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.     

狂犬病毒街毒株全长基因cDNA克隆的构建及鉴定

利用分子克隆的方法,将狂犬病毒街毒株的全基因组分为4个片段按照它们基因组上的顺序克隆到真核表达载体pVAX1上,并将G-L间隔区的非编码区替换为表达绿色荧光蛋白(GFP)的核苷酸,构建出表达绿色荧光蛋白(GFP)的重组狂犬病毒HN10株全长基因组cDNA真核表达质粒,同时,在全长cDNA的两侧嵌入锤头状核酶(HamRz)和丁型肝炎病毒核酶(HdvRz)的序列,并置于CMV启动子的控制下,为下一步拯救出该嵌合病毒提供了可直接使用的全长cDNA真核表达质粒。     

狂犬病病毒Evelyn- Rokitnicki-Abelseth疫苗株反向遗传系统的建立

摘要：【目的】建立狂犬病毒的反向遗传系统,为研制不含狂犬病病毒致病性的新型安全高效的狂犬疫苗提供技术依据。【方法】本研究采用反向遗传学方法和分子克隆技术,建立了狂犬病病毒Evelyn- Rokitnicki-Abelseth (ERA)疫苗株的CMV/ T7、T7启动子病毒拯救系统,构建了表达N、P、L蛋白的辅助质粒。【结果】成功拯救出野生型病毒rERA-VC,在Vero细胞上的生长动力学特性与父母本ERA 相同,第三代可在Vero细胞上可获得很高的生长滴度。【结论】建立了狂犬病病毒的反向遗传系统,拯救出的野生型病毒生物学特性与父母本相同。     

狂犬病病毒EveIyn-Rokitnicki-Abelseth疫苗株反向遗传系统的建立

[目的]建立狂犬病毒的反向遗传系统,为研制不含狂犬病病毒致病性的新型安全高效的狂犬疫苗提供技术依据.[方法]本研究采用反向遗传学方法和分子克隆技术,建立了狂犬病病毒Evelyn-R0kitnieki-Abelseth(ERA)疫苗株的cMV/T7、T7启动子病毒拯救系统,构建了表达N、P、L蛋白的辅助质粒.[结果]成功拯救出野生型病毒rERA-VC,在Vero细胞上的生长动力学特性与父母本ERA相同,第三代可在Vero细胞上可获得很高的生长滴度.[结论]建立了狂犬病病毒的反向遗传系统,拯救出的野生型病毒生物学特性与父母本相同.     

Mutation of Neuraminidase Cysteine Residues Yields Temperature-Sensitive Influenza Viruses

The influenza virus neuraminidase (NA) is a tetrameric, virus surface glycoprotein possessing receptor-destroying activity. This enzyme facilitates viral release and is a target of anti-influenza virus drugs. The NA structure has been extensively studied, and the locations of disulfide bonds within the NA monomers have been identified. Because mutation of cysteine residues in other systems has resulted in temperature-sensitive (ts) proteins, we asked whether mutation of cysteine residues in the influenza virus NA would yield ts mutants. The ability to rationally design tight and stable ts mutations could facilitate the creation of efficient helper viruses for influenza virus reverse genetics experiments. We generated a series of cysteine-to-glycine mutants in the influenza A/WSN/33 virus NA. These were assayed for neuraminidase activity in a transient expression system, and active mutants were rescued into infectious virus by using established reverse genetics techniques. Mutation of two cysteines not involved in intrasubunit disulfide bonds, C49 and C146, had modest effects on enzymatic activity and on viral replication. Mutation of two cysteines, C303 and C320, which participate in a single disulfide bond located in the beta5L0,1 loop, produced ts enzymes. Additionally, the C303G and C320G transfectant viruses were found to be attenuated and ts. Because both the C303G and C320G viruses exhibited stable ts phenotypes, they were tested as helper viruses in reverse genetics experiments. Efficiently rescued were an N1 neuraminidase from an avian H5N1 virus, an N2 neuraminidase from a human H3N2 virus, and an N7 neuraminidase from an H7N7 equine virus. Thus, these cysteine-to-glycine NA mutants allow the rescue of a variety of wild-type and mutant NAs into influenza virus.     

