The Hemagglutinin of Bat-Associated Influenza Viruses Is Activated by TMPRSS2 for pH-Dependent Entry into Bat but Not Human Cells

Hoffmann, Markus; Krüger, Nadine; Zmora, Pawel; Wrensch, Florian; Herrler, Georg; Pöhlmann, Stefan

We used a VSV-based vector system to study cellular entry of HAL and NAL-bearing particles. VSV pseudotypes allow convenient analysis of entry driven by diverse glycoproteins [28, 31, 61] , although one should keep in mind that due to differences in particle shape and efficiency of glycoprotein incorporation pseudotypes might not adequately mirror all aspects of cellular entry of authentic viruses [62, 63] . We found that cell lines frequently used for FLUAV propagation were not susceptible to transduction by HAL and NAL bearing particles, which is in agreement with the finding that replacement of batFLUAV-HAL and -NAL by their FLUAV counterparts is required for spread of batFLUAV in the cell lines studied so far [18, 19] . In contrast, inoculation of bat cell lines originating from five different species of micro-and megachiropteran bats revealed that three cell lines, EidNi/41, HypNi/1.1, EpoNi/22.1, were susceptible to entry mediated by batFLUAV surface proteins. EpoNi/22.1 cells showed the highest susceptibility and were thus used for further studies, while EidNi/41 cells were found to be robustly susceptible only to transduction by pseudotypes harboring the HAL18. Collectively, these findings suggest that batFLUAV surface proteins can mediate entry into certain bat cells and that entry efficiency might differ between batFLUAV subtypes. Our finding that certain bat cell lines are susceptible to pseudotypes harboring HAL of batFLUAV are in line with observations very recently documented by Maruyama et al. who found that out of a diverse panel of bat cell lines tested, cells from Miniopterus fuliginosus, Miniopterus schreibersii and Pteropus giganteus were susceptible to pH-dependent, HAL-driven entry [64] . A cell line derived from Eidolon helvum spleen cells was found to be non-susceptible in contrast to our findings with a kidney cell line established from the same species, suggesting that receptor expression might differ between organs. Somewhat more surprising, Maruyama and colleagues also observed HAL-driven entry into MDCK cells [64] , which was not detected in the present study, and these discrepant results might be attributed to use of MDCK cells from different sources or to differences in the method used to quantify pseudotype entry. Finally, it is noteworthy that cell lines from bats inhabiting different geographical locations (Africa, Asia, Europe) were found to be susceptible to HAL-driven entry, suggesting that entry is not a bottleneck for spread of batFLUAV between bat species.