Rescue of rabies virus from cloned cDNA and identification of the pathogenicity-related gene: glycoprotein gene is associated with virulence for adult mice

In order to identify the viral gene related to the pathogenicity of rabies virus, we tried to establish a reverse genetics system of the attenuated RC-HL strain, which causes nonlethal infection in adult mice after intracerebral inoculation. A full-length genome plasmid encoding the complete antigenomic cDNA of the RC-HL strain and helper plasmids containing cDNAs of the complete open reading frame of the N, P, and L genes, respectively, were constructed. After transfection of these plasmids into BHK-21 cells infected with the T7 RNA polymerase-expressing vaccinia virus, infectious rabies virus with almost the same biological properties as those of the wild-type RC-HL strain was rescued. Using this reverse genetics system of the RC-HL strain, we generated a chimeric virus with the open reading frame of the glycoprotein gene from the parent Nishigahara strain, which kills adult mice after intracerebral inoculation, in the background of the RC-HL genome. Since the chimeric virus killed adult mice following intracerebral inoculation, it became evident that the open reading frame of the glycoprotein gene is related to the pathogenicity of the Nishigahara strain for adult mice.     

麻疹病毒全长cDNA构建及其感染性的研究

为发展新型疫苗和改造目前使用的麻疹病毒疫苗,以麻疹病毒疫苗株为模板,构建了具有感染性的麻疹病毒cDNA克隆.用RT-PCR分6段扩增出麻疹病毒全长基因,通过酶切、拼接构建麻疹病毒疫苗株CC-47的全长正链cDNA序列,并精确地置于T7启动子控制下与丁型肝炎病毒核酶序列之前.克隆麻疹病毒CC-47株蛋白N、P、L编码区质粒并置于T7启动子控制下,用4个质粒共转染哺乳动物细胞,在表达T7 RNA聚合酶的重组痘苗病毒VTF7-3的作用下进行病毒拯救.经免疫荧光、PCR等方法检测证实,获得了具有感染性的麻疹病毒.所拯救的病毒在哺乳动物细胞连续传3代后,仍能检出病毒抗原和核酸.     

Rescue of measles viruses from cloned DNA.

A system has been established allowing the rescue of replicating measles viruses (MVs) from cloned DNA. On one hand, plasmids were constructed from which MV antigenomic RNAs with the correct termini are transcribed by phage T7 RNA polymerase. On the other hand, helper cells derived from the human embryonic kidney 293 cell line were generated constitutively expressing T7 RNA polymerase together with MV nucleocapsid protein and phosphoprotein. Simultaneous transfection of the helper cells with the MV antigenomic plasmid and with a plasmid encoding the MV polymerase under direction of a T7 promoter led to formation of syncytia from which MVs were easily recovered. A genetic tag comprising three nucleotide changes was present in the progeny virus. As a first application of reverse genetics, a segment of 504 nucleotides from the 5' non-coding region of the fusion gene was deleted, leading to an MV variant whose replication behaviour in Vero cells was indistinguishable from that of the laboratory Edmonston B strain. Since no helper virus is involved, this system, in principle, should be applicable to the rescue of any member of the large virus order Mononegavirales, i.e. viruses with a nonsegmented negative-strand RNA genome.     

Construction and identification of the helper plasmids for reverse genetic system of rabies virus street strain

To obtain the helper plasmids for a reverse genetics system of rabies virus, the cDNAs of the complete open reading frames of the N, P, G, and L genes of rabies street virus stain HN10 were each cloned into expression vector pVAX1, These four plasmids were identified by restriction enzyme digestion and gene sequencing. The plasmid encoding the N protein was selected to determine the expression effect of these plasmids in NA cells. The results showed that the helper plasmids for a reverse genetics system of rabies street virus strain HN10 had been successfully constructed.     

痘苗病毒/T7RNA聚合酶辅助的原核基因在真核细胞中的表达

痘苗病毒/T7RNA聚合酶这一瞬时表达系统由于具有很多优于其他表达系统的特点而被广泛地应用于表达外源蛋白.TB-Chen株轮状病毒的VP6 DNA编码片段插入到原核质粒pETL的噬菌体T7启动子和终止子之间,获得重组表达质粒pET-VP6.构建好的重组表达质粒pET-VP6通过脂质体转染到真核细胞MA104中,用携带噬...     

