Influenza remains one of the most prevalent viruses circulating amongst humans and has resulted in several pandemics. The prevention and control of H3N2 influenza is complicated by its propensity for evolution, which leads to vaccine mismatch and reduced vaccine efficacies. This study employed the strategy of serial passaging to compare the evolution of the human seasonal influenza strain A/Singapore/G2-31.1/2014(H3N2) in MDCK-SIAT1 versus primary chick embryo fibroblast (CEF) cells. Genetic analysis of the HA, NS1, NA, and PB1 gene segments by Sanger sequencing revealed the presence of specific mutations and a repertoire of viral quasispecies following serial passaging. Most quasispecies were also found in PB1, which exhibited consistently high transversion-to-transition ratios in all five MDCK-SIAT1 passages. Most notably, passage 5 virus harbored the D457G substitution in the HA2 subunit, while passage 3 virus acquired K53Q and Q69H mutations in PB1-F2. An A971 variant leading to a non-synonymous R316Q substitution in PB1 was also identified in MDCK-SIAT1 passages 2 and 4. With an increasing number of passages, the proportion of D457G mutations progressively increased and was associated with larger virus plaque sizes. However, microneutralization assays revealed no significant differences in the neutralizing antibody profiles of human-influenza-immune serum samples against pre-passaged virus and passage 5 virus. In contrast, viable virus was only detected in passage 1 of CEF cells, which gave rise to multiple viral RNA quasispecies. Our findings highlight that serial passaging is able to drive differential adaptation of H3N2 influenza in different host species and may alter viral virulence. More studies are warranted to elucidate the complex relationships between H3N2 virus evolution, viral virulence changes, and low vaccine efficacy.
Data on the viral loads in respiratory aerosols from patients infected with Delta and Omicron variants are limited. In this study, we used an exhaled breath bioaerosol collector to collect aerosol samples in coarse (> 5µm) and fine (≤ 5µm) fractions from COVID-19 patients infected with these VOCs while doing various respiratory activities. Samples were tested via SARS-CoV-2 RT-qPCR and virus culture. Nine patients (4 Delta and 5 Omicron) were included. Viral RNA was detectable in seven participants, with greater viral loads in fine aerosols. Notably SARS-CoV-2 RNA was consistently detectable in respiratory samples of all Omicron patients despite them being fully vaccinated and mostly asymptomatic in contrast with Delta patients. Singing and talking without mask generated the greatest viral loads underscoring the transmission potential of SARS-CoV-2 and its variants via respiratory aerosols. The more consistent detection of viral RNA in Omicron-infected patients may account for its greater transmissibility.
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