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In 2017, two Thoroughbred stallions, A and B in Farms A and B, respectively, in Hokkaido in Japan showed clinical signs of equine coital exanthema (ECE). In 2020, stallion C in Farm B showed clinical signs of ECE. Eighteen mares were mated within five days before stallion A developed ECE. Ten mares that mated within 3 days before onset showed clinical signs of ECE on the external genitalia. Equine herpesvirus 3 (EHV-3) was isolated from vaginal swabs from three mares that mated within 2 days before onset. Swabs from 12 mares that mated within 4 days before onset were real-time PCR (rPCR)-positive and nine of those mares had an increased EHV-3 antibody titer. The three stallions were administered valaciclovir orally and topical acyclovir ointment was applied. Treatment started on the next day after onset in stallion A and on the day of onset in stallions B and C. EHV-3 was firstly isolated from penis swabs of stallions A and B before treatment and from penis swabs of stallion C 2 days after treatment. EHV-3 was not isolated after 8, 5 and 8 days from onset in stallions A, B and C, respectively. However, swabs were rPCR-positive for at least 12, 9 and 15 days after onset of stallions A, B and C, respectively. EHV-3 was excreted from the stallions at least within 4 days before the onset of ECE, and acyclovir treatment resulted in the termination of excretion within 8 days after onset.

A 3-month-old Quarter Horse filly presented with corneal ulceration in the right eye with extensive coalescing periocular ulcerations, erosions, and cutaneous crusts. Similar periocular lesions were present around the left eye, on the gingival mucosa, and on the cutaneous and mucosal surfaces of the lips. Based on the severity of the filly's corneal lesions, expense and duration of treatment, euthanasia was elected. Histological post mortem examination revealed numerous hyperplastic and/or dysplastic epithelial cells adjacent to areas of ulceration and erosion with intranuclear viral inclusion bodies. Equine herpesvirus-3 (EHV-3) was identified by polymerase chain reaction from the right cornea and lip. The virus was isolated from the right cornea, right eyelid and lip. The dam presented with multifocal to coalescing perineal vesicles. EHV-3 was confirmed by polymerase chain reaction from the vulvar lesions and the mare recovered spontaneously. This is the first case of EHV-3 corneal infection reported in horses and emphasises that EHV-3 should be included as a differential diagnosis for vesicular lesions involving the equine periocular and oronasal epithelium.

Em agosto de 2008, um garanhão da raça Mangalarga Marchador, de quatro anos de idade, com histórico clínico de apatia, inapetência e edema de prepúcio e escroto, apresentou, ao exame clínico, exsudato purulento fluindo pelo óstio prepucial, prepúcio e mucosa peniana com inúmeras lesões circulares de bordos elevados e hiperêmicos, centro ulcerado recoberto por material amarelado de aspecto fibrinoso, com distribuição multifocal. Histologicamente, a mucosa peniana apresentou áreas de ulceração associadas a infiltrado inflamatório misto, com necrose multifocal e moderado acúmulo de fibrina, que se estendiam para o tecido conjuntivo adjacente. O diagnóstico morfológico foi de balanopostite ulcerativa fibrino-necrótica multifocal intensa, similar ao encontrado em casos de exantema coital equino (ECE), causado pelo herpesvírus equino 3 (EHV-3). Amostra de pele do prepúcio e sangue, colhido em EDTA, foram submetidos a ensaios de PCR específicos para EHV-3, observando-se a amplificação de um produto de tamanho esperado de 518pb. A detecção do EHV-3 foi confirmada por meio de seu sequenciamento, sendo a sequência de nucleotídeos depositada no GenBank sob o número GQ336877. As sequências de nucleotídeos e as de aminoácidos deduzidos apresentaram identidade de 99% e 100%, respectivamente, com a sequência de EHV-3 disponível no GenBank, número AF081188. Após 15 dias de tratamento, houve completa cicatrização das lesões, com despigmentação da pele, principalmente, no prepúcio e na bolsa escrotal. Com base nos achados clínicos, histopatológicos, PCR e sequenciamento, concluiu-se tratar de um caso de exantema coital equino, sendo o primeiro com confirmação definitiva do agente etiológico no Brasil.

