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Several hepatitis A virus (HAV) and norovirus (NV) outbreaks due to consumption of berries and vegetables have been reported during recent years. To facilitate the detection of enteric viruses that may be present on different fresh and frozen products, we developed a rapid and sensitive detection method for HAV, NV, and rotavirus (RV). Initial experiments focused on optimizing the composition of the elution buffer, improving the viral concentration method, and evaluating the performance of various extraction kits. Viruses were extracted from the food surface by a direct elution method in a glycine-Tris (pH 9.5) buffer containing 1% beef extract and concentrated by ultrafiltration. Occasionally, PCR inhibitors were present in the processed berry samples, which gave relatively poor detection limits. However, this problem was overcome by adding a pectinase treatment in the protocol, which markedly improved the sensitivity of the method. After optimization, this concentration method was applied in combination with real-time reverse transcription-PCR (RT-PCR) using specific primers in various types of berries and vegetables. The average detection limits were 1 50% tissue culture infective dose (TCID 50 ), 54 RT-PCR units, and 0.02 TCID 50 per 15 g of food for HAV, NV, and RV, respectively. Based on our results, it is concluded that this procedure is suitable to detect and quantify enteric viruses within 6 h and can be applied for surveillance of enteric viruses in fresh and frozen products.

Several hepatitis A virus (HAV) and human norovirus (HuNoV) outbreaks due to consumption of contaminated berries and vegetables have recently been reported. Model experiments were performed to determine the effectiveness of freeze-drying, freeze-drying combined with heating, and steam blanching for inactivation of enteric viruses that might be present on the surface of berries and herbs. Inactivation of HAV and inactivation of feline calicivirus, a surrogate for HuNoV, were assessed by viral culturing and quantitative reverse transcription PCR (RT-PCR), whereas HuNoV survival was determined only by quantitative RT-PCR. While freeze-drying barely reduced (<1.3 log 10 units) the amount of HAV RNA detected in frozen produce, a greater decline in HAV infectivity was observed. The resistance of HuNoV genogroup I (GI) to freeze-drying was significantly higher than that of HuNoV GII on berries. Addition of a terminal dry heat treatment at 120°C after freeze-drying enhanced virus inactivation by at least 2 log 10 units, except for HuNoV GII. The results suggest that steam blanching at 95°C for 2.5 min effectively inactivated infectious enteric viruses if they were present in herbs. Our results provide data for adjusting food processing technologies if viral contamination of raw materials is suspected.

Hepatitis A virus (HAV) and norovirus are important agents of food-borne human viral illness, with common vehicles including bivalve molluscan shellfish, soft fruit and various vegetables. Outbreaks of viral illness due to contamination of the surfaces of foods, or food preparation surfaces by for example infected food handlers are also common. Virus analysis of food matrices can contribute towards risk management for these hazards and a two-part technical specification for determination of Hepatitis A virus and norovirus in food matrices (ISO/TS 15216:2013) was published jointly by the European Committee for Standardisation and the International Organization for Standardization in 2013. As part of the European Mandate No. M381 to validate 15 standards in the field of food microbiology, an international validation study involving 18 laboratories from 11 countries in Europe was conducted between 2012 and 2014. This study aimed to generate method characteristics including limit of detection, limit of quantification, repeatability and reproducibility for ISO 15216 - Part 1, the method for quantification, in seven food matrices. The organization and results of this study, including observations that led to improvements in the standard method are presented here. After its conclusion, the method characteristics generated were added to the revised international standard, ISO 15216-1:2017, published in March 2017.

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