Equine Herpesvirus Research Articles

Voluntary Surveillance Program for Equine Influenza Virus in the United States during 2008–2021

A voluntary upper respiratory biosurveillance program in the USA received 9740 nasal swab submissions during the years 2008-2021 from 333 veterinarians and veterinary clinics. The nasal swabs were submitted for qPCR testing for six common upper respiratory pathogens:equine influenza virus (EIV), equine herpesvirus-1 (EHV-1), equine herpesvirus-4 (EHV-4), Streptococcus equi subspecies equi (S. equi), equine rhinitis A virus (ERAV), and equine rhinitis B virus (ERBV). Additional testing was performed for equine gamma herpesvirus-2 (EHV-2) and equine gamma herpesvirus-5 (EHV-5) and the results are reported. Basic frequency statistics and multivariate logistic regression models were utilized to determine the associations between risk factors and EIV positivity. The EIV qPCR-positivity rate was 9.9%. Equids less than 9 years of age with a recent history of travel and seasonal occurrence in winter and spring were the most common population that were qPCR positive for EIV. This ongoing biosurveillance program emphasizes the need for molecular testing for pathogen identification, which is critical for decisions associated with therapeutics and biosecurity intervention for health management and vaccine evaluations and development.

Detection of Equine Papillomaviruses and Gamma-Herpesviruses in Equine Squamous Cell Carcinoma.

Squamous cell carcinoma (SCC) seriously compromises the health and welfare of affected horses. Although robust evidence points to equine papillomavirus type 2 (EcPV2) causing genital lesions, the etiopathogenesis of equine SCC is still poorly understood. We screened a series of SCCs from the head-and-neck (HN), (peri-)ocular and genital region, and site-matched controls for the presence of EcPV2-5 and herpesvirus DNA using type-specific EcPV PCR, and consensus nested herpesvirus PCR followed by sequencing. EcPV2 DNA was detected in 45.5% of HN lesions, 8.3% of (peri-)ocular SCCs, and 100% of genital tumors, whilst control samples from tumor-free horses except one tested EcPV-negative. Two HNSCCs harbored EcPV5, and an ocular lesion EcPV4 DNA. Herpesvirus DNA was detected in 63.6%, 66.6%, 47.2%, and 14.2% of horses with HN, ocular, penile, and vulvar SCCs, respectively, and mainly identified as equine herpesvirus 2 (EHV2), 5 (EHV5) or asinine herpesvirus 5 (AsHV5) DNA. In the tumor-free control group, 9.6% of oral secretions, 46.6% of ocular swabs, 47% of penile samples, and 14.2% of vaginal swabs scored positive for these herpesvirus types. This work further highlights the role of EcPV2 as an oncovirus and is the first to provide information on the prevalence of (gamma-)herpesviruses in equine SCCs.

Clinical and Molecular Epidemiological Study on Herpesviruses Infection among Equid Populations in Upper Egypt

The present study was carried out to record the clinical signs of equine herpesviruses (EHVs) infection and to detect the prevalence of EHVs infection among working equids in different provinces of Egypt. A total number of 115 working equids (92 horses and 23 donkeys) were clinically examined and sampled from November 2018 till November 2019 for this study. Two samples were collected from each animal (nasal swab and blood sample) and were subjected to multiplex-PCR to detect the prevalence of different EHVs infection among equids. In the current study, the overall prevalence of EHVs infection among equid populations in Egypt was 80% by using multiplex-PCR. Moreover, the most prevalent equine herpesvirus (EHV) among equids in Upper Egypt was EHV-2 (61.74%), followed by EHV-5 (43.48%), EHV-1 (20%), and EHV-4 (13.04%). The recorded clinical signs of the examined equids harbored EHVs (PCR-positive) can be summarized as follow: a higher percentage was detected among equids with a history of acute onset (59.78%), pyrexia (57.61%), and/or systemic illness (45.65%) with or without respiratory signs (56.52%) and ocular signs (35.87%). Furthermore, 4.35% and 1.09% of EHV-1 PCR-positive equids displayed neurological signs and abortion, respectively.

The N-terminal glycine of EHV-1 UL11 is essential for the localization of UL11 and EHV-1 replication in cultured cells.

