PEDv Lab Update - Butch Baker, DVM MS, Interim Director of the Iowa Pork Industry Center, ISU Extension and Outreach, Iowa State University, from the 2014 NIAA Annual Conference titled 'The Precautionary Principle: How Agriculture Will Thrive', March 31 - April 2, 2014, Omaha, NE, USA. More presentations at http://www.trufflemedia.com/agmedia/conference/2014_niaa_how_animal_agriculture_will_thrive

1. Veterinary Diagnostic Laboratory
Iowa State University
Emerging Coronaviruses in US Swine
“Diagnostic Lab Update”
RB Baker, R Main, & K Mueller
Department of Veterinary Diagnostic and Production Animal Medicine,
Veterinary Diagnostic Lab, Iowa State University, Ames, IA 50011

2. Veterinary Diagnostic Laboratory
Iowa State University
Faculty, Staff, & Students of ISU VDL
≈22 Faculty, 100 Technical Staff, & 20 Graduate Students/Residents

3. Veterinary Diagnostic Laboratory
Iowa State University
Serving Food Animal Agriculture
Comprehensive Diagnostic Service, Teaching, and Discovery
(Iowa’s only Full-Service & Fully Accredited Veterinary Diagnostic Laboratory)
Processing 55,000+ cases/year from livestock producers
Applying world-class technology to real-world problems

4. Veterinary Diagnostic Laboratory
Iowa State University
≈ 75% Swine, 10% Bovine, 10% Poultry, + 5% Everything Else

5. Veterinary Diagnostic Laboratory
Iowa State University
Where the Pigs Are Located in US
≈ 500,000 pigs/week imported to IA to be grown

6. Total Positive PEDv Cases (NAHLN)
Seems to very closely resemble the US pig density map
reflecting PEDV’s broad dissemination across the US

7. Veterinary Diagnostic Laboratory
Iowa State University
Porcine Coronaviruses in US Swine
TGE: Transmissible Gastroenteritis
PEDV: Porcine Endemic Diarrhea Virus
PDCoV: Porcine Deltacoronavirus
* Field reports suggest it to be a pathogen
* Koch’s postulates yet to be fulfilled
-----------------------------------------------------------------------------------------------------------------------------
PRCV = Porcine Respiratory Coronavirus
* Seems to be more of a friend than a foe
Porcine enteric disease picture in the US has rapidly become more complex

8. Veterinary Diagnostic Laboratory
Iowa State University
Porcine Coronavirus Diagnostic Specimens
(TGE, PEDV, & most recently PDCoV)
• Intestine (Fresh & Fixed)
• Feces
• Fecal Swabs
• Oral Fluids
• Serum (Antibody)
Antemortem diagnostic tools are increasingly used (especially
Oral fluids & Molecular Dx) to monitor the heath status of US swine in real-time.

9. Veterinary Diagnostic Laboratory
Iowa State University
Porcine Coronavirus Serology
(Antibody Testing)
• PEDV – IFA (labor intensive, subjective, but it has been fairly robust)
• PEDV-Elisa (only routine @ UMN that I’m aware)
– Duration of detectable antibody response needs to be understood with PEDV
– Others In Development (ISU, SDSU, et al.)
– Commercially Available PEDV Elisa’s → Very Poor
• TGE-PRCV Differential Elisa
• PDCoV = No Assays Available

10. Veterinary Diagnostic Laboratory
Iowa State University
PEDV IFA
Primary PEDV Antibody Detection Assay Used Today @ ISU VDL
In-House Experimental PEDV Elisa Coming Soon
• Indirect Fluorescence Antibody Test (IFA)
– Serology / Antibody Based Assay
– Screening assay for antibody at 1:40 dilution
(positive/negative)
– 4-fold dilution (40, 80, 160, 320)
– Limitations
• Limited correlation between IFA titer and protective immunity
• IFA antibody duration is short
• Difficult to correlate IFA titer with success of feedback
Dr. Kyoung-Jin Yoon, ISU-VDL
Dr. John Johnson, ISU-VDL

11. Veterinary Diagnostic Laboratory
Iowa State University
Yoon, Madson, Magstad, Labois, et al – experimentally infected with PEDV.

12. Veterinary Diagnostic Laboratory
Iowa State University
PEDV & TGEV Diagnostics
• Immunohistochemistry (IHC)
– Valuable tool, but much less sensitive than PCR
– Detection varies based on timing of infection
• Virus Isolation (VI)
– Difficult and time consuming
– Success rate very low
Late PEDv infection (~36 hrs PI):
Severe villus atrophy & loss of
absorptive epithelium
(Source: Schwartz, Madson, Magstad et al.)
(Source: Dr. Jianqiang Zhang, ISU-VDL)
Late PEDv infection (~36 hrs PI):
Few infected cells remain (brown
stain) & absorptive cells destroyedPDCoV = Presently No IHC or Success with VI

13. Veterinary Diagnostic Laboratory
Iowa State University
Porcine Coronavirus Molecular Diagnostic Assays @ ISU VDL
Red = New Assays in Past 12-months
• TGE – PCR
• PEDV – PCR
• PEDV Differential – PCR
– Original US strain vs. more recently identified variant
• PDCoV – PCR
• Pan-Corona – PCR (Discovery – Yoon et al)
• PEDV Sequencing
– S1 Gene (Routine)
– Whole Genome (Research)
• PDCoV Sequencing
– (Research to date)
↑ Sensitive, Semi-quantitative, Same-day testing, Used Extensively

