Development of Multi-PCR for Differentiation of Vaccine Strain and Field Isolate of Porcine Pseudorabies Virus

Three pairs of primer were designed for amplification of porcine pseudorabies virus （PRV） gB, gE, and TK gene by multiplex PCR （multi-PCR） in order to differentiate vaccine strains from field isolates. Three specific bands were obtained respectively at the expected size, 427 bp （gB gene）, 298 bp （gEgene）, and 208 bp （TKgene）, Then four different gene-deleted vaccines of PRV were detected by multi-PCR. One ex- pected specific band was observed in one of samples, while two bands in the others. As shown by the detection results, the multi-PCR has high sensitivity and specificity and should be applied in pathogen diagnosis and epidemiological investigation in the future.

Development of PPA-ELISA for Detecting Antibodies against Porcine Pseudorabies Virus Using Truncated Recombinant Glycoprotein gD Expressed in E.coli

The purpose of this study was to develop a method for detecting antibodies against porcine pseudorabies virus （PRV）. According to the published genomic sequence of PRV SA strain, an approximately 1 070-bp gD gene fragment was amplified by PCR. The PCR products were cloned into the prokaryotic expression vector pET30a and the positive recombinant plasmid was transformed into E. coli BL21. Through induction with IPTG, the recombinant gD protein was expressed as inclusion bodies. As analyzed by western blot assay, the purified recombinant gD protein had good antigenicity and high specificity. Using the purified gD protein as coating antigen and horseradish peroxidase labeled staphylococcal protein A （PPA） as secondary antibody, we developed a PPA-ELISA for detecting antibodies against porcine PPV. No cross-reaction with the positive sera against seven common pathogens in pigs including classical swine fever virus, porcine parvovirus, porcine reproductive and respiratory syndrome, Japanese encephalitis virus, porcine circovirus type 2, porcine epidemic diarrhea virus, transmissible gastroenteritis virus was observed. The repeatability test showed that the intra- and inter-assay coefficients of variation were lower than 5% and 10%, respectively. Compared with the ELISA gD antibody test kit produced by IDEXX, the coincidence, sensitivity and specificity of the developed PPA-ELISA were 92.0%, 95.1% and 88.1%, respectively. The developed PPA-ELISA had good repeatability, sensitivity and specificity and was a rapid and simple serological method for surveillance of PRV antibodies in pig herds as well as for rapid diagnosis and epidemiological investigation of PRV infection.

Isolation and Identification of a Porcine Pseudorabies Virus Strain in Taizhou City

In this study, the liver, kidney and spleen tissues were collected from pigs with suspected PR in a pig farm in Jiangyan District, Taizhou City for virus isolation and identification. The isolated virus was inoculated onto PK15 monolayer cells. The virus culture was collected to extract genomic DNA for PCR assay and indirect immunoinfluscent assay. The results showed that the isolated virus was porcine pseudorabies virus, which was named TAIZ130417. The growth titer of the isolated virus reached 10 8.12 TCID 50 /ml on PK15 cells. Rabbits inoculated with the isolated virus soon exhibited pseudorabies symptoms such as itching and eventually died. The results provided reference for in-depth research and scientific prevention and control of pseudorabies.

Expression of Pseudorabies Virus gE Core Epitopes in Escherichia coli Strain BL21 and Utilization of Indirect PRV gE-ELISA

Pseudorabies virus glycoprotein E （PRV gE） has been recognized as a suitable diagnostic antigen for pseudorabies. In order to produce gE antigen in large quantities and at low cost, a gene fragment encoding PRV gE core epitopes was expressed in E. coli BL21 expression system. SDS-PAGE and Western Blotting revealed that the expression product in culture supematant of E. coli BL21 was a recombinant protein, approximately 51.8 Kd. At 5 h post-induction, protein concentration assay showed that the expression product amounted to 1.65 mg/ml, accounting for 24. 17% of total proteins in the culture supematant. An indirect PRV gE-ELISA was established by using the recombinant expression product as a coating antigen. Cross-reactivity assay showed that this antigen was PRV specific. In addition, the assay was consistently reproducible. Comparison of detection results of 240 serum samples between PRV gE-ELISA and a commercially available PRV diagnostic kit showed that there was no significant difference between these two methods （P 〉 0.05 ）.

