Deletion of the gene for the African swine fever virus BCL-2 family member A179L increases virus uptake and apoptosis but decreases virus spread in macrophages and reduces virulence in pigs.

IMPORTANCE: African swine fever virus (ASFV) causes a lethal disease of pigs with high economic impact in affected countries in Africa, Europe, and Asia. The virus encodes proteins that inhibit host antiviral defenses, including the type I interferon response. Host cells also activate cell death through a process called apoptosis to limit virus replication. We showed that the ASFV A179L protein, a BCL-2 family apoptosis inhibitor, is important in reducing apoptosis in infected cells since deletion of this gene increased cell death and reduced virus replication in cells infected with the A179L gene-deleted virus. Pigs immunized with the BeninΔA179L virus showed no clinical signs and a weak immune response but were not protected from infection with the deadly parental virus. The results show an important role for the A179L protein in virus replication in macrophages and virulence in pigs and suggest manipulation of apoptosis as a possible route to control infection.

Differential Effect of Deleting Members of African Swine Fever Virus Multigene Families 360 and 505 from the Genotype II Georgia 2007/1 Isolate on Virus Replication, Virulence, and Induction of Protection.

African swine fever virus multigene family (MGF) 360 and 505 genes have roles in suppressing the type I interferon response and in virulence in pigs. The role of the individual genes is poorly understood. Different combinations of these genes were deleted from the virulent genotype II Georgia 2007/1 isolate. Deletion of five copies of MGF 360 genes, MGF360-10L, -11L, -12L, -13L, and -14L, and three copies of MGF505-1R, -2R, and -3R reduced virus replication in macrophages and attenuated virus in pigs. However, only 25% of the immunized pigs were protected against challenge. Deletion of MGF360-12L, -13L, and -14L and MGF505-1R in combination with a negative serology marker, K145R (GeorgiaΔK145RΔMGF(A)), reduced virus replication in macrophages and virulence in pigs, since no clinical signs or virus genome in blood were observed following immunization. Four of six pigs were protected after challenge. In contrast, deletion of MGF360-13L and -14L, MGF505-2R and -3R, and K145R (GeorgiaΔK145RΔMGF(B)) did not reduce virus replication in macrophages. Following immunization of pigs, clinical signs were delayed, but all pigs reached the humane endpoint. Deletion of genes MGF360-12L, MGF505-1R, and K145R reduced replication in macrophages and attenuated virulence in pigs since no clinical signs or virus genome in blood were observed following immunization. Thus, the deletion of MGF360-12L and MGF505-1R, in combination with K145R, was sufficient to dramatically attenuate virus infection in pigs. However, only two of six pigs were protected, suggesting that deletion of additional MGF genes is required to induce a protective immune response. Deletion of MGF360-12L, but not MGF505-1R, from the GeorgiaΔK145R virus reduced virus replication in macrophages, indicating that MGF360-12L was most critical for maintaining high levels of virus replication in macrophages. IMPORTANCE African swine fever has a high socioeconomic impact and no vaccines to aid control. The African swine fever virus (ASFV) has many genes that inhibit the host's interferon response. These include related genes that are grouped into multigene families, including MGF360 and 505. Here, we investigated which MGF360 and 505 genes were most important for viral attenuation and protection against genotype II strains circulating in Europe and Asia. We compared viruses with deletions of MGF genes. Deletion of just two MGF genes in combination with a third gene, K145R, a possible marker for vaccination, is sufficient for virus attenuation in pigs. Deletion of additional MGF360 genes was required to induce higher levels of protection. Furthermore, we showed that the deletion of MGF360-12L, combined with K145R, impairs virus replication in macrophages in culture. Our results have important implications for understanding the roles of the ASFV MGF genes and for vaccine development.

Role of African Swine Fever Virus Proteins EP153R and EP402R in Reducing Viral Persistence in Blood and Virulence in Pigs Infected with BeninΔDP148R.

