Reanalysis of single-cell RNA sequencing data does not support herpes simplex virus 1 latency in non-neuronal ganglionic cells in mice.

Most individuals are latently infected with herpes simplex virus type 1 (HSV-1), and it is well-established that HSV-1 establishes latency in sensory neurons of peripheral ganglia. However, it was recently proposed that latent HSV-1 is also present in immune cells recovered from the ganglia of experimentally infected mice. Here, we reanalyzed the single-cell RNA sequencing (scRNA-Seq) data that formed the basis for that conclusion. Unexpectedly, off-target priming in 3' scRNA-Seq experiments enabled the detection of non-polyadenylated HSV-1 latency-associated transcript (LAT) intronic RNAs. However, LAT reads were near-exclusively detected in mixed populations of cells undergoing cell death. Specific loss of HSV-1 LAT and neuronal transcripts during quality control filtering indicated widespread destruction of neurons, supporting the presence of contaminating cell-free RNA in other cells following tissue processing. In conclusion, the reported detection of latent HSV-1 in non-neuronal cells is best explained using compromised scRNA-Seq datasets.IMPORTANCEMost people are infected with herpes simplex virus type 1 (HSV-1) during their life. Once infected, the virus generally remains in a latent (silent) state, hiding within the neurons of peripheral ganglia. Periodic reactivation (reawakening) of the virus may cause fresh diseases such as cold sores. A recent study using single-cell RNA sequencing (scRNA-Seq) proposed that HSV-1 can also establish latency in the immune cells of mice, challenging existing dogma. We reanalyzed the data from that study and identified several flaws in the methodologies and analyses performed that invalidate the published conclusions. Specifically, we showed that the methodologies used resulted in widespread destruction of neurons which resulted in the presence of contaminants that confound the data analysis. We thus conclude that there remains little to no evidence for HSV-1 latency in immune cells.

Increasing antigen presentation on HSV-1-infected cells increases lesion size but does not alter neural infection or latency.

CD8+ T cells have a role in the control of acute herpes simplex virus (HSV) infection and may also be important in the maintenance of latency. In this study we have explored the consequences of boosting the efficacy of CD8+ T cells against HSV by increasing the amount of an MHC I-presented epitope on the surface of infected cells. To do this we used HSVs engineered to express an extra copy of the immunodominant CD8+ T cell epitope in C57Bl/6 mice, namely gB498 (SSIEFARL). Despite greater presentation of gB498 on infected cells, CD8+ T cell responses to these viruses in mice were similar to those elicited by a control virus. Further, the expression of extra gB498 did not significantly alter the extent or stability of latency in our mouse model, and virus loads in skin and sensory ganglia of infected mice were not affected. Surprisingly, mice infected with these viruses developed significantly larger skin lesions than those infected with control viruses and notably, this phenotype was dependent on MHC haplotype. Therefore increasing the visibility of HSV-infected cells to CD8+ T cell attack did not impact neural infection or latency, but rather enhanced pathology in the skin.

Transcriptional Elongation of HSV Immediate Early Genes by the Super Elongation Complex Drives Lytic Infection and Reactivation from Latency.

The cellular transcriptional coactivator HCF-1 is required for initiation of herpes simplex virus (HSV) lytic infection and for reactivation from latency in sensory neurons. HCF-1 stabilizes the viral Immediate Early (IE) gene enhancer complex and mediates chromatin transitions to promote IE transcription initiation. In infected cells, HCF-1 was also found to be associated with a network of transcription elongation components including the super elongation complex (SEC). IE genes exhibit characteristics of genes controlled by transcriptional elongation, and the SEC-P-TEFb complex is specifically required to drive the levels of productive IE mRNAs. Significantly, compounds that enhance the levels of SEC-P-TEFb also potently stimulated HSV reactivation from latency both in a sensory ganglia model system and in vivo. Thus, transcriptional elongation of HSV IE genes is a key limiting parameter governing both the initiation of HSV infection and reactivation of latent genomes.

Robust gene expression changes in the ganglia following subclinical reactivation in rhesus macaques infected with simian varicella virus.

