Herpes B virus replication and viral lesions in the liver of a cynomolgus macaque which died from severe disease with rapid onset.

Herpes B virus (BV, Macacine alphaherpesvirus 1) infects macaques asymptomatically, with rare exceptions, but can cause fatal encephalitis in humans. Here, we report disseminated BV infection in a cynomolgus macaque that had died within 12 hour after the onset of unspecific symptoms. Multifocal lesions surrounded by viral antigen were detected in liver while other organs remained inconspicuous, indicating that the liver is a major target. Moreover, high copy numbers of viral DNA were found in feces, underlining the excrements are a potential source of transmission.

B Virus (Macacine herpesvirus 1) Glycoprotein D Is Functional but Dispensable for Virus Entry into Macaque and Human Skin Cells.

UNLABELLED: Glycoprotein D (gD) plays an essential role in cell entry of many simplexviruses. B virus (Macacine herpesvirus 1) is closely related to herpes simplex virus 1 (HSV-1) and encodes gD, which shares more than 70% amino acid similarity with HSV-1 gD. Previously, we have demonstrated that B virus gD polyclonal antibodies were unable to neutralize B virus infectivity on epithelial cell lines, suggesting gD is not required for B virus entry into these cells. In the present study, we confirmed this finding by producing a B virus mutant, BV-ΔgDZ, in which the gD gene was replaced with a lacZ expression cassette. Recombinant plaques were selected on complementing VD60 cells expressing HSV-1 gD. Virions lacking gD were produced in Vero cells infected with BV-ΔgDZ. In contrast to HSV-1, B virus lacking gD was able to infect and form plaques on noncomplementing cell lines, including Vero, HEp-2, LLC-MK2, primary human and macaque dermal fibroblasts, and U373 human glioblastoma cells. The gD-negative BV-ΔgDZ also failed to enter entry-resistant murine B78H1 cells bearing a single gD receptor, human nectin-1, but gained the ability to enter when phenotypically supplemented with HSV-1 gD. Cell attachment and penetration rates, as well as the replication characteristics of BV-ΔgDZ in Vero cells, were almost identical to those of wild-type (wt) B virus. These observations indicate that B virus can utilize gD-independent cell entry and transmission mechanisms, in addition to generally used gD-dependent mechanisms. IMPORTANCE: B virus is the only known simplexvirus that causes zoonotic infection, resulting in approximately 80% mortality in untreated humans or in lifelong persistence with the constant threat of reactivation in survivors. Here, we report that B virus lacking the gD envelope glycoprotein infects both human and monkey cells as efficiently as wild-type B virus. These data provide evidence for a novel mechanism(s) utilized by B virus to gain access to target cells. This mechanism is different from those used by its close relatives, HSV-1 and -2, where gD is a pivotal protein in the virus entry process. The possibility remains that unidentified receptors, specific for B virus, permit virus entry into target cells through gD-independent pathways. Understanding the molecular mechanisms of B virus entry may help in developing rational therapeutic strategies for the prevention and treatment of B virus infection in both macaques and humans.

Macacine Herpesvirus 1 in Long-Tailed Macaques, Malaysia, 2009-2011.

Macacine herpesvirus 1 (MaHV1; B virus) naturally infects macaques (Macaca spp.) and can cause fatal encephalitis in humans. In Peninsular Malaysia, wild macaques are abundant, and translocation is used to mitigate human-macaque conflict. Most adult macaques are infected with MaHV1, although the risk for transmission to persons who handle them during capture and translocation is unknown. We investigated MaHV1 shedding among 392 long-tailed macaques (M. fascicularis) after capture and translocation by the Department of Wildlife and National Parks in Peninsular Malaysia, during 2009-2011. For detection of MaHV1 DNA, PCR was performed on urogenital and oropharyngeal swab samples. Overall, 39% of macaques were shedding MaHV1 DNA; rates of DNA detection did not differ between sample types. This study demonstrates that MaHV1 was shed by a substantial proportion of macaques after capture and transport and suggests that persons handling macaques under these circumstances might be at risk for exposure to MaHV1.

Discovery of herpes B virus-encoded microRNAs.

Herpes B virus (BV) naturally infects macaque monkeys and is a close relative of herpes simplex virus. BV can zoonotically infect humans to cause a rapidly ascending encephalitis with approximately 80% mortality. Therefore, BV is a serious danger to those who come into contact with these monkeys or their tissues and cells. MicroRNAs are regulators of gene expression, and there have been reports of virus-encoded microRNAs. We hypothesize that BV-encoded microRNAs are important for the regulation of viral and cellular genes. Herein, we report the discovery of three herpes B virus-encoded microRNAs.