The limited knowledge on the role of many of the approximately 170 proteins encoded by African swine fever virus restricts progress toward vaccine development. Previously, the DP148R gene was deleted from the genome of genotype I virulent Benin 97/1 isolate. This virus, BeninΔDP148R, induced transient moderate clinical signs after immunization and high levels of protection against challenge. However, the BeninΔDP148R virus and genome persisted in blood over a prolonged period. In the current study, deletion of either EP402R or EP153R genes individually or in combination from BeninΔDP148R genome was shown not to reduce virus replication in macrophages in vitro. However, deletion of EP402R dramatically reduced the period of infectious virus persistence in blood in immunized pigs from 28 to 14 days and virus genome from 59 to 14 days while maintaining high levels of protection against challenge. The additional deletion of EP153R (BeninΔDP148RΔEP153RΔEP402R) further attenuated the virus, and no viremia or clinical signs were observed postimmunization. This was associated with decreased protection and detection of moderate levels of challenge virus in blood. Interestingly, the deletion of EP153R alone from BeninΔDP148R did not result in further virus attenuation and did not reduce the period of virus persistence in blood. These results show that EP402R and EP153R have a synergistic role in reducing clinical signs and levels of virus in blood. IMPORTANCE African swine fever virus (ASFV) causes a disease of domestic pigs and wild boar which results in death of almost all infected animals. The disease has a high economic impact, and no vaccine is available. We investigated the role of two ASFV proteins, called EP402R and EP153R, in determining the levels and length of time virus persists in blood from infected pigs. EP402R causes ASFV particles and infected cells to bind to red blood cells. Deletion of the EP402R gene dramatically reduced virus persistence in blood but did not reduce the level of virus. Deletion of the EP153R gene alone did not reduce the period or level of virus persistence in blood. However, deleting both EP153R and EP402R resulted in undetectable levels of virus in blood and no clinical signs showing that the proteins act synergistically. Importantly, the infected pigs were protected following infection with the wild-type virus that kills pigs.

Immunization of Pigs by DNA Prime and Recombinant Vaccinia Virus Boost To Identify and Rank African Swine Fever Virus Immunogenic and Protective Proteins.

African swine fever virus (ASFV) causes an acute hemorrhagic fever in domestic pigs, with high socioeconomic impact. No vaccine is available, limiting options for control. Although live attenuated ASFV can induce up to 100% protection against lethal challenge, little is known of the antigens which induce this protective response. To identify additional ASFV immunogenic and potentially protective antigens, we cloned 47 viral genes in individual plasmids for gene vaccination and in recombinant vaccinia viruses. These antigens were selected to include proteins with different functions and timing of expression. Pools of up to 22 antigens were delivered by DNA prime and recombinant vaccinia virus boost to groups of pigs. Responses of immune lymphocytes from pigs to individual recombinant proteins and to ASFV were measured by interferon gamma enzyme-linked immunosorbent spot (ELISpot) assays to identify a subset of the antigens that consistently induced the highest responses. All 47 antigens were then delivered to pigs by DNA prime and recombinant vaccinia virus boost, and pigs were challenged with a lethal dose of ASFV isolate Georgia 2007/1. Although pigs developed clinical and pathological signs consistent with acute ASFV, viral genome levels were significantly reduced in blood and several lymph tissues in those pigs immunized with vectors expressing ASFV antigens compared with the levels in control pigs.IMPORTANCE The lack of a vaccine limits the options to control African swine fever. Advances have been made in the development of genetically modified live attenuated ASFV that can induce protection against challenge. However, there may be safety issues relating to the use of these in the field. There is little information about ASFV antigens that can induce a protective immune response against challenge. We carried out a large screen of 30% of ASFV antigens by delivering individual genes in different pools to pigs by DNA immunization prime and recombinant vaccinia virus boost. The responses in immunized pigs to these individual antigens were compared to identify the most immunogenic. Lethal challenge of pigs immunized with a pool of antigens resulted in reduced levels of virus in blood and lymph tissues compared to those in pigs immunized with control vectors. Novel immunogenic ASFV proteins have been identified for further testing as vaccine candidates.

Deletion of the African Swine Fever Virus Gene DP148R Does Not Reduce Virus Replication in Culture but Reduces Virus Virulence in Pigs and Induces High Levels of Protection against Challenge.

