Virolysis of Feline Calicivirus and Human GII.4 Norovirus following Chlorine Exposure under Standardized Light Soil Disinfection Conditions

Transcription

1 2113 Journal of Food Protection, Vol. 74, No. 12, 2011, Pages doi: / x.jfp Copyright G, International Association for Food Protection Virolysis of Feline Calicivirus and Human GII.4 Norovirus following Chlorine Exposure under Standardized Light Soil Disinfection Conditions P. NOWAK, 1 J. R. TOPPING, 1 K. BELLAMY, 2 V. FOTHERINGHAM, 3 J. J. GRAY, 4 J. P. GOLDING, 5 G. WISEMAN, 6 AND A. I. KNIGHT 1 * 1 Leatherhead Food Research, Randalls Road, Leatherhead, Surrey KT22 7RY, UK; 2 Unilever Central Resources Ltd., Colworth Park, Sharnbrook, Bedford, Bedfordshire MK44 1LQ, UK; 3 Evans Vanodine International plc, Brierley Road, Preston, Lancashire PR5 8AH, UK; 4 Department of Microbiology, Norfolk and Norwich University Hospital, Bowthorpe Road, Norwich, Norfolk NR2 3TX, UK; 5 Health Protection Agency, Centre for Infections, Colindale Avenue, London NW9 5EQ, UK; and 6 Premier Analytical Services, The Lord Rank Centre, Lincoln Road, High Wycombe, Buckinghamshire HP12 3QS, UK MS : Received 16 February 2011/Accepted 16 June 2011 ABSTRACT The relationship between the infectivity of the feline calicivirus (FCV) vaccine strain F-9 and capsid destruction (virolysis) in response to available chlorine was investigated under standardized light soil disinfection conditions. Virolysis was measured using RNase pretreatment (in order to destroy exposed RNA following chlorine treatment) and quantitative reverse transcription PCR. A comparison between the results of plaque assays and virolysis following exposure of FCV F-9 grown in tissue culture to different concentrations of available chlorine showed a similar log-linear relationship, with.4-log reductions occurring at 48 and 66 ppm, respectively. Three non-epidemiologically linked human GII.4 noroviruses (NoVs) present in dilute clinical samples showed behavior similar to each other and were 10 times more resistant to virolysis than cultured FCV F-9. FCV F-9 when present in dilute human GII.4 samples acquired increased resistance to virolysis approaching that of human NoVs. This study represents a direct comparison between the virolysis of a surrogate virus (FCV F-9) and that of human GII.4 NoVs within the same matrix in response to available chlorine. The results support the view that matrix effects have a significant effect on virus survival. Noroviruses (NoVs) are currently recognized as the single most common cause of acute nonbacterial gastroenteritis in the industrialized world (21). Human NoVs may be divided into different genogroups and genotypes, of which genogroup II, genotype 4 (GII.4) is currently most commonly detected in association with disease. NoVs and other enterically infecting viruses are commonly spread from person to person by the fecal-oral route following contact with contaminated feces and fomites (7). Largescale outbreaks of gastroenteritis may progress across the globe and can be associated with contaminated water or food (1, 10, 26). The development of suitable control measures for NoVs is generally considered to be limited by the inability to culture human NoVs. Most data relating to the inactivation of NoVs have been obtained from the use of two related surrogate virus models, most notably, feline calicivirus (FCV) (4, 6) and, more recently, murine NoV-1 (3, 6, 33). The relevance of inactivation studies based on the behavior of surrogate viruses is unclear, and it remains to be shown that surrogate viruses can accurately reflect the behavior of the diverse human NoVs. * Author for correspondence. Tel: z44 (0) ; Fax: z44 (0) ; aknight@leatherheadfood.com. Alternative methods for predicting virus infectivity based on molecular approaches have been investigated, including quantitative studies based on quantitative reverse transcription PCR (RT-QPCR) of viral RNA as a substitute for plaque assays. However, the relationship between virus RNA survival in RT-PCR and infectivity in plaque assays is not straightforward, since infectivity may be abolished despite the persistence of residual detectable RNA (19). A number of studies have combined enzymatic pretreatments and RT-PCR either using both proteinase K and RNase pretreatment (2, 19, 20, 22) or RNase pretreatment alone (14, 18, 29) in order to provide a more exact relationship. These approaches attempt to quantify the exposure of capsid-protected RNA following a treatment or process as a measure of capsid destruction (virolysis). A number of advantages exist in the application of these approaches to noncultivable viruses, including human NoVs. First, only small quantities of dilute clinical samples are required; second, the virus can be examined in different matrices; third, the data can be directly compared with those obtained from surrogate viruses using spiking experiments; and fourth, the data can be compared with those obtained in plaque assays using surrogate viruses. Finally, absolute copy number determination is not required since the experiments measure relative differences in RNA exposure.

