Journal of Infection and Chemotherapy 28 (2022) 67–72

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Journal of Infection and Chemotherapy

journal homepage: www.elsevier.com/locate/jic

Original Article

Inactivation of human norovirus by chlorous acid water, a novel

chlorine-based disinfectant
Hisataka Goda a, b, Haruyuki Nakayama-Imaohji c, Hitoshi Yamaoka a, c, Ayano Tada c,
Tamiko Nagao d, Tomohiko Fujisawa b, A. Hajime Koyama c, Tomomi Kuwahara c, *
a
Sankei Co. Ltd., 2-2-53 Shiromi, Chuou-ku, Osaka, Japan
b
Laboratory of Food Hygiene, Faculty of Applied Life Science, Nippon Veterinary and Life Science University, 1-7-1, Kyonancho, Musashino-shi, Tokyo, 180-8602,
Japan
c
Department of Microbiology, Faculty of Medicine, Kagawa University, 1750-1, Miki, Kagawa, 761-0793, Japan
d
Department of Science for Human Health Welfare Care Major, Shikoku University Junior College

A R T I C L E I N F O A B S T R A C T

Keywords: Introduction: Human norovirus (HuNoV) is a leading cause of infectious gastroenteritis. Since HuNoV shows
Chlorous acid water resistance to alcohol, chlorine-based sanitizers are applied to decontaminate the virus on environmental surfaces.
Human norovirus Chlorous acid water (CA) has been recently approved as a novel chlorine-based disinfectant categorized as a Type
Disinfection
2 OTC medicine in Japan. In this study, we aimed to evaluate the capability of CA to inactivate HuNoV.
Organic substances
qPCR
Methods: HuNoV (genogroups GII.2 and GII.4) was exposed to the test disinfectants including CA and sodium
hypochlorite (NaClO), and the residual RNA copy was measured by reverse transcription quantitative PCR (RT-
qPCR) after pretreatment with RNase. In addition, the log10 reduction of HuNoV RNA copy number by CA and
NaClO was compared in the presence of bovine serum albumin (BSA), sheep red blood cells (SRBC), polypeptone,
meat extract or amino acids to evaluate the stability of these disinfectants under organic-matter-rich conditions.
Results: In the absence of organic substances, CA with 200 ppm free available chlorine provided >3.0 log10
reduction in the HuNoV RNA copy number within 5 min. Even under high organic matter load (0.3% each of BSA
and SRBC or 0.5% polypeptone), 200 ppm CA achieved >3.0 log10 reduction in HuNoV RNA copy number while
less than 1.0 log10 reduction was observed with 1,000 ppm sodium hypochlorite (NaClO) in the presence of 0.5%
polypeptone. CA reacted with only cysteine, histidine and glutathione while NaClO reacted with all of the amino
acids tested.
Conclusions: CA is an effective disinfectant to inactivate HuNoV under organic-matter-rich conditions.

1. Introduction
Human norovirus (HuNoV) is a major cause of foodborne viral
gastroenteritis worldwide [1], and nearly 10,000 patients are reported
annually in HuNoV food poisoning in Japan [2]. Although contaminated
food is a major route of infection, contact infection from the virus carrier
and contaminated environments play important roles in HuNoV trans­
mission. HuNoV frequently causes outbreaks in hospitals and nursing
homes because of its high transmissibility. The particle number of
HuNoV required to establish infection is as low as 100 particles [3],
while patient feces often contain more than 108 HuNoV particles per
gram [4]. As well as appropriate handwashing with soap, strict disin­
fection of environmental surfaces contaminated by the patients’ excre­
tions is critical to prevent HuNoV outbreaks [5]. Since HuNoV is a
non-enveloped virus and is resistant to a wide range of disinfectants,
appropriate selection of disinfectants and complete management of ex­
cretions are critical for infection control. According to the 2011 CDC
guidelines for HuNoV outbreak management, 1,000–5,000 ppm of so­
dium hypochlorite (NaClO) is recommended to inactivate HuNoV [6].
However, chlorine-based sanitizers such as NaClO are well known to

