Highly Pathogenic Avian Influenza A(H5N1) Virus Struck Migratory Birds in China in 2015

Abstract

Approximately 100 migratory birds, including whooper swans and pochards, were found dead in the Sanmenxia Reservoir Area of China during January 2015. The causative agent behind this outbreak was identified as H5N1 highly pathogenic avian influenza virus (HPAIV). Genetic and phylogenetic analyses revealed that this Sanmenxia H5N1 virus was a novel reassortant, possessing a Clade 2.3.2.1c HA gene and a H9N2-derived PB2 gene. Sanmenxia Clade 2.3.2.1c-like H5N1 viruses possess the closest genetic identity to A/Alberta/01/2014 (H5N1), which recently caused a fatal respiratory infection in Canada with signs of meningoencephalitis, a highly unusual symptom with influenza infections in humans. Furthermore, this virus was shown to be highly pathogenic to both birds and mammals, and demonstrate tropism for the nervous system. Due to the geographical location of Sanmenxia, these novel H5N1 viruses also have the potential to be imported to other regions through the migration of wild birds, similar to the H5N1 outbreak amongst migratory birds in Qinghai Lake during 2005. Therefore, further investigation and monitoring is required to prevent this novel reassortant virus from becoming a new threat to public health.

Figure 1. Phylogenetic analyses of the HA and PB2 gene sequences from Sanmenxia Clade 2.3.2.1c H5N1 isolates.

The reference sequences were downloaded from the GISAID and GenBank database. All of the phylogenetic analyses were performed using Raxml, with 1000 bootstrap replicates. (A) phylogenies of Clade 2.3.2 HA gene for H5N1 HPAIVs. (B) phylogenetic tree constructed using the PB2 gene sequences. Segments of the whooper swan-H5N1, A/Alberta/01/2014(H5N1), and A/tiger/Jiangsu/01/2013(H5N1) viruses are colored in red, pink and green, respectively.

Figure 2. Histopathologic analysis in lung, brain, and small intestine of the dead whooper swan and pochard naturally infected by the H5N1 virus.

Representative pictures of the lung (A,D), brain (B,E), and small intestine (C,F) of the whooper swan (A–C) and pochard (D–F) infected by the Sanmenxia Clade 2.3.2.1c H5N1 isolate. Scale bar = 200 μm. Bronchial epithelial cell desquamation, congestion and hemorrhage are found in the lungs (A,D); Inflammatory cells infiltrate around blood vessel and nerve cells are found in the brains (B,E). Intestinal villus desquamation, lymphatic tissue cells neorobiosis and disaggregation under mucosa are found in the small intestines (C,F).

Figure 3. Pathogenicity of the Sanmenxia H5N1 virus in mice.

Mice were infected by 10-fold serial dilutions of Ws/HN/SMX4/15(H5N1) virus. (A) Average percentage weight change. (B) Kaplan-Meier survival curve.

Figure 4. Receptor binding properties of the Sanmenxia H5N1 virus.

Solid-phase binding assay for (A) Ws/HN/SMX4/15(H5N1) and (B) Ws/HN/SMX9/15(H5N1) viruses to both α2,3-linked (3’SLNLN) (colored in blue) and α2,6-linked sialylglycan receptors (6’SLNLN) (colored in red), respectively. The data presented is the mean ± standard deviation (S.D.).

Figure 5. The migration routes of whooper swan (Cygnus cygnus).

The migratory routes of whooper swan in China (A) and worldwide (B) were mapped by the ArcGIS Desktop 10.2 software.

Figure 6. The migration routes of pochard (Aythya ferina).

The migratory routes of pochard in China (A) and worldwide (B) were mapped by the ArcGIS Desktop 10.2 software.