After the first H5N1 outbreak in the migratory wild bird population at Qinghai Lake (16
), China, HPAI spread to European and African poultry populations. One year after the QH05 outbreak, fatal H5N1 viruses that infected more species of wild birds were reemerging in some areas of the Qinghai Province and Tibet Autonomous Region (34
). Considering Qinghai Lake's geographical status in bird migration, the role of migratory birds, possibly as carriers in the circulation of the viruses along the flyway, has been debated extensively (11
). The H5N1 virus in migratory birds has become a grave concern. In this study, we first investigated PK virus infections in the Qinghai Province of China. The analysis of anti-AIV HI antibodies confirms that the H5 AIV infection has a high frequency in pikas. Subsequently, five isolates of the H5N1 influenza virus were identified in samples from live-caught pikas, and two kinds of PK viruses, MV-like and QH-like lineage viruses, were identified (Fig. and data not shown), demonstrating that two lineages of H5N1 viruses currently appear in pikas. Therefore, we confirm that free-ranging wild pikas are a mammal host natively infected by the H5N1 influenza A virus in the natural environment. However, whether pikas are a native mammal carrier of H5N1 virus or are a host infected by H5N1 virus from other animals needs further investigation.
Notably, in our study, seven genes of the BI virus share the greatest homology to genes of A/DK/KR/Asan5/06, which is related to the QH-like viruses responsible for the HPAI H5N1 outbreak in Eurasian and African poultry populations (1
), but its PA gene (96.0% homology) appears to have greatest identity to the MV-like PK viruses (data not shown), indicating that the BI virus is a reassortant H5N1 virus closely associated with the migration of wild birds. Thus, pikas are infected not only with the QH-like H5N1 virus but also with an H5N1 virus circulating in migratory birds that has rearranged with PK virus. These data confirm that the QH-like PK virus circulates between migratory birds and pikas. The pika, which belongs to the Ochotona
genus of the Ochotonidae family of the order Lagomorpha, is a small, wild, nonmigrating herbivorous mammal in the natural ecosystem and a major food source for all raptorial birds and carnivorous mammals (26
). The wild birds, such as bar-headed geese and ruddy shelducks, forage weeds as food. An interesting question is how the QH-like H5N1 virus circulating in wild birds transmits between pikas and wild birds. One possible hypothesis is that pikas are infected by H5N1 virus circulating in wild birds at the common weed-foraging sites. Therefore, these data imply that the H5N1 virus circulating in migratory birds has caused environmental pollution in the regions around Qinghai Lake of western China.
Unlike with the QH-like PK virus, seven of eight genes for the MV-like H5N1 viruses share high nucleotide homology to genes in the different H5N1 sublineages that have circulated in poultry in the Chinese Provinces of Guangxi, Guizhou, Hunan, and Fujian since 2004 (data not shown), confirming that the MV-like H5N1 viruses are closely related to the H5N1 viruses circulating in poultry in China's southern provinces, especially in waterfowl. Recently, reports by two research groups from the United States and China on flyways of some migratory birds at Qinghai Lake show only that wild birds migrate from Qinghai Lake to Mongolia, Eastern Asia, and the Bay of Bengal (32
). The data do not show evidence of the migration of wild birds from Qinghai to China's southern provinces. Also, the published data show no evidence of H5N1 virus transmission by migratory birds to the pika, a nonmigratory small mammal living on the plateau, and poultry raised in different regions of China's southern provinces. Therefore, why the MV-like PK viruses have a close relation to the H5N1 virus circulating in southern China needs further investigation.
Our concern is whether H5N1 viruses isolated from pikas are pathogenic to mammals and poultry. As a model, we evaluated the pathogenicity of PK virus in mice and in pika-related rabbits. After experimental infection, the QH-like PK virus is fatal to mice while the MV-like PK viruses are hardly pathogenic. However, infected rabbits showed temperature elevations and mild or moderate interstitial pneumonia, similar to the lung damage of human H5N1 virus cases with a long disease duration (8
), but suffered no clinical signs or death. Virus reisolation and immunofluorescence analysis revealed that the viral antigens were distributed mainly in turbinate bone of rabbits (Fig. ). These results indicate that the MV-like PK virus establishes an infection in the respiratory tracts of rabbits but is not pathogenic. In view of the fact that no dead pikas were found in our field investigations, we believe that the PK virus is nonpathogenic or only subtly pathogenic to pikas. Notably, in our study, the virus was shed persistently from the noses of infected rabbits at 3 to 10 dpi. Therefore, extensive surveillance of pikas and other mammalian species infected by H5N1 avian influenza virus is imperative.