title: Comparison of cell-based and PCR-based assays as methods for measuring infectivity of Tulane virus.
abstract: In this study, we used Tulane virus (TV) as a surrogate for HuNoV to evaluate for correlation between two cell-based assays and three PCR-based assays. Specifically, the cell-based plaque and TCID50 assays measure for infectious virus particles, while the PCR-based RNase exposure, porcine gastric mucin in-situ-capture qRT-PCR (PGM-ISC-qRT-PCR), and antibody in-situ-capture qRT-PCR (Ab-ISC-qRT-PCR) assays measure for an amplicon within encapsidated viral genome. Ten batches of viral stocks ranging from 3.41 × 10(5) to 6.67 × 10(6) 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 6.69 ± 2.34 TCID50 units, 9.75 ± 10.87 RNase-untreated genomic copies (GCs), 2.87 ± 3.05 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-based assays were consistent with each other, the TCID50 assay was more sensitive than the plaque assay. In contrast, the PCR-based assays were not always consistent with the cell-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-based assays.
authors.affiliation: Produce Safety and Microbiology Research Unit, Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Albany, CA 94710, USA.
authors.forename: Lei
authors.initials: L
authors.lastname: Shan
authors.affiliation: Produce Safety and Microbiology Research Unit, Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Albany, CA 94710, USA.
authors.forename: David
authors.initials: D
authors.lastname: Yang
authors.affiliation: MOST-USDA Joint Research Center for Food Safety & Bor Luh Food Safety Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, PR China.
authors.forename: Dapeng
authors.initials: D
authors.lastname: Wang
authors.affiliation: Produce Safety and Microbiology Research Unit, Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Albany, CA 94710, USA. Electronic address: peng.tian@ars.usda.gov.
authors.forename: Peng
authors.initials: P
authors.lastname: Tian
mesh_terms: D000818:Animals; D002139:Caliciviridae / Q000254:growth & development*; D002460:Cell Line; D008253:Macaca mulatta; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D019562:Viral Load / Q000379:methods*; D010948:Viral Plaque Assay / Q000379:methods*
keywords: Correlation; ISC-qRT-PCR assay; Infectivity; Plaque assay; RNase exposure assay; TCID(50) assay; Tulane virus