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.

SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion.

The B.1.617.2 (Delta) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha)1. In vitro, B.1.617.2 is sixfold less sensitive to serum neutralizing antibodies from recovered individuals, and eightfold less sensitive to vaccine-elicited antibodies, compared with wild-type Wuhan-1 bearing D614G. Serum neutralizing titres against B.1.617.2 were lower in ChAdOx1 vaccinees than in BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies to the receptor-binding domain and the amino-terminal domain. B.1.617.2 demonstrated higher replication efficiency than B.1.1.7 in both airway organoid and human airway epithelial systems, associated with B.1.617.2 spike being in a predominantly cleaved state compared with B.1.1.7 spike. The B.1.617.2 spike protein was able to mediate highly efficient syncytium formation that was less sensitive to inhibition by neutralizing antibody, compared with that of wild-type spike. We also observed that B.1.617.2 had higher replication and spike-mediated entry than B.1.617.1, potentially explaining the B.1.617.2 dominance. In an analysis of more than 130 SARS-CoV-2-infected health care workers across three centres in India during a period of mixed lineage circulation, we observed reduced ChAdOx1 vaccine effectiveness against B.1.617.2 relative to non-B.1.617.2, with the caveat of possible residual confounding. Compromised vaccine efficacy against the highly fit and immune-evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era.

Molecular modeling and docking of novel laccase from multiple serotype of Yersinia enterocolitica suggests differential and multiple substrate binding.

Multi-copper oxidases (MCOs) are widely distributed in bacteria, where they are responsible for metal homeostasis, acquisition and oxidation. Using specific primers, yacK coding for MCO was amplified from different serotypes of Yersinia enterocolitica biovar 1A. Homology modeling of the protein followed by docking with five well-known substrates for different MCO's (viz., 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid [ABTS], syringaldazine, L-tyrosine, ammonium ferrous sulfate and guaiacol), lignin monomers (Coniferyl alcohol, p-coumaryl alcohol and sinapyl alcohol) and two inhibitors i.e., kojic acid and N-hydroxyglycine was done. The docking gave maximum GoldScore i.e., 91.93 and 72.64 with ammonium ferrous sulfate and ABTS, respectively. Similarly, docking with ICM gave -82.10 and -83.61 docking score, confirming the protein to be true laccase with ferroxidase activity. Further, validation with ammonium ferrous sulfate as substrate gave laccase activity of 0.36Units/L/min. Guaiacol, L-tyrosine, and lignin monomers showed good binding affinity with protein models with GoldScores of 35.89, 41.82, 40.41, 41.12 and 43.10, respectively. The sequence study of all the cloned Yack genes showed serotype specific clade in dendrogram. There was distinct discrimination in the ligand binding affinity of Y. enterocolitica laccase, among strains of same clonal groups, suggesting it as a tool for phylogenetic studies.

Strategies used by Yersinia enterocolitica to evade killing by the host: thinking beyond Yops.

Yersinia enterocolitica is an important gastrointestinal pathogen. Its pathogenicity has been attributed primarily to the plasmid encoded Yersinia outer proteins or Yops that are known to subvert the immune system. This review, however, highlights the role of Yop-independent mechanisms that help Y. enterocolitica evade immune system and contribute significantly to its survival in the host.

Detection, distribution and characterization of novel superoxide dismutases from Yersinia enterocolitica Biovar 1A.

BACKGROUND: Superoxide dismutases (SODs) cause dismutation of superoxide radicals to hydrogen peroxide and oxygen. Besides protecting the cells against oxidative damage by endogenously generated oxygen radicals, SODs play an important role in intraphagocytic survival of pathogenic bacteria. The complete genome sequences of Yersinia enterocolitica strains show presence of three different sod genes. However, not much is known about the types of SODs present in Y. enterocolitica, their characteristics and role in virulence and intraphagocytic survival of this organism. METHODOLOGY/PRINCIPAL FINDINGS: This study reports detection and distribution of the three superoxide dismutase (sodA, sodB and sodC) genes in 59 strains of Y. enterocolitica and related species. The majority (94%) of the strains carried all three genes and constitutive expression of sodA and sodB was detected in 88% of the strains. Expression of sodC was not observed in any of the strains. The sodA, sodB and sodC genes of Y. enterocolitica were cloned in pET28a (+) vector. Recombinant SodA (82 kDa) and SodB (21 kDa) were expressed as homotetramer and monomer respectively, and showed activity over a broad range of pH (3.0-8.0) and temperature (4-70°C). SodA and SodB showed optimal activity at 4°C under acidic pH of 6.0 and 4.0 respectively. The secondary structures of recombinant SodA and SodB were studied using circular dichroism. Production of YeSodC was not observed even after cloning and expression in E. coli BL21(DE3) cells. A SodA(-) SodB(-) Escherichia coli strain which was unable to grow in medium supplemented with paraquat showed normal growth after complementation with Y. enterocolitica SodA or SodB. CONCLUSIONS/SIGNIFICANCE: This is the first report on the distribution and characterization of superoxide dismutases from Y. enterocolitica. The low pH optima of both SodA and SodB encoded by Y. enterocolitica seem to implicate their role in acidic environments such as the intraphagocytic vesicles.