委内瑞拉马脑炎

委内瑞拉马脑炎(Venezuelan equine encephalitis,VEE)是由VEE病毒复合物引起的自然疫源性疾病.1938年-1972年间,VEE在西半球周期性的暴发.此后相当长的一段时间没有该病病例的报道.近10年在墨西哥和南美洲再次出现委内瑞拉马脑炎,证明它是一种新发传染病.另外,VEE病毒复合物也是一种潜在的、可以用于战争和恐怖活动的生物武器.目前,世界对生物防御的重视,重新引起人们对VEE的关注.本文就委内瑞拉马脑炎的病原学、流行病学、病理和临床表现、以及治疗和预防等方面予以综述.     

重组委内瑞拉马脑炎病毒疫苗的研制现状

委内瑞拉马脑炎病毒引起的委内瑞拉马脑炎是一种自然疫原性疾病。该病毒可诱导感染的宿主产生细胞、体液和粘膜免疫应答,可通过分子生物学技术改造为理想的疫苗载体,从而表达病毒和细菌的多种蛋白。这些病原体包括Ⅰ型人类免疫缺陷病毒、流行性感冒病毒、人乳头瘤病毒16型、Norwalk样病毒、埃博拉病毒、苏丹病毒、马尔堡病毒、拉沙病毒、麻疹病毒、炭疽杆菌和金黄色葡萄球菌等,本文综述委内瑞拉马脑炎病毒介导的病原体疫苗的研制现状。     

实时RT-PCR法检测感染小鼠不同组织标本中的委内瑞拉马脑炎病毒

目的为委内瑞拉马脑炎病毒所致疾病的早期诊断和流行病学研究提供技术支持。方法建立了委内瑞拉马脑炎病毒特异、敏感和快速的实时RT-PCR法,并对委内瑞拉马脑炎病毒感染昆明小鼠的不同组织标本中的病毒载量进行了定量检测。结果本研究建立的实时RT-PCR法敏感(10TCID50/ml)、特异(对其它甲病毒成员,如东部马脑炎病毒和西部马脑炎病毒检测为阴性)。该方法可从委内瑞拉马脑炎病毒感染小鼠后第1d的血液、脾脏、脑组织中检测到不同含量的病毒核酸,而在感染后第3d仅从脾脏和脑组织中检测到病毒核酸。结论实时RT-PCR法不仅是一种敏感、准确的检测方法,可用来了解病毒感染量与疾病严重程度的关系和评价病毒病疫苗的免疫效果,而且也为VEEV疾病的临床诊断和流行病学监测提供工具。     

美国《Emerging Infectious Diseases》2009年第15卷第4期有关人兽共患病论文摘译

P519 墨西哥委内瑞拉马脑炎病毒人工感染潜在储存宿主的实验／／Eleanor R．Deardorff，Naomi L．Forrester,Amelia P．Travassos da Rosa等 1993年墨西哥的恰帕斯州沿海地区爆发了马脑炎疫情，125匹马受到感染，病死率达50％。此次疫情的病原体是委内瑞拉马脑炎病毒（VEEV）IE亚型，与此前的马类疾病和死亡无关。为了进一步了解该病毒株在恰帕斯州的生态学，我们人工感染了5种野生啮齿动物，     

微生物介导的麻疹病毒疫苗的研制现状

麻疹病毒引起的麻疹是一种严重危害儿童健康的病毒性疾病,采用疫苗防治该病是当前研究的热点领域之一。麻疹病毒的核壳蛋白、血凝素或融合蛋白是三种有效的疫苗候选分子,本文综述了鼠伤寒沙门氏菌、卡介苗、格式链球菌、根癌农杆菌、牛痘病毒、腺病毒血清型5、委内瑞拉马脑炎病毒、辛德巴斯病毒和人副流感病毒3型等微生物介导的麻疹病毒疫苗的研制现状。     

