流行性乙型脑炎快速血清学诊断方法研究进展

流行性乙型脑炎(简称乙脑)是由日本脑炎病毒引起的,以中枢神经系统受损病变为主的一种急性蚊媒人畜共患传染病。由于乙脑病毒的增殖宿主和贮存宿主涉及猪、马、牛和禽等多种家畜,且主要在亚洲和西太平洋地区广泛流行,不仅造成畜牧业严重经济损失,而且危害公共卫生安全。因此,对乙脑进行快速而准确的诊断具有实际意义。文章对乙脑的快速血清学诊断方法包括传统方法、新兴技术和试验特异性改良等进行较全面的综述,意在为快速、简单、准确的乙脑诊断方法研究提供参考。     

Japanese encephalitis.

Japanese encephalitis (JE) virus is a mosquito-borne flavivirus that can cause encephalitis and death in horses and humans. It is an emerging disease of international concern because it has been spreading into previously nonendemic areas. Major epidemics may occur where the virus moves into new areas, but many infections are subclinical. This article presents information on the virus, its epidemiology, and what little information has been published on the disease in horses. The methods available for the diagnosis and control of JE are described and issues raised, particularly for those countries faced with the threat of imminent incursions as the virus extends its range.     

猪日本乙型脑炎检测技术研究进展

猪日本乙型脑炎是由黄病毒科乙型脑炎病毒引起的一种人畜共患的病毒性传染病。人与多种动物均能感染,其中猪是主要的储存宿主和扩散宿主,猪感染该病毒后引发猪的繁殖障碍,使养猪业遭受重大经济损失,因此学者们一直在致力于寻找可以快速检测和诊断日本乙型脑炎的方法,为预防和治疗该病提供基础条件。文章对猪日本乙型脑炎检测技术,包括病毒分离鉴定方法、血清学方法、分子生物学方法等方面的研究进展进行了较为全面的综述。     

Knowledge and Practices of Pig Farmers Regarding Japanese Encephalitis in Kathmandu,Nepal

Japanese encephalitis (JE) is the single largest cause of viral encephalitis in the world and has been endemic in Nepal since the early 1980s. Since then, it has spread from its origins in lowland plains to the Kathmandu Valley as well as in hill and mountain districts. Pigs are amplifying hosts for the virus. The Nepal government has been encouraging the development of pig farming as a means of poverty alleviation. Whereas other countries have reduced JE through vaccination programmes and improvements in pig husbandry, these options are not economically possible in Nepal. The objective of this study was to examine the occupational risk of pig farmers in Nepal and to determine their level of knowledge and practice of JE prevention techniques. We surveyed 100 randomly selected pig farmers in the Kathmandu District and found that pig farmers were exposed to many JE risk factors including poverty and close proximity to pigs, rice paddy fields and water birds, which are the definitive hosts for the virus. Forty‐two percent of the farmers had heard of JE, 20% associated it with mosquito bites and 7% named pigs as risk factors. Few protective measures were taken. None of the farmers were vaccinated against JE nor were any pigs, despite an ongoing human vaccination campaign. This farming community had little ownership of land and limited education. JE education programmes must consider gender differences in access to public health information as there were an equal number of male and female farmers. We provide findings that can inform future JE education programmes for this vulnerable population.     

猪日本脑炎DNA疫苗的构建与鉴定

为开发猪日本脑炎新型疫苗,采用PCR方法扩增日本脑炎病毒WHe株prME和NS1基因,并将其分别克隆至DNA疫苗载体pVAX1,分别用酶切和测序分析进行鉴定。结果表明prME和NS1基因大小分别为2.1kb和1.1kb,酶切和测序结果表明,猪日本脑炎DNA疫苗pVAX1-prME和pVAX1-NS1构建成功,为进一步免疫试验奠定了基础。     

