Analysis of Next-generation Sequencing Data in Virology - Opportunities and Challenges

Viruses are the most abundant and the smallest organisms, which are relatively simple to sequence. Genome sequence data of viruses for individual species to populations outnumber that of other species. Although this offers an opportunity to study viral diversity at varying levels of taxonomic hierarchy, it also poses challenges for systematic and structured organization of data and its downstream processing. Extensive computational analyses using a number of algorithms and programs have opened exciting opportunities for virus discovery and diagnostics, apart from augmenting our understanding of the intriguing world of viruses. Unravelling evolutionary dynamics of viruses permits improved understanding of phenomena such as quasispecies diversity, role of mutations in host switching and drug resistance, which enables the tangible measurements of genotype and phenotype of viruses. Improved understanding of geno-/serotype diversity in correlation with antigenic diversity will facilitate rational design and development of efficacious vaccines against emerging and re-emerging viruses. Mathematical models developed using the genomic data could be used to predict the spread of viruses due to vector switching and the (re)emergence due to host switching and, thereby, contribute towards designing public health policies for disease management and control

Transposon libraries identify novel Mycobacterium bovis BCG genes involved in the dynamic interactions required for BCG to persist during in vivo passage in cattle

Background BCG is the most widely used vaccine of all time and remains the only licensed vaccine for use against tuberculosis in humans. BCG also protects other species such as cattle against tuberculosis, but due to its incompatibility with current tuberculin testing regimens remains unlicensed. BCG’s efficacy relates to its ability to persist in the host for weeks, months or even years after vaccination. It is unclear to what degree this ability to resist the host’s immune system is maintained by a dynamic interaction between the vaccine strain and its host as is the case for pathogenic mycobacteria. Results To investigate this question, we constructed transposon mutant libraries in both BCG Pasteur and BCG Danish strains and inoculated them into bovine lymph nodes. Cattle are well suited to such an assay, as they are naturally susceptible to tuberculosis and are one of the few animal species for which a BCG vaccination program has been proposed. After three weeks, the BCG were recovered and the input and output libraries compared to identify mutants with in vivo fitness defects. Less than 10% of the mutated genes were identified as affecting in vivo fitness, they included genes encoding known mycobacterial virulence functions such as mycobactin synthesis, sugar transport, reductive sulphate assimilation, PDIM synthesis and cholesterol metabolism. Many other attenuating genes had not previously been recognised as having a virulence phenotype. To test these genes, we generated and characterised three knockout mutants that were predicted by transposon mutagenesis to be attenuating in vivo: pyruvate carboxylase, a hypothetical protein (BCG_1063), and a putative cyclopropane-fatty-acyl-phospholipid synthase. The knockout strains survived as well as wild type during in vitro culture and in bovine macrophages, yet demonstrated marked attenuation during passage in bovine lymph nodes confirming that they were indeed involved in persistence of BCG in the host. Conclusion These data show that BCG is far from passive during its interaction with the host, rather it continues to employ its remaining virulence factors, to interact with the host’s innate immune system to allow it to persist, a property that is important for its protective efficacy.</p

Comparative Genomics of Cell Envelope Components in Mycobacteria

Mycobacterial cell envelope components have been a major focus of research due to their unique features that confer intrinsic resistance to antibiotics and chemicals apart from serving as a low-permeability barrier. The complex lipids secreted by Mycobacteria are known to evoke/repress host-immune response and thus contribute to its pathogenicity. This study focuses on the comparative genomics of the biosynthetic machinery of cell wall components across 21-mycobacterial genomes available in GenBank release 179.0. An insight into survival in varied environments could be attributed to its variation in the biosynthetic machinery. Gene-specific motifs like ‘DLLAQPTPAW’ of ufaA1 gene, novel functional linkages such as involvement of Rv0227c in mycolate biosynthesis; Rv2613c in LAM biosynthesis and Rv1209 in arabinogalactan peptidoglycan biosynthesis were detected in this study. These predictions correlate well with the available mutant and coexpression data from TBDB. It also helped to arrive at a minimal functional gene set for these biosynthetic pathways that complements findings using TraSH

Circulation and Evolution of SARS-CoV-2 in India: Let the Data Speak

The COVID-19 pandemic is a global challenge that impacted 200+ countries. India ranks in the second and third positions in terms of number of reported cases and deaths. Being a populous country with densely packed cities, SARS-CoV-2 spread exponentially. India sequenced ≈0.14% isolates from confirmed cases for pandemic surveillance and contributed ≈1.58% of complete genomes sequenced globally. This study was designed to map the circulating lineage diversity and to understand the evolution of SARS-CoV-2 in India using comparative genomics and population genetics approaches. Despite varied sequencing coverage across Indian States and Union Territories, isolates belonging to variants of concern (VoC) and variants of interest (VoI) circulated, persisted, and diversified during the first seventeen months of the pandemic. Delta and Kappa lineages emerged in India and spread globally. The phylogenetic tree shows lineage-wise monophyletic clusters of VoCs/VoIs and diversified tree topologies for non-VoC/VoI lineages designated as ‘Others’ in this study. Evolutionary dynamics analyses substantiate a lack of spatio-temporal clustering, which is indicative of multiple global and local introductions. Sites under positive selection and significant variations in spike protein corroborate with the constellation of mutations to be monitored for VoC/VoI as well as substitutions that are characteristic of functions with implications in virus–host interactions, differential glycosylation, immune evasion, and escape from neutralization

Neutralizing Antibody Response to Genotypically Diverse Measles Viruses in Clinically Suspected Measles Cases

The neutralizing antibody (Nt-Ab) response to vaccine and wild-type measles viruses (MeV) was studied in suspected measles cases reported during the years 2012–2016. The neutralization activity against MeV A, D4 and D8 genotypes was studied on sera (Panel A; n = 68 (measles-immunized) and Panel B; n = 50 (unvaccinated)) that were either laboratory confirmed or not confirmed by the presence of IgM antibodies. Additionally, the Nt-Ab response in Panel A was measured against the MeV vaccine and four wild-type viruses. Neutralization results were compared using homology modeling and molecular dynamics simulation (MDS) of MeV-hemagglutinin (H) and fusion (F) proteins. Overall, the Nt-Ab titres for MeV-A were found to be significantly lower than MeV-D4 and MeV-D8 viruses for Panel A. No major difference was noted in Nt-Ab titres between MeV-D8 viruses (Jamnagar and New Delhi), whereas MeV-D4 (Sindhudurg and Bagalkot (BGK) viruses) showed significant differences between Nt-Ab titres for Panel B. Interestingly, the substitutions observed in epitopes of H-protein, L249P and G316A are observed to be unique to MeV-BGK. MDS of H-protein revealed significant fluctuations in neutralizing epitopes due to L249P substitution. The majority of the clinically suspected cases showed Nt-Abs to MeV wild-types. Higher IgG antibody avidity and Nt-Ab titres were noted in IgM-negatives than in IgM-positives cases, indicating reinfection or breakthrough. MDS revealed reduced neutralization due to decreased conformational flexibility in the H-epitope