Improvement of PRRSV Isolation from Clinical Samples Using a ZMAC Cell Line - Dr. Jianqiang Zhang, Veterinary Diagnostic Lab, Iowa State University, from the 2018 Aptimmune Pre-AASV Symposium, March 2, 2018, San Diego, CA, USA. More presentations at http://www.swinecast.com/2018-aptimmune-symposium-aasv

1. Improvement of PRRSV Isolation from
Clinical Samples using a ZMAC Cell Line
Jianqiang (JQ) Zhang
Associate Professor
Veterinary Diagnostic Laboratory, VDPAM
College of Veterinary Medicine
Iowa State University
Aptimmune Scientific Symposium
March 3, 2018

2. Porcine Reproductive and Respiratory Syndrome
• Etiology
– PRRS Virus (PRRSV)
 2 genotypes : PRRSV-1 (European) and PRRSV-2 (North American)
• Clinical disease
– Respiratory and systemic disease
 All ages especially growing pigs
– Reproductive disorders
 Breeding swine
• Economic impact
– Loss due to PRRS in US swine industry: $664 million annually ($1.8M per day)
 Breeding herd: $302M annually
 Growing pig herd: $362M annually
Holtkamp et al, 2012

3. PRRSV Diagnostics
• Detection of viral nucleic acid, viral antigen, infectious virus
- Ante mortem
 RT-PCR (duplex for Type 1 & Type 2)
 Sequencing
 Virus isolation
• Serology
 ELISA (HerdChek-PRRS X3, IDEXX): both Type 1 and Type 2 PRRSVs -- Serum, Oral fluid
 Indirect fluorescent antibody test (IFA): Serum; could be virus-specific IFA assay
 Virus neutralization test: Serum; could be virus-specific VN test
- Post mortem
 Histopathology
 Immunohistochemistry
 Direct tissue fluorescence antibody test
 RT-PCR (duplex for Type 1 & Type 2)
 Sequencing
 Virus isolation

4. Why do we need PRRSV VI?
• Determine if a sample contains infectious (live) virus
• For vaccine development including autogenous vaccines
• For further characterization of some PRRSV strains (e.g.
pathogenesis…)
• For development and validation of some diagnostic assays
 Virus-specific indirect fluorescent antibody (IFA) assay
 Virus-specific virus neutralization test
• Other purposes …
PRRSV Virus Isolation

5. • Porcine alveolar macrophages (PAM): primary cells
- Support replication of most PRRSV strains
- Difficult to prepare reliable PAM primary culture
- Different batches of PAM are not always equally susceptible to PRRSV
- Primary cells, required to prepare periodically
• MARC-145 cell line: subclone of MA104 monkey kidney cell line
- Most commonly used cell line for PRRSV VI
- Not all PRRSVs grow in MARC-145 (especially Type I PRRSV)
• ZMAC cell line: derived from porcine alveolar macrophages
- Not evaluated for PRRSV isolation using a large number of clinical samples
Cell lines for PRRSV VI

6. Objectives
 To compare PRRSV isolation in MARC-145 and ZMAC cells using clinical
samples
 To evaluate the correlation of PRRSV concentration and specimen types
to virus isolation success
 To compare virus titers of isolates obtained in ZMAC and MARC-145 cells
 To evaluate whether PRRSV isolated in ZMAC cells can grow in MARC-145
cells and vice versa

7. PRRSV-2 VI
• 220 PRRSV-2 PCR positive specimens
- 95 serum samples with CT 13.2-36.2
- 91 lung samples with CT 13-30.2
- 34 oral fluid samples with CT 25.8-34
Specimen Number ZMAC VI + MARC-145 VI +
Serum N=95 43.15% (41/95) 8.42% (8/95)
Lung N=91 67.03% (61/91) 48.35% (44/91)
Oral fluid N=34 0 % (0/34) 0% (0/34)

8. PRRSV-2 VI
Specimen ZMAC VI + MARC-145 VI +
Serum and Lung (N=186) 54.84% (102/186) 27.95% (52/186)
Serum and Lung
MARC-145
VI +
MARC-145
VI -
Total
ZMAC VI + 45 57 102
ZMAC VI - 7 77 84
Total 52 134 186

9. PRRSV-2 VI
CT Number ZMAC VI + MARC-145 VI +
<20 N=48 87.5% (42/48) 54.17% (26/48)
20-25 N=61 70.49% (43/61) 29.7% (22/61)
25-30 N=53 30.19% (16/53) 7.55% (4/53)
30-33 N=21 4.76% (1/21) 0% (0/21)
33-37 N=3 0% (0/3) 0% (0/3)
Total N=186 54.8% (102/186) 28.0% (52/186)
Serum and lung (N=186)

