Insect-specific ascoviruses with a circular genome are distributed in the USA, France, Australia and Indonesia. Here, we report the first ascovirus isolation from Spodoptera exigua in Hunan, China. DNA-DNA hybridization to published ascoviruses demonstrated that the new China ascovirus isolate is a variant of Heliothis virescens ascovirus 3a (HvAV-3a), thus named HvAV-3h. We investigated the phylogenetic position, cell infection, vesicle production and viral DNA replication kinetics of HvAV-3h, as well as its host-ranges. The major capsid protein (MCP) gene and the delta DNA polymerase (DNA po1) gene of HvAV-3h were sequenced and compared with the available ascovirus isolates for phylogenetic analysis. This shows a close relationship with HvAV-3g, originally isolated from Indonesia, HvAV-3e from Australia and HvAV-3c from United States. HvAV-3h infection induced vesicle production in the SeE1 cells derived from S. exigua and Sf9 cells derived from S. frugiperda , resulting in more vesicles generated in Sf9 than SeE1. Viral DNA replication kinetics of HvAV-3h also demonstrated a difference between the two cell lines tested. HvAV-3h could readily infect three important insect pests Helicoverpa armigera (Hübner), Spodoptera exigua (Hübner) and Spodoptera litura (Fabricius) from two genera in different subfamilies with high mortalities.

During cell infection, the fp25k gene of baculoviruses frequently mutates, producing the few polyhedra (FP) per cell phenotype with reduced polyhedrin (polh) expression levels compared with wild-type baculoviruses. Here we report that the fp25k gene of the model baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), contains two hypermutable seven-adenine (A7) mononucleotide repeats (MNRs) that were mutated to A8 MNRs and a TTAA site that had host DNA insertions, producing fp25k mutants during Sf21 cell infection. The FP phenotype in Sf9 and Hi5 cells was more pronounced than in Sf21 cells. AcMNPV fp25k mutants produced similar levels of polyhedra or enhanced GFP, which were both under the control of the AcMNPV polh promoter for expression, in Sf21 cells but lower levels in Sf9 and Hi5 cells compared with AcMNPV with an intact fp25k gene. This correlated with the polh mRNA levels detected in each cell line. The majority of Sf21 cells infected with fp25 mutants showed high polh promoter-mediated GFP expression levels. Two cell lines subcloned from Sf21 cells that were infected with fp25k mutants showed different GFP expression levels. Furthermore, a small proportion of Hi5 cells infected with fp25k mutants showed higher production of polyhedra and GFP expression than the rest, and the latter was not correlated with increased m.o.i. Therefore, these data suggest that AcMNPV polh promoter-mediated gene expression activities differ in the three cell lines and are influenced by different cells within the cell line.

Members of the family Baculoviridae are insect-specific dsDNA viruses that have been used for biological control of insect pests in agriculture and forestry, as well as in research and pharmaceutical protein expression in insect cells and larvae. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the type species of the family Baculoviridae. During infection of AcMNPV in permissive cells, fp25k mutants are positively selected, leading to the formation of the few polyhedra (FP) phenotype with reduced yield of polyhedra and reduced virion occlusion efficiency, which leads to decreased oral infectivity for insects. Here we report that polyhedra of AcMNPV fp25k mutants produced from different insect cell lines and insects have differences in larval per os infectivity, and that these variations are due to different virion occlusion efficiencies in these cell lines and insects. Polyhedra of AcMNPV fp25k mutants produced from Sf cells (Sf21 and Sf9, derived from Spodoptera frugiperda) and S. frugiperda larvae had poorer virion occlusion efficiency than those from Hi5 cells (derived from Trichoplusia ni) and T. ni larvae, based on immunoblots, DNA isolation and larval oral infection analysis. AcMNPV fp25k mutants formed clusters of FP and many polyhedra (MP) in the fat body cells of both T. ni and S. frugiperda larvae. Transmission electron microscopy revealed that the nature of virion occlusion of AcMNPV fp25k mutants was dependent on the different cells of the T. ni fat body tissue. Taken together, these results indicate that the FP phenotype and virion occlusion efficiency of fp25k mutants are influenced by the host insect cells.

No abstract

No abstract

Background: Insect specific DNA baculoviruses have been widely used for biological control of insect pests to reduce cancers caused by chemical application in agriculture and forestry and as vectors for high yield exogenous protein expression. The most extensively studied and widely used baculovirus is the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) due to its ability to replicate in many permissive cells with high virus titer. During serial passage of AcMNPV in permissive insect cells at high MOI, the fp25k mutates at a high frequency leading to the development of the few polyhedra (FP) phenotype with reduced polyhedra production and few virion occlusions in polyhedra resulting in poor infectivity to insects. Method and results: We used site-directed mutagenesis on wild type AcMNPV (AcP3) fp25k gene to eliminate these hypermutable sites and produced a recombinant virus AcP3-FPSDM and performed restriction fragment length polymorphism (RFLP, as well as Western blot analysis to understand virion occlusion efficacy. We compared fp25k stability between AcP3 and AcP3-FPSDM in Hi5 cells. Our experimental results showed that AcP3-FPSDM performed better in virion occlusion efficiency. Conclusion: We showed that AcP3-FPSDM is a better virus for researchers to apply in agriculture and forestry to reduce chemical usage, thus cancer cases.

No abstract