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Detection and molecular characterization of norovirus from oysters
implicated in outbreaks in the US
Jacquelina W. Woods
*
, Kevin R. Calci, Joey G. Marchant-Tambone, William Burkhardt III
FDA, Division of Seafood Safety, Gulf Coast Seafood Laboratory, Dauphin Island, AL, 36528, USA
article info
Article history:
Received 25 September 2015
Received in revised form
13 May 2016
Accepted 13 May 2016
Available online 15 May 2016
Keywords:
Norovirus
Outbreak
Molecular characterization
Oysters
abstract
Human noroviruses are the leading cause of non-bacterial shellfish associated gastroenteritis. Here we
report on the detection and characterization of norovirus (NoV) in shellfish associated outbreaks. Re-
quests were received from state and federal officials for technical assistance in the analysis of shellfish for
NoV and male specific coliphage (MSC; an enteric virus surrogate) during the years 2009 thru 2014. In
outbreaks where NoV was detected, genogroup II (GII) levels ranged from 2.4 to 82.0 RT-qPCR U/g of
digestive diverticula (DD) while NoV genogroup I (GI) levels ranged from 1.5 to 29.8 RT-qPCR U/g of DD.
Murine norovirus extraction efficiencies ranged between 50 and 85%. MSC levels ranged from <6to
80 PFU/100 g. Phylogenetic analysis of the outbreak sequences revealed strains clustering with GI.8, GI.4,
GII.3, GII.4, GII.7, and GII.21. There was 100% homology between the shellfish and clinical strains
occurring in 2 of 8 outbreaks. Known shellfish consumption data demonstrated probable infectious
particles ingested as low as 12. These investigations demonstrate effective detection, quantification, and
characterization of NoV in shellfish associated with illness.
Published by Elsevier Ltd.
1. Introduction
Enteric viruses are the leading cause of foodborne infectious
diseases in the United States and of the 9.4 million cases of known
foodborne illnesses estimated annually, 58% are attributed to hu-
man noroviruses (Scallan et al., 2011). Noroviruses (NoV) are single-
stranded polyadenylated RNA viruses and they are separated into
genogroups IeVI, which includes more than 30 genotypes based on
the sequence comparison of the RNA polymerase and capsid re-
gions (Ando et al., 1995, 200 0; Caddy et al., 2013; Jiang et al., 1993;
Zheng et al., 2006). Genogroups I, II, and IV are known to infect
humans, with NoV GII.4 being the most common genotype iden-
tified within the past 8 years (Patel et al., 2009; Pringle et al., 2015).
Human norovirus is responsible for 48% of all shellfish associ-
ated outbreaks, which can result in secondary transmission where
rates can be as high as 88% (Alfano-Sobsey et al., 2012; Baker et al.,
2011; Becker et al., 2000). Virtually any food may be implicated in
NoV transmission, but bivalved molluscan shellfish present a rela-
tively high risk because of their ability to concentrate viruses from
contaminated water. Shellfish, particularly oysters, are typically
eaten raw or lightly cooked and may contain enteric viruses capable
of causing illnesses (Baker et al., 2011; Le Guyader et al., 2008).
Although the fecal indicator system has been in place for many
years, it has been very well documented that this system does not
adequately index for the presence of enteric viruses (Formiga-Cruz
et al., 2002; Hernroth et al., 2002; Kingsley, 2007; Lees, 2000). Male
specific coliphages (MSC) have been proposed as an indicator for
viral contamination in shellfish as they are accumulated like fecal
coliforms but MSC, like NoV, are not rapidly eliminated by the
shellfish (Formiga-Cruz et al., 2003; Love et al., 2010).
Various methods have been employed for the concentration of
enteric viruses from shellfish (Baert et al., 2007;Kingsley and
Richards, 2001; Le Guyader et al., 2009; Shieh et al., 2003). The
primary method for detection of enteric viruses involves molecular
based real time qPCR or RT-qPCR assays. Because viruses may be
present in very low concentrations in environmental and outbreak
samples, the level of sensitivity of real time qPCR and RT-qPCR is
advantageous for detection of low copy number. While the sensi-
tivity of PCR is beneficial, the presence of inhibitory substances (e.g.
polysaccharides), that are co-extracted during sample preparation
is of concern (Atmar et al., 1993; Cannon and Vinje, 2008; Kingsley
et al., 2002). PCR inhibition is especially relevant for the detection
of enteric viruses in shellfish and other foods. The ability to detect
viruses at low levels is critical in outbreak analysis because enteric
*Corresponding author. FDA, Gulf Coast Seafood Laboratory, 1 Iberville Dr.,
Dauphin Island, AL, 36528, USA.
