Genomic study and BEVS construction of Maruca vitrata multiple nucleopolyhedrovirus, MaviMNPV
Date Issued
2008
Date
2008
Author(s)
Chen, Yun-Ru
Abstract
A new baculovirus, Maruca vitrata multiple nucleopolyhedrovirus (MaviMNPV), is isolated from the moribund larvae. The whole MaviMNPV genome has been sequenced and analysed. The genome size is approximately 111.9 kbp, processing 126 open reading frames (ORFs). According to the phylogenetic analyses, MaviMNPV belongs to Group I NPV and is closely related to AcMNPV (Autographa californica MNPV) and BmNPV (Bombyx mori NPV). Comparing the localization of homologous ORFs, the ORFs arrangement of MaviMNPV is 100% matched to that of AcMNPV. The main objectives of this study were focused on the in vitro host range, MaviMNPV genes expression profile, and the transfer vector construction. Five cell lines, including IPLB-LD-652Y (LD), NTU-MV56, NTU-LY4 (LY4), NTU-PN-HH (HH), and Sf9, were chosen for MaviMNPV infection assays. Compared to 99% MV cells infected with egfp-MaviMNPV1, only 1% IPLB-LD-652Y cells were susceptible to MaviMNPV, and LD cells could produce virus progeny, and the virus titer could reach 105 TCID50 / ml; no virus progeny was produced in MaviMNPV-infected Sf9, LY4, and HH cells . The qPCR results also revealed that the genes of MaviMNPV could not be transcripted in the infected Sf9, LY4 and HH cells. These results supported that MaviMNPV is a distinct species of the group I lepidopteran NPVs. Based on the results of the microarray, the genes expression stages were divided into three groups: the early stage (0-6h), including 25 ORFs (19.84%); the late stage (6-15h), including 75 ORFs (59.52%); and the very late stage (15-48h), including 26 ORFs (20.64%). In the MaviMNPV-infected LD (Lymantria dispar) cells, 14 of the 25 ORFs were detectable. The ie family and several structure protein genes of MaviMNPV in LD cells showed the stable expression trend. The polyhedrin related expression levels was also detected by real-time qPCR. The results showed that MaviMNPV-infected MV cells could produce the greatest level of polyhedrin compared with that of AcMNPV-infected Sf cells. We had successively constructed two new transfer vectors, pMV-polh and pMV-polh-DE, driven by the MaviMNPV polyhedrin promoter. Two egfp recombinant MaviMNPVs, egfp-MaviMNPV1 and egfp-MaviMNPV2, were cloned. We compared the eGFP produced from intra- and extracellular levels of MaviBEV1/NTU-MV cells, MaviBEV2/NTU-MV cells, AcBEV (A. californica baculovirus expression vector, AcBEV)/Sf9 cell, and LyxyBEV (Lymantria xylina BEV, LyxyBEV)/LY cells. In the intracellular level, AcBEV/Sf9 cells produced the greatest level of eGFP while MaviBEV2/NTU-MV was the lowest BEV. In the extracellular level, MaviBEV1/NTU-MV cells was the highest production of eGFP while MaviBEV2/NTU-MV cells was the lowest BEV. Thererfore, MaviBEV1/NTU-MV has a high potential to be developed as a new BEVS for comparing with the commercial AcBEV/Sf9 cells. In conclusion, these results showed that MaviMNPV/NTU-MV cells possess a high potential not only for scientific studies but also BEVS application.
Subjects
Maruca vitrata nucleopolyhedrovirus (MaviMNPV)
baculovirus infection assay
microarray
baculovirus genes expression profile
baculovirus expression vector system (BEVS)
Type
thesis
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