A multiplex RT-PCR assay for the rapid and differential diagnosis of classical swine fever and other pestivirus infections

Classical swine fever is a highly contagious viral disease causing severe economic losses in pig production almost worldwide. All pestivirus species can infect pigs, therefore accurate and rapid pestivirus detection and differentiation is of great importance to assure control measures in swine farming. Here we describe the development and evaluation of a novel multiplex, highly sensitive and specific RT-PCR for the simultaneous detection and rapid differentiation between CSFV and other pestivirus infections in swine. The universal and differential detection was based on primers designed to amplify a fragment of the 5′ non-coding genome region for the detection of pestiviruses and a fragment of the NS5B gene for the detection of classical swine fever virus. The assay proved to be specific when different pestivirus strains from swine and ruminants were evaluated. The analytical sensitivity was estimated to be as little as 0.89 TCID₅₀. The assay analysis of 30 tissue homogenate samples from naturally infected and non-CSF infected animals and 40 standard serum samples evaluated as part of two European Inter-laboratory Comparison Tests conducted by the European Community Reference Laboratory, Hanover, Germany proved that the multiplex RT-PCR method provides a rapid, highly sensitive, and cost-effective laboratory diagnosis for classical swine fever and other pestivirus infections in swine.     

猪瘟病毒可视化RT-LAMP检测方法的建立

通过对GenBank中注册的CSFV序列进行比对分析,针对CSFV NPro/C基因中的保守区域,设计合成了一套RT-LAMP引物,应用含有Npro/C基因的阳性质粒株对RT-LAMP引物进行验证.在成功扩增出特异片段的基础上,用CSFV RNA优化RT-LAMP反应体系和条件,建立了CSFV的可视化RT-LAMP快速...     

猪瘟病毒E2蛋白在酵母中分泌表达条件的优化

影响外源基因在毕赤酵母中表达的因素很多，除了基因序列本身的内在特性外，表达条件对外源基因的表达量的高低也有着极显著的影响。本文对猪瘟病毒E2基因在毕赤酵母中不同的时间、不同的诱导型、不同的pH、不同的诱导剂量等方面表达量的差异进行了详细对比研究。结果表明，诱导后72～96h，重组E2蛋白的表达量最高。与单纯甲醇诱导相比，甘油／甲醇诱导后的表达量明显提高。在pH7．5和pH8．0表达E2蛋白是比较合适的。甲醇浓度在3％左右时E2蛋白的表达量较高。     

我国近期7株猪瘟流行野毒E2基因变异研究

应用RT PCR 和nPCR 扩增了7 株国内近期(2001 年-2003 年)流行的猪瘟野毒E2 基因,分别克隆至pGEM T 载体并对其进行了核苷酸序列测定及氨基酸序列推导,同时将其与C 株、Alfort 株、Brecsia 株进行了同源性比较及遗传进化分析,构建了CS FV的遗传发生树,并对E2 结构与功能进行了分析。所测7株野毒均包括完整的信号肽序列及部分跨膜区在内的1 170 bp,与C株、Alfort株、Brescia 株核苷酸序列同源性分别为91.6%~94.5%、89.2%~92.7%、85.9%~89.3%,氨基酸同源性分别为91.2%~95.8%、88.9%~92.0%、84.0%~90.1%;而7株野毒之间的差异很小,其核苷酸序列同源性为95.8%~99.7%,氨基酸同源性为96.3%~99. 1%。所绘制的遗传发生树分为2个组群,所测得7 株流行野毒均属于第1 群,而且可分为两亚群,与C 株在同一亚群。同时对主要抗原区氨基酸位点变异进行了分析,对其抗原决定簇的变异情况进行了推测。     

用生物信息学手段辅助分析猪瘟病毒的基因结构

借助生物信息软件,对猪瘟病毒的分子演化关系、RNA 3'端非编码区(NTR)序列及二级结构、E2基因的亲水性及抗原指数等进行了辅助分析.经检索得到CSFV全长序列23条,序列片段369条.确定了AF352565(LPC)株的碱基缺失在ORF的1174～1176位.采用基因组序列的全长ORF进行了比对并绘制了进化树,对E2基因的亲水谱与抗原指数进行计算,推测E2基因抗原性变化不大,但不同毒株之间可能存在微小差异.比较了CSFV 3'端NTR的序列并预测了3'端NTR的RNA二级结构,表明不同毒株之间存在较多差异.     

