In vitro immune serum-mediated protection of pig monocytes against African swine fever virus

Serum samples from pigs that had recovered from infection with a Dominican Republic isolate of African swine fever virus (ASFV) were mixed with dilutions of the virus, then assayed in microcultures of normal pig mononuclear leukocytes to determine whether the samples contained antibodies that protected monocytes against the virus. Protection was determined by the difference in titer (log10) between virus mixed with healthy pig serum and virus mixed with immune pig serum, using 50% cytopathogenic effect end points; protection was expressed as an immune serum-protection index. After addition of virus-serum mixtures to mononuclear leukocyte microcultures, a time-dependent decrease in protective index and production of infectious virus (determined by use of yield reduction assays) were observed. Protective effects were associated with the immunoglobulin fraction of serum, were rapidly lost on dilution, and were independent of complement. Antibody was most protective for the homologous Dominican Republic isolate of ASFV, with decreased protection against Lisbon '60 ASFV, and no protection against foot-and-mouth disease virus or bluetongue virus. Low concentrations of protective antibody were found during the acute viremic phase in infected pigs; antibody increased to maximal concentrations as the viremia decreased.     

Cytopathogenic effect of African swine fever virus for pig monocytes: characterization and use in microassay

A microculture assay is described for the titration of African swine fever virus (ASFV) using swine monocytes contained in mononuclear leucocyte (MNL) microcultures. Titration endpoints were determined by observing cytopathogenic effects (CPE) of ASFV infected monocytes with an inverted microscope at 40 X magnification. CPE was a late event following the detection of ASFV antigens in monocytes by radioimmune assay, immunofluorescence and hemadsorption. It began with the detachment, enlargement and rounding of monocytes which progressively formed into grape-like clusters of 3-20 or more cells which eventually lysed. The characteristic CPE was produced in monocyte microcultures by virulent, moderately virulent, Vero cell adapted, and nonhemadsorbing ASFV strains. The sensitivity and reproducibility of the CPE microassay was similar to that of the hemadsorption microassay.     

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.     

Cytokine mRNA expression and pathological findings in pigs inoculated with African swine fever virus (E-70) deleted on A238L

African swine fever virus (ASFV) induces a variety of immune responses and clinical forms in domestic pigs. As it is the only member of the Asfarviridae family, ASFV encodes many novel genes not encoded by other virus families. Among these genes, A238L may regulate the synthesis of pro-inflammatory cytokines, controlled mainly by NFkappaB and NFAT pathways. In this study, we inoculated two groups of pigs, one with the ASFV highly virulent E-70 isolate, deleted on A238L gene, and the other group with the parental E-70 isolate. No significant differences were observed in the clinical signs or pathology between both groups. However, the TNF-alpha mRNA expression was strongly enhanced in the PBMC from pigs inoculated with the virus deleted in A238L, reinforcing the role of the A238L gene in the inhibition of the NFkappaB pathway of expression of cytokines. No up-regulation of pro-inflammatory cytokines was observed in the PBMC of animals inoculated with the E-70 isolate, even though apoptosis and haemorrhages were evident and might be related to the presence of bystander monocyte-macrophages expressing these cytokines. Other studies using ASFV deleted in other genes inoculated in the natural hosts should be performed to gain further insight into the role of these genes in the pathogenesis of ASF.     

In vitro and in vivo association of African swine fever virus with swine erythrocytes

The association of African swine fever virus (ASFV) with swine erythrocytes in vivo, in high titers, was verified by inoculating 30 pigs with 17 ASFV isolates and assaying their plasma and washed erythrocyte fractions for residual virus. Viral antigens were specifically localized on the surface of in vitro and in vivo swine erythrocytes, using the fluorescent antibody technique and 3 monoclonal antibodies specific for ASFV. The same monoclonal antibodies immunoprecipitated virus-specific polypeptides of molecular weights 13 kd and 73 kd from ASFV-infected Vero cells. Erythrocytes from viremic swine infected with Lisbon-60, Dominican Republic, Badajoz-M98, or Cameroon isolates of ASFV were studied by transmission electron microscopy. Virus was found in membrane depressions at the surface of erythrocytes. These surface depressions resembled stages of smooth surfaced pits. Erythrocytes from viremic pigs were fragile osmotically.     

