猪瘟重组疫苗的研究进展

猪瘟是严重危害养猪业一种重要传染病,弱毒疫苗的应用一定程度地控制了该病的流行与发生,但仍存在一些缺陷,新型、高效、标记疫苗的研制一直以来成为猪瘟疫苗领域的研究热点。本文对猪瘟重组疫苗领域取得的研究研究进展进行了综述。     

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.     

猪瘟病毒FJFQ-39株的毒力鉴定

为探索一种相对客观、稳定的方法鉴定猪瘟病毒(CSFV)FJFQ-39株的毒力.本研究采用2×103TCID50的FJFQ-39株分别肌肉接种6头敏感猪,通过RT-nPCR 检测扁桃体和血液监测动物感染情况,对动物临床症状和病理变化进行系统评分,结合体温分析,判定病毒毒力.同时用相同剂量的石门株接种4头敏感猪作对照.FJFQ-39和石门接种猪的扁桃体和血液均检测到CSFV核酸;FJFQ-39接种猪的最大临床症状评分(CS)平均值为3.53.5±1.0、病理评分(PS)平均值为3.3±0.9(低于5),平均最高体温为39.3±0.2℃(低于40℃);石门接种猪的最大CS平均值为25.5±2.1、PS平均值为29.5±2.4(大于15),平均最高体温为41.8±0.2℃(高于41.0℃).实验结果表明:猪瘟病毒FJFQ-39株和石门株均成功感染了动物;评分系统结合体温测定评价CSFV毒力是可行的;FJFQ-39 属于低毒力株,而石门属于强毒株.     

Swine fever: classical swine fever and African swine fever.

Because of the clinical and pathologic similarity to common endemic diseases, introduction of CSFV or ASFV strains of moderate to low virulence represents the greatest risk to North American swine herds. Producers, veterinarians, and diagnosticians should increase their awareness of these devastating diseases and request specific diagnostic testing whenever they are suspected. Production practices that improve biosecurity will reduce the risk of introduction of CSF and ASF and limit the spread if an incursion occurs. Additional resources. The following Web sites contain excellent color photographs that will assist producers and practitioners in identifying clinical signs and gross lesions associated with CSFV and ASFV: http://www.vet.uga.edu/vpp/gray_book/FAD and http://www.pighealth.com. The latter Web site and the OIE Web site (http://www.oie.int) offer updated information on current worldwide epizootics of ASF and CSF and other swine diseases. Details of biosecurity procedures can be found at http://www.agebb.missouri.edu; see publication G2340.     

Genetic typing of classical swine fever virus

Three regions of the classical swine fever virus (CSFV) genome that have been widely sequenced were compared with respect to their ability to discriminate between isolates and to segregate viruses into genetic groups. Sequence data-sets were assembled for 55 CSFVs comprising 150 nucleotides of the 5' non-translated region, 190 nucleotides of the E2 envelope glycoprotein gene and 409 nucleotides of the NS5B polymerase gene. Phylogenetic analysis of each data-set revealed similar groups and subgroups. For closely related viruses, the more variable or larger data-sets gave better discrimination, and the most reliable classification was obtained with sequence data from the NS5B region. No evidence was found for intertypic recombination between CSFVs. A larger data-set was also analysed comprising 190 nucleotides of E2 sequence from 100 CSFVs from different parts of the world, in order to assess the extent and global distribution of CSFV diversity. Additional groups of CSFV are evident from Asia and the nomenclature of Lowings et al. (1996) [Lowings, P., Ibata, G., Needham, J., Paton, D., 1996. J. Gen. Virol. 77, 1311-1321] needs to be updated to accommodate these. A tentative assignment, adapting rather than overturning the previous nomenclature divides CSF viruses into three groups with three or four subgroups: 1.1, 1.2, 1.3; 2.1, 2.2, 2.3; 3.1, 3.2, 3.3, 3.4. The expanding data-base of CSFV sequences should improve the prospects of disease tracing in the future, and provide a basis for a standardised approach to ensure that results from different laboratories are comparable.     

Survival and inactivation of classical swine fever virus

Classical swine fever virus, like many enveloped viruses, may be regarded as moderately fragile. It shows a short but variable survival time in the environment, depending on physical conditions, but may remain viable for prolonged periods in favourable circumstances as found, for example, in stored meat. Published information is reviewed on the survival time of the virus under a variety of physical and chemical conditions, including the variable influence of environmental factors. The principles of virus inactivation by heat, pH or chemicals are discussed, with a consideration of the principles of containment for laboratories working with the virus.     

