The rapid emergence of coronavirus disease 2019 (COVID-19) as a global pandemic affecting millions of individuals globally has necessitated sensitive and high-throughput approaches for the diagnosis, surveillance, and determining the genetic epidemiology of SARS-CoV-2. In the present study, we used the COVIDSeq protocol, which involves multiplex-PCR, barcoding, and sequencing of samples for high-throughput detection and deciphering the genetic epidemiology of SARS-CoV-2. We used the approach on 752 clinical samples in duplicates, amounting to a total of 1536 samples which could be sequenced on a single S4 sequencing flow cell on NovaSeq 6000. Our analysis suggests a high concordance between technical duplicates and a high concordance of detection of SARS-CoV-2 between the COVIDSeq as well as RT-PCR approaches. An in-depth analysis revealed a total of six samples in which COVIDSeq detected SARS-CoV-2 in high confidence which were negative in RT-PCR. Additionally, the assay could detect SARS-CoV-2 in 21 samples and 16 samples which were classified inconclusive and pan-sarbeco positive respectively suggesting that COVIDSeq could be used as a confirmatory test. The sequencing approach also enabled insights into the evolution and genetic epidemiology of the SARS-CoV-2 samples. The samples were classified into a total of 3 clades. This study reports two lineages B.1.112 and B.1.99 for the first time in India. This study also revealed 1,143 unique single nucleotide variants and added a total of 73 novel variants identified for the first time. To the best of our knowledge, this is the first report of the COVIDSeq approach for detection and genetic epidemiology of SARS-CoV-2. Our analysis suggests that COVIDSeq could be a potential high sensitivity assay for the detection of SARS-CoV-2, with an additional advantage of enabling the genetic epidemiology of SARS-CoV-2.

In the last few months, there has been a global catastrophic outbreak of severe acute respiratory syndrome disease caused by the novel coronavirus SARS-CoV-2 affecting millions of people worldwide. Early diagnosis and isolation are key to contain the rapid spread of the virus. Towards this goal, we report a simple, sensitive and rapid method to detect the virus using a targeted mass spectrometric approach, which can directly detect the presence of virus from naso-oropharyngeal swabs. Using a multiple reaction monitoring we can detect the presence of two peptides specific to SARS-CoV-2 in a 2.3 min gradient run with 100% specificity and 90.5% sensitivity when compared to RT-PCR. Importantly, we further show that these peptides could be detected even in the patients who have recovered from the symptoms and have tested negative for the virus by RT-PCR highlighting the sensitivity of the technique. This method has the translational potential of in terms of the rapid diagnostics of symptomatic and asymptomatic COVID-19 and can augment current methods available for diagnosis of SARS-CoV-2.

The rapid emergence of coronavirus disease 2019 (COVID-19) as a global pandemic affecting millions of individuals globally has necessitated sensitive and high-throughput approaches for the diagnosis, surveillance and for determining the genetic epidemiology of SARS-CoV-2. In the present study, we used the COVIDSeq protocol, which involves multiplex-PCR, barcoding and sequencing of samples for high-throughput detection and deciphering the genetic epidemiology of SARS-CoV-2. We used the approach on 752 clinical samples in duplicates, amounting to a total of 1536 samples which could be sequenced on a single S4 sequencing flow cell on NovaSeq 6000. Our analysis suggests a high concordance between technical duplicates and a high concordance of detection of SARS-CoV-2 between the COVIDSeq as well as RT-PCR approaches. An in-depth analysis revealed a total of six samples in which COVIDSeq detected SARS-CoV-2 in high confidence which were negative in RT-PCR. Additionally, the assay could detect SARS-CoV-2 in 21 samples and 16 samples which were classified inconclusive and pan-sarbeco positive respectively suggesting that COVIDSeq could be used as a confirmatory test. The sequencing approach also enabled insights into the evolution and genetic epidemiology of the SARS-CoV-2 samples. The samples were classified into a total of 3 clades. This study reports two lineages B.1.112 and B.1.99 for the first time in India. This study also revealed 1,143 unique single nucleotide variants and added a total of 73 novel variants identified for the first time. To the best of our knowledge, this is the first report of the COVIDSeq approach for detection and genetic epidemiology of SARS-CoV-2. Our analysis suggests that COVIDSeq could be a potential high sensitivity assay for the detection of SARS-CoV-2, with an additional advantage of enabling genetic epidemiology of SARS-CoV-2.

