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P Britton

Publications and source records attributed to P Britton.

At least 55 records · Page 3Linked to original sources
PubMed1995

Experimental evidence of recombination in coronavirus infectious bronchitis virus.

Embryonated eggs were coinfected with two strains of the coronavirus avian infectious bronchitis virus (IBV), IBV-Beaudette and IBV-M41, to investigate whether recombination between the two strains would occur. Virions were isolated from the allantoic fluid of the coinfected eggs and putative hybrid RNAs were detected by polymerase chain reaction (PCR), using strain-specific oligonucleotides. PCR products, of the expected sizes, were obtained as predicted from potential recombination events between the nucleoprotein (N) gene and the 3'-untranslated region of the two IBV genomes. Sequencing confirmed that they corresponded to hybrid RNAs. Virus produced as a result of the mixed infection was treated with an M41-specific neutralizing monoclonal antibody and passaged in Vero cells, in which IBV-Beaudette, but not IBV-M41, replicated. Hybrid RNA was still detectable after three serial passages. Since no IBV-M41 was detectable this confirmed that infectious recombinant genomes had been produced in the embryonated eggs. These findings not only support the circumstantial evidence, from sequencing studies of IBV field strains, that recombination occurs during replication of IBV and contributes to the diversity of IBV, but also show that coronavirus RNA recombination is not limited to mouse hepatitis virus.

Animals↗
PubMed1995

Effect of tetanus toxin on basal and evoked release of 5-hydroxytryptamine and dopamine in rat hippocampus in vivo.

Tetanus toxin is a potent neurotoxin which produces seizures and usually death in mammals. In the present study, we have investigated the effect of tetanus toxin on extracellular basal and evoked release of 5-hydroxytryptamine (5-HT), dopamine (DA) and their metabolites in vivo, 7 days after toxin injection into the hippocampus of rats. Tetanus toxin decreased both basal and evoked release of 5-HT and DA. The 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA) was not significantly decreased in the extracellular space. Of the DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) concentration was decreased while that of homovanillic acid (HVA) was unchanged. These findings are considered in light of the possible contributory role that a decrease in monoamine concentration in the hippocampus might have in the effects of tetanus toxin.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1995

Characterization of the transmissible gastroenteritis virus (TGEV) transcription initiation sequence. Characterization of TGEV TIS.

The ability of the TGEV transcription initiation sequence (TIS) to produce subgenomic RNAs was investigated by placing a reporter gene, chloramphenicol acetyltransferase (CAT) under the control of either the mRNA 6 or the mRNA 7 TISs. Both constructs only produced CAT in TGEV infected cells and the amount of CAT produced from the mRNA 7 TIS was less than from the mRNA 6 TIS. Mutations were made within and around the TISs and the effect on CAT production assayed. THe results showed that the TGEV TIS acted as a initiator of transcription for CAT, though the degree of base pairing between the TIS and leader RNA was not the only factor implicated in the control transcription.

Animals↗
PubMed1995

First experimental evidence of recombination in infectious bronchitis virus. Recombination in IBV.

A high frequency of recombination has been shown to occur during replication of the coronavirus mouse hepatitis virus (MHV) in vitro as well as in vivo. Although sequencing of field strains of coronavirus infectious bronchitis virus (IBV) has indicated that IBV strains also undergo recombination, there has been no experimental evidence to support this deduction. To investigate whether recombination occurs in IBV, embryonated eggs were coinfected with IBV-Beaudette and IBV-M41. Potential recombinants were detected by strain-specific polymerase chain reaction (PCR) amplifications, using oligonucleotides corresponding to regions in the 3' end of the genome. Sequencing of the PCR products confirmed that a number of recombinations had occurred between the two strains.

Animals↗
PubMed1995

Generation of a defective RNA of avian coronavirus infectious bronchitis virus (IBV). Defective RNA of coronavirus IBV.

The Beaudette strain of IBV was passaged 16 times in chick kidney (CK) cells. Total cellular RNA was analyzed by Northern hybridization and was probed with 32P-labeled cDNA probes corresponding to the first 2 kb of the 5' end of the genome, but excluding the leader, and to the last 1.8 kb of the 3' end of the genome. A new, defective IBV RNA species (CD-91) was detected at passage six. The defective RNA, present in total cell extract RNA and in oligo-(dT)30-selected RNA from passage 15, was amplified by the reverse transcription-polymerase chain reaction (RT-PCR) to give four fragments. The oligonucleotides used were selected such that CD-91 RNA, but not the genomic RNA, would be amplified. Cloning and sequencing of the PCR products showed that CD-91 comprises 9.1 kb and has three regions of the genome. It contains 1133 nucleotides from the 5' end of the genome, 6322 from gene 1b corresponding to position 12423 to 18744 in the IBV genome and 1626 from the 3' end of the genome. At position 749 one nucleotide, an adenine residue, was absent from CD-91 RNA. By Northern hybridization CD-91 RNA was detected in virions in higher amounts than the subgenomic mRNAs.

