Assessing UV Inactivation of Adenovirus 41 Using Integrated Cell Culture Real‐Time qPCR/RT‐qPCR

Abstract

Enteric adenoviruses are among most UV-resistant viruses in water. Cytopathic effects (CPE)-based cell culture TCID50 assay as a conventional virus assessment approach has major drawbacks for enteric adenovirus since it is selective on cell lines and takes longer time to show CPE. Integrated cell culture real-time quantitative PCR (ICC-qPCR) and reverse transcriptase (RT)-qPCR were applied in this study, in comparison with TCID50, to assess UV inactivation of adenovirus type 41 (Ad41) in water. Adenovirus type 41 was exposed to UV doses of 40, 80, 160, and 320 mJ/cm2 using a collimated beam apparatus. There was no significant difference of inactivation at conducted UV doses between measurements using TCID50 assay and ICC-RT-qPCR. Both assays fitted the Chick-Watson model at 95% confidence level. The inactivation measured by ICC-qPCR did not fit the Chick-Watson model. In summary, ICC-RT-qPCR is the most appropriate alternate to CPE-based assay for assessing UV inactivation of enteric adenoviruses.

Applicability of integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) for the simultaneous detection of the four human enteric enterovirus species in disinfection studies

“…These disadvantages, inherent to the method, present critical barriers in assessing the efficacy of disinfection applications against human enteric viruses. To overcome some of these hurdles, tissue/cell culture based methods have been integrated with rapid molecularbased detection methods and have been successfully applied in disinfection studies of human adenoviruses and enteroviruses (Ding et al, 2017;Gerrity et al, 2008;Ko et al, 2005;Rodriguez et al, 2013;Ryu et al, 2015). Of particular interest, Mayer et al (2010) reported the simultaneous quantification of two serotypes of Enterovirus B (coxsackievirus B6 and echovirus 12) and one serotype of Enterovirus C (poliovirus 1) using a singular tissue culture line with three serotype specific reverse transcriptase quantitative PCR (RTqPCR) assays.…”

Recent Update on UV Disinfection to Fulfill the Disinfection Credit Value for Enteric Viruses in Water

“…(A) Standard linear regression models for LP-UV inactivation of adenovirus 2 ,,,,− + 5 , + 15 (not significantly different), AdV40, and AdV41. ,, Blue squares, orange triangles, and gray diamonds represent extracted data, while linear regressions are described with blue, orange, and gray lines for AdV2 + 5, 40, and 41, respectively. The 95% confidence interval is indicated by the shaded area.…”

“…Damage to different components of viruses caused by varying wavelengths has implications for the accuracy of detection methods. Molecular methods are a convenient proposition for enteric virus detection because infectivity assays for quantification are highly dependent on cell lines and can take days to weeks to produce cytopathic effects (CPEs). ,, Long-range qPCR (LR-qPCR) has been previously used for viral quantification after UV damage ,, and has been shown to give similar results for infectivity-based methods when using LP-UV . While molecular methods are sensitive and precise, they are only effective in detecting damage to their specific molecular target.…”

“…The data sets were selected according to the 2015 PRISMA-P meta-analysis protocols . These data sets followed the listed criteria: (1) peer-reviewed articles excluding review papers, dissertations, online documents or reports, proceedings of a conference or meeting, (2) peer-reviewed articles containing original inactivation data from original experiments conducted by the authors and published from 1984 to January 2020 and written in English, (3) publications in which an LP-UV or MP-UV lamp was used as the UV source, with the calculated fluence determined by using a collimated beam apparatus, (4) experimental data included obtained from water-related (buffer, treated water, or reclaimed water) experiments, (5) a combination of UV fluence (the product of UV irradiance and contact time) and log 10 reduction values (LRVs) from a standard inactivation study using cell culture-based experiments, preferably a plaque assay based calculation because these assays exhibit higher sensitivity than comparable cell culture data when integrated, (6) inactivation data sets excluded without clear protocol descriptions and details pertaining to the cell line host and/or qPCR, and (7) long-range qPCR or RT-PCR or a combination of integrated cell culture data only included if shown to be comparable to cell culture infectivity-based methods but excluded when the authors demonstrate significant differences between the quantification methods. , The kinetics of the virus infectivity and UV dose were calculated using the Chick–Watson model described in the following formula where Nt is the virus concentration at specific sampling times, N 0 the initial concentration of the viruses at time zero, k the inactivation rate constant by UV (cm 2 /mJ), and F the UV fluence or dose (mJ/cm 2 ).…”

“…Ninety-four articles were chosen after the initial screening. In the end, for the LP-UV data sets, we narrowed down the references to three articles on MNV, − two articles on FCV, , one article for TV, 10 articles for PV, − one article for HAV, five articles for HRV, ,,,, two articles for EV, , three articles for CVB, ,, and 14 articles for AdV. ,,,,,,− For MP-UV, five studies related to adenovirus serotype 2 ,,,, were selected for data analysis. Regarding the UV-AOP studies, one paper for inactivation by UV + H 2 O 2 for both adenovirus serotype 2 and one paper regarding UV + TiO 2 inactivation using MNV were extracted.…”