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I N Clarke

Publications and source records attributed to I N Clarke.

At least 37 records · Page 2Linked to original sources
PubMed1997

The genomic 5' terminus of Manchester calicivirus.

An enteric calicivirus showing the classic cup-shaped surface morphology was identified in a stool sample obtained from a child with symptoms of acute gastroenteritis (Portishead virus, PHV). Genomic RNA was extracted directly from the PHV stool sample and amplified by RT-PCR using primers based on the Manchester isolate of HuCV. The 3' terminus of the cDNA was defined by homopolymer tailing with dATP and revealed an additional 165 nucleotides suggesting that the previously determined Manchester HuCV (MV) genome sequence was incomplete. Homopolymer tailing of MV cDNA primed using sequence data from the 5' terminus of PHV allowed extension of the MV genome by a further 165 nucleotides thereby increasing the overall genome length to 7431 nucleotides and resulting in an additional 72 amino acids at the N-terminus of the polyprotein. A conserved sequence motif typical of other caliciviruses was also identified at the extreme 5'-terminus of the genome.

Amino Acid Sequence↗
PubMed1997

Plasmid diversity in Chlamydia.

Chlamydiae exhibit low interspecies DNA homology and plasmids from different chlamydial species can be readily distinguished by Southern blot analysis and restriction enzyme profiling. In contrast, available plasmid sequence data from within the species Chlamydia trachomatis indicate that plasmids from human isolates are highly conserved. To evaluate the nature and extent of plasmid variation, the complete nucleotide sequences were determined for novel plasmids from three diverse non-human chlamydial isolates: pCpA1 from avian Chlamydia psittaci (N352); pCpnE1 from equine Chlamydia pneumoniae (N16); and pMoPn from C. trachomatis mouse pneumonitis. Comparison of the sequence data did not identify an overall biological function for the plasmid but did reveal considerable sequence conservation (> 60%) and a remarkably consistent genomic arrangement comprising eight major ORFs and four 22 bp tandem repeats. The plasmid sequences were close to 7500 nucleotides in length (pCpA1, 7553 bp; pMoPn, 7502 bp) however the equine C. pneumoniae plasmid was smaller (7362 bp) than all other chlamydial plasmids. The reduced size of this plasmid was due to a single large deletion occurring within ORF 1; this potentially generates two smaller ORFs. The disruption of ORF 1 is the only significant variation identified amongst the chlamydial plasmids and could prove important for future vector development studies.

Amino Acid Sequence↗
PubMed1997

Capsid sequence diversity in small round structured viruses from recent UK outbreaks of gastroenteritis.

Genetic typing of small round structured viruses (SRSVs) by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing has been confined to analysis of the RNA polymerase because of the considerable genome variability outside of this region. To provide capsid sequence data for epidemiological studies and outbreak investigations, a broadly reactive capsid PCR was developed using two sets of degenerate, inosine-containing primers. Primer pairs Capla/Caplb and Caplla/Capllb specifically amplify a 223-bp region of the SRSV capsid open reading frame from SRSV genetic groups I and II, respectively. The capsid PCR was used to investigate SRSVs from nine UK outbreaks of gastroenteritis occurring between 1992 and 1995. Differential amplification by the primer pairs suggested that three strains belonged to genetic group I and six to genetic group II. The capsid amino acid sequences of the group I strains were 75.9% to 79.3% identical with Sot/91/UK (group I), while those of the group II strains were 75.9% to 98.3% identical with Bri/93/UK (group II). Phylogenetic comparison of the capsid region from the outbreak strains and 13 previously characterised SRSVs revealed clusters of strains closely related to Bri/93/UK and Tor/77/C within genetic group II. With the exception of some Bri/93/UK-like strains, there was no correlation between capsid sequence and the geographical origin of SRSVs. UK strains were found with greater than 90% capsid sequence identity to SRSVs from various locations worldwide including Australia (Cam/94/A), Canada (Tor/77/C), Hawaii (Haw/71/US), and Saudi Arabia (DSV395/90/SA) together with group I (B447/92/UK) and group II (Yat/94/UK) strains that were genetically distinct from known SRSV capsids. Three SRSVs very closely related to Bri/93/UK were from recent UK hospital outbreaks. These Bri/93/UK-like strains appear to be prevalent in the UK.

