Peg virus precipitation kit

Live P. monodon samples (~50 g body weight, n = 382), devoid of any WSSV infection, were collected from ponds and kept for acclimatization before the challenge for two days in recirculatory marine aquarium at 23–26 °C temperature and 6–8 gl⁻¹ salinity. Some essential salts were added and the marine aquarium system was standardized to provide optimal conditions in favor of growth and molting of shrimps. For maintaining appropriate healthy natural aquatic environment some chemicals that are widely used in aquaculture farms, viz., PondDtox® and PondProtect® (Novozyme, Europe) were also used in the aquarium. For the challenge experiment WSSV stock solution was prepared using PEG virus precipitation kit from ~48 g of WSSV infected tissue (hepatopancreas and gill) of shrimp (BioVision, USA). The bioassay experiment was performed to determine the virus titer responsible for 50% mortality, which was found at 10⁴ dilution of virus stock. Therefore, 40 μL of 10⁵ dilution of virus solution was injected into the tail muscle of each shrimp (n = 240) and genomic DNA was extracted from tail tissue of shrimps after 72 h of WSSV challenge experiment. Survivability as well as WSSV propagation by real-time PCR was also recorded at post 72 h.

HBV was derived from the supernatants of HepG2.2.15.7 cells^{12 (link)}, which stably expressed the complete HBV genome (genotype D). The collected supernatants were filtered through a 0.45 µm filter (Merck, #SLHV033RB) and concentrated using the PEG Virus Precipitation Kit (BioVision, #K904-50). PHHs in 24-well plates were infected with HBV (500 GEq/cell). Ten days after infection, the culture supernatants were harvested and subjected to quantification of viral DNA and HBcAg, as described above. In experiments using IFN, IFN-β (100 U/mL; Wako, #092-06061) was added to cultures 3 h before infection. Total RNA and viral DNA were extracted with RNeasy mini kit (Qiagen, #74104) and QIAamp DNA Blood Mini kit (Qiagen, #51104), respectively^{12 (link),46 (link)}. Gene quantification was done by real-time PCR as described above. The primer pairs used in this study are listed in Supplementary Table 1.

HBV stocks were derived from the supernatants of HepG2.2.15 cells, which were stably transfected with a complete HBV genome (genotype D). The collected supernatants were filtered through a 0.45-μm filter (Merck Millipore), and concentrated using PEG virus precipitation kit (BioVision, Milpitas, CA). HepG2-Tet-NTCP or its derivative cells in 24-well plates were infected with HBV (5000 GEq/cell) with or without 5 μg/ml Dox. The culture supernatants were then harvested and subjected to quantification of HBsAg and vDNA, as described above.

293 FT packaging cells were transfected with the lentivial vector plenti6.3/V5-MscL-6xHis-tag and lentiviral packaging system which is a mixture of pLP1, pLP2, and pLP/VSVG vectors using Lipofectamine3000 (Invitrogen), 48 h or 72 h later, the supernatant was collected and filtered (Millipore 0.45 µm). After being concentrated by BioVision’s PEG Virus Precipitation Kit (Catalog #K904-50, BioVision), the acquired virus was used for infection of A549 cells in the presence of polybrene (8 μg mL⁻¹, SIGMA, H-9268). After antibiotic selection for three generations (Blasticidin-S, 6 μg mL⁻¹, Solarbio, B9300), the percent of mCherry-expressed cells reached 90%, stable clones were obtained. For the doxycycline inducible lentiviral system, preparation of lentivirus and infection of A549 cells were performed as described above. Stable cell lines of A549 expressing tet-on-MscL were gained by puromycin (1 μg mL⁻¹, Solarbio) screening, the expression of MscL was confirmed by mCherry fluorescence imaging and electrophysiology recording after doxycycline (1 μg mL⁻¹, MB1088, Dalian Meilun) induction.

P0 Huh7 cells (1 × 10⁷ cells/15-cm dish) were transfected with L at 10 μg, VP35 at 0.625 μg, VP30 at 0.625 μg, NP at 5 μg, pMG at 2.5 μg, and T7 at 2.5 μg. At 72 hpt, the supernatant was harvested and clarified by centrifugation at 3,000 rpm for 5 min at 4°C. ptrVLPs were precipitated with a polyethylene glycol (PEG) virus precipitation kit (catalog number K904; BioVision), aliquoted, flash-frozen, and stored at −80°C. To determine the luciferase activity of the ptrVLP stock, P1 Huh7 target cells (3 × 10⁴ cells/well) in a 96-well format were transfected with L at 166 ng, VP35 at 10.4 ng, VP30 at 10.4 ng, and NP at 83.3 ng per well using TransIT-LT1 (Mirus Bio) (3:1 reagent-to-DNA ratio). At 24 hpt, cells were infected in duplicate with a 2-fold dilution series (50 μL to 0.02 μL). Medium was changed to fresh DMEM with 10% FBS at 24 hpi, and luciferase activity was assessed at 72 hpi using a dual-luciferase assay (Promega) and an EnVision plate reader.

HBV was derived from the supernatants of HepG2.2.15.7 cells, which stably express the HBV genome. The collected supernatants were filtered through a 0.45-μm filter (Millipore) and subsequently concentrated with a PEG Virus Precipitation Kit (BioVision, Milpitas, CA, USA). HepG2-hNTCP-C4 cells were infected with HBV (10,000 GEq/cell) in the presence of 4% PEG8000. After 16 h, the medium was replaced by medium containing 2% DMSO. Eight days after infection, the cells or cell lysates were harvested.