PhD Thesis defense by Néstor Porras González at the VISAVET Centre of the Complutense University of Madrid

April 10^th, 2026

African swine fever (ASF) is a highly lethal viral disease that affects domestic and wild suids. Its capacity for transboundary spread makes it one of the greatest threats to animal health and the global swine production industry. The emergence of ASF in Europe and its sustained circulation among Eurasian wild boar (Sus scrofa) populations have placed this species at the center of the disease’s epidemiology. This work focuses on genotype II of the ASF virus (ASFV), which is currently responsible for most outbreaks in Europe. Within this genotype, variants with different levels of virulence have been identified, ranging from highly lethal strains to attenuated ones, which may serve as a basis for immunity studies and potential vaccine candidates.

In this context, the main objective of this doctoral thesis was to conduct an in-depth analysis of the pathology associated with ASFV infection in wild boar, exploring novel histopathological and immunohistochemical aspects of the disease and their relationship to viral strain virulence. To achieve this, four specific objectives were pursued, each addressed in a dedicated scientific article.

The first objective involved the design of histopathological scoring and classification system using criteria specific to ASF in wild boar. Comparative analysis between a highly virulent strain (Arm/07) and a naturally attenuated strain (Lv17/WB/Rie1) revealed marked differences in clinical expression, viral load, and lesion development. Experimental findings demonstrated that wild boar infected with the highly virulent strain developed an acute disease form, characterized by distinctive patterns: lymphoid depletion and lymphocytolysis, especially in tonsils, spleen, and lymph nodes; vascular alterations in lymph nodes, spleen, lungs, liver, kidneys, and brain; and mononuclear inflammatory infiltrates in lungs, liver and brain. Less frequently, hemorrhagic and inflammatory lesions were also observed in other organs such as the gastrointestinal tract, bladder, adrenal glands, and pancreas, with occurrence and severity depending on the infection’s duration. No significant differences were observed between intramuscularly infected animals and those infected through contact in clinical signs, gross and histopathological findings, or tissue viral genome detection. However, contact-infected animals tended to develop more extensive late-acute lesions. In contrast, animals infected with the attenuated strain exhibited a mild clinical course, with focal and less severe lesions. Slight lymphoid depletion was observed, particularly in lymph node follicles, along with an increase in apoptotic lymphoid cells, nuclear debris, and mitotic figures in germinal centers, indicative of a prior mild lymphocytolysis process. These findings suggest a transient infection course, with early virus clearance but prolonged inflammation in the absence of evident clinical signs. This histopathological system, applied to multiple organs, proved to be a reproducible and objective tool for correlating clinical findings, viral loads, and pathological forms, with strong potential for evaluating future experimental vaccines.

The second objective focused on characterizing tertiary lymphoid organs (TLOs) formed in wild boar following inoculation/immunization with a naturally attenuated ASFV strain (Lv17/WB/Rie1) and in some cases, subsequent challenge with a highly virulent ASFV strain (Arm/07). These ectopic lymphoid aggregates exhibited a higher degree of maturation in immunized and subsequently challenged animals, with notable structural and cellular similarities to secondary lymphoid organs. TLO development was linked to preceding chronic (or transient) inflammatory processes, as indicated by positive immunolabeling for ASFV antigen in inflammatory areas adjacent to lymphoid tissue formation. Findings suggest that TLOs induced by attenuated ASFV strains, particularly near mucosal surfaces, could facilitate local protective immune responses comparable to mucosa-associated lymphoid tissue. However, their exact role, whether protective or potentially pathological, depends on multiple factors, including location, stimulus nature, and inflammation kinetics. Further research is needed to clarify cellular and humoral dynamics, as well as their protective capacity against ASFV and potential use as targets in local immunity strategies.

The third objective documented, for the first time, the presence of intrapancreatic accessory spleens (IPAS) in wild boar following inoculation/immunization with a naturally attenuated ASFV strain (Lv17/WB/Rie1) and subsequent challenge with a highly virulent strain (Arm/07). IPAS exhibited an architecture comparable to the main spleen and exhibited similar tissue alterations. Additionally, these tissues showed positive immunolabeling for ASFV antigen, demonstrating viral tropism for macrophages in splenic cords, as observed in the main spleen. Aside from chronic or persistent infection, no other common factors were identified to explain the high frequency of IPAS formation. The results suggest that persistent infection, primarily due to interactions between attenuated and highly virulent strains, may promote accessory spleen growth as a compensatory response to increased demand on the mononuclear phagocyte system’s phagocytic capacity.
The fourth objective involved immunohistochemical analysis of viral antigen distribution in combination with histopathology in wild boar inoculated with different ASFV strains, including a mutant attenuated strain (Lv17/WB/Rie1-?CD) and a highly virulent strain (Arm/07). Animals infected with the highly virulent strain exhibited hemorrhagic and inflammatory lesions, along with generalized lymphoid depletion, which correlated with extensive viral dissemination observed by day 10 post-infection. Vaccinated animals rarely displayed mild lymphoid depletion in lymph nodes. Minimal immunolabeling was observed in macrophages from tonsils and regional lymph nodes, suggesting restricted viral persistence near the oral entry point. In some vaccinated and challenged animals, infected macrophages with inclusion bodies were exclusively detected, associated with multifocal necrotic lesions in tonsillar crypts and in paracortical, trabecular, and medullary sinuses of various lymph nodes. This finding suggests effective infection control with no dissemination to other tissues, which could possibly be related to IFN-? production from resident macrophages. This supports the hypothesis that lymph nodes act as innate barriers preventing viral spread to vital organs. This protective system may be further enhanced by adaptive immunity induced by vaccination. In contrast, in animals vaccinated with two doses and subsequently challenged, neither specific viral immunoexpression nor associated histopathological lesions were observed. These animals exhibited well-developed lymphoid tissue structures, indicating the resolution of the mild alterations induced by the attenuated isolate. These results highlight that booster vaccination improves safety and provides enhanced protection against infection, supporting its value as a strategy for ASF vaccine development.

Overall, this doctoral work demonstrates that variations in the histopathological and immunohistochemical findings directly reflect the degree of virulence of genotype II strains. These results not only confirm correlations between clinical severity, tissue damage, and antigen distribution but also provide new insights into the potential use of attenuated variants as experimental vaccine candidates. These findings contribute to the improvement of diagnostic protocols, strengthen epidemiological surveillance programs in wild boar populations, and support the design of more effective control strategies for this high-impact animal health disease in Europe.