Varicella zoster virus (VZV) causes varicella during acute infection and establishes latency in the sensory ganglia. Reactivation of VZV results in herpes zoster, a debilitating and painful disease. It is believed that VZV reactivates due to a decline in cell-mediated immunity; however, the roles that CD4 versus CD8 T cells play in the prevention of herpes zoster remain poorly understood. To address this question, we used a well-characterized model of VZV infection where rhesus macaques are intrabronchially infected with the homologous simian varicella virus (SVV). Latently infected rhesus macaques were thymectomized and depleted of either CD4 or CD8 T cells to induce selective senescence of each T cell subset. After T cell depletion, the animals were transferred to a new housing room to induce stress. SVV reactivation (viremia in the absence of rash) was detected in three out of six CD8-depleted and two out of six CD4-depleted animals suggesting that both CD4 and CD8 T cells play a critical role in preventing SVV reactivation. Viral loads in multiple ganglia were higher in reactivated animals compared to non-reactivated animals. In addition, reactivation results in sustained transcriptional changes in the ganglia that enriched to gene ontology and diseases terms associated with neuronal function and inflammation indicative of potential damage as a result of viral reactivation. These studies support the critical role of cellular immunity in preventing varicella virus reactivation and indicate that reactivation results in long-lasting remodeling of the ganglia transcriptome.

Acute Simian Varicella Virus Infection Causes Robust and Sustained Changes in Gene Expression in the Sensory Ganglia.

Primary infection with varicella-zoster virus (VZV), a neurotropic alphaherpesvirus, results in varicella. VZV establishes latency in the sensory ganglia and can reactivate later in life to cause herpes zoster. The relationship between VZV and its host during acute infection in the sensory ganglia is not well understood due to limited access to clinical specimens. Intrabronchial inoculation of rhesus macaques with simian varicella virus (SVV) recapitulates the hallmarks of VZV infection in humans. We leveraged this animal model to characterize the host-pathogen interactions in the ganglia during both acute and latent infection by measuring both viral and host transcriptomes on days postinfection (dpi) 3, 7, 10, 14, and 100. SVV DNA and transcripts were detected in sensory ganglia 3 dpi, before the appearance of rash. CD4 and CD8 T cells were also detected in the sensory ganglia 3 dpi. Moreover, lung-resident T cells isolated from the same animals 3 dpi also harbored SVV DNA and transcripts, suggesting that T cells may be responsible for trafficking SVV to the ganglia. Transcriptome sequencing (RNA-Seq) analysis showed that cessation of viral transcription 7 dpi coincides with a robust antiviral innate immune response in the ganglia. Interestingly, a significant number of genes that play a critical role in nervous system development and function remained downregulated into latency. These studies provide novel insights into host-pathogen interactions in the sensory ganglia during acute varicella and demonstrate that SVV infection results in profound and sustained changes in neuronal gene expression. IMPORTANCE: Many aspects of VZV infection of sensory ganglia remain poorly understood, due to limited access to human specimens and the fact that VZV is strictly a human virus. Infection of rhesus macaques with simian varicella virus (SVV), a homolog of VZV, provides a robust model of the human disease. Using this model, we show that SVV reaches the ganglia early after infection, most likely by T cells, and that the induction of a robust innate immune response correlates with cessation of virus transcription. We also report significant changes in the expression of genes that play an important role in neuronal function. Importantly, these changes persist long after viral replication ceases. Given the homology between SVV and VZV, and the genetic and physiological similarities between rhesus macaques and humans, our results provide novel insight into the interactions between VZV and its human host and explain some of the neurological consequences of VZV infection.

Characterization of the immune response in ganglia after primary simian varicella virus infection.

Primary simian varicella virus (SVV) infection in non-human primates causes varicella, after which the virus becomes latent in ganglionic neurons and reactivates to cause zoster. The host response in ganglia during establishment of latency is ill-defined. Ganglia from five African green monkeys (AGMs) obtained at 9, 13, and 20 days post-intratracheal SVV inoculation (dpi) were analyzed by ex vivo flow cytometry, immunohistochemistry, and in situ hybridization. Ganglia at 13 and 20 dpi exhibited mild inflammation. Immune infiltrates consisted mostly of CD8(dim) and CD8(bright) memory T cells, some of which expressed granzyme B, and fewer CD11c(+) and CD68(+) cells. Chemoattractant CXCL10 transcripts were expressed in neurons and infiltrating inflammatory cells but did not co-localize with SVV open reading frame 63 (ORF63) RNA expression. Satellite glial cells expressed increased levels of activation markers CD68 and MHC class II at 13 and 20 dpi compared to those at 9 dpi. Overall, local immune responses emerged as viral DNA load in ganglia declined, suggesting that intra-ganglionic immunity contributes to restricting SVV replication.