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.     

The influence of maternal immunity on the efficacy of a classical swine fever vaccine against classical swine fever virus,genogroup 2.2, infection

In Thailand, where vaccination is routinely employed, there has been an increased incidence of chronic classical swine fever (CSF) outbreaks during the past decade. The major causative virus has been identified to be the moderate virulence, classical swine fever virus (CSFV) of the genogroup 2.2. An investigation was made into the efficacy of a CSF vaccine against this genogroup 2.2 challenge. Five-week-old pigs, grouped by their level of passive antibody titer were immunized with lapinized Chinese-strain CSF vaccine and challenged with CSFV genogroup 2.2, 13 days after vaccination. The group containing passive titers of lower than 64 at the time of immunization, had significantly higher number of CSFV-specific IFN-gamma secreting cells and was completely protected against the challenge. Interestingly, both cellular and antibody responses were inhibited in the pigs with the higher passive titer. Furthermore, following challenge, CSFV could be isolated from 50% of the pigs in this group. It was demonstrated that the CSF vaccine could induce complete protection in pigs, provided that the maternal derived titer at the time of vaccination was lower than 64. The result implied that an increase in CSFV outbreaks might be due to the inappropriate timing of vaccination as well as the nature of the CSFV genogroup 2.2.     

Evaluation of vaccine-induced maternal immunity against classical swine fever

The vaccine-induced maternal immunity against classical swine fever (CSF) was investigated in this study. Eight sows were vaccinated with the Chinese strain of classical swine fever virus (CSFV). The length of time between vaccination and farrowing was 167-217 days. Milk samples from the front, middle and back udder sections and blood samples were taken from the sows on days 3 and 14 after farrowing. Blood samples were obtained from the piglets at the age of 3, 6 and 10 weeks. The antibody level of the milk was examined by ELISA, and that of blood samples by the virus neutralization (VN) test as well. In all 3-week-old piglets and in 80% of the 6-week-old animals the neutralizing antibody level reached the titre of 1:40. In none of the 10-week-old piglets did the titre exceed the value of 1:20, but in about 25% of the piglets it reached 1:20; the half of these piglets came from two litters. In none of the piglets did the antibody level reach the negative threshold in the ELISA test during the study. No significant differences were found between the udder sections in milk antibody level by ELISA.     

The correlation of virus-specific interferon-gamma production and protection against classical swine fever virus infection.

The level of antigen-specific interferon-gamma (IFN-gamma) production can be used as an indicator of cellular immunity. In this study, we investigated the role of cellular immune response in protection against classical swine fever virus (CSFV). Pigs were vaccinated once with CSFV vaccine and challenged 6 days post-vaccination (dpv). Vaccinated animals had significantly higher CSFV-specific IFN-gamma secreting cells than the unvaccinated pigs (p<0.05) at the time of challenge and were protected against CSFV infection, whereas the control pigs died within 14 days post-infection (dpi). In the second experiment, pigs were vaccinated once with either CSFV vaccine or CSFV vaccine combined with Aujeszky's disease (AD) vaccine and challenged at 140 dpv. All vaccinated pigs developed both CSFV-specific, cellular and antibody responses and were protected against CSFV infection. However, differences in cellular, but not antibody, responses were observed in the two vaccinated groups. The group vaccinated with CSFV vaccine developed a significantly higher number of CSFV-specific, IFN-gamma secreting cells (p<0.05), exhibited a shorter fever period and less pathological changes, when compared with the group vaccinated with the combined vaccine. The kinetics of IFN-gamma production, following challenge in the two vaccinated groups, were also different. Taken together, our results indicated that CSFV-specific, IFN-gamma production could be detected early after antigen exposure and correlated with protection against CSFV challenge. Our findings highlight the role of cellular immune responses in porcine anti-viral immunity.     

猪瘟抗体间接ELISA检测试剂盒的研制和应用

对猪瘟抗体间接ELISA检测试剂盒的稳定性及保存条件等进行了摸索,确定该试剂盒具有良好的稳定性,并可在2℃～8℃环境中的保存9个月。同时,测定了本试剂盒与荧光抗体病毒中和试验的符合率为92.62%;并将该试剂盒应用于免疫动物抗体的动态监测。     

