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Ocular problems characterized by conjunctivitis, epiphora, and keratopathy were detected in 35 of 80 Thoroughbred weanling foals that also had respiratory disease. Ocular problems were determined to be caused by infection with equine herpesvirus type 2 (EHV-2) and were successfully treated with ophthalmic medication containing idoxuridine. Equine herpesvirus type 2 isolated from 3 of 5 foals from which samples were collected. The identity of the causative virus as EHV-2 was confirmed by use of electron microscopy, restriction endonuclease DNA fingerprinting, and Southern blot analysis.

Equine herpesviruses 2 and 5 (EHV-2 and EHV-5) have biological properties and genome structures that support their classification as members of the Betaherpesvirinae. In order to investigate whether this is supported by genetic data, we analysed the sequences of random DNA fragments and identified 25 EHV-2 and 28 EHV-5 genes that encode amino acid sequences with significant homology to proteins from other herpesviruses. Greatest similarity was to proteins specified by the gamma-herpesviruses Epstein-Barr virus (a gamma 1-herpesvirus) and herpesvirus saimiri (a gamma 2-herpesvirus), and the level of similarity was marginally greater to the latter. Also, like other gamma-herpesviruses, the EHV-2 and EHV-5 genomes are deficient in the CG dinucleotide, suggesting that latent genomes are methylated. EHV-2 and EHV-5 are related to each other more closely than they are to other herpesviruses, but are clearly distinct gamma-herpesviruses. The data support the establishment of at least one more subdivision of the gamma-herpesviruses (the gamma 3-herpesviruses).

A new equine herpesvirus, provisionally designated equine herpesvirus 5 (EHV5; Browning and Studdert (1987) J. Gen. Virol. 68, 1441-1447), was examined for the degree of genomic difference from equine herpesvirus 2 (EHV2) by Southern hybridizations. EHV5 and EHV2 whole genomic DNA probes were highly specific for homologous DNA only, indicating that significant genomic difference exists between the two viruses. Restriction endonuclease analysis of EHV5 strain 2-141 (EHV5.2-141) revealed that the genome is 179 kb and exists as a single isomer. Clones representing 82% of the genome were obtained and used to construct restriction maps for four restriction endonucleases. Hybridization experiments indicated that the EHV5.2-141 genome does not contain large terminal or internal repeats, although some evidence for very short repeated sequences in the genomic termini was obtained. Such a genome structure makes EHV5 unique among the equine herpesviruses but similar to the mouse, rat, and guinea pig cytomegaloviruses and the tupaiid herpesvirus. Sequence analysis of one of the genomic termini of EHV5.2-141 revealed the presence of a 30-bp sequence (pac-1; Deiss et al. (1986) J. Virol. 59, 605-618) which is highly conserved among herpesviruses.

Equine herpesvirus 4 (EHV4) glycoproteins of M(r) 63K and 250K were identified in the supernatant of infected cell cultures. The 63K glycoprotein was type-specific; that is, it reacted with monospecific sera from horses that had been immunized or infected with EHV4, but not with monospecific sera from horses immunized or infected with EHV1, a closely related alphaherpesvirus. It was postulated that the secreted protein may be the homologue of similarly secreted glycoproteins of herpes simplex virus 2 glycoprotein G (HSV2 gG) and pseudorabies virus (PRV) gX, which is the homologue of HSV2 gG. The US region of the EHV4 genome, toward the internal repeat structure, was sequenced. Four open reading frames (ORFs) were identified of which ORF4 showed 52% similarity to the gene-encoding PRV gX in a 650-nucleotide region. ORF4 coded for a primary translational product of 405 amino acids which has a predicted size of 44K. The amino acid sequence of ORF4 showed 28% identity with PRV gX and 16% identity with HSV2 gG, although significantly greater identity was observed in the N-terminal region including the conservation of 4 cysteine residues. Accordingly, we designate ORF4 as EHV4 gG. The predicted amino acid sequence of the EHV4 gG showed characteristics of an envelope glycoprotein. Expression of the entire EHV4 gG gene in the bacterial expression vector pGEX-3X produced a type-specific fusion protein of M(r) 70K of which the gG portion composes 43K. Antibody that was affinity purified from selected portions of Western blots containing the 70K gG fusion protein reacted with the 63K secreted glycoprotein. Conversely, antibody affinity purified to the 63K secreted product reacted with the 70K gG fusion protein. These results showed that the EHV4 63K secreted glycoprotein was EHV4 gG, the third alphaherpesvirus gG homologue known to be, at least in part, secreted. The type-specificity of this glycoprotein provides, for the first time, the opportunity to differentiate between antibodies present in polyclonal sera from EHV4, EHV1, and dual-infected horses and this has important implications for understanding the epidemiology of these viruses.

