Survival of viruses on fresh produce, using MS2 as a surrogate for norovirus.

AIMS: To study the survival and removal of viruses from fresh fruit and vegetables using the bacteriophage MS2 as a potential surrogate for noroviruses. METHOD AND RESULTS: Survival of MS2 in buffer and on fresh produce was studied at 4, 8 and 22 degrees C. At 4 and 8 degrees C a reduction of <1 log10 was observed after 50 days in buffer; however a reduction in excess of 1 log10 occurred within 9 days at 22 degrees C. Similar results were obtained with fresh produce with virus survival times exceeding the shelf life of the produce. In washing experiments, using a chlorine wash (100 ppm), in all but one case <1.5 log10 MS2 bacteriophage was removed from fruit and vegetables. The mean across all produce types was 0.89 log10. With potable water, reduction was lower (0.3 log mean across all produce types). CONCLUSIONS: MS2 survived for prolonged periods, both in buffer and on fresh produce, at temperatures relevant to chilled foods. It was not removed effectively by chlorine washing. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriophage MS2 has been evaluated as a potential surrogate for noroviruses on fresh produce. Experimental results together with current knowledge of norovirus resistance and survival indicate that MS2 could be used as an effective surrogate in future evaluations.

Laboratory diagnosis of SARS.

The emergence of new viral infections of man requires the development of robust diagnostic tests that can be applied in the differential diagnosis of acute illness, or to determine past exposure, so as to establish the true burden of disease. Since the recognition in April 2003 of the severe acute respiratory syndrome coronavirus (SARS-CoV) as the causative agent of severe acute respiratory syndrome (SARS), enormous efforts have been applied to develop molecular and serological tests for SARS which can assist rapid detection of cases, accurate diagnosis of illness and the application of control measures. International progress in the laboratory diagnosis of SARS-CoV infection during acute illness has led to internationally agreed World Health Organization criteria for the confirmation of SARS. Developments in the dissection of the human immune response to SARS indicate that serological tests on convalescent sera are essential to confirm SARS infection, given the sub-optimal predictive value of molecular detection tests performed during acute SARS illness.

Evaluation of a commercial ELISA for detecting Norwalk-like virus antigen in faeces.

A commercially available enzyme immunoassay, the IDEIA Norwalk-like virus (NLV) enzyme linked immunosorbent assay (ELISA; Dako Cytomation, Ely, UK) for detecting NLV antigen in faecal samples and determining the NLV genogroup was evaluated. The performance of the ELISA was compared with that of electron microscopy and the reverse transcription polymerase chain reaction by testing a panel of faecal samples collected from patients involved in outbreaks of gastroenteritis. When compared with reverse transcription-polymerase chain reaction (RT-PCR), the ELISA had a sensitivity and specificity of 55.5 and 98.3%, respectively. This compares with a sensitivity and specificity for EM of 23.9 and 99.2%, respectively. The sensitivity and specificity of the ELISA for determining the aetiology of a Norwalk virus-like outbreak, based on two or more positive samples within an outbreak, were 52.2 and 100% when two samples were collected from an outbreak and 71.4 and 100% when six or more samples were collected. The ELISA correctly identified the NLV genogroups of viruses previously characterised by partial DNA sequencing. The ELISA is a suitable alternative to the preliminary screening by EM for investigating outbreaks of gastroenteritis. Outbreaks, negative by ELISA should be examined by RT-PCR in order to detect strains non-reactive in the assay and virus strains from representative ELISA positive outbreaks should be characterised fully to allow the genetic diversity of NLVs co-circulating in the population to be described.

A microtitre plate method for isolation and typing of poliovirus using a blue-cell ELISA.

