Blijft BVDV altijd BVDV? Niets is zo veranderlijk als een RNA virus. Frank van der Meer November 30, 2016

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1 Blijft BVDV altijd BVDV? Niets is zo veranderlijk als een RNA virus. Frank van der Meer November 30, 2016

2 The first report of BVDV in the world Canadian Journal of Comparative Medicine 1946

3 Quote from Dr. Julia Ridpath Bovine Veterinarian/May-June 2006 Under the radar

4 De grote surprise

5 Mutation Rates Ch. 1 Sanjuan, R., et al, 2010 J. Virol. 5

6 Duffy et al. Nat.Rev.Genet ,

7 Why should a virus change it s gene, what are the selective forces? Quasispecies: Clouds of genotypes in a population Selective forces on viruses Immune system (+vaccination) Antivirals

8 General structure BVDV virus genome Peterhans, E. et al Veterinary Research, 41(6),

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10 Does BVDV change?

11 Phylogenetic tree of Pestivirus genus BVDV3 or atypical BVDV Bauermann F V et al. J VET Diagn Invest 2013;25:6-15

12 BVDV-3/Hobi, coming up in a farm near you Difficulty detecting using BVDV 1/2 tests, % false negatives high The clinical signs included fever ( C), cough, seromucoid nasal discharge, leukopenia, and accelerated pulse and breath. Most affected animals received support treatment and recovered within 2 weeks; however, the death of 2 animals was reported. Necropsy findings included severe tracheitis and bronchopneumonia involving the apical lung lobes. Prevalence in North America still 0%

13 Phylogenetic analysis and classification of BVDV based on Npro Peterhans, E. et al Veterinary Research, 41(6),

14 PI Prevalence 0.4% Subtypes of cattle entering Feedlots in Kansas

15 BVDV in Canada Phylogenetic tree of the BVDV 5'UTR sequences derived from feedlot cattle in Alberta that were diagnosed with Mucosal Disease on pathological examination. The branches in the tree represent the different strains of BVDV virus.

16 Transient infection with BVDV Insemination Gestation Month 1 Month 2-4 Month 5-9 Early embryonic Death Return to estrus Persistent infected Antibody negative Abortion/malformation Normal animal Antibody positive Abortion/malformation

17 Pathogenesis Oronasal infection Tonsil Regional lymphnodes Blood circulation Lymphoid tissue of the GI tract and lungs Thymus/bone marrow/spleen Lymphatic spread Lymphatic spread All other organs depending on the virulence of the virus In our experience: in PI s all organs are BVDV positive Leading to an immunosuppression

18 Biosecurity Kyle Mizen

19 UCVM Class 2014 BVDV type 2a (strain 1373) infection experiment 27 steers/5 heifers single source Weight pounds Published in CVJ

20 Can Vet J 2015;56:

21 Clinical score and weight Can Vet J 2015;56:

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23 White blood cell count (leukocytes) Days post infection Can Vet J 2015;56:

24 Drop in all fractions of the WBCs Leucocytes Granulocytes Lymphocytes Monocytes Can Vet J 2015;56:

25 Virus in blood and antibodies 2-log Can Vet J 2015;56:

26 Virus Neutralizing antibody Days post infection Can Vet J 2015;56:

27 Nasal excretion of virus Days post infection Can Vet J 2015;56:

28 Ovalbumin antibody induction following BVDV type 2 infection Optical Density (OD) BVDV 2 infected Negative control ovalbumin ovalbumin Sampling Day Can Vet J 2015;56:

29 Consequences of acute BVDV 2 infection BVDV delays the immune response Immunosuppression: white blood cells ê Weight loss Diarrhea Mortality Persistent infection induced in 1-3 month old fetuses What can you do about it? Find them and remove them

