糖基化影响H9N2亚型禽流感病毒在小鼠体内复制能力和对α-2,6唾液酸受体的亲和性
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摘要
利用反向遗传操作技术验证了H9N2亚型禽流感病毒流行株(A/Chicken/Shandong/903,简称903)HA蛋白200和295位氨基酸糖基化可以影响病毒对α-2,6唾液酸受体的亲和性,同时也能影响病毒在小鼠体内复制能力。固相ELISA结果显示,突变病毒N200Q(N→Q)和N295Q(N→Q)都能降低病毒对α-2,6唾液酸受体的亲和性。荧光定量结果显示,突变病毒N295Q主要在小鼠肺脏、脾脏和肾脏中复制(M基因拷贝分别为903野毒的56,64,39倍);而突变病毒N200Q主要在小鼠肾脏中复制(M基因拷贝分别为903野毒的936倍)。本试验为阐明H9N2亚型流感病毒跨种间传播的分子机制提供依据。
Reverse genetic manipulation technology was used to determine whether H9N2 subtype avian influenza virus strain(A/Chicken/Shandong/903,903)HA protein 200 and 295amino acid glycosylation could affect the affinity of virus toα-2,6sialic acid receptor,as well as its replication ability in mice.Solid-phase ELISA result showed that mutant viruses N200Q(N→Q)and N295Q(N→Q)could reduce the affinity of virus toα-2,6sialic acid receptor.Fluorescent quantitative results showed that mutant virus N295 Q mainly replicated in the lung,spleen,and kidney of mice(M gene copy was 56-,64-,and 39-fold of 903 wild virus,respectively),and mutant virus N200 Q mainly replicated in the kidney(M gene copy was 936-fold of 903 wild virus).The research results provided an important theoretical and experimental basis for explaining the molecular mechanism of H9N2 subtype avian influenza virus in interspecies transmission.
Reverse genetic manipulation technology was used to determine whether H9N2 subtype avian influenza virus strain(A/Chicken/Shandong/903,903)HA protein 200 and 295amino acid glycosylation could affect the affinity of virus toα-2,6sialic acid receptor,as well as its replication ability in mice.Solid-phase ELISA result showed that mutant viruses N200Q(N→Q)and N295Q(N→Q)could reduce the affinity of virus toα-2,6sialic acid receptor.Fluorescent quantitative results showed that mutant virus N295 Q mainly replicated in the lung,spleen,and kidney of mice(M gene copy was 56-,64-,and 39-fold of 903 wild virus,respectively),and mutant virus N200 Q mainly replicated in the kidney(M gene copy was 936-fold of 903 wild virus).The research results provided an important theoretical and experimental basis for explaining the molecular mechanism of H9N2 subtype avian influenza virus in interspecies transmission.
引文
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[2]郭元吉,谢健屏,王敏,等.从我国人群中再次分离到H9N2亚型流感病毒[J].中华实验和临床病毒学杂志,2000,14(3):209-212.
[3]郭元吉,谢健屏,吴昆昱,等.流感病毒A/广州/333/99(H9N2)毒株基因组特性的研究[J].中华实验和临床病毒学杂志,2002,16(2):142-145.
[4]CONG Y L,PU J,LIU Q F,et al.Antigenic and genetic characterization of H9N2swine influenza virus in China[J].J Gen Virol,2007,88(Pt 7):2035-2041.
[5]HATTA M,GAO P,HALFMANN P,et al.Molecular basis for high virulence of Hong Kong H5N1influenza A viruses[J].Science,2001,293(5536):1840-1842.
[6]LI Z,CHEN H,JIAO P,et al.Molecular basis of replication of duck H5N1influenza viruses in a mammalian mouse model[J].J Virol,2005,79:12058-12064.
[7]PU J,WANG S,YIN Y,et al.Evolution of the H9N2influenza genotype that facilitated the facilitated the genesis of the novel H7N9virus[J].Proc Natl Acad Sci USA,2014,112(2):548-553.
[8]STEVENS J,CORPER A L,BASLER C F,et al.Structure of the uncleaved human H1hemagglutinin from the extinct 1918influenza virus[J].Science,2004,303(5665):1866-1870.
[9]CONNOR R J,KAWAOKA Y,WEBSTER R G,et al.Receptor specificity in human,avian,and equine H2and H3influenza virus isolates[J].Virology,1994,205(1):17-23.
[10] MATROSOVICH M,TUZIKOV A,BOVIN N,et al.Early alterations of the receptor-binding properties of H1,H2,and H3avian influenza virus hemagglutinins after their introduction into mammals[J].J Virol,2000,74(18):8502-8512.
[11] LIU J,STEVENS D J,HAIRE L F,et al.Structures of receptor complexes formed by hemagglutinins from the Asian Influenza pandemic of 1957[J].Proc Natl Acad Sci USA,2009,106(40):17175-17180.
[12] XU R,MCBRIDE R,PAULSON J C,et al.Structure,receptor binding,and antigenicity of influenza virus hemagglutinins from the 1957 H2N2pandemic[J].J Virol,2010,84(4):1715-1721.
[13] MATROSOVICH M,TUZIKOV A,BOVIN N,et al.Early alterations of the receptor-binding properties of H1,H2,and H3avian influenza virus hemagglutinins after their introduction into mammals[J].J Virol,2000,74(18):8502-8512.
[14] GLASER L,STEVENS J,ZAMARIN D,et al.A single amino acid substitution in 1918influenza virus hemagglutinin changes receptor binding specificity[J].J Virol,2005,79(17):11533-11538.
[15] IMAI M,WATANABE T,HATTA M,et al.Experimental adaptation of an influenza H5HA confers respiratory droplet transmission to a reassortant H5HA/H1N1virus in ferrets[J].Nature,2012,486(7403):420-428.
[16] XIONG X,COOMBS P J,MARTIN S R,et al.Receptor binding by ferret-transmissible ferret-transmissible H5avian influenza virus[J].Nature,2013,497(7449):392-397.
[17] NIDOM C A,TAKANO R,YAMADA S,et al.Influenza A(H5N1)viruses from pigs,Indonesia[J].Emerg Infect Dis,2010,16(10):1515-1523.
[18] GAMBARYAN A S,MATROSOVICH T Y,PHILIPP J,et al.Receptor-binding profiles of H7subtype influenza viruses in different host species[J].J Virol,2012,86(8):4370-4378.
[19] SRINIVASAN K,RAMAN R,JAYARAMAN A,et al.Quantitative description of glycan-receptor binding of influenza A virus H7 hemagglutinin[J].PLoS One,2013,8(2):e49597.
[20] YANG H,CARNEY P J,CHANG J C,et al.Structural analysis of the hemagglutinin from the recent 2013H7N9influenza virus[J].J Virol,2013,87(22):12433-12446.