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Biomedical subjects

M J Studdert

Publications and source records attributed to M J Studdert.

At least 19 recordsLinked to original sources
PubMed1996

Equine rhinovirus 1 is more closely related to foot-and-mouth disease virus than to other picornaviruses.

Equine rhinovirus 1 (ERhV1) is a respiratory pathogen of horses which has an uncertain taxonomic status. We have determined the nucleotide sequence of the ERhV1 genome except for a small region at the 5' end. The predicted polyprotein was encoded by 6741 nucleotides and possessed a typical picornavirus proteolytic cleavage pattern, including a leader polypeptide. The genomic structure and predicted amino acid sequence of ERhV1 were more similar to those of foot-and-mouth disease viruses (FMDVs), the only members of the aphthovirus genus, than to those of other picornaviruses. Features which were most similar to FMDV included a 16-amino acid 2A protein which was 87.5% identical in sequence of FMDV 2A, a leader (L) protein similar in size to FMDV Lab and the possibility of a truncated L protein similar in size to FMDV Lb, and a 3C protease which recognizes different cleavage sites. However, unlike FMDV, ERhV1 had only one copy of the 3B (VPg) polypeptide. The phylogenetic relationships of the ERhV1 sequence and nucleotide sequences of representative species of the five genera of the family Picornaviridae were examined. Nucleotide sequences coding for the complete polyprotein, the RNA polymerase, and VP1 were analyzed separately. The phylogenetic trees confirmed that ERhV1 was more closely related to FMDV than to other picornaviruses and suggested that ERhV1 may be a member, albeit very distant, of the aphthovirus genus.

Amino Acid Sequence↗
PubMed1996

Equine gammaherpesvirus 2 (EHV2) is latent in B lymphocytes.

Peripheral blood leukocytes were collected from 5 Thoroughbred horses and examined for the presence of EHV2 in sub-populations of mononuclear cells. Peripheral blood mononuclear cells were separated on Percoll gradients and then enriched for plastic adherent cells (predominantly monocytes), surface immunoglobulin positive (sIg+) B lymphocytes and T lymphocytes, using panning techniques. The purity of each cell population was assessed by fluorescence activated cell scanning. In an infectious centre assay, each cell population was inoculated onto equine foetal kidney monolayer cell cultures which are fully permissive for the replication of EHV2. Only enrichment for sIg+ B lymphocytes resulted in a marked increase in the number of infectious centres, indicating that EHV2 is present in B lymphocytes. Freeze-thawing of sIg+ B lymphocytes, prior to inoculation onto EFK monolayer cell cultures, resulted in the complete abrogation of infectious centre formation, confirming that EHV2 is latent in B lymphocytes i.e., infectious free virus was not present in the cells. The number of EHV2 infected B lymphocytes varied considerably between horses from 4 to 780 per 10(6) cells. Evidence was also obtained that direct cell to cell contact between the epithelial cells and sIg+ B lymphocytes was necessary for the production of infectious centres. The data indicate that EHV2, like other members of the Gammaherpesvirinae, is latent within B lymphocytes.

Animals↗
PubMed1995

Application of an equine herpesvirus 1 (EHV1) type-specific ELISA to the management of an outbreak of EHV1 abortion.

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.

Abortion, Veterinary↗
PubMed1995

A type-specific serological test to distinguish antibodies to equine herpesviruses 4 and 1.

We describe a type-specific ELISA, which distinguishes antibody to equine herpesvirus 4 (EHV4; equine rhinopneumonitis) and EHV1 (equine abortion virus) thereby identifying horses that have been infected with either or both of these antigenically related viruses. The antigens used are parts of the EHV4 and EHV1 glycoprotein G (gG) homologues expressed in E. coli as fusion proteins [Crabb and Studdert, 1993: J Virol 67: 6332-6338). The expressed proteins comprise corresponding regions of the gG molecules that are highly divergent and encompass strong, typespecific epitopes. Plasma samples from 97 Thoroughbred and 174 Standardbred horses were tested, all of which were unvaccinated. All horses were strongly EHV4 ELISA positive while 30% were EHV1 ELISA positive. The type-specificity of the EHV1 gG antigen was tested in cross-absorption experiments and it was found that 96% (66 of 69) of EHV1 ELISA positive horses were true EHV1 antibody positives. It was also shown that 100% (26 of 26) horses known to have been exposed to EHV1, either by infection or immunisation with EHV1, had significant levels of antibody against the EHV1 gG antigen (i.e., all horses recognised the EHV1 epitope(s) contained within this molecule). Maintenance of EHV1 gG antibody was examined by testing sera obtained from mares four years after confirmed EHV1 abortion. Seven out of 10 of these mares remained EHV1 ELISA positive. In summary, the ELISA is highly specific and is sufficiently sensitive to detect all horses previously infected with EHV4 and most previously infected with EHV1.