2 2114 NOWAK ET AL. J. Food Prot., Vol. 74, No. 12 We have used this approach to investigate the relationship between the virolysis of FCV F-9 and infectivity in plaque assays and compared this with the virolysis of human GII.4 NoVs in response to chlorine treatment. Chlorine treatment remains the most significant and widespread control measure for maintenance of a safe water supply and disinfection. Failure in chlorine treatment can lead to large-scale outbreaks of NoV gastrointestinal disease (25). Studies utilizing the surrogate FCV F-9 in plaque assays have shown that exposure to 300 ppm of available chlorine for 10 min at room temperature in culture medium only resulted in a 1-log reduction in titer (8). In contrast, studies using purified FCV F-9 have shown that FCV F-9 infectivity was reduced by more than 4.6 log after 5 min of exposure to 3 ppm of available chlorine (28). The sensitivity to available chlorine varies depending on the purity of the virus preparation (30). The range of published data illustrates the difficulties associated with measuring inactivation by available chlorine, since the virucidal effects are dependent on the virus purity, viral aggregation, ph, protein content, other matrix components, and the presence of inorganic ions and organic matter, all of which contribute to oxidative demand. The variation in data has led to the development of European standard method EN for disinfectant testing. According to European Standard EN , effective virucidal disinfection requires a 4- log reduction in infectivity. The standard uses clean ( light soil ) conditions (0.03% protein and hard water containing calcium carbonate) in order to standardize oxidative demand between samples (9). The aims of this investigation were to determine the relationship between infectivity and virolysis for the surrogate FCV vaccine strain F-9 and examine the virolysis of human GII.4 NoVs following chlorine exposure under standardized light soil disinfection conditions. MATERIALS AND METHODS Virus samples. FCV-F9 culture conditions were as previously described (6). Stock FCV F-9 was kindly supplied by Dr. M. Carter (University of Surrey, Guildford, UK). Three different non epidemiologically linked GII.4 NoV clinical stool samples were kindly supplied by the United Kingdom Health Protection Agency reference laboratory, namely, H , H , and H (abbreviated to 249, 432, and 533 in this article). The NoV genogroup and subtype of the NoV viruses were confirmed by sequence analysis (11, UK Health Protection Agency reference laboratory, unpublished data). Stool samples were resuspended at 10% (vol/vol) in Dulbecco s phosphate-buffered saline (PBS) and briefly clarified by microcentrifugation. In common with others (6), we chose not to further purify FCV F-9 virus or purify NoV virus from stool samples, in an effort to mimic the natural state of enteric viruses as they might be found in the environment. Plaque assays. Plaque assays were performed as previously described (6) in duplicate independent experiments with triplicate determinations at each dilution. The percent survival was in comparison to the titer obtained following exposure to neutralized 0 ppm available chlorine. Preparation of chlorine test solutions. Available chlorine solutions were freshly prepared by diluting sodium hypochlorite stock solution (10 to 13% available chlorine; Sigma, Poole, Dorset, UK). Available chlorine was measured by titration (17). The available chlorine present in the stock solution was ppm. Chlorine inactivation experiments. The protocol used was based on the European standard EN for clean light soil disinfectant testing (9). Typically, FCV F-9 virus samples were prepared by diluting 5 ml of virus stock (10 9 PFU/ml) in 495 ml of hard water (1.2 mm MgCl 2, 2.5 mm CaCl 2, 3.33 mm NaHCO 3, ph , supplemented with 0.3% [wt/vol] bovine serum albumin [BSA] solution). For FCV F-9 experiments, the stock was further diluted by the addition of 500 ml of PBS to yield an FCV F-9 test solution with a final concentration of 0.15% BSA. The inactivation experiments used 20 ml of the FCV F-9 test solution, equivalent to 10 5 PFU, in a total volume of 100 ml. For GII.4 NoV samples, 495 ml of 10% (wt/vol) clarified stool in PBS (as described above) was diluted with 495 ml of