lable at ScienceDirect

Journal of Equine Veterinary Science 87 (2020) 102923

Contents lists avai

Journal of Equine Veterinary Science

journal homepage: www.j -evs.com

Review Article

Equine Herpesvirus-I Infection in Horses: Recent Updates on itsPathogenicity, Vaccination, and Preventive Management Strategies

Ameer Khusro a, Chirom Aarti a, Raymundo Rene Rivas-Caceres b, *,Alberto Barbabosa -Pliego c, **

a Research Department of Plant Biology and Biotechnology, Loyola College, Chennai, Tamil Nadu, Indiab Autonomous University of Ciudad Ju�arez, Ciudad Ju�arez, Mexicoc Facultad de Medicina Veterinaria y Zootecnia, Universidad Aut�onoma del Estado de M�exico, Toluca, Mexico

a r t i c l e i n f o

Article history:Received 4 November 2019Received in revised form7 January 2020Accepted 7 January 2020Available online 11 January 2020

Keywords:Equine herpesvirus-1HorsesManagement practicesPathogenesisVaccination

Conflict of interest statement: None declared.Statement of Animal Rights: The research was perfor

ethical standard laid down in the 1996 declaratioamendments.* Corresponding author at: Raymundo Rene Rivas-C

sity of Ciudad Ju�arez, Ciudad Ju�arez 32310, Mexico.** Corresponding author at: Alberto Barbabosa -Pliegerinaria y Zootecnia, Universidad Aut�onoma del Estad

E-mail addresses: rrivas@uacj.mx (R.R. Rivas-Cacemx (A. Barbabosa -Pliego).

https://doi.org/10.1016/j.jevs.2020.1029230737-0806/© 2020 Elsevier Inc. All rights reserved.

a b s t r a c t

Equine herpesvirus-1 (EHV-1) is one of the most common and ubiquitous viral pathogens infectingequines, particularly horses worldwide. The EHV-1 is known to induce not only humoral but also cellularimmune responses in horses. Respiratory distress, abortion in pregnant mares, neurological disorders,and neonatal foal deaths represent EHV-1 infection. Despite the limited success of inactivated, subunit,live, and DNA vaccines, over the past few decades, vaccination remains the prime preventive option tocombat EHV-1 infection in horses. However, current vaccines lack the potentiality to protect theneurological form of infections in horses. There is desperate necessity to search effectual EHV-1 vaccinesthat may stimulate not only mucosal and systemic cellular immunity but also humoral immunity in thehorses. This review highlights the state of knowledge regarding EHV-1 biology, EHV-1 pathogenesis, anddisparate vaccines studied in the past to prevent EHV-1 infection. The review also underlines the bestmanagement strategies which certainly need to be adopted by veterinarians in order to avoid andprevent EHV-1 infection and outbreak in horses in the future.

© 2020 Elsevier Inc. All rights reserved.

1. Introduction

Equine industries have substantially grown in several countriesover the past fewdecades. In the equine industry, horses represent asubstantial economic weight, which is known to provide importanteconomics to developed and developing countries. Horses aremoved at the international level for sale, breeding, and competitiveevents or races [1]. The sport horse industries have tremendouslyexpanded over the decades. For instance, the number of events

med in accordance with then of Helsinki and its later

aceres, Autonomous Univer-

o, Facultad de Medicina Vet-o de M�exico, Toluca, M�exico.res), abarbabosa@yahoo.com.

sanctioned by the Federation Equestre Internationale (FEI) hasincreased by 255% between 2004 and 2014 (from 1,483 FEI eventsorganized in 2,004e3,790 in 2014) [2].While horses playa great roleas sport animals and working animals, unfortunately, horse asso-ciated deadly diseases are a major concern for the veterinarians atpresent. Surprisingly, equine infections have increased over the pastfew years due to the overwhelming transport of animals (particu-larly horses) and relaxation strategies of veterinary regulations [3].

The transmission of equine diseases is growing rapidly, andmodern genomics techniques such as massively parallel deepsequencing have discovered deadly equine pathogens [4]. Bacteria,viruses, fungi, rickettsias, and protozoans are the prime agents tocause illness in horses. Diseases are transmitted from one animal toanother through feces, urine, air droplets inhalation, and othersecretions. Additionally, few diseases are even sexually transmitted[5]. According to the Center for Food Security and Public Health,diseases-zoonotic and otherwise, are transmitted through variousroutes, namely (1) aerosol transmission (inhalation of infecteddroplets), (2) oral transmission (consuming contaminated feed orwater, or eatable items), (3) direct contact transmission (infectionthrough an openwound, mucus membranes, blood, saliva, nose-to-

A. Khusro et al. / Journal of Equine Veterinary Science 87 (2020) 1029232

nose contact, rubbing, biting, and in utero), (4) vehicle and traffictransmission (via transport process), (5) fomite transmission(through contaminated food, water, and soil), (6) vector-bornetransmission (transmission through insects or ticks), (7) mechani-cal transmission (directly from an infected animal to a susceptibleanimal), and (8) biological transmission (when the pathogen un-dergoes a biological function within the body of the vector beforebeing transmitted to a susceptible animal) (Fig. 1) [6].

Among equine diseases, Equine herpesvirus-1 (EHV-1) infectionis highly contagious. The exposure of the virus to horses occurs veryearly in life. In order to combat the equine diseases, it is imperativenot only to understand the epidemiology, mode of transmission,and therapeutic strategies for growing pathogenesis of deadly mi-crobes but also its detrimental impact on the equine industries.Hence, a systematic review was undertaken to outline the variousaspects of Equine herpesvirus-1 (EHV-1), infecting horses globally.The information discussed herein will be useful in not only un-derstanding EHV-1 infections but also formulating unique strate-gies that would help prevent the outbreak of EHV-1 within horsepopulations in the future.