E-mail address: jacquelina.woods@fda.hhs.gov (J.W. Woods).
Contents lists available at ScienceDirect
Food Microbiology
journal homepage: www.elsevier.com/locate/fm
http://dx.doi.org/10.1016/j.fm.2016.05.009
0740-0020/Published by Elsevier Ltd.
Food Microbiology 59 (2016) 76e84
viral pathogens, unlike bacterial pathogens, cannot be enriched.
This study reports on the concentration, detection, and char-
acterization of NoV in shellfish implicated in several outbreaks that
occurred during the years 2009e2014. During this time, states’
Departments of Health and/or FDA regional shellfish specialists
requested technical assistance in the analysis of shellfish for
possible norovirus contamination. Clinical samples from ill in-
dividuals in most of the outbreaks were analyzed by the states’
Department of Health for NoV genogroup I (GI) and genogroup II
(GII). Oysters were received at FDA’s Gulf Coast Seafood Laboratory
and analyzed for the presence of norovirus using the ultracentri-
fugation protocol and real-time RT-qPCR described in this study.
Positive samples were also amplified by conventional RT-PCR using
primers from the RNA dependent RNA polymerase (RdRp) regions
and/or the capsid region of the norovirus genome.
During this study, NoV was detected and quantified from
shellfish implicated in outbreaks. In addition, NoV was character-
ized and the probable infectious particles ingested were deter-
mined based NoV levels and oyster consumption.
2. Material and methods
2.1. Clinical samples
Clinical specimens collected from ill individuals associated with
each outbreak were analyzed for norovirus GI and GII by the states’
Department of Health using RT-qPCR. Most samples were sent to
CDC for further analysis, while clinical samples from two outbreaks
were analyzed in-house after analysis of shellfish samples. In house
clinical samples were diluted with tissue culture phosphate buff-
ered saline (t.c PBS) (8.0 g NaCl, 0.2 g KCl, 0.12 g KH
2
PO
4
, 0.91 g
Na
2
PO
4
per liter), extracted using QIAamp viral RNA kit (Qiagen,
Valencia, CA), and detected by conventional RT-PCR using primers
targeting the capsid region or the RdRp region on the Cepheid
Smart Cycler II
®
(Beuret et al., 2002)(Kojima et al., 2002; Williams-
Woods et al., 2011).
2.2. Outbreak details for shellfish samples
Between 2009 and 2014 the shellfish associated norovirus
outbreaks were in the states of TN and MS in 2009 (outbreak 1), AK
in 2009 (outbreak 2), MS in 2010 (outbreak 3), WA and PA in 2011
(outbreak 4), LA in 2013 (outbreak 5), MA in 2013 (outbreak 6) LA in
2014 (outbreak 7), and WA in 2014 (outbreak 8). Corresponding
parties reported symptoms of nausea, vomiting, and/or diarrhea
within 12e48 h after consumption of the implicated oyster-
sdconsistent with norovirus infection. Oysters (Crassostrea virgin-
ica) implicated in the TN and MS outbreak in 2009 were harvested
in MS and repacked by dealers in AL and MS. Oysters were then
shipped to restaurants in LA, MS, FL, and TN. Shellfish from the AK
outbreak were harvested from a local aquafarm. After harvesting
from the farm, oysters (Crassostrea gigas) were transported to a
harbor dock and held for dock-side sale. Samples from the impli-
cated lots were received from two local retail establishments. In the
MS 2010 outbreak, the oysters (Crassostrea virginica) were har-
vested from a growing area in LA. Oysters were then shipped to
dealers in MS where the shellfish were purchased and served at a
conference. Oysters from the outbreaks in WA and PA were indi-
vidually quick frozen imported shellstock (Crassostrea gigas) har-
vested from aquafarms, while the outbreaks occurring in TN, AK,
LA, MA or MS were domestic fresh whole or shuck oysters. All
samples were shipped under frozen or refrigerated conditions and
immediately analyzed or stored at 20
C until analysis. In all
outbreaks, shellfish was consumed raw, with the exception of one
where the shellfish was partially cooked and served as Oyster
Rockefeller. The actual internal cooking temperature of the Oyster
Rockefeller was unknown.
