Spatio-temporal dynamics of intra-host variability in SARS-CoV-2 genomes.

During the course of the COVID-19 pandemic, large-scale genome sequencing of SARS-CoV-2 has been useful in tracking its spread and in identifying variants of concern (VOC). Viral and host factors could contribute to variability within a host that can be captured in next-generation sequencing reads as intra-host single nucleotide variations (iSNVs). Analysing 1347 samples collected till June 2020, we recorded 16 410 iSNV sites throughout the SARS-CoV-2 genome. We found ∼42% of the iSNV sites to be reported as SNVs by 30 September 2020 in consensus sequences submitted to GISAID, which increased to ∼80% by 30th June 2021. Following this, analysis of another set of 1774 samples sequenced in India between November 2020 and May 2021 revealed that majority of the Delta (B.1.617.2) and Kappa (B.1.617.1) lineage-defining variations appeared as iSNVs before getting fixed in the population. Besides, mutations in RdRp as well as RNA-editing by APOBEC and ADAR deaminases seem to contribute to the differential prevalence of iSNVs in hosts. We also observe hyper-variability at functionally critical residues in Spike protein that could alter the antigenicity and may contribute to immune escape. Thus, tracking and functional annotation of iSNVs in ongoing genome surveillance programs could be important for early identification of potential variants of concern and actionable interventions.

Reverse Vaccinology and Its Applications.

The application of the fields of pharmacogenomics and pharmacogenetics to vaccine design, profoundly combined with bioinformatics, has been recently termed "vaccinomics." The enormous amount of information generated by whole genome sequencing projects and the rise of bioinformatics has triggered the birth of a new era of vaccine research and development, leading to a "third generation" of vaccines, which are based on the application of vaccinomics science to vaccinology. The first example of such an approach is reverse vaccinology. Reverse vaccinology reduces the period of vaccine target detection and evaluation to 1-2 years. This approach targets the genomic sequence and predicts those antigens that are most likely to be vaccine candidates. This approach allows not only the identification of all the antigens obtained by the previous methods but also the discovery of new antigens that work on a totally different paradigm. Hence this method helps in the discovery of novel mechanisms of immune intervention. Epitope-based immune-derived vaccines (IDV) are generally considered to be safe when compared to other vectored or attenuated live vaccines. Epitope-based IDV may also provide essential T-cell help for antibody-directed vaccines. Such vaccines may have a significant advantage over earlier vaccine design approaches, as the cautious assortment of the components may diminish.

Virulence Pattern and Genomic Diversity of Vibrio cholerae O1 and O139 Strains Isolated From Clinical and Environmental Sources in India.

Vibrio cholerae is an autochthonous inhabitant of the aquatic environment. Several molecular methods have been used for typing V. cholerae strains, but there is no proper database for such scheme, including multilocus sequence typing (MLST) for V. cholerae O1 and O139 strains. We used 54 V. cholerae O1 and three O139 strains isolated from clinical and environmental sources and regions of India during the time period of 1975-2015 to determine the presence of virulence genes and production of biofilm. We devised a MLST scheme and developed a database for typing V. cholerae strains. Also, we performed pulsed-field gel electrophoresis to see the genomic diversity among them and compared it with MLST. We used the MEGA 7.0 software for the alignment and comparison of different nucleotide sequences. The advanced cluster analysis was performed to define complexes. All strains of V. cholerae, except five strains, showed variation in phenotypic characteristics but carried virulence-associated genes indicating they belonged to the El Tor/hybrid/O139 variants. MLST analysis showed 455 sequences types among V. cholerae strains, irrespective of sources and places of isolation. With these findings, we set up an MLST database on PubMLST.org using the BIGSdb software for V. cholerae O1 and O139 strains, which is available at https://pubmlst.org/vcholerae/ under the O1/O139 scheme. The pulsed-field gel electrophoresis (PFGE) fingerprint showed six fingerprint patterns namely E, F, G, H, I, and J clusters among 33 strains including strain N16961 carrying El Tor ctxB of which cluster J representing O139 strain was entirely different from other El Tor strains. Twenty strains carrying Haitian ctxB showed a fingerprint pattern classified as cluster A. Of the five strains, four carrying classical ctxB comprising two each of El Tor and O139 strains and one El Tor strain carrying Haitian ctxB clustered together under cluster B along with V. cholerae 569B showing pattern D. This study thus indicates that V. cholerae strains are undergoing continuous genetic changes leading to the emergence of new strains. The MLST scheme was found more appropriate compared to PFGE that can be used to determine the genomic diversity and population structure of V. cholerae.

Extracellular DNA builds and interacts with vibrio polysaccharide in the biofilm matrix formed by Vibrio cholerae.

Vibrio cholerae form biofilm, which is essential for their survival under harsh environmental conditions. The eDNA produced during biofilm formation and interaction with other components like vibrio polysaccharide is less studied in Vibrio cholerae despite its importance in biofilm structure and stability. In this study, we selected two strains of V. cholerae, which produced sufficient extracellular DNA in the biofilm, for characterization and studied its interaction with vibrio polysaccharide. Our data demonstrate that eDNA is present in the biofilm and interacts with VPS in V. cholerae. Our findings suggest that eDNA contributes to biofilm integrity by interacting with VPS and provides strength to the biofilm. Moreover, it might interact with other components of biofilm, which need further study.