LONG-TERM SURVEILLANCE OF LANGUR ALPHAHERPESVIRUS IN A ZOO POPULATION OF SILVERED LANGURS ( TRACHYPITHECUS CRISTATUS).

Langur alphaherpesvirus (HVL), a provisionally named alphaherpesvirus in the Simplexvirus genus, was first identified in 1991 at the Bronx Zoo in wild-origin silvered langurs ( Trachypithecus cristatus) and their descendants. HVL is closely related to B virus ( Macacine alphaherpesvirus 1) based on serologic and genetic data, but its natural history and zoonotic potential remain unknown. A cohort study was undertaken to describe the epidemiology, clinical impact, and potential management implications of this virus in a naturally infected, zoo-based population of silvered langurs. Opportunistic surveillance sampling from 1991 through 2015 resulted in 235 serum samples and 225 mucosal swabs from 75 individuals. A total of 43 individuals (57.3%) were seropositive for HVL within this period. Seroprevalence increased significantly with age, and indirect evidence suggested a peak in transmission at the onset of sexual maturity. These findings were similar to the behavior of other simplexviruses in their adapted hosts. Yearly cumulative incidence declined significantly through the study period, with zero or one new case detected each year from 2007 through 2015. The density of this population decreased within the study period for management reasons unrelated to HVL infection, and a change in age distribution or less-frequent contacts may have contributed to low transmission. In addition, clinical signs of simplexvirus infection were rare, and virus isolation was negative on all mucosal swabs, suggesting that viral shedding was infrequent. Yearly period seroprevalence remained relatively constant with a median of 45.8%, likely because of the extended survival of infected individuals within the population. Maintenance of a naturally occurring, novel virus with unknown zoonotic potential in a zoo population for over 25 yr highlights the importance of biosecurity and biosafety for management of silvered langurs and all primate species.

Identification of unique B virus (Macacine Herpesvirus 1) epitopes of zoonotic and macaque isolates using monoclonal antibodies.

Our overall aim is to develop epitope-based assays for accurate differential diagnosis of B virus zoonotic infections in humans. Antibodies to cross-reacting epitopes on human-simplexviruses continue to confound the interpretation of current assays where abundant antibodies exist from previous infections with HSV types 1 and 2. To find B virus-specific epitopes we cloned ten monoclonal antibodies (mAbs) from the hybridomas we produced. Our unique collection of rare human sera from symptomatic and asymptomatic patients infected with B virus was key to the evaluation and identification of the mAbs as reagents in competition ELISAs (mAb-CE). The analysis of the ten mAbs revealed that the target proteins for six mAbs was glycoprotein B of which two are reactive to simian simplexviruses and not to human simplexviruses. Two mAbs reacted specifically with B virus glycoprotein D, and two other mAbs were specific to VP13/14 and gE-gI complex respectively. The mAbs specific to VP13/14 and gE-gI are strain specific reacting with B virus isolates from rhesus and Japanese macaques and not with isolates from cynomolgus and pigtail macaques. The mAb-CE revealed that a high proportion of naturally B virus infected rhesus macaques and two symptomatic humans possess antibodies to epitopes of VP13/14 protein and on the gE-gI complex. The majority of sera from B virus infected macaques and simplexvirus-infected humans competed with the less specific mAbs. These experiments produced a novel panel of mAbs that enabled B virus strain identification and confirmation of B virus infected macaques by the mAb-CE. For human sera the mAb-CE could be used only for selected cases due to the selective B virus strain-specificity of the mAbs against VP13/14 and gE/gI. To fully accomplish our aim to provide reagents for unequivocal differential diagnosis of zoonotic B virus infections, additional mAbs with a broader range of specificities is critical.

Reassessing the detection of B-virus-specific serum antibodies.

B virus, a natural pathogen of macaques, can cause a fatal zoonotic disease in humans. Serologic screening of macaques by titration ELISA (tELISA, screening test) and by Western blot analysis (WBA, confirmatory test) is one of the principle measures to prevent human infection. Here we slightly modified these 2 tests and reevaluated their correlation. We developed a high-throughput tELISA and used it to screen 278 sera simultaneously against the homologous BV antigen and the heterologous antigens of Papiine herpesvirus 2 and Human herpesvirus 1. More sera (35.6%) were positive by the BV-ELISA than by the HVP2-ELISA (21.6%) or HSV1-ELISA (19.8%). The superiority of the homologous tELISA over the heterologous tELISA was prominent in low-titer sera. WBA confirmed only 21% of the tELISA-positive sera with 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.

Production of herpes B virus recombinant glycoproteins and evaluation of their diagnostic potential.

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 antigen-based 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 cross-reacted 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.

Quantitative real-time PCR for detection of monkey B virus (Cercopithecine herpesvirus 1) in clinical samples.

A TaqMan based real-time PCR assay was developed for rapid detection and quantitation of herpes B virus (Cercopithecine herpesvirus 1) in clinical samples. The assay utilizes B virus-specific primers and a probe to the non-conserved region of the gG gene to discriminate B virus from closely related alphaherpesviruses. Fifty copies of B virus DNA could be detected with 100% sensitivity with a wide range of quantitation spanning 6 logs. The assay was highly reproducible with intra- and inter-assay coefficients of variation of 0.6 and 2.4%, respectively. Clinical utility of the developed real-time PCR was evaluated by testing genomic DNA prepared from B virus clinical isolates (n=23) and human and monkey clinical specimens (n=62). This novel method was also compared with conventional cell culture with respect to sensitivity and specificity. TaqMan PCR assay was shown to be equally specific and more sensitive than culture method (culture vs. PCR sensitivity 50%) and was able to identify all B virus clinical isolates tested. Fast, reliable assessment of B virus DNA in infected cells and tissues makes real-time PCR assay a valuable tool for diagnosis and management of B virus infections.