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. 2008 Jan 29;105(4):1129-33.
doi: 10.1073/pnas.0708057105. Epub 2008 Jan 22.

Generation of biologically contained Ebola viruses

Affiliations

Generation of biologically contained Ebola viruses

Peter Halfmann et al. Proc Natl Acad Sci U S A. .

Abstract

Ebola virus (EBOV), a public health concern in Africa and a potential biological weapon, is classified as a biosafety level-4 agent because of its high mortality rate and the lack of approved vaccines and antivirals. Basic research into the mechanisms of EBOV pathogenicity and the development of effective countermeasures are restricted by the current biosafety classification of EBOVs. We therefore developed biologically contained EBOV that express a reporter gene instead of the VP30 gene, which encodes an essential transcription factor. A Vero cell line that stably expresses VP30 provides this essential protein in trans and biologically confines the virus to its complete replication cycle in this cell line. This complementation approach is highly efficient because biologically contained EBOVs lacking the VP30 gene grow to titers similar to those obtained with wild-type virus. Moreover, EBOVs lacking the VP30 gene are indistinguishable in their morphology from wild-type virus and are genetically stable, as determined by sequence analysis after seven serial passages in VP30-expressing Vero cells. We propose that this system provides a safe means to handle EBOV outside a biosafety level-4 facility and will stimulate critical studies on the EBOV life cycle as well as large-scale screening efforts for compounds with activity against this lethal virus.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Schematic diagram of EbolaΔVP30 constructs. The top row shows a schematic diagram of the EBOV genome flanked by the leader sequence (l) and the trailer sequence (t) in positive-sense orientation. Two unique restriction sites for SalI and SacI (positions 6180 and 10942 of the viral antigenome, respectively) allowed the subcloning of a fragment that spans the VP30 gene. The subgenomic fragment was then used to replace the VP30 gene with genes encoding neo or eGFP, respectively. By using the unique restriction sites, the altered subgenomic fragments were cloned back into the full-length EBOV cDNA construct.
Fig. 2.
Fig. 2.
Characterization of EbolaΔVP30-neo virus. (A) Expression of EBOV antigens by infected VeroVP30 cells. Confluent VeroVP30 cells (Left) or wild-type Vero cells (Right) were infected with EbolaΔVP30-neo for 60 min, washed, and overlaid with propagation medium with 1.5% methylcellulose. Seven days later, cells were fixed with 10% buffered formaldehyde and an immunostaining assay with an antibody to EBOV VP40 protein was performed as described in Materials and Methods. The formation of plaques in the VeroVP30 cell monolayer (Left), but not in monolayers of wild-type Vero cells (Right), illustrates that EbolaΔVP30-neo virus is biologically contained. (B) Detection of EbolaΔVP30-neo viral proteins. Supernatants derived from infected VeroVP30 (labeled +) or wild-type Vero (labeled −) cells were collected 5 days after infection and partially purified over 20% sucrose. Protein pellets were suspended in PBS and separated on polyacrylamide gels, transferred to membranes, and probed with specific antibodies to EBOV proteins.
Fig. 3.
Fig. 3.
Replication kinetics of wild-type EBOV and EbolaΔVP30-neo virus. VeroVP30 cells (Upper) and wild-type Vero cells (Lower) were infected with EBOV or EbolaΔVP30-neo at a high m.o.i. of 1.0 (Left) or a low m.o.i. of 0.01 (Right). Supernatants were harvested every 24 h after infection for 6 days. Viral titers of the respective viruses were determined by infecting confluent VeroVP30 cells or wild-type Vero cells with 10-fold dilutions of the supernatants and subsequent immunostaining as described in Materials and Methods. Virus titers for EbolaΔVP30-neo virus (filled squares) and wild-type EBOV (open circles) were comparable in VeroVP30 cells (Upper). In wild-type Vero cells (Lower), no replication was detected for EbolaΔVP30-neo virus (filled squares).
Fig. 4.
Fig. 4.
Morphology of EBOVs budding from infected cells. Vero cells infected with wild-type EBOV (Left) and VeroVP30 cells infected with EbolaΔVP30-neo virus (Right) were processed for TEM 3 days after infection as described in Materials and Methods. The pictures show virus budding from infected cells. No significant differences in morphology or budding efficiencies were observed for wild-type EBOV and EbolaΔVP30-neo virus. [Magnification: ×ばつ6,000 (Upper) and ×ばつ20,000 (Lower, boxed area from Upper).]

References

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