Inhibition of African swine fever infection in the presence of immune sera in vivo and in vitro

In assay cultures, sera from African swine fever convalescent pigs inhibited infection by homologous African swine fever virus. The infection-inhibition capacity did not correspond with the virus-neutralizing capacity. The serum did not prevent infection by heterologous virulent viruses. Sera from pigs challenge inoculated with the homologous virulent virus and later with a heterologous virulent virus inhibited the infection by different heterologous virulent viruses. These sera did not interfere with the infection by pseudorabies virus. The specificity of the reaction indicated that the infection inhibition was caused by antibody.     

Antibodies to bovine serum albumin in swine sera: implications for false-positive reactions in the serodiagnosis of African swine fever

Antibodies to bovine serum albumin were detected in swine sera by use of an immunoblotting technique. Such sera had false-positive reactions, as determined by results of African swine fever virus serodiagnostic techniques when bovine serum albumin was a contaminant in the soluble cytoplasmic antigen obtained from infected cells cultured in the presence of bovine serum. The soluble cytoplasmic antigen obtained from cell cultures infected with African swine fever virus in the presence of porcine serum did not react with the false-positive sera and, therefore, was used for African swine fever virus serodiagnostic methods, with 0% false-positive results.     

Inhibitory effect of African swine fever virus on lectin-dependent swine lymphocyte proliferation

The incubation of swine peripheral blood mononuclear cells (PBMC) with African swine fever (ASF) virus preparations strongly inhibited the proliferative response of lymphocytes to PHA and other lectins. The inhibition, which persisted after inactivation of the virus by UV radiation, was dependent upon the dose and the time that virus preparations were present in cultures. When virus preparations were fractionated by ultracentrifugation, the inhibitory activity resulted to be soluble, whereas no activity was found in the sedimented viral fraction. However, the preincubation during 4 days of this sedimented fraction with swine PBMC, before the addition of the mitogen, restored the inhibitory activity. The results obtained suggest that the inhibition is mediated by one or more soluble factors released by swine PBMC after coincubation with ASF virus in a time dependent process. These factors show a molecular weight between 40 and 80 kDa by gel filtration chromatography. The inhibitory activity described in the present paper is an indication of inhibition of lymphocyte function produced by ASF virus which can help to understand how this virus escapes from the host immune system.     

The pathogenic role of pulmonary intravascular macrophages in acute African swine fever

Recent studies of pulmonary intravascular macrophages have led to the re-examination of the mechanisms giving rise to alveolar oedema. A highly virulent isolate of African swine fever virus was replicated in pulmonary intravascular macrophages, interstitial and alveolar macrophages, fibroblasts and neutrophils. The alveolar oedema — characteristic of acute forms of African swine fever — and the vascular changes observed, which consisted of the formation of fibrin microthrombi in septal capillaries and the vacuolisation of endothelial cells, may have been due, however, to the activation of pulmonary intravascular macrophages, and not to the cytopathic effect subsequent to the replication of the African swine fever virus. Furthermore, it was observed that virus replication in cells not belonging to the mononuclear phagocyte system — such as fibroblasts and neutrophils — occurred earlier than in cells belonging to that system.     

非洲猪瘟疫苗研究的困境和展望

非洲猪瘟（ASF）目前是生猪产业最重要的猪病。因为非洲猪瘟病毒（ASFV）本身的复杂性,以及和宿主相互作用的复杂性导致ASF已经被报道一百年了,还没有商业化疫苗。理想的ASF疫苗不仅要有好的免疫保护性,更重要的是其安全性,同时如果能区分野毒感染和疫苗接种,能在适合的高质量GMP车间进行稳定低价的生产,能用于不同物种就更好了。ASF灭活疫苗研制这条道路似乎不通;亚单位疫苗、DNA疫苗、病毒活载体疫苗的免疫保护能力不足;主要包含自然缺失、传代致弱、基因工程缺失的减毒活疫苗在免疫保护方面体现了非常大的优势,但是其潜在的持续感染风险,会造成猪只的副作用,包括皮肤溃烂、关节炎,导致神经系统、呼吸系统的损伤等问题非常值得警惕。复制缺陷单周期病毒能有效地解决减毒活疫苗的安全性问题,似乎是一个值得尝试的ASF疫苗研制方向,尽管存在难以确定缺失复制相关的基因,以及难以构建能高效表达缺失基因编码蛋白,且能让单周期病毒稳定大量生产的辅助细胞系。我国针对ASFV强毒的精准剔除策略,未注册非法弱毒苗造成临床严重损失,以及一些不使用疫苗但成功从国家层面净化ASF的案例,让我们认识到针对ASF基础研究的重要性。同时至少目前阶段,ASF的防控不一定要借助疫苗,更多的要做好生物安全管控和区域ASF净化。尽管ASF疫苗研制困难重重,但针对ASF理想型疫苗的研制也应该持续进行下去,未来可能作为ASF防控的一个突破点。     

