A one-step reverse transcription quantitative real-time polymerase chain reaction (RT-QPCR) method in combination with RNase treatment and low copy number samples was developed in order to examine the effect of temperature on the ability of virus capsids to protect their RNA content. The method was applied to a non-cultivable virus (GII.4 norovirus) and Feline calicivirus vaccine strain F-9 (FCV) which is often used as a norovirus surrogate. Results demonstrated that FCV RNA is exposed maximally after 2 min at 63.3 °C and this correlated with a greater than 4.5 log reduction in infectivity as assessed by plaque assay. In contrast human GII.4 norovirus RNA present in diluted clinical specimens was not exposed maximally until 76.6 °C, at least 13.3 °C greater than that for FCV. These data suggest that norovirus possesses greater thermostability than this commonly used surrogate. Further, these studies indicate that current food processing regimes for pasteurisation are insufficient to achieve inactivation of GII.4 NoVs. The method provides a novel molecular method for predicting virus infectivity.
Bibliographical noteFunding Information:
The authors wish to thank Dr. S. Clegg for useful discussions. This work was supported under the Food LINK programme by the UK Department for Environment, Food and Rural Affairs (Defra) and by industrial support from Unilever plc, Waitrose plc, Princess Cruises plc, Atlas Genetics Ltd., Scottish Shellfish Marketing Group, Premier Foods Ltd., and Evans Vanodine plc, H.S. was a recipient of a Marie Curie Fellowship financed by the European Commission.
Copyright 2009 Elsevier B.V., All rights reserved.
- Virus infectivity