Seroprevalence Of Kaposi's Sarcoma Associated Herpesvirus (KSHV) In Hubei Province And Development Of A High-throughput Assay Targeting KSHV Latent Infection

Kaposi's sarcoma associated herpesvirus (KSHV) or Human herpesvirus 8(HHV8) is a kind of Human herpesvirus recently identified in Kaposi's sarcoma(KS) tissue. It is the pathogen of KS, Primary effusion lymphoma (PEL) andMulticentre cattleman's disease (MCD). As KS is the most common sarcoma inAIDS and in transplantation recipients, KSHV attracted more and more attention.Researches on KSHV prevalence in China were limited in XinJiang provincewhere KSHV has high seroprevalence. Little was known about prevalence of KSHVin other provinces. DNA extracted from BCBL-1 cells by Hirt assay was used astemplate for PCR to obtain off65 and orf73 genes of KSHV. These two genes werecloned into bacterial expression vector fused with His tag. ORF65 protein andC-terminus of ORF73 protein expressed in E coli were purified by Ni affinitychromatography and electroelution respectively. Using these purified proteins weoptimized ORF65-ELISA, ORF73-ELISA, ORF65-Western blot, and BCBL-1 IFAthat were used in serum screening. We tested 396 serum samples collected in Hubeiprovince from 2007 January to 2007 May from middle school students. The overallseroprevalence was 4.8%. No significant difference was observed between male andfemale.Also C-terminal of ORF73 protein was used as an antigen to immune rabbit,serum was collected, aliquoted and stored at -70℃at the sixth week until used. Itwas designated as LANA-C/ORF73-C mμlticlonal antibody. The antibody could react with C-terminal of ORF73 protein expressed in E.coli and full lenghthORF73/LANA existing in BCBL-1 cells. Typical nuclear nodular fluorescence coμldbe observed when LANA-C/ORF73-C multiclonal antibody reacted with BCBL-1 inIFA.Like other herpesviruses, the life cycle of KSHV also contains lyrical andlatent replication. KSHV latent infection was associted with pathology and could notbe cured with drμgs against KSHV DNA polymerase. Here we revealed thepossibility of LANA/ORF73 as a potential target in anti-KSHV latent replicationcompounds screening. LANA binding site (LBS) was synthized and 64 tandemLBS repeats were inserted into pGL3-basic vector without luciferase gene (pGL-64LBS). The binding between LBS and C-terminal of ORF73 protein was confirmedby EMSA. When BCBL-1 cells were transfected with recombinant plasmid pGL-64LBS, the input LBS competed with episomes for LANA to interfere LANA function.When we extracted LMW DNA from BCBL-1 cells tranfected with mμlti-LBS, wefound the copies of episomes were decreasing since 7 days after transfection. Basedon these results, we developed a high-throughput assay for screening anti-KSHVcompounds interfering binding between LANA and episomes. Ninety-six-well platewas coated with purified LANA-C/ORF73-C and biotin-LBS was then addedtogether with candidate compounds. The effect of compounds on DNA-proteinbinding was revealed by streptavidin-HRP and OPD-H₂O₂ system..