2. Equine Herpesviruses (EHVs) Infection

Equine herpesviruses (FamilyeHerpesviridae) constitute awidearray of viruses, which are known to cause severe threat to horses.Considering the pathogenicity and tissue culture behavior, Her-pesviridae is divided into three subfamilies (a, b, and g) [7]. To date,nine different species of EHVs have been reported (Table 1). Amongthem, equine herpesvirus (EHV) such as EHV-1, EHV-3, EHV-4, EHV-8, and EHV-9 belong to the genus Varicellovirus, subfamily Alpha-herpesvirinae, family Herpesviridae, and order Herpesvirales. EHV-2 and EHV-5 have been classified into genus Percavirus, subfamilyGamaherpesvirinae, family Herpesviridae, and order Herpesvirales.EHV-6 and EHV-7 have been tentatively categorized in the sub-family Alphaherpesvirinae and Gammaherpesvirinae, respectively[8]. Surprisingly, only EHV-1, EHV-2, EHV-3, EHV-4, and EHV-5 havethe potentiality to infect horses. EHV-1 and EHV-4 are known toinfect the respiratory tracts of horses [8]. Coital exanthema ismainlycaused by EHV-3. The upper respiratory tract diseases, inappetence,

Fig. 1. Transmission of diseases in

lymphadenopathy, immunosuppression, keratoconjunctivitis, gen-eral malaise, and poor performance indicate the infection of horsesby EHV-2 and EHV-5 [9].

Among pathogenic EHVs, EHV-1 is a ubiquitous pathogen of allbreeds of horses and other equids, which causes severe diseases[10]. It can cause respiratory infection, neurological disease(neurologic form), and abortion in pregnant mares (abortogenicform) [6,10]. Neurological dysfunction may also lead to EquineHerpes Myeloencephalopathy (EHM) [10]. Additionally, the virus isresponsible for major economic and welfare problems. EHV-1 in-fects almost all the horses older than 2 years. The virus is contagiousand transmits by various means, particularly by horse-to-horsecontact and contamination [11]. As a primary exposure, EHV-1 hasthe potential to cause latent infection, and thus, it does not showanykind of symptoms in the beginning. The latent infection helps thevirus to play a paramount role in the transmission. The virus can alsoremain latentwith subsequent reactivation, thereby causing diseaseduring stress factors (Fig. 2). Stress factors viz. strenuous physicalactivity, stress during transport, suppressed immunity, and fatigueinduce the virus pathogenicity [11]. Fever, nasal discharge, cough,swollen legs, red eyes, and reddish mucus membrane are the majorinitial signs of EHV-1 pathogenicity [11]. Brain dysfunction,including coma-like condition, is rare among horses due to EHV-1infection. Most of the horses infected with EHV-1 rarely developEHM. Horses infected with EHM lose coordination and experiencedifficulty in standing, urinating, anddefecating. Hind limbs aremoreseverely affected than the forelimbs. Therefore, “dog-sitting” is nolonger possible in horses. Redness of the sclera or conjunctiva andinflammation of blood vessels are also observed during EHMinfection in horses. Additionally, multifocal myeloencephalopathy,ischemic neuronal injury, hemorrhage, and thrombosis are themajor clinical symptoms of EHM [12]. The incubation period of EHMis approximately 6e10 days. The most common symptoms of res-piratory infection of EHV-1 in horses may be mild, which includehigh fever, depression, cough, and nasal discharge for about 7 days.The incubation period varies from 2 to 10 days [13]. Abortion due toEHV-1 infection occurs after 2e12 weeks in late gestation. Themare’s reproductive tract is unaffected due to the infection. How-ever, its infection in the gestation periodmay cause the death of the

horses through various routes.

Table 1Different types of EHVs and its related diseases.

Species Subfamily Genus Infections

EHV-1 a Varicellovirus Respiratory, abortion, and neurologicalEHV-2 g Percavirus Lymphadenopathy, immunosuppression, and keratoconjunctivitisEHV-3 a Varicellovirus Coital exanthmaEHV-4 a Varicellovirus RespiratoryEHV-5 g Percavirus Lymphadenopathy, immunosuppression, keratoconjunctivitis,

and poor performanceEHV-6 a Varicellovirus Coital exanthmaEHV-7 g Rhadinovirus Not knownEHV-8 a Varicellovirus RhinitisEHV-9 a Varicellovirus Neurological

A. Khusro et al. / Journal of Equine Veterinary Science 87 (2020) 102923 3

foal within a limited period after birth [14]. The incubation periodvaries from 9 to 120 days.

3. EHV-1 Structure

Fig. 3 shows the structure of EHV-1 along with various glyco-proteins. The diameter of the virus is reported about 150 nm, whichis mainly composed of an icosahedral nucleocapsid containing theviral genome. The nucleocapsid is surrounded by globular tegu-ment (space between nucleocapsid and envelope). The envelopecontains various glycoprotein peplomers [15]. Glycoprotein ismainly responsible for the virus pathogenicity by adsorption andpenetration process [15]. Unlike other herpesviruses, EHV-1 en-codes an additional glycoprotein (gp2). Glycoproteins, such as B(gB), D (gD), and M (gM), cause cell penetration and cell-to-cellspreading. Glycoprotein K (gK) helps in the cell-to-cell spreadingand virus egression. Glycoprotein C (gC) helps in the attachmentand egression. Glycoproteins E (gE) and I (gI) help in cell-to-cellspreading. Glycoprotein N (gN) helps in the processing of gM. Theviral genome contains a single linear double-stranded DNA of about150.2 kbp in size with GC content of 57%. It is composed of a uniquelong (UL) and a unique short (US) region, flanked by internal andterminal repeat sequences (IRS and TRs), respectively [10,16]. Thegenome (150,223 bp) consists of 80 open reading frames (ORFs),encoding 76 unique genes, and 4 ORFs duplicated in the TRS [16].All herpesviruses have a similar capsid structure composed of 162capsomers (12 pentons and 150 hexons). Twelve portal proteinsform a ring in the nucleocapsid, which is used by viral DNA to enterinto the capsid [17]. The tegument is composed of 12 viral proteinsand enzymes which are mainly involved in the replication mech-anism of initiation of the virus [17].