猪乙型脑炎疫苗研究进展

猪乙型脑炎（Japanese encephalitis，JE）是由乙型脑炎病毒（JEV）引起的一种经蚊虫传播的人畜共患病，且是严重威胁人畜健康的一种中枢神经系统的急性传染病。猪是JEV的重要储存宿主和扩增宿主，同时也是乙型脑炎的主要传染源，可引起母猪繁殖障碍和公猪睾丸炎。人是JEV的终末宿主，特别是儿童，感染发病后，死亡率可达25%。目前，仍无有效药物治疗乙型脑炎感染，而其流行区域的扩大及其优势基因型的改变为JEV防控策略带来了新的挑战。近年来，随着基因工程和蛋白质工程的发展，以及对病毒分子结构和功能的深入研究，一些新技术新手段被应用到JEV疫苗研发中，基因工程亚单位疫苗、病毒样颗粒疫苗、嵌合病毒减毒活疫苗、多表位疫苗、DNA疫苗等应运而生。在疫苗制备过程中，培养工艺的优化、病毒抗原的纯化、新型佐剂和耐热保护剂的应用等生产工艺的提升策略，为保障JEV疫苗的质量提供了新方向。文章简述了猪JEV疫苗的使用现状及研发进展，旨在为研制更安全、有效的猪JEV疫苗提供参考依据。     

云南省乙型脑炎病毒宿主和媒介研究

于１９７８年至１９９７年,从云南猪血液和乳猪脑组织中分离到乙型脑炎（ＪＥ）病毒５株,从蝙蝠脑组织中分得ＪＥ病毒１０株,从鸟类脑组织中分得ＪＥ病毒５株,从１５种蚊虫体内分离到ＪＥ病毒６３株,从２种蠓类中分得ＪＥ病毒２株。分析认为,猪是ＪＥ病毒的主要扩散宿主,蝙蝠和鸟类在该病毒保存和传播中起重要作用;三带喙库蚊是该病毒的主要传播媒介,伪杂鳞库蚊、霜背库蚊、蚊腿库蚊、白蚊伊和刺扰伊蚊为重要媒介,蠓类亦可     

Isolation and genetic analysis of Japanese encephalitis virus from a diseased horse in Japan

Japanese encephalitis (JE) developed in an unvaccinated half-bred horse kept in Tottori Prefecture, Japan. The animal showed ataxia with pyrexia and low appetite, and ultimately died. A viral strain was isolated from the cerebrum of the horse and was identified as JE virus (JEV) by RT-PCR using JEV specific primers. The isolated JEV was classified into genotype I by nucleotide sequence analysis of the viral envelope gene. We believe that this is the first report of the genotype I strain being isolated from a horse.     

Mosquito collection in endemic areas of Japanese encephalitis in Hokkaido, Japan

A study was conducted during 1985 and 1986 to evaluate the roles of mosquito species as possible vectors of Japanese encephalitis (JE) virus in Hokkaido. The number of Culex tritaeniorhynchus was very low among the four pig farms where outbreaks of abortion caused by JE virus were observed in swine populations. At one farm near Sapporo, only one Cx. tritaeniorhynchus was found among a total of 510 mosquitoes collected during the survey period from July to October 1985, even when JE virus activity among sentinel pigs was revealed by seroconversion. At another farm in the south, no individuals of this mosquito species were found among 987 mosquitoes collected at the time of the outbreaks of abortion. Cx. pipiens pallens, Anopheles species, Aedes vexans nipponii, and Ae. japonicus were predominant over Cx. tritaeniorhynchus. Cx. tritaeniorhynchus, almost a solve vector species of JE virus in the southern part of Japan, is probably not a vector of the virus in Hokkaido. The collected mosquitoes (2,332 from 1985 and 1,403 from 1986) were processed for virus isolation but no JE virus was isolated. More extensive field studies are necessary to provide further information on the role of mosquito species other than Cx. tritaeniorhynchus in the transmission of JE virus in the northern limits of its range including Hokkaido.     

