新疆伊犁地区动物脑组织西尼罗病毒E基因片段的检测

目的了解新疆伊犁地区动物中西尼罗病毒（West Nile virus,WNV）感染现状,为我国WNV的防治提供资料。方法采用一步法实时荧光定量反转录-聚合酶链反应（RT-PCR）,对采自新疆伊犁地区的70头驴和100只牧羊犬的脑组织进行WNV包膜蛋白（E）基因片段检测。结果 70头驴和100只牧羊犬脑组织标本的西尼罗病毒包膜蛋白基因片段检测有2例可疑阳性,但经3%凝胶电泳法再次验证后排除。故所有待检样本WNV包膜蛋白基因检测均为阴性。结论我国新疆伊犁地区的驴和牧羊犬脑组织中未检测到WNV的感染。     

西尼罗病毒空斑形成试验方法的建立与应用

目的建立西尼罗病毒的空斑形成试验方法，应用于病毒滴度测定和实验感染蚊虫及来亨鸡血液样本中病毒的定量检测。方法将稀释的样本接种常规制备的Veto细胞单层，用琼脂糖凝胶覆盖，孵育一定时间后，加中性红染色，计数空斑数，计算空斑形成单位。结果在琼脂糖凝胶中西尼罗病毒可形成直径大约1～3mm的圆形或类圆形空斑。20％病毒鼠脑悬液的病毒滴度为10^7空斑形成单位。感染蚊虫样本和来亨鸡血液样本也出现典型的空斑。结论建立了西尼罗病毒的空斑形成试验方法，该方法可应用于病毒滴度测定和实验感染蚊虫及来亨鸡血液样本中病毒的定量检测。     

病毒性脑炎患者外周血和脑脊液西尼罗病毒E基因的检测

西尼罗病毒(WNV)是单股正链RNA病毒,属于黄病毒科黄病毒属,其传播媒介和储存宿主是蚊虫[1].人感染WNV会出现感冒样症状,部分患者为脑膜脑炎、坏死性脑干炎等神经系统症状[2,3].本研究对重庆地区不明病因的78例病毒性脑炎(VE)患者血液和脑脊液进行wNV E基因片段检测.     

西尼罗病毒单克隆抗体的制备与鉴定

目的制备西尼罗病毒单克隆抗体并对其生物特性进行鉴定,为快检试剂盒的研制奠定基础。方法通过动物免疫、细胞融合、克隆和筛选等方法制备西尼罗病毒单抗,应用间接免疫荧光(IFA)、ELISA方法进行特异性、效价、亚型等的鉴定。结果获得了5株西尼罗病毒的单克隆抗体,其中4株与所检的登革病毒1-4型、黄热病毒、乙脑病毒、布尼安病毒、基孔肯雅病毒均无交叉反应,1株与乙脑病毒有交叉反应;亚型分类均为IgG2a;效价可达1∶12 800以上。结论本研究获得了5个高滴度、特异性较好的西尼罗病毒单克隆抗体。     

西尼罗病毒的蔓延及提示

西尼罗病毒是一种蚊媒病毒，能够侵袭人的中枢神经系统，引发致命性脑炎。其原发于非洲，20世纪90年代在罗马尼亚、俄罗斯和以色列流行，1999年传入北美，纽约市的脑炎大爆发首次检测到该病毒。1999～2003年，西尼罗病毒席卷北美全境，并继续在西半球大规模扩散。本文就其病原学、发病机制、流行情况、治疗及预防作了综述。     

天津市蚊虫西尼罗病毒携带调查

目的了解天津市蚊虫携带西尼罗病毒（WNV）情况。方法以灯诱法进行蚊虫采集,采用荧光定量PCR检测方法对WNV进行检测。结果天津市有候鸟栖息的湖泊、洼地周边主要蚊种为淡色库蚊（占93．68％）,同时存在凶小库蚊和三带喙库蚊。对1091只蚊虫进行西尼罗病毒检测,结果均为阴性。结论本次调查未发现蚊虫携带WNV,为预防和控制WNV传人及可能发生的流行,有必要开展对媒介蚊虫及WNV的长期监测。     