Many of the approximately 165 proteins encoded by the African swine fever virus (ASFV) genome do not have significant similarity to known proteins and have not been studied experimentally. One such protein is DP148R. We showed that the DP148R gene is transcribed at early times postinfection. Deletion of this gene did not reduce virus replication in macrophages, showing that it is not essential for replication in these cells. However, deletion of this gene from a virulent isolate, Benin 97/1, producing the BeninΔDP148R virus, dramatically reduced the virulence of the virus in vivo All pigs infected with the BeninΔDP148R virus survived infection, showing only transient mild clinical signs soon after immunization. Following challenge with the parental virulent virus, all pigs immunized by the intramuscular route (11/11) and all except one immunized by the intranasal route (5/6) survived. Mild or no clinical signs were observed after challenge. As expected, control nonimmune pigs developed signs of acute African swine fever (ASF). The virus genome and infectious virus were observed soon after immunization, coincident with the onset of clinical signs (∼106 genome copies or 50% tissue culture infective doses/ml). The levels of the virus genome declined over an extended period up to 60 days postimmunization. In contrast, infectious virus was no longer detectable by days 30 to 35. Gamma interferon (IFN-γ) was detected in serum between days 4 and 7 postimmunization, and IFN-γ-producing cells were detected in all pigs analyzed following stimulation of immune lymphocytes with whole virus. ASFV-specific antibodies were first detected from day 10 postimmunization.IMPORTANCE African swine fever (ASF) is endemic in Africa, parts of the Trans Caucasus, the Russian Federation, and several European countries. The lack of a vaccine hinders control. Many of the ASF virus genes lack similarity to known genes and have not been characterized. We have shown that one of these, DP148R, is transcribed early during virus replication in cells and can be deleted from the virus genome without reducing virus replication. The virus with the gene deletion, BeninΔDP148R, caused mild clinical signs in pigs and induced high levels of protection against challenge with the parental virulent virus. Therefore, deletion of this gene can provide a target for the rational development of vaccines.

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.

African swine fever virus NAM P1/95 is a mixture of genotype I and genotype VIII viruses.

African swine fever virus causes a lethal hemorrhagic disease of domestic pigs. The NAM P1/1995 isolate was originally described as B646L genotype XVIII; however, full genome sequencing revealed that this assignment was incorrect.

The transcriptomic insight into the differential susceptibility of African Swine Fever in inbred pigs.

African swine fever (ASF) is a global threat to animal health and food security. ASF is typically controlled by strict biosecurity, rapid diagnosis, and culling of affected herds. Much progress has been made in developing modified live virus vaccines against ASF. There is host variation in response to ASF infection in the field and under controlled conditions. To better understand the dynamics underlying this host differential morbidity, whole transcriptome profiling was carried out in twelve immunized and five sham immunized pigs. Seventeen MHC homozygous inbred Large white Babraham pigs were sampled at three time points before and after the challenge. The changes in the transcriptome profiles of infected animals were surveyed over time. In addition, the immunization effect on the host response was studied as well among the contrasts of all protection subgroups. The results showed two promising candidate genes to distinguish between recovered and non-recovered pigs after infection with a virulent African swine fever virus (ASFV) pre-infection: HTRA3 and GFPT2 (padj < 0.05). Variant calling on the transcriptome assemblies showed a two-base pair insertion into the ACOX3 gene closely located to HTRA3 that may regulate its expression as a putative genomic variant for ASF. Several significant DGEs, enriched gene ontology (GO) terms, and KEGG pathways at 1 day and 7 days post-infection, compared to the pre-infection, indicate a significant inflammation response immediately after ASF infection. The presence of the virus was confirmed by the mapping of RNA-Seq reads on two whole viral genome sequences. This was concordant with a higher virus load in the non-recovered animals 7 days post-infection. There was no transcriptome signature on the immunization at pre-infection and 1 day post-infection. More samples and data from additional clinical trials may support these findings.

Full genome sequence analysis of African swine fever virus isolates from Cameroon.

African swine fever (ASF) is a devastating disease of domestic pigs that has spread across the globe since its introduction into Georgia in 2007. The etiological agent is a large double-stranded DNA virus with a genome of 170 to 180 kb in length depending on the isolate. Much of the differences in genome length between isolates are due to variations in the copy number of five different multigene families that are encoded in repetitive regions that are towards the termini of the covalently closed ends of the genome. Molecular epidemiology of African swine fever virus (ASFV) is primarily based on Sanger sequencing of a few conserved and variable regions, but due to the stability of the dsDNA genome changes in the variable regions occur relatively slowly. Observations in Europe and Asia have shown that changes in other genetic loci can occur and that this could be useful in molecular tracking. ASFV has been circulating in Western Africa for at least forty years. It is therefore reasonable to assume that changes may have accumulated in regions of the genome other than the standard targets over the years. At present only one full genome sequence is available for an isolate from Western Africa, that of a highly virulent isolate collected from Benin during an outbreak in 1997. In Cameroon, ASFV was first reported in 1981 and outbreaks have been reported to the present day and is considered endemic. Here we report three full genome sequences from Cameroon isolates of 1982, 1994 and 2018 outbreaks and identify novel single nucleotide polymorphisms and insertion-deletions that may prove useful for molecular epidemiology studies in Western Africa and beyond.

