Herpes B Research Articles

Prevalence of antibodies to selected viruses in a long-term closed breeding colony of rhesus macaques (Macaca mulatta) in Brazil.

The rhesus macaque breeding colony of the Oswaldo Cruz Foundation (FIOCRUZ) was established in 1932 from a founding stock of 100 animals. This population has remained closed to new animal introductions for almost 70 years. A serologic survey was performed to determine the prevalence of antibodies to selected viruses as a first approach to identifying viral pathogens endemic in this population. Banked serum samples were tested for antibodies to simian immunodeficiency virus (SIV), simian T-lymphotropic virus (STLV), simian type D retrovirus (SRV), cercopithecine herpesvirus type-1 (B virus), rhesus cytomegalovirus (RhCMV), measles virus (MV), and hepatitis A virus (HAV). All samples were negative for antibodies against the simian retroviruses. The overall prevalence of antibodies was 95% for RhCMV, 45% for B virus, 35% for HAV, and 1% for MV. Prevalence was found to vary by age group.

B-virus (Cercopithecine herpesvirus 1) infection in humans and macaques: potential for zoonotic disease.

Nonhuman primates are widely used in biomedical research because of their genetic, anatomic, and physiologic similarities to humans. In this setting, human contact directly with macaques or with their tissues and fluids sometimes occurs. Cercopithecine herpesvirus 1 (B virus), an alphaherpesvirus endemic in Asian macaques, is closely related to herpes simplex virus (HSV). Most macaques carry B virus without overt signs of disease. However, zoonotic infection with B virus in humans usually results in fatal encephalomyelitis or severe neurologic impairment. Although the incidence of human infection with B virus is low, a death rate of >70% before the availability of antiviral therapy makes this virus a serious zoonotic threat. Knowledge of the clinical signs and risk factors for human B-virus disease allows early initiation of antiviral therapy and prevents severe disease or death.

Recommendations for prevention of and therapy for exposure to B virus (cercopithecine herpesvirus 1).

B virus (Cercopithecine herpesvirus 1) is a zoonotic agent that can cause fatal encephalomyelitis in humans. The virus naturally infects macaque monkeys, resulting in disease that is similar to herpes simplex virus infection in humans. Although B virus infection generally is asymptomatic or mild in macaques, it can be fatal in humans. Previously reported cases of B virus disease in humans usually have been attributed to animal bites, scratches, or percutaneous inoculation with infected materials; however, the first fatal case of B virus infection due to mucosal splash exposure was reported in 1998. This case prompted the Centers for Disease Control and Prevention (Atlanta, Georgia) to convene a working group in 1999 to reconsider the prior recommendations for prevention and treatment of B virus exposure. The present report updates previous recommendations for the prevention, evaluation, and treatment of B virus infection in humans and considers the role of newer antiviral agents in postexposure prophylaxis.

Characterization of B virus glycoprotein antibodies induced by DNA immunization.

Genes encoding glycoproteins gB, gC, gD, gE, and gG of herpes B virus (species Cercopithecine herpesvirus 1) were cloned into mammalian expression vector pcDNA3.1/V5-His. Abilities of the plasmid constructs to express recombinant glycoproteins were confirmed by Western blot analysis of transfected CHO-K1 and COS-7 cells. Antibody production was induced in rabbits by intramuscular injections with the expression constructs at four-weekly intervals. Antibodies to gB were detected after the second DNA inoculation, while it took an additional plasmid injection to induce responses to gC, gD and gE. The gG plasmid failed to stimulate antibody production. Antisera ELISA titers varied greatly depending on the gene, with gB inducing highest (21,000) and gE inducing lowest (60) antibody titer. The induced antibodies were predominantly conformation-dependent. The gB, gC, and gD antisera contained HSV cross-neutralizing antibodies, but only gB antisera contained B virus neutralizing antibodies. The gB antisera cross-reacted with HSV antigens in Western blot, ELISA, dot-blot, plaque immunostaining and immunoprecipitation assays, whereas gD and gC antisera were mostly B virus-specific. Thus, polyclonal antibodies to B virus glycoproteins can be generated by DNA immunization and used as diagnostic and research reagents.

Human exposure to herpesvirus B-seropositive macaques, Bali, Indonesia.

