Bacterial Toxins from Staphylococcus aureus and Bordetella bronchiseptica Predispose the Horse’s Respiratory Tract to Equine Herpesvirus Type 1 Infection

Respiratory disease in horses is caused by a multifactorial complex of infectious agents and environmental factors. An important pathogen in horses is equine herpesvirus type 1 (EHV-1). During co-evolution with this ancient alphaherpesvirus, the horse’s respiratory tract has developed multiple antiviral barriers. However, these barriers can become compromised by environmental threats. Pollens and mycotoxins enhance mucosal susceptibility to EHV-1 by interrupting cell junctions, allowing the virus to reach its basolateral receptor. Whether bacterial toxins also play a role in this impairment has not been studied yet. Here, we evaluated the role of α-hemolysin (Hla) and adenylate cyclase (ACT), toxins derived from the facultative pathogenic bacterium Staphylococcus aureus (S. aureus) and the primary pathogen Bordetella bronchiseptica (B. bronchiseptica), respectively. Equine respiratory mucosal explants were cultured at an air–liquid interface and pretreated with these toxins, prior to EHV-1 inoculation. Morphological analysis of hematoxylin–eosin (HE)-stained sections of the explants revealed a decreased epithelial thickness upon treatment with both toxins. Additionally, the Hla toxin induced detachment of epithelial cells and a partial loss of cilia. These morphological changes were correlated with increased EHV-1 replication in the epithelium, as assessed by immunofluorescent stainings and confocal microscopy. In view of these results, we argue that the ACT and Hla toxins increase the susceptibility of the epithelium to EHV-1 by disrupting the epithelial barrier function. In conclusion, this study is the first to report that bacterial exotoxins increase the horse’s sensitivity to EHV-1 infection. Therefore, we propose that horses suffering from infection by S. aureus or B. bronchiseptica may be more susceptible to EHV-1 infection.

Equine Herpesvirus Type 4 (EHV-4) Outbreak in Germany: Virological, Serological, and Molecular Investigations

Equine herpesvirus type 4 (EHV-4) is enzootic in equine populations throughout the world. A large outbreak of EHV-4 respiratory infection occurred at a Standardbred horse-breeding farm in northern Germany in 2017. Respiratory illness was observed in a group of in-housed foals and mares, which subsequently resulted in disease outbreak. Out of 84 horses in the stud, 76 were tested and 41 horses were affected, including 20 foals, 10 stallions, and 11 mares. Virological investigations revealed the involvement of EHV-4 in all cases of respiratory illness, as confirmed by virus isolation, qPCR, and/or serological follow-up using virus neutralization test and peptide-specific ELISA. Among infected mares, 73% (8 out of 11) and their corresponding foals shed the virus at the same time. EHV-4 was successfully isolated from four animals (including one stallion and three foals), and molecular studies revealed a different restriction fragment length polymorphism (RFLP) profile in all four isolates. We determined the complete 144 kbp genome sequence of EHV-4 isolated from infected horses by next-generation sequencing and de novo assembly. Hence, EHV-4 is genetically stable in nature, different RFLP profiles, and genome sequences of the isolates, suggesting the involvement of more than one animal as a source of infection due to either true infection or reactivation from a latent state. In addition, epidemiological investigation revealed that stress caused by seasonal changes, management practices, routine equestrian activities, and exercises contributed as a multifactorial causation for disease outbreak. This study shows the importance of implementing stress alleviating measures and management practices in breeding farms in order to avoid immunosuppression and occurrence of disease.

Recombinant Glycoprotein B of Equine herpesvirus Type 1 Elicits Protective Immune Response against Challenge in BALB/c Mouse Model

Background: Equine herpesvirus type 1 (EHV-1) is the most important viral pathogen of equines, causing respiratory illness, abortion, neonatal foal mortality and neurologic disorders. Large numbers of commercial EHV-1 vaccines are available to protect equines from the disease, but they provide only partial protection. Despite immunization with inactivated and modified live virus vaccine, mares show abortions. Present study was aimed to investigate the immunogenicity and protective efficacy of EHV-1 recombinant glycoprotein B (rgB) and gB expressing plasmid DNA against EHV-1 infection in BALB/c mice model.Methods: About 3-4 weeks old 225 female BALB/c mice were selected for the comparative study of immunization followed by challenged with EHV-1/India/Tohana/96-2 strain virus in 5 different groups of 45 animals each.Result: Following immunization, rgB vaccinated mice showed optimal stimulation of EHV-1 gB specific cell mediated and humoral mediated immunity (HMI and CMI). The gB expressing plasmid DNA vaccinated mice developed only CMI while inactivated whole virus vaccinated mice had only HMI. Upon EHV-1 challenge, all infected mice displayed variable levels of clinical signs with changes in body weight, however, vaccinated mice showed very rapid recovery with optimal protection. Positive control group mice showed severe pulmonary lesions along with persistence virus infection till 5 days post challenge (dpc) whereas vaccinated mice had less pulmonary lesion only up to 3dpc. Minimal lung lesions and early virus clearance was observed in the rgB immunized mice in comparison to the gB plasmid DNA and inactivated EHV-1 vaccine immunized mice. It has been concluded that immunization with rgB elicits optimum protective immune response against EHV-1 infection in mice model. The rgB could be a potential vaccine candidate against EHV-1 infection in equine in the future.

