Validation of an enzyme-linked immunosorbent assay kit using herpesvirus papio 2 (HVP2) antigen for detection of herpesvirus simiae (B virus) infection in rhesus monkeys

Serologic testing for antibody to monkey B virus (BV) in macaque sera is problematic due to the biohazardous nature of BV antigens. Herpesvirus papio 2 (HVP2), a herpesvirus of baboons, is nonpathogenic to humans and is genetically and antigenically more closely related to BV than is human herpes simplex virus 1. This paper describes the results of our in-house laboratory that compared a BV antigen-based enzyme-linked immunosorbent assay (ELISA) by commercial testing laboratory and an HVP2-based ELISA in our laboratory by using 447 sera from 290 rhesus monkeys. The HVP2-based ELISA identified as positive 99.11% of the sera identified as BV-positive by the BV ELISA. The BV antigen-based ELISA identified as positive 98.21% of the sera identified as BV-positive by the HVP2-based ELISA. The HVP2 ELISA also identified two BV-negative and six BV-equivocal sera as positive. Both ELISAs identified the same 85 negative and three equivocal samples as negative and equivocal, respectively. The high degree of correlation (weighted kappa coefficient, 0.94) between the two tests indicates that the HVP2 ELISA is a sensitive and reliable assay for in-house testing of the BV status of rhesus monkeys.     

Detection of B virus infection in cynomolgus monkeys by ELISA using simian agent 8 as alternative antigen.

The use of simian agent 8 (SA8) as an antigen for B virus (BV) antibody detection was evaluated in cynomolgus monkeys. Seventy-two sera judged as positive using BV antigen were all positive when the SA8 antigen was used. Out of 28 BV-negative sera 2 were positive against the SA8 antigen and one serum was classified as indeterminate. The present data indicates that detection of BV antibody can be achieved accurately and safely by enzyme-linked immunosorbent assay (ELISA) using SA8 antigen.     

Papiine herpesvirus 2 as a Predictive Model for Drug Sensitivity of Macacine herpesvirus 1 (Monkey B Virus)

