High-Pressure Inactivation of Human Norovirus Virus-Like Particles
Provides Evidence that the Capsid of Human Norovirus Is Highly
Fangfei Lou,aPengwei Huang,cHudaa Neetoo,eJoshua B. Gurtler,dBrendan A. Niemira,dHaiqiang Chen,eXi Jiang,cand
Department of Food Science and Technology, College of Food, Agricultural and Environmental Sciences,aand Division of Environmental Health Sciences, College of
Public Health,bThe Ohio State University, Columbus, Ohio, USA; Division of Infectious Diseases, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USAc;
USDA-ARS, Eastern Regional Research Center, Wyndmoor, Pennsylvania, USAd; and Department of Animal and Food Sciences, University of Delaware, Newark, Delaware,
accounts for more than 95% of nonbacterial acute gastroenteritis
(12, 30). The Centers for Disease Control and Prevention (CDC)
are sickened from food-borne illnesses each year, leading to
128,000 hospitalization and 3,000 fatalities (9). Remarkably, hu-
man NoV alone causes nearly 60% of the estimated illnesses (9).
Human NoV is classified as a category B biodefense agent by the
National Institute of Allergy and Infectious Diseases (NIAID) be-
cause it is highly contagious, extremely stable, and resistant to
with debilitating illness. Recent human volunteer studies and
mathematical modeling showed the average probability of infec-
tion for a single norovirus particle was close to 0.5 (36). Despite
the fact that human NoV causes significant health, emotional,
social, and economic burdens worldwide, no vaccines or effective
fact that human NoV cannot be grown in cell culture and there is
no small-animal model (11, 12). Consequently, research on this
biodefense agent has been seriously hampered.
Foods at high risk for human NoV contamination include
fresh produce, seafood, and ready-to-eat food (1, 13, 27). An ef-
fective food-processing technology is a key step to eliminate hu-
man NoV in high-risk foods. However, the survival, stability, and
well understood due to the lack of an appropriate cell culture
system. Two cultivable animal caliciviruses, feline calicivirus
(FCV) and murine norovirus (MNV), have been extensively used
uman norovirus (NoV) is the leading cause of acute gastro-
enteritis worldwide. It has been reported that human NoV
as human NoV surrogates (7, 39). FCV is a respiratory virus of
kittens, and unlike enteric viruses, it is susceptible to low pH and
elevated temperatures (7). MNV is genetically related to human
NoV and is considered a better surrogate (7); however, it was not
manifestations (i.e., without diarrhea and vomiting), host recep-
ceptible cell types (dendritic cells and macrophages versus diges-
tive epithelial cells), and pathogenesis (systemic infection versus
demonstrated that a number of nonthermal processing technolo-
gies, such as gamma irradiation, electron beam irradiation, and
trast, both MNV and FCV can be effectively inactivated by high-
27). However, whether these surrogates truly represent human
NoV inactivation by HPP remains unknown.
