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Applied and Environmental Microbiology, September 2007, p. 5679-5682, Vol. 73, No. 17
0099-2240/07/$08.00+0     doi:10.1128/AEM.00083-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Chlorine Sensitivity of Feline Calicivirus, a Norovirus Surrogate{triangledown}

Hiroshi Urakami,1* Kumiko Ikarashi,1 Ko Okamoto,1 Yukari Abe,1,{dagger} Tamami Ikarashi,1,§ Takeshi Kono,2 Yukifumi Konagaya,1 and Nobumasa Tanaka1

College of Applied Life Sciences,1 School of Pharmacy, Niigata University of Pharmacy and Applied Life Sciences (NUPALS), Higashijima, Akiha-ku, Niigata, Niigata 956-8603, Japan2

Received 13 January 2007/ Accepted 27 June 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 REFERENCES
 
The sensitivity to free chlorine of feline calicivirus (FCV), a norovirus surrogate, was examined relative to chlorine demand. When a crude suspension of FCV was treated with a sodium hypochlorite solution containing 10 µg/ml free chlorine, the extent of the decrease of viral infectivity clearly depended on the volume of the reaction mixture. The apparent sensitivity of FCV to free chlorine increased with the reduction of host cell debris, indicating that chlorine demand must be minimized to know the true sensitivity of the virus. We therefore partially purified the viruses from the host cell components and found that the infectivity of FCV was reduced by more than log 4.6 by 5 min of treatment with 300 ng/ml free chlorine.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
REFERENCES
 
Human norovirus is one of the most important pathogens of food-borne and waterborne gastroenteritis (12). Though chlorine disinfectants, such as sodium hypochlorite, are thought to be effective for the inactivation of norovirus, concentrations as high as 200 to 1,000 µg/ml are recommended for disinfection of an environment, kitchen utensils, and furniture soiled with patient feces and vomit (3, 13).

Several gastroenteritis outbreaks caused by recreational (2, 10) and drinking (11, 14, 17) water have been associated with the failure or ineffectiveness of chlorination. In these cases, the concentrations of free chlorine seemed to be much lower than those prescribed by the regulatory standards. The required concentrations of chlorine in recreational and drinking water are not higher than 4 µg/ml and 1 µg/ml, respectively, in the United States (22; http://www.cdc.gov/healthyswimming, 2004). These norovirus outbreaks suggest that if chlorination had been done at the standard levels, the outbreaks might not have occurred; that is, norovirus could be inactivated with chlorine levels as high as 1 µg/ml. There seems to be an obvious discrepancy between this concentration and the above-mentioned concentrations used to inactivate norovirus in an environment.

Keswick et al. (9) investigated the chlorine sensitivity of norovirus by experimental ingestion by human volunteers and reported that 3.75 to 6.25 µg/ml chlorine was insufficient to inactivate the virus. This is the only study that directly evaluated the chlorine sensitivity of norovirus, because norovirus infects only humans and there currently exists neither a cultivation nor an infection system in vivo or in vitro. Thus, similar investigations were performed using surrogate viruses. Feline calicivirus (FCV) is the most widely used surrogate, because of its phylogenetic similarity to norovirus (8) and the availability of cultivation systems in vitro. The estimates of the sensitivity of norovirus to free chlorine and other disinfectants were, therefore, determined mainly from experiments using FCV. The sensitivity of FCV to free chlorine was reported by several groups of investigators (5, 7a, 18) to be 100 to 3,000 µg/ml, and these reports are the basis for the recommended chlorine concentrations for norovirus disinfection.

The studies mentioned above were done with viral suspensions containing considerable amounts of organic matter, such as host cell components and proteins in the suspending media. It is widely accepted that free chlorine loses its virucidal and bactericidal activity in the presence of organic substances. We therefore designed experiments using partially purified viruses to minimize the chlorine demand in the present study. Our results indicated a higher FCV sensitivity to free chlorine than previously reported.