Monkey B virus (Macacine herpesvirus 1; BV) is endemic in macaques. BV (a BSL4 agent) is the primary zoonotic concern for persons working with macaques in research, and human BV infections frequently are fatal. We assessed the use of a BSL2 baboon herpesvirus (Papiine herpesvirus 1; HVP2) for predicting the drug sensitivity of BV by comparing the sensitivity of the 2 viruses to 12 antiherpetic drugs. Plaque reduction assays showed that 4 drugs (HBPG, BVdU, PFA, and BrdU) were ineffective against both viruses. Of the 8 effective drugs, both viruses were most sensitive to TFT, whereas sensitivity to the remaining 7 drugs varied between BV and HVP2 as well as between strains of HVP2. In addition, the efficacy of 5 drugs (ACV, PCV, GCV, CDV, and EDU) was tested by using a murine model. ACV and EDU were completely ineffective against both HVP2 and BV, and high doses of PCV only delayed death by a few days. GCV and CDV both protected mice against death, and CDV also prevented the development of neurologic symptoms. When the initiation of drug therapy was delayed until after virus gained access to the CNS, both GCV and CDV were ineffective. The similarity of the drug sensitivities of HVP2 and BV in both models validates the use of HVP2 as a BSL2 level model that can be used to predict drug sensitivity of BV. The greater efficacy of CDV relative to GCV suggests the potential for use of CDV in the treatment of zoonotic BV infections.Abbreviations: ACV, acyclovir; AraA, 9-β-D-arabinofuranosyl-adenine; BrdU, 5-bromo-2′-deoxyuridine; BV, monkey B virus; BVdU, (E)-5-(2-bromovinyl)-2′-deoxyuridine; CDV, cidofovir; EDU, 5-ethyl-2′-deoxyuridine; GCV, ganciclovir; HBPG, 9-(4-hydroxybutyl)-N²-phenylguanine; HSV, herpes simplex virus; HVP2, Herpesvirus papio 2; IUdR, 5-iodo-2’-deoxyuridine; PCV, penciclovir; PFA, phosphonoformic acid; TFT, trifluorothymidineMacacine herpesvirus 1 (monkey B virus; BV) is an alpha-herpesvirus of macaque monkeys and is closely related to human herpes simplex virus (HSV) types 1 and 2.^8,11,27 Although BV primarily causes asymptomatic or mild, self-limiting disease in healthy macaques, the virus is extremely neurovirulent when transmitted via bites or scratches to other nonmacaque primate species, including humans. Although human infections are not common, approximately 80% of untreated patients die of BV infection, and survivors frequently continue to suffer from neurologic sequelae. As a consequence of its lethality in humans, BV is classified as a BSL4 pathogen³ and is the single most serious zoonotic concern for veterinary and research personnel who work with macaques. The increasing popularity of ecotourism to monkey temples in Southeast Asia, where tourists and wild, BV-infected macaque populations come into direct contact, represents another potential concern for zoonotic BV infections.^9,12,13,21The antiviral drugs recommended for use in treating BV infections all were originally developed for treatment of HSV infections.^4,18 Because the genes encoding the enzymes targeted by these drugs are conserved between these viruses, BV is sensitive to many of these anti-HSV drugs. However, compared with HSV, BV is less sensitive to these drugs.^2,10,14 Although more effective drugs are needed for the treatment of BV infections, the biohazardous nature of and facility requirements associated with studying a BSL4 agent severely limit research on BV. A potential solution to this problem is using a closely related virus whose biologic and molecular properties are very similar to those of BV as a surrogate or model system in which preliminary research can be conducted safely, leaving only confirmative testing to be done with infectious BV.Baboons carry an alpha-herpesvirus (Papiine herpesvirus 2; HVP2) that is biologically and genetically very similar to BV and HSV.^7,8,15,26 In mice, most HVP2 isolates are extremely neurovirulent and closely reflect the pathogenesis of BV in mice^20,23 At the antigenic level, HVP2 and BV are so similar that HVP2 has found use as an alternative antigen for diagnostic BV serology.^17,25,28 Despite the virus''s similarity to BV, HVP2 infections have never been reported in humans. Consequently, HVP2 is rated as a BSL2 pathogen and, as such, HVP2 can be used under BSL2/ABSL2 containment. This study was conducted to assess the potential use of HVP2 as a surrogate model system for predicting the sensitivity of BV to antiviral drugs.     

Changes in the titer of anti-B virus antibody in captive macaques (Macaca fuscata, M. mulatta, M. fascicularis)

Changes in levels of antibody to B virus (Cercopithecine herpesvirus 1; BV) were examined in BV-positive macaques by ELISA. We observed increases in anti-BV IgG titers in a BV-infected cynomolgus monkey after overseas transportation by air and in a rhesus monkey after transfer from an outdoor group cage to an indoor individual cage. Although shedding of infectious virus was not examined, the increase in antibody titer suggested reactivation of BV. Interestingly, we also found an increase in anti-BV IgG levels during the breeding season in male but not female Japanese macaques kept in an enclosed outdoor colony. Further studies should be performed to investigate whether reactivation of BV led to the observed increase in the anti-BV antibody titer.     