Excretion of B virus in monkeys and evidence of genital infection.

3 different types of observation all demonstrated that B virus (Herpesvirus simiae) infection of monkeys was not confined to the mouth but was also a genital infection. (1) Latent B virus was reactivated in a seropositive female monkey, which was immunosuppressed with antilymphocyte globulin, and infectious virus was excreted in the genital tract. (2) During an epizootic in a breeding colony, B virus was isolated from 4 genital and 3 oral sites as well as from a skin lesion. (3) In cultures of sensory nerve ganglia taken from seropositive monkeys, B virus was recovered more frequently from ganglia subserving the genital region than the oral region.

Monkey B virus (Cercopithecine herpesvirus 1).

Macaques are a particularly valuable nonhuman primate model for a wide variety of biomedical research endeavors. B virus (Cercopithecine herpesvirus 1; BV) is an alpha-herpesvirus that naturally infects conventional populations of macaques. Serious disease due to BV is rare in macaques, but when transmitted to humans, BV has a propensity to invade the central nervous system and has a fatality rate greater than 70% if not treated promptly. The severe consequences of human BV infections led to the inclusion of BV in the original NIH list of target viruses for elimination by development of specific pathogen-free rhesus colonies. In macaques and especially in humans, diagnosis of BV infection is not straightforward. Furthermore, development and maintenance of true BV specific pathogen-free macaque colonies has proven dif cult. In this overview we review the natural history of BV in macaques, summarize what is known about the virus at the molecular level, and relate this information to problems associated with diagnosis of BV infections and development of BV-free macaque colonies.

In vivo experimentation with simian herpesviruses: assessment of biosafety and molecular contamination.

In vivo studies with highly pathogenic viruses prompt concerns regarding the persistence of infectious virus in pathology specimens. Although formalin fixation of tissues may inactivate infectious virus, fixation may also degrade viral nucleic acid and antigens, thereby limiting detection of virus in tissues by polymerase chain reaction (PCR) amplification or immunohistochemistry (IHC). We sought to: 1) assess the rate of inactivation of infectious virus in tissue specimens during formalin fixation, 2) assess IHC recognition of viral antigens and PCR detection of viral DNA after long-term (14 d) formalin fixation, and 3) investigate microtome contamination by DNA carry-over to subsequently sectioned tissues. Infectious baboon herpesvirus HVP2 could be recovered from fresh tissues of infected mice but not those fixed in formalin for >/=24 h. The intensity of IHC staining of viral antigen was unaffected by the duration of formalin fixation. PCR detection of viral DNA was negatively impacted by formalin fixation and/or heat inherent to paraffin processing; however, amplification of very short DNA sequences using real-time PCR was not affected. Lastly, microtome contamination by viral DNA was demonstrated by PCR screening of uninoculated control tissues that were sectioned after sectioning infected tissues. In summary, infectious virus is inactivated after only 24 h of formalin fixation whereas IHC staining remains sensitive in tissues fixed for up to 14 d. Formalin fixation does degrade DNA, but viral DNA can be detected by PCR amplification of very short DNA sequences. In addition, viral DNA can contaminate a microtome knife such that subsequently sectioned uninoculated control tissues exhibit false positive PCR amplification.

Axonal-transsynaptic spread as the basic pathogenetic mechanism in B virus infection of the nervous system.

An experimental study on the pathogenesis of B virus infection in the mouse has documented that the agent spreads in an axonal-transsynaptic manner in the nervous system. The characteristics of the spread of B virus are similar to those of other members of the herpes virus group.

Genetic and functional complementation of the HSV1 UL27 gene and gB glycoprotein by simian alpha-herpesvirus homologs.