2.3. Shellfish analysis
For virus concentration and extraction, 6 to 10 whole oysters
were shucked and the digestive diverticula (DD) were removed to
obtain a 4 g sample (Fig. 1). In some instances 12 to 20 oysters
were dissected and portioned into 4 g samples. A 100
m
l aliquot at
afinal concentration of 10
3
PFU per gram of a non-human Cal-
icivirus, San-Miguel Sea Lion Virus (provided by Alvin Smith
University of Washington) serogroup 17 (SMSV-17) or murine
norovirus (propagated in house from stocks provided by David
Kingsley) (MNV-1) was added directly to DD prior to homogeni-
zation of the DD with 40 ml of sterile Milli-Q water (Wobus et a l.,
2004). Viruses were adsorbed onto the oyster homogenate by
adjusting the pH to 4.0e5.0 using 3N HCl. After the adsorption
step, samples were then centrifuged for 15 min at 2000x gat 4
C.
Following centrifugation, the supernatant was discarded. Viruses
were eluted from the pellet by adding 40 ml of 0.75 M glycine-
0.15 M NaCl (pH 7.6) and the pH adjusted to 7.5e7.8 with 5 M
NaOH. The samples were centrifuged for 15 min at 5000x gat 4
C
followed by an additional elution with 20 ml of 0.5 M threonine-
0.15 M NaCl (pH 7.5) and centrifuged as previously described. The
glycine and threonine eluates, around 60e65 ml, were combined,
balanced, and ultracentrifuged at 170,000x g (Sorvall WX90,
Thermofisher Scientific) for 1 h at 4
C. The pellet was resus-
pended in 5-ml of tcPBS (8.0 g NaCl, 0.2 g KCl, 0.12 g KH
2
PO
4
,
0.91 g Na
2
PO
4
per liter) and transferred to a 50 ml conical tube.
Samples were then extracted first with 5 ml of chloroform, by
vortexing for 1 min and then centrifuged at 1700x gfor 15 min at
4
C. The upper aqueous phase was transferred to a clean ultra-
centrifuge tube. Th e remaining porti on was extracted with 5 -ml of
0.5 M threonine-0.15 M NaCl (pH 7.5), vortexed for 1 min, and
centrifuged as previously described. Both aqueous phases were
combined into the ultracentrifuge tube and 50 ml of tcPBS was
added to each sample, balanced, and ultracentrifuged at 170,000x
gfor 1 h at 4
C. After ultracentrifugation, the pellet was resus-
pended with 80 0
m
l of tcPBS and evenly distributed into 4 DNase/
RNase free 1.5 ml microcentrifuge tubes. One concentrate was
selected and extracted for RNA with RNeasy Mini Kit (Qiagen,
Valencia, CA) as described in (Williams-Woods et al., 2011). The
remaining three concentrates were stored at 70
C. Extracted
RNA was tested by real-time RT-qPCR and conventional RT-PCR as
described below.
Male-specific coliphage (MSC) densities in the oysters were
determined by using a modified double-agar-overlay method
described by Cabelli (Cabelli, 1988) where the Escherichia coli strain
HS(pFamp)R (ATCC # 700891) was utilized as the suitable bacterial
host strain (Cabelli, 1988; Debartolomeis and Cabelli, 1991;
Environmental Protection Agency, 2001).
2.4. Real-time RT-qPCR
2.4.1. Norovirus
Real-time RT-qPCR for NoV GI and NoV GII was completed on
two clinical samples and all shellfish samples. Positive controls
used for NoV GI and GII were in vitro RNA transcripts of sequences
cloned from positive clinical samples previously identified as NoV
(Burkhardt et al., 2006; Kageyama et al., 2003). Primers and probes
for NoV GI and GII targeted the most conserved region of the ORF1-
ORF2 junction. The real-time RT-qPCR included an RNA internal
amplification control (IAC) and was performed in a 25-
m
l reaction
using a One-Step RT-PCR Kit (Qiagen, Valencia, CA) as previously
described (DePaola et al., 2010). The inclusion of the IAC was used
J.W. Woods et al. / Food Microbiology 59 (2016) 76e84 77
Enteric Virus Concentration in Shellfish
Dissect oyster
(4g of digestive diverticula)
Add 10x wt of sample (4 grams X 10) sterile water
Blend and transfer homogenate to 50 ml conical tube
Adjust pH to 4.0 – 5.0
Shake well and centrifuge 2000 x g, 15 min., @ 4C
Discard supernatant and re-suspend pellet w/ 10x wt oyster tissue (40ml)
0.75 M glycine/0.15 M NaCl
Adjust pH to 7.5 -7.8
Centrifuge 5000 x g, 15 min., @ 4C
Decant eluate into sterile 70ml ultracentrifuge tube
Add 5 x weight oyster tissue (20ml) of 0.5M threonine/0.15M NaCl
Shake well and centrifuge 5000 x g, 15 min., @ 4C
Decant and combine eluate in 70ml ultracentrifuge tube
Spin at 170,000 x g for 1 hr, @ 4C
Resuspend pellet with 5 ml PBS
Transfer resuspended pellet to 50 ml polypropylene conical tube
Add 5ml of chloroform to suspension
Vortex 1 min
Centrifuge 1700 x g, 15 min., @ 4C
Transfer upper aqueous layer to clean 70ml ultracentrifuge tube
Add 5 ml 0.5M threonine/0.15M NaCl, pH 7.5 to chloroform tube and vortex
Centrifuge 1700 x g, 15 min., @ 4C
Transfer & combine top aqueous layer in 70 ml ultracentrifuge tube
Add 50 ml tc PBS to eluent in ultracentrifuge tube
Spin at 170,000 x g for 1 hr, @ 4C
Discard supernatant & re-suspend virus pellet in 800 μl tcPBS
Aliquot 200 μl into 4 tubes
(distribute evenly into each tube)
Fig. 1. Flow diagram of the enteric virus concentration and extraction protocol for shellfish.
