禽呼肠病毒套式RT-PCR检测方法的建立

根据GenBank中登录的禽呼肠病毒(ARV)基因组序列,设计合成了2对引物,外部引物的扩增片段大小为478 bp,内部引物的扩增片段大小为247 bp,建立了适合ARV快速检测的套式RT-PCR方法(RT-nested-PCR).采用该方法对REV毒株进行了检测,均能扩增到247 bp的条带,而禽流感病毒(H9亚型)...     

乙型脑炎病毒套式RT-PCR检测方法的建立及应用

为了建立一种快速的乙型脑炎病毒(JEV)病原检测方法,根据GenBank上登录的JEV基因组序列,设计合成内外2对引物,优化PCR反应条件,建立了检测JEV的套式RT-PCR方法。结果:该方法对猪繁殖与呼吸综合征病毒、猪瘟病毒、猪细小病毒、猪圆环病毒2型、猪伪狂犬病毒的扩增结果均为阴性;该方法第1次扩增的敏感性是100 pg,第2次扩增的敏感性是1 pg,第2次比第1次扩增的敏感性高100倍。研究表明建立的套式RT-PCR方法具有良好的特异性、敏感性,可以准确快速检测出极低含量的JEV,将为JEV感染的临床诊断、病料检测和分子流行病学调查等提供一种高效、快速、特异、灵敏的检测方法。     

H6亚型禽流感病毒套式 RT-PCR 检测方法的建立

为建立一种 H 6亚型禽流感病毒（AIV）的套式 RT-PCR检测方法,根据 GenBank 中 H 6亚型AIV HA 基因序列,设计了4条特异性引物,优化反应条件,并对所建立的方法进行特异性和敏感性的检验,用该法对154份活禽市场样品进行检测。结果表明,所建立的套式 RT-PCR 方法对其他常见禽病病原体无扩增;对 H6亚型 AIV 的最小检测限为1×102拷贝／μL,灵敏度比常规 RT-PCR 方法高100倍;154份样品的检测结果与病毒分离一致。所建立的 H6亚型 AIV 套式 RT-PCR 检测方法具有特异性强和灵敏度高的特点。     

施马伦贝格病毒套式RT-PCR检测方法的建立

根据GenBank公布的施马伦贝格病毒S基因序列,设计特异性引物,构建施马伦贝格病毒S基因重组克隆质粒作为阳性对照,经各反应条件的优化以及特异性、敏感性和重复性试验,建立了施马伦贝格病毒套式RT-PCR检测方法。结果表明,本研究建立的套式RT-PCR方法可特异性的检测施马伦贝格病毒,且与BTV、EHDV、AKV、BVDV、IBRV等其他病毒的核酸不发生交叉反应。每个反应可检测到相当于6.65×102 copies/μL施马伦贝格病毒重组克隆质粒。与传统的病毒分离及血清学方法相比较,不但耗时短(仅需5h),而且费用低廉。本研究建立的套式RT-PCR方法具有特异、敏感、重复性好、快速、费用低廉等优点,是施马伦贝格病毒快速初筛的良好方法。     

赤羽病病毒套式RT-PCR检测方法的建立与初步应用

根据GenBank中已发表的赤羽病病毒(AKAV)的S基因序列,设计了3条特异性引物,建立了检测AKAV的套式RT-PCR方法。特异性试验表明,该方法可以特异扩增出AKAV的S基因片段,但从牛病毒性腹泻病毒(BVDV)、传染性牛鼻气管炎病毒(IBRV)等13种对照病毒中均不能扩增出目的条带。敏感性试验表明,套式RT-PCR能够扩增10-5稀释度的病毒RNA(核酸含量约1.18 ng/μL),比普通RT-PCR高出1 000倍。用此方法检测130份奶牛血清样品与1份流产胎儿病料,均未检测到阳性样本,但AKAV在血清模拟样品中可被有效检出。研究结果表明,该方法灵敏、特异,为AKAV的检测提供了一个快速、有效的手段。     

猪环曲病毒套式RT-PCR检测方法的建立及应用

根据GenBank中猪环曲病毒N基因设计合成2对特异性扩增引物,成功建立了猪环曲病毒的套式RT-PCR快速检测方法。为验证本方法的特异性,以猪伪狂犬病病毒、猪传染性胃肠炎病毒、猪流行性腹泻病毒、A群猪轮状病毒、猪瘟病毒、猪嵴病毒、沙门菌及大肠杆菌作为阴性对照,结果显示只有在猪环曲病毒的核酸作为模板时才能扩增出预期的258bp和198bp 2条片段。敏感性试验显示,该方法能检测到的最低核酸含量为1pg。结果表明,建立的RT-PCR方法特异性强、灵敏度高,可用于猪环曲病毒的临床检测和流行病学调查。利用本方法对采集自四川省部分地区的50份临床样品进行检测,猪环曲病毒阳性率为30%。     

