A Bioreactor Method to Generate High-titer, Genetically Stable, Clinical-isolate Human Cytomegalovirus

Abstract

Human cytomegalovirus (HCMV) infection is a major cause of morbidity and mortality in transplant patients and a leading cause of congenital birth defects (Saint Louis, 2016). Vaccination and therapeutic studies often require scalable cell culture production of wild type virus, represented by clinical isolates. Obtaining sufficient stocks of wild-type clinical HCMV is often labor intensive and inefficient due to low yield and genetic loss, presenting a barrier to studies of clinical isolates. Here we report a bioreactor method based on continuous infection, where retinal pigment epithelial (ARPE-19) cells adhered to microcarrier beads are infected in a bioreactor and used to produce high-titers of clinical isolate HCMV that maintain genetic integrity of key viral tropism factors and the viral genome. In this bioreactor, an end-stage infection can be maintained by regular addition of uninfected ARPE-19 cells, providing convenient preparation of 107–108 pfu/ml of concentrated TB40/E IE2-EYFP stocks without daily cell passaging or trypsinization. Overall, this represents a 100-fold increase in gain of virus production of 100-times compared to conventional static-culture plates, while requiring 90% less handling time. Moreover, this continuous infection environment has the potential to monitor infection dynamics with applications for real-time tracking of viral evolution.

A molecular mechanism for probabilistic bet hedging and its role in viral latency

“…Viral recombinants TB40E IE2-YFP, HCMV AD169 pp71-EYFP, pp150-EYFP, and dual-tagged TB40E-IE-mCherry-EYFP have previously been described ( 18 , 30 , 31 , 46 , 47 )—the two-color TB40E-IE-mCherry-EYFP dual-reporter virus was cloned as an intermediate of the previously described CMDR virus ( 18 ). Viruses were propagated in MRC5 human foreskin fibroblast (HFF) cells (American Type Culture Collection; ATCC) and upon infection reaching ∼90% viral cytopathic effect or ∼90% GFP, the culture supernatant was collected and filtered through a 0.2-μm filter.…”

The HSV-1 ICP4 Transcriptional Auto-Repression Circuit Functions as a Transcriptional “Accelerator” Circuit

“…Replication kinetics of the virus were monitored at an early stage of infection in three biological replicates by infecting ARPE-19 cells with HSV-1-ICP4-YFP virus [MOI = 0.05] pretreated 24 h with HMBA (5 mM) or DMSO for three biological replicates in a 48-well plate. Cells were harvested by trypsinization at various time points post infection (0.5, 2, 8, 16, and 24 h), subjected to multiple freeze-thaws, and centrifuged, and the supernatant was used to calculate the virus titer by TCID-50 assay on MRC5 cells, as described previously (Nevels et al, 2004 ; Saykally et al, 2017 ). Titering performed in parallel on Vero cells showed almost identical trends and correlated well with ARPE and MRC5 titering but scaled by a constant value offset (i.e., quantitative, but no qualitative, titer differences were observed between ARPE, MRC5, and Vero).…”