Molecular Characterization And Pathogenicity For Poultry Of Eleven H5N2 Avian Influenza Viruses In China

Since the first emergence of H5N2 influenza virus in chickens in Pennsylvania in the United States, H5N2 avian influenza viruses have widely circulated in the world and caused many large outbreaks in poultry. During 2005-2006, eleven H5N2 viruses were isolated from mallard in jiang xi and heilongjiang province in china. To understand the characterization and pathogenicity of the avian influenza viruses (H5N2) isolated from wild birds in china, the whole genome of eleven H5N2 viruses were firstly analyzed, and then the potential threat of these wild birds isolate for domestic poultry were assessed.Sequence and phylogenetic Analysis:Viral RNAs were extracted from infection allantoic fluid by using Trizol Ls RNA extraction kit.RT-PCR for the amplification of all the gene fragments was performed by using fragment-speific primers. The PCR products were purified and sequenced on a CEQ 8000 DNA sequencer.Sequence data were compiled and analyzed by using the sequence analysis software package (DNASTAR).Sequence and phylogenetic analysis showed that the eleven strains were derived from avian influenza viruses of the Eurasian lineage, and which were divided into six viral genotypes. ten strains isolated from jiangxi province in 2005 had four to six serial basic amino acids at the hemmaglutinin cleavage site, which was considered a characteristic of influenza viruses that were highly pathogenic for chickens. Nucleotide and amino acid similarities of the HA,NA,PA and NP genes among the ten jiangxi isolates were high, and were respective 95.0%～98.7% and 93.9%～98.4%; and the highest percentages of identities between the HA,NP and NS genes of the ten jiangxi H5N2 viruses and that of DK/JX/25/04(H5N1) were 97.7%, which was the representative strain caused the outbreaks of highly pathogenic avian influenza in domestic poultry in southern province in 2004-2005. one isolated from HeiLongJiang province had a R-E-T-R motif in the connecting peptides that is typical for LPAI H5 viruses, and whose nucleotide homology was low in comparisons with jiangxi strains. Phylogenetic analysis showed that the HA,NA,PB1,PA and NS genes of the wild birds viruses clustered together, respectively, but they were descended from the different virus progenitor, and the PB2,NP and M genes divided into many branches. In addition, phylogenetic analysis showed that the HA and PA genes of ten jiangxi strains were in the same branch with influenza viruses (H5N1) isolated from both poultry and wild birds during 2004-2005 in china, and they were descended from the same virus progenitor GS/GD/1/96(H5N1).Experimental infection of chickens and ducks:4-week-old SPF chickens and ducks were inoculated intranasally with 0.2 ml allantoic fluid containing 106 EID50 viruses. The birds were sacrificed for virus isolation from the tracheal and cloacal swabs and organs at 3 and 5 days p.i and pathohistology were also performed on days 3 and 5 dpi. Pathogenicity essay:according to the recommendations of the OIE, ten of the 4-week-old birds were inoculated intravenously with 0.2 mL of a 1/10 dilution of infective allantoic fluid. The chickens were observed for clinical signs or death at 24-h intervals for 10 days.The results showed that Mallard/JX/I16/05, as a representative strain isolated from jiangxi province was highly pathogenic to chickens and ducks, and the intravenously pathogenicity index (IVPI) of the virus was 3.0 and 2.42, respectively. On the contrary, these birds showed neither clinical symptoms nor mortality after inoculation with Mallard/HLJ/134/06 virus. The results of intranasal infection showed that Mallard/JX/116/05 virus could kill much more birds including experimentally infected birds and contact birds, and cause severe neurological dysfunction. Pathoanatomical results showed that meninges, thymus, spleen and lung surface were hemorrhage, and liver and spleen were carina. Histological lesions were characterized by viral encephalitis,congestion, hemorrhage, degeneration to necrosis or heterophilis infliltration. High virus titers were detected in the all tissues and swabs, whereas All birds survived post infection with Mallard/HLJ/134/06 and did not show any clinical signs, and much lower virus titers were detected from only the bursa of fabricius and trachea, but all the experimentally inoculated and the contact control birds sero-converted on days 21 dpi.