西方马脑炎疫苗研究进展

西方马脑炎是由西方马脑炎病毒感染引起的,经蚊虫传播的急性人兽共患传染病,可引起严重的脑损伤。此外,西方马脑炎病毒也被视为潜在的生物战剂(生物恐怖剂)。目前西方马脑炎尚无获批疫苗和有效药物,研发有效疫苗用于相关人群的免疫接种很有必要。传统灭活病毒疫苗具有良好的免疫保护效能,但为了避免其制备过程中病毒培养对昂贵生物安全3级实验室的依赖以及操作者潜在感染的风险,研究者采用新的生物学技术研发西方马脑炎亚单位疫苗、DNA疫苗、嵌合体疫苗和重组活载体疫苗等新型疫苗。本文对疫苗的研究进展进行综述。     

当前传入我国风险较大的几种新发传染病

近年来,国际新发、烈性传染病疫情发生的频率大大加快,对我国的影响也日益加大。西尼罗病毒、马尔堡病毒、拉沙病毒、委内瑞拉马脑炎病毒、尼帕病毒、布氏锥虫、裂谷热病毒等多种人兽共患病原体均曾引起过人间或动物间较大规模的暴发流行。随着我国对外经贸、人员往来的日趋频繁,上述传染病疫情存在传入我国并引起国内地方性流行的巨大风险,应引起我国高度关注。     

东部马脑炎病毒分子生物学研究进展

东部马脑炎病毒 (ＥａｓｔｅｒｎＥｑｕｉｎｅＥｎｃｅｐｈａｌｏｍｙｅｌｉｔｉｓｖｉｒｕｓ ,ＥＥＥＶ)属披膜病毒科甲病毒属 ,是东部马脑脊髓炎的病原体 ,也是披膜病毒科中致病性最强的一种病毒。 1933年美国东部新泽西州和弗吉尼亚沿海地区发生马脑炎流行 ,从马脑中分离出该病毒 ,以此得名。因该病发病急 ,传染性强 ,致死率高且为人兽共患 ,一直受到国际社会的高度重视 ,在国外对ＥＥＥＶ进行了深入研究。我国研究起步较晚 ,1991年李其平等〔1〕从新疆采集的一组全沟硬蜱中分离到 1株ＥＥＥＶ ,另有人对 13个地区人血清进行抗体检测 ,阳…     

脑膜炎球菌共轭疫苗在非洲人群中的应用

该研究纳入1500例儿童及青年人，分别注射了脑膜炎球菌共轭疫苗及多聚糖四价脑炎球菌疫苗，结果显示，脑膜炎球菌共轭疫苗引出的抗体滴度为78％-96％，明显高于应用多聚糖四价脑炎球菌疫苗受试者（The New England Journal of Medicine 2011,6月16日）。     

两种甲病毒基因组序列的一步RT-PCR检测

目的　通过对不同扩增条件的优化 ,建立两种马脑炎病毒的快速RT -PCR检测方法。方法　根据东部马脑炎病毒和西部马脑炎病毒基因组相应序列设计引物 ,然后采用两步法及两种一步法分别对其基因组序列进行RT -PCR扩增 ,并用琼脂糖凝胶电泳进行观察。结果与结论　三种方法均可从两种马脑炎病毒感染的乳鼠脑和细胞上清中扩增出单一的DNA片段 ,其大小与预期的相一致。与其他两种方法相比 ,本研究所建立的一步法更为简便快速、价格低廉 ,为进一步组装这些病毒的检测试剂盒奠定了基础。     

Evaluation in humans of a new, inactivated vaccine for Venezuelan equine encephalitis virus (C-84)

