Efficacy of a live attenuated vaccine in classical swine fever virus postnatally persistently infected pigs

Classical swine fever (CSF) causes major losses in pig farming, with various degrees of disease severity. Efficient live attenuated vaccines against classical swine fever virus (CSFV) are used routinely in endemic countries. However, despite intensive vaccination programs in these areas for more than 20 years, CSF has not been eradicated. Molecular epidemiology studies in these regions suggests that the virus circulating in the field has evolved under the positive selection pressure exerted by the immune response to the vaccine, leading to new attenuated viral variants. Recent work by our group demonstrated that a high proportion of persistently infected piglets can be generated by early postnatal infection with low and moderately virulent CSFV strains. Here, we studied the immune response to a hog cholera lapinised virus vaccine (HCLV), C-strain, in six-week-old persistently infected pigs following post-natal infection. CSFV-negative pigs were vaccinated as controls. The humoral and interferon gamma responses as well as the CSFV RNA loads were monitored for 21 days post-vaccination. No vaccine viral RNA was detected in the serum samples and tonsils from CSFV postnatally persistently infected pigs for 21 days post-vaccination. Furthermore, no E2-specific antibody response or neutralising antibody titres were shown in CSFV persistently infected vaccinated animals. Likewise, no of IFN-gamma producing cell response against CSFV or PHA was observed. To our knowledge, this is the first report demonstrating the absence of a response to vaccination in CSFV persistently infected pigs.     

表达增强型绿色荧光蛋白的报告猪瘟病毒C株的构建及在家兔中生物学特性评价

猪瘟兔化弱毒疫苗C株是我国自主研发的,用于预防猪瘟（CSF）的最好弱毒活疫苗。然而,现阶段的C株不具备标记功能,无法区分野毒感染与疫苗接种。本研究旨在将增强型绿色荧光蛋白（EGFP）编码基因插入猪瘟病毒（CSFV）C株将其构建为报告病毒,为C株的基础研究提供病毒示踪工具,同时提供构建标记C株疫苗的方法。本研究中,笔者在CSFV C株中引入EGFP基因,分别构建了表达增强型绿色荧光蛋白（EGFP）的报告CSFV rHCLV-EGFP,以及用CSFV强毒Shimen（SM）株N^pro基因替换C株N^pro基因后再引入EGFP基因的嵌合报告病毒rHCLV-N^pro（SM）-EGFP。通过猪瘟抗原ELISA检测,直接观察荧光及Western blot检测EGFP的蛋白表达等方法对拯救的病毒进行鉴定,并在细胞和家兔上评价这两株病毒的生物学特性。结果显示,两株报告病毒的抗原ELISA结果均为阳性;在感染rHCLV-EGFP的细胞中并未观察到绿色荧光也未检测到EGFP蛋白,但在感染rHCLV-N^pro（SM）-EGFP的细胞中观察到EGFP的绿色荧光并检测到EGFP蛋白;细胞试验结果表明,两株报告病毒与亲本病毒具有相似的生长特性且遗传稳定;家兔试验发现,rHCLV-N^pro（SM）-EGFP保留了C株的在家兔体内的生物学特性,但rHCLV-EGFP失去了C株诱导家兔定型热反应的能力。构建的嵌合报告病毒rHCLV-N^pro（SM）-EGFP可以作为C株的报告病毒进行应用：可以对病毒示踪,用于研究C株的复制过程、病毒与细胞的相互作用;同时,该嵌合报告病毒也具备发展为标记C株疫苗的潜力。     

CP7_E2alf oral vaccination confers partial protection against early classical swine fever virus challenge and interferes with pathogeny-related cytokine responses