Only limited information about the sensitivity of human NoV
to HPP is available based on a study of human volunteer subjects
(26). Leon et al. (26) reported that HPP at 600 MPa at 6°C for 5
Received 20 February 2012 Accepted 16 May 2012
Published ahead of print 25 May 2012
Address correspondence to Jianrong Li, email@example.com.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
aem.asm.orgApplied and Environmental Microbiology p. 5320–5327 August 2012 Volume 78 Number 15
subjects receiving oysters that were treated at 600 MPa did not
have any symptoms of norovirus infection and virus shedding, as
determined by reverse transcription-PCR detection of norovirus
other laboratories demonstrated that the cultivable human NoV
surrogates (MNV and FCV) were completely inactivated at 500
MPa and 4°C with a holding time of 2 min (5, 10, 25, 27). Appar-
ently, Norwalk virus was more resistant to HPP than FCV and
ancy between the results from human volunteer and human NoV
surrogate studies. Although a human volunteer study provides
valuable information about the survival of human NoV, it is no-
table that the susceptibility of an individual to human NoV de-
pends on their preexisting immunity, blood type, and age (19, 26,
33, 36). Moreover, based on safety concerns, human volunteer
rogate to understand the survival of human NoV is urgently
Expression of the human NoV capsid protein (VP1) results in
the formation of virus-like particles (VLPs) that are antigenically
been widely used in studying the epidemiology, immunology,
structure, and host/pathogen interaction of human NoV. There
are a number of advantages to using VLPs as a model to under-
produced in large quantities by expressing VP1 in a number of
(17, 18, 20). Second, damage to VLPs can be evaluated using bio-
physical and biochemistry methods, such as electron microscopy
(EM), sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), and immunoblotting assays. Finally, VLPs possess
authentic receptor binding activity, which is essential for viral in-
effectiveness of HPP in inactivating human NoV VLPs has not
In this study, we systematically evaluated the effectiveness of
HPP in disrupting VLPs derived from a GII.4 human NoV that
currently is prevalent in many countries. Our results showed that
pressurization under 500 to 600 MPa for up to 60 min was insuf-
to be much more resistant to HPP than human NoV surrogates
and most other food- and waterborne viruses. Nevertheless, we
also discovered that the structure and function of human NoV
VLPs were completely disrupted by treatments at higher pressure
levels (700 to 900 MPa).
MATERIALS AND METHODS
Production and purification of VLPs in a baculovirus expression sys-
tem. Production and purification of VLPs derived from human NoV
sity) were described previously (13, 29). Briefly, Spodoptera frugiperda
(Sf9) cells were infected with baculovirus at a multiplicity of infection
(MOI) of 10, and the infected Sf9 cells and cell culture supernatants were
harvested at 6 days postinoculation. The VLPs were purified from cell
(wt/vol) sucrose cushion, followed by CsCl isopycnic gradient (0.39
g/cm3) ultracentrifugation. Purified VLPs were analyzed by SDS-PAGE,
the VLPs was determined using Bradford reagent (Sigma Chemical Co.,
St. Louis, MO).
High-pressure treatment of human NoV VLPs. One hundred-mi-
croliter aliquots of highly purified human NoV VLPs were packaged in
sterile polyethylene stomacher pouches (Fisher Scientific International,
Ontario, Canada) and double sealed in a larger pouch to avoid leakage
using an Impulse sealer (American International Electric, Whittier, CA).
PT-1; Avure Tchnologies, Kent, WA) using water as a hydrostatic me-
dium. Samples were treated at pressure levels above 600 MPa in a Mini
Foodlab FPG5620 high-pressure unit (Stansted Fluid Power, Essex,
United Kingdom) at the USDA Agricultural Research Service (ARS).
HPP treatments reported in this study did not include the pressure
come-up time (ca. 22 MPa/s) and release time (?4 s). All temperatures
at ?80°C after processing for further analyses.
Transmission electron microscopy. Twenty microliters of HPP-
treated and untreated human NoV VLP samples were fixed in copper
grids (Electron Microscopy Sciences, Inc., Hatfield, PA) and negatively
stained with 1% ammonium molybdate. The VLPs were visualized under
an FEI Tecnai G2 Spirit Transmission Electron Microscope (TEM) (80
kV) at the Microscopy and Imaging Facility, The Ohio State University.
Images were captured using a MegaView III side-mounted charge-cou-
pled device (CCD) camera (Soft Imaging System, Lakewood, CO).
Analysis of viral proteins by SDS-PAGE. VP1 proteins in HPP-
treated or untreated human NoV VLP samples were analyzed by SDS-
PAGE. Four microliters of each virus suspension was diluted 5-fold in
6.25 mM Tris-HCl (pH 6.8), and 5% glycerol and boiled for 5 min. Viral
proteins were separated on a 12% polyacrylamide gel, followed by Coo-
scanned using a Typhoon 9210 scanner (GE Healthcare, Piscataway, NJ),
and the intensities of protein bands were determined using ImageQuant
TL software (GE Healthcare). For each protein band, background was
subtracted, and the intensities of HPP-treated protein bands were nor-
malized to the value for untreated controls. The percentage of protein
remaining was calculated for each treatment time.
