Novel reassortment 2.3.4.4b H5N8 highly pathogenic avian influenza viruses circulating in Xinjiang, China.

In 2016, H5N8 avian influenza viruses of clade 2.3.4.4b were detected at Qinghai Lake, China. Afterwards, the viruses of this clade rapidly spread to Asia, Europe, and Africa via migratory birds, and caused massive deaths in poultry and wild birds globally. In this study, four H5N8 isolates (abbreviated as 001, 002, 003, and 004) were isolated from the live poultry market in Xinjiang in 2017. Phylogenetic analysis showed that the hemagglutinin genes of the four isolates belonged to clade 2.3.4.4b, while the viral gene segments were from multiple geographic origins. For 002, the polymerase acidic gene had the highest sequence homology (99.55 %) with H5N8 virus identified from green-winged teal in Egypt in 2016, and the remaining genes exhibited the highest sequence homologies (99.18-100 %) with those of H5N8 viruses isolated from domestic duck sampled in Siberia in 2016. The polymerase basic 1 gene clustered together with H5N8 virus identified from painted stork of India in 2016, and the remaining genes had relatively close genetic relationships with H5N8 viruses identified from the duck of Siberia in 2016 and turkey in Italy in 2017. For the other three isolates, the nucleoprotein gene of 001 had the highest sequence homology (98.82 %) and relatively close genetic relationship with H9N2 viruses identified from poultry in Vietnam and Cambodia in 2015-2017, and all the remaining genes had the highest sequence homologies (99.18 %-99.58 %) and relatively close genetic relationships with H5N8 viruses identified from poultry and waterfowl sampled in African countries in 2017 and swan sampled in China in 2016. Multiple basic amino acids were observed at cleavage sites in the hemagglutinin proteins of the H5N8 isolates, indicating high pathogenicity. In addition, the L89V, G309D, R477G, I495V, A676T and I504V mutations in the polymerase basic 2 protein, N30D and T215A mutations in the matrix 1 protein, P42S mutation, and 80-84 amino acid deletion in the nonstructural 1 protein were detected in all isolates. These mutations were associated with increased virulence and polymerase activity in mammals. Therefore, our results indicate that the H5N8 isolates involved multiple introductions of reassorted viruses, and also revealed that the wetlands of Northern Tianshan Mountain may play a key role in H5N8 AIVs disseminating among Central China, the Eurasian continent, and East African Countries.

Novel reassortant 2.3.4.4B H5N6 highly pathogenic avian influenza viruses circulating among wild, domestic birds in Xinjiang, Northwest China.

BACKGROUND: The H5 avian influenza viruses (AIVs) of clade 2.3.4.4 circulate in wild and domestic birds worldwide. In 2017, nine strains of H5N6 AIVs were isolated from aquatic poultry in Xinjiang, Northwest China. OBJECTIVES: This study aimed to analyze the origin, reassortment, and mutations of the AIV isolates. METHODS: AIVs were isolated from oropharyngeal and cloacal swabs of poultry. Identification was accomplished by inoculating isolates into embryonated chicken eggs and performing hemagglutination tests and reverse transcription polymerase chain reaction (RT-PCR). The viral genomes were amplified with RT-PCR and then sequenced. The sequence alignment, phylogenetic, and molecular characteristic analyses were performed by using bioinformatic software. RESULTS: Nine isolates originated from the same ancestor. The viral HA gene belonged to clade 2.3.4.4B, while the NA gene had a close phylogenetic relationship with the 2.3.4.4C H5N6 highly pathogenic avian influenza viruses (HPAIVs) isolated from shoveler ducks in Ningxia in 2015. The NP gene was grouped into an independent subcluster within the 2.3.4.4B H5N8 AIVs, and the remaining six genes all had close phylogenetic relationships with the 2.3.4.4B H5N8 HPAIVs isolated from the wild birds in China, Egypt, Uganda, Cameroon, and India in 2016-2017, Multiple basic amino acid residues associated with HPAIVs were located adjacent to the cleavage site of the HA protein. The nine isolates comprised reassortant 2.3.4.4B HPAIVs originating from 2.3.4.4B H5N8 and 2.3.4.4C H5N6 viruses in wild birds. CONCLUSIONS: These results suggest that the Northern Tianshan Mountain wetlands in Xinjiang may have a key role in AIVs disseminating from Central China to the Eurasian continent and East African.

