Inhibition of B virus (Macacine herpesvirus 1) by conventional and experimental antiviral compounds.

B virus infection of humans results in high morbidity and mortality in as many as 80% of identified cases. The main objective of this study was to conduct a comparative analysis of conventional and experimental antiviral drug susceptibilities of B virus isolates from multiple macaque species and zoonotically infected humans. We used a plaque reduction assay to establish the effective inhibitory doses of acyclovir, ganciclovir, and vidarabine, as well as those of a group of experimental nucleoside analogs with known anti-herpes simplex virus activity. Four of the experimental drugs tested were 10- to 100-fold more potent inhibitors of B virus replication than conventional antiviral agents. Drug efficacies were similar for multiple B virus isolates tested, with variations within 2-fold of the median effective concentration (EC(50)) for each drug, and each EC(50) was considerably lower than those for B virus thymidine kinase (TK) mutants. We observed no differences in the viral TK amino acid sequence between B virus isolates from rhesus monkeys and those from human zoonoses. Differences in the TK protein sequence between cynomolgus and pigtail macaque B virus isolates did not affect drug sensitivity except in the case of one compound. Taken together, these data suggest that future B virus zoonoses will respond consistently to conventional antiviral treatment. Further, the considerably higher potency of FEAU (2'-fluoro-5-ethyl-Ara-U) than of conventional antiviral drugs argues for its compassionate use in advanced human B virus infections.

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.

Serological evidence of alpha herpesvirus infection in sooty mangabeys.

Contact between sooty mangabeys (SMs) and a pigtailed macaque prompted the serological screening of SMs for evidence of infection with B virus. Serological tests detected SM antibodies that reacted with B virus polypeptides. Additional testing was performed with sera from SMs with no previous contact with macaques. Results from these tests indicated that 56% (33/59) of the SMs had antibodies that reacted with B virus and SA8. SM antibodies also reacted with herpesvirus papio 2 and to a lesser extent with human alpha herpesviruses (HSV-1 and HSV-2). There was an age-related increase in the presence of these antibodies in SMs that was consistent with the serological pattern of reactivity observed in other nonhuman primate species infected with alpha herpesviruses. These data suggest that SMs may be a host for a herpesvirus that is antigenically similar to those viruses present in other Old World nonhuman primates.

Antibody cross-reactivity of alphaherpesviruses as mirrored in naturally infected primates.

The extent of antibody cross-reactivity of pooled antisera from rhesus monkeys, baboons, African green monkeys, langurs, sooty mangabeys and humans to 6 alphaherpesviruses (herpes B virus, herpes papio 2, simian agent 8, langur herpes virus and herpes simplex 1 & 2) was examined by two types of enzyme linked immunosorbent assays, an antibody capture assay (tELISA) and an antigen capture assay (dELISA). Percent cross-reactivity was calculated for each serum by comparison of the homologous reaction (100%) to the reaction with heterologous viruses. Comparison of the immunological reactivity of the mangabey antiserum pool to the panel of alphaherpesviruses indicated that these antibodies were induced by a yet unidentified alphaherpesvirus. In general, monkey sera were more cross-reactive to monkey herpesviruses than to human herpesviruses.However, the extent of cross-reactivity of monkey sera to human herpesviruses was relatively lower than the cross-reactivity of human sera to monkey herpes-viruses. Because of this phenomenon of "one-way" cross-reactivity that was also observed within the group of simian herpesviruses, it was difficult to rank the immunologic distances between the viruses in absolute terms.

Fatal herpes simplex infection in a pygmy African hedgehog (Atelerix albiventris).

An adult pygmy African hedgehog developed acute posterior paresis attributed to a prolapsed intervertebral disc diagnosed by C-T scan. Corticosteroid therapy resulted in prompt resolution of the ataxia, but 2 weeks later the animal became anorexic and died. Macroscopically, the liver was stippled with punctate off-white foci which were confirmed microscopically to be foci of necrosis. Numerous hepatocytes contained intranuclear inclusions and syncytial cell formation was also present. A herpes virus was isolated and identified by fluorescent antibody and polymerase chain reaction studies as herpesvirus simplex type 1. To our knowledge, this is the first report of herpes infection in the African hedgehog and the first time herpes simplex has been identified as a cause of disease in insectivores.

Herpes B-virus specific-pathogen-free breeding colonies of macaques (Macaca mulatta): diagnostic testing before and after elimination of the infection.

