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Knowledge about African swine fever virus (ASFV) transmission and its survival in the environment is mandatory to develop rational control strategies and combat this serious disease in pigs. In this study, the risk that environmental contamination poses for infection of naïve pigs was investigated. Naïve pigs were introduced as sentinels into contaminated pens kept at ambient temperature (about 18–22˚C) either on the same day or up to 3 days after ASFV-infected pigs were removed. Three experiments were carried out in which four to six pigs per pen were inoculated with virulent ASFV isolates OURT88/1 (genotype I), Georgia 2007/1, or POL/2015/Podlaskie (genotype II), respectively. The majority of the inoculated pigs developed acute disease but with no evident haemorrhagic lesions or haemorrhagic diarrhoea and were culled at the predefined humane endpoint. The levels of ASFV DNA detected in the blood of the infected animals reached 107−9 genome copies/ml before euthanasia. Environmental swabs were taken from different surfaces in the animal rooms, as well as from faeces and urine, close to the time of introduction of the naïve animals. Relatively low quantities of virus DNA were detected in the environmental samples, in the range of 103−7 genome copies per swab or per gram and ml of faeces and urine. No infectious virus was recovered from these environmental samples. Neither clinical signs nor virus genomes were detected in the blood of any of the sentinel pigs over a period of 2 to 3 weeks after exposure, indicating that transmission from the ASFV-contaminated environment did not occur. Interestingly, viral DNA was detected in nasal and oral swabs from some of the sentinel animals at early days of exposure (ranging between 103.7−5.8 genome copies per swab), though none of them developed ASF. The results indicate a relatively low risk of ASFV transmission from a contaminated environment under the conditions provided in these experimental studies and in the absence of b

Insect production offers a sustainable source of nutrients for livestock. This comes with a risk for transmission of pathogens from the insects into the livestock sector, including viruses causing serious diseases, such as African swine fever virus (ASFV), classical swine fever virus and foot-and-mouth disease virus. ASFV is known to survive for a long time within animal meat and byproducts. Therefore, we conducted experimental exposure studies of insects to ASFV using larvae of two key insect species produced for food and feed, the mealworm; Tenebrio molitor, and the black soldier fly, Hermetia illucens. The larvae were exposed to ASFV POL/2015/Podlaskie, via oral uptake of serum or spleen material from ASFV-infected pigs. Using qPCR, the amounts of viral DNA present immediately after exposure varied from ~104.7 to 107.2 genome copies per insect. ASFV DNA was detectable in the larvae of H. illucens for up to 3 days post exposure and in T. molitor larvae for up to 9 days post exposure. To assess the presence of infectious virus within the larvae and with this, the risk of virus transmission via oral consumption, pigs were fed cakes containing larvae exposed to ASFV. Pigs that consumed 50 T. molitor or 50 H. illucens virus-exposed larvae did not become infected with ASFV. Thus, it appears, that in our experimental setting, the risk of ASFV transmission via consumption of unprocessed insect larvae, used as feed, is low.

The development of subunit vaccines against African swine fever (ASF) is mainly hindered by the lack of knowledge regarding the specific ASF virus (ASFV) antigens involved in protection. As a good example, the identity of ASFV-specific CD8+ T-cell determinants remains largely unknown, despite their protective role being established a long time ago. Aiming to identify them, we implemented the IFN ELISpot as readout assay, using as effector cells peripheral blood mononuclear cells (PBMCs) from pigs surviving experimental challenge with Georgia2007/1. As stimuli for the ELISpot, ASFV-specific peptides or full-length proteins identified by three complementary strategies were used. In silico prediction of specific CD8+ T-cell epitopes allowed identifying a 19-mer peptide from MGF100-1L, as frequently recognized by surviving pigs. Complementarily, the repertoire of SLA I-bound peptides identified in ASFV-infected porcine alveolar macrophages (PAMs), allowed the characterization of five additional SLA I-restricted ASFV-specific epitopes. Finally, in vitro stimulation studies using fibroblasts transfected with plasmids encoding full-length ASFV proteins, led to the identification of MGF505-7R, A238L and MGF100-1L as promiscuously recognized antigens. Interestingly, each one of these proteins contain individual peptides recognized by surviving pigs. Identification of the same ASFV determinants by means of such different approaches reinforce the results presented here

