital, was not included in the national serosurvey but had undergone multiple periods of high transmission in 2020 (Fig. 1A). In a district-wise stratified serosurvey conducted by the Delhi Government in January 2021, overall seropositivity was reported to be 56.1% (95% CI, 55.5-56.8%), ranging from 49.1% to 62.2% across 11 districts (2). This was expected to confer some protection from future outbreaks.Despite high seropositivity, Delhi was amongst the most affected cities during the second wave. The rise in new cases was exceptionally rapid in April, going from approximately 2000 to 20,000 between 31 March and 16 April. This was accompanied by a rapid rise in hospitalizations and ICU admissions (Fig. 1B). In this emergency situation with saturated bed occupancy by 12 April, major private hospitals were declared by the state as full COVID care-only and senior medical students, including from alternative medicine branches, were pressed into service (3). Deaths rose proportionately (Fig. 1C) and the case-fatality ratio (CFR), estimated as the scaling factor between time-advanced cases and deaths (Fig. 1D), was stable (mean, SD; 1.9, 0.3%). Population spread of SARS-CoV-2 is underestimated by test positive cases alone (1, 2). To better understand the degree of spread and the factors leading to the unexpectedly severe outbreak, we used all available data including testing, sequencing, serosurveys, and serially followed cohorts.In the absence of finely resolved or serial data from national and state surveys, we focused on data for Delhi participants of a national serosurvey of Council of Scientific and

Direct massively parallel sequencing of SARS-CoV-2 genome was undertaken from nasopharyngeal and oropharyngeal swab samples of infected individuals in Eastern India. Seven of the isolates belonged to the A2a clade, while one belonged to the B4 clade. Specific mutations, characteristic of the A2a clade, were also detected, which included the P323L in RNA-dependent RNA polymerase and D614G in the Spike glycoprotein. Further, our data revealed emergence of novel subclones harbouring nonsynonymous mutations, viz. G1124V in Spike (S) protein, R203K, and G204R in the nucleocapsid (N) protein. The N protein mutations reside in the SR-rich region involved in viral capsid formation and the S protein mutation is in the S 2 domain, which is involved in triggering viral fusion with the host cell membrane. Interesting correlation was observed between these mutations and travel or contact history of COVID-19 positive cases. Consequent alterations of miRNA binding and structure were also predicted for these mutations. More importantly, the possible implications of mutation D614G (in S D domain) and G1124V (in S 2 subunit) on the structural stability of S protein have also been discussed. Results report for the first time a bird's eye view on the accumulation of mutations in SARS-CoV-2 genome in Eastern India.

Mutational analysis of the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) template channel identified two residues, Trp(397) and His(428), which are required for de novo initiation but not for extension from a primer. These two residues interact with the Delta1 loop on the surface of the RdRp. A deletion within the Delta1 loop also resulted in comparable activities. The mutant proteins exhibit increased double-stranded RNA binding compared with the wild type, suggesting that the Delta1 loop serves as a flexible locking mechanism to regulate the conformations needed for de novo initiation and for elongative RNA synthesis. A similar locking motif can be found in other viral RdRps. Products associated with the open conformation of the HCV RdRp were inhibited by interaction with the retinoblastoma protein but not cyclophilin A. Different conformations of the HCV RdRp can thus affect RNA synthesis and interaction with cellular proteins.

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) has been proposed to change conformations in association with RNA synthesis and to interact with cellular proteins. In vitro, the RdRp can initiate de novo from the ends of single-stranded RNA or extend a primed RNA template. The interactions between the ⌬1 loop and thumb domain in NS5B are required for de novo initiation, although it is unclear whether these interactions are within an NS5B monomer or are part of a higher-order NS5B oligomeric complex. This work seeks to address how polymerase conformation and/or oligomerization affects de novo initiation. We have shown that an increasing enzyme concentration increases de novo initiation by the genotype 1b and 2a RdRps while primer extension reactions are not affected or inhibited under similar conditions. Initiation-defective mutants of the HCV polymerase can increase de novo initiation by the wild-type (WT) polymerase. GTP was also found to stimulate de novo initiation. Our results support a model in which the de novo initiation-competent conformation of the RdRp is stimulated by oligomeric contacts between individual subunits. Using electron microscopy and single-molecule reconstruction, we attempted to visualize the lowresolution conformations of a dimer of a de novo initiation-competent HCV RdRp.Polymerases undergo a series of conformational changes at different stages of nucleic acid synthesis (14). Of the templatedependent polymerases, the RNA-dependent RNA polymerases (RdRps) are the least understood in terms of their mechanism of action. RdRps are of increasing interest since cellular RdRps play important roles in the defense against nonself RNAs (44). In addition, virus-encoded RdRps are important targets for the development of antivirals. A better understanding of RNA-dependent RNA polymerases is thus important for both basic and applied science.Several model systems for biochemical study of viral RNAdependent RNA synthesis exist (4,19,20,25,37,42). Wellcharacterized RdRps include those from the hepatitis C virus (HCV) and poliovirus (5, 17). In the host, the RdRps are complexed with other viral and/or cellular proteins that are usually associated with membranous intracellular structures. The replicases are usually difficult to study biochemically, but the catalytic RdRp subunits of several viruses can be purified for functional and structural analyses (53). These recombinant proteins can reproduce some of the activities of the replicases, including the ability to initiate RNA synthesis by a de novo mechanism (22, 47-49). Furthermore, recombinant RdRps can affect the activities of other replicase subunits in vitro, suggesting that the recombinant RdRp is useful for an in-depth understanding of RNA synthesis by HCV (45, 60).RdRps form a right-hand-like structure with thumb, finger, and palm subdomains. The metal-coordinating residues important for nucleotide binding are positioned within the palm subdomain (26). An interesting feature of viral RdRps is that they tend to exist in a closed c...

