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

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

Sep-16-2013 Porcine Epidemic Diarrhea Virus 2

Sep-16-2013 3Porcine Epidemic Diarrhea Virus


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



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



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


- Spike gene(46 strains)


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


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


Based on Full-length E gene



Sep-16-2013 Porcine Epidemic Diarrhea Virus 16Sep-16-2013 Porcine Epidemic Diarrhea Virus 16

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 17Sep-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 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.



Fig. 2 Schematic representation of the PEDV genome based on the CV777 (GenBank accession No. AF353511) strain

Porcine Epidemic Diarrhea Virus 21


New variants of porcine epidemic diarrhea virus – 3 new PEDV variants were identified in vaccinated swine herds which animals exhibited severe diarrhea


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

http://dx.crossref.org/10.3201%2Feid1808.120002


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

http://dx.crossref.org/10.3201/eid1808.120002


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).


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.


DOI 10.1007/s11262-012-0794-x


PED Immune Status in Endemic Pig Farms

Questions ?PEDV Variability vs Immune Variability of Individual Pig

Distribution of SN titers among the pregnant sows


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


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


Muirhead MR & Alexander TJL, 1997

Aminopeptidase N, 150 kDa – Cellular receptor

32


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)




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.


Immune responses of serum samples from pregnant sows inoculatedwith Vero cell attenuated PEDV DR13, using IgA-specific ELISA (*P<0.05)


Antibody responses from serum samples against PEDV in 3-day-oldpiglets using IgG and IgA-specific ELISA before challenge exposure (*P<0.05)


Antibody responses from serum samples against PEDV in 3-day-oldpiglets using the SN test before challenge exposure (*P<0.05)


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.


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


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



Thank you for your attention !

Cross neutralization and new vaccine candidate ???

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