H4亚型禽流感病毒套式RT-PCR检测方法的建立

旨在建立一种快速、准确检测H4亚型禽流感病毒(AIV)的方法,针对H4亚型AIV HA基因保守序列,设计并筛选出2对特异性引物,通过优化反应条件,建立了H4亚型AIV套式RT-PCR检测方法。特异性试验结果显示,该法可特异性检测出H4亚型AIV,对其他常见禽病病原体无交叉;敏感性试验结果显示,该法对H4亚型AIV检测下限为360fg/μL;用该法对106份临床样品进行检测,临床样品检测结果与病毒分离结果一致。本研究建立的套式RT-PCR方法为H4亚型AIV的早期诊断及有效防控提供了快速、特异和敏感的检测方法。     

番鸭呼肠孤病毒套式RT-PCR检测方法的建立及应用

根据GenBank上发表的鸭呼肠孤病毒基因组序列,利用生物学软件设计合成内外2对引物,建立了检测番鸭呼肠孤病毒(Muscovy duck reovirus,MDRV)的套式RT-PCR检测方法,并运用建立的检测方法对分离病毒与其他禽病病毒进行检测。结果显示,该方法能从MDRV中扩增到与预期大小相符的特异性目的片段,检测灵敏度达到0.1 pg病毒RNA,对禽呼肠孤病毒(avian reovirus,ARV)、鸡传染性法氏囊病病毒(infectious bursal disease virus,IBDV)、番鸭细小病毒(Muscovy duck parvovirus,MDPV)、鹅细小病毒(goose parvovirus,GPV)、鸭病毒性肝炎病毒(duck hepatitis virus,DHV)等病毒样品的扩增结果均为阴性。因此,本研究为番鸭呼肠孤病毒病的快速检测及诊断研究提供参考。     

牛病毒性腹泻病毒套式RT-PCR检测方法的建立及初步应用

根据GenBank公布的牛病毒性腹泻病毒(BVDV)全基因组序列,选择保守性比较高的5'非编码区,利用Oli-go6.0软件设计了2对特异性引物,建立了检测BVDV的套式RT-PCR方法。结果表明,该方法重复性好、特异性强、敏感性高,可以准确快速检测出极低含量的BVDV,极大地提高了常规RT-PCR检测方法的特异性和敏感性。临床初步应用表明,该方法可用于动物与疫苗生物制品原辅料BVDV的快速低含量检测。     

日本脑炎病毒套式RT-PCR方法的建立与初步应用

根据GenBank上登录的日本脑炎病毒(JEV)E基因序列,设计内外2对引物.以JEV疫苗株为模板,建立了检测猪JEV的套式RT-PCR方法.应用该方法对JEV疫苗株RNA进行扩增,获得与预期大小相符,长度为228 bp的目的片段;检出JEV-RNA的灵敏度约为0.3 pg.表明所建立的套式RT-PCR方法对JEV的检测敏感性高、特异性强.     

西尼罗河病毒病的研究进展

西尼罗河病毒病是由西尼罗河病毒(West Nile Virus,WNV)引起的一种人畜共患传染病,病原是一种虫媒病毒,可导致西尼罗河热和致死性的西尼罗河脑炎。自发现以来,西尼罗河病毒病曾在世界上多个国家暴发,给畜牧业和人类生命安全造成了巨大的危害。我国目前尚无该病的发生,但是防治技术储备是必要的。本文对西尼罗河病毒病的流行病学、病原学、致病机制、临床症状和病理剖检变化、诊断方法和候选疗法进行综述,为西尼罗河病毒病的防控提供资料。     

Serologic Responses of West Nile Virus Seronegative Mature Horses to West Nile Virus Vaccines

A 42-day study was conducted to assess the impact of three West Nile virus vaccines given either as separate injections or incorporated with their counterpart equine encephalitis and tetanus vaccines on serological responses under field use conditions. Two hundred forty mature, West Nile virus seronegative (<4) horses were followed serologically pre- and postprimary and secondary vaccination with six different vaccination programs, all including West Nile virus antigens. Forty horses were unvaccinated sentinel horses. All vaccines stimulated both a primary and secondary (booster) response to vaccination that was significantly higher than that of seronegative controls. However, inclusion of West Nile virus with equine encephalitis viruses and tetanus toxoid in vaccines had a significant detrimental impact on West Nile virus serum neutralization antibody production to both the primary and secondary vaccinations.     

