A New In Situ Capture-qRT-PCR (ISC-qRT-PCR) Method for an Alternative Approach to Determine Inactivation of Human Norovirus

Introduction: Human noroviruses (HuNoVs) are the major cause of epidemic non-bacterial gastroenteritis.  Although quantitative real-time RT-PCR (qRT-PCR) is widely used for detecting HuNoVs, it only detects the presence of viral RNA and does not indicate viral infectivity.  Human blood group antigens (HBGAs) have been identified as receptors/co-receptors for both HuNoVs and Tulane Virus (TV), and are crucial for viral infection.  We propose that viral infectivity can be evaluated with a molecular assay based on receptor-captured viruses.  

Purpose: We employed TV as a HuNoV surrogate to validate the HBGA-based ISC-qRT-PCR method against the TCID₅₀ method, and applied the validated ISC-qRT-PCR method to determine the inactivation parameters for HuNoV. 

Methods: We bound HBGAs to immuno-PCR well-strips (Nunc Top-Yield), and used the immobilized receptors to concentrate TV or HuNoV.  This is followed by amplification of the captured viral genomic signal (CVGS) by in situ qRT-PCR.

Results: We first demonstrate that this ISC-qRT-PCR method could effectively concentrate and detect TV.  We then treated TV under either partial or full-inactivation conditions, and measured the remaining CVGS by ISC-qRT-PCR and tissue-culture-based amplification method (TCID₅₀).  We found that the ISC-qRT-PCR method could be used to evaluate TV inactivation caused by damage to the capsid, and study interactions between the capsid and viral receptor.  Heat, chlorine, and ethanol treatment primarily affect the capsid structure, which in turns affects the ability of the capsid to bind to viral receptors.  Inactivation of TV by these methods could be reflected by ISC-qRT-PCR method and confirmed by TCID₅₀ assay.  However, the loss of infectivity caused by damage to the viral genome (such as from UV irradiation) could not be effectively reflected by this method.  With this validated method, we further determined inactivation parameters for HuNoV.  HuNoV could be inactivated by heat at 72°C for 4 minutes, by chlorine at a final concentration of 16 ppm in less than 1 minute, and by UV irradiation at 1 J/cm².  However, ethanol had a limited effect on high-titer samples of HuNoV (> 10³copies/sample). 

Significance: We demonstrated that ISC-qRT-PCR provides an alternative approach to determine the inactivation of HuNoV.   A particular advantage of the ISC-qRT-PCR method is that it is a faster and easier method to effectively recover and detect viruses, as there is no need to extract viral RNA, nor transfer the captured virus from magnetic beads to PCR tubes for further amplification. Therefore, ISC-qRT-PCR can be easily adapted for use in automated systems for multiple samples.