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.Abbreviations: BV, B virus (Macacine herpesvirus 1); CLIA, Clinical Laboratory Improvement Amendments; EU, ELISA units; HSV, Human herpesvirus; HVL, langur herpesvirus; HVM, Herpesvirus managabey; HVP, Papiine herpesvirus; SA8, simian agent 8; tELISA, titration ELISA; UN, uninfected; WBA, Western blot analysisB virus (BV; Macacine herpesvirus 1), which is endemic in all species of macaques (natural hosts), is a member of the genus Simplexvirus, subfamily Alphaherpesvirinae and family Herpesviridae. 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, Human herpesvirus (HSV) types 1 and 2, to simian agent 8 (SA8; Cercopithecine herpesvirus 2), which is endemic in African green monkeys (Cercopithecus aethiops), and to 2 recently identified simian alphaherpesviruses, HVP2 (Papiine herpesvirus 2) in baboons (Papio spp.) and langur herpesvirus (HVL), which is endemic in langur monkeys (Presbytis spp.)^5,6,7 and which has not been officially classified or named.⁹ Recently, sera from a group of sooty mangabey monkeys (Cercocebus atys) were found to crossreact serologically with other simian simplexviruses.^6,8,11 It was assumed that crossreactive antibodies were induced by a putative alphaherpesvirus that is endemic in mangabeys. This virus was provisionally named as Herpesvirus managabey (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 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 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 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 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 is performed, including determinations of protein concentration, antigen mass, immunoreactivity with macaque serum pools, and reactivity in WBA assays.WBA was chosen as the confirmatory test for the BV-screening tELISA; in addition, WBA serves as the primary screening assay for detecting antibodies to BV, HSV1, and HSV2 in human sera collected after exposure in the workplace.^27,31 The relationship between ELISA and WBA is complex, and therefore the agreement between the tests may be lower than expected given that WBA detects only conformationally independent epitopes, whereas ELISA identifies both linear and conformational epitopes (unless the epitopes are cryptic).²⁸ In addition, the different antibodies detected by each test might be induced at distinctly different time points after infection, so that what one assay detects, the other may not.^2,8,9 Despite the difficulties encountered and due to the lack of a better alternative, both assays are required to maximize the likelihood of correctly identifying whether BV antibodies are present in a sample.Because the identification of BV-infected macaques in any colony, especially SPF colonies, is of great medical and economic significance, assays should be designed to identify low levels of antibody as soon as possible after infection.^12,31 For BV, as for other alphaherpesviruses, the presence of serum antibodies likely indicates the presence of virus that is either in an active lytic state (and actively replicating) or in an inactive, latent state. It is therefore important to have both a sensitive assay, to prevent missing low-responders, and a highly specific assay, to avoid obtaining false-negative results that can lead to costly veterinary and medical decisions.In our continuing efforts to improve the accuracy of the tELISA for BV, we recently modified and automated it.¹⁸ This process included validation experiments that enabled us to reexamine some features of the BV serologic diagnosis.Here we describe additional modifications of the tELISA and its adaptation to an automated, high-throughput 384-well format. This format enabled us to assess the sensitivities of tELISA using homologous compared with heterologous antigens in the same run. In addition, we evaluated the correlation between tELISA and WBA and discuss the effectiveness of WBA as a confirmatory test for tELISA. All assays were developed and performed inhouse in accordance with CLIA regulations,⁴ because each test either was used or could be used for identification of BV antibodies in human sera as well as macaque sera. Although CLIA does not regulate veterinary assays, we feel strongly that the tests described are superior because of the strict quality control that must be maintained for testing human samples.     

Experimental infection of baboons (Papio cynocephalus anubis) with apathogenic and neurovirulent subtypes of herpesvirus papio 2

Cercopithecine herpesvirus 16 (Herpesvirus papio 2; HVP2) is an alpha-herpesvirus of baboons (Papio spp.) that generally causes minimal to inapparent disease in the natural host species. HVP2 is very closely related genetically and antigenically to Cercopithecine herpesvirus 1 (monkey B virus; BV) of macaques, which is well known for its extreme lethality in nonmacaque species including humans. Preliminary evidence suggests that a mouse model of HVP2 would be an excellent tool for studying zoonotic BV infections. Although the pathogenicity of different BV isolates in mice spans the full range of severity from apathogenic to extremely neurovirulent, testing of multiple HVP2 isolates revealed only two distinct phenotypes in mice regardless of route of inoculation: apathogenic (HVP2ap) and highly neurovirulent (HVP2nv). For the HVP2nv mouse model to truly reflect BV infection in both its natural host and the differential pathogenicity of BV in aberrant host species, HVP2nv should not produce severe disease in its natural host. To test this, juvenile baboons were inoculated with doses of 10(6) or 10(4) plaque-forming units of HVP2ap or HVP2nv by using an oral subdermal inoculation route. Parameters followed included the appearance of lesions, shedding of infectious virus, general health, and the immune response to the infection. Regardless of the inoculum dose used, no differences were noted between the two HVP2 subtypes in baboons in any of the parameters measured. These findings further support the use of the HVP2nv mouse system as a model to elucidate and study the viral determinants associated with cross-species BV neurovirulence.     