African swine fever (ASF) is today s number one threat for the global swine industry. Neither commercial vaccine nor treatment is available against ASF and, thus far, only live attenuated viruses (LAV) have provided robust protection against lethal ASF virus (ASFV) challenge infections. Identification of ASFV proteins inducing protective immune responses is one of the major challenges to develop safer and efficient subunit vaccines. Immunopeptidomic studies recently performed in our laboratory allowed identifying ASFV antigens recognized by ASFV-specific CD8 T-cells. Here, we used data from the SLAI-peptide repertoire presented by a single set of ASFV-infected porcine alveolar macrophages to generate a complex DNA vaccine composed by 15 plasmids encoding the individual peptide-bearing ORFs. DNA vaccine priming improved the protection afforded by a suboptimal dose of the BA71∆CD2 LAV given as booster vaccination, against Georgia2007/1 lethal challenge. Interestingly, M448R was the only protein promiscuously recognized by the induced ASFV-specific T-cells. Furthermore, priming pigs with DNA plasmids encoding M488R and MGF505-7R, a CD8 T-cell antigen previously described, confirmed these two proteins as T-cell antigens with protective potential. These studies might be useful to pave the road for designing safe and more efficient vaccine formulations in the future.

African swine fever virus (ASFV) has become widespread in Europe, Asia and elsewhere, thereby causing extensive economic losses. The viral genome includes nearly 200 genes, but their expression within infected pigs has not been well characterized previously. In this study, four pigs were infected with a genotype II strain (ASFV POL/2015/Podlaskie); blood samples were collected before inoculation and at both 3 and 6 days later. During this period, a range of clinical signs of infection became apparent in the pigs. From the blood, peripheral blood mononuclear cells (PBMCs) were isolated. The transcription of the ASFV genes was determined using RNAseq on poly(A)+ mRNAs isolated from these cells. Only very low levels of virus transcription were detected in the PBMCs at 3 days post-inoculation (dpi) but, at 6 dpi, extensive transcription was apparent. This was co-incident with a large increase in the level of ASFV DNA within these cells. The pattern of the virus gene expression was very reproducible between the individual pigs. Many highly expressed genes have undefined roles. Surprisingly, some genes with key roles in virus replication were expressed at only low levels. As the functions of individual genes are identified, information about their expression becomes important for understanding their contribution to virus biology.

African swine fever (ASF) has become the major threat to the global swine industry. Lack of available commercial vaccines complicates the implementation of global control strategies. So far, only live attenuated ASF viruses (ASFV) have demonstrated solid protection efficacy at the experimental level. The implementation of molecular techniques has allowed the generation of a collection of deletion mutants lacking ASFV-specific virulence factors, some of them with promising potential as vaccine candidates against the pandemic genotype II ASFV strain currently circulating in Africa, Europe, Asia and Oceania. Despite promising results, there is room for improvement, mainly from the biosafety point of view. Aiming to improve the safety of BA71DCD2, a cross-protective recombinant live attenuated virus (LAV) lacking the ASFV CD2v gene (encoding b-glucuronidase as a reporter gene) available in our laboratory, three new recombinants were generated using BA71DCD2 as a template: The single mutant BA71DCD2f, this time containing the fluorescent mCherry reporter gene instead of CD2v, and two double recombinants lacking CD2v and either the lectin gene (EP153R) or the uridine kinase (UK) gene (DP96R). Comparative in vivo experiments using BA71DCD2f, BA71DCD2DP96R and BA71DCD2EP153R recombinant viruses as immunogens, demonstrated that deletion of either DP96R or EP153R from BA71DCD2f decreases vaccine efficacy and does not improve safety. Our results additionally confirm ASFV challenge as the only available method today to evaluate the protective efficacy of any experimental vaccine. We believe that understanding the fine equilibrium between attenuation and inducing protection in vivo deserves further study and might contribute to more rational vaccine designs in the future.

African swine fever (ASF) has become the major threat for the global swine industry. Furthermore, the epidemiological situation of African swine fever virus (ASFV) in some endemic regions of Sub-Saharan Africa is worse than ever, with multiple virus strains and genotypes currently circulating in a given area. Despite the recent advances on ASF vaccine development, there are no commercial vaccines yet, and most of the promising vaccine prototypes available today have been specifically designed to fight the genotype II strains currently circulating in Europe, Asia, and Oceania. Previous results from our laboratory have demonstrated the ability of BA71∆CD2, a recombinant LAV lacking CD2v, to confer protection against homologous (BA71) and heterologous genotype I (E75) and genotype II (Georgia2007/01) ASFV strains, both belonging to same clade (clade C). Here, we extend these results using BA71∆CD2 as a tool trying to understand ASFV cross-protection, using phylogenetically distant ASFV strains. We first observed that five out of six (83.3%) of the pigs immunized once with 106 PFU of BA71∆CD2 survived the tick-bite challenge using Ornithodoros sp. soft ticks naturally infected with RSA/11/2017 strain (genotype XIX, clade D). Second, only two out of six (33.3%) survived the challenge with Ken06.Bus (genotype IX, clade A), which is phylogenetically more distant to BA71∆CD2 than the RSA/11/2017 strain. On the other hand, homologous prime-boosting with BA71∆CD2 only improved the survival rate to 50% after Ken06.Bus challenge, all suffering mild ASF-compatible clinical signs, while 100% of the pigs immunized with BA71∆CD2 and boosted with the parental BA71 virulent strain survived the lethal challenge with Ken06.Bus, without almost no clinical signs of the disease. Our results confirm that cross-protection is a multifactorial phenomenon that not only depends on sequence similarity. We believe that understanding this complex phenomenon will be useful for designing future vacc