BACKGROUND AND PURPOSE: The National Institutes of Health's (NIH) National Center for Research Resources' (NCRR) Division of Comparative Medicine has funded the establishment of specific pathogen-free (SPF) captive macaque colonies. Herpes B-virus (Herpesvirus simiae, Cercopithecine herpesvirus type 1) has been targeted for elimination. Late seroconversion presents the greatest threat to the integrity of SPF colonies. The purpose of the study reported here was to evaluate that threat through detailed investigation of the patterns of seroreactivity and housing histories in one colony. METHODS: From 1990 through 1997, the B-virus Resource Laboratory screened macaques for B-virus, using ELISA or western immunoblot analysis. In 1993, we combined test results and housing histories to verify the seronegative status of one colony. RESULTS: Two groups of latently infected macaques were identified as to time and place of transmission. The infection was eradicated within 3 years (1990-1992), as judged by the absence of true positive seroreactivity in any screened macaques. New infections were not identified in four years of follow-up evaluation. CONCLUSION: With rigorous surveillance, the SPF status of the colony was achieved and maintained.

Outbreak of Orthoreovirus-induced meningoencephalomyelitis in baboons.

BACKGROUND AND PURPOSE: Spontaneous viral encephalitis is rare in the baboon; yet, during a 13-month period (1993-1994), eight juvenile baboons (Papio cynocephalus spp.) developed acute, progressive nonsuppurative meningoencephalomyelitis caused by an unknown agent. Clinical signs of disease included disorientation and truncal ataxia that rapidly progressed to hemiparesis or paraparesis. Clinicopathologic findings were not remarkable and appreciable gross lesions were not seen at necropsy. Microscopic examination revealed CNS lesions that were characterized by lymphoplasmacytic perivascular cuffing, microglial nodules, demyelination, axonal degeneration, vacuolization, and hemorrhage. Subsequently, a novel syncytium-inducing mammalian orthoreovirus was isolated from the brain tissue of five baboons with clinical signs of infection. METHODS: To confirm the etiologic role of the orthoreovirus, two juvenile baboons were inoculated with the virus, then were monitored for 6 weeks. RESULTS: Lesions similar to those seen in spontaneous cases were found in the CNS, and orthoreovirus was isolated from the brain of both animals. CONCLUSION: Analysis of the outbreak indicated juvenile baboons were most susceptible to disease and the virus had a possible incubation time of 46 to 66 days, but did not indicate a source of the virus or mode of transmission.

Retrospective analysis of an outbreak of B virus infection in a colony of DeBrazza's monkeys (Cercopithecus neglectus).

In 1981, an outbreak of herpetic disease developed in a colony of DeBrazza's monkeys (Cercopithecus neglectus). In seven of eight infected animals, clinical signs of infection included vesicular and ulcerative lesions on the lips, tongue, and/or palate. Histologic examination of lesions revealed intranuclear inclusion bodies, and electron microscopy revealed nucleocapsids and virions with typical herpesvirus morphology. Although a virus was isolated that appeared similar to monkey B virus, techniques available at the time did not allow precise identification of the virus. Analysis of serum from one surviving monkey collected 12 years after the outbreak revealed a pattern of reactivity characteristic of B virus-positive serum on the basis of results of ELISA and western immunoblot analysis. Polymerase chain reaction analysis of archived paraffin-embedded tissue specimens and molecular analysis of the one viral isolate obtained from a DeBrazza's monkey indicated that the virus responsible for the outbreak was a new genotype of B virus. Testing of sera from lion-tailed macaques (Macaca silenus) housed in an adjacent cage at the same zoo indicated that these animals harbored this virus and, thus, were the likely source of the virus that infected the DeBrazza's monkeys. This study documents usefulness of archiving samples from disease outbreaks for later analysis. In addition, this incident underscores the importance of considering herpes B virus infection when outbreaks of disease having characteristics of herpetic infections develop in nonhuman primates kept at institutions that also house macaques.

B-virus specific-pathogen-free breeding colonies of macaques (Macaca mulatta): retrospective study of seven years of testing.