Abbreviations: HuNoV, human norovirus; CA, chlorous acid water; CDC, Center for Disease Control and Prevention; NaClO, sodium hypochlorite; OTC, over-the-
counter; FCV, feline calicivirus; MNV, murine norovirus; RT-qPCR, reverse transcription quantitative polymerase chain reaction; BSA, bovine serum albumin; SRBC,
sheep red blood cells; TMB, 3,3′ ,5,5′ -tetramethylbenzidine.
* Corresponding author.
E-mail address: kuwahara.tomomi@kagawa-u.ac.jp (T. Kuwahara).

https://doi.org/10.1016/j.jiac.2021.10.001
Received 5 August 2021; Received in revised form 1 October 2021; Accepted 4 October 2021
Available online 9 October 2021
1341-321X/© 2021 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
H. Goda et al.
lose microbicidal activity in contact with organic substances [7,8],
leading to the risk of insufficient inactivation of HuNoV in vomitus and
feces. It is important to develop alternative disinfectants showing effi­
cient virucidal activity against HuNoV even under organic
substance-rich conditions.
In 2019, a novel chlorine-based disinfectant, chlorous acid water
(CA), which contains HClO2 as an effective component, was approved as
a Type 2 over-the-counter (OTC) medicine in Japan. A solution con­
taining 200 ppm CA was reported to inactivate feline calicivirus (FCV), a
HuNoV surrogate, to >4.0 log10 reduction under protein-rich conditions
[9]. The inactivation efficiency is superior to that of 1,000 ppm NaClO,
and thus CA is expected to inactivate HuNoV in human excretions. Due
to the lack of a conventional cell culture system for HuNoV, the HuNoV
surrogate viruses FCV and murine norovirus (MNV) are commonly used
to compare the efficacy of disinfectants against HuNoV. However,
resistance to disinfectants is known to differ between these surrogate
viruses; for example, MNV is more sensitive to alcohol than FCV [10].
Therefore, the virucidal effects of disinfectants on HuNoV should be
evaluated with HuNoV from clinical samples.
In this study, we aimed to evaluate the inactivation efficiency of CA
on HuNoV of two genogroups (GII.2 and GII.4), which were isolated
from infectious gastroenteritis patients.
2. Materials and methods
2.1. Preparation of HuNoV inoculum
Fecal samples from two HuNoV gastroenteritis patients were
collected after obtaining written informed consent. The samples were
suspended in phosphate-buffered saline (PBS [pH 7.4]) at 10% con­
centration (v/v). After large fecal material was removed by centrifuga­
tion (6,000 g, 10 min, 4 ◦ C), the supernatant was collected in fresh tubes.
Polyethylene glycol (MW 6,000) and sodium chloride were added to the
supernatant at final concentrations of 8.0% and 2.1%, respectively. After
24 h storage at 4 ◦ C, the mixture was centrifuged (8,000 g, 20 min, 4 ◦ C),
and the pellet was washed twice with PBS. Residual water was removed
with filter paper, and the pellet was dissolved in sterilized distilled
water. The resulting solutions were used as the HuNoV samples. Aliquots
were stored at − 70 ◦ C until use.
2.2. Genogrouping of HuNoV
Genogrouping of HuNoV was performed as follows. Viral RNAs were
extracted from the HuNoV samples with QIAamp Viral RNA Mini kit
(Qiagen K.K, Tokyo, Japan). RNA samples were reverse transcribed with
PrimeScript® RT reagent kit (Takara Bio Co., Ltd.) with random hex­
amers in a final volume of 20 μl at 37 ◦ C for 15 min. The reaction was
stopped by heating the sample at 85 ◦ C for 5 s. Using the cDNA as a
template, the viral genomic region encompassing ORF1 and ORF2 was
amplified with primers G2-SKF (5′ -CNTGGGAGGGCGATCGCAA-3′ ) and
G2-SKR (5′ -CCRCCNGCATRHCCRTTRTACAT-3′ ). PCR products were
purified with the QIAquick PCR purification kit (QIAGEN). The purified
amplicons were sequenced by Sanger sequencing using the primer G2-
SKF or G2-SKR. The obtained sequences were homology searched and
the genogroup determined according to the best match reference
sequence.
2.3. Disinfectants
