貉源犬瘟热病毒的分离鉴定

采集大庆某貉养殖场病貉的病料,处理后接种于非洲绿猴肾细胞(Vero)进行病毒分离。对分离毒株进行了中和试验、血凝试验、理化性质的鉴定,并用反转录-聚合酶链式反应(RT-PCR)检测感染细胞中的病毒核酸。病料接种 Vero 细胞 72 h 后产生明显的细胞病变(CPE);病毒分离株可以被犬瘟热病毒阳性血清中和,中和效价为 1∶25;分离株对氯仿、乙醚敏感,对酸和热抵抗力弱,可以凝集鸡红细胞,其凝集作用可被犬瘟热病毒阳性血清所抑制; RT-PCR检测病毒细胞培养液,扩增出的片段长287 bp,与预期设计的长度相同,经测序发现与MS01株的同源性为99%。结果表明,分离的病毒株为犬瘟热病毒,命名为CDV-DQ株。     

犬瘟热病毒山东株的分离及N蛋白基因的克隆与序列分析

本试验采用Vero细胞从临床疑似患犬瘟热的犬组织病料中分离出一株病毒,通过观察细胞病变,提取细胞培养物RNA并扩增克隆分析N蛋白基因部分序列进行了鉴定。结果表明,该毒株接种于Vero细胞后,出现了典型的细胞病变;对接种病料后的Vero细胞培养物提取总RNA进行RT-PCR扩增,得到了287 bp的目的片段;序列分析表明该分离株与犬瘟热病毒弱毒(Onderstepoort和Snyder Hill)的同源性为95.5%~96.9%,与标准强毒株(A75/17和2544/Han95)及野毒株(XJ-4、ZJ7、98-2646等)的同源性较高,为97.2%~99.0%,证明该毒株为犬瘟热病毒野毒株,命名为QD10。     

小熊猫犬瘟热病毒感染的诊断

对某动物园发病死亡小熊猫的病料进行电镜检查 ,发现了犬瘟热病毒 ( CDV)样病毒粒子 ;病料用 RT-PCR检测 ,均为 CDV阳性 ,并从部分病料中分离出了 CDV,说明小熊猫存在 CDV感染。用从小熊猫肝脏病料中分离的 CDV LP株接于幼犬 ,结果所有接种犬均发生了犬瘟热。中和试验结果表明 ,LP株是一免疫原性强的 CDV株。     

犬瘟热病的临床诊断及其病原的分离与鉴定

为了对疑似犬瘟热病例进行临床诊断与确诊,试验应用犬瘟热病毒(CDV)胶体金抗原检测试纸检测板进行检测,对死亡犬进行病理剖检,取肝脏、脾脏、肺脏、胃肠道及淋巴结等病料研磨匀浆,接种Vero细胞,观察细胞病变并应用RT-PCR技术检测感染细胞中病毒基因特异性片段。结果表明:病料接种Vero细胞24 h后出现细胞融合、形成空斑、细胞变圆及脱落等细胞病变(CPE);经RTPCR检测,扩增出病毒基因特异性片段,片段大小为335 bp。说明Vero细胞对CDV表现出良好的敏感性,是体外分离培养CDV的一个有效细胞系。     

致病性犬瘟热病毒CDV-JT1株的分离与鉴定

应用MDCK细胞从吉林某地犬瘟热(Canine distemper,CD)疑似发病犬肺脏组织中分离出1株病毒,该分离毒株经MDCK细胞传至第6代时出现典型的合胞体细胞病变(CPE)。经病毒形态观察、理化特性鉴定、RT-PCR和直接免疫荧光鉴定可知该分离毒株为犬瘟热病毒(CDV),命名为CDV-JT1;动物试验表明,试验犬接种CDV-JT1后21 d内都出现典型的犬瘟热症状;H基因序列分析表明,CDV-JT1株的H基因与中国分离株CDTaiChung株、TN株、SHLJ(07)1株、日本Hamamatsu株、Ueno株、Yanaka株和KDK-1株在基因型上同属于Asia-1型。CDV-JT1株含有包括CDV野毒株特有的309~311位在内的8个潜在的N-连接天冬酰氨糖基化位点。CDV-JT1强毒株的分离成功,对进一步开展疫苗研发、流行病学调查以及致病机理等方面的研究具有重要意义。     

