一株猪伪狂犬病病毒的分离鉴定

为了确诊山东省利津县某猪场发生的疫情是否由猪伪狂犬病病毒(PRV)感染所致,试验利用BHK-21传代细胞对疑似猪伪狂犬病死亡猪进行病毒分离,并通过PCR方法、间接免疫荧光试验及动物试验对分离病毒进行鉴定。结果表明:接种病料后48小时BHK-21细胞出现明显细胞病变,PRV荧光抗体阳性、PCR扩增片段大小为220 bp,测序结果与NCBI中伪狂犬病病毒基因组同源性为96%~98%,接种家兔后表现出明显的伪狂犬病症状,病毒毒价达到1×107.25TCID50/0.1 m L。说明该猪场的疫情由猪伪狂犬病病毒感染所致。     

猪伪狂犬病病毒LX株的分离与鉴定

从辽宁某猪场采集病料,经处理后接种BHK-21细胞进行猪伪狂犬病病毒(pseudorabies virus,PRV)分离,结果有2份病料在该细胞上盲传后可见典型的CPE,经测定其毒价为10^-7.8 TCID₅₀/mL。间接免疫荧光试验可见黄绿色荧光,用电镜对纯化的病毒进行观察,可见有囊膜包裹的圆形病毒粒子,直径为140~200 nm。该病毒对氯仿、乙醚、酸和热敏感。对有CPE的细胞培养物进行RT-PCR,结果为阳性。接种试验动物出现典型的猪伪狂犬病临床症状且死亡。结果表明,成功分离到1株PRV。     

一例集约化猪场伪狂犬的诊断及防控

<正>笔者团队开展了一个猪场暴发伪狂犬病（Pseudorabies,PR）后的确诊和防控实践。通过发病情况调查、观察临床症状和剖解组织病变,初步判断某集约化猪场暴发了伪狂犬病。采集2头典型病猪的病料经PCR鉴定为伪狂犬病毒（PRV）阳性,采集10头病猪的血清经PRVg E-ELISA检测为阳性（9/10）,确诊该猪场流行和暴发的为猪伪狂犬病。确诊后对该场猪群采取了隔离、消毒和紧急接种等综合防控措施,快速制止了该猪场PR的流行,取得显著的防控效果。     

一例猪伪狂犬病病毒的分离鉴定及综合防治措施

无菌采集疑似猪伪狂犬病病猪脑组织,进行病毒的分离鉴定。经PCR检测为阳性的病料接种PK-15细胞进行病毒分离,将所分病毒暂命名为SC株。蚀斑实验测定病毒滴度,并采用PCR和琼扩实验对其进行进一步鉴定。结果:PRV阳性病料接种PK-15细胞,传至第5代后产生稳定细胞病变,主要表现为细胞变圆、变亮、聚堆且可见典型的空斑,而对照组细胞贴壁生长良好;基于PRV gE基因的PCR检测,扩增产物为378 bp,与预期目的条带一致;用病毒蚀斑技术测定该分离株的滴度为2.65×104 PFU/mL;琼扩实验显示病毒原液至32倍稀释后均能与PRV阳性血清形成可见沉淀线。以上结果均表明所送检病猪为PRV阳性,据此为猪场提出综合防治措施。     

一例猪伪狂犬病病毒的分离鉴定及综合防治措施

无菌采集疑似猪伪狂犬病病猪脑组织,进行病毒的分离鉴定。经PCR检测为阳性的病料接种PK-15细胞进行病毒分离,将所分病毒暂命名为SC株。蚀斑实验测定病毒滴度,并采用PCR和琼扩实验对其进行进一步鉴定。结果:PRV阳性病料接种PK-15细胞,传至第5代后产生稳定细胞病变,主要表现为细胞变圆、变亮、聚堆且可见典型的空斑,而对照组细胞贴壁生长良好;基于PRV gE基因的PCR检测,扩增产物为378 bp,与预期目的条带一致;用病毒蚀斑技术测定该分离株的滴度为2.65×10~4PFU/mL;琼扩实验显示病毒原液至32倍稀释后均能与PRV阳性血清形成可见沉淀线。以上结果均表明所送检病猪为PRV阳性,据此为猪场提出综合防治措施。     