A panel of 14 monoclonal antibodies (MAbs) was used to characterize the high abundance glycoproteins of equine herpesviruses 4 (EHV-4) and 1 (EHV-1), and asinine herpesvirus 3 (AHV-3). The specificities of the MAbs, which had been determined previously for strains of EHV-4 and -1 from the U.S.A., in general were confirmed by ELISA for Australian strains of these viruses. Of the 14 MAbs seven were EHV-4 and -1 type-common and cross-reacted with AHV-3. Of the five MAbs that were EHV-1 type-specific, four cross-reacted with AHV-3, whereas neither of the EHV-4 type-specific MAbs reacted with AHV-3, providing further evidence for a closer evolutionary relationship between EHV-1 and AHV-3 than that between either of these viruses and EHV-4. By Western blot and immunoprecipitation analyses, the identity of the six major glycoproteins, gp2, gp10, gp13, gp14, gp18 and gp21/22a, of an Australian EHV-1 isolate was verified, and it was shown that AHV-3 had cross-reactive glycoproteins of very similar Mr to those of EHV-1; five homologous glycoproteins of EHV-4 were also identified. It was determined that the EHV-4 gp13 homologue had a much reduced Mr (67K) when the virus was grown in a continuous cell line than when grown in equine foetal kidney cells (95K). It is suggested that altered glycosylation by the cell line is responsible for this change in Mr. Those glycoproteins acting as major immunogens in the naturally infected host, at least in their ability to elicit antibody, were identified. It was found that gp2, gp13, gp14, gp18 and a glycoprotein at 120K (EHV-1) or 116K (EHV-4) were all important immunogens in mares following EHV-1-induced abortion, and in a specific pathogen-free foal experimentally infected with EHV-1 and later cross-challenged with EHV-4. Gp2, gp14 and gp18 were the major immunogens in the donkey in response to AHV-3 infection. The type specificity associated with these glycoproteins was also examined and it was found that although most if not all contain type-specific epitopes, gp2 and a glycoprotein at 120K, and to a lesser extent gp13 and gp18, were significantly type-specific in the serum from a mare following natural EHV-1 infection and abortion.

