Mutations arising in influenza viruses that have undergone immune pressure may promote a successful spread of mutants in nature. In order to evaluate the variability of nonpathogenic influenza virus A/duck/Moscow/4182-C/2010(H5N3) and to determine the common epitopes between it and highly pathogenic H5N1 avian influenza viruses (HPAIV), a set of escape mutants was selected due to action of MABs specific against A/chicken/Pennsylvania/8125/83(H5N2), A/Vietnam/1203/04(H5N1) and A/duck/ Novosibirsk/56/05(H5N1) viruses. The complete genomes of escape mutants were sequenced and amino acid point mutations were determined in HA, NA, PA, PB1, PB2, M1, M2, and NP proteins. Comprehensive analysis of the acquired mutations was performed using the Influenza Research Database (https://www. fludb.org) and revealed that all mutations were located inside short linear epitopes, in positions characterized by polymorphisms. Most of the mutations found were characterized as substitutions by predominant or alternative amino acids existing in nature. Antigenic changes depended only on substitutions at positions 126, 129, 131, 145 and 156 of HA (H3 numbering). The positions 126, 145 and 156 were common for HA/H5 of different phylogenetic lineages of H5N1 HPAIV (arisen from A/goose/Guangdong/1/96) and low pathogenic American and Eurasian viruses. Additionally, mutation S145P increased the temperature of HA heat inactivation, compared to wild-type, as was proved by reverse genetics. Moreover, nonpathogenic A/ duck/Moscow/4182-C/2010(H5N3) and H5N1 HPAI viruses have the same structure of short linear epitopes

The influenza A virus remains one of the most common and dangerous human health concerns due to its rapid evolutionary dynamics. Since the evolutionary changes of influenza A viruses can be traced in real time, the last decade has seen a surge in research on influenza A viruses due to an increase in experimental data (selection of escape mutants followed by examination of their phenotypic characteristics and generation of viruses with desired mutations using reverse genetics). Moreover, the advances in our understanding are also attributable to the development of new computational methods based on a phylogenetic analysis of influenza virus strains and mathematical (integro-differential equations, statistical methods, probability-theory-based methods) and simulation modeling. Continuously evolving highly pathogenic influenza A viruses are a serious health concern which necessitates a coupling of theoretical and experimental approaches to predict the evolutionary trends of the influenza A virus, with a focus on the H5 subtype.

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