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M M Binns

Publications and source records attributed to M M Binns.

At least 73 records · Page 4Linked to original sources

Alterations in DNA sequence and RNA transcription of the Bam HI-H fragment accompany attenuation of oncogenic Marek's disease herpesvirus.

The nucleotide sequences of the BamHI-H fragment of the HPRS16 strain of Marek's disease virus (MDV) and of its attenuated derivative HPRS16/att have been determined. The results show that in addition to the tandem expansion of a 132 bp sequence from two copies in HPRS16 virus to eight copies in HPRS16/att, nucleotide substitutions, deletions, and insertions were also noted. Several potential open reading frames (ORFs) were identified. One of these (ORF 13) encoded a deduced protein, mol wt 32 kD, which is likely to be the serotype-1 specific phosphoprotein expressed in tumours (1) and mapped to an EcoRI fragment within BamHI-H (2). Our results suggest that this ORF is unlikely to be the B antigen (3). ORF 4, which had some similarity to CD4 and immunoglobulin M heavy chain, was encoded by a transcript that originated within the first copy of the 132 bp repeat. ORF 21, which mapped entirely within UL, encoded a deduced protein at least 322 amino acids long that had some similarity to varicella zoster virus (VZV) alpha trans-inducing protein. None of these ORFs was altered significantly by attenuation, except ORF 4 and another small ORF (ORF 3), 5' of the 132 bp repeats, which would probably fail to be transcribed because of truncation of an RNA transcript.

Amino Acid Sequence↗

Construction and properties of a turkey herpesvirus recombinant expressing the Marek's disease virus homologue of glycoprotein B of herpes simplex virus.

A herpesvirus of turkeys (HVT) recombinant containing a 3.9 kbp fragment of Marek's disease virus (MDV) DNA encoding MDV glycoprotein B (gB), stably integrated into the thymidine kinase (TK) gene of HVT, has been constructed. The replication of the recombinant in chick embryo fibroblasts (CEF) was comparable to that of wild-type HVT. The recombinant expressed authentic MDV gB and its processed forms (110K, 65K and 48K) in CEF as shown by immunoblotting using an MDV-specific anti-peptide serum. Northern blot analysis showed that MDV gB mRNA was transcribed from MDV promoter sequences flanking the MDV gB open reading frame and also from the HVT TK promoter. However, the level of replication of the recombinant in vivo appeared to be lower than wild-type HVT as shown by the titres of HVT antibodies, determined by ELISA. Pathogenicity tests showed that the recombinant was safe and did not cause microscopic or gross Marek's disease lesions or other abnormalities. The results suggest that HVT has potential as a vector for recombinant vaccines.

Animals↗

Identification and sequence analysis of the homologues of the herpes simplex virus type 1 glycoprotein H in Marek's disease virus and the herpesvirus of turkeys.

The glycoprotein H (gH) genes of two avian herpesviruses, Marek's disease virus and the herpesvirus of turkeys, have been cloned and sequenced and the coding regions found to be of 2439 and 2424 nucleotides respectively. The predicted primary polypeptide products of these open reading frames are 813 and 808 amino acids and correspond to Mrs of 90,800 and 91,100. Both amino acid sequences exhibit characteristic glycoprotein features such as hydrophobic signal and anchor sequences and potential sites for N-linked glycosylation. Polypeptide sequence comparison to the other eight available gH sequences revealed more similarity to the alphaherpesvirus subgroup than to either beta- or gammaherpesviruses.

Amino Acid Sequence↗

Isolation and characterization of microsatellites from the canine genome.

Microsatellite sequences comprising (dC-dA)n.(dG-dT)n repeats have been isolated from canine libraries and sequenced. Oligonucleotide primers have been synthesized to the microsatellite flanking sequences and used in the polymerase chain reaction to amplify those loci from genomic DNA. The degree of polymorphism of each microsatellite was estimated in a set of unrelated dogs. It is concluded that of the 10 loci studied, nine are sufficiently polymorphic to be useful in genetic studies.

Animals↗

Characterisation of equine influenza isolates from the 1987 epizootic in India by nucleotide sequencing of the HA1 gene.

