Characterization of the glycoproteins of bat-derived influenza viruses
Text by Junki Maruyama
In 2012 and 2013, papers reported novel influenza viruses detected from bats. The bat-derived influenza viruses (BatIVs) are genetically distinct from other known influenza A viruses (IAVs), and thus have been categorized into the new subtypes. However, the biological properties of these BatIVs remain unknown since infectious strains of these viruses have not been isolated. To gain insight into the function of BatIV glycoproteins, hemagglutinin (HA) and neuraminidase (NA), we employed the vesicular stomatitis virus (VSV) pseudotype system that is often used to analyze envelope glycoproteins of highly virulent or unisolated viruses. Interestingly, VSVs carrying BatIV HAs and NAs efficiently infected particular bat cell lines but not those derived from primates, birds, and pigs—all of which are major hosts of IAVs. We further found that BatIV HAs might recognize some cellular glycoproteins as receptors rather than the sialic acids used by the other known influenza viruses.
The discovery of BatIVs was very surprising and interesting for influenza virologists who believe that natural reservoirs of IAVs were waterfowls and IAVs maintained in the reservoir hosts are the source of all IAVs infecting mammals. Thus, our group began to speculate about the potential host range of BatIVs using the VSV pseudotype system. When we first tried to generate pseudotyped VSVs, we were not sure that BatIV glycoproteins could actually be incorporated into VSV particles. But, we blindly generated VSV particles and went ahead to inoculate them into several cell lines, including some bat-derived cells established by ourselves. Twenty hours later, we observed that most of the cell lines did not show any GFP signals (pseudotyped VSVs have GFP-gene), but some bat-derived cells showed green fluorescence! This clearly told us that BatIV glycoproteins were incorporated into VSV particles and mediated virus entry into only certain cell lines.
Incorporation of BatIV glycoproteins into VSV particles should eventually be confirmed using specific antibodies. Our laboratory has an anti-HA monoclonal antibody library covering all previously known HA subtypes. Fortunately, one of the antibodies, which was broadly cross-reactive to multiple HA subtypes, also reacted with BatIV HAs so that we could successfully use it in immuno-electron microscopy and western blotting analyses. The existence of this cross-reactive antibody made us think that BatIVs are indeed related to IAVs, although they showed unique characteristics such as distinct receptor usage from the other IAVs. To be honest, I actually had doubted that BatIVs belonged to IAVs.