Selection on haemagglutinin imposes a bottleneck during mammalian transmission of reassortant H5N1 influenza viruses

Abstract

The emergence of human-transmissible H5N1 avian influenza viruses poses a major pandemic threat. H5N1 viruses are thought to be highly genetically diverse both among and within hosts, but the effects of this diversity on viral replication and transmission are poorly understood. Here we use deep sequencing to investigate the impact of within-host viral variation on adaptation and transmission of H5N1 viruses in ferrets. We show that although within-host genetic diversity in hemagglutinin (HA) increases during replication in inoculated ferrets, HA diversity is dramatically reduced upon respiratory droplet transmission, where infection is established by only 1–2 distinct HA segments from a diverse source virus population in transmitting animals. Moreover, minor HA variants present in as little as 5.9% of viruses within the source animal become dominant in ferrets infected via respiratory droplets. These findings demonstrate that selective pressures acting during influenza virus transmission among mammals impose a significant bottleneck.

Deep Sequencing Reveals Potential Antigenic Variants at Low Frequencies in Influenza A Virus-Infected Humans

“…2). We only considered SNPs if they were detected above our experimentally defined cutoff of Ն1% of sequence reads in a sample (20). In total, we detected 695 nonsynonymous and 875 synonymous SNPs from individuals infected with H3N2 viruses; in H1N1pdm-infected individuals, we detected 700 nonsynonymous SNPs and 735 synonymous SNPs (Table 4).…”

“…Single-nucleotide polymorphisms (SNPs) were detected, at nucleotide positions with coverage of at least 100 sequence reads and a central base quality score of ՆQ30, relative to a vaccine strain reference sequence (A/Victoria/361/2011 H3N2 or A/California/07/2009 H1N1pdm) using CLC Genomics Workbench version 7.5.2. SNPs were only considered for subsequent analysis if detected at a cutoff frequency of Ն1%, a validated threshold that reduces the carryover of errors introduced by reverse transcription, amplification, or basecalling (20). The nucleotide diversity statistics N and S were calculated in PoPoolation version 1.2.2 (28) using subsampled sequence mappings containing 1,000 randomly chosen sequences per nucleotide position to minimize potential coverage bias.…”

“…In contrast, deep sequencing can detect variants present at much lower frequency, revealing for example how low-frequency drug-resistant variants can rapidly emerge and become dominant after oseltamivir treatment in immunocompromised individuals (19). Moreover, we recently showed that HA variants present below the detection limit of Sanger sequencing could be transmitted via respiratory droplets (20), suggesting viral variants present at low frequency in individual hosts may have a previously unappreciated role in influenza virus biology.…”

Within-Host Evolution of Simian Arteriviruses in Crab-Eating Macaques

“…This cutoff has previously been determined as a conservative cutoff given the intrinsic error of our pipeline and instrument (45). We noted an increase in the number of low-frequency (Ͻ10%) variants called when we removed duplicate reads from our assemblies.…”

Acute Viral Escape Selectively Impairs Nef-Mediated Major Histocompatibility Complex Class I Downmodulation and Increases Susceptibility to Antiviral T Cells

“…All nonsynonymous nucleotide variants that met the criteria of being detected more than once (nonsingleton variants), having a quality score of Q30 or better, and being detected at greater than 1% at each site were tracked in follow-up studies, similarly to a previous study (23). The frequency of nonsynonymous variation at each The key to the right shows the identities of the amino acids that result from the mutations identified in the deep-sequence data.…”