Influenza A viruses (IAVs) are single stranded RNA viruses belonging to the Orthomyxoviridae family. These viruses exhibit high evolutionary rates and are present in a wide range of animal species. Host switching of IAVs is unpredictable and poses a great threat to human health. Therefore, understanding the underlying mechanisms driving such events is one of the current goals in influenza research. Among the different subtypes, H3N8 IAVs have shown a remarkable tendency to host species jumps. H3N8 equine influenza virus (EIV) is an avian-origin virus that circulated in horses for nearly 40 years before emerging in dogs in early 2000s. In the last decade H3N8 EIV has also been isolated from pigs, key hosts in influenza ecology, and other mammals. To understand the nature of host range determinants it is fundamental to study the dynamics of influenza pathogenesis at the site of infection. Respiratory explants constitute a suitable model to investigate virus replication in the target tissues of the host. This thesis aimed at thoroughly investigating the infection dynamics of host adapted and non-host adapted IAV infections in organ explants of pigs and horses.
We first described the infection phenotype of host adapted viruses such as H3N2 swine influenza (SIV) and H3N8 equine influenza in tissues of their natural hosts to build a solid background knowledge on disease pathogenesis. Swine and equine explants were infected with SIV and EIV, respectively, and accurately monitored for viral replication and structural changes at the site of infection (chapters two and three). This extensive examination has added substantial information to previous literature and confirmed that swine and equine ex vivo systems support influenza replication and are suitable to study virus pathogenesis and at the same time observe the 3Rs ethos (Reduction, Replacement and Refinement).
Next, we addressed our research interests on non-host adapted infections focusing on the H3N8 subtype. Enhancing the current knowledge on the role of evolution in the complex phenomenon of viral emergence is of utmost importance. To this end, in chapter four we compared the replication potential of phylogenetically distinct EIVs, each representing an evolutionary different period, in swine cell lines and respiratory explants. All tested EIVs replicated in cell lines whereas only the earliest EIV isolate (Uruguay/63) was able to infect swine explants with a marked tropism for the lower respiratory tract, demonstrating the presence of tissue-specific host barriers at the site of infection. The distinct phenotypes observed ex vivo support the view that evolutionary processes play important roles on host range and tropism of EIV. Nonetheless, when compared to SIV H3N2, EIV Uruguay/63 productively infected only a limited number of explants.
H3N8 EIV has originated from the avian gene pool maintained in aquatic waterfowl. To further look into host range shifts of H3N8 IAVs, in chapter five we investigated the replication dynamics of H3N8 avian influenza viruses (AIVs) in equine tracheal explants. To give an ecologically plausible context to our experiments, we tested AIVs isolated from wild birds in Mongolia, a region densely populated with wild birds and horses. H3N8 AIVs infected tracheal explants, albeit to significantly lower levels and with a different infection phenotype compared to an EIV. This difference was evidenced as lower viral titres, absence of epithelial damage and difficult viral nucleoprotein detection in the infected tissue. These findings, coupled with serological evidence of AIV infections in horses in the field, suggest that introduction of viruses from the avian reservoir can occur and that further adaptive changes may be required for a successful establishment.
Overall, by investigating the infection dynamics of IAVs in ex vivo cultures of the respiratory tract we have provided new insights into the different phenotypes displayed by host adapted and non-host adapted viruses at the site of infection. Our results support the hypothesis that viral evolution during long-term transmission of IAVs in host populations could result in dynamic changes in their host range. Such changes must be in line with ecological and epidemiological factors in order to allow the establishment of novel lineages in susceptible hosts. Finally, we have confirmed that organ explants can bridge important gaps between in vitro and in vivo experiments
