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The nucleotide and deduced amino acid sequences of the P1 region of the genomes of 10 independent equine rhinitis A virus (ERAV) isolates were determined and found to be very closely related. A panel of seven monoclonal antibodies to the prototype virus ERAV.393/76 that bound to nonneutralization epitopes conserved among all 10 isolates was raised. In serum neutralization assays, rabbit polyclonal sera and sera from naturally and experimentally infected horses reacted in a consistent and discriminating manner with the 10 isolates, which indicated the existence of variation in the neutralization epitopes of these viruses.

The antigenic relationships of four genomically divergent strains of equine herpesvirus 2 (EHV2.86/67, EHV2.5FN, EHV2.141 and EHV2.T-2) and equine herpesvirus 5 (EHV5) were examined in ELISA using a panel of EHV2.86/67 gB-specific MAbs. EHV2.86/67 and EHV2.5FN were shown to be more similar to each other than to EHV2.T-2, EHV2.141 or EHV5. Seven of nine EHV2.86/67 gB specific MAbs tested in serum neutralisation assays were shown to neutralise EHV2.86/67 and EHV2.5FN but not EHV2.141, EHV2.T-2 or EHV5. The complete nucleotide and deduced amino acid sequences of EHV2.86/67, EHV2.5FN, EHV2.141 and EHV2.T-2 gB were compared and contrasted with each other and with EHV5 gB. The four EHV2 strains were 94-96% similar at the amino acid level and variability in amino acid sequence mapped to three mains sites designated I, II and III. By contrast, the four EHV2 strains were 77-79% similar to EHV5 gB at the amino acid level. The epitope of these seven gB specific neutralising MAbs has been previously mapped to amino acids 29-74 of EHV2 gB and examination of the deduced amino acid sequence of the four sequenced strains localised the epitope of the seven MAbs to amino acids 30 to 49 located within Site I. Six other divergent strains of EHV2 were examined for variability at Site I using DNA sequencing. Examination of the deduced amino acid sequences of all ten EHV2 strains tested indicated, that based on the epitope of the neutralising MAbs the EHV2 strains formed two distinct antigenic groups, EHV2.86/67-like and EHV2.141-like. EHV5 gB showed divergence from all of the EHV2 gB sequences between amino acids 29-74.

We report the first nucleotide sequence data on equine adenovirus 2 (EAdV2) which corroborate on the molecular level that EAdV2 is distinct from equine adenovirus 1 (EAdV1). Based on sequence homology with Eadv1 the hexon gene of Eadv2 was identified. HindIII restriction fragments containing the hexon and eight other viral genes were cloned into the plasmid pUC19 and the nucleotide sequence of the hexon and the 23K proteinase genes completely determined. Amino acid (aa) comparison of sequence fragments with published adenovirus (AdV) proteins identified the genes for the E1B/19K, IVa2, DNA polymerase, terminal protein, pVI, DNA binding and 100K proteins, usually with highest similarities to human AdV. The nine EAdV2 genes appeared to be in the same relative order as homologous genes of other AdV. The EAdV2 hexon was encoded between the minor capsid precursor protein pVI upstream and the 23K proteinase gene downstream and comprised 2712 nucleotides which translated into 903 aa residues. It was more closely related to the human AdV48 hexon with 71.6% identical and 82.7% functionally similar aa than to the EAdV1 hexon gene with 69.3% aa identity and 80.7% functional similarity. The deduced aa sequence of the EAdV2 23K proteinase gene was 201 residues; it shared 59.7% identical and 75% similar aa residues with the bovine AdV3 23K proteinase as the closest relative. Phylogenetic analysis of the hexon and 23K proteinase genes indicated that EAdV2 does not share an immediate common ancestor with EAdV1 and other AdV.

The nucleotide sequence of the glycoprotein G (gG) homologue of asinine herpesvirus 3 (AHV3), a respiratory alphaherpesvirus of donkeys, was determined. The AHV3 gG gene consists of 1233 base pairs (bp) and codes for a predicted protein of 411 amino acids. This is identical in size to the equine herpesvirus 1 (EHV1) gG gene and 6 amino acids longer than the equine herpesvirus 4 (EHV4) gG gene. The predicted amino acid sequence of AHV3 gG has characteristics of a class 1 membrane protein. The amino acid sequence of AHV3 gG shows 92% and 60% identity to EHV1 gG and EHV4 gG respectively. Two regions within the gG amino acid sequences of EHV1 and EHV4 were previously defined, an N-terminal constant region and an immunodominant highly variable region located toward the C-terminus. In the corresponding constant region of AHV3 gG there was 96% and 75% amino acid identity with EHV1 and EHV4 gGs respectively. In the variable region, there was 73% and 24% identity respectively. Phylogenetic analyses using the gG nucleotide sequences indicated that AHV3 is much closer in evolutionary distance to EHV1 than either virus is to EHV4. These findings provide additional support for the view that AHV3, or another closely related virus, may be the progenitor of EHV1 and has adapted to horses in relatively recent times.

Specific serological diagnosis of equine herpesvirus 4 (EHV4; equine rhinopneumonitis virus) and EHV1 (equine abortion virus) hitherto has not been possible because of extensive antigenic cross-reactivity between these two closely related but distinct viruses. Recently, we identified EHV4 glycoprotein G (gG) and characterized it as a type-specific, secreted glycoprotein (B. S. Crabb, H. S. Nagesha, and M. J. Studdert, Virology 190:143-154, 1992). This paper shows that EHV1 gG also possesses type-specific epitopes and describes the localization of strong, type-specific epitopes to the apparently corresponding and highly variable regions comprising amino acids 287 to 382 of EHV4 gG and 288 to 350 of EHV1 gG. Fusion proteins expressing these variable regions reacted strongly and type specifically with sera from four foals, three of which were colostrum-deprived, specific-pathogen-free foals, whose history with respect to exposure to EHV4 or EHV1 was well-defined. These antigens provided the basis for the development of a single-well diagnostic enzyme-linked immunosorbent assay to distinguish horses infected with EHV4, EHV1, or both. Such a type-specific test provides for the first time the opportunity to differentiate antibodies to these viruses, and it has, therefore, important implications for understanding the epidemiology of these equine pathogens. Evidence for the existence of EHV1 in Australia 10 years prior to the first confirmed case of EHV1 abortion is presented.

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.

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.

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.

An inactivated equine adenovirus type 1 (EAdV1) vaccine was administered to 4 horses. The horses had virus-neutralizing (VN) antibody titers before they were vaccinated, but developed higher VN antibody titers in response to vaccination. Nonvaccinated control horses did not show increases in VN antibody during the study, indicating that any increase in antibody titer in vaccinated horses was a result of vaccination and not due to an EAdV1 epizootic during the study. Specific EAdV1 in vitro lymphocyte blastogenesis (LB) was evaluated, using lymphocytes from 4 vaccinated and 2 control horses. Horses were vaccinated on days 0 and 14, and the LB assays were conducted on days -4, 0, 3, 7, 10, 14, 17, 21, 24, and 28. Lymphocytes from horses were incubated for 4 days with 2 concentrations of inactivated, concentrated and purified EAdV1 antigen. The LB responses for the 2 control horses showed no significant changes during the study period (maximum stimulation indices to EAdV1 antigen for individual horses were between 2.8 and 3.6). The 4 vaccinated horses showed marked increases in stimulation indices in response to EAdV1 antigen (maximum stimulation indices, between 5.3 and 18.6). In control assays, identical lymphocyte preparations from all horses responded normally to phytohemagglutinin.

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.