A protective multiple gene-deleted African swine fever virus genotype II, Georgia 2007/1, expressing a modified non-haemadsorbing CD2v protein.

African swine fever virus is a complex DNA virus that causes high fatality in pigs and wild boar and has a great socio-economic impact. An attenuated genotype II strain was constructed by replacing the gene for wildtype CD2v protein with versions in which single or double amino acid substitutions were introduced to reduce or abrogate the binding to red blood cells and reduce virus persistence in blood. The mutant CD2v proteins were expressed at similar levels to the wildtype protein on the surface of infected cells. Three recombinant viruses also had K145R, EP153R, and in one virus DP148R genes deleted. Following immunization of pigs, the virus with a single amino acid substitution in CD2v, Q96R, induced moderate levels of replication, and 100% protection against virulent ASFV. Two additional recombinant viruses had two amino acid substitutions in CD2v, Q96R, and K108D, and induced no binding to red blood cells in vitro. In immunized pigs, reduced levels of virus in blood and strong early ASFV-specific antibody and cellular responses were detected. After challenge low to moderate replication of challenge virus was observed. Reduced clinical signs post-challenge were observed in pigs immunized with the virus from which DP148R gene was deleted. Protection levels of 83-100% were maintained across a range of doses. Further experiments with virus GeorgiaΔDP148RΔK145RΔEP153R-CD2v_mutantQ96R/K108D showed low levels of virus dissemination in tissue and transient clinical signs at high doses. The results support further evaluation of GeorgiaΔDP148RΔK145RΔEP153R-CD2v_mutantQ96R/K108D as a vaccine candidate.

Processing and localization of the african swine fever virus CD2v transmembrane protein.

The African swine fever virus (ASFV)-encoded CD2v transmembrane protein is required for the hemadsorption of red blood cells around infected cells and is also required for the inhibition of bystander lymphocyte proliferation in response to mitogens. We studied the expression of CD2v by expressing the gene with a V5 tag downstream from the signal peptide near the N terminus and a hemagglutinin (HA) tag at the C terminus. In ASFV-infected cells, a full-length glycosylated form of the CD2v protein, which migrated mainly as a 89-kDa product, was detected, as well as an N-terminal glycosylated fragment of 63 kDa and a C-terminal nonglycosylated fragment of 26 kDa. All of these forms of the protein were localized in the membrane fraction of cells. The 26-kDa C-terminal fragment was also produced in infected cells treated with brefeldin A. These data indicate that the CD2v protein is cleaved within the luminal domain and that this occurs in the endoplasmic reticulum or Golgi compartments. Confocal microscopy showed that most of the expressed CD2v protein was localized within cells rather than at the cell surface. Comparison of the localization of full-length CD2v with that of a deletion mutant lacking all of the cytoplasmic tail apart from the 12 membrane-proximal amino acids indicated that signals within the cytoplasmic tail are responsible for the predominant localization of the full-length and C-terminal 26-kDa fragment within membranes around the virus factories, which contain markers for the Golgi compartment. Processing of the CD2v protein was not observed in uninfected cells, indicating that it is induced by ASFV infection.

Primary Macrophage Culture from Porcine Blood and Lungs.

Primary cultures represent the most reliable method to isolate and propagate field isolates of African swine fever virus (ASFV ). Within the pig ASFV predominantly targets the reticuloendothelial system for replication; therefore, primary macrophage cell cultures are commonly used to isolate, propagate, and study the virus life cycle in the laboratory. In this chapter we will describe methods for the direct isolation of pulmonary alveolar macrophages by lung lavage and the culture of monocyte-derived macrophages from pig blood. We also include a method for the positive selection of CD14+ monocytes as a source for monocyte-derived macrophages from pig blood using microbeads.