hard water supplemented with 0.3% BSA and spiked with 5 ml of FCV F-9 virus stock. The GII.4 test solution therefore contained FCV F-9 as an internal control in order to examine any effects of the fecal matrix. Immediately before use, freshly prepared chlorine solution was diluted in hard water and 80 ml added to 20 ml of the virus test solution to yield the desired final available chlorine concentration in a total volume of 100 ml of hard water containing 0.03% BSA, 1.0% (vol/vol) GII.4 NoV sample, and FCV F-9 virus spike. Following the addition of fresh chlorine solution, the samples were incubated at 20uC for 30 min in order to mimic chlorine exposure during cleaning. After chlorine treatment, all samples were neutralized by the addition of 35 ml of 0.1 M sodium thiosulphate and analyzed with and without RNase pretreatment and RT-QPCR or plaque assays. RNase pretreatment. Following neutralization of chlorinetreated samples, RNase treatment was carried out by the addition of 14 ml of10 reaction buffer and 1 ml (10 U) of RNase I (RNase ONE, Promega UK Ltd., Southampton, UK) in a total volume of 150 ml. The solution was incubated at 37uC for 15 min. RNA isolation and RT-QPCR. Following neutralization and RNase treatment, viral RNA was extracted using a QIAamp viral RNA kit (Qiagen Ltd., Crawley, UK). RNA was eluted with 60 mlof the AVE buffer provided, and 2 ml (approximately RT- QPCR copies) used in each experiment. RT-QPCR was as previously described for the amplification of GII NoVs based on the amplification of a 97-bp amplicon derived from the conserved polymerase open reading frame 2 junction (12) and an equivalent 89-bp fragment of the FCV polymerase gene of FCV F-9 (29). The RT-QPCR conditions were as described previously (29), enabling the simultaneous independent PCR of both amplicons. Copy number was estimated in comparison to standard curves generated from plasmid templates with inserts containing the PCR target sequences. The percent RNA survival following neutralization, RNase treatment, RNA extraction, and RT-QPCR at x ppm available chlorine was calculated as follows: (copy number at x ppm available chlorine z Na 2 S 2 O 3 z RNase)/(starting copy number z Na 2 S 2 O 3 ) 100. RT-QPCR data for FCV F-9 were obtained from three independent experiments, each in duplicate with triplicate RT-QPCR. Results for NoVs and spiked FCV F-9 were obtained from the three non epidemiologically linked GII.4 samples, each analyzed in duplicate with triplicate RT-QPCR. The percent RNA survival was calculated from the copy number determinations obtained within each experiment. Protein determination. The addition of BSA was used to normalize the chlorine oxidative demand in all samples in

3 J. Food Prot., Vol. 74, No. 12 VIROLYSIS OF FELINE CALICIVIRUS AND HUMAN GII.4 NOROVIRUS 2115 FIGURE 1. The percent survival of the FCV F-9 RNA RT-QPCR target following exposure of FCV F-9 virus to available chlorine, chlorine neutralization, and RNase digestion (&) in comparison with the percent survival of FCV-F9 in plaque assays (n). No plaques were detected following exposure to 50 ppm of available chlorine or greater, and likewise, no RT-QPCR signal was obtained following exposure to 75 ppm of available chlorine or greater. The regression curves for the RT-QPCR data (...)(r 2 ~ ) and plaque assay data (----) (r 2 ~ ) are shown. Error bars represent the standard deviations from three independent experiments. accordance with EN (9). The protein concentration in the virus test samples prior to the addition of chlorine was confirmed as 0.15% using the Bradford assay (5) and equivalent to a final concentration of 0.03%, required by the standard testing method. Briefly, aliquots (20 ml) of the virus test solution were incubated with 250 ml of Bradford reagent (Sigma). After 45 min, the absorbance was read at 605 nm and the protein concentration determined using a BSA standard curve. Statistical analysis. All experiments were performed at least in duplicate with triplicate RT-PCR. The standard deviation from the mean, analysis of variance, and tests of significance using Student s unpaired t test were calculated using Microsoft Excel version RESULTS Requirement for RNase pretreatment. Previously published data using RT-PCR and analysis by agarose gel