Utilization of Vero Cells for Primary and Chronic BK Virus Infection

Abstract

BK virus (BKV) nephropathy has a poor prognosis for renal allograft survival with 30% to 60% risk of allograft loss over 1 year. In the past decade, BKV nephropathy has occurred in 1% to 10% of renal transplant patients, with higher rates observed in patients with increased immunosuppression exposure and renal allograft injury. Vero cells (Green monkey kidney cell origin) were optimized for BKV primary and chronic infection inclusive of culture requirements for 60-day growth and monolayer confluence. Quantification of BKV replication in the culture supernatant (SN) and cells was by real-time polymerase-chain reaction (PCR) using the Roche Lightcycler 2.0. Primary BKV infection of Vero cells is achieved by 2 hour incubation with 6.5 × 10 5 BKV copies with subsequent washing of cells leading to steady-state cellular infection of 10 2 to 10 3 BKV copies. Primary infection is demonstrated within 7 to 10 days by a >10-fold increase of BKV copies in SN. Thereafter, a BKV viral load reduction in SN to a chronic/latent level (<10 2 BKV copies in SN) is observed by 14 days. Vero cells with chronic low-level BKV infection (10 2–10 3 BKV copies in cells) exhibited reactivation (>10 5 BKV copies in SN) in >72% of late culture wells after 40 days. Vero cells can accommodate primary and chronic BKV infection followed by viral reactivation in late culture. The performance characteristics of 3 different pathogenic BKV strains obtained from patients with BKV nephropathy had infectivity profiles that correlated well the relative clinical profile in this Vero cell culture system.