Two A/Equi-2 (H3N8) isolates were obtained during the 1987 Indian equine influenza epizootic. The sequence of the Ludhiana/87 HA1 gene revealed that this isolate was very similar to recent European and North American isolates of equine influenza. In contrast, the Bhiwani/87 HA1 gene was nearly identical to the Miami/63 prototype H3 sequence. These results support the antigenic analysis previously carried out on these isolates using monoclonal antibodies. However, the finding that Bhiwani/87 is so similar to Miami/63, coupled with the finding that equine H3N8 influenza viruses have previously been shown to evolve along a single lineage, suggests that Miami/63 virus from either a vaccine or laboratory source may be the origin of the Bhiwani/87 isolate.

Amino Acid Sequence↗

Detection of antibodies against equine herpesvirus types 1 and 4 by using recombinant protein derived from an immunodominant region of glycoprotein B.

The N-terminal fragment comprising residues +1 to +50 (gB1-50) of equine herpesvirus type 1 (EHV-1) glycoprotein B was expressed as a glutathione S-transferase fusion protein in Escherichia coli. Recombinant gB1-50 (rgB1-50) was recognized in immunoblots by sera from rabbits immunized with EHV-1 and by convalescent-phase sera from horses with natural EHV-1 infections. An enzyme-linked immunosorbent assay (ELISA) for monitoring antibody levels against EHV-1 was developed by using rgB1-50, and its specificity was assessed with a panel of reference antisera against other equine viruses. A specific cross-reaction was detected with EHV-4, which was confirmed by inhibition ELISA. Convalescent-phase sera from horses with natural EHV-1 or EHV-4 infections possessed antibody titers against rgB1-50 ranging from 1:2,000 to 1:64,000, indicating the presence of an immunodominant antigenic site. The study demonstrated the potential application of rgB1-50 as a diagnostic antigen and highlights the glutathione S-transferase fusion system as a simple and effective method of producing purified milligram quantities of antigen.

Amino Acid Sequence↗

Gene translocations in poxviruses: the fowlpox virus thymidine kinase gene is flanked by 15 bp direct repeats and occupies the locus which in vaccinia virus is occupied by the ribonucleotide reductase large subunit gene.

By sequencing a fragment of 7351 bp the fowlpox virus thymidine kinase gene has been found to map to a position within the equivalent of the vaccinia HindIII I fragment. The deduced gene arrangement in fowlpox virus is I3, X, TK, I5, I6, I7, I8, G1, indicating that the homologue of the vaccinia I4 gene has been replaced by two genes X and TK. The non-essential TK gene has therefore replaced another non-essential gene, I4 (the ribonucleotide reductase large subunit) in this region. The X/TK insertion in fowlpox virus is precisely flanked by direct repeats of 15 bp suggesting that the translocation event may have involved transposition. The % identities between the fowlpox virus and vaccinia virus proteins ranged between 58.5% and 31.3%.

Amino Acid Sequence↗

Comparison of the locations of homologous fowlpox and vaccinia virus genes reveals major genome reorganization.

We have derived a restriction enzyme map for the fowlpox virus FP9 strain. Sites for BamHI, PvuII, PstI and NcoI have been mapped mainly by Southern blotting. The size of the genome derived from the restriction maps (254 kb) corresponds to the figure of 260 +/- 8 kb determined from analysis of genomic DNA by pulsed-field electrophoresis. The map can be compared with a previously published map for a different strain of fowlpox virus using the PstI digest which is common to both studies. Some 65 kb of fowlpox virus sequence, in 11 blocks, as well as individual M13 clones have been aligned with the map. Where those blocks correspond with blocks of homologous genes in vaccinia virus, it is possible to compare the genomic locations for those genes in the two viruses. This comparison reveals that, whereas there are blocks of sequence within which genes exist in the same relative position in the two viruses, the genomic location of those sequence blocks differs widely between the two viruses.

Deoxyribonuclease BamHI↗

Abortion of virologically negative foetuses following experimental challenge of pregnant pony mares with equid herpesvirus 1.

From 1988 to 1991, 51 pregnant pony mares were challenged intranasally or by aerosol with an isolate of EHV-1 (AB4) originally recovered from a quadriplegic mare. This resulted in 32 abortions, occurring from 9 to 29 days after infection. In 14 of the early abortions (Days 9-14), EHV-1 was not demonstrated in the foetal tissues by virus isolation or immunostaining despite no other non-viral cause for the abortion being evident. Application of the polymerase chain reaction to foetal tissues from 9 of these cases also proved negative. One of the 14 mares was destroyed immediately after abortion, and post-mortem examination revealed severe and widespread vasculitis, thrombosis and secondary ischaemic damage in the endometrium with replication of EHV-1 in endothelial cells. These findings suggest that EHV-1 abortion can occur due to endometrial damage without the establishment of a foetal infection.

