The effect of rolling and orbital motion on herpesvirus replication in tube cultures and shell vials

Abstract

Background: Isolation of viruses from clinical specimens remains a viable diagnostic manoeuvre but positive isolation rates may be low and time to observe cytopathic effects (CPE) may be longer than preferred for optimal management. Both shell vial (SV) centrifugation and rolling of conventional tube cultures (TC) have been used to enhance the isolation process Objectives: To determine the effect of rolling and orbital motion on the replication of herpes simplex virus (HSV) and cytomegalovirus (CMV) TC and SV. Study design: TC were inoculated with HSV or CMV at varying dilutions and subjected to rolling or incubated stationary. Samples were selected to determine the impact of motion on viral yield, time to CPE according to initial multiplicity of infection (MOI) and stage of the infection process. Similar studies were performed with SV centrifuged with virus followed by stationary incubation or not centrifuged but incubated on an orbital shaker. Results: Rolling HSV-infected TC 383 rotations per minute (RPM) (30.2 × g) for 4 days enhanced viral yields by 53-fold over TC without motion. The optimal RPM response for HSV replication occurred at 96 RPM (1.9 × g) where an 89-fold increase in viral yield was detected, P<0.01. One-step growth studies at 0,2 and 96 RPM demonstrated enhancement of HSV replication at 2 and 96 RPM. TC infected with HSV at low MOI and rolled at 96 RPM had more CPE-positive cultures after 1–3 days than controls. Late in the infection process, no differences in CPE-positive were detected between rolled and non-rolled TC. Studies with CMV and rolling at 96 RPM resulted in more positive TC and greater CPE. SV that were not centrifuged but incubated for 16 h on an orbital shaker had significantly more HSV foci than those handled by a conventional SV method. Conclusion: Our studies indicate that orbital motion and motion yielding a force near 2 × g enhances the isolation and growth of HSV and CMV in TC and SV.