FCV strain F9 was propagated in Crandell Reese feline kidney (CRFK) cells and used throughout the study. Monolayers of the cells were cultivated in Eagle's minimal essential medium (MEM; Sigma, St. Louis, MO) supplemented with 10% calf serum and were inoculated with an FCV suspension at approximately 10 tissue culture infectious doses (TCID)/cell. After the inoculation, the cells were cultivated in serum-free MEM for 1 to 2 days until they were detached from the vessel by cytopathic effects. The viruses were released from the cells by three repeats of freezing and thawing and clarified at 10,000 x g for 15 min to remove the cell debris. The viruses were recovered by centrifugation at 110,000 x g for 90 min, and the pellet was suspended in phosphate-buffered saline (PBS). This preparation is referred to as crude FCV (cFCV). A suspension of cFCV was layered on 25% sucrose and centrifuged at 160,000 x g for 4 h. The resulting pellet under the sucrose cushion was resuspended in PBS and centrifuged again at 160,000 x g for 2 h to obtain partially purified FCV (pFCV). For electron microscope observation, pFCV was applied to formvar-carbon-coated grids and negatively stained with a saturated solution of uranyl acetate.

The TCID was titrated by infecting CRFK cells in 96-well multiplates. The cells that were cultivated in MEM containing 10% calf serum for 1 to 2 days were washed and incubated at 35°C in 50 µl/well of serum-free MEM until being inoculated with FCV preparations. Viral suspensions serially diluted 10-fold in serum-free MEM were inoculated into the wells at 50 µl/well. After 3 days of incubation, the cells were washed in PBS, fixed with ethanol, and stained in 0.3% crystal violet-0.67% ammonium oxalate to distinguish the infected and intact wells by the naked eye. The infectivity was expressed as the most probable number, with 95% confidence limits, by the 5- or 10-tube method (6). All of the cultivations were carried out at 35°C in a humidified atmosphere containing 5% CO2.

For the disinfection experiments, 20 µl of FCV suspension in PBS was added to 2 ml of sodium hypochlorite solution unless otherwise noted. The disinfectant was dissolved in autoclaved distilled water, and the pHs at concentrations of 0.01, 0.1, 1, and 10 µl/ml were 7.6, 7.6, 7.8, and 8.8, respectively. After 5 min of treatment at room temperature (20 to 25°C), the same volume of 0.25 M sodium thiosulfate was added to neutralize free chlorine. The mixture was then serially diluted in MEM for the infectivity titration.

The protein concentrations of the viral suspensions were determined by using the Bio-Rad Protein Assay (Bio-Rad, CA). The concentration of free chlorine was assayed by iodometric titration (7a).

The changes in the virucidal activity of free chlorine were examined in various volumes of the reaction mixture. Twenty-microliter amounts of the cFCV suspension, containing 2.6 x 106 TCID of virus and 180 µg protein/ml, were treated in final volumes of 100, 200, 400, 1,000, and 2,000 µl containing 10 µg/ml sodium hypochlorite solution, and the infectivities were titrated after the mixtures were adjusted to the same volume (Fig. 1). No significant decrease in the infectivity was observed when the viruses were treated in a 100-µl volume of the reaction mixture, but more viruses were inactivated in the larger volume of the mixture. In the 2,000-µl suspension, the infectivity was reduced below the detection limit (4 TCID/ml), indicating inactivation of more than log 3.8. This indicated that the inactivation of FCV depended on the reaction mixture volume, probably because of the total amount of chlorine. We performed two other experiments with different batches of virus suspensions; more viruses were inactivated in the larger reaction mixture volumes. An electron microscope observation of the cFCV fraction showed a large amount of host cell components obscuring most of the FCV virions (data not shown). It is thus conceivable that more free chlorine molecules reacted with the host cell debris than with the viruses. In such a circumstance, the absolute amount of free chlorine, which is dependent on the volume of the chlorine solution added, might be critical in order for it to react with a given amount of protein and organic matter and to inactivate the viruses; hence, the extent of inactivation of FCV was dependent upon the reaction mixture volume. This observation also suggested that FCV could be inactivated by free chlorine at 10 µg/ml when chlorine demand was low enough.