某规模化猪场伪狂犬病的诊断

河南平顶山某猪场母猪出现较严重的流产和产死胎现象,且50日龄~70日龄仔猪出现神经症状,根据临床表现初步诊断为伪狂犬病。为排除猪繁殖与呼吸综合征和猪瘟,进行了实验室诊断。应用ELISA方法检测发病保育猪及母猪血清的伪狂犬病病毒野毒株gE抗体,并对发病仔猪病料进行了伪狂犬病病毒(PRV)、猪繁殖与呼吸综合征病毒(PRRSV)和猪瘟病毒(CSFV)的实时荧光定量PCR检测。结果显示,伪狂犬病病毒野毒抗体阳性,实时荧光定量PCR检测确定仔猪病料中PRV核酸阳性,PRRSV和CSFV核酸阴性。结合临床症状及实验室检测,确诊该猪场发生的是猪伪狂犬病。     

一例猪伪狂犬病病毒的分离鉴定

从临床疑似猪伪狂犬病发病仔猪的脑组织等病料中,经PCR扩增出大小为217 bp的伪狂犬病病毒gp50的基因片段,结果证实为猪伪狂犬病病毒(porcine pseudorabies virus,PRV)感染。随后采用BHK-21细胞进行猪伪狂犬病病毒的分离培养,该分离株经细胞传代培养5代后,能够产生典型的细胞病变,经PCR鉴定为伪狂犬病病毒,其病毒感染力达10^8.68 TCID₅₀/0.1mL。最后用10^7.0 TCID₅₀/mL病毒培养物接种家兔,48 h后注射部位出现典型瘙痒、皮肤破损等症状,于72 h后全部死亡。结果表明,该伪狂犬病病毒分离株对易感动物具有高致病性,为进一步开展该病毒流行病学、致病机理、疫苗免疫及诊断研究奠定了基础。     

猪伪狂犬病病毒变异毒株HeNZK-2014的分离鉴定及gE基因序列分析

为了解猪伪狂犬病病毒（PRV）变异毒株的特点,本研究采集临床疑似PRV感染发病猪的淋巴结等组织样品进行PCR鉴定,选取仅PRV阳性的组织样品经研磨除菌后取上清接种于PK-15细胞进行病毒分离培养、蚀斑纯化、PCR和间接免疫荧光法（IFA）鉴定,采用Reed-Muench法测定PRV的TCID₅₀,接种小鼠并观察临床症状,对纯化的PRV和死亡小鼠脑组织样品进行gE基因PCR扩增及测序分析。结果显示,PRV阳性病料接种于PK-15细胞24 h后出现典型细胞病变（CPE）,经3轮蚀斑纯化后PCR和IFA鉴定结果均为阳性,分离株命名为HeNZK-2014,其TCID₅₀为10^-9.77/0.1 mL;以1×10⁸个TCID₅₀病毒悬液接种小鼠22 h后可引起小鼠出现奇痒、撕咬、死亡等典型猪伪狂犬病症状,死亡小鼠脑组织样品PRV PCR检测结果为阳性;纯化病毒和死亡小鼠脑组织样品gE基因核苷酸序列同源性为100.0%,与GenBank中2011年以前登录的经典毒株位于不同分支,与2011年之后中国流行毒株位于同一分支,在第48和496位各有1个天冬氨酸（D）的插入,具有变异毒株的典型特征。本研究成功分离了1株PRV变异毒株,为进一步开展针对PRV变异毒株的疫苗及其防控研究奠定了基础。     

