Biomedical subjects

S M Boyle

Publications and source records attributed to S M Boyle.

At least 19 recordsLinked to original sources

PubMed2006

PATRIC: the VBI PathoSystems Resource Integration Center.

The PathoSystems Resource Integration Center (PATRIC) is one of eight Bioinformatics Resource Centers (BRCs) funded by the National Institute of Allergy and Infection Diseases (NIAID) to create a data and analysis resource for selected NIAID priority pathogens, specifically proteobacteria of the genera Brucella, Rickettsia and Coxiella, and corona-, calici- and lyssaviruses and viruses associated with hepatitis A and E. The goal of the project is to provide a comprehensive bioinformatics resource for these pathogens, including consistently annotated genome, proteome and metabolic pathway data to facilitate research into counter-measures, including drugs, vaccines and diagnostics. The project's curation strategy has three prongs: 'breadth first' beginning with whole-genome and proteome curation using standardized protocols, a 'targeted' approach addressing the specific needs of researchers and an integrative strategy to leverage high-throughput experimental data (e.g. microarrays, proteomics) and literature. The PATRIC infrastructure consists of a relational database, analytical pipelines and a website which supports browsing, querying, data visualization and the ability to download raw and curated data in standard formats. At present, the site warehouses complete sequences for 17 bacterial and 332 viral genomes. The PATRIC website (https://patric.vbi.vt.edu) will continually grow with the addition of data, analysis and functionality over the course of the project.

Bioterrorism↗

PubMed2001

PI3K inhibitors reverse the suppressive actions of insulin on CYP2E1 expression by activating stress-response pathways in primary rat hepatocytes.

Insulin-associated signaling pathways are critical in the regulation of hepatic physiology. Recent inhibitor-based studies have implicated a mechanistic role for phosphatidylinositol 3' kinase (PI3K) in the insulin-mediated suppression of CYP2E1 mRNA levels in hepatocytes. We investigated the dose dependence for this response and for the effects of insulin and extracellular matrix on PI3K signaling and CYP2E1 mRNA expression levels using a highly defined rat primary hepatocyte culture system. The PI3K inhibitors wortmannin and LY294002 stimulated stress-activated protein kinase/c-Jun NH(2)-terminal kinase (SAPK/JNK) and p38 mitogen-activated protein kinase (MAPK) phosphorylation in a rapid and concentration-dependent manner that paralleled the inhibition of protein kinase B (PKB) phosphorylation. Although PI3K inhibitors reversed the suppressive effects of insulin on CYP2E1 expression, these effects only occurred at concentrations well in excess of those required to achieve complete inhibition of PKB phosphorylation. These same concentrations produced cytotoxic responses as evidenced by perturbed cellular morphology and elevated release of lactate dehydrogenase. Wortmannin-mediated activation of the SAPK/JNK and p38 MAPK pathways also resulted in the mobilization of activator protein-1 complex to the nuclear compartment. We conclude that the suppression of CYP2E1 mRNA expression by insulin is not directly associated with PI3K-dependent pathway activation, but rather is linked to a cytotoxic response stemming from acute challenge with PI3K inhibitors.

Androstadienes↗

PubMed2001

Deletion of wboA enhances activation of the lectin pathway of complement in Brucella abortus and Brucella melitensis.

