Viral Load and Cell Tropism During Early Latent Equid Herpesvirus 1 Infection Differ Over Time in Lymphoid and Neural Tissue Samples From Experimentally Infected Horses

Upper respiratory tract infections with Equid Herpesvirus 1 (EHV-1) typically result in a peripheral blood mononuclear cell-associated viremia, which can lead to vasculopathy in the central nervous system. Primary EHV-1 infection also likely establishes latency in trigeminal ganglia (TG) via retrograde axonal transport and in respiratory tract-associated lymphatic tissue. However, latency establishment and reactivation are poorly understood. To characterize the pathogenesis of EHV-1 latency establishment and maintenance, two separate groups of yearling horses were experimentally infected intranasally with EHV-1, strain Ab4, and euthanized 30 days post infection (dpi), (n = 9) and 70 dpi (n = 6). During necropsy, TG, sympathetic trunk (ST), retropharyngeal and mesenteric lymph nodes (RLn, MesLn) and kidney samples were collected. Viral DNA was detected by quantitative PCR (qPCR) in TG, ST, RLn, and MesLn samples in horses 30 and 70 dpi. The number of positive TG, RLn and MesLn samples was reduced when comparing horses 30 and 70 dpi and the viral copy number in TG and RLn significantly declined from 30 to 70 dpi. EHV-1 late gene glycoprotein B reverse transcriptase PCR and IHC results for viral protein were consistently negative, thus lytic replication was excluded in the present study. Mild inflammation could be detected in all neural tissue samples and inflammatory infiltrates mainly consisted of CD3+ T-lymphocytes (T-cells), frequently localized in close proximity to neuronal cell bodies. To identify latently infected cell types, in situ hybridization (ISH, RNAScope®) detecting viral DNA was used on selected qPCR- positive neural tissue sections. In ganglia 30 dpi, EHV-1 ISH signal was located in the neurons of TG and ST, but also in non-neuronal support or interstitial cells surrounding the neuron. In contrast, distinct EHV-1 signal could only be observed in neurons of TG 70 dpi. Overall, detection of latent EHV-1 in abdominal tissue samples and non-neuronal cell localization suggests, that EHV-1 uses T-cells during viremia as alternative route toward latency locations in addition to retrograde neuronal transport. We therefore hypothesize that EHV-1 follows the same latency pathways as its close relative human pathogen Varicella Zoster Virus

Histopathologic Findings Following Experimental Equine Herpesvirus 1 Infection of Horses

Histopathological differences in horses infected with equine herpesvirus type 1 (EHV-1) of differing neuropathogenic potential [wild-type (Ab4), polymerase mutant (Ab4 N752), EHV-1/4 gD mutant (Ab4 gD4)] were evaluated to examine the impact of viral factors on clinical disease, tissue tropism and pathology. Three of 8 Ab4 infected horses developed Equine Herpesvirus Myeloencephalopathy (EHM) requiring euthanasia of 2 horses on day 9 post-infection. None of the other horses showed neurologic signs and all remaining animals were sacrificed 10 weeks post-infection. EHM horses had lymphohistiocytic vasculitis and lymphocytic infiltrates in the lungs, spinal cord, endometrium and eyes. EHV-1 antigen was detected within the eyes and spinal cord. In 3/6 of the remaining Ab4 infected horses, 4/9 Ab4 N752 infected horses, and 8/8 Ab4 gD4 infected horses, choroiditis was observed. All males had interstitial lymphoplasmacytic and/or histiocytic orchitis and EHV-1 antigen was detected. In conclusion, only animals sacrificed due to EHM developed overt vasculitis in the CNS and the eye. Mild choroiditis persisted in many animals and appeared to be more common in Ab4 gD4 infected animals. Finally, we report infiltrates and changes in the reproductive organs of all males associated with EHV-1 antigen. While the exact significance of these changes is unclear, these findings raise concern for long-term effects on reproduction and prolonged shedding of virus through semen

An Equine Herpesvirus Type 1 (EHV-1) Expressing VP2 and VP5 of Serotype 8 Bluetongue Virus (BTV-8) Induces Protection in a Murine Infection Model

Bluetongue virus (BTV) can infect most species of domestic and wild ruminants causing substantial morbidity and mortality and, consequently, high economic losses. In 2006, an epizootic of BTV serotype 8 (BTV-8) started in northern Europe that caused significant disease in cattle and sheep before comprehensive vaccination was introduced two years later. Here, we evaluate the potential of equine herpesvirus type 1 (EHV-1), an alphaherpesvirus, as a novel vectored DIVA (differentiating infected from vaccinated animals) vaccine expressing VP2 of BTV-8 alone or in combination with VP5. The EHV-1 recombinant viruses stably expressed the transgenes and grew with kinetics that were identical to those of parental virus in vitro. After immunization of mice, a BTV-8-specific neutralizing antibody response was elicited. In a challenge experiment using a lethal dose of BTV-8, 100% of interferon-receptor-deficient (IFNAR−/−) mice vaccinated with the recombinant EHV-1 carrying both VP2 and VP5, but not VP2 alone, survived. VP7 was not included in the vectored vaccines and was successfully used as a DIVA marker. In summary, we show that EHV-1 expressing BTV-8 VP2 and VP5 is capable of eliciting a protective immune response that is distinguishable from that after infection and as such may be an alternative for BTV vaccination strategies in which DIVA compatibility is of importance

