猪瘟病毒荧光定量RT—PCR检测方法的建立及其初步应用

针对CSFV基因组5＇端非编码区序列设计并合成了高度特异的一对引物和一条探针,用于猪瘟病毒实时荧光定量PCR检测方法的建立。将提取的病毒的总RNA做为模板进行反转录和PCR,将PCR产物克隆到pMDl8-T载体后进行大肠杆菌转化,提取阳性质粒做为标准品绘制标准曲线,成功地建立了特异性检测CSFV的荧光定量RT-PCR方...     

Prime-boost immunization using alphavirus replicon and adenovirus vectored vaccines induces enhanced immune responses against classical swine fever virus in mice

This study was designed to evaluate the prime-boost vaccination regimens as a novel immunization strategy for DNA vaccine against classical swine fever virus (CSFV). BALB/c mice were primed with the alphavirus replicon-vectored DNA vaccine pSFV1CS-E2-UL49 encoding the E2 protein of CSFV fused with the UL49 gene encoding the transduction protein VP22 of pseudorabies virus, followed by either homologous boosting with pSFV1CS-E2-UL49 or heterologous boosting with the recombinant adenovirus rAdV-E2 expressing the E2 protein or with the baculovirus-produced recombinant E2 protein (rE2) in adjuvant. The humoral and cell-mediated immune responses following prime-boost vaccination were assessed. The results showed that: (1) boosting with either rAdV-E2 or rE2 elicited high-level antibodies, whereas homologous boosting with pSFV1CS-E2-UL49 elicited low-level antibodies (below positive threshold); (2) heterologous boosting with rAdV-E2 resulted in stronger CD8⁺ and CD4⁺ T cells proliferation responses and higher stimulation indexes; and (3) heterologous boosting with rAdV-E2 induced more IFN-γ production. These results support the notion that a regimen of DNA prime-recombinant adenovirus boost enhances humoral and cell-mediated immune responses, and the DNA prime-protein boost regimen enhances humoral immune responses.     

牛病毒性腹泻病毒感染对猪瘟免疫的影响

猪瘟病毒（CSFV）与同属的牛病毒性腹泻病病毒（BVDV）同源性较高,抗原性上有交叉。本次调查对368份猪瘟免疫猪血清样本进行BVDV抗原检测,其中7份呈阳性,阳性率1.90%。对7份BVDV阳性血清采用ELISA和IHA两种方法检测猪瘟（CSFV）抗体水平,抗体合格率偏低,两者的结果符合率为71%。研究表明：BVDV在一定程度上干扰了猪瘟疫苗的免疫效果,影响抗体水平。     

PK-15细胞膜蛋白提取方法的比较及猪瘟病毒膜表面受体的初步鉴定

以猪肾细胞(PK-15)为材料,比较真核细胞膜蛋白提取试剂盒、超声破碎法、反复冻融法、低渗裂解法4种不同方法对PK-15细胞膜蛋白提取的影响,结果表明,通过超声破碎的膜制备方法提取的PK-15细胞膜蛋白较理想。用此方法提取PK-15细胞膜蛋白,经SDS-PAGE后转印NC膜,利用病毒铺覆蛋白印迹技术(VOPBA)鉴定CSFV病毒的细胞受体。结果发现,PK-15细胞膜上有3种能与CSFV结合的蛋白质,分子量在90～100 ku之间,暗示这些蛋白可能是CSFV受体或者受体复合物。     

Yeast-expressed classical swine fever virus glycoprotein E2 induces a protective immune response

