2. Flavi and Pestiviruses October 12, 2010

3. Flaviviridae Pestiviruses Flaviviruses Hepacviruses Hepatitis C virus BVD, Hog cholera, Border disease Yellow fever Japanese encephalitis St. Louis encephalitis Dengue West Nile virus (arthropods, biological vectors)

4. West Nile Outbreaks Israel - 1951-1954, 1957 South Africa - 1974 Romania – 1996 Italy – 1998, 2008 Russia - 1999 (human) United States –1999-2009 (equine, human) Canada - 2001-2009 (equine, human) Israel – 1998, 2000 (human) France (Rhine delta) - 2000 (equine)

5. spring early summer return from south overwinter or eggs amplification in birds late summer and fall dead-end hosts

6. Saskatchewan mosquito species shown to be capable of transmitting WNV Aedes vexans (spring to fall) Ochlerotatus flavescens, spencerii(July-August) Culex restuans*, tarsalis* (July- August) Culiseta inornata* Coquillettidia perturbans

7. How does the virus overwinter and spread? migratory birds? overwintering mosquitos? bird to bird transmission? –Komar et al. EID March 2003

8. in the mosquito 1. virus ingested with blood meal 2. virus multiplies in gut epithelial cells 3. virus leaks from gut and infects salivary glands 4. virus released in saliva during feeding (sufficient amount of virus must be ingested - > 10 5 infectious units/ml)

9. in mammals virus transmitted in mosquito saliva during probing virus deposited in extra vascular tissue replication in skin and lymph nodes amplification in extra-neural tissues viremia (secretion in milk) crosses blood/brain barrier (repl’n in vascular endothelium, exacerbated by concurrent infections) viremia terminated by immune response viral damage to neurons and glia or dysfunction inflammation perivascular infiltration (plasma cells and macs), cerebral edema. “flu” like symptoms “neurological” signs IgM, CSF IgM, pleocytosis, PCR

10. equine cases of WNV neurological disease Ataxia 86% Depression 51% Hind limb weakness 49% Difficulty or inability to rise 46% Muscle tremors 41% Fever only 24% Differentials: –rabies, EHV 1, EEE, WEE, botulism 10% to 50% of horses with neurological signs die

11. clinical signs in people most asymptomatic fever, “flu” like symptoms (fatigue, anorexia, nausea, vomiting, arthralgia, rash, lymphadenopathy) encephalitis, meningoencephalitis - ataxia, painful eyes, seizures, change in mental status (confusion) case fatality rate in hospitalized patients - 10-12% risk factor for severe disease (age 50-60 yr are 10 times and >80 yr are more than 40 times likely) Petersen and Margin, WN virus: a primer for the clinician 2002. Ann Int med 137:173

12. unusual cases in USA infant infected through breast milk 2 people infected through blood transfusion 2 laboratory workers while dissecting infected animals

13. the Canadian experience 2000 - 2,288 birds examined, 185 tested - no positives 2001 - 2,807 bird carcasses from NF to Sask tested –128 WNV infected birds from 12 health dist. in Ontario –no disease in horses or humans

14. human cases 2003-2005 MayJuneJulyAugSeptOct

15. http://www.phac-aspc.gc.ca/wnv-vwn/mon-hmnsurv-archive-eng.php

16. http://www.phac-aspc.gc.ca/wnv-vwn/mon-hmnsurv-eng.php

17. West Nile virus in domestic birds geese ducks chickens and turkeys ostriches, emus

18. testing for WNV - serology strong + cont weak + cont samples from 45 horses, Virden Manitoba (Aug-Sept, 2002) IgM capture ELISA PDS immunol. lab

19. vaccines Intervet – PreveNile Live Flavivirus chimera Fort Dodge

20. West Nile Virus – Common Client Questions Should we vaccinate our horse – is it safe and does it work? Can I catch WNV from a horse? What signs might a horse show early on? Is there a treatment? What can we do to limit the risk to our horse? Can our other pets get it? When should we vaccinate our horses?

