J Simonet1, C Gantzer. 1. Laboratoire de Chimie Physique et Microbiologie pour l'Environnement, LCPME, Equipe Microbiologie et Physique, Faculté de Pharmacie, UMR 7564 CNRS/Université Henri Poincaré Nancy I, BP, Nancy Cedex, France. julien.simonet@pharma.uhp-nancy.fr
Abstract
AIMS: This study was undertaken to gain an understanding of the factors that influence viral RNA degradation in the presence of chlorine dioxide (ClO(2)), which will be very useful in helping to define the significance of the presence of the viral genome in disinfected water. METHODS AND RESULTS: We focused our investigation on the influence of ClO(2) on extracted RNA on the one hand, and on the infectious virus on the other. Our first results show that RNA degradation, like viral inactivation, is dose dependent. The influence of the spatial organization of the targeted genomic sequence, as well as that of its size and location (and/or sequence) on degradation of the Poliovirus 1 genome by ClO(2), was studied using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The results show that the preferential sites of action of ClO(2) appear to be located in the untranslated regions, 5'- and 3'-UTR, a phenomenon influenced by both the presence of secondary structures and the genomic sequence in these regions. Our results also reveal a rapid decrease of infectious particles quantified by the cell culture for the applied dose. Comparison between cell culture and real-time PCR for viral detection reveals disagreement following disinfection treatment, even for the largest targeted fragment (a 6,989-base fragment representing the quasi-whole viral genome). CONCLUSIONS: The detection of genome fragments is insufficient to confirm the presence of the infectious virus, as each targeted fragment shows a different sensitivity. Hence, the smallest targeted fragment (76 bp) persisted throughout the analysis period, while the longest targeted fragment (6,989 bp) disappeared very rapidly. Highly sensitive regions (i.e. 5'- and 3'-UTR) should be targeted to avoid an overestimation of the risk of viral infection using molecular biology methods in water following disinfection. Further studies in this area are needed. SIGNIFICANCE AND IMPACT OF THE STUDY: To date, it has not been possible to routinely apply virological controls to drinking water because of the time-consuming nature of the gold standard technique (cell culture) and its inability to detect all serotypes (e.g. Norovirus). Molecular techniques (e.g. real-time RT-PCR) constitute a solution to the rapid and specific detection of all the serotypes. However, ignorance of the mechanisms of viral degradation prevents the validation of PCR for the measurement of the risk of infection to humans following disinfection treatment.
AIMS: This study was undertaken to gain an understanding of the factors that influence viral RNA degradation in the presence of chlorine dioxide (ClO(2)), which will be very useful in helping to define the significance of the presence of the viral genome in disinfected water. METHODS AND RESULTS: We focused our investigation on the influence of ClO(2) on extracted RNA on the one hand, and on the infectious virus on the other. Our first results show that RNA degradation, like viral inactivation, is dose dependent. The influence of the spatial organization of the targeted genomic sequence, as well as that of its size and location (and/or sequence) on degradation of the Poliovirus 1 genome by ClO(2), was studied using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The results show that the preferential sites of action of ClO(2) appear to be located in the untranslated regions, 5'- and 3'-UTR, a phenomenon influenced by both the presence of secondary structures and the genomic sequence in these regions. Our results also reveal a rapid decrease of infectious particles quantified by the cell culture for the applied dose. Comparison between cell culture and real-time PCR for viral detection reveals disagreement following disinfection treatment, even for the largest targeted fragment (a 6,989-base fragment representing the quasi-whole viral genome). CONCLUSIONS: The detection of genome fragments is insufficient to confirm the presence of the infectious virus, as each targeted fragment shows a different sensitivity. Hence, the smallest targeted fragment (76 bp) persisted throughout the analysis period, while the longest targeted fragment (6,989 bp) disappeared very rapidly. Highly sensitive regions (i.e. 5'- and 3'-UTR) should be targeted to avoid an overestimation of the risk of viral infection using molecular biology methods in water following disinfection. Further studies in this area are needed. SIGNIFICANCE AND IMPACT OF THE STUDY: To date, it has not been possible to routinely apply virological controls to drinking water because of the time-consuming nature of the gold standard technique (cell culture) and its inability to detect all serotypes (e.g. Norovirus). Molecular techniques (e.g. real-time RT-PCR) constitute a solution to the rapid and specific detection of all the serotypes. However, ignorance of the mechanisms of viral degradation prevents the validation of PCR for the measurement of the risk of infection to humans following disinfection treatment.
Authors: Kathy L Toohey-Kurth; Donna M Mulrooney; Susanne Hinkley; Mary Lea Killian; Janice C Pedersen; Mangkey A Bounpheng; Roman Pogranichniy; Steve Bolin; Roger Maes; Rebecca L Tallmadge; Laura B Goodman; Beate M Crossley Journal: J Vet Diagn Invest Date: 2020-09-30 Impact factor: 1.279