| ABSTRACT:To study the pathogenicity and molecule epidemiology H9N2 subtype avian influenza viruses(AIVs),25 isolates from China especially from Henan province during 1998～2005 were compared and analysed by crossing-hemagglutinin inhibition test(HI),EID50 test,ELD50 test,ICPI test,IVPI test and challenge infection test.The results revealed that the pathogenicity among 25 H9N2 subtype was different.Their EIDS0 were 10-6/0.2 mL～10-8.8/0.2 mL,ELD50 were 10-4.8/0.2 mL～10-8.7/0.2mL;MDT were 66.5h～103.2h;Picking up 8 strains which had different pathogenicity,continued ICPI test,IVPI test and challenge infection test.It showed that the ICPI and IVPI of most strains were zero.The peak of emiting virus were the 5th to 6th day after challenging.Among them,A/ CK/ HN/A3/98(H9N2)(3#) and A/CK/HN/02(H9N2)(18#)illustrated high pathogenicity.Not only had the highest EID50,10-8.8/0.2mL and 10-8.8/0.2mL,the highest ELD50,10-7.9/0.2mL and 10-8.7/0.2 mL,respectively;but also had the lowest MDT,66.5h and 69.9h。The ICPI were 0.238 and 0.437;IVPI were 0.34 and 0.51;In the challenge infection test the two strains emitted much more virus and lasted longer.According to these figures,all the strains can be mainly divided into three types:High pathogenicity:3#,A/CK/SQ/00(H9N2)(855),18#;Normal pathogenicity:A/CK/QX/00(H9N2)(13#),A/CK/SC/01(H9N2)(14#),A/CK/ZH/01(H9N2)(15#),A/CK/QX/01(H9N2)(17#),A/CK/HD/03(H9N2)(20#)A/CK/QX/04(H9N2)(23#);Low pathogenicity:A/Ck/HN/A1/98(H9N2) (1#),A/CK/HN/A2/98(H9N2)(2#),A/CK/HN/A4/98(H9N2)(4#),A/CK/HN/A5/98(H9N2) (5#),A/CK /GZ/99(H9N2)(6#),A/CK/HN/99(H9N2)(7#),A/CK/CG/00(H9N2)(9#),A/ CK/XC/00(H9N2)(10#),A/CK/ZM/00(H9N2)(11#),A/CK /PY/00(H9N2)(12#),A/CK /QX/01(H9N2)(16#),A/CK/ HN/02(H9N2)(19),A/CK/XZ/03(H9N2)(21#),A/CK/XZF/04 (H9N2)(22#)A/CK/KF/A1/05(H9N2)(24#)A/CK/KF/A2/05(H9N2)(25#).All the results revealed that the strain of different years and different places have different pathogenicity and their pathogenicity did not increase year by year.RT-PCR was employed to amplify the cDNA of HA gene of 3#,6#,10#,17#,18#, 20#,which can represent different pathogenicity.The result of their sequences analysis showed that:(1)cDNA of HA gene is composed of 1 683 bp nucleotides, encoding for 560 amino acids.And the pattern of cleavage sites were three types: RSSR↓GLF,RLSR↓GLF and VSSR↓GLF,typical low pathogenicity AIV strains,not the pattern of high pathogenicity AIV strains R-X-R/K-R.The receptor binding sites of 3#,6#,10#,17#,18# were YWTNVLY and excepting 3# and 18# had eight potential glycosylation sites.(2)The nucleotide homology of six strains were 82.9%～100%,the amino acid homology of them were 85.6%～99.8%.Excepting 20#,the nucleotide homology and the amino acid homology of the other five strains at least were 93%.Compared to DK/HK/G9/97 the nucleotide homology were 96.6%,95.7%,93.3%,95.7%,94.7%,82.5%;the amino acid homology were 96.4%,95.9%,94.9%,95.9%,95.1%,88.0%,they had a close homology.According to the phylogenetic tree,except 20#,others belong to Euraasian.(3)At the same time,the amino acid sequence of HA cleavage sites, potential glycosylation sites,amino acids of the receptor binding site of few strains changed.3# and 18# in 145aa～147aa increase a potential glycosylation site NGT and 20# in 141aa～143aa,305aa～307aa are NVT and NIS.Receptor binding site of 20# in 191aa and 198aa are H and E,amino acid sequence of HA cleavage site is VSSRGLF,which was different from other influenza virus isolate.(4)Compared the biology character with the sequences of HA gene,the high pathogenicity of 3# and 18# may had some relationship with the increase of potential glycosylation site in145aa～147aa,which could influence receptor binding site,increasing their pathogenicity.The change of the pathogenicity of 3# and 18# whether had some relationship with other genes need further study.The results of our study demonstrated that H9N2 subtype AIV in China during 1998～2005 had changed both in pathogenicity and HA gene,which provided scientific reference data for prevention against H9N2 AIV and the selection of vaccine strains.Keywords:AIV,HgN2 subtype,HI,RT-PCR,pathogenicity,hemagglutinin gene,clone, sequence analysis:...
|
PDF Full Text Request