一例猪伪狂犬病野毒感染的实验室诊断

为确诊衡阳市某猪场育肥猪发生的疑似猪伪狂犬病(PR)疫情,采集发病猪脾脏,运用PCR方法进行猪伪狂犬病病毒(PRV)核酸检测,利用非洲绿猴肾细胞(Vero)进行病毒分离培养,采用免疫过氧化物酶单层细胞染色法(IPMA)进行病毒血清学鉴定;用分离培养物进行家兔感染试验,观察临床症状和病例剖检变化,采用PCR检测各器官中的病毒分布情况。结果显示:病料接种Vero细胞培养3 d后,出现明显的细胞病变效应(CPE);分离培养的病毒与PRV阳性血清呈阳性反应;家兔感染后出现奇痒等神经症状并死亡,其肝、脾组织均为PRV gE核酸检测阳性。试验结果证实,分离到的病毒为PRV野毒,从而确诊了该疑似疫情。     

西藏林芝地区1例牛伪狂犬病的诊断

主要对西藏林芝地区某牛场不明原因牛猝死进行研究。通过病死牛病料组织触片镜检和分离培养皆未发现细菌;乳胶凝集试验检查血清为伪狂犬病病毒(PRV)抗体阳性;以脑组织制备DNA模板进行PCR,扩增到特异性的PRV DNA片段;对健康成年兔接种病牛脑、肺等病料,表现典型的伪狂犬病症状。综合流行病学、细菌学、血清学、分子生物学和动物试验结果,确诊为牛伪狂犬病。     

Isolation and Identification of Variant HeNZK-2014 of Pseudorabies Virus and Sequence Analysis of gE Gene

In order to learn the situation of pig pseudorabies virus (PRV) variant in this study, tissue samples such as lymph nodes which were collected from clinical pigs with suspected PRV infection were identified by PCR. PRV positive sample were inoculated on PK-15 cells after grinding and degerming, with further experiment including virus isolation, plague purification, PCR and IFA identification, TCID₅₀ confirmed by Reed-Muench method, inoculation test and observation of clinical symptoms in mice. The gE gene of purified PRV and brain tissue samples of dead mice were identified by sequencing analysis. The results showed that the virus grown on PK-15 cells could produce typical cytopathic effect (CPE) after 24 h; After three rounds of plaque purification,the isolate was PRV positive identified by PCR and IFA, and nominated as HeNZK-2014; The TCID₅₀ of the isolate was 10^-9.77/0.1 mL; The virus in 1×10⁸ TCID₅₀ inoculation was able to cause itching, tearing, death in infected mice, and PRV could be detected in tissues of dead mice; The molecular genetic variation analysis of gE gene by PCR amplification and clone sequencing indicated that the gE gene from brain tissue of infected mice shared 100.0% homology with HeNZK-2014, and located in a relatively independent branch with newly pandemic isolates in recent years after 2011, but was far from the classical strains before 2011, and both had two insertion of aspartic acid (D) at sites of 48 and 496 amino acids, which were considered to be the typical characteristics of PRV variants. This study successfully isolated a PRV variant, which laid a foundation for further research on vaccine development, prevention and control against PRV variants.     

猪伪狂犬病病毒HP-SY2022株的分离鉴定

为了解猪伪狂犬病病毒(Pseudorabies virus,PRV)野毒株的特点,本研究对沈阳某养殖场疑似感染PRV的组织病料进行PCR鉴定、病毒分离和纯化、gD、gE基因序列测定及分析、动物回归实验。结果显示：分离株能在ST细胞中产生典型的细胞病变,且PCR显示为PRV阳性;通过gD、gE基因进行序列分析发现,分离株与2011年后分离的PRV变异株位于同一进化分支;氨基酸位点分析发现分离株与PRV变异株具有相同的变异模式。进一步研究其致病性,将纯化后的病毒液接种PRV抗体阴性21日龄健康仔猪,感染仔猪出现典型的PR症状,如呼吸困难、转圈、流涎和划水动作,且在试验期内仔猪全部发病(5/5),其中4头死亡(4/5)。综上,本研究成功分离到一株PRV变异株,将其命名为HP-SY2022。这一研究为丰富我国PRV分子流行病学及后续的免疫防控提供了参考。     