Abortion, Veterinary↗

A recombinant fowlpox virus that expresses the VP2 antigen of infectious bursal disease virus induces protection against mortality caused by the virus.

The coding sequences of VP2 from a virulent strain, 52/70, of infectious bursal disease virus (IBDV) were excised from a cDNA clone and inserted into a fowlpox plasmid insertion vector. The resulting plasmid, pIBD 1, was used to construct a recombinant fowlpox virus, fpIBD 1, which expressed VP 2 as a beta-galactosidase fusion protein. Chickens vaccinated with fpIBD 1 at 1 and 14 days of age, were challenged at 28 days with either IBDV strain 52/70 or the highly virulent strain CS 89. These chickens were protected against mortality, but not against damage to the bursa of Fabricius. The protection achieved by the use of fpIBD 1 shows that VP 2 is a host protective antigen.

Amino Acid Sequence↗

Deduced amino acid sequence of the fusion glycoprotein of turkey rhinotracheitis virus has greater identity with that of human respiratory syncytial virus, a pneumovirus, than that of paramyxoviruses and morbilliviruses.

The sequence of the fusion (F) glycoprotein of turkey rhinotracheitis virus (TRTV) has been deduced from cDNA clones derived from oligo(dT)-selected infected cell RNA. The protein consists of 538 amino acids, the F2 and F1 subunits containing 102 (including the F2-F1 connecting peptide RRRR) and 436 residues, respectively. Each subunit has one potential N-linked glycosylation site. The protein has 38 to 39% amino acid identity with the F protein of respiratory syncytial virus (Pneumovirus genus) but only about half that with members of the other two genera (Paramyxovirus and Morbillivirus) in the Paramyxoviridae family. This is the first sequence evidence to support the view that TRTV is a pneumovirus, the first avian member of the genus to be described.

Amino Acid Sequence↗

Properties and evolutionary relationships of the Marek's disease virus homologues of protein kinase, glycoprotein D and glycoprotein I of herpes simplex virus.

The deduced amino acid sequences of the open reading frames (ORFs) mapping in the short unique segment (US) of Marek's disease virus (MDV) reported in the accompanying paper have been analysed using computer programs to determine their relationships to herpesvirus proteins. Analysis of the catalytic domains of protein kinases showed that the MDV kinase (MDV PK) was closely related to the alphaherpesvirus protein kinase mapping in US. The results also showed that the MDV PK was more closely related to the cellular kinases that control cell division than to the proto-oncogenes c-src and c-mos and it was predicted that the MDV PK would phosphorylate serine/threonine. The MDV homologue of herpes simplex virus (HSV) glycoprotein D (gD) contained several residues that were conserved in mammalian herpesviruses. In particular, six cysteines were perfectly aligned in all the gDs and there were numerous conservative substitutions. Although only approximately 65% of the MDV homologue of glycoprotein I (gI) of HSV has been sequenced, it was clear that a significant number of amino acid residues including four cysteines were conserved in the gI homologues of MDV and mammalian herpesviruses. Further analysis suggested that MDV gD was more closely related to the gDs of pseudorabies virus (PRV) and equine herpesvirus 1 than to the gD of HSV-1 and HSV-2. It was noted that HSV-2 glycoprotein G (gG), PRV gX and MDV gD were related and that MDV ORF4 was related to MDV gD and probably to HSV-1 gG. The results have shown a clear relationship between the genes of MDV and their counterparts in mammalian alphaherpesviruses and are consistent with the idea that MDV glycoprotein genes in US might have arisen by a process of gene duplication and independent evolution.

Amino Acid Sequence↗

DNA sequence and organization of genes in a 5.5 kbp EcoRI fragment mapping in the short unique segment of Marek's disease virus (strain RB1B).

