Clonning And Sequence Analysing Of Full-Length Genes Of High Pathogenic Avain Influenza Viruse Strains H5 Subtype From Chicken, Duck And Strich

In the present study, three avain influenza viruse (AIV) isolates (A/chick/Henan/88/02 (CK), A/duck/Henan/89/02(DK)and A/Ostrich/Henan/90/02(OR)were studied by testing their pathogenicity , infection spectrum, immunology specificity, biologic and molecular biologic specificity, genetic variation and evolution etc. The results of hemagglutination(HA) test, hemagglutination inhibition(HI) test,ELD50 assay,animal pathogenicity and experimental challenge indicated that three isolates are all high pathogenic avain influenza(HPAI) virus belonging to H5N1 subtype, and the strain OR shown lower pathogenicity to chicken than the other two strains , which reveals that AIV have great host specificityThe isolates were replicated in SPF chicken embryo.Full-length cDNAs of eight genes from each isolate were amplified by RT-PCR and then successfully cloned into vector pMDT-18. All cDNA segments of eight genes from each isolate were sequenced and compared with each other and also with some published sequences of AIVs for their homology .The sequencing result shown that all eight cDNA fragments of each strain included the completed open reading frame, initiator and terminator codon corresponding to AIV genome,respectively.Analysis of homology and genetic evolution shown that l)The sequence homologies for HA gene between Eurasia lineage strain Ire83 and isolates Ck,Dk and OR were 6.9%,86.0%and 86.1 % respectively,but only 77.5%, 76.9% and 76.7% between North American lineage strain PA83 and three isolates.All three isolates have a same deduced amino acid(AA) sequence PQRERRRKKR in HA lysing site, with more than 5 Basic amino acids, suggested that the strains are all HPAI viruses.2)The homology of NA gene between DK and OR was 98%, and 95.6% and 96% between Ck and the other two strains, respectively.A deletion of 59 nucleic acid was found at the position 165-224 both in DK and OR. implied that they have very intimate relationship.The homologic analysis of NP gene between three isolates shown great difference in nucleic acid sequenceC 91.1 %-99.5%), but less difference in amino acids homology (94%-99.4%) , suggested that AIV is type-specific.The nucleic acid sequences of M genes from all isolates shown a high homology up to 99%, especially a 99.4% homology between DK and OR.The nucleic acid sequence homology of NA genes between the isolated strains and the published strains from isotype animals was more than 98% Analysis of deduced AA sequence further confirmed that M gene is highly conservative The homologies between NS gene of the three strains and some published strains were more than 98%, suggested that NS gene is highly conservative.Cladogram analysis of PA PB1 PB2 genes suggested that the genetic evolution route of three isolates is very clear: strains DK and OR belong to the same branch different from which formed by Hongkong and North America strains.Homologies of polymerases between the strains DK and OR were 98%-99.4%.