猪瘟单克隆抗体的制备及ACI-ELISA检测猪瘟病毒的研究

本研究用猪瘟石门毒(CSFV-Shimen)免疫BALB/C小鼠,按常规单克隆抗体(McAb)技术方法制作,最终获得4株McAb,分别命名为AC9、CF8、DG5和EC9,4株McAb与基因工程CSFV E2蛋白反应结果表明:AC9、CF8和EC9是抗CS-FV E2蛋白的McAb.用AC9和CF8McAb对CSFV进行抗原捕获间接ELJSA试验(ACI-ELISA),通过一元McAb和二元McAb CAI-ELISA试验的比较,结果表明AC9与CF8两种McAb有协同作用,其捕获CSFV的能力比一元McAb显著提高.方阵试验结果表明:McAb和血清多抗(PcAb)的最佳工作稀释度分别为1:400和1:200.特异性试验和敏感性试验结果显示本法特异性强,敏感性高.最后用ACI-ELISA与PCR对30份病料的检测结果比较,表明ACI-ELISA与PCR检测结果相符.上述结果说明本研究所获得AC9和CF8可用作猪瘟诊断试剂盒的研制,是检测CSFV的有效方法.     

Introduction to classical swine fever: virus, disease and control policy

Classical swine fever virus is a spherical enveloped particle of about 40-60 nm in diameter with a single stranded RNA genome of about 12,300 bases with positive polarity, classified as a pestivirus within the family Flaviviridae. Natural hosts are domestic and wild pigs. The virus causes one of the most severe diseases in pigs world wide with grave economic consequences. The clinical picture of classical swine fever is variable, depending on the age of the affected animals and viral virulence. The virus is well characterised and reliable laboratory diagnostic procedures are available. In many parts of the world live attenuated vaccines are being used as a safe and efficient prophylactic tool. However, in EU Member States and several other countries vaccination is prohibited and CSF is controlled by a strict stamping out policy. In order to overcome the disadvantages of conventional vaccination inactivated marker vaccines have been developed that enable the distinction between vaccinated and infected animals. Whether these vaccines will be accepted as an additional tool in the framework of the stamping out policy is not yet decided.     

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。     

Prevalence of African swine fever virus and classical swine fever virus antibodies in pigs in Benue State,Nigeria

Tropical Animal Health and Production - This study investigated the prevalence of African swine fever virus (ASFV) and classical swine fever virus (CSFV) antibodies in pigs in Benue State, Nigeria....     

非典型猪瘟

猪瘟是一种致死率高、危害严重的急性传染病 ,受到各养猪国家的关注。我国对猪瘟的防制工作十分重视 ,并提出当前应采取以免疫接种为重点的综合性防制措施。近年来典型猪瘟显然不多见了 ,但在一些地区和猪场却出现了一种非典型猪瘟。由于其缺乏猪瘟的典型症状和病变 ,易被人们误诊 ,以至滥用药物 ,盲目治疗 ,既增加了药费的支出 ,又贻误了防制时机 ,给猪场带来更大的经济损失。本文就非典型猪瘟的临床诊断、病因分析和防制措施等问题 ,作简要的介绍和探讨。1　非典型猪瘟的表现形式1 1　繁殖障碍型猪瘟主要发生于生产母猪 ,其本身呈隐性感染…     

用RT-PCR技术检测盐渍猪肠衣中猪瘟病毒的研究

参考GenBank中发表的猪瘟病毒（CSFV）序列，设计一对CSFV特异性PCR引物；从CSFV感染猪盐渍小肠中提取总RNA，经逆转录后进行PCR扩增，在盐渍小肠中成功扩增出与预期大小（168bp）一致的特异性条带，而正常猪和感染猪伪狂犬病病毒的猪小肠扩增结果均为阴性。用本方法对20例不同稀释浓度的盐渍猪肠衣样本进行检测，结果显示比经典抗原检测方法（抗原捕获ELISA法）具有更高的敏感性。实验表明，本RT—PCR技术能应用于盐渍猪肠衣的CSFV检测，为快速、准确检测盐渍猪肠衣中CSFV提供了一条新途径。     

口蹄疫与经典猪瘟二联RT-PCR诊断方法的建立及其应用

根据口蹄疫病毒(FMDV)与猪瘟病毒(CSFV)基因组序列高度保守区,设计合成2对引物,以CSFV和FMDV培养物提取RNA并反转录,进行RT-PCR特异性片段扩增,扩增片段大小分别为200 bp、141 bp.结果表明,扩增产物与设计的2对引物之间的序列大小一致.通过特异性与敏感性试验,CSFV与FMDV培养物均可扩增至10-5倍稀释,最终建立的二联RT-PCR方法对上述2种病毒的敏感性亦可达10-4倍稀释,约10 pg的总RNA,证明本法对上述2种病毒具有快速、特异和高度敏感的特点.