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Title:   Author  item
                                            Richards, Garyitem
                                            Watson, Michaelitem
                                            Meade, Gloriaitem
                                            HOVAN, GREGORY - Delaware Public Health Laboratoryitem
                                            Kingsley, David
  Submitted to: Food and Environmental Virology
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 9/17/2012
                                    Publication  Date: 11/26/2012
                                    Citation: Richards, G.P., Watson, M.A., Meade, G.K., Hovan, G.L., Kingsley, D.H. 2012. Resilience of norovirus GII.4 to freezing and thawing:implications for virus infectivity. Food and Environmental Virology. 4:192-197.Interpretive Summary: Human noroviruses (NoVs) are the leading cause of food-related illness in the United States with an estimated 5.5 million cases annually.  One particular strain of NoV, referred to as genogroup II.4, is the predominant strain worldwide.  The effects of freezing and thawing on NoV survival are uncertain, but freezing and thawing would be expected to have some effect on virus infectivity in foods, environmental samples, and in laboratory cultures.  This study evaluated the effects of -80 deg C frozen storage for up to 120 days as well as the effects of repeated freeze-thaw cycles on the integrity of NoV.  NoVs contain RNA as their genetic material surrounded by a tough protein shell, referred to as their capsid.  Damage to either can inactivate the virus.  Viruses obtained from stool samples collected during outbreaks of NoV at nursing homes were subjected to either a single freeze-thaw on a weekly or bi-weekly basis for up to (120 days) or to repeated (up to 14) freeze-thaw cycles.  Results showed that NoV were resilient to freezing and thawing; no reductions in RNA levels occurred over 120 days regardless of whether the virus was frozen and thawed once or 14 times.  The effects of repeated freeze-thaw cycles on potential NoV infectivity were evaluated using a recently published porcine-mucin binding assay.  Previously, viruses inactivated by heat, UV-irradiation, and high pressure processing failed to bind to mucin-coated magnetic beads; however, we showed that virus binding to the beads was unaffected by 14 freeze-thaw cycles.  This paper provides strong evidence that freezing and thawing does not damage NoV integrity and has no effect on NoV RNA or infectivity.  We conclude that: a) freezing and thawing is not a suitable processing intervention to inactivate NoV in food or water; b) NoV may persist within the environment during winter freeze-thaws, and c) NoVs repeatedly frozen and thawed in laboratory samples will remain detectable using molecular methods.
                                Technical Abstract: Genogroup II.4 norovirus (NoV) remains the predominant NoV strain in food- and water-borne outbreaks.  Capsid integrity as well as viral RNA persistence were determined for GII.4 NoV by real-time RT-PCR after 1-14 freeze/thaw (F/T) cycles (-80 deg C/+22 deg C) or after -80 deg C storage for up to 120 days.  In both cases, capsid integrity and viral RNA titers remained stable.  RNase was exogenously added after 1-14 F/T cycles, but did not alter the amount of genomic NoV RNA detected, indicating that capsids remained intact.  Presumptive NoV infectivity was evaluated in functional studies using a porcine gastric mucin binding assay.  Viruses frozen and thawed up to 14 times bound similarly to porcine mucin, suggesting no reduction in virus infectivity.  Overall, this study shows that: a) NoV particles retain their integrity for at least14 F/T cycles, b) long-term (120 day) frozen storage does not decrease NoV titers, and c) capsid binding to receptor-like glycoprotein moieties remains unaltered after 14 F/T cycles.  This work indicates that freezing and thawing of foods or beverages would not be a practical processing intervention to reduce NoV contamination.  Likewise, repeated freezing and thawing, as might be encountered during winter months, is not expected to inactivate NoV in the environment.  Results do show that laboratory samples destined for molecular biological analyses or for use as positive controls may be repeatedly frozen and thawed without any anticipated reduction in NoV RNA titers.  This study documents the cryostability of NoV capsids and RNA to freezing and thawing and to the possible retention of virus infectivity.


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