During the period from 2001 to the following year, progenital diseases had been epidemic among the draft stallions and mares pastured together in Iwate Prefecture, the northeastern district of Japan. A stallion and 8 of 31 mares were affected in 2001, and 1 of 2 stallions and 10 of 36 mares in 2002. The clinical symptoms consisted of the formation of papules, pustules, ulcers and scabs on the progenital skin and mucosa in stallions and mares. In 2002, Equine herpesvirus 3 (EHV3) was isolated from 2 mares and the glycoprotein G gene of the virus detected from a stallion and 4 mares by polymerase chain reaction. Serum neutralizing tests showed that 12 of 38 horses, 10 clinically and 2 subclinically affected, changed to be positive for the EHV3 antibody. The results suggest that the horses were affected with equine coital exanthema (ECE) through coitus. Five mares with the antibody at the pre-pastured period may have been the possible origins of EHV3 infection in 2002, although the exact origin in 2001 remains unknown. The artificial insemination was performed for the prevention of ECE spreading through coitus on the pasture in 2003. There was no epidemic of the disease in 31 mares, although 3 mares with the antibody at the pre-pastured period showed the significant increase in the titers during the pastured period.

During a recent breeding season, ulcerative, pustular skin lesions were observed on the external genitalia of 2 mares and 1 stallion within a small herd. Based on the location and description of the skin lesions plus the clinical history, equine coital exanthema, caused by equine herpesvirus 3 (EHV3), was the primary differential diagnosis. Scrapings of skin lesions from the perineum of 2 mares were submitted for diagnostic evaluation. Virus isolation was attempted by inoculation of several cell lines of equine origin, but no cytopathic agent was detected. The skin scrapings were processed for DNA extraction, and polymerase chain reaction (PCR) amplification was performed for herpesvirus DNA polymerase and DNA-packaging protein (terminase) genes using nested, degenerate primers targeted to conserved regions of the herpesvirus genome. Products of the expected sizes were generated for both assays, and subsequent nucleotide sequencing of the amplification products established that EHV3 had been detected in DNA extracted from the skin lesions. Detection of EHV3 was confirmed using an EHV3-specific PCR assay targeted to the gC gene. Using the novel EHV3 nucleotide sequence identified in this report, a sensitive and specific PCR assay targeted to the highly conserved DNA polymerase gene was developed.

Objective To develop rapid (< 8 hour) tests using polymerase chain reaction (PCR) for the diagnosis of equine herpesvirus 3 (EHV3; equine coital exanthema virus), equine gammaherpesviruses 2 (EHV2) and EHV5, equine adenovirus 1 (EAdV1), EAdV2, equine arteritis virus (EAV), equine rhinitis A virus (ERAV; formerly equine rhinovirus 1) Design Either single round or second round (seminested) PCRs were developed and validated. Methods Oligonucleotide primers were designed that were specific for each virus, PCR conditions were defined and the specificity and sensitivity of the assays were determined. The application of the tests was validated using a number of independent virus isolates for most of the viruses studied. The PCRs were applied directly to clinical samples where samples were available. Results We developed a single round PCR for the diagnosis of EHV3, a seminested PCR for EHV2 and single round PCRs for EHV5, EAdV1, EAdV2 and RT-PCRs for EAV and ERAV. The PCR primer sets for each virus were designed and shown to be highly specific (did not amplify any recognised non-target template) and sensitive (detection of minimal amounts of virus) and, where multiple virus isolates were available all isolates were detected. Conclusion The development and validation of a comprehensive panel of PCR diagnostic tests, predominantly for viruses causing equine respiratory disease, that can be completed within 8 hours from receipt of clinical samples, provides a major advance in the rapid diagnosis or exclusion diagnosis of these endemic equine virus diseases in Australia.

EHV3 causes equine coital exanthema and has been classified as an alphaherpesvirus on the basis of its biological properties; however due to the absence of any sequence information the phylogenetic relationship has not previously been examined. The complete nucleotide sequence of the EHV3 glycoprotein G (gG) gene was determined and showed that this virus is most closely related to the alphaherpesviruses equine herpesviruses type 1 (EHV 1) and type 4 (EHV4). EHV3 gG contains conserved and variable regions which are homologous to those previously defined for EHV1 and EHV4 gG proteins. Consistent with EHV1 and EHV4 gG, the variable region of EHV3 gG was found to elicit a strong antibody response in experimentally and naturally infected horses and could be exploited for use as a diagnostic reagent.

