African swine fever virus (ASFV) is causing a worldwide pandemic affecting the porcine industry and leading to important global economic consequences. The virus causes a highly lethal hemorrhagic disease in wild boars and domestic pigs. Lack of effective vaccines hampers the control of virus spread, thus increasing the pressure on the scientific community for urgent solutions. However, knowledge on the immune components associated with protection is very limited. Here we characterized the in vitro recall response induced by immune cells from pigs intranasally vaccinated with the BA71ΔCD2 deletion mutant virus. Vaccination conferred dose-dependent cross-protection associated with both ASFV-specific antibodies and IFNγ-secreting cells. Importantly, bulk and single-cell transcriptomics of blood and lymph node cells from vaccinated pigs revealed a positive feedback from adaptive to innate immunity. Indeed, activation of Th1 and cytotoxic T cells was concomitant with a rapid IFNγ-dependent triggering of an inflammatory response characterized by TNF-producing macrophages, as well as CXCL10-expressing lymphocytes and cross-presenting dendritic cells. Altogether, this study provides a detailed phenotypic characterization of the immune cell subsets involved in cross-protection against ASFV, and highlights key functional immune mechanisms to be considered for the development of an effective ASF vaccine.

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 BA71∆CD2, a cross-protective recombinant live attenuated virus (LAV) lacking the ASFV CD2v gene (encoding β-glucuronidase as a reporter gene) available in our laboratory, three new recombinants were generated using BA71∆CD2 as a template: the single mutant BA71∆CD2f, 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 BA71∆CD2f, BA71∆CD2DP96R and BA71∆CD2EP153R recombinant viruses as immunogens, demonstrated that deletion of either DP96R or EP153R from BA71∆CD2f 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.

SUMMARYThe aim of this study was to assess the relationship between the clinical-pathologic process in rainbow trout fry (Oncorhynchus mykiss) infected with infectious pancreatic necrosis virus (IPNV) and the immunoglobulin M (IgM) levels in blood serum. Parameters were evaluated up to 45 days post infection (dpi) in fish intraperitoneally inoculated (IP) with 1X10 4 TCDI of IPNV, with minimal essential medium (MEM) and in the control group. Infected fish showed classical signs of infectious pancreatic necrosis (IPN) disease since day 19 pi, reaching 70% of accumulated mortality, and the survivor fish showed emaciation and IgM blood serum levels which progressively increased up to its maximum peak at day 31 pi; the most significant histopathological changes were the increase in melanomacrophage centers in the kidney, pancreatic necrosis and catarrhal enteritis. Viral isolation was possible only in the infected fish starting at day 3 pi up to day 45 (P > 0.5). The results suggest that even though the fish infected with IPNV can develop a humoral immune response characterized by the increase in IgM levels, this is insufficient to develop protection because while IgM levels are increasing, so does the viral title accompanied by clinical signs and histopathological injuries that are typical of the disease.Palabras clave: IPNV, IgM, virus, respuesta inmune.

The aim of the present study was to analyze the variation of esterase activity as well as the expression patterns of isozymes by isoelectric focusing on polyacrylamide gels (IF-PAGE) and conventional polyacrylamide gel electrophoresis (PAGE). Esterase activity and isoforms were analyzed on total proteins from 9 isolates of Beauveria bassiana, from different hosts and geographic areas from the State of Guerrero, Mexico. Total proteins were separated by IF-PAGE and PAGE, in order to characterize the activity and isoelectric points of the esterase isoforms detected on different isolates. Electrophoretic analysis from 9 isolates of Beauveria bassiana, revealed a polymorphic pattern in terms of esterases activity and the presence of isozymes, showing a total of 10 different isoforms with variations in both the activity and isoelectric point. Each isolate showed between 3-6 different isozymes. Cluster analysis derived from the Jaccardś coefficients showed a significant association between the presence-absence of esterase isoforms and the geographical origin of the Beauveria bassiana isolates. We conclude that the use of this methodology for functional detection combined with PAGE and IF-PAGE, can be an excellent tool for biochemical characterization of isolates and further impact evaluation of artificial epizootics derived from Biological control programs.