Detection of antibodies against African swine fever virus using infected monolayers and monoclonal antibodies

Three monoclonal antibodies, specific for porcine IgG, IgM and IgA, were used to develop isotype-specific immunoperoxidase monolayer assays for the detection of antibodies against African swine fever virus. A mixture of anti-IgM and anti-IgG monoclonal antibodies was used in an assay designed for screening sera. This test was compared with a commercially available ELISA by using experimental sera and field sera obtained after an outbreak of African swine fever on two farms in the Netherlands in 1986. Although the ELISA was less sensitive than the immunoperoxidase monolayer assay on sera taken early after infection, the tests were equally useful for screening purposes. The isotype-specific assays gave epizootiological information about the stage of infection on the two farms.     

Leukocyte-dependent platelet vasoactive amine release and immune complex deposition in African swine fever

Fifteen pigs were inoculated with African swine fever virus in a study of the pathogenesis of the disease. All pigs surviving the first two weeks developed high circulating antibody titers against African swine fever virus and persistent viremia. Hemolytic complement levels declined to 50 to 70 hemolytic complement 50 (CH50) units/ml from mean preinoculation levels of 120 CH50 units/ml. Immune deposits consisting of African swine fever antigen, host immunoglobulin G, and native C3 were found in the glomeruli of surviving pigs. All pigs surviving two weeks after inoculation developed leukocyte-bound antibody functionally characteristic of immunoglobulin E (IgE). Antigen-specific degranulation of antibody-coated leukocytes produced secondary platelet aggregation and vasoactive amine release. The results suggest that the IgE-basophil-platelet loop acting via amplification by leukocyte-derived platelet-activating factor participates in the immune complex deposition process in African swine fever.     

Extraction of viral DNA from erythrocytes of swine with acute African swine fever

The preparation of wild-type African swine fever (ASF) virus DNA from small amounts of viremic blood from acutely febrile pigs is outlined. The extracted DNA is viral and not host-cell DNA, because of specific homology with cell culture grown and purified ASF virus and because no DNA bands are obtained with an equal amount of nonviremic pig blood. Thus, in the absence of suitable serologic methods for strain identification, it is now possible to catalogue wild-type isolates by characteristic DNA restriction patterns. The wild-type virus genome contains terminal single-stranded DNA cross-links and has the largest genome size (180 kilobase pairs) reported for the ASF virus. Experimental passage of the virus in contact-infected pigs and buffy coat cultures appears to confirm the stable nature of the ASF genome in the field.     

非洲猪瘟病毒入侵细胞机制的研究进展

病毒进入宿主细胞的机制决定了病毒的趋向性和发病机理,病毒感染细胞包括吸附、穿入、脱壳、复制、组装及子代病毒颗粒的释放等步骤,对病毒入侵机制研究可以为开发有针对性的预防策略提供思路。非洲猪瘟病毒作为一种有囊膜双链DNA病毒,其入侵宿主细胞的机制与其他病毒有一定的相似之处,同样也有其特殊性。目前,人们对非洲猪瘟病毒与宿主细胞相互作用,特别是入侵机制的了解仍然非常有限。为此,本文就ASFV入侵细胞的有关研究进展进行了综述,以期为非洲猪瘟病毒致病机制相关研究提供参考。     

A highly sensitive and specific gel-based multiplex RT-PCR assay for the simultaneous and differential diagnosis of African swine fever and Classical swine fever in clinical samples

The development and standardisation of a novel, highly sensitive and specific one-step hot start multiplex RT-PCR assay is presented for the simultaneous and differential diagnosis of African swine fever (ASF) and Classical swine fever (CSF). The method uses two primer sets, each one specific for the corresponding virus, amplifying DNA fragments different in length, allowing a gel-based differential detection of the PCR products. Universal detection of ASF and CSF virus strains was achieved through selection of primers in conserved viral genome regions. The detection range was confirmed by analysis of a large collection of isolates of the two viruses. The high specificity of the assay was proven by testing related viruses, uninfected cell line cultures and healthy pig tissues. Additional confirmatory tests of the ASF and CSF virus amplicon specificity, based on restriction endonuclease analysis with BsmA I or Ban II, respectively, are also described. The analysis of whole blood and serum samples from experimentally infected animals proved the usefulness of the method for an early diagnosis of both diseases, even before the appearance of the first clinical signs. A study of 150 positive field samples from several ASF and CSF outbreaks showed the suitability of this method for a rapid (less than five hours), sensitive and specific differential diagnosis in clinical samples. In addition, a highly sensitive and specific uniplex RT-PCR for CSFV was also developed and standardised as a powerful tool for fast and early diagnosis of the disease.     

