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

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
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3. Sep-16-2013
Porcine Epidemic Diarrhea Virus
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5. Recent Outbreaks of PED and TGE in Korea
PED Outbreaks
Sep-16-2013
TGE Outbreaks
Porcine Epidemic Diarrhea Virus
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6. 4 Major Swine Diseases in Korea




Classical Swine Fever(Apr. 2009)
PCV-2
PRRS
PED
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7. Routine pattern of SN titers against PEDV
in commercial farms
SN titers(2ⁿ)
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Porcine Epidemic Diarrhea Virus
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8. 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
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9. 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
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Porcine Epidemic Diarrhea Virus
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10. - Spike gene(46 strains)
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Porcine Epidemic Diarrhea Virus
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11. 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
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12. 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
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Porcine Epidemic Diarrhea Virus
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13. Based on Full-length E gene
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16. Beginning in October 2010, the PED outbreak
overwhelmed >10 provinces in southern China,
and >1,000,000 piglets died.
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17. Sep-16-2013
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18. 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.
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Porcine Epidemic Diarrhea Virus
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19. Sep-16-2013
Porcine Epidemic Diarrhea Virus
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20. Fig. 2 Schematic representation of the PEDV genome based on the CV777
(GenBank accession No. AF353511) strain
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Porcine Epidemic Diarrhea Virus
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21. Porcine Epidemic Diarrhea Virus
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22. New variants of porcine epidemic diarrhea virus –
3 new PEDV variants were identified in vaccinated
swine herds which animals exhibited severe diarrhea
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23. Emerg Infect Dis. 2012 August; 18(8): 1350–1353.
doi: 10.3201/eid1808.120002
Sep-16-2013
Porcine Epidemic Diarrhea Virus
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24. Emerg Infect Dis. 2012
August; 18(8): 1350–1353.
doi: 10.3201/eid1808.120002
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25. 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).
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26. 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.
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27. DOI 10.1007/s11262-012-0794-x
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Porcine Epidemic Diarrhea Virus
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28. Questions ?
PEDV Variability vs
Immune Variability of Individual Pig
PED Immune Status
in Endemic Pig Farms
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29. Distribution of SN titers among the pregnant sows
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30. Case #999 farm – SN titers’ distribution
(Feb 21, 2007 by artificial immunization)
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31. Case #000 farm – SN titers’ distribution
(Feb. 22, 2007 by artificial immunization)
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32. Aminopeptidase N, 150 kDa – Cellular receptor
32
Muirhead MR & Alexander TJL, 1997

33. 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
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34. Sep-16-2013
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35. Sep-16-2013
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36. Sep-16-2013
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37. 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.
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38. Immune responses of serum samples from pregnant sows inoculated
with Vero cell attenuated PEDV DR13, using IgA-specific ELISA
(*P<0.05)
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39. Antibody responses from serum samples against PEDV in 3-day-old
piglets using IgG and IgA-specific ELISA before challenge exposure
(*P<0.05)
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40. Antibody responses from serum samples against PEDV in 3-day-old
piglets using the SN test before challenge exposure (*P<0.05)
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41. 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
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42. 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
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43. 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
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44. Thank you for your attention !
Cross neutralization and new vaccine candidate ???
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