Evidence of widespread natural recombination among field isolates of equine herpesvirus 4 but not among field isolates of equine herpesvirus 1.

Recombination in alphaherpesviruses allows evolution to occur in viruses that have an otherwise stable DNA genome with a low rate of nucleotide substitution. High-throughput sequencing of complete viral genomes has recently allowed natural (field) recombination to be studied in a number of different alphaherpesviruses, however, such studies have not been applied to equine herpesvirus 1 (EHV-1) or equine herpesvirus 4 (EHV-4). These two equine alphaherpesviruses are genetically similar, but differ in their pathogenesis and epidemiology. Both cause economically significant disease in horse populations worldwide. This study used high-throughput sequencing to determine the full genome sequences of EHV-1 and EHV-4 isolates (11 and 14 isolates, respectively) from Australian or New Zealand horses. These sequences were then analysed and examined for evidence of recombination. Evidence of widespread recombination was detected in the genomes of the EHV-4 isolates. Only one potential recombination event was detected in the genomes of the EHV-1 isolates, even when the genomes from an additional 11 international EHV-1 isolates were analysed. The results from this study reveal another fundamental difference between the biology of EHV-1 and EHV-4. The results may also be used to help inform the future safe use of attenuated equine herpesvirus vaccines.

Differentiation of respiratory and abortigenic isolates of equine herpesvirus 1 by restriction endonucleases.

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.

Isolation of caprine herpesvirus 1 from a major outbreak of infectious pustular vulvovaginitis in goats.

We describe an outbreak of infectious pustular vulvovaginitis caused by Caprine herpesvirus 1 (CpHV1) in a group of approximately 200, 8 month old virgin does that were imported to Victoria from New Zealand. CpHV1 was isolated in cell cultures from vaginal swabs from three of three affected does but not from two bucks that had been with the does. The identity of the virus as a herpesvirus was confirmed by negative stain electron microscopy. Restriction endonuclease DNA fingerprint analysis showed that the DNA fingerprints were similar, but not identical, to previously described CpHV1 isolates made in New Zealand, New South Wales, and in other parts of the world. Acute and convalescent phase sera from selected does supported the diagnosis of CpHV1 infection. It is most likely that the disease was initiated by reactivation of latent virus in at least one of four bucks that served the does, since each was positive for CpHV neutralising antibody when first tested. This is the first report of CpHV infectious pustular vulvovaginitis in goats in Victoria and to our knowledge appears to be one of the largest outbreaks recorded anywhere.

Prevalence of serum neutralising antibody to equine rhinitis A virus (ERAV), equine rhinitis B virus 1 (ERBV1) and ERBV2.

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.

Detection of viruses in nasal swab samples from horses with acute, febrile, respiratory disease using virus isolation, polymerase chain reaction and serology.

OBJECTIVE: To examine the association of viruses with acute febrile respiratory disease in horses. Design Nasal swab and serum samples were collected from 20 horses with acute febrile upper respiratory disease that was clinically assessed to have a viral origin. METHODS: Each of the samples was inoculated onto equine fetal kidney, RK13 and Vero cell cultures, and viral nucleic acid was extracted for polymerase chain reaction (PCR) or reverse transcription PCR. PCR primers were designed to amplify nucleic acid from viruses known to cause or be associated with acute febrile respiratory disease in horses in Australia. A type specific ELISA was used to measure equine herpesvirus (EHV1 and EHV4) antibody, and serum neutralisation assays were used to measure equine rhinitis A virus (ERAV) and equine rhinitis B virus 1 and 2 (ERBV1 and ERBV2) antibody titres in serum samples. RESULTS: Virus was isolated from 4 of 20 nasal swab samples. There were three isolations of EHV4 and one of ERBV2. By PCR, virus was identified in the nasal swab samples of 12 of the 20 horses. Of the 12 horses [corrected] that were positive, 17 viruses were detected as follows: there was [corrected] one triple positive (EHV4, EHV2, and EHV5), three double positives (EHV4, ERBV and EHV5, ERBV (2 horses)) and 8 [corrected] single positives (EHV4 (2 horses), EHV5 (3 horses) and ERBV (3 [corrected] horses). CONCLUSION: By virus isolation and PCR, 17 viruses were identified in nasal swab samples from 12 of 20 horses that had acute febrile respiratory disease consistent with a diagnosis of virus infection. Initial PCR identification and subsequent virus isolation led to the isolation of ERBV2 for the first time in Australia and the second time anywhere of ERBV2.

Sequence analysis of equine adenovirus 2 hexon and 23K proteinase genes indicates a phylogenetic origin distinct from equine adenovirus 1.

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.

Expression of small regions of equine herpesvirus 1 glycoprotein C in Escherichia coli.

A series of truncated equine herpesvirus 1 (EHV1) glycoprotein C (gC) molecules was examined for use as serodiagnostic antigens for EHV1 and EHV4. Small regions of EHV1 glycoprotein C, an immunodominant EHV1 glycoprotein, were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins using the bacterial expression vector pGEX-2T. Sera obtained from horses, including sera from specific-pathogen-free (SPF) foals, following exposure to either EHV1, EHV4 or both viruses were used. Several of the fusion proteins were shown to encompass EHV1 specific epitopes while others encompassed strong, cross-reactive epitopes. One clone, termed pEC-3, produced a soluble and stable fusion protein which encompassed amino acids 107-275 of EHV1 gC. Strong cross-reactive epitopes on pEC-3 were localised to a region encompassed by amino acids 137 to approximately 152 while EHV1 specific epitope(s) were identified downstream of this region, i.e., approximately amino acids 152 to 275. E. coli expressed EHV1 gC polypeptides showed clear potential for use as diagnostic reagents for the detection of cross-reactive and type-specific EHV1 and EHV4 antibodies present in convalescent equine sera.

