B virus, a natural pathogen of macaques, can cause a fatal zoonotic disease in humans. low or intermediate antibody titers. These sera might have contained antibodies to conformational epitopes that could not be detected by WBA, in which denatured antigens are used, but that could be detected by tELISA, which detects both linear and conformational epitopes. WBA confirmed 82% of the tELISA high-titer sera. However, WBA defined the remaining 18% of sera, which were negative by tELISA, as nonnegative. This finding can be attributed to the difficulties encountered with the subjective interpretation of results by WBA. Together, the current results indicate the inadequacy of WBA as a confirmatory assay for sera with low antibody titers. 1); CLIA, Clinical Laboratory Improvement Amendments; EU, ELISA units; HSV, 1), which is endemic in all species of macaques (natural hosts), is a member of the genus and family Alphaherpesviruses are characterized by the ability to establish a neurotropic, generally asymptomatic, infection in their natural hosts. Macaques spread BV within a group by contact with macaques that are shedding virus during an acute or intermittently reactivated infection. BV is closely related to 2 well-characterized human alphaherpesviruses, (HSV) types 1 and 2, to simian agent 8 (SA8; 2), which is endemic in African green monkeys (2) in baboons (spp.) and langur herpesvirus (HVL), which is endemic in langur monkeys (spp.)5,6,7 and which has not been officially classified or named.9 Recently, sera from a group of CREBBP sooty mangabey monkeys ((HVM) and is pending taxonomic evaluation. Each of the simplex viruses has remarkable host specificity in nature. However, cross-species infections with BV have been reported. BV is the only nonhuman primate alphaherpesvirus that infects humans. When it does so, BV causes an often-fatal zoonotic disease in 80% of untreated humans.7,10,21,23,32,33 BV is transmitted through bites, scratches, or contact BIIB021 with infected oral or genital body fluids. In addition, the virus can be transmitted via fomites and from human to human through contact with contaminated wounds. Virus replication occurs in epithelial or fibroblast cells at the epidermal or dermal site of virus entry; however, BV also enters the peripheral nervous system via axons without replicating locally in surrounding epithelial cells, as has been reported for other simplex viruses.20,23 Once BV enters peripheral nerves, life-long latency is established in the dorsal root of spinal ganglia or cranial ganglia of infected hosts. BV undergoes periodic reactivation in macaques as well as in humans that survive this zoonosis. In both macaques and humans, BV can be reactivated in the ganglia, generally resulting in anterograde travel of the virus and replication at the original site of infection.10,33 This event results in virus shedding from infected BIIB021 cells, an event that can be detected by PCR or virus isolation if samples are collected during this event. However, because virus shedding is unpredictable and sporadic, identification of BV infection by means of PCR or virus isolation is rare. A more practical approach to identifying infected macaques or humans is the use of serologic methods for identifying antibodies specific for BV, although the shortcomings of this approach are appreciated when screening sera from subjects that are in the midst of a primary infection but have not yet produced detectable levels of antibodies or from BV-infected subjects that lack detectable antibody because of waning levels or anergy. Because of the high lethality of BV to humans and life-long infection in survivors that lack effective strategies to clear this virus, BV antigen is produced under BSL4 conditions according to federal guidelines and under strict biosecurity regulations.3 Many laboratories in the United States, Europe, and Asia cannot produce BV antigens because of these restrictions and therefore use BIIB021 alternative crossreacting (heterologous) herpesvirus antigens such as HVP2 and HSV1 for the detection of antibodies to BV.10,19,26,29,34 Our previous studies16 indicated that using heterologous viruses in serologic assays for detecting BV antibodies contributes to increased false-negative results. Serologic diagnosis of BV infection in macaques at the National B Virus Resource Laboratory has been based on 2 principal tests that meet standards proscribed by the Clinical BIIB021 Laboratory Improvement Amendments:4 the titration ELISA (tELISA) and Western blot analysis (WBA).15,31 tELISA detects antibody in sera from most BV-infected macaques by using the complex mixture of BV antigens that is present in lysates from infected cells and adsorbed onto polystyrene microtiter plates. These infected-cell lysates are prepared by using nondenaturing detergents. Quality-control assessment of each antigen lot.