Rescue of influenza C virus from recombinant DNA

The rescue of influenza viruses by reverse genetics has been described only for the influenza A and B viruses. Based on a similar approach, we developed a reverse-genetics system that allows the production of influenza C viruses entirely from cloned cDNA. The complete sequences of the 3' and 5' noncoding regions of type C influenza virus C/Johannesburg/1/66 necessary for the cloning of the cDNA were determined for the seven genomic segments. Human embryonic kidney cells (293T) were transfected simultaneously with seven plasmids that direct the synthesis of each of the seven viral RNA segments of the C/JHB/1/66 virus under the control of the human RNA polymerase I promoter and with four plasmids encoding the viral nucleoprotein and the PB2, PB1, and P3 proteins of the viral polymerase complex. This strategy yielded between 10(3) and 10(4) PFU of virus per ml of supernatant at 8 to 10 days posttransfection. Additional viruses with substitutions introduced in the hemagglutinin-esterase-fusion protein were successfully produced by this method, and their growth phenotype was evaluated. This efficient system, which does not require helper virus infection, should be useful in viral mutagenesis studies and for generation of expression vectors from type C influenza virus.     

Sickness and recovery of dogs challenged with a street rabies virus after vaccination with a vaccinia virus recombinant expressing rabies virus N protein.

Dogs were vaccinated intradermally with vaccinia virus recombinants expressing the rabies virus glycoprotein (G protein) or nucleoprotein (N protein) or a combination of both proteins. The dogs vaccinated with either the G or G plus N proteins developed virus-neutralizing antibody titers, whereas those vaccinated with only the N protein did not. All dogs were then challenged with a lethal dose of a street rabies virus, which killed all control dogs. Dogs vaccinated with the G or G plus N proteins were protected. Five (71%) of seven dogs vaccinated with the N protein sickened, with incubation periods 3 to 7 days shorter than that of the control dogs; however, three (60%) of the five rabid dogs recovered without supportive treatment. Thus, five (71%) of seven vaccinated with the rabies N protein were protected against a street rabies challenge. Our data indicate that rabies virus N protein may be involved in reducing the incubation period in dogs primed with rabies virus N protein and then challenged with a street rabies virus and, of more importance, in subsequent sickness and recovery.     

犬瘟热病毒小熊猫株反向遗传系统的构建及其鉴定

以驯化致弱的犬瘟热病毒小熊猫株(Canine distemper virus,CDV)为模板,构建犬痘热病毒感染性cDNA克隆.对其全基因组序列测定后,用RT-PCR的方法获得组成全长基因的7个片段,通过酶切、拼接将7段CDVcDNA序列插入到真核表达载体pCI的MCS上,构建犬瘟热病毒小熊猫株的全长cDNA质粒(pCI-CDV-LP),同时分别克隆CDV小熊猫株N、P、L蛋白ORF构建三个辅助质粒.酶切鉴定和序列测定表明,pCI载体中插入的核酶及CDV cDNA序列正确无误,使用转染试剂Lipofectamine TM 2000将全长质粒和三个辅助质粒共转染中国仓鼠肾细胞(BSR),经RT-PCR、间接免疫荧光和病毒感染VERO-SLAM细胞试验鉴定,成功拯救出CDV小熊猫株,显示CDV小熊猫株反向遗传系统构建完成,为犬瘟热病毒致病机理及免疫研究奠定基础.     

狂犬病毒反向遗传学技术的研究及应用进展

反向遗传学技术是上世纪90年代在分子病毒学研究领域兴起的新技术,通常也被称为“病毒拯救”。综述了应用反向遗传学技术“拯救”狂犬病毒的主要技术体系以及反向遗传学技术在狂犬病毒致病机理、狂犬病毒疫苗及载体研究中的应用进展。     

人类及动物RNA病毒的反向遗传系统

反向遗传系统可以对RNA病毒直接进行遗传操作,为RNA病毒的分子生物学研究提供了一种强大的工具。在过去20年,特别是自90年代中期第一例负链RNA病毒感染性克隆构建成功以来,动物RNA病毒的分子生物学研究取得了长足的进展,这很大程度上归功于各种动物RNA病毒反向遗传系统的建立。这里系统总结了人类及动物非反转录RNA病毒中各类代表性成员在建立反向遗传系统时的方案设计、遇到的困难及研究者如何克服这些困难。分类讨论到的代表性病毒种属有脊髓灰质炎病毒、冠状病毒（包括SARS病毒）、黄病毒、野田村病毒、流感病毒、传染性法氏囊病病毒以及呼肠孤病毒等。     

Mokola virus glycoprotein and chimeric proteins can replace rabies virus glycoprotein in the rescue of infectious defective rabies virus particles.

