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Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria
Published on: February 23, 2014
Identification of a novel virulence factor in recombinant pneumonia virus of mice
Christine D Krempl1, Anna Wnekowicz, Elaine W Lamirande
1Department of Virology, Institute for Medical Microbiology and Hygiene, University of Freiburg, Freiburg, Germany.
Abstract:
Pneumonia virus of mice (PVM) is a murine relative of human respiratory syncytial virus (HRSV). Here we developed a reverse genetics system for PVM based on a consensus sequence for virulent strain 15. Recombinant PVM and a version engineered to express green fluorescent protein replicated as efficiently as the biological parent in vitro but were 4- and 12.5-fold attenuated in vivo, respectively. The G proteins of HRSV and PVM have been suggested to contribute to viral pathogenesis, but this had not been possible to study in a defined manner in a fully permissive host. As a first step, we evaluated recombinant mutants bearing a deletion of the entire G gene (Delta G) or expressing a G protein lacking its cytoplasmic tail (Gt). Both G mutants replicated as efficiently in vitro as their recombinant parent, but both were nonpathogenic in mice at doses that would otherwise be lethal. We could not detect replication of the Delta G mutant in mice, indicating that its attenuation is based on a severe reduction in the virus load. In contrast, the Gt mutant appeared to replicate as efficiently in mice as its recombinant parent. Thus, the reduction in virulence associated with the Gt mutant could not be accounted for by a reduction in viral replication. These results identified the cytoplasmic tail of G as a virulence factor whose effect is not mediated solely by the viral load. In addition to its intrinsic interest, a recombinant virus that replicates with wild-type-like efficiency but does not cause disease defines optimal properties for vaccine development.
Insights
Researchers created a reverse genetics system for Pneumonia virus of mice (PVM), a relative of human respiratory syncytial virus (HRSV). A G protein tail mutant showed reduced virulence without affecting viral load, suggesting its role in PVM pathogenesis.
Area of Science:
- Virology
- Immunology
- Molecular Biology
Background:
- Pneumonia virus of mice (PVM) is a murine pathogen closely related to human respiratory syncytial virus (HRSV).
- The G protein of PVM and HRSV is implicated in viral pathogenesis, but its precise role has been difficult to study.
- A reverse genetics system is crucial for dissecting viral virulence factors.
Purpose of the Study:
- To develop a reverse genetics system for PVM to study viral pathogenesis.
- To investigate the role of the PVM G protein, specifically its cytoplasmic tail, in virulence.
- To assess the potential of engineered PVM as a vaccine candidate.
Main Methods:
- Development of a reverse genetics system for PVM using a virulent strain consensus sequence.
- Generation of recombinant PVM expressing green fluorescent protein and G protein mutants (Delta G and Gt).
- In vitro and in vivo replication and pathogenesis studies in mice.
Main Results:
- Recombinant PVM and GFP-expressing PVM showed efficient in vitro replication, with significant attenuation in vivo.
- PVM mutants lacking the G gene (Delta G) or expressing a truncated G protein (Gt) were nonpathogenic in mice.
- The Delta G mutant exhibited severe replication defects in vivo, while the Gt mutant replicated efficiently but was attenuated.
- The cytoplasmic tail of the G protein was identified as a virulence factor, independent of viral load.
Conclusions:
- The PVM reverse genetics system enables the study of viral virulence factors.
- The cytoplasmic tail of the PVM G protein is a key determinant of virulence.
- Recombinant PVM with attenuated virulence but wild-type-like replication efficiency holds promise for vaccine development.
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