Rhesus monkey rhadinovirus (RRV): construction of a RRV-GFP recombinant virus and development of assays to assess viral replication.
Rhesus monkey rhadinovirus (RRV) is a gamma-2-herpesvirus that is closely related to Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8). Lack of an efficient culture system to grow high titers of virus, and the lack of an in vivo animal model system, has hampered the study of KSHV replication and pathogenesis. RRV is capable of replicating to high titers on fibroblasts, thus facilitating the construction of recombinant rhadinoviruses. In addition, the ability to experimentally infect naïve rhesus macaques with RRV makes it an excellent model system to study gamma-herpesvirus replication. Our study describes, for the first time, the construction of a GFP-expressing RRV recombinant virus using a traditional homologous recombination strategy. We have also developed two new methods for determining viral titers of RRV including a traditional viral plaque assay and a quantitative real-time PCR assay. We have compared the replication of wild-type RRV with that of the RRV-GFP recombinant virus in one-step growth curves. We have also measured the sensitivity of RRV to a small panel of antiviral drugs. The development of both the recombination strategy and the viral quantitation assays for RRV will lay the foundation for future studies to evaluate the contribution of individual genes to viral replication both in vitro and in vivo.
DeWire
Scott M
SM
Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Money
Eric S
ES
Krall
Stuart P
SP
Damania
Blossom
B
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D004274:DNA, Recombinant / Q000235:genetics*; D005814:Genes, Viral; D005818:Genetic Engineering; D016679:Genome, Viral; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008253:Macaca mulatta / Q000821:virology*; D018144:Rhadinovirus / Q000187:drug effects / Q000235:genetics* / Q000502:physiology*; D010948:Viral Plaque Assay; D014779:Virus Replication / Q000187:drug effects
D000998:Antiviral Agents; D004274:DNA, Recombinant; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins