Speaker
Description
Geographical spread and spillover events of avian influenza viruses (AIVs) are increasing in recent times. Characterizing the intra-host variation of AIVs is crucial for understanding their evolution. However, existing sequencing methods fall short in detecting rare intra-host sequence variants and reconstructing lengthy genome regions at the single-molecule level. To address this limitation, we employed a high-throughput, single-copy sequencing strategy, utilizing long-read sequencing of individually barcoded hemagglutinin (HA) cDNA segments, for a comprehensive analysis of intra-host diversity in H5N1 influenza. In non-human primates (NHP) experimentally challenged with an H5N1 Vietnam 2004 stock using an artificial aerosol, resulting in acute respiratory disease, we discovered that the H5N1 influenza virus stock, derived from an egg-passaged infectious clone, harbored multiple low-frequency variants comprising closely related minor haplotypes. The initial genetic diversity of the quasispecies of H5N1 in the NHP, resembling the challenge stock, remained high until day four. However, by day seven, a rapid and uneven decline in virus load and genetic diversity occurred, with the receptor-binding domain (RBD) along the HA segment retaining diversity. Notably, within these quasispecies, ultra-low-frequency variants shared mutations with AIV sequences recovered from mammals globally after 2004. This study highlights that an enhanced, artificial infection delivery mechanism, coupled with a challenge stock that included diverse low-frequency variants, resulted in a loose bottleneck effect. Diversification post-transmission remained constrained, likely due to robust negative selective pressure along the HA segment. Our findings underscore the significance of exploring intra-host diversity to understand virus population dynamics and emphasize the critical importance of evaluating the diversity of virus stocks in experimental challenge studies. This is essential for assessing responses to vaccines and gaining a comprehensive understanding of virus population evolution.