Subculturing cells have no effect on CRISPR/Cas9-mediated cleavage of UL30 gene in pseudorabies virus

CRISPR/Cas9-mediated genome editing can inhibit virus infection by targeting the conserved regions of the viral genomic DNA. Unexpectedly, we found previously that pseudorabies virus(PRV) could escape from CRISPR/Cas9-mediated inhibition.In order to elucidate whether the escape of PRV from Cas9-mediated inhibition was due to cell deficiencies, such as genetic instability of sgRNA or Cas9 protein, the positive cells were passaged ten times, and PRV infection in the sgRNA-expressing cells was evaluated in the present study. The results showed that subculturing cells has no effect on Cas9-mediated cleavage of PRV. Different passages of PX459-PRV cells can stably express sgRNA to facilitate Cas9/sgRNA cleavage on the UL30 gene of PRV, resulting in a pronounced inhibition of PRV infection. Studies to elucidate the mechanism of PRV escape are currently in progress.

Isolation and Identification of Sheep Pseudorabies Virus

In a sheep farm with mixed culture of pig and sheep in Shandong Province,sheep were attacked by a disease featured by foaming at the mouth,neurological symptoms and partial hair slip of legs,and the mortality of the disease was as high as 100%.In order to determine the pathogen,dead sheep were analyzed through pathogen isolation,PCR assay and direct immunofluorescence identification,and the pathogen was confirmed as pseudorabies virus（PRV）.Sequencing results showed that the g E gene of the isolated strain shared the homology of 97%-99% with the nucleotide sequence of known PRV genome in the NCBI databases,suggesting the isolate was PRV.The virus had obvious cytopathic effect through BHK cell line passage till the seventh generation,and the amount of half virus tissue cell infection（TCID50） was 1×107.5/m L following ReedMuench method.Two healthy sheep with the body weight of 20 kg were injected with the viral fluid of the isolate,and typical symptoms of pseu-dorabies（PR） were observed after 4 d.According to clinical symptoms and PCR diagnosis results,the epidemic situation of sheep farm was effec-tively controlled through comprehensive measures such as eliminating swinery in the farm,strengthening disinfection of pigsty,injecting sick sheep with pseudorabies serum,supplementing healthy sheep herb with antivirus traditional medicine Qiqing Baidu granule.

Tn7-mediated Introduction of DNA into Bacmid-cloned Pseudorabies Virus Genome for Rapid Construction of Recombinant Viruses

lacZα-mini-attTn7 was inserted into the intergenic region between the gG and gD genes in a PRV bacterial artificial chromosome (BAC) by homologous recombination in E. coli. The resulting recombinant BAC (pBeckerZF1) was confirmed by PCR and sequencing. Green fluorescent protein (GFP) gene was then transposed into pBeckerZF1 by transposon Tn7 to generate pBeckerZF2. Recombinant viruses vBeckerZF1 and vBeckerZF2 were generated by transfection with the corresponding BAC pBeckerZF1 or pBeckerZF2. The titers and cytopathic effect (CPE) observed for by vBeckerZF1 and vBeckerZF2 was comparable to that of the parental virus vBecker3. vBeckerZF2 was serial passaged for five rounds in cell culture, and the mini-Tn7 insertion was stably maintained in viral genome. These results show that recombinant viruses can be rapidly and reliably created by Tn7-mediated transposition. This technology should accelerate greatly the pace at which recombinant PRV can be generated and, thus, facilitate the use of recombinant viruses for detailed mutagenic studies.

