3.2 | AcMNPV FP had higher budding efficiency than wt AcMNPV in Hi5 cells
In order to understand why the AcMNPV FP viruses accumulated at such a high budding frequency compared to the wt AcMNPV, we compared the budding kinetics between the AcMNPV FP virus AcP13 and the wt AcMNPV AcP3 in Hi5 cells. We monitored the virus titers of the wt-virus infection in Hi5 cells during the first 72 h. It was found that AcP13 had a higher budding efficiency than that of AcP3. At 72 h post infection, there was a significant 3-fold higher BV titer of AcP13 than AcP3 (Fig. 3A).
3.3 | AcP3-FPSDM delayed FP formation and improved polyhedra production in Hi5 cells. As we displayed that serial passage of AcP3 showed rapid fp25k mutation in Hi5 cells (Fig. 2 B), we then became interested in comparing polyhedra production stabilities between AcP3 and AcP3-FPSDM that eliminated the 2 A7 MNR sites and the 10th TTAA site of the AcMNPV fp25k . We conducted 10 passages of AcP3 and AcP3-FPSDM passages in Hi5 cells. AcP3 showed a continuous reduction of polyhedra yield during the 10 passages, whereas AcP3-FPSDM showed moderate reduction of polyhedra from P0 to P2, but starting at P3 no significant reduction of polyhedra reduction was observed (Fig. 3B and D). The reduction of polyhedra yield of AcP3 at P10 compared to P0 was about 11-fold whereas the reduction of polyhedra yield of AcP3-FPSDM at P10 compared to P0 was about 2.5-fold. (Fig. 3 D).
3.4 | AcP3-FPSDM had more stable virion occlusion than AcP3 during Hi5 cell passage. Since fp25k mutations often lead to reductions in polyhedra yield and virion occlusion [17, 18], and we found polyhedra yield reduced starting at passage 3, we asked the question if there was an accompanied virion occlusion reduction of AcP3-FPSDM. Polyhedra of AcP3 and AcP3-FPSDM from the above polyhedra yield study were used for the comparison of virion occlusion between the two viruses. SDS-PAGE and Western blot were carried out with equal numbers of polyhedra used for each loading. A monoclonal antibody against AcMNPV major capsid protein VP39 was used to detect virion contents inside the polyhedra. We found that the anti-VP39 antibody detected more AcMNPV VP39 (an indication of virions in polyhedra) in the polyhedra of AcP3-FPSDM than that of AcP3 produced in Hi5 cells during passage (Fig. 4 A, B). No virion occlusion reduction of AcP3-FPSDM during the 10 passages conducted in Hi5 cells, whereas about 50% virion occlusion reduction of AcP3 was observed at P10 compared to P0 (Fig. 4 C).
3.5 | RFLP analysis of AcP3-FPSDM genome. In order to find out the genetic causes for the FP formation and polyhedra yield reduction of AcP3-FPSDM passage in Hi5 cells, EcoRI and HindIII RFLP comparison was performed using DNA purified from BVs at P10 and P0. An 8.7 kbp EcoRI fragment of AcP3-P0 appeared shifted to be 11 kbp in AcP3-P10, and a 4.98 kbp HindIII fragment in AcP3-P0 shifted to a 5.3 kbp HindIII fragment in the AcP3-P10 genome (Fig. 5A left panel). To ultimately confirm the copy number of the 287 host DNA insertion in the AcMNPV genome, Southern blot hybridization should provide the answer.