Animals↗
PubMed1995

Quantification of individual subgenomic mRNA species during replication of the coronavirus transmissible gastroenteritis virus.

A biotinylated-oligonucleotide-based method was used to isolate the subgenomic mRNAs of the coronavirus transmissible gastroenteritis virus (TGEV) to investigate the amounts of the mRNAs produced at early, middle and late times in the replication cycle. TGEV mRNA 6, which encodes the N protein, was observed to be the most abundant species throughout the replication cycle. The ratios of mRNA 6 to the other mRNAs were 1:0.11 (mRNA 2), 1:0.16 (mRNAs 3 and 4) and 1:0.37 (mRNA 5) at 12 h post-infection. All the mRNA species were differentially regulated throughout the replication cycle, although the rate of accumulation of mRNAs 4, 5 and 6, but not mRNA 3, increased markedly towards the end of the replication cycle. mRNA 7 was not detected in the system used. There was no observable correlation between the amounts of each mRNA synthesised and the potential degree of base pairing between the 3' end of the leader sequence and the transcription associated sequences on the genomic RNA at any time during the replication cycle. This indicates that the extent of base pairing was not the only factor involved in the control of subgenomic mRNA synthesis.

Animals↗
PubMed1995

Cloning and sequencing of a 8.4-kb region from the 3'-end of a Taiwanese virulent isolate of the coronavirus transmissible gastroenteritis virus.

The nucleotide sequence (8396 nucleotides) was determined, from the 3'-end of the putative polymerase gene to the poly(A) tail, for a Taiwanese virulent isolate, TFI, of transmissible gastroenteritis virus (TGEV). The TFI nucleotide sequence had very high identity to the British virulent field isolate FS772/70 (98.3%), the attenuated Purdue 115 (96.7%) and from the S gene to ORF-4 gene region, to the low passaged virulent Miller (98.3%) strains of TGEV. Comparison of the TFI S protein sequence with those determined from other TGEV strains and those of the TGEV variant, porcine respiratory coronavirus, isolated from Europe and North America showed that they had changed very little over a period of 4 decades. The two extra amino acids found to be present in the spike proteins of the virulent FS772/70 and Miller strains when compared to the avirulent Purdue strain were found to be present in the TFI strain. The genomic organisation of the TFI strain was the same as that of the other TGEV viruses.

Animals↗
PubMed1995

Liver transplantation for hepatic arteriovenous malformation in hereditary haemorrhagic telangiectasia.

Symptomatic hepatic involvement is a rare complication of hereditary hemorrhagic telangiectasia and most commonly consists of fibrosis or cirrhosis. We describe a 33-year-old woman in whom multiple hepatic arteriovenous malformations led to high output cardiac failure and liver failure due to biliary necrosis with refractory biliary sepsis, requiring orthotopic liver transplantation. Hepatic arteriovenous malformations were the first manifestation of the disease and a similar asymptomatic hepatic tumour was subsequently detected in her 60-year-old father who also had the classical cutaneous stigmata of the syndrome. Unrecognised genetic factors may determine the clinical spectrum of hereditary haemorrhagic telangiectasia including the hepatic manifestations.

Adult↗
PubMed1995

Investigation of the control of coronavirus subgenomic mRNA transcription by using T7-generated negative-sense RNA transcripts.

The subgenomic mRNAs of the coronavirus transmissible gastroenteritis virus (TGEV) are not produced in equimolar amounts. We have developed a reporter gene system to investigate the control of this differential subgenomic mRNA synthesis. Transcription of mRNAs by the TGEV polymerase was obtained from negative-sense RNA templates generated in situ from DNA containing a T7 promoter. A series of gene cassettes was produced; these cassettes comprised the reporter chloramphenicol acetyltransferase (CAT) gene downstream of transcription-associated sequences (TASs) (also referred to as intergenic sequences and promoters) believed to be involved in the synthesis of TGEV subgenomic mRNAs 6 and 7. The gene cassettes were designed so that negative-sense RNA copies of the CAT gene with sequences complementary to the TGEV TASs, or modified versions, at the 3' end would be synthesized in situ by T7 RNA polymerase. Using this system, we have demonstrated that CAT was expressed from mRNAs derived from the T7-generated negative-sense RNA transcripts only in TGEV-infected cells and only from transcripts possessing a TGEV negative-sense TAS. Analysis of the CAT mRNAs showed the presence of the TGEV leader RNA sequence at the 5' end, in keeping with observations that all coronavirus mRNAs have a 5' leader sequence corresponding to the 5' end of the genomic RNA. Our results indicated that the CAT mRNAs were transcribed from the in situ-synthesized negative-sense RNA templates without the requirement of TGEV genomic 5' or 3' sequences on the T7-generated negative-sense transcripts (3'-TAS-CAT-5'). Modification of the TGEV TASs indicated (i) that the degree of potential base pairing between the 3' end of the leader RNA and the TGEV negative-sense TAS was not the sole determinant of the amount of subgenomic mRNA transcribed and (ii) that other factors, including nucleotides flanking the TAS, are involved in the regulation of transcription of TGEV subgenomic mRNAs.