Brucella spp. are gram-negative intracellular pathogens that survive and multiply within phagocytic cells of their hosts. Smooth organisms present O polysaccharides (OPS) on their surface. These OPS help the bacteria avoid the bactericidal action of serum. The wboA gene, coding for the enzyme glycosyltransferase, is essential for the synthesis of O chain in Brucella. In this study, the sensitivity to serum of smooth, virulent Brucella melitensis 16M and B. abortus 2308, rough wboA mutants VTRM1, RA1, and WRR51 derived from these two Brucella species, and the B. abortus vaccine strain RB51 was assayed using normal nonimmune human serum (NHS). The deposition of complement components and mannose-binding lectin (MBL) on the bacterial surface was detected by flow cytometry. Rough B. abortus mutants were more sensitive to the bactericidal action of NHS than were rough B. melitensis mutants. Complement components were deposited on smooth strains at a slower rate compared to rough strains. Deposition of iC3b and C5b-9 and bacterial killing occurred when bacteria were treated with C1q-depleted, but not with C2-depleted serum or NHS in the presence of Mg-EGTA. These results indicate that (i) OPS-deficient strains derived from B. melitensis 16M are more resistant to the bactericidal action of NHS than OPS-deficient strains derived from B. abortus 2308, (ii) both the classical and the MBL-mediated pathways are involved in complement deposition and complement-mediated killing of Brucella, and (iii) the alternative pathway is not activated by smooth or rough brucellae.

Brucella abortus↗

PubMed2000

Humoral immune response of BALB/c mice to a vaccinia virus recombinant expressing Brucella abortus GroEL does not correlate with protection against a B. abortus challenge.

This work is a part of an ongoing effort to develop vaccinia virus recombinants expressing various Brucella abortus proteins. The B. abortus groEL gene encoding the antigenic heat shock protein GroEL was subcloned into vaccinia virus via homologous recombination and expression confirmed by Western blotting. Female BALB/c mice inoculated with recombinant vaccinia virus/GroEL produced GroEL and vaccinia virus specific antibodies. Mice were challenged 8 weeks post-inoculation with virulent B. abortus strain 2308 and protection measured by the rate of clearance of live Brucella from spleens. Although induction of specific immune response to GroEL and vaccinia virus was demonstrated by the appearance of antibodies in mice, no significant level of protection was demonstrable.

Animals↗

PubMed2000

Characterization of a DNA region containing 5'-(CAAT)(n)-3' DNA sequences involved in lipooligosaccharide biosynthesis in Haemophilus somnus.

Repetitive tetranucleotide sequences of 5'-(CAAT)(n)-3' have been identified at the 5' end of an open reading frame (ORF) named lob1 from Haemophilus somnus strain 738. Based on sequence analysis, lob1 has 59% DNA homology to lex2B, which is involved in lipooligosaccharide (LOS) biosynthesis in H. influenzae. We now report that the number of 5'-CAAT-3' repeats in lob1 varied from 31-35, but that 94% of colonies contained 33 repeats of 5'-CAAT-3' downstream of two potential start codons, as determined by DNA sequence analysis of the 5'-CAAT-3' region from individual colonies. If transcription began with the start codon closest to the 5'-CAAT-3' repeats, a protein of 34.5 kDa would be encoded when 33 repeats were present. However, we could not establish a correlation between the number of 5'-CAAT-3' repeats in lob1 with a specific LOS electrophoretic profile or reactivity with two LOS monoclonal antibodies, indicating multiple genes control LOS phase variation in H. somnus. Complementation of strain 129Pt with lob1 containing 33 5 '-CAAT-3' repeats in shuttle vector pLS88 resulted in transformants 129Pt(pLSlob1-33A) and 129Pt(pLSlob1-33B), both of which demonstrated the same altered LOS electrophoretic profile. Unlike strain 129Pt, both transformants underwent limited LOS phase variation, which correlated with variation in the number of 5'-CAAT-3' repeats in pLSlob1-33. Nanoelectrospray-mass spectrometry of O-deacylated LOS indicated that transformant 129Pt(pLSlob1-33A) LOS was composed of a different distribution of glycoforms than LOS of the parent strain. The ratio of glucose to galactose changed from 1:2 in strain 129Pt LOS to 2:1 in transformant 129Pt(pLSlob1-33A) LOS, as determined by gas chromatography-mass spectrometry. Nuclear magnetic resonance spectroscopy confirmed and extended these observations. Transformant 129Pt(pLSlob1-33A) was constitutively more reactive in colony immunoblotting to polyclonal antiserum made to purified strain 738 LOS, and was more susceptible to complement-mediated killing in the presence of anti-738 LOS serum than parent strain 129Pt. Based on these results, Lob1 appears to be a phase variable galactosyl transferase involved in LOS biosynthesis in H. somnus.

