African swine fever virus causes hemorrhagic disease in swine. Attenuated strains are reported in Africa, Europe, and Asia. Few studies on the diagnostic detection of attenuated ASF viruses are available. Two groups of pigs were inoculated with an attenuated ASFV. Group 2 was also vaccinated with an attenuated porcine reproductive and respiratory syndrome virus vaccine. Commercially available ELISA, as well as extraction and qPCR assays, were used to detect antibodies in serum and oral fluids (OF) and nucleic acid in buccal swabs, tonsillar scrapings, OF, and blood samples collected over 93 days, respectively. After 12 dpi, serum (88.9% to 90.9%) in Group 1 was significantly better for antibody detection than OF (0.7% to 68.4%). Group 1′s overall qPCR detection was highest in blood (48.7%) and OF (44.2%), with the highest detection in blood (85.2%) from 8 to 21 days post inoculation (dpi) and in OF (83.3%) from 1 to 7 dpi. Group 2′s results were not significantly different from Group 1, but detection rates were lower overall. Early detection of attenuated ASFV variants requires active surveillance in apparently healthy animals and is only reliable at the herd level. Likewise, antibody testing will be needed to prove freedom from disease.
The African swine fever virus (ASFV) is the cause of a recent pandemic that is threatening the global pig industry. The virus infects domestic and wild pigs and manifests with a variety of clinical symptoms, depending on the strain. No commercial vaccine is currently available to protect animals from this virus, but some attenuated and recombinant live vaccine candidates might be effective against the disease. This article describes the immunobiological characteristics of one such candidate—the laboratory-attenuated ASFV strain, Katanga-350—which belongs to genotype I. In this study, we assessed clinical signs and post-mortem changes, the levels of viremia and the presence of viral DNA caused by injection of ASF virus strains Katanga-350, Lisbon-57, and Stavropol 08/01. Intramuscular injection of this strain protected 80% of pigs from a virulent strain of the same genotype and seroimmunotype (Lisbon-57). At least 50% of the surviving pigs received protection from subsequent intramuscular infection with a heterologous (genotype II, seroimmunotype VIII) virulent strain (Stavropol 08/01). Virus-specific antibodies were detectable in serum and saliva samples between 8–78 days after the first inoculation of the Katanga-350 strain (the observational period). The results suggested that this strain could serve as a basis for the development of a recombinant vaccine against ASF viruses belonging to seroimmunotype I.
Three recombinant plasmid constructs, expressing chimeric proteins containing human ubiquitin fused to an ectodomain of one of the potentially protective proteins (p30, p54 and CD2v) of the attenuated MK-200 strain of African swine fever virus (ASFV), were created as potential inductors of specific antiviral cellular immunity. Three-time immunization of pigs with the mixture of these plasmids led to the formation of virus-specific cytotoxic T-lymphocytes (CTL), but did not induce production of virus-specific antibodies. After challenge with the homologous parental virulent ASFV strain M-78 at a dose of 103 HAD50, all five animals (four immunized pigs and one naïve) fell between the 4th and 7th days post infection. The obtained results demonstrated that induction of CTL did not protect pigs against challenge with the virulent ASFV. Balanced activation of CTL and antibody-mediated cellular mechanisms should be investigated.
A b s t r a c t Presently, due to the variety and diversity of economic and tourist ties of Russia, episodes of accidental or maybe purposeful (i.e., biological terrorism) entry of exotic infectious pathogens including Akabane disease to the Russian Federation should not be excluded. Akabane disease is a viral transmissible infection. Its recurrent outbreaks are characterized by abortions, still or premature births, or malformations (e.g. arthrogryposis and/or hydrocephaly) for calves, lambs and kids. Akabane disease virus can persist both in animal body and in vitro (e.g., in continuous cell lines). A study of the sensitivity of African green monkey kidney cell line to Akabane virus carried out earlier showed that Akabane virus caused definite cytopathic changes resulting in cell rounding followed by cytolysis and detachment of the cell monolayer within 48 hours post infection. In this paper we first reported on the cytomorphological changes caused by Akabane virus in the primary lamb synovial membrane diploid cell culture (LSMCC) prepared according to an earlier developed procedure, and on a suitability of this culture for the virus accumulation in titers sufficient for study and making diagnosis. It has been formerly determined that the lamb synovial membrane cell culture is sensitive to small ruminant lentiviruses like caprine arthritis encephalitis virus or Visna-Maedi virus in sheep. LSMCC was prepared using a method for tissue explant culture. On day 4 post inoculation of the cell monolayer with Akabane virus the cytopathic effect appeared which manifested as formation of symplasts that grew larger due to their fusion on day 5 to 6. The Akabane virus activity was 6.0±0.05 lg TCID 50 /cm 3 for strain V8935, and 5.0±0.05 lg TCID 50 /cm 3 for strain P. As many as three passages and also the primary cell culture (after freezing) kept the virus-producing activity, and the Akabane virus retained its infective properties. The lamb synovial membrane cells can be re-cultured, and excessive diploid culture can be frozen to preserve and thawed as required. It is expedient to use a strain of diploid lamb synovial membrane cells deposited and patented earlier. One more advantage of the primary LSMCC as compared to monkey cell lines is that the latter ones may be a source of simian herpes B virus.
