禽流感及汉坦病毒反向遗传操作体系建立
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摘要
禽流感是由正粘病毒科A型流感病毒属禽流感病毒引起的一种严重的人畜共患病,至2008年6月19日,共有385人感染,其中243人死亡,造成了巨大的经济损失和社会公共卫生安全问题。近几年快速发展的禽流感病毒的反向遗传学操作技术为研究禽流感病毒搭建了一个重要的研究平台。我们通过扩增Hela细胞中的人RNA聚合酶Ⅰ的启动子序列、人工合成RNA聚合酶Ⅰ的终止子序列,将启动子和终止子序列连接后插入真核表达质粒pVAX1中,构建了双向转录载体pZL2006。构建的pZL2006含有两套启动子和终止子序列,即RNA聚合酶Ⅱ的启动子(CMV启动子)和终止序列(aⅡBGH), RNA聚合酶Ⅰ的启动子(PⅠh)和鼠polⅠ终止序列(tⅠ),通过把编码vRNA的cDNA正向克隆至polⅡ启动子和终止序列之间即可实现vRNA的转录和病毒蛋白的双表达。选用2004年越南分离A/Viet Nam/1194/2004(H5N1)禽流感H5N1毒株,扩增了其基因组全部序列共8个基因片段,经过序列测定后确认扩增的基因片段与Genbank上的序列相符,将8个基因片段分别插入pZL2006载体后利用酶切鉴定筛选出阳性克隆测序后证实全部基因片段已经正确插入载体中,8个含禽流感全部基因片段的重组质粒构成禽流感病毒的反向遗传学操作系统。利用脂质体2000将8质粒系统转染进293T/MDCK细胞中,发现转染上述8质粒的细胞在36h后出现大部分细胞变圆、死亡、小块脱落并以丝相连等特征性病变,而对照组的的细胞则完全正常；利用电境在转染8质粒的细胞上清中观察到了呈球形,直径大小约80～120nm,囊膜表面具纤突,具有流感病毒典型形态的病毒粒子；血凝实验的结果表明拯救病毒的滴度为在26～27之间,病毒具有血凝活性及滴度较高；拯救的病毒重新接种MDCK细胞后在36小时可观察到细胞变圆、死亡、小块脱落并以丝相连等与野生型毒株相似的致MDCK特征病变的特性；拯救的病毒上清接种鸡胚48小时后,接种的10枚鸡胚全部死亡,而对照的鸡胚全部存活,证明重组病毒与野生型的病毒对鸡胚均具有致死作用；PCR扩增和序列测定证实拯救病毒与禽流感病毒A/Viet Nam/1194/2004(H5N1)株相同位置的序列相同,通过以上的实验结果表明成功的构建了禽流感病毒的反向遗传学操作体系。
汉坦病毒属布尼亚病毒科汉坦病毒属,是在欧亚大陆引起人类的HFRS,在美洲引起HPS的病原体,我国是受HFRS危害最为严重的国家,每年报道的病例数占世界报道病例数的90%以上。汉坦病毒株H8205株是本实验室自我国东北一HFRS病人血清中用Vero-E6细胞直接分离的毒株,同国内大部分省市的HFRS病人血清都可发生很强的免疫学反应,采用该株的NP做抗原,能较好地诊断流行于中国的汉坦病毒,已经对其生物学特性和动物致病试验等进行相关的研究,并取得了一系列的成果,但完整的基因组全序还未测定。我们通过获取含完整5'末端和3'末端的汉坦病毒H8205株L／M／S基因的全序列,与已发表的汉坦病毒的序列进行比对,发现汉坦病毒H8205株L基因与汉坦型病毒间氨基酸同源率高达95.5%～98.3%,核苷酸同源性为81.9%～88.8%；M基因与同型的代表毒株氨基酸的同源性为85.2%～99.7%,核苷酸同源性为74.5%～99.3%；S基因与同型的代表毒株氨基酸的同源性为97.2%～99.8%,核苷酸同源性为83.5%～99.4%。通过系统进化发生树发现H8205株L／M／S片段与同为汉坦型的病毒遗传距离较近,而与DOBV、SEOV、SNV、PUUV等遗传距离较远,通过参照毒株登陆国家和地区找出相近毒株,为下一步研究不同毒株的差别和抗原性变异提示方向。
汉坦病毒为负链RNA病毒,由于RNA结构特点和不稳定性使得直接在分子水平上对RNA病毒基因组难以操作,自从成功建立了以流感病毒为代表的分节段的负链RNA病毒反向遗传学操作系统,开启了流感病毒研究的新篇章,但汉坦病毒反向遗传学操作系统的建立相对滞后,使汉坦病毒研究缺少一个重要的平台,相关的研究无法进行。我们选用汉坦病毒H8205株,利用分段扩增的方法得到基因组全部片段,利用Ⅱs型限制性内切酶的特性将各个基因片段连接成L／M基因全长的cDNA,序列测定表明获得的L／M基因片段的全长基因序列完全正确,没有发生突变和缺失。将汉坦病毒的L／S基因插入真核表达质粒pCDNA-3.1(+) /pCDNA-3.1(+)-Hygro中构建重组质粒,转染真核细胞后可表达汉坦病毒的反式作用蛋白L和NP蛋白。构建含人RNA聚合酶Ⅰ启动子和鼠RNA聚合酶Ⅰ终止子的质粒pp2006,将汉坦病毒L基因的非编码区和GFP融合基因插入RNA聚合酶Ⅰ启动子(PⅠh)及其终止子(tⅠ)之间构成转录单位,转染细胞后,由细胞提供polⅠ,从PⅠh启动融合基因的合成,融合基因包含汉坦病毒L基因的5'和3'端的非编码区及GFP编码基因。汉坦病毒反式作用蛋白与融合基因结合成RNPs,GFP蛋白的表达即可启动。利用脂质体2000将汉坦病毒亚基因组感染性克隆体系(PCDNA-L/PCDNA-S/pp2006-L-GFP)转染293T细胞后24小时,观测到转染细胞中出现明显的绿色荧光蛋白,而对照细胞则无绿色荧光蛋白表达,说明汉坦病毒亚基因组感染性克隆构建成功。建立汉坦病毒亚基因组反向遗传操作系统是构建汉坦病毒反向遗传操作系统重要的环节,可以利用汉坦病毒亚基因组反向遗传操作系统评估构建的含RNA聚合酶Ⅰ质粒转录病毒基因组RNA的效率,通过检测报告基因GFP表达量,筛选出最佳的转染条件并对构建的质粒进行优化,为最终建立汉坦病毒反向遗传学操作系统奠定基础。