Experimental infection of pigs with different doses of the African swine fever virus Armenia 07 strain by intramuscular injection and direct contact

We experimentally infected pigs with the African swine fever virus (ASFV) Armenia 07 strain (genotype II) to analyze the effect of different dose injections on clinical manifestations, virus-shedding patterns, histopathology, and transmission dynamics by direct contact. Each three pigs and four pigs were injected intramuscularly with 0.1 fifty percent hemadsorbing doses (HAD₅₀)/ml, 10¹ HAD₅₀/ml and 10⁶ HAD₅₀/ml of ASFV Armenia 07 strain, respectively. Each two of three pigs injected with 0.1 HAD₅₀/ml and 10¹ HAD₅₀/ml died by 10 days post inoculation. All pigs had a gross lesion of splenomegaly. Perigastric and renal lymph nodes were enlarged and resembled blood clots in nine of ten pigs. It was revealed that 0.1 HAD₅₀/ml of this ASFV was sufficient to infect healthy pigs by intramuscular injection and caused sub-acute lethal disease. For the transmission study, two 8-week-old pigs were injected intramuscularly with 10³ HAD₅₀/ml of the same virus. Each of the experimentally inoculated pigs was co-housed with two 8-week-old naive pigs. All contact pigs exhibited clinical manifestations at 6 or 7 days after the experimentally inoculated pigs developed pyrexia. These findings suggest that this strain may spread slowly within a herd. Histologically, lymph nodes resembled blood clots were formed by severe blood absorption and followed hemorrhage result of disruption of the lymphoid sinus filling with absorbed red blood cells. The severity of the gross and histological lesions depended on duration after infection, regardless of the difference of injection doses in this study.     

Laboratory decision-making during the classical swine fever epidemic of 1997-1998 in The Netherlands

The National Reference Laboratory for classical swine fever (CSF) virus in the Netherlands examined more than two million samples for CSF virus or serum antibody during the CSF epizootic of 1997–1998. The immense amount of samples and the prevalence of border disease (BD) virus and bovine viral diarrhoea (BVD) virus infections in Dutch pig herds necessitated the diagnostic efforts of the laboratory to be focused on generating CSF specific test results throughout the eradication campaign.

Detection of 82% of the 429 outbreaks was achieved through the combined use of a direct immunofluorescence and peroxidase assay (FAT/IPA) with samples (tonsils) collected from clinically-suspected pigs. This suggests that in the majority of the outbreaks, the pigs had clinical signs that were recognised by the farmer and/or veterinarians, indicating the presence of CSF virus in a pig herd. A positive diagnosis of 74% of all the tissue samples (tonsils) collected at infected pig holdings was established by FAT. More than 140,000 heparinised blood samples were examined by virus isolation, resulting in the detection of 4.5% of the infected herds. CSF virus was isolated in approximately 29% of all the blood samples collected from pigs at infected or suspected farms.

Several serological surveys — each done within a different framework — led to the detection of 13.5% of the total number of outbreaks. The detection of CSF virus antibody in serum was carried out by semi-automated blocking ELISA. Approximately 28.5% of the sera which reacted in the ELISA were classified as CSF virus-neutralising antibody positive and 26.5% as positive for other pestiviruses following the virus neutralisation test (VNT).

We concluded that two of the CSF laboratory diagnostic methods described were determinative in the eradication campaign: first, the FAT for the screening of diseased pigs; and second, the ELISA and VNT when millions of predominantly healthy pigs needed to be screened for the presence of CSF serum antibody. Decision-making on the basis of results generated by either method can, however, be seriously hindered when samples are examined from pig herds with a high prevalence of non-CSF pestiviruses.     

