The unified proposal for classification of human respiratory syncytial virus below the subgroup level (preprint)

A globally implemented unified classification for human respiratory syncytial virus (HRSV) below the subgroup level remains elusive. Here, we formulate the global consensus of HRSV classification based on the challenges and limitations of our previous proposals and the future of genomic surveillance. From a high-quality dataset of 1,480 HRSV-A and 1,385 HRSV-B genomes submitted to NCBI and GISAID up to March 2023, we categorized HRSV-A/B sequences into lineages based on phylogenetic clades and amino acid markers. We defined 24 lineages within HRSV-A and 16 within HRSV-B, providing guidelines for prospective lineages definition. Our classification demonstrated robustness in its applicability to both complete and partial genomes. In addition, it allowed the observation of notable lineage replacements and the identification of lineages exclusively detected since the COVID-19 pandemic. We envision that this unified HRSV classification proposal will strengthen and facilitate HRSV molecular epidemiology on a global scale.

Genomic surveillance of SARS-CoV-2 and emergence of XBB.1.16 variant in Rajasthan (preprint)

Background & objectivesGenomic surveillance of positive SARS-CoV-2 samples is important to monitor the genetic changes occurring in virus, this was enhanced after the WHO designation of XBB.1.16 as a variant under monitoring in March 2023. From 5th February till 6th May 2023 all positive SARS-CoV-2 samples were monitored for genetic changes. MethodsA total of 1757 samples having Ct value <25 (for E and ORF gene) from different districts of Rajasthan were processed for Next Generation sequencing (NGS). The FASTA files obtained on sequencing were used for lineage determination using Nextclade and phylogenetic tree construction. Results and discussionSequencing and lineage identification was done in 1624 samples. XBB.1.16 was the predominant lineage in 1413(87.0%) cases while rest was other XBB (207, 12.74%) and other lineages (4, 0.2%). Of the 1413 XBB.1.16 cases, 57.47% were males and 42.53% were females. Majority (66.53%) belonged to 19-59 year age. 84.15% of XBB.1.16 cases were infected for the first time. Hospitalization was required in only 2.2% cases and death was reported in 5 (0.35%) patients. Most of the cases were symptomatic and the commonest symptoms were fever, cough and rhinorrhoea. Co-morbidities were present in 414 (29.3%) cases. Enhanced genomic surveillance helped to rapidly identify the spread of XBB variant in Rajasthan. This in turn helped to take control measures to prevent spread of virus and estimate public health risks of the new variant relative to the previously circulating lineages. XBB variant was found to spread rapidly but produced milder disease.

Chasing SARS-CoV-2 XBB.1.16 Recombinant Lineage in India and the Clinical Profile of XBB.1.16 cases in Maharashtra, India (preprint)

ABSTRACT Background: SARS-CoV-2 has evolved rapidly, resulting in emergence of lineages with competitive advantage over one another. Co-infections with different SARS-CoV-2 lineages can give rise to recombinant lineages. To date, XBB lineage is the most widespread recombinant lineage worldwide, with the recently named XBB.1.16 lineage causing a surge in the number of COVID-19 cases in India. Methodology: The present study involved retrieval of SARS-CoV-2 genome sequences from India (between 1st December 2022 and 8th April 2023) through GISAID; sequences were curated, followed by lineage and phylogenetic analysis. Demographic and clinical data from Maharashtra, India were collected telephonically, recorded in Microsoft (R) Excel, and analysed using IBM (R) SPSS statistics, version 29.0.0.0 (241). Results: A total of 2,944 sequences were downloaded from the GISAID database, of which 2,856 were included in the study following data curation. The sequences from India were dominated by the XBB.1.16* lineage (36.17%) followed by XBB.2.3* (12.11%) and XBB.1.5* (10.36%). Of the 2,856 cases, 693 were from Maharashtra; 386 of these were included in the clinical study. The clinical features of COVID-19 cases with XBB.1.16* infection (XBB.1.16* cases, 276 in number) showed that 92% of those had a symptomatic disease, with fever (67%), cough (42%), rhinorrhoea (33.7%), body ache (14.5%) and fatigue (14.1%) being the most common symptoms. Presence of comorbidity was found in 17.7% of the XBB.1.16* cases. Among the XBB.1.16* cases, 91.7% were vaccinated with at least one dose of vaccine against COVID-19. While 74.3% of XBB.1.16* cases were home-isolated; 25.7% needed hospitalization/institutional quarantine, of these, 33.8% needed oxygen therapy. Out of 276 XBB.1.16* cases, seven (2.5%) cases succumbed to the disease. Majority of XBB.1.16* cases who died belonged to an elderly age group (60 years and above), had underlying comorbid condition/s, and needed supplemental oxygen therapy. The clinical features of COVID-19 cases infected with other co-circulating Omicron variants were similar to XBB.1.16* cases. Conclusion: The study reveals that XBB.1.16* lineage has become the most predominant SARS-CoV-2 lineage in India. The study also shows that the clinical features and outcome of XBB.1.16* cases were similar to those of other co-circulating Omicron lineage infected cases in Maharashtra, India. Keywords: XBB.1.16, XBB.1.16.1, XBB.1.16*, XBB, Omicron variant, COVID-19, SARS-CoV-2, Clinical features

