Replication Of Oryctes Nudivirus (Ornv) In Insect Cell Line DSIR-HA-1179 And Its Infectivity On Neonates Of Rhinoceros Beetle, Oryctes Rhinoceros

Abstract

The Oryctes nudivirus (OrNV) or previously known as Oryctes baculovirus is a biocontrol agent for rhinoceros beetle, Oryctes rhinoceros. The replication of three types of OrNV, the types A, B, and C in the insect cell line DSIR-HA-1179 was studied. The cells were grown in PS100 media containing 10% fetal bovine serum. The signs of infection in cells by all types of OrNV were clearly seen at 10 to 14 days after treatment (DAT). The infected cells experienced cytopathic effects (CPE) involving formation of high number of vesicle or globule like-structures around the cells. The density of viable cells in flasks inoculated by the nudivirus decreased over time. At 16 DAT, the viable cell density in flasks inoculated with OrNV types A, B, and C was 1.49 × 105, 0.29 × 105, and 4.78 × 105 cells ml-1 respectively, significantly lower (P<0.05) compared to control flask of 7.98 × 105 cells ml-1. Based on the Tissue Culture Infectious Dose 50% (TCID50), it was found that the OrNV type B was more virulent than the OrNV types A and C as it produced high infectious virus particles. The TCID50 values for the OrNV types A and C were 3.00 × 107 infectious unit (IU ml-1) and 8.03 × 105 IU ml-1 as compared to type B which was 7.09 × 107 IU ml-1. The PCR analysis showed that all types of OrNV had successfully replicated in the cells as early as 4 DAT. Pathogenicity study on neonates using cells-propagated OrNV has again showed that the OrNV type B was more pathogenic than the other types of OrNV. The LT50 value for the OrNV type B was 33.47 days, shorter than the OrNV type A (49.35 days) and the OrNV type C (48.58 days). The DNA profiles of OrNV extracted from dead infected neonates in the bioassay were the same as those from infected gut tissues collected from field population. Thus, it is suggested that the Malaysian OrNV types A, B and C were successfully produced in cell line DSIR-HA-1179.