甘肃省四种母猪繁殖障碍性疫病抗体血清学检测

运用乳胶凝集试验(LAT)和ELISA方法对酒泉、张掖、金昌、武威、兰州、临夏、白银、定西、天水和平凉10个市州38个规模化和农村个体养猪场送检的619份血样和血清进行了PPV、PR、PRRS和JE免疫抗体检测,3年度平均阳性率PPV为80.83%、PR为60.70%、PRRS为43.39%、JE为47.57%,免疫抗体平均阳性率以PPV最高,PRRS和JE免疫抗体平均阳性率偏低。对8市州24个猪场采送的132份血清进行的PPV、PR、PRRS和JE感染抗体检测表明,PPV感染抗体平均阳性率为18.57%、PR感染抗体平均阳性率为15.28%、PRRS感染抗体平均阳性率为23.28%J、E感染抗体平均阳性率为24.14%。     

A survey of antibodies to arthropod-borne viruses in Japanese cattle

Sera collected from the southern parts of Japan were subjected to serological tests for antibodies to 24 arthropod-borne or suspected arthropod-borne viruses. A high incidence (82%) of hemagglutination-inhibiting (HI) antibodies was found with Japanese encephalitis (JE) virus. HI antibodies to other Flaviviruses, Murray Valley encephalitis, Apoi, Kunjin, Stratford and Kokobera, were also found in some of the sera, but seemed to be due to cross reaction with JE virus. High neutralizing (NT) antibody incidences were obtained with Akabane (60%) and Aino (30%) viruses known to be endemic in Japan. NT antibodies were also found for Bunyaviruses, Batai and Wongla; Reoviridae viruses, D'Aguilar, Warrego, and Mitchell River; and Kowanyama and Belmont viruses. Complement fixing antibodies were found for Reoviridae viruses bluetongue type 1 and Ibaraki; Picornavirus Nodamura and Rhabdovirus bovine ephemeral fever. No antibodies were detected with Reoviridae viruses Corriparta and Eubenangee; Bunyavirus Trubanaman; and Alfavirus Chikungunya.     

日本乙誓脑炎病毒NS1基因及其疫苗的研究进展

日本乙型脑炎是由日本乙型脑炎病毒引起的严重的人畜共患传染病。乙型脑炎病毒的三种糖基化蛋白PrM、E和NS1是其主要免疫保护性抗原,其中PrM和E蛋白是日本乙型脑炎病毒的结构蛋白,其结构和相关疫苗研究的报道较多。NS1蛋白是日本乙型脑炎病毒的非结构蛋白,其主要参与病毒复制的早期阶段,推测可能参与病毒组装和释放。可诱导补体依赖性溶细胞反应,不能产生中和抗体,能诱发机体在非中和性抗体存在的情况下产生保护性免疫。在接种乙脑病毒的细胞的细胞内、细胞表面及上清液中均含有大量的NS1蛋白。对小鼠接种NS1蛋白或NS1蛋白特异性抗体,均能让小鼠产生对乙脑病毒感染的保护性免疫作用。本文主要就乙型脑炎病毒非结构基因NS1及其免疫疫苗的研究进展进行综述,为其更进一步深入研究IEV NS1基因及其相关疫苗奠定基础。     

Prevalence and risk factors of Japanese encephalitis virus (JEV) in livestock and companion animal in high-risk areas in Malaysia