30 Is it that easy!

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35 Serum Ear notch

36 Heart, lung, liver, kidney, spleen, duodenum, muscle, mes Lnn, lung lnn, urine bladder, gall bladder, bile, feces, urine, ovary/testicle, obex, jejunum, ileum, milk udder tissue, colon, ceacum, skin (=ear), nasal swab, Tonsil, esophagus, serum, white blood cells (PBL) And the same from the fetus (if present and possible) Everything PCR positive except bile and feces

37 Biotype CP and NCP Non-cytopathogenic cytopathogenic

38 Example BVDV: Rearrangements as genetic basis for mucosal disease In short: The genomic rearrangements generating cp viruses: 1. Duplication of BVDV sequences 2. Insertion of cellular sequences ot the NS2 and NS3 junction Just to be complete also: 1. In frame deletions affecting the NS2-3 polypeptide 2. Introduction of pointmutations at various places in the genome 3. Recombinations between different BVDV viruses End result: Production of the NS3 protein next to NS2-3 polyprotein

39 Mucosal disease

40 MD lesion in the mouth of a PI.

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42 MD cases in feedlot animals

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44 BRD project: October market-derived mixed-breed beef calves mean arrival weight 712 lbs. 548 (73.45%) calves pulled within 35 days on feed IDEXX HerdchekBVBVAb Interpretation S/P value <0.2 = negative = suspect ³ 0.3 = positive Results: 506 (67.83%) negative 76 (10.19%) suspect 164 (21.98%) positive IDEXX Herdchek* IBRgB Interpretation blocking %: <45% = negative = suspect ³ 55 = positive Results: 398 (53.35%) negative 52 (6.97%) suspect 296 (39.68 %) positive Courtesy of Barbara Wolfger and Karin Orsel used with permission

45 The BVDV containing vaccines: (Jan, 2015) M=modified live K=killed

46 Does biotype matter? Why CP in a vaccine? Animals first exposed or primed with NCP BVDV biotype had significantly higher antibody titres But a low secondary proliferative response against the virus whatever the biotype used in the secondary inoculation. Animals initially inoculated with the CP BVDV biotype: high T cell reactivity during the secondary exposure despite the lower level of neutralizing activity seen in these calves. This distinction between humoral and cell-mediated responses would suggest the existence of distinct Th1 and Th2 subpopulations in the regulation of specific immune responses to BVDV Lambot et al. Journal of General Virology (1997), 78,

47 T lymphocytes are induced when vaccination takes place IFOMA Pentavalent MLV vaccine was Evaluated for stimulation of CD4, CD8, and other T cell responses to BVDV types 1 and 2, BHV1, BRSV, and PI3 Calves with maternal antibody The vaccine did not stimulate an antibody response Did stimulate all three T cell types to respond to BVDV types I and II in vitro Protected the calves from challenge with virulent BVDV type 2. No evidence of antibody or T cell responses to BHV-1, BRSV, or PI-3 in calves with maternal antibody. Induction of T lymphocytes specific for BVD virus in calves with maternal antibody. Viral Immunol, 17(1):13-23, 2004 Biologicals Volume 31, Issue 2, June 2003, Pages

48 BVDV MLV vaccinated calves with maternal antibodies A single dose of a commercial MLV vaccine containing BVDV type 1 (Singer strain), BVDV type 2 (Bolin 296 strain), BHV-1 (Colorado strain), BRSV (proprietary strain) and PI-3 (proprietary strain) with proprietary adjuvant (Express 5, Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO) was administered subcutaneously (2 ml). 4 calves per control group, 8 calves per vaccinated group 1V/C= 1-2 weeks old; 2V/C= 4-5 weeks and 3V/C = 7-8 weeks Platt et al Vaccine 27 (2009)

49 Effect of BVDV vaccination on pregnancy risk Vaccination with BVDV vaccine does NOT change pregnancy rates Theriogenology 83 (2015)

50 Effect of BVDV vaccination on abortion fetal protection Theriogenology 83 (2015)

51 In more detail: Fetal protection Theriogenology 83 (2015)

52 10 PI s analyzed PI PI 2 PI 3 Farm 1 Farm 2 Farm 3 Farm 4 Farm PI 4 NADL (M31182) PI 8 Oregon (AF091605) PI 5 Osloss (M96687) 100 PI 6 PI 7 PI 9 BVDV 1A BVDV 1B 84 PI