含RGD受体结合位点口蹄疫病毒Asia1/JS/China/2005株的拯救及初步鉴定

摘要：【目的】构建含有RGD受体结合位点口蹄疫病毒（FMDV）Asia1/JS/China/2005株的全长感染性cDNA克隆。【方法】采用定点突变方法,构建Asia1型FMDV含有预期突变的全长cDNA克隆pFMDV-RGD。pFMDV-RGD重组质粒经NotI线化后,与表达T7 RNA聚合酶的真核质粒pcDNAT7P共转染BHK-21细胞,进行FMDV-RGD病毒拯救。【结果】序列测定结果表明成功构建了FMDV含有RGD受体位点的Asia1/JS/China/2005全长cDNA克隆。共转染实验获得拯救病毒,对拯救的病毒分别进行序列测定、间接免疫荧光、电子显微镜观察和乳鼠致病性分析,表明成功拯救了含有RGD受体结合位点的Asia1/JS/China/2005株FMDV。【结论】该实验为进一步研究含有RGD和RDD受体结合位点2个拯救病毒生物学特性的差异奠定了基础。     

用反向遗传技术致弱基因VIId型鹅源新城疫病毒ZJI株

将新城疫病毒ZJI株基因组cDNA全长分成7个片段,依次连接并克隆至TVT7R转录载体中,构建了含ZJI株全基因组cDNA的转录载体(pNDV/ZJI),pNDV/ZJI与3个辅助表达质粒pCI-NP、pCI-P和pCI-L共转染BSR-T7/5细胞,成功拯救出了具有感染性的新城疫病毒粒子。设计两对引物,经overlapPCR方法将该毒株F蛋白裂解位点的112、115和117位碱性氨基酸突变成弱毒株特征的非碱性氨基酸后,替换pNDV/ZJI上的对应序列,构建了转录载体pNDV/ZJIFM,将pNDV/ZJIFM与3个辅助表达质粒共转染BSR-T7/5细胞,成功拯救出了致弱的基因VIId型鹅源新城疫病毒NDV/ZJIFM,获救病毒的鸡胚最小致死剂量平均死亡时间(MDT)大于120h,同时该病毒的脑内接种致病指数(ICPI)为0.16,上述结果表明,获救病毒的毒力已被致弱,是一个较为理想的疫苗候选株。     

含RGD受体结合位点口蹄疫病毒Asial/JS/China/2005株的拯救及初步鉴定

[目的]构建含有精氨酸.甘氨酸.天冬氨酸(RGD)受体结合位点口蹄疫病毒(FmDV)Asial/JS/Chind2005株的全长感染性cDNA克隆.[方法]采用定点突变方法,构建Asial型FMDV含有预期突变的全长cDNA克隆pFMDV-RGD.pFMDV-RGD重组质粒经NotI线化后,与表达T7 RNA聚合酶的真核质粒peDNATIP共转染BHK-21细胞,进行FMDV-RGD病毒拯救.[结果]序列测定结果表明成功构建了FMDV含有RGD受体位点的Asial/JS/China/2005全长cDNA克隆.共转染试验获得拯救病毒,对拯救的病毒分别进行序列测定、间接免疫荧光、电子显微镜观察和乳鼠致病性分析,表明成功拯救了含有RGD受体结合位点的Asial/JS/China/2005株FMDV.[结论]该试验为进一步研究含有RGD和RDD受体结合位点2个拯救病毒生物学特性的差异奠定了基础.     