Proteins of purified virions of equine herpesvirus 4 (EHV-4; equine rhinopneumonitis), EHV-1 (equine abortion virus) and asinine herpesvirus 3 (AHV-3) were compared by metabolic labelling with [35S]methionine or [14C]glucosamine during growth of low passage virus in natural host cells (horse or donkey) and high passage virus in an appropriate cell line and analysis by SDS-PAGE. Approximately 25 different proteins (Mr 300K to 21.5K) were clearly resolved for each virus. The three viruses had similar profiles although significant differences were found. The proteins of the cell line-grown viruses were similar to their precursor viruses grown in natural host cells although some small differences, probably related to differences in glycosylation by the various cell types, were noted. Six or seven high abundance glycoproteins were identified for EHV-4, EHV-1 and AHV-3. The profile of seven glycoproteins of AHV-3 was more similar to EHV-1 than to EHV-4. Antigenic relationships of the proteins of the three viruses were examined using radioimmunoprecipitation (RIP) and Western blot analyses and a series of polyclonal sera raised in colostrum-deprived, specific pathogen-free (SPF) foals which were immunized with inactivated EHV-4 (foal 3) or EHV-1 (foal 1), challenged and cross-challenged; a polyclonal donkey serum to AHV-3 was also used. The ontogeny of the antibody response in the SPF foals was studied and the major immunogenic proteins, as determined by RIP, were correlated with previously determined serum neutralizing antibody titres. Antibodies were first detected 14 days after primary immunization and were directed to EHV-4 proteins of Mr 113K, 75K and 56K or EHV-1 proteins of 110K, 78K, 60K and 58K. Antibodies to these same three (EHV-4) or four (EHV-1) proteins, together with antibodies to the major capsid protein and proteins of 67K (EHV-4) and 87K (EHV-1) were detected in response to primary infection (control foal 2) and these sera had high neutralizating antibody titres. The antigens of the three viruses were extensively cross-reactive with immunodominant proteins in the Mr ranges 150K to 110K and 62K to 56K. However, cross-absorption of EHV-4 and EHV-1 SPF foal antisera indicated the presence of significant amounts of type-specific antibody.

A clone library of 11 of 15 BamHI fragments representing 81% of the 140 kilobase DNA genome of the prototype bovine encephalitis herpesvirus strain N569 (BEHV.N569) was constructed. The clones were used to verify the BamHI, BstEII, EcoRI, and HindIII genomic maps for BEHV.N569 published by Engels et al. [Virus Res 6: 57-73 (1986)] for the same virus although some amendments/variations to the BamHI map were found in that 3 previously unidentified restriction sites were identified. Restriction site maps for BglII and KpnI were also derived for BEHV.N569. Southern blot analysis using 32P-labelled BEHV DNA as probe indicated that bovine herpesvirus 1 (BHV1), buffalo herpesvirus 1 (BuHV1) and caprine herpesvirus 1 (CaHV1) were similar and that the similarity occurred throughout the entire length of the genomes; CaHV1 was more distantly related to the other 3 viruses. Because of the similarities BEHV.N569 and BHV1. Cooper cloned DNA fragments were used to construct BamHI, BglII, BstEII, EcoRI, KpnI, and HindIII restriction site maps for the genome of BuHV1 and BamHI, BglII, and KpnI maps for the genome of BHV1.V155, a genital strain.

DNA was prepared from 43 equine herpesvirus type 1 (EHV 1) isolates, 11 of which were from horses with respiratory disease, 22 from aborted equine foetuses, and 10 from foals that died perinatally. The restriction endonuclease DNA fingerprints of 10 of the 11 respiratory isolates, known with certainty to have been recovered from horses with respiratory disease, were entirely different from all but 3 of the 32 foetal or perinatal foal isolates. The exceptional respiratory isolate, EHV 1 Army 183, had a foetal (F) strain fingerprint but this virus cannot be said with certainty to have been isolated from the respiratory tract. The 3 exceptional foetal isolates, had respiratory (R) strain fingerprints, and were recovered from single sporadic abortions. There are no exceptions to the view that only R strains have been isolated from naturally occurring respiratory disease. Also it is clear that major epizootics of abortion (abortion storms) and of perinatal foal mortality are caused by F strains. The data together with an analysis of the epidemiological patterns, particularly in Australia, strongly support the view that F and R strains be regarded as separate species, EHV 1 and 4 respectively.

The genomes of 51 isolates of slowly cytopathic equine herpesviruses were examined by digestion with restriction endonucleases. Forty-seven of the isolates showed considerable fragment pattern heterogeneity although common fragments were evident, especially when any two isolates were compared or when they were digested with SalI. Fifteen of the 47 viruses, selected for their diverse fragment patterns, showed a high degree of homology in Southern blot hybridization. In contrast, four viruses, representing three epidemiologically distinct isolations, shared few, if any, comigrating fragments with the 47 equine herpesvirus 2 (EHV-2) isolates, although they shared comigrating fragments with each other. These four viruses showed reduced homology to a representative EHV-2 isolate by Southern blot hybridization under stringent conditions. Although not sharply delineated from EHV-2, these four viruses grew very slowly and had low yields in vitro, and preliminary data suggested they had a significantly smaller genome than EHV-2 (148 +/- 12 kb compared to 190 kb). These four viruses may be prototypic of a novel equine betaherpesvirus.