西尼罗病毒输血传播和献血者筛检

2002年．西尼罗病毒(WNV)可以通过输血途径传播(TAT)已得到证实。虽然蚊虫叮咬仍然是西尼罗病毒的主要传播途径,但也说明了西尼罗病毒在输血方面的筛检工作还做得不够。本文主要阐述2003年美国血液采集机构(BCAs)运用核酸扩增技术(NATs)对西尼罗病毒血样筛检的结果及发现6例输血感染者。结果表明通过对献血者的血样进行西尼罗病毒的筛检可以大大提高输血的安全性。     

西尼罗病毒感染的抗体检测方法研究进展

病毒核酸编码3种结构蛋白和7种非结构蛋     

致纽约脑炎的新病毒:西尼罗病毒

本文介绍了在美国纽约爆发的圣路易脑炎的流行情况、传播途径和临床表现,并提出导致的原因为西尼罗病毒.作者针对旅行者提出预防措施.     

西尼罗病毒研究现状

西尼罗病毒病（West Nile virus disease）是由西尼罗病毒（West Nile virus,WNV）感染引起的一种人畜共患的急性传染病。该病毒最初是1937年从乌干达西尼罗地区一名发热的妇女血液中分离发现而得名。WNV感染人类后可引起西尼罗热和WNV性脑炎。鸟类是WNV的贮存宿主,人和哺乳动物是偶然宿主,传播媒介主要为蚊。     

A recombinant West Nile virus envelope protein vaccine candidate produced in Spodoptera frugiperda expresSF+ cells

In this study, a recombinant truncated West Nile virus envelope protein antigen (rWNV-E) was produced in serum-free cultures of the expresSF+ insect cell line via baculovirus infection. This production system was selected based on its use in the production of candidate human and animal vaccine antigens. A defined fermentation and purification process for the rWNV-E antigen was established to control for purity and immunogenicity of each protein batch. The material formulated with aluminum hydroxide was stable for greater than 8 months at 4 °C. The recombinant vaccine candidate was evaluated for immunogenicity and protective efficacy in several animal models. In mouse and hamster WNV challenge models, the vaccine candidate induced viral protection that correlated with anti-rWNV-E immunogenicity and WNV neutralizing antibody titers. The rWNV-E vaccine candidate was used to boost horses previously immunized with the Fort Dodge inactivated WNV vaccine and also to induce WNV neutralizing titers in naïve foals that were at least 14 weeks of age. Furthermore, the vaccine candidate was found safe when high doses were injected into rats, with no detectable treatment-related clinical adverse effects. These observations demonstrate that baculovirus-produced rWNV-E can be formulated with aluminum hydroxide to produce a stable and safe vaccine which induces humoral immunity that can protect against WNV infection.     

Matrix-M™ adjuvanted envelope protein vaccine protects against lethal lineage 1 and 2 West Nile virus infection in mice

West Nile virus (WNV) is a mosquito-transmitted flavivirus and an emerging pathogen in many parts of the world. In the elderly and immunosuppressed, infection can progress rapidly to debilitating and sometimes fatal neuroinvasive disease. Currently, no WNV vaccine is approved for use in humans. As there have been several recent outbreaks in the United States and Europe, there is an increasing need for a human WNV vaccine. In this study, we formulated the ectodomain of a recombinant WNV envelope (E) protein with the particulate saponin-based adjuvant Matrix-M™ and studied the antigen-specific immune responses in mice. Animals immunized with Matrix-M™ formulated E protein developed higher serum IgG1 and IgG2a and neutralizing antibody titers at antigen doses ranging from 0.5 to 10 μg compared to those immunized with 3 or 10 μg of E alone, E adjuvanted with 1% Alum, or with the inactivated virion veterinary vaccine, Duvaxyn^® WNV. This phenotype was accompanied by strong cellular recall responses as splenocytes from mice immunized with Matrix-M™ formulated vaccine produced high levels of Th1 and Th2 cytokines. Addition of Matrix-M™ prolonged the duration of the immune response, as elevated humoral and cellular responses were maintained for more than 200 days. Importantly, mice vaccinated with Matrix-M™ formulated E protein were protected from lethal challenge with both lineage 1 and 2 WNV strains. In summary, Matrix-M™ adjuvanted E protein elicited potent and durable immune responses that prevented lethal WNV infection, and thus is a promising vaccine candidate for humans.     