Disinfectants used in this study were chlorous acid water (CA, 8,000
ppm of available chlorine, Sankei Co. Ltd.), sodium hypochlorite
(NaClO, 60,000 ppm of available chlorine, OYALOX Co. Ltd.), chlorine-
releasing disinfectant cleaner (RST, Kyorin Pharmaceutical Co.), 80%
ethanol (Kenei Pharmaceutical Co) and 0.1% benzalkonium chloride
(Showa Pharmaceutical Co.).
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Journal of Infection and Chemotherapy 28 (2022) 67–72
2.4. Virolysis test for HuNoV
The effect of different types of disinfectants on HuNoV virolysis was
evaluated by reverse transcription quantitative PCR (RT-qPCR) in
conjunction with RNase treatment (in order to destroy any exposed
RNA) as described by Nowak et al. [11]. HuNoV samples (100 μl) were
mixed with 900 μl of the test disinfectants or sterilized distilled water
(control) and incubated with agitation at 25 ◦ C. HuNoV in the reaction
was adjusted to 107 order of RNA copy/ml. Bovine serum albumin (BSA,
Sigma), polypeptone (Nihon Pharmaceutical Co. Ltd.), meat extract
(Nacalai Tesque, Inc.), or sheep red blood cells (SRBC, Japan Bio Serum)
was added to the reaction mixture to a final concentration of 0.3% or
0.5% when necessary. After a setting time, the reaction samples were
diluted 10-fold with Difco D/E neutralizing broth (Becton Dickinson and
Company) to inactivate the disinfectants. After the neutralized samples
were treated with 20 μg/ml of DNase-free ribonuclease (NIPPON GENE
Co. Ltd.) at 25 ◦ C for 30 min, viral RNA was extracted from 140 μl of the
mixture using QIA viral RNA extraction kit (QIAGEN). HuNoV RNA copy
number was measured by qPCR using Norovirus GI/GII kit (Takara Bio
Co., Ltd.) and a Thermal Cycle Dice Real Time System III (TP970,
Takara).
2.5. Measurement of free available chlorine
Free available chlorine was measured by the tetramethylbenzidine
(TMB) method as described previously [12]. 3,3′ ,5,5′ -tetrame­
thylbenzidine (0.01% in 50% acetone) was used as a color indicator. 1.6
M phosphate buffer (pH 6.5) was made by mixing 1.6 M each of po­
tassium dihydrogen phosphate and dipotassium hydrogen phosphate.
After the test samples (9.5 ml) were buffered with 0.5 ml of the phos­
phate buffer, 1.0 ml of TMB indicator was added to the buffered samples.
The absorbance of the samples at 655 nm was then immediately
measured. Free available chlorine levels in the samples were calculated
from a standard curve made with NaClO.
2.6. Statistical analysis
Data were collected from three independent experiments and
expressed as mean ± standard deviation (SD). Statistical differences of
the data were evaluated with either Student’s t-test or one-way ANOVA
followed by Tukey’s post-hoc test or Dunnett’s test. Differences were
considered to be significant when p-values were less than 0.05.
3. Results
3.1. Genogrouping of HuNoVs
RT-PCR with primers G2-SKF and G2-SKR was performed with two
fecal samples from HuNoV gastroenteritis patients. The 344-bp sequence
obtained from each sample was homology searched by BLAST and
applied to the Norovirus Typing Tool version 2.0 [13]. One of the se­
quences was genogrouped into the GII.4 Sydney_2012 subcluster and
best matched to the norovirus GII isolate Hu/SP/GII.4 Valencia
[P31]/3935 (99.71% identity). Another was genogrouped into the
unassigned subcluster of GII.2 and best matched to the norovirus GII
isolate NoV/0170FB/17/TZ (99.12% identity). We used these two
HuNoV strains.
3.2. Effect of CA on HuNoV virolysis
We selected disinfectants that are widely used for environmental
sanitation in Japan to evaluate the virolytic effect on HuNoV under
organic-matter-free conditions. As shown in Fig. 1, chlorine-based dis­
infectants such as chlorous acid (CA, 200 ppm) and sodium hypochlorite
(NaClO, 200 ppm and 500 ppm) achieved more than a 3-log10 reduction
of HuNoV RNA copy number of both genogroups (GII.4 and GII.2)
H. Goda et al. Journal of Infection and Chemotherapy 28 (2022) 67–72

