Distribution of herpes simplex virus types 1 and 2 genomes in human spinal ganglia studied by PCR and in situ hybridization

Clinical data indicate that the recurring herpes simplex virus (HSV) from oro-labial lesions is HSV subtype 1 and that the virus from genital lesions is HSV-2. This suggests that HSV-1 and HSV-2 reside in latent forms in the trigeminal ganglia and sacral ganglia, respectively. However, the distribution of latent HSV-1 and HSV-2 infections in human spinal ganglia has not been fully examined. This report concerns the application of polymerase chain reaction (PCR) and in situ hybridization (ISH) to such a study. By using PCR and employing the respective primers, HSV-1 and HSV-2 DNAs were detected in 207 of 524 samples from 262 spinal ganglia (from the cervical to the sacral ganglia) examined on both sides. The percentages of HSV-1 and HSV-2 detected in a given set of ganglia were similar, indicating an absence of site preference. By ISH, few but positive hybridization signals were detected evenly in sacral ganglia sections. The data suggest that regional specificity of recurrent HSV infections is not due to regional distribution of latent virus, but that local host factors may be important for recurrences. J. Med. Virol. 52:136–142, 1997. © 1997 Wiley-Liss, Inc.     

Factors affecting herpes simplex virus reactivation from the explanted mouse brain

The majority of encephalitis induced by herpes simplex virus type I (HSV-1) is due to viral reactivation from latency, but few studies have investigated the factors influencing viral reactivation in the brain due to the lack of a sensitive assay. We have established an ex vivo explant assay, which induced efficient viral reactivation in the dissociated mouse brain. Applying this assay, we investigated the infection of four HSV-1 strains with varying degrees of neurovirulence in three mouse strains with different levels of susceptibility to HSV-1 infection. We found that virulent HSV-1 strains and susceptible mouse strains exhibited prolonged viral growth during acute infection, increased latent viral genomes, and efficient explant reactivation in the brain stem. Collectively, both viral neurovirulence and host susceptibility positively correlate with HSV-1 reactivation from the explanted mouse brain.     

Either a CD4(+)or CD8(+)T cell function is sufficient for clearance of infectious virus from trigeminal ganglia and establishment of herpes simplex virus type 1 latency in mice

Following ocular infection of normal mice, herpes simplex virus type 1 (HSV-1) establishes a latent infection in the trigeminal ganglia (TG) with the complete absence of detectable infectious virus. In this study, the role of CD4(+)and CD8(+)T cell dependent immune responses is examined in relation to clearing infectious virus from the TG following HSV-1 ocular challenge. Nude mice, which lack T cells, and MHC(o/o)mice, which lack both MHC class I and MHC class II, were challenged ocularly with wild-type HSV-1. Over 70% of the TG from mice surviving the infection contained infectious virus, indicative of a chronic infection in these TG, rather than a latent infection. No infectious virus was detected in TGs from infected C57BL/6 parental mice. Ocular challenge of CD4(o/o)A(beta(o/o, CD8(o/o)or beta(2)m(o/o)mice resulted in latent rather than chronic infection. Similarly, when C57BL/6 mice were depleted for CD4(+)or CD8(+)T cells from 4 days before ocular challenge to 26 days after ocular challenge, no free virus was detected in TGs of challenged mice. In contrast, when mice were depleted of both their CD4(+)and CD8(+)T cells, over 90% of TGs were positive for free virus, suggesting that the lack of virus clearance was due to the combined lack of both CD4(+)T cells and CD8(+)T cells (i.e. in the presence of either CD4(+)T cells or CD8(+)T cells alone all of the infectious virus was cleared and latency was established).))Copyright 1999 Academic Press.     

