| The 1994 discovery of Kaposi Sarcoma-associated herpesvirus (KSHV) was an important finding, especially as the sharp rise in HIV infections in the early 1990's led to an explosive Kaposi Sarcoma (KS) epidemic in men who have sex with men. This cancer-causing herpesvirus is the causative agent of three diseases in addition to KS: Multicentric Castlemen Disease (MCD), Primary Effusion Lymphoma (PEL), and the newly described condition KSHV Inflammatory Cytokine Syndrome (KICS). By understanding viral gene expression in human patients, we hope to better understand KSHV infection and its diseases, with the hope to shed light on novel therapeutic techniques.;In order to quantify KSHV viral gene expression, I devised methods to profile human clinical specimens using high-throughput qPCR. This technique was used to profile microRNA expression and was published as a methods video journal in the Journal of Visualized Experiments (Chugh, Tamburro, and Dittmer, 2010). The accompanying manuscript is found in the Appendix chapter. This same high-throughput technique was employed in Chapter II to determine KSHV viral load and to determine expression of viral kinases, and in Chapter III to determine KSHV gene expression profiles.;In Chapter II, we hypothesized that HIV patients with newly diagnosed KS in Malawi could potentially benefit from treatment with the antiherpesviral drug Ganciclovir (GCV). This was addressed through a pilot study with the AIDS Malignancy Consortium (AMC). By using qPCR to assess gene expression of two viral kinases critical to drug responsiveness, we found evidence that 78% of patients express a viral kinase and would potentially benefit from this therapy. This finding is important, as it indicates that patients can be stratified for treatment based on kinase expression in a tumor biopsy, which will maximize potential effectiveness and limit waste of drugs in a resource limited setting. This work will lead to upcoming treatment studies in this population.;Next, we used whole-genome KSHV transcriptome profiling to understand whether all KS patients from the Malawian cohort express similar gene expression profiles or if subgroups can be distinguished. Our results indicate the gene expression profiles vary significantly between patients. Specifically, two subpopulations were discovered- those with and those without high levels of genome-wide transcription. Patients with high transcription levels demonstrated significantly higher CD4 counts and KSHV viremia in plasma, although HIV viremia, number of tumors, or size of biopsied lesion were not significantly associated with KSHV transcription. This work is described in Chapter III.;In Chapter IV, we employed high-throughput DNA sequencing as a method to diagnose co-infections in an end-stage AIDS patient. This was published in the Journal Virology (Tamburro et al. 2012). The patient had minimal KS skin lesions despite having one of the highest viral loads on record- leading to the diagnosis of the newly described condition KSHV Inflammatory Cytokine Syndrome (KICS). Additionally, sequencing uncovered a co-infection with HHV6a, which may have accounted for Systemic Inflammatory Response Syndrome (SIRS) that ultimately led to the patient's death. Notable to the research community, this work resulted in the first complete sequence of KSHV using direct methods, rather than relying on pre-sequencing cloning or cell passage methods. |
