Study On Characterization Of Mythimna Separata Nuclear Polyhedrosis Virus In Vitro

Multiple enveloped nuclear polyhedrosis viruses(MNPV) belong to the family Bac-uloviridae and are characterized by the multiply rod-shaped virions surrounded by a viral envelope. The MNPV show considerable potential as viral insecticides for use against ma -ny major insect pest species. For their full potential as microbial pest control agents to be realized, the molecular biology, genetics and mass production of MNPVs must investigated. The major breakthrough is the establishement of virus-cell culture system.The replication of Mythimna separata multiple nuclear polyhedrosis virus (Ms MNPV) in a new Lepidopteran cell line,NEAU-Ms-7311(Ms-7311), is presented. Virus growth curve for MsMNPV in Ms7311 cells indicated that infectious budded virus(BV) was present in the medium 20 hr pi and maximum titer of 2.57*106 TCID50/ml was attained at 144 hr. The first serial passage of virus which was obtained from the hemolymph of infected larvae was established following infection of the cells at a m.o.i. of 0.5 TCID50/cell. The effects of serial passage of the virus on OB or BV production demonstrated that the relative BV titer from 1st to 5th passage at approximately2.57><105 TCIDso/ml. The titers decreased with increae of the passage. Occlusion body production in the early passage was high, with level of 12.35 OBs/cell. After 6th passage, OB production decreased dramatically about 2.35 OBs/cell. Bioassays were performed using OBs generated from different viral passage to examine the relationship between mortality and passage number. Using the same OB concentration(l*106 OBs/cell), early passage virus produced constant larval mortality( 67.27%~63.05%) and there was a rapid decrease with late passage virus in mortality. The twenty six clones by plaque purification from early or late passages of virus were obtained. Two clones,C4 and C9, of them from early passages were isolated by bioassay with higher motality(77.21%, 70.20%) than wild virus(66.72%). The titer of the clone4 and clone9 was 1.79xl06 TCID50/ml and391.38* 106 TCID5o/ml respectively; OB production in clone4 and clone9 were higher with level of 14.92 OBs/cell and 13.56 OBs/cell separately than wild virus. LC50 values of 1.16x104OBs/ml and 1.04 * 104 OBs/ml,respectively, were calculated.