Fig. 1. Effects of the selected disinfectants on HuNoV RNA copy number

reduction. HuNoVs of the GII.4 and GII.2 genogroups were exposed to the
indicated concentrations of the respective disinfectant for 30 min. After
neutralization and RNase treatment, intact HuNoV particles were quantified by
RT-qPCR. The data are shown as mean ± SD of the log10 reduction in HuNoV
RNA copy number. Statistical analysis was performed in each genogroup. Col­
umns marked with different letters indicate significant differences (p < 0.01).

within 30 min. Effective log10 reduction of the HuNoV RNAs (3.43 ±

0.47 and 2.80 ± 0.07 for GII.4 and GII.2, respectively) was observed
with RST, a chlorine-releasing sanitizer containing detergents, but the
effect was less than those of CA and NaClO. On the other hand, only
limited log10 reduction of the viral RNA (1.02 ± 0.33 and 0.09 ± 0.03 for
GII.4 and 0.05 ± 0.21 and 0.05 ± 0.10 for GII.2) was detected with 80%
ethanol and 0.1% benzalkonium chloride (BZC), respectively. Based on
these results, we carried out further analysis of CA and NaClO.

3.3. Effect of CA on HuNoV virolysis in the presence of 0.3% BSA

We determined the treatment time to provide >3.0 log10 reduction of Fig. 2. Log10 reduction of HuNoV RNA copy number after treatment with
HuNoV RNA copy number by CA and NaClO, each containing 200 ppm CA and NaClO in the absence (A) or presence of 0.3% BSA (B). HuNoV GII.4
free available chlorine. Without 0.3% BSA, 200 ppm CA achieved >3.0- and GII.2 were treated with CA or NaClO with the free-chlorine level adjusted
to 200 ppm, for the indicated times. After neutralization and RNase treatment,
log10 reduction of HuNoV GII.4 and GII.2 within 5 min and 1 min,
intact HuNoV particles were quantified by RT-qPCR. The data are shown as
respectively (Fig. 2A). NaClO of 200 ppm provided equivalent effects on
mean ± SD of the log10 reduction in HuNoV RNA copy number. Significant
both HuNoV GII.4 and GII.2 within 1 min. In the presence of 0.3% BSA, differences (p < 0.01) between CA and NaClO are indicated by asterisks (*).
200 ppm CA needed 30 min to achieve >3.0-log10 reduction of HuNoV
GII.4 (Fig. 2B). Of note, the HuNoV GII.4 virolysis with 200 ppm CA
3.5. HuNoV virolysis by CA under conditions simulating highly
increased in a time-dependent manner even in the presence of 0.3% BSA.
contaminated environments
These results indicate that the virucidal effect of CA is long-acting even
in the presence of organic matter. HuNoV GII.2 used in this study was
Guidelines for the evaluation of environmental sanitizers published
more susceptible to 200 ppm CA with >99.9% of the viral particles being
by the Japanese Society for Infection Prevention and Control recom­
damaged within 1 min even in the presence of 0.3% BSA. On the other
mend 0.3% BSA plus 0.3% SRBC as an organic matter load to simulate
hand, >90% of both HuNoV GII.4 and GII.2 remained intact even after
highly contaminated conditions [15]. On the other hand, the Japan Food
30-min treatment with 200 ppm NaClO in the presence of 0.3% BSA.
Hygiene Association requests to add 0.5% polypeptone into in vitro
assays to evaluate the virucidal activity of disinfectants in human ex­