Cost-effectiveness of measures to prevent classical swine fever introduction into The Netherlands

Recent history has demonstrated that classical swine fever (CSF) epidemics can incur high economic losses, especially for exporting countries that have densely populated pig areas and apply a strategy of non-vaccination, such as The Netherlands. Introduction of CSF virus (CSFV) remains a continuing threat to the pig production sector in The Netherlands. Reducing the annual probability of CSFV introduction (P_CSFV) by preventive measures is therefore of utmost importance. The choice of preventive measures depends not only on the achieved reduction of the annual P_CSFV, but also on the expenditures required for implementing these measures. The objective of this study was to explore the cost-effectiveness of tactical measures aimed at the prevention of CSFV introduction into The Netherlands. For this purpose for each measure (i) model calculations were performed with a scenario tree model for CSFV introduction and (ii) its annual cost was estimated. The cost-effectiveness was then determined as the reduction of the annual P_CSFV achieved by each preventive measure (ΔP) divided by the annual cost of implementing that measure (ΔC). The measures analysed reduce the P_CSFV caused by import or export of pigs. Results showed that separation of national and international transport of pigs is the most cost-effective measure, especially when risk aversion is assumed. Although testing piglets and breeding pigs by a quick and reliable PCR also had a high cost-effectiveness ratio, this measure is not attractive due to the high cost per pig imported. Besides, implementing such a measure is not allowed under current EU law, as it is trade restrictive.     

猪瘟病毒Erns和E2蛋白重组表达及抗体间接ELISA检测方法的建立

本研究将CSFV Erns和E2基因的主要抗原区进行克隆,构建pET32a-Erns和pET32aEK-E2重组质粒,将其转化大肠杆菌BL21(DE3)进行诱导表达和纯化,以纯化的rErns、rE2重组蛋白为包被抗原,经条件优化,分别建立CSFV rErns和rE2抗体间接ELISA检测方法.SDS-PAGE结果显示,...     

A novel spatial and stochastic model to evaluate the within and between farm transmission of classical swine fever virus: II validation of the model

A new, recently published, stochastic and spatial model for the evaluation of classical swine fever virus (CSFV) spread into Spain has been validated by using several methods. Internal validity, sensitivity analysis, validation using historical data, comparison with other models and experiments on data validity were used to evaluate the overall reliability and consistency of the model. More than 100 modifications in input data and parameters were evaluated. Outputs were obtained after 1000 iterations for each new scenario of the model. As a result, the model was shown to be consistent, being the probability of infection by local spread, the time from infectious to clinical signs state, the probability of detection based on clinical signs at day t after detection of the index case outside the control and surveillance zones and the maximum number of farms to be depopulated at day t the parameters that have more influence (>10% of change) on the magnitude and duration of the epidemic. The combination of a within- and between-farm spread model was also shown to give significantly different results than using a purely between-farm spread model. Methods and results presented here were intended to be useful to better understand and apply the model, to identify key parameters for which it will be critical to have good estimates and to provide better support for prevention and control of future CSFV outbreaks.     

猪瘟病毒和不同型猪繁殖与呼吸综合征病毒的多重RT-PCR检测方法的建立

为建立能同时检测猪瘟病毒(CSFV)和猪繁殖与呼吸综合征病毒(PRRSV)的方法,针对CSFV和PRRSV的基因序列设计3对特异性引物,第1对引物扩增CSFV毒株NS2基因508 bp片段,第2对引物扩增PRRSV美洲型经典毒株和变异毒株Nsp2基因338 bp/248 bp片段,第3对引物扩增PRRSV欧洲型毒株ORF5基因614 bp片段.经过反应条件的优化,建立了能同时检测并区分CSFV毒株和PRRSV美洲型经典毒株、变异毒株及欧洲型毒株的多重RT-PCR方法.该方法可以特异扩增CSFV和PRRSV,而与猪口蹄疫病毒(FMDV)、猪伪狂犬病病毒(PRV)、猪细小病毒(PPV)、猪圆环病毒2型(PCV-2)均无交叉反应;对CSFV和PRRSV 4种重组质粒标准品的检出下限均为1.67×103拷贝/μL.对采集的106份临床疑似病料进行检测,结果CSFV和PRRSV变异株混合阳性4份,占3.77 ％(4/106);CSFV阳性7份,占6.60％(7/106);PRRSV变异株阳性17份,占16.04 ％(17/106).结果表明,建立的多重RT-PCR检测方法可以用于CSFV和PRRSV的临床快速鉴别诊断和流行病学调查.     