electrophoresis have suggested that treatment of FCV F-9 with available chlorine alone can be sufficient to abolish RNA signals from exposed RNA (19). In order to investigate the requirement of the RNase step using RT- QPCR, we first investigated the protection of exposed FCV F-9 viral RNA following chlorine treatment at different concentrations in preliminary experiments. Following exposure to low concentrations (10 ppm) of available chlorine, RNase pretreatment was required in order to maximize degradation of exposed RNA (P, 0.001, data not shown). At 100 ppm, RT-QPCR signals could be significantly reduced, to,0.03% of the starting copy number, without added RNase; however, the addition of RNase resulted in elimination (.4-log reduction) of the RT- QPCR signal. The RNase pretreatment step was therefore retained in order to maximize degradation of exposed RNA and avoid false-positive signals. There was no significant difference between the copy numbers in controls digested with RNase and undigested samples in the absence of available chlorine, showing that no free exposed RNA was present in the samples. Comparison of plaque assays and virolysis in response to available chlorine for surrogate FCV F-9. Figure 1 shows a comparison between the loss of infectivity measured by plaque assays and reductions in the RT-QPCR target survival measured by virolysis. The data show a close agreement between the two assays, although the correlation coefficient within the RNase RT-QPCR assay was not as good as that in the plaque assay (r 2 ~ and , respectively). Extrapolation of the slope of the RNase RT- QPCR data was consistent with,0.01% survival of the RT- QPCR target at 66 ppm, and this was in agreement with a lack of detectable RT-QPCR signal at 75 ppm of available chlorine or greater. Extrapolation of the slope of the plaque assay data predicted,0.01% virus survival at 48 ppm, i.e., a.4-log reduction in infectivity, and this was in agreement with the lack of detectable plaques at 50 ppm of available chlorine or greater. Based on a comparison of the two approaches, the predicted concentration of available chlorine required to produce a.4-log reduction in infectivity for FCV F-9 was only 18 ppm greater (66 ppm) by the RNase RT-QPCR assay than that obtained from the plaque assay (48 ppm). Virolysis of human GII.4 NoVs in clinical isolates in response to available chlorine. Figure 2 shows the mean percent survival of the GII.4 NoV RNA RT-QPCR target from the three different dilute NoV GII.4 samples in response to available chlorine. All samples were also spiked with the FCV F-9 virus. The three non epidemiologically linked GII.4 NoV samples yielded very similar data (Fig. 2), resulting in a log-linear exponential fit with an r 2 value of The slope of the graph was used to estimate the available chlorine concentration required in order to reduce the survival of the NoV RT-QPCR target to,0.01% (equivalent to a.4-log reduction in FCV F-9 infectivity as shown in Fig. 1). This equivalent reduction required exposure of NoV GII.4 samples to 610 ppm of available chlorine.

4 2116 NOWAK ET AL. J. Food Prot., Vol. 74, No. 12 FIGURE 2. The percent survival of the NoV GII.4 RT-QPCR target following exposure of dilute GII.4 samples to available chlorine, chlorine neutralization, and RNase digestion for three different non epidemiologically linked GII.4 clinical samples: 249 (%), 432 (n), and 533 (#). The mean value for all isolates is shown ) with the standard deviation and (N regression curve ( ) (r 2 ~ ). Results of spiking FCV F-9 virus into the dilute human GII.4 NoV samples. The results of the virolysis assay showed that the resistance to available chlorine of FCV F-9 virus when spiked into the dilute NoV GII.4 samples varied depending on the particular NoV sample but was always greater than that of cultured FCV F-9 (Fig. 3). DISCUSSION The data show a similarity between the results obtained for FCV F-9 in plaque assays and those obtained for FCV F- 9 using the RNase RT-QPCR assay. All assays were performed using a standardized method for measuring the efficacy of disinfectants that takes into account chlorine oxidative demand by standardizing the diluent (hard water) and protein concentration (0.03% BSA). Available chlorine is a powerful oxidant and can oxidize