A simple, sensitive, specific and rapid procedure for isolating and typing polioviruses is described. Specimens are inoculated onto confluent monolayers of cell lines (Hep-2C, L20B or RD) seeded into microtitre plates. After 24-48 h, the infected cells are stained with monoclonal antibodies specific for poliovirus types 1,2,3 or a blend of the three antibodies followed by an anti-mouse IgG-horseradish peroxidase conjugate. On addition of substrate, infected cells stain an intense blue colour and are easily distinguished from uninfected cells by light microscopy. Poliovirus infection can be detected before the appearance of cytopathic effects (CPE). This Blue-Cell ELISA test was evaluated against conventional culture and seroneutralisation on a range of polio isolates and clinical specimens. The sensitivity and specificity of the Blue-Cell ELISA compared to neutralisation was 100% (87/87) on culture supernatants of poliovirus isolates sent to our reference laboratory for confirmation. All the poliovirus isolates were typed within 24 h of specimen inoculation using the new method compared to 6-10 days by conventional culture and neutralisation. The method proved to be more sensitive than conventional culture when clinical specimens were examined. Of 43 clinical specimens from which poliovirus had been previously isolated by various laboratories in the U.K., 30/43 (69.8%) were positive for poliovirus by the Blue-Cell ELISA compared to 29/43 (67.4%) by conventional culture and neutralisation. Neutralisation of specimens exhibiting CPE indicated that all of the polioviruses were correctly typed with the new method. CPE was not observed by conventional culture in any specimen that was negative in the Blue-Cell ELISA. There were no cross-reactions with a range of other enteroviruses.

Control of food-borne viruses.

There are two main food-borne virus infections. These are viral gastroenteritis caused by small round structured viruses (SRSV) of the Norwalk group and hepatitis A. Both infections are normally transmitted directly from person-to-person, but on occasions they may also be food-borne or water-borne. Viruses do not multiply or produce toxins in foods, and foods merely act as vehicles for their passive transfer. Foods may be contaminated by infected food-handlers, and outbreaks frequently involve cold foods that require much handling during preparation. Foods may also be contaminated in their growing and harvesting areas by sewage polluted water, and molluscan shellfish have been particularly implicated. PCR and ELISA based methods are being developed for detection and typing of viruses in patients and also in food samples. Sensitive detection methods should facilitate the design of improved food processing methods to ensure virus-free food.

Feline calicivirus as a model system for heat inactivation studies of small round structured viruses in shellfish.

Commercial heat treatment procedures for molluscan shellfish are based on data obtained for the inactivation of hepatitis A virus (HAV) in cockles. However, the most frequently reported illness associated with consumption of bivalve molluscs is gastroenteritis caused by small round structured viruses (SRSVs) of the Norwalk group. Conditions for inactivation of SRSVs are unknown. In this study a feline calicivirus was used as a model for the SRSV group and conditions for its heat inactivation determined. Experiments showed that feline calicivirus is more readily inactivated in shellfish than HAV, and confirmed that current heating recommendations to the UK shellfish industry are adequate. A reverse transcription polymerase chain reaction (RT-PCR) assay for the detection of calicivirus in shellfish was developed and results compared with isolation in cell culture. The RT-PCR detected virus in some samples that failed to yield virus on culture. This has important implications if molecular virology techniques are to be used in the design and monitoring of shellfish treatment procedures and for routine testing of food samples.

Viral meningitis in Cyprus and England : summer 1996.

An outbreak of viral meningitis began in Cyprus on 5 July 1996. By 28 August a total of 316 cases had been admitted to hospital, most of whom were infants and young children; 55 (17%) were less than 1 year of age, 117 (37%) were aged 1 to 4 years, 103 (33

Detection and characterisation of bisegmented double-stranded RNA viruses (picobirnaviruses) in human faecal specimens.

The prevalence of picobirnaviruses (PBVs) in human stools was investigated by polyacrylamide gel electrophoresis (PAGE) analysis of 832 faecal specimens collected between 1982 and 1993 from patients in various clinical groups. Similar prevalences (9-13%) were detected in patients with or without gastroenteritis and throughout the age range of 3 to > 65 years. Two methods for the extraction of nucleic acid, a phenol/chloroform method and a guanidinium thiocynate (GTC)/silica method, were compared. Detection of PBVs by PAGE was three times more sensitive following RNA extraction by the GTC/silica method. Characterisation of three strains was carried out. Segment sizes ranged from 1.625 to 1.95 kilo base pairs (Kbp) and 2.2 to 2.5 Kbp for the fast and slow migrating bands, respectively. The nuclic acid was shown to be double-stranded RNA (dsRNA) by nuclease digestion. PBV-like particles were detected by electron microscopy in two PAGE-positive stools. Virion diameters ranged from 35 to 41 nm and a buoyant density of 1.38-1.4 g/ml in caesium chloride (CsCl) was demonstrated. These findings suggest that PBVs are widespread in humans in the United Kingdom. However, no disease association could be demonstrated.