The PAN-INDIA 1000 SARS-CoV-2 RNA Genome Sequencing Consortium has achieved its initial goal of completing the sequencing of 1000 SARS-CoV-2 genomes from nasopharyngeal and oropharyngeal swabs collected from individuals testing positive for COVID-19 by Real Time PCR. The samples were collected across 10 states covering different zones within India. Given the importance of this information for public health response initiatives investigating transmission of COVID-19, the sequence data is being released in GISAID database. This information will improve our understanding on how the virus is spreading, ultimately helping to interrupt the transmission chains, prevent new cases of infection, and provide impetus to research on intervention measures. This will also provide us with information on evolution of the virus, genetic predisposition (if any) and adaptation to human hosts. One thousand and fifty two sequences were used for phylodynamic, temporal and geographic mutation patterns and haplotype network analyses. Initial results indicate that multiple lineages of SARS-CoV-2 are circulating in India, probably introduced by travel from Europe, USA and East Asia. A2a (20A/B/C) was found to be predominant, along with few parental haplotypes 19A/B. In particular, there is a predominance of the D614G mutation, which is found to be emerging in almost all regions of the country. Additionally, mutations in important regions of the viral genome with significant geographical clustering have also been observed. The temporal haplotype diversities landscape in each region appears to be similar pan India, with haplotype diversities peaking between March-May, while by June A2a (20A/B/C) emerged as the predominant one. Within haplotypes, different states appear to have different proportions. Temporal and geographic patterns in the sequences obtained reveal interesting clustering of mutations. Some mutations are present at particularly high frequencies in one state as compared to others. The negative estimate Tajimas D (D = -2.26817) is consistent with the rapid expansion of SARS-CoV-2 population in India. Detailed mutational analysis across India to understand the gradual emergence of mutants at different regions of the country and its possible implication will help in better disease management.

Hepatitis C virus (HCV) infection leads to acute and chronic liver diseases, and new classes of anti-HCV therapeutics are needed. Cyclosporine A (CsA) inhibits HCV replication and CsA derivatives that lack the immunosuppressive function are currently in clinical trials as candidate anti-HCV drugs. Here we characterize several independently derived HCV replicons with varying levels of CsA resistance due to mutations in nonstructural protein 5B (NS5B), the HCV-encoded polymerase. Mutant HCV replicons engineered with these mutations showed resistance to CsA. The mutations reside in two distinct patches in the polymerase: the template channel and one face of a concave surface behind the template channel. Mutant NS5B made by cells expressing the HCV replicon had increased ability to bind to RNA in the presence of CsA. Purified recombinant NS5B proteins containing the mutations were better at de novo initiated RNA synthesis than the wild-type control. Furthermore, the mutant proteins were able to bind RNA with Ϸ8-fold higher affinity. Last, mutation near the template channel alleviated the lethal phenotype of a mutation in the concave patch, P540A. This intramolecular compensation for the HCV replicase function by amino acid changes in different domains was further confirmed in an infectious cell culture-derived virus system. C yclosporine A (CsA), a commonly used immunosuppressant for transplant patients, has recently emerged as a potential new anti-hepatitis C virus (HCV) therapeutic. CsA and its derivatives potently inhibit HCV replication both in cell-culture systems and in mice with transplanted human liver, 1-3 although no consensus has emerged on the in vivo benefits of using CsA over Tacrolimus (FK506), a compound that lacks anti-HCV effect in vitro, for HCV-infected liver-transplant patients. [4][5][6][7][8][9] More recent clinical trials with a CsA derivative, DEBIO-025, yield promising results in human immunodeficiency virus (HIV) and HCV-coinfected patients, but drug resistance in vivo has not been studied. 10 Studies of the antiviral effect of CsA on HCV replicons has led to the identification of cyclophilin (CyP) as an essential cofactor for HCV replication [11][12][13] ; point mutations in nonstructural protein 5B (NS5B) and NS5A are associated with CsA resistance in vitro. 14,15 In addition, NS5B interacts with both CyPA and CyPB both in vitro and in vivo. 12,13 These data establish NS5B as an indirect viral target for the CsA-mediated inhibition of HCV replication. The CyPA-NS5B interaction has also been shown to be the principal mediator of cyclosporine resistance in vitro. 12

Persistence of hepatitis C virus (HCV) infection is observed only in a subset of infected individuals and among them only some respond to treatment. Genome-wide association studies (GWAS) carried out around the world identified single nucleotide polymorphisms (SNPs) in the IL28B locus that are strongly associated with both HCV clearance and treatment response. The functional significance of these associations however, is not clear. In this report we show that an SNP rs28416813 in the distal promoter region of IL28B that is in close proximity to a non-consensus NF-κB-binding site affects downstream reporter gene expression. The effect is likely due to differential binding of NF-κB at the non-consensus site. The non-protective allele showed a reduction in luciferase reporter gene expression compared to the protective allele in HEK293T cells under different experimental conditions including treatment with tumor necrosis factor alpha (TNF-α) and 5′ triphosphorylated dsRNA. Furthermore, the HCV RNA polymerase was able to induce transcription from the IL28B promoter in a RIG-I-dependent manner. This induction was influenced by the alleles present at rs28416813. We also demonstrate strong linkage disequilibrium between rs28416813 and another important SNP rs12979860 in two ethnic populations. These results suggest possible mechanisms by which SNPs at the IL28B locus influence spontaneous clearance and treatment response in chronic HCV infections.