Plasmid CDS5 influences infectivity and virulence in a mouse model of Chlamydia trachomatis urogenital infection.

The native plasmid of both Chlamydia muridarum and Chlamydia trachomatis has been shown to control virulence and infectivity in mice and in lower primates. We recently described the development of a plasmid-based genetic transformation protocol for Chlamydia trachomatis that for the first time provides a platform for the molecular dissection of the function of the chlamydial plasmid and its individual genes or coding sequences (CDS). In the present study, we transformed a plasmid-free lymphogranuloma venereum isolate of C. trachomatis, serovar L2, with either the original shuttle vector (pGFP::SW2) or a derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene (pCDS5KO). Female mice were inoculated with these strains either intravaginally or transcervically. We found that transformation of the plasmid-free isolate with the intact pGFP::SW2 vector significantly enhanced infectivity and induction of host inflammatory responses compared to the plasmid-free parental isolate. Transformation with pCDS5KO resulted in infection courses and inflammatory responses not significantly different from those observed in mice infected with the plasmid-free isolate. These results indicate a critical role of plasmid CDS5 in in vivo fitness and in induction of inflammatory responses. To our knowledge, these are the first in vivo observations ascribing infectivity and virulence to a specific plasmid gene.

Chlamydia trachomatis clinical isolates identified as tetracycline resistant do not exhibit resistance in vitro: whole-genome sequencing reveals a mutation in porB but no evidence for tetracycline resistance genes.

Chlamydia trachomatis is the most common bacterial sexually transmitted infection worldwide and the leading cause of preventable blindness in developing countries. Tetracycline is commonly the drug of choice for treating C. trachomatis infections, but cases of antibiotic resistance in clinical isolates have previously been reported. Here, we used antibiotic resistance assays and whole-genome sequencing to interrogate the hypothesis that two clinical isolates (IU824 and IU888) have acquired mechanisms of antibiotic resistance. Immunofluorescence staining was used to identify C. trachomatis inclusions in cell cultures grown in the presence of tetracycline; however, only antibiotic-free control cultures yielded the strong fluorescence associated with the presence of chlamydial inclusions. Infectivity was lost upon passage of harvested cultures grown in the presence of tetracycline into antibiotic-free medium, so we conclude that these isolates were phenotypically sensitive to tetracycline. Comparisons of the genome and plasmid sequences for the two isolates with tetracycline-sensitive strains did not identify regions of low sequence identity that could accommodate horizontally acquired resistance genes, and the tetracycline binding region of the 16S rRNA gene was identical to that of the sensitive control strains. The porB gene of strain IU824, however, was found to contain a premature stop codon not previously identified, which is noteworthy but unlikely to be related to tetracycline resistance. In conclusion, we found no evidence of tetracycline resistance in the two strains investigated, and it seems most likely that the small, aberrant inclusions previously identified resulted from the high chlamydial load used in the original antibiotic resistance assays.

Deduced amino acid sequences of class 1 protein (PorA) from three strains of Neisseria meningitidis. Synthetic peptides define the epitopes responsible for serosubtype specificity.

The previously determined nucleotide sequence of the porA gene, encoding the class 1 outer membrane protein of meningococcal strain MC50, has been used to clone and sequence the porA gene from two further strains with differing serosubtype specificities. Comparison of the predicted amino acid sequences of the three class 1 proteins revealed considerable structural homology with major variation confined to two discrete regions (VR1 and VR2). The high degree of structural homology between the sequences gave predicted secondary structures that were almost identical, with the variable domains located in hydrophilic regions that are likely to be surface located and hence accessible to antibody binding. The predicted amino acid sequences have been used to define the epitopes recognized by mAbs with serosubtype specificity. A series of overlapping decapeptides spanning each of the class 1 protein sequences have been synthesized on solid-phase supports and probed with mAbs. Antibodies with P1.16 and P1.15 subtype specificity reacted with sequences in the VR2 domain, while antibodies with P1.7 subtype specificity reacted with sequences in the VR1 domain. Further peptides have been constructed to define the minimum epitopes recognized by each antibody. Thus we have been able to define linear peptides on each class 1 protein molecule that are responsible for subtype specificity and that represent targets for a protective immune response.

