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M J Studdert

Publications and source records attributed to M J Studdert.

At least 37 records · Page 2Linked to original sources
PubMed1998

Equine herpesvirus-4 glycoprotein G is secreted as a disulphide-linked homodimer and is present as two homodimeric species in the virion.

Glycoprotein G (gG) homologues have been found in most alphaherpesviruses although little is known about their structure or function. In this study, three species of equine herpesvirus-4 (EHV-4) gG were identified: a full-length 68 kDa virion-associated species (gGVL), a 12 kDa virion-associated species (gGVS) and a 60 kDa secreted species (gGS), detected in the medium of infected cells. gGS and gGVS appear to be proteolytic cleavage products of gGVL and correspond to the N- and C-terminal regions, respectively. It was shown that gGS and gGVL are similarly glycosylated possessing mostly N-linked complex-type carbohydrate side chains. Western blots of proteins separated under nonreducing conditions established that gGS is secreted as a 120 kDa glycoprotein while the virion-associated species, gGVL and gGVS, are present in the virion as 140 and 20 kDa proteins, respectively. As gGS and gGVL do not appear to associate stably with other viral proteins, it is most likely that each species exists as a disulphide-linked homodimer. Pulse-chase experiments indicated that gGVL is rapidly assembled as a homodimer prior to both carbohydrate side-chain maturation in the Golgi and proteolytic cleavage. Proteolytic cleavage of full-length gG occurs during or immediately after passage through the Golgi. Secreted and virion-associated species of gG were identified in the closely related virus EHV-1 and were of similar molecular masses to the corresponding EHV-4 gG species.

Animals↗
PubMed1998

Characterization of glycoprotein B of the gammaherpesvirus equine herpesvirus-2.

Twenty-two monoclonal antibodies (MAbs) were generated to the gammaherpesvirus equine herpesvirus-2 (EHV-2). Using Western blot analysis, eight MAbs recognized an Escherichia coli glutathione S-transferase (GST)-glycoprotein B (gB) fusion protein and, using overlapping GST-gB fusion proteins, a neutralization epitope was mapped to amino acids 29-74. One of the gB-specific MAbs was used to characterize the glycosylation and kinetics of synthesis of EHV-2 gB. EHV-2 gB is synthesized as a 97 kDa polypeptide that is co-translationally modified to a 130 kDa high-mannose precursor that forms a 260 kDa dimer shortly after synthesis. Each 130 kDa precursor is endoproteolytically cleaved to disulphide-linked subunits of 75 and 58 kDa prior to further processing to complex oligosaccharide-containing subunits of 89 and 65/62 kDa. The 89 and 65/62 kDa subunits of EHV-2 gB contain 39 and 17 kDa of N-linked oligosaccharides, respectively, and do not contain any O-linked oligosaccharides. Western blot analysis of purified EHV-2 virions established that gB exists as a 320 kDa dimer in the virion envelope.

Amidohydrolases↗
PubMed1998

Seroprevalence of equine herpesvirus 1 in thoroughbred foals before and after weaning.

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.

Animals↗
PubMed1997

Identification, cloning and sequence analysis of the equine adenovirus 1 hexon gene.

Based on sequence homology with human adenovirus 2 (HAdV2), the hexon gene of equine adenovirus 1 (EAdV1) was identified. HindIII restriction fragments containing the hexon and other viral genes were cloned into the plasmids pUC19 and pBlueScript SK(-) and sequenced. The nucleotide sequence of the hexon gene was completely determined and partial sequence data were obtained for seven other EAdV1 genes. Amino acid (aa) sequence comparison with published adenovirus (AdV) proteins identified the genes for the IIIa, penton, pVII, PVI, 23K proteinase, DNA binding and 100K proteins. The eight EAdV1 genes appeared to be in the same relative order as homologous genes of other AdV. The EAdV1 hexon protein was encoded between the hexon-associated pVI upstream and the 23K proteinase gene downstream and comprised 2742 nucleotides which translated into 913 aa. Similar to other members of the genus Mastadenovirus the EAdV1 hexon yielded two highly conserved genome segments at the N- and C-termini which flanked intermediate variable and hypervariable regions. The majority of the residue differences between EAdV1 and other AdV hexons occurred in two loops that are known for other AdV to protrude from the surface of the nucleocapsid. Amino acid comparisons with other AdV hexons revealed highest homology with HAdV12 hexon with 72% identical and 83% functionally similar residues, followed by bovine AdV3 hexon with 71% identities and 82% functional residue conservation. Phylogenetic analysis suggested that EAdV1 and other AdV do not have an immediate common ancestor.

Amino Acid Sequence↗
PubMed1997

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.

Adenoviridae↗
PubMed1997

Identification of noncytopathic equine rhinovirus 1 as a cause of acute febrile respiratory disease in horses.

Equine rhinovirus 1 (ERhV1) is a recognized cause of acute febrile respiratory disease in horse, although the virus is rarely isolated from such animals, despite seroprevalence rates as high as 50% in some horse populations. Recently, ERhV1 has been shown to be most closely related to foot-and-mouth disease virus, raising questions as to its disease associations in horses. We report that ERhV1 infection was the likely cause of two separate outbreaks of severe febrile respiratory disease which involved more than 20 horses. Attempts to isolate ErhV1 from nasopharyngeal swabs by conventional cell culture methods were unsuccessful, in that cytopathology was not observed. Viral antigen was detected by immunofluorescence assay in the cytoplasm of cells infected with 10 of 15 nasopharyngeal swab samples, indicating the presence and presumably replication of ERhV1. A rise in serum neutralizing antibody titer between acute- and convalescent-phase sera confirmed that ERhV1 was causatively associated with one of the outbreaks. ERhV1 RNA was detected in nasopharyngeal swabs collected from all horses during the acute phase of disease by reverse transcription-PCR. Nucleotide sequencing of amplified products showed that within each outbreak a single strain of ERhV1 was involved but that distinct viruses were involved in each outbreak. A retrospective study of samples from nine other outbreaks of respiratory disease in horses suggested ERhV1 etiology in at least two of these. We conclude that the relative importance of ERhV1 as a cause of acute febrile respiratory disease in horses has been underestimated due to failure in many instances to isolate virus by conventional cell culture methods.

Acute Disease↗
PubMed1997

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.

Alphaherpesvirinae↗
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↗
PubMed1996

Genomic location and nucleotide sequence of a porcine adenovirus penton base gene.

The putative penton base gene of a porcine adenovirus serotype 3 (PAV3) has been identified, cloned and sequenced. The genomic location of the PAV3 penton base was deduced by probing a Southern blot with a polymerase chain reaction generated product containing the human adenovirus type 2 (HAV2) penton base gene. Sequencing revealed an open reading frame (ORF) of 1527 nucleotides coding for a polypeptide of 509 amino acids. However, cDNA analysis indicated an acceptor splice site one nucleotide upstream of the second ATG in the ORF. This produced an ORF of 1452 nucleotides coding for a polypeptide of 484 amino acids with a calculated molecular weight of 54.5 kDa. Comparison with the HAV2 penton base amino acid sequence revealed the putative PAV3 penton base homologue to be 87 amino acids shorter with an overall amino acid homology of approximately 65%. Comparison with the penton base proteins of other HAV types revealed a region between amino acid positions 283 and 379 with no similarity.

Adenoviridae↗
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↗