Charting Latency Transcripts in Kaposi's Sarcoma-Associated Herpesvirus by Whole-Genome Real-Time Quantitative PCR

Department of Microbiology and Immunology, The University of Oklahoma Health Science Center, Oklahoma City, Oklahoma 73104, USA.
Journal of Virology (Impact Factor: 4.44). 07/2002; 76(12):6213-23. DOI: 10.1128/JVI.76.12.6213-6223.2002
Source: PubMed


The division into a latent or lytic life cycle is fundamental to all herpesviridae. In the case of Kaposi's sarcoma-associated
herpesvirus (KSHV) (human herpesvirus 8), latent genes have been implicated in cell autonomous transformation, while certain
lytic genes procure a tumor friendly milieu through paracrine mechanism. To query KSHV transcription, we devised and validated
a high-throughput, high-specificity, high-sensitivity, real-time quantitative reverse transcription-PCR array. This novel
methodology is applicable to many human pathogens. Its first use demonstrated that the mRNA levels for KSHV LANA, v-cyclin,
and v-FLIP do not increase at any time after viral reactivation. The mRNA for LANA-2/vIRF-3 is similarly resistant to viral
reactivation. In contrast, every other latent or lytic message was induced. Hence, LANA, v-FLIP, v-cyclin, and LANA-2 constitute
a group of uniquely regulated transcripts in the KSHV genome.

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    • "This platform is flexible such that individual QPCR assays can easily be substituted according to the needs of a particular experiment and expanded to a 96 Â 96 format if screening for additional transcripts is required in the future. While PCR-based array profiling of KSHV and EBV transcripts in primary Kaposi sarcoma lesions and virus-positive cell lines has been reported previously (Dittmer, 2003; Fakhari and Dittmer, 2002; Kurokawa et al., 2005; Wang et al., 2009; Whitehurst et al., 2013), the present work provides two novel features. First, the Fluidigm system requires very little starting material (less than 25 ng RNA) making it suitable for analyzing small clinical samples. "
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    • "Persistent HHV8 infection is characterized by both latency and lytic replication, and diseases associated with HHV8 infection appear to be the result of both long-term latent infection and viral reactivation. Latent infection is associated with the absence of virus production and the expression of a limited number of viral genes, many of which provide a survival advantage (Dittmer et al., 1998; Ensoli et al., 2001; Fakhari and Dittmer, 2002). Lytic replication is characterized by the timely expression of immediate early, early, and late genes resulting in the release of mature virions. "
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    • "Moreover, this dual high-throughput technology also can collect network data from the experiments and validate the network model. For instance, Fakhari and Dittmer created the polymerase chain reaction (PCR) chip technology to detect the gene expression [30]. The results demonstrated that the technology was convenient for the high-throughput studies. "
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