Identification of Host Factors Associated with the Development of Equine Herpesvirus Myeloencephalopathy by Transcriptomic Analysis of Peripheral Blood Mononuclear Cells from Horses

Equine herpesvirus-1 is the cause of respiratory disease, abortion, and equine herpesvirus myeloencephalopathy (EHM) in horses worldwide. EHM affects as many as 14% of infected horses and a cell-associated viremia is thought to be central for EHM pathogenesis. While EHM is infrequent in younger horses, up to 70% of aged horses develop EHM. The aging immune system likely contributes to EHM pathogenesis; however, little is known about the host factors associated with clinical EHM. Here, we used the “old mare model” to induce EHM following EHV-1 infection. Peripheral blood mononuclear cells (PBMCs) of horses prior to infection and during viremia were collected and RNA sequencing with differential gene expression was used to compare the transcriptome of horses that did (EHM group) and did not (non-EHM group) develop clinical EHM. Interestingly, horses exhibiting EHM did not show respiratory disease, while non-EHM horses showed significant respiratory disease starting on day 2 post infection. Multiple immune pathways differed in EHM horses in response to EHV-1. These included an upregulation of IL-6 gene expression, a dysregulation of T-cell activation through AP-1 and responses skewed towards a T-helper 2 phenotype. Further, a dysregulation of coagulation and an upregulation of elements in the progesterone response were observed in EHM horses

Antibody and cellular immune responses following DNA vaccination and EHV-1 infection of ponies

Equine herpesvirus-1 (EHV-1) is the cause of serious disease with high economic impact on the horse industry, as outbreaks of EHV-1 disease occur every year despite the frequent use of vaccines. Cytotoxic T-lymphocytes (CTLs) are important for protection from primary and reactivating latent EHV-1 infection. DNA vaccination is a powerful technique for stimulating CTLs, and the aim of this study was to assess antibody and cellular immune responses and protection resulting from DNA vaccination of ponies with combinations of EHV-1 genes. Fifteen ponies were divided into three groups of five ponies each. Two vaccination groups were DNA vaccinated on four different occasions with combinations of plasmids encoding the gB, gC, and gD glycoproteins or plasmids encoding the immediate early (IE) and early proteins (UL5) of EHV-1, using the PowderJect XR research device. Total dose of DNA/plasmid/vaccination were 25 μg. A third group comprised unvaccinated control ponies. All ponies were challenge infected with EHV-1 6 weeks after the last vaccination, and protection from clinical disease, viral shedding, and viremia was determined. Virus neutralizing antibodies and isotype specific antibody responses against whole EHV-1 did not increase in either vaccination group in response to vaccination. However, glycoprotein gene vaccinated ponies showed gD and gC specific antibody responses. Vaccination did not affect EHV-1 specific lymphoproliferative or CTL responses. Following challenge infection with EHV-1, ponies in all three groups showed clinical signs of disease. EHV-1 specific CTLs, proliferative responses, and antibody responses increased significantly in all three groups following challenge infection. In summary, particle-mediated EHV-1 DNA vaccination induced limited immune responses and protection. Future vaccination strategies must focus on generating stronger CTL responses.15 page(s

Equid herpesvirus-1 Distribution in Equine Lymphoid and Neural Tissues 70 Days Post Infection

Equid herpesvirus-1 (EHV-1) causes respiratory disease, abortion and myeloencephalopathy in horses worldwide. As member of the Alphaherpesvirinae, latency is key to EHV-1 epidemiology. EHV-1 latent infection has been detected in the trigeminal ganglion (TG), respiratory associated lymphoid tissue (RALT) and peripheral blood mononuclear cells (PBMC) but additional locations are likely. The aim of this study was to investigate the distribution of viral DNA throughout the equine body. Twenty-five horses divided into three groups were experimentally infected via intranasal instillation with one of three EHV-1 viruses and euthanized on Day 70, post infection. During necropsy, TG, various sympathetic/parasympathetic ganglia of head, neck, thorax and abdomen, spinal cord dorsal root ganglia, RALT, mesenteric lymph nodes, spleen and PBMC of each horse were collected. Genomic viral loads and L-(late) gene transcriptional activity in each tissue and PBMC were measured using qPCR. In addition, immunohistochemistry (IHC) was applied on neural parenchyma tissue sections. EHV-1 DNA was detected in many neural and lymphoid tissue sections, but not in PBMC. L-gene transcriptional activity was not detected in any sample, and translational activity was not apparent on IHC. Tissue tropism differed between the Ab4 wild type and the two mutant viruses