A consensus primer PCR approach was used to (i) investigate the presence of herpesviruses in wild and zoo equids (zebra, wild ass, tapir) and to (ii) study the genetic relationship of the herpesvirus of pigeons (columbid herpesvirus 1) to other herpesvirus species. The PCR assay, based on degenerate primers targeting highly conserved regions of the DNA polymerase gene of herpesviruses, was modified by using a mixture of degenerate and deoxyinosine-substituted primers. The applicability of the modification was validated by amplification of published DNA polymerase genes of 16 herpesvirus species and of the previously uncharacterized DNA polymerase genes of equine herpesvirus 3 (EHV-3) and equine herpesvirus 5 (EHV-5). The modified assay was then used for partial amplification of the polymerase of columbid herpesvirus 1 which is presently classified as a β-herpesvirus based on biological criteria. Sequence analysis of amplicons obtained from four different viral strains revealed a close relationship of columbid herpesvirus 1 to members of the subfamily Alphaherpesvirinae, especially to Marek’s disease herpesvirus. This was confirmed by characterization of additional 1.6 kb of the columbid herpesvirus 1 polymerase. Consensus PCR analysis of blood samples from zebras, a wild ass and a tapir revealed amplicons showing high percentages (>50%) of sequence identity to DNA polymerases of γ-herpesviruses. In particular, the zebra and the wild ass sequence were closely related to each other and to the polymerases of the equine γ-herpesviruses EHV-2 and EHV-5 with sequence identities of >80%. This is a first indication that novel γ-herpesviruses are present in wild and zoo equids.

Equine coital exanthema (ECE), caused by equid herpesvirus 3 (EHV-3), has been recognized as an economically significant venereal disease for years. However, no infection models on the natural host have been established. In order to set up an experimental infection protocol, seronegative and seropositive mares were topically inoculated in the perineal region with 4 × 10 6 TCID 50 /ml of EHV-3. Clinical signs were then evaluated by means of a designed scoring system, and body temperature was recorded daily. Virological, and serological studies were also performed. Typical ECE lesions, with clinical scores of 90, 92, 160 and 172, were observed in the four seronegative animals. Only mild ECE lesions were observed in the two seropositive mares, being the clinical scores 53 and 41. Both groups of mares shed the virus, but the duration of virus shedding was shorter and its intensity was lower in seropositive mares than in seronegative ones. Moreover, EHV-3 antibody response was detected in both seronegative and seropositive mares after experimental infection and re-infection, being more moderate in seropositive ones. As a conclusion, EHV-3 infection of mares was experimentally achieved in a reproducible manner. The typical lesions of ECE were observed after topical EHV-3 infection in seronegative mares, in association with virus excretion and neutralizing antibody kinetics.

Summary State of latency, well known for several herpesviruses, has been proposed for equine herpesvirus-3 (EHV-3) and supported by epidemiological observations. No detailed assessment about reactivation, patterns of excretion and re-excretion has been formally reported. An experimental reactivation study by corticosteroid treatment in previously naturally infected horses was therefore carried out. Two polo mares with clinical and virologically confirmed history of equine coital exanthema were injected with dexamethasone and prednisolone on 3 successive days. Clinical signs, body temperature and clinical samples for virological and serological studies were obtained daily. Mares did not show any systemic clinical signs or hyperthermia. EHV-3 shedding, seroconversion and the presence of a small lesion were observed in one of the mares under study 2 weeks after corticosteroid treatment. The results demonstrate that this virus exhibits a latency-reactivation behaviour similar to that of other alpha herpesviruses. Reactivation of latency may have an important bearing on the appearance of clinical signs in mares and/or stallions during the breeding season without the actual evidence of transfer from mare to stallion or vice versa.

