潍坊某猪场一起伪狂犬病的诊治

2014年9月山东潍坊某猪场发生疑似猪伪狂犬病疫情,采集病死仔猪的脑组织等,利用Vero细胞做病毒分离,并设计一对伪狂犬病病毒(PRV)g E基因片段的特异性引物对分离病毒进行PCR鉴定及家兔接种试验。结果表明:脑组织上清液接种Vero细胞后有典型的细胞病变;PCR扩增产物电泳后显示出990bp长的目的片段,目的片段基因序列与6株PRV毒株的g E基因序列核苷酸同源性在97.7%~100%之间,证实该病毒为PRV;分离病毒接种家兔出现典型的伪狂犬病症状。临床诊断结合实验室鉴定以及动物接种试验,确诊该病例为猪伪狂犬病。     

猪伪狂犬病病毒北京株的分离鉴定

从北京某猪场疑似猪伪狂犬病发病死亡仔猪脑及内脏中分离到1株病毒并进行了鉴定。该分离毒株接种ST细胞24 h后出现圆缩、聚集、脱落等典型的细胞病变(CPE);分离毒株能够被伪狂犬病病毒标准阳性血清中和;分离病毒接种家兔后,引起家兔出现奇痒等典型的伪狂犬病临床症状;同时根据GenBank公布的PRV的gD基因设计引物并扩增出特异性的目的片段,扩增产物经过测序比较,表明扩增产物序列为猪伪狂犬病病毒基因序列。以上结果证实该病毒为猪伪狂犬病病毒,依据分离地点命名为猪伪狂犬病病毒北京株。     

猪伪狂犬病病毒的分离和鉴定

从病猪脑部分离到1株病毒.该病毒接种Balb/c小鼠出现神经症状,病死率为60% ,接种PK-15细胞出现拉网病变.伪狂犬病阳性血清能特异性的中和该分离毒.根据基因库(GenBank)的PRV gE基因设计的引物能扩增出特异性片段,证实该病毒为伪狂犬病病毒.     

2株伪狂犬病毒株的分离鉴定及生物学特性分析

为了解伪狂犬病病毒(PRV)变异情况，从山东和江苏两地的某发病猪场分别采集阳性病料接种BHK-21细胞，连续传代进行病毒分离，观察细胞病变；对细胞培养物进行间接免疫荧光与蛋白印迹鉴定，通过对病毒滴度的测定绘制了分离病毒株的一步生长曲线；进一步动物试验鉴定分离株的毒力，并对其gE基因进行遗传进化分析。结果显示：利用PCR成功扩增出PRV的gB、gC和gE特异性基因，病料接种BHK-21细胞后产生了特异性细胞病变；病毒感染细胞后表达了gE蛋白，并与抗PRV单抗特异性结合，显示成功分离到2株分离毒株，分别命名为SDWF2019和JSNT2019;通过序列分析，2个分离株与我国新流行的PRV毒株属于同一遗传进化分支。本研究为我国华东区猪伪狂犬病的疫情监测提供了参考。     

一株水貂源伪狂犬病毒株的分离鉴定

从辽宁大连疑似水貂伪狂犬病发病死亡水貂脑、内脏中及饲喂的猪肝中分离到1株病毒并进行了鉴定。该分离毒株接种BHK-21细胞24 h后出现圆缩、聚集、脱落等典型的细胞病变(CPE);分离毒株能够被伪狂犬病病毒标准阳性血清中和;分离病毒接种家兔后,引起家兔出现奇痒等典型的伪狂犬病临床症状;同时根据Gen Bank公布的PRV的Tg ET基因设计引物并扩增出特异性的目的片段,扩增产物经过测序比较,表明扩增产物序列为猪伪狂犬病毒g E基因序列。以上结果表明,该病毒为伪狂犬病毒,依据来源确定为水貂源性伪狂犬病病毒株。     

