Final Report Summary - INFLUENZA TROPISM (Evolutionary Determinants of Influenza Virus Pathogenesis and Tissue Tropism) The aim of IIF 302060 project “Influenza Tropism” was to characterize the selective pressures that shape influenza virus tissue tropism along the human respiratory tract. Influenza A viruses can cause isolated cases of zoonotic infections, when they are transmitted from animals to humans, as well as pandemics and seasonal epidemics, spreading through the global human population. Infection by influenza viruses results in more or less severe disease, from life-threatening pneumonia to mild trachea-bronchitis. The range of diseases caused by these viruses, and their ability to spread or not from human to human, are associated with their tissue tropism along the respiratory tract. Here we hypothesize that transmission constraints on the one hand, and pre-existing immunity on the other, shape the influenza virus tissue tropism patterns, and in turn drive the evolution of novel influenza virus variants.To evaluate whether transmission constraints affect influenza virus tissue tropism, experimental evolution of influenza virus H1N1 was assessed under different selective pressures. Viruses originating from the upper respiratory tract (nose) of experimentally inoculated ferrets were passaged along a chain of infection. In parallel, viruses originating from the lower respiratory tract (lungs) of experimentally inoculated ferrets were passaged along another chain of infection. The tropism patterns, and associated genetic changes, of the passaged viruses were compared at the end of the chains of infection.To evaluate whether the presence of pre-existing herd immunity affects influenza virus tissue tropism, the tropism patterns of pandemic influenza viruses H3N2 (1968) and H1N1 (2009) in experimentally inoculated ferrets were compared to that of their seasonal descendants (H3N2 of 1971 and H1N1 of 2013). By definition, pandemic viruses circulating during their first year in the human population are faced with little pre-existing immunity. In contrast, seasonal influenza viruses can encounter significant levels of pre-existing immunity resulting from the pandemic and subsequent epidemics. Any phenotypic differences in tissue tropism between pandemic and seasonal viruses are expected to result from the selective pressure caused by the building-up of herd immunity in the human population.To further assess the link between tissue tropism and evolution of novel influenza variants, mathematical models informed by published and above experimental work were developed. The models focused on the within-host dynamics of the virus in the human respiratory tract. The outcome of evolution and competition between variants during infection was mathematically determined.The experiments designed to evaluate the role of transmission constraints and herd immunity on influenza virus tropism and associated evolutionary changes have been completed. Their results and analyses are being prepared for publication. Analyses revealed rapidly occurring changes in the tissue tropism of viruses passaged in the upper respiratory tract of ferrets. These tended to cause more severe pulmonary lesions than viruses passaged in the lower respiratory tract of ferrets, which exhibited little changes along the experimental chain of infection. Strikingly, pandemic influenza viruses were found to cause more severe pulmonary lesions and infected significantly more alveolar epithelial cells than their seasonal descendants. The building-up of herd immunity may thus selectively drive seasonal influenza viruses out of the human lungs. Interestingly, mathematical models of strain competition in the human respiratory tract suggested that immunity may strongly limit the emergence of novel influenza strains during infection, further supporting the major role immunity likely plays in the evolution of influenza virus tissue tropism. The results of these models have been published in The Lancet 2014 384(9959):2077-81. Probabilistic models, described in a manuscript currently under review, indicate that reassortment occurring upon co-infection may represent a more likely route for the evolution of novel pandemic viruses than the accumulation of three or more mutations, the latter being hampered by host immunity. Overall, IIF 302060 project “Influenza Tropism”, by combining experimental approach and mathematical modeling, aimed at a more comprehensive understanding of influenza virus within-host evolution to better predict influenza virus population-level emergence and spread, and associated morbidity and mortality burdens. Such understanding is essential to support the implementation of pandemic preparedness plans and policy decisions enabling informed assessment of the public health threat continuously posed by influenza A viruses.