Clinical Characteristics and Outcomes of Laboratory-Confirmed SARS-CoV-2 Cases Infected with Omicron subvariants and XBB recombinant variant (preprint)

Background: The SARS-CoV-2 has evolved to produce new variants causing successive waves of infection. Currently, six variants are being monitored by the World Health Organization that are replacing BA.5. These include BQ.1*, BA.5 with one or several of five mutations (R346X, K444X, V445X, N450D, N460X), BA.2.75*, XBB*, BA.4.6*, and BA.2.30.2*. BQ.1 and XBB variants are more immune evasive and have spread quickly throughout the world. With the concern of the potential severity of infections caused by these variants, the present study describes the clinical characteristics and outcomes of these major variants in Maharashtra. Material and Methods: A total of 1039 Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) positive SARS-CoV-2 samples, with a cycle threshold value (Ct) less than 25, were processed for SARS-CoV-2 whole genome sequencing between 10th July 2022 and 10th December 2022. All corresponding demographic and clinical data were recorded and analyzed using MicrosoftTM ExcelTM and Epi InfoTM. Results: Out of 1039 samples sequenced, 829 (79.79%) were assigned Pango lineages, of which BA.2.75 (67.31%) was the predominant Omicron variant, followed by the XBB* (17.13%), BA.2.38* (5.43%), BA.2.10* (3.62%) and BA.5* (3.50%). A total of 494 cases were contacted telephonically, of which 455 (92.11%) were symptomatic with mild symptoms. Fever (78.46%) was the most common symptom, followed by rhinorrhoea (46.37%), cough (42.20%), myalgia (19.56%) and fatigue (18.24%). Of the 494 cases, 379 (76.72%) cases recovered at home, and 115 (23.28%) were institutionally quarantined/ hospitalized. Among the home-isolated and hospitalized cases, 378 (99.74%) and 101 (87.83%) recovered with symptomatic treatment, whereas 01 (0.26%) and 14 (12.17%) succumbed to the disease, respectively. Of the 494 cases, 449 (90.89%) were vaccinated with at least one dose of the COVID-19 vaccine, 40 (8.10%) were unvaccinated, and for 05 (1.01%) cases, vaccine data was not available. Conclusion: The current study indicates that the XBB* variant is causing mild disease in India. However, as XBB* possess both immune-escape and infectivity-enhancing mutations, it has the potential to spread to other parts of the world rapidly.

Substantial immune response in Omicron infected breakthrough and unvaccinated individuals against SARS-CoV-2 variants of concerns (preprint)

