Food Safety Magazine, February/March 2013

SEAFOOD(infectious and noninfectious virus particles) in foods; however,there are no internationally recognized standard methods todate. In an effort to develop standardized procedures, the EuropeanCommittee for Standardization established a TechnicalAdvisory Group for Viruses to develop and publish standard virusextraction and assay procedures for food surfaces, soft fruitand salad vegetables, bottled water and bivalve molluscan shellfish.11 After many years of work, their results should be availablesoon. Virus isolation from foods, either through a rinsingprocedure or by extraction, must be followed by analysis of theInadequacies of Norovirus SurrogateResearchThe quest for an assay to detect infectious noroviruses infood and water has been hampered by the inability to propagatehuman noroviruses in cell or tissue culture systems and theinability to infect common laboratory animals. Consequently,other viruses that can be assayed for infectivity are often used asnorovirus surrogates. One of the earliest viruses to stake a claimas a norovirus surrogate was feline calicivirus, which produceseasily quantified plaques in feline kidney cell culture. A morerecent entry in the search for a surrogate is murine norovirus,which is genetically more similar to human norovirus than isfeline calicivirus and produces plaques in a mouse macrophagecell line. To date, over 400 papers have been published on the“Several methods have been developed to extract and test fortotal norovirus contamination...in foods; however, there are nointernationally recognized standard methods to date.”viruses. In spite of improvements in our ability to extract virusesfrom foods, the analysis of rinses and extracts leaves muchto be desired. Assay methods are almost exclusively based onreverse-transcription polymerase chain reaction (RT-PCR), amolecular-based procedure that amplifies viral RNA into complementaryDNA (cDNA) copies. RT-PCR-based assays have anumber of limitations. Perhaps the most significant is that theydetect total virus presence (both infectious and noninfectiousvirus particles). Thus, viruses inactivated by chlorine, other disinfectants,sunlight, heat, high pressure, etc. can still test positiveby RT-PCR. Other limitations of RT-PCR are that the assayis subject to laboratory contamination and is frequently inhibitedby compounds in the extracts of shellfish or other foods.Various controls must be included in both virus extraction andassay to demonstrate the effectiveness of the extraction and thelack of inhibitors or contaminants in the assay. Such controlsincrease the time and complexity of the procedures but are necessaryfor accurate interpretation of the results.Determining Norovirus Infectivity. For over 40 years, researchershave attempted to propagate noroviruses in culture. Unlikemany human viruses, which can be assayed and quantified incell culture, norovirus propagation has not been successful, inspite of some reports to the contrary. Plaque assays and cytopathogenicityassays are the basis for quantitative assessmentfor many viruses, but are ineffective for human noroviruses.Animal models are another way to monitor the infectivity ofsome viruses, but human noroviruses are incapable of replicationin laboratory animals. Recent advances suggest it will soonbe possible to separate inactive from potentially active norovirususing magnetic beads coated with molecules that mimic thecellular receptors to which noroviruses bind. It would then be12, 13possible to extract potentially infectious norovirus particles.use of feline calicivirus and murine norovirus to determine theeffectiveness of chemical disinfectants and processing technologieson norovirus inactivation. Other norovirus surrogates havebeen proposed; however, it has become abundantly clear thatnone of the surrogates tested to date perfectly mimics humannorovirus. 14 In many cases, human noroviruses may be morepersistent than the surrogates. Such variability in inactivationrates between the surrogate and the pathogen itself might havebeen anticipated, because different strains of even the same viruscan have widely varying inactivation kinetics. For instance,different strains of feline calicivirus showed differences in theirinactivation by chemicals, heat and pH, 15, 16 whereas differentstrains of hepatitis A virus showed substantial differences in inactivationby heat and HPP. 17 Since human norovirus illnessesare caused by any of a wide variety of norovirus strains, it isunlikely that surrogate testing in itself will provide accurate dataor data useful for the promulgation of regulations for the foodindustry. Currently, only human volunteer studies with the actualpathogens can definitively determine norovirus infectivityor the efficacy of sanitation interventions. 14The Need for Clinical TrialsClinical trials have been performed on human norovirusfor decades, but the perceptions that they may be too risky orexpensive have dissuaded some governments from fundingsuch trials. In the United States, clinical trials are still fundedby some agencies, and the information provided in respect tonorovirus inactivation is essential to developing methods applicableto food processing. However, as a practical matter, thehigh cost and complexity of human trials limit the scope of thistype of research. As previously mentioned, the results to date ofsurrogate-based studies are of limited value. Human clinical trialsare needed to evaluate the effectiveness of disinfectants andprocessing technologies on human norovirus inactivation andto identify true norovirus surrogates. 14 Only then will definitiveinformation be available to properly evaluate the retention anddisinfection of infectious noroviruses in foods.60 F o o d S a f e t y M a g a z i n e