Recent advances in the development of recombinant vaccines against classical swine fever virus: cellular responses also play a role in protection

Classical swine fever virus (CSFV) is the causative agent of one of the most devastating porcine haemorrhagic viral diseases, classical swine fever (CSF). CSFV mainly infects endothelial cells and macrophages and at the same time promotes bystander apoptosis of the surrounding T cells, causing strong immune suppression and high mortality rates. Most animals experience acute infection, during which they either die or survive by producing neutralising antibodies to the virus. However, in a few cases, the impaired immune system cannot control viral progression, leading to chronic infection. Efficient live attenuated vaccines against CSFV exist and are routinely used only in endemic countries. The ability of these vaccines to replicate in the host, even at very low rates, makes it extremely difficult to distinguish vaccinated from infected animals, favouring a restricted policy regarding vaccination against CSFV in non-endemic countries. There is a clear need for efficient and safer marker vaccines to assist in the control of future CSF outbreaks. In this review article, some of the most recent advances in the field of recombinant vaccines against CSFV are presented and the nature of the protective immune responses they induce is discussed.     

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.     

猪瘟疫苗研究进展

猪瘟是猪的一种高度接触性传染病 ,由于其造成巨大的经济损失 ,故而为清除本病作了很多工作。猪瘟疫苗的接种是控制猪瘟的重要手段。猪瘟疫苗包括灭活苗、弱毒苗及基因工程疫苗。猪瘟兔化弱毒苗因其优良的免疫原性是目前猪瘟防治应用最为广泛的疫苗之一。基因工程疫苗是目前猪瘟疫苗研究的热点领域 ,其包括亚单位疫苗、合成肽苗、活载体苗、核酸疫苗等。值得一提的是猪瘟病毒标记疫苗研究近年来发展迅速 ,目前已出现商品化的亚单位标记疫苗 ,可使猪瘟疫苗接种和自然感染区别更为简单、快捷。本文就猪瘟疫苗的发展及应用作了简要介绍     

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.     

Monitoring of classical swine fever in wild boar (Sus scrofa) in Slovenia

Classical swine fever (CSF) is a highly contagious multi-systemic haemorrhagic viral disease of pigs. Not only domestic pigs, but also wild boar appear to play a crucial role in the epidemiology of CSF. Spleen (n = 739) and blood coagulum (n = 562) sampled from wild boars (Sus scrofa) shot in 2002, and serum samples from 746 wild boar shot in 2003 and 2004, were tested throughout Slovenia. In 2002, 17 samples were positive on enzyme-linked immunosorbent assay (ELISA) test for antibodies against classical swine fever virus (CSFV). Positive ELISA test was confirmed by a virus neutralization test. All other samples were negative. This is the first report that describes the epidemiology of CSFV from 2002 on, and the monitoring of the wild boar population in Slovenia at present.     

Classical swine fever (CSF) in wild boar: the role of the transplacental infection in the perpetuation of CSF

Thirty-four pregnant wild sows and their unborn progeny derived from an endemically infected population in the district of Nordvorpommern (Mecklenburg-Western Pomerania) were investigated for classical swine fever virus (CSFV) and antibodies. During the last 2.5 years of the epidemic, 20 out of 34 pregnant wild sows investigated were serologically positive. No CSFV or viral RNA was detected in organs derived from these animals and their progeny. This indicates that young wild boars persistently infected by transplacental virus transmission do not play a crucial role in the perpetuation of CSFV in wild boar. Other factors seem to be more important for the establishment of CSF as well as for virus perpetuation in the population.     

分子生物学技术在猪瘟诊断中的应用

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有效的控制.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.早已宣布消灭了猪瘟的一些欧洲国家又报道有猪瘟发生.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,给养猪业造成极大的经济损失.论文综述了猪瘟分子生物学诊断技术的研究进展,为猪瘟的及时诊断提供参考.     

Isolation and characterization of immortalized porcine aortic endothelial cell lines

Primary porcine endothelial cells have a limited life span in culture. After four to five passages, they tend to de-differentiate and eventually reach senescence. The aim of this work was to establish immortalized porcine aortic endothelial cell lines (AOCs) to facilitate in vitro studies of different pathological process involving the endothelium. Primary porcine aortic endothelial cells (PAECs) were transfected with a plasmid containing the SV40 genome and selected on the basis of morphological and phenotypical features. Flow cytometry analysis demonstrated uptake of acetylated low density lipoproteins (Ac-LDL) and constitutive expression of SLA class I, CD29, CD31, CD41/61, CD80/86, CD46, SWC3, and LAMP-1 antigens by all analyzed lines and showed little differences to primary cells. The functional similarity between primary and immortalized endothelial cells was demonstrated in a cytotoxicity assay using a human natural killer cell line (NKL) as effector. The AOCs cell lines should be valuable tools for in vitro study of the human immune response against pig endothelial cells. In addition, they would be very useful to gain insight in the pathogenesis of some viral haemorrhagic diseases of pig such as African swine fever (ASF) or classical swine fever (CSF).     