14. Veterinary Diagnostic Laboratory
Iowa State University

15. 0
50
100
150
200
250
300
350
#PEDvPositiveCases
Date by Week
Positive PEDv Cases per Week by Farm Type
(Data Collated by USDA NAHLN)
Unknown
Grow-Finish
Sow
**black lines represent the total number of positive cases for that week

16. 0
1000
2000
3000
4000
5000
6000
#PEDvPositiveCases
Date by Week
Cumulative Positive PEDv Cases by Farm Type
Unknown
Grow-Finish
Sow

17. Veterinary Diagnostic Laboratory
Iowa State University
PEDV: Diagnostics
Polymerase chain reaction (PCR)
• Target: Nucleoprotein (ISU) or Matrix
– Targets the conserved portion of the genome
• PCR tests are very sensitive and specific
– Detect small amounts of target (virus) in the sample
– Does not require much fecal content for detection
• Same day testing
• Sample types = Intestine, Feces, Fecal Swabs, Oral Fluids
• Tests require interpretation
– Based on clinical presentation, sample type, location, etc.
– Is the test outcome what you expected?
3’
ORF1a
ORF1b
S ORF3 M
N
frameshift
E
L AAA
Dr. Jianqiang Zhang, ISU-VDL
Cycle threshold values:
Range: 8-12 in acutely
affected, neonatal piglets
Lots of PEDV!

18. Veterinary Diagnostic Laboratory
Iowa State University
PEDV: Diagnostics
PEDV Sequencing
• Spike protein sequence
– Variable portion of genome
– Determine genetic relatedness of PED viruses
– Evaluate genetic evolution
• Sequencing at ISU-VDL
– S1 gene: 2.2 kb
• Highly variable portion of spike gene being used for routine sequencing for epidemiological purposes.

19. Dr. Jianqiang Zhang, ISU-VDL
Two Distinctly Different Groups
of PEDVs Circulating in US
• January 2014 – ISU VDL S1 Sequencing
– 15 PEDV positive cases
– 10 cases shared 99.1-100% nucleotide
identities to PEDV strains identified in
US swine since April 2013
– 5 cases shared 99.6-100% nucleotide
identities to each other but only 93.4-
94.6% nucleotide identities to the
originally identified US strain

20. Veterinary Diagnostic Laboratory
Iowa State University
PEDV Variant: Epidemiology
• January 2014 – ISU VDL retrospective testing
– 183 random PEDV PCR positive fecal samples from ISU VDL cases
submitted May to September 2013
– 6 of 183 cases were positive “PEDV variant”
– Five of the 6 PEDV variant positive cases were submitted in May 2013
and the other in early June
• In retrospect, both PEDV strains have been present and
circulating in US since April/May 2013.
– Earliest PEDV positive case identified in the US was submitted to the
ISU VDL on April 15, 2013.

21. Veterinary Diagnostic Laboratory
Iowa State University
PDCoV
• PDCoV not PEDV or TGEV or PRCV
– Delta vs. Alpha Coronaviruses
– Doesn’t appear to be any cross-protection
• Vomiting and diarrhea (Field/Diagnostic Reports)
– Similar clinical signs and lesions to PEDV/TGE
– Reportedly more transient &/or less severe
– Koch’s postulates yet to be fulfilled
• ISU-VDL Available Assays
– PDCV PCR is currently offered
– PDCV sequencing becoming available
• Limited retrospective testing on PDCoV completed to date
Alphacoronavirus: PEDV
Deltacoronavirus: PDCV

22. Veterinary Diagnostic Laboratory
Iowa State University
0
2
4
6
8
10
12
14
16
18
#PDCoVPositiveCases
Date by Week
Positive PDCoV Cases @ ISU VDL per Week by Farm Type
Unknown
Grow-Finish
Sow
2-17-14
Began PDCV PCR
Testing at ISU VDL

23. Veterinary Diagnostic Laboratory
Iowa State University
0
10
20
30
40
50
60
70
80
#PDCoVPositiveCases
Date by Week
Cumulative Positive PDCoV Cases by Farm Type
Unknown
Grow-Finish
Sow
2-17-14
Began PDCV PCR
Testing at ISU VDL

24. Veterinary Diagnostic Laboratory
Iowa State University
ISU VDL PDCoV Positive Swine Cases
Week Received
@ VDL
Total # PCR
Positive Cases
Farm State
IA IL IN MI MN MO NC NE OH OK PA ?
12/22/13 1 1
12/29/13
1/5/14
1/12/14
1/19/14 2 1 1
1/26/14
2/2/14 2 1 1
2/9/14 6 4 1 1
2/16/14 10 6 1 1 1 1
2/23/14 12 2 1 5 1 1 1 1
3/2/14 14 1 5 1 3 1 1 1
3/9/14 17 8 1 1 1 3 1 1 1
3/16/14 8 3 1 3 1
Total 71 26 10 7 1 3 9 4 3 3 1 3 1
**totals include biological samples only--no environmental or feed samples are included in the this data

25. Veterinary Diagnostic Laboratory
Iowa State University
Emerging Coronaviruses in US Swine
“Diagnostic Lab Update”
Lots New, Lots Going On!
Thank You
Questions ?