International Journal of Food Microbiology 273 (2018) 28–32

Contents lists available at ScienceDirect

International Journal of Food Microbiology

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

Evaluation of gaseous chlorine dioxide for the inactivation of Tulane virus T

on blueberries☆
⁎
David H. Kingsleya, , Rafael E. Pérez-Péreza, Brendan A. Niemirab, Xuetong Fanc
a
USDA ARS ERRC Food Safety & Intervention Technologies Research Unit, Delaware State University, Dover, DE, United States
b
USDA ARS ERRC Food Safety & Intervention Technologies Research Unit, Wyndmoor, PA, United States
c
USDA ARS ERRC Chemical Residue and Predictive Microbiology Research Unit, Wyndmoor, PA, United States

A R T I C LE I N FO A B S T R A C T

Keywords: To determine the effectiveness of gaseous chlorine dioxide (gClO2) against a human norovirus surrogate on
Gaseous produce, gClO2 was generated and applied to Tulane virus-coated blueberries in a 240 ml-treatment chamber.
Chlorine dioxide gClO2 was produced by an acidifying sodium chlorite solution. Initial assessments indicated that blueberries
Norovirus treated with gClO2 generated from ≤1 mg acidified sodium chlorite in the small chamber appeared unaffected
Blueberries
while gClO2 generated from ≥10 mg of acidified sodium chlorite solution altered the appearance and quality of
the blueberries. Treatments of inoculated blueberries with gClO2 generated from 0.1 mg sodium chlorite reduced
the virus populations by > 1 log after exposure for 30 to 330 min. For the 1 mg sodium chlorite treatments, the
virus populations were reduced by > 2.2 log after 15 min exposure and to non-detectable levels (> 3.3 logs
reductions) after 180 min exposure. Measured concentrations of gClO2 peaked in the treatment chamber at
0.9 μg/l after 10 min for 0.1 mg treatments and 600 μg/l after around 20 min for 1 mg treatment. Overall results
indicate that gClO2 could be a feasible waterless intervention for blueberries and other produce.

1. Introduction
Human norovirus (HuNoV) is the leading cause of epidemic non-
bacterial gastroenteritis (Glass et al., 2009; Hall et al., 2013), and
causes approximately half of all foodborne illnesses in the United States
annually (Scallan et al., 2011). Transmission is through the fecal-oral
route or via exposure to aerosolized vomitus (Atmar et al., 2014; Kirby
et al., 2016). Preharvest HuNoV contamination of produce can occur
via exposure to feces, contaminated soil, nonpotable irrigation water, as
well as fungicides and insecticides made with contaminated water
(Olaimat and Holley, 2012). Postharvest human handling, as well as
contaminated harvest and processing equipment, rinse water and ice, or
even vomiting events within the proximity of produce (Kirby et al.,
2016) are all subsequent potential sources of HuNoV contamination
(Olaimat and Holley, 2012). As a result, produce, such as berry fruits
that are eaten uncooked, are prone to transmit foodborne norovirus
(Kniel and Shearer, 2009; Tavoschi et al., 2015). Therefore a practical
non-thermal means of inactivating HuNoV is highly desirable.
Unfortunately, foodborne viruses are predominately non-enveloped,
and therefore are generally resistant to most common aqueous disin-
fectants, including ethanol, and quaternary ammonium compounds
(Fraisse et al., 2011; Gulati et al., 2001; Jean et al., 2003; Kingsley
et al., 2014; Nowak et al., 2011; Tung et al., 2013). Although HuNoV
has recently been propagated on a limited basis (Ettayebi et al., 2016;
Jones et al., 2015), research evaluating the inactivation of human
norovirus remains hindered by the challenges of assessing virus viabi-
lity in vitro. Therefore, routinely cultivable surrogates, such as Tulane
virus (TV; Farkas, 2015) which is closely related to HuNoV, are com-
monly used to assess inactivation. While all human surrogates have
slightly different properties and cannot be directly compared to non-
propagable HuNoV since its properties remain largely undefined, Tu-
lane virus was chosen for this study based on its genetic similarities to
human norovirus, its ease of propagation, its robustness and property
similarities to murine norovirus (Cromeans et al., 2014; Hirneisen and
Kniel, 2013; Tian et al., 2013), as well as its ability to bind to porcine
gastric mucin in an manner analogous manner to human norovirus
(Dancho et al., 2012; Li and Chen, 2015).
One potential disinfectant is gaseous chlorine dioxide (gClO2).
Recent meta-analysis of literature identifies gClO2 as a highly effective
sanitizer for inactivation of key bacterial pathogens such as Escherichia
coli O157:H7, Salmonella and Listeria monocytogenes (Prado-Silva et al.,
2015). gClO2 has several advantages. First, it can inactivate bacterial