An Automated ELISA Using Recombinant Antigens for Serologic Diagnosis of B Virus Infections in Macaques

B virus (Macacine herpesvirus 1) occurs naturally in macaques and can cause lethal zoonotic infections in humans. Detection of B virus (BV) antibodies in macaques is essential for the development of SPF breeding colonies and for diagnosing infection in macaques that are involved in human exposures. Traditionally, BV infections are monitored for presence of antibodies by ELISA (a screening assay) and western blot analysis (WBA; a confirmatory test). Both tests use lysates of infected cells as antigens. Because WBA often fails to confirm the presence of low-titer serum antibodies detected by ELISA, we examined a recombinant-based ELISA as a potential alternative confirmatory test. We compared a high-throughput ELISA using 384-well plates for simultaneous antibody screening against 4 BV-related, recombinant proteins with the standard ELISA and WBA. The recombinant ELISA results confirmed more ELISA-positive sera than did WBA. The superiority of the recombinant ELISA over WBA was particularly prominent for sera with low (<500 ELISA units) antibody titers. Among low-titer sera, the relative sensitivity of the recombinant ELISA ranged from 36.7% to 45.0% as compared with 3.3% to 10.0% for WBA. In addition, the screening and confirmatory assays can be run simultaneously, providing results more rapidly. We conclude that the recombinant ELISA is an effective replacement for WBA as a confirmatory assay for the evaluation of macaque serum antibodies to BV.Abbreviations: BV, B virus (Macacine herpesvirus 1); EU, ELISA units; g, glycoprotein; HSV, herpes simplex virus; tELISA, titration ELISA; UN, uninfected; WBA, western blot analysisB virus (BV; Macacine herpesvirus 1) is a member of the genus Simplexvirus, subfamily Alphaherpesvirinae and family Herpesviridae. The virus occurs naturally in macaques (Macaca spp.) and causes a lethal zoonotic infection in 80% of untreated humans. Because biomedical professionals working with macaques, their cells, or tissues are at risk for becoming infected with BV, it is important to know the status of macaques involved in potential BV exposures. Although cases of BV infection after encounters between tourists and macaques have not been reported, any event that involves direct or fomite-associated contact with macaques has inherent risks. Identification of zoonotic BV infection through the detection of antibodies enables timely antiviral intervention, which is critical to reduce or prevent morbidity and mortality. Similarly rapid detection is important to maintain the biointegrity of SPF captive macaque colonies. The identification of BV in clinical specimens is achieved by using cell culture, PCR, or antibody detection methods. Because BV is shed only rarely from peripheral sites, the identification of BV infection in monkeys and humans currently is based on antibody detection (serology).^14,23,28In our laboratory, current serological diagnosis for B virus infections has been based on 2 principal tests: a titration-based (that is, traditional) ELISA (tELISA) as a screening test and western blot analysis (WBA) as a confirmatory test. Each test uses quality-controlled BV antigens that are prepared from lysates of infected cells.^20,22,23 Because BV is the only simplex virus in the Alphaherpesvirinae subfamily that is known to infect macaques,^14,28 antibodies interacting with BV antigens are used to indicate BV infection and not an infection due to a crossreacting virus. In practice, tELISA has identified numerous BV antibody-positive sera, the majority of which are low-titer sera from SPF colonies, which fail to be confirmed by WBA, and therefore, are classified as false positives.²³ We, therefore, searched for other approaches that could be used for confirmation of tELISA results. One reasonable option was the use of BV recombinant proteins as antigens. Numerous investigators have used recombinant-based assays for routine diagnosis of infections with viruses, including cytomegalovirus,³⁶ Epstein–Barr,⁶ herpes simplex (HSV1 and HSV2)^{2,3,17,31,32,34} Crimean–Congo hemorrhagic fever,¹⁰ HIV,³⁶ dengue,^5,11,27 hepatitis C,²⁴ hepatitis B,⁸ West Nile,²⁶ influenza,¹⁶ Ebola, and Marburg³³ viruses.Screening for the presence of serum IgG molecules against an array of defined and purified recombinant antigens has distinct advantages over assays that use the entire complement of viral antigens that are present in virus-infected cells. This is particularly true for pathogens that require BSL4 laboratories.^28,33 The pattern of reactivity obtained against each individual recombinant protein may have diagnostic value, by enabling identification of the stage of infection and the prediction of the prognosis of the disease.^3,4,18 However, using a single or only a few recombinant proteins as ELISA antigens can lead to a false-negative result if the antibody repertoire produced after BV infection reacts with other antigenic determinants that are not represented by the particular recombinant antigens used in the test.^{3,18,28,31,34}Several laboratories have examined the efficacy of using a single BV recombinant antigen (that is, glycoprotein D [gD]) for diagnosing BV infections in macaques^25,37 and humans,¹⁵ and we previously reported the diagnostic potential of an ELISA that incorporated several recombinant BV antigens.²⁸ We chose 4 recombinant BV glycoproteins as candidate antigens: peptides corresponding to the full-length extracellular domain of gB, gC, and gD and the membrane-associated segment of gG (gGm). Among these antigens, gGm was the most BV-specific, because it failed to crossreact with antibodies induced by HSV1 and HSV2. To validate the use of the recombinant BV antigens for the purpose of BV antibody detection, a panel of antibody-negative (n = 40) and antibody-positive (n = 75) macaque sera that were confirmed to be positive by tELISA and WBA were tested against the panel of the 4 B virus recombinant antigens, all of which showed fairly high sensitivity for detecting antibodies to BV.²⁸Here, we examine the performance of the recombinant-based ELISA (rELISA) for BV detection by using numerous (>1000) macaque sera, which have a broad range of antibody titers as determined by tELISA. Because manual ELISA to identify antibodies against an array of antigens are too laborious to be cost-effective, we adapted a previously described high-throughput automated single-antigen ELISA performed in 384-well plates to detect antibodies in macaque sera to multiple BV antigens.²³ This assay format has been adapted to include antigens from other alphaherpesviruses²³ and can be easily modified further for other viruses. We then compared the performance of the rELISA with that of whole-virus tELISA and WBA. The main goal of this study was to determine whether the 384-well rELISA is an effective alternative to WBA as a confirmatory assay for tELISA.     