Avian Influenza (AI) is a serious zoonotic disease caused by Type A Avian Influenza Virus (AIV) of the family Orthomyxoviridae. By June 19,2008, totally 385 persons were infected by this virus, and 243 of them died. This disease caused huge economic losses, and public health and social security issues. In recent years, the newly developed reverse genetics technique of AIV provides an important platform for the study on AIV. We constructed a bi-direction transcription plasmid pZL2006 by amplifying human RNA polymeraseⅠpromoter sequence from Hela cells, synthesizing RNA polymeraseⅠterminator sequence, and inserting the combinations of promter and terminator sequence into eukaryotic expression plasmid pVAX1. The constructed plasmid pZL2006 contained two sets of promoter and termination sequences, namely RNA polymeraseⅡpromoter (CMV promoter) and termination sequences (aⅡBGH), RNA polymeraseⅠpromoter (PⅠh) and murine polymeraseⅠterminator sequences (tⅠ). The positive cDNA encoding vRNA was inserted between polymeraseⅡpromoter and termination sequence to achieve vRNA transcription and viral protein expression. In this study, we amplified the complete genome sequence of H5N1 strain A/Viet Nam/1194/2004(H5N1) isolated from Vietnam in 2004, and obtained eight gene fragments. The eight gene fragments were sequenced and their sequences compositions matched those in GenBank. These fragments were inserted into vector pZL2006, which was confirmed by sequencing positive clones from restriction digestion. The eight recombinant plasmids containing the complete gene fragment of AIV constructed the reverse genetics system of AIV. The eight-plasmid system was transfected into the 293 T/MDCK cells by using Liposome 2000. After 36 h of this transfection, most of the infected cells became round, died, and small pieces of cells fell off etc, while the control cell group was in normal state. The spherical particle, about 80-120 nm in diameter, having capsule surface with spike was observed in the supernatant of transfected cells with eight plamids by electron microscope, which was characterisitic morphology of influenza viron. The results of hemagglutination test (HA) showed that the titer to rescue virus was between 26 to 27, which means that the rescued virus has hemagglutinin activity and a higher titer. After 36 h of reinoculation of the rescued virus in MDCK cells, the infected cells became round, died, small