B virus (cercopithecine herpesvirus 1) is the only deadly alphaherpesvirus that is zoonotically transmissible from macaques to humans. The detection of humoral immune responses is the method of choice for the rapid identification of B virus-infected animals. We evaluated the diagnostic potential of recombinant B virus glycoproteins for the detection of immunoglobulin G (IgG) antibodies in monkey and human sera. Glycoproteins B, C, and E and secreted (sgG) and membrane-associated (mgG) segments of glycoprotein G (gG) were expressed in the baculovirus expression system, while gD was expressed in CHO cells. We developed recombinant protein-based IgG enzyme-linked immunosorbent assays (ELISAs) and compared their diagnostic efficacies by using B virus antibody-negative (n ؍ 40) and -positive (n ؍ 75) macaque sera identified by a whole antigenbased ELISA and Western blotting. The diagnostic sensitivities of the gB-, gC-, gD-, and mgG-ELISAs were 100, 97.3, 88.0, and 80.0%, respectively. The specificities of the gB-, gC-, and gD-ELISAs and of the mgG-ELISA were 100 and 97.5%, respectively. In contrast, the sensitivities and specificities of sgG-and gE-ELISAs were low, suggesting that sgG and gE are less effective diagnostic antigens. Sera from nonmacaque monkeys crossreacted with gB, gC, and gD, and only baboon sera reacted weakly with mgG. Human herpes simplex virus type 1 (HSV-1)-and HSV-2-positive sera pools reacted with gB and gD, whereas sera from B virus-infected individuals reacted with all four antigens. These data indicate that gB, gC, gD, and mgG have a high diagnostic potential for B virus serodiagnosis in macaques, whereas mgG may be a valuable antigen for discrimination between antibodies induced by B virus and those induced by other, closely related alphaherpesviruses, including HSV-1 and -2.Human infection with B virus (also called cercopithecine herpesvirus 1, monkey B virus, and herpes B virus) is the most feared occupational hazard among individuals working with macaque monkeys, since fatality is often the outcome of infection, which proceeds in the absence of effective antiviral therapy (25,56). The use of macaques in research has been steadily growing over the last decade and is expected to rise quickly in the near future due to the increasing demands for these animals for use in HIV/AIDS investigations, vaccine trials, drug testing, and research into bioterrorism agents. As macaque usage increases, frequencies of human exposures to B virus are increasing as well. Rapid and accurate diagnostic tests are urgently needed to aid in the early identification of clinical cases, which is essential for a timely initiation of antiviral therapies in zoonotically infected humans. In addition to human diagnostics, enhanced assays are required for monitoring specific-pathogen-free (SPF) macaque colonies established by the National Institutes of Health for the breeding of B virusfree animals (55), as these animals often demonstrate only very low levels of antibody.Unfortunately, a direct diagnosis of infectio...