The DNA sequence of a 5.5 kbp EcoRI fragment located in the short unique region (US) of the 'highly oncogenic' strain RB1B of Marek's disease virus (MDV) was determined. The sequence contained six open reading frames (ORFs), four of which were homologous to proteins mapping in the US region of herpes simplex virus type 1 (HSV-1). These include the homologues of HSV-1 protein kinase, glycoprotein D (gD), glycoprotein I (gI) and US2 which is of unknown function. The MDV ORFs had a marked bias for A or T in the third codon position and analysis of the dinucleotide frequencies showed a marginal deficit in ApG/CpT but no overall deviation of CpG from random expectations. Comparison of genes in the US region of MDV to herpesvirus proteins confirmed and extended our previous observation that MDV is more closely related to alphaherpesviruses than to gamma-herpesviruses. We also showed that MDV possessed a homologue of HSV-1 gD which is lacking in varicella-zoster virus (VZV) but that MDV probably lacked homologues of US4 and US5 of HSV-1. These results show that in contrast to the genes in the long unique region which were grossly collinear in HSV, VZV and MDV, those mapping in US show greater diversity.

Amino Acid Sequence↗

A recombinant fowlpox virus expressing the hemagglutinin-neuraminidase gene of Newcastle disease virus (NDV) protects chickens against challenge by NDV.

The hemagglutinin-neuraminidase (HN) gene from the Beaudette C strain of Newcastle disease virus (NDV) has been expressed in a recombinant fowlpox virus vector. The HN gene, under the control of the vaccinia p7.5 promoter, was inserted into a nonessential gene in the terminal inverted repeats of fowlpox virus. Expression was demonstrated in tissue culture, a protein of the correct size for fully glycosylated HN protein being recognized by an HN-specific monoclonal antibody on Western blots. When the recombinant fowlpox virus was inoculated into chickens by intravenous or wing-web routes, antibody which recognizes HN from purified NDV virions was produced. Protective immunity to NDV was generated in the chickens; at the highest dose of vaccine 100% of the chickens tested were protected against challenge with a virulent strain of NDV.

Animals↗

A fowlpox virus vaccine vector with insertion sites in the terminal repeats: demonstration of its efficacy using the fusion gene of Newcastle disease virus.

In this paper we report the development and testing of a fowlpox virus vector system. Insertion sites in non-essential regions within the terminal inverted repeats of the virus have been characterised. Foreign genes inserted into these sites are shown to be present in two copies in the resultant recombinant virus. To test the potential use of this vector as a live vaccine the fusion gene of Newcastle disease virus (NDV) has been inserted into a vaccine strain of fowlpox virus, and inoculated into chickens. The experiments demonstrate the ability of the recombinant to protect chickens against challenge by a virulent strain of NDV and to elicit the formation of anti-fusion protein antibody.

Animals↗

Identification of a new membrane-associated polypeptide specified by the coronavirus infectious bronchitis virus.

Nucleotide sequences from the third open reading frame of mRNA D (D3) of infectious bronchitis virus (IBV) were expressed in bacteria as part of a fusion protein with beta-galactosidase. Antiserum raised in rabbits against this fusion protein immunoprecipitated from IBV-infected chick kidney or Vero cells a polypeptide of 12.4K, the size expected for a D3-encoded product. The D3 polypeptide is apparently non-glycosylated, and appears to be associated with the membrane fraction of infected cells, as judged by cell fractionation and immunofluorescence.

Amino Acid Sequence↗

Analysis of the fowlpox virus genome region corresponding to the vaccinia virus D6 to A1 region: location of, and variation in, non-essential genes in poxviruses.

The DNA sequence of the fowlpox virus genome corresponding to the vaccinia virus D6 to A1 region has been determined. Translation of this sequence reveals fowlpoxvirus gene homologues corresponding to the D6, D7, D9, D10, D11, D12, D13 and A1 genes of vaccinia virus. In contrast, no gene homologue for the non-essential vaccinia virus D8 gene was present in fowlpox virus. Instead, a gene transcribed from the opposite strand to the vaccinia virus D8 gene showing no homology to any previously sequenced poxvirus gene was present. The amino terminus of the fowlpox virus D9 homologue had undergone substantial changes, including frameshifts which would be predicted to inactivate the gene. Insertion of a gene cartridge composed of the vaccinia virus p7.5 promoter and the lacZ gene into the fowlpox virus D8, D9 and D10 genes in vitro, followed by recombination into fowlpox virus, was carried out. Stable insertion mutants with the correct genotype were obtained for D8 and D9 which, when tested in chickens did not appear to have been attenuated. No stable insertion mutants were obtained for D10, indicating that this gene probably encodes a function which is essential for virus replication. The D8 and D9 genes of fowlpox virus represent useful insertion sites for the construction of recombinant fowlpox virus vaccines.

Amino Acid Sequence↗