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The objective of this study was to determine the incidence of serum neutralising (SN) antibody to ERAV, ERBV1 and ERBV2 in a population of horses from birth to 22 years of age. The prevalences of ERAV, ERBV1 and ERBV2 SN antibodies in 381 sera obtained from 291 horses were 37%, 83% and 66%, respectively. ERAV, ERBV1 and ERBV2 maternal antibody was present in foals 12 h postsuckling but by 10-12 months, ERAV SN antibody was not detected in any of the horses, while ERBV1 and ERBV2 SN antibodies were common (83% and 100%, respectively). Sera were obtained from 44 Thoroughbred horses when they were newly introduced into a training centre when their average age was 23 months and a second sample was obtained approximately 7 months later. ERAV SN antibody was present in 8 (18%) when first bled and in 27 (61%) when tested 7 months later. Accordingly 19 of the 44 horses (43%) seroconverted to ERAV within 7 months of entering the training stable. Among all the horses the average ERAV SN antibody titre was relatively high (3796) and in contrast, ERBV1 and ERBV2 titres were relatively low (average 84 and 45, respectively) and often fell to below detectable levels over time and at a rate comparable to new seroconversions in the same group of horses.

Objective: To develop and validate specific, sensitive and rapid diagnostic tests using RT-PCR for the detection of Ross River virus (RRV), Kunjin virus (KV) and Murray Valley encephalitis virus (MVEV) infections in horses., Methods: Primer sets based on nucleotide sequence encoding the envelope glycoprotein E2 of RRV and on the nonstructural protein 5 (NS5) of KV and MVEV were designed and used in single round PCRs to test for the respective viruses in infected cell cultures and, in the case of RRV, in samples of horse blood and synovial fluid., Results: The primer pairs designed for each of the three viruses amplified a product of expected size from prototype viruses that were grown in cell culture. The identity of each of the products was confirmed by nucleotide sequencing indicating that in the context used the RT-PCRs were specific. RRV was detected in serums from 8 horses for which there were clinical signs consistent with RRV infection such that an acute-phase serum sample was taken and submitted for RRV serology testing. The RRV RT-PCR was analytically sensitive in that it was estimated to detect as little as 50 TCID50 of RRV per mL of serum and was specific in that the primer pairs did not amplify other products from the 8 serum samples. The RRV primers also detected virus in three independent mosquito pools known to contain RRV by virus isolation in cell culture. Samples from horses suspected to be infected with KV and MVEV were not available., Conclusion: Despite much anecdotal and serological evidence for infection of horses with RRV actual infection and associated clinical disease are infrequently confirmed. The availability of a specific and analytically sensitive RT-PCR for the detection of RRV provides additional opportunities to confirm the presence of this virus in clinical samples. The RT-PCR primers for the diagnosis of KV and MVEV infections were shown to be specific for cell culture grown viruses but the further validation of these tests requires the availability of appropriate clinical samples from infected horses.

