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J P Clewley

Publications and source records attributed to J P Clewley.

At least 19 recordsLinked to original sources
PubMed1995

Sequence distances between env genes of HIV-1 from individuals infected from the same source: implications for the investigation of possible transmission events.

Previously described transmission studies have shown that HIV strains isolated from individuals infected from a common source are more homogeneous than HIV strains isolated from individuals with unrelated infections. This has been the basis, in at least four instances, for deciding whether apparently epidemiologically related cases represent actual transmissions. To date, HIV transmission studies have usually included sequence data from the most likely source of infection, and the probability of transmission from the donor to the recipient has been assessed by measuring sequence similarity against control data using likelihood analysis. We have recently studied a putative transmission involving a UK health care worker (CPHL1), a patient of CPHL1 (CPHL2), and CPHL3, a member of the same "sex circle" as CPHL2. We have used sequence distance and neighbour joining methods as well as likelihood analysis as means of determining genetic relatedness. Though no other source of infection was available our findings did not support the possibility that CPHL1 had infected CPHL2. Strain CPHL3 was closer to CPHL2 than to CPHL1. It is shown that control data from documented transmission events can be used to establish the source of infection in the absence of an index case. It is also shown that the C2-V3 region analysed in previous transmission studies is unreliable for accurate phylogenetic analysis. The results indicated that gp120 is a more informative region than C2-V3 for molecular transmission studies. Sequence distances between the env genes of related and unrelated infections have been derived in this work.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Genetic and antigenic variation in the haemagglutinin of recently circulating human influenza A (H3N2) viruses in the United Kingdom.

Variation in the haemagglutinin (HA) gene of influenza A (H3N2) viruses isolated in the U.K. and abroad from 1992-1994, was determined by nucleotide sequencing of the HA1 domain of the HA gene. Viruses isolated in the U.K. early in the 1992-93 season were from the A/Beijing/353/89 lineage and were replaced later that season by viruses from the A/Beijing/32/92 lineage. Viruses from the new lineage continued to be isolated during the 1993-94 season, but were heterogeneous. Most of these isolates were more closely related to an A/Beijing/32/92 variant, A Hong Kong/23/92, but could be distinguished into three groups by serology (of which one group was circulating during the previous season) and four groups based on sequence variation in the HA gene. However, phylogenetic analysis of antigenically-distinct isolates showed that the HA gene is evolving along one lineage. Sequence analysis identified mainstream, subgroup and strain specific amino acid substitutions. There was a broad correlation between the observed amino acid changes and the antigenic sites of the HA. The results of this study highlight the value of regular molecular analysis of circulating viruses.

Antigenic Variation↗
PubMed1995

Macintosh sequence analysis software. DNAStar's LaserGene.

The analysis of information in nucleotide and amino acid sequence data from an investigator's own laboratory, or from the ever-growing worldwide databases, is critically dependent on well planned and written software. Although the most powerful packages previously have been confined to workstations, there has been a dramatic increase over the last few years in the sophistication of the programs available for personal computers, as the speed and power of these have increased. A wide choice of software is available for the Macintosh, including the LaserGene suite of programs from DNAStar. This review assesses the strengths and weaknesses of LaserGene and concludes that it provides a useful and comprehensive range of sequence analysis tools.

Minicomputers↗
PubMed1995

Analysis and genotyping of PCR products of the Amplicor HIV-1 kit.

The Roche Amplicor PCR kit was used to detect HIV-1 DNA in UK patients of known serostatus. Four false-negative and/or equivocal results were obtained from patients who were known to be anti HIV seropositive (Tosswill et al., 1994). Cells from the blood of these patients were shown to contain HIV DNA after extraction, concentration and amplification by nested PCR using primers flanking those in the kit. To determine whether DNA sequence divergence was the cause of these discrepancies, the gag region targeted by the primers in the kit was sequenced for specimens giving positive, equivocal and false-negative results. No greater degree of sequence divergence was found within the primer and probe target regions among the equivocals and false-negatives than among the positive control specimens. The few misleading results were probably attributable to low copy numbers of proviral DNA in these specimens. Sequences obtained from the target and flanking regions of the kit were sufficient to allow the genotype of the virus to be determined.

Base Sequence↗
PubMed1995

Morphology and antigenicity of recombinant B19 parvovirus capsids expressed in transfected COS-7 cells.