2013-2018年福建省部分规模化猪场猪伪狂犬病病毒野毒感染的流行病学调查

为了解2013-2018年福建省西南地区规模化猪场猪群伪狂犬病病毒(PRV)野毒感染情况,本研究分别采用PCR和ELISA检测方法,对2013-2018年该地区787个规模化猪场送检的1320份疑似发病猪群组织样品、3041场次的猪场67660份猪血清样品进行PRV病原学、血清学检测.结果显示:送检病例猪组织样品PRV...     

利用棉绳采集口腔液监测猪伪狂犬病方法的应用分析

为分析使用棉绳采集口腔液监测猪场伪狂犬病(PR)方法的科学性,本研究拟通过实验室水平、动物试验及临床应用3个方面对使用棉绳采集口腔液监测猪场PR这一方法开展应用分析。首先对采样条件进行优化,并通过模拟试验测定棉绳对伪狂犬病病毒(PRV)的释放能力,通过动物试验测定病毒感染后检出时间,最后对临床样品进行检测分析。结果表明:使用直径为1.0 cm的棉绳,在早上喂料前采集口腔液样品,采样时间为20~30 min,可采集到更能满足试验要求的口腔液。病毒释放能力测定显示,棉绳对PRV的释放能力为50%左右,使用棉绳采集口腔液可检测到1个TCID50/0.1 mL的病毒含量。动物试验检测发现,猪群在感染后28 d中除第5天外,口腔液中病原含量均高于鼻拭子中病原含量,口腔液检测效果优于鼻拭子,且口腔液中病毒的检出时间(感染后第1天)早于血液中抗体转阳时间(感染后第7天)。临床样品检测分析结果表明,PRV疫苗免疫后也可通过口腔液检测到疫苗毒,并且存在无法通过口腔液检测感染PRV野毒后稳定猪中带毒的情况,因此口腔液检测方法应结合gE抗体检测,才可综合判断猪群是否为感染群体。综上,本研究优化了口腔液采集方法,并测定了棉绳对口腔液的释放能力和感染后检出时间,表明口腔液可作为较好的监测猪群PR的手段;口腔液监测方法需结合gE抗体检测来综合判断猪群是否为感染群体。     

利用棉绳采集口腔液监测猪伪狂犬病方法的应用分析

为分析使用棉绳采集口腔液监测猪场伪狂犬病（PR）方法的科学性,本研究拟通过实验室水平、动物试验及临床应用3个方面对使用棉绳采集口腔液监测猪场PR这一方法开展应用分析。首先对采样条件进行优化,并通过模拟试验测定棉绳对伪狂犬病病毒（PRV）的释放能力,通过动物试验测定病毒感染后检出时间,最后对临床样品进行检测分析。结果表明：使用直径为1.0 cm的棉绳,在早上喂料前采集口腔液样品,采样时间为20~30 min,可采集到更能满足试验要求的口腔液。病毒释放能力测定显示,棉绳对PRV的释放能力为50%左右,使用棉绳采集口腔液可检测到1个TCID₅₀/0.1 mL的病毒含量。动物试验检测发现,猪群在感染后28 d中除第5天外,口腔液中病原含量均高于鼻拭子中病原含量,口腔液检测效果优于鼻拭子,且口腔液中病毒的检出时间（感染后第1天）早于血液中抗体转阳时间（感染后第7天）。临床样品检测分析结果表明,PRV疫苗免疫后也可通过口腔液检测到疫苗毒,并且存在无法通过口腔液检测感染PRV野毒后稳定猪中带毒的情况,因此口腔液检测方法应结合gE抗体检测,才可综合判断猪群是否为感染群体。综上,本研究优化了口腔液采集方法,并测定了棉绳对口腔液的释放能力和感染后检出时间,表明口腔液可作为较好的监测猪群PR的手段;口腔液监测方法需结合gE抗体检测来综合判断猪群是否为感染群体。     

