New in situ capture quantitative (real-time) reverse transcription-PCR method as an alternative approach for determining inactivation of tulane virus

Abstract

Human noroviruses (HuNoVs) are the major cause of epidemic nonbacterial gastroenteritis. Although quantitative (real-time) reverse transcription-PCR (qRT-PCR) is widely used for detecting HuNoVs, it only detects the presence of viralRNAand 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.Wepropose that viral infectivity can be evaluated with a molecular assay based on receptor- captured viruses. In this study, we employed TV as an HuNoV surrogate to validate the HBGA-based capture qRT-PCR method against the 50% tissue culture infectious dose (TCID50) method.Weemployed type BHBGAon an immuno-well module to concentrate TV, followed by amplification of the captured viral genome by in situ qRT-PCR. We first demonstrated that this in situ capture qRT-PCR (ISC-qRT-PCR) method could effectively concentrate and detect TV.Wethen treated TV under either partial or full inactivation conditions and measured the remaining infectivity by ISC-qRT-PCR and a tissue culture-based amplification method (TCID50). Wefound that the ISC-qRT-PCR method could be used to evaluate virus inactivation deriving from 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 the virus by these methods could be reflected by the ISCqRT- PCR method and confirmed by TCID50 assay. However, the loss of the infectivity caused by damage to the viral genome (such as that fromUVirradiation) could not be effectively reflected by this method. Despite this limitation, the ISC-qRT-PCR provides an alternative approach to determine inactivation of Tulane virus. A particular advantage of the ISC-qRT-PCR method is that it is also a faster and easier method to effectively recover and detect the viruses, as there is no need to extract viralRNAor to 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. © 2014, American Society for Microbiology.