Autographa californica multiple nuclear polyhedrosis virus

For the generation of recombinant baculoviruses was used the expression vector pFastbac™1 of Bac-to-Bac® baculovirus expression system (Thermo Fisher, USA, cat. no.10359–016). Under the promoter of polyhedrin (polh), the genetic sequence of the first 330 bp of the N-terminal region of the polyhedrin was cloned and the genetic sequence of the GFP was ligated into its C-terminal in an open reading frame to generate a fusion protein called PH_(1–110)GFP [28 (link)]. The polh promoter and the polyhedrin sequence were taken from Autographa californica multiple nucleopolyhedrovirus virus (AcMNPV). For the generation of the PH-WT-GFP chimeric polyhedra, the pFastbac™ Dual expression vector (Thermo Fisher, USA, cat. No.10712024) was used, the WT polyhedrin was cloned under the p10 promoter and the PH_(1–110)GFP under polh promoter. The baculoviruses were amplified, purified and titrated by following the recommendations and protocols provided by the supplier (Thermo Fisher, USA).

Insects used in this report included three species, S. exigua, H. armigera, S. litura. Except H. armigera that was provided by Dr. Xuelian Sun of Wuhan Institute of Virology, the other two species were collected from cotton fields. Insects were reared on artificial diets in incubators with temperature at 27±1°C, relative humidity 70-80% and photoperiod (L/D, 16∶8). Insect cells included SeE1 received from Dr. A. H. McIntosh of Biological Control of Insects Research Laboratory, Missouri [32] and maintained in HyQ® SFX-Insect MP™ (HyClone, Utah) serum free media supplemented with 5% fetal bovine serum [32] and Sf9 purchased from Invitrogen (Carlsbad, California) and maintained in TNM-FH supplemented with 10% fetal bovine serum. Insect cells were cultured in an incubator with temperature controlled at 27±0.5°C. Viral DNA included Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) E2, SfAV-1a, HvAV-3g, HvAV-3f and TnAV-2d prepared previously in the lab [27] (link), [33] (link). HvAV-3h virions were purified from hymolymph of S. exigua larvae infected with HvAV-3h in this report.

All plasmids used in this study were constructed using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, Whitby, ON, Canada) according to manufacturer’s directions. Primers used for construction of all plasmids were synthesized by Integrated DNA Technologies (IDT; Coralville, IA, USA) and are given in Table 1. The spacer sequences for the sgRNA are given in Table 2.
The plasmid p6.9GFP-sgRNA, which encodes the p6.9GFP reporter cassette and SfU6-sgRNA for targeting Cas9, has been described previously [10 (link)]. Briefly, to construct the p6.9-GFP-encoding CRISPR transfer plasmids, first the coding region of the p10 gene, including upstream and downstream sequences to include its endogenous promoter and 3’ UTR, was amplified from AcMNPV genomic DNA and inserted into pACUW51. The p10 ORF was then replaced with the gfp gene, and the SfU6-sgRNA fragment was inserted downstream to derive p10GFP-sgRNA. Finally, the p6.9 promoter region was amplified from AcMNPV genomic DNA and inserted in place of the p10 promoter sequence in p10GFP-sgRNA to yield p6.9GFP-sgRNA. Inverse PCR was used to exchange the spacer sequence region on plasmid p6.9GFP-sgRNA with those specific to the gp64 or vp80 ORF [11 (link)]. To generate the transfer plasmids encoding the HIV-1 gag gene, the gfp ORF was replaced with the gag gene from the plasmid pAdCMV5-gagGFP [12 (link)] using PCR and NEBuilder HiFi DNA Assembly as described previously [10 (link)].