新疆地区蚊类携带西尼罗病毒情况调查研究

为查明新疆地区蚊体内携带西尼罗病毒的情况,从2005年-2007年每年的7月~10月采集新疆不同地区的蚊子共计16000余只,并提取总RNA,通过RT-nest PCR的方法检测样品中西尼罗病毒。结果表明,调查样品中没有检测到西尼罗病毒,但不能就此推测新疆是否存在西尼罗病毒,至少对新疆地区和全国制定针对不同动物和人的积极防御政策有重要的公共卫生学意义。     

West Nile virus infection in horses,Indian ocean

The circulation of West Nile virus (WNV) in horses was investigated in the Southwest Indian ocean. In 2010, blood samples were collected from a total of 303 horses originating from Madagascar, Mauritius, Reunion and the Seychelles and tested for WNV-specific antibodies. An overall seroprevalence of 27.39% was detected in the Indian Ocean with the highest WNV antibody prevalence of 46.22% (95% CI: [37.4–55.2%]) in Madagascar. The age and origin of the horses were found to be associated with the WNV infection risk. This paper presents the first seroprevalence study investigating WN fever in horses in the Southwest Indian Ocean area and indicates a potential risk of infection for humans and animals. In order to gain a better understanding of WN transmission cycles, WNV surveillance needs to be implemented in each of the countries.     

West Nile virus: North American experience

West Nile virus, a mosquito‐vectored flavivirus of the Japanese encephalitis serogroup, was first detected in North America following an epizootic in the New York City area in 1999. In the intervening 11 years since the arrival of the virus in North America, it has crossed the contiguous USA, entered the Canadian provinces bordering the USA, and has been reported in the Caribbean islands, Mexico, Central America and, more recently, South America. West Nile virus has been reported in over 300 species of birds in the USA and has caused the deaths of thousands of birds, local population declines of some avian species, the clinical illness and deaths of thousands of domestic horses, and the clinical disease in over 30 000 Americans and the deaths of over 1000. Prior to the emergence of West Nile virus in North America, St. Louis encephalitis virus and Dengue virus were the only other known mosquito‐transmitted flaviviruses in North America capable of causing human disease. This review will discuss the North American experience with mosquito‐borne flavivirus prior to the arrival of West Nile virus, the entry and spread of West Nile virus in North America, effects on wild bird populations, genetic changes in the virus, and the current state of West Nile virus transmission.     

Equine encephalomyelitis outbreak caused by a genetic lineage 2 West Nile virus in Hungary

Background: The spread of lineage 2 West Nile virus (WNV) from sub‐Saharan regions to Europe and the unpredictable change in pathogenicity indicate a potential public and veterinary health threat and requires scientific awareness. Objectives: To describe the results of clinical and virological investigations of the 1st outbreak of a genetic lineage 2 WNV encephalomyelitis in horses. Animals: Seventeen horses with neurologic signs. Methods: Information regarding signalment, clinical signs, and outcome was obtained for each animal. Serology was performed in 15 cases, clinicopathological examination in 7 cases, and cerebrospinal fluid was collected from 2 horses. Histopathology was carried out in 4 horses, 2 of which were assessed for the presence of WNV in their nervous system. Results: WNV neutralizing antibody titers were between 10 and 270 (median, 90) and the results of other serological assays were in agreement with those of the plaque reduction neutralization test. Common signs included ataxia, weakness, asymmetric gait, muscle tremors, hypersensitivity, cranial nerve deficits, and recumbency. Twelve animals survived. Amplicons derived from the infection‐positive specimens allowed molecular characterization of the viral strain. Conclusions and Clinical Importance: From our results, we conclude that this outbreak was caused by a lineage 2 WNV strain, even though such strains often are considered nonpathogenic. Neurological signs and survival rates were similar to those reported for lineage 1 virus infections. The disease occurrence was not geographically limited as had been the typical case during European outbreaks; this report describes a substantial northwestern spread of the pathogen.     

West Nile virus and its emergence in the United States of America

Zoonotic West Nile virus (WNV) circulates in natural transmission cycles involving certain mosquitoes and birds, horses, humans, and a range of other vertebrates are incidental hosts. Clinical infections in humans can range in severity from uncomplicated WNV fever to fatal meningoencephalitis. Since its introduction to the Western Hemisphere in 1999, WNV had spread across North America, Central and South America and the Caribbean, although the vast majority of severe human cases have occurred in the United States of America (USA) and Canada. By 2002-2003, the WNV outbreaks have involved thousands of patients causing severe neurologic disease (meningoencephalitis and poliomyelitis-like syndrome) and hundreds of associated fatalities in USA. The purpose of this review is to present recent information on the epidemiology and pathogenicity of WNV since its emergence in North America.     

西尼罗病毒的基因进化分析

为更好的了解西尼罗病毒在世界各地的流行特征,从GenBank中随机选取45株西尼罗病毒的全基因或部分基因序列,进行同源性分析构建了基因进化树.发现西尼罗病毒可以分为两个谱系,1系病毒广泛分布在非洲西部、中东、东欧、印度、美国和澳大利亚等地,2系病毒分布在非洲亚撒哈拉、马达加斯加等地.1系的西尼罗病毒可进一步分为3个分支.对其中的22株西尼罗病毒部分基因序列进行同源性比较,发现1系病毒之间核苷酸同源在76.8%以上,氨基酸同源性在78.3%以上,而2系病毒与1系病毒核苷酸同源性约为64.7%～76.2%,氨基酸同源性约为64.7%～93.0%.