COS-7 cells transfected with parvovirus B19-simian virus 40 (SV40) hybrid vectors have previously been shown to express B19 structural proteins. In this study the morphology and antigenicity of B19 proteins expressed in these cells were investigated. At 84 h after transfection, approximately 10% of the COS-7 cells expressed B19 antigen, and the yield was equivalent to 2 x 10(3) to 2 x 10(5) B19 particles/transfected cell. The B19 proteins self-assembled into capsids that were morphologically and antigenically similar to native B19 virions, and could substitute for native antigen in a B19 IgM assay. Recombinant capsids lacking the recently described 11 kDa protein also resembled native virions.

Animals↗
PubMed1995

A simple and sensitive DNA hybridization assay used for the routine diagnosis of human parvovirus B19 infection.

A dot blot hybridization assay for parvovirus B19 diagnosis was developed by using a PCR-generated probe, digoxigenin labelling, and chemiluminescence detection. Different labelling techniques and hybridization solutions were evaluated. From this analysis a protocol was devised for routine diagnostic use. The protocol enabled 1 pg of B19 DNA to be detected. The results of applying this method to 8,369 diagnostic samples collected during 1994 and 1995 are given.

Base Sequence↗
PubMed1994

An intervening sequence (IVS) in the 16S rRNA gene of the eubacterium Helicobacter canis.

PCR amplicons enlarged by approximately 250bp were generated from the 16S rRNA (rrs) genes of certain strains of the recently described Helicobacter species, H. canis. The DNA sequence of the rrs gene of one such strain was determined, and it was shown that an intervening sequence (IVS) of 235bp followed nucleotide 199 in the rrs sequence. In four other H. canis strains, identical or similar IVSs were found, always at the same location in the rrs gene. The secondary structures of the RNA transcripts of the IVSs were predicted. They were characterised by the presence of a conserved stem-loop structure, a potential recognition site for RNA processing enzymes. Ribosomal RNA was compared from a strain of H. canis with and without the IVS-containing rrs gene. In the former 16S rRNA appeared as two fragments, whose sizes were consistent with cleavage at either side of the IVS, and which were not subsequently religated. The IVS sequence was not represented elsewhere in the H. canis genome. Its evolutionary significance is discussed.

Base Sequence↗
PubMed1994

Polymerase chain reaction and sequencing for typing rhinovirus RNA.

Primers were designed and tested for their ability to distinguish rhinoviruses from enteroviruses. A primer set derived from the 5'-UTR/VP coding region junction was able to amplify all the rhinovirus serotypes tested. Enteroviruses were either not amplified by these primer pairs or produced a band of larger size that could easily be discriminated from the rhinovirus-specific product. In contrast, primers embedded in the 5'-UTR region alone were able to amplify both rhinovirus and enterovirus RNA. It is shown that rhinoviruses could be specifically typed by sequencing the amplicon derived from the 5'-UTR set. The sequences of the 5'-UTR region of ten previously unsequenced rhinoviruses were derived. The sequences obtained cluster into two groups: 1B, 41, 15, 30, 63, 31, 56, and 44; and 17, 69, and 70. Amplicons from serotypes 17,69, and 70 also group by sequence with the equivalent region of HRV14 from the genetic database, while the others group with 2 and 89.

Animals↗
PubMed1994

Two types of 16S rRNA gene are found in Campylobacter helveticus: analysis, applications and characterization of the intervening sequence found in some strains.

In the recently described species Campylobacter helveticus, two sizes of PCR amplicon were detected with primers homologous to conserved regions of the 16S rRNA gene. A conventionally sized gene was sequenced from the type strain, NCTC 12470, placing the new species as phylogenetically related to C. upsaliensis and the thermotolerant campylobacters. This nucleotide sequence enabled PCR primers to be designed for use in rapid molecular identification of C. helveticus and its closest phylogenetic relative, C. upsaliensis. When this assay was employed to characterize 22 'C. upsaliensis-like' isolates, twelve were identified as C. helveticus and nine as C. upsaliensis, in agreement with data obtained with a C. helveticus-specific DNA probe. A 550 bp amplicon internal to the 16S rRNA gene of C. helveticus was used to determine restriction fragment length polymorphisms (RFLPs) in genomic Southern blots, confirming that the copy number of the C. helveticus gene was three, and identifying nine 16S rRNA gene profiles. In 5/12 C. helveticus isolates identified by PCR, an enlarged amplicon was detected. The enlarged 16S rRNA gene of one of these strains, NCTC 12838, was sequenced and shown to contain an atypical intervening sequence (IVS) of 148 nucleotides. The position and size of such an IVS was inferred in the other four isolates by PCR with primers 5' and 3' to its position in NCTC 12838. This is a first report of an IVS in the 16S rRNA gene of a eubacterium.