A new, formalin-inactivated vaccine for Venezuelan equine encephalitis (VEE) virus (C-84), prepared from an attenuated vaccine strain of virus (TC-83), was tested in humans. Only occasional, mild, local and systemic reactions were noted in 28 volunteers; no meaningful changes in clinical laboratory values occurred. The vaccine augmented preexisting titers of serum neutralizing antibody to VEE virus in seropositive recipients of TC-83 vaccine, and it induced high titers of neutralizing antibody in nonimmune subjects after one primary and two booster vaccinations. Circulating antibody persisted for at least 14 months in these persons. The neutralizing antibody produced after one dose of C-84 vaccine in immune subjects and after booster doses in nonimmune subjects had broad cross-reactivity within the VEE virus complex. The C-84 vaccine induced a VEE virus-specific lymphocyte transformation response. The vaccine was safe, and immunologic results showed it to be highly antigenic in healthy immune and nomimmune adults.     

Persistence in humans of antibody after immunization with four alphavirus vaccines.

Immunization with attenuated VEE virus vaccine resulted in persistence of neutralizing antibody for 12 years. Immunization with inactivated WEE vaccine converted 83% of the subjects, killed EEE vaccine converted 27% and killed Chikungunya vaccine induced no significant titers. Antibody formed as a result of immunization with inactivated vaccines was of short duration, i.e. less than 1 year. Attenuated VEE was responsible for some heterologous antibody rises to the other three alphaviruses. Among the inactivated vaccines WEE and Chikungunya vaccines produced one heterologous rise each to EEE virus.     

Limited potential for mosquito transmission of genetically engineered, live-attenuated Venezuelan equine encephalitis virus vaccine candidates.

In an attempt to improve the current live-attenuated vaccine (TC-83) for Venezuelan equine encephalitis (VEE), specific mutations associated with attenuation of VEE virus in rodent models were identified. These mutations were inserted into full-length cDNA clones of the Trinidad donkey strain of VEE virus by site-directed mutagenesis, and isogenic virus strains with these mutations were recovered after transfection of baby hamster kidney cells with infectious RNA. We evaluated 10 of these strains for their ability to replicate in and be transmitted by Aedes taeniorhynchus, a natural vector of epizootic VEE virus. Two vaccine candidates, one containing a deletion of the PE2 furin cleavage site, the other a combination of three separate point mutations in the E2 glycoprotein, replicated in mosquitoes and were transmitted to hamsters significantly less efficiently than was either parental (wild type) VEE virus or TC-83 virus. Although the attenuated strains were transmitted to hamsters by mosquitoes, after intrathoracic inoculation, there was no evidence of reversion to a virulent phenotype. The mutations that resulted in less efficient replication in, or transmission by, mosquitoes should enhance vaccine safety and reduce the possibility of environmental spread to unintentional hosts.     

Immune protection against staphylococcal enterotoxin-induced toxic shock by vaccination with a Venezuelan equine encephalitis virus replicon

A candidate vaccine against staphylococcal enterotoxin B (SEB) was developed using a Venezuelan equine encephalitis (VEE) virus vector. This vaccine is composed of a self-replicating RNA, termed "replicon," containing the VEE nonstructural genes and cis-acting elements and a gene encoding mutagenized SEB (mSEB). Cotransfection of baby hamster kidney cells with the mSEB replicon and 2 helper RNA molecules resulted in the release of propagation-deficient mSEB-VEE replicon particles (mSEB-VRPs). Mice inoculated subcutaneously with mSEB-VRPs were protected (15 of 20 mice) from a challenge with 5 median lethal dose units of wild-type (wt) SEB. T cells from mice vaccinated with mSEB-VRP responded normally both in vitro to wt SEB and in recall response to the inactivated mSEB polypeptide. The profile of cytokines measured after challenge with wt SEB suggested that the mode of protection was predominantly Th1 dependent. Our results suggest that the VEE replicon is a practical and convenient model system for evaluating efficacy of vaccines for the control of bacterial diseases.     

