Chlorine And Chlorine Dioxide Disinfection Law Of Human Rotavirus And Genome Damage Research

ObjectiveDespite the health risks posed by waterborne human rotavirus （HRV）, littleinformation is available concerning the effectiveness of chlorine or chlorine dioxide（ClO₂）, against HRV，and the results are inconsistent due to the differences in theexperimental conditions （e.g. pH, temperature, type of water, the aggregation status ofvirus and the detection method）. Moreover, measuring the efficiency of virusdisinfection with PCR has been criticized as inadequate due to the production offraught signals. Such a claim, however, presupposes an understanding of thetheoretical PCR response. Many previous studies have assumed that the loss in PCRsignal upon disinfection should equal the loss in infectivity, without accounting forthe fact that different positions of the virus nucleic acid may have different levels ofresistance and only a fraction of the viral genome was measured.In the present research, we determined the disinfection efficacy of chlorine andClO₂on HRV in typical water treatment using E.coli25922and coliphage MS2asbacterial and viral surrogates, respectively. Moreover, primer sets covering almost theentire viral genome were used to ensure a full capture of genome damage of HRVafter chlorine and ClO₂disinfection. In particular, we focused on （i） evaluating thedisinfection efficacy of chlorine and ClO₂,（ii） relating the loss of genomicamplification to the loss of infectivity after disinfection, and （iii） determining ifqRT-PCR assays can be used to monitor the infectivity of HRV, in particular, upondisinfection that causes inactivation via single genomic damage.Methods1. The disinfection efficacy of chlorine and ClO₂on HRV in typical water treatmentwas determined using E.coli25922and coliphage MS2as bacterial and viralsurrogates, respectively. E.coli15597was enumerated by membrane filtrationfollowed by growth on sodium sulfite agar, coliphage MS2was assayed by thedouble agar layer plaque technique on host E.coli15597, and the titration ofHRV was determined by the TCID50assay as described previously. Thedisinfection experiments were conducted in ODF buffer using two differentdisinfectants （chlorine and ClO₂） at a constant20°C. Two flasks, representing experimental replicates, were prepared for each disinfection condition. Theresidual disinfectant concentrations Ctfor each experiment were fit separately toa first-order rate equation, and the natural log values of the survival ratio for eachexperiment were fit to the efficiency factor Hom （EFH） model.2. A total of16primer sets covering almost the entire viral genome were designedwith Oligo v7.0software according to the HRV strain WA genomic sequencetaken from the NCBI GenBank database. The primers were validated by RT-PCRbefore disinfection, and the sensitivity of all the primer sets were determined byusing RT-PCR as the reference test. Efficacy of chlorine and ClO₂disinfection onHRV in typical water treatment were evaluated using both standard cell culturefor infectivity analysis and RT-PCR assays for genome stability to relate the lossin genomic amplification to the loss in infectivity after inactivation. Thepersistence of different genomic regions corresponding to inactivation levelsfrom fully infective to about1,2,3,4and>5logs （inactivation beyond thedetection limit of TCID50assay） were explored by utilizing time courseexperiments of different doses of disinfectants （chlorine:0.2,0.4,0.6mg/L andClO₂:0.05,0.1,0.2mg/L）. However, there were a mixture of inactivated andintact viruses even after60min of contact time by the largest dose of disinfectant,and we further detected the genome integrity of completely inactivated HRVafter60min of exposure to1.2mg/L chlorine and0.5mg/L ClO₂.3. A total of4specific primers were designed in the1227-2354bp of the VP4geneof HRV, which play an important role in the infection of cells. Primers in thisstudy were designed using Oligo v7.0. To prepare the cDNA standard forqRT-PCR assay, the product was separated on agarose gel, purified with the GelExtraction Kit and then cloned into vector. The sequence in the plasmid wasconfirmed by sequencing, and the quantification of the cDNA standard wascarried out by UV spectrophotometry. Efficacy of0.6mg/L chlorine disinfectionon HRV in typical water treatment were evaluated using both standard cellculture for infectivity analysis and qRT-PCR assays for genome stability tocompare the evalution results of the two methods.Results1. Systematic examination of the sensitivity of E.coli25922, MS2and HRV to chlorine and chlorine dioxide: Both chlorine-and ClO₂-induced inactivation rateof E.coli25922, MS2and HRV were dose-dependent, and inactivation curvesshowed non-liner inactivation for all tested conditions. According to theefficiency factor Hom model, Ct value ranges required for a4-log reduction ofE.coli25922, MS2and HRV at20°C by chlorine were0.61⁰.71,2.06³.19and5.55⁵.59mg/L min, respectively. Ct value ranges required for a4-log reductionof E.coli25922, MS2and HRV at20°C by ClO₂were0.37⁰.72、0.83⁰.95�'�1.21².47mg/L min, respectively.2. Relationship between loss of infectivity and genome damage for HRV afterchlorine and ClO₂disinfection: For chlorine, VP1（2012-3302bp） and VP3（21-1656bp） regions were the most susceptible genomic regions, and VP1（15-1376bp）, VP4（4-1260bp）, VP6（1-1356bp） and VP7（7-1062bp） weredetectable even when HRV was completely inactivated. Damage to VP4（1227-2354bp） was correlated with the disapperence of infectivity. For ClO₂, theregions amplified by primer sets6and12（VP3（21-1656bp） and NSP1（8-1523bp）） were found to be the most susceptible, and （VP3（1215-2569bp）） wasconsidered less susceptible. Primer sets1,2,3,4,5,8,9,10,11,13,14,15and16showed the strongest resistance, and amplification products of VP1（15-3302bp），VP2（4-2702bp），VP4（4-2354bp），VP6（1-1356bp），VP7（7-1062bp）,NSP2（4-1043bp）, NSP3（1-1050bp）, NSP4（6-748bp） and NSP5（20-664bp）were detectable even when HRV was completely inactivated.3. Chlorine inactivation and resistance of HRV evaluated by cell culture andqRT-PCR assay: Inactivation of HRV with0.6mg/L chlorine were assessed usingboth qRT-PCR and cell culture assays by chorine. Due to the detection limits ofcell culture assay, the residual virus could not be detected any more by cellculture method after a contact time of20min. In contrast, the qRT-PCR methodcan detect as low as100.12TCID50/ml of HRV in water samples. Despite thesedifferences, the chlorine inactivation of HRV detected by the cell culture assaywas comparable to those detected by qRT-PCR in this study, and we postulatethat qRT-PCR can track viral infectivity after chlorine disinfection.ConclusionsThe disinfection efficacy of ClO₂is higher than that of chlorine under the tested condition for E.coli25922, MS2and HRV.E.coli25922is the most sensitive of the three tested microorganisms, and HRVis less sensitive to both chlorine and ClO₂inactivation than MS2, which is oftentaken as being representative of viruses.The current chlorine treatment strategy may be inadequate to manage the risk ofwaterborne HRV transmission.Damage to the VP4gene （1227-2354bp） is associated with the disappearance ofHRV infectivity by chorine.No correlation between the genome damage and the loss of infectivity isobserved with the ClO₂-treated HRV.Given the fact that decreases in infectivity are always accompanied bydose-dependent decreases in qRT-PCR signal, we demonstrate that qRT-PCRcan be used to track HRV infectivity after chlorine.