Crystallization and preliminary X-ray diffraction analysis of the protease from Southampton norovirus complexed with a Michael acceptor inhibitor.

Noroviruses are the predominant cause of human epidemic nonbacterial gastroenteritis. Viral replication requires a cysteine protease that cleaves a 200 kDa viral polyprotein into its constituent functional parts. Here, the crystallization of the recombinant protease from the Southampton norovirus is described. Whilst the native crystals were found to diffract only to medium resolution (2.9 Å), cocrystals of an inhibitor complex diffracted X-rays to 1.7 Šresolution. The polypeptide inhibitor (Ac-EFQLQ-propenyl ethyl ester) possesses an amino-acid sequence designed to match the substrate specificity of the enzyme, but was synthesized with a reactive Michael acceptor group at the C-terminal end.

Sequence and genome organization of a human small round-structured (Norwalk-like) virus.

Small round-structured viruses (SRSVs), also known as Norwalk or Norwalk-like viruses, are the major worldwide cause of acute, epidemic nonbacterial gastroenteritis in humans. These viruses, which contain a single-stranded RNA genome, have remained refractory to molecular characterization because of the small amounts of virus in clinical samples and the absence of an animal model and an in vitro culture system. The complete genomic nucleotide sequence of an SRSV, Southampton virus, was determined. The 7696-nucleotide RNA genome encodes three open reading frames whose sequences and organization strongly support proposals that SRVSs are members of the Caliciviridae.

Population-based analysis of ocular Chlamydia trachomatis in trachoma-endemic West African communities identifies genomic markers of disease severity.

BACKGROUND: Chlamydia trachomatis (Ct) is the most common infectious cause of blindness and bacterial sexually transmitted infection worldwide. Ct strain-specific differences in clinical trachoma suggest that genetic polymorphisms in Ct may contribute to the observed variability in severity of clinical disease. METHODS: Using Ct whole genome sequences obtained directly from conjunctival swabs, we studied Ct genomic diversity and associations between Ct genetic polymorphisms with ocular localization and disease severity in a treatment-naïve trachoma-endemic population in Guinea-Bissau, West Africa. RESULTS: All Ct sequences fall within the T2 ocular clade phylogenetically. This is consistent with the presence of the characteristic deletion in trpA resulting in a truncated non-functional protein and the ocular tyrosine repeat regions present in tarP associated with ocular tissue localization. We have identified 21 Ct non-synonymous single nucleotide polymorphisms (SNPs) associated with ocular localization, including SNPs within pmpD (odds ratio, OR = 4.07, p* = 0.001) and tarP (OR = 0.34, p* = 0.009). Eight synonymous SNPs associated with disease severity were found in yjfH (rlmB) (OR = 0.13, p* = 0.037), CTA0273 (OR = 0.12, p* = 0.027), trmD (OR = 0.12, p* = 0.032), CTA0744 (OR = 0.12, p* = 0.041), glgA (OR = 0.10, p* = 0.026), alaS (OR = 0.10, p* = 0.032), pmpE (OR = 0.08, p* = 0.001) and the intergenic region CTA0744-CTA0745 (OR = 0.13, p* = 0.043). CONCLUSIONS: This study demonstrates the extent of genomic diversity within a naturally circulating population of ocular Ct and is the first to describe novel genomic associations with disease severity. These findings direct investigation of host-pathogen interactions that may be important in ocular Ct pathogenesis and disease transmission.

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.

Molecular cloning and sequence analysis of a developmentally regulated cysteine-rich outer membrane protein from Chlamydia trachomatis.

Two 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.

Sulfur-rich proteins of Chlamydia trachomatis: developmentally regulated transcription of polycistronic mRNA from tandem promoters.