Ⅰ类新城疫病毒NDV08-004株病毒拯救体系的建立

根据Ⅰ类新城疫病毒NDV08-004基因组序列(GenBank登录号FJ794269),设计7对引物,采用RT-PCR将NDV08-004基因组分段扩增(片段A～G)并分别克隆入pGEM-Teasy载体,然后采用单一的酶切位点将7个片段依次亚克隆到转录载体pOLTV5中,构建了含NDV08-004全基因组cDNA的转录载体NDV08-004-pO。采用构建的三个辅助质粒(pCI-NP、pCI-P和pCI-L)与基因组NDV08-004-pO按照2:1:1:2的比例共转染BSR T7/5细胞,结果成功拯救出具有血凝性的NDV08-004,初代分离物血凝价为4.33log2±0.58。Ⅰ类NDV反向遗传操作体系的建立为开展Ⅰ类NDV的致病机制奠定了基础。     

Rescue and Preliminary Application of a Recombinant Newcastle Disease Virus Expressing Green Fluorescent Protein Gene

Based on the complete genome sequence of Newcastle disease virus (NDV) ZJI strain, seven pairs of primers were designed to amplify a cDNA fragment for constructing the plasmid pNDV/ZJI, which contained the full-length cDNA of the NDV ZJI strain. The pNDV/ZJI, with three helper plasmids, pCIneoNP, pCIneoP and pCIneoL, were then cotransfected into BSR-T7/5 cells expressing T7 RNA polymerase. After inoculation of the transfected cell culture supernatant into embryonated chicken eggs from specific-pathogen-free (SPF) flock, an infectious NDV ZJI strain was successfully rescued. Green fluorescent protein (GFP) gene was amplified and inserted into the NDV full-length cDNA to generate a GFP-tagged recombinant plasmid pNDV/ZJIGFP. After cotransfection of the resultant plasmid and the three support plasmids into BSR-T7/5 cells, the recombinant NDV, NDV/ZJIGFP, was rescued. Specific green fluorescence was observed in BSR-T7/5 and chicken embryo fibroblast (CEF) cells 48h post-infection, indicating that the GFP gene was expressed at a relatively high level. NDV/ZJIGFP was inoculated into 10-day-old SPF chickens by oculonasal route. Four days post-infection, strong green fluorescence could be detected in the kidneys and tracheae, indicating that the recombinant GFP-tagged NDV could be a very useful tool for analysis of NDV dissemination and pathogenesis.     

Rescue and preliminary application of a recombinant newcastle disease virus expressing green fluorescent protein gene

Based on the complete genome sequence of Newcastle disease virus (NDV) ZJI strain, seven pairs of primers were designed to amplify a cDNA fragment for constructing the plasmid pNDV/ZJI, which contained the full-length cDNA of the NDV ZJI strain. The pNDV/ZJI, with three helper plasmids, pCIneoNP, pCIneoP and pCIneoL, were then cotransfected into BSR-T7/5 cells expressing T7 RNA polymerase. After inoculation of the transfected cell culture supernatant into embryonated chicken eggs from specific-pathogen-free (SPF) flock, an infectious NDV ZJI strain was successfully rescued. Green fluorescent protein (GFP) gene was amplified and inserted into the NDV full-length cDNA to generate a GFP-tagged recombinant plasmid pNDV/ZJIGFP. After cotransfection of the resultant plasmid and the three support plasmids into BSR-T7/5 cells, the recombinant NDV, NDV/ZJIGFP, was rescued. Specific green fluorescence was observed in BSR-T7/5 and chicken embryo fibroblast (CEF) cells 48h post-infection, indicating that the GFP gene was expressed at a relatively high level. NDV/ZJIGFP was inoculated into 10-day-old SPF chickens by oculonasal route. Four days post-infection, strong green fluorescence could be detected in the kidneys and tracheae, indicating that the recombinant GFP-tagged NDV could be a very useful tool for analysis of NDV dissemination and pathogenesis.     