Fig. 2. Transmission cycle of EHV-1 in horses.

4. EHV-1 Pathogenicity

The EHV-1 infection in horses via respiratory routs is mainlydue to the inhalation of infected aerosols, as well as contact withinfective secretion. The inhalation of EHV-1 causes its replicationinside the epithelia of the upper respiratory tract, includingpharynx, turbinates, soft palate, and tracheal epithelium [18](Fig. 4). The viral infection initiates with the interaction of EHV-1with the putative glycoprotein D receptor(s) [19]. As a matter offact, after the attachment process, the virus enters by either of thetwo processes, by receptor-mediated endocytosis or direct fusionof the virus envelope with the plasma membrane [20]. EHV-1utilizes multiple endocytic pathways in disparate cell types inorder to initiate the pathogenicity [20]. The caveolar endocytosisis known to infect neural microvascular endothelial cells, whileequine dermal cells involve energy-and pH-dependent endocy-tosis. After fusion, the naked virus particles move to the cytoplasmand initiate the replicationmechanism. EHV-1 infects the immunecells by penetrating the epithelium layer and infects further theinner tissues of the respiratory tract and lymph nodes [21].

The presence of the EHV-1 virus in the lymph nodes isconsidered as the second stage of infection (Fig. 4). In fact, theinfected lymphocytes move from lymph nodes to the blood,thereby causing viremia [21]. The virus spreads throughout thehost body via infected peripheral blood mononuclear cells(PBMC) [21]. Further, the virus migrates to the uterus. In theuterus, the infected lymphocytes adhere to the endothelial cellsin small blood vessels supplying the placenta (Fig. 4). Theinfected endothelial cell forms the protein clots, causing throm-bosis and vascular damage [22]. It leads to the inflammation ofblood vessels, thereby damaging the placenta, and thus, the virusmigrates to the fetus and consequently results in the abortion[23]. Surprisingly, all EHV1 isolates are not responsible forcausing abortion [24]. EHV1 induced abortion does not affect themare’s subsequent reproductive efficacy [25]. In case, the virusinfects in the later stages of pregnancies, the fetus may be bornalive, but soon after the birth, the respiratory infections lead toits death [25].

When the virus infected PBMC reaches to the nervous systemand infects vascular endothelia of small arterioles and venules, itindicates the initiation of a neurological disorder (Fig. 4) [26]. Ac-cording to Edington et al [27], EHV-1 can infect endothelial cells ofthe blood vessels of the nervous system, causing vasculitis,thrombosis, hypoxia, and secondary ischemic damage, thus, lead-ing to the nervous system disorders [28]. Mori et al [29] demon-strated the promising role of an inbred mouse as a valuable modelfor investigating the pathogenic attributes of EHV-1-inducedmyeloencephalopathy in horses. Currently, El-Habashi et al [30]depicted that EHVs invade the brain of the suckling hamster modelvia the trigeminal nerve besides the abducens, oculomotor, andfacial nerves, thereby suggesting the neuronal spread of neuro-pathogenic viruses to the brain.

Fig. 3. EHV-1 structure along with various glycoproteins.

Fig. 4. Schematic representation of EHV-1 pathogenesis.

A. Khusro et al. / Journal of Equine Veterinary Science 87 (2020) 1029234

A. Khusro et al. / Journal of Equine Veterinary Science 87 (2020) 102923 5

5. Vaccination Against EHV-1

Vaccination helps animals’ combat deadly infections. The im-mune response is stimulated by vaccines by tricking the body intobelieving that they are under the influence of infection so thatprotective antibodies and other immune mechanisms are induced[10]. In order to vaccinate large populace, immunization is consid-ered to be an effective approach. Interestingly, vaccination hashelped to get rid of a few infectious diseases in horses. However, atpresent, vaccination is considered as an important strategy tocombat EHV-1 infection [10]. Thewhole inactivated EHV-1 vaccinesare available in the market, which induces antibodies, and thus,provides distinct patterns of protection against the infection [3].Currently, either inert or live vaccines are being administrated. Inertvaccines consist of killed whole virus, subunit proteins, or DNA,while live vaccines correspond to the attenuated virus or livingvirus-based vector vaccines [3].

Both the humoral and cellular immune responses are essentialfor the successful vaccination against EHV-1 infection [26]. Thevaccine protects against the stimulation of respiratory infection, thesystemic dissemination of EHV-1 through a cell-associated viremia,and reactivation of the virus [26]. In general, foals are vaccinatedover 3e5 months of age. Further, the second immunization is rec-ommended within 4e6 weeks. In order to enhance immunity, asingle dose of vaccination is recommended every 3 or 6 months. Itis also suggested to vaccinate pregnant mares at the fifth, seventh,and ninth months of pregnancy in order to avoid virus-inducedabortion [3].

