Veterinary Diagnostic LaboratoryIowa 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

Veterinary Diagnostic LaboratoryIowa State University

Faculty, Staff, & Students of ISU VDL

≈22 Faculty, 100 Technical Staff, & 20 Graduate Students/Residents

Veterinary Diagnostic LaboratoryIowa 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 producersApplying world-class technology to real-world problems

Veterinary Diagnostic LaboratoryIowa State University

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

Veterinary Diagnostic LaboratoryIowa State University

Where the Pigs Are Located in US

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

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

Veterinary Diagnostic LaboratoryIowa 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

Veterinary Diagnostic LaboratoryIowa 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.

Veterinary Diagnostic LaboratoryIowa 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

Veterinary Diagnostic LaboratoryIowa State University

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

Veterinary Diagnostic LaboratoryIowa State University

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

Veterinary Diagnostic LaboratoryIowa 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 destroyed PDCoV = Presently No IHC or Success with VI

Veterinary Diagnostic LaboratoryIowa State University

Porcine Coronavirus Molecular Diagnostic Assays @ ISU VDLRed = 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

Veterinary Diagnostic LaboratoryIowa State University

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Positive PEDv Cases per Week by Farm Type(Data Collated by USDA NAHLN)

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**black lines represent the total number of positive cases for that week

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Veterinary Diagnostic LaboratoryIowa State University

PEDV: DiagnosticsPolymerase 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

ORF1bS ORF3 M

N

frameshift

E

L AAA

Dr. Jianqiang Zhang, ISU-VDL

Cycle threshold values: Range: 8-12 in acutely

affected, neonatal pigletsLots of PEDV!

Veterinary Diagnostic LaboratoryIowa 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.

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

Veterinary Diagnostic LaboratoryIowa 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.

Veterinary Diagnostic LaboratoryIowa 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

Veterinary Diagnostic LaboratoryIowa State University

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Positive PDCoV Cases @ ISU VDL per Week by Farm Type

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2-17-14Began PDCV PCR

Testing at ISU VDL

Veterinary Diagnostic LaboratoryIowa State University

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Cumulative Positive PDCoV Cases by Farm Type

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2-17-14Began PDCV PCR

Testing at ISU VDL

Veterinary Diagnostic LaboratoryIowa 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

Veterinary Diagnostic LaboratoryIowa State University

Emerging Coronaviruses in US Swine“Diagnostic Lab Update”

Lots New, Lots Going On!

Thank YouQuestions ?