Hazel Appleton

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.

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.

A possible virus aetiology in outbreaks of food-poisoning from cockles.

In a series of outbreaks of food-poisoning associated with the consumption of cockles, no bacterial pathogens were demonstrable either in faeces of patients or in cockles. However, small round virus-like particles have been detected in a high proportion of the faecal specimens in three of the outbreaks. These particles are similar in size, morphological features and density to particles seen in outbreaks of winter vomiting and non-bacterial gastroenteritis although in preliminary tests they are serologically distinctive.

VIRUS-LIKE PARTICLES IN WINTER VOMITING DISEASE

In an outbreak of winter vomiting disease affecting both pupils and staff in a primary school, virus-like particles were found in 7 out of 8 faecal specimens examined by electron microscopy. The particles measured 26 nm in diameter and had a buoyant density of 1-38--1-40 g/cm3 in caesium chloride. They could not be cultured in tissue-culture or organ-culture. In immune electron microscopy tests the particles appeared to differ antigenically from the Norwalk and Hawaii agents. Two out of three patients examined more than one month after their illness were still excreting the particles.

A search for faecal viruses in new-born and other infants

Faecal specimens were collected at weekly intervals over the winter months from 141 new-born infants without diarrhoea. Contrary to the findings in other studies, no viruses were detected by electron micriscopy or culture in any of these specimens. Over the same period faecal specimens were collected from 84 infants up to four years of age admitted to hospital. Rotaviruses or adenoviruses were found in 48% of infants with gastroenteritis. Enteroviruses and other small round virus-like particles were found in infants both with and without gastroenteritis. No viruses or pathogenic bacteria could be found in 34% of specimens from infants with gastroenteritis.

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.

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.

Outbreaks of viral gastroenteritis associated with foods

There are few animal viruses that threaten human health through the food chain, but such a dissemination of viruses to livestock constitutes a major threat to animal health as numerous incidents witness. Fresh frozen meat and offal constitute the main hazard, although other products unless properly treated could also be involved. Such treatments are often difficult to evaluate as to their efficacy to inactivate different viruses, unless at least one step is included which in itself produces the desired effect. Amongst the measures taken to minimize the danger only a total ban on imports of certain animal products can be considered fully effective. However, such a measure is frequently not feasible for economical reasons. Other measures such as regulations on the recirculation of waste food, prescribing heat treatment prior to their use for feeding animals generally do not yield the desired results.