Western blotting. VP1 protein was separated by 12% SDS-PAGE, as
described above, and further transferred onto a Hybond ECL nitrocellu-
lose membrane (Amersham, Piscataway, NJ) in a Trans-Blot Semi-Dry
electrophoretic transfer cell (Bio-Rad, Hercules, CA). The blots were first
(PBST) containing 5% skim milk and then incubated with a polyclonal
antibody against human NoV VP1 protein at a dilution of 1:5,000. After
being washed with PBST three times (15 min per wash), the blot was
incubated with a horseradish peroxidase (HRP)-conjugated anti-mouse
IgG secondary antibody (1:100,000 dilution; Invitrogen). After being
tific, Pittsburg, PA) and exposed to Kodak BioMax MR film (Kodak,
Receptor binding assays. Saliva samples from healthy adult volun-
by the Institutional Review Board at the Cincinnati Children’s Hospital
Medical Center. The receptor binding capabilities of pressure-treated or
untreated VLPs to HBGA types A, B, and O were measured using saliva-
binding assays as described by Huang et al. (18). Briefly, microtiter plates
were coated with saliva samples at a dilution of 1:5,000 in PBS (pH 7.4).
?g/ml in PBS were added. The bound human NoV VLPs were detected
High-Pressure Processing of Human Norovirus VLPs
August 2012 Volume 78 Number 15 aem.asm.org 5321
the addition of HRP-conjugated goat anti-guinea pig IgG (Invitrogen) at
a dilution of 1:5,000. The plates in each step were incubated at 37°C for 1
h and washed 5 times with PBS. The enzyme signals were detected with a
TMB kit (Kirkegard & Perry Laboratories), and the optical density at 450
(ELISA) plate reader. The binding affinity of VLPs was determined using
an endpoint dilution method in which the VLPs were diluted serially to
reach a cutoff point of an optical density at 450 nm of 0.1.
tistical analysis was performed by one-way multiple comparisons using
?0.05 was considered statistically significant.
Effect of high pressure on the integrity of human NoV VLPs.
Previous studies have demonstrated that human NoV surrogates
(MNV and FCV) and most other food- and waterborne viruses
(such as hepatitis A virus [HAV] and human rotavirus [HRV])
human NoV under HPP using VLPs as a model was tested under
the same conditions. As shown in Fig. 1A, the expression of hu-
man NoV VP1 in insect cells formed small, round, structured
particles that were structurally similar to native virions. Consis-
tent with previous observations (31, 37), two sizes of VLPs with
diameters of 30 to 38 nm and 20 to 23 nm were found under EM
(Fig. 1A). Surprisingly, the integrity of the capsid structure was
the number of 38-nm particles observed was notably reduced,
while the 23-nm particles remained unaffected (data not shown).
The pressure was then increased to a level of 600 MPa at 4°C for 5
to 60 min, but the results were essentially similar to those at 500
detectable (Fig. 1C). As the holding time increased to 60 min, the
38-nm VLPs disappeared, whereas the 23-nm VLPs were still in-
tact (Fig. 1D).
38-nm VLPs were reduced in number with a commensurate in-
crease in the treatment time (i.e., from 5 to 30 min); however,
ment (Fig. 1E and F). As the treatment time increased to 45 min,
ber of 38-nm VLPs was notably reduced after 15 min (Fig. 1H),
and the 38-nm particles were undetectable after a 30-min treat-
ment (Fig. 1I). In addition, the number of 23-nm particles also
at 4°C for various holding times (5, 15, 30, 45, and 60 min). VLPs were also treated at 900 MPa at 4°C for 1, 2, 5, and 8 min. HPP-treated and untreated samples
were negatively stained with ammonium molybdate and visualized by transmission electron microscopy. The 38-nm VLPs are indicated by arrows. At each
pressure level, selected treatment time points are shown.