Representative strains of EHV isolated from an aborted foetus and from a horse with rhinopneumonitis in New Zealand had restriction endonuclease DNA fingerprints typical of those usually associated with these syndromes elsewhere and now designated EHV1 and 4 respectively. EHV1 was isolated from the brain and spinal cord of a 4-year-old gelding that died of myeloencephalitis. A mare on the same farm, at about the same time as the gelding developed myeloencephalitis, aborted and EHV1 was isolated from the tissues of the aborted foetus. Restriction endonuclease DNA fingerprints of the viruses isolated from myeloencephalitis and abortion were indistinguishable by Bam HI but were distinguishable using Bgl I, Pvu II, Xho I and Hind III. The restriction endonuclease DNA fingerprints of 3 EHV1 strains known to cause myeloencephalitis were compared with each other and with EHV1 strains not known to be associated with myeloencephalitis. The Bgl I Pvu II and Hind III DNA fingerprints of the 3 myeloencephalogenic strains appear distinguishable from non-myeloencephalogenic strains. Abortion was induced in a mare by intrauterine inoculation of EHV4. The Bam HI, Bgl I, Pvu II, Xho I and Hind III restriction endonuclease DNA fingerprints of the inoculum virus were indistinguishable from virus recovered from the foetus. It was concluded that passage of the virus through the foetus did not detectably alter the restriction endonuclease DNA fingerprint.

Restriction endonuclease DNA fingerprints of herpesviruses isolated from 3 unrelated epidemics of bovine encephalitis are similar to each other and totally different from bovine herpesvirus 1 (BHV1). Herpesviruses, antigenically related to BHV1, isolated from goats and buffalo have distinct DNA fingerprints. We propose that bovine encephalitis herpesvirus is prototypic of a new bovine herpesvirus type and that alpha herpes viruses from individual ruminant species are species specific.

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.

The immunological and virological status of 3 foals in respect of equine herpesviruses (EHV) was established and the foals were sequentially infected with EHV2, EHV3 and EHV1. Following experimental infection with EHV2, no clinical signs of disease were observed in any foal. The inoculation of EHV3 into the genital tract resulted in lesions of the mucous membrane and perineal skin that were considered typical of equine coital exanthema. Following intransal inoculation of EHV3 extensive ulceration and pustule formation on the nasal mucosa was observed by day 5 accompanied at day 7 by a profuse, mucopurulent nasal discharge; on day 8 pustular lesions of the skin about the external nares appeared. Signs of disease after inoculation with EHV1 were milder than expected in 2 of the 3 foals. The possibility that recent, prior infection with EHV2 and/or 3 may protect against EHV1 was considered.

The number of plaques produced in a feline embryo (FEmb) cell line and in three independently derived kitten kidney (KK) cell cultures varied in a consistent and reproducible manner when each was inoculated with the same number of feline herpesvirus 1 (FHV1) plaque forming units (PFU); the three KK cells produced 2-9 times more plaques than FEmb cells. One of the three KK cells produced FHV1 plaques that were smaller in diameter than those FEmb cells. Each of the three KK cell cultures inoculated with the same number of FEmb cell PFU of a strain of feline calicivirus (FCV) produced different numbers of plaques; two of the three KK cell cultures produced 2-3 times more plaques than FEmb cells. The plaque diameter of FCV in the three KK cells was 30-50% smaller than the plaque diameter in FEmb cells.