The recent emergence of highly mutated SARS-CoV-2 Omicron variant has debilitating effect on public health system of the affected countries worldwide. Currently India is facing third wave of COVID-19 pandemic and going through a severe crisis. Within short span of time, the variant has shown high transmissibility and capability of evading the immune response generated against natural infection and vaccination. The immune escape potential of Omicron is a serious concern and further needs to be explored. In the present study, we have assessed the IgG and neutralizing antibody (NAb) response in breakthrough individuals vaccinated with two doses ChAdOx1 nCoV-19 vaccine (n=25), breakthrough individuals vaccinated with two doses of BNT162b2 mRNA vaccine (n=8) and unvaccinated individuals (n=6). All these individuals were infected with Omicron variant. The IgG antibody activity in the sera of the ChAdOx1 nCoV-19 and BNT162b2 mRNA breakthrough individuals was comparable with S1-RBD, while it was lesser in BNT162b2 mRNA breakthrough individuals with N protein and inactivated whole antigen IgG ELISA. BNT162b2 mRNA breakthrough individuals showed moderate reduction in NAb GMTs compared to ChAdOx1 nCoV-19 against Alpha, Beta and Delta. However, 3-fold higher reduction was observed with omicron variant in BNT162b2 mRNA than ChAdOx1 nCoV-19. Apparently, Alpha variant was modestly resistant to the sera of unvaccinated individuals than Beta, Delta and Omicron. Our study demonstrated substantial immune response in that the individuals infected with Omicron. The neutralizing antibodies could effectively neutralize the Omicron and other VOCs including the most prevalent Delta variant.

An in vitro and in vivo approach for the isolation of Omicron variant from human clinical specimens (preprint)

Due to failure of virus isolation of Omicron variant in Vero CCL-81 from the clinical specimens of COVID-19 cases, we infected Syrian hamsters and then passage into Vero CCL-81 cells. The Omicron sequences were studied to assess if hamster could incorporate any mutation to changes its susceptibility. L212C mutation, Tyrosine 69 deletion, and C25000T nucleotide change in spike gene and absence of V17I mutation in E gene was observed in sequences of hamster passage unlike human clinical specimen and Vero CCL-81 passages. No change was observed in the furin cleavage site in any of the specimen sequence which suggests usefulness of these isolates in future studies.

Respiratory Viruses Among Ethnic Nicobarese During COVID-19 Pandemic (preprint)

Background: Acute respiratory infections (ARIs) and severe acute respiratory illness (SARI) are public health burdens globally. The percentage of non-SARS CoV-2 respiratory viruses among patients having ARI and SARI who visit Car Nicobar's hospital settings is undocumented. Changes in the epidemiology of other respiratory viruses during COVID19 pandemic is being reported worldwide. Methods: : Inpatient and outpatient settings at BJR hospital, Car Nicobar Island, India, were used to conduct prospective monitoring for ARI and SARI among Nicobarese tribal members. The patients with ARI and SARI were enlisted in BJR hospital from June 2019 to May 2021. At the ICMR-NIV in Pune, duplex qRTPCR assays were used to test the presence of respiratory viruses. The prevalence of non- SARS CoV-2 respiratory viruses was measured by comparing here between pandemic and pre-pandemic periods. Results: : During the COVID19 pandemic, Influenza A (H3N2) (55.7%), and rhinovirus (12.5%) were predominantly reported non-SARS CoV-2 respiratory viruses while Human metapneumovirus (48.1%) and influenza A (H1N1)pdm09 (18.5%) were most commonly reported in the prepandemic period. This result indicates the altered circulation of non-SARS CoV-2 during pandemic. Conclusions: : A considerable proportion of respiratory infection was correlated with respiratory viruses. Prevalence of non-SARS CoV-2 respiratory viruses was high at the time of infection when compared with pre-pandemic period, at Car Nicobar Island. This study enlightened the change in circulation of other respiratory viruses among the indigenous Nicobarese tribes. Clinicians and allied medical staff should be more prudent of these respiratory infections.

Delta Variant SARS-CoV-2 infections in pediatric cases during the second wave in India (preprint)

The aim of this study was to identify the SARS-CoV-2 lineages circulating in the pediatric population of India during the second wave of the pandemic. Clinical and demographic details linked with the nasopharyngeal/oropharyngeal swabs (NPS/OPS) collected from SARS-CoV-2 cases (n=583) aged 0-18 year and tested positive by real-time RT-PCR were retrieved from March to June 2021.Symptoms were reported among 37.2% of patients and 14.8% reported to be hospitalized. The E gene CT value had significant statistical difference at the point of sample collection when compared to that observed in the sequencing laboratory. Out of these 512 sequences 372 were VOCs, 51 were VOIs. Most common lineages observed were Delta, followed by Kappa, Alpha and B.1.36, seen in 65.82%, 9.96%, 6.83% and 4.68%, respectively in the study population. Overall, it was observed that Delta strain was the leading cause of SARS-CoV-2 infection in Indian children during the second wave of the pandemic. We emphasize on the need of continuous genomic surveillance in SARS-CoV-2 infection even amongst children.

