Dr. Darin Madson - Porcine Epidemic Diarrhea Virus - Research & Diagnostics

Porcine Epidemic Diarrhea Virus

Research & Diagnostics

Darin Madson

Iowa State University

[email protected]

2015 Iowa Pork Congress


• U.S. introduction quick Facts

1. First confirmed infection = April 16, 2013 (OH)

2. Diagnosed in over 30 states

3. Over 9,000 confirmed cases

4. Over 40,000 cases tested (Nov. 2013 - Nov. 2014)

5. ~22.5% of cases tested are positive (1/5)

6. Two distinct viruses (prototype and variant)


• Virus characteristics quick facts

1. Coronavirus (alphacoronavirus)

2. First reported in England (1971)

3. Officially recognized/isolated in 1978

4. Similar to TGE but there is no antibody cross protection

5. Currently endemic in Asian countries

6. 2010/11 reported increased virulence


• Virus strains quick facts1. PEDV strains can be divided into group 1 and group 2

2. PEDV strains have two S gene patterns

3. Circulating PEDV strains appear to have been introduced into the US concurrently

4. In one year, no significant changes occurred in the variant strain, but prototype strain has undergone genetic changes

5. Prevalence of US variant strain is lower than US

prototype strain.


• Pathogenesis quick facts

1. Relatively stable virus

2. Fecal-oral transmission; few particles needed

3. Small intestine infection malabsorptive issues

4. Variable diarrhea +/- vomiting age dependent

5. Fecal shedding beyond clinical signs


Villous to crypt ratio 7:1


Villous to crypt ratio 3:1




• Diagnostic tests

– Polymerase Chain Reaction (PCR)– Detection of viral genomic material in fresh samples– High sensitivity and specificity

– Immunohistochemistry– Detection of antigen in fixed tissues– High specificity and low sensitivity

– Virus isolation

– Serology– Antibody production post infection – Maternal antibody– Vaccination


• PCR – Multiple available

• S gene, N gene, and nested labs using different PCRs

• ISU is currently running

–N gene real-time– M gene for tetra-core (PEDv/TGEV multiplex)

– S gene gel based

– Nested

• PCR samples– Feces, intestine, oral fluids, feed, Swiffer

0

5

10

15

20

25

30

35

40

45

0 1 2 3 4 5 6 7 10 14 17 21 24 28 31 35

PC

R C

ycle

Th

resh

old

Days Post-infection (DPI)

DPI N # positive % positive

0 36 0 0

1 36 8 22%

2 32 32 100%

3 28 28 100%

4 24 24 100%

5 20 20 100%

6 20 20 100%

7 20 20 100%

10 16 11 69%

14 16 14 88%

17 12 9 75%

21 12 5 42%

24 8 5 63%

28 8 0 0

31 4 0 0

35 4 0 0

1

Fecal shedding and PCR detection; acute infection


0.1

1

10

100

1000

10000

100000

DPI 1 DPI 2 DPI 3 DPI 4 DPI 5 DPI 6 DPI 7 DPI 10 DPI 14 DPI 17 DPI 21 DPI 24 DPI 28 DPI 31 DPI 35

ave

rage

PFU

/mL

eq

uiv

ale

nts

Days Post-Inoculation

Individual FecalSwabs Average

Group Oral FluidsAverage

25% WD

30% WD

50% WD

85%

normal

feces

Slide courtesy of Dr. Yoon


Feces vs oral fluids in acute infection

% positives Virus level (Ct)

0

20

40

60

80

100

2 4 7 10 14 21

1st time 2nd time

0

5

10

15

20

25

30

35

40

45

Day 2 Day 4 Day 7 Day 10 Day 14 Day 21

1st time 2nd time

Ct

valu

e

Days post inoculation

Fecal shedding; repeat infection



• Differential PCR

– Prototype and variant strains

Whole genomeidentity

S1 nucleotideidentity

Prototype VS variant

98.6%-99.2% 91.9%- 93.8%

Within variant 99.4%-99.9% 99.5%- 100%

Within prototype 99.6%-100% 97.5%- 100%


PEDV sequencing

European

0.01

USA 2013-2014 (Blue)Mexico 2014 (Bright blue)Korea 2013-2014 (Purple)China 2011-2014 (Red)

China 2011-2014

China 2011-2013 Korea 2008-2012Thailand 2008-2013 (Green)Japan (Year?) (Yellow) Vietnam 2013

USA 2013-2014 Mexico 2014 China 2004, 2008, 2010-2013 Korea 2009 (Purple)European 1970-80s (Black)Japan (Year?)