The specificity of selected immune responses to equine herpesvirus type 1 (EHV-1) and type 4 (EHV-4) was examined in 3 colostrum-deprived specific-pathogen-free foals. Single foals were vaccinated with inactivated EHV-1, inactivated EHV-4, or control cell lysate plus adjuvant followed by successive intranasal challenge exposures with EHV-1 and EHV-4 or with EHV-4 and EHV-1. Vaccination with inactivated virus preparations elicited cellular immune responses and antibody which were augmented by subsequent challenge exposures. Cellular immune responses, as measured by in vitro lymphocyte blastogenesis, were cross-reactive after foals were given either EHV-1 or EHV-4. Serum virus-neutralizing antibody responses were type-specific for foals given EHV-1, but were cross-reactive after EHV-4 administrations. It was concluded that diseases caused by EHV-1 and EHV-4 may be more effectively controlled with a bivalent vaccine containing both EHV-1 and EHV-4 than with the presently used monovalent vaccines based on EHV-1 alone.

The restriction endonuclease DNA fingerprints of 20 low passage, epidemiologically unrelated isolates of equine herpesvirus 4 (equine rhinopneumonitis virus) showed considerable heterogeneity in certain fragments, the positions of which were assigned to quite restricted positions on the 141 kilobase (kb) genome. We note that the heterogeneity observed in the restriction endonuclease DNA fingerprints of EHV 1 (equine abortion virus) and of pseudorabies virus also tend to map to these same restricted regions. The restriction endonuclease DNA fingerprints of an EHV 1 strain was invariant using low (less than 1) multiplicity of infection during 20 passages in equine cells but when adapted to hamster cells developed an approximately 0.8 kb deletion in the unique short region of the genome between 8 and 11 passages.

Viruses classified by immunologic criteria as equine herpesvirus 1 cause respiratory disease and abortion in horses. Restriction endonuclease analyses of the DNA's of viruses from animals with respiratory disease and from aborted fetuses show that the patterns for respiratory viruses, while similar to each other, are entirely different from the patterns for fetal viruses. It is therefore proposed that the DNA restriction endonuclease patterns of fetal and respiratory viruses analyzed in this study be designated as prototypic of equine herpesvirus 1 and 4, respectively.

Some kinetic data on the inactivation of equine herpesvirus type 1 (EHV1) and equine rhinovirus type 1 (ERhV1) by betapropiolactone (BPL) and ultraviolet (UV) irradiation are reported. 0.25% BPL at 37 degrees C for 1 h reduced the titre of EHV1 by greater than 10(3 . 4) and of ERhV1 by greater than 10(4 . 1) TCID50/ml. UV irradiation (334 microW/cm2) produced similar reductions in titre after 2 min. These data were used as a basis for inactivating EHV1 and ERhV1 by the combined action of BPL and UV irradiation. Viruses were exposed to 0.1% BPL for 1 h at 4 degrees C with constant stirring, followed by UV irradiation for 2 min, followed by incubation for 3 h at 37 degrees C. Inactivated EHV1 elicited secondary immune responses only in horses whereas ERhV1 produced primary immune responses in mice (including athymic nu/nu mice), rabbits and probably in horses.

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.

The epidemiology of equine herpesvirus 2 was examined by using restriction endonuclease DNA fingerprints to distinguish viruses isolated from two groups of horses. The first group consisted of three yearlings isolated from other horses but in contact with each other for 418 days, whereas the second comprised seven mares and their foals, which were sampled at monthly intervals from parturition until the foals were about 180 days old. There was a complex pattern of transmission, with 15 different viruses isolated from both groups. Four distinguishable viruses were isolated from the three yearlings by day 16 of quarantine, and by day 141 an additional two viruses were isolated. Up to five different viruses were isolated from one yearling. Although four repeat isolations of one virus from the nasal cavity of one yearling over 54 days indicated that equine herpesvirus 2 established persistent infection with constant shedding, most repeat isolations yielded distinguishable viruses. Identical viruses were isolated from the nasal cavity and leukocytes of one yearling and the nasal cavity and vagina of another, indicating that a particular equine herpesvirus 2 strain was not site specific. Although seven different viruses were isolated from the three yearlings throughout the quarantine period, two appeared to establish latent infections; one virus was not isolated until 141 days after quarantine, whereas the second was first isolated 16 days after quarantine and then for the second time, from the same horse, 402 days later. Multiple concurrent local infections were demonstrated by the isolation of two or more viruses from the same nasal swab.