☆
Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by
the U.S. Department of Agriculture (USDA). The USDA is an equal opportunity provider and employer.
⁎
Corresponding author.
E-mail address: David.Kingsley@ars.usda.gov (D.H. Kingsley).

https://doi.org/10.1016/j.ijfoodmicro.2018.01.024
Received 27 November 2017; Received in revised form 19 January 2018; Accepted 30 January 2018
Available online 01 February 2018
0168-1605/ Published by Elsevier B.V.
D.H. Kingsley et al.
pathogens, yeasts, mold and bacillus spores on berry fruits (Han et al.,
2004; Popa et al., 2007; Shirasaki et al., 2016; Sy et al., 2005, Wu and
Kim, 2007). Second, it can be applied without substantially damaging
the organoleptic properties of produce (Han et al., 2004; Goméz-Lopéz
et al., 2008; Goméz-Lopéz et al., 2009). gClO2 can potentially reach
areas that liquid ClO2 cannot, or areas that are impenetrable to other
liquid disinfectants due to hydrophobic coatings (Olaimat and Holley,
2012). Furthermore, dry technologies are preferred by the berry in-
dustry since water can potentially encourage molds and spoilage. Also
unlike other chlorine-based sanitizers, gClO2 does not form undesirable
compounds by reacting with ammonia to form chloramine or trihalo-
methane compounds, the latter of which is considered carcinogenic
when reacting with proteins and other organic compounds (Di Cristo
et al., 2013, Fan and Sokorai, 2015). High humidity seems to be a key
aspect of gClO2 efficacy since attenuated results have been observed
below 30–40% humidity (Shirasaki et al., 2016). The mechanism of
action of ClO2 against enteric viruses is thought to be via simultaneous
damage to both the capsid and the 5′ end of the virus RNA (Li et al.,
2004; Yeap et al., 2016). The objective of the present study was to
determine the feasibility of gClO2 for inactivation of TV on the surface
of blueberries. Here, the kinetics of gClO2-mediated TV inactivation
were characterized as a function of concentration and treatment time.
2. Materials and methods
2.1. Virus and cell culture
Tulane virus (TV) was obtained from Dr. Haiqiang Chen (University
of Delaware) and Dr. Xi Jiang (University of Cincinnati College of
Medicine). TV was propagated in confluent monolayers of the monkey
kidney cell line MK2-LLC. MK2-LLC cells were cultured in low serum
Eagle's minimum essential medium (Opti-MEM; Gibco, Paisley PA),
supplemented with 2% fetal bovine sera (FBS; Atlanta Biologicals,
Flowery Branch GA); penicillin/streptomycin (100 U/ml) (Gibco) and
Glutamax (Gibco), at 37 °C under a 5% CO2 atmosphere. Virus stocks
were prepared by infection of MK2-LLC cells for 3 days followed by
freezing and thawing, pelleting of debris by centrifugation at 1000 ×g
for 30 min, followed by 0.2 μM filtration.
2.2. Blueberries contamination and virus extraction
Blueberries were obtained from a local supermarket. The con-
tamination ratio was 100 μl of TV (4 × 107 pfu/ml) per blueberry; the
sample size was five blueberries. Contamination was performed in a
50 ml conical tube, gentle agitation with a vortex was applied for 1 min
and then allowed to dry inside a biosafety cabinet for 1 h. After gClO2
treatment, the TV was eluted from the blueberries using 3 mL of the
elution buffer (100 mM Tris-HCl, 50 mM glycine, 50 mM MgCl2 and 1%
soy protein at pH 9.5) and gently agitating the blueberries for 1 min.
Later, the eluted virus was removed and the pH was neutralized to 7.0
with 2 N HCl. Then, ten-fold dilutions in Earle's balanced salt solution
(EBSS, Gibco) were prepared for all samples.
2.3. Chlorine dioxide treatment
For the gClO2 treatment, five TV-infected blueberries were placed
inside an adapted Mason jar (240 ml) containing a 10-ml glass beaker
(Fig. 1). The interior beaker contained a small stir bar and 1 ml of
distilled H2O containing different amounts (0.1, 1, 2.5, 5 or 10 mg) of
sodium chlorite (Natriumchlorit, Fluka Analytical, Italy). The jar was
sealed with an airtight lid equipped with a septum, and placed onto a
stir plate (Thermix Model 120MR, Fisher Scientific Inc., Fair Lawn NJ)