RNA. However, the FIGURE 3. The percent survival of the FCV F-9 RT-QPCR target following exposure of FCV F-9 virus spiked into GII.4 NoV samples to available chlorine, chlorine neutralization, and RNase digestion. FCV F-9 was spiked in GII.4 samples 249 (%), 432 (n), and 533 (#). The mean for FCV F-9 in all spiked samples is shown ). The mean data for unspiked FCV F-9 grown in tissue (N culture are also shown for comparison ( ). inclusion of RNase within the assay was required in order to maximize the degradation of exposed RNA. RNase pretreatment was required at low (10-ppm) concentrations of available chlorine in order to maximize exposure of RNase-sensitive RNA from damaged capsids. Therefore, the primary mode of chlorine inactivation appears to be via the virus capsid, resulting in the exposure of RNase-susceptible viral RNA, concomitant with loss of infectivity. We chose not to include a simultaneous proteinase K and RNase A pretreatment step such as previously described (19), owing to the potential of proteinase K to digest the virus capsid and the reported diverse effects of proteinase K and related proteases on RNase A activity. These effects can range from complete inactivation (32) to a limited proteolysis resulting in the conversion of RNase A to active RNase S (15, 24). This makes the data from combined enzymatic approaches difficult to evaluate and could account for the contrasting results obtained with murine NoV-1 in temperature and high pressure processing studies (2, 27). Additionally, RNase I as used in this study possesses a broad substrate specificity for the more efficient degradation of exposed RNA (16). The resistance of FCV F-9 to available chlorine as determined in this study is less than that reported previously (8, 23). However, the higher levels of chlorine resistance observed by others may result from the treatment of virus preparations containing tissue culture medium. This should be avoided since published data show that tissue culture medium has significant and fast-acting antioxidant activity (approximately equivalent to the addition of 10% or more added serum (13)), and this will significantly reduce the available chlorine. Although the relationship between the results from the FCV F-9 plaque assays and the RNase RT-QPCR data was not exact, the data were similar over a narrow range of available chlorine. The predicted available chlorine concentrations obtained by the different methods resulting in equivalent 4-log reductions in infectivity and the RT-QPCR target were 48 and 66 ppm, respectively. In contrast, the predicted available chlorine concentration required for the equivalent reduction in NoV GII.4 RT-QPCR target survival was 10 times greater (610 ppm). This represents a

5 J. Food Prot., Vol. 74, No. 12 VIROLYSIS OF FELINE CALICIVIRUS AND HUMAN GII.4 NOROVIRUS 2117 conservative estimate in comparison with the observed reductions in RT-QPCR target survival and infectivity for FCV F-9 that would predict an equivalent NoV reduction in infectivity at 450 ppm of available chlorine. All comparative studies using human NoVs reported to date have only been based on single NoV isolates, and therefore, the relevance of different studies is uncertain. Perhaps surprisingly, our data revealed little difference in the behavior of the three non epidemiologically linked GII.4 NoV samples in response to available chlorine. This lack of similarity of the data also suggests that gross clumping or aggregation of particles did not occur, since this would be expected to produce outlying data. A comparison of the virolysis data (obtained from the RNase pretreatment and RT-QPCR) using GII.4 NoV samples and the same samples spiked with FCV F-9 virus allowed a direct comparison for the first time between the behavior of a spiked surrogate virus and that of GII.4 NoV isolates in the same matrix in response to available chlorine. Under these conditions, both viruses were simultaneously and equally exposed to chlorine, neutralized, and treated with RNase and the RNA coextracted. This was then followed by independent RT-QPCR using the same cycling conditions and similar-size amplicons derived from equivalent genomic regions. Overall, when the FCV F-9 virus was spiked into the different GII.4 samples, the FCV F-9 virus then