Animal models of herpes simplex virus immunity and pathogenesis.

Herpes simplex viruses are ubiquitous human pathogens represented by two distinct serotypes: herpes simplex virus (HSV) type 1 (HSV-1); and HSV type 2 (HSV-2). In the general population, adult seropositivity rates approach 90% for HSV-1 and 20-25% for HSV-2. These viruses cause significant morbidity, primarily as mucosal membrane lesions in the form of facial cold sores and genital ulcers, with much less common but more severe manifestations causing death from encephalitis. HSV infections in humans are difficult to study in many cases because many primary infections are asymptomatic. Moreover, the neurotropic properties of HSV make it much more difficult to study the immune mechanisms controlling reactivation of latent infection within the corresponding sensory ganglia and crossover into the central nervous system of infected humans. This is because samples from the nervous system can only be routinely obtained at the time of autopsy. Thus, animal models have been developed whose use has led to a better understanding of multiple aspects of HSV biology, molecular biology, pathogenesis, disease, and immunity. The course of HSV infection in a spectrum of animal models depends on important experimental parameters including animal species, age, and genotype; route of infection; and viral serotype, strain, and dose. This review summarizes the animal models most commonly used to study HSV pathogenesis and its establishment, maintenance, and reactivation from latency. It focuses particularly on the immune response to HSV during acute primary infection and the initial invasion of the ganglion with comparisons to the events governing maintenance of viral latency.

Type I IFN suppresses Cxcr2 driven neutrophil recruitment into the sensory ganglia during viral infection.

Infection induces the expression of inflammatory chemokines that recruit immune cells to the site of inflammation. Whereas tissues such as the intestine and skin express unique chemokines during homeostasis, whether different tissues express distinct chemokine profiles during inflammation remains unclear. With this in mind, we performed a comprehensive screen of the chemokines expressed by two tissues (skin and sensory ganglia) infected with a common viral pathogen (herpes simplex virus type 1). After infection, the skin and ganglia showed marked differences in their expression of the family of Cxcr2 chemokine ligands. Specifically, Cxcl1/2/3, which in turn controlled neutrophil recruitment, was up-regulated in the skin but absent from the ganglia. Within the ganglia, Cxcl2 expression and subsequent neutrophil recruitment was inhibited by type I interferon (IFN). Using a combination of bone marrow chimeras and intracellular chemokine staining, we show that type I IFN acted by directly suppressing Cxcl2 expression by monocytes, abrogating their ability to recruit neutrophils to the ganglia. Overall, our findings describe a novel role for IFN in the direct, and selective, inhibition of Cxcr2 chemokine ligands, which results in the inhibition of neutrophil recruitment to neuronal tissue.

Analysis of T cell responses during active varicella-zoster virus reactivation in human ganglia.

UNLABELLED: Varicella-zoster virus (VZV) is responsible for both varicella (chickenpox) and herpes zoster (shingles). During varicella, the virus establishes latency within the sensory ganglia and can reactivate to cause herpes zoster, but the immune responses that occur in ganglia during herpes zoster have not previously been defined. We examined ganglia obtained from individuals who, at the time of death, had active herpes zoster. Ganglia innervating the site of the cutaneous herpes zoster rash showed evidence of necrosis, secondary to vasculitis, or localized hemorrhage. Despite this, there was limited evidence of VZV antigen expression, although a large inflammatory infiltrate was observed. Characterization of the infiltrating T cells showed a large number of infiltrating CD4(+) T cells and cytolytic CD8(+) T cells. Many of the infiltrating T cells were closely associated with neurons within the reactivated ganglia, yet there was little evidence of T cell-induced neuronal apoptosis. Notably, an upregulation in the expression of major histocompatibility complex class I (MHC-I) and MHC-II molecules was observed on satellite glial cells, implying these cells play an active role in directing the immune response during herpes zoster. This is the first detailed characterization of the interaction between T cells and neuronal cells within ganglia obtained from patients suffering herpes zoster at the time of death and provides evidence that CD4(+) and cytolytic CD8(+) T cell responses play an important role in controlling VZV replication in ganglia during active herpes zoster. IMPORTANCE: VZV is responsible for both varicella (chickenpox) and herpes zoster (shingles). During varicella, the virus establishes a life-long dormant infection within the sensory ganglia and can reawaken to cause herpes zoster, but the immune responses that occur in ganglia during herpes zoster have not previously been defined. We examined ganglia obtained from individuals who, at the time of death, had active herpes zoster. We found that specific T cell subsets are likely to play an important role in controlling VZV replication in ganglia during active herpes zoster.

