Dr. Rodger Main - Porcine Epidemic Diarrhea Virus (PEDv) Diagnostics

Veterinary Diagnostic LaboratoryIowa State University

Emerging Coronaviruses in US Swine Diagnostic Update

Rodger Main, DVM, PhDDirector of Operations

ISU VDL


ISU-VDL is housed at the ISU College of Vet Med

≈ 145 Veterinary Students / Class


Veterinary Diagnostic & Production Animal Medicine (VDPAM) Organizational Chart

www.vdpam.iastate.eduUpdated 2/15/2012

Veterinary Extension:G. Dewell: Beef ExtensionJ. McKean: Swine ExtensionJ. Shearer: Dairy Extension

D. Trampel: Poultry Extension Veterinarian and Diagnostician

P. HalburChair of VDPAM, Executive Director of ISU VDL

515-294-8791, [email protected]

Veterinary Diagnostic Laboratory (VDL): R. Main, Director of Operations 515-294-1950, [email protected]

Bacteriology & Clinical Microbiology: T. Frana*, J. Kinyon, PhAST: Pharmacology Analytical Support Team) H. Coetzee* L.Wulf, D. BortzMolecular & Viral Diagnostics: P. Gauger*, K. Harmon, KJ. Yoon, J. ZhangPathology: K. Schwartz*, P. Arruda,

E. Burrough, V. Cooper, P. Gauger, B. Janke, D. Madson, T. Opriessnig, G. Stevenson, D. Trampel (poultry)

Serology: J. Johnson*, L. Lirola. Toxicology/Nutrition: S. Ensley*,

P. Imerman, W. Rumbeiha, D. SniderEpidemiology/Disease Ecology: J. ZimmermanInformatics: R. BergheferQuality Assurance: K Boesenberg-SmithAdministration: J Holdredge

Food Supply Veterinary Medicine (FSVM): P. Gorden, Director 515-294-3837, [email protected]

Beef: T. Engelken*, H. Coetzee, G. Dewell, R. Dewell, A. O’Connor, P. Plummer, S. Sweiger, J. Thompson, J. Thomson, B. Vander LeyDairy: P. Gorden*, D. Bickett-Weddle, A. Kreuder, A. Krull, B. Leuschen, P. Plummer, J. Shearer, J. Thompson, L. Timms, B. Vander Ley, J. WestEmbryo Transfer: J. West*, M. JahnkeEpidemiology: G. Dewell, R. Dewell, S. Hurd, A. O’Connor, J. Thomson, E. Uhlenhopp, C. Wang, J. ZimmermanFood Animal and Camelid Hospital andField Services: P. Gorden*, G. Dewell, T. Engelken, A. Kreuder, A. Krull, B. Leuschen, P. Plummer, J. Schleining, J. Thompson, B. Vander LeySmall Ruminants: C. Plummer*, P. Plummer, J. Schleining, J. Thompson, L. TimmsSwine: L. Karriker*, R. Baker, H. Harris, D. Holtkamp, J. McKean, A. RamirezTheriogenology: M. Jahnke B. Leuschen, C. Plummer, J. West

Swine Medicine Education Center (SMEC):L. Karriker (Director), B. Baker, T. Ellingson, P. Halbur, J. Hocker, D. Madson, D. Olsen

Animal Welfare:S. Millman*, H. Coetzee, R. Dewell, J. Shearer

http://www.vetmed.iastate.edu/departments/vdpam/extension.aspx?id=2288

http://www.vetmed.iastate.edu/departments/vdpam/default.aspx?id=2288

http://www.vetmed.iastate.edu/departments/VDPAM/vdl.aspx

http://www.vetmed.iastate.edu/departments/vdpam/foodsupply.aspx


Faculty, Staff, & Students of ISU VDL

≈22 faculty,100 technical staff, & 20 graduate students/residents


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 - Applying world-class technology to real-world problems


≈ 75% Swine, 10% Bovine, 10% Poultry, 5% Everything Else


Location of Pigs in the US

≈ 500,000 pigs/week imported to Iowa to be grown


Serving Veterinarians & Producers Throughout the US

FY 2014 = Number of VDL Case Accessions by State of Origin


PEDv UpdateInformation up to July 31, 2014


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Unknown Grow-Finish

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Positive PEDv Cases per Week by Farm Type