3.4. Effect of CA on HuNoV virolysis in the presence of other types of
cretions [14]. According to these guidelines, we examined the HuNoV
organic substances
virolytic activity of CA in these conditions.
As shown in Fig. 4, 200 ppm CA provided >3.0 log10 reduction in
Polypeptone and meat extract are proposed as surrogates for vomitus
HuNoV RNA copy number of both GII.4 and GII.2 within 60 min in the
and feces, respectively, to evaluate virucidal activities of disinfectants in
presence of 0.3% each of BSA and SRBC or 0.5% polypeptone, and the
human excretions [14]. As shown in Fig. 3, 200 ppm CA provided >3.0
effects were superior to 1,000 ppm NaClO. However, more than 30-min
log10 reductions in both HuNoV GII.4 and GII.2 within 30 min in the
contact was needed to achieved >3.0 log10 reduction in HuNoV RNA
presence of 0.3% polypeptone (Fig. 3A) or 0.3% meat extract (Fig. 3B).
copy number even with 200 ppm CA in the case of GII.4. Although a
On the other hand, >90% of both HuNoV GII.4 and GII.2 remained
>3.0 log10 reduction in HuNoV RNA copy was observed in GII.2 in the
intact after 30-min treatment with NaClO (both 200 ppm and 500 ppm)
presence of 0.3% each of BSA and SRBC, the HuNoV virolysis was
in the presence of 0.3% polypeptone or 0.3% meat extract. In consistent
reduced to a 2.37 ± 0.29 log10 reduction in GII.4 when the free available
with the CDC guideline [6], 1,000 ppm NaClO induced effective virol­
chlorine level of CA was decreased to 100 ppm, the effect being equiv­
ysis on HuNoV GII.2 in the presence of these organic matters while the
alent to 1,000 ppm NaClO (Fig. 4A). In the presence of 0.5% poly­
effect was weak on GII.4 genogroup.
peptone, HuNoV virolysis by 60-min contact with 100 ppm CA was
reduced to 0.96 ± 0.20 and 0.64 ± 0.02 log10 reduction in GII.4 and
GII.2, respectively, but the effects were superior to 1,000 ppm NaClO

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H. Goda et al. Journal of Infection and Chemotherapy 28 (2022) 67–72

Fig. 3. Log10 reduction of HuNoV RNA copy number after treatment with
CA and NaClO in the presence of 0.3% polypeptone (A) or 0.3% meat
extract (B). HuNoV GII.4 and GII.2 were treated with CA (200 ppm) or NaClO
(200 ppm, 500 ppm or 1,000 ppm) for 30 min. After neutralization and RNase
treatment, intact HuNoV particles were quantified by RT-qPCR. The data are
shown as mean ± SD of the log10 reduction in HuNoV RNA copy number.
Columns marked with different letters indicate significant differences (p
< 0.05). Fig. 4. Log10 reduction of HuNoV RNA copy after treatment with CA and
NaClO in the presence of 0.3% BSA plus 0.3% SRBC (A) or 0.5% poly­
peptone (B). HuNoV GII.4 and GII.2 were treated with CA (200 ppm or 100
(Fig. 4B). These results indicate that the HuNoV virolytic effect of CA is
ppm) or NaClO (1,000 ppm) for indicated time. After neutralization and RNase
relatively stable even in the presence of organic substances. treatment, intact HuNoV particles were quantified by RT-qPCR. The data are
shown as means ± SD of the log10 reduction in HuNoV RNA copy. Columns
marked with different letters indicate significant differences (p < 0.01).
3.6. Reactivity of CA to amino acids