Virus-specific cellular blastogenesis and interleukin-2 production in swine after recovery from African swine fever

Animals recovered from viral diseases represent an important model to study the host cellular and humoral immune responses to the etiologic agents. This is particularly important for African swine fever virus (ASFV) infections in which antibodies have little or no virus-neutralizing effect. Pigs surviving experimental infection with the naturally occurring low-virulent, nonhemadsorbing ASFV/NH/P68 (NHV) isolate did, however, exhibit virus-specific T-cell activities, as measured by a variety of assays. A strong virus-induced, antigen-specific blastogenic response was observed only with blood mononuclear cells (BMC) from ASF-recovered swine, whereas cells from recovered and naive swine responded similarly to the mitogens concanavalin A and phytohemagglutinin. The ASFV-induced blastogenesis was dependent on virus dose and on the presence of adherent cells. Blood mononuclear cells cultured with antigenically related hemadsorbing ASFV isolates of different virulence characteristics, the highly virulent L60 isolate and moderately virulent DRII isolate, exhibited a similar magnitude of blastogenesis to cells infected with the low-virulent NHV isolate. Virus-infected cells proved to be an efficient inducer of interleukin-2 (IL-2) activity to cells from recovered swine, but not from naive swine, whereas T-cell-specific lectins induced production of similar amounts of IL-2 activity from cells of naive and recovered swine. Correlated with the appearance of virus-induced IL-2 activity in the culture supernatant was the induction of promiscuous killing in cells exposed to prolonged (7 days) virus stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Entry of African swine fever virus into Vero cells and uncoating

African swine fever virus (ASFV) enters Vero cells by adsorptive endocytosis [Valdeira, M.L., Geraldes, A., 1985. Morphological study on the entry of African swine fever virus into cells, Biol. Cell. 55, 35–40]. Electron microscopy of a lysosomotropic drug-controlled penetration indicated that this step takes place in the endosomes, after fusion between the viral envelope and the limiting membrane of the endosome. Inhibition studies with colcemid, cytochalasin B, sodium azide, dinitrophenol, lysosomotropic weak bases, and the ionophore monensin, showed that the virus uptake is largely independent of cytoskeletal and lysosomal function, but dependent on oxidative phosphorylation. Some protease inhibitors inhibited viral replication at an early step, indicating that the initiation of infection depends on a viral proteolytic cleavage.     

Interferon status and white blood cells during infection with African swine fever virus in vivo

African swine fever virus (ASFV) is the causative agent of African swine fever that is the significant disease of domestic pigs, with high rates of mortality. ASFV is double-stranded DNA virus whose genes encode some proteins that are implicated in the suppression of host immune response. In this study, we have modeled in vivo infection of ASFV for determination of interferon (IFN) status in infected pigs. We measured the level of IFN-α, -β and -γ by enzyme-linked immunosorbent assay and showed that the level of IFN-α sharply decreased during infection. Unlike IFN-α, the level of IFN-β and -γ increased from the 2nd and 4th days post-infection, respectively. Also, we analyzed the population dynamics of peripheral white blood cells of infected pigs due to their important role in host immune system. We showed that the atypical lymphocytes appeared after short time of infection and this result is in accordance with our previous study done in vitro. At the last day of infection about 50% of the total white blood cells were destroyed, and the remaining cells were represented mainly by small-sized lymphocytes, reactive lymphocytes and lymphoblasts.     

Detection of African swine fever virus antibodies by immunoblotting assay.

An immunoblotting assay has been adapted to detect antibodies against African swine fever virus. The electrophoretic transfer of proteins and the immunoreaction conditions were optimized, using 4 mA/cm2 of current intensity and 10 micrograms of soluble cytoplasmic antigen of infected cells per strip. Filters of polyvinylidene difluoride showed the highest capacity for protein absorption, but nitrocellulose filters showed lower backgrounds. The specificity and the pattern of the proteins induced by African swine fever virus that react with the antisera were determined in immunoblotting assay, IP30 being the most reactive protein.     

Diversity of African swine fever virus

An African swine fever virus is an heterogeneous population, consisting of clones having different biological characteristics in respect to hemadsorption, virulence, infectivity, plaque size, and antigenic determinants. The following observations were made: Nonhemadsorbing virus (NHV) have been segregated from field isolates from Haiti (HT-1) and a bone marrow- and buffy coat-passaged Portuguese isolate (L'60BM89BC1) and appear as a major, minor, or equal mixture with hemadsorbing viruses in the virus population. Biological characteristics of the virus inoculated into pigs often differed from viruses isolated later from the same pigs. Virulence and nonhemadsorbing characteristics of isolated clones were genetically stable. The lethal effect of 2 NHV clones of L'60BM89BC1 virus was dose-dependent; small doses of virus induced immunologic deaths or recoveries from the clinical disease in pigs, and large doses induced acute deaths. The NHV of Lisbon isolate of 1960 (L'60) and HT-1 isolate share the same antigenic determinants for inducing protection. Tengani isolate contained clones of distinctly different antigenic determinants, not shared by L'60 or HT-1 isolate that enabled it to overcome the protection induced by the other clones. Passaging of an African swine fever virus isolate in pigs or cell cultures may readily alter the proportions of the different clones in the population and thereby change its overall characteristics. A new virus population with atypical hemadsorption was found in HT-1 field isolate and L'60BM89BC1 virus.     