Equine herpesvirus type 1 (EHV-1) UL11 is a 74-amino-acid (aa) protein encoded by ORF51. UL11 is modified by acylation including myristoylation and palmitoylation. Myristoylation of EHV-1 UL11 is assumed to occur on the N-terminal glycine, while palmitoylation is assumed to occur on the seventh and ninth cysteines. ORF51, which encodes the first 24 aa, overlaps ORF50 encoding UL12. We previously demonstrated that UL11 was essential for EHV-1 replication in cultured cells and that UL11 was localized at the Golgi apparatus where herpesviruses obtain their final envelope. It is unclear whether the acylation is related to the localization of EHV-1 UL11 and viral replication. In this study, we investigated the role of UL11 acylation in the intracellular localization and viral growth and replication of EHV-1. We constructed seven UL11 acylation mutant plasmids and seven UL11 acylation mutant BAC DNAs; then, we analysed the localizations of the mutant UL11s and attempted virus rescue. We found that both the N-terminal glycine and the seventh or ninth cysteine, especially N-terminal glycine, were involved in the localization of UL11 and viral replication. Taken together, these results suggest that EHV-1 viral growth requires that UL11 is modified by myristoylation of an N-terminal glycine and by palmitoylation of at least one of the cysteines, and that UL11 is localized at the Golgi apparatus. This study shows that a single amino acid in EHV-1 can determine the fate of viral replication.

How do we prevent and control future outbreaks of equine herpesvirus myeloencephalopathy at equestrian gatherings?

How do we prevent and control future outbreaks of equine herpesvirus myeloencephalopathy at equestrian gatherings?

Attenuation of equine herpesvirus 1 through deletion of gE gene and its pathological evaluation in murine model.

Equine herpesvirus 1 (EHV1) infection is aglobal health problem in equines and the virus is responsible for abortions, respiratory disease and myeloencephalitis in horses. Disease management requires proper biosecurity and immunoprophylactic measures. Vaccines strengthening both arms of immunity are essential for proper control and there has been acontinuous focus in this area for generation of better vaccines. Here we report construction of bacterial artificial chromosome (BAC) clone of EHV-1 strain Tohana for mutagenesis of the virus and generation of gE gene deletion mutant EHV1. The BAC clone was generated by inserting the mini-F plasmid replacing ORF71 of EHV1 and transforming into E. coli for generation of EHV1-BAC. The infectious virus was regenerated from EHV-1 BAC DNA in RK13 cells. To check utility of EHV1-BAC, we have generated mutant EHV1 by deleting the virulence-associated gE gene. The mutant virus (vToHΔgE) showed significantly reduced plaque size without affecting replication efficiency. Pathological evaluation of lesions in BALB/c mice infected with vToHΔgE revealed reduction in clinical signs and pathology in comparison to the wild-type virus. Generation of infectious BAC of EHV1 and its usage in construction of attenuated viruses shows potential of the technology for development of indigenous modified live vaccine for EHV1. Keywords: quine herpesvirus 1; bacterial artificial chromosome (BAC); mutation; glycoprotein E; vaccine.

Double and quadruple deletion mutant of EHV-1 is highly attenuated and induces optimal immune response

Equid alphaherpesvirus 1 (EHV-1) infection causes significant health problems in equines. The EHV-1 infection leads to abortion storm in mares, respiratory disease and myeloencephalopathy. Despite the wide use of vaccines, the outbreaks of EHV-1 infections keep occurring globally, suggesting the need for the development of improved vaccines. Gene deletion attenuated mutant viruses could be a good candidate for the development of modified live vaccines. Here, we report the generation of mutant EHV-1 by deleting virulence (glycoprotein E & internal repeat 6; IR6) and immune evasive (pUL43 & pUL56) associated genes either individually or in combinations; and comprehensive evaluation of mutants through in vitro characterization followed by in vivo study in murine model to adjudge the attenuation of the virus and immune responses generated by mutants vis-à-vis wild type (wt) virus. The EHV-1 mutants with deletion of IR6 and gE genes (vToH-DMV) and four genes (i.e., gE, IR6, pUL43 and pUL56) (vToH-QMV) revealed a significant reduction in plaque size with minimal loss in replication efficiency in comparison to the wt virus. Further, in vivo studies showed virus attenuation adjudged through significant reduction in clinical signs, weight loss, gross and histopathological lesions in comparison to wt virus also revealed improved immune responses estimated through serum neutralization and flow cytometric analysis of CD4 + and CD8 + cell populations. Thus it can be concluded that EHV-1 mutants viz. vToH-DMV and vToH-QMV (novel combination) are promising vaccine candidates and qualify to be studied for adjudging the protective efficacy with wt virus challenge.