Amino Acid Sequence↗
PubMed1997

Parkville virus: a novel genetic variant of human calicivirus in the Sapporo virus clade, associated with an outbreak of gastroenteritis in adults.

This report describes the characterization of Parkville virus, the etiologic agent of an outbreak of foodborne gastroenteritis, that has the morphology of a calicivirus and genetic properties that distinguish it from previously identified strains in the Sapporo/Manchester virus clade. Sequence analysis of the Parkville virus genome showed it contained the RNA-dependent RNA polymerase motifs GLPSG and YGDD characteristic of members of the family Caliciviridae with an organization identical to that reported for the Manchester virus where the capsid region of the polyprotein is fused to the RNA polymerase. Parkville virus however, demonstrates considerable sequence divergence from both the Manchester and Sapporo caliciviruses, providing the first indications that genetic diversity exists within caliciviruses of this previously homogeneous clade. On the basis of recent advances in the genetic characterization of members of the family Caliciviridae, we propose a new interim phylogenetic classification system in which Parkville virus would be included with Manchester and Sapporo virus as a separate group distinct from the small round-structured viruses (Norwalk-like viruses) that also cause diarrhea in humans.

Adult↗
PubMed1996

Expression of Neisseria meningitidis class 1 porin as a fusion protein in Escherichia coli: the influence of liposomes and adjuvants on the production of a bactericidal immune response.

High level expression of meningococcal class 1 protein was achieved in Escherichia coli using the p-GEMEX-1 vector, in which the protein was expressed in inclusion bodies (IB), as a fusion with the bacteriophage T7 gene 10 capsid protein. The fusion protein (FP) was engineered with a factor Xa protease site between the gene 10 and class 1 protein, but treatment with the enzyme resulted in cleavage at additional sites within the class 1 protein. Since it was not possible to remove the leader protein, the intact FP provided an alternative antigen for immunization. Antisera raised to FP, solubilized from IB and incorporated into liposomes, generated a subtype-specific response which was weakly bactericidal for meningococci. In order to remove any possible effect of E. coli LPS present in IB, the FP was further purified by SDS-PAGE and incorporated into liposomes, either alone or in combination with the adjuvants monophosphoryl lipid A or muramyl dipeptide. The incorporation of adjuvants in liposomes resulted in stimulation of the overall immune response to FP, but the resulting antisera were not bactericidal. However an effective bactericidal response was obtained with the purest preparation of FP in liposomes, without any additional adjuvants, revealing that attempts to increase further the immunogenicity of such antigens must not be at the expense of interfering with optimal protein folding.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1996

The VP3 gene of human group C rotavirus.

The complete nucleotide sequence of genome segment 4 from the human group C rotavirus (Bristol strain) was determined. Comparison of the nucleotide sequences of the genome termini with the consensus 5' and 3' terminal non-coding sequences of the human group C rotavirus genome revealed characteristic 5' and 3' sequence motifs. Human group C rotavirus genome segment 4 is 2,166bp long and encodes a single open reading frame of 2,082 nucleotides (693 amino acids) starting at nucleotide 55 and terminating at nucleotide 2,136 giving a 3' untranslated region of 30 nucleotides. Alignment with the porcine group C VP3 equivalent gene showed the human gene is one amino acid longer, and that the proteins have 84.1% amino acid sequence identity. A conserved potential nucleotide binding motif shared with the porcine VP3 sequence was identified. Analogy with the group A rotaviruses suggested that the genome segment 4 encodes the group C rotavirus guanylyltransferase.

Amino Acid Sequence↗
PubMed1996

Polyprotein processing in Southampton virus: identification of 3C-like protease cleavage sites by in vitro mutagenesis.

