New immunofluorescence assays for detection of human herpesvirus 8-specific antibodies

Several assays have been developed for detection of immunoglobulin G antibo dies to Human herpesvirus 8 (HHV-8), including immunofluorescence assays (I FAs) and enzyme-linked immunosorbent assays (ELISAs), However, the specific ity and sensitivity of these assays are not completely defined due to the l ack of a "gold standard." Although IFAs based on primary effusion lymphoma (PEL) cell lines are used widely, the assays can be confounded by nonspecif ic reactions against cellular components and potential cross-reaction with anti bodies against other herpesviruses. To provide more reliable IFAs, we established recombinant Semliki Forest viruses (rSFVs) expressing the HHV-8 -specific proteins ORF73 and K8.1 and used BHK-21 cells infected with these rSFVs For IFA (ORF73-IFA and K8.1-IFA), Expression of the HHV-8-specific p roteins at very high levels by the rSFV system allowed easy scoring for IFA and thereby increased specificity, The rSFV system also allowed detection of antibodies against glycosylation-dependent epitopes of K8.1. Titers meas ured by rSFV-based IFAs and PEL-based IFAs correlated well (correlation coe fficients of >0.9), and concordances of seroreactivities between rSFV-based and PEL-based IFAs were >97% (kappa > 0.93). K8.1-IFA was more sensitive t han either ORF73-IFA or peptide ELISAs. Using PEL-based lytic IFA as a refe rence assay, the sensitivity and specificity of K8.1-IFA were estimated to be 94 and 100%, respectively. HHV-8 prevalences determined by K8.1-IFA amon g the human immunodeficiency virus (HIV)-positive (HIV+) Kaposi's sarcoma ( KS) patients, HIV+ KS- patients, and healthy controls were 100, 65, and 6.7 %, respectively, which were consistent with prior reports. Therefore, our r SFV based IFAs may provide a specific and sensitive method for use in epide miology studies. in addition, they will provide a basis for further develop ment of diagnostic tests for HHV-8 infection.