Experimental challenge of horses after prime-boost immunization with a modified live equid alphaherpesvirus 1 vaccine administered by two different routes.

The immune response and protective efficacy of a modified equid alphaherpesvirus 1 (EHV-1) vaccine administered by two different routes were tested in horses. Horses that received intramuscular (IM) priming and an intranasal (IN) booster with a 28-day interval (IM-IN group [n = 6]), IN priming and IM booster (IN-IM group [n = 5]), or no vaccination (control group [n = 6]) were challenged with EHV-1 strain 10-I-224 28 days after the second vaccination. Both vaccinated groups had significantly higher serum virus-neutralizing titers than the control group, with increased levels of serum IgGa, IgGb, and IgA antibodies (p < 0.05). The nasal antibody response was dominated by the IgGa and IgGb subclasses in both vaccinated groups, with no IgA antibody response. After challenge infection, three of six control horses were pyretic for 1-4 days post-inoculation (dpi), whereas none in the vaccinated groups were pyretic during this period. The number of horses that were pyretic at 5-10 dpi was 4 out of 6 for the controls, 3 out of 6 for the IM-IN group, and 2 out of 5 for the IN-IM group. Nasal virus replication in the IN-IM group (3-4 dpi) and IM-IN group (3 dpi) was significantly lower than in the control group (p < 0.05). All of the control horses showed viremia, whereas two horses in the IM-IN group and one in the IN-IM group did not. In conclusion, although IM-IN or IN-IM vaccination did not elicit a mucosal IgA response, it provided partial protection at a level similar to that of the conventional program, likely due to systemic antibodies and mucosal IgG subclass responses.

Occurrence of the selected parasites and viral infections in horses and donkeys in Turkey

The present study aimed to investigate the prevalence of certain protozoa (B. caballi, T. equi, T. gondii, Neospora sp.) and viral agents (equine influenza, equine viral arteritis, equine herpesviruses) of equids in Balikesir and its surroundings, in Turkey. Plasma and serum samples were collected from 66 horses and 96 donkeys. Babesia caballi, T. equi and Neospora sp. antibodies were detected with c-ELISA, whereas T. gondii antibodies were revealed by the Sabin Feldman Dye test. Viral agents were detected by the PCR technique. The prevalence rates of the protozoa in horses were 12.12% for B. caballi, 34.84% for T. equi, 9.09% for T. gondii, and 10.6% for Neospora sp. The molecular prevalence of the viral agents amounted to 3.03% for equine influenza virus and 6.06% for equine herpesvirus 5. Equine viral arteritis virus and other herpesviruses (1, 2 and 4) were not detected in any of the samples. The rates of seropositivity in donkeys were 1.69% for B. caballi, 71.87% for T. equi, 90.62% for T. gondii, 23.95% for Neospora sp., 1.04% for equine influenza virus, 0% for equine viral arteritis virus, 3.12% for equine herpesvirus 5, and 0% for other herpesviruses. This study is the first to report the existence of anti-Neospora sp. antibodies in donkeys and the seroprevalence of T. gondii in horses and donkeys in Western Anatolia, Turkey.

