PhD Thesis defense by Aleksandra Kosowska at the VISAVET Centre of the Complutense University of Madrid

June 30^th, 2025

The rapid and widespread outbreaks of African swine fever (ASF) across multiple continents have underscored the urgent need for effective control strategies to mitigate its devastating impact. ASF is a highly contagious hemorrhagic disease affecting domestic pigs and wild boar, with severe consequences for animal health, international trade, and regional economies. Caused by a large, complex double-stranded DNA virus, ASF virus (ASFV) can persist in the environment for extended periods, facilitating indirect transmission and long-term circulation. As a notifiable disease under the World Organization for Animal Health (WOAH), ASF requires immediate reporting and coordinated control efforts to prevent its spread.

Wild boar (Sus scrofa) play a key role in ASF persistence and transmission in Europe and Asia, acting as long-term reservoirs of the virus in endemic areas. Their wide-ranging movement, social behavior, and interaction with contaminated environments facilitate ASFV circulation, making eradication challenging. Moreover, ASFV environmental persistence in carcasses, soil, and fomites sustains indirect transmission cycles. Given the limited effectiveness of conventional control measures, vaccination strategies have been proposed as a promising approach, particularly for managing ASF in wild boar populations.

Among the available options, live attenuated vaccines (LAVs) have demonstrated the greatest potential for inducing protective immunity in wild boar and domestic pigs. However, due to their inherent nature, concerns related to viral shedding, transmission dynamics, and environmental contamination risks must be thoroughly evaluated before LAVs can be safely implemented.

This doctoral thesis, titled Characterization of shedding, transmission, and environmental contamination of different strains of African swine fever virus, aims to address these knowledge gaps by evaluating the Lv17/WB/Rie1 isolate, a naturally attenuated genotype II ASFV isolate, as a potential LAV candidate. Through a series of experimental studies, this doctoral thesis provides critical insights into viral shedding, environmental contamination, and transmission risks, contributing to the development of science-based strategies for ASF control.

To achieve these goals, this thesis is divided into three main objectives.
The first objective is to characterize ASFV shedding patterns in wild boar infected with isolates of varying virulence. Shedding is a key factor in virus transmission and persistence, making its evaluation crucial in assessing vaccine safety. The study examines viral excretion in different biological fluids, comparing highly virulent and attenuated ASFV isolates. Particular focus is placed on shedding routes, viral loads, and differences between directly inoculated and in-contact animals, as these factors determine the potential spread of ASFV within wild boar populations. The findings from this objective will offer critical insights into how the attenuated Lv17/WB/Rie1 isolate behaves compared to virulent field strains, aiding in its evaluation as a potential LAV candidate.

The second objective is to evaluate the environmental contamination patterns associated with different ASFV isolates and improve virus detection in contaminated environments. ASFV can persist on surfaces, acting as a potential indirect transmission source for susceptible animals.

The first aspect of this objective investigates contamination levels across various environmental surfaces following experimental infections with a virulent field strain and an attenuated vaccine candidate (Lv17/WB/Rie1). The study examines where viral DNA persists, providing essential data to compare the environmental risks posed by virulent versus attenuated ASFV isolates. Understanding these contamination patterns is crucial for evaluating whether LAVs introduce additional environmental risks compared to field isolates.

The second aspect focuses on validating a novel sampling methodology to enhance ASFV environmental surveillance. Since traditional sampling methods require high-level biosafety conditions, their application in field settings is limited. This study assesses the effectiveness of pre-hydrated sponges with a surfactant liquid, which enables ASFV DNA detection while ensuring virus inactivation, providing a safe and efficient approach for field diagnostics.

The third objective is to assess the transmission risk of the attenuated Lv17/WB/Rie1 isolate at the wildlife-livestock interface. One of the primary concerns regarding LAV implementation is the potential for spillover transmission to domestic pigs, which could raise biosafety and regulatory concerns. This study examines the likelihood of transmission between infected wild boar and domestic pigs, evaluating whether the vaccine candidate poses a risk to pig farms. A controlled fencing interaction model simulates real-world contact scenarios, generating valuable data on the potential spread of the attenuated isolate to susceptible domestic pigs. The objective aims to determine whether the Lv17/WB/Rie1 isolate can be safely implemented in wild boar populations without compromising domestic swine biosecurity, ensuring that LAVs do not introduce unintended transmission risks.

While the Lv17/WB/Rie1 isolate presents strong potential as a vaccine candidate, further refinements—such as targeted gene deletions—are necessary to enhance its safety profile and meet the European Medicines Agency (EMA) regulatory requirements for commercialization. Additionally, continued biosafety evaluations, transmission risk assessments, and environmental monitoring are essential to ensure that the benefits of vaccination outweigh any potential risks.

The findings of this doctoral thesis contribute valuable scientific evidence supporting the safe and effective use of LAVs for ASF control, particularly in wild boar populations. By improving our understanding of viral shedding, environmental contamination, and transmission dynamics, this research provides a solid foundation for developing ASF vaccination strategies. Ultimately, these insights are expected to aid in designing evidence-based ASF control programs, mitigating the impact of the disease on wildlife, domestic pigs, and the global swine industry.