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Are Viruses Alive?

Noroviruses and hepatitis A virus are the most common pathogens in viral infections transmitted by these kinds of foods. To improve microbiological detection and monitoring and to increase insights into the contribution of fruits and vegetables to foodborne viral transmission, sensitive, reliable, and standardized methods are needed. More studies on virus detection methods for foods are being published, but validated consensus protocols are not yet available.

In this paper, different methodologies are reviewed critically. The use of process controls and internal amplification controls is discussed. Soft fruits and vegetables consumed raw are sources of fairly large and growing numbers of viral infections in various countries Reid and Robinson ; Seymour and Appleton Among the foodborne enteric viruses, noroviruses NoVs are the most common cause of illness Koopmans and Duizer , but other viruses such as hepatitis A and E virus HAV and HEV , rotavirus, and enterovirus are implicated in foodborne outbreaks as well Dalton et al.

Frozen raspberries have been linked to at least two outbreaks of HAV Reid and Robinson ; Ramsay and Upton and two outbreaks of caliciviruses Le Guyader et al. In one of these outbreaks, the same batch of raspberries caused a series of sub-outbreaks in which multiple strains of NoV infected 1, people in Denmark during summer Falkenhorst et al.

In , caliciviruses were detected in different lots of Serbian frozen raspberries as reported in the EU Rapid Alert Systems for food and feed. At least three outbreaks of hepatitis A have been attributed to frozen strawberries, and lettuce and raw blueberries were associated with an outbreak of hepatitis A in New Zealand Calder et al.

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In September , during a big outbreak of hepatitis A, which occurred in the USA and involved approximately persons, three people died. Preliminary analysis of a case—control study implicated raw green onions as the source of the infection Anonymous ; Wang and Moran In the UK, These products, which are grown for relatively brief periods and are normally eaten raw e.

Contamination can occur during growth of vegetables as a result of the use of contaminated fertilizers or wastewater Steele and Odumeru It is possible that enteric viruses in soil can be transferred to the whole plant through damaged roots; however, this way of contaminating fruits and vegetables was inefficient and has been observed only during experiments performed with large numbers of virus particles Oron et al.

When account is taken of the ban on the use of sewage sludge as a fertilizer for production of fruits and vegetables, such internal contamination should not occur naturally.

On the other hand, the surface of fruits and vegetables may well be exposed to fecal contamination during irrigation with contaminated water Ward et al. During the preparation and distribution of these food types, viruses can be transmitted by infected food handlers especially when hygienic norms are not followed Dalton et al. Some studies have shown that approximately 9. Moreover, these products are processed without the inclusion of a step for inactivation or removal of bacteria and viruses.

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To improve microbiological monitoring of food quality and assess the true role of food in viral transmission, standardized methods need to be developed for use in reference laboratories and for the monitoring of foodstuffs. Various studies have dealt with the development of standardized methods for detection of enteric viruses in foods. Some of these studies were carried out as part of European projects such as S eafood plus www. However, a single, detailed, and internationally validated protocol may not be suitable for all fruits and vegetables.

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Rather, various modifications of a basic protocol for groups of fruit and vegetables are thought to be needed to take account of the differences in the morphology and hydrophobic interactions of fruit and vegetable surfaces, differences in tissue compositions that affect the amounts of PCR inhibitory substances in, the pH of, and other qualities of, samples from fruits and vegetables , and the processing conditions to which the produce were subjected before they were sampled Dalton ; Cliver et al. In this paper, current methodologies are reviewed and various strategies are proposed for the choice of controls for procedures for detection of viruses.

The use of an internal amplification control IAC , which is necessary for international standardization, is discussed. Before molecular methods were common use, methods for detecting human enteric viruses in foods were based on the inoculation of suitable food extracts into cell cultures. The technique is extremely sensitive and specific but it is unable to distinguish between infectious and non-infectious virus particles Richards Also, the amplification of the genetic material can often be inhibited by substances that are present in the foods and therefore the removal or inactivation of potential inhibitors is a very important step in this methodology Atmar et al.

Most methods are designed to concentrate and purify viruses and remove inhibitors before performing the RT-PCR. For these purposes, multi-step elution and extraction procedures are employed using a combination of several reagents. Using this approach, significant progress has been made in the detection of enteric viruses in shellfish Le Guyader et al.

But much still needs to be done before protocols for use with shellfish are suitable for other foods. Typically, a method for detection of viruses in food involves the three different steps of: 1 elution and clarification of the virus from the foodstuff, 2 concentration of the virus particles, and 3 nucleic acid extraction and detection. Various strategies have been proposed for the performance of each step. Detection of virus in fruits and vegetables starts with the elution of the virus particles from the surface of the product.

