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 Laboratory
Iowa State University
Faculty, Staff, & Students of ISU VDL
≈22 Faculty, 100 Technical Staff, & 20 Graduate Students/Residents 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
Veterinary Diagnostic Laboratory
Iowa State University
≈ 75% Swine, 10% Bovine, 10% Poultry, + 5% Everything Else
Veterinary Diagnostic Laboratory
Iowa State University
Where the Pigs Are Located in US
≈ 500,000 pigs/week imported to IA to be grown
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Iowa State University
Total Positive PEDv Cases (NAHLN)
Seems to very closely resemble the US pig density map
reflecting PEDV’s broad dissemination across the US
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
Veterinary Diagnostic Laboratory
Porcine enteric disease picture in the US has rapidly become more complex
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
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Iowa State University
Oral fluids & Molecular Dx) to monitor the heath status of US swine in real-time.
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 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
Veterinary Diagnostic Laboratory
Iowa State University
Yoon, Madson, Magstad, Labois, et al – experimentally infected with PEDV.
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
Late PEDv infection (~36 hrs PI):
PDCoV = Presently No IHC or Success with VI
(Source: Schwartz, Madson, Magstad et al.)
(Source: Dr. Jianqiang Zhang, ISU-VDL)
Few infected cells remain (brown
stain) & absorptive cells destroyed
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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)
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↑ Sensitive, Semi-quantitative, Same-day testing, Used Extensively
Iowa State University
Veterinary Diagnostic Laboratory
Iowa State University
Positive PEDv Cases per Week by Farm Type
(Data Collated by USDA NAHLN)
350
# PEDv Positive Cases
300
250
Unknown
Grow-Finish
Sow
200
150
100
50
0
Date by Week
**black lines represent the total number of positive cases for that week
Cumulative Positive PEDv Cases by Farm Type
6000
# PEDv Positive Cases
5000
Unknown
Grow-Finish
4000
Sow
3000
2000
1000
0
Date by Week
PEDV: Diagnostics
Polymerase chain reaction (PCR)
• Target: Nucleoprotein (ISU) or Matrix
Cycle threshold values:
Range: 8-12 in acutely
affected, neonatal piglets
Lots of PEDV!
– 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?
frameshift
L
ORF1a
S
M
ORF3
ORF1b
N
AAA
E
Dr. Jianqiang Zhang, ISU-VDL
Veterinary Diagnostic Laboratory
Iowa State University
3’
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.
Veterinary Diagnostic Laboratory
Iowa State University
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 93.494.6% nucleotide identities to the
originally identified US strain
Dr. Jianqiang Zhang, ISU-VDL
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.
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Iowa State University
PDCoV
• PDCoV not PEDV or TGEV or PRCV
Alphacoronavirus: PEDV
– 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
Deltacoronavirus: PDCV
Veterinary Diagnostic Laboratory
Iowa State University
Positive PDCoV Cases @ ISU VDL per Week by Farm Type
18
16
# PDCoV Positive Cases
14
12
Unknown
2-17-14
Began PDCV PCR
Testing at ISU VDL
Grow-Finish
Sow
10
8
6
4
2
0
Veterinary Diagnostic Laboratory
Date by Week
Iowa State University
Cumulative Positive PDCoV Cases by Farm Type
80
# PDCoV Positive Cases
70
60
50
Unknown
Grow-Finish
Sow
2-17-14
Began PDCV PCR
Testing at ISU VDL
40
30
20
10
0
Date by Week
Veterinary Diagnostic Laboratory
Iowa State University
ISU VDL PDCoV Positive Swine Cases
Farm State
Week Received Total # PCR
@ VDL
Positive Cases
IA
12/22/13
1
IL
IN
MI
MN
MO
NC
NE
OH
OK
PA
?
1
12/29/13
1/5/14
1/12/14
1/19/14
2
1
1
2/2/14
2
1
2/9/14
6
4
2/16/14
10
6
1
2/23/14
12
2
1
3/2/14
14
1
5
3/9/14
17
8
1
3/16/14
8
3
1
Total
71
26
10
1/26/14
1
1
1
5
1
1
1
1
1
1
1
1
1
3
1
1
3
1
1
3
7
1
3
9
1
1
1
1
4
3
3
1
3
**totals include biological samples only--no environmental or feed samples are included in the this data
Veterinary Diagnostic Laboratory
Iowa State University
1
Emerging Coronaviruses in US Swine
“Diagnostic Lab Update”
Lots New, Lots Going On!
Thank You
Questions ?
Veterinary Diagnostic Laboratory
Iowa State University