J.W. Woods et al. / Food Microbiology 59 (2016) 76e8478
to access inhibition in the RT-qPCR reactions. Inhibition was
determined by comparing the IAC C
t
value of the RT-qPCR negative
control and the IAC Ct value of the sample. A one to two C
t
differ-
ence between the IAC of the negative control and the IAC of the
sample is defined as minimal to no inhibition. Greater than 3.33 C
t
difference between the IAC of the negative control and the IAC of
the sample is considered significant inhibition. Briefly, thermal
cycling was run using a Smart Cycler II
®
(Cepheid, Sunnyvale, CA)
with the following conditions: 50
C for 3000 s, 95
C for 900 s
followed by 50 cycles of 95
C for 10 s, 53
C for 25 s, 62
C for 70 s.
Fluorescence was read at the end of the 62
C elongation step.
2.4.2. MNV-1 and SMSV-17 extraction controls
Real-time RT-qPCR withthe inclusion of an RNA IAC was utilized
for detection of MNV-1 or SMSV-17 in a 25-
m
l reaction using One-
Step RT-PCR Kit. Primers and probe sequences for SMSV-17 and
MNV targeted ORF1 and ORF2 of the genome, respectively (DePaola
et al., 2010; Hewitt et al., 2009; Smith et al., 1998). The MNV-1 and
SMSV-17 extraction and RT-qPCR positive controls were propa-
gated in house from stock virus utilizing RAW 264.7 (MNV-1) (ATCC
TIB-71) and Vero cells (SMSV-17) (Smith et al., 1998; Wobus et al.,
2006). The primer concentration for MNV-1 and the IAC were
200 nM and 75 nM, respectively, while the 5
0
nuclease probe
concentrations for MNV-1 and the IAC targets were 100 and
150 nM, respectively. The final concentration of MgCl
2
in the RT-
qPCR reaction was 4 mM. Thermal cycling was run using a Smart
Cycler II
®
under the following conditions: 50
C for 3000 s, 95
C for
900 s followed by 45 cycles of 95
C for 15 s, 55
C for 20 s, 62
C for
60 s. Fluorescence was read at the end of the 62
C elongation step
Smart Cycler II
®
. Default analysis parameters were used, except the
manual threshold fluorescence units were set to 10.
Real-time RT-qPCR utilized for detection of SMSV-17 was
analyzed as previously described (DePaola et al., 2010). Thermal
cycling was run under the following conditions: 50
C for 3000 s,
95
C for 900 s followed by 45 cycles of 94
C for 10 s, 62
C for 20 s,
72
C for 40 s. Fluorescence was read at the end of 72
C elongation
step.
2.5. Conventional RT-PCR for NoV detection
Detection by conventional RT-PCR was completed on all RT-
qPCR positive samples for NoV GI or GII with previously
described primers, MON (Beuret et al., 2002) and GSK (Kojima et al.,
2002). Conventional RT-PCR conditions for NoV MON primers were
RT (reverse transcription) at 50
C for 50 min, Taq polymerase
activation at 95
C for 15 min followed by 50 cycles of 94
C for 30 s,
52
C for 1 min 30 s, and 60
C for 30 s with a of hold 72
C for 7 min.
Conventional RT-PCR conditions for NoV GSK primers were RT at
50
C for 50 min, Taq polymerase activation at 95
C for 15 min
followed by 50 cycles of 95
C for 30 s, 55
C for 30 s, and 62
C for
60 s with a of hold 72
C for 7 min. Conventional RT-PCR assays
were analyzed using Smart Cycler II
®
and PCR amplicons were
detected by electrophoresis using 2% agarose gels and visualized
using ethidium bromide (0.5
m
g/ml).
