猪瘟病毒、猪繁殖与呼吸障碍综合征病毒和乙型脑炎病毒多重RT-PCR检测方法的建立

根据猪瘟病毒(CSFV)的E2基因保守序列、猪繁殖与呼吸障碍综合征病毒(PRRSV)的ORF7基因及部分ORF6和3-UTR基因序列和猪乙型脑炎病毒(JEV)的E基因保守序列,设计了3对引物,扩增大小分别为288 bp、430 bp和1 015 bp目的片段.以纯培养病毒抽提RNA,制备cDNA模板,利用3对引物,通过条件的优化,建立了同时检测这3种病毒的多重RT-PCR.CSFV、PRRSV和JEV的RNA最小检出量分别为0.270 ng、0.049 ng、0.067 ng,其它的RNA病毒如TGEV以及DNA病毒如PPV、PrV、PCV-2检测结果为阴性.以β-actin为对照,利用本方法检测了174份临床样品(血清、肺脏和胎儿),其结果为:PRRSV、CSFV和JEV的阳性率分别为30.4%、4.6%和1.7%;CSFV和PRRSV混合感染阳性率为1.7%.同时对临床样品中CSFV、JEV和PRRSV阳性PCR产物进行测序分析,发现CSFV、PRRSV和JEV的PCR产物序列和NCBI数据库中目标基因的同源性分别为92%、90%和89%,这证实了多重PCR的阳性结果为上述3种病毒.结果证明,所建立的多重RT-PCR可用于临床检测.     

复合RT-PCR检测猪繁殖与呼吸综合征和猪瘟及猪流感等3种病毒方法的建立

为了快速准确地对猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)和猪流感病毒(SIV)进行诊断与鉴别诊断,特进行试验。根据GenBank上已发表的PRRSV的ORF7基因序列、CSFV的C基因序列和SIV的M基因序列,设计合成了3对特异引物,开展RTPCR检测方法的研究。分别建立了PRRSV、CSFV和SIV的单项RT-PCR诊断方法,并最终建立了PRRSV、CSFV和SIV的复合RT-PCR诊断方法,可分别及同时扩增PRRSV 430 bp、CSFV 775 bp和SIV 225 bp的特异性片段。试验证明该方法适用于PRRSV、CSFV和SIV的诊断及鉴别诊断。     

猪繁殖与呼吸综合征病毒SYBR Green I荧光定量RT-PCR方法的建立

根据文献合成1对针对PRRSV毒株ORF6基因高度保守序列的引物,建立用于检测PRRSV的SYBR Green I荧光定量RT-PCR诊断方法.提取PRRSV-LC病毒RNA,经所建立的荧光定量RT-PCR方法扩增,可获得特异的扩增曲线,而日本乙型脑炎病毒(JEV)、猪伪狂犬病毒(PRV)、猪细小病毒(PPV)和猪圆环病毒(PCV)进行同条件检测,结果为阴性;方法可检测到1 TCID_(50) PRRSV,比琼脂糖凝胶电泳敏感度高10～100倍.结果表明,所建立的PRRSV荧光定量RT-PCR诊断方法特异性好、敏感性高,可进一步应用于猪繁殖与呼吸综合征的临床诊断和科学研究.     

猪繁殖与呼吸综合症病毒江西株的分离与鉴定

试验从江西省爆发疑似PRRS临床症状的某猪场病猪血清中分离到一株致Marc-145细胞病变的病毒。负染电镜观察结果可见直径约50nm大小的有囊膜的球形病毒粒子；超薄切切片可见胞浆内有大量直径约40－80nm球形或椭圆形病毒粒子。间接免疫荧光试验结果表明该分离与美洲型PRRS阳性血清反应发出强特异性荧光，而与欧洲型阳性血清只出现弱的荧光效应，初步鉴定分离株为美洲型PRRS病毒，暂命名为JX株。     

