Cells, viral strains and plasmids

The Sf9 insect cell line, originally obtained from the fall armyworm Spodoptera frugiperda, was cultured at 27 ℃ in IB serum-free cell culture medium (YSK BIO, Zhejiang, China). AcMNPV was used as the parent virus in this study. The modified Bac-to-bac baculovirus expression system was obtained from Prof. Zhihong Hu of the Wuhan Institute of Virology, Chinese Academy of Sciences. The chemically competent E. coli AcBac-syn carries the helper plasmid and the AcMNPV bacmid (shuttle vector). The donor plasmid pBacT contains three different promoters vp39, p6.9 and op166.

Preparation of recombinant baculoviruses

The recombinant AcMNPVs were generated by following the manufacturer’s instructions for the Bac-to-Bac® baculovirus expression system. The pBacT was used as the backbone to construct the donor vectors. The bacmids were then purified and used to transfect one million Sf9 cells in the 12-well plate by using Cellfectin II Reagent (Thermo Fisher Scientific). After a 4-day incubation at 27 ℃, the cell culture supernatant was collected and centrifuged at 500 × g for 10 min to obtain the passage zero (P0) recombinant AcMNPVs.

Virus titer determination and gene copy number determination

For virus titer determination, we targeted the DNA polymerase (DNApol) gene (a conserved and abundant baculoviral marker), along with two exogenous genes (eGFP and EV71-P1), using qPCR with specific primers. The standard curve for qPCR was established using serial dilutions of known concentrations of Bacmid containing the DNApol, eGFP, or EV71-P1 gene. Cycle threshold (Ct) values obtained from the qPCR reactions were compared to the standard curve to calculate the gene copy numbers. The ratio of the eGFP gene or EV71-P1 gene relative to the DNApol gene in each passage of the virus was further calculated. Briefly, Total viral DNA was prepared from the supernatants of each passage using the FastPure Viral DNA/RNA Mini Kit V2 (Vazyme, Nanjing, China). The qPCR was conducted with ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The primers designed for DNApol, eGFP and EV71-P1 were as follows: DNApol-F, 5’-CAATGCCTAGCCACCGTAAT-3’; DNApol-R, 5’-AATCGTTGACCGACTACAGC-3’; eGFP-F, 5’-TATCATGGCCGACAAGCAGA-3’; eGFP-R, 5’-ATGCCGAGAGTGATCCCG-3’; EV71-F, 5’-GGACCTTGAATACGGAGCCT-3’; EV71-R, 5’-GAGTCGTGATGGCAGTTCG-3’.

Gene expression detection

Expression levels of DNApol and lef5 genes of the recombinant viruses were analyzed at days 1,2 and 3 post-infection. The total RNA was purified by FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme, Nanjing, China). Reverse transcription was then conducted with the HiScript II Q RT SuperMix for qPCR (Vazyme, Nanjing, China). SYBR Green-based qPCR was performed using Pro Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). Tubulin gene expression from Sf9 cells was used as a reference. The primers designed for tubulin, DNApol and lef5 were as follows: tubulin-F, 5’-GGGCATGGACGAGATGGA-3’; tubulin-R, 5’-GGACACCAGGTCGTTCATGTT-3’; DNApol-F, 5’-CAATGCCTAGCCACCGTAAT-3’; DNApol-R, 5’-AATCGTTGACCGACTACAGC-3’; lef5-F, 5’-CCATTTGCTTTGAAGCGAGG-3’; lef5-R, 5’-GCGCTCTTTCACCAAATCAC-3’. Gene expression was calculated relative to tubulin using the formula 2[Ct (β−tubulin) – Ct (DNApol/lef5)]. The relative expression of lef5 to DNApol was further calculated.

Cell infection and serial passages

The titers of the P0 generation viruses were determined using qPCR to ensure successful rescue and nearly identical titers of each recombinant AcMNPV. Sf9 cells were seeded in 6-well plates at a density of 2 × 106 cells per well. In the first experimental setup, cells were infected with recombinant viruses using a volume ratio of 0.5%. After incubation at 27 °C for 4 days, samples were collected. The medium containing multiplied viruses was centrifuged at 3000 rpm for 5 min to remove cells. Budded viruses (BVs) in the supernatant were stored at 4 °C for viral quantification and subsequent infection passages. A total of 15 infection passages were completed for each recombinant AcMNPV, using two independently rescued viruses per recombinant. An additional experiment was conducted using a volume ratio of 2.5%, following the same methodology.

Immunofluorescence Assay (IFA)

Sf9 cells were cultured in 24-well plates and inoculated with recombinant AcMNPVs. For immunofluorescence assay, cells were fixed with 4% paraformaldehyde in PBS for 30 min. Subsequently, cells were washed with PBS and permeated with 0.2% (wt/vol) Triton X-100-PBS solution for 10 min at room temperature. Then, cells were washed and blocked with 3% bovine serum albumin (BSA) in PBS at 37℃ for 30 min. After washing, cells were incubated with rabbit anti-EV71 polyclonal antibody for 2 h at 37℃, followed by Alexa Fluor 594-conjugated donkey anti-rabbit IgG H+L for 1 h at 37°C.

Western blotting

Cells were harvested and lysed in SDS lysis buffer for 30 min on ice, followed by centrifugation at 14,000 × g for 15 min at 4°C. The supernatant was boiled in 1 × loading buffer at 100°C for 10 min and separated through a 4 to 12% gradient gel sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred onto a nitrocellulose membrane and incubated with rabbit anti-EV71 polyclonal antibody or mouse anti-GP64 monoclonal antibody after blocking with 5% BSA in PBST. Proteins were detected using horseradish peroxidase-conjugated secondary antibodies and chemiluminescence.

Statistical analysis

Graphs were plotted and analyzed by using GraphPad Prism 8.0 software (GraphPad, CA, USA). Experimental data are presented as the mean ± standard deviation (SD) for a minimum of three biological replicates. The study utilized an unpaired t-test to examine the significant differences between each group. Differences were denoted by *, p < 0.05; **, p < 0.01 and ***, p < 0.001.