The complete DNA sequence of herpes B virus (Cercopithecine herpesvirus 1) strain E2490, isolated from a rhesus macaque, was determined. The total genome length is 156,789 bp, with 74.5% G؉C composition and overall genome organization characteristic of alphaherpesviruses. The first and last residues of the genome were defined by sequencing the cloned genomic termini. There were six origins of DNA replication in the genome due to tandem duplication of both oriL and oriS regions. Seventy-four genes were identified, and sequence homology to proteins known in herpes simplex viruses (HSVs) was observed in all cases but one. The degree of amino acid identity between B virus and HSV proteins ranged from 26.6% (US5) to 87.7% (US15). Unexpectedly, B virus lacked a homolog of the HSV ␥ 1 34.5 gene, which encodes a neurovirulence factor. Absence of this gene was verified in two low-passage clinical isolates derived from a rhesus macaque and a zoonotically infected human. This finding suggests that B virus most likely utilizes mechanisms distinct from those of HSV to sustain efficient replication in neuronal cells. Despite the considerable differences in G؉C content of the macaque and B virus genes (51% and 74.2%, respectively), codons used by B virus are optimal for the tRNA population of macaque cells. Complete sequence of the B virus genome will certainly facilitate identification of the genetic basis and possible molecular mechanisms of enhanced B virus neurovirulence in humans, which results in an 80% mortality rate following zoonotic infection.Comparative genome analyses of closely related viruses offer insight into the protein coding capacities of viral genomes (18, 69), a means to biologically classify viruses (2, 19) and identify viral genes involved in virulence and pathogenicity (1,37,67). The number of completely sequenced viral genomes has been increasing rapidly and now exceeds 1,000, including 29 herpesvirus genomes (GenBank data, http://www.ncbi.nlm.nih.gov /PMGifs/Genomes/viruses.html).B virus (Cercopithecine herpesvirus 1, monkey B virus) is a member of the subfamily Alphaherpesvirinae, which together with human herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) constitutes the genus Simplexvirus. B virus generally causes only mild localized or asymptomatic infections in its natural hosts, Asian monkeys of the genus Macaca (33,34,74). In contrast, B virus infections in foreign hosts, humans or monkey species other than macaques, often result in encephalitis, encephalomyelitis, and death (53,73,74).The genome organization of B virus is similar to that of HSV-1 and HSV-2: the unique long (U L ) and unique short (U S ) segments flanked by inverted long (R L ) and short (R S ) repeat sequences are covalently joined in four possible isomeric configurations (26). Sequence analysis of the partial U S regions of B virus and simian agent 8 virus demonstrated that human and primate viruses are colinear in this genomic segment (51). However, only a limited number of B virus gene sequences from the U L region have b...

The mapping of linear epitopes of B virus (Cercopithecine herpesvirus 1 and herpes B virus) glycoprotein D (gD) was accomplished by screening the constructed gD epitope library with serum from B virus-infected macaques. The immunodominant epitope, gD (362-370), was identified within the C-terminal region of B virus gD that was highly conserved among 19 B virus clinical isolates but was not present in either herpes simplex virus (HSV)-1 or HSV-2 gD. A substantial percentage of serum samples from macaques (95%) and humans (80%) infected with B virus contained antibodies to this epitope. Antibodies against HSV types 1 or 2 did not react with this epitope; thus, gD (362-370) has unique potential to detect B virus-specific antibody responses in human serum, even in the presence of antibodies to HSV-1 and HSV-2.

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 antigenbased 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 crossreacted 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.Human infection with B virus (also called cercopithecine herpesvirus 1, monkey B virus, and herpes B virus) is the most feared occupational hazard among individuals working with macaque monkeys, since fatality is often the outcome of infection, which proceeds in the absence of effective antiviral therapy (25,56). The use of macaques in research has been steadily growing over the last decade and is expected to rise quickly in the near future due to the increasing demands for these animals for use in HIV/AIDS investigations, vaccine trials, drug testing, and research into bioterrorism agents. As macaque usage increases, frequencies of human exposures to B virus are increasing as well. Rapid and accurate diagnostic tests are urgently needed to aid in the early identification of clinical cases, which is essential for a timely initiation of antiviral therapies in zoonotically infected humans. In addition to human diagnostics, enhanced assays are required for monitoring specific-pathogen-free (SPF) macaque colonies established by the National Institutes of Health for the breeding of B virusfree animals (55), as these animals often demonstrate only very low levels of antibody.Unfortunately, a direct diagnosis of infectio...

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.