Animals↗

PubMed2000

Characterization of specific immune responses of mice inoculated with recombinant vaccinia virus expressing an 18-kilodalton outer membrane protein of Brucella abortus.

Using the shuttle vector pMCO2 and the vaccinia virus wild-type WR strain, we constructed a recombinant virus expressing an 18-kDa outer membrane protein of Brucella abortus. BALB/c mice inoculated with this virus produced 18-kDa protein-specific antibodies, mostly of immunoglobulin G2a isotype, and in vitro stimulation of splenocytes from these mice with purified maltose binding protein-18-kDa protein fusion resulted in lymphocyte proliferation and gamma interferon production. However, these mice were not protected against a challenge with the virulent strain B. abortus 2308. Disruption of the 18-kDa protein's gene in vaccine strain B. abortus RB51 did not affect either the strain's protective capabilities or its in vivo attenuation characteristics. These observations suggest that the 18-kDa protein plays no role in protective immunity.

Animals↗

PubMed2000

Overexpression of protective antigen as a novel approach to enhance vaccine efficacy of Brucella abortus strain RB51.

Brucella abortus strain RB51 is an attenuated rough strain that is currently being used as the official live vaccine for bovine brucellosis in the United States and several other countries. We reasoned that overexpression of a protective antigen(s) of B. abortus in strain RB51 should enhance its vaccine efficacy. To test this hypothesis, we overexpressed Cu/Zn superoxide dismutase (SOD) protein of B. abortus in strain RB51. This was accomplished by transforming strain RB51 with a broad-host-range plasmid, pBBR1MCS, containing the sodC gene along with its promoter. Strain RB51 overexpressing SOD (RB51SOD) was tested in BALB/c mice for its ability to protect against challenge infection with virulent strain 2308. Mice vaccinated with RB51SOD, but not RB51, developed antibodies and cell-mediated immune responses to Cu/Zn SOD. Strain RB51SOD vaccinated mice developed significantly (P < 0.05) more resistance to challenge than those vaccinated with strain RB51 alone. The presence of the plasmid alone in strain RB51 did not alter its vaccine efficacy. Also, overexpression of SOD did not alter the attenuation characteristic of strain RB51.

Animals↗

PubMed2000

Brucella abortus strain RB51 as a vector for heterologous protein expression and induction of specific Th1 type immune responses.

Brucella abortus strain RB51 is a stable, rough, attenuated mutant widely used as a live vaccine for bovine brucellosis. Our ultimate goal is to develop strain RB51 as a preferential vector for the delivery of protective antigens of other intracellular pathogens to which the induction of a strong Th1 type of immune response is needed for effective protection. As a first step in that direction, we studied the expression of a foreign reporter protein, beta-galactosidase of Escherichia coli, and the 65-kDa heat shock protein (HSP65) of Mycobacterium bovis in strain RB51. We cloned the promoter sequences of Brucella sodC and groE genes in pBBR1MCS to generate plasmids pBBSODpro and pBBgroE, respectively. The genes for beta-galactosidase (lacZ) and HSP65 were cloned in these plasmids and used to transform strain RB51. An enzyme assay in the recombinant RB51 strains indicated that the level of beta-galactosidase expression is higher under the groE promoter than under the sodC promoter. In strain RB51 containing pBBgroE/lacZ, but not pBBSODpro/lacZ, increased levels of beta-galactosidase expression were observed after subjecting the bacteria to heat shock or following internalization into macrophage-like J774A.1 cells. Mice vaccinated with either of the beta-galactosidase-expressing recombinant RB51 strains developed specific antibodies of predominantly the immunoglobulin G2a (IgG2a) isotype, and in vitro stimulation of their splenocytes with beta-galactosidase induced the secretion of gamma interferon (IFN-gamma), but not interleukin-4 (IL-4). A Th1 type of immune response to HSP65, as indicated by the presence of specific serum IgG2a, but not IgG1, antibodies, and IFN-gamma, but not IL-4, secretion by the specific-antigen-stimulated splenocytes, was also detected in mice vaccinated with strain RB51 containing pBBgroE/hsp65. Studies with mice indicated that expression of beta-galactosidase or HSP65 did not alter either the attenuation characteristics of strain RB51 or its vaccine efficacy against B. abortus 2308 challenge.

