We could demonstrate that 4% formaldehyde fixation followed by acetone: methanol (1:1) treatment allowed the immunofluorescence detection of both the Ebola virus proteins NP, VP35, VP40 and the cellular PML protein in the virus infected cells. In fact, the fixation procedure was fully comparable with fixation in acetone alone (data not shown). This method provides an easier, and in routine laboratory procedure more reliable, alternative to the protocols of van der Groen  and Kurata  where the epitope recovery in formaldehyde fixed cells required controlled enzymatic treatment with trypsin or pronase. It gives a comparable result to paraformaldehyde fixation followed by Triton X-100 permeabilization .
Our staining data suggest that for routine detection of infectious virus or titration of virus stocks, immunofluorescence staining of NP protein (since staining of this protein gave rise to the most easily recognizable pattern, clearly differing from existing cellular structures) on formaldehyde fixed and subsequently methanol:acetone treated cells, is a useful tool.
The presence of NP-containing viral inclusions throughout the cell cycle indirectly suggests that Ebola virus does not induce an immediate re-programming of the cells by abruptly shutting down host protein synthesis but prepares for virus replication in a much more stealth-like fashion. The development of huge cytoplasmic inclusions without apparent cellular distress is in line with previous observations that filoviruses induced cytopathic effects may be absent or require several days to manifest .
VP35 is an essential component of the Ebola nucleocapsid . Here we show that VP35 accumulates in cytoplasmic inclusions that are morphologically indistinguishable from NP-containing viral inclusions. Ebola virus replicates in the cytoplasm through double stranded RNA intermediates and it appears that it has evolved a mechanism that interferes with dsRNA induced interferon response . VP35 was shown to be able to rescue NS1 mutant influenza A virus replication arguing that it is a type I IFN antagonist. VP35 could also block dsRNA or virus-mediated induction of IFN responsive promoters . Negative stranded RNA viruses often sequester their dsRNA intermediate into ribonucleoprotein complexes that form cytoplasmic inclusions. The presence of VP35 in the Ebola virus inclusions suggests that it intercepts the dsRNA induced response in a close spatial proximity to the viral RNA.
VP40 showed a completely different sub-cellular localization from NP and VP35. It was primarily associated with the cell membrane. Biochemical studies earlier showed that VP40 is a membrane binding protein that oligomerizes upon interaction with lipid membranes . The oligomerization leads to self assembly into membrane-containing particles  and subsequent release into the cell culture medium . Unlike the Marburg virus encoded VP40 that localized both to the viral inclusions as well as to clusters of intracellular membrane or to plasma membrane protrusions in infected macrophages , Ebola VP40 was primarily associated with the cell membrane. It also entered the nucleus, similarly to the Marburg encoded VP40, raising the possibility that VP40 may play additional, e.g. gene expression regulatory role beside the involvement in virus assembly and budding.
Importantly, the staining with anti-VP40 antibodies on formaldehyde fixed and acetone:methanol treated cells allowed the direct immunofluorescence detection of extracellular virions and/or filamentous virus-like particles. The size of most viruses is regularly below the resolution of the light microscope. Although the spatial resolution of the fluorescence microscope is constrained by the same physical laws as any other light microscopes, e.g. it does not allow the reliable separation of two points that are closer than 0.2 μm, it still allows the detection of light emitting particles on dark background that are far much smaller, e.g. individual protein molecules. Ebola Zaire virions with average length of 1.3 ± 0.7 μm and occasional length up to 14 μm  are well within the limits of fluorescence microscopy. The possibility to carry out 3D reconstitution of fluorescence images picturing nascent virions in combination with staining for additional two or three viral and/or cellular proteins should provide ample amount of information about the molecular mechanism of the egress of Ebola virus.
IFN-alpha appears to play a crucial role in the anti-viral defense in Ebola virus infected experimental animals. Mice lacking the type I IFN receptor, but not wild type mice, died upon subcutaneous inoculation of the virus . The adenosine analogue 3-Deazaneplanocin A was recently shown to protect from Ebola virus induced death of infected mice through massive induction of IFN-alpha .
We found that IFN-alpha treatment decreased Ebola virus protein production in vitro and inhibited the development of cytopathic effects. These data are in line with the findings that IFN-alpha could reduce infection 10 to 100-fold in cell cultures and also delay the death of infected monkeys . One important anti-viral mechanism that is induced as part of the interferon response is the increased expression of the components of the subnuclear organelle ND10 or PML body. A variety of DNA and RNA viruses encode proteins that specifically interact with the PML bodies and regularly destroy them. Rhabdoviruses share several molecular features with filoviruses. The rabies virus encoded P phosphoprotein is a distant homologue of the Ebola VP35. It interacts with and reorganizes PML bodies . Here we show that Ebola virus infection did not dissolve PML bodies. On the contrary, individual cells expressing viral proteins regularly showed increased expression of PML. Moreover, although Ebola virus interferes with the IFN response it could not inhibit the IFN-alpha mediated induction of PML. VP35, a possible candidate for interference with PML function, never entered the nucleus but remained exclusively cytoplasmic. Importantly, Vero E6 cells, that supported Ebola replication very efficiently, expressed low levels of PML and showed much smaller virus-dependent and IFN-mediated increase in PML expression than MCF7 cells that served as a poor host for the virus. Our data suggest that Ebola virus lacks effective anti-PML mechanism. The data are also consistent with the possibility that increased PML expression might hinder virus infection, similarly to what was previously demonstrated for vesicular stomatitis virus (VSV) [32, 33], influenza virus , human foamy virus (HFV)  and lymphocytic choriomeningitis virus (LCMV) . Our results lead to the testable hypothesis that introduction of constitutively expressed PML cDNA into Vero E6 cells should inhibit, and inhibitory RNA (RNAi) mediated downregulation of PML in MCF7 cells should promote, the propagation of the Ebola virus in vitro.