Dynamics of African swine fever virus shedding and excretion in domestic pigs infected by intramuscular inoculation and contact transmission

African swine fever virus (ASFV) is a highly virulent swine pathogen that has spread across Eastern Europe since 2007 and for which there is no effective vaccine or treatment available. The dynamics of shedding and excretion is not well known for this currently circulating ASFV strain. Therefore, susceptible pigs were exposed to pigs intramuscularly infected with the Georgia 2007/1 ASFV strain to measure those dynamics through within- and between-pen transmission scenarios. Blood, oral, nasal and rectal fluid samples were tested for the presence of ASFV by virus titration (VT) and quantitative real-time polymerase chain reaction (qPCR). Serum was tested for the presence of ASFV-specific antibodies. Both intramuscular inoculation and contact transmission resulted in development of acute disease in all pigs although the experiments indicated that the pathogenesis of the disease might be different, depending on the route of infection. Infectious ASFV was first isolated in blood among the inoculated pigs by day 3, and then chronologically among the direct and indirect contact pigs, by day 10 and 13, respectively. Close to the onset of clinical signs, higher ASFV titres were found in blood compared with nasal and rectal fluid samples among all pigs. No infectious ASFV was isolated in oral fluid samples although ASFV genome copies were detected. Only one animal developed antibodies starting after 12 days post-inoculation. The results provide quantitative data on shedding and excretion of the Georgia 2007/1 ASFV strain among domestic pigs and suggest a limited potential of this isolate to cause persistent infection.     

Moderately virulent African swine fever virus infection: blood cell changes and infective virus distribution among blood components

Blood samples of pigs infected with a moderately virulent African swine fever virus (ASFV) isolate, obtained from the Dominican Republic (DR-II), were monitored temporally for viremia, infective ASFV association with major blood components, differential changes in blood cell composition, and plasma antibodies to ASFV. After intranasal/oral virus inoculation, pigs underwent acute infection and illness that resolved. Acute illness began on postinoculation day (PID) 4 and continued to PID 11, and pigs were febrile, with maximal infective ASFV titers detected in blood. By PID 11, initial antibody titers to ASFV antigens were detected in plasma. The WBC numbers were maintained near preinoculation counts; however, lymphocyte counts decreased slightly with a compensatory increment in neutrophil and monocyte numbers. From PID 11 to PID 25, rectal temperatures gradually returned to preinoculation values, titers of viremia began to decrease, plasma antibody to ASFV antigens increased to peak titers, and WBC numbers increased slightly. Percentages of lymphocytes returned to preinoculation values, neutrophil percentages decreased to slightly below preinoculation values, monocyte percentages were mildly increased, and eosinophil percentages were unaffected. From PID 25 to PID 46, titers of viremia further decreased, and plasma titers of antibodies to ASFV antigens remained high. In pigs with DR-II viremia (PID 4 to PID 46), most viral infectivity (greater than 95%) was RBC associated. Plasma contained less than 1% infectivity, and less than 0.1% of virus was in the WBC fraction (monocytes, lymphocytes, and granulocytes). After PID 46, viremia was no longer detectable.     

不同非洲猪瘟病毒株j5R基因及其编码蛋白的比较

根据非洲猪瘟病毒ＭａｌａｗｉＬＩＬ２０／１株ｊ５Ｒ阅读框Ｃ端抗原决定簇序列制备的合成肽能被不同毒株感染康复猪的免疫血清识别。多聚酶链反应研究表明,９个不同毒株的ｊ５Ｒ基因长度略有差异;蛋白转印杂交试验证明,这种基因长度的差异导致相应蛋白多肽的长度多样性。这些结果进一步证明,长度多样性是非洲猪瘟病毒基因及其编码蛋白较为普遍的特点。     

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.     

Quantitative aspects of the transmission of African swine fever

The contagiousness of pigs during different stages of infection with African swine fever virus was assessed by measuring the amount of virus excreted and the amounts of virus in the blood and other tissues, as well as determining the infectious dose of the virus by various routes. The virus was present in substantial amounts in secretions and excretions of acutely infected pigs for only 7 to 10 days after the onset of fever and was present in the greatest amount in the feces. Virus persisted in the blood of some recovered and clinically normal pigs for 8 weeks and in the lymphoid tissues for at least 12 weeks. The intranasal/oral ID50, and the IV/IM ID50 of a moderately virulent isolate of African swine fever virus were determined to be 18,500 and 0.13, 50% headsorbing units, respectively. A highly virulent isolate required approximately 10-fold more virus to cause infection by the intranasal/oral route.     