Domestic goose as a model for West Nile virus vaccine efficacy

West Nile virus (WNV) is an emergent pathogen in the Americas, first reported in New York during 1999, and has since spread across the USA, Central and South America causing neurological disease in humans, horses and some bird species, including domestic geese. No WNV vaccines are licensed in the USA for use in geese. This study reports the development of a domestic goose vaccine efficacy model, based on utilizing multiple parameters to determine protection. To test the model, 47 geese were divided in two experiments, testing five different vaccine groups and two sham groups (challenged and unchallenged). Based on the broad range of results for individual metrics between the Challenged-Sham and Unchallenged-Sham groups, the best parameters to measure protection were Clinical Pathogenicity Index (CPI), plasma virus positive geese on days 1–4 post-inoculation and plasma virus titers, and brain histological lesion rates and severity scores. Compared to the Challenged-Sham group, the fowlpox virus vectored vaccine with inserts of WNV prM and E proteins (vFP2000) provided the best protection with significant differences in all five metrics, followed by the canarypox virus vectored vaccine with inserts of WNV prM and E proteins (vCP2018) with four metrics of protection, recombinant vCP2017 with three metrics and WNV E protein with one. These data indicate that domestic geese can be used in an efficacy model for vaccine protection studies using clinical, plasma virological and brain histopathological parameters to evaluate protection against WNV challenge.     

A plant-produced vaccine protects mice against lethal West Nile virus infection without enhancing Zika or dengue virus infectivity

West Nile virus (WNV) has caused multiple global outbreaks with increased frequency of neuroinvasive disease in recent years. Despite many years of research, there are no licensed therapeutics or vaccines available for human use. One of the major impediments of vaccine development against WNV is the potential enhancement of infection by related flaviviruses in vaccinated subjects through the mechanism of antibody-dependent enhancement of infection (ADE). For instance, the recent finding of enhancement of Zika virus (ZIKV) infection by pre-exposure to WNV further complicates the development of WNV vaccines. Epidemics of WNV and the potential risk of ADE by current vaccine candidates demand the development of effective and safe vaccines. We have previously reported that the domain III (DIII) of the WNV envelope protein can be readily expressed in Nicotiana benthamiana leaves, purified to homogeneity, and promote antigen-specific antibody response in mice. Herein, we further investigated the in vivo potency of a plant-made DIII (plant-DIII) in providing protective immunity against WNV infection. Furthermore, we examined if vaccination with plant-DIII would enhance the risk of a subsequent infection by ZIKV and Dengue virus (DENV). Plant-DIII vaccination evoked antigen-specific cellular immune responses as well as humoral responses. DIII-specific antibodies were neutralizing and the neutralization titers met the threshold correlated with protective immunity by vaccines against multiple flaviviruses. Furthermore, passive administration of anti-plant DIII mouse serum provided full protection against a lethal challenge of WNV infection in mice. Notably, plant DIII-induced antibodies did not enhance ZIKV and DENV infection in Fc gamma receptor-expressing cells, addressing the concern of WNV vaccines in inducing cross-reactive antibodies and sensitizing subjects to subsequent infection by heterologous flavivirus. This study provides the first report of a WNV subunit vaccine that induces protective immunity, while circumventing induction of antibodies with enhancing activity for ZIKV and DENV infection.     

利用VSV△G＊伪型病毒建立安全、快速的西尼罗病毒抗体检测方法的尝试

目的为探讨利用水泡性口炎病毒（vesicular stomatitis virus,VSV）假病毒粒子系统包装西尼罗病毒（West Nile virus,WNV）的E蛋白可行性,构建3种E蛋白基因的真核表达质粒。以期用于WNV感染的流行病学调查。方法将表达WNN囊膜蛋白E的质粒体外转染293T细胞,然后感染VSV△G＊G,并对E蛋白表达质粒进行改造,分别在基因序列前加入信号肽,在终止密码子之前加入VSV△G蛋白的细胞质尾序列。结果包装出的VSV△G＊-WNVE滴度没有明显升高。提示WNV E蛋白与VSV的囊膜匹配性不佳,VSV△G＊-WNVE作为病毒中和抗原代替野毒进行抗体筛查还需要进一步提高转染和包装效率。结论VSV作为高效、安全的新型载体、活病毒疫苗载体和肿瘤基因治疗载体以及高危病毒的研究工具具有巨大的研究价值和应用潜力。     

