1,720,995 research outputs found
Viral gastroenteritis: the human caliciviruses
No full textThe caliciviruses are a major cause of viral gastroenteritis but the lack of diagnostic procedures has resulted in their role being under-rated. Norwalk-like viruses are detected by negative stain electron microscopy and their appearance results in their being called small round structured viruses. Spread is by food and contamination of the environment by vomitus. There are many genetic variants and only small numbers of virus particles are shed during infection. Sapporo-like viruses have a 3-fold axis of symmetry and are usually associated with paediatric gastroenteritis. Cell culture systems are lacking. There is no long-lived immunity following infection. Hand washing and protective clothing are essential during outbreaks
Stable cloning of the amino terminus of the 60k outer membrane protein of Chlamydia trachomatis serovar L1
No full textA mixed twenty-five base oligonucleotide probe was synthesized to the amino terminus of the 60 kDa cysteine-rich outer membrane protein of Chlamydia trachomatis serovar F. This probe hybridized to a single band in Southern blots of C. trachomatis serovar L1 DNA. However, repeated attempts at screening plasmid-based genomic libraries failed to yield positive signals. As an alternative approach, bacteriophage M13 was used as the primary cloning vector. With this vector two identical stable recombinants were isolated carrying a 2.2 kb Pst1 insert that hybridized to the oligonucleotide probe. Attempts to re-clone this fragment in plasmid-based vectors yielded very small unstable colonies. DNA encoding the amino terminus of the 60 kDa outer membrane protein was further localised to a 274 bp Sau3A fragment which was sub-cloned in both orientations in m13. DNA sequence analysis of this fragment from C. trachomatis serovar L1 demonstrated a perfect 15/15 amino acid match to the amino terminus of the 60 kDa outer membrane protein from C. trachomatis serovar F. On the basis of these results we suggest bacteriophage M13 as a general cloning vector to avoid the toxic effects of the amino termini of foreign outer membrane proteins in Escherichia coli
Molecular cloning and sequence analysis of a developmentally regulated cysteine-rich outer membrane protein from Chlamydia trachomatis.
No full textTwo overlapping genomic fragments have been cloned from Chlamydia trachomatis serovar L1 DNA. Sequence determination of 2530 bp has revealed two open reading frames coding for 'cysteine-rich' (Cr) proteins. One of these proteins was confirmed, by analysis of the inferred amino acid sequence, as the 60-kDa Cr outer membrane protein associated with differentiation of reticulate bodies (RBs) into elementary bodies (EBs). The other smaller 15-kDa protein contained a high percentage of methionine and cysteine and may correspond to a reported smaller and co-ordinately synthesised Cr outer-membrane protein also associated with RB to EB differentiation. Sequencing showed three potential stem-loop structures within the 5', 3' and intergenic regions of the cloned fragment. Southern-blot analysis revealed that the cloned fragment is conserved in ten serovars of C. trachomatis and that a strongly cross-hybridising fragment is also present in Chlamydia psittaci
The nucleotide sequence of the 60 kDa cysteine rich outer membrane protein of Chlamydia psittaci strain EAE/A22/M
Full text linkGenome organization in the caliciviridae
No full textCaliciviruses 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
Genetic diversity and identification of human infection by amplification of the chlamydial 60-kilodalton cysteine-rich outer membrane protein gene
No full textThe 60-kDa cysteine-rich outer membrane protein genes of Chlamydia psittaci, Chlamydia pneumoniae, and Chlamydia trachomatis have very different 5' ends, but two areas flanking this variable region show absolute sequence conservation. This observation permitted differentiation of the three species of Chlamydia by the polymerase chain reaction (PCR), forming the basis of a diagnostic test for chlamydial infections. The PCR product containing the variable region of the respective 60-kDa CrP genes was also subjected to restriction endonuclease digestion, enabling differentiation of individual type strains of C. psittaci. Differentiation was possible between lymphogranuloma venereum and trachoma isolates of C. trachomatis. The PCR-based diagnostic test was successful with all strains of chlamydiae studied. The PCR primers showed high specificity and did not produce any product with common bacterial pathogens that may share the same sites of infection
A conserved sequence motif at the 5' terminus of the Southampton virus genome is characteristic of the Caliciviridae
No full textWe 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
Polyprotein processing in Southampton virus: identification of 3C-like protease cleavage sites by in vitro mutagenesis
No full textA 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
Cloning of noncultivatable human rotavirus by single primer amplification
No full textA novel, sequence-independent strategy has been developed for the amplification of full-length cDNA copies of the genes of double-stranded RNA (dsRNA) viruses. Using human (Bristol) group C rotavirus as an example, a single amino-linked modified oligonucleotide (primer 1) was ligated to either end of each dsRNA genome segment by using T4 RNA ligase. Following reverse transcription, annealing, and repair of cDNA strands, amplification of the viral dsRNA genome was accomplished by polymerase chain reaction using a single complementary oligonucleotide (primer 2). Northern (RNA) hybridization of cDNA to virus dsRNA indicated that it was possible to generate cDNA representing the complete genome from very small clinical samples. This technique was used to determine the complete nucleotide sequence (728 bp) and coding assignment of gene 10, which revealed an open reading frame of 212 amino acids with limited homology to NS26 from human group A rotavirus. In contrast to previous tailing methods, the addition of one defined primer allowed unequivocal identification of terminal nucleotides and should be generally applicable to viruses with segmented dsRNA genomes and especially for analysis of clinical samples, for which very limited quantities of biological material are available
- …
