▼ Baculoviruses are promising for bio-control of insect pests in agriculture and have been used for exogenous protein expression. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has been widely studied for baculovirus early and late gene transcription in insect cell lines such as Sf21, Sf9 derived from Spodoptera frugiperda and High Five™ (Hi5) from Trichoplusia ni. AcMNPV expresses high levels of the polyhedrin (polh) protein which can occlude virions into polyhedra for protection from UV irradiation in the field. The AcMNPV fp25k gene is prone to mutation during high multiplicity of infection (MOI) of insect cells and fp25k mutation can lead to the development of few polyhedra (FP)/cell phenotype in contrast to a multiple polyhedra (MP)/cell phenotype of the wild type fp25k mutant viruses have been reported to have reduced polyhedrin expression and reduced virion occlusion which leads to low expression of foreign gene, low yield and poor quality of bio-insecticide. Here, we found that AcMNPV fp25k contains two 7 adenine (A7) mononucleotide repeats (MNRs) that were mutated to A8 MNRs and the 10th TTAA site contains a 287 bp cellular DNA insertion during passage in Sf21 cells to form fp25k mutants. Sf9 and Hi5 cell lines infected with the mutant viruses only showed the typical FP whereas many Sf21 cells showed MP. The mutant viruses had reduced polh mRNA and protein expression levels in Sf9 and Hi5 but not in Sf21 cell line. Clusters of MP were observed in the fat body cells of T. ni and S. frugiperda larvae infected with mutant viruses. Although MP were produced in both insects, virion occlusion occurred in T. ni larval fat body and Hi5 cells but not in S. frugiperda fat body, Sf21 and Sf9 cell lines. Transmission electron microcopy revealed that some fat body cells of T. ni larvae produced MP filled with virions. Therefore, insect cells that showed better protein expression and/or virion occlusion should be cloned for improved viral insecticide development and for improved protein expression. Further evidence demonstrated that elimination of the mutation sites of AcMNPV fp25k delayed FP development and improved polyhedra production for better viral insecticide production. Advisors/Committee Members: Cheng, Xiao-Wen.