Canine infectious respiratory disease complex (CIRDC) causes contagious respiratory disease in dogs and encompasses several etiologic agents. Viral pathogens associated with CIRDC include canine adenovirus type 2 (CAV-2), canine distemper virus (CDV), canine herpesvirus-1 (CaHV-1) and canine influenza virus (CIV). Vaccine availability and efficacy vary and there are currently no antivirals available for treatment. Interferons (IFNs) are at the frontline of the immune defense against many viral infections and interferon lambda (IFNL) has shown to be a potent antiviral. We hypothesized that canine interferon lambda-3 (IFNL3) treatment of canine respiratory epithelial cells (ALI-CRECs) will reduce replication of kennel cough viruses.ALI-CRECs were isolated, cultured and characterized morphologically and immunologically prior to infection with CDV, CaHV-1, CIV and CAV-2. The value of canine IFNL3 treatment for reducing viral titers as well as induction of interferon responses were evaluated for each virus.Treatment of ALI-CRECs with IFNL3 induced the expression of interferon stimulated genes. In addition, prophylactic IFNL3 administration resulted in a reduction in viral DNA with CT value changes of up to 13 intracellularly and up to 9 extracellularly. In contrast, for CDV and CaHV-1 a reduction in viral RNA/DNA was observed only intracellularly with CT value changes of up to 11 and up to 4 respectively. Prophylactic IFNL3 treatment also significantly reduced viral titers 1.2 to 3 log-fold for CAV-2, 0.9-2.5 log-fold for CDV, and 1.1-1.7 log-fold for CaHV-1. No effect of IFNL3 was observed for CIV RNA and CIV viral titers. Coinciding with a reduction in viral replication, interferon stimulated genes were differentially expressed in interferon treated and infected cells when compared with controls.In conclusion, ALI-CRECs have shown to be excellent systems to study respiratory viruses and evaluate the potential antiviral effect of IFNL3. However further in-depth mechanistic studies are needed to fully understand how IFNL3 stimulates/modulates local immune responses to induce antiviral effects. Moreover, while this study highlights the potential antiviral efficacy of IFNL3 for several canine respiratory viruses, determination of safety, effective doses in vivo as well as therapeutic potential will have to be evaluated in dogs.

BackgroundEquine herpes virus type 1 (EHV-1) infection in horses is associated with upper respiratory disease, neurological disease, abortions, and neonatal death.Review QuestionDoes pharmacological therapy decrease either the incidence or severity of disease or infection caused by EHV-1 in domesticated horses?MethodsA systematic review was preformed searching AGRICOLA, CAB Abstracts, Cochrane, PubMed, Web of Science, and WHO Global Health Index Medicus Regional Databases to identify articles published before February 15, 2021. Selection criteria were original research reports published in peer reviewed journals, and studies investigating in vivo use of therapeutic agents for prevention or treatment of EHV-1 in horses. Outcomes assessed included measures related to clinical outcomes that reflect symptomatic EHV-1 infection or virus infection. We evaluated risk of bias and performed a GRADE evaluation of the quality of evidence for interventions.ResultsA total of 7009 unique studies were identified, of which 9 met the inclusion criteria. Two studies evaluated valacyclovir or small interfering RNAs, and single studies evaluated the use of a Parapoxvirus ovis-based immunomodulator, human alpha interferon, an herbal supplement, a cytosine analog, and heparin. The level of evidence ranged between randomized controlled studies and observational trials. The risk of bias was moderate to high and sample sizes were small. Most studies reported either no benefit or minimal efficacy of the intervention tested.Conclusions and Clinical ImportanceOur review indicates minimal or limited benefit either as a prophylactic or post-exposure treatment for any of the studied interventions in the mitigation of EHV-1-associated disease outcome.

Equine herpesvirus-1 (EHV-1) is a highly prevalent and frequently pathogenic infection of equids. The most serious clinical consequences of infection are abortion and equine herpesvirus myeloencephalopathy (EHM). The previous consensus statement was published in 2009 and considered pathogenesis, strain variation, epidemiology, diagnostic testing, vaccination, outbreak prevention and control, and treatment. A recent survey of American College of Veterinary Internal Medicine large animal diplomates identified the need for a revision to this original consensus statement. This updated consensus statement is underpinned by 4 systematic reviews that addressed key questions concerning vaccination, pharmaceutical treatment, pathogenesis, and diagnostic testing. Evidence for successful vaccination against, or effective treatment of EHV-1 infection was limited, and improvements in experimental design and reporting of results are needed in future studies of this important disease. This consensus statement also updates the topics considered previously in 2009.

