New Avian Influenza Virus (H5N1) in Wild Birds, Qinghai, China

Highly pathogenic avian influenza virus (H5N1) (QH09) was isolated from dead wild birds (3 species) in Qinghai, China, during May–June 2009. Phylogenetic and antigenic analyses showed that QH09 was clearly distinguishable from classical clade 2.2 viruses and belonged to clade 2.3.2.

I n May 2005, highly pathogenic avian infl uenza (HPAI) virus (H5N1) caused a disease outbreak in wild birds in the Qinghai Lake region of the People's Republic of China (1). Subsequently, this virus (QH05, clade 2.2) disseminated from Asia to Europe and Africa, which has led to great concern and energetic debates about the role of migratory birds in infl uenza epidemics (1)(2)(3)(4)(5). In 2006, this virus was detected in migratory birds in Qinghai (6,7). In 2007, viruses similar to QH05 were isolated from surveyed anseriformes in Qinghai and showed only a short evolutionary distance from earlier viruses (8). Genetic diversity of avian infl uenza viruses (H5N1) was not detected in wild birds in Qinghai before 2008 (7,8). We report evidence that a second lineage of viruses, in addition to clade 2.2, has emerged in wild birds in Qinghai.
To determine the pathogenesis of this outbreak, we obtained organs, including lung and brain, and cloacal swabs from 13 birds at different times. Tissue samples were inoculated into 10-day-old, embryonated, specifi c pathogen-free eggs for virus isolation. Hemagglutinin and neuraminidase subtypes were determined as described (9). Eleven avian infl uenza viruses (H5N1) were isolated from 3 species of wild birds: 4 from great crested grebes, 5 from great blackheaded gulls, and 2 from brown-headed gulls. Results of virus isolation for samples from a bar-headed goose and a shelduck were negative. Samples from great cormorants and a common coot were not obtained and tested.
We sequenced genomes of the 11 viruses and found that the viruses (QH09) were closely related and showed 100% homology at the nucleotide level. Sequences of the entire genome of QH09 (representative strain A/great crested grebe/Qinghai/1/09) are available in GenBank (accession nos. CY063315-CY063322).
Hemagglutinin of QH09 virus had a series of basic amino acids (PQRERRRKR) at the cleavage site. Neuraminidase of this virus had a deletion of 20 aa at residues 49-68 in the stalk region. No amino acid substitutions conferred resistance to adamantane or neuraminidase inhibitors. Unlike many isolates related to Qinghai Lake strains of clade 2.2, QH09 virus does not have an E627K substitution in basic polymerase. Nonstructural protein 1 had a deletion of 5 aa at residues 80-84, which is commonly observed in HPAI viruses (H5N1) that are circulating in Southeast Asia.

Conclusions
Genetic and antigenic characterization of HPAI viruses (H5N1) from wild birds in Qinghai in 2009 suggests that these viruses are closely related to clade 2.3.2 and are clearly distinguishable from the classical QH05 clade 2.2. Previously reported QH05-like virus represented >4 genotypes, and no 2 QH05-like viruses were identical at the amino acid or nucleotide sequence levels (1,6). Although isolates of QH09 were obtained from 3 species at different times, 100% homology of the 11 isolates of QH09 was observed, which suggests that wild birds in Qinghai in 2009 were newly infected by 1 strain of virus. Clade 2.3.2 viruses from wild nonpasserine bird species were reported in Hong Kong in 2007-2008 (10). The fact that a similar virus was isolated from a whooper swan (order Anseriformes) in Japan in 2008 showed that clade 2.3.2 was dispersed by migration of wild birds (11 (14). However, whether the 2 virus clades have propagated in other mammalian hosts is unknown.
Qinghai Lake is located near multiple avian fl yways. Although there are no reports of detection of clade 2.3.2 virus in wild birds near Qinghai Lake, the fi nding of clade 2.3.2 virus in the Gengahai wetlands of Qinghai increases concerns about a potential pandemic and the likelihood that avian infl uenza virus (H5N1) will again spread and increase its genetic diversity. Therefore, determining movements of wild migratory waterfowl from Qinghai Lake and their virologic status is needed to assess potential avian vectors of HPAI virus (H5N1).