2.6. Sequence and phylogenetic analysis
For sequence analysis, positive NoV GI and/or GII RT-PCR
products of corresponding size were cut and purified with Qiagen
Gel Extraction kit (Qiagen, Valencia, CA). Purified PCR products
were amplified using M13 tailed MON or GSK primers (Williams-
Woods et al., 2011). Gel purified products were sequenced
directly utilizing M13 primers. Sequencing was conducted with the
DTCS Quick Start kit (Beckman Coulter, Fullerton, CA) on the CEQ
8000 (Beckman Coulter, Fullerton, CA) sequence analyzer.
Sequences from the NoV RNA-dependent RNA polymerase (RdRp)
region (213 bp) or the capsid region (329 or 343 bp) were analyzed
using nucleotide BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and
were edited using BioEdit 7.0.9 (Hall, 1999). Sequence alignment
and comparison was performed using the NCBI bl2seq and MEGA
6.0 alignment program (Tamura and S., 2013). Phylogenetic trees of
the 213, 329, and 343 nucleotides (nt) for NoV were constructed
based on neighbor-joining methods using MEGA 6.0 (Tamura and
S., 2013).
2.7. Quantification
NoV levels were determined using standard curve (r2 >0.99) or
Most Probable Number (MPN). Standard curves were based upon
the end-point dilutions of NoV RNA and the endpoint was assigned
a value of 1 RT-PCR unit for the last dilution where only 2 of 3 re-
actions were positive (Katayama et al., 2008; Sanchez et al., 2012).
For the standard curve quantification, reactions with C
t
values of
zero were described as non-detects. MPN values were determined
using a MPN table with RT-qPCR reactions at 0.08, 0.06, 0.04, and
0.02 g equivalents per reactionproducing a positive C
t
value. For the
MPN quantification, non-detected samples were described as less
than values derived from the MPN calculator (http://www.
i2workout.com/mcuriale/mpn/). Determination of infectious par-
ticles ingested was based on the average size of the digestive
diverticula per individual outbreak investigation, RT-qPCR units
(derived from standard curve or MPN), and the number of oysters
consumed (in cases where the amount consumed was unknown, a
value of 1 was assigned). Infectious particles ingested were also
based on average extraction efficiencies of 60% and the assumption
of a ratio of noninfectious versus infectious NoV particles of 40:1
(Burkhardt et al., 2002). These levels were derived using the
following formula:
Infectious particles ingested ¼[(RT-PCR units/gram average
size of DD) (oysters consumed) ¼Level consumed þ(levels
consumed 0.40) ÷40].
2.8. Extraction efficiency
Samples were spiked with non-human Caliciviruses SMSV-17 or
MNV-1 at a final concentration of 10
3
PFU/gram to determine the
extraction efficiency. The amount of SMSV-17 or MNV-1 recovered
was based on the extracted virus standard curve generated with
spiked levels of SMSV-17 or MNV-1. The ratio of RT-PCR units to
infectious units of SMSV-17 and MNV-1 was 40:1 to 100:1
respectively. Both enteric viruses were propagated and titered in-
house by plaque assay and end-point titration. Analysis of out-
breaks prior to 2011 utilized SMSV-17 as the extraction control but
due to proprietary issues, analysis of outbreaks occurring after 2011
utilized MNV-1 as the extraction control.
3. Results
3.1. Clinical analysis and shellfish outbreak details
Clinical and shellfish samples from outbreak 1 (TN and MS,
2009) were positive for NoV GII (additional analysis of clinical
samples done in-house) (Table 1). Shellfish consumed during these
outbreaks were from the same lot and harvested from the same
area. During outbreak 2 (AK, 2009), NoV GI and GII were detected in
clinical samples while only NoV GII was detected in the shellfish.
NoV GI and GII were detected in the shellfish samples of outbreak 4
(WA and PA, 2011) and outbreak 8 (WA, 2014). In addition to
multiple genogroups, multiple genotypes were also detected in the
clinical and shellfish samples for outbreak 4 (additional analysis of
J.W. Woods et al. / Food Microbiology 59 (2016) 76e84 79
clinical samples done in-house). Only NoV GII was detected for
outbreaks 3 (MS, 2010), 5 (LA, 2010), and 7 (LA, 2014) while in
outbreak 6 (MA, 2013) only GI was detected in the shellfish and
clinical samples. Multiple genogroups were detected in the shell-
fish during outbreak 8 (WA, 2014) and clinical samples were not
analyzed.