Similarities in nucleotide sequence between serum and faecal human parvovirus DNA.

Two DNA clones were obtained from faecal specimens containing a parvovirus-like small round virus from a 1977 outbreak of gastroenteritis, and their nucleotide sequences were determined and found to be essentially identical with parts of the published sequence of serum parvovirus B19 and with a B19 isolate (JB) partially sequenced in this study. The clones corresponded mainly to genome regions coding for non-structural proteins, but also include a sequence of some 160 bp coding for structural proteins. Southern blotting experiments with a full-length B19 probe revealed a virion-sized 5 x 5 kbp DNA band in specimens from gastroenteritis cases in both 1977 and 1986. Thus the nucleotide sequence and hybridization results suggest that the virus seen in these studies is very similar to B19. Further work is necessary to clarify the antigenic relationship of these viruses.

Studies on heat inactivation of hepatitis A virus with special reference to shellfish. Part 1. Procedures for infection and recovery of virus from laboratory-maintained cockles.

The consumption of bi-valve molluscan shellfish has been associated with outbreaks of viral gastroenteritis and hepatitis A. Investigations were undertaken to determine the heat inactivation conditions necessary to render shellfish such as cockles safe for the consumer. Conditions for the laboratory maintenance of live cockles are described. In preliminary experiments either poliovirus (10(6) TCID50/ml seawater) or hepatitis A virus (HAV) (approx. 10(4) RFU/ml seawater) was introduced into the shellfish tank. Following 48 h filter feeding, virus was recovered from cockles using an adsorption-elution extraction procedure. Titres of virus recovered ranged from 10(4) to 10(5) TCID50/ml of shellfish extract for poliovirus and from 10(3) to 10(5) RFU/ml of shellfish extract for HAV. Active ingestion of the virus from the seawater was demonstrated by recovering virus from within cockle guts. To quantify recovered HAV, end-point dilutions and an adaptation of a radioimmunofocus assay (RIFA) were compared. The tests were of similar sensitivity but the RIFA has the advantage of being relatively rapid, shortening the time taken to complete an experiment by as much as 4 weeks.

Small round viruses: classification and role in food-borne infections.

Since the first observation of Norwalk virus in the electron microscope in 1972, many different small virus particles in the size range 20-40 nm have been described world-wide in association with outbreaks of gastroenteritis. Progress characterizing these agents has been hampered by the relatively small numbers of particles present in clinical material and the lack of success in culturing them. Although the relationship between some of these viruses remains confusing, a number of distinct groups has emerged, based on morphological features and limited physical data. Immuno-electron microscopy has proved valuable in detecting viruses but the addition of antibody can mask surface morphological features. Examination of viruses in negatively stained preparations without added antibody has revealed distinct morphological differences and viruses previously thought to be simply antigenic variants within the Norwalk group of viruses clearly belong to other groups. Preliminary evidence suggests that one human virus unrelated to Norwalk has a single-stranded DNA genome and is a parvovirus. Some groups have been implicated in outbreaks of food-borne gastroenteritis, particularly after the consumption of shellfish, and their role in other food-borne and water-borne outbreaks is being increasingly recognized.

Red-bellied tamarins (Saguinus labiatus): Hematologic reference values and hemopathologic responses.

Hematologic reference values have been established for captive adult red-bellied tamarins (Saguinus labiatus) by carrying out full blood counts and fibrinogen estimations on 25 clinically normal animals. The only significant sex difference detected was in the reticulocyte count which was higher in females than in males. The reference values were used to identify abnormal changes in the blood of nine clinical cases. Hypochromic anemia, neutrophilia, and raised fibrinogen levels were found in animals with self-inflicted injuries, dermatitis, and ileocecal intussusception. Target cells and jaundiced plasma were noted in a case of yersiniosis. Two animals in which generalized muscle wasting was the main abnormal clinical sign were severely anemic, and in one of these cases a significant number of Heinz bodies was present. The findings in these two animals were compared with those in common marmosets (Callithrix jacchus) with possible wasting marmoset syndrome.

