Recombinant baculovirus genome that absence part of ORF1629 has used to enhance efficiency of recombinant virus selection because a role of ORF1629 is believed to essential for viral replication in insect cells. It can be recovered by recombination with a transfer vector containing a complete ORF1629. Some recombinant bacmids were generated for this purpose. Recombinant bacmid GOZA, one of them, has a mini-F replicon for Escherichia coli and the partial ORF1629 gene. By accident, we could observe the replication of ApGOZA singly in Sf9 cells. Produced virus from ApGOZA was investigated for the existence of truncated ORF1629 not intact ORF1629 by PCR amplification and genomic sequencing. Also, we could observe the replication of AcGOZA alone in Sf9 cells. To analyze the influence of truncated ORF1629, the viral growth, BV production and polyhedral formation were conducted comparing to wild type virus and recombinat virus containing intact ORF1629. The results suggested the probability that ORF1629 is not essential for replication.

ORF78 (ac78) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a baculovirus core gene of unknown function. To determine the role of ac78 in baculovirus life cycle, an ac78-deleted mutant AcMNPV, Ac78KO, was constructed. Quantitative PCR analysis revealed that ac78 is a late gene in the viral life cycle. After transfection into Spodoptera frugiperda cells, Ac78KO produced a single-cell infection phenotype indicating that no infectious budded viruses (BVs) were produced. The defection in BV production was also confirmed by both viral titration and Western blot. However, viral DNA replication is unaffected. Analysis of BV and occlusion derived virus (ODV) revealed that AC78 is associated with both forms of the virions and is a structural protein located to viral envelope. Electron microscopy showed that ac78 also plays an important role in embedding of ODV into occlusion body. This study therefore demonstrates that AC78 is a late virion associated protein and is essential for the viral life cycle.

ORF11 (ac11) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved gene of unknown function. To determine the role of ac11 in baculovirus life cycle, an ac11-knockout mutant AcMNPV, Ac11KO, was constructed. qPCR analysis revealed that ac11 is an early gene in the life cycle. After transfection into Spodoptera frugiperda cells, Ac11KO produced a single cell infection phenotype indicating that no infectious budded viruses (BVs) were produced. The defection in BV production was confirmed by both viral titration and Western blot. However, viral DNA replication is unaffected. Electron microscopy showed that ac11 is required for nucleocapsids envelopment to form ODV and their subsequent embedding into OB. This study therefore demonstrates that ac11 is an early gene which is essential for the viral life cycle.

ORF11 (ac11) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved baculovirus gene whose homologs are found in all lepidoteran Group I NPV, but its function is unknown so far. To determine the role of ac11 in baculovirus life cycle, ac11 knock-out mutant, Ac11KO, was constructed using the plasmid capture system (PCS). Real-Time PCR analysis showed that ac11 transcript was first detected at 6 h post-infection (p.i.) and accumulated to maximum at 48 h p.i., indicating that ac11 is belong to late gene. When the genomic DNA of Ac11KO was transfected into Sf9 cells, viral replication was restricted to a cell transfected originally. While viral transmission of the Ac11KO was not observed in Sf9 cells, production of budded virus (BV) in Sf9 cells transfected with Ac11KO was observed by transmission electron microscopy (TEM). These results suggest that the ac11 is essential for AcMNPV to produce infective BV.

Among 154 putative ORFs of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), ac78 and ac79 are highly conserved genes in baculovirus, but their functions in the virus life cycle have been unknown so far. To determine their roles in AcMNPV replication, knockout mutants, ac78KO and ac79KO, were constructed using the plasmid capture system (PCS). Real-Time PCR analysis showed that both of ac78 and ac79 transcripts were first detected at 6 hours post-infection, and accumulated to maximum at 24 hours post-infection, suggesting that both of ac78 and ac79 are belong to late gene. When the genomic DNA of ac78KO was transfected into Sf9 cells, viral replication was restricted to a single cell infection. These results demonstrated that the ac78 play an important role in BV production, and therefore is essential for AcMNPV to mount a successful infection. Whereas Sf9 cells infected with the ac79KO showed normal viral symptoms such as rounding and swelling, OBs were not observed from majority of infected cells. These results suggested that the ac79 might play an important role in OB production.

