Reassortant H9N2 Influenza Viruses Containing H5N1-Like PB1 Genes Isolated from Black-Billed Magpies in Southern China

Guoying Dong1,2#, Cong Xu3#, Chengmin Wang1, Bin Wu1, Jing Luo1, Hong Zhang1, Dale Louis Nolte4, Thomas Jude Deliberto4, Mingxing Duan5, Guangju Ji2*, Hongxuan He1*

1 Key Laboratory of Animal Ecology and Conservation Biology, National Research Center For Wildlife Born Diseases, Institute of Zoology, Chinese Academy of Sciences, Beijing, China, 2 National Laboratory of Biomacromolecules, Institute of Biophysics of Chinese Academy of Sciences, Beijing, China, 3 College of Food Science, Southwest University, Chongqing, China, 4 National Wildlife Disease Program, USDA/APHIS/Wildlife Services, United States Department of Agriculture, Fort Collins, Colorado, United States of America, 5 State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Life Sciences, Tsinghua University, Beijing, China


Abstract

H9N2 influenza A viruses have become endemic in different types of terrestrial poultry and wild birds in Asia, and are occasionally transmitted to humans and pigs. To evaluate the role of black-billed magpies (Pica pica) in the evolution of influenza A virus, we conducted two epidemic surveys on avian influenza viruses in wild black-billed magpies in Guangxi, China in 2005 and characterized three isolated black-billed magpie H9N2 viruses (BbM viruses). Phylogenetic analysis indicated that three BbM viruses were almost identical with 99.7 to 100% nucleotide homology in their whole genomes, and were reassortants containing BJ94-like (Ck/BJ/1/94) HA, NA, M, and NS genes, SH/F/98-like (Ck/SH/F/98) PB2, PA, and NP genes, and H5N1-like (Ck/YN/1252/03, clade 1) PB1 genes. Genetic analysis showed that BbM viruses were most likely the result of multiple reassortments between co-circulating H9N2-like and H5N1-like viruses, and were genetically different from other H9N2 viruses because of the existence of H5N1-like PB1 genes.

Genotypical analysis revealed that BbM viruses evolved from diverse sources and belonged to a novel genotype (B46) discovered in our recent study.

Molecular analysis suggested that BbM viruses were likely low pathogenic reassortants.

However, results of our pathogenicity study demonstrated that BbM viruses replicated efficiently in chickens and a mammalian mouse model but were not lethal for infected chickens and mice.

Antigenic analysis showed that BbM viruses were antigenic heterologous with the H9N2 vaccine strain.

Our study is probably the first report to document and characterize H9N2 influenza viruses isolated from black-billed magpies in southern China. Our results suggest that black-billed magpies were susceptible to H9N2 influenza viruses, which raise concerns over possible transmissions of reassortant H9N2 viruses among poultry and wild birds.