Aims: To identify the respiratory viruses that are present among foals in New Zealand and to establish the age at which foals first become infected with these viruses., Methods: Foals were recruited to the study in October/ November 1995 at the age of 1 month (Group A) or in March/ April 1996 at the age of 4-6 months (Groups B and C). Nasal swabs and blood samples were collected at monthly intervals. Nasal swabs and peripheral blood leucocytes (PBL) harvested from heparinised blood samples were used for virus isolation; serum harvested from whole-blood samples was used for serological testing for the presence of antibodies against equine herpesvirus (EHV)-1 or -4, equine rhinitis-A virus (ERAV), equine rhinitis-B virus (ERBV), equine adenovirus 1 (EAdV-1), equine arteritis virus (EAV), reovirus 3 and parainfluenza virus type 3 (PIV3). Twelve foals were sampled until December 1996; the remaining 19 foals were lost from the study at various times prior to this date., Results: The only viruses isolated were EHV-2 and EHV-5. EHV-2 was isolated from 155/157 PBL samples collected during the period of study and from 40/172 nasal swabs collected from 18 foals. All isolations from nasal swabs, except one, were made over a period of 2-4 months from January to April (Group A), March to April (Group B) or May to July (Group C). EHV-5 was isolated from either PBL, nasal swabs, or both, from 15 foals on 32 occasions. All foals were positive for antibodies to EHV-1 or EHV-4, as tested by serum neutralisation (SN), on at least one sampling occasion and all but one were positive for EHV-1 antibodies measured by enzyme-linked immunosorbent assay (ELISA) on at least one sampling occasion. Recent EHV-1 infection was evident at least once during the period of study in 18/23 (78%) foals for which at least two samples were collected. SN antibodies to ERBV were evident in 19/23 (83%) foals on at least one sampling occasion and 15/23 foals showed evidence of seroconversion to ERBV. Antibodies to ERAV were only detected in serum samples collected from foals in Group A and probably represented maternally-derived antibodies. Haemagglutination inhibition (HI) antibody titres 1:10 to EAdV-1were evident in 21/23 (91%) foals on at least one sampling occasion and 16/23 foals showed serological evidence of recent EAdV-1 infection. None of the 67 serum samples tested were positive for antibodies to EAV, reovirus 3 or PIV3. There was no clear association between infection with any of the viruses isolated or tested for and the presence of overt clinical signs of respiratory disease., Conclusions: There was serological and/or virological evidence that EHV-1, EHV-2, EHV-5, EAdV-1 and ERBV infections were present among foals in New Zealand. EHV-2 infection was first detected in foals as young as 3 months of age. The isolation of EHV-2 from nasal swabs preceded serological evidence of infection with other respiratory viruses, suggesting that EHV-2 may predispose foals to other viral infections.

Aim: To identify viruses associated with respiratory disease in young horses in New Zealand., Methods: Nasal swabs and blood samples were collected from 45 foals or horses from five separate outbreaks of respiratory disease that occurred in New Zealand in 1996, and from 37 yearlings at the time of the annual yearling sales in January that same year. Virus isolation from nasal swabs and peripheral blood leukocytes (PBL) was undertaken and serum samples were tested for antibodies against equine herpesviruses (EHV-1, EHV-2, EHV-4 and EHV-5), equine rhinitis-A virus (ERAV), equine rhinitis-B virus (ERBV), equine adenovirus 1 (EAdV-1), equine arteritis virus (EAV), reovirus 3 and parainfluenza virus type 3 (PIV3)., Results: Viruses were isolated from 24/94 (26%) nasal swab samples and from 77/80 (96%) PBL samples collected from both healthy horses and horses showing clinical signs of respiratory disease. All isolates were identified as EHV-2, EHV-4, EHV-5 or untyped EHV. Of the horses and foals tested, 59/82 (72%) were positive for EHV-1 and/or EHV-4 serum neutralising (SN) antibody on at least one sampling occasion, 52/82 (63%) for EHV-1-specific antibody tested by enzyme-linked immunosorbent assay (ELISA), 10/80 (13%) for ERAV SN antibody, 60/80 (75%) for ERBV SN antibody, and 42/80 (53%) for haemagglutination inhibition (HI) antibody to EAdV-1. None of the 64 serum samples tested were positive for antibodies to EAV, reovirus 3 or PIV3. Evidence of infection with all viruses tested was detected in both healthy horses and in horses showing clinical signs of respiratory disease. Recent EHV-2 infection was associated with the development of signs of respiratory disease among yearlings [relative risk (RR)=2.67, 95% CI=1.59-4.47, p=0.017]., Conclusions: Of the equine respiratory viruses detected in horses in New Zealand during this study, EHV-2 was most likely to be associated with respiratory disease. However, factors other than viral infection are probably important in the development of clinical signs of disease.

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.