Animals↗

PubMed2000

Complementation of Brucella abortus RB51 with a functional wboA gene results in O-antigen synthesis and enhanced vaccine efficacy but no change in rough phenotype and attenuation.

Brucella abortus RB51 is a stable rough, attenuated mutant vaccine strain derived from the virulent strain 2308. Recently, we demonstrated that the wboA gene in RB51 is disrupted by an IS711 element (R. Vemulapalli, J. R. McQuiston, G. G. Schurig, N. Srirauganathan, S. M. Halling, and S. M. Boyle, Clin. Diagn. Lab. Immunol. 6:760-764, 1999). Disruption of the wboA gene in smooth, virulent B. abortus, Brucella melitensis, and Brucella suis results in rough, attenuated mutants which fail to produce the O polysaccharide (O antigen). In this study, we explored whether the wboA gene disruption is responsible for the rough phenotype of RB51. We complemented RB51 with a functional wboA gene, and the resulting strain was designated RB51WboA. Colony and Western blot analyses indicated that RB51WboA expressed the O antigen; immunoelectron microscopy revealed that the O antigen was present in the cytoplasm. Crystal violet staining, acryflavin agglutination, and polymyxin B sensitivity studies indicated that RB51WboA had rough phenotypic characteristics similar to those of RB51. Bacterial clearance studies of BALB/c mice indicated no increase in the survival ability of RB51WboA in vivo compared to that of RB51. Vaccination of mice with live RB51WboA induced antibodies to the O antigen which were predominantly of the immunoglobulin G2a (IgG2a) and IgG3 isotypes. After in vitro stimulation of splenocytes with killed bacterial cells, quantitation of gamma interferon in the culture supernatants indicated that RB51WboA immunization induced higher levels of gamma interferon than immunization with RB51. Mice vaccinated with RB51WboA were better protected against a challenge infection with the virulent strain 2308 than those vaccinated with RB51. These studies indicate that in addition to the disruption of the wboA gene there is at least one other mutation in RB51 responsible for its rough phenotype. These studies also suggest that the expressed O antigen in RB51WboA is responsible either directly or indirectly for the observed enhancement in the T-cell response.

Animals↗

PubMed1999

Identification of an IS711 element interrupting the wboA gene of Brucella abortus vaccine strain RB51 and a PCR assay to distinguish strain RB51 from other Brucella species and strains.

Brucella abortus vaccine strain RB51 is a natural stable attenuated rough mutant derived from the virulent strain 2308. The genetic mutations that are responsible for the roughness and the attenuation of strain RB51 have not been identified until now. Also, except for an assay based on pulsed-field gel electrophoresis, no other simple method to differentiate strain RB51 from its parent strain 2308 is available. In the present study, we demonstrate that the wboA gene encoding a glycosyltransferase, an enzyme essential for the synthesis of O antigen, is disrupted by an IS711 element in B. abortus vaccine strain RB51. Exploiting this feature, we developed a PCR assay that distinguishes strain RB51 from all other Brucella species and strains tested.

Animals↗

PubMed1999

Genetic characterization of a Tn5-disrupted glycosyltransferase gene homolog in Brucella abortus and its effect on lipopolysaccharide composition and virulence.