4株高致病性猪繁殖与呼吸障碍综合征病毒的分离与鉴定

2007年7月从江苏某些猪场采集的具有明显高热症状的病料组织中分离出4株病毒,经对病毒的TCID50测定、血清学试验、病毒基因鉴定,确认这4株为美洲型猪繁殖与呼吸综合征病毒.病毒基因序列分析发现:4株病毒的NSP2基因均存在2处共30个氨基酸缺失,缺失氨基酸分别位于481位、532～560位.氨基酸序列同源性分析可见:分离株与VR2332株、MLV株的同源性很低,在60.3%至68.6%之间;与国内疫苗株CH-1a的同源性为83.1%～83.7%;与国内分离的变异株JXAI株、HUN4株、HB-1(sh)/2002株同源性很高,为91.2%～98.5%;同时4株分离株之间的同源性很高,为99.3%～100.0%.系统发育树表明:分离的4个毒株与JXAI毒株和HUN4株有较近的亲缘关系,与其他株的亲缘关系较远.动物回归试验表明,4株病毒均可以引起仔猪明显的临床高热症状.     

Characterization of Porcine Reproductive and Respiratory Syndrome Virus Deletion Mutant

The genome of the isolate of porcine reproductive and respiratory syndrome virus （PRRSV） from China, designated HPBEDV, was sequenced and analyzed. The size of the genome of HPBEDV was 15 334 nucleotides （nt）. Comparative analysis of HPBEDV with the genomic sequences of the domestic and other isolates （JXA 1, HUN4, CH-1 a, B J-4, VR2332, and LV） revealed that HPBEDV shared 98.4, 98.0, 89.0, 88.7, and 88.6% identity with the American strain JXA1, HUN4, CH- 1a, BJ-4, and VR2332, respectively, but only 54.7% identity with the European reference strain Lelystad virus. The NSP2 gene had 2 850 nt and encoded 950 amino acids （aa）, with two discontiguous deletions of 1 aa and 29 aa at positions 482 and 534-562, respectively, relative to VR-2332. Also, phylogenetic analysis with the published PRRSV genomic sequences indicated that the newly emerging isolate form a clade with the VR-2332 isolates. Therefore, HPBEDV was a novel strain with deletions in NSP2 gene.     

猪繁殖与呼吸综合征RT-PCR诊断方法的建立

为建立一种方便快捷的检测猪繁殖与呼吸综合征的RT-PCR方法,根据GenBank中已发表的猪繁殖与呼吸综合征病毒GP5基因设计一对引物,并以合成的cDNA为模板,初步建立了检测猪繁殖与呼吸综合征的RT-PCR方法,进而对对照病毒及采集的病料组织进行了特异性、敏感性和重复性检测。结果显示,该研究建立的RT-PCR方法敏感性高,最低检测阈为10 TCID50/mL,扩增产物的特异性良好,重复性良好,可用于猪繁殖与呼吸综合征的临床诊断及流行病学调查。     

Influence of Hypericum perforatum Extract on Piglet Infected with Porcine Respiratory and Reproductive Syndrome Virus

To study the influence of Hypericum perforatum extract （HPE） on piglets infected with porcine respiratory and reproductive syndrome virus （PRRSV）, enzyme-labeled immunosorbent assay （ELISA） and cytopathic effect （CPE） were used to determine in vitro whether HPE could induce swine pulmonary alveolar macrophages （PAMs） to secrete IFN-γ, and whether PRRSV titers in PAMs were affected by the levels of HPE-induced IFN-γ. HPE （200 mg·kg-1） was administrated by oral gavage to piglets infected with the PRRSV in vivo to observe whether HPE affected the viremia, lung viral titers, and weight gain of piglets infected with PRRSV. The results showed that HPE was capable of inducing PAMs to produce IFN-γ in a dose dependent manner and HPE pretreatment was capable of significantly reducing PRRSV viral titers in PAMs （P〈 0.01）. Administration of HPE to the PRRSV-infected animals significantly （P〈 0.05） reduced viremia over time as compared with the PRRSV-infected animals. But there was not significant decrease in lung viral titers at day 21 post-infection between the HPE- treated animals and the PRRSV-infected control piglets. There were no significant differences in weight gain over time among the HPE-treatment animals, the normal control, and the HPE control animals. The PRRSV-infected animals caused significant （P〈 0.01） growth retardation as compared with the HPE controls and the normal piglets. It suggested that HPE might be an effective novel therapeutic approach to diminish the PRRSV-induced disease in swine.     