10. PRRSV-1 VI
Specimen Number ZMAC VI + MARC-145 VI +
Serum N=21 23.81% (5/21) 0% (0/21)
Lung N=8 75% (6/8) 50% (4/8)
Oral fluid N=22 0 % (0/22) 0% (0/22)
• 51 PRRSV-1 PCR positive specimens
- 21 serum samples with CT 18.4-36.8
- 8 lung samples with CT 17.4-35.4
- 22 oral fluid samples with CT 31-36.8

11. PRRSV-1 VI
Specimen ZMAC VI + MARC-145 VI +
Serum and Lung (N=29) 37.93% (11/29) 13.79% (4/29)
Serum and Lung
MARC-145
VI +
MARC-145
VI -
Total
ZMAC VI + 4 7 11
ZMAC VI - 0 18 18
Total 4 25 29

12. PRRSV-1 VI
CT Number ZMAC VI + MARC-145 VI +
<20 N=3 66.67% (2/3) 66.67% (2/3)
20-25 N=8 75% (6/8) 25% (2/8)
25-30 N=8 37.5% (3/8) 0% (0/8)
30-37 N=5 0% (0/5) 0% (0/8)
33-37 N=5 0% (0/5) 0% (0/8)
Total N=29 37.9% (11/29) 13.8% (4/29)
Serum and lung (N=29)

13. Methods : Virus titration
Cytopathogenic effect (CPE), Fluorescent antibody staining (FA-staining)
and real-time RT PCR
20 type II positive PRRSV samples
Inoculate into MARC-145 Inoculate into ZMAC
Titrate into MARC-145 Titrate into ZMAC

14. Results : Virus titration in ZMAC and Marc-145 cells
virus titration in MARC-145 (TCID50/mL) virus titration in ZMAC (TCID50/mL)
TCID50/mL
Sample
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

15. Re-isolation of PRRSV-2 VI isolates in different cell lines
84 PRRSV-2 VI isolates in ZMAC MARC-145 cells
• 45 (53.57%) virus isolates from ZMAC cells grew in MARC-145 cells.
• 39 (46.43%) virus isolates from ZMAC cells did not grow in MARC-145 cells.
• 43 (100%) virus isolates from MARC-145 cells grew in ZMAC cells.
43 PRRSV-2 VI isolates in MARC-145 ZMAC cells

16. Conclusions
• PRRSV was not isolated from any oral fluid samples (Ct 25.8-34) in this study
regardless of using ZMAC or MARC-145 cells.
• Higher success rate of PRRSV isolation in ZMAC from serum and lung samples with
CT<30 compared to MARC-145
• For samples with VI positive in both MARC-145 and ZMAC cells, majority of the
isolates had similar titers.
• Not all of the PRRSV isolates obtained in ZMAC cells can grow in MARC-145 cells.
• ZMAC cell line provides an additional tool for PRRSV VI and characterization.

17. 1. Veterinarian submits diagnostic samples to the ISU VDL
2. PRRSV positive detection through RT-PCR
– Veterinarian requests sequencing
– Veterinarian requests virus isolation
3. Virus isolation performed in MARC-145 cells
– Positive status confirmed by IFA and sequencing
– Negative status: VI performed in ZMAC cells
4. Virus isolation in ZMAC cells
– Cases that were PRRSV VI negative in MARC-145 cells
– Requested by veterinarian regardless of PRRSV VI in MARC-145 cells or not
– Positive status confirmed by RT-PCR and sequencing
– Negative status, second passage in ZMAC; if negative again the case is closed
ISU VDL: Diagnostic Submission Flow

18. 5. Veterinarian requests to forward isolate to Diamond Labs
6. Diamond Labs documentation
- Veterinarian must sign and submit Transfer Form to ISU VDL
7. Isolate tested for purity
- An aliquot of the isolate is confirmed negative for pathogens below by PCRs
• Influenza A virus, porcine circovirus type 2
• M. hyopneumoniae, M. hyorhinis, M. hyosynoviae
- Aptimmune is responsible for diagnostic expenses
8. ISU VDL documentation
- Material transfer agreement signed by Diamond Labs
ISU VDL: Isolate Submission to Ancillary Lab
PRRSV isolate is forwarded to Diamond Labs

19. Acknowledgements
Iowa State University
• Wannarat Yim-Im
• Dr. Phil Gauger
• Dr. Rodger Main
• Dr. Karen Harmon
• Dr. Jie Park
• Haiyan Huang
Aptimmune Biologics
• Dr. Gabriela Calzada
• Dr. Steve Berger
• Aaron Gilbertie
Funding:
• American Association of Swine
Veterinarians Foundation
Fellowship:
• W. Yim-im sponsored by CPF (Thailand) PCL.