猪流行性腹泻病毒与猪伪狂犬病病毒二重RT-PCR检测方法的建立及应用

为建立猪流行性腹泻病毒（PEDV）与猪伪狂犬病病毒（PRV）的快速鉴别检测方法,本研究根据GenBank已登录的PEDV膜蛋白M基因和PRV gE基因保守区域序列设计了2对特异性引物,以PRV和PEDV混合总RNA为反转录模板,初步建立了PRV和PEDV的二重RT-PCR检测方法,并进行了特异性、敏感性、重复性验证和临床应用检测。结果显示,该方法对两种病毒的最低检测限均为10 TCID₅₀/mL病毒含量,重复性好,特异性强,可特异性地扩增PEDV和PRV细胞培养物,但对其他7种病原对照扩增不出任何条带,对26份临床疑似PEDV和PRV感染样品检测结果与测序鉴定结果完全一致。本研究成功建立了PEDV和PRV的二重RT-PCR检测方法,为临床上猪流行性腹泻和猪伪狂犬病的快速鉴别诊断提供了方法。     

伪狂犬病病毒FJMH1907b株的分离和gD基因的演化分析

为探究福建省新流行的猪伪狂犬病病毒(Pseudorabies virus,PRV)gD基因的遗传进化情况,根据GenBank已公布的PRV gD基因序列设计1对特异性扩增引物,对新分离PRV FJMH1907b株的gD基因进行PCR扩增、测序,与国内外已发表的15个毒株进行同源性比对分析,并构建遗传进化树.新分离FJM...     

猪伪狂犬病病毒的分离鉴定及其gE基因序列分析

从福建省某猪场疑似伪狂犬病的发病3日龄仔猪的脑、肺脏中分离到1株病毒.该病毒接种家兔出现了典型的伪狂犬病症状,接种PK-15细胞36 h后出现了圆缩、集聚、脱落等典型的细胞病变,猪伪狂犬病病毒阳性血清能特异性中和该分离病毒.根据已发表的伪狂犬病病毒(PRV)gE基因的序列,设计并合成了一对引物,采用PCR方法可扩增特异性298 bp的DNA片段.测序结果与GenBank中有代表性的6株参考毒株相应基因序列比较,核苷酸和氨基酸序列同源性分别为95.5%~99%和91.8%~98%.系统进化树结果表明分离株与湖北分离株Ea株亲缘关系最近.     

猪伪狂犬病病毒套式PCR检测方法的建立与应用

对伪狂犬病病毒(PRV)国标PCR方法进行优化,建立一种高灵敏度的套式PCR方法。根据伪狂犬病病毒gD基因序列,设计并合成了2对引物,通过反应体系和条件的优化建立套式PCR方法。通过灵敏性试验、特异性试验、病料检测对比试验等验证建立方法的适用性。结果表明,建立的方法检测伪狂犬病病毒DNA的极限为2.6×10-7 ng/mL,灵敏度比国家标准方法提高了1 000倍,从疑似PRV感染组织病料中能大幅度提高阳性检出率。本研究成功建立了一种灵敏度高、特异性好的快速检测猪伪狂犬病病毒的套式PCR检测方法。     

高致病性猪繁殖与呼吸综合征病毒和猪伪狂犬病病毒双重PCR检测方法的建立及初步应用

为快速鉴别诊断高致病性猪繁殖与呼吸综合征和猪伪狂犬病,根据GenBank中登录的高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)Nsp2基因缺失区和猪伪狂犬病病毒(PRV) gB基因保守区序列,分别设计并合成了2对特异性扩增引物,通过对反应条件的优化,建立了能够同时检测HP-PRRSV和PRV的双重PCR诊断方法.利用建立的双重PCR方法对30份临床可疑样品进行检测,并与商品化单项PCR试剂盒进行对比验证.结果表明,本试验建立的双重PCR方法具有较高的灵敏度和特异性,与商品化单项PCR试剂盒检测结果完全一致.因此,该双重PCR方法能够用于HP-PRRSV和PRV单项或混合感染的临床样品快速鉴别检测.     

应用PCR技术检测伪狂犬病病毒

根据伪狂犬病病毒(PRV)的gE基因序列,设计并合成了一对引物,以闽A株DNA为模板,建立了检测PRV的PCR方法。该方法能从猪细小病毒闽A株和FB株中扩增出一条长度为1 808 bp的片段,对Bartha株、gE-株检测为阴性;而以猪圆环病毒、猪繁殖与呼吸综合征病毒、猪伪狂犬病病毒、猪瘟病毒和正常细胞的核酸为模板的均为阴性;敏感性试验表明,该体系可检测到10pg的猪细小病毒基因组DNA。表明该方法适用于检测猪伪狂犬病病毒野毒株或非gE基因缺失弱毒株。     

