Reassortant H9N2 influenza viruses containing H5N1-like PB1 genes isolated from black-billed magpies in Southern 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.

Figure 1. Phylogenetic relationships for HA (A) and NA (B) genes of the BbM viruses and analyzed H9N2 reference viruses.

Neighbor-joining phylogenetic trees were generated using the MEGA program. Nucleotides 82 to 1396 for HA and 41 to 1363 for NA genes were analyzed. BbM viruses were underlined in red, while representative viruses from each lineage were highlighted in other colors and marked using bold black arrow. Abbreviations: BbM, black-billed magpie; CHU and CU, chukkar; CK, chicken; DK, duck; GF, guinea fowl; GS, goose; PA, Parakeet; PG, pigeon; PH, pheasant; QA, quail; SP, sparrow; SW, swine; TY, turkey; WD, wild duck; AH, Anhui; BJ, Beijing; CA, California; DE, Germany; FJ, Fujian; GD, Guangdong; GX, Guangxi; HB, Hubei; HK, Hong Kong; HLJ, Heilongjiang; HeN, Henan; IE, Ireland; JS, Jiangsu; KR, Korea; LN, Liaoning; NC, Nanchang; NJ, Nanjing; PK, Pakistan; SD, Shandong; SH, Shanghai; ST, Shantou; TB, Tibet; TJ, Tianjin; WI, Wisconsin; and YU, Yunnan.

Figure 2. Phylogenetic relationships of PB1 (A), PB2 (B), and PA (C) genes of the BbM viruses and analyzed H9N2 reference viruses.

Neighbor-joining phylogenetic trees were generated using the MEGA program. Analysis was based on the following nucleotides: PB1, 229 to 2233; PB2, 52 to 2255; and PA, 58 to 2094. Viruses were labeled using the same way as Figure 1. Virus abbreviations are listed in the legend of Figure 1.

Figure 3. Phylogenetic relationships of NP (A), M (B), and NS (C) genes of the BbM viruses and analyzed H9N2 reference viruses.

With MEGA, neighbor-joining phylogenetic trees were constructed based on nucleotides 65 to 1398 for NP, 78 to 784 for M, and 54 to 680 for NS genes. Virus abbreviations are listed in the legend of Figure 1. Viruses were highlighted using the same colors and method as Figure 1.

Figure 4. The genetic analysis of BbM viruses and H9N2 reference strains from various lineages.

Schematic diagrams represent the origins of genes of BbM viruses and H9N2 reference strains. Eight gene segments in each of the schematic virus particles are arranged from top to bottom to represent PB2, PB1, PA, HA, NP, NA, M, and NS genes, and are indicated in same color with representative viruses for each lineage. Virus names and abbreviations are listed in the legend of Figure 1.