Characterization and Application of a New Monoclonal Antibody with High Specificity for Helicobacter hepaticus

Background and Aims:  Infection with Helicobacter hepaticus is suggested to play a role in the pathogenesis of chronic liver disease in humans. However, reactive antigens among Helicobacter species make the development of an H. hepaticus ELISA test with high specificity difficult. A new monoclonal antibody from a hybridoma clone (HRII-51) showed high specificity to H. hepaticus without cross-reaction to other gastrointestinal bacteria.
Methods:  The molecular weight of HRII-51 immunoreactive antigen was examined by Western blot of H. hepaticus probed with the monoclonal antibody HRII-51. A HRII-51-immunoreactive antigen capture ELISA was prepared in which the specific antigen was anchored by HRII-51-immobilized ELISA plate. Accuracy of HRII-51 antigen capture ELISA was examined using sera obtained from mice inoculated with Helicobacter species. Specificity of HRII-51 antigen capture ELISA was compared to that of H. hepaticus antigen-based ELISA using human sera with absorption by H. pylori cell lysate.
Results:  HRII-51 immunoreactive antigen had a molecular weight of 15 kDa. Sensitivity and specificity of HRII-51 antigen capture ELISA were 87.0% and 97.6% in mice inoculated with Helicobacter species. In human sera, modification of the results by absorption with H. pylori lysate was smaller in HRII-51 antigen capture ELISA comparing with H. hepaticus -antigen-based ELISA.
Conclusion:  Use of the HRII-51 antigen capture ELISA would be a useful approach for the serodiagnosis of H. hepaticus infection in both experimental animals and humans.     