表达猪瘟E2基因的重组伪狂犬病毒构建及其 生物学特性分析

以构建的TK、gI、gE、US9、部分gN基因缺失的绿色荧光标记猪伪狂犬病毒rPRV-BE为亲本株,通过同源重组构建了表达猪瘟病毒(CSFV C株)主要免疫原性基因E2的重组伪狂犬病毒rPRV-CSFV PE2SC。经PCR、间接免疫荧光试验(IFA)鉴定证实构建正确,并能成功表达E2蛋白。同时,对重组病毒在PK15细胞中的遗传稳定性、增殖特性等进行了研究,结果显示第5、10、15、20代重组病毒经PCR均能扩增出与原代重组病毒相同大小的约为3469 bp含猪瘟病毒E2基因的重组片段;第20代重组病毒感染细胞孔中出现明显的针对E2蛋白的荧光,表明重组病毒在体外连续传20代后仍能稳定遗传。一步生长曲线测定结果表明,与野毒PRV(SX株)、亲本毒rPRV-BE相比,重组病毒rPRV-CSFV PE2SC前期增殖速度较快,病毒滴度到达峰值的时间早,峰值病毒滴度低于野毒,与亲本毒相当,最高病毒滴度依次为10~(6.7)TCID_(50)/mL、10~(8.0)TCID_(50)/mL、10~(7.0)TCID_(50)/mL。结果显示构建的重组病毒具有良好的遗传稳定性及体外复制能力,为进一步对其免疫效果的评价以及PRV基因缺失重组疫苗的研制奠定基础。     

Efficacy of E2-sub-unit marker and C-strain vaccines in reducing horizontal transmission of classical swine fever virus in weaner pigs

At present, two types of vaccines against classical swine fever (CSF) virus are commercially available: E2 sub-unit marker vaccines and the conventional attenuated live C-strain vaccines. To evaluate the reduction of the horizontal virus transmission, three comparable experiments were carried out in which groups of weaner pigs (vaccinated with a marker vaccine or a C-strain vaccine) were challenged with CSF virus at 0, 7, and 14 days post-vaccination (dpv). Virus transmission was prevented totally when the challenge occurred at 14 dpv with an E2-marker vaccine (0/12 contact pigs positive in virus isolation (VI); R = 0 (0; 1.5)). At 7 dpv, transmission was reduced slightly (5/12 contact pigs positive in VI; R = 1.0 (0.3; 3.0)), whereas at 0 dpv, vaccination had no effect on transmission (10/12 contact pigs positive in VI; R = 2.9 (1.5; 10.8)). In the C-strain-vaccinated pigs, no virus transmission was detected even when the challenge was performed at the same day as the vaccination (0/12 contact pigs positive in VI; R = 0 (0; 1.5)).     

检测CSFV、JEV、PRRSV三种RNA病毒多重RT-PCR方法的建立

猪瘟病毒(CSFV)、流行性乙型脑炎病毒(JEV)和猪繁殖与呼吸综合征病毒(PRRSV)是引起严重的种猪繁殖障碍的病原,而且经常混合感染,及时准确诊断是防治的前提。根据GenBank发表序列选取3对引物建立检测CSFV、JEV和PRRSV病毒的多重RT-PCR方法,扩增产物分别为508 bp、380 bp、263 bp。经与IDEXX商品化的检测CSW抗原试剂盒比较,二者的符合率为96.7%;扩增JEV和PRRSV PCR产物分别经EcoR V和Sau3A I酶切得到预期的片段。建立的多重RT-PCR检测JEV、PRRSV和CSFV敏感度分别为12.5个TCID_(50)、10个TCID_(50)和10~(-3)ng总RNA。结果表明该多重RT-PCR方法具有很好的特异性和敏感性,可用于临床三种病毒核酸的检测。     

新乡市某猪场猪瘟病毒的鉴别检测

新乡市某猪场发生疑似猪瘟病例,为了鉴别其为疫苗毒还是野毒,采用细胞免疫化学方法和RT-PCR,并对其NS5B基因进行序列测定,序列比对并构建进化树,结果表明,猪只为猪瘟野毒感染,且分离的猪瘟病毒与石门(Shimen)株在基因序列上未发生大的变异。     

