Quantitative analysis of Kaposi sarcoma-associated herpesvirus (KSHV) in KSHV-associated diseases

Background. Accurate numbers of copies of Kaposi sarcoma-associated herpesvirus ( KSHV) and numbers of virus-infected cells in lesions caused by KSHV-associated diseases are unknown. Methods. Quantitative polymerase chain reaction (PCR) and computerized imaging of immunohistochemical analysis were performed on pathologic sections of samples from persons with KSHV-associated diseases. Results. Real-time PCR and semiquantitative PCR-Southern blotting demonstrated that DNA extracted from biopsy samples of KS lesions contained similar to 1-2 viral copies/cell. KSHV-associated lymphoma contained 10-50 viral copies/cell. Computerized-image analysis demonstrated that similar to 49% of cells expressed KSHV-encoded latency-associated nuclear antigen in KS biopsy samples. On the basis of results of real-time PCR and computerized-image analysis, the predicted number of viral copies was 3.2 viral copies/cell in KS lesions. Computerized-image analysis also revealed that the expression of open-reading frame (ORF)-50 protein, an immediate early protein of KSHV, was very rare in KS lesions, which implies that they were mainly composed of proliferating cells latently infected with KSHV. In multicentric Castleman disease lesions, 25% of virus-infected cells expressed ORF50 protein, which suggests the frequent lytic replication of KSHV. Conclusions. Numbers of viral copies and of virus-positive cells vary among KSHV-associated diseases, which suggests different mechanisms of viral pathogenesis. The combination of real-time PCR and computerized-image analysis provides a useful tool for the assessment of the number of viral copies in KSHV-associated diseases.