Mecanismos de persistencia de virus de hepatitis A en bivalvos

1. Mecanismos de persistencia de virus de hepatitis A en bivalvos David H. Kingsley USDA ARS FSIT Dover Laboratory

2. Bioconcentration vs. Persistence Bivalve shellfish bioconcentrate water-borne pathogens to high levels (1000x) Fecal bacteria are readily purged; fecal viruses are not Some viruses persist in shellfish better than others (?)

3. Infectious HAV in Oysters at 3 Weeks Postinoculation Single oysters 9 x 104 pfu overnight

4. Weeks Post-Contamination bp Mr NC1 NC2 1 2 3 4 5 6 PC A 300- -267 HAV 100- Weeks Weeks B bp Mr - NC1 0 1 2 3 PC Mr NC 4 5 6 PC 300- -267 HAV 100- Weeks Post-Contamination bp C Mr NC1 0 1 2 3 4 5 6 NC2 300- -267 HAV 100-

5. Why and how do viruses persist in shellfish?

6. Literature on viruses and shellfish • Indications of variable persistence with different virus types • Dissemination of the virus throughout the virus • Radiolabeled virus appears to be intracellular • Hemocytes??

7. Hemocytes are involved in: Wound repair Nutrient transport Innate immune defense Digestion of food particles http://www.mdsg.umd.edu/issues/chesapeake/oysters/education/oysblood.htm Waste ejected

8. Hemocyte Hypothesis • Does virus acidic pH sensitivity correlate with persistence in shellfish? • Does persistence in hemocytes correlate with persistence in the oyster meat ? • Can we remove hemocytes and transfer to another oyster? Does virus still persist? poliovirus; hepatitis A virus, murine norovirus; and feline calcivirus

9. Sequestration within hemocytes Is virus associated with hemocytes? 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. MW f-HL H-pell HL-unf + (-) Virus associated with hemocytes not hemolymph

10. Virus Persistence within hemocyte (H) and whole oyster (W) Does persistence in hemocytes correlate with persistence in the oyster meat? (PV) 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 107 pfu/ml, and depuration for varying periods. (+) (-) MW H5 W5 H9 W9 H15 W15

11. Persistence within hemocytes (H) and whole oyster (W) 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 post-exposure. 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. Does persistence in hemocytes correlate with persistence in the oyster meat? HAV (+) (-) MW H29 W29 (+)

12. Exposure of oysters to 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

13. pH Sensitivity *30 min treatment Most Acid Resistant HAV, >MNV, >PV, >FCV Least Acid Resistant

14. 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)

15. Adoptive Transfer • Oysters have no histocompatibilty nonself/self recognition –no rejection • Remove hemocytes from HAV-exposed oyster (abductor muscle) transfer to non-exposed oyster…bypass the digestive tract • Detection of virus up to 2 weeks later

16. Testing whole oysters after transfer of HAV-contaminated hemocytes 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 RT-PCR control,. Lane 9 is a negative RT-PCR Can we remove hemocytes and transfer to another oyster? Does virus still persist? MW 1W 2W 3W H (+) (-) C oy Whole oysters test positive for HAV two weeks after hemocyte transfer

17. Can we use shellfish hemocytes to make better virus extraction and RT-PCR testing methods?

18. HAV & MNV distribution in laboratory-contaminated oysters Viral RNA is detected in oyster hemocytes as well as in tissues of HAV and MNV laboratory-contaminated oysters.

19. Detection of HAV & MNV in seeded oysters Viral RNA extracted from seeded oyster hemocytes had the same detection sensitivity for HAV and better sensitivity for MNV than in RNA extracted from seeded whole oyster homogenates.

20. Detection of HAV & MNV in laboratory-contaminated oysters Viral RNA extracted from hemocytes and tissues is detected at similar levels in laboratory-contaminated oysters.

21. Conclusion • Virus is associated with hemocytes • Relationship between low pH tolerance and persistence within shellfish meats • Virus can remain within hemocytes for extended periods • Oyster hemocytes can be used instead of whole oyster tissues for virus extraction.