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G Stanway

Publications and source records attributed to G Stanway.

At least 37 records · Page 2Linked to original sources
PubMed1994

Molecular and biological characteristics of echovirus 22, a representative of a new picornavirus group.

Recent sequence analysis revealed that the human pathogen echovirus 22 (EV22) is genetically distant from all the other picornaviruses studied to date (T. Hyypiä, C. Horsnell, M. Maaronen, M. Khan, N. Kalkkinen, P. Auvinen, L. Kinnunen, and G. Stanway, Proc. Natl. Acad. Sci. USA 89:8847-8851, 1992). We have further characterized the biological properties of the virus and show here that the virion has properties similar to those of other picornaviruses. However, the protein composition is unique, in that most copies of one of the three major capsid proteins, VP0, do not undergo the further processing to VP2 and VP4 observed during the maturation of the virus in previously studied picornaviruses. Alignment of the capsid protein sequences with those of other picornaviruses revealed, furthermore, that the VP3 polypeptide contains an apparent insertion of approximately 25 amino acids at its amino terminus. An arginine-glycine-aspartic acid (RGD) motif is found in VP1, and by using synthetic peptides, it was shown that this sequence plays a role in cell surface receptor recognition. Finally, EV23 was shown to share remarkable identity with EV22 in certain parts of the genome and also belongs to this previously unrecognized picornavirus group.

Amino Acid Sequence↗
PubMed1993

Preliminary crystallographic analysis of coxsackievirus A9.

Coxsackievirus A9 has been crystallized as small rhombic dodecahedra of maximum dimension 0.3 mm. These crystals have been shown, using synchrotron radiation, to diffract X-rays to beyond 3 A, and to have a stability in the beam comparable to that of other related virus crystals. The unit cell is tetragonal with dimensions a = b = 495 A, c = 695 A and alpha = beta = gamma = 90 degrees, with a space group of P4n22. A substantial body of diffraction data has been collected and this crystal form appears to be suitable for structure determination. Phasing of these data will be attempted using molecular replacement.

Animals↗
PubMed1993

Pathogenetic differences between coxsackie A and B virus infections in newborn mice.

Coxsackieviruses are divided into A and B subgroups on the basis of their pathogenicity in newborn mice. Although used in the classification of these viruses, our understanding of the details of the infection is incomplete due to the lack of sensitive and specific techniques to localize the viruses in affected tissue. We have used in situ hybridization to detect coxsackievirus genomes in tissues of newborn mice after infection by five serotypes (A2, A9, A21, B3 and B4) through different administration routes. Our results indicate that coxsackie A viruses are able to affect both skeletal and heart muscle while the coxsackievirus B subgroup infects a wide range of tissues. In addition to striated muscle these include central nervous system, liver, exocrine pancreas and brown fat. This model will make it possible to analyze molecular factors determining tissue tropism.

Animals↗
PubMed1992

A distinct picornavirus group identified by sequence analysis.

Although echovirus 22 is presently classified as a member of the enterovirus group in the family of picornaviruses, it has been reported to have exceptional biological properties when compared with other representatives of the group. We have determined the complete nucleotide sequence of the echovirus 22 (Harris strain) genome, which appears to be significantly different from all the other studied picornaviruses. However, the organization of the genome [7339 nucleotides, excluding the poly(A) tract] is similar to that of previously sequenced picornaviruses. This genome includes a 5' untranslated region, relatively well-conserved when compared with aphtho- and cardioviruses, followed by an open reading frame coding for a 2180-amino acid-long polyprotein. The amino termini of capsid polypeptides VP1 and VP3 were determined by direct sequencing, and the other proteolytic cleavage sites in the polyprotein were predicted by comparison with other picornavirus proteins. The amino acid identities of echovirus 22 polypeptides with the corresponding proteins of other picornaviruses are in the 14-35% range, similar to those percentages seen when representatives of the five picornavirus groups (entero-, rhino-, cardio-, aphtho-, and hepatoviruses) are compared. Our results suggest that echovirus 22 belongs to an independent group of picornaviruses.

Amino Acid Sequence↗
PubMed1992

The nucleotide sequences of wild-type coxsackievirus A9 strains imply that an RGD motif in VP1 is functionally significant.

We have shown previously that, compared to other enteroviruses, the coxsackievirus A9 (CAV-9) prototype strain, Griggs, contains a C-terminal extension to the capsid protein VP1 and that within this extension there is an RGD (arginine-glycine-aspartic acid) motif. To determine whether these features are found in other CAV-9 strains and therefore analyse whether they are likely to be functionally important, we have determined the nucleotide sequence of the appropriate region from five strains, isolated over a 25 year period. The results indicate that there is considerable diversity between the strains and there is little correlation between nucleotide sequence identity and date of isolation. All isolates exhibit the VP1 extension and although its amino acid sequence is otherwise variable, the RGD motif is common to all. This conservation of sequence, within a region which can otherwise vary, implies that the RGD sequence must be functionally significant. The VP1 extension shows similarity to sequences found in foot-and-mouth-disease virus strains and to part of the precursor of the cellular protein, human transforming growth factor beta, and the possible significance of these observations is discussed.

