Genetic differentiation of infected from vaccinated animals after implementation of an emergency vaccination strategy against classical swine fever in wild boar

Oral emergency vaccination against classical swine fever is a powerful tool to control disease outbreaks among European wild boar and thus to safeguard domestic pigs in affected regions. In the past, when virus detection was mainly done using virus isolation in cell culture or antigen enzyme-linked immunosorbent assays, modified live vaccine strains like C-strain "Riems", were barely detectable after oral vaccination campaigns. Nowadays, the use of highly sensitive molecular techniques has given rise to an increase in vaccine virus detections. This was also the case during the 2009 outbreak among German wild boar and the subsequent vaccination campaigns. To guarantee a rapid differentiation of truly infected from C-strain vaccinated animals, a combination of differentiating multiplex rRT-PCR assays with partial sequencing was implemented. Here, we report on the rational and use of this approach and the lessons learned during execution. It was shown that positive results in the recently developed vaccine strain (genotype) specific rRT-PCR assay can be taken as almost evidentiary whereas negative results should be confirmed by partial sequencing. Thus, combination of multiplex rRT-PCR assays as a first line differentiation with partial sequencing can be recommended for a genetic DIVA strategy in areas with oral vaccination against classical swine fever in wild boars.     

Protective efficacy of a Classical swine fever virus C-strain deletion mutant and ability to differentiate infected from vaccinated animals

Classical swine fever (CSF) continues to be the most economically damaging pig disease in the world. The disease can be effectively controlled by vaccination with the live C-strain vaccine. This vaccine, however, does not enable the serological differentiation between infected and vaccinated animals (DIVA) and its use can therefore impose severe trade restrictions. CSF-specific diagnostic ELISAs detect antibodies directed against the conserved and immunodominant A domain of the E2 structural glycoprotein. We previously reported the production of a C-strain virus in which the immunodominant TAVSPTTLR epitope of the A domain is stably mutated with the aim to render the virus suitable as a DIVA vaccine. We here report that a single vaccination with this vaccine virus protected pigs from a lethal challenge dose of the highly virulent Brescia strain. Analysis of the sera, however, demonstrated that a commercially available E2 ELISA was unsuitable as an accompanying DIVA test.     

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。     

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.     

Classical swine fever virus Strain 'C'. How long is it detectable after oral vaccination?

To determine the persistence period of C-strain vaccine virus in immunized animals, domestic pigs and wild boars were vaccinated orally and killed on different days post vaccinationem (dpv). Tissue samples were taken at necropsy from both species for detection of C-strain virus. From domestic pigs nasal swabs and faeces were also collected. During the investigation period (2-12 dpv) vaccine virus could never be detected in nasal secretions and in faeces of vaccinated domestic pigs. In contrast, C-strain virus was found in organs until day 8 pv in domestic pigs and until day 9 pv in wild boars. Whereas in domestic pigs virus was detected in tonsils, Ln. mandibularis or in spleen, in wild boar it only was found in tonsils. We conclude that C-strain vaccine virus is not detectable in wild boars longer than 10-12 days after intake of the vaccine baits.     

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.     

Oral immunisation of swine with a classical swine fever vaccine (Chinese strain) and transmission studies in rabbits and sheep

Seven experiments including a total of 47 pigs, 11 wild boars, 26 rabbits, 10 hares and 16 sheep were carried out to assess the efficacy, safety and transmission of the Chinese vaccine strain of the classical swine fever virus (CSFV) administrated by the oral route. Within 3 weeks after oral vaccination, a clear seroconversion occurred in the pigs. Six weeks after vaccination, vaccinated pigs were fully protected against a virulent challenge. The C-strain was not isolated from tonsils, spleen, lymph nodes, thymus, saliva, urine and faeces of pigs within 4 days after oral vaccination. In one experiment, susceptible pigs were placed in direct contact with vaccinated pigs. None of these contact-exposed pigs became serologically positive for CSFV antibodies. It is concluded that the C-strain induces protection in pigs when administrated by the oral route and is not shed by vaccinated pigs. Serum anti-CSFV antibodies developed in seven out of eight wild boars vaccinated by the oral route. No vaccine virus was detected in the spleen and tonsils of these animals. The results in wild boar were in accordance with those obtained in domestic pigs. Sheep did not show any clinical signs after oral vaccination while rabbits had moderate hyperthermia and growth retardation. No clinical response to oral immunisation in hares was detected. At the end of the experiment, no sheep had detectable serum antibodies against CSFV, whereas a few vaccinated rabbits and hares became seropositive. None of the contact-exposed rabbits and hares seroconverted. These data indicate that the C-strain is safe for sheep and as expected, moderately or not pathogenic for rabbits and hares. These efficacy and safety studies on oral vaccination with the C-strain under experimental conditions provide essential information for further studies in wild boars under experimental and field conditions, including assays with baits to control a CSF epidemic.     