Comparative lung pathology of equine herpes virus 1 and equine influenza infection in BALB/c mouse model

Comparative lung pathology of equine herpes virus 1 and equine influenza infection in BALB/c mouse model

Exploring the potential of invariable surface glycoprotein (ISG65) as promising antigen for diagnosis of Trypanosoma evansi infection

Trypanosoma evansi, a hemoflagellate protozoan, leads to wasting disease, surra in livestock animals causing huge economic losses. Currently, the preferred assay for surra diagnosis is whole cell lysate (WCL) based ELISA, which requires the use of rodents for WCL preparation. To avoid use of laboratory animals, we used recombinant DNA technology to express T. evansi invariable surface glycoprotein (ISG) in E. coli. The potential of recombinant ISG65 (rISG65) as a diagnostic antigen was investigated in immunoblot and indirect ELISA using experimentally infected equine serum samples from 0 to 84 days post infection. The results indicated that rISG65 reacted with horse T. evansi positive serum giving two bands of approximately 48 kDa and 96 kDa. T. evansi-specific antibodies were detected as early as 10 and 14 days post infection using immunoblot and indirect ELISA, respectively using rISG65 antigen. No cross-reactivity was observed in ELISA and immunoblot with different serum samples of equines positive for Equine herpesvirus 1, Burkholderia mallei, and Theileria equi infections. Several immunoreactive regions were observed between 30 and 100 kDa in T. evansi isolate of horse origin indicating the existence of multiple copies of ISG protein in a single trypanosome. The recombinant ISG has proven to be good candidate antigen to be used in ELISA for serodiagnosis of T. evansi infection in different animals.

Equine Herpesvirus Type 1 Myeloencephalitis in the Brazilian Amazon

An investigative and epidemiological study was carried out for equine herpesvirus type 1 (HVE-1) in 10 outbreaks of neurological disease from different farms in the state of Pará, Brazil. 25 horses were studied: six male and 19 females, aged between one and 13 years. A necropsy of six horses was performed, and the others recovered either with or without treatment (T1-vitamin B1 + dexamentasone; T2-vitamin B1 + flunixim meglumine). Animals that received treatment recovered after eight days. The main clinical signs observed were motor incoordination, progressive paresis, thoracic and/or pelvic limbs abducted after induction of clinical examination, knuckling of the hind fetlocks, sagging and swaying of the hindquarters while standing or walking and paresis. All animals were positive: 88% (22/25) in nested PCR and 72% (18/25) in serum neutralization (including three negatives in serology). Focal brownish areas compatible with hemorrhage were found in the white and gray matter of the spinal cord of two animals. On histological analysis, there were perivasculitis and neutrophilic vasculitis in the gray matter of the spinal cord and brain. Based on the evidence, this work proves the circulation of HVE-1 in the Amazon biome, mainly in the state of Pará, Brazil.

Role of evolutionary epidemiology in the determination of the risk factors associated with some equine viral diseases

The evolutionary epidemiology is crucial as it does not only help in tracking the origin, spreading, prediction, and control of viruses but also explains the failure causes of some vaccines and serological diagnostic tools. To keep animal welfare, it is essential to raise awareness of the multiple risk factors associated with the different epidemics. Arthropod-borne viruses like African horse sickness virus (AHSV) and equine infectious anaemia virus (EIAV) are related to vectors multiplication. Accordingly, their seasonal occurrence was attributed to the environmental climatic conditions. While equine influenza virus (EIV) and equine herpes virus (EHV) were found to occur in winter and spring (foaling seasons), respectively. The management risk factors resulted in the occurrence and reactivation of latently infected cases. The RNA viruses are characterized by genetic assortment which results in increasing pathogenicity, and failure of the used vaccines. The EHVs able to establish infection in different host tissues adding to their immune evasion strategies. Most of the diseases occurred at the age over 2 years although the EIAV takes long time to appear. The hard work of males and other stress factors render them more liable for infection with equine viral arteritis (EVA), EIAV, and EHV. Genetically, some breeds of horses were at risk of AHSV, EVA, and EHV infection. Most of the donkeys, mules, and zebra develop subclinical forms that magnifies their role in the epidemiological situation. Different phenomena like overwintering in AHSV, hard work in EIV, virus hidden nature and latency in EHV should be more analysed.