Equine rhinitis A virus (ERAV) is a respiratory pathogen of horses and is classified as an Aphthovirus, the only non-Foot-and-mouth disease virus (FMDV) member of this genus. In FMDV, virion protein 1 (VP1) is a major target of protective antibodies and is responsible for viral attachment to permissive cells via an RGD motif located in a distal surface loop. Although both viruses share considerable sequence identity, ERAV VP1 does not contain an RGD motif. To investigate antibody and receptor-binding properties of ERAV VP1, we have expressed full-length ERAV VP1 in Escherichia coli as a glutathione S-transferase (GST) fusion protein (GST-VP1). GST-VP1 reacted specifically with antibodies present in serum from a rabbit immunized with purified ERAV virions and also in convalescent-phase sera from horses experimentally infected with ERAV. An antiserum raised in rabbits to GST-VP1 reacted strongly with viral VP1 and effectively neutralized ERAV infection in vitro. Using a flow cytometry-based binding assay, we found that GST-VP1, but not other GST fusion proteins, bound to cell surface receptors. This binding was reduced in a dose-dependent manner by the addition of purified ERAV virions, demonstrating the specificity of this interaction. A separate cell-binding assay also implicated GST-VP1 in receptor binding. Importantly, anti-GST-VP1 antibodies inhibited the binding of ERAV virions to Vero cells, suggesting that these antibodies exert their neutralizing effect by blocking viral attachment. Thus ERAV VP1, like its counterpart in FMDV, appears to be both a target of protective antibodies and involved directly in receptor binding. This study reveals the potential of recombinant VP1 molecules to serve as vaccines and diagnostic reagents for the control of ERAV infections.

Objective: To develop and validate specific, sensitive and rapid (< 8 hour) diagnostic tests using polymerase chain reaction (PCR) for the diagnosis of abortion and respiratory disease caused by equine herpesvirus 1 (EHV1; equine abortion virus) and EHV4 (equine rhinopneumonitis virus)., Design: Primer sets based on nucleotide sequences encoding glycoprotein H (gH) of EHV1 and gB of EHV4 were designed and used in single round and second round (seminested) PCRs, and in a multiplex PCR for the diagnosis of EHV1 and EHV4 infections., Methods: Oligonucleotide primers were designed for each virus, PCR conditions were defined and the specificity and sensitivity of the assays were determined. The tests were applied to tissue samples from aborted equine foetuses and to nasopharyngeal swabs from horses with acute febrile respiratory disease., Results: Individual single round and a second round (seminested) EHV1 and EHV4 PCRs were specific in that EHV1 primers amplified all (n = 30) EHV1 isolates and did not amplify EHV4. Similarly EHV4 primers amplified all (n = 6) EHV4 isolates and did not amplify EHV1. Both PCRs were sensitive in that the first round EHV1 PCR detected 1220 molecules of EHV1 plasmid DNA and the first round EHV4 PCR detected 7280 molecules of EHV4 plasmid DNA. The EHV1 second round PCR was 100 times more sensitive in that it detected 12 molecules of EHV1 DNA and the EHV4 second round PCR was 1000 times more sensitive in that it detected 8 molecules of EHV4 DNA. There was a high correlation between detection of EHV1 by virus isolation and PCR when tissue samples from 71 aborted foetuses were examined; all samples positive by virus isolation were positive by PCR. Similarly the EHV4 PCR was at least as sensitive as virus isolation when applied to nasaopharyngeal swabs from horses with respiratory disease in that all samples positive by virus isolation were also positive by PCR., Conclusion: Individual single round and second round (seminested) PCRs and a seminested multiplex PCR were developed that enabled reliable, rapid detection of EHV1 and EHV4 in aborted foetal tissues and nasopharyngeal swab samples.

The International Committee on Taxonomy of Viruses (ICTV) has recently approved several proposals submitted by the present Caliciviridae Study Group. These proposals include the division of the family into 4 new genera designated Lagovirus, Vesivirus, "Norwalk-like viruses (NLVs), and "Sapporo-like viruses (SLVs); the latter 2 genera were assigned temporary names until acceptable names can be determined by the scientific community. The genera have been further divided into the following species: Feline calicivirus and Vesicular exanthema of swine virus (genus Vesivirus), Rabbit hemorrhagic disease virus and European brown hare syndrome virus (genus Lagovirus), Norwalk virus (genus NLV), and Sapporo virus (genus SLV). In addition, the ICTV approved a proposal to remove the hepatitis E virus from the Caliciviridae into an "unassigned classification status.