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Seems to very closely resemble the US pig density map reflecting PEDV’s broad dissemination across the US



Apr-13 May-13 Jun-13 Jul-13 Aug-13 Sep-13 Oct-13 Nov-13 Dec-13 Jan-14 Feb-14 Mar-14 Apr-14 May-14 Jun-14 Jul-140

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# Tested# PositivePercent positive

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PEDv Positive Cases by Month (including percent)


PDCoV• PDCoV not (PEDV, TGEV or PRCV)

– Delta vs. Alpha Coronaviruses– Doesn’t appear to be cross-protection

• Vomiting and diarrhea (Field/Diagnostic Reports)

– Similar clinical signs and lesions to PEDv/TGE– Reportedly more transient and/or less severe

• ISU-VDL Assays– Only PDCoV PCR is currently offered– PDCoV sequencing (NGS – Research)– No Serology Test Currently Available

Alphacoronavirus: PEDV

Deltacoronavirus: PDCV


Retrospective Testing Found 1st Few Cases in August 2013

22-Dec

28-Dec

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9-Jan15-Ja

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n27-Ja

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Cumulative PDCoV Positive Cases

Date by Week

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(Scale = Hundreds vs. Thousands)


22-Dec 5-Jan 19-Jan 2-Feb 16-Feb 2-Mar 16-Mar 30-Mar 13-Apr 27-Apr 11-May 25-May 8-Jun 22-Jun 6-Jul 20-Jul0

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Retrospective Testing found 1st Case in Aug 2013


Distribution Seems to Be Noticeably Different than PEDV


Real-Time PCR Primary PEDV & PDCoV Diagnostic Tool of Choice

– Same day testing – Highly sensitive…(Doesn’t take much feces… more not always better)– Sample types = Intestine, Feces, Fecal Swabs, Oral Fluids

• Qualitative & Semi-Quantitative Assessment of Shedding


Screening PCR’sScreening = N-Gene or Matrix, More Conserved

Courtesy of Dr. Jianqang Zhang, ISU VDL


Oral Fluids proving to be a very effective sample type for ante-mortem PEDV PCR testing

Sample type of choice group-housed growing pig / growing replacement gilt surveillance


PEDv Swab Submissions

Starplex vs Copan


ISU VDL’s Molecular & Viral Diagnostic Services


Jun-13 Jul-13 Aug-13 Sep-13 Oct-13 Nov-13 Dec-13 Jan-14 Feb-14 Mar-14 Apr-14 May-14 Jun-14 Jul-140

2,000

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ISU VDL PEDv Total PCR Tests

Date by Month

# P

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ests


ISU VDL Porcine Coronavirus Molecular Diagnostic AssaysRed = New in Past Year

• TGE – PCR• PanCorona – PCR (Discovery Yoon et al)

• PEDv – PCR (& now quantitative PCR)

• PEDv Differential – PCR– Original US strain vs. more recent variant

• PDCoV – PCR

• PEDv Sequencing– S1 Gene (Routine)– Whole Genome (Research)

• PDCov Sequencing– Whole Genome (Research)

↑ Sensitivity, Semi-quantitative, Same-day testing, Used Extensively


PEDV SequencingPEDv Sequencing• Spike protein sequence

– Variable portion of genome– Determine genetic relatedness – 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.


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 (≈ 6% different) to the originally identified US strain.

(Source: Dr. Jianqiang Zhang, ISU-VDL)


PEDv Full-length S gene

USA/IA/2013/18984(KF804028)

USA/IA/2013/22038homo(KF650373)

USA/IA/2013/IA2(KF468754)

USA/IN/2013/17846(KF452323)

USA/IN/2013/19338Ehomo(KF650370)

USA/MN/2013(KF468752)

USA/IA/2013/IA1(KF468753)

USA/CO/2013(KF272920)

USA/IA/2013/16465(KF452322)_S

China/2012/AH2012(KC210145)

China/2012/CH-FJZZ(KC140102)

China/2012/GD-A(JX112709)

SKorea-SM98(GU937797)