猕猴血清B病毒抗体三种检测方法的比较

目的对B病毒（B virus）抗体检测的3种方法进行比较,寻求准确、可靠、经济的检疫方法。方法对以HSV-1为抗原的玻片酶免疫法、B病毒为抗原的玻片酶法（EIA）和酶联免疫吸附法（ELISA）的猕猴血清B病毒抗体检测结果进行比较。结果HSV-1为抗原的EIA与B病毒为抗原的EIA、ELISA检测结果符合率分别为97.7%和95.5%。结论HSV-1为抗原EIA的检测结果与B病毒抗原EIA和ELISA的检测结果一致性较好,可以做为初筛手段,且检测效果较好,投入资金相对最低,达到节约成本的目的。     

Mothers of children with Down syndrome have higher herpes simplex virus type 2 (HSV-2) antibody levels

Summary The antibody response to herpes simplex virus (HSV) was studied in 53 mothers of children with Down syndrome (Ds) and compared with that in 154 controls, using sera sampled during pregnancy or at delivery. Conventional analysis of HSV complement fixing antibodies showed the same frequency of positivity for the two groups (70%). When the levels of IgG antibodies to an HSV-1 and an HSV-2 antigen preparation were determined by an enzyme-linked immunosorbent assay (ELISA) technique, it was found that the Ds and control mothers had similar levels of IgG antibodies to HSV-1, whereas the level of IgG antibodies to HSV-2 was significantly (P<0.001) higher in Ds mothers. The ratio of HSV-2 to HSV-1 ELISA IgG was calculated for each mother and the distribution of these ratios also differed significantly between the control and Ds mothers. The differences found were not due to differences in age distribution in the control and Ds groups. For comparison a third procedure, measurement of thymidine kinase blocking antibody (TK ab), was used. With this procedure the mothers were divided into groups estimated to be positive for HSV-1, HSV-2, or both. Statistical analyses showed a good correlation between the type found in TK ab analyses and the ratio found in the ELISA HSV test. The results clearly demonstrated an overrepresentation of HSV-2 antibody positivity among Ds mothers, though not of sufficient magnitude to imply that HSV-2 can be the major cause of Ds. It is discussed whether HSV-2 might be related to the recently increased birthrate of children with Ds among young mothers in Sweden or to localized geographical clustering of Ds births, or whether the increased HSV-2 antibody positivity merely indicates that factors following the same epidemiological pattern are involved in the aetiology of Ds.     

Serum Antigen and Antibody Detection in Echinococcosis: Application in Serodiagnosis of Human Hydatidosis

Diagnosis of hydatidosis is based on immunodiagnostic methods along with radiological and ultrasound examinations. The objectives of the present study were to develop a specific and simple antigen-based ELISA method for diagnosis of hydatidosis and compare it with antibody detection method. The subjects in this study included 89 patients in the following groups: surgically confirmed hydatidosis patients (35 cases), control with other parasitic diseases (29 cases), and healthy controls (25 cases). Hyperimmune serum was raised against hydatid cyst fluid in rabbits. Anti-hydatid cyst IgG was purified by affinity chromatography using protein A column and labeled with horseradish peroxidase. Collected sera were assessed for hydatid cyst antigens and antibody by ELISA. Circulating hydatid antigen was found in 9 out of 35 patients with surgically confirmed hydatidosis. A sensitivity of 25.7% and a specificity of 98.0% were calculated for the antigen detection assay. Antibody detection by indirect ELISA, using antigen B, showed that 94.2% of patients (33 cases) have anti-hydatid cyst antibodies in their serum while cross reaction was noted in a few of non-hydatidosis patients. A sensitivity of 94.2% and specificity of 81.6% were found for the antibody detection assay. Findings of this study indicated that antibody detection assay is a sensitive approach for diagnosis of hydatid cyst while antigen detection assay might be a useful approach for assessment of the efficacy of treatment especially after removal of the cyst.     

A new microplate neutralization test for typing of herpes simplex virus.

