Bioligical Characterization And Pathogenicity Of 2 Strains Of H5N6 Subtype Avian Influenza Viruses

Human infectionby a novel H5N6 avian influenzavirus?AIV? was first reported in Sichuan province, China, in April 2014. It recurred in Guangdong province, in December 2015. The recurrence of human infections with H5N6 viruses has raised concerns over their source and evolution. As the regions of emergence of H5N6 infections in poultry were of great distance from each other, and the human infections showed this same pattern, we assessed the role of wild birds in the spread of H5N6 and the pathogenicity and transmission in different birds. For this, we conducted epidemic surveillance of avian influenza among wild birds in Guangdong province, China, during 2014-2015. Two H5N6 viruses, A/Oriental Magpie-robin/Guangdong/SW8/2014 and A/Pallas' s Sandgrouse/Guangdong/ZH283/2015 were isolated from wild birds.Genetic and phylogenetic analysis of virus sequences revealed they were originated from multiple reassortments of 2.3.4.4 H5N1 and H6N6 avian influenza viruses belonged to ST192-like of Eurasian lineage.The virus isolated in February 2015?ZH283? was highly identical to a human isolated H5N6 in December 2015?A/Guangdong/ZQ874/2015?, while the other viruse isolated in 2014 have high identity with another human-isolated H5N6?A/Guangzhou/39715/2014?. Thus, wild birds might be the source of human H5N6 infections. They might transmit the H5N6 virus to domestic poultry, which then infect humans. Molecular evolution analysis of whole genome sequence found that the amino acid sequence of HA genes had the cleavage site-RRRKR?G-, which represents the molecular basis of high pathogenic avian influenza virus strain. The position I155 T of ZH283 virus was mutated, indicating that there have a affinity of avian influenza viruses for human-type receptors.The PB2 gene of the two viruses had no mutation at position E627 K or D701 N. The mutation of A184 k in the NP protein and the mutation of D92 E in the NS1 protein.To investigate the pathogenicity of the H5N6, pathogenic test was carried on SPF chickens, mice, guinea pigs, ducks and quails.The SW8 and ZH283 viruses replicated systemically in chickens, which could be detected from all the tested organs, including the lung, kidney, spleen, cecal tonsils, bursa of fabricius, trachea, pancreas, liver, heart and brain. The SW8 and ZH283 viruses replicated highly in lung; the mean titers were 7.83 log₁₀EID₅₀ and 10.08 log₁₀EID₅₀, respectively The two novel viruses could also replicate in brain; the mean titers were from 6.33log₁₀EID₅₀ to 8.67 log₁₀EID₅₀. SW8 and ZH283 viruses shedding from the inoculated chickens were detected in oropharyngeal and cloacal swabs. Babl/c mice pathogenic test,all the mice inoculated with SW8 and ZH283 were resulting in 100% lethality and mortality. Mice infected with SW8 viruse caused an average body weight loss of 12.7 %. While infection with ZH283 virus exhibited body weight loss of 23 %.The SW8 and ZH283 viruses replicated highly in lung; the mean titers were 8.75 log₁₀EID₅₀ and 9.08 log₁₀EID₅₀ at 3 DPI and 9.08 log₁₀EID₅₀ and 10.00 log₁₀EID₅₀ at 5 DPI, respectively. Guinea pigs pathogenic test, the SW8 and ZH283 viruses replicated systemically in guinea pigs, which could be detected from all the tested tissues,such as thenasal turbinate, trachea and lung, the mean titers were from 3.75 log₁₀EID₅₀ to 6.42 log₁₀EID₅₀. The SW8 and ZH283 viruses replicated highly in nasal turbinate; the mean titers were 6.33 log₁₀EID₅₀ and 6.42 log₁₀EID₅₀, respectively. The two viruses could also replicate in lung; the mean titers were 6.25 log₁₀EID₅₀ and 5.75 log₁₀EID₅₀. SW8 and ZH283 viruses shedding were detected in nasal wash samples and cloacal swabs.The pathogenicity of ducks test, the SW8 and ZH283 viruses replicated systemically in ducks, which could be detected from all the tested organs. The native contact quails, housed with the ducks inoculated with SW8 or ZH283 virus, resulting in 1/3 or 2/3 lethality and mortality.The native contact quails virus shedding could be tested from both oropharyngeal and cloacal swabs and the virus titers was higher than the native contact duck. The pathogenicity of quail test, the quails inoculated with SW8 or ZH283 virus, resulting in 100% lethality and mortality. The native contact quails inoculated with SW8 or ZH283 virus, resulting in 1/3 or 2/3 lethality and mortality. The native contact ducks inoculated with SW8 or ZH283 virus have 3/3 and 2/3 survival. The SW8 and ZH283 viruses replicated systemically in quails, which could be detected from all the tested organs, whose virus titers was from 7.25 log₁₀EID₅₀ to 9.5 log₁₀EID₅₀. All the quails and ducks were detected in oropharyngeal and cloacal swabs.In conclusion, our results indicated that H5N6 from different birds show different host ranges and transmission, and pathogenicity of H5N6 in different birds may be associated the replication ability of virus. H5N6 could infect SPF chickens, mice, guinea pigs and quails, not only caused death in chickens and mice but with high virus load. H5N6 was low pathogenic to ducks. It could transmit to the quails and ducks. These results suggest a possible role of wild birds in the spread of H5N6 and its potential threat for worldwide circulation of H5N6 along wild bird migratory flyways and thus posing threats to the poultry industry and public health.