口蹄疫病毒O/QYYS/s/06株感染性克隆的构建

本研究在完成FMDVO/QYYS/s/06株全基因组序列测定的基础上,分3段对全基因组进行克隆,其后将各片段克隆到载体P43中,从而获得携带O/QYYS/s/06株基因组全长cDNA的重组质粒P43C。将重组质粒P43C与表达RNA聚合酶的质粒T7共转染BHK-21细胞,48h后收获培养液接种2~3d乳鼠,取经乳鼠传代后的第4代病毒液,经反向间接血凝、中和试验和测序等方法证明拯救的病毒为O型FMDV。以上结果表明,O/QYYS/s/06株全长cDNA分子克隆的构建成功。     

Characterization of M gene-deficient rabies virus with advantages of effective immunization and safety as a vaccine strain

Matrix (M) protein of rabies virus is known to play an important role in assembly and budding of the progeny virus. We generated an M gene-deficient rabies virus, RC-HLDeltaM, using a reverse genetics system of rabies virus RC-HL strain to develop a novel type of vaccine. RC-HLDeltaM infection was confined within a single cell in mouse neuroblastoma cells. This deficient virus failed to generate the progeny virus in the cells. In contrast, RC-HLDeltaM propagated in BHK cells inductively expressing M protein. Suckling and adult mice inoculated intracerebrally with the parental RC-HL strain showed lethal infection and transient body weight loss, respectively, whereas both suckling and adult mice inoculated with RC-HLDeltaM showed no symptoms. The neutralizing antibody against rabies virus was successfully induced by intramuscular immunization with 10(5) focus-forming units of RC-HLDeltaM but not UV-inactivated RC-HL. Intranasal immunization with RC-HLDeltaM resulted in almost the same antibody titer to rabies virus as that in the case of immunization with live RC-HL strain. These findings indicate that RC-HLDeltaM is a candidate for a novel rabies vaccine that is safer and more effective than are current vaccines.     

重组新城疫病毒Anhinga株与TRAIL蛋白协同杀伤肿瘤细胞

将新城疫病毒（Newscastle disease virus, NDV）Anhinga株的全 长基因组cDNA克隆质粒、pTM1-L、pTM1-P、pTM1-NP表达质粒共转染稳定表 达T7 RNA聚合酶的BSRT7/5细胞,得到拯救NDV病毒．通过PCR、酶切法、测 序证明拯救病毒中存在引入的分子标签.通过血凝实验、蚀斑测定证明成功 拯救病毒.并研究了该重组病毒对4种不同人肿瘤细胞的体外杀伤效果．首 次证明重组Anhinga株对SMMC-7721细胞、A549细胞、HepG2细胞和SH-SY5Y 细胞均有杀伤作用．该重组病毒主要诱导SMMC-7721细胞和A549细胞凋亡, 诱导HepG2细胞和SH-SY5Y细胞坏死．TNF家族成员TRAIL蛋白可以显著增强 NDV杀伤肿瘤细胞的效果.本实验为进一步研究重组NDV用于肿瘤治疗奠定基 础．     

Recovery of pathogenic measles virus from cloned cDNA

Reverse genetics technology so far established for measles virus (MeV) is based on the Edmonston strain, which was isolated several decades ago, has been passaged in nonlymphoid cell lines, and is no longer pathogenic in monkey models. On the other hand, MeVs isolated and passaged in the Epstein-Barr virus-transformed marmoset B-lymphoblastoid cell line B95a would retain their original pathogenicity (F. Kobune et al., J. Virol. 64:700-705, 1990). Here we have developed MeV reverse genetics systems based on the highly pathogenic IC-B strain isolated in B95a cells. Infectious viruses were successfully recovered from the cloned cDNA of IC-B strain by two different approaches. One was simple cotransfection of B95a cells, with three plasmids each encoding the nucleocapsid (N), phospho (P), or large (L) protein, respectively, and their expression was driven by the bacteriophage T7 RNA polymerase supplied by coinfecting recombinant vaccinia virus vTF7-3. The second approach was transfection with the L-encoding plasmid of a helper cell line constitutively expressing the MeV N and P proteins and the T7 polymerase (F. Radecke et al., EMBO J. 14:5773-5784, 1995) on which B95a cells were overlaid. Virus clones recovered by both methods possessed RNA genomes identical to that of the parental IC-B strain and were indistinguishable from the IC-B strain with respect to growth phenotypes in vitro and the clinical course and histopathology of experimentally infected cynomolgus monkeys. Thus, the systems developed here could be useful for studying viral gene functions in the context of the natural course of MeV pathogenesis.