The conventional C-strain vaccine induces early protection against classical swine fever (CSF), but infected animals cannot be distinguished from vaccinated animals. The CP7_E2alf marker vaccine, a pestivirus chimera, could be a suitable substitute for C-strain vaccine to control CSF outbreaks. In this study, single oral applications of CP7_E2alf and C-strain vaccines were compared for their efficacy to induce protection against a CSF virus (CSFV) challenge with the moderately virulent Bas-Rhin isolate, in pigs as early as two days post-immunization. This work emphasizes the powerful potential of CP7_E2alf vaccine administered orally by a rapid onset of partial protection similar to that induced by the C-strain vaccine. Furthermore, our results revealed that both vaccinations attenuated the effects induced by CSFV on production of the pig major acute phase protein (PigMAP), IFN-α, IL-12, IL-10, and TGF-β1 cytokines. By this interference, several cytokines that may play a role in the pathogeny induced by moderately virulent CSFV strains were revealed. New hypotheses concerning the role of each of these cytokines in CSFV pathogeny are discussed. Our results also show that oral vaccination with either vaccine (CP7_E2alf or C-strain) enhanced CSFV–specific IgG2 production, compared to infection alone. Interestingly, despite the similar antibody profiles displayed by both vaccines post-challenge, the production of CSFV-specific IgG1 and neutralizing antibodies without challenge was lower with CP7_E2alf vaccination than with C-strain vaccination, suggesting a slight difference in the balance of adaptive immune responses between these vaccines.     

采用SYBR Green I实时荧光定量RT-PCR方法对猪瘟强毒与疫苗弱毒的鉴别研究

为建立一种能够区分猪瘟病毒(CSFV)强毒与弱毒疫苗C株的SYBR Green Ⅰ荧光定量RT-PCR结合熔解曲线分析方法,本研究对GenBank中登录的25株CSFV强毒株和兔化弱毒疫苗C株全基因组序列进行比较分析,设计一对共用下游引物以及分别针对CSFV强毒株与弱毒疫苗的特异性上游引物,其Tm值分别为84.5±0.5℃和88.5±0.5℃,熔解曲线分析显示为单特异峰.检测结果显示本实验建立的鉴别CSFV强毒感染与弱毒疫苗的荧光定量RT-PCR结合熔解曲线分析方法特异性强,对其他相关病毒无特异性扩增;敏感性高,最低检出量为5×RID50的细胞疫苗基因组拷贝;重复性好;并且扩增效率高、线性范围广、检测时间短,可对免疫猪群中CSFV强毒感染做出快速准确的鉴别检测,为有效防制猪瘟提供依据.     

Classical swine fever virus strain "C" protects the offspring by oral immunisation of pregnant sows

The aim of this study was to evaluate if oral immunisation of wild sows protects the fetuses from transplacental infection. Two experiments were carried out with gilts vaccinated orally with C-strain virus approximately 5 weeks after insemination. They were challenged at mid-gestation with highly virulent classical swine fever virus (CSFV) or moderately virulent field virus. The results revealed that oral vaccination has no negative impact on the pregnancy, and all vaccinated sows developed neutralising antibodies. After infection no symptoms were detected in the six vaccinated-infected sows. Challenge virus could neither be found in blood, nasal and fecal swabs or saliva nor in organs sampled at necropsy. Likewise, all fetuses originating from vaccinated sows were virologically and serologically negative. In contrast, the controls developed a short viremia and as a result of the transplacental infection all fetuses were CSFV positive. In addition, 22 serologically positive wild sows of an endemically infected area, where oral vaccination had also been carried out, and their offspring were free from CSFV or viral RNA. Our results confirm that oral immunisation of pregnant wild sows with C-strain vaccine may protect the fetuses against CSF.     

猪瘟病毒E2蛋白主要抗原区编码基因的原核表达及抗原性研究

本试验旨在分析猪瘟病毒E2蛋白的免疫反应,应用RT-PCR方法扩增了编码猪瘟病毒兔化弱毒疫苗株C株囊膜糖蛋白E2基因主要编码区,并定向克隆到表达载体pET-30a-c(+)中,获得重组表达载体pET30a-E2,将其转化大肠杆菌Rosetta(DE3)感受态细胞,经IPTG诱导表达。SDS-PAGE和Western blottin分析结果表明,E2基因获得高效融合表达,且具有免疫反应活性。本试验为建立E2抗原的检测试剂盒奠定基础。     

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.     

A promising multiple-epitope recombinant vaccine against classical swine fever virus

Classical swine fever (CSF) is a highly contagious and often fatal disease of swine. It is caused by classical swine fever virus (CSFV), one of the members of the genus Pestivirus of the Flaviviridae family. The development of a safe and effective vaccine against the CSF is critical to pandemic control, this article shows a tandem-repeat multiple-epitope recombinant vaccine can protect pigs from CSFV challenge. That was composed as following: two copies each of glycoprotein E2 residues 693–707, 241–276 and 770–781, and two copies amino acid residues 1446–1460 of the non-structural protein NS2-3. In the challenge test, all of the swine vaccinated with Chinese vaccine strain (C-strain) were fully protected from a challenge with CSFV. However, after three successive vaccinations with the multiple-epitope recombinant vaccine, three out of five pigs were protected from challenge with CSFV (in terms of both clinical signs and viremia). These results demonstrate that multiple-epitope recombinant vaccine which carrying the major CSFV epitopes can induce a high level of epitope-specific antibodies and exhibit a protective capability that parallels induced by C-strain to a certain extent.     