pieces of cells fell off etc., which were very similar to the cytopathic characteristics of MDCK cells infected by the wild strain. After 48 h of the inoculation of the supernatant of rescued virus in 10 chicken embryo eggs, all of them died, while the control chicken embryo eggs survived. This indicated that both the recombinant virus and the wild virus have lethality to chicken embryo eggs. PCR amplification and sequencing confirmed that the rescued virus is identical to the A/Viet Nam/1194/2004(H5N1) strain in the sequence composition from the same region. Based on the experimental results mentioned above, it can be concluded that the reverse genetics operating system of AIV was successfully constructed in this study.
Hantaviruses belonging to Hantavirus, Bunyaviridae, can cause human hemorrhagic fever with renal syndrome (HFRS) in Asia and Europe, and hantavirus pulmonary syndrome (HPS) in America. HFRS is the most serious in China, and more than 90 percent cases were reported in our country. Hantavirus strain H8205 was isolated in this lab from the serum of one HFRS patient in Heilongjiang province by using Vero-E6 cells. This strain can produce strong immunological responses to the serum of HFRS patients from most of China. The NP of this strain was used as antigen, and was very useful in the diagnosis of Hantavirus prevalent in China. The biological characteristics and animal pathogenic test were investigated in this study, and a series of results were obtained, but the complete genome has not been sequenced. Currently, the sequences of the genes L, M, S of Hantavirus H8205 Strain including the complete 5' and 3'ends were obtained by using RT-PCR and RACE. Compared with the published sequences of Hantavirus representative strains, H8205 has 95.5%-98.3% amino acid homology and 81.9%-88.8% nucleic acid homology in L gene,85.2%-99.7% amino acid homology and 74.5%-99.3% nucleic acid homology in M gene, and 97.2%-99.8% amino acid homology and 83.5%-99.4% nucleic acid homology in S gene. The phylogenetic tree based on the L/M/S gene sequence analysis indicated that H8205 is genetically closer to other strains belonging to the same Hantavirus type, while distantly related to DOBV, SEOV, SNV, PUUV. By reference strains, we can find similar strains from different countries or regions, which provides information for the further study on the differences among strains and their antigen variation.