辣蓼黄酮正丁醇部位对PRV感染RAW264.7细胞氧化应激的干预作用及组蛋白乙酰化水平的影响

试验旨在探究辣蓼黄酮正丁醇部位(FNB)对猪伪狂犬病毒(PRV)感染小鼠单核巨噬细胞(RAW264.7)氧化应激反应的干预作用及组蛋白乙酰化水平的影响。采用CCK-8法检测FNB对细胞的毒性作用,采用不同浓度的FNB(25、50、100 mg/L)处理PRV感染的RAW264.7细胞4、8、12 h,测定氧化应激相关因子和组蛋白乙酰化水平。结果显示,与PRV感染组相比,25 mg/L FNB作用于PRV感染的RAW264.7细胞12 h后,细胞内诱导性一氧化氮合酶(iNOS)活性及一氧化氮(NO)、丙二醛(MDA)水平降低,谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、小鼠血红素氧合酶1(HO-1)和小鼠醌氧化还原酶1(NQO1)活性升高,iNOS mRNA的表达水平极显著下调(P<0.01),HO-1 mRNA的表达水平显著上调(P<0.05),核因子E2相关因子2(Nrf2)、NQO1的表达水平极显著上调(P<0.01)。与PRV感染组相比,25 mg/L FNB组处理12 h时后,细胞内组蛋白乙酰化酶(HAT)的活性降低,组蛋白去乙酰化酶(HDAC)的活性升高,细胞内HAT mRNA的表达水平下调,HDAC mRNA的表达水平上调。研究表明,FNB对PRV感染RAW264.7细胞诱导的氧化应激具有一定的调节作用,可通过提高细胞内多种抗氧化酶的基因表达水平发挥抗氧化作用,调节细胞内的乙酰化水平,降低PRV感染导致的氧化应激损伤。

Pseudorabies virus VHS protein abrogates interferon responses by blocking NF-κB and IRF3 nuclear translocation

Herpesviruses antagonize host antiviral responses through a myriad of molecular strategies culminating in the death of the host cells.Pseudorabies virus(PRV)is a significant veterinary pathogen in pigs,causing neurological sequalae that ultimately lead to the animal's demise.PRV is known to trigger apoptotic cell death during the late stages of infection.The virion host shutdown protein(VHS)encoded by UL41 plays a crucial role in the PRV infection process.In this study,we demonstrate that UL41 inhibits PRV-induced activation of inflammatory cytokine and negatively regulates the cGAS-STING-mediated antiviral activity by targeting IRF3,thereby inhibiting the translocation and phosphorylation of IRF3.Notably,mutating the conserved amino acid sites(E192,D194,and D195)in the RNase domain of UL41 or knocking down UL41 inhibits the immune evasion of PRV,suggesting that UL41 may play a crucial role in PRV's evasion of the host immune response during infection.These results enhance our understanding of how PRV structural proteins assist the virus in evading the host immune response.

Comparison on Detection Results of PRV Wild Virus Antibody Provided by Different Institutions

The detection results from different institutions were performed at the first stage of PRV wild virus antibody supervision in swine breeding. The serum samples were collected from 71 individuals and each individual was sampled twice at one week interval. The results showed that the positive coincidences for gE antibody between two institutions were 35.71% and 45.45 % ：espectively, with the total detection coincidences of 87.32% and 91.55% correspondingly. The positive coincidences for gE antibody between the two detections of each institution were 40.00% and 75.00% respectively, with the total detection coincidences of 87.32% and 97.18% correspondingly. It indicates that it is very necessary to screen the detection institution at the first supervision stage of PRV wild vires for swine population.

