The Effect of a Small Conotoxin-Like ctx Gene from Autographa californica Nuclear Polyhedrosis Virus (AcMNPV) on Insect Hemolymph Melanization.

The conotoxin-like (ctx) gene encodes a small cysteine-rich polypeptide in various baculoviruses. Previous research has demonstrated that the product of the ctx gene could be purified from insect cells infected by Autographa californica nuclear polyhedrosis virus (AcMNPV), but its function was unknown. In this paper, we compared the conserved cysteine motif structure (CX3GX2CX5CCX3CX6C) of the ctx gene in baculoviruses and generated recombinant Bombyx mori nuclear polyhedrosis virus (BmNPV) with the BmNPV bacmid system. The recombinant BmNPV contained the ctx gene from AcMNPV or a fusion gene of ctx with eGFP, respectively. Fluorescence in CTX-eGFP-positive cells was mainly observed on the cell membrane. To gain insight into CTX function, two methods were used to elucidate the affect CTX had on hemolymph melanization in vivo and in vitro in insect larvae and pupae. The results indicated that CTX abrogates hemolymph melanization; however, the mechanisms require further evaluation.

Genomic sequence, organization and characteristics of a new nucleopolyhedrovirus isolated from Clanis bilineata larva.

BACKGROUND: Baculoviruses are well known for their potential as biological agents for controlling agricultural and forest pests. They are also widely used as expression vectors in molecular cloning studies. The genome sequences of 48 baculoviruses are currently available in NCBI databases. As the number of sequenced viral genomes increases, it is important for the authors to present sufficiently detailed analyses and annotations to advance understanding of them. In this study, the complete genome of Clanis bilineata nucleopolyhedrovirus (ClbiNPV) has been sequenced and analyzed in order to understand this virus better. RESULTS: The genome of ClbiNPV contains 135,454 base pairs (bp) with a G+C content of 37%, and 139 putative open reading frames (ORFs) of at least 150 nucleotides. One hundred and twenty-six of these ORFs have homologues with other baculovirus genes while the other 13 are unique to ClbiNPV. The 30 baculovirus core genes are all present in ClbiNPV. Phylogenetic analysis based on the combined pif-2 and lef-8 sequences places ClbiNPV in the Group II Alphabaculoviruses. This result is consistent with the absence of gp64 from the ClbiNPV genome and the presence instead of a fusion protein gene, characteristic of Group II. Blast searches revealed that ClbiNPV encodes a photolyase-like gene sequence, which has a 1-bp deletion when compared with photolyases of other baculoviruses. This deletion disrupts the sequence into two small photolyase ORFs, designated Clbiphr-1 and Clbiphr-2, which correspond to the CPD-DNA photolyase and FAD-binding domains of photolyases, respectively. CONCLUSION: ClbiNPV belongs to the Group II Alphabaculoviruses and is most closely related to OrleNPV, LdMNPV, TnSNPV, EcobNPV and ChchNPV. It contains a variant DNA photolyase gene, which only exists in ChchNPV, TnSNPV and SpltGV among the baculoviruses.

Expression of Bombyx mori nucleopolyhedrovirus ORF76 in permissive and non-permissive cell lines by a novel Bac-to-Bac/BmNPV baculovirus expression system.

Open reading frame 76 of Bombyx mori nucleopolyhedrovirus (BmNPV), designated as Bm76, is a gene whose function is completely unknown. With EGFP fused to the 3' terminal of Bm76 as the reporter gene and BmNPV bacmid as the expression vector, a recombinant bacmid was successfully constructed expressing Bm76-EGFP fusion protein under the control of polyhedrin promoter in Bombyx mori cells (Bm cells), BmNPV's permissive cell line, laying the foundation for rescue experiment of Bm76 deletion mutant. Moreover, the supernatant from Bm cells transfected with the recombinant bacmid was used to infect Trichoplusia Ni cells (Tn cells), BmNPV's non-permissive cell line. Unexpectedly, the expression of Bm76-EGFP fusion protein in some Tn cells was detected, implying that viral DNA was replicated in these cells. The causes are being studied for the inability of BmNPV to produce enough viable budded viruses in Tn cells despite of viral DNA replication.