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Summary. -Infl uenza A viruses (IAVs) cause acute respiratory infections of humans, which are repeated yearly. Human IAV infections are associated with signifi cant morbidity and mortality and therefore they represent a serious health problem. All human IAV strains are originally derived from avian IAVs, which, aft er their adaptation to humans, can spread in the human population and cause pandemics with more or less severe course of the disease. Presently, however, the potential of avian IAV to infect humans and to cause the disease cannot be predicted. Many studies are therefore focused on factors infl uencing the virulence and pathogenicity of IAV viruses in a given host. Th e virus-host interaction starts by virus attachment via the envelope glycoprotein hemagglutinin (HA) to the receptors on the cell surface. In addition to receptor binding, HA mediates also the fusion of viral and endosomal membranes, which follows the virus endocytosis. Th e fusion potential of HA trimer, primed by proteolytic cleavage, is activated by low pH in endosomes, resulting in HA refolding into the fusion-active form. Th e HA conformation change is predetermined by its 3-D structure, is pH-dependent, irreversible and strain-specifi c. Th e process of fusion activation of IAV hemagglutinin is crucial for virus entry into the cell and for the ability of the virus to replicate in the host. Here we discuss the known data about the characteristics of fusion activation of HA in relation to IAV virulence and pathogenicity.Keywords: infl uenza A viruses; pH optimum of fusion; IAV virulence and pathogenicity; HA conformation change; IAV interspecies transmission; adaptation changes * Corresponding author. E-mail: viruevar@savba.sk; phone: 421-2-59302427. Abbreviations: HA = hemagglutinin; HA0 = HA precursor; HA1 = heavy chain of HA; HA2 = light chain of HA; HPAI = highly pathogenic avian infl uenza; IAV(s) = infl uenza A virus(es); LPAI = low pathogenic avian infl uenza; M1 protein = matrix protein; mRNA = messenger RNA; NA = neuraminidase; NEP protein = nuclear export protein; NS1 protein = nonstructural protein 1; PA = polymerase acidic protein; PB1 = polymerase basic protein 1; PB2 = polymerase basic protein 2; RBS = receptor binding site; RNA = ribonucleic acid; RNP = ribonucleoprotein; SA = sialic acid; vRNA = viral RNA Content: 1. Introduction 2. Structure and function of hemagglutinin 2.1 Receptor binding activity and IAV host tropism 2.2 Fusion activity and structural rearrangements of HA during the membrane fusion 3. Th e HA cleavability by host proteases and HA fusion activation pH as factors of IAV virulence and pathogenicity 3.1 Cleavage activation of HA as a determinant of IAV pathogenicity 3.2 Fusion activation pH as a determinant of IAV virulence 4. Th e role of fusion activation pH and stability of HA in the adaptation of IAV to the new host 5. Other viral proteins infl uencing the IAV virulence 6. Conclusion

Influenza A viruses (IAVs) enter into cells by receptor-dependent endocytosis. Subsequently, conformational changes of haemagglutinin are triggered by low environmental pH and the N terminus of HA2 glycoprotein (gp) is inserted into the endosomal membrane, resulting in fusion pore formation and genomic vRNA release into the cytoplasm. However, the pH optimum of membrane fusion is host-and virus-specific and can have an impact on virus pathogenicity. We prepared mutants of neurotropic IAV A/WSN/33 (H1N1) with aa substitutions in HA2 gp at the site of HA1/HA2 interaction, namely T64 2 H (HA2 numbering position 64, H1 numbering position HA407; referred to as mutant '64'), V66 2 H ('66') (HA409); and a double mutant ('D') with two aa substitutions (T64 2 H, V66 2 H). These substitutions were hypothesized to influence the pH optimum of fusion. The pH optimum of fusion activity was measured by a luciferase assay and biological properties of viruses were monitored. The in vitro and in vivo replication ability and pathogenicity of mutants were comparable (64) or lower (66, D) than those of the wild-type virus. However, the HA2 mutation V66 2 H and double mutation T64 2 H, V66 2 H shifted the fusion pH maximum to lower values (ranging from 5.1 to 5.3) compared to pH from 5.4 to 5.6 for the wild-type and 64 mutant. The decreased replication ability and pathogenicity of 66 and D mutants was accompanied by higher titres in late intervals post-infection in lungs, and viral RNA in brains compared to wild-type virus-infected mice. These results have implications for understanding the pathogenicity of influenza viruses.