Japanese encephalitis (JE) is vector-borne zoonotic disease which causes encephalitis in humans and horses. Clinical signs for Japanese encephalitis virus (JEV) infection are not clearly evident in the majority of affected animals. In Malaysia, information on the prevalence of JEV infection has not been established. Thus, a cross-sectional study was conducted during two periods, December 2015 to January 2016 and March to August in 2016, to determine the prevalence and risk factors in JEV infections among animals and birds in Peninsular Malaysia. Serum samples were harvested from the 416 samples which were collected from the dogs, cats, water birds, village chicken, jungle fowls, long-tailed macaques, domestic pigs, and cattle in the states of Selangor, Perak, Perlis, Kelantan, and Pahang. The serum samples were screened for JEV antibodies by commercial IgG ELISA kits. A questionnaire was also distributed to obtain information on the animals, birds, and the environmental factors of sampling areas. The results showed that dogs had the highest seropositive rate of 80% (95% CI: ±?11.69) followed by pigs at 44.4% (95% CI: ±?1.715), cattle at 32.2% (95% CI: ±?1.058), birds at 28.9% (95% CI: ±?5.757), cats at 15.6% (95% CI: ±?7.38), and monkeys at 14.3% (95% CI: ±?1.882). The study also showed that JEV seropositivity was high in young animals and in areas where mosquito vectors and migrating birds were prevalent.     

流行性乙型脑炎病毒E蛋白的截短表达与间接ELISA诊断方法的建立

为建立一种快速检测流行性乙型脑炎病毒抗体的方法,本研究参照已发表的JEV基因组序列,应用RT-PCR扩增了长约1000bp的E基因片段,连接pET30a表达载体中,经诱导后获得了以包涵体形式表达的重组E蛋白。重组蛋白纯化后,经免疫印迹检测证明其具有良好的抗原性和特异性。以该蛋白作为诊断抗原,建立了检测流行性乙型脑炎病毒抗体的E-ELISA诊断方法。该诊断方法具有良好的敏感性、特异性和重复性,为JEV的快速诊断、免疫猪群抗体监测和JEV流行病学调查提供了一种快速、简便的血清学诊断方法。     

猪乙型脑炎病毒在Vero细胞上的繁殖特性及其灭活疫苗的免疫原性研究

试验旨在对猪乙型脑炎病毒（JEV,SA14-14-2株）在传代细胞上的繁殖培养特性及其制备的灭活疫苗免疫原性进行研究,确定猪JEV在细胞培养瓶中培养的关键技术参数及其灭活疫苗的免疫原性。以Vero细胞培养病毒,通过接种时间、接毒量、吸附时间、吸附温度、维持液pH、维持培养温度及培养时间7个条件的优化,将繁殖的病毒液冻融一次,采用病毒蚀斑数测定方法测定病毒滴度。按照优化好的条件繁殖一批毒液,经β-丙内酯灭活,与双相佐剂混合,制备成猪乙型脑炎灭活疫苗。两次免疫（间隔14 d）接种乙型脑炎抗体阴性仔猪,首次免疫前（0 d）、免疫后第7、14、21、28、35、42天采集血清,检测血清中和抗体。二免后第28天进行乙型脑炎P3强毒的攻击,攻毒前（0 d）、攻毒后第1、2、3、5、7、9天采集血浆,攻毒后第14天剖杀免疫猪,采集脑组织,检测血浆和脑组织中JEV。结果显示,用细胞培养瓶进行培养,将Vero细胞培养至48 h进行病毒接种,接种量为1 000 PFU/mL,病毒吸附温度为37℃,吸附时间为90 min,吸附后用pH 7.6~8.8维持液继续培养,培养温度为35℃,培养96 h后收获毒液,冻融一次,可获得较高滴度的病毒。仔猪免疫制备的灭活疫苗后血清抗体水平迅速升高,血浆和脑组织中均未检测出JEV,免疫组试验猪能抵抗强毒攻击,可获得有效免疫保护。本试验结果为猪乙型脑炎疫苗的生产提供了参考依据。     

5种脑炎人兽共患病病毒多重RT-PCR检测方法的建立

为建立同时检测流行性乙型脑炎病毒(JEV)、森林脑炎病毒(TBEV)、东方马脑炎病毒(EEEV)、西方马脑炎病毒(WEEV)和基孔肯雅病毒(CHIKV)5种人兽共患脑炎病病毒的多重RT-PCR方法,本研究根据GenBank登录的相关病毒基因序列设计特异引物,通过优化引物组合及PCR反应条件,建立可同时检测5种病毒的方法,扩增片段长度分别为411 bp(JEV)、945 bp(TBEV)、193 bp(EEEV)、545 bp(WEEV)和769 bp(CHIKV);该方法具有良好的特异性,对病毒核酸最低检测拷贝数分别为7.1×103、3.6×103、2.2×103、5.6×103和5.1×103.该方法具有特异性强、灵敏度高、操作简便等优点,为以上5种人兽共患脑炎病病毒提供快速检测手段.     