African swine fever virus (ASFV) is an extremely genetically and phenotypically heterogeneous pathogen. Previously, we have demonstrated that experimental inoculation of pigs with an attenuated strain, Katanga-350 (genotype I, seroimmunotype I) (ASFV-Katanga-350), can induce protective immunity in 80% of European domestic pigs against the homologous virulent European strain Lisbon-57. At least 50% of the surviving pigs received protection from subsequent intramuscular infection with a heterologous virulent strain, Stavropol 01/08 (genotype II, seroimmunotype VIII) (ASFV-Stavropol 01/08). In this study, we assessed clinical signs, the levels of viremia, viral DNA, anti-ASFV antibodies and post-mortem changes caused by subsequent intramuscular injection with ASFV-Katanga-350 and heterologous ASFV-Stavropol 01/08. Inoculation of pigs with the ASFV-Katanga-350 did not protect animals from the disease in the case of the subsequent challenged ASFV-Stavropol 01/08. However, 40% of pigs were protected from death. Moreover, the surviving animals showed no pathomorphological changes or the presence of an infectious virus in the organs after euthanasia at 35 days post challenging. The ability/inability of attenuated strains to form a certain level of protection against heterologous isolates needs a theoretical background and experimental confirmation.
A b s t r a c tThe causal agent of African swine fever (ASF) is a DNA virus belonging to Asfaviridae family which affects both wild boar Sus scrofa and domestic pig Sus scrofa domestica. Special features of the course of (ASF and its forms should be considered for the effective use of various ASF diagnostic methods aimed at the pathogen or specific antibody identification. ASF diagnosis in the wild boar is of special importance. The wild boar susceptibility to ASF virus is well known, and the disease has been repeatedly reproduced in experiments and detected in European wild boars in natural conditions. It is not unfrequently that when shooting wild boars, only organ samples are delivered to laboratories, so we decided to estimate the diagnostic value of the antibodies detected in tissues and evaluate an earlier developed test system for its efficacy when used for the assay. This report represents the results of validation of an indirect ELISA (a commercial kit «VNIIVViM ASF-ELISA Ab/Ag») ASF virus specific antibody detection in blood serum and spleen tissue extracts. For comparison, an indirect immunofluorescence assay (indirect IFA) was used. To estimate the obtained results, ROC analysis was applied. Examination of positive (n = 66) and negative (n = 410) porcine blood serum samples using indirect ELISA showed high sensitivity and specificity of the method with reference to IFA. Among the 476 serum samples examined in indirect ELISA, only 8 sera (1.6 %) were within the positive/negative cutoff area. The highest sensitivity (100 %) and specificity (99.27 %) for indirect ELISA when examining both domestic pig and wild boar blood sera were determined at a cutoff value of 0.264. The samples of spleen extracts to be used for the study were collected from clinically healthy wild boars in ASF-affected Smolensk region in 2013 to 2014. In view of an acute form of the disease, we can suspect that the animals exhibiting positive reaction were in a latency period of the pathogeny. When examining the positive (n = 59) and negative (n = 678) spleen extracts, we also determined high sensitivity and specificity levels of indirect ELISA with reference to indirect IFA. Among the samples examined (n = 737), 10 (1.3 %) samples of spleen extracts were within the positive/negative cutoff area. The highest sensitivity (100 %) and specificity (98.82 %) were observed at a cutoff value of 0.284. Thus, we confirmed the assay to be effective for porcine blood sera with 100 % sensitivity (94.6 to 100 %) and 99.27 % specificity (97.90 to 99.80 %) and for spleen extracts with 100 % sensitivity (93.90 to 100 %) and 98.82 % specificity (97.70 to 99.50 %).
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