Investigating equine host barriers to infection with influenza A viruses

Influenza A viruses (IAVs) are significant pathogens of humans and animals whose main natural host is considered to be wild waterfowl. IAVs have jumped the species barrier on multiple occasions, sometimes with devastating consequences. Successful infection and onward transmission (i.e. viral emergence) requires highly specific interactions between virus and host proteins. However, how an avian virus adapts to a mammalian host to establish as a novel pathogen after initial interspecies transmission is not yet clear. It was hypothesized that adaptation of an avian virus to mammals would involve changes in virus-host interactions that would result in more efficient viral replication and counteraction of immune responses. To test this hypothesis this thesis firstly describes the characterization of an equine dermal cell line (E.Derm) for the study of infection with EIVs. A panel of H3N8 AIVs was selected to investigate how equine host barriers affect the replication kinetics of distinct viruses. Finally, the transcriptome of the equine cells was investigated after infection with two evolutionary distinct H3N8 equine influenza viruses (H3N8 EIVs), and treatment with interferon-alpha (IFN-α). H3N8 EIV is an avian-origin virus that emerged in 1960s and has been circulating in horses for over 50 years, thus providing a natural model system to study the interspecies transmission and post-transfer adaptation of an avian influenza virus to a mammalian host. To examine the cellular response to infection, equine dermal cells (E.Derm) were infected with either A/equine/Uruguay/63 or A/equine/Ohio/2003. Total RNA was extracted at 4 and 24 hours post-infection for RNA sequencing and downstream transcriptomics analysis. Mock-infected cells and interferon-treated cells were also included for comparison purposes. RNA-seq data were analysed using CuffDiff2 to identify differentially expressed (DE) genes between samples. Ingenuity Pathway Analysis was used to determine the intracellular pathways in which DE genes were involved. The results showed clear differences on the intracellular pathways affected between the viruses, which were especially evident during the eclipse phase of virus replication. Distinct intracellular pathways were identified as important for EIV adaptation to the horse, which in turn could be employed by other avian influenza viruses to establish in mammals

Long-term adaptation following influenza A virus host shifts results in increased within-host viral fitness due to higher replication rates, broader dissemination within the respiratory epithelium and reduced tissue damage

The mechanisms and consequences of genome evolution on viral fitness following host shifts are poorly understood. In addition, viral fitness -the ability of an organism to reproduce and survive- is multifactorial and thus difficult to quantify. Influenza A viruses (IAVs) circulate broadly among wild birds and have jumped into and become endemic in multiple mammalian hosts, including humans, pigs, dogs, seals, and horses. H3N8 equine influenza virus (EIV) is an endemic virus of horses that originated in birds and has been circulating uninterruptedly in equine populations since the early 1960s. Here, we used EIV to quantify changes in infection phenotype associated to viral fitness due to genome-wide changes acquired during long-term adaptation. We performed experimental infections of two mammalian cell lines and equine tracheal explants using the earliest H3N8 EIV isolated (A/equine/Uruguay/63 [EIV/63]), and A/equine/Ohio/2003 (EIV/2003), a monophyletic descendant of EIV/63 isolated 40 years after the emergence of H3N8 EIV. We show that EIV/2003 exhibits increased resistance to interferon, enhanced viral replication, and a more efficient cell-to-cell spread in cells and tissues. Transcriptomics analyses revealed virus-specific responses to each virus, mainly affecting host immunity and inflammation. Image analyses of infected equine respiratory explants showed that despite replicating at higher levels and spreading over larger areas of the respiratory epithelium, EIV/2003 induced milder lesions compared to EIV/63, suggesting that adaptation led to reduced tissue pathogenicity. Our results reveal previously unknown links between virus genotype and the host response to infection, providing new insights on the relationship between virus evolution and fitness

Absence of adaptive evolution is the main barrier against influenza emergence in horses in Asia despite frequent virus interspecies transmission from wild birds

Virus ecology and evolution play a central role in disease emergence. However, their relative roles will vary depending on the viruses and ecosystems involved. We combined field studies, phylogenetics and experimental infections to document with unprecedented detail the stages that precede initial outbreaks during viral emergence in nature. Using serological surveys we showed that in the absence of large-scale outbreaks, horses in Mongolia are routinely exposed to and infected by avian influenza viruses (AIVs) circulating among wild birds. Some of those AIVs are genetically related to an avian-origin virus that caused an epizootic in horses in 1989. Experimental infections showed that most AIVs replicate in the equine respiratory tract without causing lesions, explaining the absence of outbreaks of disease. Our results show that AIVs infect horses but do not spread, or they infect and spread but do not cause disease. Thus, the failure of AIVs to evolve greater transmissibility and to cause disease in horses is in this case the main barrier preventing disease emergence.Peer Reviewe