with stir speed setting of 2. Then, 1 mL of 10% HCl (Alfa Aesar, Ward
Hill MA) was injected, through the septum with a long-needle syringe,
into the beaker to instantaneously produce gClO2.
29
International Journal of Food Microbiology 273 (2018) 28–32
2.4. Virus quantification
TV was quantified using confluent monolayers of MK2-LLC cells on
six well dishes inoculated with serially-diluted virus in EBSS. Infection
was performed for 2 h, rocking plates every 15 min at 37 °C followed by
overlay with a 1:1 mix of 2× Opti-MEM (37 °C) and 3% low melting
agarose at 42 °C (Fisher Inc.). Plates were incubated for 4 days in a
tissue culture incubator at 37 °C, followed by staining with 1 ml of a
1:10 dilution of a 0.33% neutral red (Fisher) stock. After 4 h of in-
cubation with the stain, plaques were counted. Log reductions were
calculated based on the amount of TV extracted from inoculated sam-
ples without gClO2 treatment. In cases where no TV plaques were ob-
served, the maximum reduction was assumed by subtracting the
amount of TV extracted from untreated berries minus the limit of de-
tection for this assay (20 pfu).
2.5. Gas concentration measurements
Two commercial protocols were used to measure the concentrations
of chlorine dioxide inside the jars during the treatment periods. For
higher concentration treatments of gClO2 (≥1 mg sodium chlorite),
5 mL of headspace was withdrawn from the sealed jar using a glass
syringe. The 5 mL gas sample was injected through a Teflon-lined
septum into a sealed glass sampling cell (Hach Inc., Loveland, CO)
containing 10 mL of cold (4 °C) dH2O and 0.1 g DPD (N,N-diethyl-p-
phenylenediamine; Hach Inc.). The sampling cell was shaken vigor-
ously, then read using a DR/890 Colorimeter (Hach Inc.). For the jars
with 0.1 mg of sodium chlorite, the DPD method was not sensitive
enough to detect the low ClO2 concentrations. The Gastec Detector
Tube System (Gastec Inc., Fukayanaka, Japan) was therefore employed
to measure the gClO2 concentrations inside the jars with 0.1 mg of so-
dium chlorite. The system is composed of the Gastec Pump and detector
tubes (detection range: 0.025–1.2 ppm). To measure gClO2 concentra-
tions in the jars, 50 ml of headspace from the jars was withdrawn into
the detector tubes connected to the jars (through a septum and a
needle) at one end and the pump at the other end. Concentrations were
read using the scale printed on the tubes as gClO2 caused color changes
of the tube content. All measurements were repeated at least three
times (n = 3).
2.6. Humidity measurements
The same jar set up for ClO2 measurement was used to measure
relative humidity. The sensor of the Traceable Alarming Hygrometer/
Thermometer (Control Company, Friendswood, TX) was placed inside
the treatment jar through an opening in the lid, and the jar and opening
were sealed with an airtight lid and a rubber stopper. Humidity was
measured for up to 5 h, which was the longest chlorine dioxide treat-
ment time used on the blueberries. The numbers were recorded every
10 s for the first min, every min for 35 min, every half hour for 3 h and
every h for 5 h.
2.7. Statistical analysis
Experiments were repeated three or four times. Effects of sodium
chlorite amounts, and treatment times were analyzed with SAS version
9.4 (SAS Institute, Cary, NC) using the Duncan Multiple Range test of
General Linear Model procedure. Only significant (P < 0.05) results
are discussed unless stated otherwise.
3. Results
gClO2 was applied to TV-coated blueberries in a mason jar chamber,
as shown in Fig. 1, by acidifying solutions containing quantities of so-
dium chlorite ranging from 0.1 to 10 mg. Initial results indicated that a
30-min treatment with 2.5, 5 and 10 mg of acidified sodium chlorite
D.H. Kingsley et al. International Journal of Food Microbiology 273 (2018) 28–32

Fig. 1. Scheme of gaseous chlorine dioxide treatment system. Chambers with sodium chlorite were sealed with five Tulane virus contaminated-blueberries and chlorine dioxide was
generated by adding 1 ml of 10% HCl to various amounts of sodium chlorite.