5.1. Inactivated Whole EHV-1 Vaccines and Subunit Vaccines

The vaccination for EHV-1 initiated in the 1960s, the majority ofwhich is the inactivated whole virus or subunit vaccines [10]. Thelack of pathogenic traits and virus replication is the leading bene-ficial aspects of these vaccines. On the other hand, local tolerance,pyrexia, risk of incomplete inactivation, and manipulation ofpathogens in production are the major concerns of these vaccines[10]. At present, inactivated whole EHV-1 vaccines are being uti-lized at a larger scale. For example, Duvaxyn EHV-1/4 is an inacti-vated whole EHV-1 and EHV-4 vaccine, adjuvanted with carbomer.Carbomer, an emulsifying agent, is a polyacrylic acid with anextremely high molecular weight. Duvaxyn EHV-1 (an inactivatedwhole EHV-1 vaccine) induces antibodies responses in foals andpregnant mares after few vaccinations [31].

The vaccination is known to reduce not only the clinicalsymptoms of infections but also virus shedding in foals. Addition-ally, the abortion in the vaccinated pregnant mares is observed tothe reduced as compared to the unvaccinated mares. Latestresearch showed that the vaccination of ponies twice with a con-ventional whole EHV-1 inactivated vaccine adjuvanted withcarbomer induced a strong humoral response [32]. The previousstudy showed the seroconversion of 159mares and 101 foals after 3vaccinations with an inactivated whole EHV-1 vaccine. Less than30% of mares and 50% of foals responded to vaccination [33]. Inspite of the wide use of inactivated virus vaccines in severalcountries, abortion is still being reported [34].

Whole inactivated virus vaccines are being replaced with anumber of subunit vaccines. The purified virions are being dis-integrated using varied detergents or ether to obtain a split vaccine[10]. The composition of split vaccines is more or less similar to theinactivated whole virus vaccines and show similar drawbacks.Subunit vaccines consist of one ormore pure or semi-pure antigens,purified from the pathogen or produced by baculovirus-expressionsystems using insect cells [10]. The identification of appropriateproteins for the subunit vaccine is rather difficult because of the

wide expression of proteins by the virus. The recognition of cellreceptors and attachment to the target cells is carried out mainly byglycoproteins that contain antibody epitopes [35]. The antigenicityof these vaccines is being tested in a rodent model. Baculovirusesinfected insect cells have been inoculated into mice, either indi-vidually or in combination. Serum antibodies to the virus are stim-ulated by recombinant gD and gH of baculoviruses. Immunizationwith gD produces antibody and enhances the EHV-1 clearance fromthe respiratory tract of mice [36]. Similar observations have beenrecorded with baculovirus recombinant gC [35] and gB [37].

5.2. Immunostimulating Complex-based Vaccines

The presence of strong adjuvants to induce an immune responseis essential for immunization with proteins [10]. Membrane pro-tein/antigens are generally adjuvanted with Quillaia saponin (QuilA), integrated into immuno-stimulating complexes (ISCOM), ormixed with Iscomatrix to induce their antigenicity. ISCOM orIscomatrix-based vaccines have been used successfully against EIV-1 infection in the horse [38e40]. A subunit vaccine, constitutinggp2, gp10, gB, gC, gD, and gM, was purified from EHV-1 strain V592and formulated with the adjuvant Iscomatrix-induced antibodyresponse and protected hamsters from viral infection [41]. Theintramuscular immunization containing subunit vaccines inducedthe humoral immune responses in ponies. Ponies vaccinated threetimes and subsequently infected with EHV-1 strain (a few weeksafter the third immunization) showed a reduction in the clinicalsymptoms [42]. According to the recent study, ponies, pregnantmares, and foals were intramuscularly administrated the inacti-vated Sf9 insect cells infected by a recombinant baculovirus codingfor EHV-1 gD and mixed with the adjuvant Iscomatrix [43]. Maresand foals vaccinated twice with a subunit vaccine containing gly-coproteins mixed with the Iscomatrix adjuvant have showndecreased virus shedding. However, the vaccination did not affectviremia [44].

5.3. DNA Vaccines

DNA vaccines have advantages compared to the inactivatedwhole virus or subunit vaccines [3]. Over the past few years,plethoras of in vivo studies have demonstrated the antigenicity, aswell as the efficiency of this vaccine against EHV-1 pathogenicity.The intramuscular immunization of DNA vaccine encoding gDexhibited to elicit humoral and cell-mediated immunity in horses,which showed an enhanced concentration of gD-specific antibody[45]. However, in the study of Foote et al [43], no stimulation ofantibody was observed after immunizing the horses with DNAcoding for gD. In another report, the antibody responsewas inducedafter vaccinating the ponies with the cationic lipid DNA/DMRIE-DOPE vaccine. Further, DNA vaccine adjuvanted with aluminumphosphate reduced the duration of nasopharyngeal virus excretion.The vaccination did not affect viremia [32]. Thus, DNA vaccinationinduces EHV-1 specific antibody response in the horses.

5.4. Noninfectious EHV-1 Light-particles

EHV-1 light-particles (EHV-1 L-particles) have been identified aspotent immunizing agents. The immunization of mice with non-replicative EHV-1 L-particles based on the administration routestimulated the antibody response [46]. The immunization withEHV-1 L-particles stimulated antibody response in specific-pathogen-free (spf) foals. Clinical symptoms of infection, virusshedding, and cell-associated viremia subsequent to EHV-1 strainAb4 infection were milder in immunized ponies [47].

