1. Viral reproductive disorders in sows an update 2011 H. Nauwynck Laboratory of Virology Faculty of Veterinary Medicine Ghent University, Belgium

2. 1. CL (progesterone) Normalgestation in the sow 2. Cytokine/growth factor cross talk ? 3. Immune suppression

3. How do viruses cause reproductive problems? General disease (fever) ~ pro-inflammatory cytokines causing distortion of cytokine balance -> swine influenza virus, … 2. Replication of pathogens in reproductive tract/embryo/fetus ~ damaging the placenta and/or cytokine balance ~ direct negative impact on embryo/fetus -> DNA viruses: Aujeszky’s disease virus, parvovirus, porcine circovirus 2 -> RNA viruses: porcine reproductive and respiratory syndrome virus, porcine enteroviruses, encephalomyocarditis virus, classical swine fever virus

4. How do virusesreachembryos/fetuses? 1. Contaminatedsperm

5. 2. Viremia + transplacental spread How do virusesreachembryos/fetuses?

6. 1. Via contaminatedsperm Virus excretion in sperm ~ viremia DNA-viruses ADV (<14 dPI) PPV (<14 dPI) PCV2 (<50 dPI;>) RNA-viruses PRRSV (<50 dPI;>) PEV (<14 dPI) CSFV (<14 dPI) Embryo Virus rep & † Virus rep & † Virus rep & † No virus rep (onlyinfectionsow) Virus rep & † Virus rep & † repeat breeding repeat breeding

7. At what stage can embryos be infected ? 2-8 cells morula blastocyst hatched blastocyst (0-4dpins) (4-5dpins) (5-7dpins) (>7dpins) - - - - ? ? - - - - ? ? - - - - ? ? + + + - + + ADV PPV PCV2 PRRSV PEV CSFV Virus should resist aggressive uterine environment!!! 1° difficult to inactivate: PPV, PCV2, PEV (no envelope) 2° intracellular: ADV, CSFV

8. Zona pellucida is an ideal barrier 1° Physical barrier Channels in ZP : ± 30 nm (inner ø) Viruses > 30nm (ADV, PRRSV, CSFV) 2° Chemical barrier Viruses < 30nm (PPV, PCV2, PEV)

9. Outcome of embryonic PPV-infection Uninfected PPV-infected

10. Outcome of embryonic PCV2-infection Surgical ET HB 6 dpins PCV2/mock Inspection of ovaries (follicles/CL) Euthanasia D14 post ET Collection & analysis of embryos

11. Outcome of embryonic PCV2-infection Sow n° A B C D E F G Embryos at transfer n° status 26 mock 26 mock 20 PCV2 23 PCV2 20 PCV2 18 PCV2 29 PCV2 Ovaries CL CL CL CL follicles follicles follicles Embryos at euthanasia n° 19 17 13 3 0 0 0 normal 19 15 7 0 0 0 0 degenerated 0 2 6 3 0 0 0 mock normal embryo PCV2 degenerated embryo

12. Outcome of embryonic PCV2-infection Sow n° A B C D E F G Embryos at transfer n° status 26 mock 26 mock 20 PCV2 23 PCV2 20 PCV2 18 PCV2 29 PCV2 Ovaries CL CL CL CL follicles follicles follicles Embryos at euthanasia n° 19 17 13 3 0 0 0 normal 19 15 7 0 0 0 0 degenerated 0 2 6 3 0 0 0 0 0 0 4 6 3 IHC-PCV2

13. Outcome of embryonic PCV2-infection Sow n° A B C D E F G Embryos at transfer n° status 26 mock 26 mock 20 PCV2 23 PCV2 20 PCV2 18 PCV2 29 PCV2 Embryos at euthanasia Ovaries CL CL CL CL follicles follicles follicles n° 19 17 13 3 0 0 0 normal 19 15 7 0 0 0 0 degenerated 0 2 6 3 0 0 0 0 0 0 4 6 3 PCV2 infection of embryos leads to • embryonic death • return to oestrus of sow (regular/irregular) • both PCV2a and PCV2b IHC-PCV2

14. 2. Viremia + transplacental spread DNA-viruses (PPV, PCV2, ADV) RNA-viruses (PRRSV, CSFV, PEV 1, 3, 6 & 8, EMCV) • Cell-free: PPV, PEV, PRRSV, EMCV • Cell-associated: ADV, PCV2, CSFV

15. Transplacental spread - cell free virus PRRSV (> 70dpi) PPV, PEV, EMCV * * * * * *

16. Normal placenta fetal site maternal site

17. Transplacentalspread- PRRSV - 1. cell free virus + free mø

18. Transplacental spread - PRRSV - 2. cell free virus + migrating mø

19. Transplacental spread - PRRSV - 3. infection of migrating mø

20. Transplacental spread - PRRSV - 4. entry of fetal placenta by migrating mø

21. Transplacental spread - PRRSV - 5. explosive replication in fetal placental mø

22. Transplacental spread - PRRSV - 6. apoptosis in infected mø and bystander cells

23. Transplacental spread - PRRSV - 7. apoptosis in infected mø and bystander cells/necrosis?

24. Transplacental spread - PRRSV - 8. dysfunction placenta

25. Transplacental spread- cell-associated virus * * * * PCV2, ADV, CSFV * * * * * * * * * * * * * * * * * *

26. Cell-associated transplacental spread of ADV Maternal placenta Fetal placenta Maternal placenta

27. Interfetal virus spread PPV/PEV/PRRSV ADV/CSFV PCV2 *

28. Clinical outcome - fetus Infection time (days of gestation) 0-30 † † † - † † 30-60 † † † (†) † †/IT terato 60-90 † †/IR †/IR † †/IR †/IR 90-113 † IR IR †/IR IR †/IR ADV PPV PCV2 PRRSV PEV CSFV IR immune response IT immune tolerance

