Biology subjects

Gunalan, V.

Publications and source records attributed to Gunalan, V..

3 recordsLinked to original sources

bioRxiv2026

Targeted metagenomic recovery of coronaviruses from wildlife samples

Coronaviruses (CoVs) are diverse RNA viruses infecting a wide range of hosts, with significant implications for public health, animal production and welfare. Bats are key reservoirs of mammalian CoVs and have contributed to the emergence and circulation of several zoonotic viruses in a wildlife context, while mustelids represent important hosts at the human-animal interface, as highlighted by SARS-CoV-2 outbreaks in farmed mink. Using metagenomic next-generation sequencing, we screened bats and wild mustelids and recovered and characterized full-length CoV genomes. Building on previous findings in Danish wildlife, including alpha-CoVs and a MERS-related beta-CoV in bats, this study expands current knowledge of coronavirus diversity and evolution in wildlife, emphasizing their relevance for zoonotic emergence.

microbiology↗

bioRxiv2024

SARS-CoV-2 sequencing artifacts associated with targeted PCR enrichment and read mapping

Protocols and pipelines for SARS-CoV-2 genome sequencing were rapidly established when the COVID-19 outbreak was declared a pandemic. The most widely used approach for sequencing SARS-CoV-2 includes targeted enrichment by PCR, followed by shotgun sequencing and reference-based genome assembly. As the continued surveillance of SARS-CoV-2 worldwide is transitioning towards a lower level of intensity, it is timely to re-visit the sequencing protocols and pipelines established during the acute phase of the pandemic. In the current study, we have investigated the impact of primer scheme and reference genome choice by sequencing samples with multiple primer schemes (Artic V3, V4.1 and V5.3.2) and re-processing reads with multiple reference genomes. We have also analysed the temporal development in ambiguous base calls during the emergence of the BA.2.86.x variant. We found that the primers used for targeted enrichment can result in recurrent ambiguous base calls, which can accumulate rapidly in response to the emergence of a new variant. We also found examples of consistent base calling errors, associated with PCR artifacts and amplicon drop-out. Similarly, misalignments and partially mapped reads on the reference genome resulted in ambiguous base calls, as well as defining mutations being omitted from the assembly. These findings highlight some key limitations of using targeted enrichment by PCR and reference-based genome assembly for sequencing SARS-CoV-2, and the importance of continuously monitoring and updating primer schemes and bioinformatic pipelines.

bioinformatics↗

bioRxiv2021

Predicting potentially permissive substitutions that improve the fitness of A(H1N1)pdm09 viruses bearing the H275Y NA substitution

Oseltamivir-resistant influenza viruses arise due to amino-acid mutations in key residues, but these changes often reduce their replicative and transmission fitness. Widespread oseltamivir-resistance has not yet been observed in A(H1N1)pdm09 viruses. However, it is known that permissive mutations in the neuraminidase (NA) of former seasonal A(H1N1) viruses from 2007-2009 buffered the detrimental effect of the NA H275Y mutation, resulting in fit oseltamivir-resistant viruses that circulated widely. This study explored two approaches to predict permissive mutations that may enable a fit H275Y A(H1N1)pdm09 variant to arise. A computational approach used phylogenetic and in silico protein stability analyses to predict potentially permissive mutations, which were then evaluated by in vitro NA enzyme activity and expression analysis, followed by in vitro replication. The second approach involved the generation of a virus library which encompassed all possible individual 2.9 x 104 codon mutations in the NA whilst keeping H275Y fixed. To select for variant viruses with the greatest fitness, the virus library was serially passaged in ferrets (via contact and aerosol transmission) and resultant viruses were deep sequenced. The computational approach predicted three NA permissive mutations, and even though they only offset the in vitro impact of H275Y on NA enzyme expression by 10%, they could restore replication fitness of the H275Y variant in A549 cells. In our experimental approach, a diverse virus library (97% of 8911 possible single amino-acid substitutions were sampled) was successfully transmitted through ferrets, and sequence analysis of resulting virus pools in nasal washes identified three mutations that improved virus transmissibility. Of these, one NA mutation, I188T, has been increasing in frequency since 2017 and is now present in 90% of all circulating A(H1N1)pdm09 viruses. Overall, this study provides valuable insights into the evolution of the influenza NA protein and identified several mutations that may potentially facilitate the emergence of a fit H275Y A(H1N1)pdm09 variant.

microbiology↗