Bacterial artificial chromosome derived simian varicella virus is pathogenic in vivo.

BACKGROUND: Varicella zoster virus (VZV) is a neurotropic alphaherpesvirus that infects humans and results in chickenpox and herpes zoster. A number of VZV genes remain functionally uncharacterized and since VZV is an obligate human pathogen, rigorous evaluation of VZV mutants in vivo remains challenging. Simian varicella virus (SVV) is homologous to VZV and SVV infection of rhesus macaques (RM) closely mimics VZV infection of humans. Recently the SVV genome was cloned as a bacterial artificial chromosome (BAC) and BAC-derived SVV displayed similar replication kinetics as wild-type (WT) SVV in vitro. METHODS: RMs were infected with BAC-derived SVV or WT SVV at 4x10(5) PFU intrabronchially (N=8, 4 per group, sex and age matched). We collected whole blood (PBMC) and bronchoalveolar lavage (BAL) at various days post-infection (dpi) and sensory ganglia during latent infection (>84 dpi) at necropsy and compared disease progression, viral replication, immune response and the establishment of latency. RESULTS: Viral replication kinetics and magnitude in bronchoalveolar lavage cells and whole blood as well as rash severity and duration were similar in RMs infected with SVV BAC or WT SVV. Moreover, SVV-specific B and T cell responses were comparable between BAC and WT-infected animals. Lastly, we measured viral DNA in sensory ganglia from both cohorts of infected RMs during latent infection. CONCLUSIONS: SVV BAC is as pathogenic and immunogenic as WT SVV in vivo. Thus, the SVV BAC genetic system combined with the rhesus macaque animal model can further our understanding of viral ORFs important for VZV pathogenesis and the development of second-generation vaccines.

Broadening the repertoire of functional herpes simplex virus type 1-specific CD8+ T cells reduces viral reactivation from latency in sensory ganglia.

A large proportion of the world population harbors HSV type 1 (HSV-1) in a latent state in their trigeminal ganglia (TG). TG-resident CD8(+) T cells appear important for preventing HSV-1 reactivation from latency and recurrent herpetic disease. In C57BL/6J mice, half of these cells are specific for an immunodominant epitope on HSV-1 glycoprotein B, whereas the other half are specific for 18 subdominant epitopes. In this study, we show that the CD8(+) T cell dominance hierarchy in the TG established during acute infection is maintained during latency. However, CD8(+) T cells specific for subdominant epitopes lose functionality, whereas those specific for the immunodominant epitope exhibit increased functionality in latently infected TG. Furthermore, we show that IL-10 produced by 16.4 ± 2.8% of TG-resident CD4(+) T cells maintains the immunodominance hierarchy in part through selective inhibition of subdominant CD8(+) T cell proliferation. Upon systemic anti-IL-10R Ab treatment, we observed a significant expansion of functional subdominant CD8(+) T cells, resulting in significantly improved protection from viral reactivation. In fact, systemic anti-IL-10R Ab treatment prevented viral reactivation in up to 50% of treated mice. Our results not only demonstrate that HSV-1 reactivation from latency can be prevented by expanding the repertoire of functional TG-resident CD8(+) T cells, but also that IL-10R blockade might have therapeutic potential to reduce or eliminate recurrent herpetic disease.

Age and immune status of rhesus macaques impact simian varicella virus gene expression in sensory ganglia.

Simian varicella virus (SVV) infection of rhesus macaques (RMs) recapitulates the hallmarks of varicella-zoster virus (VZV) infection of humans, including the establishment of latency within the sensory ganglia. Various factors, including age and immune fitness, influence the outcome of primary VZV infection, as well as reactivation resulting in herpes zoster (HZ). To increase our understanding of the role of lymphocyte subsets in the establishment of viral latency, we analyzed the latent SVV transcriptome in juvenile RMs depleted of CD4 T, CD8 T, or CD20 B lymphocytes during acute infection. We have previously shown that SVV latency in sensory ganglia of nondepleted juvenile RMs is associated with a limited transcriptional profile. In contrast, CD4 depletion during primary infection resulted in the failure to establish a characteristic latent viral transcription profile in sensory ganglia, where we detected 68 out of 69 SVV-encoded open reading frames (ORFs). CD-depleted RMs displayed a latent transcriptional profile that included additional viral transcripts within the core region of the genome not detected in control RMs. The latent transcriptome of CD20-depleted RMs was comparable to the latent transcription in the sensory ganglia of control RMs. Lastly, we investigated the impact of age on the establishment of SVV latency. SVV gene expression was more active in ganglia from two aged RMs than in ganglia from juvenile RMs, with 25 of 69 SVV transcripts detected. Therefore, immune fitness at the time of infection modulates the establishment and/or maintenance of SVV latency.

