Rapid detection of hepatitis a virus
and murine norovirus in hemocytes of
contaminated oysters.
B.A. Dancho and D.H. Kingsley
USDA, ARS, FSIT
Eastern Regional Research Center
Delaware State University
Epidemiology of foodborne disease
outbreaks- U.S. 2006
2009. MMR Weekly 58:609-615.
• Norovirus- most common
cause (54% of outbreaks
and 11,879 cases)
• Hepatitis A virus- second
most common viral cause
(<1% of outbreaks and 50
cases)
• The economic burden of foodborne illness in the U.S. is
estimated at $152 billion in health-related costs each year
(2010. Sharff, R.L. Produce Safety Project at Georgetown University).
Virus Overview
Norovirus
• Caliciviridae
• Non-enveloped, icosahedral,
38-40 nm in diameter
• 7-8kb ssRNA(+) genome
• Fecal-oral transmission
• Incubation- 12-48 hours
• Symptoms- acute-onset
vomiting, diarrhea, abdominal
cramps, and nausea
Hepatitis A virus
• Picornaviridae
• Non-enveloped, spherical, 30
nm in diameter
• 7.5kb ssRNA(+) genome
• Fecal-oral transmission
• Incubation- 28 days
• Symptoms- nausea, anorexia,
malaise, jaundice, abdominal
cramps
Human norovirus and hepatitis A virus
contaminate shellfish
2008. Bosh, A., et. al. Curr Opin Biotechnol 19(3): 295-301.
Human norovirus and hepatitis A virus
contaminate shellfish
• Recent oyster-associated outbreaks
– 2010. Westrell, T., et. al. Norovirus outbreaks linked to oyster consumption in the
United Kingdom, Norway, France, Sweden and Denmark, 2010. Euro Surveill 15.
– 2009. Guillois-Becel, Y., et al. An oyster-associated hepatitis A outbreak in France in
2007. Euro Surveill 14.
– 2008. Le Guyader, F. S., et. al. Aichi virus, norovirus, astrovirus, enterovirus, and
rotavirus involved in clinical cases from a French oyster-related gastroenteritis
outbreak. J Clin Microbiol 46:4011-7.
– 2007. Bialek, S. R., et. al. Use of molecular epidemiology to confirm a multistate
outbreak of hepatitis A caused by consumption of oysters. Clin Infect Dis 44:838-40.
• Viral presence in 5-55% of random market samples of
oysters perfomed in the U.S. and Europe
– 2010. Depaola, A., et. al. Bacterial and Viral Pathogens in Live Oysters: U.S. Market
Survey 2007. Appl Environ Microbiol. 76:2754-68.
– 2010. Terio, V., et. al. Norovirus in retail shellfish. Food Microbiol 27:29-32.
Goals
Part 1
• To determine whether oyster hemocytes
could be utilized in cell-based viral extraction
procedures with commercially available RNA
extraction methods.
Part 2
• To determine the D10 value (the radiation
dose required for one log reduction of viral
load) of MNV in PBS and oyster homogenate.
Need for rapid viral detection in shellfish
• FDA and USDA diagnostics for viral contamination of
shellfish are time-consuming (1-2 days)
•
•
2010. Depaola, A., et. al. Appl Environ Microbiol. 76:2754-68.
2001. Kingsley, D. H., and G. P. Richards. Appl Environ Microbiol 67:4152-7.
–
Glycine, polyethylene glycol, TRI-reagent, poly dT bead (GPTT) viral RNA extraction protocol
• Methods using commercial RNA extraction kits are more
rapid
– 2009. Gentry, J., et. al. J Virol Methods 156:59-65.
– 2007. de Roda Husman, A. M., et. al. J Food Prot 70:967-74.
Oyster hemocytes are a site of viral
persistence
• Enteric viruses persist in oysters for
extended periods
– Hepatitis A virus persists for 6 weeks
(Kingsley, D. H., and G. P. Richards. 2003. J Food Prot 66:331-4.)
– Hemocytes are a site of viral persistence
• Oysters have an open circulatory
system
– Circulate hemolymph
– Hemocytes -oyster multifunctional blood cells
(immunity & digestion)
– Hemolymph can easily be drawn out with a
pipette, and the hemocytes can be separated
by centrifugation.