Changes in macrophages in spleen and lymph nodes during acute African swine fever: expression of cytokines

To gain further insight into the pathogenesis of African swine fever (ASF), the cytokine expression by macrophages in spleen and lymph nodes were examined. Twenty-one piglets were inoculated with the highly virulent isolate Spain-70 of ASF virus and killed in groups at 1-7 post-inoculation days (pid). An increase in the immunohistochemical detection of proinflammatory monokines in spleen and renal and gastrohepatic lymph nodes is reported, along with an increase in the serum levels of TNF-alpha and IL-1 beta. The expression of these cytokines is detected simultaneously in time and space with the viral protein 73 (vp 73) of the ASF virus detection. Our results demonstrate that mononuclear phagocyte system cell activation results in the release of several cytokines that could induce apoptosis of lymphocytes and haemodynamic changes.     

非洲猪瘟病毒入胞过程相关蛋白及分子机制的研究进展

非洲猪瘟病毒(ASFV)是非洲猪瘟(ASF)的病原,可引发家猪和野猪急性、传染性出血死亡,目前尚无有效的疫苗和抗病毒药物。ASFV是囊膜病毒,其囊膜蛋白的结构和功能可能是影响病毒入侵和细胞嗜性的重要因素。病毒入胞是病毒感染细胞的第一步,通常是通过细胞表面特定的分子与病毒蛋白相结合吸附于宿主细胞表面,对ASFV入胞过程的病毒蛋白或宿主因子为靶点或可有效抑制ASFV的复制。本文从ASFV入胞过程中起重要作用的囊膜蛋白出发,对ASFV的入胞分子机制进行综述,为ASFV入胞深入研究以及治疗性药物和疫苗的研发提供参考。     

The pathogenesis of vesicular exanthema of swine virus and San Miguel sea lion virus in swine

Vesicular exanthema of swine virus type A48 or San Miguel sea lion virus type 2, when inoculated intradermally into swine, resulted in fluid-filled vesicles at the sites of inoculation in the snout, coronary band, and tongue. Pigs that developed vesicles also had fevers. Secondary vesicle formation varied, depending on virus serotype. Viremia was found in one pig infected with San Miguel sea lion virus five days after infection. Virus was recovered from nasal-oral passages for up to five days after infection in both groups of pigs and from the gastrointestinal and urinary tracts of pigs infected with San Miguel sea lion virus. Neutralizing antibodies began to increase three days after inoculation and reached peak titers in seven to ten days. In the absence of secondary bacterial infection, healing was well advanced by ten days after inoculation. Lesions usually were limited to nonhaired portions of the integument and tongue. Individual epithelial cells became infected when a break in the skin allowed virus access to susceptible epithelial cells from either exogenous or endogenous sources. Individual infected cells ruptured and adjacent cells were infected, resulting in the formation of multiple microvesicles. Centrifugal coalescence of microvesicles led to formation of grossly visible macrovesicles. Lesions rarely developed from viral contamination of intact hair follicles. A mild virus-induced encephalitis was seen in pigs infected with vesicular exanthema of swine virus, and virus was recovered from brain tissue of pigs infected with San Miguel sea lion virus.     

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.     

The Antibody Response in Pigs Inoculated with Attenuated African Swine Fever Virus

Pigs were inoculated with a modified isolate of African swine fever virus (ASFV). Complement-fixing (CF) and agar gel diffusion precipitin (AGDP) antibodies could be detected in the serums of most pigs from 14-days postinoculation (DPI) until their immunity was challenged with virulent ASFV at 117 DPI.

Reductive cleavage with 2-mercaptoethanol showed that serums collected at 14 to 35 DPI contained 19S antibody, but that the 7S antibody was dominant at 35 and 117 DPI. This distribution of antibody was confirmed by sucrose-gradient centrifugation. Nearly all of the early serums also contained 7S antibodies which fixed complement and reacted in the AGDP test. Pigs whose serums contained both CF and AGDP antibodies at time of challenge failed to develop acute disease while pigs without CF antibodies were usually not protected.