showed an increased protection from available chlorine that approached that of the GII.4 samples. Spiked FCV F-9 was always more resistant than cultured FCV F-9. The data were consistent with those obtained from temperature inactivation studies that have shown that dilute NoV clinical samples also conferred increased thermostability to virolysis on spiked FCV F-9 virus (29). This protection was independent of protein concentration, since all samples were standardized to the same protein load according to the standard method. Protection may be conferred by other stool components, such as gastric mucin. This increased resistance and differences in the behavior of spiked FCV F-9 virus may represent differences in the affinity and survival of FCV F-9 for the different stool matrices. The data suggest that the observed increased resistance of GII.4 samples compared with that of cultured FCV F-9 results from protection by the dilute (1.0%, vol/vol) fecal matrix and is not a property of the NoV per se. To our knowledge, this study represents the first direct comparison between the stability of a surrogate virus (FCV F-9) and that of human GII.4 NoVs within the same matrix in response to available chlorine. The data obtained support the view that studies based on the infectivity of surrogate viruses can lead to underestimations of conditions required for virus infectivity (23, 30). Although standardized light soil conditions were used in this study, it is likely that NoVs present in natural samples or adherent to surfaces may be considerably more resistant when present in heavy soil conditions, emphasizing the need for adequate cleaning followed by appropriate disinfection in order to reduce the risks of contamination. ACKNOWLEDGMENTS This independent collaborative research was supported under the Food LINK programme by the UK Department for Environment, Food and Rural Affairs (Defra), by academic support from the University of Surrey, by the UK Health Protection Agency, by industrial support from Unilever Central Resources Ltd., Waitrose plc, Carnival Cruises plc, Atlas Genetics Ltd., McDonalds Europe Ltd., Scottish Shellfish Marketing Group Ltd., Premier Foods Ltd., and Evans Vanodine plc, and by the Leatherhead Food Research food safety forum. REFERENCES 1. Ayers, L. T., I. T. Williams, S. Gray, and P. M. Griffin Surveillance for foodborne disease outbreaks United States Morb. Mortal. Wkly. Rep. 58: Baert, L., C. E. Wobus, C. E., E. Van Coille, L. B. Thackray, J. Debevere, and M. Uyttendaele Detection of murine norovirus 1 by using plaque assay, transfection assay, and real-time reverse transcription-pcr before and after heat exposure. Appl. Environ. Microbiol. 74: Belliot, G., A. Lavaux, D. Souihel, A. Agnello, and P. Pothier Use of murine norovirus as a surrogate to evaluate resistance of human norovirus. Appl. Environ. Microbiol. 74: Bidawid, S., N. Malik, O. Adegbunrin, S. A. Sattar, and J. M. Farber Norovirus cross-contamination during food handling and interruption of virus transfer by hand antisepsis: experiments with feline calicivirus as a surrogate. J. Food Prot. 67: Bradford, M. M A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: Cannon, J. L., E. Papafragkou, G. W. Park, J. Osborne, L.-A. Jaykus, and J. Vinjé Surrogates for the study of norovirus stability and inactivation in the environment: a comparison of murine norovirus and feline calicivirus. J. Food Prot. 69: Carter, M. J Enterically infecting viruses: pathogenicity, transmission and significance for food and waterborne infection. J. Appl. Microbiol. 98: Duizer, E., P. Bijkerk, B. Rockx, A. De Groot, F. Twisk, and M. Koopmans Inactivation of caliciviruses. Appl. Environ. Microbiol. 70: European Standard EN Chemical disinfectants and antiseptics. Virucidal quantitative suspension test for chemical disinfectants and antiseptics used in human medicine. Test method and requirements (phase 2, step 1). ISBN: Falkenhorst, G., L. Krusell, M. Lisby, S. B. Madsen, B. Böttiger, and K. Mølbak Imported frozen raspberries cause a series of norovirus outbreaks in Denmark, Euro Surveill. 10:E Gray, J Personal communication. 12. Kageyama, T., S. Kojima, M. Shinohara, K. Uchida, S. Fukushi, F. B. Hoshino, N. Takeda, and K. Katayama Broadly reactive and highly sensitive assay for Norwalk-like viruses based on real-time quantitative reverse transcription-pcr. J. Clin. Microbiol. 