Animals↗
PubMed1995

Monitoring interval cancers in breast screening programmes: the east Anglian experience. Quality Assurance Management Group of the East Anglian Breast Screening Programme.

Interval cancer rates are a major determinant of the success of a screening programme. In the Swedish two county study, on which the United Kingdom programme is based, a 39% reduction in mortality was observed in screened women aged 50-64. Using data from the Swedish study, the relationship between interval cancer incidence and the likely future effect on breast cancer mortality was quantified. In East Anglia, as elsewhere in the United Kingdom, interval cancers rates are nearly double those obtained in Sweden: interval cancer rates in the first, second, and third years respectively, after a negative screen were 24%, 59%, and 79% of the expected underlying incidence in the absence of screening. The corresponding figures from the two county study were 17%, 30%, and 56%. From these it was estimated that the mortality reduction in East Anglia will be 21%, which is lower than the 35% observed in invited women in this age group in the Swedish two county study and the 25% specified in the Health of the Nation target. In a rereading exercise, using screening mammograms from women who were screen normal, who had screen detected cancers, or who subsequently developed interval cancers, four out of five radiologists recommended recall for around 70% of the original mammograms (classed as screen normal at time of screening) from 33 interval cancers. This suggests that sensitivity is a contributory factor to the higher interval cancer rates in East Anglia.

Aged↗
PubMed1994

Characterization of a replicating and packaged defective RNA of avian coronavirus infectious bronchitis virus.

The Beaudette strain of IBV was passaged 16 times in chick kidney cells. Total cellular RNA was analyzed by Northern hybridization and was probed with 32P-labeled cDNA probes corresponding to the first 2 kb of the 5' end of the genome, but excluding the leader, and to the last 1.8 kb of the 3' end of the genome. A new, defective IBV RNA species (CD-91) was detected at passage 6. The defective RNA, present in total cell extract RNA and in oligo-(dT)30-selected RNA from passage 15, was amplified by the reverse transcription-polymerase chain reaction (RT-PCR) to give four fragments. The oligonucleotides used were selected such that CD-91 RNA, but not the genomic RNA, would be amplified. Cloning and sequencing of the PCR products showed that CD-91 comprises 9.1 kb and has three regions of the genome. It contains 1133 nucleotides from the 5' end of the genome, 6322 from gene 1b corresponding to position 12,423 to 18,744 in the IBV genome, and 1626 from the 3' end of the genome. At position 749 one nucleotide, an adenine residue, was absent from CD-91 RNA. By Northern hybridization CD-91 RNA was detected in virions in higher amounts than the subgenomic mRNAs.

Base Sequence↗
PubMed1994

Cloning and expression of bovine and porcine interleukin-2 in baculovirus and analysis of species cross-reactivity.

The cDNAs encoding bovine and porcine interleukin-2 (IL-2) have been expressed using the baculovirus Autographa californica nuclear polyhedrosis virus as a vector in insect cells. Insect cells infected with recombinant viruses secreted bovine and porcine IL-2 into the culture medium, with biological activities for maintaining the proliferation of homologous cells. When the activities of these two IL-2 proteins and commercially available human IL-2 were tested on heterologous cells differences were found. Recombinant bovine (rb)IL-2 only supported the growth of bovine lymphocytes and was not active on human, mouse or porcine lymphocytes. Recombinant porcine (rp)IL-2 and recombinant human (rh)IL-2 supported the proliferation of human, bovine, porcine and murine cells. However, the proliferative response of human lymphocytes to rpIL-2 was only 50% of that seen with rhIL-2. Sequence differences at the predicted p55 and p75 contact binding sites may explain this.

Amino Acid Sequence↗
PubMed1993

Control of TGEV mRNA transcription.