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For this purpose, cucumbers Cucumis sativum were experimentally contaminated with poliovirus. The virus was eluted from samples by two consecutive treatments with 1,ml of 0.

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However, many other eluting buffers have been used such as phosphate buffer, pH 7. Dubois et al. The proposed washing fluid breaks the electrostatic and hydrophobic interactions between fruit or vegetable surfaces and viruses. The supplementing of the buffer with Tris favors the elution of virus particles from acidic berries and provides a satisfactory medium when the eluate is to be used in RT-PCR, but it decreases viral infectivity. To limit the decrease in viral infectivity caused by the solution, it was supplemented with 50mM MgCl 2. Previous studies had shown that enteric viruses are more resistant to inactivation in the presence of MgCl 2 Parry and Mortimer Le Guyader et al.

Four of the methods included the use of a different elution buffer. For method C, the addition of a cationic agent CatFloc TL polydiallyldimethylammonium chloride and a chloroform—butanol mixture to enhance the flocculation and clarification of the eluate was proposed. The data suggested that methods C and E provided better results, irrespective of the differences between subsequent concentration steps.

After elution, it is necessary to remove food particles from the eluate containing the viruses in suspension. This clarification should be performed at high pH to prevent the adsorption of viruses to the fruit or vegetable matter. The pellet containing fruit or vegetable matter could then be discarded while viruses remained in the aqueous phase. Filtration through large porosity filters, previously treated for non-adsorption of viruses, has also been used to remove food particles.

Without the addition of pectinase, jellies can form in eluates containing juices from soft fruits such as raspberries and frozen fruits. If contaminated at all, foods are likely to contain very low levels of enteric viruses. Considering this and the low infectious dose of viruses, the concentration step is very important for realistic detection of enteric viruses in food products. Various strategies have been proposed for the concentration of viruses. Virus concentration methods always have to be adapted to be compatible with the eluant used and the type of food that is being analyzed.

For example, juices released from soft fruits might interfere with virus concentration through filtration by clogging the filters. In such a case, precipitation of viruses would be preferable. Viruses can be precipitated by the addition of polyethylene glycol PEG to the clarified eluate, which is a compound that reduces the solubility of macromolecules Lewis and Metcalf Salad vegetables are more resistant to disruption by washing treatments than are soft fruits.

Viruses in clarified eluates from salad vegetables can therefore be concentrated by filtration, which is more rapid than concentration by precipitation. The viruses adsorbed on membrane or cartridge filters can be eluted with the same eluants as used with fruit and vegetable surfaces, but with smaller volumes of eluate. This last method requires expensive equipment and can be used only with eluates free of vegetable matter that were obtained from hard fruits or vegetables. Ward et al. To determine the efficiency of virus recovery by this method, poliovirus was seeded onto cabbage, celery, spinach, and lettuce.

After elution of samples with 2l of phosphate buffer, pH 9.

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Gilgen et al. In this paper, concentration was achieved by a three-phase isolation procedure that included filtration of eluate through a positively charged nylon membrane ultrafilter and cleanup of the viral RNA with a silica-based membrane. The percentages of recovery of enteric viruses can be variable depending on the method and the vegetable used, e. A rapid method for the concentration of enteric viruses by filtration was recently validated by an intralaboratory study Dubois et al.

These procedures avoid the use of filtration or virus flocculation by removing food debris by centrifugation then concentrating virus particles through sedimentation by ultracentrifugation. Initial details of the methods were published by Kurdziel et al. Various soft fruits and salad vegetables were artificially contaminated with poliovirus, then stomached or pulsified with 1M sodium bicarbonate to facilitate removal of viruses from the vegetable surfaces. Infectious polioviruses were enumerated before contamination of, and after extraction from, the produce to determine the efficiency of recovery.

The methods were useful for studies of virus survival Kurdziel et al. Soya protein powder was incorporated in the alkaline extractant to facilitate desorption of virus particles from surfaces.

Instead of stomaching the fruits, they were gently rolled for 15min to facilitate removal of virus from the surfaces without producing too much debris. Also, pectinase was added prior to the centrifugation step to produce a smaller and more easily resuspendable pellet after ultracentrifugation. The ultracentrifugation time was increased to 2h, as this was found to increase recovery. Recovery of NoV from raspberries was estimated by diluting the suspensions used to contaminate the fruit and the extracts, extracting nucleic acids, and applying an RT-PCR assay.

The limiting factors for the efficiency of recovery were considered to be the volume of fruit extract used for nucleic acid extraction and the volume of nucleic acid extract used in the RT-PCR. Another concentration method proposed by various authors is ultrafiltration, in which virus is entrapped in a sample because of its molecular size rather than by particle charge.

Pores in the membrane, varying from 10 to kDa, permit passage of liquids and low molecular mass particles in solution and exclude viruses and macromolecules.