RNA was extracted at various times from cells infected with Chlamydia trachomatis serovar L1. Northern-blot analysis showed that transcription of the CrP gene encoding the 60-kDa cysteine-rich outer membrane protein (CrP) produces a temporally controlled polycistronic mRNA. Primer extension analysis indicated the presence of tandem promoters separated by 66 nt with transcriptional start points (tsp) located 577 and 643 nt upstream from the start codon of the mature 60-kDa CrP. Nucleotide (nt) sequencing of this region revealed a small open reading frame (SORF) with coding potential for an 88-amino acid protein containing 13 cysteine residues. This SORF is transcribed as both a polycistronic 2300-nt mRNA together with the CrP gene, and as a separate 480-nt mRNA. Analysis of the upstream sequences, around the tsp for these mRNAs, revealed the presence of three inverted repeat structures that might act as binding domain(s) for a regulatory protein.

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.

The molecular biology of human caliciviruses.

Within the last decade molecular analyses of the genome of Norwalk-like viruses (NLVs) have confirmed that this important group of infectious agents belongs to the Caliciviridae family. NLVs have a positive-sense, single-stranded RNA genome of approximately 7700 nucleotides excluding the polyadenylated tail. The genome encodes three open reading frames: ORF 1 is the largest (approximately 1700 amino acids) and is expressed as a polyprotein precursor that is cleaved by the viral 3C-like protease; ORF 2 encodes the viral capsid (550 amino acids); and ORF 3 encodes a small basic protein of unknown function. Comparative sequencing studies of human caliciviruses have revealed a second distinct group of viruses known as Sapporo-like viruses (SLVs). SLVs also have a single-stranded, positive-sense RNA genome of approximately 7400 nucleotides and the small 3' terminal ORF (NLV-ORF3 equivalent) is retained. Phylogenetic analyses of NLV and SLV genomic sequences have assigned these viruses to two different genera with each genus comprised of two distinct genogroups. The fundamental difference in genome organization between NLVs and SLVs is that the polyprotein and capsid ORFs are contiguous and fused in SLVs. Progress in understanding the molecular biology of human caliciviruses is hampered by the lack of a cell culture system for virus propagation. Studies on viral replication and virion structure have therefore relied on the expression of recombinant virus proteins in heterologous systems. Norwalk virus capsid expressed in insect cells assembles to form virus-like particles (VLPs). Structural studies have shown that Norwalk virus VLPs are comprised of 90 dimers of the capsid protein.

National epidemic of Lordsdale Norovirus in the UK.

BACKGROUND: In early 2002 reports of outbreaks of gastroenteritis reached unprecedented levels in the UK. Forty five Norovirus outbreaks were reported in January 2002. OBJECTIVES: The objective of the study was to determine whether the outbreaks were Noroviral in origin and if so whether they represented a homogeneous or heterogeneous collection of Noroviruses by applying EIA and sequence analysis to representative faecal samples. STUDY DESIGN: Faecal specimens were collected during the week of highest incidence from 21 outbreaks in a variety of health care settings including hospitals and nursing homes. The outbreaks occurred in geographically distinct regions of the UK and samples were collected by reference laboratories in Glasgow, Manchester, Bristol and Southampton. RESULTS: The samples were all positive for Noroviruses by negative stain electron microscopy (EM) and Lordsdale virus (LV) EIA, therefore reverse transcriptase polymerase chain reaction (RT-PCR) amplification and nucleotide sequencing of the Norovirus RNA polymerase gene was performed on amplicons from samples of each of the 21 outbreaks to investigate the nature and extent of diversity. All samples were very closely related to the reference Lordsdale virus genome sequence. LV was first discovered during an hospital outbreak of gastroenteritis in Southampton General Hospital in March 1993. CONCLUSIONS: Noroviruses are a major cause of outbreaks of gastroenteritis in health care settings. LV is the predominant Norovirus in the UK and was detected in outbreaks that occurred during the national peak of gastroenteritis reports in January 2002.

Viral zoonoses and food of animal origin: caliciviruses and human disease.

