Characterization of Novel Reassortant Influenza A (H5N2) Viruses Isolated from Poultry in Eastern China, 2015

Abstract

Recently, novel variants of H5 highly pathogenic avian influenza viruses (AIVs) have been frequently isolated from poultry and wild birds in Asia, Europe and North America. Live poultry markets (LPMs) play an important role in the dissemination of influenza viruses. Four H5N2 AIVs were isolated from poultry during surveillance of AIVs in LPMs in Eastern China, in 2015. Whole-genome sequencing, combined with phylogenetic and antigenic analyses were performed to characterize these viruses. These H5N2 viruses had undergone extensive reassortment resulting in two genetic groups of viruses in poultry. These viruses exhibited slightly pathogenicity in mice, and replicated without prior adaptation. The continued circulation of these novel H5N2 viruses may represent a threat to human health.

Keywords: avian influenza viruses; eastern China; poultry; reassortant; subtype H5N2.

FIGURE 1

Phylogenetic analysis of the HA (positions 1–1,704), NA (positions 40–1,341), PB2 (positions 1–2,280), PB1 (positions 16–2,268), PA (positions 1–2,151), NP (positions 1–1,436), M (positions 29–923), and NS (positions 28–768) of H5N2 avian influenza viruses compared to reference influenza viruses obtained from the Influenza Virus Resource (http://www.ncbi.nlm.nih.gov). The phylogenetic tree was created by the maximum likelihood method and bootstrapped with 1,000 replicates using the MEGA software version 6.0. Chinese avian influenza viruses from poultry in this study are highlighted by triangles, and the novel 2014–2015 H5N6 influenza virus, which caused human infection, is indicated by a dot. Scale bar represents the distance unit between sequence pairs.

FIGURE 2

Comparison of deduced amino acid sequences of HA using the MegAlign program. Identical amino acids are shown as dots. The blue box represents the HA sequence at the cleavage site, black boxes represent potential N-linked glycosylation sites, and red/yellow boxes indicate residues involved in the receptor-binding site.

FIGURE 3

Weight variation of BALB/c mice infected with the H5N2 avian influenza viruses. Each mouse in a group was infected intranasally with 10^6.0 EID₅₀ of each virus. The survival rates and body weight of mice were measured daily from the date of challenge to 14 days after challenge (^∗∗P < 0.05; one-way ANOVA).

FIGURE 4

Histology and immunohistochemistry of mice infected with ZJ-1026109 at 6 days post-infection. (A) Histology of lung sections from inoculated mice stained with hematoxylin and eosin. (B) Immunohistochemical detection of viral nucleoprotein in lungs from inoculated mice. Triangles indicate alveolar edema containing fibrin, erythrocytes, and inflammatory cells. Arrows indicate positively stained lung alveolar epithelial cells.