抗伪狂犬病病毒gC抗体制备及其gC蛋白表达

用纯化的重组蛋白His-gCN813制备抗PRV-gC抗体,并分析伪狂犬病病毒（pseudorabies virus,PRV）gC蛋白在真核细胞的表达情况。本研究以提取的病毒基因组为模板,PCR克隆PRV gC全长基因,构建gC真核表达质粒p3xFLAG-gC,在真核细胞中表达gC基因;纯化蛋白His-gCN813制备的抗体,Western blotting和间接免疫荧光（IFA）检测到p3xFLAG-gC转染真核细胞和PRV感染细胞中的gC蛋白。结果表明本试验成功构建了PRV gC基因真核表达系统,获得了特异性抗PRV gC抗体,重组gC真核表达质粒p3xFLAG-gC转染Vero细胞,表达的重组蛋白gC为72 ku,PRV感染Vero细胞,表达的gC蛋白为55、72和94 ku,主要定位在细胞浆,gC蛋白可以作为PRV感染的指示分子,为下一步研究PRV和宿主相互作用及PRV的复制奠定了基础。     

Replication and immunosuppressive effects of Pseudorabies virus on swine peripheral blood mononuclear cells.

The infectivity and potential immunosuppressive effects of Pseudorabies virus (PRV) was evaluated in swine peripheral blood mononuclear cells (PBMC). Virus progeny titers and viral DNA synthesis at various intervals post-inoculation revealed the replication of PRV in both peripheral blood monocytes and lymphocytes; however, replication in lymphocytes was restricted compared with monocytes. PRV infection resulted in the damage and death of monocytes. Although PRV did not appear to affect the viability of the lymphocytes, PRV infection suppressed lymphocyte functions such as proliferation and interleukin-2 (IL-2) synthesis in response to Concanavalin A. This immunosuppression was dependent upon the multiplicity of infection (MOI) of infectious PRV. UV-inactivated PRV was not immunosuppressive. There was no effect of PRV on natural killer (NK) cell activity. The reduction of lymphocyte proliferation by PRV was not reversible by the addition of supernatant containing porcine IL-2 and non-infected monocytes to the infected cultures. The results from these in vitro studies demonstrate that PRV can infect and cause immunosuppressive effects on swine PBMC. These effects may explain the potential role of PRV in predisposing infected pigs to secondary infection and support the hypothesis that PRV can spread systemically by infected PBMC in blood and lymph.     

Role of the raccoon in the transmission of pseudorabies: a field and laboratory investigation

Serum samples were collected from 42 raccoons trapped in a pseudorabies enzootic area of Missouri. All samples were negative for neutralizing antibodies to pseudorabies virus (PRV). Raccoons were orally exposed with one of four dose levels of PRV. The raccoons were found to be susceptible to moderately large doses of PRV tissue culture infective dose (10(4) - 10(5) TCID50). All raccoons given 10(3) TCID50 of PRV survived and did not develop clinical signs. The PRV was consistently isolated from tonsillar swabs collected from raccoons that died after exposure. The PRV was also isolated from the brain, tonsils, lungs, and salivary glands of raccoons that died. It was isolated from samples of the brain and tonsils collected from a raccoon 5 days after death. Neutralizing antibodies to PRV could not be detected in the serum of raccoons that survived PRV exposure. Tonsillar swabs and tissue samples collected from these raccoons were free of PRV. The results indicated that the raccoon may serve as a short-term reservoir for PRV, but it is unlikely to have an ipizootiologic role as a long-term, subclinical carrier of the virus.     

猪伪狂犬病病毒多克隆抗体的制备及免疫学活性研究

本研究旨在获得抗猪伪狂犬病病毒(PRV)闽A株的多克隆抗体,为PRV的治疗与检测提供理论基础.本研究在PK-15细胞上进行PRV的增殖,测定其TCID₅₀为10^-7.372,粗提蛋白后,测定PRV蛋白浓度为3.6 mg/mL.试验选用25只健康、雄性、体重为2.5 kg±0.2 kg的新西兰大白兔为试验动物,用获得的PRV为抗原免疫后,获得抗PRV多克隆抗体.测定其抗血清效价为1:32 000,抗原包被稀释度为1:40,最佳包被条件为4 ℃ 12 h,最佳封闭时间为1 h,酶标二抗最佳工作稀释度为1:8 000.细胞病变中和试验结果表明,本研究制备的PRV抗血清在1:16的稀释情况下能保护50%的PK-15细胞免受PRV的攻击,而阴性血清不能保护PK-15细胞免受PRV的感染.结果表明本研究成功制备了PRV多克隆抗体.     