Spread of herpes simplex virus type 1 in the central nervous system during experimentally reactivated encephalitis

Because many of the features of reactivated herpes simplex virus type 1 (HSV-1) central nervous systems (CNS) infections in vivo are incompletely understood, we used an animal model to study the development of the morphological, ultrastructural, radiological and immunological changes which occurred during acute and experimentally reactivated diseases. Rabbits were intranasally inoculated with HSV-1, and their latent trigeminal ganglionic and CNS infections were reactivated by intravenous injection of cyclophosphamide and dexamethasone. Technetium brain scans were performed to localize areas of blood-brain barrier breakdown, and cerebrospinal fluid (CSF) was analysed for IgG content by radial immunodiffusion assays. Nervous system tissues were studied by in situ hybridization and by immunofluorescent, light and electron microscopic techniques. Diffuse uptake of technetium was observed as HSV-1 spread transsynaptically into the brain during the acute phase of infection, and viral antigens and nucleic acids were detected in both the CNS olfactory and trigeminal systems. During latency, viral RNA was detected in the nuclei of neurons within the CNS olfactory cerebral and entorhinal cortices, indicating that HSV-1 became latent within the same CNS structures that were involved during the acute phase of infection. Following drug-induced reactivation, the brain scans revealed a more focal breakdown of the blood-brain barrier, and both neurons and neuronal processes in the entorhinal and olfactory cortices contained viral nucleic acids which correlated with the ultrastructural presence of HSV-1 virions. During the reactivated phase of infection a marked increase in the CSF IgG index occurred without an increase in the CSF: serum albumen ratio indicating a prompt intrathecal response in infected rabbits as compared to controls. To some extent, the CSF IgG index reflected the degree of histopathological damage.     

Herpes simplex type 1 infects and establishes latency in the brain and trigeminal ganglia during primary infection of the lip in cotton rats and mice

Summary.  The majority of the human population has been infected with herpes simplex virus type 1 (HSV-1). During a typical primary episode, HSV-1 spreads from the oral pharynx to the trigeminal ganglia, where a latent HSV-1 infection is established. Cold sores at the mucocutaneous junction of the lip are the typical manifestation of recurrent HSV-1. We investigated whether HSV-1 also infects the brain during the primary infection. We used HSV-1 infected BALB/c mice and inbred cotton rats as models. While both species were susceptible to HSV-1 infection, the time course of lesion formation and healing in the cotton rat more closely reflected that seen in humans. In both species, HSV-1 replicated in the brainstem and cerebellum, as well as the trigeminal ganglia during a primary infection of the lip. The brain infection was produced by a low inoculation dose, and did not cause observable neurologic signs or mortality. Using PCR and RT-PCR techniques, we demonstrated HSV-1 thymidine kinase in the absence of infectivity in the brains of both species 30–40 days after primary infection. Received January 8, 2001 Accepted May 3, 2001     

The vomeronasal chemosensory system as a route of neuroinvasion by herpes simplex virus

We have investigated the potential of neurotropic microbes to invade the central nervous system (CNS) via the peripheral nervous system. Herpes simplex virus type 1 (HSV-1) strain KH6 and herpes simplex virus type 2 (HSV-2) strain 186 were found to infect chemosensory neurons in the vomeronasal organ (the pheromone detector) following intranasal inoculation of mice. HSV-1 strain KH6 infection was further transmitted to the accessory olfactory bulb (first relay), the medial amygdala (second relay), and the bed nucleus of the stria terminalis and the ventromedial hypothalamus (third relay). HSV-1 strain KH6 also targeted the olfactory and trigeminal systems. HSV-2 strain 186 predominantly attacked the brainstem including the trigeminal system. While both viruses did not induce apoptosis in infected chemosensory neurons, they did in infected brain tissue. These results suggest that neurotropic viruses can invade the brain by infecting vomeronasal chemosensory neurons and that the restrained induction of apoptosis in the infected neurons may facilitate viral transmission to the CNS.     

Detection of viral DNA and activation of latent herpes simplex virus in the rabbit neural tissue

Both trigeminal ganglia, brain stem, and cornea from rabbits with established latent herpes simplex virus type 1 (HSV-1) infection were examined by explantation and by spot blot hybridization using strain 17 Kpn I fragments i, d, and h and the DNA extracted from above mentioned tissues. Correlation between positive hybridization and reactivation of infectious virus in the cultured explants was documented by enhanced hybridization with the DNA extracts from explanted ganglion samples. In addition, we found positive hybridization in some noncultured ganglion and brain stem samples which did not yield infectious virus by explantation. Keeping in mind the pitfalls of false positive hybridization, the results may indicate during latency the presence in neural tissues of HSV DNA sequences which did not spontaneously reactivate in culture.     