Lou et al.
aem.asm.org Applied and Environmental Microbiology
imum pressure level that can be achieved in our pressure unit.
Remarkably, it was found that only a short holding time was suf-
a 1-min treatment (Fig. 1K), the number of 38-nm VLPs was
detected and the number of 23-nm VLPs was dramatically re-
duced (Fig. 1L). Moreover, the integrity of the 23-nm VLPs was
Taken together, these results demonstrated that (i) the 38-nm
VLPs can be disrupted at 500 to 600 MPa held for 45 to 60 min,
whereas the 23-nm VLPs are still intact after a 60-min holding
time; (ii) the 38-nm and the 23-nm VLPs can be disrupted at 700
5 min, respectively.
Effect of high pressure on human NoV capsid protein. To
determine whether HPP degraded the human NoV capsid pro-
teins, we analyzed the untreated and pressurized VLPs by SDS-
PAGE. As shown in Fig. 2, two protein bands with molecular
masses of 58 and 52 kDa, corresponding to the native full-length
VP1 and the cleaved form of VP1 (cVP1) of the baculovirus-de-
rived VLPs, were detected in both treated and untreated samples.
At 500-MPa pressure, the abundance of native VP1 and cVP1
of human NoV was not degraded at 500 MPa at a holding time of
samples treated at 600 MPa for up to 60 min (data not shown).
However, degradation of VP1 was observed when pressure was
of VP1 protein bands was reduced to 60 to 80% of the untreated
level when the treatment time increased from 30 to 60 min at 700
MPa. At 800 MPa for 15 min, only 30 to 40% of VP1 and cVP1
reduction of VP1 was observed even when the holding time was
increased to 30, 45, and 60 min. Remarkably, the majority of VP1
and cVP1 proteins were degraded after 900-MPa treatment for
only 1 min (Fig. 2D, top). Quantitative analysis showed that only
about 10% of human NoV VP1 protein remained undamaged
2 and 5 min. Western blotting was also performed to determine
whether the undegraded viral proteins still reacted with a human
NoV-specific antibody. As shown in Fig. 2 (middle row of gels),
the amounts of native and cVP1 proteins detected by Western
blotting were indistinguishable from those detected by SDS-
PAGE, demonstrating that pressurized VLPs were still able to re-
act with norovirus-specific antibody. Therefore, these results
demonstrated that pressure levels of 500 to 600 MPa held for 60
min did not degrade viral proteins whereas higher pressure levels
from 700 to 900 MPa significantly reduced the viral proteins.
FIG 2 Analysis of human NoV capsid protein by SDS-PAGE and Western blotting. (A) Effect of 500-MPa pressure on human NoV capsid protein. (Top)
Analysis of VP1 proteins by SDS-PAGE. The purified VLPs were pressurized at 500 MPa at 4°C for 5, 15, 30, 45, and 60 min. Two micrograms of untreated and
treated VP1 proteins was analyzed by 12% SDS-PAGE, followed by Coomassie staining. VP1, native full-length capsid protein; cVP1, cleaved VP1. (Middle)
Western blot analysis of VP1 proteins using guinea pig anti-human NoV antiserum. (Bottom) Quantitative analysis of remaining VP1 proteins detected by
SDS-PAGE after HPP treatment. (B) Effect of 700-MPa pressure on human NoV capsid protein. One microgram of untreated and treated VP1 proteins was
analyzed by 12% SDS-PAGE. (C) Effect of 800-MPa pressure on human NoV capsid protein. Two micrograms of untreated and treated VP1 proteins was
analyzed by 14% SDS-PAGE. (D) Effect of 900-MPa pressure on human NoV capsid protein. VLPs were treated under 900 MPa at 4°C for 1, 2, and 5 min. Five
micrograms of untreated and treated VP1 proteins was analyzed by 12% SDS-PAGE.