I型犬腺病毒黑龙江株(CAV-H)的分离、鉴定及致弱的研究

为研究犬腺病毒(CAV)致弱疫苗的免疫原性,本研究将病犬的组织样品接种于MDCK细胞内,进行病毒分离及鉴定。鉴定结果显示,分离的病毒在MDCK细胞内产生明显的细胞病变(CPE),电镜下可见典型的腺病毒粒子;回归动物试验显示,该分离株可导致犬出现明显的CAV感染症状;PCR鉴定结果表明分离的病毒为CAV,命名为CAV-H株;将CAV-H株接种MDCK细胞连续传代至110代,在传代过程中,病毒滴度由初始105.0 TCID50/0.1 mL上升至107.0 TCID50/0.1 mL,而CAV对犬的致病力逐渐降低;免疫效力试验结果表明,CAV-H可对同源强毒攻击的犬提供完全的免疫保护作用,可作为理想的疫苗候选毒株。     

犬瘟热病毒强毒株的分离与鉴定

选取来自黑龙江省病犬的肺脏、胰脏、肝脏、脾脏、肾、膀胱、淋巴结等组织病料,研磨上清接种非洲绿猴肾细胞（Vero）进行病毒的分离。对分离毒株进行了形态学、血清学、回归动物试验鉴定,并用反转录-聚合酶链式反应（RT-PCR）检测感染细胞中的病毒核酸。分离得到的病毒在Vero细胞上可产生明显的细胞病变（CPE）,病毒形态学、血清学、回归动物试验、RT-PCR鉴定均为阳性,确定所分离的病毒为犬瘟热病毒,命名为CDV-HLJ株。     

一株I型犬腺病毒的分离与鉴定

为给I型犬腺病毒(CAV-I)后续研究提供基础材料,采集疑似犬传染性肝炎患犬粪便,用PCR方法进行CAV-I核酸检测,并运用犬肾传代细胞(MDCK)进行病毒分离培养,采用PCR和免疫过氧化物酶单层细胞染色法(IPMA)检测病毒在细胞内的繁殖状态。结果显示:患犬粪便样品PCR检测为CAV-I核酸阳性,病料接种MDCK细胞培养24 h后出现明显的"葡萄串样"细胞病变效应(CPE);培养物血清学鉴定为CAV抗原阳性,不同代次病毒培养物均为CAV-I核酸强阳性;第9代病毒核酸序列与Gen Bank中CAV-I毒株核苷酸相似性在99.0%以上,与CAV-Ⅱ相似性仅为65.3%;该病毒与CAV-I毒株处于同一遗传分支;病毒接种MDCK后8 h内均无抗原检出,12 h后细胞核中分布大量的CAV抗原,24 h后细胞出现典型的"葡萄串样"病变,抗原在细胞中呈弥散分布。上述结果表明,分离到的毒株为CAV-I,命名为CAV-ZY180711。     

水貂犬瘟热病毒LD-1株的分离与鉴定

取山东某貂场疑似犬瘟热病毒（canine distemper virus,CDV）感染的水貂肝脏等组织病料,通过RT-PCR检测呈CDV阳性,且无水貂细小病毒存在,将病料接种原代CEF细胞、传代系Vero细胞和DF1细胞3种细胞进行病毒分离,通过优化细胞培养条件,最终在Vero细胞上传代培养成功,出现露珠状典型细胞病变（CPE）。分离毒应用RT-PCR、PCR产物测序、抗体中和试验（SN）、间接免疫荧光试验（IFA）及病毒包涵体检查等多种方法进行了CDV的鉴定,结果显示CDV阳性,表明分离到的病毒为水貂CDV,并将其命名为CDV LD-1株。     

