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Influenza A viruses (IAV) originating from birds can cause severe disease in humans. Among the several subtypes transmitted to humans, subtypes H5N1 and H7N9 are of particular interest as they caused outbreaks in humans with high mortality rates. These viruses continue to occasionally transmit from birds to humans representing a pandemic risk. During infection IAV have to overcome host-specific cellular defence mechanisms of the innate immunity, which are not fully defined. In this work, we wanted to investigate interaction of IAV with two factors of innate immunity, namely, airway mucus and IFN-induced antiviral proteins.
In humans, IAV replicate in the respiratory epithelium of airways. Airway epithelium is covered by the so-called mucous blanket. Mucins, highly glycosylated sialylglycoproteins, are the major component of this blanket. They serve as decoy receptors for many pathogens, including viruses. It is generally believed, that human mucins mainly express α2-3-linked sialic acids and that avian viruses are more sensitive to neutralization by mucus than human viruses (Couceiro et al., 1993). However, more recent reports show higher sensitivity of human viruses to neutralization by mucus than avian viruses.
In the first part of this thesis, we wanted to address this discrepancy and study inhibitory function of mucus secretions from human tracheo-bronchial epithelial (HTBE)-cultures. We found that inhibition depends on the cell system used. In standard laboratory MDCK cells human seasonal virus showed high sensitivity to mucus while infection of avian virus was not inhibited. However, in HTBE-cultures human and avian virus showed comparable sensitivity to mucus. The finding that avian and human viruses bound to mucus to similar extend supported our data. To identify the inhibitory components in mucus we investigated properties of mucus samples. Our data show that mucus samples express α2-6- and α2-3-linked sialic acids and that inhibition of influenza infection is dependent on sialic acids in mucus. In summary, we show that avian and human viruses are inhibited by human bronchial mucus which contradicts the general hypothesis that avian viruses are more sensitive to mucus than human viruses.
In the second part, we investigated properties of HA and NA affecting virus sensitivity to the IFN-β-induced antiviral state by constructing recombinant viruses with HA and NA of representative avian, zoonotic and human influenza viruses and internal genes of the lab strain A/Puerto Rico/8/1934. IFN-β is secreted during initial phase of virus infection and induces expression of several hundred so-called interferon-stimulated genes (ISGs). These proteins can have antiviral activity like the interferon induced transmembrane protein family (IFITM), which inhibits viral fusion.
Virus sensitivity to the antiviral state correlated with the pH-optimum of fusion. Viruses with high pH-optimum of fusion, like viruses containing HA and NA of the zoonotic viruses H5N1 or H7N9, infected IFN-β-stimulated cells more efficiently than viruses with low pH-optimum of fusion. These findings were confirmed by testing recombinant mutant viruses with single point mutations in the HA of A/Hong Kong/1/1968 affecting the pH-optimum of fusion.
Concordant with the data from recombinant viruses, wildtype isolates of H5N1, H7N9 and pandemic virus from 2009 showed a higher pH-optimum of fusion and lower sensitivity to the antiviral state than pandemic 1968 and seasonal human virus.
IFITM proteins were already described as a potent inhibitor of influenza virus fusion. We show that inhibition of viruses by IFITM proteins is dependent on the pH-optimum of viral-induced fusion. Viruses with a high pH-optimum of fusion, such as H5N1 and H7N9, showed lower restriction in MDCK cells stably expressing human IFITM2 and IFITM3 than viruses with a low pH-optimum of fusion.
Our results demonstrate for the first time that the pH-optimum of fusion affects virus sensitivity to IFN-induced effector molecules, including IFITM. These data imply that the high pH-optimum of fusion of zoonotic viruses, such as H5N1 and H7N9, may contribute to their ability to infect and cause severe disease in humans.