Laboratory Diagnosis and Quantification of African Swine Fever Virus Using Real-Time Polymerase Chain Reaction.

Real-time polymerase chain reaction (PCR) for the detection of African swine fever virus (ASFV) is the tool of choice for the diagnostic laboratory and is a robust and easily scalable method for the researcher analyzing viral replication both in vitro and in vivo. In this chapter, we describe protocols for both quantitative real-time polymerase chain reactions (qPCR) and non-quantitative real-time polymerase chain reactions (real-time PCR) for the detection of African swine fever virus genome in a range of samples.

In vitro and in vivo antiviral activity of nucleoside analogue cHPMPC against African swine fever virus replication.

African swine fever virus (ASFV) causes a haemorrhagic disease affecting wild boar and domestic pigs which can result in morbidity and fatality rates of up to 100%. ASFV is a large double-stranded DNA virus which replicates predominantly in the cell cytoplasm and codes for its replication and transcription machinery. No vaccine is widely available and control depends on early detection, culling of infected herds and adherence to biosecurity measures. In this study the small molecule nucleoside analogue, cyclic cidofovir (cHPMPC), was evaluated for its ability to inhibit replication of four different ASFV genotypes in primary porcine macrophages. Time of addition studies demonstrated that cHPMPC effectively inhibits ASFV replication and late gene expression when added pre-infection or early post-infection but not when added at late times, suggesting the drug target may be the virus DNA polymerase, or the RNA polymerase involved in late transcription. Oral administration of cHPMPC delayed onset of clinical signs and significantly reduced viral titres in blood and tissues of treated pigs. These results indicate that cHPMPC is a promising compound for further development to control ASFV outbreaks.

Cellular and Humoral Immune Responses after Immunisation with Low Virulent African Swine Fever Virus in the Large White Inbred Babraham Line and Outbred Domestic Pigs.

African swine fever virus is currently present in all of the world's continents apart from Antarctica, and efforts to control the disease are hampered by the lack of a commercially available vaccine. The Babraham large white pig is a highly inbred line that could represent a powerful tool to improve our understanding of the protective immune responses to this complex pathogen; however, previous studies indicated differential vaccine responses after the African swine fever virus challenge of inbred minipigs with different swine leukocyte antigen haplotypes. Lymphocyte numbers and African swine fever virus-specific antibody and T-cell responses were measured in inbred and outbred animals after inoculation with a low virulent African swine fever virus isolate and subsequent challenge with a related virulent virus. Surprisingly, diminished immune responses were observed in the Babraham pigs when compared to the outbred animals, and the inbred pigs were not protected after challenge. Recovery of Babraham pigs after challenge weakly correlated with antibody responses, whereas protective responses in outbred animals more closely correlated with the T-cell response. The Babraham pig may, therefore, represent a useful model for studying the role of antibodies in protection against the African swine fever virus.

Deletion of the K145R and DP148R Genes from the Virulent ASFV Georgia 2007/1 Isolate Delays the Onset, but Does Not Reduce Severity, of Clinical Signs in Infected Pigs.

African swine fever virus causes a frequently fatal disease of domestic pigs and wild boar that has a high economic impact across 3 continents. The large double-stranded DNA genome codes for approximately 160 proteins. Many of these have unknown functions and this hinders our understanding of the virus and host interactions. The purpose of the study was to evaluate the role of two virus proteins, K145R and DP148R, in virus replication in macrophages and virulence in pigs. To do this, the DP148R gene, alone or in combination with the K145R gene, was deleted from the virulent genotype II Georgia 2007/1 isolate. Neither of these deletions reduced the ability of the viruses to replicate in porcine macrophages compared to the parental wild-type virus. Pigs infected with GeorgiaΔDP148R developed clinical and post-mortem signs and high viremia, typical of acute African swine fever, and were culled on day 6 post-infection. The additional deletion of the K145R gene delayed the onset of clinical signs and viremia in pigs by 3 days, but pigs showed signs of acute African swine fever and were culled on days 10 or 13 post-infection. The results show that the deletion of DP148R did not attenuate the genotype II Georgia 2007/1 isolate, contrary to the results obtained with the genotype I Benin97/1 isolate. Additional deletion of the K145R gene delayed clinical signs, but infected pigs reached the humane endpoint. The deletion of additional genes would be required to attenuate the virus.

Transmission pathways of foot-and-mouth disease virus in the United Kingdom in 2007.

