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Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates
Published on: July 6, 2013
Murine cytomegalovirus with a transposon insertional mutation at open reading frame M35 is defective in growth in
Ada Tam1, Jiaming Zhu, Rong Hai
1Division of Infectious Diseases, School of Public Health, University of California-Berkeley, Berkeley, CA 94720, USA.
Abstract:
We had previously constructed a pool of murine cytomegalovirus (MCMV) mutants that contained a Tn3-based transposon sequence randomly inserted in the viral genome. In the study reported here, one of the mutants, RvM35, which contains the transposon insertion at open reading frame M35, was characterized both in vitro in tissue cultures and in immunocompetent Balb/c and immunodeficient SCID mice. Our results provide the first direct evidence to suggest that M35 is not essential for viral replication in vitro in NIH 3T3 cells. Compared to the wild-type strain and a rescued virus that restored the M35 region, the viral mutant was attenuated in growth in both the intraperitoneally infected Balb/c and SCID mice. At 21 days postinfection, the titers of the mutant in the salivary glands, lungs, spleens, livers, and kidneys of the SCID mice were lower than the titers of the wild-type Smith strain and the rescued virus by 50,000-, 100-, 10-, 100-, and 50-fold, respectively. Moreover, the growth of RvM35 is severely attenuated in the salivary glands. The virulence of the mutant virus also appears to be attenuated, because no death was observed in SCID mice infected with RvM35 until 35 days postinfection, while all the animals infected with the wild-type and rescued viruses died 27 days postinfection. Our results suggest that M35 is important for MCMV virulence in killing SCID mice and is required for optimal viral growth in vivo, including in the salivary glands.
Insights
Murine cytomegalovirus (MCMV) open reading frame M35 is not essential for in vitro replication but is crucial for viral virulence and growth in SCID mice.
Area of Science:
- Virology
- Immunology
- Genetics
Background:
- Murine cytomegalovirus (MCMV) is a model pathogen for studying herpesvirus biology.
- Random transposon mutagenesis is a tool for identifying viral genes essential for replication and virulence.
Purpose of the Study:
- To characterize the role of MCMV open reading frame M35 in viral replication and virulence.
- To investigate the in vitro and in vivo effects of a transposon insertion in M35.
Main Methods:
- Construction and characterization of a murine cytomegalovirus (MCMV) mutant (RvM35) with a transposon insertion in open reading frame M35.
- In vitro replication assays in NIH 3T3 cells.
- In vivo studies in immunocompetent Balb/c and immunodeficient SCID mice, assessing viral titers and survival rates.
Main Results:
- MCMV M35 is not essential for viral replication in vitro.
- The RvM35 mutant showed significantly attenuated viral growth in multiple organs (salivary glands, lungs, spleen, liver, kidneys) of SCID mice compared to wild-type and rescued viruses.
- RvM35 exhibited reduced virulence, with delayed mortality in SCID mice.
Conclusions:
- MCMV open reading frame M35 is important for viral virulence in SCID mice.
- M35 is required for optimal MCMV growth in vivo, particularly in the salivary glands.
