狂犬病病毒弱毒疫苗株反向遗传操作系统的建立

目的构建狂犬病病毒弱毒疫苗株SRV9全长cDNA感染性克隆,并建立其反向遗传操作系统。方法通过DNAStar对狂犬病病毒SRV9株全长基因组序列进行分析,利用单一的酶切位点,将SRV9全长cDNA分为4段,根据每段重叠区域的酶切位点拼接全长,分别连入pCI和pCDNA3.1(+)载体,并通过PCR方法分别在全长序列的3′端和5′端引入核酶HamRZ和HdvRZ序列,构建全长真核表达质粒pCI-SRV9和pD-SRV9。同时构建能表达狂犬病病毒核蛋白(N)、磷蛋白(P)、糖蛋白(G)和聚合酶蛋白(L)的4个辅助质粒。分别将pCI-SRV9或pD-SRV9与辅助质粒通过脂质体共转染BSR细胞,拯救重组病毒。结果两种质粒表达系统均可拯救到有感染活性的病毒粒子,但pD-SRV9表达质粒的拯救效率(8/8)较pCI-SRV9表达质粒拯救效率3/30高;重组病毒与母本野生病毒的体外生长动力学曲线相一致,相同培养时间的病毒滴度差异无统计学意义(P>0.05)。结论成功构建了狂犬病病毒弱毒疫苗株的反向遗传操作系统,为进一步研究狂犬病病毒致病机理、筛选新型狂犬病疫苗或开发基于狂犬病病毒载体的其他疾病疫苗奠定了基础。     

4株狂犬病街毒的分离及其核蛋白和糖蛋白序列分析

目的以4株狂犬病街毒分离株及我国南北方不同地区狂犬病病毒街毒为研究对象,通过其核蛋白及糖蛋白基因的同源性及进化分析,比较我国狂犬病流行毒株与人用及兽用狂犬病疫苗株间的基因及抗原性差异,为相关疫苗的研究奠定理论基础。方法以直接免疫荧光法(DFA)检测疑似狂犬病的犬脑标本,以乳鼠颅内接种法(MIT)和细胞培养分离技术(CIT)分离狂犬病病毒,并进行TCID50和LD50测定;以RT-PCR法扩增其核蛋白(N)和糖蛋白(G)基因,克隆入T载体并测序后,下载GenBank内已有的狂犬病病毒数据资源,以(Clustal和MEGA3软件)进行基因同源性比对及系统发生分析。结果从广东、河北两省分离到狂犬病病毒街毒4株,分别命名为GN07、WJ07-1、WJ07-2、GC07;序列分析表明4株狂犬病病毒均为基因1型,其N基因与G基因核苷酸的同源性分别为89.5%~99.9%和87.9%~99.9%,其中GN07与WJ07-1、WJ07-2、GC07的同源性较低。与己知的基因1型狂犬病毒相比,WJ07-1、WJ07-2、GC07与湖南、湖北、云南、浙江等地分离株及印度尼西亚分离株核苷酸同源性最高,分别为97.8%~99.4%和92.2%;GN07与CTN疫苗株核苷酸同源性最高,为94.5%。系统发育分析表明,4株病毒与CTN疫苗株同源性较高,与人用狂犬病疫苗3aG、PV株及兽用狂犬病疫苗ERA株和Flury疫苗株的同源性相对较远。适应细胞培养后,四株街毒的LD50和TCID50最高者为GN07,而WJ07-1较低,差别较大。结论4株狂犬病分离株与我国南北方不同地区狂犬病流行毒株的基因和氨基酸序列存在一定差异,在进化关系上分属于不同的分支。     

关于狂犬病病毒的最新报道

<正> 1881年Pasteur已开始对狂犬病进行研究,把人的狂犬病病毒接种于家兔而获得成功。他揭示了病毒的固定化、神经系统内的分布及生存时间与温度的关系等重要成果,这为现在进行狂犬病病毒研究和疫苗制备确立了良好的基础。但是本病毒有强烈的神经组织亲和性,在非神经组织来源的细胞培养物中不容易纯化增殖,而敏咸细胞与病毒株的结合是有条件性限制。虽然很早以前已着手研究本病毒,但其进展则较其他病毒缓慢。在近十年间有了细胞水平和分子生物学研究的成果,对重新研究狂犬病是有意义的。1980年世界卫生组织报告。在狂犬病今后的研究课题中,应确定使用近代的单克隆抗体及遗传基因操作技术,对狂犬病病毒的病原性与增殖、变异及疫苗作为重点进行全面研讨。     

