New immunofluorescence assays for detection of Human herpesvirus 8-specific antibodies.
Several assays have been developed for detection of immunoglobulin G antibodies to Human herpesvirus 8 (HHV-8), including immunofluorescence assays (IFAs) and enzyme-linked immunosorbent assays (ELISAs). However, the specificity and sensitivity of these assays are not completely defined due to the lack of a "gold standard." Although IFAs based on primary effusion lymphoma (PEL) cell lines are used widely, the assays can be confounded by nonspecific reactions against cellular components and potential cross-reaction with antibodies 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 proteins 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 measured by rSFV-based IFAs and PEL-based IFAs correlated well (correlation coefficients 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 than either ORF73-IFA or peptide ELISAs. Using PEL-based lytic IFA as a reference 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 among 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 rSFV-based IFAs may provide a specific and sensitive method for use in epidemiology studies. In addition, they will provide a basis for further development of diagnostic tests for HHV-8 infection.
Inoue
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Division of Viral and Rickettsial Diseases, STD, and TB Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA. nai0@cdc.gov
Mar
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Dollard
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Pau
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Zheng
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Pellett
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PE
D000818:Animals; D000914:Antibodies, Viral / Q000032:analysis; D002460:Cell Line; D006224:Cricetinae; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000592:standards; D000939:Epitopes / Q000276:immunology / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015967:Gene Expression Regulation, Viral; D006023:Glycoproteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D006031:Glycosylation; D006566:Herpesviridae Infections / Q000175:diagnosis* / Q000453:epidemiology / Q000276:immunology; D019288:Herpesvirus 8, Human / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D007668:Kidney / Q000166:cytology; D008967:Molecular Biology / Q000379:methods / Q000592:standards; D009687:Nuclear Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D010750:Phosphoproteins; D011817:Rabbits; D012672:Semliki forest virus; D012680:Sensitivity and Specificity; D016036:Seroepidemiologic Studies; D014764:Viral Proteins
D000914:Antibodies, Viral; D000939:Epitopes; D006023:Glycoproteins; C113454:K8.1 protein, Human herpesvirus 8; D009687:Nuclear Proteins; D010750:Phosphoproteins; D014764:Viral Proteins; C118485:latent nuclear antigen (LNA)