Epidemiological aspects of some equine viral diseases

Although different equine viruses’ outbreaks have been recorded. However, the most important ones in are the African horse sickness virus (AHSV), equine influenza virus (EIV), equine viral arteritis (EVA), Equine infectious anaemia virus (EIAV), and equine herpes viruses (EHV). To combat these diseases, it is imperative to understand their epidemiological aspects. So, the current review aims to highlight some epidemiological aspects including; causative agents, clinical forms, history, prevalence and geographical distribution, source of infection, and methods of transmission. The AHSV mainly causes pulmonary, and cardiac forms with high morbidity and mortality rates in Africa. The EIV is found all over the world and results in respiratory signs. The EVA has low morbidity and mortality rates and is mainly found in the Americas and Europe, its significance is due to the reproductive problems as abortion in mares and subfertility in stallions. The EIAV has low morbidity and mortality rates and causes long time course disease mainly of fever, and chronic anaemia or death. The EHVs are the current most important pathogens due to their endemicity all over the world and their high morbidity. It causes respiratory, abortion, neonatal, and sometimes neurological manifestations. Aerosols and body excretions are the main sources of infection with EIV, EVA, and EHV. Venereal EVA transmission occurs through natural breeding or artificial insemination with the semen of infected or carrier stallions. The spreading of arboviruses is greatly affected by the vector activity like the AHSV which transmitted by the the Culicoide. imicola biting midges, and the EIAV by family Tabanidae. In general, it is recommended to take all epidemiological measures, including vaccinations and vector control, to limit the spread of such diseases and reduce economic losses.

Prevention of fatal equine herpesvirus type 1 encephalitis in mice by immunization with a limited-replication cycle virus

Equine herpesvirus type 1 (EHV-1) is a devastating pathogen of horses, their natural hosts, and causes fatal encephalitis in non-natural hosts. We previously demonstrated that acylation of the tegument protein UL11 is required for viral replication in cultured cells. We created a mutant virus (EHV-1 UL12 trunc UL11 G2AC7AC9A), in which glycyl and cysteinyl residues at positions 2, 7 and 9 of UL11 that are normally acylated were replaced with alanyl residues. This virus, designated the 2/7/9 mutant, has a limited-replication cycle (LRC), in which replication stops after just a few cycles. Here, we tested whether the 2/7/9 mutant could be used as a vaccine against fatal encephalitis in a mouse model. A virulence test showed that the 2/7/9 mutant was not pathogenic in mice and elicited an antibody response. We also attempted to use the 2/7/9 mutant to immunize mice against a zebra-borne EHV-1, 94–137. Two trials were conducted, each with five immunized mice, five non-immunized and five control mice. In both trials, clinical signs and fatalities were much lower in the immunized mice than in the non-immunized mice. In addition, none of the mice in either trial developed neutralizing antibodies, indicating that the immunity induced by the 2/7/9 mutant was not due to neutralizing activity. The results indicate that the 2/7/9 LRC mutant has promise as a vaccine against EHV-1 infection non-natural hosts.

Epidemiological Aspects of Equid Herpesvirus-Associated Myeloencephalopathy (EHM) Outbreaks.

Equid Herpesvirus Myeloencephalopathy (EHM) is a multifactorial disease following an EHV-1 infection in Equidae. We investigated a total of 589 horses on 13 premises in Europe in search of risk factors for the development of EHM. We found that fever (p < 0.001), increasing age (p = 0.032), and female sex (p = 0.042) were risk factors for EHM in a logistic mixed model. Some breeds had a decreased risk to develop EHM compared to others (Shetland and Welsh ponies; p = 0.017; p = 0.031), and fewer EHV-1-vaccinated horses were affected by EHM compared to unvaccinated horses (p = 0.02). Data evaluation was complex due to high variability between outbreaks with regards to construction and environment; viral characteristics and the virus's transmissibility were affected by operational management. This study confirms earlier suspected host-specific risk factors, and our data support the benefit of high vaccine coverage at high-traffic boarding facilities.

Molecular detection of equid herpesvirus in bronchoalveolar lavage fluid from asymptomatic horses in Southern Brazil.