Europe/CV777(AF353511)

SKorea/DR13virulent(JQ023161)

China/2008/JS2008(KC109141)

SKorea/DR13attenuated(JQ023162)

China/2004/JS2004-2(AY653204)

China/2011/JLGZL(JQ638923)

China/2012/FJXM-2(JX070672)

China/2012/JS120103(KF453513)

China/2010/HBQX-10(JX501318)

2014002116A_S

2014000087-2_S

2013059573-5_S

2014003293_S

2014000084-2_S

0.005

PEDV viruses identified in US starting from April 2013 (Original US Strains)

PEDV variants identified in US

≈ 3- 4% different on whole S-gene


PED Variant: Epidemiology(same as PEDV IN-DEL)

• 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.


Porcine Coronavirus Serology (Antibody Testing)

• PEDv – IFA (labor intensive, subjective, proven to be robust assay)

• PEDv-ELISA (Whole-virus ELISA starting ≈September 1, 2013 at ISU)

- UMN VDL – PEDV Elisa– Duration of detectable antibody response needs to be understood with PEDv– Commercially Available PEDV ELISA’s → Very Poor

• PEDv FFN (SDSU)

• TGE-PRCv Differential ELISA• PDCoV = No Assays Available


PEDV IFAPrimary 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-VDLDr. John Johnson, ISU-VDL


PEDV IFA test

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Development of IFA antibody over time

(Source: Yoon, Sun)


Yoon, Madson, Magstad, Labois, et al – experimentally infected with PEDV.


PED WV ELISAPrimary PEDv Antibody Detection Assay

(Starting ≈ September 1, 2014 as routinely available)

• Enzyme-Linked Immunosorbent Assay (ELISA)– Serology / Antibody Based Assay– Currently developing testing for milk and colostrum

• Detects antibody for a longer duration on the response curve than IFA and NP ELISAs (original and variant strain)

• Sample to positive ratio of 0.8 established as the cutoff (S:P ratio range from 0.5 – 0.8 as suspect)• Advantages

– Objectively evaluated– High sample throughput potential – Same day results


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 destroyed

*PDCoV = Presently No IHC or Success with VI


Immunohistochemistry (IHC)

Normal neonatal pig: Healthy, long

intestinal villi

Early PEDv infection (~8 hrs PI): Infected cells (brown stain)

line the villi

Late PEDv infection (~36 hrs PI): Severe villus atrophy &

loss of absorptive epithelium

Late PEDv infection (~36 hrs PI): Few infected cells

remain (brown stain) & absorptive cells destroyed

1

4

2

3

(Source: Schwartz, Madson, Magstad et al.)


Questions ?

PEDV or PDCoV Diagnostics


Diagnostic Information • Significant Efforts to Improve:

– Quality (Identity Preservation)– Value – Utility – Connectivity– Convenience– Systems of Traceability


Stepwise Template for Improvement(Development in 2013, Rolling Out in 2014)

1. Submission Forms– Site / Premises / Flow / Group … & Affiliates– Improved test selection

2. Laboratory Information Management System– Capture, Manage, Store, & Report– Pre-Population F(x), and Site-Level Affilliates