猪瘟与高致病性蓝耳病混合感染的诊断及病原特性分析

[目的]诊断由猪瘟病毒（CSFV）、猪繁殖与呼吸综合征病毒（PRRSV）和猪伪狂犬病毒（PRV）引发的猪疫病,并分析病原。[方法]采集福建某猪场发病猪的组织器官,提取病毒DNA或RNA,PCR检测病毒感染。采用ELISA方法检测CSFV、PRRSV和PRV的感染情况。[结果]测序分析和ELISA结果表明,猪场存在PRRSV、CSFV、PRV3种病毒感染。[结论]该次猪病疫情主要是由CSFV和高致病性PRRSV混合感染引起。     

猪伪狂犬病、猪瘟、猪繁殖与呼吸综合症、圆环病毒2型的相关性研究

2003-2004年对广东省猪伪狂犬病(PR)、猪瘟(CSF)、猪繁殖与呼吸综合症(PRRS)、圆环病毒2型(PCV2)病原或野毒抗体调查发现,当前猪场这4种病的混合感染严重.对PR、PCV2、PRRS、CSF的动态流行情况进行相关性分析,结果表明:PR与PCV2之间存在较强的正相关性(P=0.012),相关系数为0.648,互谱图显示,PR与PCV2之间存在同时发生的关系.PCV2与CSF存在较弱的相关性,它们的互谱图表现为:PCV2的流行会微弱影响着未来7个月(负相关)、4个月(正相关)时CSF的流行情况,但这种影响是不显著的(P>0.05).     

猪瘟和猪繁殖与呼吸综合征病毒混合感染的诊断

2009年3月贵州省某猪场送检病猪3例,为确诊病因,进行了流行病学调查,实验室剖检病变观察,细菌学、荧光抗体和RT-PCR检测。经流行病学调查和剖检病变观察,该病例为疑似猪繁殖与呼吸综合征病毒、猪瘟病毒感染所致;该病例细菌分离鉴定结果为阴性,猪瘟直接荧光抗体检测结果为阳性,猪繁殖与呼吸综合征病毒和猪瘟病毒RT-PCR核酸检测为阳性。表明,该病例为猪瘟病毒和猪繁殖与呼吸综合征病毒混合感染。     

猪乙脑快速诊断方法的建立与应用

根据乙脑病毒基因组3′末端保守区设计一对特异性引物,建立了乙脑病毒不同株的RT-PCR诊断技术.该技术可以从乙脑病毒WHe株、SA14-14-2株、P3株、SA14-14-2VS株接种的鼠脑组织中扩增出486bp的核酸片段,检出的灵敏度约为2pg.对20份临床样品的检测结果与小鼠脑内接种试验(MIT)的测定结果完全吻合,但RT-PCR诊断技术可在3h内直接对组织进行诊断,具有敏感、快速、稳定的优点.     

猪繁殖与呼吸综合征病毒新疆株的分离鉴定

从新疆阿克苏地区某规模化猪场发病仔猪体内分离到1株病毒,该病毒能在Marc-145细胞上增殖并产生特征性的细胞病变,而在Vero与BHK-21细胞上不出现细胞病变.病毒能被猪繁殖与呼吸综合征病毒阳性血清特异性地中和,用RT-PCR反应能扩增出369 bp的特异性片段.将病毒接种30日龄血清阴性仔猪,可检测到血清中出现病毒特异性抗体,将分离株命名为XJPRRSV1/03株.     