21. Pestiviruses Systemic haemorrhagic disease - pigs (USA) Enteric disease - calves (USA) Congenital, neurological “hairy-shakers” (England/Wales) 1940s HCV BVDV BDV

22. Flaviviruses Pestiviruses Hepatitis C Louping ill Yellow fever Dengue Pestiviruses of Artydactyla bovine viral diarrhoea (BVDV) hog cholera (HCV) border disease (BDV) Japanese encephalitis Murray Valley St. Louis encephalitis West Nile Wide host range (camelids, deer etc)

23. BVDV genome and gene products UTR structural nonstructural Regions that show the most variation gp53 p125 (p54+p80)

24. Biotypes of BVDV NON-cytopathic (natural state) Cytopathic (mutant) Based on effect on cells in tissue-culture

25. CP vs NCP – genetic differences UTR structural nonstructural p125 (p54+p80)

26. Biotypes - implications Non-cytopathic –Implications for vaccines and research Cytopathic –Mucosal disease

27. BVDV “types” Not different serotypes!! BVDV-1 BVDV-2 Based on: 1. sequence differences In non-translated region of genome Does not imply differences in pathogenicity 2. Antigenic differences UTR structural nonstructural

28. Antigenic differences between BVDV-1 and BVDV-2 Serum against BVDV-1 strains BVDV-2 strains VN titre against BVDV-1 strains BVDV-2 strains 800->12,800100->3,200 50->4003,200->51,200 Pellerin et al.(1994) Virology 203:260-268

29. Pathogenesis - infection Intra-species –PI carriers Inter species Vaccine related? Artificial breeding programs Blood Persistence in acutely infected animals

30. Pathogenesis

31. Pathogenesis - disease in imm.competent, non-pregnant animals Sub-clinical Mild fever, leukopenia, decreased milk production Mild BVD - mild erosive lesions, ulcerative stomatitis, diarrhoea, respiratory Severe disease - lesions mimic MD, thrombocytopenia, haemorrhagic syndrome, hyphemia

32. Pathogenesis - pregnant animals All syndromes described above Embryonic death Abortions Birth defects Persistently infected calves

33. Pathogenesis - PI animals Healthy, normal “poor-doers” Mucosal disease

34. Guess which one is persistently infected? Calves of the same age. From Lee et al. CVJ 38:29

35. consequences of having a PI animal Loneragan et al. JAVMA, Feb 15, 2005 –PI animals more likely to be ill, require treatment or die –Incontact animals more likely to be sick, require treatment Dieguez et al. Res Vet Sc, Aug 2009 Correlation BVD status and respiratory disorders, mortality

36. Mucosal disease NCP->CP (p80) Infection with antigenically related CP virus (mutation, herd-mate, vaccination?) Low morbidity high mortality High fever, depression, anorexia, diarrhoea Ulcerative mucosal lesions Death 2 days -> 3 weeks

37. Mucosal Disease (by mutation) MD

38. Mucosal Disease (by infection or injection) MD

39. No Mucosal Disease if CP virus antigenically different Immune response No MD

40. Diagnostic procedures Virus isolation –Small numbers ($31.50) –Herd screening - microtitre ($10 -2->10, $6 >10) Antigen capture ELISA –Herd screening Serology –VN ($14) –ELISA ($5/animal) PCR –genotyping ($62) –pooled samples ($30)

41. Diagnostic procedures PCR (BVDV-1 vs BVDV-2) Immunohistochemistry or IFA –Abortions ($45) –PI animals (approx. $6/animal) BVDV-2 BVDV-1a BVDV-1b

42. Immunohistochemistry - PI vs acute Persistently infected calf: Antigen in hair follicle epithelium Acutely infected calf: Antigen in superficial layer of epidermis (foci) From Brad Njaa et al. 2000. J. Vet. Diag. Invest. 12:393-399.

43. Diagnostic Parameters (acute infection) infection incubation period clinical disease virus infect. virus detectable in serum infect. virus detectable in WBC Virus det. by PCR antigens in biopsy antibody 5-7 days

44. Diagnostic parameters (abortion) Fetus –Often no infectious virus –Submit liver, kidney, spleen, thymus for IH or IFA –Fetal antibody if late term

45. Diagnostic parameters (PI) Large amounts of virus in blood, serum and secretions (10 3 -10 7 TCID 50 /ml ) Maternal antibody interferes with isolation <3 months - submit blood >3 months - serum Repeat isolation in 3 months Immunohistochemistry - follicle epithelium

46. Herd screening

47. Management of BVD Test and remove PI animals Test all new born calves for 9 months For 9-12 months segregate age groups Quarantine replacements Vaccination with MLV BVDV BVD infections may persist for some time after removal of PI animals (Collins et al. 2009)

48. Vaccines Inactivated or attenuated Most (all) contain CP BVDV

49. http://www.cattlenetwork.com/content.asp?contentid=119624

50. Interspecies transfer Sheep Wild ruminants –Natural infections (caribou: 40-100%) –Transfer (llamas, alpacas)