Variable sensitivity of a feline embryo cell line and of three kitten kidney cell cultures to feline herpes- and caliciviruses.

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.

Restriction enzyme maps for equine adenovirus 1 genome.

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.

Immunogenicity of equine herpesvirus type 1 (EHV1) and equine rhinovirus type 1 (ERhV1) following inactivation by betapropiolactone (BPL) and ultraviolet (UV) light.

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.

Epidemiological studies of equine herpesvirus 1 (EHV-1) in Thoroughbred foals: a review of studies conducted in the Hunter Valley of New South Wales between 1995 and 1997.

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.

Epidemiology of EHV-1 and EHV-4 in the mare and foal populations on a Hunter Valley stud farm: are mares the source of EHV-1 for unweaned foals.

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.

Reconstitution of primary, severe, combined immunodeficiency in man and horse.

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.

Nylon wool column fractionation and characterisation of feline lymphocyte subpopulations.

Feline separated mononuclear cells (SMC) were obtained from peripheral blood by ficoll-diatrizoate gradient separation. SMC were further fractionated on nylon wool columns into nylon wool adherent cells (NWAC) and nylon wool effluent cells (NWEC). The three cell populations, SMC, NWAC and NWEC, were characterised using direct immunofluorescent staining for surface immunoglobulin (sIg) as a B cell marker and neuramidase treated guinea pig erythrocyte-rosette formation (E-rosettes) and mitogen-induced lymphocyte blastogenesis (LB) as possible T-cell markers. Feline SMC consisted of 30.1 +/- 4.0% sIg+ cells 36.6 + 5.4% E-rosette forming cells and 33.3% null cells i.e. cells which were sIg- and non E-rosette forming. Fractionation of SMC on nylon wool columns yielded NWEC which were significantly enriched for T cells in that they contained 68.6 +/- 2.9% E-rosette forming. Fractionation of SMC on nylon wool columns yielded NWEC which were significantly enriched for T cells in that they contained 68.6 +/- 2.9% E-rosette forming cells. NWAC were 51.0% +/- 10.8% sIg+, approximately 20% of cells were lost. The LB responsiveness of NWEC to concanavalin A (Con A) and phytonaemagglutinin-P (PHA-P) was enhanced compared to SMC. NWAC were non-responsive to Con A and PHA-P at all concentrations tested. It was concluded that nylon wool column fractionation of feline SMC was an efficient procedure for T cell enrichment and that the enriched cells retained the properties of E-rosette formation and blastogenesis by mitogens.

Optimal conditions for in vitro blastogenesis of feline peripheral blood lymphocytes.

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.

Attempted reconstitution of a foal with primary severe combined immunodeficiency.

A foal with primary severe combined immunodeficiency, diagnosed within the first two weeks of life, was maintained with its dam in semi-isolation. The foal received continuous prophylactic antibiotic therapy, plasma from a sibling hyperimmunised with equine adenovirus vaccine, and intensive general nursing care. A full sibling female was selected as a bone marrow donor on the basis of red blood cell cross-matching and mixed lymphocyte reactions. Cyclophosphamide was given before two bone marrow transfusions at 35 and 73 days of age. To prevent graft versus host disease graft versus host disease the foal was maintained on methotrexate therapy. Reconstitution was not achieved nor were there signs of graft versus host disease. The foal died suddenly four days after the second bone marrow transfer when 77 days old. It had remained clinically free of any life threatening infectious disease and at necropsy a remarkable degree of freedom from infectious disease was confirmed. The most notable necropsy findings were bilateral nephrosis and myocardial degeneration and fibrosis. The likely cause of death was an electrolyte imbalance, particularly hypokalaemia, which secondarily affected the myocardium. Renal toxicity caused by the cytotoxic drugs, especially cyclophosphamide, may have contributed to the electrolyte imbalance.

Nucleotide sequence of canine herpesvirus homologues of herpes simplex virus type 1 US2, US3, glycoproteins I and E, US8.5 and US9 genes.

The partial nucleotide sequence of two BamHI fragments that span the unique short region (US), terminal repeat region (TR) and internal repeat region (IR) of canine herpesvirus (CHV) has been determined. Data obtained revealed several open reading frames (ORF's) identified as the US2, US3, gI, gE and US9 homologues of herpes simplex virus type 1 (HSV1). The CHV homologues also show significant identity in amino acid sequence with those encoded by feline herpesvirus type 1 (FHV1), bovine herpesvirus (BHV1) and equine herpesvirus (EHV1). Translation of another ORF showed little amino acid identity with the gene products of other alpha-herpesviruses. Its genomic position relative to the other CHV homologues would suggest it is the US8.5 gene of CHV.

Isolation of an adenovirus antigenically distinct from equine adenovirus type 1 from diarrheic foal feces.

Adenovirus was isolated in equine fetal kidney cell cultures from the feces of 2 foals with diarrhea that also had large numbers (greater than 10(6)/g) of rotavirus particles in their feces. Unlike equine adenovirus type 1 (EAdV1), the fecal EAdV did not hemagglutinate human O, rhesus macaque, or equine RBC. By serum neutralization, the fecal viruses were identical with each other, but showed no relationship to EAdV1. Antiserum prepared against the fecal viruses did not contain hemagglutination-inhibiting antibody to EAdV1. It is proposed that the fecal viruses be considered prototypic of EAdV2. The frequency of neutralizing antibody to EAdV2 in 339 equine serum samples was 77%. Neither EAdV1 nor EAdV2 is related by serum neutralization to any of 30 human adenovirus serotypes.

In vitro blastogenesis of equine lymphocytes by inactivated equine adenovirus type 1 antigen.

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.