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Establishment of fruit bat cells (Rousettus aegyptiacus) as a model system for the investigation of filoviral infection.

Krähling V, Dolnik O, Kolesnikova L, Schmidt-Chanasit J, Jordan I, Sandig V, Günther S, Becker S - PLoS Negl Trop Dis (2010)

Bottom Line: This was not possible with other cell lines previously tested.Moreover, we established protocols to reliably rescue recombinant Marburg viruses from R06E cells.These data indicated that R06E cells are highly suitable to investigate the biology of filoviruses in cells derived from their presumed reservoir.

View Article: PubMed Central - PubMed

Affiliation: Institut für Virologie, Philipps-Universität Marburg, Marburg, Germany.

ABSTRACT

Background: The fruit bat species Rousettus aegyptiacus was identified as a potential reservoir for the highly pathogenic filovirus Marburg virus. To establish a basis for a molecular understanding of the biology of filoviruses in the reservoir host, we have adapted a set of molecular tools for investigation of filovirus replication in a recently developed cell line, R06E, derived from the species Rousettus aegyptiacus.

Methodology/principal findings: Upon infection with Ebola or Marburg viruses, R06E cells produced viral titers comparable to VeroE6 cells, as shown by TCID(50) analysis. Electron microscopic analysis of infected cells revealed morphological signs of filovirus infection as described for human- and monkey-derived cell lines. Using R06E cells, we detected an unusually high amount of intracellular viral proteins, which correlated with the accumulation of high numbers of filoviral nucleocapsids in the cytoplasm. We established protocols to produce Marburg infectious virus-like particles from R06E cells, which were then used to infect naïve target cells to investigate primary transcription. This was not possible with other cell lines previously tested. Moreover, we established protocols to reliably rescue recombinant Marburg viruses from R06E cells.

Conclusion/significance: These data indicated that R06E cells are highly suitable to investigate the biology of filoviruses in cells derived from their presumed reservoir.

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Related in: MedlinePlus

Expression of viral proteins in different cell lines.(A) 4×105 HUH7, VeroE6 or R06E cells were infected with 0.1 TCID50/cell MARV or ZEBOV. Cell lysates were prepared at 48 and 72 h p.i., subjected to Western blot analysis to detect cellular tubulin and VP40 of MARV and ZEBOV using mouse monoclonal antibodies. The total amount of cellular proteins in the samples was quantified by separating cell lysates on SDS PAGE, which were then stained with Coomassie Blue. Using the Odyssey Infrared Imaging Application Software, the protein signals were quantified. VP40 levels normalized to total cell protein are shown in (B).
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pntd-0000802-g002: Expression of viral proteins in different cell lines.(A) 4×105 HUH7, VeroE6 or R06E cells were infected with 0.1 TCID50/cell MARV or ZEBOV. Cell lysates were prepared at 48 and 72 h p.i., subjected to Western blot analysis to detect cellular tubulin and VP40 of MARV and ZEBOV using mouse monoclonal antibodies. The total amount of cellular proteins in the samples was quantified by separating cell lysates on SDS PAGE, which were then stained with Coomassie Blue. Using the Odyssey Infrared Imaging Application Software, the protein signals were quantified. VP40 levels normalized to total cell protein are shown in (B).

Mentions: The three cell lines were infected with 0.1 TCID50 per cell and Western blot analysis was performed with lysed cells at 48 and 72 h p.i. using monoclonal antibodies specific for MARV or ZEBOV VP40 or the cellular cytoskeleton protein tubulin. These analyses revealed that the fruit bat cells accumulated considerably more viral protein than the other cell lines tested (Fig. 2A). To further support this result, we quantified total protein levels of the different cell lines by Coomassie staining of cell lysates separated by SDS gels at 48 h and 72 h p.i. which were scanned using the Odyssey Analyzer (The 72 h gels are shown beneath the Western blots in Fig. 2A) The amount of VP40 was then normalized against the total cell protein (Fig. 2B). Quantification clearly shows that at 48 as well as 72 h p.i. the highest amount of viral protein can be found in R06E cells when compared to HUH7 or VeroE6 cells (Fig. 2B). However, our data also indicate that R06E cells support filoviral propagation to an extent comparable with VeroE6 or HUH7 cell lines. Furthermore, EM analysis of ultrathin sections of R06E cells revealed morphological signs of filoviral infection similar to those detected previously in cells originating from monkeys or humans [35], [36] (Fig. 1C). Taken together, these observations suggest that R06E cells may not release viral particles with a comparable efficiency to that with which they produce structural proteins. Further experiments to examine the viral budding efficiency and interaction of viral proteins with proteins of the endosomal sorting complex required for transport (ESCRT) in fruit bat-derived cells will be very important, as it has been shown that interaction of MARV VP40 with TSG101 or interaction of ZEBOV VP40 with TSG101 and Nedd4 plays an important role during the budding process [37]–[39].


