Influenza H7N9 and H9N2 viruses: coexistence in poultry linked to human H7N9 infection and genome characteristics

Abstract

Avian influenza virus A of the novel H7N9 reassortant subtype was recently found to cause severe human respiratory infections in China. Live poultry markets were suspected locations of the human H7N9 infection sources, based on the cases' exposure histories and sequence similarities between viral isolates. To explore the role of live poultry markets in the origin of the novel H7N9 virus, we systematically examined poultry and environmental specimens from local markets and farms in Hangzhou, using real-time reverse transcription-PCR (RT-PCR) as well as high-throughput next-generation sequencing (NGS). RT-PCR identified specimens positive for the H7 and N9 genomic segments in all of the 12 poultry markets epidemiologically linked to 10 human H7N9 cases. Chickens, ducks, and environmental specimens from the markets contained heavily mixed subtypes, including H7, N9, H9, and N2 and sometimes H5 and N1. The idea of the coexistence of H7N9 and H9N2 subtypes in chickens was further supported by metagenomic sequencing. In contrast, human H7N9 infection cases (n = 31) were all negative for H9N2 virus according to real-time RT-PCR. The six internal segments were indistinguishable for the H7N9 and H9N2 viruses. The H9, N2, and internal-segment sequences were very close to the sequence of the H9N2 virus circulating in chickens in China recently. Our results provide direct evidence that H9N2 strains coexisted with the novel human-pathogenic H7N9 influenza virus in epidemiologically linked live poultry markets. Avian influenza A virus of the H9N2 subtype likely made a recent contribution to the evolution of the H7N9 virus and continues to do so.

Importance: Our results suggest that avian influenza A virus of the H9N2 subtype likely made a recent contribution to the evolution of the H7N9 virus, a novel reassortant avian influenza virus A subtype, and continues to do so. The finding helps shed light on how the H7N9 virus emerged, spread, and transmitted to humans. It is of considerable interest for assessing the risk of the possible emergence of novel reassortant viruses with enhanced transmissibility to humans.

FIG 1

The locations of the live poultry markets epidemiologically linked to the H7N9 patients in Hangzhou, China. The abbreviations on the map at the upper right represent the names of the live markets epidemiologically linked to the H7N9 patients in Hangzhou. The market of JSC is not shown on the right upper portion of the map due to its location at a village 77 kilometers west of Hangzhou.

FIG 2

Subtypes of influenza A virus found in the poultry specimens. The numbers under circles represent the total reads of NGS mapping to H7, H9 and H5, or N9, N2, and N1.

FIG 3

Phylogenetic analyses of hemagglutinin genes and neuramidinase genes of H7N9 viruses and H9N2 viruses. The viruses reported in this paper were highlighted using red frames.

FIG 4

Phylogenetic analyses of six internal genes of H7N9 viruses and H9N2 viruses. The viruses reported in this paper were highlighted using red frames.