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The following series of concise summaries addresses the evolution of infectious agents in relation to sex in animals and humans from the perspective of three specific questions: (1) what have we learned about the likely origin and phylogeny, up to the establishment of the infectious agent in the genital econiche, including the relative frequency of its sexual transmission; (2) what further research is needed to provide additional knowledge on some of these evolutionary aspects; and (3) what evolutionary considerations might aid in providing novel approaches to the more practical clinical and public health issues facing us currently and in the future?

We know surprisingly little about the evolutionary origins of Chlamydia trachomatis. It causes both ocular (trachoma) and sexually transmitted infections in humans, it is an obligate intracellular pathogen, and there are only a few "isolates" that have been well characterized. From the first few genomes analyzed, it seems that the C. trachomatis genome is highly conserved. The genomes possess high synteny and, in some cases, the sequence variation between genomes is as little as 20 SNPs. Recent indications from partial genome analyses suggest that recombination is the mechanism for generating diversity. There is no accurate molecular clock by which to measure the evolution of C. trachomatis. The origins of both sexually transmitted and ocular C. trachomatis are unclear, but it seems likely that they evolved with humans and shared a common ancestor with environmental chlamydiae some 700 million years ago. Subsequently, evolution within mammalian cells has been accompanied by radical reduction in the C. trachomatis genome.

Chlamydiae are widespread bacterial pathogens responsible for a broad range of diseases, including sexually transmitted infections, pneumonia and trachoma. To validate the existence of hitherto hypothetical proteins predicted from recent chlamydial genome sequencing projects and to examine the patterns of expression of key components at the protein level, we have surveyed the expressed proteome of Chlamydia trachomatis strain L2. A combination of two-dimensional gel analysis, multi-dimensional protein identification (MudPIT) and nanocapillary liquid chromatography-tandem mass spectrometry allowed a total of 328 chlamydial proteins to be unambiguously assigned. Proteins identified as being expressed in the metabolically inert form, elementary body, of Chlamydia include the entire set of predicted glycolytic enzymes, indicating that metabolite flux rather than de novo synthesis of this pathway is triggered upon infection of host cells. An enzyme central to cell wall biosynthesis was also detected in the intracellular form, reticulate body, of Chlamydia, suggesting that the peptidoglycan is produced during growth within host cells. Other sets of proteins identified include 17 outer membrane-associated proteins of potential significance in vaccine studies and 67 proteins previously annotated as hypothetical or conserved hypothetical. Taken together, >/=35% of the predicted proteome for C. trachomatis has been experimentally verified, representing the most extensive survey of any chlamydial proteome to date.

Reverse transcriptase polymerase chain reaction (RT-PCR), electron microscopy (EM) and a genotype II specific antigen capture enzyme immunoassay (EIA), (Lordsdale strain) were used to establish the prevalence of Norwalk-like viruses (NLV) among sporadic cases of childhood gastroenteritis in South West England over a winter season. Samples of 3,172 stools from cases of gastroenteritis in children aged under 7 years sent to the Bristol Public Health Laboratory over the 1999/2000 winter 'season' were tested prospectively by EM, EIA and RT-PCR. The results from sporadic cases were compared with 1,360 samples from 285 outbreaks of gastroenteritis which were sent to the laboratory over the same period. In total NLV was established as the causal agent in 326 cases (10.3%) of sporadic gastroenteritis by one or more of the tests (EM 30 (0.9%), EIA 132 (4.2%) and RT-PCR 276 (8.7%)). The presence of other enteric viruses was established using EM and rotavirus EIA. Rotaviruses were the most common cause of viral gastroenteritis with 684 cases (21.6%). Other viruses detected included, adenovirus 124 cases (3.9%), astrovirus 97 cases (3.1%) and calicivirus in 7 cases (0.2%). NLV was the second most common viral agent indicating a significant role in cases of sporadic childhood gastroenteritis.

Human enteric caliciviruses have been assigned to two distinct genera: the Norwalk-like viruses (NLVs) and the Sapporo-like viruses (SLVs). During a 3-year surveillance of gastroenteritis in the South West of England during November 1997-2000, a total of 27 clinical samples containing SLVs were collected. PCR amplicons covering a region of the RNA polymerase gene were obtained from 18 of the SLV samples. Sequence analysis of the PCR products indicated that the SLV isolates could be assigned to one of the two major genetic groups represented by Sapporo and London/92 caliciviruses. One of these isolates belonging to the London/92 group (Bristol/98) was subjected to a complete genome sequence analysis. The full genomic sequence of the Bristol/98 isolate was determined from RNA extracted from a single stool sample and consists of 7490 nucleotides, excluding the poly(A) tail. The genome is organised into two open reading frames (ORFs), similar to that of Manchester SLV although the small ORF overlapping the region encoding the capsid protein observed in Manchester SLV is absent in Bristol/98 SLV. The polyprotein (ORF1) of Bristol/98 SLV consists of 2,280 amino acids and, as observed in all SLVs, the structural protein is encoded in frame and contiguous with the 3' terminus of the ORF1. Phylogenetic studies based on complete capsid sequences and genome arrangements within the SLVs indicate that the human enteric viruses within the "Sapporo-like" virus clade should be divided into two distinct genetic groups analogous to the assignment of the Norwalk-like viruses.

