Molecular genetics of recent
PEDV isolates and Understanding
the variability of PEDV
BongKyun Park
DVM, MSc, PhD
Dept. of ...
Sep-16-2013

Porcine Epidemic Diarrhea Virus

2
Sep-16-2013

Porcine Epidemic Diarrhea Virus

3
Recent Outbreaks of PED and TGE in Korea
PED Outbreaks

Sep-16-2013

TGE Outbreaks

Porcine Epidemic Diarrhea Virus

5
4 Major Swine Diseases in Korea





Classical Swine Fever(Apr. 2009)
PCV-2
PRRS
PED

Sep-16-2013

Porcine Epidemic Di...
Routine pattern of SN titers against PEDV
in commercial farms

SN titers(2ⁿ)
Sep-16-2013

Porcine Epidemic Diarrhea Virus
...
PED(Porcine Epidemic Diarrhea)








A devastating enteric disease with acute diarrhea,
vomiting, dehydration and si...
Details in samples tested, PEDV isolation and
sequencing
Prov

Jan. 2002 – Aug. 2005: Positive 267/ 737 tested
Sequencing
...
- Spike gene(46 strains)

Sep-16-2013

Porcine Epidemic Diarrhea Virus

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
...
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
...
Based on Full-length E gene

Sep-16-2013

Porcine Epidemic Diarrhea Virus

13
Sep-16-2013

Porcine Epidemic Diarrhea Virus

14
Sep-16-2013

Porcine Epidemic Diarrhea Virus

15
Beginning in October 2010, the PED outbreak
overwhelmed >10 provinces in southern China,
and >1,000,000 piglets died.

Sep...
Sep-16-2013

Porcine Epidemic Diarrhea Virus

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 referen...
Sep-16-2013

Porcine Epidemic Diarrhea Virus

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

21
New variants of porcine epidemic diarrhea virus –
3 new PEDV variants were identified in vaccinated
swine herds which anim...
Emerg Infect Dis. 2012 August; 18(8): 1350–1353.
doi: 10.3201/eid1808.120002

Sep-16-2013

Porcine Epidemic Diarrhea Virus...
Emerg Infect Dis. 2012
August; 18(8): 1350–1353.
doi: 10.3201/eid1808.120002

Sep-16-2013

Porcine Epidemic Diarrhea Virus...
Phylogenetic analysis of the complete M gene showed that
all south China PEDV strains have a close relationship
with most ...
Phylogenetic analysis based on S1 or sM gene, which
have high levels of variations, indicated that each sample
was related...
DOI 10.1007/s11262-012-0794-x

Sep-16-2013

Porcine Epidemic Diarrhea Virus

27
Questions ?
PEDV Variability vs
Immune Variability of Individual Pig

PED Immune Status
in Endemic Pig Farms

Sep-16-2013
...
Distribution of SN titers among the pregnant sows

Sep-16-2013

Porcine Epidemic Diarrhea Virus

29
Case #999 farm – SN titers’ distribution
(Feb 21, 2007 by artificial immunization)

Sep-16-2013

Porcine Epidemic Diarrhea...
Case #000 farm – SN titers’ distribution
(Feb. 22, 2007 by artificial immunization)

Sep-16-2013

Porcine Epidemic Diarrhe...
Aminopeptidase N, 150 kDa – Cellular receptor

32

Muirhead MR & Alexander TJL, 1997
Table 1. Reverse transcriptase-polymerase chain reaction results in
suckling pigs orally(Oral) or intramuscularly(IM) infe...
Sep-16-2013

Porcine Epidemic Diarrhea Virus

34
Sep-16-2013

Porcine Epidemic Diarrhea Virus

35
Sep-16-2013

Porcine Epidemic Diarrhea Virus

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 day...
Immune responses of serum samples from pregnant sows inoculated
with Vero cell attenuated PEDV DR13, using IgA-specific EL...
Antibody responses from serum samples against PEDV in 3-day-old
piglets using IgG and IgA-specific ELISA before challenge ...
Antibody responses from serum samples against PEDV in 3-day-old
piglets using the SN test before challenge exposure (*P<0....
RT-PCR detection of virus shedding in serial passages of
colostrum-deprived 3-day-old pigs
Level of pig passage

Pig
No.

...
Survival rate (%) of piglets born from vaccinated sows
after challenge with wild-type PEDV

90
80
70
60
50
40
30
20
10
0

...
Acknowledgements





Dr. Jin-Sik Oh, DVM, MSc, PhD
Dr. Dae-Sop Song, DVM, PhD
Dr. Seong-Jun Park, BS, PhD
Green Cross...
Thank you for your attention !
Cross neutralization and new vaccine candidate ???

Sep-16-2013

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

  1. 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. 2. Sep-16-2013 Porcine Epidemic Diarrhea Virus 2
  3. 3. Sep-16-2013 Porcine Epidemic Diarrhea Virus 3
  4. 4. Recent Outbreaks of PED and TGE in Korea PED Outbreaks Sep-16-2013 TGE Outbreaks Porcine Epidemic Diarrhea Virus 5
  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. 6. Routine pattern of SN titers against PEDV in commercial farms SN titers(2ⁿ) Sep-16-2013 Porcine Epidemic Diarrhea Virus 7
  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. 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. 9. - Spike gene(46 strains) Sep-16-2013 Porcine Epidemic Diarrhea Virus 10
  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. 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. 12. Based on Full-length E gene Sep-16-2013 Porcine Epidemic Diarrhea Virus 13
  13. 13. Sep-16-2013 Porcine Epidemic Diarrhea Virus 14
  14. 14. Sep-16-2013 Porcine Epidemic Diarrhea Virus 15
  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. 16. Sep-16-2013 Porcine Epidemic Diarrhea Virus 17
  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. 18. Sep-16-2013 Porcine Epidemic Diarrhea Virus 19
  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. 20. Porcine Epidemic Diarrhea Virus 21
  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. 22. Emerg Infect Dis. 2012 August; 18(8): 1350–1353. doi: 10.3201/eid1808.120002 Sep-16-2013 Porcine Epidemic Diarrhea Virus 23
  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. 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. 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. 26. DOI 10.1007/s11262-012-0794-x Sep-16-2013 Porcine Epidemic Diarrhea Virus 27
  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. 28. Distribution of SN titers among the pregnant sows Sep-16-2013 Porcine Epidemic Diarrhea Virus 29
  29. 29. Case #999 farm – SN titers’ distribution (Feb 21, 2007 by artificial immunization) Sep-16-2013 Porcine Epidemic Diarrhea Virus 30
  30. 30. Case #000 farm – SN titers’ distribution (Feb. 22, 2007 by artificial immunization) Sep-16-2013 Porcine Epidemic Diarrhea Virus 31
  31. 31. Aminopeptidase N, 150 kDa – Cellular receptor 32 Muirhead MR & Alexander TJL, 1997
  32. 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. 33. Sep-16-2013 Porcine Epidemic Diarrhea Virus 34
  34. 34. Sep-16-2013 Porcine Epidemic Diarrhea Virus 35
  35. 35. Sep-16-2013 Porcine Epidemic Diarrhea Virus 36
  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. 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. 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. 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. 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. 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. 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. 43. Thank you for your attention ! Cross neutralization and new vaccine candidate ??? Sep-16-2013 Porcine Epidemic Diarrhea Virus 44

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