3.2. Analysis of shellfish
NoV was detected in all outbreak samples analyzed from 2009
to 2014; with the exception of one outbreak that occurred in 2010
where the samples tested were not from the implicated lot (results
not shown). The size of each DD in the 4 g sample analyzed varied
from 0.7 g to 1.0 g. When examining the IAC, there was minimal to
no inhibition present in the RT-qPCR reactions. Extraction effi-
ciency ranged from 50 to 85% for all outbreaks (Table 1). The LOD
(limit of detection) and LOQ (limit of quantification) for NoV were
33 and 150 genomic copies respectively. For the extraction control,
the LOQ was 5 PFU/g and the LOD was 0.5 PFU/g. The ranges of RT-
qPCR units per gram of DD for NoV genotype I outbreaks were <10
to 29.8 (Table 1). For NoV GII detection, the ranges were 2.4e82 RT-
qPCR units. Levels of NoV GII RT-qPCR units detected were the
highest in shellfish outbreaks associated with the imported product
(outbreak 4 WA, 2011). For outbreak 2 (AK), outbreak 3 (MS) and
outbreak 6 (MA) MSC levels were 17, 10, and 80 PFU/100 g
respectively. In all other outbreaks, MSC levels were below detec-
tion limits (Table 1).
In three of the outbreaks, NoV GI and GII were detected. In
outbreak 1 and 6, the same genotype was detected in the shellfish
and clinical sample (Table 1). For outbreak 1 (MS), outbreak 4 (PA),
and outbreak 5 (LA), the number of oysters consumed was known,
and for the other outbreaks, the number consumed was unknown
and was assumed to be at least one (Table 2). The total ingested
particles ranged from 1.0 to 2572.5, with the highest levels occur-
ring in the outbreak where there was a documented consumption
of 35 oysters. The number of infectious particles ingested ranged
from 1.0 to 64.3.
3.3. Phylogenetic analysis
The phylogenetic trees were constructed utilizing the neighbor-
joining method based on the nucleotide sequence from the RdRp
region (213 nt for GI and GII) or capsid region (329nt for GI and
343nt for GII). In outbreak 1, the shellfish and clinical nucleotide
sequence demonstrated 100% similarity and were associated with
GII.7 (Fig. 2). In outbreaks 2 and 3, NoV sequence was closely
associated with GII.7 and GII.4 Minerva, respectively (Fig. 2). The
clinical and oyster strains from outbreak 4 (WA) showed 10 0%
similarity with Genebank reference strains EU085529 and
GU299761 which corresponds to GI.1 and GI. 8, respectively (Fig. 3).
For outbreak 4, NoV GII for the clinical and oyster strains showed
100% similarity with Genebank reference strains DQ438972 and
JX439803 which corresponds to GII.17 and GII.3 respectively
(Fig. 4). The nucleotide sequence identities for outbreak 8 showed
100% homology between the clinical and oyster nucleotide
sequence which were characterized as GI.4 (Fig. 3). Only partial
sequences were obtained for outbreaks 2, 4 (PA), 5 and 7, therefore
genotypes could not be accurately determined. Sequences for
outbreaks 1, 2, 3, 4, 6 and 8 were deposited into GeneBank with the
following accession numbers KT277938, KT277939, KT277940,
KT277941, KT277942, KT277943, KT277944, KT277947, KT277948,
KT277946, and KT277949.
Table 1
NoV and indicator levels in outbreaks.
Outbreak Year Avg size DD(g) MPN or RT-qPCR units/g Genogroup/genotype shellfish MSC PFU/100 g Genotype clinical Extraction eff.
g
1
a
2009 1.0 52.5 GII.7 <6 GII.7 69
2 2009 1.0 65.3 GII.7 17 GI.4, GII.4 62
3 2010 0.9 13.8 GII.4 Minerva/Den Haag 10 GII.4 New Orleans 70
4
c
2011 1.0 1.5, 5.5 GI.8, GII.3 <7.3 GI.1, GII.17 68
4
d
2011 1.0 82 GII
V
<7.0 GII
V
60
5 2013 0.9 5.9 GII
b
<9.9 GII
V
68
6 2013 0.7 29.8 GI.4 80 GI.4 85
7 2014 1.0 4.8, 28 GI
b
, GII.12 <11.4 GII.7 68
8
c
2014 0.9 <LOQ∞, 2.4 GI∞, GII.21 <10 not tested* 50
Table represents comparison of detectable RT-qPCR units per gram of oyster.
a
Results from analysis of oysters from TN.
c
Outbreak from imported oysters in WA.
d
Outbreak
from imported oysters in PA. VGenogroup determined by RT-qPCR, no genotyping was performed.
b
Outbreaks 5 and 7 yielded partial sequences therefore accurate genotyping
could not be determined. ∞Below limit for quantification or sequencing. *No clinical specimen was collected for testing.
g
Extraction efficiency using SMSV-17 or MNV as an
extraction control.
