Rhesus monkey rhadinovirus (RRV): construction of a RRV-GFP recombinant virus and development of assays to assess viral replication

Abstract

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.

Disruption of LANA in Rhesus Rhadinovirus Generates a Highly Lytic Recombinant Virus

“…We previously reported the kinetics of gene expression with regard to immediate-early, early, and late genes during RRV lytic infection and have reported that they are similar to those seen during KSHV reactivation (17,25). We also generated a RRV-green fluorescent protein (GFP) recombinant virus and devised plaque assays and real-time quantitative PCR (QPCR) assays for the determination of virus titers (18). This validates the experimental methods used here.…”

“…Our lab has previously published the generation of an RRV recombinant virus expressing GFP (RRV-GFP) using homologous recombination (18). We used a similar homologous recombination approach to construct a RRV⌬LANA/GFP recombinant virus by inserting a GFP expression cassette into the R-LANA ORF to disrupt R-LANA transcription and translation.…”

Tumor-targeted gene transfer in vivo via recombinant Newcastle disease virus modified by a bispecific fusion protein

“…Despite the fact that our bispecific fusion protein ·HN-IL-2 was constructed from the human IL-2 sequence, FACS analysis Several studies (20,25,26) employed real-time PCR to quantify EGFP or GFP transgene expression. To quantify EGFP expression in vivo, we also applied RRT-PCR in this study.…”

Molecular Imaging of Pulmonary Disease In Vivo

“…Nonetheless, a variety of microbes have been transformed with GFP variants and have shown signifi- cant promise for the study of pathogenesis (70). Thus, the latest generation of GFP reporters allows the investigation of gene expression in individual bacterial cells within specific environments (70)(71)(72)(73)(74).…”