Figure 1
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  		FIG. 1. Infectivity of crude FCV after treatment with 10 µg/ml free chlorine in different volumes of reaction mixture. Filled circle, infectivity without treatment. Vertical bars represent 95% confidence limits. *, lower limit is <log 0; #, infectivity is below detection limit (<4 TCID/ml).

 
The experiment described above suggested that the virucidal activity of free chlorine was reduced in the presence of host components, so we purified the viruses and examined the effect of free chlorine on FCV in each fraction from the purification procedure (Table 1). The titer of FCV-infected cells after freezing and thawing and the titer of supernatant from these infected cells after sedimentation were not affected by chlorine treatment at 1 µg/ml for 5 min. cFCV was harvested from the supernatant by centrifugation at 110,000 x g and suspended in PBS. The FCV in this fraction was highly sensitive to chlorine at 1 µg/ml; the infectivity was reduced by log 5.6 but still detected. After centrifugation on a sucrose cushion (pFCV), infectious viruses were not detected after free chlorine treatment, indicating that the loss of infectivity was greater than log 4.3. However, when cFCV was suspended in serum-free MEM, the decrease in infectivity from the same treatment was approximately log 1.1 (data not shown). This discrepancy is probably because MEM contains reducing reagents, such as cysteine, which interfere with the action of sodium hypochlorite. This and two other similar experiments showed that the virucidal activity of free chlorine was strengthened by decreasing the chlorine demand in the course of purification.


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  		TABLE 1. Changes in infectivity of increasingly purified FCV after treatment with free chlorinea

 
To investigate the chlorine sensitivity of FCV under chlorine demand-free conditions, the protein concentration of the viral suspensions was minimized by purification through the sucrose cushion. We prepared two batches of pFCV with protein concentrations of 1.0 and 12 µg/ml, containing viral titers of 3.4 x 105 and 8.0 x 106 TCID/ml, respectively. Electron microscope observations of pFCV revealed that the FCV virions were highly concentrated, with some amount of host cell debris (data not shown). Amounts of 20 µl of the pFCV fraction were mixed with various concentrations of 2 ml of sodium hypochlorite, such that the final protein concentrations were 10 and 120 ng/ml, and incubated for 5 min. When the batch with 10 ng/ml protein was treated in the reaction mixture with 0.1 µg/ml sodium hypochlorite, the infectivity was decreased below the detection limit (1.8 TCID/ml), showing an inactivation greater than log 3.2 (Fig. 2). When the batch with 120 ng/ml protein was examined, infectivity was still detected after treatment with 100 ng/ml free chlorine (log 3.5 decrease), but 300 ng/ml free chlorine reduced the infectivity below the detection limit; that is, the reduction was greater than log 4.6 (Fig. 2). Other than these, some other experiments with pFCV also showed a marked decrease of viral titers at between 0.1 and 1 µg/ml of chlorine. These results show that FCV can be inactivated by free chlorine in amounts as low as 0.3 µg/ml, if the chlorine demand is minimal.


Figure 2
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  		FIG. 2. Infectivity of partially purified FCV suspensions containing different concentrations of protein after treatment with various concentrations of free chlorine. Circles, results with 10 ng/ml protein. Triangles, results with 120 ng/ml protein. Vertical bars represent 95% confidence limits.

 
An outbreak of norovirus gastroenteritis occurred at a swimming club in Vermont because of chlorination failure (2). The outbreak terminated after the water was hyperchlorinated to 3.5 µg/ml. Podewils et al. (16) analyzed this outbreak epidemiologically and suggested that norovirus is not as resistant to free chlorine as expected from previous studies using surrogates. A study by Keswick et al. (9) showed that a viral suspension treated at a concentration between 3.75 and 6.25 µg/ml free chlorine for 30 min still had infectivity equivalent to that of the untreated control suspension for human volunteers. They prepared samples of norovirus from patient stool samples, suspended in a broth that contained proteins. In such circumstances, the virucidal activity of free chlorine is expected to decline (7, 21).