JEV、PRRSV、PPV及PRV多重PCR检测方法的建立及应用

为了建立一种能快速鉴别猪日本脑炎病毒(JEV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪细小病毒(PPV)和猪伪狂犬病病毒(PRV)的准确、高效的多重常规PCR检测方法,针对JEV、PRRSV、PPV、PRV基因保守区域合成特异性引物,优化后获得最优反应条件,对该方法特异性、敏感性、重复性进行了测定,并应用该方法对临床样品进行初步应用。结果显示,该方法对JEV、PRRSV、PPV、PRV可进行特异性扩增,对混合阳性质粒检测下限达2×10^(6)copies/μL,对猪圆环病毒2型、猪瘟病毒、猪流行性腹泻病毒、猪丁型冠状病毒、猪传染性胃肠炎病毒等相关病毒均无扩增,且重复性良好。用该方法检测51份2021年采集的广西区内组织样品,检出JEV、PRRSV、PPV、PRV阳性率分别为7.84%、50.98%、5.88%、23.53%,且存在多重混合感染的情况。所建立的多重PCR方法具有良好的特异性、敏感性及重复性,可应用于临床常见猪繁殖障碍性病毒病的快速诊断和监测预警,为猪繁殖障碍性病毒病提供了诊断技术支持。

ASFV、PRV和PCV2多重荧光定量PCR检测方法的建立和应用

非洲猪瘟病毒(ASFV)、伪狂犬病毒(PRV)、猪圆环病毒2型(PCV2)是危害养猪业的3种重要病毒,但目前国内外尚无可同时检测这3种病原的方法。为建立一种可同时检测上述3种病原的方法,本研究以ASFV的B646L基因、PRV的gE基因及PCV2的rep基因为靶基因,采用Prime 3 web软件分别设计特异性引物和探针。采用相应引物分别构建上述3种靶基因的重组质粒,经测序鉴定后分别作为质粒标准品,并均稀释到1×105拷贝/μL后按照体积比1∶1∶1混匀后作为模板,采用方阵法对引物、探针、酶和各反应条件等的优化,初步建立ASFV、PRV和PCV2多重荧光定量PCR检测方法。结果显示,该方法标准曲线Ct值与相应质粒标准品浓度均存在良好的线性关系,相关系数(R2)均大于0.99。利用该方法同时检测猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、口蹄疫病毒(FMDV)、猪细小病毒(PPV)、牛病毒性腹泻病毒(BVDV)、传染性胃肠炎病毒(TGEV)等病原,结果显示,本实验建立的该方法仅特异性扩增ASFV、PRV和PCV2,与其他病原均无交叉反应,特异性强;分别利用该方法与已公布的中华人民共和国农业农村部公告第172号中ASFV荧光定量PCR检测方法、PCV2荧光定量PCR方法(GB/T 35901-2018)、PRV荧光定量PCR检测方法(GB/T 35911-2018)对10倍倍比稀释(1.0×10^(4)拷贝/μL~1.0×10_(0)拷贝/μL)后的pUC57-ASFV-B646L、pUC57-PRV-gE、pGEMT-PCV2-rep质粒标准品1∶1∶1的混合物检测,结果显示该方法对3种质粒标准品的最低检测限均为10拷贝/μL,相同或略高于上述各病原的单一qPCR检测结果,表明本实验建立的该方法敏感性较高;选取3个稀释度的质粒标准品分别按1∶1∶1比例混合后进行组内和组间重复性试验,结果显示组内组间变异系数均小于3%,重复性好。利用本实验建立的方法和上述3种病原标准检测方法对114份临床样品检测,结果显示,本实验建立的方法对ASFV、PRV和PCV2检测的的阳性样品数分别为10份、10份和42份,未发现混合感染现象,与3种病原的标准方法相比符合率均为100%。以上结果表明,本研究建立的ASFV、PRV和PCV2多重荧光定量PCR方法特异性强、敏感性高、重复性好,可用于这3种常见猪病的鉴别诊断和流行病学调查。

Construction and immunogenicity of recombinant pseudorabies virus expressing the modified GP5m protein of porcine reproduction and respiratory syndrome virus