Enzootic and epizootic Venezuelan equine encephalomyelitis virus in horses infected by peripheral and intrathecal routes

Forty-five horses were infected peripherally or intrathecally with enzootic or epizootic strains of Venezuelan equine encephalomyelitis (VEE) virus. Low titers of virus appeared in cerebrospinal fluid (CSF) after peripheral inoculation of enzootic or epizootic VEE virus strains. Intrathecal infection with either epizootic or enzootic VEE virus produced higher titers of virus in CSF than did peripheral infection. In contrast to peripheral infections with enzootic strains, intrathecal infections with these strains caused death. The animals that died had widespread histopathologic changes and large amounts of virus in brain tissue. The attenuated VEE virus vaccine strain, TC-83, also multiplied in the brain of horses inoculated intrathecally but caused no clinical disease and little histopathologic damage.     

Genetic evidence for the origins of Venezuelan equine encephalitis virus subtype IAB outbreaks.

Epizootics of Venezuelan equine encephalitis (VEE) involving subtype IAB viruses occurred sporadically in South, Central and North America from 1938 to 1973. Incompletely inactivated vaccines have long been suspected as a source of the later epizootics. We tested this hypothesis by sequencing the PE2 glycoprotein precursor (1,677 nucleotides) or 26S/nonstructural protein 4 (nsP4) genome regions (4,490 nucleotides) for isolates representing most major outbreaks. Two distinct IAB genotypes were identified: 1) 1940s Peruvian strains and 2) 1938-1973 isolates from South, Central, and North America. Nucleotide sequences of these two genotypes differed by 1.1%, while the latter group showed only 0.6% sequence diversity. Early VEE virus IAB strains that were used for inactivated vaccine preparation had sequences identical to those predicted by phylogenetic analyses to be ancestors of the 1960s-1970s outbreaks. These data support the hypothesis of a vaccine origin for many VEE outbreaks. However, continuous, cryptic circulation of IAB viruses cannot be ruled out as a source of epizootic emergence.     

Assessment of assays for the serodiagnosis of Venezuelan equine encephalitis.

An enzyme-linked immunosorbent assay (ELISA), an immunofluorescence assay (IFA), a plaque-reduction neutralization (PRN) assay and an immunoblot assay, all by means of an antigen prepared from the attenuated Venezuelan equine encephalitis (VEE) vaccine strain of virus, were compared with the conventional haemagglutination-inhibition (HAI) assay for the serodiagnosis of VEE. The HAI assay, which includes the use of wild type virus antigen, was less sensitive than the other assays when known-positive samples of serum from an epidemic of VEE were tested. The superior sensitivity of the IgG ELISA was confirmed by assaying both VEE epidemic samples and a bank of samples from VEE vaccinees. Samples with antibody specific for other Alphaviruses, however, cross reacted weakly in this assay. The PRN, immunoblot and HAI assays, although less sensitive than the ELISA, proved more specific. Experimental infection of guinea-pigs demonstrated the value of the IgM ELISA in the early detection of VEE virus infection. Immunoglobulin M was first found at 4 days post-inoculation (p.i.) during the viraemic phase of infection. Immunoglobulin G was detected by ELISA, PRN assay and IFA at 6 days p.i. Immunoblot and HAI assays, however, did not give positive results until 10 days p.i. The results support the diagnostic use of ELISA for detecting VEE virus-specific IgM and IgG, and the use of the specific PRN assay for confirming the diagnosis.     

Limited potential for mosquito transmission of genetically engineered,live-attenuated western equine encephalitis virus vaccine candidates