Other studies using surrogate viruses, such as FCV, have provided experimental data to deduce the effective levels of chlorine for norovirus disinfection. An amount of more than 100 µg/ml was required when an equal amount of the disinfectant was added to FCV suspended in culture fluid containing 10% serum (4). An amount of 3,000 µg/ml chlorine reduced the infectivity by more than log 5 in a reaction medium consisting mainly of serum-free MEM (5). Approximately 400 µg/ml was needed to inactivate FCV by more than log 3.95 (18). On the other hand, Thurston-Enriquez et al. (19) purified FCV by using polyethylene glycol and chloroform and estimated that an amount of free chlorine of less than 0.2 µg/ml inactivated FCV by log 2 or more, depending on the pH of the reaction medium and the aggregation of the viruses. We tried using chloroform-treated cFCV to avoid aggregation of the virions, but found that chloroform itself significantly decreased the viral infectivity. We therefore used partially purified viruses in the present study, obtained by employing a sucrose cushion in centrifugation, and found that pFCV minimized the protective effect of chlorine demand. As a result, it was revealed that more than log 4.6 FCV could be inactivated by an amount of free chlorine of 300 ng/ml, suggesting that the chlorine sensitivity of FCV is much lower than previously estimated. An investigation of the disinfection of wastewater with high biochemical oxygen demand (20) revealed that FCV strain F9 was much more sensitive than poliovirus to free chlorine. When 23 strains of poliovirus were treated under a condition of minimal chlorine demand, the decrease of the viral infectivity was greater than log 2 with treatment with 400 ng/ml free chlorine for 10 min (15). These observations also suggest that FCV is not so resistant to free chlorine as is widely accepted. However, when instruments or furniture are contaminated with patient feces or vomit, a higher concentration of free chlorine is still needed, because chlorine demand is much higher in such circumstances than in those in the present study.

Although widely used as a surrogate, FCV is thought to have a stability in conditions of low pH and heat that is different from that of norovirus (1); thus, some other viruses, such as canine calicivirus (5) and murine norovirus (1), are also used as surrogates. We used a laboratory-adapted strain of FCV in the present study; the adaptation may reduce resistance to disinfectants. Payment et al. (15) compared the chlorine sensitivities of 32 strains of poliovirus and coxsackievirus but could not find differences between laboratory and wild strains. The exact sensitivity of human norovirus is impossible to determine without establishing a stable cultivation system for the virus. However, if the chlorine sensitivity of human norovirus is similar to that of FCV, the regulation of standard levels of free chlorine for drinking and recreational water, as low as 1 µg/ml, may be effective in norovirus disinfection in circumstances with low chlorine demand.


   ACKNOWLEDGMENTS
 
We thank M. Okada of Chiba Prefectural Institute of Public Health, Chiba, Japan, for providing FCV F9.

This study was supported by the Promotion and Mutual Aid Corporation for Private Schools of Japan and by the Ministry of Education, Science, Sports and Culture.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Food Science, School of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences (NUPALS), 265-1 Higashijima, Akiha-ku, Niigata, Niigata 956-8603, Japan. Phone: 81(250) 25-5148. Fax: 81(250) 25-5021. E-mail: urakami{at}nupals.ac.jp Back

{triangledown} Published ahead of print on 6 July 2007. Back

{dagger} Present address: Katayama Shokuhin Co. Ltd., Shibata, Niigata 957-0293, Japan. Back

§ Present address: Faculty of Marine Science, Tokyo University of Marine Science and Technology, Minato-ku, Tokyo 108-8477, Japan. Back


   REFERENCES
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 ABSTRACT
 INTRODUCTION
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Applied and Environmental Microbiology, September 2007, p. 5679-5682, Vol. 73, No. 17
0099-2240/07/$08.00+0     doi:10.1128/AEM.00083-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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