Protection of red-legged partridges (Alectoris rufa) against West Nile virus (WNV) infection after immunization with WNV recombinant envelope protein E (rE)

West Nile virus (WNV) is maintained in nature in an enzootic transmission cycle between birds and mosquitoes, although it occasionally infects other vertebrates, including humans, in which it may result fatal. To date, no licensed vaccines against WNV infection are available for birds, but its availability would certainly benefit certain populations, as birds grown for restocking, hunting activities, or alimentary purposes, and those confined to wildlife reservations and recreation installations. We have tested the protective capability of WNV envelope recombinant (rE) protein in red-legged partridges (Alectoris rufa). Birds (n = 28) were intramuscularly immunized three times at 2-weeks interval with rE and a control group (n = 29) was sham-immunized. Except for 5 sham-immunized birds that were not infected and housed as contact controls, partridges were subcutaneously challenged with WNV. Oropharyngeal and cloacal swabs and feather pulps were collected at several days after infection and blood samples were taken during vaccination and after infection. All rE-vaccinated partridges elicited anti-WNV antibodies before challenge and survived to the infection, while 33.3% of the sham-immunized birds succumbed, as did 25% of the contact animals. Most (84%) unvaccinated birds showed viremia 3 d.p.i., but virus was only detected in 14% of the rE vaccinated birds. WNV-RNA was detected in feathers and swabs from sham-immunized partridges from 3 to 7 d.p.i., mainly in birds that succumbed to the infection, but not in rE vaccinated birds. Thus, rE vaccination fully protected partridges against WND and reduced the risk of virus spread.     

Cost-effectiveness of West Nile virus vaccination

West Nile virus (WNV) was first detected in the Western Hemisphere in 1999 in New York City. From 1999 through 2004, >16,600 cases of WNV-related illnesses were reported in the United States, of which >7,000 were neuroinvasive disease and >600 were fatal. Several approaches are under way to develop a human vaccine. Through simulations and sensitivity analysis that incorporated uncertainties regarding future transmission patterns of WNV and costs of health outcomes, we estimated that the range of values for the cost per case of WNV illness prevented by vaccination was US 20,000 dollars-59,000 dollars(mean 36,000 dollars). Cost-effectiveness was most sensitive to changes in the risk for infection, probability of symptomatic illness, and vaccination cost. Analysis indicated that universal vaccination against WNV disease would be unlikely to result in societal monetary savings unless disease incidence increases substantially over what has been seen in the past 6 years.     

Pre-clinical development of a hydrogen peroxide-inactivated West Nile virus vaccine

West Nile virus (WNV) is a mosquito-transmitted pathogen with a wide geographical range that can lead to long-term disability and death in some cases. Despite the public health risk posed by WNV, including an estimated 3 million infections in the United States alone, no vaccine is available for use in humans. Here, we present a scaled manufacturing approach for production of a hydrogen peroxide-inactivated whole virion WNV vaccine, termed HydroVax-001 WNV. Vaccination resulted in robust virus-specific neutralizing antibody responses and protection against WNV-associated mortality in mice or viremia in rhesus macaques (RM). A GLP-compliant toxicology study performed in rats demonstrated an excellent safety profile with clinical findings limited to minor and transient irritation at the injection site. An in vitro relative potency (IVRP) assay was developed and shown to correlate with in vivo responses following forced degradation studies. Long-term in vivo potency comparisons between the intended storage condition (2–8 °C) and a thermally stressed condition (40 ± 2 °C) demonstrated no loss in vaccine efficacy or protective immunity over a 6-month span of time. Together, the positive pre-clinical findings regarding immunogenicity, safety, and stability indicate that HydroVax-001 WNV is a promising vaccine candidate.