We constructed a rough mutant of Brucella abortus 2308 by transposon (Tn5) mutagenesis. Neither whole cells nor extracted lipopolysaccharide (LPS) from this mutant, designated RA1, reacted with a Brucella O-side-chain-specific monoclonal antibody (MAb), Bru-38, indicating the absence of O-side-chain synthesis. Compositional analyses of LPS from strain RA1 showed reduced levels of quinovosamine and mannose relative to the levels in the parental, wild-type strain, 2308. We isolated DNA flanking the Tn5 insertion in strain RA1 by cloning a 25-kb XbaI genomic fragment into pGEM-3Z to create plasmid pJM6. Allelic exchange of genomic DNA in B. abortus 2308 mediated by electroporation of pJM6 produced kanamycin-resistant clones that were not reactive with MAb Bru-38. Southern blot analysis of genomic DNA from these rough clones revealed Tn5 in a 25-kb XbaI genomic fragment. A homology search with the deduced amino acid sequence of the open reading frame disrupted by Tn5 revealed limited homology with various glycosyltransferases. This B. abortus gene has been named wboA. Transformation of strain RA1 with a broad-host-range plasmid bearing the wild-type B. abortus wboA gene resulted in the restoration of O-side-chain synthesis and the smooth phenotype. B. abortus RA1 was attenuated for survival in mice. However, strain RA1 persisted in mice spleens for a longer time than the B. abortus vaccine strain RB51, but as expected, neither strain induced antibodies specific for the O side chain.

Animals↗

PubMed1999

Association with the cellular export receptor CRM 1 mediates function and intracellular localization of Epstein-Barr virus SM protein, a regulator of gene expression.

Splicing and posttranscriptional processing of eukaryotic gene transcripts are linked to their nuclear export and cytoplasmic expression. Unspliced pre-mRNAs and intronless transcripts are thus inherently poorly expressed. Nevertheless, human and animal viruses encode essential genes as single open reading frames or in the intervening sequences of other genes. Many retroviruses have evolved mechanisms to facilitate nuclear export of their unspliced mRNAs. For example, the human immunodeficiency virus RNA-binding protein Rev associates with the soluble cellular export receptor CRM 1 (exportin 1), which mediates nucleocytoplasmic translocation of Rev-HIV RNA complexes through the nuclear pore. The transforming human herpesvirus Epstein-Barr virus (EBV) expresses a nuclear protein, SM, early in its lytic cycle; SM binds RNA and posttranscriptionally activates expression of certain intronless lytic EBV genes. Here we show that both the trans-activation function and cytoplasmic translocation of SM are dependent on association with CRM 1 in vivo. SM is also shown to be associated in vivo with other components of the CRM 1 export pathway, including the small GTPase Ran and the nucleoporin CAN/Nup214. SM is shown to be present in the cytoplasm, nucleoplasm, and nuclear envelope of transfected cells. Mutation of a leucine-rich region (LRR) of SM inhibited CRM 1-mediated cytoplasmic translocation and SM activity, as did leptomycin B, an inhibitor of CRM 1 complex formation. Surprisingly, however, leptomycin B treatment and mutation of the LRR both led to SM becoming more tightly attached to intranuclear structures. These findings suggest a model in which SM is not merely a soluble carrier protein for RNA but rather is bound directly to intranuclear proteins, possibly including the nuclear pore complex.

Animals↗

PubMed1998

Cytochromes P450 in crustacea.