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.

The prevalence of EHV-1 and EHV-4 antibody-positive horses was determined using a type specific ELISA on serum samples collected from 229 mares and their foals resident on a large Thoroughbred stud farm in the Hunter Valley of New South Wales in February 1995. More than 99% of all mares and foals tested were EHV-4 antibody positive, while the prevalence of EHV-1 antibody positive mares and foals were 26.2 and 11.4%, respectively. Examination of the ELISA absorbance data for the individual mares and foals suggested that the EHV-1 antibody positive foals had been infected recently with EHV-1 and that a sub-group of the mare population was the likely source of infectious virus for the unweaned foals.

Sero-epidemiological studies conducted between 1995 and 1997 on two large Thoroughbred stud farms in the Hunter Valley of NSW showed clear evidence of EHV-1 infection in foals as young as 30 days of age. Similarly, serological evidence suggested that these foals were infected with EHV-1 from their dams or from other lactating mares in the group, with subsequent foal to foal spread of infection prior to weaning. These studies also provided evidence of EHV-1 infection of foals at and subsequent to weaning, with foal to foal spread of EHV-1 amongst the weanlings. These data indicated that the mare and foal population was a reservoir of EHV-1, from which new cases of infection propagated through the foal population both before and after weaning. The results of these studies support the long standing management practices of separating pregnant mares from other groups of horses to reduce the incidence of EHV-1 abortion. Also, these results have important implications for currently recommended vaccination regimens, as the efficacy of vaccination in already latently infected horses is unknown.

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.

The complete nucleotide sequence of the gammaherpesvirus equine herpesvirus 5 (EHV5) glycoprotein B (gB) was determined and the deduced amino acid sequence compared with that of the second equine gammaherpesvirus EHV2. EHV5 gB is an 870 amino acid protein and is 79% similar and 66% identical with EHV2 gB at the amino acid level. EHV5 gB like EHV2 gB is a disulphide linked heterodimer with subunits of 92 and 68 kDa. EHV5 gB is an integral membrane glycoprotein containing only N-linked oligosaccharides and contains a putative endoproteolytic cleavages site at amino acids 422-485. The EHV5 gB amino acid sequence showed greatest homology with other members of the Rhadinovirus genus of the subfamily Gammaherpesvirinae. Alignment of EHV5 gB sequence with the gB sequence of seven other gammaherpesviruses showed conservation of 10 cysteine residues as well as conservation of three predicted sites of N-linked glycosylation; the highest degree of conservation of the predicted sites of N-linked glycosylation was observed between EHV5 and the other members of the Rhadinovirus genus. Phylogenetic analysis confirmed EHV2 and EHV5 were most closely related to each other and equally distant from other members of the Rhadinovirus genus included in the analysis.

The putative hexon gene of a porcine adenovirus serotype 3 (PAV3) has been identified, cloned and the nucleotide sequence determined. The genomic location of the PAV3 hexon gene was determined and an open reading frame (ORF) encoding a polypeptide of 939 amino acids identified. Comparison of the nucleotide sequence of the putative PAV3 hexon gene with the sequence of the HAV2 hexon gene returned an overall identity of approximately 63%. A stop codon 144 nucleotides upstream and a start codon 18 nucleotides downstream of the ORF were identified and comparison with the HAV genome demonstrated that their positions corresponded to the stop site of the pVI gene and start site of the 23K gene, respectively. To confirm the correct start codon of the putative PAV3 hexon gene, the acceptor splice site for the putative PAV3 hexon gene was determined from cDNA and found to be between the two guanines immediately upstream of the first ATG in the ORF. Comparison with the HAV2 hexon protein showed overall identity of approximately 65%, with higher identity in the carboxy-terminus of approaching 76% over 380 amino acids. Multiple alignment of the PAV3 hexon amino acid sequence with other known HAV and animal adenovirus hexon sequences indicated that conservation is generally maintained but that identity is much lower within the loop structures of the protein.