The Pathogenesis and Immune Evasive Mechanisms of Equine Herpesvirus Type 1

Equine herpesvirus type 1 (EHV-1) is an alphaherpesvirus related to pseudorabies virus (PRV) and varicella-zoster virus (VZV). This virus is one of the major pathogens affecting horses worldwide. EHV-1 is responsible for respiratory disorders, abortion, neonatal foal death and equine herpes myeloencephalopathy (EHM). Over the last decade, EHV-1 has received growing attention due to the frequent outbreaks of abortions and/or EHM causing serious economical losses to the horse industry worldwide. To date, there are no effective antiviral drugs and current vaccines do not provide full protection against EHV-1-associated diseases. Therefore, there is an urgent need to gain a better understanding of the pathogenesis of EHV-1 in order to develop effective therapies. The main objective of this review is to provide state-of-the-art information on the pathogenesis of EHV-1. We also highlight recent findings on EHV-1 immune evasive strategies at the level of the upper respiratory tract, blood circulation and endothelium of target organs allowing the virus to disseminate undetected in the host. Finally, we discuss novel approaches for drug development based on our current knowledge of the pathogenesis of EHV-1.

Evaluation of Colostral Immunity Against Equine Herpesvirus Type 1 (EHV-1) in Martina Franca's Foals

Eight Martina Franca pregnant jennies were selected in order to evaluate the transfer of colostral antibodies against equine herpesvirus type 1 in their relative foals after immunization with a commercial inactivated vaccine, compared with an unvaccinated group. Samples of serum and colostrums/milk were collected from jennies and foals under study starting from 10 min before and up to 21 days after the foaling. Specific anti-EHV-1 antibody titers were evaluated by means of a serum neutralization test, and the results obtained from both groups were analyzed. The serological titers in the vaccinated jennies was significantly higher (p < 0.01). No significant differences were found in the specific time-point intervals in both groups examined (p > 0.05). The antibody titers in milk at the time of delivery and subsequent withdrawal (T0 and T1) were very high in both groups, but no significant differences were found between the two groups (p > 0.05). In the foal sera, a significant difference was found between foals in the vaccinated group compared with those in the unvaccinated group (p < 0.05). Finally, a significant correlation (p < 0.05) was observed between the antibody titers found in serum and colostrum of jennies and the foal titers in the first time-point sampling (up to 12 h after foaling). The results confirm a substantial homology in the antibody production compared with other most investigated equids, highlighting the efficacy of the vaccination against EHV-1 of the jennies to ensure the protective immunity to their foals during the first weeks after delivery.

Equine Herpesvirus Type 1 Modulates Cytokine and Chemokine Profiles of Mononuclear Cells for Efficient Dissemination to Target Organs

Equine herpesvirus type 1 (EHV-1) causes encephalomyelopathy and abortion, for which cell-associated viremia and subsequent virus transfer to and replication in endothelial cells (EC) are responsible and prerequisites. Viral and cellular molecules responsible for efficient cell-to-cell spread of EHV-1 between peripheral blood mononuclear cells (PBMC) and EC remain unclear. We have generated EHV-1 mutants lacking ORF1, ORF2, and ORF17 genes, either individually or in combination. Mutant viruses were analyzed for their replication properties in cultured equine dermal cells, PBMC infection efficiency, virus-induced changes in the PBMC proteome, and cytokine and chemokine expression profiles. ORF1, ORF2, and ORF17 are not essential for virus replication, but ORF17 deletion resulted in a significant reduction in plaque size. Deletion of ORF2 and ORF17 gene significantly reduced cell-to-cell virus transfer from virus-infected PBMC to EC. EHV-1 infection of PBMC resulted in upregulation of several pathways such as Ras signaling, oxidative phosphorylation, platelet activation and leukocyte transendothelial migration. In contrast, chemokine signaling, RNA degradation and apoptotic pathways were downregulated. Deletion of ORF1, ORF2 and ORF17 modulated chemokine signaling and MAPK pathways in infected PBMC, which may explain the impairment of virus spread between PBMC and EC. The proteomic results were further confirmed by chemokine assays, which showed that virus infection dramatically reduced the cytokine/chemokine release in infected PBMC. This study uncovers cellular proteins and pathways influenced by EHV-1 after PBMC infection and provide an important resource for EHV-1 pathogenesis. EHV-1-immunomodulatory genes could be potential targets for the development of live attenuated vaccines or therapeutics against virus infection.