High-Pressure Processing of Human Norovirus VLPs
August 2012 Volume 78 Number 15aem.asm.org 5323
NoV VLPs. One of the major advantages of using the VLP as a
a native virion (18, 20, 33), which is an important indicator for
norovirus survival. Based on this fundamental premise, the effect
of HPP on the binding of human NoV VLPs to three types of
VLPs efficiently bound to all three types of HBGAs in a dose-
dependent manner (data not shown). There were no significant
reductions of receptor binding to all three types of saliva when
VLPs were pressurized at 500 MPa (Fig. 3A) and 600 MPa (data
not shown), with holding times ranging from 5 to 60 min. Inter-
estingly, the receptor binding of VLPs to each type of saliva was
3B). The binding of VLPs to type A saliva was not significantly
treatment at 700 MPa for 5 and 15 min, respectively. When the
holding time was increased to 30 min, receptor binding to type A,
B, and O saliva was reduced to 60%, 40%, and 41%, respectively.
types of receptor binding after treatment for 45 and 60 min (P ?
0.05). After treatment for 45 min, the ability of VLPs to bind to
type A, B, and O saliva was reduced to 40%, 30%, and 42%, re-
spectively. After a 60-min treatment, receptor binding to type A,
B, and O saliva was further reduced to 15 to 25% compared to
untreated VLPs (Fig. 3B). The receptor binding ability of VLPs
was significantly reduced at 800 MPa held for 5 to 15 min (Fig.
3C). At 800 MPa for 5 min, VLPs retained 85%, 60%, and 65% of
their binding ability to type A, B, and O receptors, respectively
(Fig. 3C), demonstrating that type A receptor binding was more
resistant to HPP than binding to types B and O (P ? 0.05). Inter-
estingly, the receptor binding activities of all three blood types
were reduced to 15 to 20% when the treatment time increased to
15 min. The inactivation kinetics of all three receptor binding
activities exhibited a flat tailing effect, retaining 10 to 15% when
the treatment time was increased to 15 to 60 min (Fig. 3C). The
receptor binding activity of human NoV VLPs was dramatically
reduced after pressurization at 900 MPa for 1 min (Fig. 3D). Less
than 15% of receptor binding activity remained for all three types
as the treatment time increased from 2 to 5 min. After an 8-min
treatment, the receptor binding activity of human NoV VLPs was
abolished (less than 3%). Taken together, these results demon-
pressures of 700, 800, and 900 MPa require 60, 15, and 1 min,
and (iii) receptor binding of types O and B were more sensitive to
HPP than that of type A.
To date, the survival of human NoV is not well understood, be-
VLPs as a model to determine the stability of human NoV under
HPP. We found that the structure and receptor binding function
MPa for 15 min, or 900 MPa for 2 min. To our knowledge, this is
the first report on the stability of human NoV capsid under HPP.
Evidence that human norovirus is more stable than its sur-
been extensively used as surrogates to study human NoV (7, 27,
39). Previously, we and others have shown that MNV and FCV
of human NoV than previously reported surrogates because it
of 500 to 600 MPa were not sufficient to destroy human NoV
VLPs. Although extending the holding time increases conforma-
tional alteration and functional impairment of VLPs, the effec-
tiveness of HPP in damaging VLPs appears to be more pressure
dependent, as there is more prominent disruption of VLPs with
MPa, 45 min was required to disrupt the structure and receptor
severe at 800 and 900 MPa, which required only 15 and 2 min to
disrupt VLPs, respectively. Therefore, our results suggest that hu-
MNV and FCV.
To date, the most convincing data regarding the stability of
human NoV has come from the human volunteer study (26).
genomic copies of Norwalk virus (human NoV G I.1) with or
without HPP treatment. It was found that HPP at 600 MPa and
6°C for 5 min completely inactivated Norwalk virus in seeded
oysters, based on the lack of infection and virus shedding in the
challenged volunteers. In contrast, treatment at 400 MPa (at 6°C
or 25°C) for 5 min was insufficient to prevent norovirus infection
FIG 3 Receptor binding activities of human NoV VLPs after high-pressure
processing. Purified human NoV VLPs were pressurized at levels from 500 to
900 MPa at 4°C for various holding times. The receptor binding activities of
untreated and treated VLPs to three types of HBGA saliva (type A, B, and O
plate reader. The binding affinity of VLPs was determined using an endpoint
dilution method in which VLPs were diluted serially to reach a cutoff point of
an OD450of 0.1. The percentage of receptor binding compared to untreated
samples was calculated for each treatment time. (A) Receptor binding after
500-MPa treatment. (B) Receptor binding after 700-MPa treatment. (C) Re-
Lou et al.