Attenuation of the adaptive immune response in rhesus macaques infected with simian varicella virus lacking open reading frame 61.

Varicella zoster virus (VZV) is a neurotropic alphaherpesvirus that causes chickenpox during primary infection and establishes latency in sensory ganglia. Infection of rhesus macaques (RM) with the homologous simian varicella virus (SVV) recapitulates hallmarks of VZV infection. We have shown that an antisense transcript of SVV open reading frame 61 (ORF61), a viral transactivator, was detected most frequently in latently infected RM sensory ganglia. In this study, we compared disease progression, viral replication, immune response, and the establishment of latency following intrabronchial infection with a recombinant SVV lacking ORF61 (SVVΔORF61) to those following infection with wild-type (WT) SVV. Varicella severity and viral latency within sensory ganglia were comparable in RMs infected with SVVΔORF61 and WT SVV. In contrast, viral loads, B and T cell responses, and plasma inflammatory cytokine levels were decreased in RMs infected with SVVΔORF61. To investigate the mechanisms underlying the reduced adaptive immune response, we compared acute SVV gene expression, frequency and proliferation of dendritic cell (DC) subsets, and the expression of innate antiviral genes in bronchoalveolar lavage (BAL) samples. The abundance of SVV transcripts in all kinetic classes was significantly decreased in RMs infected with SVVΔORF61. In addition, we detected a higher frequency and proliferation of plasmacytoid dendritic cells in BAL fluid at 3 days postinfection in RMs infected with SVVΔORF61, which was accompanied by a slight increase in type I interferon gene expression. Taken together, our data suggest that ORF61 plays an important role in orchestrating viral gene expression in vivo and interferes with the host antiviral interferon response.

Immunohistochemical detection of intra-neuronal VZV proteins in snap-frozen human ganglia is confounded by antibodies directed against blood group A1-associated antigens.

Varicella-zoster virus (VZV) causes chickenpox, establishes latency in trigeminal (TG) and dorsal root ganglia (DRG), and can lead to herpes zoster upon reactivation. The VZV proteome expressed during latency remains ill-defined, and previous studies have shown discordant data on the spectrum and expression pattern of VZV proteins and transcripts in latently infected human ganglia. Recently, Zerboni and colleagues have provided new insight into this discrepancy (Zerboni et al. in J Virol 86:578-583, 2012). They showed that VZV-specific ascites-derived monoclonal antibody (mAb) preparations contain endogenous antibodies directed against blood group A1 proteins, resulting in false-positive intra-neuronal VZV staining in formalin-fixed human DRG. The aim of the present study was to confirm and extend this phenomenon to snap-frozen TG (n=30) and DRG (n=9) specimens of blood group genotyped donors (n=30). The number of immunohistochemically stained neurons was higher with mAb directed to immediate early protein 62 (IE62) compared with IE63. The IE63 mAb-positive neurons always co-stained for IE62 but not vice versa. The mAb staining was confined to distinct large intra-neuronal vacuoles and restricted to A1(POS) donors. Anti-VZV mAb staining in neurons, but not in VZV-infected cell monolayers, was obliterated after mAb adsorption against blood group A1 erythrocytes. The data presented demonstrate that neuronal VZV protein expression detected by ascites-derived mAb in snap-frozen TG and DRG of blood group A1(POS) donors can be misinterpreted due to the presence of endogenous antibodies directed against blood group A1-associated antigens present in ascites-derived VZV-specific mAb preparations.

Simian varicella virus gene expression during acute and latent infection of rhesus macaques.