BACKGROUND:Equine herpes virus type 1 (EHV-1) infection in horses is associated with respiratory and neurologic disease, abortion, and neonatal death.HYPOTHESIS:Vaccines decrease the occurrence of clinical disease in EHV-1-infected horses.METHODS:A systematic review was performed searching multiple databases to identify relevant studies. Selection criteria were original peer-reviewed research reports that investigated the in vivo use of vaccines for the prevention of disease caused by EHV-1 in domesticated horses. Main outcomes of interest included pyrexia, abortion, neurologic disease, viremia, and nasal shedding. We evaluated risk of bias, conducted exploratory meta-analyses of incidence data for the main outcomes, and performed a GRADE evaluation of the quality of evidence for each vaccine subtype.RESULTS:A total of 1018 unique studies were identified, of which 35 met the inclusion criteria. Experimental studies accounted for 31/35 studies, with the remainder being observational studies. Eight vaccine subclasses were identified including commercial (modified-live, inactivated, mixed) and experimental (modified-live, inactivated, deletion mutant, DNA, recombinant). Risk of bias was generally moderate, often because of underreporting of research methods, and sample sizes were small leading to imprecision in the estimate of the effect size. Several studies reported either no benefit or minimal vaccine efficacy for the primary outcomes of interest. Meta-analyses revealed significant heterogeneity was present, and our confidence in the quality of evidence for most outcomes was low to moderate.CONCLUSIONS AND CLINICAL IMPORTANCE:Our review indicates that commercial and experimental vaccines minimally reduce the incidence of clinical disease associated with EHV-1 infection.

Abstract Background Equine herpesvirus type 1 (EHV‐1) infection is associated with upper respiratory disease, EHM, abortions, and neonatal death. Research Questions Are nasal secretions a more sensitive biological sample compared to blood for the detection of EHV‐1 infection? How long is EHV‐1 detectable after primary infection by PCR? Methods MedLine and Web of Science searches identified original peer‐reviewed reports evaluating nasal shedding and viremia using virus isolation methods or PCR published in English before October 9, 2023. Results Sixty experimental and 20 observational studies met inclusion criteria. EHV‐1 detection frequency by qPCR in nasal secretions and blood from naturally‐infected horses with fever and respiratory signs were 15% and 9%, respectively; qPCR detection rates in nasal secretions and blood from horses with suspected EHM were 94% and 70%, respectively. In experimental studies the sensitivity of qPCR matched or exceeded that seen for virus isolation from either nasal secretions or blood. Detection of nasal shedding typically occurred within 2 days after EHV‐1 inoculation with a detection period of 3 to 7 days. Viremia lasted 2 to 7 days and was usually detected ≥1 days after positive identification of EHV‐1 in nasal secretions. Nasal shedding and viremia decreased over time and remained detectable in some horses for several weeks after inoculation. Conclusions and Clinical Importance Under experimental conditions, blood and nasal secretions have similar sensitivity for the detection of EHV‐1 when horses are sampled on multiple consecutive days. In contrast, in observational studies detection of EHV‐1 in nasal secretions was consistently more successful.

BACKGROUND:Equine herpes virus type 1 (EHV-1) infection in horses is associated with upper respiratory disease, neurological disease, abortions, and neonatal death. OBJECTIVE:To determine if there is an association between the level and duration of EHV-1 viremia and either abortion or equine herpesvirus myeloencephalopathy (EHM) in domesticated horses? METHODS:A systematic review was performed searching numerous databases to identify peer reviewed reports that evaluated viremia and EHM, or viremia and abortion published before January 19, 2021. Randomized controlled trials and observational studies were assessed for risk of bias or publication quality. RESULTS:A total of 189 unique studies were identified, of which 34 met the inclusion criteria. Thirty studies evaluated viremia and neurologic outcomes including 4 observational studies. Eight experimental studies examined viremia and abortion, which used the Ab4 and OH03 virus strains or recombinant Ab4 derivatives. Incidence rates for both EHM and abortion in experimental studies varied among the studies as did the level of evidence. Viremia was generally detectable before the onset of either EHM or abortion. Risk of bias was generally low to moderate, sample sizes were small, and multiple studies reported negative outcome data. CONCLUSIONS AND CLINICAL IMPORTANCE:The results of this study support that viremia is regularly present before EHM or abortion occurs. However, no inferences could be made about the relationship between the occurrence of either neurological signs or abortion and the magnitude or duration of viremia.