河南省九株狂犬病毒的糖蛋白基因序列分析

目的 了解河南省狂犬病毒与人用、兽用狂犬病疫苗株在糖蛋白基因核苷酸和氨基酸序列水平上的差异,为有效控制狂犬病疫情提供初步的科学依据.方法 以反转录-半套式聚合酶链反应(RT-heminested-PCR)扩增2006年12月分离自河南省信阳市的9株狂犬病毒街毒株,经纯化、克隆、测序后获得9条糖蛋白基因全长序列,采用生物信息学软件构建基因系统发育树,对糖蛋白基因序列和氨基酸序列进行分析.结果 9株狂犬病毒糖蛋白在核苷酸及氨基酸序列水平上彼此的同源性分别为97.6%～98.9%和99.2%～99.8%;9株病毒与CTN疫苗的同源性最高,其核苷酸及氨基酸同源性分别为85.6%～93.0%和91.9%～92.9%;9株病毒与其他疫苗株相比,其核苷酸及氨基酸同源性分别为80.4%～83.3%和87.7%～92.5%;与已知的基因1型狂犬病毒比较,9株病毒糖蛋白氨基酸序列发生了若干位点的氨基酸取代.结论 9株河南省狂犬病毒街毒株均属基因1型,CTN疫苗株可能为目前我国河南省所流行的狂犬病提供较好的保护效果.     

狂犬病固定毒Vero细胞适应株3aG-V的生物学特性研究

对狂犬病固定毒Vero细胞适应株 3aG V的生物学特性进行了研究 ,包括病毒的形态、抗原结构、培养条件、致病性、免疫原性、纯毒试验及其在中枢神经系统是否形成尼氏小体检查。结果表明狂犬病病毒 3aG V株具有抗原性好 ,培养产毒量高 ,保持有aG固定株弱毒性、传代稳定、无变异 ,可作为替代地鼠肾细胞狂犬病疫苗aG毒株 ,用于Vero细胞培养病毒生产出毒液毒力高 ,灭活后效力高 ,安全性好的纯化Vero细胞狂犬病疫苗的生产用疫苗株。     

狂犬病mRNA疫苗的应用和研究进展

狂犬病是一个古老而又传统的疾病,其公共安全威胁持续存在,人类需要新型、快速和价格更为低廉的疫苗技术对其进行防治。mRNA疫苗作为一种新型的核酸疫苗, 在感染性疾病的预防控制和癌症的治疗等领域已经取得阶段性进展,因此狂犬病mRNA疫苗有望成为抗击狂犬病的新生力量。本文将从狂犬病mRNA疫苗的结构、递送系统和临床前及临床研究等方面进行简要综述,阐明狂犬病mRNA疫苗目前的研究现状。     

反向遗传学技术在狂犬病病毒研究中的应用

狂犬病是一种重要的人兽共患病,病死率几乎达100%,是全球性的重要公共卫生问题。近年来,尽管对狂犬病的防控取得了很大进展,但狂犬病病毒致病机理的研究仍有待深化。反向遗传学是一门新兴学科,近年来已广泛应用于狂犬病病毒研究中的各个领域。本文着重介绍反向遗传学技术在狂犬病病毒基因结构、致病机理、新型载体和疫苗研发中的应用,并对其未来发展方向进行了展望。     

狂犬病毒在Vero细胞上的传代适应

我国目前使用的是原代地鼠肾细胞狂犬疫苗(PHKC-RV),它是狂犬病毒北京株经原代地鼠肾细胞和豚鼠脑相继传代后制备的。尽管无明显的副反应,但就目前应用的情况看,其效力远不及HDCV,疫苗的制备需大量的地鼠,病毒产量也低。若用传代细胞生产狂犬病疫苗,可以降低成本,缓解地鼠供求紧张的状况,缓解人用狂犬病疫苗供不应求的状况,而且可以带来较高的经济效益。 Vero细胞是经WHO检定合格并推荐作为生产人用灭活狂犬病疫苗的传代细胞。它不但来源方便,     

狂犬病毒HEP-dG株的拯救

目的筛选免疫原性高、致病性低的狂犬病疫苗候选株。方法应用反向遗传技术,在狂犬病毒弱毒株HEP-Flury株全基因组3'-N-P-M-G-L-5'的假基因区域(G与L之间),插入一个G基因,构建携带双G基因的全长感染性克隆,其与辅助质粒共转染BHK-21细胞,盲传十代,用荧光抗体和RT-PCR鉴定病毒。结果成功获得携带双G基因的狂犬病毒HEP-dG株,该病毒在BHK-21细胞中稳定生长,其病毒滴度达到1010FFU/mL。结论HEP-dG株会为研制高效的狂犬病病毒基因工程口服疫苗提供了良好的疫苗候选株。     