Table 2
Approximate levels of NoV particles ingested.
Outbreak Year NoV
b
Oysters consumed* Levels in oysters
b
Total ingested Approximate infectious particles ingested
1
a
2009 GII 35 1837.5 2572.5 64.3
2 2009 GII 1 65.3 91.42 2.28
3 2010 GII 1 12.7 17.6 1.0
4
c
2011 GI, GII 1 1.5, 55.2 77.28 1.0, 1.9
4
d,e
2011 GII 1 82 114.28 2.9
5 2013 GII 9 368.5 515.9 12.9
6 2013 GI 1 29.8 41.7 1.0
7 2014 GI, GII 1 3.4, 41.1 4.7, 58.0 1.0, 1.4
8 2014 GI, GII 1 1, 2,1 1, 2.3 1.0, 1.0
b
Comma separates GI and GII genotype in RT-qPCR units/g. *Oyster consumption assumed to be 1 unless otherwise known.
a
Results from analysis of oysters from MS.
c
Outbreak from imported oysters in WA.
d
Outbreak from imported oysters in PA.
e
Documented case of ingestion of only 1 oyster caused NoV illness.
J.W. Woods et al. / Food Microbiology 59 (2016) 76e8480
4. Discussion
Enteric viruses, unlike pathogenic bacteria, require a specific
host cell to replicate (Benton and Ward, 1982; Gonzalez-Hernandez
et al., 2014; Guix et al., 2005). Therefore methods employed for
detection of enteric viruses, such as NoV and hepatitis A virus must
demonstrate minimal inhibition and the capability of detecting
viruses at very low levels. During these investigations, NoV was
detected in shellfish implicated in outbreaks where multiple ge-
notypes were detected. Typically when multiple genotypes are
detected, sewage contamination is thought to be the cause and for
outbreaks 4 and 7, it was determined that shellfish harvested from
these growing areas were directly impacted by human sewage. In
outbreak 1, only one genotype (GII.7) was associated with the
clinical and shellfish samples, although it was later determined that
the growing area was directly impacted by sewage discharge. In
addition, phylogenetic analysis demonstrated 100% homology be-
tween the two clinical and shellfish samples in outbreak 1, which
were collected in two different states. For outbreak 2, only NoV
GII.7 was detected in the shellfish while GI.4 and GII.4 was detected
in the clinical isolate. After the State’s investigation, it was deter-
mined that the shellfish implicated in outbreak 2 had been har-
vested and held in the water dockside near a waste water treatment
plant (WWTP) outfall. During outbreak 6, GI.4 was detected in the
clinical and shellfish samples and phylogenetic analysis revealed
100% homology between the clinical and shellfish samples. Over-
board discharge was suggested during outbreak 6, but it was never
officially confirmed. In the US during the years 2009e2012, less
than 2% of reported foodborne outbreaks were attributed to
molluscan shellfish and non GII.4s were the most common geno-
types identified in foodborne outbreaks (Hall et al., 2014). During
these shellfish outbreak investigations, GII.4 was only associated
with outbreak 3. The levels detected in shellfish were the highest
for outbreak 4 (82 RT-qPCR units/g DD) which had a documented
NoV infection with the ingestion of only one oyster. The lowest
levels of GI and GII detected were in outbreaks 4 and 8, respectively.
The implicated shellfish consumed in these outbreaks were from
growing areas that were impacted by ‘fresh’fecal contamination or
non-point sources. Viruses in that instance were minimally
exposed to environmental stressors, such as sunlight and temper-
ature, which can cause inactivation of viruses and render them
non-infectious (Chang et al., 1985; Hill, et al., 1970; Nuanualsuwan
et al., 2002).
In all eight outbreak investigations, the levels detected were
lower than levels found a shellfish associated outbreaks occurring
in France where a C
t
value of 33 was detected. The difference in this
lower C
t
value is equivalent to 2 logs higher NoVlevels in the French
outbreak compared to the outbreaks in these investigations (Le
Guyader et al., 2006; Le Guyader et al., 2008; Wang et al., 2015).
In another shellfish associated outbreak which occurred in New
South Wales, NoV GII was detected with Ct values ranging from 37
to 39, which was comparable to levels in these outbreak in-
vestigations; although the NoV detected in the Wales outbreak was
unable to be characterized (Fitzgerald et al., 2014).