Feline herpesvirus-1 (FHV-1) is responsible for approximately 50% of diagnosed viral upper respiratory tract disease in cats. The virus infects and replicates in the epithelial cells located in upper respiratory tract. Commercial vaccines do not protect cats from the infection itself or development of latency. Previously, our lab developed a cell culture model using primary feline respiratory epithelial cells (pFRECs) to study respiratory innate immunity to FHV-1 and FHV-1 deletion mutants. However, the numbers of pFRECs that can be obtained per cat is limited. To improve the usage of respiratory epithelial 3D cultures in FHV-1 research, the present study immortalized feline respiratory epithelial cells (iFRECs) and characterized them morphologically and immunologically and evaluated the response to FHV-1 infection. Immortalization was achieved by transduction with Lenti-SV40T and Lenti-HPV E6/E7. Immortalized FRECs could be successfully subcultured for >20 passages, with positive gene expression of SV40T and HPV E6/E7. Immortalized FRECs expressed similar innate immunity-associated genes compared to pFRECs, including genes of Toll-like receptors (TLR1-9), interferon induced genes (OAS1, OAS3, IFI44, IFITM1, IFIT1), chemokines (CCL2, CCL3, CXCL8), pro-inflammatory and regulatory cytokines (IL-6, IL-4, IL-5, IL-12, and IL-18), and antimicrobials (DEFβ10, DEFβ4B). Finally, FHV-1 inoculation resulted in characteristic cytopathic effects starting at 24 hpi, with more than 80% cells detached and lysed by 72 hpi. Overall FHV-1 growth kinetics in iFRECs resembled the kinetics observed in pFRECs. In conclusion, we demonstrated that iFRECs are a useful tool to study feline respiratory disease including but not limited to FHV-1.

Equine herpesvirus-1 (EHV-1) myeloencephalopathy (EHM) is a devastating consequence of EHV-1 infection that has significant economic consequences. However, clinical EHM is relatively rare and occurs in only approximately 10% of infected horses. While there is a positive correlation between the duration and magnitude of viremia and incidence of EHM, it is likely that a combination of host and viral factors determine whether EHM occurs. The identification of these factors is of high interest for the equine community and has been the topic of much research for vaccine development and to predict which horses might be most at risk for developing EHM. The aim of this review is to highlight host immunity contributions to EHM pathogenesis at different sites of EHV-1 infection to shed light on the different aspects and interdependence of the response to EHV-1 in the time course of infection.

Equine myeloencephalopathy (EHM), an uncommon manifestation of equine herpesvirus 1 (EHV-1) infection, can cause devastating losses on individual farms, boarding stables, veterinary hospitals, and show and racing venues. An improved understanding of EHM has emerged from experimental studies and from data collected during field outbreaks at riding schools, racetracks, horse shows, and veterinary hospitals throughout North America and Europe. These outbreaks have highlighted the contagious nature of EHV-1 and have prompted a reevaluation of diagnostic procedures, treatment modalities, preventative measures, and biosecurity protocols for this disease. This article focuses on recent data related to the cause, epidemiology, pathogenesis, immunity, diagnosis, treatment, and prevention of EHV-1 infection with emphasis on EHM.

Felid herpesvirus-1 (FeHV-1) is an important respiratory and ocular pathogen of cats and current vaccines are limited in duration and efficacy because they do not prevent infection, viral nasal shedding and latency. To address these shortcomings, we have constructed FeHV-1 gE-TK- and FeHV-1 PK- deletion mutants (gE-TK- and PK-) using bacterial artificial chromosome (BAC) mutagenesis and shown safety and immunogenicity in vitro. Here, we compare the safety and efficacy of a prime boost FeHV-1 gE-TK- and FeHV-1 PK- vaccination regimen with commercial vaccination in cats. Cats in the vaccination groups were vaccinated at 3-week intervals and all cats were challenge infected 3 weeks after the last vaccination. Evaluations included clinical signs, nasal shedding, virus neutralizing antibodies (VN), cytokine mRNA gene expression, post-mortem histology and detection of latency establishment. Vaccination with gE-TK- and PK- mutants was safe and resulted in significantly reduced clinical disease scores, pathological changes, viral nasal shedding, and viral DNA in the trigeminal ganglia (the site of latency) following infection. Both mutants induced VN antibodies and interferons after immunization. In addition, after challenge infection, we observed a reduction of IL-1β expression, and modulation of TNFα, TGFβ and IL10 expression. In conclusion, this study shows the merits of using FeHV-1 deletion mutants for prevention of FeHV-1 infection in cats.

