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Mutation Profiles of eGFP-Tagged Small Ruminant Morbillivirus During 45 Serial Passages in Ribavirin-Treated Cells.

ABSTRACT: Small ruminant morbillivirus (SRMV), formerly known as peste-des-petits-ruminants virus, classified into the genus Morbillivirus in the family Paramyxoviridae. Its L protein functions as the RNA-dependent RNA polymerases (RdRp) during viral replication. Due to the absence of efficient proofreading activity in their RdRps, various RNA viruses reveal high mutation frequencies, making them evolve rapidly during serial passages in cells, especially treated with a certain mutagen, like ribavirin. We have previously rescued a recombinant enhanced green fluorescence protein-tagged SRMV (rSRMV-eGFP) using reverse genetics. In this study, the rSRMV-eGFP was subjected to serial passages in ribavirin-treated cells. Due to the ribavirin-exerted selective pressure, it was speculated that viral progenies would form quasispecies after dozens of passages. Viral progenies at passage-10, -20, -30, -40, and -50 were separately subjected to next-generation sequencing (NGS), consequently revealing a total of 34 single-nucleotide variations, including five synonymous, 21 missense, and one non-sense mutations. The L sequence was found to harbor eight missense mutations during serial passaging. It was speculated that at least one high-fidelity variant was present in viral quasispecies at passage-50. If purified from the population of viral progenies, this putative variant would contribute to clarifying a molecular mechanism in viral high-fidelity replication in vitro.

SUBMITTER: Liu F

PROVIDER: S-EPMC8333274 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Genetic and phylogenetic analyses suggest that the pandemic H1N1/2009 virus was derived from well-established swine influenza lineages; however, there is no convincing evidence that the pandemic virus was generated from a direct precursor in pigs. Furthermore, the evolutionary dynamics of influenza virus in pigs have not been well documented. Here, we subjected a recombinant virus (rH1N1) with the same constellation makeup as the pandemic H1N1/2009 virus to nine serial passages in pigs. The severity of infection sequentially increased with each passage. Deep sequencing of viral quasispecies from the ninth passage found five consensus amino acid mutations: PB1 A469T, PA 1129T, NA N329D, NS1 N205K, and NEP T48N. Mutations in the hemagglutinin (HA) protein, however, differed greatly between the upper and lower respiratory tracts. Three representative viral clones with the five consensus mutations were selected for functional evaluation. Relative to the parental virus, the three viral clones showed enhanced replication and polymerase activity in vitro and enhanced replication, pathogenicity, and transmissibility in pigs, guinea pigs, and ferrets in vivo. Specifically, two mutants of rH1N1 (PB1 A469T and a combination of NS1 N205K and NEP T48N) were identified as determinants of transmissibility in guinea pigs. Crucially, one mutant viral clone with the five consensus mutations, which also carried D187E, K211E, and S289N mutations in its HA, additionally was able to infect ferrets by airborne transmission as effectively as the pandemic virus. Our findings demonstrate that influenza virus can acquire viral characteristics that are similar to those of the pandemic virus after limited serial passages in pigs. Importance: We demonstrate here that an engineered reassortant swine influenza virus, with the same gene constellation pattern as the pandemic H1N1/2009 virus and subjected to only nine serial passages in pigs, acquired greatly enhanced virulence and transmissibility. In particular, one representative pathogenic passaged virus clone, which carried three mutations in the HA gene and five consensus mutations in PB1, PA, NA, NS1, and NEP genes, additionally was able to confer respiratory droplet transmission as effectively as the pandemic H1N1/2009 virus. Our findings suggest that pigs can readily induce adaptive mutational changes to a precursor pandemic-like virus to transform it into a highly virulent and infectious form akin to that of the pandemic H1N1/2009 virus, which underlines the potential direct role of pigs in promoting influenza A virus pathogenicity and transmissibility.

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Mutation Profiles of eGFP-Tagged Small Ruminant Morbillivirus During 45 Serial Passages in Ribavirin-Treated Cells.

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