Foot-and-mouth disease (FMD) virus causes an acute vesicular disease of domesticated and wild ruminants and pigs. Identifying sources of FMD outbreaks is often confounded by incomplete epidemiological evidence and the numerous routes by which virus can spread (movements of infected animals or their products, contaminated persons, objects, and aerosols). Here, we show that the outbreaks of FMD in the United Kingdom in August 2007 were caused by a derivative of FMDV O(1) BFS 1860, a virus strain handled at two FMD laboratories located on a single site at Pirbright in Surrey. Genetic analysis of complete viral genomes generated in real-time reveals a probable chain of transmission events, predicting undisclosed infected premises, and connecting the second cluster of outbreaks in September to those in August. Complete genome sequence analysis of FMD viruses conducted in real-time have identified the initial and intermediate sources of these outbreaks and demonstrate the value of such techniques in providing information useful to contemporary disease control programmes.

A porcine macrophage cell line that supports high levels of replication of OURT88/3, an attenuated strain of African swine fever virus.

The main target cells for African swine fever virus (ASFV) replication in pigs are of monocyte macrophage lineage and express markers typical of the intermediate to late stages of differentiation. The lack of a porcine cell line, which accurately represents these target cells, limits research on virus host interactions and the development of live-attenuated vaccine strains. We show here that the continuously growing, growth factor dependent ZMAC-4 porcine macrophage cell line is susceptible to infection with eight different field isolates of ASFV. Replication in ZMAC-4 cells occurred with similar kinetics and to similar high titres as in primary porcine bone marrow cells. In addition we showed that twelve passages of an attenuated strain of ASFV, OURT88/3, in ZMAC-4 cells did not reduce the ability of this virus to induce protection against challenge with virulent virus. Thus, the ZMAC-4 cells provide an alternative to primary cells for ASFV replication.

Deletion of the Gene for the Type I Interferon Inhibitor I329L from the Attenuated African Swine Fever Virus OURT88/3 Strain Reduces Protection Induced in Pigs.

Live attenuated vaccines are considered to be the fastest route to the development of a safe and efficacious African swine fever (ASF) vaccine. Infection with the naturally attenuated OURT88/3 strain induces protection against challenge with virulent isolates from the same or closely related genotypes. However, adverse clinical signs following immunisation have been observed. Here, we attempted to increase the OURT88/3 safety profile by deleting I329L, a gene previously shown to inhibit the host innate immune response. The resulting virus, OURT88/3ΔI329L, was tested in vitro to evaluate the replication and expression of type I interferon (IFN) and in vivo by immunisation and lethal challenge experiments in pigs. No differences were observed regarding replication; however, increased amounts of both IFN-ß and IFN-α were observed in macrophages infected with the deletion mutant virus. Unexpectedly, the deletion of I329L markedly reduced protection against challenge with the virulent OURT88/1 isolate. This was associated with a decrease in both antibody levels against VP72 and the number of IFN-γ-producing cells in the blood of non-protected animals. Furthermore, a significant increase in IL-10 levels in serum was observed in pigs immunised with OURT88/3ΔI329L following challenge. Interestingly, the deletion of the I329L gene failed to attenuate the virulent Georgia/2007 isolate.

A Pool of Eight Virally Vectored African Swine Fever Antigens Protect Pigs Against Fatal Disease.

Classical approaches to African swine fever virus (ASFV) vaccine development have not been successful; inactivated virus does not provide protection and use of live attenuated viruses generated by passage in tissue culture had a poor safety profile. Current African swine fever (ASF) vaccine research focuses on the development of modified live viruses by targeted gene deletion or subunit vaccines. The latter approach would be differentiation of vaccinated from infected animals (DIVA)-compliant, but information on which viral proteins to include in a subunit vaccine is lacking. Our previous work used DNA-prime/vaccinia-virus boost to screen 40 ASFV genes for immunogenicity, however this immunization regime did not protect animals after challenge. Here we describe the induction of both antigen and ASFV-specific antibody and cellular immune responses by different viral-vectored pools of antigens selected based on their immunogenicity in pigs. Immunization with one of these pools, comprising eight viral-vectored ASFV genes, protected 100% of pigs from fatal disease after challenge with a normally lethal dose of virulent ASFV. This data provide the basis for the further development of a subunit vaccine against this devastating disease.