Equine herpesviruses (EHVs) are a group of nine agents that cause diseases ranging in severity from a neurological disease, which can be fatal in more than 50% of infected animals (equid herpesvirus 1, EHV-1), to mild or subclinical infection of B-lymphocytes (equid herpesvirus 5, EHV-5). Among the nine equid herpesviruses, three are referred to as asinine herpesviruses, which primarily infect donekys. The six ‘true’ equid herpesviruses (EHV-1, EHV-2, EHV-3, EHV-4, EHV-5, and EHV-9) infect horses primarily and generally have a very narrow host range in vivo. However, EHV-9 is able to infect many different species and often causes lethal encephalitis in nonequine hosts. EHVs represent two subfamilies, Alphaherpesvirinae and Gammaherpesvirinae, and two genera, Varicellovirus and Rhadinovirus, within the family Herpesviridae. The most important EHVs clinically and economicaly are EHV-1 and EHV-4, whose genomes have been sequenced in their entirety. Mainly for these reasons, most research worldwide is focused on EHV-1, for which detailed studies on gene regulation, replication cycle in cultured cells, pathogenesis, and vaccine development have been conducted, while detailed knowledge on these functions in other EHVs is scarce.

Publisher Summary The horse (Equus caballus) is unusual, in that it is host to three distinct alphaherpes viruses, designated as equine herpesvirus 1 (EHV1; equine abortion virus), EHV4 (equine rhinopneumonitis virus), and EHV3 (equine coital exanthema virus). EHV4 and EHVl are closely related viruses that, prior to 1981, were considered a single virus type or subtypes of a single virus. There was an assumption that this single virus, “EHV1,” caused an acute respiratory tract disease called “rhinopneumonitis,”which established a latent infection and then, as an unpredictable sequel in the case of a pregnant mare, underwent reactivation followed by viremic spread to the fetus, causing abortion. This chapter is concerned primarily with EHV4 and EHV1, with brief consideration of AHV3, highlighting its relationship to EHV1. Earlier, the reviews of “EHV1” may be consulted for a historic perspective and earlier references. However, each of these reviews carries ambiguities related to a belief that only a single virus type “EHV1,” rather than two distinct viruses, was involved. This chapter mainly focuses on the advances in our understanding of the molecular biology, including genetic and antigenic characteristics, of EHV4 and EHVl since 1981. A brief overview of the clinical syndromes and pathogenesis, epidemiology, laboratory diagnosis, vaccines, and immunity of EHV4 and EHVl is also given in this chapter.

The transcription of equine herpesvirus type 3 (EHV-3; equine coital exanthema virus) has been examined and found to be temporally regulated into three classes: immediate early (IE), early (E), and late (L). Hybridization of in vivo 32PO4-labeled transcripts revealed that IE transcript(s) are derived exclusively from the inverted repeat segments (IRs) of the viral genome, while E and L transcripts are not restricted to any specific region of the genome. Northern blot analysis of EHV-3 IE RNA revealed a single transcript of approximately 5.7 kb (3.8 MDa). We have previously shown that transcription of equine herpesvirus type 1 (EHV-1) DNA is temporally regulated and produces a single 6 kb IE RNA which is derived from the IRs segments. In this paper, we show that the EHV-1 and EHV-3 IE RNA species are homologous, reflecting the colinearity of the genomes of these two related viruses. While four IE polypeptides are synthesized in EHV-1 infected cells in the presence of actinomycin D following the removal of a cycloheximide block, only one major IE polypeptide (180 kDa) is detectable in EHV-3 infected cells under these conditions. However, immunoprecipitation of EHV-3 infected cell extracts with polyvalent rabbit antisera to IE1 of EHV-1 revealed at least two other viral specific IE polypeptides.

Equine herpesvirus type 3 (EHV-3) DNA, isolated from purified virions of the large-plaque strain, was digested with the restriction endonucleases XbaI, Bg/II, EcoRI, and HindIII. Several lines of evidence indicated that the DNA extracted from purified virions was composed of long (L) and short (S) components and was present as two isomeric forms, P and IS. The evidence included: (i) after electrophoresis on agarose gels, the summed molecular weights of the digestion products exceeded that expected from intact, unit size DNA; (ii) quantitative measurements of radioactivity (molar ratios) indicated 'minor bands' (0.5 M) interspersed among the major (1.0 M) bands; and (iii) a brief digestion with lambda-5'-exonuclease, prior to digestion with restriction endonuclease, resulted in the loss of some submolar and molar ratio bands, indicative of three termini. A preliminary fragment linkage map of the XbaI digestion products revealed EHV-3 DNA to contain only one recognition site in the unique sequence of the S component. From this linkage map, the size of the S component was deduced to be (22.3 +/- 5) X 10(6) molecular weight.