A genomic clone of the small, round-structured virus Southampton virus (SV) was constructed from a set of overlapping PCR amplicons. Sequence analysis confirmed the absence of mutations and accurate ligation of the PCR products. The SV cDNA was cloned into a vector for in vitro production of RNA and subsequent translation by rabbit reticulocyte lysate. Two polypeptides corresponding to the N-terminal and C-terminal regions of the viral polyprotein were expressed in Escherichia coli and used to produce murine antisera for detection of translation products. Three major translation products of 113, 48, and 41 kDa were identified in a coupled transcription-translation system. The large 113-kDa protein reacted with antisera raised against the C-terminal region of the polyprotein and represents a precursor of the viral RNA polymerase. The 48-kDa protein detected in vitro reacted specifically with antisera raised against the polyprotein N terminus, showing that translation was initiated in SV at the three tandem in-frame AUG codons at the 5' end of the genome. A series of nested 3' deletions of the large open reading frame encoding the viral polyprotein was used to define the translation initiation site and genomic location of the viral protease. The results are consistent with a model in which translation of the viral genome is initiated at one of the three in-frame AUG codons starting at nucleotide position 5 and in which active viral protease is produced following translation of a region located between NheI (nucleotide 3052) and SphI (nucleotide 4056), resulting in rapid cleavage of a large precursor protein. Abolition of the viral 3C-like protease activity by site-directed mutagenesis of the putative active-site cysteine (Cys-1238) resulted in production of a large protein of approximately 200 kDa which reacted with both N-terminal and C-terminal antisera. Two potential polyprotein cleavage sites containing the preferred picornaviral QG recognition site were identified on either side of the putative 2C-like helicase region of the polyprotein. Proteolysis at these positions would give rise to products with relative molecular masses identical to those of the products detected in the rabbit reticulocyte system. Site-directed mutagenesis was used to introduce a single base change which resulted in the substitution of glutamine residues with proline residues at amino acids 399 and 762. These mutations completely abolished cleavage of the polyprotein at these positions and gave rise to alternative products with molecular masses which matched the predicted sizes for a single cleavage at either Q-399 or Q-762. These data indicate that the small, round-structured virus Southampton virus produces a 3C-like protease which has two primary cleavage sites at positions 399 and 762. Proteolytic cleavage at these positions releases the putative viral 2C-like helicase.

Amino Acid Sequence↗
PubMed1995

Polymerase chain reaction detection of small round-structured viruses from two related hospital outbreaks of gastroenteritis using inosine-containing primers.

Two outbreaks of gastroenteritis in the UK which occurred nine days apart at Lymington and Southampton hospitals were investigated. The clinical and epidemiological features of both outbreaks were characteristic of small round-structured virus (SRSV) infection with rapid onset of diarrhoea and/or nausea and vomiting and propagation of the outbreaks by secondary spread. SRSV particles were observed by immune electron microscopy (EM) in 60% of faecal samples from both outbreaks and no other pathogens were detected. The index case for the second outbreak was a patient who was admitted with diarrhoea and vomiting after being discharged from Lymington hospital during the first outbreak. The possibility that the two outbreaks were caused by the same strain of SRSV was investigated by the polymerase chain reaction (PCR). New inosine-containing PCR primers were designed to amplify the RNA polymerase region of SRSV cDNA from genetic groups I and II. The PCR using the group II primers achieved a higher detection rate for SRSVs in faecal samples (68% of samples positive from both outbreaks) than immune EM. SRSVs were not detected using the group I primers or using conventional degenerate PCR primers. The nucleotide sequences of PCR amplicons from both outbreaks were identical providing molecular epidemiological evidence for the involvement of a single SRSV strain. Comparison of the RNA polymerase region of this virus with the equivalent regions of genetic group I (69.4-75.0% amino acid identify) and genetic group II (88.9-100% amino acid and 77.1-88.1% nucleotide identity) SRSVs revealed that the causative SRSV was a distinct member of genetic group II.

Amino Acid Sequence↗
PubMed1995

Human enteric caliciviruses have a unique genome structure and are distinct from the Norwalk-like viruses.