新疆南疆猪繁殖与呼吸综合征病毒分子流行病学研究

【目的】确定新疆南疆猪养殖场是否存在PRRSV感染,以及感染的PRRSV毒株类型及基因特点,从而针对性的有效防制PRRS。【方法】设计7对引物,对疑似PRRS发病猪场组织样品进行RT-PCR、克隆、测序和序列分析。【结果】47例样品,35例PRRSV阳性,阳性率高达74.47%（35/47）;35株均为美洲型毒株,其中有10株属于高致病性PRRSV,占总检测样品的21.28%（10/47）,占阳性样品的28.57%（10/35）;综合分析本研究采集样品猪养殖场不存在欧洲型毒株。【结论】新疆南疆存在美洲型PRRSV感染,且存在高致病性毒株。     

猪繁殖与呼吸综合征病毒重组N蛋白间接ELISA检测方法的建立

在最佳条件下,通过对重组质粒pGEX-6P-N进行诱导表达,并利用Glutathione Sepharose 4B亲和吸附柱对表达产物（重组N蛋白）进行纯化。用重组N蛋白作为包被抗原,通过对相关条件进行优化（如抗原包被量,血清稀释度,酶标二抗最佳工作浓度,底物作用时间等）,确定判定标准,最终初步建立了检测PRRSV抗体的间接ELISA方法。用建立的间接ELISA方法检测360份血清样品,并与IDEXX公司研制的ELISA试剂盒检测的结果相比较,批内重复性试验变异系数均值为1.52%,批间变异系数为6.17%,符合率达92.5%,试验表明,建立的间接ELISA检测方法具有良好的重复性和特异性,可用于PRRSV血清抗体的检测。     

Evaluation of the Pathogenicity of a Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Variant in Piglets

Since May 2006,a highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) variant characterized by 30 amino acids deletion within its NSP2-coding region emerged and caused extensive economic losses to China's pig industry.To investigate the in vivo pathogenicity and immune responses of the newly emerging PRRSV,3 groups of 60-d-old conventional piglets were inoculated intranasally with a representative strain of the HP-PRRSV variant HuN4 with 3 different infection doses (3× 103-3× 105 TCID50).The results revealed that the virus variant caused severe disease in piglets and the significant clinical characteristics consisted of persistently high fever (41.0-41.9℃) and high morbidity and mortality (60-100％),the marked clinical signs of PRRS and severe histopathogenic damages in multiple organs.It induced rapid and intense humoral immune responses and seroconversion was detected in most infected pigs at 7 d post-infection (DPI).The virus vigorously replicated in vivo and the highest virus average titer was 9.7 log copies mL-1 serum at 7 DPI.Elevated levels of IFN-y and IL- 10 cytokine production in serum in this study were also observed.Taken together,our results demonstrated that the HP-PRRSV variant HuN4 strain is highly pathogenic for piglets and suitable to be a reference strain of highly virulent PRRSV for evaluating the efficacy of the new vaccines.     

Review for Porcine Reproductive and Respiratory Syndrome Virus

Abstract Porcine reproductive and respiratory syndrome(PRRS)is the severest disease of pigs worldwide,caused by a highly genetically diverse RNA virus,called Porcine reproductive and respiratory syndrome virus(PRRSV).The research summarized the genome characteristics of PRRSV particles and the most updated knowledge of structure protein function,and introduced the intellectual of PRRSV transmission and host immune response,which is very important for prevention and control for PRRS.A report showed that mass vaccination can stabilize the immunity of the entire herd,and this is the first required step for a PRRS eradication plan.However,the attenuated live vaccines may not achieve a valid prevention.The final goal of the EU project is to develop new generation,efficacious and safe maker vaccines that can be adapted to temporary changes and geographical differences.Robinson reported that broadly antibodies could neutralize all rapidly evolving typeⅠand typeⅡviruses,while further studies are expected to elucidate mechanisms of neutralizing antibody production and maturation and to investigate conserved epitope targets of cross-neutralization in this rapidly evolving virus.     

高致病性猪蓝耳病、猪瘟和沙门氏菌混合感染的诊断

通过流行病学调查、病猪剖检病变观察和实验室诊断,得出贵州省某猪场发病猪为高致病性蓝耳病、猪瘟和沙门氏菌混合感染.其中,高致病性蓝耳病感染率为100%,猪瘟感染率50%,沙门氏菌感染率25%.