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TDK conference 2023Oláh Barbara - year 5 University of Veterinary Medicine Budapest, Department of Microbiology and Infectious Diseases Supervisors: Dr. Tenk Miklós, Dr. Zádori Zoltán, Dr. Mészáros István Influenza A virus is an endemic disease in Europe, mainly in poultry, wild birds and exotic pet birds, often asymptomatic but sometimes associated with high fever, severe lethargy, respiratory and sometimes neurological symptoms, hemorrhagy and diarrhea. Due to its high mutation rate and broad host specificity, it can infect and adapt to a wide range of bird and mammalian species, and a better understanding of this can help us to control it more effectively. The aim of our studies was to investigate the in vitro adaptation of the avian influenza virus and the unknown function of the NEG8 protein, which has not been previously studied in avian influenza. As a first step, we investigated the susceptibility of the duck-derived cell line DuO to seven different serotypes of avian influenza strains. We selected two isolates of different serotypes (H3N8 and H5N9), characterized in detail their relevant biological properties (cytopathogenicity, infectious titer (fluorescent focus unit (FFU)/ml), growth curve, copy number) and performed serial passages on the DuO cell lines, monitoring the changes in their biological properties. During adaptation, the final titer of H5N9 stocks decreased until the third pass (105 FFU/ml) and then fluctuated between 105-106 FFU/ml until the ninth pass. It then increased to 107 FFU/ml and reached 108 FFU/ml after the 15th pass. With the H3N8 serotype, we are currently in the 7th pass and have similar data on the change in final titer. In this case, we also observed a significant decrease (from 107 to 105 FFU/ml) until pass 3, followed by a fluctuation between 105 and 106 FFU/ml. Along with the change in end titer, we also observed significant changes in the slope of the viral growth curve and the number of apoptotic nuclei that appeared as a result of infection. Our high-coverage sequence data obtained by new-generation (Illumina) sequencing showed that the frequency of SNPs (small nuclear polymorphisms) in virus samples decreased in both serotypes during passages, suggesting homogenization of quasi species and, in our opinion, a consequence of selection of less adapted variants. In several cases we observed intrinsic quasi species heterogeneity in triplets of amino acids known to influence virulence, pathogenicity and host specificity (e.g. amino acids 126, 138 and 159 of the hemagglutinin protein). The role of the NEG8 protein in the viral infection cycle is currently unknown and has only been investigated in human studies. Our in silico analyses revealed the presence of a transmembrane domain in the protein, and our transfection assays confirmed the localization of the protein in the endoplasmic reticulum, which was unchanged in infected cells (in the presence of other viral proteins). Some of our results suggest that expression of the protein induces cell death in transfected cells, but this requires further confirmation. List of lectures |