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.

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.

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.

Genotype 1 and genotype 2 bovine noroviruses are antigenically distinct but share a cross-reactive epitope with human noroviruses.

The bovine enteric caliciviruses Bo/Jena/1980/DE and Bo/Newbury2/1976/UK represent two distinct genotypes within a new genogroup, genogroup III, in the genus Norovirus of the family Caliciviridae. In the present study, the antigenic relatedness of these two genotypes was determined for the first time to enable the development of tests to detect and differentiate between both genotypes. Two approaches were used. First, cross-reactivity was examined by enzyme-linked immunosorbent assay (ELISA) using recombinant virus-like particles (VLPs) and convalescent-phase sera from calves infected with either Jena (genotype 1) or Newbury2 (genotype 2). Second, cross-reactivity was examined between the two genotypes with a monoclonal antibody, CM39, derived using Jena VLPs. The two genotypes, Jena and Newbury2, were antigenically distinct with little or no cross-reactivity by ELISA to the heterologous VLPs using convalescent calf sera that had homologous immunoglobulin G titers of log10 3.1 to 3.3. CM39 reacted with both Jena and heterologous Newbury2 VLPs. The CM39 epitope was mapped to nine amino acids (31PTAGAQIAA39) in the Jena capsid protein, which was not fully conserved for Newbury2 (31PTAGAPVAA39). Molecular modeling showed that the CM39 epitope was located within the NH2-terminal arm inside the virus capsid. Surprisingly, CM39 also reacted with VLPs from two genogroup II/3 human noroviruses by ELISA and Western blotting. Thus, although the bovine noroviruses Jena and Newbury2 corresponded to two distinct antigenic types or serotypes, they shared at least one cross-reactive epitope. These findings have relevance for epidemiological studies to determine the prevalence of bovine norovirus serotypes and to develop vaccines to bovine noroviruses.

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.

Host range of chlamydiaphages phiCPAR39 and Chp3.

The host range of phiCPAR39 is limited to four Chlamydophila species: C. abortus, C. caviae, C. pecorum, and C. pneumoniae. Chp3 (a newly discovered bacteriophage isolated from C. pecorum) shares three of these hosts (C. abortus, C. caviae, and C. pecorum) but can additionally infect Chlamydophila felis. The ability to support replication was directly correlated with the binding properties of the respective bacteriophages with their host species. Binding studies also show that phiCPAR39 and Chp3 use different host receptors to infect the same host cells: cell binding is sensitive to proteinase K treatment, confirming that the chlamydiaphage receptors are proteinaceous in nature.

Studies of epidemiology and seroprevalence of bovine noroviruses in Germany.

Jena virus (JV) is a bovine enteric calicivirus that causes diarrhea in calves. The virus is approximately 30 nm in diameter and has a surface morphology similar to the human Norwalk virus. The genome sequence of JV was recently described, and the virus has been assigned to the genus Norovirus of the family CALICIVIRIDAE: In the present study, the JV capsid gene encoded by open reading frame 2 was cloned into the baculovirus transfer vector pFastBac 1, and this was used to transform Escherichia coli to generate a recombinant bacmid. Transfection of insect cells with the recombinant baculovirus DNA resulted in expression of the JV capsid protein. The recombinant JV capsid protein undergoes self-assembly into virus-like particles (VLPs) similar to JV virions in size and appearance. JV VLPs were released into the cell culture supernatant, concentrated, and then purified by CsCl equilibrium gradient centrifugation. Purified JV VLPs were used to hyperimmunize laboratory animals. An antigen capture enzyme-linked immunosorbent assay (ELISA) was developed and characterized initially with clinical specimens containing defined human noroviruses and bovine diarrheal samples from calves experimentally infected with JV; the ELISA was specific only for JV. The ELISA was used to screen 381 diarrheal samples collected from dairy herds in Thuringia, Hesse, and Bavaria, Germany, from 1999 to 2002; 34 of these samples (8.9%) were positive for JV infection. The unexpectedly high prevalence of JV was confirmed in a seroepidemiological study using 824 serum or plasma samples screened using an anti-JV ELISA, which showed that 99.1% of cattle from Thuringia have antibodies to JV.

Biological properties and cell tropism of Chp2, a bacteriophage of the obligate intracellular bacterium Chlamydophila abortus.