Since the last review of this topic, further insight has been gained into the presence and functions of cytochrome P450 proteins in the hepatopancreas and other organs of aquatic crustacean species, although progress has been slow relative to the advances in other species. Recent studies with several lobster, shrimp, crab and crayfish species suggest that cytochromes P450 in the 2 and 3 families are the most abundant forms in hepatopancreas microsomes. Substrates normally metabolized by CYP2 and CYP3 family members are monooxygenated more rapidly by crustacea than substrates normally metabolized by CYP1 family enzymes, e.g. erythromycin, testosterone and aminopyrine are much more rapidly monooxygenated than ethoxyresorufin. Some progress has been made in cloning and sequencing crustacean P450 forms. CYP2L1 and CYP2L2 cDNA sequences have been cloned from spiny lobster hepatopancreas libraries, and there was evidence for at least two more cytochromes P450 in spiny lobster hepatopancreas. An area of continued interest, but of no consensus or general findings, relates to the presence and inducibility of CYP1 family members in crustacea. Some studies indicate weak induction of total cytochrome P450 and increased turnover of substrates normally associated with CYP1, while others show no effect of the classic inducers that act at the Ah receptor in vertebrates. A few studies of the roles of cytochromes P450 in the biosynthesis and degradation of steroids, including ecdysteroids, have been published. Further studies are needed to understand the regulation and normal function of the crustacean cytochromes P450.

Animals↗

PubMed1998

Cloning and sequencing of yajC and secD homologs of Brucella abortus and demonstration of immune responses to YajC in mice vaccinated with B. abortus RB51.

To identify Brucella antigens that are potentially involved in stimulating a protective cell-mediated immune response, a gene library of Brucella abortus 2308 was screened for the expression of antigens reacting with immunoglobulin G2a antibodies from BALB/c mice vaccinated with B. abortus RB51. One selected positive clone (clone MCB68) contained an insert of 2.6 kb; nucleotide sequence analysis of this insert revealed two open reading frames (ORFs). The deduced amino acid sequences of the first and second ORFs had significant similarities with the YajC and SecD proteins, respectively, of several bacterial species. Both the YajC and SecD proteins were expressed in Escherichia coli as fusion proteins with maltose binding protein (MBP). In Western blots, sera from mice vaccinated with B. abortus RB51 recognized YajC but not SecD. Further Western blot analysis with purified recombinant YajC protein indicated that mice inoculated with B. abortus 19 or 2308 or B. melitensis RM1 also produced antibodies to YajC. In response to in vitro stimulation with recombinant MBP-YajC fusion protein, splenocytes from mice vaccinated with B. abortus RB51 were able to proliferate and produce gamma interferon but not interleukin-4. This study demonstrates, for the first time, the involvement of YajC protein in an immune response to an infectious agent.

Amino Acid Sequence↗

PubMed1997

Phase variation and conservation of lipooligosaccharide epitopes in Haemophilus somnus.

The bovine-specific pathogen Haemophilus somnus is capable of undergoing structural and antigenic phase variation in its lipooligosaccharide (LOS) components after in vivo and in vitro passage. However, commensal isolates from the reproductive tract have not been observed to vary in phase (T. J. Inzana, R. P. Gogolewski, and L. B. Corbeil, Infect. Immun. 60:2943-2951, 1992). We now report that specific monoclonal antibodies (MAbs) to the LOSs of Haemophilus aegyptius, Neisseria gonorrhoeae, and Haemophilus influenzae, as well as H. somnus, reacted with some phase-variable epitopes in H. somnus LOS. All reactive MAbs bound to LOS components of about 4.3 kDa in the same H. somnus isolates, including a non-phase-varying strain. Following in vitro passage of a clonal variant of strain 738 that was nonreactive with the MAbs, 11.8% of young colonies shifted to a reactive phenotype. A digoxigenin-labelled 5'-CAATCAATCAATCAATCAATCAATCAAT-3' oligonucleotide probe hybridized to genomic DNA from strain 738 but did not react with DNA from a non-phase-varying strain. Sequence analysis of the gene containing 5'-CAAT-3' tandem sequences revealed 48% amino acid homology with the lex-2B gene-encoded protein of H. influenzae type b. Our results indicate that some LOS epitopes are conserved between H. somnus and other Haemophilus and Neisseria species, that LOS phase variation may occur at a high rate in some strains of H. somnus, and that phase variation may, in part, be due to 5'-CAAT-3' tandem sequences present in H. somnus genes.

