title: Evaluation of a Porcine Gastric Mucin and RNase A Assay for the Discrimination of Infectious and Non-infectious GI.1 and GII.4 Norovirus Following Thermal, Ethanol, or Levulinic Acid Plus Sodium Dodecyl Sulfate Treatments.
abstract: Human noroviruses (NoVs) are a major source of foodborne illnesses worldwide. Since human NoVs cannot be cultured in vitro, methods that discriminate infectious from non-infectious NoVs are needed. The purpose of this study was to evaluate binding of NoV genotypes GI.1 and GII.4 to histo-blood group antigens expressed in porcine gastric mucin (PGM) as a surrogate for detecting infectious virus following thermal (99 °C/5 min), 70% ethanol or 0.5% levulinic acid (LV) plus 0.01 or 0.1% sodium dodecyl sulfate (SDS) sanitizer treatments and to determine the limit of detection of GI.1 and GII.4 binding to PGM. Treated and control virus samples were applied to 96-well plates coated with 1 µg/ml PGM followed by RNase A (5 ng/µl) treatment for degradation of exposed RNA. Average log genome copies per ml (gc/ml) reductions and relative differences (RD) in quantification cycle (Cq) values after thermal treatment were 1.77/5.62 and 1.71/7.25 (RNase A) and 1.73/5.50 and 1.56/6.58 (no RNase A) for GI.1 and GII.4, respectively. Treatment of NoVs with 70% EtOH resulted in 0.05/0.16 (GI.1) and 3.54/10.19 (GII.4) log reductions in gc/ml and average RD in Cq value, respectively. LV (0.5%) combined with 0.1 % SDS provided a greater decrease of GI.1 and GII.4 NoVs with 8.97 and 8.13 average RD in Cq values obtained, respectively than 0.5% LV/0.01 % SDS. Virus recovery after PGM binding was variable with GII.4 > GI.1. PGM binding is a promising surrogate for identifying infectious and non-infectious NoVs after capsid destruction, however, results vary depending on virus strain and inactivation method.
authors.affiliation: Department of Food Science and Technology, Center for Food Safety, University of Georgia, 1109 Experiment Street, Griffin, GA, 30223, USA.
authors.forename: Olamide T
authors.initials: OT
authors.lastname: Afolayan
authors.affiliation: Department of Food Science and Technology, Center for Food Safety, University of Georgia, 1109 Experiment Street, Griffin, GA, 30223, USA. ccwebb@uga.edu.
authors.forename: Cathy C
authors.initials: CC
authors.lastname: Webb
authors.affiliation: Department of Food Science and Technology, Center for Food Safety, University of Georgia, 1109 Experiment Street, Griffin, GA, 30223, USA. jcannon@uga.edu.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Cannon
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D017250:Caliciviridae Infections / Q000821:virology*; D000431:Ethanol / Q000494:pharmacology*; D005752:Gastric Mucins / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D007982:Levulinic Acids / Q000494:pharmacology*; D029322:Norovirus / Q000737:chemistry / Q000145:classification / Q000187:drug effects / Q000302:isolation & purification*; D012259:Ribonuclease, Pancreatic / Q000032:analysis; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology*; D013552:Swine
chemical_list: D005752:Gastric Mucins; D007982:Levulinic Acids; D012967:Sodium Dodecyl Sulfate; D000431:Ethanol; D012259:Ribonuclease, Pancreatic; C032246:levulinic acid
keywords: Ethanol; Levulinic acid; Norovirus; Porcine gastric mucin; Sodium dodecyl sulfate; Surrogate; Thermal inactivation