Establishment of fruit bat cells (Rousettus aegyptiacus) as a model system for the investigation of filoviral infection.

Krähling V, Dolnik O, Kolesnikova L, Schmidt-Chanasit J, Jordan I, Sandig V, Günther S, Becker S - PLoS Negl Trop Dis (2010)

Expression of viral proteins in different cell lines.(A) 4×105 HUH7, VeroE6 or R06E cells were infected with 0.1 TCID50/cell MARV or ZEBOV. Cell lysates were prepared at 48 and 72 h p.i., subjected to Western blot analysis to detect cellular tubulin and VP40 of MARV and ZEBOV using mouse monoclonal antibodies. The total amount of cellular proteins in the samples was quantified by separating cell lysates on SDS PAGE, which were then stained with Coomassie Blue. Using the Odyssey Infrared Imaging Application Software, the protein signals were quantified. VP40 levels normalized to total cell protein are shown in (B).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2927428&req=5

pntd-0000802-g002: Expression of viral proteins in different cell lines.(A) 4×105 HUH7, VeroE6 or R06E cells were infected with 0.1 TCID50/cell MARV or ZEBOV. Cell lysates were prepared at 48 and 72 h p.i., subjected to Western blot analysis to detect cellular tubulin and VP40 of MARV and ZEBOV using mouse monoclonal antibodies. The total amount of cellular proteins in the samples was quantified by separating cell lysates on SDS PAGE, which were then stained with Coomassie Blue. Using the Odyssey Infrared Imaging Application Software, the protein signals were quantified. VP40 levels normalized to total cell protein are shown in (B).
Mentions: The three cell lines were infected with 0.1 TCID50 per cell and Western blot analysis was performed with lysed cells at 48 and 72 h p.i. using monoclonal antibodies specific for MARV or ZEBOV VP40 or the cellular cytoskeleton protein tubulin. These analyses revealed that the fruit bat cells accumulated considerably more viral protein than the other cell lines tested (Fig. 2A). To further support this result, we quantified total protein levels of the different cell lines by Coomassie staining of cell lysates separated by SDS gels at 48 h and 72 h p.i. which were scanned using the Odyssey Analyzer (The 72 h gels are shown beneath the Western blots in Fig. 2A) The amount of VP40 was then normalized against the total cell protein (Fig. 2B). Quantification clearly shows that at 48 as well as 72 h p.i. the highest amount of viral protein can be found in R06E cells when compared to HUH7 or VeroE6 cells (Fig. 2B). However, our data also indicate that R06E cells support filoviral propagation to an extent comparable with VeroE6 or HUH7 cell lines. Furthermore, EM analysis of ultrathin sections of R06E cells revealed morphological signs of filoviral infection similar to those detected previously in cells originating from monkeys or humans [35], [36] (Fig. 1C). Taken together, these observations suggest that R06E cells may not release viral particles with a comparable efficiency to that with which they produce structural proteins. Further experiments to examine the viral budding efficiency and interaction of viral proteins with proteins of the endosomal sorting complex required for transport (ESCRT) in fruit bat-derived cells will be very important, as it has been shown that interaction of MARV VP40 with TSG101 or interaction of ZEBOV VP40 with TSG101 and Nedd4 plays an important role during the budding process [37]–[39].

Bottom Line: This was not possible with other cell lines previously tested.Moreover, we established protocols to reliably rescue recombinant Marburg viruses from R06E cells.These data indicated that R06E cells are highly suitable to investigate the biology of filoviruses in cells derived from their presumed reservoir.

View Article: PubMed Central - PubMed

Affiliation: Institut für Virologie, Philipps-Universität Marburg, Marburg, Germany.

ABSTRACT

Background: The fruit bat species Rousettus aegyptiacus was identified as a potential reservoir for the highly pathogenic filovirus Marburg virus. To establish a basis for a molecular understanding of the biology of filoviruses in the reservoir host, we have adapted a set of molecular tools for investigation of filovirus replication in a recently developed cell line, R06E, derived from the species Rousettus aegyptiacus.

Methodology/principal findings: Upon infection with Ebola or Marburg viruses, R06E cells produced viral titers comparable to VeroE6 cells, as shown by TCID(50) analysis. Electron microscopic analysis of infected cells revealed morphological signs of filovirus infection as described for human- and monkey-derived cell lines. Using R06E cells, we detected an unusually high amount of intracellular viral proteins, which correlated with the accumulation of high numbers of filoviral nucleocapsids in the cytoplasm. We established protocols to produce Marburg infectious virus-like particles from R06E cells, which were then used to infect naïve target cells to investigate primary transcription. This was not possible with other cell lines previously tested. Moreover, we established protocols to reliably rescue recombinant Marburg viruses from R06E cells.

Conclusion/significance: These data indicated that R06E cells are highly suitable to investigate the biology of filoviruses in cells derived from their presumed reservoir.

Show MeSH
Related in: MedlinePlus