Persistence of Enteric Viruses
within Oysters (Crassostrea
virginica)
David Kingley1 and Keleigh Provost2
1USDA ARS FSIT Dover Laboratory
2College of Agric. and Related Sciences,
Delaware State Univ.
USDA Microbial Safety of
Aquaculture Products Center of
Excellence
 Located within Department of Agriculture and
Related Sciences, Delaware State University,
Dover DE
DHK’s USDA Laboratory Research
 Enhance shellfish safety by developing rapid
methods for detection of viruses
 Understanding of how and why viruses
persist within bivalve tissues (hemocytes)
 Identify and evaluate potential intervention
technologies to eliminate or inactivate
pathogenic viruses from shellfish
Virus Transmission
Fecal-oral route: Person-to-Person,
Contaminated Water, Food handlers,
Shellfish
Do Not Replicate in Food Items
Cold, Dark, and Wet concept
Small infectious dose (10-100 virions for
HAV and Norwalk?)
Sources of Enteric Viruses




Septic systems
Raw/Treated Sewage
Overboard waste disposal
Floods
Variable HPP results
HAV 3-log10 reduction at 400 MPa in oysters
MNV 400 MPA in oysters reduces 3-log10
HuNoV 600 MPa required inactivate 10,000
GEC units (Human volunteers) in oysters
(400 MPa unsuccessful)
High Pressure Processing and
Viruses: Take Home Messages
 HPP can kill HAV and NV – requires
pressures above commercial range
 Pressure level is predominate; time
secondary
 Temperature is important: For NV
surrogates-cold enhances; for HAV cold is
protective
 Food Matrix effects are important
 Some food-borne viruses may be resistant
 Exceptions are the rule (pH and temp)
Bioconcentration vs. Persistence
Bivalve shellfish bioconcentrate water-borne
pathogens to high levels (1000x)
Fecal bacteria are readily purged; fecal
viruses are not (why not?)
Some viruses persist in shellfish better than
others (?)
IntroductionWhy do enteric viruses persist in shellfish?
Sequestration within hemocytes
Relationship between virus testing for whole
oysters and hemocytes
Demonstrate virus persistence within
hemocytes-relationship with acid tolerance
HAV persists in shellfish
Hemocytes are involved in:
http://www.mdsg.umd.edu/issues/chesapeake
/oysters/education/oysblood.htm
Wound repair
Nutrient transport
Innate immune defense
Digestion of food particles
Hemocytes are phagocytic
Phagolysosomes are acidic
Waste ejected
Sequestration within hemocytes
MW
f-HL
H-pell
HL-unf
+
(-)
Virus associated with hemocytes not hemolymph
Separation of hemocytes from hemolymph. Oysters were contaminated with 4.3 x 107 PFU of HAV for O/N and
then hemocytes and hemolymph were extracted. Hemocytes were separated from hemolymph by centrifugation and
the supernatant was tested or filtered. Testing by extraction of the viral RNA and RT-PCR.
Persistence within hemocytes (H) and whole oyster (W)
MW
H5
W5
H9
W9 H15 W15
(+) (-)
Days 5-15. This is a representative gel for three trials. Whole shucked oysters and hemocyte samples were obtained
after exposure to poliovirus, 1 ml of 1.12 x 108 pfu/ml, and depuration for varying periods.
Persistence within hemocytes (H) and whole oyster (W)
MW
H29
W29
(+)
(+)
(-)
Persistence of HAV in extracted tissues and hemocytes. Whole shucked oyster meat or hemocytes were extracted
after exposure to HAV (4.28 x 107 pfu/ml) and depuration for varying lengths of time. Lane 1 is a 100 Bp DNA ladder.
Lane 3 is a hemocyte sample from 29 days post-exposure, lane 4 is a whole oyster sample from 29 days postexposure. Lane 5 is a positive control, with 1 μl of denatured virus, 8 μl of water and 1 μl of RNAse inhibitor. Lane 6 is
also a positive RT-PCR virus control with 1 μl of column run HAV RNA, and 9 μl of water. Lane 6 is a negative RT-PCR
control with 10 μl of water.
Exposure of all 4 viruses at once
 Same 3 x 106 RT-PCR units of virus o/n; two separate
trials; individual oysters




FCV detected at day 0 only
PV detected at day 1 only
MNV detected at day 3 and day 12
HAV beyond 21 days
 Most persistent HAV >MNV >PV >FCV least persistent

Caveat: GPTT extraction probably not equivalent for all viruses
pH Sensitivity
*30 min treatment
2
PV
HAV
HAV
0
FCV
Log Reduction
MNV
FCV
-2
FCV
MNV
PV
-4
FCV
-6
FCV
Polio
HAV
MNV
-8
PV
pH1
pH2
pH3
pH4
pH5
pH Treatments
Most Acid Resistant HAV, >MNV, >PV, >FCV Least Acid Resistant
Persistence within hemocytes
 Viable HAV and PV was isolated from hemocytes
 Hemocytes (+) whole oysters (+)
 Relationship between acid tolerance and
persistence
 Slow release to hemocytes (?)
 Transfer experiments (no adaptive immune system)
Testing whole oysters after transfer of HAV-contaminated hemocytes
MW
C oy
1W
2W 3W
H
(+)
(-)
Whole oysters test positive for HAV two weeks after hemocyte transfer
HAV persistence after transfer of contaminated hemocytes to naïve oysters. (several oysters 4 x107 pfu o/n pooled
then transfer ) Lane 1 is a 100 bp DNA ladder. Lane 3 is a control of the naïve oyster, before the transfer. Lane 4 is a whole
oyster sample 1 week post-transfer. Lane 5 is whole oyster sample 2 weeks post-transfer. Lane 6 is a whole oyster sample
3 weeks post-transfer. Lane 7 is a subsample of the contaminated hemocytes before injection. Lane 8 is a positive RTPCR control,. Lane 9 is a negative RT-PCR
Conclusion
 Relationship between low pH tolerance and
persistence within shellfish meats
 Virus is associated with hemocytes
 HAV can remain within hemocytes for
extended periods
Hemocytes: Research Directions
and Implications
 More detailed research-visualize within
hemocytes (phagolysosomes) …track virus
movement thru the oyster
 Hemocytes appear to be a good detection
target
 Design new intervention strategies??