Objective: To investigate the seroprevalence of equine herpesvirus 1 in foals around weaning and after weaning on two large Thoroughbred farms using a type-specific enzyme-linked immunosorbent assay to determine exposure to infection., Design: A longitudinal population study in groups of Thoroughbred weanling foals., Study Population: Two hundred weanling Thoroughbred foals from a population of about 380 foals were enrolled on two adjacent stud farms in the Hunter Valley of New South Wales. Foals on both farms were weaned from February to May 1995 into randomly selected groups of 10 to 15 foals. Farms were selected because of their willingness to cooperate in the survey and because their detailed records of foals and their movements. They were representative of well-managed large Thoroughbred stud farms in New South Wales. Both studs had upper respiratory tract disease among weanling foals around weaning each year although the seroprevalence of viral respiratory disease on either farm was not known before the study., Procedure: Serum was collected from foals within each group at fortnightly intervals from 9th February until 1st June 1995, and at a single follow-up period in August 1995. Each sample was tested in triplicate using an antibody-detection ELISA which is type-specific for EHV-1 and EHV-4 antibodies., Results and Conclusions: There was serological evidence of EHV-1 infection both before and after weaning. The prevalence of EHV-1 antibody in the sample population increased during the study and individual cases of EHV-1 infection were identified. The increase was caused both by the seroconversion of foals within the groups and by the recruitment into the study of foals with pre-existing EHV-1 antibody. Evidence of EHV-1 infection in Thoroughbred foals after weaning has not been reported previously in Australia and this has implications for vaccination regimens.

Sera from 33 Australian thoroughbred mares were tested during an outbreak of equine herpesvirus 1 (EHV1) abortion with an enzyme-linked immunosorbant assay (ELISA) for the presence of EHV1-specific antibodies. The ELISA used a recombinant EHV1 antigen derived from glycoprotein G (gG) and distinguished antibodies to EHV1 from those of the antigenically related and widespread herpesvirus EHV4. Sera were obtained from most of the mares on three occasions, three, 13 and 67 days after the first abortion. Mares which were negative in the ELISA were kept separate from mares which were positive. A second abortion occurred two days after the first and two more abortions and one perinatal death occurred later. Sera from these last three mares showed a significant increase in EHV1-specific antibody on day 13 indicating a recent infection with EHV1. Ten other mares did not have antibodies to EHV1 on day 13 but had seroconverted to EHV1 by day 67. Despite the EHV1 infection, these mares foaled normally, possibly because the infection had occurred either late in gestation or after foaling. Seven mares that remained negative in the ELISA throughout the testing period did not abort, and neither did 11 mares that were positive in the ELISA when they were first tested.

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.

Physical maps were constructed for the genome of equine adenovirus 1 (EAV1) using the restriction enzymes; DraI, EcoRV, NotI and SfiI. The total size of the EAV1 genome was 34.4 kb estimated by comparison with known DNA standards and the polarity of the fragment order, with respect to the left and right molecular ends, was determined by hybridization with known regions of the human adenovirus 2 (HAV2) genome.

The restriction endonuclease DNA fingerprints of 57 isolates of equine herpesvirus 1 (EHV1; equine abortion virus) from abortion, perinatal foal mortalities and encephalitis from 15 epidemics that occurred in Australasia between 1975 and 1989 were examined using the enzymes Bam HI, EcoRI and Bgl II. There was a remarkable degree of uniformity in the restriction patterns; mobility differences were observed in only 14 of 52 (27%) of the fragments. Twelve of these 14 fragments were located within the repeat structures that bracket the unique short region of the genome or were located at the left terminus of the 150 kilobase pair genome. Based on the Bam HI fingerprints the commonest virus identified in our study was EHV1.IP (P is for prototype strain). There was a single notable exception in that the Bam HI fingerprints of all 8 isolates from one of 3 Victorian farms that experienced abortion in 1989 resembled a variant EHV1.IB that was identified as a cause of abortion in Central Kentucky in 1970 to 1974. We present evidence that EHV1.IB caused abortion in California in 1964 and has remained unaltered in its Bam HI restriction pattern. No antigenic differences were found among 4 distantly related EHV1 isolates, including the variant IB, using a panel of 5 monoclonal antibodies to glycoprotein C (gC), a glycoprotein recognised to be highly variable. The uniformity of these unrelated EHV1 isolates is further evidence for a recent origin for EHV1 and may help to explain the natural history of this virus in the horse in which it seems to be a cause of serious epidemics of abortion and perinatal mortality, and less commonly of encephalitis.

