Methods for recovery of hepatitis A virus (HAV) and other viruses from processed foods and detection of HAV by nested RT-PCR and TaqMan RT-PCR David C. Love ⁎ , 1 , Michael J. Casteel 2 , John S. Meschke 3 , Mark D. Sobsey Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, United States ABSTRACT ARTICLE INFO Article history: Received 14 November 2007 Received in revised form 26 April 2008 Accepted 23 May 2008 Keywords: Hepatitis A virus Coliphage Processed foods Reverse transcription-PCR Enteric viruses are important agents of foodborne disease. Unfortunately, robust, quantitative methods for sampling and analysis of enteric and other viruses in processed or complex foods are not well-established. As a result, epidemiologically determined etiologies or pathogen sources in foodborne outbreaks are rarely confirmed by virological analysis. In this study, an acid-adsorption elution concentration (AEC) method previously used to monitor virus occurrence and investigate enteric virus outbreaks in shellfish was adapted for examination of processed food items, namely tomato sauce and blended strawberries. Hepatitis A virus (HAV), poliovirus, and coliphage MS2 (MS2) were seeded in 10 or 30 g samples of tomato sauce or blended strawberries, recovered by AEC, and quantified by cell culture infectivity assay. In addition, nested reverse transcription-polymerase chain reaction (RT-PCR) and TaqMan RT-PCR assays were used to detect HAV RNA. Viruses were efficiently adsorbed to foods as an initial concentration step, with infectious HAV and MS2 adsorption of 67% and 93%, respectively, to tomato sauce, and 89% and 99%, respectively, to blended strawberries. Forty-three to 65% of HAV and poliovirus were subsequently eluted and recovered from tomato sauce using 0.5 M threonine, pH 7.2. The lower limits of HAV detection were at initial seeding levels of 14 PFU/g of tomatosauce and 33 PFU/g of blended strawberries. Unlike TaqMan RT-PCR, nested RT-PCR was not inhibited by undiluted final RNA extracts of tomato sauce or blended strawberries. The successful adaptation of the AEC method for enteric and other virus recovery, quantitation and detection in processed foods demonstrates its potential for use in the investigation of foodborne outbreaks of viral etiology and for validation of virus disinfection and sanitary processing procedures used by the food industry. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Norovirus, hepatitis A virus (HAV), rotavirus, and astrovirus are responsible for an estimated 9.2 million acute foodborne gastro- intestinal illnesses annually in the United States (Mead et al., 1999). Virological analysis of foods, including retrospective testing of food implicated in disease outbreaks, is rarely performed and few such analyses are successful in conclusively linking suspected foodborne viral diseases to their source (Daniels et al., 2000; Schwab et al., 2000). This is because virological testing of foods is labor intensive, technically demanding, expensive, and few sensitive methods are available (Dubois et al., 2002, 2006; Le Guyader et al., 2004). Furthermore, foodborne viral pathogens occur in low numbers and tend to be non-randomly dispersed (Cliver et al., 1983). However, small amounts of fecal contamination may become widely distributed and incorporated throughout a food source during processing, and the scale of consumption and production of such foods on a global basis is large. Processed foods in which fecal viruses have been incorporated, such as fruit and vegetable-based sauces, purées, pastes, and blended fruit desserts and drinks are major sources of outbreaks and cases of disease (Niu et al., 1992; Anonymous, 1997, 1999, 2002, 2003, 2004, 2006; Hutin et al., 1999). Such foods pose additional challenges for virological testing because of their relatively complex matrices. While effective virus recovery and detection methods are needed for processed foods, regulators and industry currently lack such tools. The goal of the present study was to demonstrate that techniques such as elution and chemical manipulation of electrostatic forces used in the past to recover viruses from shellfish meats (Sobsey et al., 1975; Jaykus et al., 1996; Mullendore et al., 2001) could be modified and used for HAV recovery from other types of foods, such as strawberry purée (i.e., halved, strawberries in sauce blended for consistency) and tomato sauce. The other viruses used in this study were coliphage MS2 and a vaccine strain of poliovirus, because they have been proposed for use as surrogates and positive controls in method development studies in food and environmental virology. Infectivity assays were International Journal of Food Microbiology 126 (2008) 221–226 ⁎ Corresponding author. Tel.: +1 510 643 0355; fax: +1 510 643 9714. E-mail address: davelove@berkeley.edu (D.C. Love). 1 Current address: Department of Civil and Environmental Engineering, 207 O'Brien Hall, University of California at Berkeley, Berkeley, CA 94720, United States. 2 Current address: Microbial Intelligence Group, L.L.C., P.O. Box 1292, San Bruno, CA 94066, United States. 3 Current address: Department of Environmental and Occupational Health Sciences, University of Washington, 4225 Roosevelt Way NE, Suite 100, Seattle, WA 98195-6099, United States. 0168-1605/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2008.05.032 Contents lists available at ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro