Dr. Bong Kyun Park - Research View of Porcine Epidemic Diarrhea Virus
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Research View of Porcine Epidemic Diarrhea Virus - Dr. Bong Kyun Park, Professor and Swine Virologist, Veterinary Virology Laboratory, Seoul National University, from the 2013 Allen D. Leman Swine ...

Research View of Porcine Epidemic Diarrhea Virus - Dr. Bong Kyun Park, Professor and Swine Virologist, Veterinary Virology Laboratory, Seoul National University, from the 2013 Allen D. Leman Swine Conference, September 14-17, 2013, St. Paul, Minnesota, USA.

More presentations at http://www.swinecast.com/2013-leman-swine-conference-material

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Dr. Bong Kyun Park - Research View of Porcine Epidemic Diarrhea Virus Presentation Transcript

  • 1. Molecular genetics of recent PEDV isolates and Understanding the variability of PEDV BongKyun Park DVM, MSc, PhD Dept. of Veterinary Microbiology College of Veterinary Medicine Seoul National University Sep-16-2013 Porcine Epidemic Diarrhea Virus
  • 2. Sep-16-2013 Porcine Epidemic Diarrhea Virus 2
  • 3. Sep-16-2013 Porcine Epidemic Diarrhea Virus 3
  • 4. Recent Outbreaks of PED and TGE in Korea PED Outbreaks Sep-16-2013 TGE Outbreaks Porcine Epidemic Diarrhea Virus 5
  • 5. 4 Major Swine Diseases in Korea     Classical Swine Fever(Apr. 2009) PCV-2 PRRS PED Sep-16-2013 Porcine Epidemic Diarrhea Virus 6
  • 6. Routine pattern of SN titers against PEDV in commercial farms SN titers(2ⁿ) Sep-16-2013 Porcine Epidemic Diarrhea Virus 7
  • 7. PED(Porcine Epidemic Diarrhea)     A devastating enteric disease with acute diarrhea, vomiting, dehydration and significant mortality in swine, incurring heavy economic losses In many swine-raising countries, notably in Europe, North America, southeast Asian countries including China. Extremely difficult to differentiate PED from TGE clinically The family Coronaviridae, serologically unrelated Sep-16-2013 Porcine Epidemic Diarrhea Virus 8
  • 8. Details in samples tested, PEDV isolation and sequencing Prov Jan. 2002 – Aug. 2005: Positive 267/ 737 tested Sequencing PEDV isolation (Passage No.) No. RT-PCR PEDV 2007: Positive 84 / 1,195 tested Sample Positive Inoculatio S ORF3 M 0 3 5 7 9 10 GG 822 135 23 21 9 15 15/15 10/15 8/15 7/15 6/15 6/15 GW 59 16 2 2 1 2 2/2 2/2 0/2 0/2 0/2 0/2 GB 57 13 7 2 0 2 2/2 2/2 2/2 2/2 2/2 2/2 GN 186 31 1 2 1 2 2/2 1/2 0/2 0/2 0/2 0/2 JB 215 43 7 6 6 4 4/4 2/4 0/4 0/4 0/4 0/4 JN 83 8 0 0 0 1 1/1 0/1 0/1 0/1 0/1 0/1 CB 181 26 5 4 3 3 3/3 2/3 0/3 0/3 0/3 0/3 CN 329 79 24 10 5 6 6/6 5/6 3/6 2/6 2/6 2/6 8 1,932 351 69 47 25 35 35/35 24/35 13/35 11/35 10/35 10/35 Sep-16-2013 Porcine Epidemic Diarrhea Virus 9
  • 9. - Spike gene(46 strains) Sep-16-2013 Porcine Epidemic Diarrhea Virus 10
  • 10. PEDV ORF3 gene e3991 CPF531 CPF259 CPF872 6 3 M4758 V2501 M4759 5 6 M2227 BIF118 26 PFF1051 VF1016 CPF63 9 9 MF78 VF131 17 22 M2366 88 66 64 26 PF4275 e1642 90 e2541 e1834 e2540 DBI825 33 46 94 28 23 BI1108 M2503 e2539 Parent DR13 BI2804 98 69 38 49 51 M1763 M1764 BI981 e942 BI976 M1595 BI1166 66 32 93 99 100 CPF299 CPF1195 PFF514 CPF1173 PFF513 64 6 6 CPF39 CPF106 BIF256 4 2 PFF188 PFF381 CPF1074 PFF285 CPF1043 CPF193 CPF935 KPED-9 Attenuated DR13 P-5V LZC CV777 Br1/87 0.01 Sep-16-2013 Porcine Epidemic Diarrhea Virus 11