Varicella zoster virus (VZV) is a neurotropic α-herpesvirus that causes chickenpox during primary infection and establishes latency in sensory ganglia. Reactivation of VZV results in herpes zoster and other neurological complications. Our understanding of the VZV transcriptome during acute and latent infection in immune competent individuals remains incomplete. Infection of rhesus macaques with the homologous simian varicella virus (SVV) recapitulates the hallmarks of VZV infection. We therefore characterized the SVV transcriptome by quantitative real-time reverse transcriptase PCR during acute infection in bronchial alveolar lavage (BAL) cells and peripheral blood mononuclear cells, and during latency in sensory ganglia obtained from the same rhesus macaques. During acute infection, all known SVV open reading frames (ORFs) were detected, and the most abundantly expressed ORFs are involved in virus replication and assembly such as the transcriptional activator ORF 63 and the structural proteins ORF 41 and ORF 49. In contrast, latent SVV gene expression is highly restricted. ORF 61, a viral transactivator and latency-associated transcript, is the most prevalent transcript detected in sensory ganglia. We also detected ORFs A, B, 4, 10, 63, 64, 65, 66, and 68 though significantly less frequently than ORF 61. This comprehensive analysis has revealed genes that potentially play a role in the establishment and/or maintenance of SVV latency.

Neutralizing anti-gH antibody of Varicella-zoster virus modulates distribution of gH and induces gene regulation, mimicking latency.

The anti-glycoprotein H (gH) monoclonal antibody (anti-gH-MAb) that neutralizes varicella-zoster virus (VZV) inhibited cell-to-cell infection, resulting in a single infected cell without apoptosis or necrosis, and the number of infectious cells in cultures treated with anti-gH-MAb declined to undetectable levels in 7 to 10 days. Anti-gH-MAb modulated the wide cytoplasmic distribution of gH colocalized with glycoprotein E (gE) to the cytoplasmic compartment with endoplasmic reticulum (ER) and Golgi markers near the nucleus, while gE retained its cytoplasmic distribution. Thus, the disintegrated distribution of gH and gE caused the loss of cellular infectivity. After 4 weeks of treatment with anti-gH-MAb, no infectious virus was recovered, even after cultivation without anti-gH-MAb for another 8 weeks or various other treatments. Cells were infected with Oka varicella vaccine expressing hepatitis B surface antigen (ROka) and treated with anti-gH-MAb for 4 weeks, and ROka was recovered from the quiescently infected cells by superinfection with the parent Oka vaccine. Among the genes 21, 29, 62, 63, and 66, transcripts of gene 63 were the most frequently detected, and products from the genes 63 and 62, but not gE, were detected mainly in the cytoplasm of quiescently infected cells, in contrast to their nuclear localization in lytically infected cells. The patterns of transcripts and products from the quiescently infected cells were similar to those of latent VZV in human ganglia. Thus, anti-gH-MAb treatment resulted in the antigenic modulation and dormancy of infectivity of VZV. Antigenic modulation by anti-gH-MAb illuminates a new aspect in pathogenesis in VZV infection and the gene regulation of VZV during latency in human ganglia.

Differentiated neuroblastoma cells provide a highly efficient model for studies of productive varicella-zoster virus infection of neuronal cells.

Varicella-zoster virus (VZV) is a highly species-specific herpesvirus that targets sensory ganglionic neurons. This species specificity has limited the study of many aspects of VZV pathogenesis, including neuronal infection. We report development of a highly efficient neuroblastoma cell model to study productive VZV infection of neuronal cells. We show that differentiation of SH-SY5Y neuroblastoma cells yields a homogenous population of neuron-like cells that are permissive to the full VZV replicative cycle. These cells supported productive infection by both laboratory and clinical VZV isolates, including the live varicella vaccine. This model may enable rapid identification of genetic determinants facilitating VZV neurotropism.

Naturally-occurring canine herpesvirus-1 infection of the vestibular labyrinth and ganglion of dogs.

Although the involvement of herpesviruses in vestibular disease of humans has been recognised for many years, knowledge of such a link in companion animal species is restricted to cats. This study was conducted to assess the prevalence of canine herpesvirus-1 (CaHV-1) infection of the vestibular labyrinth (VL) and vestibular ganglion (VG) of dogs by PCR. 'Field' herpesvirus was detected in the VL of 17% and in the VG of 19% of 52 dogs, respectively. None of the 11 dogs with infected VG and/or VL exhibited signs of vestibular disease, whereas clinical signs in the remaining three animals were attributable to intra-cranial neoplasia. As reported for other species, the putative role of herpesvirus infection in canine vestibular disease requires further elucidation.