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.     

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.     

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.     

新型猪瘟疫苗的研究进展

猪瘟是危害猪健康的重要传染病之一,具有急性、热性和高度接触性等特性,给世界养猪业造成了巨大的经济损失。目前,传统疫苗接种仍是预防猪瘟的主要手段,虽然传统疫苗（如C株）在防控猪瘟方面发挥着巨大的作用,但也存在一些缺陷,如无法区分野毒感染和疫苗免疫动物,猪瘟免疫失败时有发生等。因此,研制新型猪瘟疫苗具有重要意义。随着分子生物学技术与重组DNA技术的不断发展及基因工程疫苗研究的不断深入,亚单位疫苗、核酸疫苗、活载体重组疫苗、基因缺失疫苗、全长感染性cDNA标记疫苗和合成肽疫苗6种新型猪瘟疫苗被相继开发。与传统疫苗相比,新型疫苗拥有廉价、安全、高效、易于运输与保存、能区分野毒感染和疫苗免疫等优点。作者对6种新型猪瘟疫苗的研究进展作一综述,以期为猪瘟的防控提供参考。     

Characterization of C-strain "Riems" TAV-epitope escape variants obtained through selective antibody pressure in cell culture

ABSTRACT: Classical swine fever virus (CSFV) C-strain "Riems" escape variants generated under selective antibody pressure with monoclonal antibodies and a peptide-specific antiserum in cell culture were investigated. Candidates with up to three amino acid exchanges in the immunodominant and highly conserved linear TAV-epitope of the E2-glycoprotein, and additional mutations in the envelope proteins ERNS and E1, were characterized both in vitro and in vivo.It was further demonstrated, that intramuscular immunization of weaner pigs with variants selected after a series of passages elicited full protection against lethal CSFV challenge infection. These novel CSFV C-strain variants with exchanges in the TAV-epitope present potential marker vaccine candidates. The DIVA (differentiating infected from vaccinated animals) principle was tested for those variants using commercially available E2 antibody detection ELISA. Moreover, direct virus differentiation is possible using a real-time RT-PCR system specific for the new C-strain virus escape variants or using differential immunofluorescence staining.     

Differential quantification of cloned CVI988 vaccine strain and virulent RB-1B strain of Marek’s disease viruses in chicken tissues, using real-time PCR

The ‘gold standard’ vaccine against Marek’s disease in poultry is the CVI988/Rispens virus, which is not easily distinguishable, antigenically or genetically, from virulent Marek’s disease herpesvirus. Accurate differential measurement of the CVI988 vaccine and virulent viruses is important to investigate mechanisms of vaccinal protection. Minimal sequence differences between CVI988 and virulent MDV strains restrict the application of molecular diagnostic methods such as real-time PCR to distinguish between these viruses. The use of bacterial-artificial-chromosome (BAC) cloned CVI988 virus, which carries the BAC vector sequences in place of the U_s2 gene, allows its differential quantification from virulent strains using real-time PCR assays that target the BAC vector sequence and the U_S2 gene respectively. These novel assays allowed investigation of replication of both serotype-1 vaccine virus (cloned CVI988) and challenge virus (RB-1B strain) in tissues of individual chickens in an experimental vaccination-challenge model of Marek’s disease.     

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)).     

两种荧光定量PCR方法检测猪瘟病毒的比较及应用

根据猪瘟病毒（Classical swine fever virus,CSFV）5端非编码区保守区域,设计猪瘟病毒特异性的引物和探针,建立了基于SYBR Green和TaqMan探针的两种荧光定量PCR检测方法。灵敏性和特异性试验结果表明,两种方法能够特异性地检测出不同型的猪瘟病毒,而对同属的牛病毒性腹泻病病毒以及其它一些主要猪病病原检测结果均为阴性。两种方法对猪瘟病毒C株的检测下限均为101TCID50,均高于常规的套式PCR。用携带该基因片段的重组质粒为模板进行检测,SYBR Green方法的检测下限为3×100copies,比TaqMan探针方法更加灵敏（3×101copies）。应用建立的SYBR Green方法对2009年采集于浙江地区不同猪场的20份病猪样品进行CSFV检测,有8份样品为阳性,与套式PCR方法检测结果一致。进一步应用SYBR Green方法对不同感染阶段细胞内病毒RNA复制水平进行检测,与病毒滴度的结果基本一致。因此,本研究建立的SYBRGreen荧光定量PCR检测方法能够应用于组织和培养细胞中猪瘟病毒的检测。     

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.     