ORF43 (ac43) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved baculovirus gene whose function is unknown. To determine the role of ac43 in baculovirus life cycle, we used a new AcMNPV bacmid (bAc-MK) and generated ac43 deletion virus (ac43KO) by using the plasmid capture system (PCS). After transfection into Spodoptera frugiperda cells, ac43KO produced significantly different OBs which with much larger size; and especially had much single nucleocapsids compared to Ac-MK. Furthermore, ac43KO bacmid led to defect in transcription and expression of polyhedrin, which result in less OBs production. However, ac43KO didn't affect BV production since there's no remarkable difference of BV titer in both ac43KO and Ac-MK. These results demonstrate that ac43 play an important role in polyhedrin expression, OB formation, and virion assembly.

Autographa californica nucleopolyhedrovirus (AcMNPV) has a large doublestrand DNA genome of approximately 134 kbp and harbors 156 open reading frames (ORFs). To elucidate DNA replication cascade of AcMNPV, we developed a novel baculovirus genome that can be maintained in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. This genome, named bAc-MK, contains a mini-F replicon and a kanamycin resistance marker. Using a convenient Tn7 transposon-based system, pPCS-S, 55 single ORF-truncated mutants were generated by random insertion into bAc-MK genome. These single ORF-truncated mutants were independently transfected into Sf9 cells, 16 of them were found affecting viral replication since they defected in producing polyhedra. Furthermore, to verify the pathogenicity of the single ORF-truncated mutants, the remaining 39 mutants were subjected to bioassay to Spodoptera exigua 3rd instar larvae. Among them, ac9-, ac49-, ac103- and ac105-knockout mutants showed higher mortality compared to that of bAc-MK. These results suggested that these ORFs could be related to pathogenicity of AcMNPV.

Autographa californica nucleopolyhedrovirus (AcMNPV) has a large doublestrand DNA genome of approximately 134 kbp and comprises 156 open reading frames (ORFs). To elucidate DNA replication cascade of AcMNPV, we developed a novel baculovirus genome that can be maintained in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. This genome, named bAc-MK, contains a mini-F replicon and a kanamycin resistance marker. Using a convenient Tn7 transposon-based system, pPCS-S, which contains an ampicillin resistance gene, 56 single ORF-truncated mutants were generated by random insertion into bAc-MK genome. These single ORF-truncated mutants were independently transfected into Sf9 cells to verify viral replication. Interestingly, both lef-1 and p48 knockout mutants showed normal viral replication in infected cells, which are reported to essential for viral replication. These results suggest that these single ORF-truncated mutants are useful for elucidation of viral replication cascade.

This study was conducted to investigate the insecticidal capacity of several recombinant baculoviruses to P. xylostella and S. exigua larvae. NeuroBactrus was constructed as follows: the cry1-5 of Bacillus thuringiensis 2385-1 was inserted into Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) genome by fusion of polyhedrin-cry1-5-polyhedrin under the control of polyhedrin gene promoter, and insect-specific neurotoxin from the scorpion Androctonus australis (AaIT) under the control of early promoter from Cotesia plutellae bracovirus was introduced by fusion of orf603 partial fragment in the opposite direction of polyhedrin gene, respectively. Other recombinant baculoviruses derived from the NeuroBactrus - NBt-DelA (deleted AaIT), NBt-Del5 (deleted cry1-5), and NBt-DelA5 (deleted AaIT and cry 1-5) - were manufactured in serial passages in vitro. The data were analyzed by SPSS. The value of LC50 was lower when P. xylostella larvae fed on cabbage coated with NeuroBactrus (4068.4) than when it fed on cabbage coated with AcMNPV (4.5x106). Survival time (ST50) of P. xylostella larvae (2.54days) was shorter when it fed on cabbage coated with NeuroBactrus than when it fed on cabbage coated with other recombinant baculoviruses (7.54days, 7.68days, and 8.26days) and AcMNPV (9.67days). S. exigua larvae presented the same results.

The baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV), a large circular double-stranded DNA virus whose genome encodes at least 155 open reading frames (ORFs), is highly pathogenic to a number of lepidopteran insects and widely used to transduce various cells for exogenous gene expression. Although many genes of AcMNPV have been identified, the genome-wide study related to viral replication has not been well announced. In this study, to elucidate DNA replication cascade of AcMNPV, we firstly developed a novel baculovirus genome that can be maintained in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. This genome, named bAc-MK, contains a mini-F replicon and a kanamycin resistance marker. Using a convenient Tn7 transposon-based system, pPCS-S, which contains an ampicillin resistance gene, ORF knock-out mutants were generated by random insertion into bAc-MK genome. These mutants will be suffered DNA microarray to elucidate AcMNPV replication cascade.