Effects of a porcine reproductive and respiratory syndrome virus infection on the development of the immune response against pseudorabies virus

The aim of this study was to investigate the effects of a porcine reproductive and respiratory syndrome virus (PRRSV) infection on the development of the immune response after pseudorabies virus (PRV) vaccination in pigs. Pigs were intranasally inoculated with the European PRRSV strain, Lelystad virus ter Huurne, and were vaccinated intramuscularly with PRV 2 weeks later (LV-PRV group). Control pigs were vaccinated with PRV only (PRV group). Eight weeks after PRV vaccination, pigs from both groups were challenged intranasally with wild-type PRV. We measured the lymphoproliferative, and the cytolytic responses to PRV of peripheral blood mononuclear cells (PBMC), isolated from blood samples. In addition, serum samples were examined for antibodies against PRV and LV. One week after PRV vaccination, PBMC proliferated abundantly to PRV in both groups. However, in the LV-PRV group the lymphoproliferative response declined after 1 week, whereas, in the PRV group, the lymphoproliferative response was high for 3 weeks and declined thereafter (P<0.05). After challenge, the lymphoproliferative response was 1 week earlier and was consistently and significantly higher in the PRV group than in the LV-PRV group. The PRV-specific killing was higher at 3 weeks after PRV vaccination and 5 weeks after PRV challenge 19+/-3 and 24+/-6%, respectively, in the PRV group, compared to 7+/-4 and 6+/-9%, respectively, in the LV-PRV group (P<0.05). However, later after vaccination and challenge the cytolytic response was identical in both groups. The antibody titre against PRV developed equally in both groups. After challenge, no PRV virus was isolated from both groups. From these results we conclude that, although PRRSV infection did cause changes in the time course of the T-lymphocyte response after PRV vaccination, PRRSV infection did not inhibit the development of vaccine-induced protection after PRV.     

Preparation of Polyclonal Antibodies against Swine Pseudorabies Virus and Study on the Immunological Activity

This study was aimed to obtain polyclonal antibody against swine pseudorabies virus (PRV) Min A strain,and provide a theoretical basis for the study of the treatment and detection of PRV.This study was performed on PK-15 cell and proliferation of PRV was measured as TCID₅₀ 10^-7.372,the protein concentration of PRV was measured as 3.6 mg/mL.Choosing five healthy male rabbits (2.5 kg±0.2 kg) as experimental animals and using PRV obtained as the antigen,we got polyclonal antibody against PRV.Antiserum titer was 1:32 000,antigen coating dilution was 1:40,the best coating conditions was 4 ℃ 12 h,the best blocking time was 1 h,the best working dilution of enzyme labled antibody was 1:8 000,the result of cell lesions neutralization test showed that PRV antiserum prepared in this assay at 1:16 dilution could protect 50% of PK-15 cells from being infected by PRV,and negative serum couldn't protect PK-15 cells from being infected by PRV.The study successfully prepared polyclonal antibodies against PRV.     

抗猪伪狂犬病病毒gH抗体的制备及gH在感染细胞中表达分析

为检测伪狂犬病病毒(PRV)在体外细胞中糖蛋白H(gH)的表达情况,本研究构建原核表达重组质粒pET-gHN660,并在大肠杆菌中诱导表达重组蛋白,纯化的gH重组蛋白免疫实验动物制备抗PRV gH抗体,经western blot和IFA检测到病毒感染细胞中gM蛋白的表达,病毒感染细胞后表达的gH蛋白大小为95 ku,定位于细胞浆中,gH蛋白在病毒感染细胞4 h可以检出,随PRV的复制gH蛋白表达增加,gH蛋白可以作为PRV复制的指示蛋白.本研究利用制备的抗体分析感染细胞中gH蛋白的表达情况,初步探讨感染细胞中PRV的复制,为PRV和宿主相互作用的研究奠定基础.     

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。