Methylenetetrahydrofolate reductase (MTHFR) links the folate cycle to the methionine cycle in one-carbon metabolism. The enzyme is known to be allosterically inhibited by S-adenosyl methionine (SAM) for decades, but the importance of this regulatory control to one carbon metabolism has never been adequately understood. To shed light on this issue, we exchanged selected amino acid residues in a highly conserved stretch within the regulatory region of yeast MTHFR to create a series of feedback-insensitive, deregulated mutants. These were exploited to investigate the impact of defective allosteric regulation on one carbon metabolism. We observed a strong growth defect in the presence of methionine. Biochemical and metabolite analysis revealed that both the folate and methionine cycles were affected in these mutants, as was the transulfuration pathway, leading also to a disruption in redox homeostasis. The major consequences, however, appeared to be in the depletion of nucleotides. 13C isotope labelling and metabolic studies revealed that the deregulated MTHFR cells undergo continuous transmethylation of homocysteine by methyltetrahydrofolate (CH3THF) to form methionine. This reaction also drives SAM formation and further depletes ATP reserves. SAM was then cycled back to methionine, leading to futile cycles of SAM synthesis and recycling, and explaining the necessity for MTHFR to be regulated by SAM. The study has yielded valuable new insights into the regulation of one carbon metabolism, and the mutants appear as powerful new tools to further dissect out the intersection of one carbon metabolism with various pathways both in yeasts and in humans.

To understand the spread of SARS-CoV2, in August and September 2020, the Council of Scientific and Industrial Research (India), conducted a sero-survey across its constituent laboratories and centers across India. Of 10,427 volunteers, 1058 (10.14%) tested positive for SARS CoV2 anti-nucleocapsid (anti-NC) antibodies; 95% of which had surrogate neutralization activity. Three-fourth of these recalled no symptoms. Repeat serology tests at 3 (n=607) and 6 (n=175) months showed stable anti-NC antibodies but declining neutralization activity. Local sero-positivity was higher in densely populated cities and was inversely correlated with a 30 day change in regional test positivity rates (TPR). Regional seropositivity above 10% was associated with declining TPR. Personal factors associated with higher odds of sero-positivity were high-exposure work (Odds Ratio, 95% CI, p value; 2∙23, 1∙92–2∙59, <0.0001), use of public transport (1∙79, 1∙43–2∙24, <0.0001), not smoking (1∙52, 1∙16–1∙99, 0∙0257), non-vegetarian diet (1∙67, 1∙41–1∙99, <0.0001), and B blood group (1∙36,1∙15-1∙61, 0∙001).

In the last few months, there has been a global catastrophic outbreak of severe acute respiratory syndrome disease caused by the novel corona virus SARS-CoV-2 affecting millions of people worldwide. Early diagnosis and isolation is key to contain the rapid spread of the virus. Towards this goal, we report a simple, sensitive and rapid method to detect the virus using a targeted mass spectrometric approach, which can directly detect the presence of virus from naso-oropharyngeal swabs. Using a multiple reaction monitoring we can detect the presence of two peptides specific to SARS-CoV-2 in a 2.3 minute gradient run with 100% specificity and 90.4 % sensitivity when compared to RT-PCR. Importantly, we further show that these peptides could be detected even in the patients who have recovered from the symptoms and have tested negative for the virus by RT-PCR highlighting the sensitivity of the technique. This method has the translational potential of in terms of the rapid diagnostics of symptomatic and asymptomatic COVID-19 and can augment current methods available for diagnosis of SARS-CoV-2.

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Background: SARS-CoV-2 infection has caused 64,469 deaths in India, with 7, 81, 975 active cases till 30th August 2020, lifting it to 3rd rank globally. To estimate the burden of the disease with time it is important to undertake a longitudinal seroprevalence study which will also help to understand the stability of anti SARS-CoV-2 antibodies. Various studies have been conducted worldwide to assess the antibody stability. However, there is very limited data available from India. Healthcare workers (HCW) are the frontline workforce and more exposed to the COVID-19 infection (SARS-CoV-2) compared to the community. This study was conceptualized with an aim to estimate the seroprevalence in hospital and general population and determine the stability of anti SARS-CoV-2 antibodies in HCW. Methods: Staff of a tertiary care hospital in Delhi and individuals visiting that hospital were recruited between April to August 2020. Venous blood sample, demographic, clinical, COVID-19 symptoms, and RT-PCR data was collected from all participants. Serological testing was performed using the electro-chemiluminescence based assay developed by Roche Diagnostics, in Cobas Elecsys 411. Seropositive participants were followed- upto 83 days to check for the presence of antibodies. Results: A total of 780 participants were included in this study, which comprised 448 HCW and 332 individuals from the general population. Among the HCW, seroprevalence rates increased from 2.3% in April to 50.6% in July. The cumulative prevalence was 16.5% in HCW and 23.5% (78/332) in the general population with a large number of asymptomatic individuals. Out of 74 seropositive HCWs, 51 were followed-up for the duration of this study. We observed that in all seropositive cases the antibodies were sustained even up to 83 days. Conclusion: The cumulative prevalence of seropositivity was lower in HCWs than the general population. There were a large number of asymptomatic cases and the antibodies developed persisted through the duration of the study. More such longitudinal serology studies are needed to better understand the antibody response kinetics.