豆莢螟核多角體病毒 (MaviNPV) 在 NTU-MV 細胞株系之體外增殖及其 MV74 蛋白特徵分析

Abstract

A new cell line, NTU-MV, derived from pupal tissues of an economically important pest, the legume pod borer Maruca vitrata, was established. This cell line contained four major cell types: polymorphic cells, round cells, spindle-shaped cells, and comma cells. The doubling time of MV cells in TNM-FH medium supplemented with 8% FBS at 28 ℃ was 27 h. The chromosome numbers of MV cells varied widely from 16 to 268. Compared to other insect cell lines, the MV cell line produced distinct isozyme patterns with esterase, malate dehydrogenase, and lactate dehydrogenase. Confirmation that NTU-MV was derived from M. vitrata was demonstrated by showing that the sequence of the internal transcribed spacer regions (ITS) of the MV cells was 98% identical to that of M. vitrata larvae. Two NTU-MV cell strains, NTU-MV1 and NTU-MV56, were selected based on susceptibility to MaviNPV (M. vitrata nucleopolyhedrovirus). NTU-MV, MV1, and MV56 cells showed a high susceptibility to MaviNPV and produced high yields of polyhedra (47-50 OBs/cell, 4 × 107-5.96 × 107 OBs/ml) and extracellular virus (3.83 × 107-2.51 × 108 TCID50/ml) after 2 weeks of MaviNPV infection. The NTU-MV cell line and its strains are useful tools for studying the molecular biology of MaviNPV. The MaviNPV genome is relatively small (111, 953 bp) and it contains only 126 putative open reading frames (ORFs). Except MaviNPV mv74, 125 homologous genes of MaviNPV were found in Autographa californica multiple nucleopolyhedrovirus (AcMNPV). MaviNPV mv74 homologs were also found in other NPVs, but mv74 only shared 21 to 26% identities with other NPV homologs in aminoacid sequences. MaviNPV mv74 consists of 675 base pairs encoding 224 amino acids. mv74 contains an early promoter motif, which is defferent from the traditional early promoter motif, and characterized by TA-rich region followed by a CAGT motif 29 bp downstream. MaviNPV mv74 RNA expression could be at first detected at 2 h postinfection, and expression levels reached the peack at 72 h postinfection. The MV74 protein was detected initially at 48 h postinfection with the peak at 72 h postinfection. The MV74 presented in both the cytoplasm and nucleus of the infected cells, especially co-localized with the occlusion bodies (OBs). MV74 was found on the surface of the OBs, but not on the extracellular virus or occlusion-derived virus. We conclude that MV74 is an occlusion body (OB)-associated protein.