利用随机肽库鉴定猪瘟病毒E2蛋白抗原表位

利用噬菌体随机12肽库对抗猪瘟病毒（classical swine fever virus CSFV）糖蛋白E2特异的单抗A11进行表位鉴定，经过4轮筛选后，随机挑取10个噬菌体克隆作竞争ELISA检测。结果表明，10个克隆中除4号克隆外，其余9个均能抑制原核表达的E2蛋白和A1l单抗之间的抗原抗体反应，抑制率在35％～64％；DNA测序表明，所有产生竞争抑制作用的8个噬菌体克隆的12肽序列均舍有XXWRXXXL核心序列，而没有抑制作用的克隆则不含该核心序列；Western-blot试验证明，所挑阳性克隆均能被单抗A11识别。多序列比较发现，该核心序列与猪瘟病毒E蛋白的28～35位氨基酸TTWKEYSH有一定的同源性，人工合成的含有部分核心序列氨基酸的多肽可以与单抗A11反应，表明单抗A11所针对的抗原表位位于CSFVE2蛋白的28～35位氨基酸。     

斑点免疫金渗滤法检测猪瘟抗体的研究

以猪瘟抗原包被硝酸纤维素膜，然后用胶体金标记SPA，建立检测猪瘟抗体水平的斑点免疫金渗滤法检测试纸盒。通过胶体金标记金黄色葡萄球菌A蛋白(SPA)直接显色，阳性者出现红色斑点，结果易于判断。整个试验过程仅需5min，操作简单，与猪兰耳病、猪伪狂犬病、猪细小病毒、新城疫和禽流感等阳性血清不发生交叉反应。同时将该法与目前猪瘟的常规检测方法Dot—ELISA法和间接血凝试验同时对200份猪血清进行猪瘟抗体检测比较，符合率达98．4％和98．9％。说明该法微量、特异、敏感可靠，检测时间短，效果直观，非常适用于猪瘟的早期诊断和普查以及疫苗免疫效果后抗体水平的检测。     

Phylogenetic analysis of recent classical swine fever virus (CSFV) isolates from Assam, India

Classical swine fever (CSF), a highly contagious viral disease of pigs, is endemic in India. As there is no information concerning the accurate genetic typing of classical swine fever virus (CSFV) isolates in India, 16 CSF viruses isolated during 2005-2007 from domestic pigs in different districts of Assam were typed in 5′ UTR (150 nucleotides). To confirm the genetic typing results and to study the genetic variability, selected viruses were also analyzed in E2 (190 nt) and NS5B gene (409 nt) regions. Phylogenetic analysis revealed that all the 16 CSFV isolates analyzed belonged to group 1 and subgroup 1.1 in contrast to the situation in other Asian countries. Additionally, analysis in E2 and NS5B region placed the Indian isolates in a clearly separated clade within subgroup 1.1. The results suggest that subgroup 1.1 CSF viruses are currently circulating in India, which is important for epidemiology and control of CSF.     

Improved sero-monitoring assay for classical swine fever (CSF) using the recombinant E2 protein of a recent Korean isolate

Classical swine fever (CSF) is a highly contagious disease of pigs that causes fever, diarrhea and paralysis, often resulting in death. E2 is the major structural protein of the CSF virus (CSFV) and mediates the entrance of the virus, subsequently inducing a neutralizing immune response. In this study, the E2 gene of a recent Korean isolate of CSF, SW03, was cloned and the DNA sequence was compared to other strains via phylogenetic analysis. With the purified E2 protein, an enzyme-linked immunosorbent assay (ELISA) was developed for the serodiagnosis of CSFV infection. The sensitivity and specificity of the E2-ELISA were 96.1% and 94.8%, respectively. A total of 17 out of 485 field-collected pig sera tested demonstrated conflicting results between two ELISA methods, a commercial kit and the E2-ELISA. Of these sera, 60% were determined to be CSFV positive by a virus neutralization test (VNT), suggesting involvement of different immune responses in the cases of CSFV infection. As the E2-ELISA was developed using a recent Korean isolate, SW03, this assay is capable of rapidly identifying newly emerging CSFV strains.