Equine herpesvirus 1 (EHV-1) affects horses worldwide and causes respiratory disease, abortions, and equine herpesvirus myeloencephalopathy (EHM). Following infection, a cell-associated viremia is established in the peripheral blood mononuclear cells (PBMCs). This viremia is essential for transport of EHV-1 to secondary infection sites where subsequent immunopathology results in diseases such as abortion or EHM. Because of the central role of PBMCs in EHV-1 pathogenesis, our goal was to establish a gene expression analysis of host and equine herpesvirus genes during EHV-1 viremia using RNA sequencing. When comparing transcriptomes of PBMCs during peak viremia to those prior to EHV-1 infection, we found 51 differentially expressed equine genes (48 upregulated and 3 downregulated). After gene ontology analysis, processes such as the interferon defense response, response to chemokines, the complement protein activation cascade, cell adhesion, and coagulation were overrepresented during viremia. Additionally, transcripts for EHV-1, EHV-2, and EHV-5 were identified in pre- and post-EHV-1-infection samples. Looking at micro RNAs (miRNAs), 278 known equine miRNAs and 855 potentially novel equine miRNAs were identified in addition to 57 and 41 potentially novel miRNAs that mapped to the EHV-2 and EHV-5 genomes, respectively. Of those, 1 EHV-5 and 4 equine miRNAs were differentially expressed in PBMCs during viremia. In conclusion, this work expands our current knowledge about the role of PBMCs during EHV-1 viremia and will inform the focus on future experiments to identify host and viral factors that contribute to clinical EHM.

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.

Toxoplasma gondii is hypothesized to manipulate the behavior of warm-blooded hosts to promote trophic transmission into the parasite's definitive feline hosts. A key prediction of this hypothesis is that T. gondii infections of non-feline hosts are associated with costly behavior toward T. gondii's definitive hosts; however, this effect has not been documented in any of the parasite's diverse wild hosts during naturally occurring interactions with felines. Here, three decades of field observations reveal that T. gondii-infected hyena cubs approach lions more closely than uninfected peers and have higher rates of lion mortality. We discuss these results in light of 1) the possibility that hyena boldness represents an extended phenotype of the parasite, and 2) alternative scenarios in which T. gondii has not undergone selection to manipulate behavior in host hyenas. Both cases remain plausible and have important ramifications for T. gondii's impacts on host behavior and fitness in the wild. The parasite causing toxoplasmosis can manipulate prey to behave in ways that promote transmission to the parasite's definitive feline hosts. The first study consistent with this extended phenotype in the wild finds that infected hyena cubs approach lions more closely than uninfected peers and have higher rates of lion mortality.

Equine herpesvirus 1 (EHV-1) ubiquitously infects horses worldwide and causes respiratory disease, abortion, and equine herpesvirus myeloencephalopathy. Protection against EHV-1 disease is elusive due to establishment of latency and immune-modulatory features of the virus. These include the modulation of interferons, cytokines, chemokines, antigen presentation, and cellular immunity. Because the modulation of immunity likely occurs at the site of first infection—the respiratory epithelium, we hypothesized that the mucosal influenza vaccine Flu Avert ® I.N. (Flu Avert), which is known to stimulate strong antiviral responses, will enhance antiviral innate immunity, and that these responses would also provide protection from EHV-1 infection. To test our hypothesis, primary equine respiratory epithelial cells (ERECs) were treated with Flu Avert, and innate immunity was evaluated for 10 days following treatment. The timing of Flu Avert treatment was also evaluated for optimal effectiveness to reduce EHV-1 replication by modulating early immune responses to EHV-1. The induction of interferons, cytokine and chemokine mRNA expression, and protein secretion was evaluated by high-throughput qPCR and multiplex protein analysis. Intracellular and extracellular EHV-1 titers were determined by qPCR. Flu Avert treatment resulted in the modulation of IL-8, CCL2, and CXCL9 starting at days 5 and 6 post-treatment. Coinciding with the timing of optimal chemokine induction, our data also suggested the same timing for reduction of EHV-1 replication. In combination, our results suggest that Flu Avert may be effective at counteracting some of the immune-modulatory properties of EHV-1 at the airway epithelium and the peak for this response occurs 5–8 days post-Flu Avert treatment. Future in vivo studies are needed to investigate Flu Avert as a prophylactic in situations where EHV-1 exposure may occur.