Molecular approaches to diagnose respiratory viruses have provided an opportunity for early and subclinical pathogen detection, particularly in samples from the upper respiratory tract. This study aimed to investigate the presence of herpesviruses, particularly equid herpesvirus (EHV)-2 and EHV-5, in samples from the lower respiratory tract of healthy racehorses from Southern Brazil. Samples from the lower respiratory tract (i.e., bronchoalveolar lavage fluid [BALF]) were assessed by video endoscopy, cytological evaluation of BALF, and tracheal aspirates (TA), along with quantitative polymerase chain reaction (qPCR), to detect equine herpesvirus infection in the lower respiratory tract samples and compare corresponding cytological and endoscopic findings. At least one abnormality per horse during endoscopy examination was observed, including, but not limited to, mucous secretion in the airways and pharyngeal lymphoid hyperplasia. The presence of EHV-2 and/or EHV-5 was detected by qPCR in 3/10 animals. One horse was positive for EHV-2 alone, one for EHV-5 alone, and one for both. To the authors' knowledge, this is the first molecular detection of EHV-2 and EHV-5 in Brazilian thoroughbred horses. These findings may provide new insights into the epidemiology of EHV-2 and EHV-5 in Brazilian horses, evidencing the importance of the molecular investigation, early detection, and prevention of respiratory diseases.

A case of malignant transformation of equine immune‐mediated keratitis to B‐cell lymphoma

SummaryA 13‐year‐old Thoroughbred/Oldenburg gelding presented as a tertiary referral for a 6‐year history of immune‐mediated keratitis (IMMK) after the development of corneal and limbal nodules in the presence of medical therapy. A keratectomy extending to the limbus was performed to remove the affected cornea and limbal nodules. Two corneal‐limbal samples were submitted for histopathology and initially diagnosed as marked lympho‐histiocytic stromal keratitis with epithelial hyperplasia, keratinisation and keratinocyte apoptosis consistent with IMMK. Additional testing with immunohistochemical staining for CD3, CD20, PAX5 and IBA1 revealed large, atypical B‐lymphocytes on a background of histiocytes and mature T‐lymphocytes, with concern for T‐cell‐rich large B‐cell lymphoma. PCR antigen receptor rearrangement clonality testing was also performed and was diagnostic for large B‐cell lymphoma. Polymerase chain reaction (PCR) for equine herpesvirus 5 (EHV‐5) was positive. Following keratectomy, a short course of neomycin/polymyxin B/dexamethasone ophthalmic ointment was prescribed; no other treatment nor adjunct therapy was performed. Eighteen months post‐operation, the case remains visual and comfortable on no medications.

Frequency of Detection of Respiratory Pathogens in Clinically Healthy Show Horses Following a Multi-County Outbreak of Equine Herpesvirus-1 Myeloencephalopathy in California.

Actively shedding healthy horses have been indicated as a possible source of respiratory pathogen outbreak, transmission, and spread. Using nasal swabs from clinically healthy sport horses submitted for qPCR testing after an outbreak of equine herpesvirus-1 (EHV-1) myeloencephalopathy (EHM) in the spring of 2022, this study aimed to identify the rate of clinically healthy horses shedding common and less characterized respiratory pathogens within the sport horse population to better understand their role in outbreaks. Swabs were collected during a required quarantine and testing period, according to the United States Equestrian Federation (USEF), and showed return-to-competition requirements. Common respiratory pathogens, such as equine influenza virus (EIV), EHV-4, and equine rhinitis B virus (ERBV), were found at low but stable frequencies within previously reported ranges, whereas EHV-1 and Streptococcus equi subspecies equi (S. equi) were found at or above previously reported frequencies. Less characterized respiratory pathogens, such as EHV-2, EHV-5, and S. equi subspecies zooepidemicus (S. zooepidemicus), were found within previously reported ranges. Common respiratory pathogens, especially EHV-1 following the multiple EHM outbreaks, were found to be circulating in clinically healthy sport horse populations, reflecting their silent transmission. The strategy of quarantine and EHV-1 qPCR testing of clinically healthy horses was successful at eliminating additional EHM outbreaks and facilitating safe return to competition with no reported respiratory disease outbreaks following the subsequent shows in California.

Detection of Local Isolates of Equine Herpes virus Type 1 Associated with Abortion and Respiratory Manifestation in Horses in Giza

Detection of Local Isolates of Equine Herpes virus Type 1 Associated with Abortion and Respiratory Manifestation in Horses in Giza