CELL-BASED SCREENING ASSAY FOR INHIBITORS OF PORCINE CIRCOVIRUS TYPE (PCV2) REPLICATION CARLA BIANCA LUENA VICTORIO YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE & SWISS TROPICAL AND PUBLIC HEALTH INSTITUTE UNIVERSITY OF BASEL 2010 CELL-BASED SCREENING ASSAY FOR INHIBITORS OF PORCINE CIRCOVIRUS TYPE (PCV2) REPLICATION CARLA BIANCA LUENA VICTORIO (BSc. Molecular Biology and Biotechnology) University of the Philippines Diliman A THESIS SUBMITTED FOR THE DEGREE OF JOINT MASTER OF SCIENCE IN INFECTIOUS DISEASES, VACCINOLOGY AND DRUG DISCOVERY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE & SWISS TROPICAL AND PUBLIC HEALTH INSTITUTE UNIVERSITY OF BASEL 2010 Acknowledgements This research project wouldn’t have been possible without the support and funding from Temasek Life Science Laboratories (TLL) and the expertise of the researchers at the Animal Health Biotechnology Group headed by Prof. Jimmy Kwang. My special thanks to my supervisor, Prof. Kwang, co‐ supervisor, Prof. Justin Chu, and mentor Mr. Anbu Karuppannan for giving me guidance and direction in this research endeavor. This Joint Msc program had been a wonderful, albeit stressful, experience and I am grateful for this opportunity bestowed to me by Novartis Institute of Tropical Diseases (NITD), Swiss Tropical and Public Health Institute (STPH), and National University of Singapore (NUS). My special thanks to Ms. Christine Mensch for all the much‐needed assistance during my stay in Basel, and to Ms. Susie Soh for the constant gentle reminders in Singapore. I would like to acknowledge the program lecturers, most especially Prof. Reto Brun, for providing the inspiration to delve into the field of drug discovery. To the JIBES, thank you for making life outside the lab and lecture memorable. To Patricia, who has been my life raft these past 2 years; to Casey, Mad, Ed, Sukriti, Hanwern, Neisha, and Ashley, thank you for all the fond memories. I wish to express my heartfelt thanks to the people of TLL who showed me how to make stress a bearable part of daily life. To Shaz, Peypey, Lu ting, Keiko, Adi, Ivan, and Ranjay, thanks for all your support; and to Vin, who has been my source of respite during the crucial moments, thank you for the companionship and strength. To my friends back home in the Philippines and in Basel, who still give me constant emotional support, thank you for all your wonderful efforts. Lastly, I would like to express my whole‐hearted gratitude to my family for being my constant refuge. Thank you for the selfless love, caring support, and understanding and for allowing me to pursue my dreams independently. Cell‐Based Screening Assay for Inhibitors of Porcine Circovirus Type 2 (PCV2) Replication Page | 1 Table of contents Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 List of Tables and Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 List of Abbreviations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 1. Introduction 1.1. Porcine Circoviruses (PCV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 1.1.1. PCV Taxonomy, Morphology and Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 1.1.2. Pathogenesis and Replication Cycle of PCV2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 1.2. PCV2‐Associated Diseases (PCVAD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 1.2.1. Postweaning Multisystemic Wasting Syndrome (PMWS) . . . . . . . . . . . . . . . . . . . 17 1.2.2. PDNS and other PCVAD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 1.2.3. Treatment of PCVAD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 1.3. Assay Development and Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 1.3.1. Cell‐based and Cell‐free Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 1.3.2. Signal Detection Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 1.3.3. Assay Development for HTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 1.4. Objectives of the Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 2. Materials and Methods 2.1. Maintenance of cell lines 2.1.1. Culturing PK15‐C1 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 2.1.2. Culturing 3D4/31 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 2.1.3. Culturing #4 Clone Hybridoma Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 2.1.4. Cryopreservation of Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 2.1.5. Establishment of Cell Growth Curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Cell‐Based Screening Assay for Inhibitors of Porcine Circovirus Type 2 (PCV2) Replication Page | 2 2.2. Production of High Titer PCV2 Stock 2.2.1. Infecting Cells and