Classic human enteric caliciviruses (HuCVs) have a distinctive morphology and are primarily associated with pediatric acute gastroenteritis. Although morphologically distinct from the small round structured viruses (SRSVs), the classic HuCVs are thought to be closely related and were anticipated to have a similar genome organisation. We report the first genome sequence and molecular characterisation of a classic human enteric calicivirus associated with a case of acute vomiting and diarrhoea in an infant. The RNA genome (7266 nt) is smaller than the genome of SRSVs from the two genetic groups and has a unique arrangement of open reading frames. Further analysis of the 3' terminal 3 kb from a second unrelated isolate confirmed this genomic organisation. Analysis of capsid and RNA polymerase sequences together with the unique genomic organisation of classic HuCV suggest these viruses are more closely related to the animal caliciviruses than the enteric SRSV group of viruses.

Amino Acid Sequence↗
PubMed1995

Molecular characterization of the 11th RNA segment from human group C rotavirus.

The complete nucleotide sequence of genome segment 11 from the noncultivatable, human group C rotavirus (Bristol strain) was determined. Comparison of the nucleotide sequence of the segment termini with the consensus 5' and 3' terminal noncoding sequences of the human group C rotavirus genome revealed characteristic 5' and 3' sequences. Human group C rotavirus genome segment 11 is 613 bp long and encodes a single open reading frame of 450 nucleotides (150 amino acids) starting at nucleotide 39 and terminating at nucleotide 489, leaving a long 3' untranslated region of 124 nucleotides. The predicted translation product has a calculated molecular weight of 17.7 kD and contains four potential N-linked glycosylation sites. No significant homologies to other viral proteins were found in database searches. Hydropathy analysis predicted the human group C rotavirus genome segment 11 translation product has a hydrophilic carboxy terminus (amino acids 54-150) and a hydrophobic amino terminus (amino acids 1-53) that can be further subdivided into three short hydrophobic sequences--H1, H2, and H3. These features are analogous to the integral membrane glycoprotein NSP4 encoded by group A rotavirus gene 10.

Amino Acid Sequence↗
PubMed1995

A conserved sequence motif at the 5' terminus of the Southampton virus genome is characteristic of the Caliciviridae.

We have determined the 5'terminal cDNA sequence for the genome of Southampton virus, a recently characterized, human, small round-structured virus (SRSV). Genomic RNA was extracted directly from a stool sample and amplified by RT-PCR by homopolymer tailing of the 3' terminus of the cDNA. The additional sequence increases the overall length of the Southampton virus genome by 12 nucleotides, resulting in a significant change to the genome organization by extending the first large open reading frame (ORF) by 51 amino acids. The 5'terminal bases pGpT and the presence of conserved genome and putative subgenomic RNA terminal motifs are now prominent features shared between the human SRSV Southampton virus and the animal caliciviruses rabbit hemorrhagic disease virus and feline calicivirus.

Amino Acid Sequence↗
PubMed1995

Capsid diversity in small round-structured viruses: molecular characterization of an antigenically distinct human enteric calicivirus.

Studies of antigenic variation between small round-structured viruses (SRSVs) using immune electron microscopy have revealed 3 antigenic types currently circulating in the UK represented by the strains SRSV/Bri/93/UK, SRSV/Sot/91/UK and SRSV/Mel/89/UK. Mel/89/UK RNA was isolated from a 1989 school outbreak of gastroenteritis. The 3'-terminal 3435 nucleotides (excluding the poly(A) tail) were determined by RT-PCR and cDNA sequencing, completing our molecular characterization of antigenically diverse SRSVs. Coding regions for the calicivirus RNA polymerase and capsid protein were found together with a 3' open reading frame of unknown function. The polymerase region was most highly conserved between Mel/89/UK and the other two SRSVs while the 3' open reading frame exhibited extreme variation. Phylogenetic analysis of SRSV capsids showed that Mel/89/UK differed significantly from Bri/93/UK and Sot/91/UK (62 and 39% identity, respectively) and was distinct from 6 other non-UK SRSVs that had been previously characterized. This was consistent with the designation of Mel/89/UK as a novel antigenic variant. Comparison of the capsid amino acid sequences of the 3 UK strains together with the antigenically distinct SRSV/Nor/68/US revealed a hypervariable region that could be surface-exposed and contain the SRSV antigenic determinants.

Amino Acid Sequence↗
PubMed1995

Genome organization in the caliciviridae.