3.5
a a a
3.0
a

2.5 ** ** **
*
Log reduction

2.0

1.5

1.0 b

0.5

0.0
0.1 1 2.5 5 10
Amount of NaClO2 (mg)

Fig. 2. Reduction of Tulane virus on blueberry surfaces after 30 min treatments with
gaseous chlorine dioxide generated from different amounts of sodium chlorite. Four trials
were performed (N = 4; n = 12). Error bars represent S.D. Bars with same letters are not
significantly different (P < 0.05). ⁎⁎Denotes treated samples where no virus was detected
for all trials. ⁎Denotes samples where no virus was detected for two trials.

reduced viable TV to levels below the limit of detection (≥2.7 log10),

while 1 and 0.1 mg resulted in inactivation averaging 2.5 and 0.6 log10,
respectively (Fig. 2). Based on visual observation, concentrations at
≥10 mg of acidified sodium chlorite clearly altered the appearance and
quality of the blueberries, while treatments ≤5 mg appeared less da-
maging, and blueberries treated with ≤1 mg appeared unaffected. So-
lutions of 0.1 and 1 mg were chosen for further optimization and eva-
luation of virus inactivation.
Since there was no observed damage to blueberries observed after
treatments with ≤1 mg of sodium chlorite, the effects of variable Fig. 3. Changes in headspace chlorine dioxide in the treatment chamber as a function of
treatment time. Measurements were performed in triplicate (n = 3). Vertical bars re-
treatment times for 0.1 and 1 mg gClO2-treated blueberry samples were
present standard deviations. Chlorine dioxide was generated from 0.1 mg (A) and 1 mg
subsequently evaluated. In addition, temporal humidity and gClO2
(B) sodium chlorite.
concentrations within the chamber were characterized. As shown in
Fig. 3A for 0.1 mg of sodium chlorite, a peak gClO2 concentration of
0.9 μg/l was observed after 10 min with a rapid decline to negligible

30
D.H. Kingsley et al. International Journal of Food Microbiology 273 (2018) 28–32

100 even though the amount of chlorite differed only by a factor of 10.
While humidity was low initially within the chamber, high humidity
was rapidly achieved. For example > 60% and > 80% humidity was
80 observed at 7 and 17 min, respectively (Fig. 4), which was maintained
for up to 5 h. As shown in Fig. 5A, 0.1 mg samples treated for 5 to
120 min had limited inactivation. No inactivation was observed after
Humidity (%)

60 5 min but inactivation observed ranged from 0.7–1.2 logs for 15 min
treatments and beyond. There were no significant differences in the
reductions of the virus among the treatment times ranging from 15 to
40
330 min (P < 0.05). For 1 mg chlorite (Fig. 5B), no virus was detected
(> 3.3- log reduction) after 180 min or more, with ≥2.2-log reduction
observed for 15 min and beyond.
20
4. Discussion
0
0 100 200 300 The goal of this study was to determine the feasibility of using gClO2
to inactivate a norovirus surrogate on blueberries. Here we demonstrate
Time (min) that gClO2 is an effective intervention for virus-contaminated blue-
Fig. 4. Changes in relative humidity (%) in the treatment chamber as a function of berries with sodium chlorite amounts between 0.1 and 1 mg readily
treatment time. Measurements were performed in triplicate (n = 3). Vertical bars re- inactivating TV without obvious damage to blueberries. Further, we
present standard deviations. characterized the humidity profile and temporal gClO2 concentrations
within the chamber during these experiments.
For gClO2 concentrations, it was noted that the differences in the
4 A gas concentrations were disproportionally larger than the ten-fold dif-
ference between 0.1-mg and 1-mg treatments. Presumably this differ-
ence was due to absorbance of gClO2 by blueberries (and the treatment
chambers); reducing the headspace gClO2 in the treatment jars. When a
3
lower amount of chlorite was used, the gas generated from chlorite
probably could not keep up with the gClO2 consumption by the berries,
thereby reducing headspace concentrations. Also it is important to note
2 a a that we used two different methods to determine ClO2 concentrations in
a a
the treatment chamber. The DPD method which was used for measuring
1 ab ClO2 in the 1 mg chlorite chamber gave higher readings (6 times
higher) than the gas tube method which was used for the 0.1 mg
Log reduction