Rapid Emergence of the Reassortant 2.3.4.4b H5N2 Highly Pathogenic Avian Influenza Viruses in a Live Poultry Market in Xinjiang, Northwest China.

Live poultry markets (LPMs) play a key role in reassorting and spreading avian influenza viruses (AIVs). In 2018, four strains of H5N2 AIVs were isolated from domestic ducks (Anas platyrhynchos) during AIV surveillance from the LPM in Urumqi, Xinjiang, China. All gene segments of the isolates were amplified by reverse transcription-PCR and sequenced; then, the viral genetic mutations, reassortant, and origin were analyzed. Higher nucleotide identities were observed among each gene of the isolates, indicating a common ancestor. The hemagglutinin (HA) genes of the isolates all classified into the clade 2.3.4.4b; the HA, matrix protein (MP), and nonstructural protein (NS) genes were all clustered together with the local H5N6 highly pathogenic AIVs (HPAIVs) identified in the same LPM of Urumqi in July 2017; the neuraminidase albumen, polymerase basic proteins 1 and 2, polymerase acidic protein, and nucleocapsid protein genes (NA, PB1, PB2, PA, and NP) all had close phylogenetic relationships with the local H9N2 AIVs identified in the same LPM from September to October 2018. Multiple basic amino acids were present at the cleavage site of the HA protein, which was associated with HPAIVs. These results indicated that the reassortant clade 2.3.4.4b H5N2 HPAIVs were rapidly generated from reassortment between the H5N6 and H9N2 AIVs in the local LPM of Urumqi in 2018.

Rápida aparición de los virus de influenza aviar altamente patógenos H5N2 reacomodados 2.3.4.4b en un mercado de aves vivas en Xinjiang, en el noroeste de China. Los mercados de aves vivas desempeñan un papel clave en el reacomodo y en la propagación de los virus de la influenza aviar. En el año 2018, se aislaron cuatro cepas del virus de influenza aviar H5N2 de patos domésticos durante los procedimientos de vigilancia para influenza aviar en mercados de aves vivas en Urumqi, Xinjiang, China. Todos los segmentos de genes de los aislados se amplificaron mediante transcripción reversa y PCR y se secuenciaron; posteriormente, se analizaron las mutaciones genéticas virales, el reacomodamiento y el origen. Se observaron altas identidades de nucleótidos entre cada gene de los aislados, lo que indica un ancestro común. Todos los genes de hemaglutinina (HA) de los aislamientos se clasificaron en el clado 2.3.4.4b; los genes de la proteína HA, la proteína de matriz (MP) y la proteína no estructural (NS) se agruparon junto con los virus de influenza altamente patógenos locales H5N6 identificados en el mismo mercado de aves vivas de Urumqi en julio de 2017; la albúmina de la neuraminidasa, las proteínas básicas de la polimerasa 1 y 2, la proteína ácida de la polimerasa y los genes de la proteína de la nucleocápsida (NA, PB1, PB2, PA y NP) tenían relaciones filogenéticas cercanas con virus de influenza locales H9N2 identificados en el mismo mercado de aves vivas de septiembre a octubre del 2018. Hubo múltiples aminoácidos básicos presentes en el sitio de disociación de la proteína HA, que se asoció con virus de influenza de alta patogenicidad. Estos resultados indicaron que los virus de influenza de alta patogenicidad H5N2 del clado reacomodado 2.3.4.4b se generaron rápidamente a partir del reacomodo entre los virus de influenza H5N6 y H9N2 en el mercado de aves vivas local de Urumqi en el año 2018.