U.S. prototype strain

U.S. variant strain

570 Global PEDV S1

Group II

Group I


• Immunohistochemistry (IHC)

– Multiple primary antibodies

– Fixed intestine only

– Need good quality tissue

• In situ hybridization• Dr. Eric Burrough

Average IHC Score/Intestinal Segment/DPI

Scoring system 0=negative 1=<10% 2=10-50% 3=>50%

0

0.5

1

1.5

2

2.5

3

1 2 3 4 7 14 21

Duodenum

Prox. Jejunum

Mid Jejunum

Distal Jejunum

Ileum

Days Post Inoculation

Score

IHC Score

Avg. PCR Ct

2+ 15.011 25.17

Neg 32.9



Neonate IHC detection post infection; hours

• Virus Isolation

– Difficult and time consuming

• 15% success rate, or less, and unpredictable

• Intestine and feces– Baby pig intestines not a great sample milk content

• Vero cells in presence of 5 µg/ml trypsin

Dr. Jianqiang Zhang, ISU-VDL


Detectable anti-PEDV Antibody response between 7-14 dpi ELISA (IgG, IgA) & IFA

Coinciding with resolution of clinical diarrhea and subsequent declining of number of virus-shedding pigs –Madson et al.2014-

Max. IFA IgG antibody titer response on dpi 21Antibody titers declining markedly thereafter

The declining = decrease no of virus-shedding pigs

Porcine Epidemic Diarrhea VirusWeaned pigs

Slide information courtesy of Dr. Gimenez-Lirola



AssayDays post-inoculation (DPI)

0 7 10 14 21 28 35 42 49 56

IFA 0 0 85% 95% 100% 100% 100% 95% 90% 60%

WV ELISA 0 15% 85% 95% 100% 100% 100% 100% 100% 100%

4-weeks-old pigs (n=20) over time after inoculation with PEDV under experimental conditions

Cut-off:

PEDV WV ELISA: S/P ratio > 0.8

PEDV IFA: Titer ≥ 40

Comparative sensitivity PEDV WV ELISA vs PEDV IFA

(Assay performance on experimental samples)



Detection rate (%) over-time

DPC IFA WV ELISA

41 100 100

61 100 100

91 85.71 89.29

100 46.67 73.33

114 16.67 73.33

Serum samples (n=270) from a PEDV naïve sow farm between 6-25 weeks following a PEDV outbreak

Overtime detection on field samples




• Serology quick facts

1. Antibody detection between 7-10 days post infection similar for ELISA and IFA

2. Peak titer around 21 days post infection

3. Both tests will detected prototype and variant antibodies

4. The WV ELISA will detect antibodies longer

• Sow immunity studies overview (in progress)

– Sow herd infected with PEDV; April 2014

– 9 sows relocated to ISU in Septmember

• Negative control: n=3

• Treatment: n=5

– 3 challenge control sows


Piglet Challenge24-48 hrs of age

Sample Collection

DPI Serum Feces Milk

0 X X X

Farrowing(5-6) X X X

11 (T) X X X

14 (F) X X X

19 (T) X X X

22 (F) X X X

26 (T) X X X

29 (F) X X X

DPI Serum Feces Weight

Birth (0) X X X

4 (T) X X

7 (F) X X X

11 (T) X

14 (F) X X X

18 (T) X

21 (F) X X X

Sow Samples Piglet Samples

Treatment

Results

GroupPED Clinical

SignsLitter Mortality

Negative Control Sows

0/3 -

Negative Control Piglets/Litters

0/3 6/34 (17.6%)

Challenge Control Sows

3/3 -

Challenge Control Piglets/Litters

3/3 31/31 (100%)

Treatment Sows 0/5 -

TreatmentPiglets/Litters

1/5 9/70 (12.9%)

Treatment

Conclusions

• Protective immunity was present in sows previously infected with PEDV when challenged with homologous PEDV

• Piglets born to sows previously exposed to PEDV had protective immunity when exposed to and challenged with homologous PEDV within 48 hours post-birth

• Additional information through diagnostics will offer diagnostic evidence supporting field observations thus far

Questions???

Acknowledgments

Dr. Drew MagstadtDr. Paulo ArrudaDr. YoonDr. GaugerDr. ZhangDr. Gimenez-Lirola

Health & Medicine

Dr. Darin Madson - Porcine Epidemic Diarrhea Virus - Research & Diagnostics