Hantavirus is negative-sense RNA virus. RNA structure characteristics and instability makes it very difficult to operate the genome of RNA virus at the molecular level. The successful establishment of reverse genetics operating system of segmented negative-sense RNA virus, e.g. AIV, is a new start point for the study of Influenza Virus. However, the construction of Hantavirus reverse genetics systems has not established yet, which prevented from performing the related studies of Hantavirus due to lacking an important study platform. In this study, the Hantavirus H8205 strain was chosen. The complete gene fragments of this strain were obtained by segment amplification. The gene fragments were ligated into the full-length cDNA of L/M genes by usingⅡs restriction endonucleases. The sequencing results showed that the obtained full-length cDNA was completely correct, without any mutations or deletions. Two recombinant plasmids, PCDNA-L/PCDNA-S, were constructed by inserting the L/S genes of H8205 into the eukaryotic expression vector pcDNA-3.1(+)/pcDNA-3.1-Hygro(+). Two trans-acting proteins L and NP of Hantavirus were expressed by transfecting eukaryotic cells with the RNA polymeraseⅡpromoter (PCMV) and terminator sequences in the eukaryotic expression vector. The plasmid pp2006 containing human RNA polymerase I promoter and murine RNA polymeraseⅠterminator was constructed. The non-coding region of Hantavirus L gene and GFP fusion gene were inserted between RNA polymerase I promoter (PⅠh) and the terminator (TⅠ) to form a transcription unit. After the transfection into cells, the fusion gene containing the 5'and 3'non-coding regions of L gene of Hantavirus and GFP coding gene, was expressed by using the polymerase I in cells and the promoter PⅠh. The trans-acting protein of Hantavirus combined the fusion gene into RNPs and the expression of GFP protein was activated. After 24 h of the transfection of Hantavirus subgenomic infectious clone (PCDNA-L/PCDNA-S/ PP2006-L-GFP) into the 293 T cells by using Liposome 2000, obvious green fluorescent protein was observed in the transfected cells, while there is no green fluorescent protein observed in the control cells. This means that the subgenomic infectious clone of Hantavirus was successfully established, which is an important part to establish the reverse genetics system of Hantavirus. The system of subgenome can be used to evaluate RNA transcription efficiency of the plasmid containing RNA polymerase I, and find the optimal transfection conditions and optimized the constructed plasmid by detecting the expression quantity of the reporter gene GFP, which will lay a solid foundation for the final establishment of Hantavirus reverse genetics system.
汉坦病毒属布尼亚病毒科汉坦病毒属,是在欧亚大陆引起人类的HFRS,在美洲引起HPS的病原体,我国是受HFRS危害最为严重的国家,每年报道的病例数占世界报道病例数的90%以上。汉坦病毒株H8205株是本实验室自我国东北一HFRS病人血清中用Vero-E6细胞直接分离的毒株,同国内大部分省市的HFRS病人血清都可发生很强的免疫学反应,采用该株的NP做抗原,能较好地诊断流行于中国的汉坦病毒,已经对其生物学特性和动物致病试验等进行相关的研究,并取得了一系列的成果,但完整的基因组全序还未测定。我们通过获取含完整5'末端和3'末端的汉坦病毒H8205株L／M／S基因的全序列,与已发表的汉坦病毒的序列进行比对,发现汉坦病毒H8205株L基因与汉坦型病毒间氨基酸同源率高达95.5%～98.3%,核苷酸同源性为81.9%～88.8%；M基因与同型的代表毒株氨基酸的同源性为85.2%～99.7%,核苷酸同源性为74.5%～99.3%；S基因与同型的代表毒株氨基酸的同源性为97.2%～99.8%,核苷酸同源性为83.5%～99.4%。通过系统进化发生树发现H8205株L／M／S片段与同为汉坦型的病毒遗传距离较近,而与DOBV、SEOV、SNV、PUUV等遗传距离较远,通过参照毒株登陆国家和地区找出相近毒株,为下一步研究不同毒株的差别和抗原性变异提示方向。
