Minimal essential media

VZV Parent of Oka (pOka), a varicella isolate that was the parent strain used to derive the current VZV vaccine strain, was used in all this work. pOka VZV was previously shown to induce chronic pain indices in the rat model [15 (link)]. Virus was used at less than 12 passes beyond receipt from M. Takahashi (Osaka University, Japan) and propagated as detailed previously [26 (link), 27 (link)] on the VZV permissive MeWo human cell line (ATCC, Manassas, VA). Cells and virus were grown in Minimal Essential Media (MEM) supplemented with 10% fetal bovine serum (FBS) and antibiotic and antimycotic mixture (Sigma, St. Louis, MO). VZV was prepared to high titer as previously detailed [15 (link)] on confluent monolayers of MeWo cells infected at ~0.1 plaque forming units (pfu)/cell. Virus infected cells incubated at 35 °C for 48–72 h post infection were harvested by trypsin digestion, and optimal VZV cell viability was maintained by slow freezing aliquots in MEM containing 20% FBS and 10% dimethyl sulfoxide (Sigma, St. Louis, MO), followed by storage under liquid nitrogen. Virus titers of frozen stocks were assessed by an infectious center assay. Uninfected cell equivalents were prepared using similar treatments. All virus and cellular stocks were provided by Dr. Kinchington’s laboratory.

The rainbow trout gill (RTGill-W1) cell line and the rainbow trout liver (RTL-W1) cell line were a gift from Sylvie Bony from the University of Lyon, France. CTV (Heenan88 isolate) was kindly provided by Yannik Debing from the Rega Institute for Medical Research, Department of Microbiology and Immunology, Leuven, Belgium. The cells were maintained in Leibovitz’s L-15 media (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal calf serum (Amimed, London, UK) and 50 U/mL penicillin/streptomycin (Biochrom/Millipore, Berlin, Germany) at 18 °C. For virus infection, 4 × 10⁴ cells were added in 200 µL of medium to a 96-well plate (TPP, Trasadingen, Switzerland). Once confluent, the cells were inoculated overnight with 4 × 10⁶ genome equivalents in 50 µL Minimal Essential Media (MEM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 2% heat-inactivated fetal calf serum, and 50 U/mL penicillin/streptomycin. At 5 h post-infection, the cells were washed three times with MEM and then incubated in MEM at 18 °C over a period of several weeks.