A microplate serum neutralization test for estimation of complement-requiring neutralizing (CRN) antibody was established as the first step for simplification of typing of herpes simplex virus (HSV). When guinea pigs were immunized with type 2 HSV, the late sera could mostly differentiate the types of HSV better than hyperimmune rabbit sera, the CRN titer against the heterologous type 1 HSV being much lower than the homologous titer. Sera of guinea pigs immunized with type 1 HSV showed about the same level of cross reaction against type 2 HSV as did rabbit antisera. Guinea pig sera having minimal levels of cross reaction were selected, and their high dilution (1:160) and complement were added to serial 10-fold dilutions of virus in the microplate titration of virus infectivity. Selective reduction of virus titer by either antiserum could determine the type of HSV. No equivocal intermediate case was found among a number of stock strains including many fresh isolates. The typing result coincided with that determined by a modification of Yang et al's method based on virus titers obtained with Vero and primary chick embryo cells. The typing based on plaquing in chick embryo cells sometimes failed to identify type 1 HSV.     

Anti-CeHV1 antibodies of two cynomolgus macaques cross-react with HSV2 but not HSV1 antigens in ELISA

BACKGROUND: The aim of the study was to compare the cross-reactivity of macaque anti-CeHV1 antibodies with type 1 and type 2 human herpes simplex viruses (HSV1 and HSV2). METHODS: We studied the serum of 344 animals which had been tested either positive (n = 39) or negative (n = 305) for the presence of CeHV1 antibodies by expert laboratories. Macaque serums were studied by means of two ELISA: one based on HSV1 antigen-coated wells, the other on polystyrene beads coated with HSV1 and HSV2 antigens in approximately equal proportions. RESULTS: In the serum of two animals originating from Vietnam, we found anti-CeHV1 antibodies cross-reacting with HSV2 but not with HSV1 antigens. For the serum with the highest titer, inhibition by soluble antigens confirmed the high affinity of the antibodies for HSV2 antigens. CONCLUSIONS: Tests using HSV1 and HSV2 in a combined way are better suited to macaque screening than tests using only HSV1 antigens.     

A phage-linked immunoabsorbant system for the detection of pathologically relevant antigens

This report describes a novel system for the immunological detection of immobilized antigen. The detection of herpes simplex virus (HSV) antigen was used as an example. Bacteriophage M13, containing the E. coli lac Z gene, was used as the "reporter" molecule in an immunoassay which is otherwise analogous to the enzyme-linked immunoabsorbant assay (ELISA). Briefly, HSV infected cells were incubated with a mouse monoclonal antibody specific for HSV antigen, followed by rabbit anti-mouse serum and mouse anti-M13 serum. Immune complexes were incubated with viable M13 phage. M13 binding was due to the presence of M13 antibodies, whose presence ultimately depended on the binding of monoclonal antibody to HSV. Phage was recovered by elution in pH = 11. Recovered phage was used to infect E. coli. M13 was quantitated by either plaque assay or by an assay for phage-induced beta-galactosidase activity in appropriate E. coli strains. The amount of M13 recovered was proportional to the number of HSV infected cells probed. Therefore, M13 served as a "bio-amplifiable tag" to antibody, as enzymes do in the ELISA. Since M13 is viable, its signal can be amplified by infection of susceptible bacteria, and the promise for an enormously sensitive immunoassay exists. The sensitivity of the assay described here is compared to the ELISA in the detection of HSV infection cells, as an example of the novel assay's potential. Significantly, the novel assay was more sensitive than the ELISA when samples were tested under identical circumstances. This technique is called the phage-linked immunoabsorbant assay (PHALISA), by analogy to the ELISA.     

Sequence and Genetic Arrangement of the US Region of the Monkey B Virus (Cercopithecine Herpesvirus 1) Genome and Comparison with the US Regions of Other Primate Herpesviruses

The sequence of the unique short (U(S)) region of monkey B virus (BV) was determined. The 13 genes identified are arranged in the same order and orientation as in herpes simplex virus (HSV). These results demonstrate that the BV U(S) region is entirely colinear with that of HSV type 1 (HSV-1), HSV-2, and simian agent 8 virus.     