A. Khusro et al. / Journal of Equine Veterinary Science 87 (2020) 1029236

5.5. Live Attenuated Vaccines

The exogenous and endogenous pathways play a critical role inpresenting the antigens to the immune system, and vaccinesstimulate the immune response similar to those induced by infec-tion [15]. The intranasal or intramuscular administration of a liveattenuated/modified EHV-1 is an imperative approach for vacci-nation against EHV-1 [15]. However, there is a possibility to revertthe virulence through this vaccination strategy. EHV-1 mutants viz.thymidine kinase negative (TK-) and temperature-sensitive (Ts)mutants are mainly used as live attenuated EHV-1 vaccines [15].The intramuscular or intravenous administration of TK- EHV-1 toEHV-1 unprimed ponies did not stimulate any symptoms. Theantibody response was observed in vaccinated ponies after EHV-1infection. Significant reduction in the clinical symptoms of dis-ease and virus shedding were also found to be reduced in TK- EHV-1 immunized ponies [48]. Another TK- EHV-1 mutant was evalu-ated in specific pathogen-free foals [49]. While it was able toreplicate in foals, it was found less pathogenic than the wild typeEHV-1. Vaccination with the TK- mutant partially reduced theclinical signs of infection stimulated by challenge infectionwith thewild type virus but did not prevent cell-associated viremia [49]. Inanother study, the intranasal immunization of 11 pregnant mareswas carried out. Few of them developed a cell-associated viremia.However, most of the mares were protected against abortion wheninfected with EHV-1, 4e6 months after the inoculation of the Tsvirus [50]. In another report, the intranasal administration of a liveattenuated EHV-1 vaccine did not stimulate mucosal antibodies,which indicated the failure of attenuated EHV-1 vaccines to preventvirus shedding [51].

5.6. Poxvirus-based Vector Vaccine for EHV-1

Recombinant poxviruses have been extensively implied forvaccination [52]. As a matter of fact, poxviruses are geneticallystable and allow the insertion of a large segment of foreign DNAcoding for selected antigens [10]. Vaccinia or avipoxvirus (i.e.,canarypox or fowlpox virus) associated recombinant poxviruses arecommercially available, and a number of recombinant canarypoxbased vaccines have been developed for the horse [32]. Recently, arecombinant poxvirus vector coding for the IE protein, a knowncytotoxic T-lymphocytes (CTL) target protein, has been used forvaccination in horses [53]. The study illustrated that vaccinia-basedvaccines could induce CMI in the horse, and thus, it may contributeto protect against EHV-1 infection [53].

6. Vaccination Against EHV-1 Infection: Recent Updates

Saleh [54] demonstrated the safety and potentiality of EHV-1inactivated vaccine adjuvanted with Montanide pet gel A. Afterinoculation, the immune responses of horses were monitored up toeight months. Horses inoculated with the prepared vaccine showeda lack of postvaccinal reaction. In another study, Abdelwahab et al[55] showed the immune response of the EHV-1 inactivated vac-cine in horses. Three of the horses were vaccinated through theintramuscular route by 2 doses of the vaccine with a one-monthinterval, as well as another three horses were kept as control.Serum samples were collected from all vaccinated and control an-imals every 2 weeks until the 26th week postvaccination (WPV),which weremonitored for EHV-1 antibodies using ELISA, AGPT, andVNT. None of the vaccinated horses showed nasal charge, swellingat the site of injection, or an increase in body temperature post-vaccination. Vaccination of horses exhibited an increased antibodyresponse against EHV-1 at 2WPV in horses. After bolstering, anti-bodies titer continued to increase until reaching its peak at the 12

WPV with mean antibodies titer 1,269.7 in horses; then, the anti-bodies titer decreased gradually until the 26th WPV with aconsiderable protective antibody level. In addition, AGPT revealed aprecipitin line in collected samples with high antibodies. Resultsalso exhibited an increase in the virus-neutralizing antibody titer at2WPV with the mean antibodies titer 1 in horses. After bolstering,antibodies titer continued to increase until reaching its peak at the12 WPV with mean antibodies titer 3.1 in horses, and then, theantibodies titer decreased gradually until the 26th WPV with aconsiderable protective antibody level.

Humoral and cellular immune responses of horses vaccinatedwith inactivated EHV-1 vaccine adjuvanted with Montanide ISA70were examined and compared with immune responses of horsesvaccinated with a vaccine adjuvanted with Saponin-rehydragelmixture [56]. The virus was inactivated by binary ethyleneimineand residual virus infectivity was carried out on VERO cells andchorioallantoic membrane of ECE. Cell-mediated immune re-sponses in horses by lymphocyte blastogenesis assay depictedincreased CTL at the 5th day postvaccination and decreased at 10th

day; then, begins to increase at 5th day postbolstering till 30 dayspostvaccination. Further, Saponin-rehydragel adjuvanted vaccineincreased CTL. The vaccine adjuvanted by Montanide ISA 70induced more immune potentiation with high VN index and ELISAantibody titers than vaccine adjuvanted by Saponin-rehydrageladjuvant [56].

Bannai et al [57] performed epizootiological investigation ofEHV-1 infection among Japanese racehorses before and after thereplacement of an inactivated vaccine with a modified live vaccine.Three-year-old horses that received the first dose of the live vaccineshowed higher geometric mean (GM) VN titers (205 and 220) thanthose that received inactivated vaccine. Four-year-old horses from2015 to 2017 that had received the live vaccine in the previousepizootic periods had higher GM titers (205e246) than those from2011 to 2014, which had received the inactivated vaccine. Accord-ing to the report, the live EHV-1 vaccine is highly immunogenic andprovides greater VN antibody responses than the inactivated vac-cine. Results showed the replacement of an inactivated vaccinewith a live vaccine.

7. Vaccination Against EHV-1 Infection: Future Strategies

An ideal vaccine should not only protect against respiratoryinfections but also prevent the viremia. The induction of potentialmucosal and cellular immune responses is required as a futurestrategy for vaccine development. Recombinant vaccines withenhanced immune responses should be the prime focus for vacci-nation in horses. Detailed research is essential to identify EHV-1virulent genes that might be promising candidates for developingefficacious vaccines. Most importantly, in vivo studies need to beundertaken in existing equine models for investigating the potencyof new vaccines developed.

