Studies of H5N1 influenza virus infection of pigs by using viruses isolated in Vietnam and Thailand in 2004

To determine whether avian H5N1 influenza viruses associated with human infections in Vietnam had transmitted to pigs, we investigated serologic evidence of exposure to H5N1 influenza virus in Vietnamese pigs in 2004. Of the 3,175 pig sera tested, 8 (0.25%) were positive for avian H5N1 influenza viruses isolated in 2004 by virus neutralization assay and Western blot analysis. Experimental studies of replication and transmissibility of the 2004 Asian H5N1 viruses in pigs revealed that all viruses tested replicated in the swine respiratory tract but none were transmitted to contact pigs. Virus titers from nasal swabs peaked on day 2, and low titers were detected in the liver of two of the four pigs tested. Our findings indicate that pigs can be infected with highly lethal Asian H5N1 viruses but that these viruses are not readily transmitted between pigs under experimental conditions.     

Lethality to ferrets of H5N1 influenza viruses isolated from humans and poultry in 2004

The 2004 outbreaks of H5N1 influenza viruses in Vietnam and Thailand were highly lethal to humans and to poultry; therefore, newly emerging avian influenza A viruses pose a continued threat, not only to avian species but also to humans. We studied the pathogenicity of four human and nine avian H5N1/04 influenza viruses in ferrets (an excellent model for influenza studies). All four human isolates were fatal to intranasally inoculated ferrets. The human isolate A/Vietnam/1203/04 (H5N1) was the most pathogenic isolate; the severity of disease was associated with a broad tissue tropism and high virus titers in multiple organs, including the brain. High fever, weight loss, anorexia, extreme lethargy, and diarrhea were observed. Two avian H5N1/04 isolates were as pathogenic as the human viruses, causing lethal systemic infections in ferrets. Seven of nine H5N1/04 viruses isolated from avian species caused mild infections, with virus replication restricted to the upper respiratory tract. All chicken isolates were nonlethal to ferrets. A sequence analysis revealed polybasic amino acids in the hemagglutinin connecting peptides of all H5N1/04 viruses, indicating that multiple molecular differences in other genes are important for a high level of virulence. Interestingly, the human A/Vietnam/1203/04 isolate had a lysine substitution at position 627 of PB2 and had one to eight amino acid changes in all gene products except that of the M1 gene, unlike the A/chicken/Vietnam/C58/04 and A/quail/Vietnam/36/04 viruses. Our results indicate that viruses that are lethal to mammals are circulating among birds in Asia and suggest that pathogenicity in ferrets, and perhaps humans, reflects a complex combination of different residues rather than a single amino acid difference.