Amino Acid Sequence↗
PubMed1991

RGD-dependent entry of coxsackievirus A9 into host cells and its bypass after cleavage of VP1 protein by intestinal proteases.

The recently reported nucleotide sequence of coxsackievirus A9 (CAV-9) showed that unlike other enteroviruses, CAV-9 has an insertion of about 17 amino acids at the C-terminal end of VP1 (K. H. Chang, P. Auvinen, T. Hyypiä, and G. Stanway, J. Gen. Virol. 70:3269-3280, 1989). This sequence includes the RGD (arginine-glycine-aspartic acid) motif which is known to be important in certain protein-protein interactions. We studied the inhibitory effect of RGD-containing peptides in the attachment of CAV-9 to African green monkey kidney cells. A peptide corresponding to the RRGDM sequence derived from the inserted segment of CAV-9 was found to block virus attachment effectively, and the inhibition was dose dependent. Substitution of glutamic acid for the homologous aspartic acid completely abolished the inhibitory effect, indicating great specificity of the action. During replication in the gut, all enteroviruses are exposed to host proteolytic enzymes. Exposure of CAV-9 to purified trypsin or human intestinal fluid resulted in selective cleavage of the VP1 capsid protein. Intact and trypsin-cleaved VP1 proteins gave identical N-terminal sequences, indicating that cleavage of VP1 takes place near the C terminus. Attachment of proteolytically cleaved infectious CAV-9 to green monkey kidney cells was not prevented by RGD-containing peptides, indicating that cleaved CAV-9 is able to bypass RGD-dependent entry. The altered receptor specificity of proteolytically cleaved viruses may have important consequences in the pathogenesis of enteric infections.

Amino Acid Sequence↗
PubMed1990

A novel method of typing rhinoviruses using the product of a polymerase chain reaction.

At present rhinoviruses are detected and serotyped in tissue cultures, a slow and laborious process. Previously we have described how the polymerase chain reaction can be used as a rapid method for detecting the presence of a rhinovirus, or enterovirus, in clinical samples without the need to culture. Here we describe a new method which uses the product of the polymerase chain reaction to determine the type of the rhinovirus. The technique is rapid and simple and should eventually greatly facilitate studies on rhinovirus infections.

Gene Amplification↗
PubMed1990

Identification of rhinoviruses by cDNA probes.

We have used nucleic acid hybridization for the detection and grouping of human rhinoviruses (HRV) according to their genetic relationships. Fifteen rhinovirus reference strains, seventy-one clinical isolates and four enteroviruses were propagated in cell cultures, spotted onto membrane filters and hybridized with radioactively labelled cDNA probes covering different parts of the genomes of HRV-1B, HRV-2, HRV-14, HRV-85 and HRV-89. When the rhinovirus and enterovirus reference strains were tested, the 5' probe of HRV-2 hybridized with thirteen of the fifteen HRV reference strains, with poliovirus type 3 and with ECHO virus 11. The HRV-14 5' probe reacted with eleven HRV reference strains and with all the enteroviruses studied. Sixty-nine of the 71 clinical isolates were recognised by the HRV-2 5' probe, whereas the HRV-14 probe from the same part of the genome hybridized with 54 field isolates. One of the two isolates that remained negative with the HRV-2 5' probe was detected with the HRV-2 probe that derived from the P2 region of the genome, and the other isolate was not detected by any of the probes. Probes from other parts than the 5' end of the genome were generally more specific, and clusters could be formed based on the reactivity of the HRV strains with these probes.

DNA Probes↗
PubMed1990

Detection and differentiation of picornaviruses in clinical samples following genomic amplification.

A polymerase chain reaction (PCR) assay was used to detect and differentiate picornaviruses (PVs), using primers homologous to the 5' non-coding and VP2 regions of the PV genome. The PCR resulted in a 530 bp PCR product for human rhinoviruses (HRVs) and a 650 bp product for polioviruses, coxsackieviruses (CV) or echoviruses. The PCR assay could detect as little as 1 p.f.u. of virus in either cerebrospinal fluid (CSF) or stool, using ethidium bromide-stained gels. Standard strains of poliovirus, CV, echovirus and HRV were detected, with the exception of echovirus type 22. In contrast, heterologous viruses, such as herpes simplex virus, human cytomegalovirus, adenovirus, influenza virus and rotavirus, as well as human and monkey cell DNA, were not amplified. In nasal swabs taken from patients with respiratory infections, the PCR detected 27 of 28 HRV isolation-positive specimens. All specimens from which viruses other than HRVs were isolated were negative by PCR. The PCR definitively identified poliovirus and CVs from the CSF or stool of patients with aseptic meningitis, as well as CV in the pericardial fluid of a patient who had suffered a myocardial infarction. Specimens taken from patients with similar pathologies, and from which heterologous viruses were isolated, were uniformly negative by PCR.

Base Sequence↗
PubMed1989

Amplification of rhinovirus specific nucleic acids from clinical samples using the polymerase chain reaction.