BACKGROUND AND PURPOSE: National Institutes of Health's Division of Comparative Medicine has sponsored a multi-institutional program for the establishment of specific-pathogen-free (SPF) macaque colonies. B virus (Herpesvirus simiae, Cercopithecine herpesvirus type 1) has been targeted in this surveillance. Participating institutions have established individual timetables for frequency of testing and types of monitoring and husbandry techniques, all with the common goal of producing pathogen-free monkeys for research. The greatest biosecurity threat to the program comes from failure to detect seronegative latent infections, either in first-year macaques or macaques introduced in subsequent years, although these are supposed to operate as closed colonies. METHODS: From January 1990 through December 1996, we screened macaques for B virus, using enzyme-linked immunoabsorbant assay (ELISA) and Western blot analysis. RESULTS: During the first year, 1,097 macaques from six colonies were tested, and 88.4% tested negative for B virus. During the seventh year, 1,843 were tested, of which 99.7% tested negative. Seropositive macaques were detected as late as the seventh year. CONCLUSIONS: An aggressive program to establish an SPF colony of captive breeding macaques can be effective in reducing the risk of B-virus exposure.

Clinical disease associated with simian agent 8 infection in the baboon.

Simian agent 8 (SA8) is an alphaherpesvirus that was first reported as a spontaneous natural infection in a captive baboon colony in 1988. It was first isolated from an African vervet monkey in 1958 and was classified as a simian agent. Simian agent 8 was later isolated from a baboon rectal swab specimen in 1969 and from an oral lesion in a vervet monkey in 1972. Restriction endonuclease analysis was used to identify the virus as SA8. In a 1-year period, 70 baboons housed in two outside 6-acre breeding corrals developed lesions principally on the genitalia and oral cavity. The incidence was the same for males and females, with recurrence rate, severity of the lesions, and duration for the lesions to resolve being greater in the female baboons. Lesions involving the mouth, tongue, and lips were most commonly observed in the juvenile population. The lesions tended to start as small multiple papules or vesicles, which advanced to large pustular or ulcerative areas. Using an every-other-day treatment regimen consisting of Nolvasan cleaning and procaine penicillin G injections, it took an average of 14 to 21 days for the lesions to resolve totally. Thirty-seven percent of the baboons with herpetic lesions experienced another episode of SA8 infection, usually within 1 year of development of the primary lesion. Several complications have been documented to be associated with SA8 infections. Partial or total vaginal obstruction is most common, leading to impaired breeding performance and pyelonephritis. A vaginal corrective surgical procedure has been developed to allow these females to return to productive breeding status within the colony. Penile urethral obstruction, also causing pyelonephritis, was observed in the male baboons. A case of sciatic neuritis was reported in a baboon that presented with self mutilation of the foot; viral isolation revealed the etiologic agent to be SA8. Four female baboons with chronic SA8 infections went on to develop perineal neoplasms. This is an economically important disease entity in captive baboons because it causes severe morbidity, decreased reproductive performance, and ultimately death in 1% of the baboon colony each year. The baboon is a promising animal model in which to study genital herpes as it relates to disease in human beings.

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. The B virus Working Group.

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.

Herpesvirus papio 2, an SA8-like alpha-herpesvirus of baboons.

Several SA8 isolates obtained from baboons were compared to the prototype SA8 herpesvirus of African green monkeys. SDS-PAGE and restriction enzyme analyses revealed definite differences between green monkey and baboon isolates. DNA and amino acid sequences of the gB, gD and gJ glycoprotein genes exhibited substantial differences in variable regions. For the gB and gD, the amount of amino acid substitutions between SA8 and the baboon viruses was comparable to levels observed between analogous genes of SA8 & B virus or HSV1 & HSV2. Although a high degree of antigenic cross-reactivity was apparent, virus-specific antigenic determinants were also readily detected. Phylogenetic analyses supported separation of the baboon isolates and SA8 as distinct viruses. Taken together these results suggest that although closely related to SA8, the baboon viruses represent a distinct simian alpha-herpesvirus which we propose be designated Herpesvirus papio 2.

Occurrence of human papillomavirus and p53 gene mutations in Kaposi's sarcoma.

Epidemiological evidence indicates that a sexually transmitted agent might be involved in the etiopathogenesis of Kaposi's sarcoma (KS). The prevalence of human papillomaviruses (HPV) in KS has been the focus of several investigations that have reported conflicting data. In addition, mutations of the p53 gene, which are the most frequent genetic changes found in human tumors, are absent in HPV-positive cervical carcinomas leading to the hypothesis that the function of p53 in HPV-positive tumors is inactivated through binding to the E6 viral gene product. Thus, the present study was designed to investigate the presence of HPV and p53 gene mutations in 17 formalin-fixed, paraffin-embedded KS [7 acquired immunodeficiency syndrome-KS (AIDS-KS) and 10 classic KS] specimens. HPV 6 DNA was detected in an AIDS-KS specimen, and HPV 16 DNA was found in 3 classic KS specimens. Heterozygous mutations of the p53 gene were detected in five (24%) KS samples. No p53 mutations were detected in HPV-positive KS. The p53 mutations were mainly transversions (four of five). These data indicate that HPV may contribute to the pathogenesis of some cases of KS and that p53 alteration may represent a key event in the progression of the malignancy.

