Molecular Characteristics Analysis And Pathogenicity Study Of H5N1/H7N8/H10N4 Avian Influenza Virus

Avian influenza is an infection and syndrome caused by a type A virus whichbelongs to orthomyxoviridae. In this research, we conducted a survey of avianinfluenza virus of migratory bird in Qinghai Lake in spring 2006, and used sandwichELISA for screening, the antigen positive samples were inoculate into embryos inorder to isolate avian influenza virus, then three highly pathogenic H5N1 avianinfluenza virus were isolated from dying bar-headed goose, black-headed gull andwhooper swan. The three isolates were named A/Bar-headedGoose/Qinghai/F/2006(H5N1), A/Black-headed Gull/Qinghai/3/2006(H5N1) andWhooper Swan/Qinghai/01/2006(H5N1). The research results were, nucleotidesequence analysis revealed that three isolates shared a high homology with each other(99%) except PB1 genes of Bar-headed Goose/Qinghai/F/200 and Whooperswan/Qinghai/01/2006, which shared 94% identity. The results of genome sequencinganalysis of the three virus isolates suggested that they were closed to strains isolatedfrom Qinghai and Siberia in 2005. The HA genes of the three isolates had the samemultiple amino acid sequence (-RRRKKR-) at the connecting peptide between HA1and HA2, which is considered to be a distinguishing characteristic of highlypathogenic influenza viruses. The three isolates did not have a mutation of Glu92 inthe NS1 protein, and had acquired a Lys residue at position 627 of PB2 protein, whichhas been suggested to be associated with increased virulence of H5N1 viruses. Thethree virus isolates also had a 20-amino-acid deletion in the NA stalk (residues 49 to68), but did not have mutations at the amino acids (Leu26, Va127, Ala30, Ser31) in the transmembrane region of the M2 protein, which has been suggested to be associatedwith amantadine resistance. Nucleotide sequence and phylogenetic analysis revealedthat the three Qinghai viruses were very similar to each other and were closest toisolates obtained from Qinghai and Siberia in 2005, but distinct from other poultryviruses found in Southeast Asia. In experimental infection, all three viruses werehighly pathogenic to chicken and mice, as the OIE standard.In addition, subtype characterization, gene cloning, sequencing analysis andpathogenicity study of two avian influenza viruses, which isolates from duck in 1979,were also did. Two rare subtypes AIV were confirmed, they were H7N8 and H10N4,and the two viruses were named A/DK/Hubei/137/83(H10N4) andA/DK/Hubei/216/83(H7N8). HA,NA,M,NS,PA,PB1 and PB2, seven full-lengthgenes of each viruses, were amplified by using the universal primers. The amplifiedsegments were cloned into the pMD19-T vector, respectively. The results showed that,the NA sequence of the two viruses had no 20 amino acid missing in 48-68 sites, andno residue lost in NS gene in 263-277 sites. The motif of HA cleavage site wereP-E-V-V-Q-G-R and P-E-I-P-K-R. A/DK/Hubei/216/83(H7N8) shared 96%～98%homology with different subtypes avian influenza viruses which isolated from HongKong during 1972～1997, in addition, evolutionary analysis showed that the remainingfive gene except NA and PB2, had close phylogenetic relationship with 1972～1997strains. A/DK/Hubei/137/83(H10N4), sequence similarity analysis revealed that theNS, PA, PB1, PB2 gene shared 98%～99% homology with the 1997～2004 H5N1subtype avian influenza virus strains, and except NA and M gene, the other five geneformed independence branch with their own, and the distance between the otherisolates were far. The experimental infection result showed that the two viruses wereall low pathogenic avian influenza virus.