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Existing real-time reverse transcriptase PCR (RT-qPCR) has certain limitations for the point-of-care detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since it requires sophisticated instruments, reagents and skilled laboratory personnel. In this study, we evaluated an assay termed the reverse transcriptase-polymerase spiral reaction (RT-PSR) for rapid and visual detection of SARS-CoV-2.The RT-PSR assay was optimized using RdRp gene and evaluated for the detection of SARS-CoV-2. The time of 60min and a temperature of 63°C was optimized for targeting the RNA-dependent RNA polymerase gene of SARS-CoV-2. The sensitivity of the assay was evaluated by diluting the in-vitro transcribed RNA, which amplifies as low as ten copies.The specific primers designed for this assay showed 100% specificity and did not react when tested with other lung infection-causing viruses and bacteria. The optimized assay was validated with 190 clinical samples in two phases, using automated RTPCR based TrueNat test, and the results were comparable.The RT-PSR assay can be considered for rapid and sensitive detection of SARS-CoV-2, particularly in resource-limited settings. To our knowledge, there is as yet no RT-PSR-based kit developed for SARS-CoV-2.

In this study we report the whole genome sequencing (WGS) based analysis of blood-borne Campylobacter fetus subsp. fetus MMM01 isolated from a diabetic patient to obtain deeper insights in to the virulence and host adaptability. The sequenced genome of C. fetus subsp. fetus MMM01 along with reference genomes retrieved from NCBI was subjected to various in-silico analysis including JSpecies, MLST server, PATRIC server, VFanalyzer, CARD, PHASTER to understand their phylogenetic relation, virulence and antimicrobial resistance profile. The genome had a size of 1,788,790 bp, with a GC content of 33.09%, nearly identical to the reference strain C. fetus subsp. fetus 82-40. The MLST based phylogenetic tree constructed revealed the polyphyletic branching and MMM01 (ST25) was found to be closely related to ST11, both belong to the sap-A serotype which are more common in human infections. VFanalyzer identified 88 protein-coding genes coding for several virulence factors including Campylobacter adhesion to fibronectin, flagellar apparatus, cytolethal distending toxin operons and Campylobacter invasion antigen proteins which enhance the virulence of bacteria along with resistance genes against antibiotics including fluoroquinolone, chloramphenicol, tetracycline, and aminoglycoside in MMM01, which points to enhanced survival and pathogenicity of this zoonotic pathogen. It was interesting to find that MMM01 lacked FGI-II island found in most of the clinical isolates, which encoded CRISPR Cas and prophage II regions. More details about the complexity and evolution of this zoonotic pathogen could be learned from future studies that concentrate on comparative genome analysis using larger genome datasets.

Pan genomic analysis of the global SARS-CoV-2 isolates has resulted in the identification of several regions of increased genetic variation but there is absence of research on its association with the clinical outcome. The present study fills the vacuum and does mutational analysis of genomic sequence of Indian SARS-CoV-2 isolates. Results reveal the existence of non-synonymous G614 spike protein mutation in 61.45% of the total study genome along with three other mutations. Further, temporal variation in the frequencies of G614 genotype in the country is observed. The examination of the probable association of G614 genotype with COVID-19 severity shows that CFR G614 genotype in India is positively and strongly correlated. It appears that the clinical outcome of the COVID-19 cases in India are significantly and adversely affected by the increasing trend in the G614 genotype; which needs to be addressed combining both laboratory experiments and epidemiological investigations.

It has been observed that not all strains of Vibrio vulnificus are virulent. Determining the virulence of strains that are frequently present in seafood is of significance for ensuring seafood safety. This study is an attempt to predict the virulence of seafood-borne V. vulnificus isolated along the Mangaluru Coast, India. The isolates tested possessed a vcgC gene sequence with high similarity to that in the clinical strain. Transcriptional analysis of core virulence genes in seafood isolate E4010 showed the phenomenon of contact-mediated expression of rtxA1 which correlated well with the actin disintegration and cytotoxicity. These results suggest that the seafood isolates tested in this study possess a functional RtxA1 which could help in initiating the infection. However, other putative virulence genes such as vvpE encoding an extracellular protease, vvhA encoding hemolysin, flp encoding tad pilin and ompU encoding fibronectin-binding protein were also constitutively expressed. Virulence-associated attributes such as cytotoxicity and adherence matched the response of the clinical strain (p &gt, 0.05). On the other hand, the environmental strains showed higher serum sensitivity compared with the clinical strain. These findings show that the part of virulence attributes required for the disease process might be intact in these isolates.

Highlights • First observation on the extensive deletion of amino acid residues in the C-terminal region of the E protein in some of the SARS-CoV-2 sequenced from the India. . • The deletion is outside the primer binding region of RTqPCR targeting E gene used in India in isolates from various states except Odisha, which had deletion in primer binding region. • Characteristic in-frame deletion in the E protein map to the homopentameric interface and PDZ binding motif (PBM) present in the C-terminal region of E protein. • The isolates with deletions belonged to different clades., The envelope glycoprotein (E) is the smallest structural component of SARS-CoVs; plays an essential role in the viral replication starting from envelope formation to assembly. The in silico analysis of 2086 whole genome sequences from India performed in this study provides the first observation on the extensive deletion of amino acid residues in the C-terminal region of the envelope glycoprotein in 34 Indian SARS-CoV-2 genomes. These amino acid deletions map to the homopentameric interface and PDZ binding motif (PBM) present in the C-terminal region of E protein as well as immediately after the reverse primer binding region as per Charité protocol in 26 of these genomes, hence, their detection through RT-qPCR may not be hampered and therefore E gene-based RT-qPCR would still detect these isolates. Eight genomes from the State of Odisha had deletion even in the primer binding site. It is possible that the deletions in the C-terminal region of E protein of these genomes are a result of adapting to a newer geographical area and host. The information on the clinical status was available only for 9 out of 34 cases and these were asymptomatic. However, further studies are indispensable to understand the functional consequences of amino acid deletion in the C terminal region of SARS-CoV-2 envelope protein in the viral pathogenesis and host adaptation.