8. EHV-1 Infection: Management Considerations

Several factors and strategies are required to cover farm safetyand biosecurity, especially horse industries. In spite of the vacci-nation strategies, various management practices or suggestions areessential to prevent EHV-1 infection and outbreak in horses.

� A closed herd can be a significant management practice. Addi-tionally, the farm can be divided into various sections, and thenthe nontransient animals can be kept in areas away from thehorses that are constantly coming and going.

A. Khusro et al. / Journal of Equine Veterinary Science 87 (2020) 102923 7

� Separating the broodmares from the show horses can decreasethe possibility of the mares contracting a disease from thetransient horses.

� Investigating the horse’s background, health certificate, nega-tive infectious test results, and the vaccination status can beideal approaches while introducing new horses into theoperation.

� In order tominimize the risk of viral infection onto the farm, it isessential to quarantine any new horse away from the otherhorses for a minimum of 30 days. The quarantine period can bereduced if the health status and history of the horse are known.

� The transportation equipment need to be disinfected regularly.� Sharing feeders or watering tanks and grooming the equipmenton new horses should be avoided completely.

� New horses should be vaccinated during the quarantine periodas per the vaccination program.

� Personnel should clean their bodies, footwear, and clothes whilehandling quarantined horses. The horses that leave and comeback to the farm should be categorized as a new arrival group.When the animals are away from the farm, their exposure toother animals should be limited.

� The lead ropes, saddle pads, and blankets, belonging to otherhorse owners, should be avoided. The consumption of contami-nated feed or drinkingwater should not be allowed to the horses.

� The number of visitors should be limited, which can reduce therisk of infections. A logbook for all the visitors needs to bemaintained too.

� Horses infected with EHV-1 should be kept in their existing sta-bles and segregated from other horses during exercise periods.

� If horses develop fever, respiratory infections, and neurologicalsymptoms, the veterinarian should be notified, and the move-ment of other horses in that area should be avoided until theinfection is diagnosed by molecular techniques.

� Horses indicating the clinical symptoms of EHV-1 should beremoved immediately and kept in an isolated area.

� Since stress plays a pivotal role in eliciting the onset of clinicalsymptoms, horses kept in the infected area should not be sub-jected to strenuous physical exercise or long-distance trans-portation until their health status can be confirmed.

� Horses that have been identified EHV-1 positive within thedesignated quarantine areas should be retested periodically.

9. Conclusions and Future Perspective

The EHV-1 infection is the leading cause of tremendous eco-nomic losses to the equine industries worldwide. Over the past fewdecades, the incidence of EHV-1-associated complications in horseshas increased at an alarming rate. Vaccines are widely used amongracehorses and broodmares in order to reduce virus shedding andduration of viremia. Currently, no commercially available vaccineshave the potentiality to exhibit complete protection against EHV-1infection. However, commercial vaccines are known to reducesymptoms only in the respiratory and abortive form of EHV-1infection. Current vaccines lack the ability to protect the neuro-logical form of the infection in horses. There is a desperate neces-sity to identify effectual EHV-1 vaccines that may stimulate bothcellular and humoral immunity in the horses, alongwith a safer andeffective administration route. Recent research works have createda ray of hope among veterinarians considering the effectiveness offew inactivated, as well as live vaccines. Research is in progress toidentify the best agents for preventing and controlling the EHV-1infection and outbreak. Till then, the transmission of EHV-1 in-fections among equines can certainly be avoided by adopting andfollowing strictly disparate best management practices.

References

[1] Powell DG. The international movement of horses and its influence on thespread of infectious diseases. Rev Sci Tech 1985;5:155e61.

[2] Federation Equestre Internationale (FEI). Personal Communication. FEI eventgrowth update 2014. Lausanne, Switzerland: FEI Veterinary Department;2015.

[3] Minke JM, Audonnet JC, Fischer L. Equine viral vaccines: the past, present andfuture. Vet Res 2004;35:425e43.

[4] Brosnahan MM, Brooks SA, Antczak DF. Equine clinical genomics: a clinician’sprimer. Equine Vet J 2010;42:658e70.

[5] Samper JC, Tibary A. Disease transmission in horses. Theriogenology 2006;66:551e9.

[6] Loh EH, Zambrana-Torrelio C, Olival KJ, Bogich TL, Johnson CK, Mazet JA,Karesh W, Daszak P. Targeting transmission pathways for emerging zoonoticdisease surveillance and control. Vector Borne Zoonotic Dis 2015;15:432e7.

[7] Davison AJ. Evolution of the herpesviruses. Vet Microbiol 2002;86:69e88.[8] Davison AJ, Eberle R, Ehlers B, Hayward GS, McGeoch DJ, Minson AC, et al. The

order Herpesvirales. Arch Virol 2009;154:171e7.[9] Bel�ak S, P�alfi V, Tuboly S, Bartha L. Passive immunization of foals to prevent

respiratory disease caused by equine herpesvirus type 2. Zentralbl Veter-inarmed B 1980;27:826e30.

[10] Kapoor S, Sharma H, Singh M, Kumar P, Ranjan K, Kumari A, et al. Equineherpesviruses: a brief review. Adv Anim Vet Sci 2014;2:46e54.

[11] Oladunni FS, Horohov DW, Chambers TM. EHV-1: a constant threat to thehorse industry. Front Microbiol 2019;10:2668.

[12] Wilson WD. Equine herpesvirus 1 myeloencephalopathy. Vet Clin North AmEquine Pract 1997;13:53e72.

[13] Allen GP, Bryans JT. Molecular epizootiology, pathogenesis and prophylaxis ofequine herpesvirus 1 infections. Prog Vet Microbiol Immunol 1986;2:78e144.