鉴别犬瘟热病毒强弱毒株SYBR Green Ⅰ荧光定量RT-PCR检测方法的建立

为建立犬瘟热强弱毒株SYBR Green Ⅰ荧光定量RT-PCR检测方法,根据Gen Bank公布的犬瘟热毒株的全基因序列并参考相关文献,选择M基因保守区域合成了1对通用引物M1/M4及鉴别犬瘟热强弱毒株的特异引物M2和M3。试验证明,该方法重复性良好;检测强弱毒株的最低检出限度在10拷贝;对新城疫病毒、水貂肠炎病毒、阿留申病毒、犬腺病毒、犬细小病毒5种病毒的特异性检测均为阴性;对175份犬瘟热疑似病料进行荧光定量RT-PCR,检出127份阳性样品,其中强毒株98份,弱毒疫苗株29份,常规RT-PCR检出112份阳性样品。结果表明,该方法的敏感性高于常规RT-PCR检测,并能有效的鉴别强弱毒株。     

犬瘟热病毒在不同组织细胞上增殖特性的研究

将犬瘟热病毒(CDV)分别接种于长满单层的鸡胚成纤维细胞(CEF)、非洲绿猴肾细胞系(Ve-ro)、犬肾细胞系(MDCK),观察其病变时间、病变特征,并对其进行RT-PCR鉴定和TCID50测定。结果表明,CDV在这3种细胞上均能增殖,并产生明显的细胞病变(CPE),但在Vero细胞上产生病变的时间短,病变明显,且TCID50最高;RT-PCR检测CDV在不同细胞上的病毒培养液,均能扩增出337bp的片段;CDV在Vero细胞上的增殖滴度明显高于在CEF、MDCK细胞上的增殖滴度。表明本试验所选用的3种细胞中,Vero细胞最适宜培养CDV,所获得的CDV的毒价最高,病变最明显。     

犬瘟热病毒强毒株HBF-1株的培育及致病性研究

为研究从北极狐病料样品中分离的一株强毒的致病性,本实验采用病例复制、RT-PCR检测、间接免疫荧光检测(IFA)和电镜观察等方法证实分离得到犬瘟热病毒(CDV),并命名为HBF-1。对该分离株H基因的核苷酸序列比对显示,HBF-1与疫苗株的同源性为91.0%～91.5%,与国内外分离株的同源性为93.5%～99.9%。病毒传代培育试验结果显示HBF-1已适应在北极狐、貉、水貂和犬体内繁殖,具有较广的感染范围。但各种动物的临床症状和剖检病理变化存在不同程度的差异,表明HBF-1分离株对北极狐、貉、水貂和犬的致病力不同;毒力测定结果显示其半数感染量分别为102.46 ID50/mL、102.95 ID50/mL、102.46 ID50/mL和102.58 ID50/mL,表明HBF-1为一株CDV强毒株,可以在不同的经济动物间进行水平传播。本研究结果为开发新的CDV疫苗提供了实验基础。     

Pathogenesis of two strains of lion (Panthera leo) morbillivirus in ferrets (Mustela putorius furo)

Canine distemper virus (CDV) was previously considered to have a host range restricted to the canid family. In 1994, the virus was associated with sporadic outbreaks of distemper in captive felids. However, after severe mortality occurred in the Serengeti lions (Panthera leo), attention became focused on the pathogenesis of the virus and a concerted effort was made to identify the virus as CDV or a closely related feline morbillivirus. The present study was designed to explore the susceptibility of ferrets to challenge with two morbilliviruses isolated from lions and the protective effects of a modified-live mink distemper vaccine. Because mortality in ferrets infected with pathogenic CDV approaches 100%, the ferret was selected as a test animal. Two strains of lion morbillivirus were used as a challenge, A92-27/20 (California lion isolate) and A94-11/13 (Serengeti lion isolate). The two strains of lion morbillivirus were antigenically related to CDV (Rockborn strain), and ferrets were susceptible to both of the viruses when inoculated intraperitoneally. The inoculated ferrets were anorectic at 5-6 days postinoculation (PI), exhibited oculonasal discharge at 9-12 days PI, and became moribund at 12-22 days PI. Severe bilateral conjunctivitis was the typical clinical sign. Inclusion bodies characteristic of morbillivirus (eosinophilic, intranuclear, and intracytoplasmic) were distributed in many epithelial cells, including those of the skin, conjunctiva, gallbladder, liver, pancreas, stomach, trachea, lung, urinary bladder, and kidney. Virus was reisolated from selected lung tissues collected at necropsy and identified by CDV-specific immunofluorescence. Ferrets vaccinated with the mink distemper vaccine (Onderstepoort strain) were protected from challenge with the two lion strains, adding further support to the premise that the viruses are closely related to CDV.     