浅谈非洲猪瘟防控措施

非洲猪瘟是一种高度接触性的传染病，对养猪业造成了巨大的损失；随着人们对非洲猪瘟的认识逐步深化，猪场防控措施也逐步在完善；构建完善的生物安全体系是防控的核心，将病毒阻挡在猪场之外；对于有问题的猪场，全面排查和“精准拔牙”，结合抗体检测能够很好地控制住病毒的传播，使猪群稳定下来。     

Mechanism of thrombocytopenia in African swine fever

Pigs were inoculated with an African swine fever (ASF) isolate of moderate virulence, and the changes in the number of circulating blood platelets during infection were correlated with the appearance of antiviral antibody and fluctuations in total plasma hemolytic complement concentrations. Thrombocytopenia was detected by postinoculation days (PID) 7 and 8, and antiviral antibody was detected by PID 7, using an indirect immunofluorescence technique. The total hemolytic complement concentration was moderately and transiently decreased from PID 5 to 9, but was consistently low from PID 18 to 26. Pigs inoculated with an ASF virus isolate of greater virulence had a decrease in platelet counts on PID 6 and 7, and the total plasma hemolytic complement levels decreased in all pigs by PID 6 to 7. Antibody to ASF virus was not detected in pigs inoculated with the more virulent isolate. Pigs sensitized to ASF viral antigen with an inactivated-virus vaccine or by previous infection with ASF were challenge exposed. Sensitized pigs became clinically ill and thrombocytopenic by 24 to 72 hours earlier than did inoculated, nonsensitized pigs. Vaccinated pigs inoculated with homologous virus had lower blood virus concentrations than did nonvaccinated pigs. African swine fever virus-sensitized pigs inoculated with heterologous virus had a higher fatality rate than did nonsensitized pigs, and the pigs died peracutely, with only a few gross lesions in evidence. In vitro experiments demonstrated that ASF virus antigen induced platelet aggregation in platelet-rich plasma from recovered, nonviremic pigs. Viral antigen, antibody, or complement was not demonstrable on the surface of platelets from pigs inoculated with ASF virus isolate, by direct immunofluorescence testing.     

Approaches to the identification of non-essential genes of African swine fever virus.

It is poorly understood why vaccines could not be developed for the control and prevention of African swine fever (ASF) virus infection. The aim of our study was to identify genes non-essential for ASF virus replication because there were indications that certain viral gene products, which apparently are non-essential for viral replication, conferred protection from death due to ASF. A cosmid library representing the genome of ASF virus strain France 64 was established and characterized. Then, in order to inactivate viral genes by insertion, the beta-galactosidase (beta-gal) gene was introduced either randomly or at specific locations of selected cloned DNA fragments. These constructions were transfected into cells which had been previously infected with a cell-culture-adapted viral strain in order to allow the generation of recombinant progeny virus. Viable recombinant progeny was identified by at least one of the following means: (1) expression of beta-gal; (2) detection of beta-gal specific DNA by plaque hybridization, and (3) absence of a functional product of the inactivated gene. Presently, we are characterizing a recombinant virus with an insertionally inactivated thymidine kinase gene.     

Cytotoxic lymphocytes induced by African swine fever infection

The generation of lymphocytes cytotoxic to African swine fever virus infected testis cells during in vivo infection is described. Peripheral blood lymphocytes from 16 pigs developed cytotoxicity seven to eight days after infection but the lysis was not restricted to autologous cells.     

African swine fever: microplaque assay by an immunoperoxidase method.

A microplaque assay for Vero cell-adapted Lisborn '60 strain of African swine fever virus (L'60-uncloned) and a large plaque-forming strain cloned from the L'60-uncloned strain was developed by an immunoperoxidase method. The immunoperoxidase method can be used to stain microplaques of 3 days after inoculation, whereas the conventional plaque assay requires 5 to 7 days to develop visible plaques. A linear relationship between viral concentration in the inoculum and plaque numbers was observed. Viral titers obtained by both microplaque assay and conventional plaque assay were comparable, and both methods were reproducible and reliable. The viral titer obtained by either one of the plaque assay methods was approximately 0.9 log10 lower than that obtained by the hemadsorption test.