Pseudorabies virus(PRV),an alpha-herpesvirus,has been developed as a live viral vector for animal vaccines.However,the PRV recombinant virus TK^(-)/gE^(-)/GP5^(+)expressing GP5 of porcine reproductive and respiratory syn-drome virus(PRRSV),based on the PRV genetically depleted vaccine strain TK^(-)/gE^(-)/LacZ^(+),scarcely stimulated the vaccinated animals to produce neutralizing antibodies against PRRSV.To develop a booster-specific immune response of such PRV recombinants,the ORF5m gene(the modified ORF5 gene having better immune responses)was substituted for the ORF5 gene and introduced into PRV TK^(-)/gE^(-)/LacZ^(+),resulting in a PRV recombinant named TK^(-)/gE^(-)/GP5m^(+),which expressed the modified GP5m protein.The recombinant virus was confirmed using PCR,Southern blotting and Western blotting.TK^(-)/gE^(-)/GP5m^(+)and TK^(-)/gE^(-)/GP5^(+)expressing the authentic GP5 protein were inoculated into Balb/c mice to evaluate their immune responses.The results indicated that the protecting neutralization antibodies(the 3/6 vaccinated mice obtained 1:16)and cell immune responses induced by TK^(-)/gE^(-)/GP5m^(+)against PRRSV were higher than that induced by TK^(-)/gE^(-)/GP5^(+).Thus,the development of the new PRV recombinant expressing the modified GP5m protein as a candidate vaccine established the basis for the study of bivalent genetic engineering vaccines against PRRSV and PRV.

黄芩多糖对PRV感染猪睾丸细胞的作用及其差异基因表达分析

为研究黄芩多糖对伪狂犬病毒(PRV)感染猪睾丸(ST)细胞的作用并筛选与调控相关的差异基因,本试验建立PRV感染ST细胞模型,将其分为预防、治疗和杀毒模式,分别使用不同浓度的黄芩多糖溶液进行处理,每个模式下设立空白对照组和PRV对照组。采用CCK-8法检测黄芩多糖对ST细胞存活率的影响,显微镜观察各组细胞形态,ELISA检测IFN-γ、IFN-α和TNF-α细胞因子含量;在24 h时提取RNA进行转录组测序,分析其差异表达基因并进行GO功能和KEGG通路分析。结果显示,PRV感染ST细胞出现病变。与PRV对照组相比,黄芩多糖浓度为97.66μg/mL时,3种模式中细胞存活率均极显著升高(P<0.01),细胞病变减轻,IFN-γ和IFN-α含量均极显著提高(P<0.01),TNF-α含量极显著降低(P<0.01),治疗指数(TI)分别为2、4和4。治疗模式下,97.66μg/mL黄芩多糖处理组、空白对照组和PRV对照组比较共交集差异基因74个,其中SOCS3、VEGFA、ZBTB18、CLCN6、RSBN1、RBM47和TET2基因差异表达较为显著,差异表达基因显著富集到15条信号通路,PRV对照组与空白对照组和PRV对照组与黄芩多糖治疗组比较,差异表达基因主要分别涉及核糖体、内吞、MAPK、TNF和MAPK、mTOR、TLR等相关通路。结果表明,黄芩多糖对PRV感染ST细胞具有保护作用,其作用可能是通过调节TNF信号通路和TLR信号通路中SOCS3、CCL5、FOS、PIK3R1和MAP3K8等相关基因的表达来实现。本试验结果将为进一步探索黄芩多糖调控PRV感染宿主的作用机制提供理论依据。