Utilizing co-transfection of DNA from glycoprotein gB- strain of HSV1 and cloned fragments of several simian alpha-herpesviruses containing the UL26, UL27 (gB glycoprotein), and UL28 gene homologs, replication-competent recombinant viruses were produced. Genetic analysis of one HSV1/SA8 recombinant (HSV1/SgB) demonstrated the presence of SA8 DNA comprising the entire UL27 (gB) gene and parts of the UL28 and UL26 ORFs in an otherwise HSV1 genome. The recombinant was shown to express the SA8 gB and p40 proteins (UL27 & UL26.5 gene products, respectively); all other proteins were indistinguishable from those of HSV1. The recombinant behaved like SA8 in gB-specific virus neutralization and cell surface antibody binding assays, while plaque morphology and replication kinetics were very similar to HSV1. Despite its overwhelming HSV1 genetic constitution, the recombinant displayed a pathogenic phenotype in mice very different from the parental HSV1. While HSV1 produced corneal disease in ocularly infected mice and readily spread to the nervous system. HSV1/SgB was markedly impaired in both respects. These results demonstrate the functional equivalency of the cercopithecine monkey virus gB glycoproteins and genes (including transcriptional regulatory elements) in HSV1, the functional nature of HSV1/SA8 chimeric UL28 and UL26 genes/proteins, and that UL28, gB and/or p40 proteins may effect the pathogenicity of HSV1.

Comparison of the EBNA1 proteins of Epstein-Barr virus and herpesvirus papio in sequence and function.

The EBNA1 protein of Epstein-Barr virus (EBV) supports replication and maintenance of the circularized viral chromosome in cells that are latently infected. We have isolated, sequenced, and functionally characterized the EBNA1 gene of herpesvirus papio (HVP), an EBV-like virus that infects baboons. The amino acid sequences of EBNA1 of HVP and EBV are 56% identical, if the difference in the length of the glycine and alanine containing repetitive region, which is much shorter for HVP EBNA1, is omitted for the calculation. The key structural features of the DNA-binding/dimerization domain (the carboxyl-terminal domain) appear to have been conserved, as have amino acids in the two regions thought to be most critical for DNA binding. Most of the salient features of the amino-terminal two-thirds of EBNA1 (the amino-terminal domain), including a dearth of sequences predictive of alpha-helical or beta-sheet structures, are shared by the two sequences, although numerous gaps in this region were needed for alignment of the sequences. The amino-terminal fifty amino acids of EBNA1 of both EBV and HVP weakly resemble the amino terminus of rat ribosomal protein S2. Plasmids carrying oriP of either virus replicated stably in mammalian cells and supported efficient outgrowth of colonies under selection when supported by EBNA1 from either virus, although with each oriP there was a noticeable preference for EBNA1 to be from the same virus. HVP EBNA1 was less effective than EBV EBNA1 at activating the enhancer function of EBV oriP and under certain conditions was less effective than EBV EBNA1 at supporting maintenance of plasmids carrying EBV oriP. Results obtained with hybrid EBNA1 molecules indicated that differences in the amino-terminal and carboxyl-terminal domains, respectively, are primarily responsible for the differences in transcriptional activation and plasmid maintenance, respectively. The results showed that changes within EBNA1 can differentially alter its transcriptional and replicational activities.

Naturally acquired simian varicella virus infection in African green monkeys.

Simian varicella virus (SVV) infection of primates shares clinical, pathological, immunological, and virological features with varicella-zoster virus infection of humans. Natural varicella infection was simulated by exposing four SVV-seronegative monkeys to monkeys inoculated intratracheally with SVV, in which viral DNA and RNA persist in multiple tissues for more than 1 year (T. M. White, R. Mahalingam, V. Traina-Dorge, and D. H. Gilden, J. Neurovirol. 8:191-205, 2002). The four naturally exposed monkeys developed mild varicella 10 to 14 days later, and skin scrapings taken at the time of the rash contained SVV DNA. Analysis of multiple ganglia, liver, and lung tissues from the four naturally exposed monkeys sacrificed 6 to 8 weeks after resolution of the rash revealed SVV DNA in ganglia at multiple levels of the neuraxis but not in the lung or liver tissue of any of the four monkeys. This animal model provides an experimental system to gain information about varicella latency with direct relevance to the human disease.

Biology of B virus in macaque and human hosts: a review.

B virus is a zoonotic alpha-herpesvirus enzootic in Asian monkeys of the genus Macaca. At least 25 cases of human disease caused by B virus have occurred to date, leading to death in 16 instances. Advances in the technology available for the diagnosis of B virus infection and in the agents for its treatment are improving the prognosis for cases in human beings. Efforts are under way at several institutions in the United States to establish B virus-free colonies of rhesus macaques for use in biomedical research. Unfortunately, the epidemiology of B virus in group-housed macaques is poorly understood. The elucidation of factors important in the transmission of B virus between monkeys will greatly enhance efforts to eradicate this virus and may help to minimize further human exposure to the agent.