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Isolation, Identification and Molecular Analysis of the Main of Genes Avian Influenza Virus Isolates from Different Hosts

Author: WangWeiLi
Tutor: QianAiDong
School: Jilin Agricultural University
Course: Preventive Veterinary Medicine
Keywords: avian influenza virus isolation and identification sequence analysis inherit evolution antigen distribution
CLC: S852.65
Type: PhD thesis
Year: 2006
Downloads: 724
Quote: 2
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Abstract


In this study, 17 strains of H5 and H9 subtype avian influenza virus (AIV)were isolated fromtrachea, cloacal swabs and tissue of chickens, gooses, ducks, pigeons and swines. They were found asglobular enveloped virion under TEM. The hemagglution inhibition (HI), Neuraminidase inhibition (NI)test and fluorescence RT-PCR indicated that these isolates belong to H5 or H9 subtype of influenza Avirus.The results of the 50 percent embryo infectious doses (EID50),Intravenous pathogenicity index(IVPI), iutraeerebral pathogenicity index (ICPI) showed that 15 (4 from chickens, 8 from gooses, 1fromducks, 1from pigeons and 1 from swines) of the HSN1 AIV isolates are highly pathogenic avianinfluenza and 2 of the H5N2 and H9N2 AIV isolates from chockens are low pathogenicity. AIV areunstable to heat, and are inactivated under 56℃for 30min,60℃for 10min and 65℃~70℃for 3min.AIV are sensitive to ether, chloroform and acid.Based on published gene sequences in Genebank, 6 pairs of specific primers which is used toamplify HA and NA genes of H5N1,H5N2 and H9N2 AIV isolates were designed and synthesised.First the viral RNAs of all the isolates were extracted and used in reverse transcription-polymerasechain reaction (RT-PCR)for amplifying the full-length cDNAs of HA and NA genes of the isolates.The cDNAs were then cloned to vector pMD18-T, the gained recombinants were transferred into JM 109bacteria and identified by enzyme digestion, PCR confirmation and then sequenced. The results ofsequence analysis showed that the cloned HA genes of 16 H5 subtype AIV isolates from different hostscontain whole open reading frame, consist of 1707 bp and encode 568 amino acids residues. There are 7(or 6)potential glycosylation sites and 6 basic amino acids (R-R-R-K-K-R) insert at the cleavage sites inHA genes of the AIV isolates which is a symbol of highly pathogenic avian influenza. Amino acids ofreeeptor-binding sites. are YWIHELY, amino acids of left edge of receptor-binding sites are SGVSS,amino acids of right edge of receptor-binding sites are NGQSG; amino acids of receptor-binding sites226 and 228 were of binding to Avian. The cloned NA genes consist of 1350 (G7, 1410) bp and encode450(469) amino acid residues. There are 3(G7,4) potential glycosylation sites in NA genes of the 15H5N1 influenza virus isolates, There are 20 amino acids missing in NA genes after place 48 (except G7)which is of prevalence virus. The cloned HA genes of H5N2 and H9N2 AIV isolates from chickenconsist of 1695,1683bp and encode 564,560 amino acid residues respectively and contains whole open reading frame without basic amino acids insert at the cleavage site in HA genes of the influenza virusisolates which is a symbol of lowly pathogenic avian influenza ,the cloned NA genes of H5N2 andH9N2 AIV isolates from chicken consist of 1401, 1410 bp and encode 467, 470 amino acid residuesand contain whole open reading frame. Amino acids of receptor-binding sites of H9N2 AIV isolate areYWTNVLY, amino acids of left edge of receptor-binding sites are NGQQG, amino acids of right edgeof receptor-binding sites are GTSKA.HA and NA genes of AIV isolates and HA and NA genes of the isolates and reference isolates areanalysiscd by software. The results of phylogenetic analysis showed homology of nucleotides andamino acids of HA genes is 95.4%~99.6%and 95.2%~99.5%among all the isolates; homology ofnucleotides and amino acids of NA genes is 95.9%~100%and 95.3%~100%among all the isolates.Homology of nucleotides and amino acids of HA genes is 94.2%~99.9%, 94.2%~99.6%betweensome isolates and the reference ones, homology of nucleotides and amino acids of NA genes is86.0%~100%,86.6%~100%between some isolates and the reference ones. The results ofphylogenetic analysis showed homology is high in same subtype and doesn’t have obvious differencebecause of hosts, and genes of the isolates come from different source, so genes of the isolates arevariety.Based on published gene sequences in Genebank, 11 pairs of specific primers of 8 genes weredesigned and synthesised.The viral RNAs were extracted and used in RT-PCR for amplifying the eightfull-length cDNAs of the 2 goose and 1 pigeon isolates. The cDNAs were then cloned to vector andsequenced. The results show that each of 8 genes cDNA fragments contain the whole open readingframes, Full-length cDNAs of each genes are 1730bp、1371 bp (1410bp)、1542 bp、2322 bp、2330 bp、2233 bp、1020 bp and 884 bp and encode 568、449 (G7 469)、499、757、759、716、351and 230 aminoacids residues respectively, The results of phylogenetic analysis showed the isolates were divided intotwo different genetic groups,G7 is a sylnbol of one group with no amino acids missing in NA and NSgenes. High homology exists among each genes of the isolates with A/GS/HK/ww28/00, 98.1%~99.6%, for HA、NA、NPand PB1 4 genes, homology above 99.1%, myble the 4 genes of G7 wereprovided by A/GS/HK/ww28/00, they all come from the ancestor, A/GS/GD/1/96, and belong to thesame allele.Group of G8 and P belong to same allele with 20 amino acids missing after 48 of NAgene,and 5 amino acids missing after 79 compared with other virus.Virus isolation and viral antigen distribution of a goose AIV isolate in different tissues of infectedchickens by nose and eye drop application, intravenous injection were studied. The results showed thatthe AIV can be isolated from trachea, cloacal swabs at postinnoculation 12h-192h by virus isolation,the AIV can be detected from heart, liver, spleen, lung, kidney, brain and muscle at postinnoculation 24h-216h by fluorescence RT-PCR and imnunofluorecence. The results show that the isolate is highlypathogenic to chicken, and the time of antigen distribution is different with different tissues.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology
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