Herpesvirus B (Cercopithecine herpesvirus 1) has been implicated as the cause of approximately 40 cases of meningoencephalitis affecting persons in direct or indirect contact with laboratory macaques. However, the threat of herpesvirus B in nonlaboratory settings worldwide remains to be addressed. We investigated the potential for exposure to herpesvirus B in workers at a “monkey forest” (a temple that has become a tourist attraction because of its monkeys) in Bali, Indonesia. In July 2000, 105 workers at the Sangeh Monkey Forest in Central Bali were surveyed about contact with macaques (Macaca fascicularis). Nearly half of those interviewed had either been bitten or scratched by a macaque. Prevalence of injury was higher in those who fed macaques. Serum from 31 of 38 Sangeh macaques contained antibodies to herpesvirus B. We conclude that workers coming into contact with macaques at the Sangeh Monkey Forest are at risk for exposure to herpesvirus B.

Detection of B virus antibody in monkey sera using glycoprotein D expressed in mammalian cells.

The gene encoding glycoprotein D (gD) of the monkey B virus (Cercopithecine herpesvirus 1) was cloned into a mammalian expression vector, pcDNA3.1(-), and the recombinant plasmid DNA was transfected into COS7 cells. The expression of gD in transfected COS7 cells was detected by indirect immunofluorescence assay or radioimmunoprecipitation analysis (RIPA). Although the expressed gD protein was revealed to react well with sera from monkeys naturally infected with B virus by RIPA, some sera showed reduced reactivity when analyzed by the Western blotting (WB) method. Some sera also showed relatively high background when the WB was performed using gD expressed from recombinant plasmid. The mutant gD protein lacking the transmembrane domain (TM) and cytoplasmic tail (CT) was next expressed in COS7 cells. The mutant protein was secreted into culture medium without apparent loss of the antigenicity. Using the secretory form of the gD protein as antigen in dot blot analysis, sera from B virus-infected monkeys were shown to react with the mutant protein without nonspecific reaction. Since the recombinant gD or its derivative lacking TM and CT could be expressed in mammalian cells with proper antigenicity, these antigens appeared to be useful for serological detection of B virus infection in monkeys.

Immunogenicity of a DNA vaccine against herpes B virus in mice and rhesus macaques

Herpes B virus ( Cercopithecine herpesvirus 1) is endemic in captive macaque populations and poses a serious threat to humans who work with macaques or their tissues. A vaccine that could prevent or limit B virus infection in macaques would lessen occupational risk. To that end, a DNA vaccine plasmid expressing the B virus glycoprotein B (gB) was constructed and tested for immunogenicity in mice and macaques. Intramuscular (IM) or intradermal (ID) immunization in mice elicited antibodies to gB that were relatively stable over time and predominately of the IgG2a isotype. Five juvenile macaques were immunized by either IM+ID ( n=2) or IM ( n=3) routes, with two booster immunizations at 10 and 30 weeks. All five animals developed antibodies to B virus gB, with detectable neutralizing activity in the IM+ID immunized animals. These results demonstrated that DNA immunization can be used to generate an immune response against a B virus glycoprotein in uninfected macaques.

Characterization of Gross and Histological Lesions in Balb/c Mice Experimentally Infected with Herpesvirus saimiri 1 (HVS1)

Accidental B virus (Herpesvirus simiae) infection of human beings working with macaques is frequently fatal. However, the pathogenic potential of other similar simian alphaherpesviruses, such as the squirrel monkey virusHerpesvirus saimiri (HVS1), is virtually unknown. As part of an effort to develop a murine model for infections with these agents, Balb/c mice were inoculated intramuscularly in the left hindlimb with 10 to 106plaque forming units (PFU) of HVS1. After observation for clinical signs of infection for 21 days, mice were killed and specimens collected for serology and histopathology. Mice receiving 5103PFU of HVS1 exhibited severe, pruritic, ulcerative skin lesions near the site of inoculation and developed unilateral or bilateral hindlimb paralysis with severe muscle atrophy. Histological lesions were characterized by a necrotizing dermatitis and folliculitis. Spinal cord lesions consisted of a non-suppurative myelitis affecting primarily the ipsilateral dorsal horn of the thoracolumbar spinal cord with occasional extension to ventral and contralateral spinal cord regions. Immunohistochemical labelling confirmed the presence of viral antigen within the lesions, and anti-HVS1 IgG concentrations were related to the occurrence of disease. HVS1 infection in some mice extended from the ipsilateral dorsal horn and funiculus into the ventral and contralateral grey and white matter, resulting in bilateral hindlimb paralysis. Thoracolumbar spinal cord lesions resolved without continued spread of the virus to cranial nervous system structures, i.ecervical spinal cord and brain.

