Tnm fh medium

AnpeNPV L2 (GenBank accession number: EF207986) was propagated in A. pernyi pupae and viral DNA was extracted as previously described (Fan et al. 2007 (link)). Tn-Hi5 cells (BTI-TN-5B1-4) purchased from Thermo Fisher Scientific (Waltham, MA) were cultured in TNM-FH medium (GE Healthcare Life Sciences, Chicago, IL) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco, Billings, MT) containing 0.5% penicillin–streptomycin solution (Gibco) at 27°C. Restriction enzymes were purchased from TaKaRa (Dalian, China).

Alphanodavirus-free Tn-Hi5 cell line (BTI-TN-5B1-4) was purchased from Thermo Fisher Scientific (United States). Cells were cultured in TNM-FH medium (GE Healthcare Life Sciences) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco) containing 0.5% penicillin-streptomycin solution (Gibco) at 27°C. ApNPV-Δph/egfp⁺ (Wang et al. 2010 (link)) was propagated in A. pernyi pupae and used to infect the cell line. Viral titer was determined by an end-point dilution assay with Tn-Hi5 cells according to a published method (Cha et al. 1997 (link)). For the infection, 1 × 10⁶ Tn-Hi5 cells were seeded into wells of a 6-well tissue culture plate (Falcon) and infected with ApNPV-Δph/egfp⁺ (multiplicity of infection: 10). The ApNPV-Δph/egfp⁺ inoculum was removed after 1 h. The cells were rinsed with SF-900TM II medium (Gibco) and cultured in TNM-FH medium supplemented with 10% FBS at 27°C. The time at which the inoculum was removed was considered as 0 hpi. Cells were collected at 6, 12, 18, 24, 36, and 48 hpi. Uninfected cells (mock infection) were collected at 48 h as the control. Three independent biological replicates were prepared at each time point.

Antheraea pernyi pupae were obtained from the Sericultural Research Institute of Liaoning Province (Fengcheng, China). AnpeNPV L2 (GenBank: EF207986) and a linearized derivative of AnpeNPV (Zhao et al. 2020 ) were available in our lab and propagated in A. pernyi pupae. The AnpeNPV genomic DNA was extracted as per previously described methods (Fan et al. 2007 (link)). Tn-Hi5 cells (BTI-TN-5B1-4) were purchased from Thermo Fisher Scientific (Waltham, MA) and cultured in TNM-FH medium (GE Healthcare Life Sciences, Chicago, IL) supplemented with 10% (v/v) fetal bovine serum (Gibco, Billings, MT) containing 0.5% penicillin–streptomycin solution (Gibco) at 27°C. SF-900 II serum-free medium (Cat. no. 10902-088) and Cellfectin II (Cat. no. 10362100) were purchased from Thermo Fisher Scientific.