dr. bong kyun park - research view of porcine epidemic diarrhea virus
DESCRIPTION
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-materialTRANSCRIPT
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
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PED Outbreaks TGE Outbreaks
Recent Outbreaks of PED and TGE in Korea
4 Major Swine Diseases in Korea
Classical Swine Fever(Apr. 2009) PCV-2 PRRS PED
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Sep-16-2013 Porcine Epidemic Diarrhea Virus 7
Routine pattern of SN titers against PEDV in commercial farms
SN tite rs(2ⁿ)
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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Details in samples tested, PEDV isolation and
sequencing Jan. 2002 – Aug. 2005: Positive 267/ 737 tested
2007: Positive 84 / 1,195 testedProvNo.
SampleRT-PCRPositive
SequencingPEDV
Inoculatio
PEDV isolation (Passage No.)
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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- Spike gene(46 strains)
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PEDV ORF3 gene e3991
CPF531
CPF259
CPF872
M4758
V2501
M4759
M2227
BIF118
PFF1051
VF1016
CPF63
MF78
VF131
M2366
PF4275
e1642
e2541
e1834
e2540
DBI825
BI1108
M2503
e2539
Parent DR13
BI2804
M1763
M1764
BI981
e942
BI976
M1595
BI1166
CPF299
CPF1195
PFF514
CPF1173
PFF513
CPF39
CPF106
BIF256
PFF188
PFF381
CPF1074
PFF285
CPF1043
CPF193
CPF935
KPED-9
Attenuated DR13
P-5V
LZC
CV777
Br1/87
100
98
90
99
64
99
66
88
94
4938
69
66
23
46
42
64
66
93
32
51
33
28
26
22
17
26
56
63
0.01
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PEDV M gene MF78
VF131
M2227
BIF118
M1595
V2501
BI1108
CPF259
CPF531
PFF1051
BI981
Parent DR13
BI976
M1763
QH
LJB/03
JS-2004-2
PFF285
PFF513
PFF514
PFF381
PFF188
CPF193
CPF299
BIF256
CPF1074
KPED-9(f ield)
KPED-9(cell)
JMe2
Cjinju99
CV777
Br1/87
Attenuated DR13
M2366
e1642
e1697
9984
91
95
72
72
93
57
99
85
73
88
95
49
55 68
72
40
63
34
39
43
37
0.01
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Based on Full-length E gene
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Beginning in October 2010, the PED outbreak overwhelmed >10 provinces in southern China, and >1,000,000 piglets died.
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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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Fig. 2 Schematic representation of the PEDV genome based on the CV777 (GenBank accession No. AF353511) strain
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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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Emerg Infect Dis. 2012 August; 18(8): 1350–1353. doi: 10.3201/eid1808.120002
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Emerg Infect Dis. 2012 August; 18(8): 1350–1353. doi: 10.3201/eid1808.120002
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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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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 predictionanalysis.
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DOI 10.1007/s11262-012-0794-x
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PED Immune Status in Endemic Pig Farms
Questions ?PEDV Variability vs Immune Variability of Individual Pig
Distribution of SN titers among the pregnant sows
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Case #999 farm – SN titers’ distribution (Feb 21, 2007 by artificial immunization)
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Case #000 farm – SN titers’ distribution (Feb. 22, 2007 by artificial immunization)
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Muirhead MR & Alexander TJL, 1997
Aminopeptidase N, 150 kDa – Cellular receptor
32
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Group DPI* Tonsil Lung Liver Spleen Kidney Intestines Lymph nodesSmall Large Submandibular Mesenteric Inguinal
Oral 2 0/2 0/2 0/2 1/2 0/2 1/2 1/2 1/2 1/2 0/2
4 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
IM 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 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2
Control 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
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)
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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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Immune responses of serum samples from pregnant sows inoculatedwith Vero cell attenuated PEDV DR13, using IgA-specific ELISA (*P<0.05)
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Antibody responses from serum samples against PEDV in 3-day-oldpiglets using IgG and IgA-specific ELISA before challenge exposure (*P<0.05)
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Antibody responses from serum samples against PEDV in 3-day-oldpiglets using the SN test before challenge exposure (*P<0.05)
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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 2 3 4 5 6 7
1 1-A − +/+a +/+ +/+ +/+ +/+ +/+ −
1-B − +/+ +/+ +/+b
Control − − − − − − − −
2 2-A − +/+ +/+ +/+ +/+ − − −
2-B − +/+ +/+ +/+b
Control − − − − − − − −
3 3-A − +/+ +/+ +/+ − − − −
control − − − − − − − −a 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.
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0
10
20
30
40
50
60
70
80
90
Oral(20/ 23) IM(6/ 15) Control(14/ 14)
Survival rate (%) of piglets born from vaccinated sows after challenge with wild-type PEDV
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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
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Thank you for your attention !
Cross neutralization and new vaccine candidate ???