|XU, SHUXIA - Henan Agricultural University|
|WANG, DAPENG - Shanghai Jiaotong University|
Submitted to: Food Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/12/2014
Publication Date: 6/1/2015
Citation: Xu, S., Wang, D., Yang, D., Tian, P. 2015. Alternative methods to determine infectivity of Tulane virus: a surrogate for human norovirus. Food Microbiology. DOI: 10.1016/j.fm.2014.12.004.
Interpretive Summary: Culturable animal caliciviruses are widely-used as surrogates for human norovirus (HuNoV), the latter of which cannot yet be grown in tissue culture. Traditionally, the infectivity of a culturable virus was determined by plaque and/or 50% tissue culture infectious dose (TCID50) assay, both of which are relatively time-consuming and labor-intensive. Although molecular approaches such as quantitative real time RT-PCR (qRT-PCR) and RT-PCR have been used for the detection of viral genome, they are incapable of determining viral infectivity. In this study, we evaluated alternative assays for determinating the infectivity of the Tulane virus (TV), a surrogate for HuNoV. Treated with heat, UV irradiation, or chlorine to points where TV was inactivated partially or fully, the treated virus was measured for infectivity using a variety of assays, which is then compared with the TCID50 assay as the reference. In the RNase exposure assay, RNase was used to remove the non-infectious free viral RNA from detection in qRT-PCR. In short and long RT-PCR assays, longer target size in viral genome were used. CRMC-qRT-PCR and ISC-qRT-PCR assays utilize virus receptors from cell culture and human saliva (respectively) to capture and segregate viable viral particles from everything else, and is then quantitated by qRT-PCR. Finally, culture-mediated qRT-PCR assay (CMA-qRT-PCR) allows captured viruses to replicate for 24 hours in cell culture, and is similarly followed by qRT-PCR. The results suggest that RNase exposure assay, CMRC-qRT-PCR, and LT-RT-PCR have limited value toward the evaluation of TV inactivation. ISC-qRT-PCR could be used as an alternative method for evaluating viral inactivation from heat and chlorine, but not for UV. Overall, the CMA-qRT-PCR method takes significantly less time and labor than the current plaque and TCID50 assays (5 days) and exhibits high sensitivity in distinguishing the infectivity of TV.
Technical Abstract: Culturable animal caliciviruses are widely-used as surrogates for human norovirus (HuNoV), which can not replicate in cells. 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. TV was treated with heat, UV irradiation, or chlorine toward partial or full inactivation. Treated TV was measured for infectivity by RNAse exposure assay, cellular-receptor-mediated capture qRT-PCR (CRMC-qRT-PCR) assay, receptor-mediated in situ capture qRT-PCR assay (ISC-qRT-PCR), cell-culture-mediated amplification qRT-PCR (CMA-qRT-PCR), and TCID50 assay. RNase exposure assay had limited value in identification of inactivation status of TV, and was only useful for measuring TV inactivation caused by heat. CRMC-qRT-PCR assay exhibited low sensitivity and specificity for the evaluation of virus infectivity. Less than 2 logs reduction in the captured viral genomic signals (CVGS) were observed when TV was treated at full inactivation conditions for heat, chlorine, and UV irradiation. The ISC-qRT-PCR method could be used to evaluate virus inactivation deriving from damage to viral capsid caused by heat and chlorine. However, the ISC-qRT_PCR method underestimates the loss of infectivity caused by damage to the viral genome (such as from UV irradiation). CMA-qRT-PCR, which combines the molecular approaches with the traditional tissue culture based assay, could be used as an alternative method to rapidly determine the infectivity of TV. An infection kinetics study demonstrated that viral amplification could be detected in cells as early as 12 hours post-infection (hpi) and anplification would reach a plateau at 48 hpi. There was a good correlation between the inactivation status of TV as measured by the CMA-qRT-PCR method, and TCID50 assay. Overall, the CMA-qRT-PCR method takes significantly less time and labor than the current plaque and TCID50 assays (5 days), and exhibits high sensitivity in distinguishing infectivity of TV.