Caliciviruses are important veterinary and human pathogens. The viruses gain their name from characteristic cup-shaped structures seen on the virion surface by negative stain electron microscopy. In humans caliciviruses are a major cause of diarrhoeal disease. There are two fundamentally different genome structures amongst human caliciviruses. The Norwalk-like or small round structured viruses (SRSVs) are viruses that have an amorphous structure when viewed by EM, they have a genome composed of 3 major open reading frames (ORFs). These viruses cause epidemic gastroenteritis amongst all age groups. In contrast, the 'classic' human caliciviruses (HuCVs) display the typical calicivirus surface structure and have their capsid ORF fused to and contiguous with the non structural proteins forming one giant polyprotein. HuCVs are predominantly associated with paediatric infections and are only a minor cause of disease in humans. Spread of disease for both SRSVs and HuCVs is usually by faecal oral transmission. SRSVs are a major cause of foodborne gastroenteritis especially linked to the consumption of sewage-contaminated shellfish. However, there is no evidence that these viruses replicate in shellfish or that they originate from an animal source.

Chlamydia trachomatis 60 kDa cysteine rich outer membrane protein: sequence homology between trachoma and LGV biovars.

An 820 bp AccI-PstI fragment of the 60 kDa cysteine rich outer membrane protein (CrP) gene from C. trachomatis serovar L1 was used as a probe to locate the 60 kDa CrP gene of a recent serovar B trachoma isolate (Jali 20/OT). The probe hybridized to a single 1.8 kb SpeI fragment in Southern blot analyses of different restriction endonuclease digests of C. trachomatis serovar B DNA. This fragment was ligated into Lambda Zap II arms and Bluescript SK(-) recombinants, released by infection with the helper phage R408, were used as template for DNA sequence determination. Sequence analysis demonstrated a very high level of homology between the Jali 20/OT 60 kDa CrP and the previously published serovar L1 60 kDa CrP with only 8 out of 507 amino acid substitutions between the two proteins.

Construction of an epitope vector utilising the diphtheria toxin B-subunit.

An immunogenic loop within the diphtheria toxin has been deleted from the B-subunit by a modification of the inverse polymerase chain reaction (IPCR) and replaced by a unique restriction endonuclease site. An oligonucleotide encoding an identified epitope sequence from the major outer membrane protein of Neisseria meningitidis of similar size and structure to that deleted has been introduced into the restriction site. Expression of the resulting chimeric B-subunit from Escherichia coli yielded a protein that was recognised by a panel of antibodies specific for the meningococcal epitope. Initial immunisation data suggest that this protein could elicit an antibody response against both diphtheria toxin and meningococcal proteins.

A sensitive method for the production of diagnostic fingerprints of the genome segments of field isolates of rotavirus.

The 3'-terminal labelling procedure for analyzing the genome segment profile of field isolates of rotavirus (Clarke and McCrae, 1981, J. Virol. Methods 2, 203) has been further developed to produce a rapid and sensitive method for generating diagnostic fingerprints from individual species of double-stranded RNA. The fingerprints were obtained by a one-dimensional resolution of overlapping terminally labelled oligonucleotides produced by partial nuclease digestion with a base-specific nuclease. This fingerprinting method should be of great value in characterizing the molecular details of genome segment variation and will facilitate detailed molecular epidemiological studies of this important virus group.

Intracellular parasitism of chlamydiae: specific infectivity of chlamydiaphage Chp2 in Chlamydophila abortus.

The obligate intracellular nature of chlamydiae presents challenges to the characterization of its phages, which are potential tools for a genetic transfer system. An assay for phage infectivity is described, and the infectious properties of phage Chp2 were determined.

Serotype 1 and 2 bovine noroviruses are endemic in cattle in the United kingdom and Germany.

The genomically and antigenically distinct bovine noroviruses Bo/Jena/1980/DE and Bo/Newbury2/1976/UK have been associated with calf diarrhea. In the present seroprevalence study, both were found to be endemic in cattle from Germany and the United Kingdom, a finding in contrast to previous virus prevalence studies. They were less common than group A rotaviruses, particularly in calves, suggesting a different epidemiology.