chlorite treatments.
b
The results of these experiments suggest several ways to further
0 improve the utility of gClO2 treatments. First, humidity is a critical
4 5 B 15 30 60 180
a 330
a parameter for gClO2 effectiveness (Han et al., 2002; Morino et al., 2009;
a Goméz-Lopéz et al., 2009) that can dramatically effect inactivation. For
a example, Shirasaki et al. (2016) demonstrated that gClO2 at a con-
3 ** ** centration of 1 ml/m3 reduced populations of Staphylococcus aureus and
ab * Escherichia coli by < 2 logs in low humidity (30–40%), and by > 3 logs
* in high humidity (75–85%). Presumably establishing high humidity
2 levels prior to, and during, gClO2 application would have enhanced
inactivation of TV. In particular we note that initial 30-min time
b treatments with 0.1 and 1 mg sodium chlorite gave less inactivation
1 than subsequent 30-min sample treatments evaluated as part of the
330 min time course. One possible explanation for this discrepancy
might be variable atmospheric humidity at initial treatment. Second in
0 this experimental system, it is clear that a bolus of gClO2 gas is gen-
5 15 30 60 180 330 erated which peaks after a short period, and then declines. Presumably
a more constant (low) level of gClO2 would enhance virus inactivation
Treatment time (min) while limiting concomitant organoleptic damage.
Future studies will involve larger commercial scale gClO2 treat-
Fig. 5. Reduction of Tulane virus on blueberry surfaces by gaseous chlorine dioxide with
ments to demonstrate commercial feasibility utilizing industrial
variable treatment times. Blueberries with inoculated virus were treated for 5–330 min
with chlorine dioxide generated from A. 0.1 mg sodium chlorite (N = 3; n = 9) and B. equipment that provides constant levels of gClO2. We envision that
1 mg sodium chlorite (N = 4; n = 12). Error bars represent S.D. Bars with same letters are large scale treatments could be performed within temperature-control
not significantly different (P < 0.05). ⁎⁎Denotes treated samples where no virus was walk-in storage units after berries are packed in perforated plastic
detected for all trials. ⁎Denotes samples where no virus was detected for one trial. containers. Furthermore formal evaluation of sensory quality of berries
has not yet been performed on treated berries. Once optimal con-
concentrations at 35 min. For 1 mg chlorite treatments, a 600 μg/l peak centrations of constant levels of gClO2 are determined, it will be im-
was observed at about 20 min with tailing to limited concentrations at portant to compare the organoleptic qualities of berries after treatment
120 min (detection limit 30 μg/l) (Fig. 3B). Our results indicated that and after extended cold storage and to evaluate sensory quality of cold-
the differences in gClO2 concentrations in the treatment chambers be- stored blueberries treated with gClO2 levels that are sufficient to in-
tween the 0.1 and 1 mg sodium chlorite treatments were > 100 fold activate viruses. Also it should be noted that although gaseous ClO2 is
effective in inactivating Tulane viruses and other human pathogens on

31
D.H. Kingsley et al.
fresh produce items, ClO2 treatments may lead to formation of by-
products such as chlorite and chlorate (Trinetta et al., 2010; Smith
et al., 2014). It has been shown that these disinfection byproducts are
mainly found on the stem scar of tomatoes and the rind of cantaloupes
after ClO2 treatments (Smith et al., 2014). Possible formation of ClO2
byproducts on blueberries will need further investigation.
Curiously, aqueous ClO2 appears to be somewhat less effective than
gClO2. For example, for experiments with murine norovirus (MNV),
aqueous chlorine dioxide was reportedly less effective than peracetic
acid or sodium hypochlorite (chlorine) for inactivating MNV on
strawberries and blueberries (Girard et al., 2016). For HuNoV itself,
limited capsid disruption was noted after treatment with 200 ppm so-
lutions of chlorine dioxide (Nowak et al., 2011). Similar results were
noted by Kingsley et al. (2014) who tested specific liquid disinfectants
against HuNoV stool suspensions using the porcine gastric mucin
magnetic bead assay; essentially an assay that assesses capsid damage
based on the virus particle's ability, or inability, to bind to its receptor.
Results showed that GI.1 HuNoV was rapidly inactivated by chlorine
but 350 ppm chlorine dioxide in water required treatments for about
1 h to inactivate norovirus stool suspensions (~3 log10). Why gClO2 is a
more effective sanitizer than aqueous ClO2 remains to be determined.
Overall, our results demonstrate that gaseous ClO2 should be a suitable
intervention method for virus-contaminated blueberries and possibly
other produce as well.
Acknowledgments
We wish to thank Gary P. Richards for critical reading of the
manuscript. Funding for Rafael Perez-Perez was provided by USDA
NIFA grant #2015-69003-23410.
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