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 destroyed PDCoV = 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

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! 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 2-17-14 Began PDCV PCR Testing at ISU VDL

23. Veterinary Diagnostic Laboratory Iowa State University 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 IAILINMIMNMONCNEOHOKPA? 12/22/131 1 12/29/13 1/5/14 1/12/14 1/19/1421 1 1/26/14 2/2/1421 1 2/9/1464 1 1 2/16/141061 1 1 1 2/23/1412215 1 1 11 3/2/141415 13111 3/9/14178111 311 1 3/16/14831 3 1 Total7126107139433131 ** 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 ?