41: Lewinska, A., M. Wnuk, E. Slota, and G. Bartosz Total antioxidant capacity of cell culture medium. Clin. Exp. Pharmacol. Physiol. 34: Liu, P., Y. Yuen, H.-M. Hsiao, L.-A. Jaykus, and C. Moe Effectiveness of liquid soap and hand sanitizers against Norwalk virus on contaminated hands. Appl. Environ. Microbiol. 76: Makert, Y., J. Koditz, J. Mansfeld, U. Arnold, and R. Ulbrich- Hofmann Increased proteolytic resistance of ribonuclease A by protein engineering. Protein Eng. 14: Meador, J., B. Cannon, V. J. Cannistraro, and D. Kennel Purification and characterisation of Escherichia coli RNase I. Comparisons with RNase M. Eur. J. Biochem. 187: Mendham, J., R. C. Denney, J. D. Barnes, and M. J. K. Thomas Available chlorine in hypochlorites, p In Vogel s textbook of quantitative chemical analysis, 6th ed. Pearson Education Ltd., Harlow, England. ISBN

6 2118 NOWAK ET AL. J. Food Prot., Vol. 74, No Mormann, S., M. Dabish, and B. Becker Effects of technological processes on the tenacity and inactivation of norovirus GGII in experimentally contaminated foods. Appl. Environ. Microbiol. 76: Nuanualsuwan, S., and D. O. Cliver Pretreatment to avoid positive RT-PCR results with inactivated viruses. J. Virol. Methods 104: Nuanualsuwan, S., and D. O. Cliver Capsid functions of inactivated human picornaviruses and feline calicivirus. Appl. Environ. Microbiol. 69: Patel, M. M., A. J. Hall, J. Vinjé, and U. D. Parashar Noroviruses: a comprehensive review. J. Clin. Virol. 44: Pecson, B. M., L. Valerio Martin, and T. Kohn Quantitative PCR for determining the infectivity of bacteriophage MS2 upon inactivation by heat, UV-B radiation, and singlet oxygen: advantages and limitations of an enzymatic treatment to reduce false positives. Appl. Environ. Microbiol. 75: Poschetto, L. F., A. Ike, T. Papp, U. Mohn, R. Bohm, and R. E. Marsschang Comparison of the sensitivities of noroviruses and feline calicivirus to chemical disinfection under field-like conditions. Appl. Environ. Microbiol. 73: Richards, F. M On the enzymatic activity of subtilisin modified ribonuclease. Proc. Natl. Acad. Sci. USA 44: Scaracella, C., S. Carasi, F. Cadoria, L. Macchi, A. Pavan, M. Salamana, G. L. Alborali, M. M. Losio, P. Boni, A. Lavazza, and T. Seyler An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June Euro. Surveill. 14:pii: Siebenga, J. J., H. Vennema, D. P Zheng, J. Vinjé, B. E. Lee, X. L. Pang, E. C. Ho, W. Lim, A. Choudekar, S. Broor, T. Halperin, N. B. Rasool, J. Hewitt, G. E. Greening, M. Jin, Z. J. Duan, Y. Lucero, M. O Ryan, M. Hoehne, E. Schreier, R. M Ratcliff, P. A. White, N. Iritani, G. Reuter, and M. Koopmans Norovirus illness is a global problem: emergence and spread of norovirus GII.4 variants, J. Infect. Dis. 200: Tang, Q., D. Li, J. Xu, J. Wang, Y. Zhao, Z. Li, and C. Xue Mechanism of inactivation of murine norovirus-1 by high pressure processing. Int. J. Food Microbiol. 137: Thurston-Enriquez, J. A., C. N. Haas, J. Jacangelo, and P. C. Gerba Chlorine inactivation of adenovirus type 40 and feline calicivirus. Appl. Environ. Microbiol. 69: Topping, J. R., H. Schnerr, J. Haines, M. Scott, M. Carter, M. M. Willcocks, K. Bellamy, D. Brown, J. Gray, C. Gallimore, and A. I. Knight Temperature inactivation of feline calicivirus vaccine strain F-9 in comparison with human noroviruses using an RNA exposure assay and reverse transcribed quantitative polymerase chain reaction a novel method for predicting infectivity. J. Virol. Methods 156: Tree, J. A., M. R. Adams, and D. N. Lees Disinfection of feline calicivirus (a surrogate for norovirus) in wastewaters. J. Appl. Microbiol. 98: Urakami, H., K. Ikarashi, K. Okamoto, Y. Abe, T. Ikarashi, T. Kono, Y. Konagaya, and N. Tanaka Chlorine sensitivity of feline calicivirus, a norovirus surrogate. Appl. Environ. Microbiol. 73: Wiegers, U. J., and H. Hilz A new method using proteinase K to prevent mrna degradation during isolation from HeLa cells. Biochem. Biophys. Res. Commun. 44: Wobus, C. E., S. M. Karst, L. B. Thackray, K. O. Chang, S. V. Sosnovtsev, G. Belliot, A. Krug, J. M. Mackenzie, K. Y. Green, and H. W. Virgin Replication of norovirus in cell culture reveals a tropism for dendritic cells and macrophages. PLoS Biol. 2: