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Biomedical subjects

M J Studdert

Publications and source records attributed to M J Studdert.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1992

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.

Adenoviridae↗
PubMed1992

Cloning and restriction endonuclease mapping of the genome of an equine herpesvirus 4 (equine rhinopneumonitis virus), strain 405/76.

Purified virion DNA of an Australian isolate of equine herpesvirus 4(EHV 4.405/76) was digested with restriction enzymes and the DNA fragments were cloned into pUC19. The resulting recombinant plasmid library, representing 92% of the virus genome, was used in hybridization analyses to construct restriction maps for BamHI, EcoRI, and SalI for the EHV4 genome. The results show that the genome of EHV 4.405/76 was approximately 145 kb and comprised a unique long (UL) region of 112 kb and a unique short (US) region of 12.4 kb. US is flanked by an internal and terminal repetitive sequence (IRS and TRS) of about 10.3 kb. The BamHI and EcoRI restriction maps are similar to those previously published for an English isolate EHV 4.1942 strain although some differences such as location of an additional fragment and changes in positions of two other small fragments were found.

Animals↗
PubMed1992

Equine herpesvirus 5: comparisons with EHV2 (equine cytomegalovirus), cloning, and mapping of a new equine herpesvirus with a novel genome structure.

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.

Animals↗
PubMed1992

Identification of equine herpesvirus 4 glycoprotein G: a type-specific, secreted glycoprotein.

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.

Amino Acid Sequence↗
PubMed1992

The molecular epidemiology of equine herpesvirus 1 (equine abortion virus) in Australasia 1975 to 1989.

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.

Abortion, Veterinary↗
PubMed1991

Characterization of the major glycoproteins of equine herpesviruses 4 and 1 and asinine herpesvirus 3 using monoclonal antibodies.

A panel of 14 monoclonal antibodies (MAbs) was used to characterize the high abundance glycoproteins of equine herpesviruses 4 (EHV-4) and 1 (EHV-1), and asinine herpesvirus 3 (AHV-3). The specificities of the MAbs, which had been determined previously for strains of EHV-4 and -1 from the U.S.A., in general were confirmed by ELISA for Australian strains of these viruses. Of the 14 MAbs seven were EHV-4 and -1 type-common and cross-reacted with AHV-3. Of the five MAbs that were EHV-1 type-specific, four cross-reacted with AHV-3, whereas neither of the EHV-4 type-specific MAbs reacted with AHV-3, providing further evidence for a closer evolutionary relationship between EHV-1 and AHV-3 than that between either of these viruses and EHV-4. By Western blot and immunoprecipitation analyses, the identity of the six major glycoproteins, gp2, gp10, gp13, gp14, gp18 and gp21/22a, of an Australian EHV-1 isolate was verified, and it was shown that AHV-3 had cross-reactive glycoproteins of very similar Mr to those of EHV-1; five homologous glycoproteins of EHV-4 were also identified. It was determined that the EHV-4 gp13 homologue had a much reduced Mr (67K) when the virus was grown in a continuous cell line than when grown in equine foetal kidney cells (95K). It is suggested that altered glycosylation by the cell line is responsible for this change in Mr. Those glycoproteins acting as major immunogens in the naturally infected host, at least in their ability to elicit antibody, were identified. It was found that gp2, gp13, gp14, gp18 and a glycoprotein at 120K (EHV-1) or 116K (EHV-4) were all important immunogens in mares following EHV-1-induced abortion, and in a specific pathogen-free foal experimentally infected with EHV-1 and later cross-challenged with EHV-4. Gp2, gp14 and gp18 were the major immunogens in the donkey in response to AHV-3 infection. The type specificity associated with these glycoproteins was also examined and it was found that although most if not all contain type-specific epitopes, gp2 and a glycoprotein at 120K, and to a lesser extent gp13 and gp18, were significantly type-specific in the serum from a mare following natural EHV-1 infection and abortion.

Animals↗