Coronavirus proteins are translated from a nested set of subgenomic mRNAs which have common 3' termini with unique 5' extensions. Evidence suggests that coronavirus mRNAs are generated by a mechanism of leader primed transcription. Leader RNA binds to consensus sequences upstream of each gene on full length negative strand viral RNA and transcription proceeds to the 5' end of the negative strand to produce the nested set of mRNAs. Even though this gives rise to polycistronic mRNA species only the 5' extension of each mRNA is translated to give the viral proteins. The leader RNA for TGEV is about 90 nucleotides long and contains the sequence which recognises the leader binding sites on the negative strand RNA. Evidence suggests that the length of the leader binding sequence may be involved in transcriptional control of individual mRNAs. In order to investigate this a virus specific mRNA isolation method was developed to measure the relative amounts of mRNAs synthesised during an infection of LLC-PK1 cells with TGEV (strain FS772/70). Thus the relative quantity of each mRNA can be determined and correlated with the variation in size of the leader binding site.

Animals↗
PubMed1993

Genomic organisation of a virulent Taiwanese strain of transmissible gastroenteritis virus.

Transmissible gastroenteritis (TGE) infection causes 65% of infectious piglet diarrhoea in Taiwan. A virulent Taiwanese strain, TFI, of transmissible gastroenteritis virus (TGEV) from a field outbreak was isolated in cell culture and plaque purified. Phenotypic differences were observed in the ability of TFI to infect certain cell lines. TGEV strains TLM-83 (PRCV Belgium), TO-163 (TGEV Japan) and Purdue-115 (TGEV USA) infected both ST (swine testis) and RPTG (pig kidney) cell lines whereas TFI infected ST but not RPTG cells. To investigate this phenotypic variation cDNA was generated from TFI genomic and amplified by PCR with oligonucleotides derived from published TGEV sequence data. An 8.4kb cDNA derived from the 3'-end of the TFI genome was sequenced. Eight ORFs, corresponding to the three structural protein genes, four potential genes and the 3'-end of an incomplete ORF whose amino acid sequence corresponded to the carboxyl end of the 1b subunit of the polymerase gene, were identified on the TFI sequence. The overall sequence similarity of TFI with the other TGEV strains was over 97%. However, several deletions, insertions and point mutations were found on the TFI sequence when compared with other TGEV strains. The TFI S protein was found to contain 1449 amino acids, as also identified for the FS772/70 and Miller TGEV strains, but two amino acids longer than the Purdue S protein. The TFI ORF-3a gene encodes 72 amino acids, however, a 37 nucleotide deletion was found 16 nucleotides downstream of the TFI ORF-3a stop codon.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

The use of PCR genome mapping for the characterisation of TGEV strains.

Previous studies on different transmissible gastroenteritis virus (TGEV) strains, including porcine respiratory coronavirus (PRCV), have identified regions within the genome that are polymorphic as regards insertions and deletions. For example the 672 base deletion within the S gene and multiple deletions 5', within and 3' of the ORF-3a gene were detected in strains of PRCV. The presence of deletions may be associated with a change in the virulence, attenuation or tissue tropism of the isolate. The Nouzilly (188-SG) TGEV vaccine strain was attenuated by passage of a cell culture adapted virulent isolate D-52 188 times through swine testis cells after treatment with gastric juice. PCR amplification with oligonucleotides, corresponding to known TGEV sequences, were used to analyse D-52 and 188-SG for genetic variation. Results with several pairs of oligonucleotides within the first 1565 nucleotides of the S gene did not identify a deletion within this region of the genome from either strain. However, oligonucleotides directed against the ORF-3a/3b region detected a deletion of about 250 nucleotides within the 188-SG genome but not in the D-52 genome. Since all the attenuated TGEV strains so far sequenced, PRCV, Miller SP and 188-SG, contained deletions within the ORF-3a/3b, it would suggest that this region of the TGEV genome is involved in regulating viral virulence.

Amino Acid Sequence↗
PubMed1992

Tetanus toxin alters 5-hydroxytryptamine, dopamine, and their metabolites in rat hippocampus measured by in vivo microdialysis.

Tetanus toxin is a potent neurotoxin, the mode of action of which is unknown, although it is widely considered to be mediated by loss of inhibitory neurotransmission. In the present study we have investigated the effect of a single unilateral injection of tetanus toxin in the hippocampus on extracellular concentrations of 5-hydroxytryptamine (5-HT), dopamine and their metabolites in freely moving rats. Tetanus toxin treatment caused a progressive decline in hippocampal dialysate 5-hydroxytryptamine over the 3-day period following the injection. After 3 days the level of 5-HT on the injected side was only 20% of the control side. On the second day of observation the level of dopamine was found to be elevated by more than 2-fold but this was not the case for either of the other 2 days studied. The 5-hydroxytryptamine metabolite, 5-hydroxyindoleacetic acid declined in a manner similar to that of the parent transmitter, but to a lesser extent. Of the dopamine metabolites, 3,4-dihydroxyphenylacetic acid was decreased on the second and third days of observation, whilst homovanillic acid was not significantly altered during the period of observation.

3,4-Dihydroxyphenylacetic Acid↗