PubMed Health⌕ Search

Biomedical subjects

M A McCrae

Publications and source records attributed to M A McCrae.

At least 37 records · Page 2Linked to original sources
PubMed1991

Outer capsid glycoprotein vp7 is recognized by cross-reactive, rotavirus-specific, cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTLs) generated in mice orally inoculated with rotaviruses lyse target cells infected with different rotavirus serotypes (cross-reactive CTLs). Using vaccinia virus recombinants expressing individual rotavirus proteins from two different rotavirus serotypes, we found that cross-reactive CTLs recognize target cells expressing outer capsid protein vp7 better than those expressing outer capsid protein vp4 or inner capsid protein vp6. These findings may be relevant to vaccine strategies which include immunization with reassortant rotaviruses or viral or bacterial vectors expressing individual rotavirus proteins. The region or regions of vp7 which are antigenically conserved among different rotavirus serotypes and recognized by cross-reactive CTLs remain to be determined.

Animals↗
PubMed1991

Sequence of the gene encoding the major neutralization antigen (VP7) of serotype 10 rotavirus.

The sequence of the gene encoding the major neutralization antigen (VP7) of the type member (bovine virus strain B223) of the proposed serotype 10 of rotavirus has been determined. This was done using a rapid new strategy involving direct sequencing by primer extension of cDNA generated from the relevant virus gene (gene 8) using a combined reverse transcription/polymerase chain reaction. The sequence obtained is 1062 bp in length and contains a single long open reading frame capable of encoding a protein of 326 amino acids. Comparison of the sequence to that of the corresponding gene of the other major virus serotype found in cattle, serotype 6, showed that it was approximately 25% divergent at the nucleotide level and 18% divergent at the amino acid level.

Amino Acid Sequence↗
PubMed1991

Detection of group B and C rotaviruses by polymerase chain reaction.

We adapted the polymerase chain reaction (PCR) to detect the noncultivatable group B and C rotaviruses and introduced a simple and convenient technique to purify viral RNA from stool specimens. Double-stranded RNA present in stool extracts was purified by adsorption to hydroxyapatite and was used as the template for reverse transcription and polymerase amplification. Primer pairs specific for group B (gene 8) and group C (gene 6) rotaviruses were selected to amplify group-characteristic sizes of cDNA copies readily identifiable in ethidium bromide-stained agarose gels. These primer pairs were used separately in individual PCR assays or were pooled with a primer pair specific for group A rotavirus (gene 9) in a combined PCR assay for the simultaneous detection of all three rotavirus groups. The method was very sensitive and was used to identify both human and porcine strains of group B and C rotaviruses in stool specimens. A second PCR amplification with internal group-specific primers served to increase further the sensitivity of the test and to confirm the diagnostic results obtained in the first amplification.

Base Sequence↗
PubMed1990

Sequence analysis of the gene (6) encoding the major capsid protein (VP6) of group C rotavirus: higher than expected homology to the corresponding protein from group A virus.

Two overlapping c-DNA clones, which hybridized in Northern blots to RNA segment 6 of the prototype strain (Cowden) of group C rotavirus, were selected from a c-DNA library in pBR322 and sequenced. The gene 6 sequence obtained was 1349 nucleotides and contained a single long open reading frame encoding a protein of 394 amino acids (total MW, 44,479) which is in line with the size of the major capsid protein VP6. Comparison of the group C sequence with that of the corresponding group A rotavirus gene revealed homology levels of 55 and 42% for nucleotides and amino acids, respectively. These values were surprisingly high in view of previous immunological and nucleic acid hybridization data which failed to show any cross-reaction between group A and non-group A rotaviruses. The epidemiological implications of these observations are discussed.

Amino Acid Sequence↗
PubMed1990

The application of polymerase chain reaction to the detection of rotaviruses in faeces.

An assay protocol based on exploiting the polymerase chain reaction (PCR) for the detection of rotavirus in infected faeces is described. The assay is 100,000 times more sensitive than the standard electropherotype method that is widely used. It also gives a 5000-fold increase in sensitivity over the hybridisation based assay previously developed (Pedley and McCrae, 1984) and does not require the use of radioisotopes. The amplified product is a full length c-DNA copy of the gene encoding the major neutralisation antigen of the virus whose molecular cloning and sequence analysis will allow detailed information on the molecular basis of epidemiological variation to be rapidly collected.