The residual free available chlorine of 100 ppm each of CA and
NaClO after 10-min contact with the indicated concentrations of amino
acids or glutathione was monitored by the TMB method. As shown in
Fig. 5 and Table S1, the free available chlorine of CA was reduced dose-
dependently in contact with cysteine, histidine and glutathione while no
reduction was observed with other amino acids such as glycine, threo­
nine, leucine, lysine and glutamic acid. In the case of NaClO, free
available chlorine was decreased to under the detection limit after
contact with all of the amino acids tested. These results indicate that CA
has reaction selectivity to amino acids, while this is not the case for
NaClO.
4. Discussion
CA is a novel type of chlorine-based disinfectant which has been
approved as a Type 2 OTC medicine in Japan. Previous reports
demonstrated that CA shows virucidal and sporicidal activity equivalent
to NaClO [9,16]. NaClO is a powerful and rapidly-acting disinfectant
that is recommended to decontaminate non-enveloped viruses including
HuNoV [5]. However, the free chlorine level of NaClO rapidly declines
in contact with organic substances because of its high reactivity [8,17].
We have reported so far that CA maintains its free chlorine level even
in the presence of organic matter and efficiently inactivates feline cal­
icivirus (FCV, HuNoV surrogate) in solutions containing 0.3% BSA [9,
12,16]. Therefore, CA is suitable for the sanitation of environmental
surfaces contaminated by feces or vomit from patients infected with
HuNoV. However, the susceptibilities of HuNoV surrogates such as FCV,
MNV and Tulane virus to disinfectants are considered to be different
from that of HuNoV [10,18]. In this study, we investigated the ability of
CA to inactivate two HuNoV strains of the GII genogroup (GII.2 and
GII.4) isolated from patients with acute gastroenteritis. Due to the lack
of an authorized assay system using in vitro cell culture for HuNoV, we
evaluated the damaging effect of CA against HuNoV by measuring the
viral RNA copy number employing RT-qPCR combined with RNase
treatment as described by Nowak et al. [11].
The results showed that 200 ppm CA reduced the number of intact
HuNoV GII.2 and GII.4 particles by > 3.0 log10 within 5 min in the
absence of organic substances (Fig. 2A). In the presence of 0.3% each of
BSA, polypeptone or meat extract, 200 ppm CA provided >3.0 log10
reduction in HuNoV RNA copy number within 30 min. On the other
hand, >90% of the virus remained intact after treatment with NaClO
(200 and 500 ppm) under the same conditions. Even with 1,000 ppm
NaClO, only 1.16 to 1.20 log10 reduction in HuNoV GII.4 RNA copy
number was observed (Figs. 2B and 3). Although CA was more effective
to reduce intact HuNoV than NaClO under organic-matter-rich condi­
tions, it should be noted that even with 200 ppm CA, more than 30-min
treatment is needed to inactivate HuNoV in highly contaminated envi­
ronments (Fig. 4). Therefore, direct use of CA to vomits or feces must be
avoided. In clinical setting, it is important to use CA after removing the
human excretions according to the authorized infection control manual.
Interestingly, the virolytic effects of CA and NaClO on HuNoV were
impaired differently depending on the organic matter. CA was more
affected by BSA, while NaClO was more affected by polypeptone and
meat extract. These findings indicated that the reaction mode of CA and

