【作者】 王秀荣； 邓国华； 于康震； 乔传玲； 刘丽玲； 陈化兰； 姜永萍；

【Author】 WANG Xiu-rong; DENG Guo-hua, YU Kang-zhen, QIAO Chuan-ling, LIU Li-ling, CHEN Hua-lan, JIANG Yong-ping(1. National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Harbin 150001,china; 2. Animal Influenza research Center of Harbin Veterinary Research Institute of CAAS, Harbin, 15000I,china)

【机构】 中国农业科学院哈尔滨兽医研究所农业部动物流感重点开放实验室及兽医生物技术国家重点实验室；

【摘要】 流感病毒是属于正粘病毒科的RNA病毒,只有A型流感病毒可以在自然条件下感染鸟类。A型流感病毒基因组含有8个单股负链RNA病毒节段,从禽类中分离到15个血凝素、9个神经氨酸酶亚型。A型流感病毒感染家禽后根据致病力不同可以分为高致病力和低致病力病毒,高致病力毒株对禽有高度致死力,死亡率可高达100%,这些高致病力毒株均属于H5和H7亚型,但不是所有的H5和H7亚型流感都是高致病力的毒株。低致病力毒株大多数属于H9N2亚型,在全世界范围引起广泛的疾病综合症,H9N2病毒引起的病症比H5和H7禽流感引起的病症要温和得多,主要局限于呼吸道症状,但是在其他疾病病原合并感染或环境因素的协同下,临床症状可能严重得多。我们在基因芯片平台上建立了一种可靠地检测H5、H7、H9亚型禽流感病毒(AIV)的快速检测技术,实验中使用的毒株为A/Goose/Guangdong/1/96(H5N1)、A/African starling/983/79(H7N1)and A/Turkey/Wisconsin/1/66(H9N2)。本研究设计的DNA芯片可以用来鉴别禽流感病毒的亚型,通过RT-PCR获得大的500bp的禽流感病毒基因cDNAs片段,克隆,从重组质粒扩增DNA片段,并点到玻璃载体上,制成芯片。在病毒RNA反转录过程中,用Cy5标记做靶cDNAs。靶cDNAs是一个包括禽流感病毒HA、NS和M基因的混合物。依据M、NS基因鉴别型,依据HA基因鉴别亚型。扫描芯片上探针结合位点,杂交信号与预期设想基本一致。结果显示,DNA芯片技术可以提供一种有效的AIV诊断方法。更多还原

【Abstract】 Abstract: Influenza viruses, which belong to the orthomyxoviridae family, enveloped RNA viruses. Only type A influenza viruses are known to cause natural infections in birds. Influenza A viruses contains a genome of eight single-stranded RNA segments of negative-polarity. But viruses of all 15 haemagglutinin and all 9 neuraminidase influenza A subtypes in the majority of possible combinations have been isolated from avian species. Influenza A viruses infecting poultry can be divided into two distinct groups on the basis of their ability to cause disease. The very virulent viruses cause highly pathogenic avian influenza (HPAI), in which mortality may be as high as 100%. These viruses have been restricted to subtypes H5 and H7, although not all viruses of these subtypes cause HPAI. However, H9N2 viruses have recently been responsible for disease in poultry in various parts of the world. The virus causes a much milder, primarily respiratory disease, which maybe exacerbated by other infections or environmental conditions. We have developed a rapid microarray-based assay for the reliable detection of H5, H7 and H9 subtypes of avian influenza virus (AIV). The strains used in the experiment were A/Turkey/England/N28/73 (H5N2), A/African starling/983/79 (H7N1) and A/Turkey/Wiscosin/1/66 (H9N2) .The current DNA micro array has been shown to identify the subtypes of AIV. The cDNAs clone which encoding approximately 500-bp influenza virus gene fragments were obtained by Reverse transcription PCR technique, the DNA fragments which were reamplified from recombinant plasmids, were spotted on a glass-bound microarray. Cy5-labeled fluorescent probes were hybridized to these target cDNAs, which generated from vRNA during reverse transcription. These target cDNAs contained multiple fragments of AIV including the hemagglutinin gene, nonstructural protein gene and matrix protein gene. The type of AIV were detected with the nonstructural and matrix protein genes, and the subtype of AIV were differentiated with hemagglutinin gene. The arrays were scanned to determine the probe binding sites. The hybridization pattern agreed perfectly with the known grid location of each target. The results show that DNA microarray technology provides a useful diagnostic method for AIV.更多还原