Role of dendritic cells in enhancement of herpes simplex virus type 1 latency and reactivation in vaccinated mice

Ocular infection with herpes simplex virus type 1 (HSV-1) frequently leads to recurrent infection, which is a major cause of corneal scarring. Thus, the prevention of the establishment of latency should be a primary goal of vaccination against HSV-1. To this end, we have examined the contribution of dendritic cells (DCs) to the efficacy of a vaccine against ocular HSV-1 infection. Transgenic mice (expressing a CD11c-diphtheria toxin receptor-green fluorescent protein construct) with a BALB/c background were immunized with a vaccine consisting of DNA that encodes five HSV-1 glycoproteins or were immunized with vector control DNA. The vaccinated mice were then depleted of their DCs through the injection of diphtheria toxin before and after ocular challenge with HSV-1. Analyses of HSV-1 replication in the eye, blepharitis, corneal scarring, and the survival of the infected mice upon primary infection indicated that DC depletion neither promoted nor compromised the efficacy of the vaccine. In contrast, DC depletion was associated with an approximately fivefold reduction in the level of latent virus in the trigeminal ganglia (TGs) of latently infected mice, as well as a significant reduction in the reactivation rate of latent virus. The possibility that DCs enhance the latency of HSV-1 in the TGs of ocularly infected mice suggests for the first time that DCs, rather than acting as “immune saviors,” can exacerbate disease and compromise vaccine efficacy by enhancing viral latency and reactivation.     

Carriage of herpes simplex virus and human papillomavirus in oral mucosa is rare in young women: A long-term prospective follow-up

BackgroundFollowing the primary oral infection, herpes simplex virus (HSV) establishes latency in the ganglia of sensory neurons. Episodically induced by stress, HSV is able to cause recurrent infection at the primary infection site, accompanied by virus shedding. The oral shedding of HSV contributes to mother–child-transmission of HSV. Human papillomavirus (HPV) is associated with oral malignancies, and its interaction with oral HSV should be studied further.ObjectivesTo analyze the prevalence of HSV-1 and HSV-2-infection in oral mucosal scrapings of women during and after pregnancy and to elucidate the prevalence of HPV and HSV-co-carriage in oral mucosa.Study designA longitudinal cohort study of 304 mothers in the Finnish Family HPV study followed-up for 6 years after pregnancy, with 7 serial samplings. Mothers’ oral brush samples were analyzed with quantitative PCR for HSV-1 and -2 DNA and the findings were compared with their HPV DNA status.ResultsAltogether, 2.2% of all 1873 collected epithelial brush samples were HSV-1 DNA positive, while none tested HSV-2 DNA positive. Of the 304 mothers, 11.8% were HSV-1 DNA positive at least once. Most of the women who tested HSV-1 DNA positive before delivery remained HSV-1 DNA positive also after pregnancy. HSV-1 positive women were almost invariably HPV-negative; only four (0.2%) samples were detected with HSV-HPV co-carriage.ConclusionsThis is the first prospective follow-up study on oral HSV shedding and its association with coexistent HPV, analyzed in the same oral mucosal scrapings. HSV and HPV co-carriage is rare in oral mucosa of healthy young mothers.     

Colonization of murine ganglia by a superinfecting strain of herpes simplex virus

We report on the colonization of murine trigeminal ganglia after sequential infection of mice by herpes simplex viruses (HSVs). In preliminary studies, we have established that whereas the HSV-1(F) strain efficiently colonizes ganglia when inoculated by either the ear or eye routes, the HSV-1 X HSV-2 recombinant C7D colonizes ganglia when inoculated by the eye route only. The experimental design consisted of inoculating the right eye with C7D on day 1 and with HSV-1(F) in both left and right eyes on day 26. Both right and left trigeminal ganglia were removed and analyzed independently for latent virus on day 52. Our studies indicate that HSV-1(F) viruses were recovered from all left trigeminal ganglia but from only a small number of right trigeminal ganglia. Some right trigeminal ganglia yielded no viruses, whereas others yielded both C7D and HSV-1(F) viruses identified on the basis of plaque morphology and restriction enzyme cleavage patterns of viral DNA. The results indicate that more than one virus may colonize the same ganglion and that trigeminal ganglia may be protected from colonization by a superinfecting virus by determinants acting at a local level in the absence of demonstrable virus.     