Enzyme-linked immunosorbent assay for efficient detection of antibody to bluetongue virus in pronghorn (Antilocapra americana)

An indirect enzyme-linked immunosorbent assay (ELISA), using cell-associated viral antigen, was developed for detection of antibody to bluetongue virus (BTV) in field-collected pronghorn (Antilocapra americana) sera. To test the applicability of the ELISA to seroepizootiologic studies, pronghorn serum samples from three Wyoming counties (USA) were tested. Bluetongue virus ELISA results were compared to those of the bluetongue immunodiffusion assay. Discrepant serum samples were retested for reaction to either BTV or epizootic hemorrhagic disease virus. The pronghorn BTV ELISA gave rapid, quantitative, objective results and should facilitate testing large numbers of sera for BT diagnostic and seroepizootiologic studies.     

Diagnosis of Corynebacterium pyogenes infection in pigs by immunodiffusion test with protease antigen.

A protease antigen was prepared from the culture supernatant of Corynebacterium pyogenes by concentrating with a flash evaporator and ultrafiltration. It was adjusted to the concentration of 32 units by the single radial immunodiffusion with a tentative standard serum. In the immunodiffusion test, the antigen of 4 units reacted enough with sera having an antibody titer ranging from 1 to 128. As a result, it was decided that the antigen of 4 units should be used in the immunodiffusion test for the detection of protease antibody. By the immunodiffusion test, protease antibody was demonstrated in about 35% of 443 sera from pigs collected at random. The antibody titer showed the distribution of 2 peaks. The summits of the two peaks were seen at 4 and 32 of antibody titer, respectively. The valley between the two peaks was seen at 16 of titer. From the result, a diagnostic criterion of the immunodiffusion test was decided provisionally as follows: above 16 of antibody titer is positive, 1 to 8 suspect, and less than 1 negative. On the other hand, protease antibody was demonstrated in sera from 13 of 14 pigs carrying abscesses from which C. pyogenes had been isolated. Its titer was 8 (in 2 pigs), 16 (in 1), 32 (in 3), 64 (in 6), and 128 (in 1). From these results, it was proposed that the immunodiffusion test with protease antigen be used for the diagnosis of C. pyogenes infection in pigs.     

A micro-neutralization system for detection of s-CRN antibody to herpes simplex virus

It was learned that the ordinary micro-neutralization system with herpes simplex virus (HSV) gave a composite result of the initial neutralization and the effect of antibody on subsequent growth of unneutralized virus. In the case of slow-reacting complement-requiring neutralizing (s-CRN) antibody, which was detected by incubating virus-serum mixtures at 4 C for 3 days before addition of complement, the titer obtained was lower than expected from the result of the plaque reduction test. This was thought ascribable to its low ability to prevent viral breakthrough caused by growth of unneutralized virus. This was overcome by adding an appropriate amount of hyperimmune antibody at 3 hr after addition of cells. The endpoint of s-CRN antibody so determined was but slightly lower than that obtained by the plaque reduction test. Early (1-week) rabbit sera, which were negative in the ordinary micro-neutralization test, titered 1:2,560 to 1:5,120 when tested by this method. When the 3-day sensitization in the cold was substituted by 5-hr incubation at 37 C, the titer obtained was 2 to 4-fold lower; in this case, however, the whole process could be finished within 3 days. Also, s-CRN antibody reactive with type 2 HSV in homologous and heterologous sera could be detected by the same method using type 1 hyperimmune serum as the additional antibody.     

EB病毒DNA聚合酶基因的表达与纯化鼻咽癌病人诊断中的应用

以Ｅｐｓｔｅｉｎ－Ｂａｒｒ病毒（ＥＢＶ）ＤＮＡ聚合酶为抗原,建立了简便、快速、敏感和特异的鼻咽癌诊断方法。构建原表达载体ｐＲＳＥＴ－ＤＮＡ聚合酶及其亚克隆ＰＲＳＥＴ－Ａ１和ＢＬ２１（ＤＥ３）中表达的产物,经Ｗｅｓｔｅｒｎ－ｂｌｏｔ检测其抗原性并用于检测鼻咽癌（ｎａｓｏｐｈａｒｙｎｇｅａｌ ｃａｒｃｉｎｏｍａ,ＮＰＣ）病人血清中的抗体。在ＤＰＣ病人血清中存在抗ＥＢ病毒ＤＮＡ聚合酶的ＩｇＧ抗体,并证明