Isolation and characterization of novel reassortant H6N1 avian influenza viruses from chickens in Eastern China

Abstract

Background: The H6N1 subtype of avian influenza viruses (AIVs) can infect people with an influenza-like illness; the H6N1 viruses possess the ability for zoonotic transmission from avians into mammals, and possibly pose a threat to human health.

Methods: In 2017, live poultry markets (LPMs) in Zhejiang Province were surveyed for AIVs. To better understand the genetic relationships between these strains from Eastern China and other AIVs, all gene segments of these strains were sequenced and compared with sequences available in GenBank. In this study, we analyzed the receptor-binding specificity, antigenic characteristics, and pathogenicity of these two H6N1 viruses.

Results: In 2017, two H6N1 AIVs were isolated from chickens during surveillance for AIVs in LPMs in Eastern China. Phylogenetic analysis showed that these strains shared genetic characteristics from H6, H10, H1, and H4 AIVs found in ducks and wild birds in East Asia. These AIV strains were able to replicate in mice without prior adaptation.

Conclusions: In this study, we report the discovery of new strains of H6N1 viruses from chickens with novel gene reassortments. Our results suggest that these chickens play an important role generating novel reassortments in AIVs, and emphasize the need for continued surveillance of AIV strains circulating in poultry.

Keywords: Avian influenza viruses; Chickens; Eastern China; Reassortant; Subtype H6N1.

Fig. 1

Phylogenetic trees of HA (positions 1–1701), NA (positions 1–1410), PB2 (positions 1–2280), PB1 (positions 1–2274), PA (positions 1–2151), NP (positions 1–1497), M (positions 1–982), and NS (positions 1–838) segments from the novel reassortant H6N1 avian influenza viruses, ZJ-1664 and ZJ-1667. The tree was created by the maximum likelihood method and bootstrapped with 1000 replicates using the MEGA6 software version 6.0. The H6N1 viruses characterized are indicated by a triangle in red, and the H6N1 influenza virus responsible for human infection in 2013 is indicated by a dot in red. The scale bar represents the distance unit between sequence pairs

Fig. 2

A simplified schematic showing the putative genomic composition of the novel reassortant H6N1 avian influenza viruses, ZJ-1664 and ZJ-1667. The eight gene segments (from top to bottom) in each virus are polymerase basic protein 2 (PB2), polymerase basic protein 1(PB1), polymerase acidic protein (PA), hemagglutinin (HA), nucleocapsid protein (NP), neuraminidase (NA), matrix protein (M), and nonstructural protein (NS). Each color represents a separate virus background. Mongolia/520/2015, A/duck/Mongolia/520/2015(H1N1); Mongolia/66/2015, A/duck/Mongolia/66/2015(H10N2); Hubei/ZYSYG15/2015, A/duck/Hubei /ZYSYG15/2015(H6N2); Korea/SK14/2014, A/wild bird/Korea/SK14/2014(H1N1); Wuhan/WHHN16/2014, A/wild bird/Wuhan/WHHN16/2014(H1N1); Mongolia/543/2015, A/duck/Mongolia/543/2015(H4N6). The illustration is based on the nucleotide-distance comparison and phylogenetic analysis

Fig. 3

In vitro growth properties of the H6N1 avian influenza viruses. Growth curves were determined for each virus in MDCK or A549 cells after inoculation at an MOI of 0.1. Each point on the curve indicates the mean and standard deviation from three independent experiments

Fig. 4

Weight variation of BALB/c mice infected with the H6N1 avian influenza viruses. Each mouse in a group was infected intranasally with 10^6.0 EID50 of each virus (50 ul). A group inoculated with PBS (50 ul) as control was included. The body weight of mice were measured daily from the date of challenge to 14 days after challenge

Fig. 5

Histology and immunohistochemistry of mice infected with ZJ-1664 at 6 days post–infection. Histology of lung sections stained by hematoxylin and eosin from inoculated mice (a) or negative control (b). Immunohistochemical detection of virus nucleoprotein in lungs from inoculated mice (c) or negative control (d). Arrows indicate positively stained lung alveolar epithelial cells