Classical swine fever (CSF) is an economically important swine disease worldwide. The glycoprotein E2 of classical swine fever virus (CSFV) is a viral antigen that can induce a protective immune response against CSF. A recombinant E2 protein was constructed using the yeast Pichia pastoris expression system and evaluated for its vaccine efficacy. The yeast-expressed E2 (yE2) was shown to have N-linked glycosylation and to form homodimer molecules. Four 6-week-old specified-pathogen-free (SPF) piglets were intramuscularly immunized with yE2 twice at 3-week intervals. All yE2-vaccinated pigs could mount an anamnestic response after booster vaccination with neutralizing antibody titers ranging from 1:96 to 1:768. Neutralizing antibody titers at 10 weeks post booster vaccination ranged from 1:16 to 1:64. At this time, the pigs were subjected to challenge infection with a dose of 1 × 10⁵ TCID₅₀ (50% tissue culture infective dose) virulent CSFV strain. At 1 week post challenge infection, all of the yE2-immunized pigs were alive and without symptoms or signs of CSF. Neutralizing antibody titers at this time ranged from 1:4,800 to 1:12,800 and even to 1:51,200 one week later. In contrast, the control pigs continuously exhibited signs of CSF and had to be euthanized because of severe clinical symptoms at 6 days post challenge infection. All of the yE2-vaccinated pigs were E^rns antibody negative and had seroconverted against E^rns by post challenge day 11, suggesting that yE2 is a potential DIVA (differentiating infected from vaccinated animals) vaccine. The yeast-expressed E2 protein retains correct immunogenicity and is able to induce a protective immune response against CSFV infection.     

种公猪精液中猪瘟和蓝耳病病毒混合感染的快速检测

参考GenBank公布的猪蓝耳病病毒(PRRSV)VL2332株、LV株以及猪瘟兔化弱毒(CSFV)C株的基因序列,各设计合成了一对引物,建立了在相同PCR扩增条件下能同时检测PRRSV和CSFV的RT-PCR方法。对2003~2004年期间江苏、浙江、安徽、福建、上海等省市的17个大中型猪场送检的186份种公猪精液进行了检测,结果18份呈PRRSV阳性,24份呈CSFV阳性,其中有11份为PRRSV和CSFV的混合感染,约占送检精液样品的5.91%。试验结果表明,所建立的RT-PCR方法可用于精液中这2种野毒感染的快速鉴定和分子流行病学调查。     

CSFV的RT-PCR检测在控制与净化猪瘟中的应用

应用反转录-聚合酶链反应(RT-PCR)检测健康猪扁桃体猪瘟病毒以监控与净化猪瘟.2006年用RT-PCR对广西某存栏250头种猪场的母猪扁桃体连续进行3次猪瘟病毒检测,检出并清除带毒猪,猪群中猪瘟病毒的带毒猪明显下降.结果表明,RT-PCR检测猪扁桃体可应用于猪场猪瘟的控制与净化.     

Generation and efficacy evaluation of a recombinant adenovirus expressing the E2 protein of classical swine fever virus

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), which causes significant economic losses to the pig industry worldwide. The E2 glycoprotein of CSFV is the main target for neutralizing antibodies. This study was aimed to develop a recombinant human adenovirus type 5 expressing the CSFV E2 gene (rAdV-E2) and evaluate its efficacy in rabbits and pigs. The results showed that the rabbits and the pigs immunized with the rAdV-E2 developed high-level CSFV-specific neutralizing antibodies. The rAdV-E2-immunized rabbits were protected from fever induced by infection with C-strain, which is pathogenic to the rabbit, and the rAdV-E2-immunized pigs were protected from lethal challenge with highly virulent Shimen strain. This indicates that the recombinant adenovirus can be an attractive candidate vaccine for preventing CSF.     

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.     

Factors critical for successful vaccination against classical swine fever in endemic areas

Classical swine fever (CSF) or hog cholera, caused by the classical swine fever virus (CSFV), is one of the most important viral diseases that cause serious economic loss to the swine industry worldwide. During the past 5 years, several techniques for measuring porcine cell-mediated immunity (CMI) were applied, in conjunction with other conventional techniques, to study factors that influence the induction of CSFV-specific immunity. Information, obtained from a series of experiments, demonstrated cell-mediated immune responses in providing protective immunity against CSF infection. Although it has been confirmed that commercially available modified live CSF vaccines are able to induce complete protection in vaccinated pigs, several factors including maternal immunity, the age of primary vaccination, vaccination protocol and complications caused by other pathogens, can greatly affect the effectiveness of CSF vaccines in the field.     

