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.

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.

Astrovirus-associated gastroenteritis in children.

In a small astrovirus-associated outbreak of gastroenteritis in a ward of a local children's hospital two out of five children with symptoms excreted astrovirus particles. No astrovirus particles were found in faeces from the remaining asymptomatic child, and no other viral or bacterial pathogens were found in any of the children. Virus excretion persisted for only a few days. Rising antibody titres to the astrovirus particles were demonstrated in one child, and IgM was also demonstrated in this patient's serum.

Comparison of ELISA with virus isolation for the diagnosis of genital herpes.

An enzyme linked immunosorbent assay (ELISA) system which detects and simultaneously types herpes simplex virus antigens in clinical specimens from patients with genital herpes has been compared with standard tissue culture isolation. Although more sensitive than a similar method previously described and also more sensitive than electron microscopy and immunofluorescence, ELISA did not detect all the viruses isolated in tissue culture. Costs were comparable. The speed of obtaining the result together with knowledge of the type causing infection are useful when antiviral chemotherapy is envisaged and when considering the likelihood of recurrences.

Comparison of the continuous cell line 293 with human embryo kidney cells and human embryo fibroblast cells for the cultivation of ocular viruses.

The continuous cell line 293 was evaluated as a replacement for primary human embryo kidney (HEK) cells in the cultivation of ocular viruses. The 293 cells were found to be as sensitive as HEK cells and human embryo fibroblast (HEF) cells for the cultivation of adenoviruses and herpes simplex virus (HSV) respectively. As a continuous cell line, 293 cells are preferable to HEK and HEF cells for the routine isolation of ophthalmic viruses.

Comparison of a commercial ELISA system with restriction endonuclease analysis for typing herpes simplex virus.

Isolates of herpes simplex virus which had previously been typed by restriction enzyme analysis were typed again with a commercial ELISA system using polyclonal antibodies. There was complete correlation between the two techniques. Although restriction is more precise and definitive, when typing only is required the simplicity of ELISA makes it the preferred technique.

Potassium tartrate-glycerol as a density gradient substrate for separation of small, round viruses from human feces.

Cesium chloride density gradients are frequently used for virus concentration or purification in the preparation of human feces for examination by electron microscopy, Disruption of some of the fecal viruses occurs if they are pelleted from the density gradient in an additional concentration step. This report highlights an important limitation imposed by the use of cesium chloride as a density gradient substrate in attempting to recover small, round, virus-like particles from feces and suggests an alternative substrate which preserves virus morphology without the use of additional protective agents.

Identification of the elementary bodies of Chlamydia trachomatis in the electron microscope by an indirect immunoferritin technique.

An indirect immunoferritin (IIF) technique is described for recognizing the elementary bodies (EB) of Chlamydia trachomatis in unsectioned preparations. Both the EB of a genital strain of C. trachomatis grown in irradiated McCoy cells and EB in clinical specimens obtained from patients attending a venereal disease clinic were identified by the IIF test in the electron microscope. Cell culture-grown EB were detected by ferritin staining for up to 4 weeks after the organisms had lost their infectivity for tissue cultures. The IIF test was of comparable sensitivity to isolation methods in detecting chlamydiae in clinical specimens. Other possible applications of the IIF technique are discussed.

Group C rotavirus associated with fatal enteritis in a family outbreak.

A family outbreak of gastroenteritis involving three adults and three children is described in which diarrhoea and vomiting were the main clinical features. One infant died in whom no pathogens could be detected in either small or large intestinal postmortem samples. Stool samples from two symptomatic siblings contained rotaviruses as demonstrated by electron microscopy. Both of these faecal samples were negative when assayed in a group A specific rotavirus enzyme-linked immunosorbent assay (ELISA) and subsequent genomic analysis of these rotaviruses was suggestive of group C rotavirus. Serological evidence showed that these atypical rotaviruses were members of serogroup C. Other atypical rotaviruses in faecal samples from sporadic cases in symptomatic children were detected over a similar time period and location. These had electrophoretic RNA profiles similar to those in the family outbreak. Furthermore, seroepidemiological studies detected group C rotavirus antibody in blood donors resident in the location of the family outbreak.

An outbreak of gastroenteritis in young children caused by adenoviruses.

During October and November, 1978, gastroenteritis developed in 17 of 24 young children aged between eight months and two years from an R.A.F. station in the U.K. The illness, in which diarrhoea was always the predominant symptom, had an incubation period of eight to ten days and lasted about a week. It seemed to be transmitted from child to child, and in all but one instance parents and older siblings remained well. Stool specimens from 14 of the affected children were examined bacteriologically and virologically, and a highly significant association was found between the presence of adenovirus particles in stools, identified by electron microscopy, and the acute stage of the illness. This evidence suggests that an adenovirus was the cause of this outbreak of gastroenteritis.

Human enteric Caliciviridae: a new prevalent small round-structured virus group defined by RNA-dependent RNA polymerase and capsid diversity.

Sequence comparison of the RNA-dependent RNA polymerases of small round-structured viruses (SRSVs) from 10 recent U.K. outbreaks of gastroenteritis revealed significant genetic variation. Computer analyses indicated that these viruses can be divided into two discrete groups. SRSV group I contains the previously characterized antigenic type 1 Norwalk and type 3 Southampton viruses. The amino acid sequences of the RNA polymerase, capsid and ORF3 of these two viruses are relatively similar (about 92%, 69% and 72% amino acid identity, respectively). A representative member of group II SRSVs, Bristol virus, was subjected to a detailed genetic analysis. Bristol virus is a recent antigenic type 2 isolate from a U.K. hospital outbreak of gastroenteritis. Using a single clinical sample the 3'-terminal 3881 nucleotide cDNA sequence [excluding the poly(A) tail] of this virus was determined. Analysis of the sequence revealed significant differences from those of group I viruses with the RNA polymerase region, capsid and ORF3 showing only about 62%, 43% and 30% amino acid identity respectively with the equivalent proteins of the Norwalk and Southampton viruses. These data suggest that the morphologically identical SRSVs belong to at least two genetically distinct groups.

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.

Polymerase chain reaction detection of small round-structured viruses from two related hospital outbreaks of gastroenteritis using inosine-containing primers.

Two outbreaks of gastroenteritis in the UK which occurred nine days apart at Lymington and Southampton hospitals were investigated. The clinical and epidemiological features of both outbreaks were characteristic of small round-structured virus (SRSV) infection with rapid onset of diarrhoea and/or nausea and vomiting and propagation of the outbreaks by secondary spread. SRSV particles were observed by immune electron microscopy (EM) in 60% of faecal samples from both outbreaks and no other pathogens were detected. The index case for the second outbreak was a patient who was admitted with diarrhoea and vomiting after being discharged from Lymington hospital during the first outbreak. The possibility that the two outbreaks were caused by the same strain of SRSV was investigated by the polymerase chain reaction (PCR). New inosine-containing PCR primers were designed to amplify the RNA polymerase region of SRSV cDNA from genetic groups I and II. The PCR using the group II primers achieved a higher detection rate for SRSVs in faecal samples (68% of samples positive from both outbreaks) than immune EM. SRSVs were not detected using the group I primers or using conventional degenerate PCR primers. The nucleotide sequences of PCR amplicons from both outbreaks were identical providing molecular epidemiological evidence for the involvement of a single SRSV strain. Comparison of the RNA polymerase region of this virus with the equivalent regions of genetic group I (69.4-75.0% amino acid identify) and genetic group II (88.9-100% amino acid and 77.1-88.1% nucleotide identity) SRSVs revealed that the causative SRSV was a distinct member of genetic group II.