永生化猪视网膜色素上皮细胞的建立及初步应用

【目的】建立永生化猪视网膜色素上皮细胞系,为病毒学研究提供一种新的细胞材料。【方法】合成猪腺病毒3型E1基因,将其克隆至真核表达质粒pCI-neo中,构建E1基因真核表达质粒pCI-neo-E1,进行PCR和测序鉴定。提取pCI-neo-E1质粒,转染原代猪视网膜色素上皮细胞,用新霉素G418筛选猪永生化视网膜色素上皮细胞系(RPECs)。检测RPECs的角蛋白18和19,通过细胞计数测定其生长曲线,流式细胞仪检测细胞周期,并对其进行染色体核型分析。分别将猪流行性腹泻病毒(PEDV)CV77毒株、猪圆环病毒2型(PCV2)DBN-SX07株和猪伪狂犬病毒(PRV)Bartha-K61株接种RPECs,同时将PEDV CV77毒株接种绿猴肾细胞(Vero细胞),将PCV2 DBN-SX07株接种猪肾细胞(PK15细胞),将PRV Bartha-K61株接种仓鼠肾成纤维细胞(BHK细胞),观察细胞病变效应(CPE),并采用间接免疫荧光法测定病毒的滴度。【结果】PCR和测序结果显示,真核表达质粒pCI-neo-E1构建成功。将pCI-neo-E1转染原代猪视网膜色素上皮细胞,经G418筛选得到永生化细胞株,该细胞株表达角蛋白18和19,传代50代仍保持上皮样细胞形态,增殖活性良好,细胞周期及染色体核型特征与正常的二倍体细胞一致。PEDV CV77毒株感染猪RPECs 24 h后,CPE明显,滴度为10^(5.25)TCID_(50)/mL,低于其在Vero细胞中的滴度(10^(8.125) TCID_(50)/mL)。PCV2 DBN-SX07毒株感染猪RPECs后可产生明显CPE,病毒滴度为10^(6.125)TCID_(50)/mL,略高于其在PK15细胞上的滴度(106.0 TCID_(50)/mL)。PRV Bartha-K61毒株感染猪RPECs后,CPE明显,病毒滴度为10^(8.75)TCID_(50)/mL,略低于其在BHK上的滴度(108.875 TCID_(50)/mL)。【结论】成功构建了永生化猪视网膜细胞系,可用于猪源病毒的培养,尤其是培养PCV2可产生明显CPE,易于观察,为后续开展疫苗工艺的提升提供了一种新的细胞材料。

伪狂犬病病毒研究进展

伪狂犬病是由伪狂犬病病毒(pesudorabies virus,PRV)感染引起的一种严重的传染性病毒病,主要引起猪的繁殖失败、生长停滞和呼吸系统障碍,2周龄以下仔猪死亡率高达100%。伪狂犬病属于多种动物共患传染病,主要经口、鼻感染,通过在上呼吸道上皮细胞中的复制,进一步感染神经系统和内脏器官。PRV有11种糖蛋白分子,在病毒的毒力、感染入侵和致病过程中发挥重要作用。临床上采用疫苗免疫和鉴别诊断相结合的方式进行PRV的防控,起到了一定的效果,但是仍然给养猪业造成巨大损失。因此,本文综述和讨论了猪伪狂犬病的临床症状以及病原的分子特征、致病机制和疫苗研究的最新进展,以期为该病疫苗研究和诊断试剂开发提供参考。

基于模式病毒评价商品化消毒剂对非洲猪瘟病毒的消杀效果

为了解常用商品化消毒剂对非洲猪瘟病毒(ASFV)的消杀效果,为其消毒剂的筛选提供参考。该研究利用伪狂犬病毒(pseudorabies virus,PRV)与ASFV病毒理化特征的相似性,以PRV作为替代标识毒株,建立了消毒剂细胞评价模型,于体外检测了3种市售消毒剂对生长在BHK-21细胞中PRV的灭活效果。分别从3类消毒剂的不同方面对消毒剂抗病毒作用的影响进行消杀效果的评估。研究显示,有效含量为0.125 g·L^(-1)的戊二醛癸甲溴铵溶液,以及有效含量为0.5 g·L^(-1)的过氧乙酸溶液和二氯异氰脲酸钠,在室温作用30 min,均可有效灭活PRV。其中,过氧乙酸综合评价最优;戊二醛癸甲溴铵性价比最高,但所需消杀时间较长,受低温影响比较严重。该研究可为养殖场防控ASFV的感染提供消毒剂筛选的理论依据,并对ASFV的消毒标准提供参考。