[14] Perkins G, Ainsworth DM, Erb HN, Del Piero F, Miller M, Wilkins PA, et al.Clinical, haematological and biochemical findings in foals with neonatalequine herpesviruse1 infection compared with septic and premature foals.Equine Vet J 1999;31:422e6.

[15] Paillot R, Case R, Ross J, Newton R, Nugent J. Equine herpes virus-1: virus,immunity and vaccines. Open Vet Sci J 2008;2:68e91.

[16] Telford EA, Watson MS, McBride K, Davison AJ. The DNA sequence of equineherpesvirus-1. Virology 1992;189:304e16.

[17] Baker TS, Newcomb WW, Booy FP, Brown JC, Steven AC. Three-dimensionalstructures of maturable and abortive capsids of equine herpesvirus 1 fromcryoelectron microscopy. J Virol 1990;64:563e73.

[18] Kydd JH, Smith KC, Hannant D, Livesay GJ, Mumford JA. Distribution of equidherpesvirus-1 (EHV-1) in the respiratory tract of ponies: implication forvaccination strategies. Equine Vet J 1994;26:466e9.

[19] Azab W, Osterrieder N. Type 1 (EHVe1) and EHVe4 determine glycoproteinsD of equine herpesvirus cellular tropism independently of integrins. J Virol2012;86:2031e44.

[20] Hasebe R, Sasaki M, Sawa H, Wada R, Umemura T, Kimura T. Infectious entryof equine herpesviruse1 into host cells through different endocytic pathways.Virology 2009;393:198e209.

[21] Kamel M, Pavulraj S, Osterrieder K, Azab W. EHV-1 pathogenesis: currentin vitro models and future perspectives. Front Vet Sci 2019;6:251.

[22] Smith KC,Whitwell KE, Binns MM, Dolby CA, Hannant D, Mumford JA. Abortionof virologically negative foetuses following experimental challenge of pregnantpony mares with equid herpesvirus 1. Equine Vet J 1992;24:256e9.

[23] Smith KL, Allen GP, Branscum AJ, Frank Cook R, Vickers ML, Timoney PJ, et al.The increased prevalence of neuropathogenic strains of EHV-1 in equineabortions. Vet Microbiol 2010;141:5e11.

[24] Mumford JA, Hannant D, Jesset DM, O’Neill T, Smith KC, Ostlund E. Abortigenicand neurological disease caused by experimental infection with equidherpesviruse1. In: Proceedings 7th international conference on equine in-fectious diseases, Tokyo; 1994. p. 261e75.

[25] Crabb BS, Studdert MJ. Rhinopneumonitis (equine herpesvirus 4) and equineabortion (equine herpesvirus 1), in virus infections of equines. In: Studdert MJ,editor. Virus infections of Vertebrates, 6. Amsterdam: Elsevier; 1996.p. 11e37. Chapter 2.

[26] Rusli ND, Mat KB, Harun HC. A review: interactions of equine herpesvirus-1with immune system and equine lymphocyte. Open J Vet Med 2014;4:294e307.

[27] Edington N, Bridges CG, Patel JR. Endothelial cell infection and thrombosis inparalysis caused by equid herpesvirus-1: equine stroke. Arch Virol 1986;90:111e24.

[28] Schenkel AR, Goehring LS, Hussey GS, Ashton LV, Lunn DP. Infection of centralnervous system endothelial cells by cell-associated EHV-1. Vet Microbiol2011;148:389e95.

[29] Mori CMC, Mori E, Favaro LL, Santos CR, Lara MC, Villalobos EM, et al. Equidherpesvirus typee1 exhibits neurotropism and neurovirulence in a mousemodel. J Comp Path 2012;146:202e10.

[30] EleHabashi N, Kato Y, ELeNahass E, Fukushi H, Hirata A, Sakai H, et al. Anocular infection model using suckling hamsters inoculated with equineherpesvirus 9 (EHVe9): kinetics of the virus and timeecourse pathogenesis ofEHVe9einduced encephalitis via the eyes. Vet Pathol 2013;50:56e64.

[31] Heldens JG, Hannant D, Cullinane AA, Prendergast MJ, Mumford JA, Nelly M,Kydd JH, Weststrate MW, van der Hoven R. Clinical and virological evaluation

A. Khusro et al. / Journal of Equine Veterinary Science 87 (2020) 1029238

of the efficacy of an inactivated EHV1and EHV4 whole virus vaccine (DuvaxynEHV1,4). Vaccination/challenge experiments in foals and pregnant mares.Vaccine 2001;19:97e114.

[32] Minke JM, Fischer L, Baudu P, Guigal PM, Sindle T, Mumford JA, et al. Use ofDNA and recombinant canarypox viral (ALVAC) vectors for equine herpesvirus vaccination. Vet Immunol Immunopathol 2006;111:47e57.

[33] Foote CE, LoveDN, Gilkerson JR,Whalley JM. Serological responses ofmares andweanlings following vaccination with an inactivated whole virus equineherpesvirus 1 and equine herpesvirus 4 vaccine. VetMicrobiol 2002;88:13e25.

[34] van Maanen C, Willink DL, Smeenk LA, Brinkhof J, Terpstra C. An equineherpesvirus 1 (EHV1) abortion storm at a riding school. Vet Q 2000;22:83e7.

[35] Tewari D, Nair SV, De Ungria MC, Lawrence GL, Hayden M, Love DN, et al.Immunization with glycoprotein C of equine herpesvirus-1 is associated withaccelerated virus clearance in a murine model. Arch Virol 1995;140:789e97.