Two previously unknown and distinct herpesviruses were isolated from donkeys. One, with the characteristics of a betaherpesvirus, was isolated from the leukocytes of an apparently healthy donkey, while the second, an alphaherpesvirus, was recovered from the nasal cavity of donkeys given high doses of corticosteroids, and caused rhinitis in two seronegative weanling donkeys when they were intranasally infected. Few, if any, restriction endonuclease fragments were shared by the donkey betaherpesvirus, equine herpesvirus 2 (EHV 2) or EHV 5, a second distinctly different equine betaherpesvirus, nor by the donkey alphaherpesvirus, EHV 1, EHV 4, or EHV 3. In Southern blot analysis the donkey betaherpesvirus showed low levels of sequence similarity to both EHV 2 and EHV 5, while the donkey alphaherpesvirus and EHV 1 shared a moderate degree of sequence similarity, less similarity with EHV 4 and very low level of sequence similarity with EHV 3. These two isolates appear prototypic of two previously unrecorded herpesviruses for which the names asinine herpesvirus 2 and 3 are suggested for the betaherpesvirus and the alphaherpesvirus respectively.

Infection of nonpermissive primary hamster embryo cells with equine herpesvirus type 3 (EHV-3; multiplicity of infection = 10 pfu/cell) resulted in an abortive infection and the development of several hundred foci of rapidly growing cells. Five of these foci were chosen at random for the establishment of transformed cell lines, designated EVD-1 (equine venereal disease) through 5. These transformed cell lines exhibited altered biological properties typical of transformed cells, including immortality, growth to high saturation density, colony formation in soft agar, reduced serum requirements, aneuploid karyotype, and oncogenicity in syngeneic animals. Subsequently, five corresponding tumor cell lines (EVD-1T through 5T) with similar biological properties were established. All EHV-3 transformed and tumor cell lines have been shown to express EHV-3-specific proteins by indirect immunofluorescence assays employing rabbit antisera to EHV-3 infected equine cells. None of the transformed cell lines were found to release infectious virus by infectious center or cocultivation assay or to contain viral particles by electron microscopy.

This chapter provides an overview of the classification and description of the equine coital exanthema virus, its hosts, the key developments regarding the virus, and the diagnostic techniques and reagents. The equine coital exanthema virus causes a venereally transmitted disease characterized by vesicles, pustules, and ulcers on the penis, prepuce of stallions, vagina, vulva, and perineal area of mares. The hosts that are infected by the equine coital exanthema virus include horses. A worldwide distribution for the equine coital exanthema virus is suspected. A microplaque virus neutralization (VN) assay system is the most sensitive diagnostic technique for the equine coital exanthema virus. Complement-fixing (CF) antibodies indicate infection within 60 days.

Equine herpesvirus 3 (EHV-3)-infected equine cells display a kinetics of infected cell polypeptide (ICP) synthesis at 34 degrees C that is typical of coordinate cascade gene regulation of herpesviruses. In contrast, when infected cell cultures are incubated at the restricted temperature of 39 degrees C, the shift from early (beta) gene expression to late (gamma) gene expression is perturbed, i.e., there is an accumulation of early (beta) gene products and a decrease in, or absence of, late (gamma) gene products. Some of the affected late (gamma) gene products may be glycoproteins since these ICPs co-migrated with radiolabeled bands from infected cells incubated with [3H] glucosamine, separated by polyacrylamide gel electrophoresis. These findings are consistent with previous findings (Jacob, 1986), indicating that the growth restriction is in a late viral function(s) and possibly involves envelopment of nucleocapsids into infectious virions.

Six of 6 equine herpesvirus type 3 (EHV3) isolates, 5 of which were epidemiologically unrelated, produced a mixture of small and large plaque variants in equine foetal kidney cells under methylcellulose. In 4 of 4 instances the cleavage site(s) generating the Bam HI A fragment of large plaque variants was distinct from the site(s) for the same fragment of small plaque variants.