29. Clinical outcome - sow Infection time (days of gestation) 60-90 abortion msw msw late abortion/msw msw abortion/msw 90-113 abortion/msw normal normal late abortion/msw normal abortion/msw 0-30 repeat br. repeat br. repeat br. - repeat br. repeat br. 30-60 abortion msw msw - msw abortion/msw ADV PPV PCV2 PRRSV PEV CSFV msw: mummies, stillborn and weakborn piglets at birth

30. ADV

31. PPV Inoculated po at 47 days of gestation Euthanized at 81 days of gestation Natural case At birth

32. Inoculated at 43 days of gestation Euthanized at 98 days of gestation PPV

33. Virus + mummified + + autolytic normal PCV2 Aspect at birth Inoculated (days of gestation) Intra-uterine spread Interruption of gestation Abs 57 mummified - + - stillborn autolytic 75 + - - 92 normal + - -

34. mummified Myocarditis PCV2 inoculated at 57 days of gestation collected at 21 days PI Ascites, congestion of internal organs At birth

35. PRRSV

36. PEV Inoculated in fetus 8 at 59 days of gestation Hysterectomy at 113 days of gestation 7 living fetuses

37. CSFV Inoculation of CSFV (low virulence) po at 43 days of gestation Collection at parturition

38. Reproductive failure - Diagnosis? Main complaint: abortion ≠ stages of gestation Fetuses fresh, rigor mortis (firm) Fetuses not fresh, partially mummified Lungs/spleen fetuses (esp. with necrotic foci in liver) No serum sows!!! Immunofluorescence Virus isolation/PCR ADV, CSFV Sera sow Seroconversion SIV or other pathogen causing general disease fever

39. Reproductive failure - Diagnosis? Main complaint: SMEDI Fetuses/piglets at birth Mummies (≠ stages), stillborn and normal piglets 1) Heart/spleen/lungs fetuses (<70 dpins; <17cm) 2) Body fluid/serum fetuses (>70dpins; >17cm) No serum sows!!! 1) Virus isolation/PCR 2) Antibody detection (BE CAREFULL WITH PCV2!!!) PPV, PCV2, PEV, EMCV

40. Reproductive failure - Diagnosis? Main complaint: late abortion/early farrowing Fetuses at late abortion/early farrowing Early stages of mummification (brown), stillborn and normal piglets Placenta/lungs/spleen fetuses Serum stillborn/piglets before colostrum uptake No serum sows!!! Virus isolation(±)/PCR PRRSV

41. Control 1° Eradication ADV √ CSFV √ Other viruses in the future? PRRSV?

42. Control 2° Vaccination/”controlled” immunisation PPV PCV2 PEV PRRSV

43. Gilts OK PPV √ 40-100 kg 10-20 kg Sows Sows + offspring OK 20-40 kg OK

44. Gilts PPV √ 40-100 kg 10-20 kg Sows ! ! ! Sows + offspring 20-40 kg

45. Gilts Gilts V V V PPV OK √ 40-100 kg 10-20 kg Sows OK OK Sows + offspring 20-40 kg V

46. Gilts PCV2 OK √ 40-100 kg 10-20 kg Sows Sows + offspring OK 20-40 kg OK

47. Gilts PCV2 √ 40-100 kg 10-20 kg Sows ! ! ! Sows + offspring 20-40 kg

48. Gilts Gilts V V PCV2 V OK √ 40-100 kg 10-20 kg Sows OK OK Sows + offspring 20-40 kg V

49. Gilts PRRSV OK √ 40-100 kg 10-20 kg Sows Sows + offspring OK 20-40 kg OK

50. Gilts PRRSV √ 40-100 kg 10-20 kg Sows ! ! ! Sows + offspring 20-40 kg

51. Gilts Gilts V V PRRSV V OK? √ 40-100 kg 10-20 kg Sows OK OK Sows + offspring 20-40 kg V

52. Gilts PRRSV √ 40-100 kg 10-20 kg Sows ! ! ! ! Sows + offspring 20-40 kg

53. Gilts PEV OK √ 40-100 kg 10-20 kg Sows Sows + offspring 20-40 kg

54. Laboratory of Virology & Laboratory of ReproductiveTechnology Faculty of VeterinaryMedicine GhentUniversity, Belgium H. Nauwynck Van Soom G. Labarque R. Sanchez G. Vanroose S. Tanghe B. Mateusen P. Meerts D. Lefebvre D. Saha U. Karniychuk E. Bussalleu J. Beek Virus-embryo/fetus work