Latent varicella-zoster viral DNA in human trigeminal and thoracic ganglia

BACKGROUND. Some human herpesviruses become latent in dorsal-root ganglia. Primary infection with the varicella-zoster virus causes chickenpox, followed by latency, and subsequent reactivation leading to shingles (zoster), but the frequency and distribution of latent virus have not been established. METHODS. Using the polymerase chain reaction, we performed postmortem examinations of trigeminal and thoracic ganglia of 23 subjects 33 to 88 years old who had not recently had chickenpox or shingles to identify the presence of latent varicella-zoster viral DNA. Oligonucleotide primers representing the origin of replication of the varicella-zoster virus and varicella-zoster virus gene 29 were used for amplification. RESULTS. Among the 22 subjects seropositive for the antibody to the virus, both the viral origin-of-replication and gene-29 sequences were detected in 13 of 15 subjects (87 percent) in whom trigeminal ganglia were examined and in 9 of 17 (53 percent) in whom thoracic ganglia were examined. Viral DNA was not detected in brain or mononuclear cells from the seropositive subjects. None of three thoracic ganglia from the one seronegative subject contained varicella-zoster viral DNA. CONCLUSIONS. These findings indicate that after primary infection with varicella-zoster virus (varicella), the virus becomes latent in many ganglia--more often in the trigeminal ganglia than in any thoracic ganglion--and that more than one region of the viral genome is present during latency.     

Herpes simplex virus type 1 infection induces upregulation of interleukin-23 (p19) mRNA expression in trigeminal ganglia of BALB/c mice.

We investigated the expression kinetics of several cytokines in trigeminal ganglia (TG) and in brains of BALB/c mice during the course of ocular herpes simplex virus type 1 (HSV-1) infection. All mice recovered from the infection within 2 weeks. The quantitative rapid real-time RT-PCR method was used to analyze interleukin-4 (IL-4), interferon-gamma (IFN-gamma), IL-12p35, IL-12p40, and the recently described IL-23 (p19) mRNA in TG, brain, and splenocyte samples. In TG, we found elevated expression of mRNA for IL-23 (p19) from early acute infection (day 3) to the beginning of the latent phase (day 14). The increase was not detected in brain or in the spleen. IL-4 expression occurred in both TG and brain from the beginning of the experiment to the latent phase. During the latent phase (days 14 and 31), IL-4 expression was significantly elevated in the brain when compared with the uninfected controls (p < 0.05). Considerable expression of IFN-gamma mRNA was detected in TG of mice during acute HSV-1 infection. The expression of IL-23 was detected also in the brains of the mice, even though no significant changes were found during the acute HSV-1 infection. This is, to our knowledge, the first report to show elevated expression of IL-23 (p19) mRNA (p < 0.05) during viral infection in TG of mice.     

Suitability and clinical application of a multiplex nested PCR assay for the diagnosis of herpes simplex virus infections

A novel multiplex nested polymerase chain reaction (PCR) assay was designed and evaluated for routine diagnosis of herpes simplex virus (HSV) infections in patients with either putative HSV infection of the central nervous system or suspected HSV keratitis. Single-tube amplification of HSV type 1 (HSV-1) or type 2 (HSV-2) DNA extracted from cerebrospinal fluid (CSF) or from keratectomy specimens was followed by differentiation of the virus type-specific PCR products either by agarose gel analysis or by DNA enzyme immunoassay. Among 417 CSF specimens obtained from 395 consecutive patients with clinically suspected HSV infection, 11 (2.6%) were positive for HSV-1 DNA and four (1.0%) probes were positive for HSV-2 DNA. None of the specimens was positive for both HSV-1 and HSV-2 DNA. The genome of HSV-2 was detected in a CSF sample obtained from a woman with meningoencephalitis and genital herpes. The presence of PCR inhibitors was detected in six of 111 (5.4%) reconstructed CSF samples. Inhibition could be removed following extraction with a commercial kit. HSV-1 DNA, but no HSV-2 DNA, was detected in corneal buttons obtained from patients with suspected herpetic keratitis. No contamination has been recorded during the 2-year routine use of this test, which has met the specific requirements of a diagnostic laboratory. © 1996 Wiley-Liss, Inc.