Harvesting Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 2.2.2. Increasing PCV2 Titer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 2.2.3. Detection of Infection by Western Blot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 2.2.4. Detection of Infection by PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 2.2.5. Virus titration by IFA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 2.3. Cell‐Based Screening Assay Optimization 2.3.1. Downscaling IFA to 384‐well Plate Format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 2.3.2. Cell‐Based ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 2.4. Testing Efficacy of Reference Drugs Against PCV2 Replication 2.4.1. Establishment of Standard Curve for FI at Various . . . . . . . . . . . . . . . . . . . . . . . 34 Seeding Densities 2.4.2. Evaluating Drug Cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 2.4.3. Inhibition of PCV2 Replication with Reference Drugs . . . . . . . . . . . . . . . . . . . . . . 35 2.5. Generation of Graphs and Statistical Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 3. Results 3.1. Preparation of materials needed for the screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 3.1.1. Finding the best cell line for the screening assay . . . . . . . . . . . . . . . . . . . . . . . . 37 3.1.2. Growth dynamics of PK15‐C1 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 3.1.3. Generation of high titer PCV2 stock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 3.1.4. Large‐scale production of monoclonal antibodies . . . . . . . . . . . . . . . . . . . . . . . . . 40 (clone #4) 3.2. Scaling down of Assay to 384‐well plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 3.2.1. Comparison of infection rates between glucosamine‐treated . . . . . . . . . . . . . . . 42 and untreated cells 3.2.2. Determining optimum cell seeding density, MOI, and . . . . . . . . . . . . . . . . . . . . . 42 duration of infection Cell‐Based Screening Assay for Inhibitors of Porcine Circovirus Type 2 (PCV2) Replication Page | 3 3.2.3. Infection at higher MOI to induce 50% infection rates. . . . . . . . . . . . . . . . . . . . . . 47 3.2.4. Comparison of FITC with Alexa Fluor 546 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 3.3. Testing efficacy of reference drugs against PCV2 replication . . . . . . . . . . . . . . . . . . . . . .50 3.3.1. Standardizing the alamar blue cytotoxicity assay. . . . . . . . . . . . . . . . . . . . . . . . . . 50 3.3.2. Determining cell tolerance for CAPE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 3.3.3. Efficacy of CAPE against PCV2 replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 3.3.4. Development of screening assay using cell‐ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 6.1. Effect of glucosamine treatment on infection rates at . . . . . . . . . . . . . . . . . . . . . . . . . . 79 various seeding densities 6.2. Standard curves for FI and absorbance with alamar blue . . . . . . . . . . . . . . . . . . . . . . . . 82 Cell‐Based Screening Assay for Inhibitors of Porcine Circovirus Type 2 (PCV2) Replication Page | 4 Summary PVC2 is a small non‐enveloped virus that causes a wide array of porcine diseases categorized under the umbrella term PCV‐Associated Diseases (PCVAD). To date, the only available antiviral strategy, albeit ineffective against diseased pigs, is prevention via vaccination. Thus, treatment of affected pigs requires discovery of drugs that inhibit viral replication. The focus of this MSc project was to develop a suitable primary screening assay for inhibitors of PCV2 replication and subsequently perform a proof of concept trial using reference drugs. PK15‐ C1, a cell line previously shown to be highly permissive to PCV2 infection (Zhu et al., 2007), was used in the study to grow PCV2 to a high titer (106 TCID50/ml) and exhibited infection rates > 50% at 15 MOI in a 96‐well plate format. The assay was subsequently scaled down to 384‐well plates for better amenability to HTS, and the major part of the study was aimed at optimizing this. Infection with PCV2 was done both at low ( 10) MOI but neither succeeded in inducing minimum of 50% infection rate. Even with proof of concept trials performed in 96‐well plates employing reference drugs CAPE, U0126, and MPA at 15 MOI resulted to infection rates lower than 50%. This sudden and unexpected drop in infectivity precluded further testing, so a cell‐based ELISA, which does not require minimum infection rate, was tested instead. Assay sensitivity was assessed by S/N and S/B ratios. Although S/N ratios were promising, S/B values were