A reverse genetics approach which allows the generation of infectious defective rabies virus (RV) particles entirely from plasmid-encoded genomes and proteins (K.-K. Conzelmann and M. Schnell, J. Virol. 68:713-719, 1994) was used to investigate the ability of a heterologous lyssavirus glycoprotein (G) and chimeric G constructs to function in the formation of infectious RV-like particles. Virions containing a chloramphenicol acetyltransferase (CAT) reporter gene (SDI-CAT) were generated in cells simultaneously expressing the genomic RNA analog, the RV N, P, M, and L proteins, and engineered G constructs from transfected plasmids. The infectivity of particles was determined by a CAT assay after passage to helper virus-infected cells. The heterologous G protein from Eth-16 virus (Mokola virus, lyssavirus serotype 3) as well as a construct in which the ectodomain of RV G was fused to the cytoplasmic and transmembrane domains of the Eth-16 virus G rescued infectious SDI-CAT particles. In contrast, a chimeric protein composed of the amino-terminal half of the Eth-16 virus G and the carboxy-terminal half of RV G failed to produce infectious particles. Site-directed mutagenesis was used to convert the antigenic site III of RV G to the corresponding sequence of Eth-16 G. This chimeric protein rescued infectious SDI-CAT particles as efficiently as RV G. Virions containing the chimeric protein were specifically neutralized by an anti-Eth-16 virus serum and escaped neutralization by a monoclonal antibody directed against RV antigenic site III. The results show that entire structural domains as well as short surface epitopes of lyssavirus G proteins may be exchanged without affecting the structure required to mediate infection of cells.     

Oral vaccination of raccoons (Procyon lotor) with an attenuated (SAD-B19) rabies virus vaccine

Unlike previous reports to the contrary, raccoons (Procyon lotor) were successfully vaccinated against rabies with a liquid SAD-B19 attenuated virus vaccine administered per os and given in vaccine-laden baits. There was neither evidence of vaccine-induced rabies in raccoons nor in a limited safety trial with opossums (Didelphis virginiana) given SAD-B19. Protection from lethal street rabies virus infection was not absolute: only three of nine raccoons given 1 x 10(6.0) TCID/ml were protected versus five of 10 raccoons given 1 x 10(7.0) TCID/ml of SAD-B19 and challenged 4 mo after consumption of vaccine-laden baits. Six of eight raccoons consuming 1 x 10(8.8) TCID/ml of SAD-B19 vaccine in baits survived street rabies virus challenge 2 mo postvaccination. Raccoon survivorship was not wholly dependent upon rabies virus-neutralizing antibody titer on the day of challenge. Vaccinated raccoons demonstrated a prominent anamnestic response within 1 wk following challenge. Surviving raccoons were observed for a minimum of 3 mo following street rabies virus challenge with neither clinical nor pathologic evidence of rabies. The SAD-B19 rabies vaccine administered within baits in captivity appears less effective for raccoons than for its demonstrated efficacy in the immunization of free-ranging foxes (Vulpes vulpes) in Europe.     

Rescue of recombinant Thogoto virus from cloned cDNA

Thogoto virus (THOV) is a tick-transmitted orthomyxovirus with a genome consisting of six negative-stranded RNA segments. To rescue a recombinant THOV, the viral structural proteins were produced from expression plasmids by means of a vaccinia virus expressing the T7 RNA polymerase. Genomic virus RNAs (vRNAs) were generated from plasmids under the control of the RNA polymerase I promoter. Using this system, we could efficiently recover recombinant THOV following transfection of 12 plasmids into 293T cells. To verify the recombinant nature of the rescued virus, specific genetic tags were introduced into two vRNA segments. The availability of this efficient reverse genetics system will allow us to address hitherto-unanswered questions regarding the biology of THOV by manipulating viral genes in the context of infectious virus.     

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.     

Rescue of Influenza A Virus from Recombinant DNA

We have rescued influenza A virus by transfection of 12 plasmids into Vero cells. The eight individual negative-sense genomic viral RNAs were transcribed from plasmids containing human RNA polymerase I promoter and hepatitis delta virus ribozyme sequences. The three influenza virus polymerase proteins and the nucleoprotein were expressed from protein expression plasmids. This plasmid-based reverse genetics technique facilitates the generation of recombinant influenza viruses containing specific mutations in their genes.