aem.asm.org Applied and Environmental Microbiology
and shedding in human subjects. In addition, Sánchez et al. (32)
compared the stabilities of human NoV GII.4 and MNV under
HPP by quantification of viral genomic RNA using real-time re-
ment at 500 MPa for 15 min, whereas 2.5-log-unit reduction was
their results is that human NoV may be more stable than MNV.
The higher stability of human NoV led to less release of its
genomic RNA when exposed to environmental RNase. As a con-
sequence, more genomic-RNA copies (or less reduction) of hu-
man NoV were detected by real-time RT-PCR. These observa-
tions, coupled with our finding that the human NoV capsid is
stable under HPP than animal caliciviruses, such as MNV and
Comparative pressure resistance of native human NoV viri-
ons and VLPs. The recent human volunteer study indicated that
Norwalk virus was completely inactivated under 600 MPa at 6°C
for 5 min (26). In comparison, this study showed that a holding
time of more than 60 min at this pressure level was required to
disrupt the structure and function of human NoV capsid. How
might we account for this apparent discrepancy? First, the VLPs
used in our study were derived from human NoV strain GII.4,
whereas human NoV strain GI.1 Norwalk virus was used in the
supported by the fact that some viruses within the same genus or
family have been known to exhibit distinct barosensitivities to
HPP (16, 22, 28). For example, poliovirus, a picornavirus, is ex-
tremely resistant to HPP (16, 22, 38). In contrast, HAV, another
picornavirus, is quite sensitive to HPP (6, 16, 24). Similarly, cox-
sackievirus A9 is highly pressure sensitive whereas coxsackievirus
B5 is highly pressure resistant (22). Second, the susceptibility of
human volunteers to human NoV infection remains to be deter-
mined. The infectivity of human NoV is dependent on the preex-
isting human NoV immunity, blood type, and age of the human
subjects (19, 26, 33, 36). The minimum infectious dose for each
individual may be different. Third, we grant the possibility that
human NoV VLPs may be more stable than native virions. VLPs
virions contain major (VP1) and minor (VP2) capsids and
genomic RNA (4, 37, 39). For native virions, the interaction of
capsid proteins with genomic RNA may play a role in the proper
RNA-protein interaction, which may impair virus infectivity.
Fourth, it is possible that loss of infectivity may occur earlier than
out that human NoV may be even more stable under pressure
than VLPs under HPP. It has been reported that one role for VP2
disassembly and degradation (4, 39).
Another interesting finding from this study is that the 23-nm
this study, it was found that HPP first disrupted the 38-nm VLPs,
followed by the 23-nm VLPs. Since both the 23- and 38-nm VLPs
are composed predominantly of the 58,000-molecular-weight
(58K) capsid protein and they share similar biochemical proper-
susceptibilities to HPP could be ascribed to different icosahedral
symmetries of capsids, numbers of copies of the VP1 protein,
tains 180 units, or 90 dimers, of VP1 that assemble into icosahe-
is formed by 60 units of the VP1 protein arranged as 30 dimers
exhibiting T ? 1 symmetry (21, 28). Interestingly, many animal
and human caliciviruses have particles of two sizes. For example,
particles of two sizes (35 to 40 and 15 to 20 nm in diameter) were
rhagic disease virus and European brown hare syndrome virus,
native infectious small virus particles are always more stable than
the large ones.
New mechanistic insights into virus inactivation by HPP.