人用狂犬病疫苗和被动免疫制剂研发进展

狂犬病(Rabies)是由狂犬病毒(Rabies virus)引起的一种人兽共患病。人一旦发病致死率几乎100%。目前预防狂犬病的人用疫苗为狂犬病毒灭活疫苗。随着基因工程技术的进步,新一代以狂犬病毒G蛋白(Glycoprotein)为免疫原的疫苗在开发中,包括不同表达系统的G蛋白亚单位疫苗,G蛋白DNA疫苗,G蛋白病毒载体疫苗等。同时本文还探讨了未来可能取代狂犬病免疫球蛋白的被动免疫制剂(单克隆抗体)的研发进展。     

Characterization of an antigenic determinant of the glycoprotein that correlates with pathogenicity of rabies virus.

The pathogenicity of fixed rabies virus strains for adult mice depends on the presence of an antigenic determinant on the viral glycoprotein. Two virus-neutralizing monoclonal antibodies have been used to identify this determinant. All pathogenic strains of fixed rabies virus bind to these antibodies and are neutralized by them, whereas nonpathogenic strains fail to react with these monoclonal antibodies and are not neutralized by them. Antigenic variants of the rabies virus with altered glycoprotein were selected by growing virus in the presence of one monoclonal antibody, 194-2. All variants that lost their ability to react with this antibody and an additional antibody, 248-8, were found to be nonpathogenic for adult mice. Analysis of tryptic peptides of the glycoproteins of pathogenic parent virus and nonpathogenic variants and the amino acid sequence of a specific variant tryptic peptide revealed that the change in pathogenicity corresponded to an amino acid substitution at position 333 of the glycoprotein molecule. The nucleotide sequence of the nonpathogenic variant glycoprotein gene contained a base change that confirmed the single amino acid substitution in the tryptic peptide replacing arginine-333 in the parental glycoprotein. We conclude that arginine-333 is essential for the integrity of an antigenic determinant and for the ability of rabies viruses to produce lethal infection in adult mice.     

A recombinant rabies virus expressing vesicular stomatitis virus glycoprotein fails to protect against rabies virus infection

To investigate the importance of the rabies virus (RV) glycoprotein (G) in protection against rabies, we constructed a recombinant RV (rRV) in which the RV G ecto- and transmembrane domains were replaced with the corresponding regions of vesicular stomatitis virus (VSV) glycoprotein (rRV-VSV-G). We were able to recover rRV-VSV-G and found that particle production was equal to rRV. However, the budding of the chimeric virus was delayed and infectious titers were reduced 10-fold compared with the parental rRV strain containing RV G. Biochemical analysis showed equal replication rates of both viruses, and similar amounts of wild-type and chimeric G were present in the respective viral particles. Additional studies were performed to determine whether the immune response against rRV-VSV-G was sufficient to protect against rabies. Mice were primed with rRV or rRV-VSV-G and challenged with a pathogenic strain of RV 12 days later. Similar immune responses against the internal viral proteins of both viruses indicated successful infection. All mice receiving the rRV vaccine survived the challenge, whereas immunization with rRV-VSV-G did not induce protection. The results confirm the crucial role of RV G in an RV vaccine.     

Oral immunization and protection of raccoons (Procyon lotor) with a vaccinia-rabies glycoprotein recombinant virus vaccine.

Animal rabies control has been frustrated by the existence of multiple wildlife reservoirs and the lack of efficacious oral vaccines. In this investigation, raccoons fed a vaccinia-rabies glycoprotein recombinant virus in a sponge bait developed rabies virus-neutralizing antibody (0.6-54.0 units) and resisted street rabies virus infection 28 and 205 days after feeding. Additional raccoons immunized by oral infusion with attenuated antigenic variants of rabies virus strains CVS-11 and ERA failed to develop rabies virus-neutralizing antibody. This work demonstrates the feasibility of a recombinant virus vaccine containing the rabies glycoprotein gene for immunization of raccoons, and possibly other wildlife, to obtain long-term protection against rabies.     

Protection from rabies by a vaccinia virus recombinant containing the rabies virus glycoprotein gene.