Animals↗
PubMed1995

Diagnosis of equine gammaherpesvirus 2 and 5 infections by polymerase chain reaction.

Nested polymerase chain reaction (PCR) assays were developed for the detection of equine herpesvirus 2 (EHV2) and equine herpesvirus 5 (EHV5) using the nucleotide sequences from the glycoprotein B (gB) gene of EHV2 and the thymidine kinase (TK) gene of EHV5. The simultaneous use of EHV2 specific and EHV5 specific primers in one nested amplification assay (multiplex PCR) enabled a rapid, specific and sensitive diagnosis for each virus. PCR was found to be 10(3) times more sensitive than virus isolation by cell culture for EHV2 and 10(6) for EHV5. In separate PCR assays, the routine detection limit after ethidium bromide staining was 0.6 fg for EHV2 plasmid DNA and 2.3 fg for EHV5 plasmid DNA, equivalent for both viruses to approximately 100 genome copies. The detection limits in multiplex PCR were 6 pg for EHV2 and 2.3 fg for EHV5, respectively. PCR assays were applied to studies of the epidemiology of EHV2 and EHV5 infections of racehorses and breeding mares in Victoria and New South Wales, Australia. Peripheral blood leukocytes from 31% of horses were positive for EHV2, 16% positive for EHV5, 8% positive for both viruses and 63% negative for both viruses. EHV2 PCR was also successfully used to detect EHV2 DNA in nasal secretions from horses. The multiplex PCR assay proved to be a rapid and reliable method for the simultaneous detection and differentiation of 2 related equine gammaherpesviruses.

Animals↗
PubMed1995

The nucleotide sequence of asinine herpesvirus 3 glycoprotein G indicates that the donkey virus is closely related to equine herpesvirus 1.

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.

Amino Acid Sequence↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1994

Isolation of equine herpesvirus type 2 (equine gammaherpesvirus 2) from foals with keratoconjunctivitis.

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.

Animals↗
PubMed1994

Comparative studies of the structural proteins and glycoproteins of equine herpesviruses 2 and 5.

The structural proteins of equine herpesvirus 2 (EHV-2) and EHV-5, recently shown to be gammaherpesviruses, were identified and compared. Labelled proteins and glycoproteins were separated by SDS-PAGE and although EHV-2 and EHV-5 had similar protein profiles, bands in some positions were virus-specific. Six glycoproteins, with distinct profiles, were identified for both EHV-2 and EHV-5. Rabbit antisera to EHV-2 and EHV-5 and horse antiserum to EHV-2 were used in radioimmunoprecipitations, Western blot analysis and ELISA to investigate the immunogenicity and cross-reactivity of virus proteins. These analyses revealed that while EHV-2 and EHV-5 proteins share many common epitopes, they also possess type-specific epitopes. A 0.71 kb region of the EHV-2 glycoprotein B (gB) gene was expressed as a fusion protein in Escherichia coli. Antiserum raised in a rabbit to the EHV-2 fusion protein was used to identify a 64K EHV-2 protein as EHV-2 gB. Antiserum to EHV-2 gB was used to identify a 66K EHV-5 protein as EHV-5 gB. These proteins, which may represent subunits of gB rather than the entire molecule, appear the most immunodominant of the structural virion proteins as identified by Western blot.

Animals↗
PubMed1993

Equine herpesviruses 2 and 5 are gamma-herpesviruses.

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).

Amino Acid Sequence↗
PubMed1993

Analysis of the nucleotide sequence of five genes at the left end of the unique short region of the equine herpesvirus 4 genome.

Eco RI fragment G of equine herpesvirus 4 strain 405/76 (EHV 4.405/76) is located at the left end of the unique short region close to or extending into the internal repeat region of the prototypic arrangement of the genome. The nucleotide sequence of two subclones designated HS and G 19, contiguous within Eco RI fragment G, was determined for each strand by obtaining a nested set of deletion clones of these double-stranded DNA plasmids. Analysis of the nucleotide sequence revealed that the two subclones contain 5449 base pairs with four complete open reading frames (ORFs) and part of a fifth ORF. Comparison of the predicted amino acid sequences of these reading frames showed that they correspond to ORFs 67, 68, 69, 70, and 71 of equine herpesvirus type 1 (EHV 1) [41], of which ORFs 68, 69, and 70 are homologous to human herpes simplex virus (HSV) genes in the unique short (US) region, i.e., US 2, US 3, and US 4. ORF 67' of EHV 4 and ORF 67 of EHV 1 are homologous (65.7%) but these genes have no homologue in HSV 1.

Amino Acid Sequence↗
PubMed1993

Epitopes of glycoprotein G of equine herpesviruses 4 and 1 located near the C termini elicit type-specific antibody responses in the natural host.

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.

Abortion, Veterinary↗