Comparison of Cell-based and PCR-based Assays as Methods for Measuring Infectivity of Tulane Virus

Introduction: Human noroviruses (HuNoV) are not culturable viruses. The detection of HuNoV mainly depends on RT-PCR-based assays which cannot measure the infectivity of the virus. We propose that the detection of encapsidated viral genome could be used as an indicator for viral infectivity.

Purpose: Tulane virus was used as a surrogate for HuNoV to compare correlations between the cell-based and the PCR-based assays for measuring viral infectivity. 

Methods: Plaque assay and tissue culture 50% infectious dose (TCID₅₀) assay were used as cell-based assays. RNase exposure, porcine-gastric-mucin-mediated in-situ-capture qRT-PCR (PGM-ISC-qRT-PCR), and antibody-mediated in-situ-capture qRT-PCR (Ab-ISC-qRT-PCR) assays were used as the PCR-based assays to measure for encapsidated viral genome. 

Results: Ten batches of viral stocks ranging from 3.41 x 10⁵ to 6.67 x 10⁶ plaque forming units (PFUs) were used for side by side comparison with PFU as a reference. The results indicate that one PFU was equivalent to 6.7 ± 2.3 TCID₅₀ units, 9.8 ± 10.9 RNase-untreated genomic copies (GCs), 2.8 ± 3.1 RNase-treated GCs, 0.07 ± 0.07 PGM-ISC-qRT-PCR GCs, and 0.52 ± 0.39 Ab-ISC-qRT-PCR GCs. We observed that while the cell culture-based assays were consistent with each other, the TCID₅₀ assay was 6.7-times more sensitive than the plaque assay. However, the PCR-based assays were not always consistent with the cell culture-based assays. The very high variations in GCs as measured by both ISC-RT-qPCR assays made them difficult to correlate against the relatively small variations (<20-fold) in the PFUs or TCID₅₀ units as measured by the cell culture-based assays.  

Significance: Two cell-based assays were consistent with each other, with the TCID₅₀ assay being 6.7-times more sensitive than the plaque assay. However, there was no significant correlation between the cell-based assays and the PCR-based assays.