DWV infection and replication at the early stagein vitrousing honey bee pupal cells

Abstract

Deformed wing virus (DWV) has been best characterized among honey bee viruses; however, very little is known about the mechanisms of viral infection and replication due to the lack of honey bee cell lines. To resolve this problem, we established in vitro system to reconstitute DWV binding and entry to the host cell followed by translation of the genome RNA and the polyprotein processing with honey bee pupal cells. Using this system, P-domain of VP1 was found to be essential for DWV infection/replication but not binding/entry to the cell. DWV efficiently infects/replicates in cells derived from early but not late pupa, suggesting that the undifferentiated cells are targeted for the viral infection/replication. Furthermore, we found that inhibitors for mammalian picornavirus 3C-Protease, Rupintrivir and Quercetin suppress DWV infection/replication, indicating that this in vitro system is also useful for screening a compound to modify the viral infection/replication. Our in vitro system should help to understand the mechanisms of DWV infection and replication at the early stage.ImportanceRecent decline of managed honey bee colonies has been driven by the pathogens and parasites. However, studying the mechanisms of pathogen infection and replication in honey bee at molecular and cellular levels has been challenging. DWV is the most prevalent virus in honey bee across the globe and we established in vitro system to reconstitute the viral infection and replication with the primary pupal cells. Using RNA-dependent RNA polymerase (RdRP) and the negative strand of DWV genome RNA as markers, we show that the pupal cells can support DWV infection and at least replication at the early stage. The results shown in this report indicate that our in vitro system helps to uncover the mechanisms of DWV infection and replication. Furthermore, it is also feasible to conduct a large scale screening for compounds to inhibit or stimulate DWV infection/replication.

DWV Infection in vitro Using Honey Bee Pupal Tissue

“…The head was sagittally dissected to approximately half and each tissue was suspended in 100 μL of Grace medium containing 10% FBS and antibiotics (penicillin and streptomycin) with or without DWV (9.4 × 10 8 copy) in 24-well plate at 33°C for 1 h. Fresh culture medium (400 μL) was then added and incubated at 33°C for 16 h. DWV was pre-incubated with either anti-VP1 P-domain ( Wu et al, 2020a ) or anti-VP1 (524–750) antibody at the indicated amount of protein for 30 min, and then added to the pupal head tissue for infection as above. The dissected pupal head tissue was pre-incubated with either purified VP1 P-domain protein ( Wu et al, 2020a ) or BSA at the indicated amount of protein in Grace culture medium at room temperature for 1 h. DWV was then added for infection as above. Rupintrivir, Quercetin, or DMSO was added to the pupal head tissue at the indicated concentration together with DWV as above.…”

“…After centrifugation, the supernatants were applied to 10% SDS-PAGE and the proteins were transferred to a nitrocellulose membrane (Pall ® Life Sciences). The membrane was then blocked with PBST (PBS with 0.1% Tween-20) containing 5% BSA at room temperature for 1 h followed by incubating with 1,000-fold diluted anti-RdRP antibody ( Wu et al, 2020a ) at 4°C overnight. The membrane was washed three times with PBST (5 min each), and then incubated with 10,000-fold diluted IRDye ® 680RD donkey anti-rabbit IgG (H+L) (LI-COR Biosciences) in PBST containing 5% skim milk at room temperature for 1.5 h. The membrane was washed as above, and then visualized using Odyssey Imaging System (LI-COR Biosciences).…”