The electron microscopical and physical characteristics of small round human fecal viruses: an interim scheme for classification.

Many of the small round human fecal viruses implicated in outbreaks of nonbacterial gastroenteritis have been collected together and examined under the electron microscope. Negatively stained preparations without the addition of antibody were used so that the surface morphology of the virus particles remained clearly visible. It was apparent that several viruses, previously thought to be simply antigenic variants within the Norwalk group of viruses, show distinct morphological differences and quite clearly belong to other virus groups. By comparing the features of all the viruses examined in this study, both with each other and with standard cell culture strains of enterovirus, parvovirus, and calicivirus, it has been possible to propose an interim classification scheme, based primarily on the morphological appearance of the particles and supported by estimations of size and buoyant density.

Foodborne gastroenteritis of unknown aetiology: a virus infection?

In almost a quarter of outbreaks of gastroenteritis reported to the Public Health Laboratory service by medical officers of environmental health and environmental health officers as possible foodborne infection in 1980 food poisoning organisms were not isolated. In a third of this group the incubation period was longer than the usual range for bacterial food poisoning organisms, and possibly some of the outbreaks were viral in origin. Viruses were detected by electron microsocpy in 88% of faecal specimens from similar outbreaks associated with shellfish but in only 23% of specimens from outbreaks associated with other foods. Recommendations are made for future investigation of such outbreaks including the collection of epidemiological data and specimens for virological study.

Diagnosis of recent hepatitis A infection: a comparison of two methods for detecting specific IgM.

Radioimmunoassay (RIA) tests for anti-hepatitis A virus (HAV) IgM were carried out on 728 sera: 283 were tested by both a method using an anti-mu serum bound to a solid phase and a method involving preliminary separation of igM by sucrose density gradient (SDG) centrifugation, 354 by the anti-mu method alone and two by the SDG method alone. Similar proportions of sera were found to be positive by each method (42.5%, 41.7%), but equivocal results were commoner by the SDG method (4.7% compared with 1.5%). There were 21 (5.5%) discrepant results from the sera tested by both methods, 20 of which could have been due to the higher sensitivity of the anti-mu method. The SDG method generally gave unequivocal results on sera collected within six weeks of the onset of jaundice. Separation of the IgM fraction by re-orientation centrifugation was quick, but otherwise offered no special advantage over separation on a swing-out rotor. The use of 2 mercaptoethanol (2 ME) reduction to assess the purity of the IgM fraction increased confidence in the specificity of the test. It led, however, to the exclusion of 16 reactive sera (4.2%), all of which were found to be positive in the anti-mu test. The anti-mu method gave better discrimination between positive and negative sera than the SDG method and detected IgM both earlier and later in infection. The results of tests designed to check the specificity of the anti-mu procedure were satisfactory. As it is potentially cheaper and easier to perform, the anti-mu method seems, in all respects, to be superior to the SDG method.

Creatinine: a review.

Measurement of creatinine has many applications. We review the determination of urinary creatinine as a valid index of completeness of 24-h urine collection, the clinical utility of the determination of creatinine clearance ratios, and measurement of the ratio of the clearance of specific analytes, such as amylase, to the ratio of clearance of creatinine. The chemistry and variables that affect the Jaffé reaction are reviewed, and attempts at improvement of specificity are discussed. We also review and assess techniques other than the Jaffé reaction for measurement of creatinine.

The biochemical and histochemical demonstration of lactase induction in fetal rat intestine by intra-amniotic injection of lactose.

A single dose of 20 mg beta-D-lactose injected into the amniotic sac of rats on day 17 of pregnancy induced an increase in lactase activity in fetal jejunum. This effect was first noted two days after injection and lasted for at least two additional days. Histoenzymatic investigation indicated that this enzyme was located on the surface of the absorptive cells lining the villi and thus corresponds to the "dietary" form of beta-galactosidase. A much smaller increase, based presumably on progressive increase in fetal size (age) was found in control fetuses which had received glucose or no injections. Peak lactase values in fetuses receiving lactose were substantially higher than peak values in control fetuses. In both lactose-injected and non-injected rats which were allowed to deliver, there was a sharp drop in lactase values coincident with birth.