Small round structured viruses (SRSVs) are the major cause of outbreaks of gastroenteritis in the UK. Diagnosis is problematic due to insensitive electron microscopy (EM) or technically demanding reverse transcription polymerase chain reaction (RT-PCR) techniques. We have studied outbreaks of non-bacterial gastroenteritis using an EIA based upon recombinant capsid protein from the currently prevalent circulating strain of SRSV (Lordsdale Genotype II) and compared its performance against EM and RT-PCR assays. Faecal specimens sent to the Bristol Public Health Laboratory for outbreak investigation from December 1996 to December 1997 were applied retrospectively to the SRSV EIA and results compared with the routine EM and RT-PCR that had been carried out prospectively. Overall, the three tests identified SRSVs in specimens from 70% of the outbreaks (213/305) investigated. Of the 213 total positive outbreaks, the EIA identified 71%, that compared favourably with EM (63%) and RT-PCR (84%). The Lordsdale Genotype II SRSV EIA provides a simple cost-effective assay that will for the first time make detection of currently circulating SRSV strains associated with UK outbreaks available to all routine laboratories. The EIA format makes the assay widely applicable to non-specialist laboratories, unlike the RT-PCR assay, and the improved sensitivity over EM will allow successful screening of UK outbreaks alongside commercial EIAs currently available for adenovirus, astrovirus and rotavirus. Furthermore, the assay will allow rapid identification of emerging SRSV strains.

Southampton virus (SV) and Lordsdale viruses (LV) are small round structured viruses characterised recently and belong to two separate genogroups. The capsid genes of these viruses were expressed in insect cells using recombinant baculoviruses. Both SV (genogroup 1) and LV (genogroup 2) capsid proteins self-assembled to form virus-like particles (VLPs). The VLPs were used in a standard enzyme-linked immunosorbent assay (ELISA) to screen for antibodies to SV and LV in 1,729 age-stratified human sera collected in Verona, Italy between January and November 1996. SV VLPs were labile compared with LV VLPs. There was a large difference in the prevalence of SV (28.7%) compared with LV (91.2%). However, presentation of SV VLPs using chicken egg yolk antibody-coated wells (IgY capture ELISA) with a subset of serum samples from patients (0-19 years) increased the number of positive sera significantly (50.5%), indicating that SV antigen integrity is an important factor in the assay. Recent reverse transcription-polymerase chain reaction (RT-PCR) studies have shown that LV is circulating currently and analysis of IgY capture ELISA data showed greater reactivity for LV than SV, reflecting a genuinely lower rate of recent infection by this genogroup 1 virus.

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.

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.

The gene coding for the major inner capsid protein VP6 of human group C rotavirus was cloned into baculovirus using the pBlueBac2 vector and expressed in insect cells. When cultured in High Five cells, VP6 was expressed at a high level and exported to the cell culture medium. Purified VP6 was used to immunise rabbits. Hyperimmune rabbit serum, which reacted with native human group C rotavirus in infected cells, was used to develop and optimise an EIA for the detection of antibodies to group C rotavirus using the recombinant VP6 as a source of antigen. In a local epidemiological survey of 1000 sera grouped by age, an average of 43% of samples were found to have antibodies to human group C rotavirus with the highest proportion (66%) in the 71-75 year age group. In comparison, 97% of adults and 85% of children had antibodies to recombinant VP6 from the bovine RF strain of group A rotavirus. These results suggest that infection with human group C rotavirus is a common occurrence despite the apparent rarity of reports of human group C rotavirus in clinical samples from patients with gastroenteritis.

The major outer membrane protein (MOMP) is the prime candidate for the development of a chlamydial vaccine. Antibodies to the subspecies-specific epitope neutralize chlamydial infection. Monoclonal antibodies (MAbs) to this epitope were prepared either by immunization with whole chlamydiae or with a 16 amino acid synthetic peptide. The critical binding site on the subspecies epitope for these MAbs was determined to single amino acid resolution using several hundred solid-phase peptides. A frame shift of just one amino acid in critical binding site completely prevented antibody binding to viable chlamydiae. A single MAb to whole organisms was capable of spanning both the surface-exposed, conformation-dependent, subspecies epitope and a buried, conformation-independent species epitope some 10 A distant. Immunization with peptide generated an MAb with reduced binding constraints which permitted the antibody to bind with broadened species-specificity at the subspecies binding site. The results show for the first time the importance of both critical binding site and conformation at the subspecies epitope. We suggest that the conformational flexibility of short, epitopic peptide vaccines may in some cases be advantageous, giving rise to extended specificity not attained with the natural protein.