Pathogenicity of a modified-live pseudorabies vaccine virus in lambs

Subcutaneous injections of modified-live pseudorabies virus (PRV) vaccine into lambs caused clinical signs and death within 1 week after injection in 4 of 5 inoculated lambs. The clinical signs included depression, high fever, muscle fasciculations and convulsions, occasionally followed by death within 48 hours of the initial clinical signs. Histologic examinations and virus isolation procedures demonstrated PRV in the CNS of infected lambs. Sera from sick lambs remained negative for PRV antibodies. Two subsequent serial passages of the vaccine virus in lambs resulted in similar clinical signs and death in 6 of 10 inoculated lambs. Again, PRV was isolated from tissues of sick lambs, and the histopathologic findings were characteristic of the disease. Affected lambs remained seronegative to PRV, as did lambs that remained clinically normal after inoculation. There was no evidence of PRV transmission to uninoculated lambs and pigs housed with the infected lambs.     

某规模化猪场猪伪狂犬病的诊断

试验采用流行病学调查、临床观察和实验室检查对华北地区某疑似伪狂犬病发病猪场进行了诊断。猪场发病情况主要为母猪流产,新生仔猪死亡率较高,肥猪死亡率低。发病或死亡仔猪扁桃体充血出血、溃疡,肺脏出血、水肿,心肌色淡、心包积液,脑膜充血;表现为坏死性扁桃体炎、出血性淋巴结炎、出血性间质性肺炎、间质性心肌炎和非化脓性脑炎。发病猪扁桃体、肺脏、脑组织匀浆上清液接种家兔后出现奇痒等典型症状并很快死亡。发病猪脑组织可检出猪伪狂犬病毒gD基因特异性目的片段。猪场伪狂犬病野毒感染抗体阳性率为63%（63/100）。本试验结果表明,确诊该猪场发生了伪狂犬病。     

The response of pigs inoculated with a thymidine kinase-negative (TK-) pseudorabies virus to challenge infection with virulent virus

12 Large-White-Landrace piglets were subdivided in four groups of 3 and housed in separate units. The piglets of three groups were inoculated with the 86/27V 6C2 thymidine kinase negative (TK-) mutant of pseudorabies virus (PRV), by different routes. A second inoculation with the same mutant was given to the pigs 21 days later. The animals of a fourth group were left as uninoculated controls. 21 days following the second inoculation with the TK- mutant all pigs were challenge infected with the virulent PRV. On post challenge day (PCD) 30 all pigs were killed and samples for virus detection and histology were taken from several organs. The inoculated TK- mutant of PRV did not induce any ill effects in the pigs except a transient febrile reaction in some animals. Virus was recovered from nasal swabbings from one pig 2 days after the first inoculation of the mutant. After challenge exposure with virulent PRV, the TK- mutant-inoculated pigs were apparently protected, whereas the control pigs all were severely affected and recovered very slowly over 3 weeks. Virus was isolated from the nasal swabbings from the TK- mutant-inoculated pigs on PCDs 2 and 4, whereas the nasal swabbings from the control piglets were all positive for virus from PCD 2 through PCD 10. DNA analysis of the virus recovered showed a pattern identical to that of the virulent PRV. Histologic lesions were found in the respiratory and the central nervous systems, however, the lesions in the TK- mutant-inoculated pigs were much milder compared to those registered for the control pigs. Virus was not isolated from any of the tissue samples that were tested, but viral DNA with sequences typical of PRV genome was detected by PCR in all samples of trigeminal ganglia from either the TK- mutant-inoculated pigs or from the controls.