[Background]: African swine fever (ASF) is a re-expanding devastating viral disease currently threatening the pig industry worldwide. MicroRNAs are a class of 17–25 nucleotide non- coding RNAs that have been shown to have critical functions in a wide variety of biological processes, such as cell differentiation, cell cycle regulation, carcinogenesis, apoptosis, regulation of immunity as well as in viral infections by cleavage or translational repression of mRNAs. Nevertheless, there is no information about miRNA expression in an ASFV infection., [Methods]: In this proof-of-concept study, we have analyzed miRNAs expressed in spleen and submandibular lymph node of experimentally infected pigs with a virulent (E75) or its derived attenuated (E75CV1) ASFV strain, as well as, at different times post-infection with the virulent strain, by high throughput sequencing of small RNA libraries., [Results]: Spleen presented a more differential expression pattern than lymph nodes in an ASFV infection. Of the most abundant miRNAs, 12 were differentially expressed in both tissues at two different times in infected animals with the virulent strain. Of these, miR-451, miR-145-5p, miR-181a and miR-122 presented up-regulation at late times post-infection while miR-92a, miR-23a, miR-92b-3p, miR-126-5p, miR-126-3p, miR-30d, miR-23b and miR-92c showed down-regulation. Of the 8 differentially expressed miRNAs identified at the same time post-infection in infected animals with the virulent strain compared with animals infected with its attenuated strain, miR-126-5p, miR-92c, miR-92a, miR-30e-5p and miR-500a-5p presented up-regulation whereas miR-125b, miR-451 and miR-125a were down-regulated. All these miRNAs have been shown to be associated with cellular genes involved in pathways related to the immune response, virus-host interactions as well as with several viral genes., [Conclusion]: The study of miRNA expression will contribute to a better understanding of African swine fever virus pathogenesis, essential in the development of any disease control strategy.

African swine fever virus (ASFV) is the causal agent of African swine fever, a hemorrhagic and often lethal porcine disease causing enormous economical losses in affected countries. Endemic for decades in most of the sub-Saharan countries and Sardinia, the risk of ASFV-endemicity in Europe has increased since its last introduction into Europe in 2007. Live attenuated viruses have been demonstrated to induce very efficient protective immune responses, albeit most of the time protection was circumscribed to homologous ASFV challenges. However, their use in the field is still far from a reality, mainly due to safety concerns. In this study we compared the course of the in vivo infection caused by two homologous ASFV strains: the virulent E75 and the cell cultured adapted strain E75CV1, obtained from adapting E75 to grow in the CV1 cell-line. Interestingly, the kinetics of both viruses not only differed on the clinical signs that they caused and in the virus loads found, but also in the immunological pathways activated throughout the infections. Furthermore, E75CV1 confirmed its protective potential against the homologous E75 virus challenge and allowed the demonstration of poor cross-protection against BA71, thus defining it as heterologous. The in vitro specificity of the CD8+ T-cells present at the time of lethal challenge showed a clear activation against the homologous virus (E75) but not against BA71. These findings will be of utility for a better understanding of ASFV pathogenesis and for the rational designing of safe and efficient vaccines against this virus.

One of the main criticisms to DNA vaccines is the poor immunogenicity that they confer on occasions, at least in large animals. Confirming this theory, immunization with plasmid DNA encoding two African swine fever virus genes in frame (pCMV-PQ), failed in inducing detectable immune responses in pigs, while it was successful in mice. Aiming to improve the immune responses induced in swine, a new plasmid was constructed, encoding the viral genes fused in frame with a single chain variable fragment of an antibody specific for a swine leukocyte antigen II (pCMV-APCH1PQ). Our results clearly demonstrate that targeting antigens to antigen professional cells exponentially enhanced the immune response induced in pigs, albeit that the DNA vaccine was not able to confer protection against lethal viral challenge. Indeed, a viremia exacerbation was observed in each of the pigs that received the pCMV-APCH1PQ plasmid, this correlating with the presence of non-neutralizing antibodies and antigen-specific SLA II-restricted T-cells. The implications of our discoveries for the development of future vaccines against African swine fever virus and other swine pathogens are discussed. © 2011 Elsevier Ltd.