HAV & MNV distribution in laboratorycontaminated oysters
Laboratory-contamination with HAV and MNV (NoV surrogate)
Separate hemocytes from oyster tissues
Extract RNA with the Rneasy Mini Kit (Qiagen)
Real-time qRT-PCR analysis
HAV & MNV distribution in laboratorycontaminated oysters
Viral RNA is detected in oyster hemocytes as well as in tissues of HAV and
MNV laboratory-contaminated oysters.
Detection of HAV & MNV in laboratorycontaminated oysters
Laboratory-contamination with HAV and MNV
Prepare hemocyte and whole oyster samples
Extract RNA:
Hemocytes- Dynabeads Oligo(dT)25 (Invitrogen)
Rneasy Mini Kit (Qiagen)
Whole Oysters- GPTT
Real-time qRT-PCR analysis
Detection of HAV & MNV in laboratorycontaminated oysters
Viral RNA extracted from hemocytes and tissues is detected at similar
levels in laboratory-contaminated oysters.
Detection of HAV & MNV in seeded oysters
Prepare hemocyte and whole oyster samples
Seed with HAV and MNV
Extract RNA:
Hemocytes- Dynabeads Oligo(dT)25 (Invitrogen)
Rneasy Mini Kit (Qiagen)
Whole Oysters- GPTT
Real-time qRT-PCR analysis
Detection of HAV & MNV in seeded oysters
Viral RNA extracted from hemocytes has lower LODs than in tissues in
virus-seeded oysters.
Detection of HAV & MNV in seeded oysters
Viral RNA extracted from seeded oyster hemocytes had a higher recovery
for both HAV and MNV than in RNA extracted from seeded whole oyster
homogenates.
Summary & Conclusions-Part 1
• Viral RNA is detected in oyster hemocytes as well as in
tissues of HAV and MNV laboratory-contaminated
oysters.
• Viral RNA extracted from hemocytes had a lower LOD
than in whole oyster homogenates in laboratorycontaminated oysters.
• Viral RNA extracted from seeded oyster hemocytes
had a higher recovery than in RNA extracted from
whole oyster homogenates.
• Oyster hemocytes can successfully be used instead of
whole oyster tissues to expedite viral extraction from
oysters.
Goals
Part 1
• To determine whether oyster hemocytes
could be utilized in cell-based viral extraction
procedures with commercially available RNA
extraction methods.
Part 2
• To determine the D10 value (the radiation
dose required for one log reduction of viral
load) of MNV in PBS and oyster homogenate.
Electron Beam
• The National Center for Electron Beam
Research, Texas A&M University System
• The current maximum FDA-approved
irradiation dose for shellfish is 5.5 kGy
D10 value of MNV in PBS
Dilute MNV in PBS or oyster homogenate
Electron-beam inactivation (0-5.5 kGy)
MNV plaque assay
Inactivation of MNV in PBS - Combined
6.00
5.00
y = -0.3985x + 5.3463
D10 = 2.51
Log PFU/ml
4.00
3.00
2.00
1.00
0.00
0.00
1.00
2.00
3.00
E-beam Dose (kGy)
4.00
5.00
6.00
MNV Inactivation in Oyster Homogenate -Combined
5.8
5.6
y = -0.1563x + 5.441
R² = 0.9808
D-10 = 6.3979 kGy
5.4
Log PFU/ ml of OH
5.2
5
4.8
4.6
4.4
4.2
4
0
1
2
3
E-Beam Dose (kGy)
4
5
6
Summary-Part 2
• D10 value of MNV in PBS is 2.5 kGy
• D10 value of MNV in oyster
homogenate is 6.4 kGy
• Studies need to be performed in live
oysters to determine whether doses
below 5.5 kGy will be effective in
reducing the viral load.
Acknowledgements
USDA, ARS, FSIT
Microbial Safety of Aquaculture
Products Center of Excellence
David Kingsley, Ph.D.
Gary Richards, Ph.D
Michael Watson
Gloria Meade