Cell-Based screening assay for inhibitors of porcine circovirus Type2 (PCV2) replication

Abstract

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) and high (> 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 < 2 and sensitivity was found insufficient for further assay development. This was probably due to low infection rates (< 5%) resulting from stringent blocking and washing, which were necessary to reduce background signals in ELISA. Thus, significantly improving infection rates above 50% is necessary to optimize these cell-based screening assays for inhibitors of PCV2 replication.