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H. Goda et al.
Fig. 5. Effect of amino acids on the free available chlorine level of CA and
NaClO. Amino acids were added to the indicated concentrations with 100 ppm
each of CA (A) or NaClO (B). After 10-min incubation, the residual free avail­
able chlorine level was measured by the TMB method [12]. The data are
expressed as means of percentages of the initial free available chlorine level
from three independent repeats. Refer to Table S1 for the date with statistical
analysis, which includes the results from 15 kinds of amino acids tested.
NaClO with organic substances is different.
To address the reason why CA maintains its sanitizing effect in the
presence of organic matter, we monitored the free available chlorine
level of CA and NaClO after contact with selected amino acids at con­
centrations ranging from 0.1 to 10 mM. As shown in Fig. 5, CA showed
selective reactions with cysteine, histidine and glutathione, while NaClO
reacted with all of the amino acids tested. These results indicate that
reaction selectivity to microbial components might make CA more
tolerant to organic matter load than NaClO.
Hatanaka et al. reported that CA effectively killed Campylobacter
jejuni even in the presence of 0.5% BSA [19]. They found that CA
aggregated outer membrane proteins of C. jejuni, but damage was not
found in its genomic DNA, concluding that the mechanism of CA to kill
C. jejuni is deterioration of proteins with essential functions. In line with
these findings, we observed that the residual HuNoV RNA copy number
after exposure to 200 ppm CA without RNase treatment was nearly 1.8
log10 greater than that with RNase treatment before RT-qPCR (data not
shown). Thus, it is plausible that CA damages HuNoV by attacking
specific amino acids in the viral capsid protein. Recently, we identified a
novel reactive oxygen species, the chloroperoxyl radical (ClOO・), in
acidified sodium chlorite (ASC), and this radical is long-lived and a main
source of oxidizing power of ASC [20]. Since CA is a stabilized form of
ASC and a long-lived disinfectant, it is plausible that ClOO・ is a main
active component of CA. Analysis of the reaction mode of ClOO・ with
microbial components is expected to reveal the disinfection mechanism
of CA in future studies. Rachmadi et al. reported that MNV became less
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Journal of Infection and Chemotherapy 28 (2022) 67–72
susceptible to free chlorine through repeated exposure [21]. The
reduced susceptibility to free chlorine is correlated with a non­
synonymous mutation at (nt) 7280 in the minor capsid protein (VP2)
gene. The correlation of amino acid substitution and susceptibility to
chlorine indicates that virucidal activity of chlorine-based disinfectants
to HuNoV originates from the deterioration of viral capsid protein
function.
In this study, we evaluated the virolytic effect of CA on HuNoV based
on the reduction in the viral RNA copy number after treatment with
RNase, which distinguishes the damaged viral particles from intact vi­
rions. At present, RT-qPCR is a standard procedure to quantify HuNoV
due to the lack of an in vitro cell culture system to propagate HuNoV.
However, Costantini et al. reported that the reduction of HuNoV RNA by
test disinfectants did not correlate with the loss of viral infectivity [22].
They showed that 50 ppm free chlorine completely abrogated infectivity
of HuNoV. Nonetheless, a substantial amount of viral RNA was still
detected by RT-qPCR without RNase treatment. This finding indicates
that simple RT-qPCR might underestimate the potential of disinfectants
to inactivate HuNoV. Recently, an in vitro method for HuNoV propa­
gation employing human intestinal organoids has been developed [23]
and introduced to evaluate the inactivation capability of several disin­
fectants on HuNoV [22,24]. Although this method is still challenging
and not standardized to compare the decontamination efficiency of
disinfectants on HuNoV, a propagation assay using human intestinal
organoids will help to understand the mechanism of CA to inactivate
HuNoV.
In conclusion, CA effectively damages HuNoV even in the presence of
organic substances. This stability of CA is considered to originate from
the selective reactivity against specific microbial molecules. This char­
acteristic is advantageous not only for sanitation of environmental sur­
faces that are contaminated with human excretions containing HuNoV,
but also for reducing the risk of genotoxicity by chlorine.
Statement of ethics
The experimental protocol was approved by the institutional
research ethical review board of Kagawa University (approval number
2019-266). Fecal sampling was performed after informed consent in
compliance with the Helsinki Declaration was obtained from the
patients.
Author disclosure statement
This study was supported by collaboration fund from Kagawa Uni­
versity and Saikei Co. Ltd.
Author contribution
HG, TF and TK conceived and designed the study. HG, HNI, HY and
AT collected data. HG, TN, HK, TF and TK performed analysis and
interpreted the collected data. HG, TF and TK wrote the manuscript.
Declaration of competing interest
This study was performed as collaborative research of Kagawa Uni­
versity and Saikei Co. Ltd. All authors have no conflict of interest to
declare other than collaborative research funding from the company.
Acknowledgements
We thank Ms. Yukiko Nogami in Tokushima Health COOP, Tokush­
ima Kensei Hospital, for her assistance to collect the patients’ feces and
Ms. Sayuri Nagashima for her technical assistance.
H. Goda et al.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jiac.2021.10.001.
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