  • 11. PEDV M gene 88 37 MF78 VF131 M2227 BIF118 43 M1595 39 55 49 95 V2501 BI1108 40 CPF259 34 72 CPF531 PFF1051 63 BI981 68 Parent DR13 BI976 M1763 QH LJB/03 73 JS-2004-2 PFF285 PFF513 PFF514 PFF381 99 PFF188 CPF193 CPF299 BIF256 CPF1074 85 KPED-9(field) 91 72 KPED-9(cell) 93 JMe2 Cjinju99 95 CV777 Br1/87 57 Attenuated DR13 72 84 M2366 e1642 99 e1697 0.01 Sep-16-2013 Porcine Epidemic Diarrhea Virus 12
  • 12. Based on Full-length E gene Sep-16-2013 Porcine Epidemic Diarrhea Virus 13
  • 13. Sep-16-2013 Porcine Epidemic Diarrhea Virus 14
  • 14. Sep-16-2013 Porcine Epidemic Diarrhea Virus 15
  • 15. Beginning in October 2010, the PED outbreak overwhelmed >10 provinces in southern China, and >1,000,000 piglets died. Sep-16-2013 Porcine Epidemic Diarrhea Virus 16
  • 16. Sep-16-2013 Porcine Epidemic Diarrhea Virus 17
  • 17. Sequences of the S genes from this outbreak were 99.1%– 100.0% homologous and had 88.7%–98.9% nt identity with all reference strains, 98.5%–98.9% with Thailand strains, and 94.5%–95.1% with vaccine strain CV777. The partial S gene deduced amino acid sequences were compared and also showed a high degree of homology (98.0%–100.0%); they had 85.3%–98.7% identity with all reference strains, 98.0%–98.7% with Thailand strains, and 93.3%–94.7% with vaccine strain CV777. Sep-16-2013 Porcine Epidemic Diarrhea Virus 18
  • 18. Sep-16-2013 Porcine Epidemic Diarrhea Virus 19
  • 19. Fig. 2 Schematic representation of the PEDV genome based on the CV777 (GenBank accession No. AF353511) strain Sep-16-2013 Porcine Epidemic Diarrhea Virus 20
  • 20. Porcine Epidemic Diarrhea Virus 21
  • 21. New variants of porcine epidemic diarrhea virus – 3 new PEDV variants were identified in vaccinated swine herds which animals exhibited severe diarrhea Sep-16-2013 Porcine Epidemic Diarrhea Virus 22
  • 22. Emerg Infect Dis. 2012 August; 18(8): 1350–1353. doi: 10.3201/eid1808.120002 Sep-16-2013 Porcine Epidemic Diarrhea Virus 23
  • 23. Emerg Infect Dis. 2012 August; 18(8): 1350–1353. doi: 10.3201/eid1808.120002 Sep-16-2013 Porcine Epidemic Diarrhea Virus 24
  • 24. Phylogenetic analysis of the complete M gene showed that all south China PEDV strains have a close relationship with most of the strains in Korea and Thailand, but differ genetically from the vaccine strain (CV777). Sep-16-2013 Porcine Epidemic Diarrhea Virus 25
  • 25. Phylogenetic analysis based on S1 or sM gene, which have high levels of variations, indicated that each sample was related to the specific reference strain, and this finding was consistent with the protein characterization prediction analysis. Sep-16-2013 Porcine Epidemic Diarrhea Virus 26
  • 26. DOI 10.1007/s11262-012-0794-x Sep-16-2013 Porcine Epidemic Diarrhea Virus 27
  • 27. Questions ? PEDV Variability vs Immune Variability of Individual Pig PED Immune Status in Endemic Pig Farms Sep-16-2013 Porcine Epidemic Diarrhea Virus 28
  • 28. Distribution of SN titers among the pregnant sows Sep-16-2013 Porcine Epidemic Diarrhea Virus 29
  • 29. Case #999 farm – SN titers’ distribution (Feb 21, 2007 by artificial immunization) Sep-16-2013 Porcine Epidemic Diarrhea Virus 30
  • 30. Case #000 farm – SN titers’ distribution (Feb. 22, 2007 by artificial immunization) Sep-16-2013 Porcine Epidemic Diarrhea Virus 31
  • 31. Aminopeptidase N, 150 kDa – Cellular receptor 32 Muirhead MR & Alexander TJL, 1997