Mass mortality in seals caused by a newly discovered morbillivirus

During a recent disease outbreak among harbour seals (Phoca vitulina) in the North and Baltic seas, more than 17,000 animals have died. The clinical symptoms and pathological findings were similar to those of distemper in dogs. Based on a seroepizootiological study, using a canine distemper virus (CDV) neutralization assay, it was shown that CDV or a closely related morbillivirus (phocid distemper virus-PDV) was the primary cause of the disease. The virus was isolated in cell culture from the organs of dead seals and characterized as a morbillivirus by serology (immunofluorescence neutralization and enzyme-linked immunosorbent assays) and by negative contrast electron microscopy. Experimental infection of SPF dogs resulted in the development of mild clinical signs of distemper and CDV-neutralizing antibodies. The disease was reproduced in seals by experimental inoculation of organ material from animals that had died during the outbreak. However, seals that had been vaccinated with experimental inactivated CDV vaccines were protected against this challenge. This fulfilled the last of Koch's postulates, confirming that the morbillivirus isolated from the seal organs, was the primary cause of the disease outbreak. The recent demonstration of the presence of a similar virus in Lake Baikal seals (Phoca sibirica), which infected these Siberian seals 1 year before the northwestern European seals were infected, raises new questions about the origin of this infectious disease in pinnipeds.     

犬瘟热病毒贵州分离株H基因的克隆及其序列分析

根据GenBank中犬瘟热病毒Onderstepoort株序列设计特异性引物,以犬瘟热病毒贵州分离株(CDV-GZ1)RNA为模板,应用RT-PCR对病毒H蛋白编码基因进行了扩增、克隆及序列分析.测序结果表明,CDV-GZ1 H基因ORF由1 824 bp组成,可编码607个氨基酸;与已发表23株其它CDV H基因核苷...     

急性犬瘟热感染组织脏器的研究

临床中犬瘟热多分为急性和亚急性,3月龄犬多发本病,急性病例和亚急性病例的临床症状和病毒复制的脏器与慢性病例有很大区别。临床上急性病例的病变主要集中在肺脏和淋巴组织,组织病理学可观察到淋巴结炎和多核巨细胞性肺炎。根据免疫组织化学染色结果发现,在犬瘟热急性病例的淋巴结皮质中犬瘟热抗原呈强阳性,且密度很高。而病犬虽然表现出严重的肺炎症状,但在犬的肺脏中犬瘟热病毒抗原阳性反应弱,仅在支气管上皮和少量的巨噬细胞胞浆中观察到。犬瘟热病毒首先在犬的淋巴组织中进行复制、增殖,破坏被膜下淋巴窦和淋巴小节中的T淋巴细胞和B淋巴细胞,影响淋巴组织输出到外周血液循环中的淋巴细胞数量。本研究结果表明,犬瘟热虽然临床上以肺脏病变最严重,但在急性和亚急性病例,犬瘟热病毒主要在淋巴组织的T淋巴细胞、B淋巴细胞和巨噬细胞中增殖,故应以淋巴组织作为犬瘟热病毒的主要分离脏器。     

犬瘟热免疫预防研究进展

犬瘟热是由犬瘟热病毒感染引起的急性、高度接触性传染病.病犬通过眼、鼻分泌物、唾液、尿和粪便排出病毒,污染饲料、水源及用具等,经消化道感染其他易感动物.也可通过飞沫经呼吸道传播,偶尔也可见到胎盘感染,引起流产和死胎.康复犬尿液中也长期带毒.一旦发生CD,除早期应用抗血清及对症疗法有效外,中晚期病例多预后不良,因此,必须依靠免疫预防控制该病的发生.文章对其流行病学、发病机理、机体免疫和免疫程序进行了综述,特别是对犬瘟热的免疫类型、疫苗种类以及免疫程序进行了详细阐述.