基因Ⅰ型乙型脑炎病毒病毒样颗粒的制备及其对小鼠的免疫原性分析

降低猪场的流行性乙型脑炎（Japanese encephalitis，JE）感染率对于阻断人的JE暴发至关重要，鉴于我国JE防控形势的紧迫性和现有基因Ⅲ型商品化乙型脑炎病毒疫苗对部分基因Ⅰ型乙型脑炎病毒（genotype Ⅰ Japanese encephalitis virus，GⅠ JEV）流行毒株免疫保护的不确定性，急需研制安全高效、成本低廉的新型猪用GI JEV疫苗。本研究在杆状病毒载体pFastBac^TM多克隆酶切位点插入GⅠ JEV的prME基因表达盒，测序验证后的阳性重组质粒转染DH10Bac^TM感受态细胞后获得Bacmid-prME重组杆粒，进一步将其转染至Sf9昆虫细胞后，获得Bac-prME重组杆状病毒。Western blot、间接免疫荧光及电镜观察GⅠ JEV病毒样颗粒组成蛋白表达及病毒样颗粒形成情况，通过小鼠免疫试验初步评价GⅠ JEV病毒样颗粒的免疫原性。Sf9昆虫细胞中prME基因表达盒编码的蛋白得到高效表达，且组装形成病毒样颗粒。病毒样颗粒免疫原性评估结果表明：GⅠ JEV病毒样颗粒可诱导免疫小鼠产生高滴度的乙型脑炎病毒中和抗体。本研究利用杆状病毒昆虫细胞表达系统获得了GⅠ JEV病毒样颗粒，为研制安全高效的乙型脑炎病毒病毒样颗粒亚单位疫苗提供物质基础。     

引起猪繁殖障碍综合征的六种传染病的血清学监测

作者采用试剂盒7套，监测了六种能引起猪繁殖障碍综合征的传染病，对来自有繁殖障碍症状的182场次进行猪瘟（HC）抗原检测，并对包括这182场次在内的335个猪场病例的血清样品1790份及田间血清样品28950份进行HC、猪繁殖与呼吸综合征（PRRS）、伪狂犬病（PR）、日本乙型脑炎（JE）、细小病毒感染（PPV）、布鲁氏菌病（Bruc）等六种传染病的抗体监测。结果表明，在有繁殖障碍症状猪场，HC抗原检出率高达69．8％，并且在不使用疫苗的情况下，PRRS、PR、JE和PPV的抗体阳性均分别为46．9％、33％、10．5％和52．6％。不使用疫苗时，血清样品的PRRS和PR的抗体阳性率也高。PPV较为普遍，它与JE也不容忽视。HC与PRRS、PR、JE和PPV中的一种或几种混合感染可能是引起繁殪障碍造成严重损失的主要原因。Bruc，在湖南省的猪群中已得到控制。要控制猪繁殖障碍综合征，必须注重综合防制，优化免疫程度、把握引种关、加强生物安全措施是防制的关键。     

猪乙型脑炎疫苗的研究现状与发展方向

猪乙型脑炎是一种嗜神经性虫媒病毒引起的人兽共患传染病。疫苗的免疫接种是控制猪乙型脑炎发生的主要手段。阐述了猪乙型脑炎常规疫苗和新型疫苗的研究进展,为进一步研究猪乙型脑炎疫苗提供参考。