Safety Assessment Studies in Nonhuman Primates

Worldwide regulatory guidelines for drug safety evaluations recommend testing in both a rodent and a nonrodent species. Non-human primates, which are phylogenetically close to man, are often thought of as the “ideal” nonrodent species. Historically, because of conservation issues, biosafety concerns, and price and supply issues, use of nonhuman primates in toxicology programs has been restricted to special cases. Recently, however, biopharmaceutical scientists have turned to primates as the only nonhuman species in which the biological activities of some drugs are expressed. Also, as the cost of test article for toxicology studies has increased, there has been a corresponding increase in the use of primates because of their smaller body size compared to dogs. With increasing demand, primatologists have solved the price, supply, and conservation issues by worldwide development of breeding centers that produce adequate numbers of well-characterized rhesus and cynomolgus macaques. New international personnel protective standards, shipping and quarantine requirements have addressed some of the public health concerns. However, tuberculosis, Herpesvirus simiae (B virus) and the “Ebola-like” hemorrhagic primate viruses remain of concern to primate researchers. With new requirements for specialized training, housing, equipment, and procedures for primate research and husbandry, many pharmaceutical companies now outsource their primate toxicology work to contract research organizations. The effort to “harmonize” international regulatory requirements for nonclinical toxicology has led to more uniformity in nonhuman primate study design. Typical study designs and procedures are discussed.

Mechanisms that regulate Epstein-Barr virus EBNA-1 gene transcription during restricted latency are conserved among lymphocryptoviruses of Old World primates.

Epstein-Barr virus (EBV), the only known human lymphocryptovirus (LCV), displays a remarkable degree of genetic and biologic identity to LCVs that infect Old World primates. Within their natural hosts, infection by these viruses recapitulates many key aspects of EBV infection, including the establishment of long-term latency within B lymphocytes, and is therefore a potentially valuable animal model of EBV infection. However, it is unclear whether these LCVs have adopted or maintained the same mechanisms used by EBV to express essential viral proteins, such as EBNA-1, in the face of cell-mediated repression of EBV gene expression that occurs upon establishment of the asymptomatic carrier state. To address this issue, we determined whether the endogenous LCVs of baboon (Cercopithecine herpesvirus 12) and rhesus macaque (Cercopithecine herpesvirus 15) have the functional equivalent of the EBV promoter Qp, which mediates exclusive expression of EBNA-1 during the restricted programs of EBV latency associated with the carrier state. Our results indicate that (i) both the baboon and rhesus macaque LCVs have a genomic locus that is highly homologous to the EBV Qp region, (ii) key cis-regulatory elements of Qp are conserved in these LCV genomes and compose promoters that are functionally indistinguishable from EBV Qp, and (iii) EBNA-1 transcripts identical in structure to EBV Qp-specific EBNA-1 mRNAs are present in nonhuman LCV-infected cells, demonstrating that these Qp homologs are indeed utilized as alternative EBNA-1 promoters. These observations indicate that the molecular mechanisms which regulate EBV gene expression during restricted latency have been conserved among the LCVs. The contribution of these mechanisms to viral persistence in vivo can now be experimentally tested in nonhuman primate models of LCV infection.

Development of a competitive ELISA for detection of primates infected with monkey B virus ( Herpesvirus simiae)

Two competitive ELISAs (C-ELISAs) are described that allow detection of antibodies against monkey B virus (BV, Cercopithecine herpesvirus 1). The assays utilize monoclonal antibodies (MABs) directed against the BV glycoprotein B (gB). Two of these MABs specifically recognize BV gB while a third MAB also reacts with the gB homologues of other primate α-herpesviruses (herpes simplexvirus-1, HSV-1; HSV-2; simian agent-8, SA8; and Herpesvirus papio-2, HVP2). A C-ELISA using the single cross-reactive MAB 3E8 allowed detection of host antibodies against HSV-1, HSV-2, SA8, HVP2 or BV, thus proving to be a sensitive assay for the detection of infection by any of these primate α-herpesviruses. The C-ELISA using BV-specific MABs was less sensitive but did allow some discrimination between infection by BV versus other α-herpesviruses. It was also shown that a C-ELISA using HVP2 as antigen and the cross-reactive MAB 3E8 was as sensitive for detection of BV antibody in macaque sera as an assay employing BV antigen. This test format allows detection of BV-infected primates without the biohazards associated with preparation and use of BV antigen.

