## 4 Discussion  
Maintenance of a virus within its natural reservoir requires its persistence in the animal population and/or in individual animals, its tolerance by the host, and its replication to high enough levels to allow for transmission to naive animals. Our results from investigating replication kinetics, tolerance, and persistence of EBOV in primary cells from _M. condylurus_ provide further evidence that this microbat species may be important in the maintenance of ebolaviruses and be a likely reservoir host. The integral membrane protein NPC1 has been described as a key component in the divisorial entry process [28; 31; 74; 75], shown to be essential for EBOV entry [28; 29; 30] and MARV susceptibility [31]. While the absence of the required NPC1 receptor, as demonstrated in NPC1-knockout HEK293 cells, reliably prevents EBOV infection, NPC1 receptor expression levels correlated with the amounts of EBOV released in the supernatant in the majority of the tested cell isolates. Consequently, primary cell isolates with particularly low NPC1 receptor expression levels (MoTra Prim, MoLu Prim, MoSp Prim Early, and MoKi Prim cells) supported only low EBOV replication rates. In contrast, cells with particularly high NPC1 receptor expression levels (HeLa, HEK293, and Vero) correlated with very high virus replication rates. Comparing primary spleen cells at passage 5 (MoSp Prim Early), which have low NPC1 receptor expression levels, to the same spleen cells at passage 29 (MoSp Prim Late), which have increased NPC1 expression levels, the same correlation was observed with low and high EBOV replication rates, respectively. In two cell isolates, this correlation was not observed; MoSk Prim and NyKi Prim cells revealed moderate EBOV replication rates, but low or high NPC1 receptor expression, respectively. These results underline that the NPC1 receptor is a key component that influences tropism and replications kinetics, but not the only host cell determinant for EBOV replication. Availability of attachment factors and different efficiency of viral replication, transcription, translation, assembly, and budding of virus progeny may also influence EBOV replication rates in different cell types. In addition, the percentage of infected cells in a monolayer influences the amount of detectable viral RNA in cell culture supernatants. Additionally, the amount of virus entering cells of different cell isolates during infection may be different and would influence calculations, so that the discussed replication rates can only serve as an approximation. Future NPC1 knockdown or knockout experiments for selected cell types could confirm the correlation between NPC1 expression and EBOV replication efficiency. Nearly all human cell types tested, and a very broad range of other mammalian cells are susceptible to EBOV infection. Only a few cell types have been described to be refractory to EBOV infection. Those that are known to be refractory are cells with D502F NPC1 polymorphism from _Eidolon helsum_ fruit bats [34], cells from patients with Niemann-Pick C1 disease [31], cells of lymphoid origin [27; 76; 77] including human B-, T-, and NK cells, murine lymphoid cell lines or mosquito cells [78]. Despite the moderate NPC1 receptor expression, no distinct EBOV replication could be observed in supernatants of MoLi Prim cells within the first 4 days of infection. Using EBOV-GFP, we demonstrated that infection of MoLi Prim cells is inefficient, but cells were not refractory, with only few cells in the monolayer being infected, suggesting a possible impaired virus entry by insufficiently expressed cellular attachment factors. Several EBOV-infected MoLi Prim cells in the monolayer developed into small clusters of infected foci, indicating a very inefficient infection of cells, followed by strong replication, but no detectable release of infectious virus particles. Instead, infection of neighboring cells seemed to result exclusively from cell-to-cell transmission, which has not been described for filoviruses.