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.     

表达猪瘟病毒E2蛋白重组猪繁殖与呼吸道综合征病毒的研究

为构建表达猪瘟病毒(CSFV)E2蛋白重组猪繁殖与呼吸道综合征病毒(PRRSV),本研究首先利用高致病性PRRSV弱毒疫苗HuN4-F112株的感染性分子克隆作为平台,构建了一个在nsp2区有缺失的感染性分子克隆,命名为pHuN4-F112-△480-620。以pHuN4-F112-△480-620作为载体,采用突变PCR的方法将CSFV的主要保护性抗原E2基因1 bp～9 99 bp,1 bp～600 bp,1 bp～330 bp及256 bp～330 bp基因片段分别插到nsp2中aa 480～aa 620位氨基酸缺失编码区域。结果显示,插入完整E2基因或较大E2基因片段的重组PRRSV cDNA质粒均未能拯救出病毒,只有插入较小的E2基因片段(256 bp～330 bp)的重组病毒cDNA质粒成功地拯救出了重组病毒rPRRSV-F112-E2(256-330),拯救的病毒能够在MARC-145细胞上引起明显的细胞病变,而且生长速度明显高于其亲本病毒,间接荧光检测表明该重组病毒能够表达外源基因。     

Comparing the epidemiological and economic effects of control strategies against classical swine fever in Denmark

In 2006, total Danish pork exports were valued at €3.8 billion, corresponding to approximately 5% of the total Danish exports, and an outbreak of a notifiable disease would have dramatic consequences for the agricultural sector in Denmark. Several outbreaks of classical swine fever (CSF) have occurred in Europe within the last decade, and different control strategies have been suggested. The objective of this study was to simulate the epidemiological and economic consequences of such control strategies in a CSF epidemic under Danish conditions with respect to herd demographics and geography and to investigate the effect of extra biosecurity measures on farms. We used InterSpread Plus to model the effect of nine different control strategies: the minimum measures required by the EU plus depopulation of contact herds (EUplus), extra depopulation of neighbouring herds, extra surveillance within the protection and surveillance zones, extra biosecurity in SPF herds—or in all herds, vaccination of all pigs in the 1 or 2 km zones using live vaccine as a protective measure (vaccination-to-kill), vaccination of all weaners and finishers in the 1 or 2 km zones using an E2 marker vaccine as a suppressive measure (vaccination-to-live). Each epidemic was simulated to start in four different index herds: production herds located in low, medium and high pig density areas, respectively; and a nucleus herd in an area of high pig density. For each control strategy and index case, we calculated the size and duration of the epidemic, the number of depopulated and/or vaccinated herds and animals, the control costs borne by the public and the pig industry, respectively, as well as the loss of exports associated with the epidemic.The simulations showed that the EUplus strategy is the most effective of the evaluated strategies with respect to limiting the size, duration and cost of the epidemic, regardless of the index case. However, regarding the number of slaughtered animals, the vaccination-to-live strategies appeared to be more effective.Epidemics become larger and last longer if the index case is a nucleus herd. This implies that biosecurity in nucleus herds is extremely important to avoid transmission of CSF to these herds.Simulations showed that a Danish CSF epidemic will be moderate in most cases and will include fewer than 10 cases and last less than 2 weeks on average. However, for some iterations, long-lasting and large epidemics were observed. Irrespective of the size and duration, an epidemic is expected to be very costly due to the export losses.     

