Immunization of Pigs with Recombinant Plasmids Containing Genes of Ubiquitinated p30, p54 and CD2v Proteins of African Swine Fever Virus

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.Kao potencijalni induktori specifičnog antivirusnog celularnog imuniteta, kreirane su 3 rekombinantne plazmidne konstrukcije, koje predstavljaju himerne proteine, koje sadrže humani ubikvitin, na čijem ektodomenu je inkorporiran po jedan od sledećih potencijalno zaštićenih proteina (p30, p54 i CD2v), a koji potiču od atenuiranog soja afričke kuge svinja MK - 200. Navedenim plazmidima je izvršena trostruka imunizacija svinja, koja je kod njih dovela do posledičnog formiranja virus specifičnih citotoksičnih T limfocita (CTL), ali bez uticaja na indukciju sinteze virus specifičnih antitela. Nakon veštačke infekcije ovih imunizovanih svinja i to homolognim virulentnim sojem virusa afričke kuge svinja M - 78, u dozi od 103 HAD50, svih 5 svinja (4 imunizovane svinje i 1 neimunizovana - kontrolna) su uginule između 4 i 7 dana nakon infi ciranja. Dobijeni rezultati su pokazali da indukcija samo celularnog imunog odgovora, odnosno citotoksičnih T limfocita, ne štiti svinje od infekcije virulentnim sojem virusa afričke kuge svinja. Izbalansiranu aktivaciju citotoksičnih T limfocita i antitelima posredovane ćelijske imunske mehanizme trebalo bi dalje istražiti

Imunizacija svinja sa rekombinantnim plazmidima koji sadrže gene ubikvitarnih p30, p54 i cd2v proteina virusa afričke kuge svinja

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.Kao potencijalni induktori specifičnog antivirusnog celularnog imuniteta, kreirane su 3 rekombinantne plazmidne konstrukcije, koje predstavljaju himerne proteine, koje sadrže humani ubikvitin, na čijem ektodomenu je inkorporiran po jedan od sledećih potencijalno zaštićenih proteina (p30, p54 i CD2v), a koji potiču od atenuiranog soja afričke kuge svinja MK - 200. Navedenim plazmidima je izvršena trostruka imunizacija svinja, koja je kod njih dovela do posledičnog formiranja virus specifičnih citotoksičnih T limfocita (CTL), ali bez uticaja na indukciju sinteze virus specifičnih antitela. Nakon veštačke infekcije ovih imunizovanih svinja i to homolognim virulentnim sojem virusa afričke kuge svinja M - 78, u dozi od 103 HAD50, svih 5 svinja (4 imunizovane svinje i 1 neimunizovana - kontrolna) su uginule između 4 i 7 dana nakon infi ciranja. Dobijeni rezultati su pokazali da indukcija samo celularnog imunog odgovora, odnosno citotoksičnih T limfocita, ne štiti svinje od infekcije virulentnim sojem virusa afričke kuge svinja. Izbalansiranu aktivaciju citotoksičnih T limfocita i antitelima posredovane ćelijske imunske mehanizme trebalo bi dalje istražiti

Inoculation with ASFV-Katanga-350 Partially Protects Pigs from Death during Subsequent Infection with Heterologous Type ASFV-Stavropol 01/08

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

Inoculation with ASFV-Katanga-350 Partially Protects Pigs from Death during Subsequent Infection with Heterologous Type ASFV-Stavropol 01/08

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

Immunobiological Characteristics of the Attenuated African Swine Fever Virus Strain Katanga-350

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

An Assessment of Diagnostic Assays and Sample Types in the Detection of an Attenuated Genotype 5 African Swine Fever Virus in European Pigs over a 3-Month Period

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