Peer-rearing influences subsequent maternal behavior and infant survival in a newly formed herpes B-virus negative rhesus macaque group

The maternal behavior of primiparous rhesus macaques (Macaca mulatta), peer-reared since 1/2 years(s) of age as part of aHerpesvirus simiae (herpes B-virus) screening protocol, was examined and compared to a control group of conspecifics reared in their natal group. Infant survival was significantly higher in control groups as compared to the test group, a result attributed to the high incidence of infant kidnapping/abandonment in the test group. Among the test subjects, infant survival rate increased as the birth season progressed, thus it is possible that exposure to mothers/infants helped in the maternal success of those females who gave birth later in the season. Test group infants were touched by group members significantly more than the infants of control subjects, whereas these infants were groomed by their mothers and in a ventral position for a greater time relative to the infants of the test subjects. This study suggests that females, partially reared in peer groups, may be at early risk for maternal incompetence and consequent greater infant mortality, and that exposure to mother-infant dyads may augment the proficiency of maternal skills.

Prevalence of Herpes B Virus Antibody in Nonhuman Primates reared at the National University of Japan.

A serological investigation by means of an enzyme immuno assay test for herpes B virus (cercopithecine herpesvirus 1) was performed on 961 sera of healthy nonhuman primates reared in laboratory animal facilities which belong to the Association of Laboratory Animal Facilities of the National University of Japan. An antibody prevalence of 40% (384/ 961) was demonstrated. The antibody titer was shown to be higher among macaques (60% of cynomolgus monkeys, 53% of rhesus monkeys, and 34% of Japanese monkeys) than among non-macaque species (21%). These data indicate that nonhuman primates reared in animal facilities may present an occupational health problem and a potential zoonotic biohazard as demonstrated in limited cases in the United States.

Threat to Humans from Virus Infections of Non-human Primates.

Several hundred distinct non human primate species are recognised, and they are likely to harbour a similar range of viruses to humans. Simians such as cynomolgus and rhesus macaques, African green monkeys, and marmosets are widely used for biomedical research, but despite this extensive close contact very few simian viruses have been shown to pose a threat of infection or illness to humans. Herpesvirus Simiae is the best recognised zoonotic hazard of simians. It is an alphaherpes virus of Asiatic macaques, which causes a mild or subclinical primary infection followed by latency in its natural host. It can be acquired by humans following a bite and causes an ascending meningoencephalitis. Less than 40 human cases have been described and the mortality rate in untreated human infections is 70%. The infection is treatable with acyclovir and extensive guidelines for managing simians and potential exposures have been developed. Ebola virus and Marburg virus have caused epizootics in cynomolgus macaques and vervet monkeys respectively, which have resulted in human infection and fatalities. However, non human primates are unlikely to be their natural host. More recently simian immunodeficiency virus and simian foamy virus have infected researchers, but infection has not been linked to illness. Simian viruses also pose a direct threat to humans through the use of primary monkey tissue cultures in laboratory work and vaccine manufacture, indeed a significant exposure of the human population occurred when cells contaminated with SV40 a polyomavirus of rhesus monkeys were used for polio vaccine production. New medical interventions such as xenotransplantation using primate organs pose a potential risk which requires careful assessment. Copyright 1997 by John Wiley & Sons Ltd.

Characterization of virus-specific and cross-reactive monoclonal antibodies to Herpesvirus simiae (B virus).

A panel of 13 monoclonal antibodies (MAbs) was produced that detect B virus (BV) proteins. Several of these MAbs were highly specific for BV, while the remainder cross-reacted in varying degrees with the other primate alphaherpesviruses. Utilizing western blot and radioimmunoprecipitation analysis, the MAbs were found to detect at least four distinct BV-infected cell antigens, several of which were composed of multiple polypeptides. One target antigen has been identified as the BV glycoprotein B (gB) homologue and was recognized by both virus-specific and cross-reactive MAbs. Although gB is an essential protein, none of the anti-gB MAbs neutralized infectious virus.

Complete nucleotide sequence of the herpesvirus simiae glycoprotein G gene and its expression as an immunogenic fusion protein in bacteria.