3. Web-reporting Tool– User Access to Case Reports (Current/Historical)

4. Web-Submission Tool


Updated Submission Forms & Data Capture

```

Species Specific = Test Request Form


Necropsy = All Species

Updated Submission Forms & Data Capture


How to Pre-Populate LIMS1. Go to the ISU VDL website2. Click “Pre-populate ISU VDL’s LIMS”3. Download “LIMS Pre-Population Spreadsheet”4. Populate fields 5. Email spreadsheet to [email protected]


Implications of Pre-Population


One sheet of Premises ID barcode labels per site will be designed, printed, and mailed to the address indicated in the email

Place premises ID barcode label in the designated area on the submission form

One premises ID per submission form

Use premises ID barcodes on VDL submissions

How to Use Premises ID Barcode Labels


Implications of Pre-Population

& Most Recently Added Ability to Affiliate by Site vs by Case


How to Order Premises ID Barcode Labels

1. Go to the ISU VDL website

2. Click “Premises ID Barcode Label Information”

3. Download “Premises ID Label Spreadsheet”

4. Populate fields

5. Email spreadsheet to [email protected]: Clinic Name:Clinic Address:

Premise ID Official Site Name/Code Owner00ABC12 Bob's Finisher Bob 00ABC13 Bob's Nursery Bob00ABC14 Bob's Sow Bob00ABC15 Bob's Farrowing Bob00ABC16 Bill's Finisher Bob00ABC17 Bill's Nursery Bob00ABC18 Bill's Sow Bob00ABC19 Bill's Farrowing Bob

Dr. SmithSmith's Vet Clinic111 Main Street, Paradise, IA 55555

Veterinarian: Clinic Name:Clinic Address:

Premise ID Official Site Name/Code Owner

Outcome:ISU VDL has bar-code labels pages printed at remote location and mails back to client


New Web Submission Tool


Stepwise Template for Improvement(Development in 2013, Rolling Out in 2014)

1. Submission Forms– Site / Premises / Flow / Group … & Affiliates– Improved test selection

2. Laboratory Information Management System– Capture, Manage, Store, & Report

3. Web-reporting Tool– User Access to Case Reports (Current/Historical)

4. Web-Submission Tool (Field Beta-Testing)

Making Progress, Step-by-Step


Diagnostic Information • Significant Efforts to Improve:

– Quality (Identity Preservation)– Value – Utility – Connectivity– Convenience– Systems of Traceability

Goal = Positioned to Support Needs of Next Generation of Food Animal Agriculturalists


Katie Woodard, DVMClient Outreach and Education

[email protected]

Iowa State University Veterinary Diagnostic Laboratory

Kate Mueller, DVMDiagnostic Database Tools & Connectivity

[email protected]


PEDV/PDCoV Reporting – USDA Federal Order

• June 5, 2014• Required :

– Premises ID – Date of collection – Type of farm site– Test method – Test result

Veterinarian

VDL’s USDA

StateOr

USDA VMO

Samples

Results

Answers

Questions

Results

ISU VDL’s Role = Test & Report (Streamline As Much Possible for Our Clients)


Emerging Coronaviruses in US Swine Diagnostic Update

Thank You & Questions ?




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Comparing Alternative Fecal Swabs

Copan-5:1

Starplex-5:1

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CT V

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PEDv Dendrogram based on full-length genomes

USA/IA/2013/49379

USA/IA/2013/IA2(KF468754)

USA/IA/2013/22038(KF650373)

USA/MN/2013(KF468752)

USA/IA/2013/18984(KF804028)

USA/IN/2013/19338(KF650370)

USA/IN/2013/17846(KF452323)

USA/IA/2013/16465(KF452322)

USA/NC/2013/49469

USA/NC/2013/35140

USA/CO/2013/019349(KF267450)

USA/IA/2013/IA1(KF468753)

USA/CO/2013(KF272920)

China/2012/AH2012(KC210145)

USA/IL/2013/59573-5

USA/MO/2014/3293

USA/OH851

China/2011/ZMDZY(KC196276)

China/2011/BJ-2011-1(JN825712)

China/2012/JS-HZ2012(KC210147)

China/2012/GD-B(JX088695)

China/2012/FJZZ-9(KC140102)

China/2011/FJND-3(JQ282909)

China/2011/AJ1102(JX188454)

China/2011/LC(JX489155)

China/2011/ZJCZ4(JX524137)

China/2011/CHGD-01(JX261936)

China/2012/GDGZ(KF384500)

China/2012/GD-A(JX112709)

China/2011/GD-1(JX647847)

SKorea/2009/DR13virulent(JQ023161)

China/1986/CH-S(JN547228)

Europe/CV777(AF353511)

China/LZC(EF185992)

SKorea/SM98(GU937797)

China/2008/JS2008(KC210146)

SKorea/DR13attenuated(JQ023162)

China/2012/SD-M(JX560761)

China(KC189944)

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PEDv viruses identified in US starting from April 2013 (original strains)

PEDv variant identified in US

≈ 1% based whole genome

Health & Medicine

Dr. Rodger Main - Porcine Epidemic Diarrhea Virus (PEDv) Diagnostics