猪源Rab基因shRNA文库与稳定细胞株的构建及其对猪伪狂犬病病毒增殖的影响

【目的】探究Rab家族蛋白在猪伪狂犬病病毒(Porcine pseudorabies virus,PRV)感染宿主细胞中的作用。【方法】以猪肾上皮细胞(PK15)为试验模型,构建Rab基因shRNA文库,并用嘌呤霉素筛选获得稳定的能够抑制猪源Rab基因表达的细胞株。用实时荧光定量PCR检测细胞敲减效率,通过流式细胞术检测敲减Rab基因对PRV增殖的影响;用Western blotting检测PRV-gB蛋白表达量,使用实时荧光定量PCR检测敲减Rab基因后PRV-gB、PRV-TK基因以及相关细胞炎性因子表达量。【结果】试验成功构建包含13种Rab基因敲减细胞系的shRNA文库。流式细胞术检测结果显示敲减Rab基因能显著抑制PRV-GFP增殖(P<0.05);病毒滴度与Western blotting检测结果表明,敲减Rab基因极显著抑制子代病毒的产生以及PRV-gB蛋白的表达(P<0.01)。实时荧光定量PCR检测结果显示,敲减Rab基因会显著或极显著抑制PRV-gB、PRV-TK基因表达(P<0.05;P<0.01);敲减Rab14和Rab27基因后细胞中IFN-β、ISG15、IL-1β、IL-18基因表达量均显著或极显著升高(P<0.05;P<0.01)。【结论】敲减Rab基因能够抑制PRV增殖。研究结果为进一步研究Rab基因在PRV生活周期中发挥的作用奠定基础。

猪伪狂犬病毒荧光重组酶介导核酸扩增快速检测方法的建立与应用

【目的】基于荧光重组酶介导核酸扩增(Recombinase-aided amplification,RAA)技术,建立一种猪伪狂犬病毒(Porcine pseudorabies virus,PRV)快速检测方法。【方法】根据PRV gE基因序列,设计特异性引物及探针,优化扩增体系,建立PRV荧光重组酶介导核酸扩增检测方法,检验其特异性、敏感性和重复性,应用该方法对临床样品进行检测。【结果】该方法在43℃恒温反应23 min即可完成PRV核酸扩增,最低检出限为111 copies·μL^(-1);与猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)、猪流行性腹泻病毒(Porcine epidemicdiarrheavirus,PEDV)、猪轮状病毒(Porcinerotavirus,PoRV)、猪传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)、猪圆环病毒2型(Porcine circovirus 2,PCV2)、猪圆环病毒3型(Porcine circovirus 3,PCV3)均无交叉反应。重复性试验显示,组内和组间变异系数均小于5%;40份临床样品检测结果显示PRV阳性率为15%(6/40),检测结果与常规聚合酶链式反应(PCR)一致。【结论】成功建立了简便快速、高效准确的PRV实时荧光RAA检测方法,为PRV的快速检测和流行病学调查提供了新的检测手段。

云南地区部分猪场猪呼吸道疾病综合症(PRDC)患病猪群中PRRSV,PCV-2,CSFV,PRV混合感染调查

针对云南省不同地区不同季节猪呼吸道疾病综合征进行流行病学调查,根据其临床特征,在不同规模养殖场共采集1 164份血清样品,利用ELISA和RT-PCR方法检测其猪瘟抗原CSFV,猪繁殖与呼吸综合征抗原PRRSV,猪伪狂犬病PRV gE抗体及猪圆环病毒2型PCV-2特异抗体。结果表明:各季节和不同生长阶段猪群存在一种及两种以上的病毒混合感染,四重感染相对较少。从全年看单独感染占39.10%,PCV-2感染率最高,其次是PRV,PRRSV和CSFV;二重感染占26.13%,以PCV-2+PRV最常见,其次是PCV-2+PRRSV、PRV+PRRSV;三重感染占8.05%,PCV-2+PRRSV+PRV最常见;有四重感染出现,仅占1.57%。从季节来看,春季和夏季混合感染最高,分别为76.53%和60.74%,冬季和秋季的混合感染率分别为59.50%和53.55%。从年龄和性别看,育肥猪的混合感染率高于仔猪,分别为68.66%和61.11%,种公猪的感染率高于母猪,分别为70.00%和55.51%。说明4种病毒有单独感染或混合感染,推测其中PCV-2在混合感染中可能充当免疫抑制的角色,混合感染使PRDC症状更明显,死亡率增高。