Efficacy, safety, and lot to lot immunogenicity of an inactivated SARS-CoV-2 vaccine (BBV152): a double-blind, randomised, controlled phase 3 trial (preprint)

BackgroundWe report the clinical efficacy against COVID-19 infection of BBV152, a whole-virion inactivated SARS-CoV-2 vaccine formulated with a Toll-like receptor 7/8 agonist molecule adsorbed to alum (Algel-IMDG). MethodsWe did a double-blind, randomised, multicentre, phase 3 clinical trial in 25 Indian hospitals to evaluate the efficacy, safety, and immunological lot consistency of BBV152. Healthy adults (age 18-98 years) randomised 1:1 using a computer-generated randomisation scheme received two intramuscular doses of vaccine or placebo administered four weeks apart. The primary outcome was laboratory-confirmed symptomatic COVID-19, occurring at least 14 days after the second dose. Secondary outcomes were efficacy in sub-groups for age (18-< 60 years and [≥] 60 years) and in participants with pre-existing stable medical conditions. We also evaluated safety, reactogenicity, and consistency of immune responses for three consecutive manufacturing lots. FindingsBetween November 16, 2020 and January 7, 2021 we recruited 25,798 participants who were randomised to BBV152 or placebo groups; 24,419 received two doses of BBV152 (n = 12,221) or placebo (n = 12,198). In a case-driven analysis, 130 cases of symptomatic COVID-19 were reported in 16,973 (0{middle dot}77%) participants with follow-up at least two weeks after the second vaccination; 24 occurred in the vaccine group and 106 in placebo recipients giving an overall vaccine efficacy of 77{middle dot}8% (95% CI: 65{middle dot}2-86{middle dot}4). Sixteen cases, one vaccinee and 15 placebo recipients, met the severe symptomatic COVID-19 case definition giving a vaccine efficacy of 93{middle dot}4% (57{middle dot}1-99{middle dot}8). Efficacy against asymptomatic COVID-19 was 63{middle dot}6% (29{middle dot}0-82{middle dot}4). BBV152 conferred 65{middle dot}2% (95% CI: 33{middle dot}1-83{middle dot}0) protection against the SARS-CoV-2 Variant of Concern, B.1.617.2 (Delta). BBV152 was well tolerated with no clinically or statistically significant differences in the distributions of solicited, unsolicited, or serious adverse events between vaccine and placebo groups. No cases of anaphylaxis or vaccine-related deaths were reported. InterpretationBBV152 was immunogenic and highly efficacious against symptomatic and asymptomatic COVID-19 variant associated disease, particularly against severe disease in adults. Vaccination was well tolerated with an overall incidence of adverse events observed over a median of 146 days that was lower than that observed with other COVID-19 vaccines. FundingThis work was supported and funded by Bharat Biotech International Limited and partly co-funded by the Indian Council of Medical Research. Clinicaltrials.gov: NCT04641481

Isolation of SARS-CoV-2 B.1.1.28.2 P2 variant and pathogenicity comparison with D614G variant in hamster model (preprint)

Background: Considering the potential threat from emerging SARS-CoV-2 variants and the rising COVID-19 cases, SARS-CoV-2 genomic surveillance is ongoing in India. We report herewith the isolation of the P.2 variant (B.1.1.28.2) from international travelers and further its pathogenicity evaluation and comparison with D614G variant (B.1) in hamster model. Methods: Virus isolation was performed in Vero CCL81 cells and genomic characterization by next generation sequencing. The pathogenicity of the isolate was assessed in Syrian hamster model and compared with B.1 variant. Results: B.1.1.28.2 variant was isolated from nasal/throat swabs of international travelers returned to India from United Kingdom and Brazil. The B.1.1.28.2 variant induced body weight loss, viral replication in the respiratory tract, lung lesions and caused severe lung pathology in infected Syrian hamster model in comparison, with B.1 variant infected hamsters. The sera from B.1.1.28.2 infected hamsters efficiently neutralized the D614G variant virus whereas 6-fold reduction in the neutralization was seen in case of D614G variant infected hamsters sera with the B.1.1.28.2 variant. Conclusions: B.1.1.28.2 lineage variant could be successfully isolated and characterization could be performed. Pathogenicity of the isolate was demonstrated in Syrian hamster model and in comparison, with B.1 variant was found more pathogenic. The findings of increased disease severity and neutralization reduction is of great concern and point towards the need for screening the vaccines for efficacy.

