First isolation of SARS-CoV-2 from clinical samples in India.
Ontology highlight
ABSTRACT:
SUBMITTER: Sarkale P
PROVIDER: S-EPMC7366528 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundGiven the persistence of viral RNA in clinically recovered coronavirus disease 2019 (COVID-19) patients, subgenomic RNAs (sgRNAs) have been reported as potential molecular viability markers for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, few data are available on their longitudinal kinetics, compared with genomic RNA (gRNA), in clinical samples.MethodsWe analyzed 536 samples from 205 patients with COVID-19 from placebo-controlled, outpatient trials of peginterferon Lambda-1a (Lambda; n = 177) and favipiravir (n = 359). Nasal swabs were collected at 3 time points in the Lambda (days 1, 4, and 6) and favipiravir (days 1, 5, and 10) trials. N-gene gRNA and sgRNA were quantified by quantitative reverse transcription polymerase chain reaction. To investigate the decay kinetics in vitro, we measured gRNA and sgRNA in A549ACE2+ cells infected with SARS-CoV-2, following treatment with remdesivir or dimethylsulfoxide control.ResultsAt 6 days in the Lambda trial and 10 days in the favipiravir trial, sgRNA remained detectable in 51.6% (32/62) and 49.5% (51/106) of the samples, respectively. Cycle threshold (Ct) values for gRNA and sgRNA were highly linearly correlated (marginal R 2 = 0.83), and the rate of increase did not differ significantly in the Lambda trial (1.36 cycles/d vs 1.36 cycles/d; P = .97) or the favipiravir trial (1.03 cycles/d vs 0.94 cycles/d; P = .26). From samples collected 15-21 days after symptom onset, sgRNA was detectable in 48.1% (40/83) of participants. In SARS-CoV-2-infected A549ACE2+ cells treated with remdesivir, the rate of Ct increase did not differ between gRNA and sgRNA.ConclusionsIn clinical samples and in vitro, sgRNA was highly correlated with gRNA and did not demonstrate different decay patterns to support its application as a viability marker.
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Project description:Background & objectivesSince 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.MethodsThroat 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.ResultsThree 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 & conclusionsThe 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.
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Project description:ObjectivesA novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is responsible for the current coronavirus disease 2019 global pandemic. Only a few laboratories routinely isolate the virus, which is because the current co-culture strategy is highly time-consuming and requires a biosafety level 3 laboratory. This work aimed to develop a new high-throughput isolation strategy using novel technologies for rapid and automated isolation of SARS-CoV-2.MethodsWe used an automated microscope based on high-content screening (HCS), and we applied specific image analysis algorithms targeting cytopathic effects of SARS-CoV-2 on Vero E6 cells. A randomized panel of 104 samples, including 72 that tested positive by RT-PCR and 32 that tested negative, were processed with our HCS strategy and were compared with the classical isolation procedure.ResultsThe isolation rate was 43% (31/72) with both strategies on RT-PCR-positive samples and was correlated with the initial RNA viral load in the samples, in which we obtained a positivity threshold of 27 Ct. Co-culture delays were shorter with the HCS strategy, where 80% (25/31) of the positive samples were recovered by the third day of co-culture, compared with only 26% (8/30) with the classic strategy. Moreover, only the HCS strategy allowed us to recover all the positive samples (31 with HCS versus 27 with classic strategy) after 1 week of co-culture.ConclusionsThis system allows the rapid and automated screening of clinical samples with minimal operator workload, which reduces the risk of contamination and paves the way for future applications in clinical microbiology, such as large-scale drug susceptibility testing.
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