53 O Cluster O1 86 O4 O2 O PI PI PI 2 PI 10 PI 5 97 Cluster 2.1 O7 O PI 4 PI PI 7 71 PI PI 5 Colon Ileum Milk MLN Obex Serum Tonsil PI Cluster Cluster Cluster PI 7 PI 8 PI 9 PI 10 Cluster 8.2 Cluster 9.1 Diversity of BVDV in PI s Dow et al. PLOS ONE DOI: /journal.pone July 1, 2015 Colon

54 BVDV clusters by Tissue E1-E2 NS5B Green=colon, blue=mln, purple=obex, orange=serum, teal=ileum, red=tonsil Dow et al. PLOS ONE DOI: /journal.pone July 1, 2015

55 Clustering is by Tissue is Significant E1-E2 NS5B Dow et al. PLOS ONE DOI: /journal.pone July 1, 2015

56 Dow et al. PLOS ONE DOI: /journal.pone July 1, 2015

57 O Cluster O1 86 O4 O2 O PI PI PI 2 PI 10 PI 5 97 Cluster 2.1 O7 O PI 4 PI PI 7 71 PI PI 5 Colon Ileum Milk MLN Obex Serum Tonsil PI Cluster Cluster Cluster PI 7 PI 8 PI 9 PI 10 Cluster 8.2 Cluster 9.1 Diversity of BVDV in PI s Colon

58 PI Colon Ileum Milk MLN Obex Serum Tonsil PI 3 PI 4 80 Lactating dairy cow (mother) Four month old fetus 0.002

59 Viruses through a genetic funnel Barriers in the placenta?

60 Practice: BVDV strains Western Canada Chernick, Godson, van der Meer: Infection, Genetics and Evolution 28 (2014)

61 When did BVDV 1a came to Western Canada? Chernick, Godson, van der Meer: Infection, Genetics and Evolution 28 (2014)

62 BVDV Phylogeny (E2) 50.0 V051_E2 V050_E2 V012_E2 V010_E2 V015_E2 V045_E2 V026_E2 V070_E2 V079_E2 V040_E2 V020_E2 V059_E2 V099_E2 V083_E2 V027_E2 V085_E2 V057_E2 V089_E2 V042_E2 V036_E2 V073_E2 V081_E2 V088_E2 V033_E2 V060_E2 V014_E2 V054_E2 V023_E2 V076_E2 V002_E2 V024_E2 V046_E2 V065_E2 V008_E2 V028_E2 V022_E2 V005_E2 V029_E2 V019_E2 V047_E2 V001_E2 V100_E2 V097_E2 V009_E2 V052_E2 V039_E2 V075_E2 V044_E2 V035_E2 V086_E2 V062_E2 V098_E2 V037_E2 V095_E2 V034_E2 V031_E2 V043_E2 V056_E2 V077_E2 V017_E2 V082_E2 V067_E2 V048_E2 V092_E2 V021_E2 V025_E2 V041_E2 V074_E2 V013_E2 V006_E2 V091_E2 V063_E2 V080_E2 V049_E2 V078_E2 V087_E2 V007_E2 V016_E2 V011_E2 1a 1b 2 ~100 years ~200 years ~500 years 50 Chernick et al. Submitted

63 Next generation sequencing RT with random primers RT with BVDV specific primers <1% BVDV reads >95% BVDV reads

64 Next generation sequencing approach

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67 Diversity of BVDV 1a on amino acid level Chernick et al. Submitted

68 Contemporary strains vs. reference strains E2

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70 Influenza H7N7 crossing the species barrier Chicken Turkey Bataille A, van der Meer F, Stegeman A, Koch G (2011) PLoS Pathog 7(6): e

71 Automated Image Processing Eschbaumer, M., et al J. Virol. Methods

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73 Questions?