Characterization of the low-pathogenic H7N7 avian influenza virus in Shanghai, China

Abstract

H7N7 avian influenza virus (AIV) can divided into low-pathogenic AIV and high-pathogenic AIV groups. It has been shown to infect humans and animals. Its prevalence state in wild birds in China remains largely unclear. In this study, a new strain of H7N7 AIV, designated CM1216, isolated from wild birds in Shanghai, China, was characterized. Phylogenetic and nucleotide sequence analyses of CM1216 revealed that HA, NA, PB1, NP, and M genes shared the highest nucleotide identity with the Japan H7 subtype AIV circulated in 2019; the PB2 and PA genes shared the highest nucleotide identity with the Korea H7 subtype AIV circulated in wild birds in 2018, while NS gene of CM1216 was 98.93% identical to that of the duck AIV circulating in Bangladesh, and they all belong to the Eurasian lineage. A Bayesian phylogenetic reconstruction of the 2 surface genes of CM1216 showed that multiple reassortments might have occurred in 2015. Mutations were found in HA (A135 T, T136S, and T160 A [H3 numbering]), M1 (N30D and T215 A), NS1 (P42S and D97 E), PB2 (R389 K), and PA (N383D) proteins; these mutations have been shown to be related to mammalian adaptation and changes in virulence of AIVs. Infection studies demonstrated that CM1216 could infect mice and cause symptoms characteristic of influenza virus infection and proliferate in the lungs without prior adaption. This study demonstrates the need for routine surveillance of AIVs in wild birds and detection of their evolution to become a virus with high pathogenicity and ability to infect humans.

Keywords: H7N7; avian influenza virus; mouse; pathogenicity; phylogenetics.

Figure 1

Phylogenetic tree of HA and NA genes of CM1216. The neighbor-joining tree was constructed using the Kimura 2-parameter model in the MEGA-X software (http://www.megasoftware.net/). Bootstrap values were calculated for 1,000 replicates; values less than 75% are not shown. Numbers indicate neighbor-joining bootstrap values. The virus (CM1216) characterized in this study is indicated by a filled triangle, the Korean H7N7 virus is indicated by a filled circle.

Figure 2

Phylogenetic tree of other 6 internal genes of CM1216.

Figure 3

Weight loss, pulmonary index, and virus titer in mice. (A) Weight loss of infected mice (filled circle) and control mice (filled square). (B) Pulmonary index of infected mice (filled circle) and control mice (filled square). (C) The titers of virus replication in lungs at 3, 5, and 7 d.p.i. of infected mice (black bars) and control mice (gray bars). Mice were infected intranasally with H7N7 virus (50 μL of 10⁶ EID50/100 μL). Samples were collected at corresponding days after infection, and viral titers were determined by TCID50. ∗P < 0.05, ∗∗P < 0.01.

Figure 4

Histopathologic analyses of lung tissue from CM1216 infected mice. Histology of lung sections stained with hematoxylin and eosin (H&E) from inoculated mice at 3 (A), 5 (B), 7 (C) d.p.i., and control mice (D). Black arrows indicate representative areas with infiltration of inflammatory cells, edema, and mild detachment of bronchial epithelial cells.

Figure 5

Immunohistochemical (IHC) analysis of lung tissue from CM1216 infected mice. IHC staining was performed on lung sections of 3 (A), 5 (B), 7 (C) d.p.i., and control mice (D). The black arrow indicates the virus area.