  • 32. Table 1. Reverse transcriptase-polymerase chain reaction results in suckling pigs orally(Oral) or intramuscularly(IM) infected with highly attenuated porcine epidemic diarrhea virus, DR13 (* Days postinfection) Group Oral DPI* Tonsil Lung Liver Spleen Kidney Intestines Small Large Lymph nodes Submandibular Mesenteric Inguinal 0/2 0/2 1/2 0/2 1/2 1/2 1/2 1/2 0/2 0/2 0/2 0/2 2/2 1/2 1/2 2/2 2/2 2/2 0/2 6 0/2 0/2 1/2 1/2 1/2 1/2 1/2 1/2 1/2 0/2 13 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 20 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 4 0/2 1/2 2/2 0/2 1/2 1/2 1/2 1/2 1/2 0/2 6 0/2 2/2 2/2 2/2 1/2 1/2 1/2 2/2 2/2 0/2 13 0/2 1/2 0/2 0/2 0/2 0/2 1/2 0/2 0/2 0/2 20 Control 0/2 4 IM 2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 4 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 6 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 13 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 20 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 Sep-16-2013 Porcine Epidemic Diarrhea Virus 33
  • 33. Sep-16-2013 Porcine Epidemic Diarrhea Virus 34
  • 34. Sep-16-2013 Porcine Epidemic Diarrhea Virus 35
  • 35. Sep-16-2013 Porcine Epidemic Diarrhea Virus 36
  • 36. Fecal shedding of PEDV DR13 after oral inoculation in pigs   RT-PCR & RFLP For up to 6 days in 3-day-old piglets, 9 days in 2-week-old pigs, and 3 days in late-term pregnant sows. * All 3-day (n=2) and 2-week-old (n=4) piglets were inoculated with 5 mL of virus suspension, and sows (n=16) with 1 mL. Srain DR13 was harvested from intestinal mucosa of infected pigs and either administered as a 10% suspension (parent strain; 0 passage) or propagated in Vero cells. Sep-16-2013 Porcine Epidemic Diarrhea Virus 37
  • 37. Immune responses of serum samples from pregnant sows inoculated with Vero cell attenuated PEDV DR13, using IgA-specific ELISA (*P<0.05) Sep-16-2013 Porcine Epidemic Diarrhea Virus 38
  • 38. Antibody responses from serum samples against PEDV in 3-day-old piglets using IgG and IgA-specific ELISA before challenge exposure (*P<0.05) Sep-16-2013 Porcine Epidemic Diarrhea Virus 39
  • 39. Antibody responses from serum samples against PEDV in 3-day-old piglets using the SN test before challenge exposure (*P<0.05) Sep-16-2013 Porcine Epidemic Diarrhea Virus 40
  • 40. RT-PCR detection of virus shedding in serial passages of colostrum-deprived 3-day-old pigs Level of pig passage Pig No. Days after oral inoculation 0 1 5 6 7 − +/+a +/+ +/+ +/+ +/+ +/+ − − +/+ +/+ +/+b Control − − − − − − − − 2-A − +/+ +/+ +/+ +/+ − − − 2-B − +/+ +/+ +/+b Control − − − − − − − − 3-A 3 4 1-B 2 3 1-A 1 2 − +/+ +/+ +/+ − − − − control − − − − − − − − PEDV detection in feces using RT-PCR/RFLP patterns (HindIII andXhoII) of the virus. b Pigs 1B and 2B were sacrificed at day 3 after oral inoculation, and small intestines collected for preparing the next oral inoculum. a Sep-16-2013 Porcine Epidemic Diarrhea Virus 41
  • 41. Survival rate (%) of piglets born from vaccinated sows after challenge with wild-type PEDV 90 80 70 60 50 40 30 20 10 0 Oral(20/ 23) Sep-16-2013 IM(6/ 15) Control(14/ 14) Porcine Epidemic Diarrhea Virus 42
  • 42. Acknowledgements     Dr. Jin-Sik Oh, DVM, MSc, PhD Dr. Dae-Sop Song, DVM, PhD Dr. Seong-Jun Park, BS, PhD Green Cross Veterinary Products Co. Ltd Sep-16-2013 Porcine Epidemic Diarrhea Virus 43
  • 43. Thank you for your attention ! Cross neutralization and new vaccine candidate ??? Sep-16-2013 Porcine Epidemic Diarrhea Virus 44