Amino Acid Sequence↗

PubMed1996

cDNA and protein sequence of a major form of P450, CYP2L, in the hepatopancreas of the spiny lobster, Panulirus argus.

A P450 fraction was previously isolated from spiny lobster hepatopancreas microsomes and shown in reconstitution experiments to be efficient in catalyzing the monooxygenation of benzphetamine, aminopyrine, benzo(a)pyrene, progesterone, and testosterone. In this study, N-terminal sequence information up to residue 39 was obtained from this P450 and used to design degenerate primers for screening a cDNA library constructed from hepatopancreas mRNA. Clones were obtained that contained part of the coding region of a P450 protein. Further exact primers were designed that permitted the isolation of clones containing coding information for other parts of the P450 sequence, as well as a clone that coded for the complete P450 protein sequence. The open reading frame of the complete coding region corresponded to a protein of 492 amino acids. The deduced amino acid sequence of this P450 was about 36% similar to individual mammalian P450s in the 2 family and did not show strong matches with other proteins in the data base. Based on sequence and the previously determined function, this spiny lobster P450 was assigned by the P450 nomenclature committee to a new P450 subfamily, CYP2L. This is the first description of a P450 primary sequence from a marine crustacean species and the first assignment of an invertebrate P450 into the 2 family.

Amino Acid Sequence↗

PubMed1996

Detailed physical and deletion mapping of 8p with isolation of YAC clones from tumour suppressor loci involved in colorectal cancer.

Loss of heterozygosity (LOH) of markers at chromosome 8p is frequently noted in many different tumour types, including colorectal cancer. Numerous investigations indicate the presence of more than tumour suppressor gene (TSG) located on 8p. In this study, we describe a detailed LOH map in colorectal cancer and relate this to physical mapping data from reduced radiation 8p hybrids, yeast artificial chromosome (YAC) co-localisation of markers and fluorescence in situ hybridisation data. These data indicate the presence of two regions harbouring putative TSG's between the polymorphic markers for the LPL gene-D8S298 (approximately 4 Mb) and the markers D8S136-D8S137 (approximately 8 Mb). Yeast Artificial Chromosomes (YAC) have been isolated from these regions of interest to aid the localisation of the putative TSG's.

Chromosome Mapping↗

PubMed1996

Characterization of the haemorrhagic enteritis virus genome and the sequence of the putative penton base and core protein genes.

Haemorrhagic enteritis virus (HEV) is a member of a genetically ill-defined group within the genus Aviadenovirus which causes significant clinical disease in gallinaceous fowl. Using DNA obtained from a low virulence isolate of HEV passed in turkeys, we developed a genomic restriction map and estimated an apparent genomic length of 25.5 kb. No evidence for extensive DNA hybridization was found between the HEV genome and either the hexon or penton base genes of human adenovirus 2 (HAdV-2) and fowl adenovirus 10 (FAdV-10). The HEV penton base gene was identified by PCR using primers based on conserved adenoviral DNA sequences. The penton base gene was expressed in Escherichia coli as a fusion protein and detected by anti-HEV serum in both colony and denaturing gel immunoblots. DNA sequencing revealed a putative penton base ORF with a predicted amino acid sequence showing approximately 39.0%, 53.0% and 44.2% similarity with the penton base of HAdV-2, human adenovirus 40 (HAdV-40) and FAdV-10, respectively. The penton base gene was located at 43.3-48.6 m.u. on the HEV genome and had a remarkably low G+C content (33.8%). DNA sequencing also revealed ORFs for putative core proteins resembling pVII, p-mu and a partial ORF similar to pVI (hexon-associated protein) of HAdV-2 and HAdV-40. The results support the claim that HEV represents a distinct group of viruses within the genus Aviadenovirus.

Amino Acid Sequence↗