Characterization of the H5N1 highly pathogenic avian influenza virus derived from wild pikas in China

Abstract

The highly pathogenic H5N1 avian influenza virus emerged from China in 1996 and has spread across Eurasia and Africa, with a continuous stream of new cases of human infection appearing since the first large-scale outbreak among migratory birds at Qinghai Lake. The role of wild birds, which are the natural reservoirs for the virus, in the epidemiology of the H5N1 virus has raised great public health concern, but their role in the spread of the virus within the natural ecosystem of free-ranging terrestrial wild mammals remains unclear. In this study, we investigated H5N1 virus infection in wild pikas in an attempt to trace the circulation of the virus. Seroepidemiological surveys confirmed a natural H5N1 virus infection of wild pikas in their native environment. The hemagglutination gene of the H5N1 virus isolated from pikas reveals two distinct evolutionary clades, a mixed/Vietnam H5N1 virus sublineage (MV-like pika virus) and a wild bird Qinghai (QH)-like H5N1 virus sublineage (QH-like pika virus). The amino acid residue (glutamic acid) at position 627 encoded by the PB2 gene of the MV-like pika virus was different from that of the QH-like pika virus; the residue of the MV-like pika virus was the same as that of the goose H5N1 virus (A/GS/Guangdong [GD]/1/96). Further, we discovered that in contrast to the MV-like pika virus, which is nonpathogenic to mice, the QH-like pika virus is highly pathogenic. To mimic the virus infection of pikas, we intranasally inoculated rabbits, a species closely related to pikas, with the H5N1 virus of pika origin. Our findings first demonstrate that wild pikas are mammalian hosts exposed to H5N1 subtype avian influenza viruses in the natural ecosystem and also imply a potential transmission of highly pathogenic avian influenza virus from wild mammals into domestic mammalian hosts and humans.

FIG. 1.

Pika sampling sites (A) and phylogenetic trees (B) of HA, NA, M, PA, PB1, NP, PB2, and NS genes of H5N1 virus. (A) Circles indicate dates and locations of animal-trapping sites. The animals were sampled from the following sites: Bird Island (36°59′N, 99°50′E), Garila (36°53′N, 99°38′E), Shenhekou (36°57′N, 99°47′E), Quanwan (36°57′N, 99°37′E), Heimahe (36°45′N, 99°46′E), Xingxinghai (33°50′N, 96°50′E), and Longbaotan (27°35′N, 89°35′E). (B) The numbers near the branches are the bootstrap values of 1,000 replicates. The sublineage of MV-like PK virus comprising the strains QW, SHK, GRL, and HMH is shown in red. The sublineage of QH-like PK virus comprising the BI strain is marked in blue.

FIG. 2.

Weight change (A) and mortality (B) of mice inoculated with PK virus strains BI, SHK, HMH, GRL, and QW.

FIG. 3.

Changes in body temperature and the microscopic lesions of rabbits infected with the PK virus. (A) Elevated body temperatures in H5N1 virus-infected rabbits from 4 to 8 dpi. (B) Antigen detection and histopathological observations. (a) Viral antigens (indicated by arrows) detected in the turbinate epithelial cells of the infected rabbits by indirect immunofluorescence; (b) corresponds to subpanel a and shows the desquamation of turbinate epithelial cells (indicated by an arrow); (c) desquamation of the tracheal epithelial cells (indicated by an arrow); (d) mild interstitial pneumonitis in the lungs (indicated by an arrow); (b, c, and d) hematoxylin- and eosin-stained specimens. Magnification, ×20.