我国近期猪瘟分子流行病学动态

利用反转录聚合酶链式反应 (RT PCR)及测序技术 ,测定 2 0 0 2年分离于我国甘肃、湖南、海南、青海等省的 9株猪瘟野毒的E2基因序列 ,并与C 株疫苗毒进行同源性比较 ,绘制CSFV系统发生树。结果表明 ,近期分离于我国的 9株野毒与C 株核苷酸序列间的同源性在80 .7%～ 99.1 %,氨基酸同源性在 89.3%～98.9%。9株野毒分属于 2个不同的组群 ,其中 5株与我国经典的石门强毒和C 株疫苗毒同属于Subgroups1 .1 ,另 4株属于与C 株有较大差异的Subgroup2 .1。     

猪瘟兔化弱毒疫苗株基因组的遗传变异分析

参照已发表的猪瘟病毒基因组序列设计了9对引物，用RT-PCR从猪瘟兔化弱毒疫苗株细胞培养物中扩增得到了覆盖猪瘟病毒基因组全长的9个cDNA片段，将所得cDNA片段分别克隆至pMD18-T载体中，经测序和拼接后，获得了猪瘟兔化弱毒疫苗株基因组全序列。序列分析表明，猪瘟兔化弱毒疫苗株基因组全长12310个碱基，其5’非编码区（5＇-NCR）和3＇-NCR分别由373和239个碱基组成，在3’末端有富含T的碱基插入，其间为1个大的开放阅读框架，编码3898个氨基酸残基的多聚蛋白，与国内外已发表的另外7个猪瘟兔化弱毒疫苗株基因组全序列相比，核苷酸同源性为98．7％～99．9％，氨基酸同源性为98．6％～99．9％。基因组全序列比较显示，猪瘟兔化弱毒疫苗株基因组在遗传上相当稳定。     

Efficacy of lyophilised C-strain vaccine after oral immunisation of domestic pigs and wild boar against classical swine fever: first results

The aim of this study was to evaluate the efficacy of lyophilised C-strain vaccine in domestic pigs and wild boar after oral application. A new spherical bait form (diameter 3 cm) containing lyophilised vaccine virus and the recent vaccine baits were used for animal experiments. Four vaccination groups were established in experiment 1 (group 1: recent liquid bait vaccine; group 2: spherical baits containing one dose of the lyophilised vaccine; groups 3 (domestic pigs) and 4 (wild boar): spherical baits containing two doses of the lyophilised vaccine) and two groups in experiment 2 (group 1: recent liquid bait vaccine; group 2: spherical baits with two doses of the lyophilised vaccine). Challenge was carried out with the highly virulent virus strain "Alfort 187" (using 100 TCID50 in the first and 1.000 TCID50 in the second experiment). Our results showed that the animals vaccinated with lyophilised C-strain vaccine developed high neutralising antibody titres comparable to those obtained after vaccination with the recent bait vaccine. All pigs which picked up the baits remained healthy after challenge. Neither clinical symptoms nor viremia or virus shedding were observed after infection except in one pig (group 2, experiment 2) which had not consumed the vaccine bait. The surviving domestic pigs and wild boar were tested negative for CSFV and viral RNA at the end of the study. This result demonstrates that lyophilised vaccine may become an effective vaccine formulation for oral immunisation of wild boar against CSF in the near future.     