Animals↗
PubMed1989

Detailed structural analysis of a genome rearrangement in bovine rotavirus.

A genome rearrangement involving RNA segment 11 of a bovine rotavirus has been analysed by molecular cloning and sequencing. This revealed that the rearranged genome segment was generated by a head to tail concatemerisation of two almost full length copies of segment 11. The upstream copy of the gene has lost its 3' end and the downstream copy its 5' end. The truncation of the upstream copy of the gene occurs within the termination codon for VP11 converting it from a UAG to a UGA, the rearranged gene is therefore still able to encode a normal VP11. The possible mechanisms by which this rearrangement may have been generated are discussed.

Amino Acid Sequence↗
PubMed1989

Molecular biology of rotaviruses. VIII. Quantitative analysis of regulation of gene expression during virus replication.

A sensitive and quantitative solution hybridization assay recently developed in this laboratory has been applied to the study of the regulation of viral gene expression in rotavirus-infected cells. Measurement of the cumulative level of viral plus-strand (mRNA) synthesis at hourly intervals throughout the growth cycle has provided evidence for both quantitative and qualitative regulation of transcription. Qualitative control was found only when cycloheximide was used to block protein synthesis in infected cells, when transcription of four of the viral genes (genes 5, 6, 7, and 9) was independent of protein synthesis. Quantitative regulation was demonstrated by the accumulation of mRNA to much higher levels for some of the viral genes (e.g., genes 2 and 7) relative to others (e.g., genes 4 and 6). In addition to quantitative control at the level of transcription, measurement of the relative molar amounts of the various viral proteins at 6.5 h postinfection showed that their levels did not directly reflect those of their encoding RNAs, indicating the existence of translational control of gene expression in the rotavirus system. Analysis of the levels of minus strand synthesized for each viral gene showed that they were not all accumulated to the same level. The significance of this result in the light of the presumed similarities in replication strategy to that of the mammalian reoviruses is discussed.

Animals↗
PubMed1988

Molecular cloning and sequencing of the gene (M2) encoding the major virion structural protein (mu 1-mu 1C) of serotypes 1 and 3 of mammalian reovirus.

Full-length c-DNA copies of the M2 gene from the Lang strain of type 1 and the Dearing strain of type 3 reovirus have been cloned in the Escherichia coli plasmid pAT153. DNA sequencing of these clones showed that the type 3 gene was 2207 nucleotides long and the single long open reading frame encoded a primary translation product (mu 1) of 709 amino acids with a molecular weight of 76,000. The type 1 gene was three nucleotides shorter at 2204 with the deletions occurring near the center of the coding sequence so that the primary translation product of this gene was one amino acid shorter at 708. Sequence homology between the two genes had an overall value of 85%, rising to 95% when only the noncoding sequences were compared. The 334 nucleotide changes between the two genes were distributed throughout the sequence with no apparent areas of concentration. Comparison of the predicted amino acid sequences showed that there were 24 differences between the two giving a homology of 96.6% at the protein level. The amino acid changes of which only 9 were nonconservative were again spread fairly evenly throughout the coding sequence although there was one small patch of 5 changes in a stretch of 10 amino acids near the carboxyl terminus. The post-translational cleavage to convert mu 1 to the major virion protein mu 1C is revealed as involving the removal of 42 amino acids exclusively from the amino terminus of mu 1. Simple addition of trypsin-sensitive cleavage sites or predicted secondary structure failed to show the cause of the large difference known to exist in the protease sensitivities of virions carrying these two proteins.

Amino Acid Sequence↗
PubMed1988

A rapid and sensitive solution hybridisation assay for the quantitative determination of specific viral RNA sequences.

A solution hybridisation assay has been developed which allows the quantitation of specific viral (RNA) sequences in infected cells. The assay makes use of single-stranded (ss) RNA probes of known polarity synthesised at high specific activity in vitro from cDNA clones of the relevant viral gene by the SP6 or T7 RNA polymerase. These probes are used together with samples containing the RNA to be detected at a known concentration to construct a calibration curve to relate RNase resistant radioactivity following solution hybridisation to amount of RNA. The amount of RNA in experimental samples is then determined using the calibration curve that is produced each time the assay is performed. The UKtc strain of Rotavirus growing in BSC-1 cells was used to develop this method but with the substitution of suitable cDNA clones it could be applied to any viral system.

DNA↗
PubMed1988

Analysis of the structural polypeptides of a porcine group C rotavirus.