[36] Tewari D, Whalley JM, Love DN, Field HJ. Characterization of immune re-sponses to baculovirus-expressed equine herpesvirus type 1 glycoproteins Dand H in a murine model. J Gen Virol 1994;75(Pt7):1735e41.

[37] Kukreja A, Walker C, Fitzmaurice T, Awan A, Love DN, Whalley JM, et al.Protective effects of equine herpesvirus-1 (EHV-1) glycoprotein B in a murinemodel of EHV-1-induced abortion. Vet Microbiol 1998;62:303e11.

[38] Crouch CF, Daly J, Hannant D, Wilkins J, Francis MJ. Immune responses andprotective efficacy in ponies immunised with an equine influenza ISCOM vac-cine containing an ’American lineage’ H3N8 virus. Vaccine 2004;23:418e25.

[39] Crouch CF, Daly J, Henley W, Hannant D, Wilkins J, Francis MJ. The use of asystemic prime/mucosal boost strategy with an equine influenza ISCOMvaccine to induce protective immunity in horses. Vet Immunol Immunopathol2005;108:345e55.

[40] Paillot R, Grimmett H, Elton D, Daly JM. Protection, systemic IFN and antibodyresponses induced by an ISCOM-based vaccine against a recent equineinfluenza virus in its natural host. Vet Res 2008;39:21.

[41] Cook RF, O’Neill T, Strachan E, Sundquist B, Mumford JA. Protection againstlethal equine herpes virus type 1 (subtype 1) infection in hamsters by im-mune stimulating complexes (ISCOMs) containing the major viral glycopro-teins. Vaccine 1990;8:491e6.

[42] Hannant D, Jessett DM, O’Neill T, Dolby CA, Cook RF, Mumford JA. Responsesof ponies to equid herpesvirus-1 ISCOM vaccination and challenge with virusof the homologous strain. Res Vet Sci 1993;54:299e305.

[43] Foote CE, Love DN, Gilkerson JR, Rota J, Trevor-Jones P, Ruitenberg KM, et al.Serum antibody responses to equine herpesvirus 1 glycoprotein D in horses,pregnantmares and young foals. Vet Immunol Immunopathol 2005;105:47e57.

[44] Foote CE, Raidal SL, Pecenpetelovska G, Wellington JE, Whalley JM. Inoculationof mares and very young foals with EHV-1 glycoproteins D and B reduces

virus shedding following respiratory challenge with EHV-1. Vet ImmunolImmunopathol 2006;111:97e108.

[45] Ruitenberg KM, Love DN, Gilkerson JR, Wellington JE, Whalley JM. Equineherpesvirus 1 (EHV-1) glycoprotein D DNA inoculation in horses with pre-existing EHV-1/EHV-4 antibody. Vet Microbiol 2000;76:117e27.

[46] Mohd-Azmi ML, Field HJ, Rixon FJ, McLauchlan J. Protective immune re-sponses induced by non-infectious L-particles of equine herpesvirus type-1:implication of cellular immunity. J Microbiol 2002;40:11e9.

[47] Mohd-Azmi ML, Gibson JS, Rixon FJ, McLauchlan J. Protection of specificpathogen free (Spf) foals from severe equine herpesvirus type-1 (EHV-1)infection following immunization with non-infectious Lparticles. J Microbiol2002;40:183e92.

[48] Cornick J, Martens J, Martens R, Crandell R, McConnell S, Kit S. Safety andefficacy of a thymidine kinase negative equine herpesvirus-1 vaccine in younghorses. Can J Vet Res 1990;54:260e6.

[49] Slater JD, Gibson JS, Field HJ. Pathogenicity of a thymidine kinase-deficientmutant of equine herpesvirus 1 in mice and specific pathogen-free foals.J Gen Virol 1993;74(Pt 5):819e28.

[50] Patel JR, Bateman H, Williams J, Didlick S. Derivation and characterisation of alive equid herpes virus-1 (EHV-1) vaccine to protect against abortion andrespiratory disease due to EHV-1. Vet Microbiol 2003;91:23e39.

[51] Breathnach CC, Yeargan MR, Sheoran AS, Allen GP. The mucosal humoralimmune response of the horse to infective challenge and vaccination withequine herpesvirus-1 antigens. Equine Vet J 2001;33:651e7.

[52] Moss B. Genetically engineered poxviruses for recombinant gene expression,vaccination, and safety. Proc Natl Acad Sci U S A 1996;93:11341e8.

[53] Paillot R, Ellis SA, Daly JM, Audonnet JC, Minke JM, Davis-Poynter N, et al.Characterisation of CTL and IFN-g-synthesis in ponies following vaccinationwith a NYVAC based construct coding for EHV-1 immediate early gene, fol-lowed by challenge infection. Vaccine 2006;24:1490e500.

[54] Saleh NS. Study on inactivated equine herpesvirus-1 vaccine adjuvanted withmontanide pet gel A. Benha Vet Med J 2015;28:26e33.

[55] Abdelwahab MG, El Moghazy HM, Ibrahim EM, Hamouda FI, Warda SMA.Immune response against inactivated equine herpes virus vaccine preparedfrom local isolate in horses and donkeys in Egypt. J Am Sci 2018;14:74e83.

[56] Abdel-Hady HA, Khalil SA, ELkabbany MMA. Studies on cellular and humoralimmune responses of horses vaccinated with equine herpesvirus-1 vaccineusing different adjuvants. Alexandria J Vet Sci 2016;51:82e9.

[57] Bannai H, Tsujimura K, Nemoto M, Ohta M, Yamanaka T, Kokado H, et al.Epizootiological investigation of equine herpesvirus type 1 infection amongJapanese racehorses before and after the replacement of an inactivated vac-cine with a modified live vaccine. BMC Vet Res 2019;15:280.