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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

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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.

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Acknowledgments

We thank Amy Mann and Alison Payton for their laboratory assistance throughout this project. This work was funded by the USDA-NIFA Food Virology Collaborative, NoroCORE (2011-68003-30395).

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Authors and Affiliations

  1. Department of Food Science and Technology, Center for Food Safety, University of Georgia, 1109 Experiment Street, Griffin, GA, 30223, USA

    Olamide T. Afolayan, Cathy C. Webb & Jennifer L. Cannon

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  1. Olamide T. Afolayan
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  2. Cathy C. Webb
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  3. Jennifer L. Cannon
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Correspondence to Jennifer L. Cannon.

Electronic supplementary material

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12560_2015_9219_MOESM1_ESM.docx

Supplementary material 1 Supplementary Fig. 1 Experimental design for PGM plus RNase A assay for thermal treated and non-treated GI.1 and GII.4 (Sydney) viruses. PGM, CBS, and RNase A added to wells of microtiter plate as listed in rows A-H. Sample/controls added to wells as listed in columns 1-5. Shaded wells were not used in the assay. Three replicate trials were performed. (DOCX 42 kb)

12560_2015_9219_MOESM2_ESM.docx

Supplementary material 2 Supplementary Fig. 2. Experimental design for PGM plus RNase A assay for LV/SDS treated and non-treated GI.1 and GII.4 (New Orleans) viruses. PGM, CBS, and RNase A added to wells of microtiter plate as listed in rows A-H. Sample/controls added to wells as listed in columns 1-12. Shaded wells were not used in the assay. Three replicate trials were performed. (DOCX 68 kb)

12560_2015_9219_MOESM3_ESM.docx

Supplementary material 3 Supplementary Fig. 3. Experimental design for PGM plus RNase A assay for EtOH treated and non-treated GI.1 and GII.4 (Sydney) viruses. PGM, CBS, and RNase A added to wells of microtiter plate as listed in rows A-H. Sample/controls added to wells as listed in columns 1-12. Shaded wells were not used in the assay. Two replicate trials were performed. (DOCX 41 kb)

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Afolayan, O.T., Webb, C.C. & Cannon, J.L. 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. Food Environ Virol 8, 70–78 (2016). https://doi.org/10.1007/s12560-015-9219-z

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