Isolation and characterization of SARS-CoV-2 VOC, 20H/501Y.V2, from UAE travelers (preprint)

Multiple SARS-CoV-2 variants have been emerged and created serious public health in the affected countries. The variant of Concern associated with high transmissibility, disease severity and escape mutations is threat to vaccination program across the globe. Travel has been important factor in spread of SARS-CoV-2 variants worldwide. India has also witnessed the dreadful effect of these SARS-CoV-2 variants. Here, we report the Isolation and characterization of SARS-CoV-2 VOC, 20H/501Y.V2 (B.1.351), from UAE travelers to India. The virus isolate would be useful to determine the efficacy of the currently available vaccines in India.

SARS CoV-2 variant B.1.617.1 is highly pathogenic in hamsters than B.1 variant (preprint)

Background: The recent emergence of new SARS-CoV-2 lineage B.1.617 in India has been associated with a surge in the number of daily infections. This variant has combination of specific mutations L452R, E484Q and P681R reported to possibly enhance the transmissibility with likelihood of escaping the immunity. We investigated the viral load and pathogenic potential of B.1.617.1 in Syrian golden hamsters. Methods: Two groups of Syrian golden hamsters (9 each) were inoculated intranasally with SARS CoV-2 isolates, B.1 (D614G) and B.1.617.1 respectively. The animals were monitored daily for the clinical signs and body weight. The necropsy of three hamsters each was performed on 3, 5- and 7-days post-infection (DPI). Throat swab (TS), nasal wash (NW) and organ samples (lungs, nasal turbinate, trachea) were collected and screened using SARS-CoV-2 specific Real-time RT-PCR. Results: The hamsters infected with B.1.617.1 demonstrated increased body weight loss compared to B.1 variant. The highest viral load was observed in nasal turbinate and lung specimens of animals infected with B.1.167.1 on 3 DPI. Neutralizing antibody (NAb) and IgG response in hamsters of both the groups were observed from 5 and 7 DPI respectively. However, higher neutralizing antibody titers were observed against B.1.167.1. Gross pathology showed pronounced lung lesions and hemorrhage with B.1.671 compared to B.1. Conclusions: B.1617.1 and B.1 variant varied greatly in their infectiousness, pathogenesis in hamster model. This study demonstrates higher pathogenicity in hamsters evident with reduced body weight, higher viral load in lungs and pronounced lung lesions as compared to B.1 variant.

Convergent evolution of SARS-CoV-2 spike mutations, L452R, E484Q and P681R, in the second wave of COVID-19 in Maharashtra, India (preprint)

As the global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic expands, genomic epidemiology and whole genome sequencing are being constantly used to investigate its transmissions and evolution. In the backdrop of the global emergence of variants of concern (VOCs) during December 2020 and an upsurge in a state in the western part of India since January 2021, whole genome sequencing and analysis of spike protein mutations using sequence and structural approaches was undertaken to identify possible new variants and gauge the fitness of current circulating strains. Phylogenetic analysis revealed that the predominant clade in circulation was a distinct newly identified lineage B.1.617 possessing common signature mutations D111D, G142D, L452R, E484Q, D614G and P681R, in the spike protein including within the receptor binding domain (RBD). Of these, the mutations at residue positions 452, 484 and 681 have been reported in other globally circulating lineages. The structural analysis of RBD mutations L452R and E484Q along with P681R in the furin cleavage site, may possibly result in increased ACE2 binding and rate of S1-S2 cleavage resulting in better transmissibility. The same two RBD mutations indicated decreased binding to selected monoclonal antibodies (mAbs) and may affect their neutralization potential. Experimental validation is warranted for accessing both ACE2 binding and the effectiveness of commonly elicited neutralizing mAbs for the strains of lineage B.1.617. The emergence of such local variants through the accumulation of convergent mutations during the COVID-19 second wave needs to be further investigated for their public health impact in the rest of the country and its possibility of becoming a VOC.