For each outbreak investigation the approximate level of infec-
tious particles ingested was determined. These levels were based
Fig. 2. Phylogenetic analysis of outbreak sequences generated using NoV GII primers (213 nt) from the RNA-dependent RNA-polymerase region utilizing MUSCLE alignment. Tree
contains sequences from outbreaks 1, 2, and 3 and was constructed using the neighbor-joining method (MEGA 6.0). Scale bar represents 0.05 substitutions per base position.
Bootstrap values are indicated as 500 replicates.
J.W. Woods et al. / Food Microbiology 59 (2016) 76e84 81
on 100% recovery (average recovery was 60%) of the virus with
estimated 40:1 ratio of non-infectious to infectious particles. It was
interesting that the number of ingested particles per individual
oyster was the highest for outbreak 4 (PA) at levels of 82. The level
of infectious particles ingested calculated for this outbreak was 2.9,
which is well below the estimated levels of 18 particles that can
cause NoV illness, but the genetic predisposition was not known for
the individual therefore the infected person could have been sus-
ceptible to one infectious particle (Atmar et al., 2014; Teunis et al.,
2008; Thebault et al., 2013). For outbreak 1, the average number of
oysters consumed was 35, with 64.3 approximate infectious par-
ticles ingested. This outbreak had the highest approximate infec-
tious particles ingested and NoV was detected in the clinical
specimens 3 weeks after the onset of illness. The total levels
ingested in outbreak 5 were 515.9 with the consumption of 9
oysters with estimated infectious particles of 12.9. The C
t
values for
the outbreaks in these studies ranged from 38 to 42 (data not
shown). Given the high C
t
values or low copies detected, ability or
inability to genotype the sequences was not directly related to the
genomic copies detected during each outbreak.
The MSC levels were below the detection limit for the majority
of the outbreaks, demonstrating that these indicator microorgan-
isms did not index for the presence of enteric viruses during
outbreak analyses. However, in outbreak 2, where the source of
contamination was identified as human wastewater associated,
MSC levels were detected. In outbreak 3, human sewage discharge
was also suspected as there were 2 WWTP and septic systems
within a few miles of the growing area where the implicated lot
was harvested. The MSC levels for outbreak 6 cannot be easily
explained as this outbreak was suspected overboard discharge,
although there was a WWTP in close proximity of the growing area.
For outbreaks 1, 4, and 7 the lack of MSC detection could be
explained by sewage impact from small land based sources or other
non-point sources where MSC contribution would be minimal.
Further investigation of the growing area may help identify other
potential sources of MSC.
5. Conclusion
In these studies, an ultracentrifugation protocol which in-
corporates extraction controls was used for the detection and
subsequent characterization of NoV in several oyster associated
outbreaks. Characterization of NoV revealed 100% identity between
clinical and shellfish samples in 2 of the 8 outbreaks and the C
t
values of NoV detected were not indicative of the ability to
sequence NoV RT-PCR products. This is the first study to include the
probable infectious particles ingested with the addition of MSC
(enteric virus surrogate) levels in shellfish associated outbreak
samples. Although the calculation of infectious particle ingested is
an estimate, this can be useful as a reference once effective prop-
agation of norovirus has been achieved.
In addition, the protocol in this study has been used for
extraction and detection of enteric viruses in clams and mussels
with average extraction efficiencies of 46% (data not shown). The
Fig. 3. Phylogenetic analysis of outbreak sequences generated using NoV GI primers (329 nt) from the capsid region utilizing MUSCLE alignment. Tree contains sequences from
outbreaks 4 and 8 using the neighbor-joining method (MEGA 6.0). Scale bar represents 0.05 substitutions per base position. Bootstrap values are indicated as 1000 replicates.
J.W. Woods et al. / Food Microbiology 59 (2016) 76e8482
concentrate generated from this protocol can also be used for
propagation of culturable enteric viruses, if desired (Woods, 2010).
Acknowledgments
For their assistance during the outbreak investigations, the au-
thors would like to thank John Dunn, Tennessee Department of
Health, Sara W. Longan, Alaska Department of Health, Erica Busby,
Massachusetts Department of Health, Laura Johnson, Washington
State Department of Health, John Veazey, FDA Southeast Regional
Shellfish Specialist, Gary Wolfe, FDA Regional Shellfish Specialist,
Kristina Phelps, FDA Pacific Regional Shellfish Specialist, Michael
Antee, FDA Pacific Regional Shellfish Specialist, and Amy Fitzpa-
trick, FDA Northeast Regional Shellfish Specialist. We would like to
thank David Kingsley for the murine norovirus stock and we would
also like to thank the CDC Calicinet team.
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