Equid herpesviruses are a group of nine infectious agents, which can cause conditions ranging from neurological disease with fatality rates of more than 50% of infected animals to mild or subclinical infection. Among the nine equid herpesviruses, three are referred to as asinine herpesviruses (EHV-6, EHV-7, EHV-8), because they primarily infect donkeys. Five equid herpesviruses (EHV-1, EHV-2, EHV-3, EHV-4, EHV-5) primarily infect horses. The definitive host of EHV-9 remains enigmatic and may represent an ancient perissodactyl herpesvirus for which evidence of infection can also be found in rhinoceros. In addition and much as EHV-1, EHV-9 can infect other mammals with fatal outcomes. Equine herpesviruses represent two subfamilies, the Alpha- and Gammaherpesvirinae, and two genuses, the Varicello- and Percaviruses, within the Herpesviridae family. Notably, an equid betaherpesvirus has not yet been identified. The clinically and economically most important equine herpesviruses are EHV-1 and EHV-4, which explains the focus of most research worldwide on those two closely related viruses. There are numerous studies on gene regulation, replication in cultured cells, pathogenesis, and vaccine development for EHV-1 and EHV-4, while comparable knowledge for other equid herpesviruses is limited.

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.

EDITORIAL article Front. Vet. Sci., 10 January 2020Sec. Veterinary Experimental and Diagnostic Pathology Volume 6 - 2019 | https://doi.org/10.3389/fvets.2019.00492

Toxoplasma gondii is widely reported to manipulate the behavior of its non-definitive hosts in ways that promote lethal interactions with the parasite’s definitive feline hosts. Nonetheless, there is a lack of data on the association between T. gondii infection and costly behavioral interactions with felids in nature. Here, we report that three decades of field observations reveal T. gondii infected hyena cubs approach lions more closely than uninfected peers and have higher rates of lion mortality. Our findings support the hypothesis that T. gondii’s manipulation of host boldness is an extended phenotype that promotes parasite transmission from intermediate hosts to feline predators. While upregulating hyena boldness toward lions might achieve this, it may also reflect a collateral influence of manipulative traits that evolved in other hosts (e.g., rodents). In either case, our findings corroborate the potential impacts of a globally distributed and generalist parasite ( T. gondii ) on fitness-related interaction with felids in a wild host. One Sentence Summary Wild hyenas infected with the parasite T. gondii show evidence of costly behavioral manipulation when interacting with lions.

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.

Feline herpesvirus-1 (FHV-1) is the primary cause of viral respiratory and ocular disease in cats. While commercial vaccines can provide clinical protection, they do not protect from infection or prevent latency. Moreover, they are not safe for intranasal administration. Our overall objective is to develop a new mucosal vaccine against FHV-1 disease to address these shortcomings. Feline herpesvirus-1 deletion mutants of glycoprotein C (gC-), gE (gE-), US3-encoded serine/threonine protein kinase (PK-), and both gE and thymidine kinase (gE-TK-) were generated by bacterial artificial chromosome (BAC) mutagenesis. Tracheal tissue explants from eight cats were used to compare the pattern of viral infection and associated tissue damage, as well as virus spread through the basement membrane following inoculation with wild-type virus (WT), and gE-, gE-TK-, PK-, and gC- mutants. Tissues were collected at 24, 48, or 72 hours post-inoculation (hpi) followed by immunohistochemistry (IHC) for FHV-1. Histological changes were graded based on the distribution of virus infected cells and the severity of tissue damage. Inoculations with the WT virus resulted in maximal scores at 72 hpi both at a multiplicity of infection (MOI) of 1 and 0.1. Inoculation with the gE- mutant produced scores similar to scores of explants inoculated with the WT virus at 24 and 48 hpi, but scores were significantly decreased at 72 hpi. Explants inoculated with the gE-TK- mutant showed significantly decreased scores at all time points. Further, the majority of explants inoculated with the PK- mutant resulted in scores of zero at all time points, regardless of MOI. Finally, inoculation with WT resulted in significant stromal invasion below the infected epithelium, while stromal invasion was observed in less than 50 % of the samples following inoculation with gE-, gE-TK-, PK-, or gC- mutants and confined closely to the area surrounding the infected epithelium. In conclusion, the gE-TK- and PK- mutants exhibited significantly reduced virulence, tissue damage and spread to the underlying stroma, suggesting that they may be good vaccine candidates for in vivo testing.