Serum antibodies directed against classical swine fever virus and other pestiviruses in wild boar (Sus scrofa) in the Republic of Croatia

The presence of serum antibodies directed against classical swine fever (CSF) virus and other pestiviruses among the wild boar (Sus scrofa) population in Croatia was investigated. During 2003, serum samples from 214 wild boars were collected in 10 hunting areas in the continental part of the country.The sera were examined by enzyme immunoassay (ELISA) and in the virus neutralization test (VNT). Out of 214 sera tested 111 (51.87 %) were positive by ELISA and regarding neutralising antibodies, against CSFV 75 (35.05 %) samples were positive. In the VNT with the C-strain (conventional live vaccine strain China) and the strain Uelzen were used. Samples were also tested for neutralizing antibodies against border disease virus (BDV) using the strain 137/4 and against bovine viral diarrhoea virus (BVDV) using the NADL strain. Neutralizing antibodies against the C-strain were detected in 36 sera (16.82 %), against strain Uelzen in 17 sera (7.94 %) and in 22 sera (10.28 %) against both strains. In five sera (2.33 %) neutralizing antibodies against BVDV and BDV were found.     

Rabies Vaccination: Higher Failure Rates in Imported Dogs than in those Vaccinated in Italy

The current European Union (EU) legislation decrees that pets entering the EU from a rabies‐infected third country have to obtain a satisfactory virus‐neutralizing antibody level, while those moving within the EU require only rabies vaccination as the risk of moving a rabid pet within the EU is considered negligible. A number of factors driving individual variations in dog vaccine response have been previously reported, including a high rate of vaccine failure in puppies, especially those subject to commercial transport. A total of 21 001 observations collected from dogs (2006–2012) vaccinated in compliance with the current EU regulations were statistically analysed to assess the effect of different risk factors related to rabies vaccine efficacy. Within this framework, we were able to compare the vaccination failure rate in a group of dogs entering the Italian border from EU and non‐EU countries to those vaccinated in Italy prior to international travel. Our analysis identified that cross‐breeds and two breed categories showed high vaccine success rates, while Beagles and Boxers were the least likely to show a successful response to vaccination (88.82% and 90.32%, respectively). Our analysis revealed diverse performances among the commercially available vaccines, in terms of serological peak windows, and marked differences according to geographical area. Of note, we found a higher vaccine failure rate in imported dogs (13.15%) than in those vaccinated in Italy (5.89%). Our findings suggest that the choice of vaccine may influence the likelihood of an animal achieving a protective serological level and that time from vaccination to sampling should be considered when interpreting serological results. A higher vaccine failure in imported compared to Italian dogs highlights the key role that border controls still have in assessing the full compliance of pet movements with EU legislation to minimize the risk of rabies being reintroduced into a disease‐free area.     

Comparative evaluation of live marker vaccine candidates "CP7_E2alf" and "flc11" along with C-strain "Riems" after oral vaccination

Due to the tremendous socio-economic impact of classical swine fever (CSF) outbreaks, emergency vaccination scenarios are continuously under discussion. Unfortunately, all currently available vaccines show restrictions either in terms of marker capacities or immunogenicity. Recent research efforts were therefore directed at the design of new modified live marker vaccines. Among the most promising candidates the chimeric pestiviruses "CP7_E2alf" and "flc11" were identified. Within an international research project, these candidates were comparatively tested in challenge experiments after a single oral vaccination. Challenge infection was carried out with highly virulent CSF virus strain "Koslov", 14 or 21 days post vaccination (dpv), respectively. Safety, efficacy, and marker potential were addressed. All assessments were done in comparison with the conventional "gold standard" C-strain "Riems" vaccine. In addition to the challenge trials, multiple vaccinations with both candidates were performed to further assess their marker vaccine potential. All vaccines were safe and yielded full protection upon challenge 21 days post vaccination. Neither serological nor virological investigations showed major differences among the three vaccines. Whereas CP7_E2alf also provided clinical protection upon challenge at 14 days post vaccination, only 50% of animals vaccinated with flc11, and 83% vaccinated with C-strain "Riems" survived challenge at this time point. No marked differences were seen in protected animals. Despite the fact that all multiple-vaccinated animals stayed sero-negative in the accompanying marker test, the discriminatory assay remains a weak point due to delayed or inexistent detection of some of the vaccinated and subsequently infected animals. Nevertheless, the potential as live marker vaccines could be confirmed for both vaccine candidates. Future efforts will therefore be directed at the licensing of "Cp7_E2alf" as the first live marker vaccine for CSF.