猪瘟病毒E2基因的扩增及序列分析

本研究利用RT—PCR技术，对流行于国内部分地区的9株猪瘟病毒（CSFV）E2基因进行扩增，与C-株等4株参考毒株做了同源性比较，绘制了遗传进化关系发生树，结果表明，所测序列共由442个氮基酸残基组成。可将13株CSFV分为两个组群，其E2基因间的核苷酸序列同源性为81．9％～99．7％，所推导氨基酸序列同源性为88．2％～99．5％，其中9株CSFVE2基因的长度均为1324bp，编码的氨基酸序列均包括完整信号肽序列和跨膜区序列，9株流行毒株与C-株之间核苷酸序列同源性为81．9％～95．3％。所推导氨基酸序列同源性为88．2％～95．5％。说明近期流行毒株的变异呈现一定的多样性，并且多数毒株已向远离疫菌株方向变异。     

Evaluation of a multiplex real-time RT-PCR for quantitative and differential detection of wild-type viruses and C-strain vaccine of Classical swine fever virus

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), one of OIE listed diseases. Most of the currently available detection methods do not allow discrimination between wild-type CSF viruses and the vaccine strains. This study was designed to develop a multiplex real-time RT-PCR for the quantitative and differential detection of wild-type viruses and C-strain vaccine widely used in China. CSFV specific primers and two differently labeled TaqMan probes for the differentiation of wild-type viruses from C-strain vaccine were designed in the 5'-untranslated region of the viral genome of CSFV. The two TaqMan probes specifically hybridize wild-type viruses of different subgroups and C-strain vaccine, respectively, in the multiplex real-time RT-PCR, with no cross-reaction to a number of non-CSFV porcine viruses. The sensitivity of the assay for detecting wild-type and C-strain-type vaccine viruses was determined to be 41.8 and 81.5copies/microL viral RNA, respectively. Completely correct differentiation of wild-type viruses from C-strain vaccine was achieved when testing reference strains and characterized field isolates of CSFV in China. The multiplex real-time RT-PCR was able to detect the viral RNA in the whole blood samples of experimentally infected pigs as early as 2 days post-infection, 3 to 4 days prior to the onset of clinical signs in co-housed pigs. The agreements between the multiplex real-time RT-PCR and a multiplex RT-nested PCR for detection of wild-type and C-strain-type viruses were 96.9% and 100%, respectively, when detecting 106 different field samples. There is a positive correlation between the titers of C-strain vaccines titrated in rabbits and RNA copies quantitated by the multiplex real-time RT-PCR. The novel assay described here is rapid and sensitive, and is useful for differentiating field strains and C-strain of CSFV in China.     

Genetic diversity of the envelope glycoprotein E2 of classical swine fever virus: recent isolates branched away from historical and vaccine strains

The envelope glycoprotein E2 of classical swine fever virus (CSFV) plays multiple roles in the viral life cycle, interaction with host and pathogenesis. We sequenced the E2 gene of 34 CSFV isolates from southeastern China for analysis of genetic diversity in this particular region. Phylogenetic analysis revealed that genotype 2.1b viruses became predominant in southeastern China with 33 isolates clustered in 2.1b and only 1 isolate belonged to 2.2. Pairwise comparisons demonstrated isolates in this study showed a homology of 78.4-82.6% to Chinese C-strain in the 190nt of the E2 fragment. The minimum similarity within the 2.1b isolates was 91.1%. Variability of the full length E2 amino acid sequences of 45 CSFV isolates representative of three main groups of CSFV including 19 from southeastern China during 2004--2007 and 26 from other parts of China and other countries was analyzed by the differences between non-synonymous (dN) and synonymous (dS) rates and the entropy values. Two variable and three conserved regions as well as some specific positions under positive selection were defined. Our results suggest that recent isolates from southeastern China have shifted away from historical CSFV isolates including the vaccine strains probably as a result of their adaptive abilities to the selection forces within the host.     

可溶性E2蛋白作为抗原的检测猪瘟病毒血清抗体间接ELISA方法的建立

以可溶性重组E2蛋白作为抗原,建立了猪瘟病毒(CSFV)血清抗体间接ELISA诊断方法(rE2-ELISA).将猪瘟疫苗毒株E2基因主要抗原区(A-D)基因克隆到表达栽体pGEX-6p-1上,转化E.coli,降低诱导温度至20℃,获得48 000大小E2融合蛋白,部分目的蛋白以可溶性形式表达.Western blotting试验证实,E2融合蛋白可以和CSFV阳性血清发生特异性结合.亲和层析纯化后的E2融合蛋白作为抗原,建立了检测CSFV血清抗体的间接rE2-ELISA方法.该方法的特异性试验结果表明,与PRRSV、PCV2、PPV和PRV阳性血清之间不存在交叉反应;用rE2-ELISA和国外同类试剂盒(CSF-Ab-Kit)检测142份田问血清样品,2种试剂盒的阳性检测率分别为83.81%和88.73%.因此,rE2-ELISA猪瘟抗体检测试剂盒具有良好的敏感性和特异性,适合应用在大规模的CSFV血清抗体的检测工作中.     

