title: Alternative methods to determine infectivity of Tulane virus: a surrogate for human nororvirus.
abstract: Culturable animal caliciviruses are widely-used as surrogates for human norovirus (HuNoV). The infectivity of a culturable virus was traditionally determined by plaque assay and/or 50% tissue culture infectious dose (TCID50) assay, both of which are time-consuming and labor-intensive. Molecular approaches, such as quantitative real time RT-PCR (qRT-PCR) and RT-PCR, could be used for detection of the viral genome but yet fail to determine the infectivity of a virus. In this study, we evaluated different assays for determination of infectivity of Tulane virus (TV), a surrogate for HuNoV. The infectivity of TV was measured by RNase exposure assay, RT-PCR assays, cellular-receptor-mediated capture qRT-PCR assay, receptor-mediated in situ capture qRT-PCR assay, cell-culture-mediated amplification qRT-PCR, and confirmed by TCID50 assay. RNase exposure assay was only useful for measuring TV inactivation caused by heat. Short template RT-PCR assay did not reflect inactivation status of TV. Partial reduction in viral RNA signal could be measured by long-template RT-PCR only when TV was inactivated by thermal or chlorine treatments at full-inactivation levels. Cellular-receptor-mediated capture qRT-PCR exhibited low sensitivity and specificity for the evaluation of virus infectivity. The in situ capture qRT-PCR assay could be used to evaluate virus inactivation deriving from damage to viral capsid caused by heat and chlorine. The cell-culture-mediated amplification qRT-PCR could be used as an alternative method to rapidly determine the infectivity of TV.
authors: College of Agronomy, Henan Agricultural University, No.63, Nongye Road, Zhengzhou, 450002, China; Produce Safety and Microbiology Research Unit, Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Albany, CA 94710, USA. Electronic address: shuxiaxu97@gmail.com.
authors: Shuxia
authors: S
authors: Xu
authors: Produce Safety and Microbiology Research Unit, Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Albany, CA 94710, USA; Dept. of Food Science and Technology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, China.
authors: Dapeng
authors: D
authors: Wang
authors: Produce Safety and Microbiology Research Unit, Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Albany, CA 94710, USA.
authors: David
authors: D
authors: Yang
authors: College of Agronomy, Henan Agricultural University, No.63, Nongye Road, Zhengzhou, 450002, China.
authors: Huashan
authors: H
authors: Liu
authors: 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: Peng
authors: P
authors: Tian
mesh_terms: D002139:Caliciviridae / Q000254:growth & development / Q000502:physiology*; D017250:Caliciviridae Infections / Q000821:virology*; D002460:Cell Line; D006801:Humans; D050296:Microbial Viability; D029322:Norovirus / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D038301:Virus Inactivation
keywords: Cellular culture mediated amplification qRT-PCR (CMA-qRT-PCR); Human norovirus (HuNoV); In situ capture qRT-PCR (ISC-qRT-PCR); Inactivation status; RNase exposure assay; Rapid assay; TCID(50); Tulane virus (TV)