We describe a novel method for the detection of human rhinoviruses in clinical samples, using the polymerase chain reaction. Two synthetic oligonucleotide primers were produced that bind in the 5' noncoding region of all rhinovirus serotypes tested, about 350 nucleotides apart, and were used to prime polymerase chain reaction amplification of the intervening stretch of DNA. The product of this reaction, which can be clearly visualized by gel electrophoresis, is a discrete 380 bp band, the occurrence of which is diagnostic of the presence of a rhinovirus in the clinical sample analysed. The technique, which is rapid, sensitive, and reliable, has been used successfully for all the different rhinovirus serotypes tested to date in our laboratory. However, the sensitivity of detection is greatly dependent on the inclusion of both tRNA and vanadyl complexes during the viral RNA extraction process. Using this technique, under optimal conditions, we were able to detect virus in clinical samples with titres as low as TCID50 10(2.5).

Base Sequence↗
PubMed1989

Rhinovirus detection using probes from the 5' and 3' end of the genome.

This study investigated the abilities of cDNA probes from the 5' and 3' ends of the genome of human rhinoviruses (HRV-) 14, 9, and 1B to detect RNA from 59 rhinovirus serotypes. The results show that probes from the 5' end of the genomes of HRV-14, 9, and 1 B detected a large number of serotypes but the detection rate was variable and depended on the degree of homology with the particular probe. In contrast, all the 3' end probes were specific for the homologous virus. However, a long HRV-9 probe detected a large number of serotypes. It was concluded that such cDNA probes would not detect all serotypes with equal efficiency. Synthetic oligonucleotides corresponding to short but highly conserved regions in the 5' non coding region may overcome this problem.

DNA Probes↗
PubMed1989

Genetic diversity of enterovirus subgroups.

Enterovirus serotypes were studied using nucleic acid hybridization and nucleotide sequence analysis. A great majority of enteroviruses could be roughly divided into two larger subgroups the first consisting of poliovirus and certain coxsackievirus A serotypes. The second subgroup included coxsackie B viruses, most ECHO viruses, enterovirus 71 and representatives of coxsackie A viruses. Enterovirus 70 showed low homology to the viruses in both groups. Interestingly, ECHO virus 22 failed to react with any of the hybridization probes indicating a relatively distant relationship. The close relationship between coxsackie B and ECHO viruses as well as between polio and certain coxsackie A viruses was also evident when nucleotide sequences of the 3' end noncoding parts were compared.

Base Sequence↗
PubMed1989

Detection of enteroviruses using cDNA and synthetic oligonucleotide probes.

This study compares the detection of enterovirus RNA by cDNA probes prepared from both the 5' and 3' end of the genome of coxsackie A21 and B4 with the use of synthetic oligonucleotides prepared from short but highly conserved sequences in the 5' end non-coding region of the picornavirus genome. The cDNA probes detected enteroviruses with a variable level of sensitivity which presumably depended on the degree of genomic homology with the detecting probes. Generally probes from coxsackievirus A21 detected more enteroviruses than did similar probes from coxsackievirus B4. Probes from the 5' end of the genome of both viruses were more sensitive than 3' end probes. In contrast, synthetic oligonucleotides detected all enteroviruses efficiently suggesting that these probes could be useful as 'universal' probes to detect any enterovirus. This paper discusses the application of these probes in the diagnosis and differentiation of enteroviruses.

Base Sequence↗
PubMed1989

Sequences in the 5' non-coding region of human rhinovirus 14 RNA that affect in vitro translation.

A subgenomic cDNA clone from human rhinovirus 14 (HRV-14), comprising the 5' non-coding region and the first 1182 nucleotides of the coding sequence, has been inserted into a vector under the control of the T7 promoter, and RNA was transcribed. Deletions in the 5' non-coding sequence modulated viral polyprotein synthesis significantly in a reticulocyte lysate system. Removal of the first 491 nucleotides had little effect, but deletion of a further 55 nucleotides (491 to 546) significantly increased the efficiency of the translation process. Further deletion to nucleotide 621 almost abolished translation, suggesting an essential role for the 546 to 621 nucleotide sequence. The efficiency of the translation process can also be influenced by the addition of ribosomal salt wash prepared from uninfected HeLa cells.

DNA↗
PubMed1989

The complete nucleotide sequence of coxsackievirus A21.

We have determined the complete nucleotide sequence of coxsackievirus A21 (CAV-21), the first member of this enterovirus subgroup to be analysed in molecular detail. The sequence, which is 7401 nucleotides long, encodes an open reading frame of 2206 codons, preceded by a 5' non-coding region of 711 nucleotides and followed by a 3' non-coding region of 72 nucleotides plus a poly(A) tract. The most striking feature is the remarkable homology to the poliovirus (greater than 90% at the amino acid level) in the 3' part of the genome. The rest of the genome is much less homologous, suggesting that CAV-21 is a recombinant virus. Rhinovirus-like characteristics, including the length of the 5' non-coding region and a slight --U/--A imbalance in codon usage, may be related to the fact that CAV-21, like rhinoviruses, infects the upper respiratory tract. However, the sequence sheds little light on the molecular basis of the shared receptor specificity.

Amino Acid Sequence↗