Characterization Of Caged Pet Birds H3N8 Influenza A Virus And Generation Of A Reassortant Virus By Reverse Genetics

The studies characterize H3N8 influenza viruses from songbirds at the point of entry to Hong Kong, from the "Bird Garden" markets in Kowloon Hong Kong and from different region of the world. The majority of these isolates were from the water bowls on the cages. Three different sampling studies from Hong Kong different sites yielded a total of 18 H3N8 influenza isolates from 321 samples (5.6%) and two Newcastle Disease Virus isolates were obtained (0.6%).Antigenic analysis in hemagglutinin inhibition (HI) tests showed that 18 H3N8 isolates from caged pet bird markets in Hong Kong and were very homogenous with no detectable antigenic variation between isolations, but A/Shama/Indonesia/99, and A/Parakeet/ Netherland/33/01 isolates from Canada and from Alexander reacted with different mode compared to songbird's viruses from Hong Kong 2001 (more than 4 fold drop). All the caged pet birds H3N8 influenza viruses reacted with A/Nanchang /13/00 virus except A/parakeet/Netherland/33/01. The songbird's viruses antigenically closely related to A/Equine/Jinlin/89.Phylogenetic analysis showed that each genes of H3N8 caged pet birds from different region of the world are all grouped together and highly related to each other, with 95.9-100% similarity. Especially, the songbird's viruses are more closely to each other, with 99.2-100% identity to each other. Seven of eight genes of caged pet birds viruses belong to Eurasia lineage except NA gene, which belong to North America avian lineage with respectively 92.8-94.2% and 75.9-76.4% identity to mat of A/Marllard / Edmonton / 220/90(H3N8) [MLEDM90] and A/Duck/Hokkaido/8/80 (H3N8)[DKHOK80]. The HA gene of caged pet birds viruses were highly related to A/Aquatic Bird/Hong Kong/399/99(H3N8)[ABHK99] and A/Equine/Jilin/89(H3N8) [EQJL89], respectively with93.0-94.1% and 92.9-93.6% similarity. The caged pet birds viruses NS gene showed a high degree of identity with the NS "A" allele. The NP gene of the caged pet birds influenza viruses showed 95.6-97.9% similarity to A/Aquatic bird/Hong Kong/ m603 / 98(H11N1)[ABHK98]. The most closely related viruses to the M, PA, PB2, and PB1 genes of caged pet birds influenza viruses are respectively A/Duck/Hong Kong/698/79(H5N3), A/Duck/Hong Kong/ Y439 /97 (H9N2), A/Environment/Hong Kong/437-6/99(H5Nl), and A/Duck/Anyang/AVL-1/01(H5N1); respectively with 97.0, 96.9, 95.5, and 96.7% homology.Replication test showed that all quail and chickens tested had substantial levels of viruses (3.5-5.01og10/ml and 1.8-4.51og10/ml respectively) in the trachea on day 3-post infection with less at day 5. In contrast no virus were shed in the cloacal in quail but only 2 chickens shed virus infects with low titer (0.0-1.51og10/ml). A notable feature of the studies was the amount of viruses detected in the drinking water both at 3 and 5 days post infection. None of the inoculated birds showed any signs of disease. The songbird H3N8 virus replicated more efficiently in quail than in chickens. Transmission test showed that only directly contact quails could detect the virus in the trachea on day 5 after infection. There are no detectable virus found in chickens and quail by fecal contact, aerosol contact routine. The above studies indicated that the H3N8 viruses are transmitted through the water and the viruses were still detectable in the water troughs after the poultry had stopped shedding. The stability test of the H3N8 virus in drinking water supported that the avian species might transmit the virus among the caged pet birds.In general, the caged pet birds H3N8 viruses were antigenically and molecularly closely related to each other and to earlier isolates of H3N8 influenza viruses from different regions of the world indicating that these viruses are highly genetically conserved and that a stable lineage occurs in passerine birds. All the caged pet bird H3N8 influenza viruses came from a common precursor.