Inoculation of rabbits and mice with a vaccinia-rabies glycoprotein recombinant (V-RG) virus resulted in rapid induction of high concentrations of rabies virus-neutralizing antibodies and protection from severe intracerebral challenge with several strains of rabies virus. Protection from virus challenge also was achieved against the rabies-related Duvenhage virus but not against the Mokola virus. Effective immunization by V-RG depended on the expression of a rabies glycoprotein that registered proline rather than leucine as the eighth amino acid from its NH2 terminus (V-RGpro8). A minimum dose required for effective immunization of mice was 10(4) plaque-forming units of V-RGpro8 virus. beta-propiolactone-inactivated preparations of V-RGpro8 virus also induced high levels of rabies virus-neutralizing antibody and protected mice against intracerebral challenge with street rabies virus. V-RGpro8 virus was highly effective in priming mice to generate a secondary rabies virus-specific cytotoxic T-lymphocyte response following culture of lymphocytes with either ERA or PM strains of rabies virus.     

福建省首例狂犬病街毒分离株基因组序列测定及分析

目的 对福建省首次分离的狂犬病毒街毒株进行全基因组序列测定分析,了解病毒序列及进化特征,为进一步研究街毒的遗传变异规律积累理论资料。方法 从病毒细胞培养液中直接提取病毒RNA,采取分段RT-PCR的方法进行序列扩增,将获得的各基因片段进行分子克隆后测序,运用生物学软件进行拼接得到全基因组序列,并进行分析。结果 应用自行设计的引物,获得了狂犬病毒街毒株的全基因组核苷酸序列,编码区没有插入和缺失的发生,对毒株在N蛋白主要抗原位点发生的突变为在B细胞表位发生了379位V→L的突变;糖蛋白抗原位点I(231位)的氨基酸为L;在抗原位点II 199位点发生了R→G的突变,抗原位点III 333位为精氨酸(R)。与国内河北一街毒株BD06无论在G基因的进化上还是N基因的分型上都有着很近的亲缘关系。结论 获得狂犬病毒株全基因组序列,了解病毒重要的氨基酸位点特征,为进一步探讨福建省狂犬病毒变异、传播机制等奠定基础。     

优化密码子以提高狂犬病病毒糖蛋白基因在原核细胞的表达

目的探讨提高狂犬病病毒(RV)HEP-Flury株糖蛋白(GP)基因在原核细胞中表达的水平和与抗体的反应水平。方法以RVHEP-Flury株的全长质粒为模板,采用PCR方法扩增获得去除信号肽基因的GP(G1)、GP的膜外区基因(G2)。选取膜外区基因中抗原表位富集区基因,对氨基酸密码子进行修饰并合成其序列(G3)。分别将G1、G2及G3基因亚克隆到表达载体pET32a(+),构建融合表达质粒pET32a-G1、pET32a-G2及pET32a-G3,转化表达宿主菌BL21,并利用IPTG诱导表达。经SDS-PAGE,Western-blotting和薄层扫描分析,比较重组蛋白的表达量。结果 G3基因的表达量比G1、G2基因明显提高。结论通过密码子优化,能显著提高G基因在原核细胞中的表达水平。     

Applications of pox virus vectors to vaccination: an update.

Recombinant pox viruses have been generated for vaccination against heterologous pathogens. Amongst these, the following are notable examples. (i) The engineering of the Copenhagen strain of vaccinia virus to express the rabies virus glycoprotein. When applied in baits, this recombinant has been shown to vaccinate the red fox in Europe and raccoons in the United States, stemming the spread of rabies virus infection in the wild. (ii) A fowlpox-based recombinant expressing the Newcastle disease virus fusion and hemagglutinin glycoproteins has been shown to protect commercial broiler chickens for their lifetime when the vaccine was administered at 1 day of age, even in the presence of maternal immunity against either the Newcastle disease virus or the pox vector. (iii) Recombinants of canarypox virus, which is restricted for replication to avian species, have provided protection against rabies virus challenge in cats and dogs, against canine distemper virus, feline leukemia virus, and equine influenza virus disease. In humans, canarypox virus-based recombinants expressing antigens from rabies virus, Japanese encephalitis virus, and HIV have been shown to be safe and immunogenic. (iv) A highly attenuated vaccinia derivative, NYVAC, has been engineered to express antigens from both animal and human pathogens. Safety and immunogenicity of NYVAC-based recombinants expressing the rabies virus glycoprotein, a polyprotein from Japanese encephalitis virus, and seven antigens from Plasmodium falciparum have been demonstrated to be safe and immunogenic in early human vaccine studies.