【作者】 朱于敏； 于晓明； 黄耀伟； 黄芬芬； 于瑞嵩； 董世娟； 李震；

【Author】 Zhu Yumin,Yu Xiaoming,Huang Fenfen,Yu Ruisong,Dong Shijuan,Li Zhen

【机构】 上海市农业科学院畜牧兽医研究所； 美国弗吉尼亚理工学院暨州立大学； 江西农业大学动物医学院；

【摘要】 基因4型戊型肝炎病毒(HEV)是流行于我国的主要基因型,本研究旨在建立基因4型猪HEV本土分离株(SAAS-FX17)的感染性克隆,为HEV的复制及致病机理研究提供技术平台。采用套式RT-PCR及RACE方法,将SAAS-FX17株的全长cDNA分10段进行扩增,得到全基因序列。经序列分析,找到均匀分布在全基因序列上的4个单一酶切位点,将全基因序列分成5段进行重新扩增,5’端和3’端加上2个酶切位点,得到5个两端都含有单一酶切位点的片段,分别克隆至pJET1.3载体中。将pGEM-9Zf(-)载体的多克隆位点替换为构建全长cDNA所需的6个酶切位点,经5次双酶切、连接,构建成SAAS-FX17株的全长cDNA克隆。经体外转录,转染Huh-7细胞及肝内注射SD大鼠进行体内外病毒拯救。细胞转染实验经IFA检测到ORF2蛋白的表达,证实RNA转录物可以在Huh-7细胞内增殖。动物实验结果发现2(2/5)只实验组大鼠在注射RNA后第23天粪便HEVRNA呈阳性,第30天5只大鼠粪便HEVRNA全部为阳性,一直持续至第45天。3(3/5)只大鼠在感染后第30天血清抗体阳转,在第38天抗体水平达到峰值。2(2/5)只大鼠抗体阳转时间较迟,出现在感染后的第40d,在第45天达到峰值。所有实验组大鼠血清抗体水平在感染后第52d恢复到感染前水平。本研究成功构建了第一株本土基因4型HEV的感染性克隆,证明SD大鼠可作为HEV研究的动物模型。更多还原

【Abstract】 Genotype 4 hepatitis E virus(HEV)is the major genotype prevalent in China.This study aims to construct the infectious cDNA clone of a native genotype HEV strain(SAAS-FX17).Ten overlapping fragments spanning the entire genome were amplified by nested reverse transcription-PCR and Race methods.The full-length genomic sequence was obtained by sequence analysis of the ten overlapping fragments.4 unique restriction sites were found across the genome and an alian restriction site was added to the 5’ and 3’ end,respectively.5 overlapping fragments covering the full-length viral genome flanked by unique restriction enzyme sites were re-amplified and cloned into pJET1.3 vector.The plasmid pGEM-9Zf(-)vector was modified by replacing the polyclonal sites with a stuffer fragment which contained the above-mentioned 6 restriction enzyme sites.The five overlapping fragments were sequencially cloned into the pGEM-9Zf(-)vector to get the full-length cDNA clone of SAAS-FX17 strain.Capped RNA transcripts of the cDNA clone was transfected to human liver Huh-7 cells and inoculated intrahepatically into SD rats to rescue the virus.ORF2 capsid proteins was identified in the tranfected Huh7 cells,indicating that the cDNA clones were replication competent.The fecal virus shedding by 2(2/5)inoculated SD rats were firstly detected on the 23 days after inoculation(dpi)and for all 5(5/5)on the 30dpi.The fecal virus shedding lasted until 45 dpi.Seroconversion to anti-HEV for 3(3/5)rats were firstly detected on the 30 dpi and peaked on the 38dpi.Whereas,2(2/5)rats had a delayed seroconversion on the 40dpi and peaked on the 45dpi.The seroconversion in all the rats returned to the background level as negative control on the 52dpi.This is the first report of construction of infectious cDNA clone of genotype 4 HEV,which also demonstrated that SD rats can be used as animal model in HEV research.更多还原