Molecular determinants associated with temporal succession of SARS-CoV-2 variants in Uttar Pradesh, India.

The emergence and rapid evolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) caused a global crisis that required a detailed characterization of the dynamics of mutational pattern of the viral genome for comprehending its epidemiology, pathogenesis and containment. We investigated the molecular evolution of the SASR-CoV-2 genome during the first, second and third waves of COVID-19 in Uttar Pradesh, India. Nanopore sequencing of the SARS-CoV-2 genome was undertaken in 544 confirmed cases of COVID-19, which included vaccinated and unvaccinated individuals. In the first wave (unvaccinated population), the 20A clade (56.32%) was superior that was replaced by 21A Delta in the second wave, which was more often seen in vaccinated individuals in comparison to unvaccinated (75.84% versus 16.17%, respectively). Subsequently, 21A delta got outcompeted by Omicron (71.8%), especially the 21L variant, in the third wave. We noticed that Q677H appeared in 20A Alpha and stayed up to Delta, D614G appeared in 20A Alpha and stayed in Delta and Omicron variants (got fixed), and several other mutations appeared in Delta and stayed in Omicron. A cross-sectional analysis of the vaccinated and unvaccinated individuals during the second wave revealed signature combinations of E156G, F157Del, L452R, T478K, D614G mutations in the Spike protein that might have facilitated vaccination breach in India. Interestingly, some of these mutation combinations were carried forward from Delta to Omicron. In silico protein docking showed that Omicron had a higher binding affinity with the host ACE2 receptor, resulting in enhanced infectivity of Omicron over the Delta variant. This work has identified the combinations of key mutations causing vaccination breach in India and provided insights into the change of [virus's] binding affinity with evolution, resulting in more virulence in Delta and more infectivity in Omicron variants of SARS-CoV-2. Our findings will help in understanding the COVID-19 disease biology and guide further surveillance of the SARS-CoV-2 genome to facilitate the development of vaccines with better efficacies.

Phase III, Randomized, Double-Blind, Placebo Controlled Trial of Efficacy, Safety and Tolerability of Antiviral Drug Umifenovir vs Standard Care of Therapy in Non-Severe Covid-19 Patients (preprint)

SummaryBackground: SARS-Cov2 has caused millions of deaths worldwide and effective antivirals are not yet available. We evaluated the efficacy, tolerability and safety of Umifenovir, a drug used originally against influenza in Russia and China, in non-severe COVID19 adult patients from India. Method : A double-blind placebo controlled Phase III trial spread over 3 hospitals in Lucknow, India viz. King George’s Medical University, ERAs Lucknow Medical College and Ram Manohar Lohia Institute of Medical Sciences was carried out. RT-PCR confirmed COVID19 patients in the age group from 18-75 yrs. were recruited and further stratified into Mild-asymptomatic and Moderate cases. Exclusions included, pregnant or lactating women, severe patients, those suffering from Acute respiratory distress syndrome (ARDS), sepsis shock, requiring invasive ventilator support, ECMO or shock requiring vasopressor support, severe liver disease, severe renal impairment, comorbid conditions like asthma, diabetes with second-third line medicines as defined in the WHO guidance document. Patients were randomized 1:1 on placebo with standard care of therapy or Umifenovir (800 mg BID, maximum 14 days) with standard care of therapy respectively. Computerized randomization was carried out for each group separately through sequentially numbered, opaque, sealed envelopes (SNOSE) in block sizes of six and was administered independently through clinical-site coordinators and to have sufficient number of Mild-asymptomatic and moderate patients. The primary endpoint for Asymptotic-mild patients was time to nasopharyngeal swab negativity by two RT-PCR tests for SARS COV2 antigens taken 24 hrs apart from the date of randomization. For Moderate patients, the average change in the ordinal scale from the baseline scores from randomization on the eight-point ordinal scale as defined by WHO was calculated as the primary endpoint. This trial is registered with the Clinical Trial Registry of India (CTRI) Number: CTRI/2020/09/027535.Findings: A total of 132 patients were recruited in the trial after screening between 3rd October 2020 to 28th April 2021. Of these, 9 patients withdrew consent/ stopped medication on their own. The remaining 123 patients were almost equally distributed into Asymptomatic (35%), Mild (32%) and Moderate (33%) symptoms groups respectively. No Serious adverse events were noted in any of the patients. Only few minor events like headache, stomach ache and nausea were reported and this also was observed almost equally between the Umifenovir and Standard-of-care arms respectively. In the Primary endpoint corresponding to the Mild-asymptomatic patient group (n=82), we found that 73% patients in the Umifenovir arm were RT-PCR negative on the 5th day (P=0.004), while only 40% patients in the placebo arm were negative. In the moderate group, the WHO scores for the Umifenovir arm corresponded to faster clinical improvement as compared to the Placebo arm (P=0.125 on day 3). In the Mild-asymptomatic group, the clinical improvement assessed by the WHO score on day 5 was statistically significant (P=0.019) in the Umifenovir arm compared to the placebo arm and agrees well with the primary endpoint results. Interpretation: Umifenovir is efficacious for Mild-asymptomatic patients and meets the primary and secondary endpoint criteria of the trial. The drug is safe and well tolerated at a dosage of 800 mg BID, maximum 14 days, in adult patients. It exhibits faster time to cure with median 5 days (95%CI, 5-14) in Umifenovir group and median 7 days (95%CI, 5-14) for the placebo group. In moderate patients, administration of Umifenovir co-relates with faster clinical recovery, albeit with less statistical significance. In future studies, the drug should be evaluated as a prophylactic in high-risk adults, and for efficacy in children and pregnant/lactating women.

Severe Acute Respiratory Syndrome Coronavirus-2 genome sequence variations relate to morbidity and mortality in Coronavirus Disease-19 (preprint)

Outcome of infection with Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) may depend on the host, virus or the host-virus interaction related factors. Complete SARS-CoV-2 genome was sequenced using Illumina and Nanopore platforms from naso-/oro-pharyngeal ri-bonucleic acid (RNA) specimens from COVID-19 patients of varying severity and outcomes, including patients with mild upper respiratory symptoms (n=35), severe disease admitted to intensive care with respiratory and gastrointestinal symptoms (n=21), fatal COVID-19 outcome (n=17) and asymptomatic (n=42). Of a number of genome variants observed, p.16L>L (Nsp1), p.39C>C (Nsp3), p.57Q>H (ORF3a), p.71Y>Y (Membrane glycoprotein), p.194S>L (Nucleocapsid protein) were observed in similar frequencies in different patient subgroups. However, seventeen other variants were observed only in symptomatic patients with severe and fatal COVID-19. Out of the latter, one was in the 5UTR (g.241C>T), eight were synonymous (p.14V>V and p.92L>L in Nsp1 protein, p.226D>D, p.253V>V, and p.305N>N in Nsp3, p.34G>G and p.79C>C in Nsp10 protein, p.789Y>Y in Spike protein), and eight were non-synonymous (p.106P>S, p.157V>F and p.159A>V in Nsp2, p.1197S>R and p.1198T>K in Nsp3, p.97A>V in RdRp, p.614D>G in Spike protein, p.13P>L in nucleocapsid). These were completely absent in the asymptomatic group. SARS-CoV-2 genome variations have a significant impact on COVID-19 presentation, severity and outcome.