Specific mutations associated with attenuation of Venezuelan equine encephalitis (VEE) virus in rodent models were identified during efforts to develop an improved VEE vaccine. Analogous mutations were produced in full-length cDNA clones of the Cba 87 strain of western equine encephalitis (WEE) virus by site-directed mutagenesis in an attempt to develop an improved WEE vaccine. Isogenic viral strains with these mutations were recovered after transfection of baby hamster kidney cells with infectious RNA. We evaluated two of these strains (WE2102 and WE2130) for their ability to replicate in and be transmitted by Culex tarsalis, the principal natural vector of WEE virus in the United States. Each of the vaccine candidates contained a deletion of the PE2 furin cleavage site and a secondary mutation in the E1 or E2 glycoprotein. Both of these potential candidates replicated in mosquitoes significantly less efficiently than did either wild-type WEE (Cba 87) virus or the parental clone (WE2000). Likewise, after intrathoracic inoculation, mosquitoes transmitted the vaccine candidate strains significantly less efficiently than they transmitted either the wild-type or the parental clone. One-day-old chickens vaccinated with either of the two vaccine candidates did not become viremic when challenged with virulent WEE virus two weeks later. Mutations that result in less efficient replication in or transmission by mosquitoes should enhance vaccine safety and reduce the possibility of accidental introduction of the vaccine strain to unintentional hosts.     

Host immune responses after administration of inactivated Venezuelan equine encephalomyelitis virus vaccines. II. Kinetics of neutralizing antibody responses in donors and adoptively immunized recipients.

Lymphoid cell responses to immunization with various formalin-inactivated Venezuelan equine encephalomyelitis (VEE) virus vaccines were monitored in mice by assessment of the development of both the neutralizing antibody response in sera of spleen cell donors and the adoptive neutralizing antibody response induced by spleen cell transfer in recipients. Donors immunized intraperitoneally with formalin-inactivated VEE vaccine (a single dose or a dose on three consecutive days) developed early serum neutralizing antibody responses (larger than or equal to 1:88-1:100) by seven days after immunization. Recipients of spleen cells from such mice were, however, incapable of eliciting a neutralizing antibody response (less than or equal to 1:10). Only spleen cells from donors immunized with inactivated VEE vaccine plus adjuvants (particularly complete Freund's adjuvant and Bordetella pertussis) were consistently capable of producing early, high-titer serum neutralizing antibody responses in adoptively immunized recipients (larger than or equal to 1:50-1:120 on day 4). The magnitude of neutralizing antibody responses of donors to inactivated VEE vaccines did not serve as a useful indicator of whether spleen cells from such mice could adoptively induce antibody responses in recipients. Finally, treatment of immune spleen cells with rabbit antiserum to mouse thymocytes, but not with rabbit antiserum to mouse gamma-globulin or normal rabbit serum, abolished the capacity of such cells to transfer an antibody response adoptively.     

Characterization and selection of HIV-1 subtype C isolates for use in vaccine development

HIV-1 genetic diversity among circulating strains presents a major challenge for HIV-1 vaccine development, particularly for developing countries where less sequence information is available. To identify representative viruses for inclusion in candidate vaccines targeted for South Africa, we applied an efficient sequence survey strategy to samples from recently and chronically infected persons residing in potential vaccine trial sites. All 111 sequences were subtype C, including 30 partial gag, 26 partial pol, 27 V2-V3 env, and 28 V5-partial gp41 sequences. Of the 10 viruses cultured from recently infected individuals, 9 were R5 and 1 was R5X4. Two isolates, Du151 and Du422, collected within 2 months of infection, were selected as vaccine strains on the basis of their amino acid similarity to a derived South African consensus sequence The selection of recently transmitted R5 isolates for vaccine design may provide an advantage in a subtype C R5-dominant epidemic. The full-length Du422 gag and Du151 pol and env genes were cloned into the Venezuelan equine encephalitis (VEE) replicon particle (VRP) expression system. Du422 Gag protein expressed from the VRP accumulated to a high level and was immunogenic as demonstrated by cytotoxic T lymphocyte responses in mice vaccinated with gag-VRPs. Optimization of codon use for VRP expression in human cells did not enhance expression of the gag gene. The cloned Du151 env gene encoded a functional protein as demonstrated by fusion of VRP-infected cells with cells expressing CD4 and CCR5. Genes identified in this study have been incorporated into the VEE VRP candidate vaccines targeted for clinical trial in South Africa.