Previously, we found that disruption of the viral envelope and/or
capsid structure, but not degradation of the viral protein or ge-
nome, was the primary mechanism underlying HPP-induced vi-
rus inactivation (27, 28). This is consistent with the fact that HPP
generally alters the quaternary and tertiary structures, but not the
primary and secondary structures, of proteins, since HPP gener-
ally has no adverse effect on covalent bonds (2, 15). However, in
tural proteins, such as VP1, VP2, and VP3, were significantly re-
duced at 450 MPa for 2 min (28). These viral proteins may be
degraded by trypsin, since rotavirus requires it for growth (3, 28).
In this study, we found that the VP1 protein was significantly
diminished at 800 MPa for 10 min and at 900 MPa for 1 min, but
not at pressure levels of 500 to 600 MPa even after 60 min. Since
these VLPs were expressed from insect cells without the addition
of trypsin and protease inhibitors were included in all solutions
used for VLP purification, the degradation of VLPs at 800 to 900
MPa is likely due to HPP. It is possible that some covalent bonds
may be susceptible to HPP at ultrahigh pressure levels and/or for
extended holding times, which leads to protein degradation. Fur-
thermore, we also found that impairment of the receptor binding
activity is one of the mechanisms of viral inactivation by HPP.
HPP is able to destabilize the tertiary structure of VLPs, which
leads to disruption of the receptor binding domain(s), thus im-
pairing its receptor binding. Interestingly, HPP differentially dis-
rupted the binding of human NoV VLPs to different types of HB-
GAs. Binding activities to types B and O were more sensitive to
HPP than that to type A. Therefore, it is likely that the binding
domain(s) of human NoV VLPs for types B and O is more easily
altered by HPP than that for type A. Previously, it has been sug-
gested that HPP affected the receptor binding of MNV to RAW
264 cells based on quantifying the genomic copies of MNV being
did not directly measure the binding of MNV to sialic acid, the
functional receptor for MNV. Taken together, mechanisms of vi-
ral inactivation by HPP may include disruption of the viral enve-
lope and capsid structure, receptor binding activity of the attach-
ment protein, and perhaps degradation of some viral proteins at
ultrahigh pressure levels.
High-Pressure Processing of Human Norovirus VLPs
August 2012 Volume 78 Number 15 aem.asm.org 5325
Optimization of HPP parameters for the inactivation of
pressure-resistant viruses. It should be noted that human NoV
VLPs were eluted in PBS solution (pH 7.0), and all pressure treat-
ments were performed at 4°C. Under these conditions, human
NoV VLPs were highly resistant to HPP. Intriguingly, HPP at
pressures of 800 to 900 MPa efficiently disrupted human NoV
VLPs in a rather short time (2 to 15 min). Since pH and temper-
parameters, such as pH, temperature, and salt, to enhance the
inactivation of human NoV VLPs at commercially acceptable
pressure levels (usually less than 700 MPa). An important techni-
of pressure and temperature because an increase in pressure level
leads to an increase in temperature. Although our current study
human NoV VLPs, preliminary data showed that VLP inactiva-
tion was significantly enhanced at an initial temperature of 20°C
compared to that at an initial temperature of 4°C under 700 MPa
pressurization at 600 MPa for 5 min. Whether other HPP-resis-
tant viruses can be effectively inactivated at higher pressure levels
(700 to 900 MPa) may also be investigated based on the human
process termed “temperature-assisted HPP” may be a prospective
In summary, the present study highlights a major gap in our
understanding of the stability of the capsid of human NoV under
HPP. Our results demonstrate that HPP is capable of inactivating
the capsid of human NoV at 800 to 900 MPa with a short holding
time and that the capsid of human NoV is more stable under
pressure than reported surrogate viruses. While human NoV
VLPs may not fully equate to viable human NoV, destruction of
the VLP capsid is highly suggestive of a representative response
that would take place in viable human NoV.
and X.J., a food safety challenge grant (2011-68003-30005) from the
J.B.G., and B.A.N., and a NoroCORE project grant (2011-68003-30395)
from USDA NIFA to J.L. and X.J.
We thank Linda Saif for providing the VP1 gene of human NoV GII.4
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