The nucleotide sequence of a 2384 bp portion within the unique short (Us) region of the herpesvirus simiae (simian herpes B virus; SHBV) genome is presented. A partial and a complete open reading frame (ORF) were found within this nucleotide sequence. The partial ORF encodes the C terminus (147 amino acids) of a protein kinase which is highly conserved in the herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and simian agent 8 (SA8) Us regions. The complete ORF is located 3' to the partial ORF within the 2384 bp sequence and encodes a 593 amino acid glycoprotein which appears to be closely related to the SA8 glycoprotein G (gG), but shares little amino acid similarity with gG of HSV-1 and -2. However, the complete ORF shares certain features conserved among most alphaherpesvirus gGs, notably three highly conserved cysteine residues and an adjacent N-glycosylation site. Therefore, it was concluded that this complete ORF encodes the SHBV gG. The 358 amino acid C-terminal portion of SHBV gG was expressed in Escherichia coli as a fusion protein and this was detected by immunoblotting with sera from cynomolgus monkeys which were either experimentally or naturally infected with SHBV. The purified fusion protein was inoculated into rabbits to raise an antiserum which recognized a number of apparently SHBV gG-specific protein bands in extracts from SHBV-infected simian cells.

Risk of Venereal B Virus (Cercopithecine Herpesvirus 1) Transmission in Rhesus Monkeys Using Molecular Epidemiology

The importance of venereal modes of B virus (cercopithecine herpesvirus 1) transmission was evaluated in 49 rhesus monkeys tested at necropsy. Antibodies to B virus were demonstrated in 19 monkeys, but no active viral shedding was detected in mucosal swabs collected at death. The polymerase chain reaction demonstrated presence of the ICP 18.5 (UL28) gene of B virus in neuronal tissues of 15 monkeys presumed to be latently infected, including 12.8% of trigeminal and 22.9% of lumbosacral ganglia pools. Two monkeys tested positive at both sites. Breeding history was predictive of B virus seropositivity (odds ratio, 1.64; 95% confidence interval, 1.21-2.23; P < .05). The population attributable risk of B virus seropositivity due to breeding was 22.7%, similar to the proportion of monkeys with B virus DNA in neuronal tissues subserving the genital region. Sexual contact is a significant, but not predominant, mode of B virus transmission between monkeys.

Guidelines for the Prevention and Treatment of B-Virus Infections in Exposed Persons

Cercopithecine herpesvirus 1 (B virus), enzootic among monkeys of the genus Macaca, causes minimal morbidity in its natural host. In contrast, human B-virus infection presents as rapidly ascending encephalomyelitis with a fatality rate of approximately 70%. This infection remains an uncommon result of macaque-related injuries, although the increase in the use of macaques for research on simian retrovirus infection and hepatitis has expanded the number of opportunities for human exposure. In response to this situation, Emory University and the Centers for Disease Control and Prevention jointly sponsored a B Virus Working Group to formulate a rational approach to the detection and management of human B-virus infection. The resulting guidelines are presented herein and are based upon information from published cases, unpublished cases managed by working-group members, knowledge of the behavior of herpes simplex virus, and--in the absence of hard data--the collective judgment of the group. Although consensus among the co-authors existed on the major points covered by these guidelines, opinions varied widely regarding specific recommendations.

Diagnosis and Management of Human B Virus (Herpesvims simiae) Infections in Michigan

Three men who had worked at the same animal research facility and had had contact with macaque monkeys were infected with B virus (Herpesvirus simiae). Their clinical presentations varied from self-limited aseptic meningitis syndrome to fulminant encephalomyelitis and death. Patient 1 was treated only after a respiratory arrest and other signs of advanced brain stem dysfunction had occurred. He died 8 days after hospital admission, despite treatment with acyclovir. Patient 2 presented with subtle signs and symptoms of brain stem encephalitis. He received antiviral therapy with intravenous ganciclovir. Patient 3 had a headache without meningismus and was also treated with acyclovir. Both patients 2 and 3 survived and did not have objective sequelae. Viral culturing, ELISA and western blot antibody testing, and magnetic resonance imaging all proved useful in the diagnosis of these patients' conditions.

Rapid Detection of B Virus (Herpesvirus Simiae) DNA by Polymerase Chain Reaction

Rapid diagnosis of B virus (herpesvirus simiae) infection in humans followed by early antiviral treatment is essential for the patient's survival. To improve laboratory diagnosis of B virus infections, a polymerase chain reaction (PCR)-based test using synthetic oligonucleotide primers and probe was developed to detect B virus DNA in clinical samples. After the specificity of the PCR was assessed for detection of several B virus isolates, the method was used to investigate human and monkey specimens, and results were compared with those obtained by viral culture. PCR appeared to be more sensitive than conventional virus isolation and thus of practical use for a rapid identification of B virus infection when conventional viral cultures are negative.