Base Sequence↗
PubMed1993

BK virus antigenic variants: sequence analysis within the capsid VP1 epitope.

DNA sequences for the VP1 gene which codes for the major capsid protein of BK virus (BKV) and may be responsible for antigenic variability were determined for seven BKV isolates. The observed sequence differences and those previously reported correlate with the typing of isolates into four groups by haemagglutination inhibition. Amino acid coding alterations were found to be clustered within residues 61 to 83. Each antigenic group was found to have a characteristic amino acid sequence between residues 61 and 83. Several clones originating from a single isolate, although differing slightly in restriction enzyme digestion patterns, were found to be identical in this region. The VP1 sequences of three of the four groups were analysed by hydropathy plots and two hydrophilic areas of high antigenicity were identified. One of these corresponds to residues 61 to 83 and it is postulated that this region is the epitope responsible for serotypic differences between BK isolates.

Amino Acid Sequence↗
PubMed1993

Genomic typing of BK virus in clinical specimens by direct sequencing of polymerase chain reaction products.

Two hundred and twelve urine specimens, from several clinical groups, were examined for BK virus (BKV) using the polymerase chain reaction (PCR) to detect the VP1 region of BKV DNA. Positive results were obtained on 14 specimens from 44 post-transplant patients (31.8%), 10 specimens from 39 pregnant women (25.6%), and 5 specimens from 100 children (5%) but not on any specimens from 29 laboratory staff. Twenty-eight of the amplified BKV genomes, 19 from urine specimens, eight from culture fluid of inoculated tissue, and also one from a throat washing were directly sequenced from single-stranded templates immobilized via a biotinylated primer; it was possible to assign all to one of the four subtypes of BKV which had previously been identified on the basis of variation in nucleotide sequence of the VP1 region. Serological subgroup classification correlated with the genomic subtyping results in 21 of the isolates. The distribution of the BKV subtypes and the clinical status of the infected individuals are discussed.

BK Virus↗
PubMed1993

Polymerase chain reaction for detection of herpesvirus simiae (B virus) in clinical specimens.

A polymerase chain reaction (PCR) was designed which is specific to Macaca fascicularis (cynomolgus monkey) isolates of B virus. The PCR primers produced the expected 188 basepair product from the Cyno 2 strain and seven other cynomolgus monkey isolates of B virus. Oligomer hybridization with a 31-mer oligonucleotide was used to confirm the origin of this product. The PCR failed to amplify DNA of Epstein-Barr virus, cytomegalovirus, varicella-zoster virus, and other alphaherpesviruses (herpes simplex virus types 1 and 2, four SA 8 isolates and three rhesus isolates of B virus). PCR testing of swabs obtained from four orally-infected cynomolgus monkeys confirmed the presence of B virus DNA in samples previously shown to be positive by culture. In addition, PCR detected B virus in several swabs from infected monkeys that were culture negative. Total DNA extracts from the trigeminal and sacral ganglia of these animals were tested by nested PCR and B virus DNA was detected in the trigeminal ganglia of 3 of the 4 orally-infected cynomolgus monkeys. Nested PCR did not detect B virus DNA in total DNA extracts obtained from the brains of the four monkeys.

Animals↗
PubMed1993

Detection of HIV-1 in serum, using reverse transcription and the polymerase chain reaction (RT-PCR).

We have used a simple method for detecting HIV-1 in the serum of infected individuals using the polymerase chain reaction (PCR). This is useful if only serum, or other specimens which would not be expected to harbour proviral DNA, is available for diagnostic testing. Viral RNA present in serum is bound to silica particles in the presence of a high concentration of guanidinium thiocyanate (GuSCN) which denatures any proteins present, specifically ribonucleases. After washing the RNA/silica pellet, the RNA is eluted in water and reverse transcribed using random primers and Moloney murine leukaemia virus reverse transcriptase in the presence of a modified PCR buffer. The resultant cDNA is amplified using nested PCR and the products of amplification are detected by gel electrophoresis and ethidium bromide staining. The identity of bands on the gel is confirmed using a digoxigenin-labelled oligomer probe. The method is a general one applicable to amplification of any RNA species.

Base Sequence↗