Of 467 cat serums tested for antibody to feline immunodeficiency virus (FIV) 120 (26%) were positive. The average age of positive cats was 7.5 years (range 1 to 16 years), and 67% were male. Of 110 serums collected in 1980, 27 (24.5%) were positive. A wide variety of clinical signs including oral cavity disease, anorexia, weight loss, lethargy, depression, fever, respiratory and urinary tract disease, conjunctivitis, abscesses, anaemia and lymphadenopathy were observed in the cats with serum antibody. There was often a history of chronic disease or recurrence of particular or various clinical signs in these cats. FIV was isolated from 4 of 8 FIV antibody positive cats by cocultivation of patient lymphocytes with donor lymphocytes in the presence of interleukin 2.

Tests for T- and B-cell quantitation and immune function were developed, and their application in the diagnosis of primary severe combined immunodeficiency disease (CID) in Arabian foals was investigated. Foals with CID had severe lymphopenia and had small or zero numbers of B cells, as shown by immunofluorescence of surface immunoglobulin (Ig), erythrocyte-antibody-complement rosetting, and staphylococcal protein A rosetting. Serum IgM was undetectable in four CID foals 25 to 71 days old. Demonstrable antibody responses were not elicited in CID foals by phage phi X-174, a potent antigen in normal foals. Nonspecific esterase (NSE) staining in the pattern of a single vesicle was investigated as a possible marker for equine T cells. For normal foals, 64.0% of peripheral blood lymphocytes stained NSE positive. The CID foal 1 had only 4.0% NSE-positive lymphocytes, whereas CID foals 2, 3, and 4 had 75%, 68%, and 77.5%, respectively. In an in vitro T-cell function test, lymphocytes from 12 normal foals did not show a response. In normal foals, intradermal injection of 50 micrograms of phytohemagglutinin induced visible reactions, and skin grafting induced a pronounced mononuclear cell response at the base of the graft. In contrast, there was little or no response in the foals with CID.

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.

Severe combined immunodeficiency disease (SCID) in foals is the only known animal model for the autosomal recessive form of primary SCID in man. A major requirement in the treatment of SCID is the maintenance of the patient in a disease free state until definitive therapy can be undertaken. This paper reviews the current status of prophylactic and definitive therapy in man and the horse. Particular emphasis is placed on the methods of reconstitution available, involving foetal tissues and bone marrow.

A microculture technique was developed for the in vitro blastogenesis of feline lymphocytes. Blastogenesis of ficoll-diatriazoate gradient separated mononuclear cell, washed blood and whole blood were compared. In general the whole blood cultures yielded higher stimulation indices (SI) than the washed blood or separated mononuclear cell cultures. The effect of several variables on the stimulation of lymphocyte cultures was examined. A cell concentration of 3 x 10(5) cells/well and a 1:20 dilution of washed and unwashed whole blood gave optimal stimulation with concanavalin A (Con A). Phytohaemagglutinin-P (PHA-P) did not give significant levels of stimulation. Inactivated fetal calf serum (FCS) at levels of 2.5% (for washed blood) and 5% (for separated mononuclear cell and whole blood) gave highest SI. Supplementation with FCS was preferable to autologous, homologous or horse sera for all cultures. Optimal SI was obtained in all cultures incubated for 3 days and labelled with 1 microCi tritiated thymidine (3H-TdR) for the last 16 hours. The highest SI were in the range of 70 to 105 (18,764 to 42,681 counts per minute (CPM) for separated mononuclear cell culture, 100 to 165 (28,403 to 45,334 CPM) for washed blood culture and 105 to 186 (41,076 to 69,999 CPM) for whole blood culture.

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.