应用假病毒和假型病毒制备抗猪瘟病毒E2蛋白中和性单克隆抗体的初步研究

为制备抗猪瘟病毒(CSFV)单克隆抗体(MAb),本实验以表达CSFV E2囊膜糖蛋白的水泡口炎假病毒免疫BALB/c小鼠,取其脾细胞与骨髓瘤细胞SP2/0进行融合;利用间接ELISA方法和携带荧光素酶(Luciferase)报告基因的HIV-luc/CSFV-E1E2假病毒系统筛选分泌中和性E2MAb的杂交瘤细胞;测定MAb亚型并纯化后,间接ELISA方法测定MAb的效价;采用western blot鉴定MAb的特异性亲和力;利用HIV-luc/CSFV-E1E2假病毒进行体外中和试验,分析MAb抑制病毒感染的能力。结果表明本实验获得了1株分泌中和性MAb的杂交瘤细胞9C8,该MAb能与E2蛋白特异性结合,而且体外抑制试验中和效价大于1∶25600。     

猪瘟野毒感染抗体阻断ELISA检测方法的建立与初步评价

以猪瘟野毒E2蛋白为包被抗原、辣根过氧化物酶标记的猪瘟野毒单抗作为酶标抗体,建立检测猪瘟野毒抗体的阻断ELISA方法。猪瘟野毒E2最适包被浓度为0.03μg/mL,待检血清最适稀释度为1∶4,酶标猪瘟野毒单抗稀释度为1∶1 000。用建立的阻断ELISA方法检测369份临床阴性血清,计算阻断率,确定临界值,阻断率>40%为猪瘟野毒抗体阳性,阻断率≤40%为猪瘟野毒抗体阴性。用建立的ELISA方法检测84份血清,其中78份为免疫猪瘟疫苗的血清,6份为猪瘟病毒感染血清。结果显示,78份免疫血清均检测为猪瘟野毒抗体阴性,6份猪瘟感染血清均检测为猪瘟野毒抗体阳性。因此可初步判定该方法可用于鉴别诊断猪瘟病毒自然感染动物和C株疫苗免疫动物的血清抗体,并为临床检测猪瘟野毒抗体提供便捷、快速,精准的检测工具,对猪瘟的临床诊断、预防以及猪瘟净化工作具有非常重要的参考意义。     

猪瘟兔化弱毒E2基因重组杆状病毒的构建及抗体制备

为制备猪瘟特异性抗体,将猪瘟兔化弱毒E2基因序列修饰后克隆到pFastBac1质粒载体,经转座、转染后构建重组杆状病毒,并用纯化的重组猪瘟E2蛋白免疫大耳白兔制备特异性抗体。实验结果表明成功获得能分泌重组猪瘟E2蛋白的杆状病毒,使用纯化的重组猪瘟E2蛋白制备的猪瘟抗体具有良好的特异性和敏感性,为猪瘟病毒荧光抗体染色液的制备奠定了基础。     

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.     

单克隆抗体捕捉猪瘟病毒抗原ELISA方法的建立

分别用原核表达的猪瘟病毒（CSFV）主要抗原E2蛋白和猪瘟基因疫苗免疫BALB/c小鼠,通过细胞融合与克隆筛选出5株稳定分泌CSFV抗体的杂交瘤细胞株1E2、1G7、3A2、3B7和4B6.间接免疫荧光和Western-blotting试验结果表明,5株单抗均与CSFV E2蛋白和全病毒抗原反应.将筛选的CSFV特异性单抗1E2、3B7和4B6纯化后等量混合后包被酶标板（捕捉抗体）,与兔抗CSFV IgG（检测抗体）联合应用,建立起CSFV抗原捕捉ELISA（AC-ELISA）方法.随后采用方阵滴定法确定了单抗与多抗的最适工作浓度及判定检测结果的OD450临界值.最后以建立的AC-ELISA检测CSFV细胞培养物、CSFV攻毒死亡猪的病料和临床猪瘟组织样品,结果表明,该方法敏感、特异、重复性好,与病毒分离和RT-PCR方法符合率分别为86.2%和90.3%.