The inoculation of equine herpesvirus type 3 (EHV3) strain 65/61 into the amniotic cavity of a mare 6-7 months pregnant resulted in abortion 11 days later. Following abortion typical lesions of coital exanthema were not observed in the genital tract of the mare, nor was EHV3 isolated from her. Serological evidence, however, indicated that the mare was infected with EHV3 following inoculation. Grossly the foetal disease was characterised by placentitis, focal ulcerative dermatitis, focal necrosis of the lungs and a striking diptheritic gastritis. Histological findings were interstitial pneumonia, diffuse hepatitis, generalised myositis, extensive vascular necrosis and degeneration of a range of epithelial cells. EHV3 was isolated from the placenta and placental fluids, stomach fluid, pooled thoracic and abdominal fluid, skin, lung, spleen and small intestine of the foetus.

The genome of a low equine cell passage equine herpesvirus 2 was partially cloned and physical maps for the restriction endonucleases BamHI, EcoRI, HindIII, and SalI determined. The genome length was estimated to be 192 kilobase pairs (kbp) and no evidence of isomerization was found. Two separate repeat structures were detected: 18 kbp direct terminal repeats; and an unrelated second pair of short internal, indirect repeats at 0.20 and 0.75 map units. Such a genomic structure does not appear to have been reported amongst the herpesviruses--all the genomes that do not isomerize either have repeat structures only at the termini, or if present internally, have only direct repeats.

The BamHI, EcoRI, and HindIII physical maps of the genomes of 14 isolates of equine herpesvirus 2 (EHV 2) were determined by Southern blot analysis using DNA fragments of a previously mapped EHV 2 strain 86/67. No two isolates had identical maps for all 3 enzymes, the number of differing cleavage sites between pairs of isolates varying from 3 to 21. Overall 75 cleavage sites were mapped, of which 40 were variable. Cleavage sites occurred throughout the genome, including within the terminal repeat regions. Additionally, fragment length polymorphisms, independent of cleavage site loss or gain, were mapped to 5 regions of the genome, 4 of which occurred within the terminal repeat regions.

Equine herpesviruses with a deoxyribonucleic acid density of 1.716 to 1.717 g/cm(3) were compared with one another by the plaque-reduction test and by the rate of development of cytopathic effect as indicated by plaque size in rabbit kidney cultures. Of the 19 isolates studied, the 9 which had already been tentatively labeled equine abortion viruses were serologically similar to one another; each of them grew more quickly than did any of the other 10 isolates although the mean plaque sizes formed a series of gradations with no clear hiatus which would permit the unequivocal delineation of the abortion viruses from the slowly growing strains. The 10 slowly growing isolates showed antigenic heterogeneity even though complement was present; the neutralizing capacity of an antiserum against the heterologous strains was, in most instances, markedly less than against the homologous strains, the range of the 50% endpoints being much greater than that observed among the equine abortion viruses, or among isolates of herpes simplex type 1. There was no cross neutralization between the equine abortion viruses and any of the 10 slowly growing isolates. An extra band of deoxyribonucleic acid, at 1.723 to 1.725 g/cm(3), was present in two of the slowly growing strains when originally grown in rabbit cells, but was no longer present after passage in cat cells. This band occupied the same position as one reported in the hamster-passaged strain of equine abortion virus, and had a density similar to that of the equine genital herpesvirus. Although the taxonomic demarcation of the equine abortion viruses and the slowly growing herpesviruses from one another is still open to question, they can be conveniently labeled equine herpesviruses 1 and 2, respectively; the genital virus would be termed equine herpesvirus 3.