逆向遗传学技术在猪瘟病毒研究中的应用

猪瘟是猪的最严重疾病之一,其病原 是猪瘟病毒,基因组为单股正链RNA。猪瘟 弱毒疫苗在猪瘟的免疫防治中发挥了巨大作 用,但由于弱毒疫苗免疫的动物不能从血清 学上与自然感染动物区分,使其应用受到很 大限制,研制标记疫苗是解决这个问题的重 要途径。由于RAN的结构不稳定成为阻碍 RNA病毒研究的主要障碍,所以病毒分子生 物学是新型猪瘟疫苗研究的必要手段。近年 来兴起的逆向遗传研究将RNA病毒的基因 组转化为cDNA,文章就猪瘟病毒逆向遗传 研究的意义、方法、概况及其在猪瘟新型疫苗 研究中的应用进行了全面综述。     

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.     

上海地区规模化猪场牛病毒性腹泻病毒感染状况的血清学调查

为调查猪瘟疫苗是否会引起牛病毒性腹泻的发生及上海地区规模化猪场该病的流行情况,本试验采用酶联免疫吸附试验方法,对分别免疫接种3种猪瘟疫苗的57头试验猪以及上海地区2005年-2009年10个区(县)共740份血清进行了牛病毒性腹泻病毒抗原和抗体的检测.经检测,所有样品抗原和抗体均为阴性.结果表明,免疫猪瘟脾淋苗不能使猪产生BVDV交叉抗体,猪群因猪瘟疫苗感染BVDV的可能性比较低,该病近几年在上海市猪群中并没有出现.     

The lesional changes and pathogenesis in the kidney in african swine fever

African swine fever is a viral haemorrhagic disease of pigs which has been used as a model for the study of viral haemorrhagic diseases in man. The acute course of the disease is characterized by acute proliferative glomerulonephritis, with viral replication in mesangial cells and occasional focal necrosis of the renal tubular system; hyperplasia of the collecting ducts is associated with evident virus replication. Haemorrhages have been attributed to endothelial dysfunction, aggravated by virus replication in endothelial cells in the final stages of the disease. The renal interstitium displays intense oedema and an infiltrate largely composed of macrophages. Virus replication has also been observed in fibroblasts and in the smooth0muscle cells of arterioles and venules.In subacute-chronic forms of the disease, various types of glomerulonephritis are observed, ranging from mesangial proliferative glomerulonephritis to focal and segmental hyalinosis associated with immune-mediated phenomena. No striking changes are reported in the renal tubular system. Interstitial haemorrhages are associated with diapedesis due to immunologically mediated events. The interstitium has also been found to contain a lymphohistiocytic infiltrate with abundant plasma cells. No evidence has been reported of viral replication in any cell population.Abbreviations ASF African swine fever - ASFV African swine fever virus - C complement component - DNA deoxyribonucleic acid - HAD₅₀ 50% haemoadsorbing doses - Ig immunoglobulin - MPS mononuclear phagocytic system - PAS periodic acid-Schiff reagent     

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.     

A review of viral diseases of the European wild boar: effects of population dynamics and reservoir rôle

There has been a worldwide increase in the number and geographical spread of wild boar populations in recent decades leading to an increase in both the circulation of disease agents and greater contact with domestic animals and humans. Diseases affect the population dynamics of wildlife but the effects of most viral diseases on the European wild boar are largely unknown. Many viral diseases present in domestic pig populations are also present in wild boars where they can provide a disease reservoir, as is clearly the case with classical swine fever, but little is known about other viral diseases such as porcine circovirus diseases or hepatitis E. This review considers the current scientific knowledge of the effects of viral diseases on wild boar populations and their r?le as potential disease reservoirs. The focus is on those viral diseases of domestic swine and wild boars that are included as notifiable by the Office International des Epizooties (OIE).     