用荧光定量RT-PCR方法检测猪瘟病毒

为了建立能特异检测不同基因型猪瘟病毒(Classical swine fever virus,CSFV),同时又能区分其他瘟病毒的基因检测方法,本实验针对CSFV基因组5′端非编码区设计并合成了简并引物和TaqMan探针,在优化反应条件的基础上,成功地建立了特异检测CSFV的荧光定量RT-PCR检测方法。再以已知滴度的CSFV石门株血毒总RNA反转录产物建立标准品,该标准品可以用于定量临床样品中的CSFV滴度,所建立的荧光定量PCR方法可以灵敏地检测出10~(-0.82)个TCID_(50)病毒含量。最后用建立的方法对108份临床样品进行检测并同时进行病毒分离,荧光定量PCR方法检测出73份阳性样品且与病毒分离的符合率为100%,而常规RT-PCR只检测出54份阳性样品,表明本荧光定量RT-PCR法在检测猪瘟病料上具有潜在的应用价值。     

猪瘟病毒持续感染与猪瘟预防控制

研究证明,在自然条件下,猪瘟病毒持续感染通常是在免疫力较低的情况下,由于环境中的病毒反复感染产生的,由于感染猪还有一定的免疫力,病毒虽然可以在其体内局部存留,但还不足以引起猪发病.一般情况下,感染猪虽然持续带毒,但不表现临床症状,然而却可以不断向外排毒,再次感染其他猪,污染环境;带毒的种猪可以通过母猪的胎盘和公猪的精液传播给仔猪,造成仔猪的先天免疫耐受,导致疫苗免疫失败.实验还证明,即使在良好的免疫状况下,反复多次人工感染猪瘟强毒也可造成持续感染.由此可见,猪瘟病毒持续感染是一个复杂的问题.     

猪瘟诊断方法的研究进展

猪瘟是由猪瘟病毒引起的一种急性、发热性、接触性传染病,可引起各种年龄猪发病。随着对猪瘟病毒研究的深入,猪瘟在一定程度上得到了有效控制。但是近年来,世界各国流行的猪瘟在流行病学、临床症状和病理变化等方面出现了一些新的变化,猪瘟的防控出现了许多新的情况。我国猪瘟的发病率亦呈上升趋势,严重威胁着我国养猪业的发展,给养猪业造成了极大的经济损失。因此,建立准确的实验室诊断方法,对于预防和控制猪瘟有重要意义。本文综述了猪瘟诊断技术方面的研究进展,为猪瘟的及时诊断提供参考。     

三个厂家猪瘟活疫苗免疫效果比较试验

在同一条件下对3个厂家生产的猪瘟细胞源活疫苗进行了免疫效果评价试验,并与猪瘟传代细胞苗免疫效果进行比较。结果发现3个厂家生产的猪瘟细胞源活疫苗存在一定差异,2个厂家的免疫效果较好,1个厂家的免疫效果较差。猪瘟传代细胞苗免疫效果优于猪瘟细胞源活疫苗,猪瘟传代细胞苗免疫1次,抗体合格率高,持续时间长,猪瘟细胞源活疫苗要免疫两次才能获得比较好的效果。同时,我们发现高致病性猪蓝耳病活疫苗（JXA1-R株）对猪瘟抗体产生有一定影响。     

猪瘟病毒野毒株RT-LAMP可视化检测方法的建立

本研究旨在建立一种可视化检测猪瘟病毒(CSFV)野毒株的反转录.环介导等温扩增方法(RT-LAMP).根据CSFV的NS5B基因序列设计一套RT-LAMP引物,以样品的cDNA为模板,利用Bsf DNA聚合酶,在62℃恒温条件下进行扩增,扩增产物中加入sYBR Green I染料直接或在紫外光下观察判定扩增结果.该方法可检测出不同基因型的CSFV厂野毒株,其检出极限为2.5 TCID_(50)的CSFV,与实时荧光定量RT.PCR方法的敏感性相当;特异性试验表明,该方法对猪瘟免化弱毒疫苗株(HCLV)、牛病毒性腹泻病毒以及其它常见猪源病毒均无扩增反应;通过对126份不同样品进行检测比较,该方法与实时荧光定量RT-LAMP检测方法的符合率达100%.与引物.探针能量转移PCR方法的符合率为98.4%.该方法无需特殊仪器,是一种适用于基层的快速、简便的CSFV野毒鉴别检测方法.