汉坦病毒为负链RNA病毒,由于RNA结构特点和不稳定性使得直接在分子水平上对RNA病毒基因组难以操作,自从成功建立了以流感病毒为代表的分节段的负链RNA病毒反向遗传学操作系统,开启了流感病毒研究的新篇章,但汉坦病毒反向遗传学操作系统的建立相对滞后,使汉坦病毒研究缺少一个重要的平台,相关的研究无法进行。我们选用汉坦病毒H8205株,利用分段扩增的方法得到基因组全部片段,利用Ⅱs型限制性内切酶的特性将各个基因片段连接成L／M基因全长的cDNA,序列测定表明获得的L／M基因片段的全长基因序列完全正确,没有发生突变和缺失。将汉坦病毒的L／S基因插入真核表达质粒pCDNA-3.1(+) /pCDNA-3.1(+)-Hygro中构建重组质粒,转染真核细胞后可表达汉坦病毒的反式作用蛋白L和NP蛋白。构建含人RNA聚合酶Ⅰ启动子和鼠RNA聚合酶Ⅰ终止子的质粒pp2006,将汉坦病毒L基因的非编码区和GFP融合基因插入RNA聚合酶Ⅰ启动子(PⅠh)及其终止子(tⅠ)之间构成转录单位,转染细胞后,由细胞提供polⅠ,从PⅠh启动融合基因的合成,融合基因包含汉坦病毒L基因的5'和3'端的非编码区及GFP编码基因。汉坦病毒反式作用蛋白与融合基因结合成RNPs,GFP蛋白的表达即可启动。利用脂质体2000将汉坦病毒亚基因组感染性克隆体系(PCDNA-L/PCDNA-S/pp2006-L-GFP)转染293T细胞后24小时,观测到转染细胞中出现明显的绿色荧光蛋白,而对照细胞则无绿色荧光蛋白表达,说明汉坦病毒亚基因组感染性克隆构建成功。建立汉坦病毒亚基因组反向遗传操作系统是构建汉坦病毒反向遗传操作系统重要的环节,可以利用汉坦病毒亚基因组反向遗传操作系统评估构建的含RNA聚合酶Ⅰ质粒转录病毒基因组RNA的效率,通过检测报告基因GFP表达量,筛选出最佳的转染条件并对构建的质粒进行优化,为最终建立汉坦病毒反向遗传学操作系统奠定基础。
Avian Influenza (AI) is a serious zoonotic disease caused by Type A Avian Influenza Virus (AIV) of the family Orthomyxoviridae. By June 19,2008, totally 385 persons were infected by this virus, and 243 of them died. This disease caused huge economic losses, and public health and social security issues. In recent years, the newly developed reverse genetics technique of AIV provides an important platform for the study on AIV. We constructed a bi-direction transcription plasmid pZL2006 by amplifying human RNA polymeraseⅠpromoter sequence from Hela cells, synthesizing RNA polymeraseⅠterminator sequence, and inserting the combinations of promter and terminator sequence into eukaryotic expression plasmid pVAX1. The constructed plasmid pZL2006 contained two sets of promoter and termination sequences, namely RNA polymeraseⅡpromoter (CMV promoter) and termination sequences (aⅡBGH), RNA polymeraseⅠpromoter (PⅠh) and murine polymeraseⅠterminator sequences (tⅠ). The positive cDNA encoding vRNA was inserted between polymeraseⅡpromoter and termination sequence to achieve vRNA transcription and viral protein expression. In this study, we amplified the complete genome sequence of H5N1 strain A/Viet Nam/1194/2004(H5N1) isolated from Vietnam in 2004, and obtained eight gene fragments. The eight gene fragments were sequenced and their sequences compositions matched those in GenBank. These fragments were inserted into vector pZL2006, which was confirmed by sequencing positive clones from restriction digestion. The eight recombinant plasmids containing the complete gene fragment of AIV constructed the reverse genetics system of AIV. The eight-plasmid system was transfected into the 293 T/MDCK cells by using Liposome 2000. After 36 h of this transfection, most of the infected cells became round, died, and small pieces of cells fell off etc, while the control cell group was in normal state. The spherical particle, about 80-120 nm in diameter, having capsule surface with spike was observed in the supernatant of transfected cells with eight plamids by electron microscope, which was characterisitic morphology of influenza viron. The results of hemagglutination test (HA) showed that the titer to rescue virus was between 26 to 27, which means that the rescued virus has hemagglutinin activity and a higher titer. After 36 h of reinoculation of the rescued virus in MDCK cells, the infected cells became round, died, small pieces of cells fell off etc., which were very similar to the cytopathic characteristics of MDCK cells infected by the wild strain. After 48 h of the inoculation of the supernatant of rescued virus in 10 chicken embryo eggs, all of them died, while the control chicken embryo eggs survived. This indicated that both the recombinant virus and the wild virus have lethality to chicken embryo eggs. PCR amplification and sequencing confirmed that the rescued virus is identical to the A/Viet Nam/1194/2004(H5N1) strain in the sequence composition from the same region. Based on the experimental results mentioned above, it can be concluded that the reverse genetics operating system of AIV was successfully constructed in this study.