古典猪瘟病毒基因组及ORF编码蛋白的结构和功能

古典猪瘟是严重危害养猪业发展的重要疫病之一,对古典猪瘟病毒的分子生物学研究能揭示病毒复制和致病机理,寻找免疫保护关键位点,为新型疫苗开发奠定基础。文章综述了近年对古典猪瘟病毒基因组和相关蛋白的研究成果,对以后的研究方向作了展望。     

A histopathologic,immunohistochemical, and ultrastructural study of the intestine in pigs inoculated with classical swine fever virus

The aim of this study was to report on the lesions occurring in the intestine during experimental classical swine fever (CSF) and to clarify the nature of infected cells and the distribution of viral antigen. Thirty-two pigs were inoculated with the virulent CSF virus (CSFV) isolate Alfort 187 and slaughtered from 2 to 15 postinoculation days; four animals of similar background served as a control group. Immunohistochemistry, electron microscopy, and the transferase-mediated deoxyuridine triphosphate nick-end labeling method were used to detect viral antigens and apoptosis. The results showed progressive lymphoid depletion and mucosal necrosis. The lymphoid depletion could have been caused by apoptosis of lymphocytes but could not be directly attributed to the effect of CSFV on these cells. Vascular changes, pathogenic bacteria, and viral infection of epithelial cells were ruled out as causes of necrotic lesions. However, large virally infected monocytes-macrophages with ultrastructural changes indicative of activation were observed in the intestine. This suggests that monocytes-macrophages play an important role in the pathogenesis of intestinal lesions. An understanding of the function of these cells will require additional study.     

Hygienic Applications of Biotechnical Measures in Pigs*

A key factor in the hygienic application of embryo transfer (ET) and its associated technologies is effective risk management. This depends on knowing what the risks of infectious disease transmission are and how they can be reduced or removed. Risk assessment depends on a knowledge of the pathogenesis of the disease, the procedures used in ET and its associated technologies and the results from research into infectious disease transmission by embryos or through ET. Two approaches can be taken to ensure the safe health status of the embryo: 1) by determining that the donor animals (male and female) are free from specific diseases, or 2) by ensuring that the zona pellucida (ZP)-intact embryo is collected in a sanitary manner and handled (washed, treated, evaluated, etc.) according to recommended procedures. The former approach is applicable to diseases on which no or insufficient research has been done to determine the risk of their being transmitted by embryos or where the results of research done indicate a risk of the disease being transmitted. The latter approach is a preferred option with diseases where results of research done indicate that the risk of their being transmitted by embryos collected from infected or recovered donors is negligible. Infectious diseases for which research results indicate negligible risk of transmission by ZP-intact (ZP-I) embryos collected from infected or recovered donors include: pseudorabies (Aujesrky's disease), hog cholera (swine fever), foot and mouth disease, and swine vesicular disease. Infectious diseases on which insufficient research has been done include: African swine fever, vesicular stomatitis, enterovirus disease, parvouirus disease and leptospirosis. A problem associated with this approach is ensuring that procedures shown to be effective under experimental conditions are properly carried out under field conditions: the use of nationally accredited embryo collection teams may solve this problem. Healthy recipients and good record keeping are also critical factors in the hygienic application of ET. The health status of an embryo collected from a specific disease-free donor will not be adversely affected by damage to its ZP. Where research has shown that ZP-I embryos that have been exposed to particular pathogens are not infected or contaminated after proper washing or washing and treatment, it can be assumed that the risks of transmission of the diseases caused by these pathogens will not be increased by deliberate (micromanipulation) or accidental damage to the ZP after the washing/treatment procedures have been carried out. The risks of infectious disease transmission when embryos are produced by in vitro fertilization or blastomere transplantation to mature oocytes, followed by culture, have yet to be determined.     

猪瘟抗体胶体金快速定量检测卡的研制

应用胶体金免疫层析技术,结合胶体金定量读数仪研制出一种快速定量检测猪血清中猪瘟抗体水平的检测卡。该检测卡以胶体金标记高灵敏的猪瘟重组E2蛋白,同时在NC膜上包被同一蛋白,经正交实验确定最适条件,同时通过与对照线的颜色对比,应用胶体金定量读数仪,可对样本中猪瘟抗体水平进行定量。该检测卡检测方法简便、快速、稳定性好。与猪圆环病毒病、猪伪狂犬病、猪繁殖与呼吸综合征等常见猪疫病抗体均无交叉反应。检测71份猪血清样本结果与ELISA结果的符合率达90.1%。结果表明,实验研制的猪瘟抗体胶体金快速定量检测卡可用于基层兽医站和养殖企业的检测。