Diagnosis and management of human B virus (Herpesvirus 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.

B virus-specific pathogen-free (SPF) breeding colonies of macaques: issues, surveillance, and results in 1992.

The NIH's National Center for Research Resources, Comparative Medicine Program has sponsored a multi-institutional program for the establishment of specific pathogen-free (SPF) macaque colonies. Herpes B virus (Cercopithecine herpesvirus I) has been targeted as part of this surveillance. Participating institutions have established individual timetables for frequency of testing, types of monitoring, and husbandry techniques, all with the common goal of producing pathogen-free monkeys for research. From January 1990 through December 1992, we screened animals for evidence of B virus infection, using ELISA and immunoblot to detect humoral antibodies. A total of 984 animals were tested during the first year of the program. At the start of the third year, 631 animals remained in our testing program. Of the 36.9% eliminated for all causes over a 3-year period, B virus screening accounted for 12.1, 1.2, and 0.5% during years 1, 2, and 3, respectively. The greatest threat to the success of the program comes from failure to detect seronegative animals with latent infections, if they do indeed exist, either in first-year animals or animals introduced in subsequent years. The best assurance that a colony is SPF comes from negative results of repeated testing. Introducing new animals into an established SPF colony should be done only after careful screening. Simulations using mathematical models suggest that the best way to detect seronegative animals with latent infections is monthly or bimonthly testing separated by a waiting period. Duration of the waiting period cannot be defined precisely until more is known about the reactivation potential of putative seronegative animals with latent infections.

Infrequent shedding and transmission of herpesvirus simiae from seropositive macaques.

The epizootiologic properties of Herpesvirus simiae (B virus) were studied in singly housed macaques (Macaca mulatta and M. fascicularis) in a biomedical vivarium to determine whether commonly encountered environments and procedures such as quarantine, breeding, Caesarean section, parturition, and social stress induced virus shedding and transmission. Macaques were tested serologically and for infectious virus. Oral, conjunctival, and vaginal swab samples were obtained repeatedly. Virus excretion was not detected during a 7-week quarantine of 32 newly acquired, singly housed animals tested every other week for 6 weeks, and none of 19 seronegative animals from this group seroconverted during 7 weeks in quarantine. No virus shedding was detected in 16 seropositive animals tested weekly for 3 weeks after Caesarean section or normal parturition or in 11 seropositive animals following introduction of new males to animals rooms. One animal seroconverted after repeated breeding of seropositive animals to seronegative partners. Fifty-three singly housed offspring remained seronegative for up to 10 years, even if born to seropositive dams, and only 1 of 86 singly housed animals less than 7 years old was seropositive. These results suggest that shedding of B virus from seropositive macaques is uncommon, when subjected to common laboratory procedures or environments, and that transmission is rare in singly housed animals. These results may be useful in establishing B virus-free colonies of macaques.

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.

Epidemiology of cercopithecine herpesvirus 1 (B virus) infection and shedding in a large breeding cohort of rhesus macaques.

The epidemiology of B virus infection in a large (n = 157) cohort of rhesus macaques at the California Regional Primate Research Center was evaluated prospectively from September 1989 through January 1991 by serial physical examinations, a behavioral substudy (n = 51), and repeated diagnostic testing. Half were B virus antibody-positive at baseline; subsequently, incident cases of infection were documented through serology alone (42) or with virus isolation (5). Eight recurrent infections and a single symptomatic (primary) case were observed. Risk of B virus infection increased as monkeys aged, with few > 3 years old remaining uninfected. Postpubertal monkeys and those entering sexual adolescence (2-3 years) were at greatest risk, although wounding by cagemates and breeding history (for females) were both significant predictors of time to infection. B virus was isolated from oral or conjunctival and genital tissues in equal proportions. Transmission occurred only during the breeding season, possibly coinciding with an elevation in social stressors in the population.