A number of bacteriophages belonging to the Microviridae have been described infecting chlamydiae. Phylogenetic studies divide the Chlamydiaceae into two distinct genera, Chlamydia and Chlamydophila, containing three and six different species, respectively. In this work we investigated the biological properties and host range of the recently described bacteriophage Chp2 that was originally discovered in Chlamydophila abortus. The obligate intracellular development cycle of chlamydiae has precluded the development of quantitative approaches to assay bacteriophage infectivity. Thus, we prepared hybridomas secreting monoclonal antibodies (monoclonal antibodies 40 and 55) that were specific for Chp2. We demonstrated that Chp2 binds both C. abortus elementary bodies and reticulate bodies in an enzyme-linked immunosorbent assay. Monoclonal antibodies 40 and 55 also detected bacteriophage Chp2 antigens in chlamydia-infected eukaryotic cells. We used these monoclonal antibodies to monitor the ability of Chp2 to infect all nine species of chlamydiae. Chp2 does not infect members of the genus Chlamydia (C. trachomatis, C. suis, or C. muridarum). Chp2 can infect C. abortus, C. felis, and C. pecorum but is unable to infect other members of this genus, including C. caviae and C. pneumoniae, despite the fact that these chlamydial species support the replication of very closely related bacteriophages.

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.

Organization and expression of calicivirus genes.

The application of molecular techniques to the characterization of caliciviruses has resulted in an extensive database of sequence information. This information has led to the identification of 4 distinct genera. The human enteric caliciviruses have been assigned to 2 of these genera. This division is reflected not only in sequence diversity but in a fundamental difference in genome organization. Complete genome sequences are now available for 5 enteric caliciviruses and demonstrate that human and animal enteric caliciviruses are phylogenetically closely related. Currently, there is no cell culture system for the human viruses; therefore, studies have relied on heterologous expression and in vitro systems. These studies have shown that in both human and animal viruses the viral nonstructural proteins are produced from a polyprotein precursor that is cleaved by a single viral protease. The purpose of this article is to provide an overview of the current knowledge of genome structure and gene expression in the enteric caliciviruses.

A diagnostic EIA for detection of the prevalent SRSV strain in United Kingdom outbreaks of gastroenteritis.

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.

Molecular characterization of a bacteriophage (Chp2) from Chlamydia psittaci.

Comparisons of the proteome of abortifacient Chlamydia psittaci isolates from sheep by two-dimensional gel electrophoresis identified a novel abundant protein with a molecular mass of 61.4 kDa and an isoelectric point of 6.41. C-terminal sequence analysis of this protein yielded a short peptide sequence that had an identical match to the viral coat protein (VP1) of the avian chlamydiaphage Chp1. Electron microscope studies revealed the presence of a 25-nm-diameter bacteriophage (Chp2) with no apparent spike structures. Thin sections of chlamydia-infected cells showed that Chp2 particles were located to membranous structures surrounding reticulate bodies (RBs), suggesting that Chp2 is cytopathic for ovine C. psittaci RBs. Chp2 double-stranded circular replicative-form DNA was purified and used as a template for DNA sequence analysis. The Chp2 genome is 4,567 bp and encodes up to eight open reading frames (ORFs); it is similar in overall organization to the Chp1 genome. Seven of the ORFs (1 to 5, 7, and 8) have sequence homologies with Chp1. However, ORF 6 has a different spatial location and no cognate partner within the Chp1 genome. Chlamydiaphages have three viral structural proteins, VP1, VP2, and VP3, encoded by ORFs 1 to 3, respectively. Amino acid residues in the phiX174 procapsid known to mediate interactions between the viral coat protein and internal scaffolding proteins are conserved in the Chp2 VP1 and VP3 proteins. We suggest that VP3 performs a scaffolding-like function but has evolved into a structural protein.

Interaction of primary human endometrial cells with Neisseria gonorrhoeae expressing green fluorescent protein.

Infection of the endometrium by Neisseria gonorrhoeae is a pivotal stage in the development of pelvic inflammatory disease in women. An ex vivo model of cultures of primary human endometrial cells was developed to study gonococcal-host cell interactions. To facilitate these studies, gonococci were transformed with a hybrid shuttle vector containing the gfp gene from Aequoria victoria, encoding the green fluorescent protein (GFP), to produce intrinsically fluorescent bacteria. The model demonstrated that both pili and Opa proteins were important for both mediating gonococcal interactions with endometrial cells and inducing the secretion of pro-inflammatory cytokines and chemokines. Pil+ gonococci showed high levels of adherence and invasion, regardless of Opa expression, which was associated with increased secretion of IL-8 chemokine and reduced secretion of IL-6 cytokine. Gonococcal challenge also caused increased secretion of TNF-alpha cytokine, but this did not correlate with expression of pili or Opa, suggesting that release of components from non-adherent bacteria may be involved in TNF-alpha induction. Thus, the use of cultured primary endometrial cells, together with gonococci expressing green fluorescent protein, has the potential to extend significantly our knowledge, at the molecular level, of the role of this important human pathogen in the immunobiology of pelvic inflammatory disease.