Hantaviruses belonging to Hantavirus, Bunyaviridae, can cause human hemorrhagic fever with renal syndrome (HFRS) in Asia and Europe, and hantavirus pulmonary syndrome (HPS) in America. HFRS is the most serious in China, and more than 90 percent cases were reported in our country. Hantavirus strain H8205 was isolated in this lab from the serum of one HFRS patient in Heilongjiang province by using Vero-E6 cells. This strain can produce strong immunological responses to the serum of HFRS patients from most of China. The NP of this strain was used as antigen, and was very useful in the diagnosis of Hantavirus prevalent in China. The biological characteristics and animal pathogenic test were investigated in this study, and a series of results were obtained, but the complete genome has not been sequenced. Currently, the sequences of the genes L, M, S of Hantavirus H8205 Strain including the complete 5' and 3'ends were obtained by using RT-PCR and RACE. Compared with the published sequences of Hantavirus representative strains, H8205 has 95.5%-98.3% amino acid homology and 81.9%-88.8% nucleic acid homology in L gene,85.2%-99.7% amino acid homology and 74.5%-99.3% nucleic acid homology in M gene, and 97.2%-99.8% amino acid homology and 83.5%-99.4% nucleic acid homology in S gene. The phylogenetic tree based on the L/M/S gene sequence analysis indicated that H8205 is genetically closer to other strains belonging to the same Hantavirus type, while distantly related to DOBV, SEOV, SNV, PUUV. By reference strains, we can find similar strains from different countries or regions, which provides information for the further study on the differences among strains and their antigen variation.
Hantavirus is negative-sense RNA virus. RNA structure characteristics and instability makes it very difficult to operate the genome of RNA virus at the molecular level. The successful establishment of reverse genetics operating system of segmented negative-sense RNA virus, e.g. AIV, is a new start point for the study of Influenza Virus. However, the construction of Hantavirus reverse genetics systems has not established yet, which prevented from performing the related studies of Hantavirus due to lacking an important study platform. In this study, the Hantavirus H8205 strain was chosen. The complete gene fragments of this strain were obtained by segment amplification. The gene fragments were ligated into the full-length cDNA of L/M genes by usingⅡs restriction endonucleases. The sequencing results showed that the obtained full-length cDNA was completely correct, without any mutations or deletions. Two recombinant plasmids, PCDNA-L/PCDNA-S, were constructed by inserting the L/S genes of H8205 into the eukaryotic expression vector pcDNA-3.1(+)/pcDNA-3.1-Hygro(+). Two trans-acting proteins L and NP of Hantavirus were expressed by transfecting eukaryotic cells with the RNA polymeraseⅡpromoter (PCMV) and terminator sequences in the eukaryotic expression vector. The plasmid pp2006 containing human RNA polymerase I promoter and murine RNA polymeraseⅠterminator was constructed. The non-coding region of Hantavirus L gene and GFP fusion gene were inserted between RNA polymerase I promoter (PⅠh) and the terminator (TⅠ) to form a transcription unit. After the transfection into cells, the fusion gene containing the 5'and 3'non-coding regions of L gene of Hantavirus and GFP coding gene, was expressed by using the polymerase I in cells and the promoter PⅠh. The trans-acting protein of Hantavirus combined the fusion gene into RNPs and the expression of GFP protein was activated. After 24 h of the transfection of Hantavirus subgenomic infectious clone (PCDNA-L/PCDNA-S/ PP2006-L-GFP) into the 293 T cells by using Liposome 2000, obvious green fluorescent protein was observed in the transfected cells, while there is no green fluorescent protein observed in the control cells. This means that the subgenomic infectious clone of Hantavirus was successfully established, which is an important part to establish the reverse genetics system of Hantavirus. The system of subgenome can be used to evaluate RNA transcription efficiency of the plasmid containing RNA polymerase I, and find the optimal transfection conditions and optimized the constructed plasmid by detecting the expression quantity of the reporter gene GFP, which will lay a solid foundation for the final establishment of Hantavirus reverse genetics system.
引文
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