Caliciviruses cause a wide spectrum of important diseases. These viruses have a positive-sense single-stranded RNA genome; recently, the complete genome sequences of several caliciviruses have been determined. This review outlines the genome organization and phylogenetic relationships of the animal and candidate human caliciviruses.

Animals↗
PubMed1995

Human enteric Caliciviridae: the complete genome sequence and expression of virus-like particles from a genetic group II small round structured virus.

Comparisons of the RNA polymerase and capsid sequences of small round structured viruses (SRSVs) have recently shown these are genetically diverse viruses which fall into two distinct groups. The genomes of two group I viruses, Southampton and Norwalk viruses have been characterized; however, similar data for the genetic group II SRSVs have not been available until now. We report here the complete genome sequence of a recent group II SRSV, Lordsdale virus. The Lordsdale virus genome is 7555 nt in length and has a similar organization to the group I SRSVs. The large ORF in the 5' half of the genome (5100 nt) is shorter than the group I SRSV ORF1 (5367 nt), but has the characteristic 2C helicase, 3C protease and 3D RNA polymerase enzyme motifs. ORF2, encoding the structural protein is of a similar size to the group I viruses but the small 3'-terminal ORF is significantly larger in group II. A highly conserved sequence of 28 nt was identified at the start of Lordsdale virus ORF1 and repeated at the start of ORF2. These conserved motifs are typical of the animal caliciviruses. Comparison of the 150 N-terminal amino acids in the ORF1 protein revealed little identity between the two SRSV genetic groups, reflecting the shorter ORF1 in the group II virus. Recombinant baculoviruses containing ORF2 and ORF3 sequences were constructed and used to express large quantities of the group II Lordsdale virus structural protein. The capsid protein formed virus-like particles by self assembly which resembled 'empty' SRSVs.

Amino Acid Sequence↗
PubMed1995

The CrP operon of Chlamydia psittaci and Chlamydia pneumoniae.

One of the critical developmental events during the unique intracellular life cycle of Chlamydiae is their differentiation from a metabolically active, replicative form or reticulate body (RB) to an infectious extracellular form of the organism (elementary body or EB). This process is characterized by the expression of two extraordinarily cysteine-rich envelope proteins of molecular masses 9 kDa and 60 kDa. We describe the molecular cloning and sequence determination of the 9 kDa cysteine-rich proteins (CrPs) of C. pneumoniae and C. psittaci. Comparison of these 9 kDa CrP amino acid sequences with those of C. trachomatis showed regions of structural variation and conservation. Transcription of the 9 kDa CrP genes occurred as both a monocistronic message and as a bicistronic message which included the 60 kDa CrP gene. Transcription of the 9 kDa and 60 kDa CrP genes was tightly linked to the chlamydial growth cycle with synthesis of their mRNAs and consequent translation of the 60 kDa CrPs occurring as RBs differentiated to form EBs. The maximal rate of transcription occurred late in the growth cycle from a single but highly conserved promoter which had close similarity with the Escherichia coli consensus promoter sequences. A stem and loop structure which could be involved in regulating translation of mRNA occurred in all three species between the transcriptional start point and the ribosome binding site. Although transcription is initiated from a single promoter in all three chlamydial species, transcriptional termination points for the monocistronic and bicistronic mRNAs differ in both number and position.

Amino Acid Sequence↗
PubMed1994

Sequence conservation of the major outer capsid glycoprotein of human group C rotaviruses.

Several outbreaks of Group C rotavirus infection have occurred in the United Kingdom, in one instance infection was associated with the death of a 4-month-old infant in the Bristol area. The origin of human group C rotavirus is unknown although there has been some speculation that porcine species may be a possible source of human infection. Direct reverse transcription-polymerase chain reaction sequencing of VP7 genes from two UK outbreaks (Bristol and Preston) and sequence analysis from a sporadic case of infection from Brazil (Belém) showed that each of these genes was identical in size (1,063 bp) and has revealed a surprising level (97.8-99.8%) of gene sequence conservation. Sequence comparisons with an isolate from Japan imply that the human group C rotaviruses so far characterised originate from a recent common ancestor with a worldwide distribution.

Amino Acid Sequence↗