The seroepidemiology of genogroup 1 and genogroup 2 Norwalk-like viruses in Italy.

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.

Identification of further proteolytic cleavage sites in the Southampton calicivirus polyprotein by expression of the viral protease in E. coli.

Southampton virus (SV) is a human enteric calicivirus with a positive-sense RNA genome which encodes a protease as part of a large precursor polyprotein. Expression vectors based on pRSET were constructed carrying the entire 3C-like viral protease (3Cpro) sequence together with flanking sequences from a region of the viral genome 3'-distal to the putative helicase-encoding region. Expression from these vectors in E. coli resulted in discrete protein products with smaller than expected molecular sizes. This confirmed that an active viral protease was produced in E. coli and that the protease was capable of cleaving the expressed protein at defined sites. Expressed cleavage products surrounding the protease region of the viral polyprotein were separated by SDS-PAGE, transferred to PVDF membranes and subjected to N-terminal sequence analysis. Cleavage occurred at an EG dipeptide at the N terminus of the putative VPg (961E/GKNKG) and also at the protease/polymerase boundary (1280E/GGDKG). The N terminus of the protease was released from the VPg C terminus at an EA dipeptide in the sequence 1099E/APPTL. These studies demonstrate that SV enteric calicivirus encodes a 3C-like protease with a specificity similar to the picornaviral 3C protease and that the SV polyprotein is cleaved into at least six mature products.

Molecular characterization of a bovine enteric calicivirus: relationship to the Norwalk-like viruses.

Jena virus (JV) is a noncultivatable bovine enteric calicivirus associated with diarrhea in calves and was first described in Jena, Germany. The virus was serially passaged 11 times in colostrum-deprived newborn calves and caused diarrheal disease symptoms at each passage. The complete JV genome sequence was determined by using cDNA made from partially purified virus obtained from a single stool sample. JV has a positive-sense single-stranded RNA genome which is 7,338 nucleotides in length, excluding the poly(A) tail. JV genome organization is similar to that of the human Norwalk-like viruses (NLVs), with three separate open reading frames (ORFs) and a 24-nucleotide sequence motif located at the 5' terminus of the genome and at the start of ORF 2. The polyprotein (ORF 1) consists of 1,680 amino acids and has the characteristic 2C helicase, 3C protease, and 3D RNA polymerase motifs also found in the NLVs. However, comparison of the N-terminal 100 amino acids of the JV polyprotein with those of the group 1 and group 2 NLVs showed a considerable divergence in sequence. The capsid protein (ORF 2) at 519 amino acids is smaller than that of all other caliciviruses. JV ORF 2 was translated in vitro to produce a 55-kDa protein that reacted with postinfection serum but not preinfection serum. Phylogenetic studies based on partial RNA polymerase sequences indicate that within the Caliciviridae JV is most closely related to the group 1 NLVs.

Enzyme-linked immunosorbent assay based on recombinant human group C rotavirus inner capsid protein (VP6) To detect human group C rotaviruses in fecal samples.

A recent study showed that 43% of a population in the United Kingdom were seropositive for group C rotavirus. The higher than expected incidence may be due to limited diagnosis of acute human group C rotavirus infections because no routine test is available. Human group C rotavirus infections are routinely diagnosed by electron microscopy (EM) and a negative group A rotavirus enzyme-linked immunosorbent assay (ELISA) result. An antigen-detection ELISA was developed with hyperimmune antibodies raised to human group C rotavirus recombinant VP6 (Bristol strain) expressed in insect cells. The assay was used to screen fecal samples to determine the prevalence of group C rotavirus infection. Samples positive by ELISA were confirmed by EM, polyacrylamide gel electrophoresis of double-stranded RNA, or detection of the VP6 gene by reverse transcription-PCR. Retrospective analysis indicated a 1 to 2% detection rate of positivity among samples from patients with acute diarrhea.

Parkville virus: a novel genetic variant of human calicivirus in the Sapporo virus clade, associated with an outbreak of gastroenteritis in adults.

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.

Capsid sequence diversity in small round structured viruses from recent UK outbreaks of gastroenteritis.

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.

Seroepidemiology of human group C rotavirus 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.