Pigs surviving challenge with virulent virus showed no increase in antibody titers, or reversion to 19S antibody.

     

Patterns of alveolar macrophage activation upon attenuated and virulent African swine fever viruses in vitro

The pattern of porcine alveolar macrophage (AM) activation upon classical stimuli of two strains of African swine fever (ASF) viruses, an attenuated ASFV-BA71V and virulent ASFV-Georgia2007 were investigated. In an in vitro experiment ASFV-Georgia2007-infected AM showed M1 polarization pattern different from the one induced by classical stimuli. Altered morphology, appearance of binuclear cells, decreased synthesis of IFN-alpha as well as IFN-epsilon was observed compared with attenuated ASFV-BA71V, and decreased synthesis of IFN-omega compared with intact cells. However, CD68 level did not significantly differ between alveolar macrophage populations infected by ASFV-Georgia2007 and control group, while both LPS/IFN-gamma stimulation and non-pathogenic ASFV-BA71V virus increased the level of CD68 soluble receptor.AM infection with ASFV-Georgia2007 resulted in remarkable DNA proliferation whereas LPS/IFN-gamma and ASFV-BA71V induced less expressed DNA proliferation in activated cells. The higher value of nitric oxide was obvious in the cells infected with ASFV-BA71V, compared to ASFV-Georgia2007 and LPS/IFN-gamma activated cells.In conclusion, pattern of activation of alveolar macrophages induced by ASFV-Georgia2007 virus differs from the one expressed in LPS/IFN-gamma- and ASFV-BA71V-activated cells. ASFV-BA71V and LPS/IFN-gamma share similar antiviral response of porcine AM. Therefore we assume that wild type virulent ASFV can partially down regulate antiviral response of AM and conclude that evolutionary decrease of virulence in ASFV is related to alterations of control of the host cell antiviral response.     

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.     

Effect of infections with swine fever virus on immune functions. III. Antibody response to lipopolysaccharide and sheep red blood cells

In order to determine the effect of infections with low-virulent swine fever virus (SFV) on antibody responses, pigs were administered lipopolysaccharide (LPS) or sheep red blood cells (SRBC), 2 days after infection. Infected pigs showed an enhanced primary response to LPS late during infection. The secondary response to LPS seemed to be unaffected. Both the primary and secondary antibody response to SRBC appeared to be enhanced rather than depressed in infected pigs. These in vivo findings suggest that pigs infected with low-virulent SFV do not develop a depression of B lymphocyte function.     

In vivo and in vitro effects of moderately virulent African swine fever virus on mitogenesis of pig lymphocytes

Six pigs were infected oro-nasally with a moderately virulent African swine fever (ASF) virus from the Dominican Republic (DR II). The effect of virus infection on the pig's immune system was tested by measuring peripheral leucocyte numbers and the ability of mononuclear leucocytes (MNL) to respond by lymphocyte proliferation (LP) to the mitogens phytohemagglutinin-P (PHA-P), concanavalin-A (Con-A), and pokeweed mitogen (PWM). All 6 pigs developed high viremias between 4 and 18 days post-inoculation (DPI) which became undetectable by 32 to 46 DPI. Virus was found in erythrocytes, plasma, and mononuclear leucocytes from peripheral blood. Overall, virus infection had only minor effects on the number of circulating leucocytes, lymphocytes, monocytes and granulocytes. At the early acute phase of infection slight neutrophilia and lymphocytopenia were observed with mildly elevated monocyte numbers and slightly depressed neutrophil numbers that continued from the time of evident reduction in viremia to beyond the period of viral clearance. The infected pigs readily produced high titers of ASF virus antibody shortly after the onset of viremia. No significant differences in LP responses of MNL from the 6 pigs to PHA-P, Con-A and PWM were observed after infection when compared to those obtained with MNL from normal pigs. The in vitro addition of infectious ASF virus to MNL from normal pigs did not affect LP responses to any of the three mitogens. These results do not support the hypothesis that immunosuppression is a consequence of ASFV infection of pigs.