The Spodoptera frugiperda-derived SF-21 cells were cultured at 27 °C in Grace’s Insect Medium (Invitrogen), containing 10% fetal bovine serum, penicillin G (60 μg/mL), and streptomycin sulfate (200 μg/mL). The Spodoptera frugiperda-derived Sf9 (clonal isolate 9 from cell line IPLB-SF21-AE) cells were cultured at 27 °C in TC-100 medium (Gibco), containing 10% fetal bovine serum (Atlanta Biologicals/R&D Systems, Minneapolis, MN), penicillin G (60 μg/mL), streptomycin sulfate (200 μg/mL), and amphotericin B (0.5 μg/mL). The E2 strain of AcMNPV was the parental strain, from which AcEGFP was derived by homologous recombination, as previously described [19 (link)]. The single chiA-reprogrammed virus Acp6.9-chiA/v-cath (abbreviated Acp6.9-chiA hereafter), AcMNPV-repaired with its native intergenic chiA/v-cath promoter sequence restored (simply called AcMNPV-Rep hereafter), and the dual chiA/v-cath reprogrammed virus Acp6.9-chiA/polh-cath, with the p6.9 promoter driving chiA and the polh promoter driving v-cath, were all derived from AcEGFP by homologous recombination, as described elsewhere [19 (link)]. Briefly, the AcMNPV-Rep virus (i.e., a virus in which the native 45 bp intergenic chiA/v-cath promoter region was swapped with the polh-egfp cassette in AcEGFP) was derived from AcEGFP, using a donor plasmid (pBSK.CCnative; [21 (link)]) and methods [19 (link)] described earlier. The polh-promoter-based v-cath-reprogrammed gene construct was isolated as an XbaI/EcoRI fragment from the pBSK.chiA/ph-CA plasmid [17 (link)] and cloned into pBSK.p6.9.chiA/v-cath [19 (link)] to produce pBSK.p6.9-chiA/polh-cath. The pBSK.p6.9-chiA/polh-cath plasmid was co-transfected with AcEGFP genomic DNA into SF-21 cells, and Acp6.9-chiA/polh-cath was isolated, and the intergenic promoter sequences and genome structures were assessed, as described previously [19 (link)]. Budded viruses were replicated and titrated by end-point dilution [22 (link)], using SF-21 cells (when used for cell culture experiments) or Sf9 cells (when used for insect experiments).
T. ni larvae (3rd instar) were obtained from Benzon Research (Carlisle, PA) and synthetic insect diet was from Southland Products, Inc. (Lake Village, AR). Viruses were constructed in complete (with antibiotics and 10% serum) TC-100 medium. Fifth instar T. ni larvae were injected, using a gauge 22 Hamilton syringe, with 10 µL of virus suspensions, containing 5 × 10⁶ TCID₅₀ units per µL (total dose of 5 × 10⁴ TCID₅₀ units) of each virus and placed in individual containers containing artificial diet and maintained at 27 °C. Control insects were injected with 10 µL of complete TC-100 growth medium lacking virus. Insects were assessed every 8 h for responsiveness to prodding with a blunt glass rod, in order to determine the time of death. To assess insect liquefaction, insects were inspected and photographed every 24 h. Insects were maintained at 27 °C on a weighing dish, set in a sealed plastic container humidified with a damp tissue and were photographed daily. Experiments to assess insect pathology and mortality were repeated three times. Insects that died or pupated within the first 48 h were not included in the studies; about thirty larvae per virus/treatment were analyzed in each replicate. Insects were photographed with an Olympus digital camera, model F4000. Larval survival data were analyzed with GraphPad Prism 9.2.0.

Recombinant baculoviruses expressing influenza NA from A/California/07/09 (H1N1)pdm09 (N1-Bac) were prepared and propagated in the Sf21 insect cell line. Specifically, a synthetic DNA sequence encoding the transmembrane domain and ectodomain of A/California/07/09 (H1N1)pdm09 NA, followed by a hexameric Histidine tag, was inserted into a pAB-pEG-hhp10-HMc-6MC vector [14 (link)], from which the 6H-6MC vector element had been removed, with the NA sequence placed downstream of the honeybee melittin (HM) signal peptide. Recombinant AcMNPV (i.e., NA-Bac) was generated by co-transfecting the expression vector plasmids with a modified baculovirus genomic DNA, flashBac^TM ULTRA (Mirus Bio LLC, Madison, WI, USA), into Sf21 cells by TransIT^®-Insect Transfection Reagent (Mirus Bio LLC, Madison, WI, USA). The resulting recombinant baculovirus was amplified in the Sf21 cells and isolated through end-point dilution, as described previously [14 (link),15 (link)]. We used a wild-type baculovirus (wt-Bac) lacking recombinant protein expression, which has been well characterized in previous studies as a negative control [13 (link),15 (link)].