Polyacrylamide gel analysis of the structural polypeptides of purified group C virions allowed six major proteins to be identified. Of these, two (52,000- and 39,000-molecular-weight polypeptides) were shown to be in the outer virion shell as judged by the ability to strip them from virions by treatment with EDTA. Treatment of purified particles with endo-beta-N-acetylglucosaminidase F showed that the 39,000-molecular-weight outer shell polypeptide is probably posttranslationally glycosylated. Serological cross-comparison of groups A and C by using Western blotting (immunoblotting) extended the previously demonstrated lack of cross-reaction for the group antigen to show that none of the structural polypeptides cross-reacted. Possible implications of these findings for the epidemiology of rotaviruses are discussed.

Animals↗
PubMed1987

Nucleic acid-based analyses of non-group A rotaviruses.

Simple genome profile studies on polyacrylamide gels allow all non-group A rotaviruses isolated so far to be recognized by the absence of the tight triplet (7-9) of RNA segments seen in all group A viruses. However, reliance solely on genome profile studies for rotavirus grouping can be misleading and, for virus group definition, additional corroborating nucleic acid and serological studies are essential. Terminal fingerprint analysis was the first generation of nucleic acid-based assays that allowed discrimination between the various rotavirus groups. By means of this technique the clear definition of five rotavirus groups (A-E), correlating exactly with those found by a serological assay, has been possible, with preliminary evidence for at least two additional groups. The technical sophistication of fingerprinting techniques prevents their widespread use in epidemiological studies; the development of a second generation of nucleic acid-based assays is therefore under way. These employ molecularly cloned cDNA probes to the genomes of non-group A viruses which can be widely distributed for use in 'dot-blot' screening of faecal samples and, if expressed as protein in Escherichia coli, should provide a ready source of viral antigen for use in surveying viral prevalence through the screening of serum antibody levels.

DNA↗
PubMed1987

Expression of a major bovine rotavirus neutralisation antigen (VP7c) in Escherichia coli.

Sequences from genomic RNA segment 8 of the United Kingdom tissue-culture (t.c.)-adapted bovine rotavirus encoding a major viral neutralisation antigen VP7c have been expressed in Escherichia coli. Expression under the regulated control of the bacteriophage lambda pR promoter was as a C-terminal extension to E. coli beta-galactosidase (beta Gal). Following temperature induction, high levels of the fusion protein were synthesised and accumulated in induced cells, making up 5%-15% of total bacterial cell protein after 2 h of induction. Immunisation of sero-negative rabbits and mice with gel-purified fusion-protein raised antibodies, which gave specific immunofluorescence with virus-infected cells and were able to immunoprecipitate proteins of the VP7 complex from such cells. Hyperimmune sera also gave a virus-type-specific reaction in a solid-phase enzyme-linked immunoabsorbant assay and neutralised virus infectivity in standard plaque-reduction assays.

Animals↗
PubMed1986

A new type of atypical rotavirus in pigs.

Two independent isolates of an unusual type of rotavirus were obtained from over 900 faecal samples obtained from piglets with diarrhoea. Genome profiles, containing eleven segments, were not characteristic of group A electropherotypes and corresponded to an atypical virus. The profiles were, however, distinct from those of other previously described atypical porcine rotaviruses. Serological comparisons showed that the two isolates were related, but unrelated to other known groups of porcine rotavirus. The new isolates were passaged in gnotobiotic piglets in which they caused mild clinical signs of enteritis. A survey of porcine sera from the United Kingdom indicated a widespread distribution of antibody to this virus in pigs aged 10 weeks and over.

Animals↗
PubMed1986

Definition of two new groups of atypical rotaviruses.

Comparative antigenic and nuclei acid analyses were carried out on two new atypical rotavirus isolates coming respectively from chickens (D/132) and pigs (E/DC-9). Indirect immunofluorescence showed that each virus carried different group antigens which were also distinct from those of previously described rotavirus groups. By genome profile analysis each virus had a pattern of genomic RNAs clearly distinct from those of the other rotavirus groups. Comparative terminal fingerprinting of corresponding genome segments from the two viruses showed large differences between them, indicating that all of their genomic RNAs had significant differences in sequence both from each other and from the three previously defined rotavirus groups. On the basis of these results, extension of the number of rotavirus groups from three to five is proposed, with isolates D/132 and E/DC-9 being the type members of groups D and E respectively.

Animals↗