Characterization of growth and Oryctes rhinoceros nudivirus production in attached cultures of the DSIR-HA-1179 coleopteran insect cell line

Abstract

The DSIR-HA-1179 coleopteran cell line is a susceptible and permissive host to the Oryctes rhinoceros nudivirus (OrNV), which has been used as a biocontrol agent against the coconut rhinoceros beetle (Oryctes rhinoceros); a pest of palms in the Asia-Pacific region. However, little is known about growth and metabolism of this cell line, knowledge of which is necessary to develop an in vitro large-scale OrNV production process. The strong anchoragedependent characteristics of the cell line, its particular fragility and its tendency to form dense clumps when manipulated, are the most likely reasons that have precluded further development of the cell line. In order to characterize DSIR-HA-1179 cells, there was first a need for a reliable technique to count the cells. A homogenous cell suspension suitable for enumeration could be produced by treatment with TrypLE Express TM with optimum mean time for cell release calculated as 30 min. The cell line was adapted to grow in four serum-supplemented culture media namely TC-100, IPL-41, Sf-900 II and Sf-900 III and cell growth, glucose consumption, lactate and ammonia production were assessed from static-batch cultures. The maximum viable cell density was reached in Sf-900 II (17.9 9 10 5 cells/ml), with the maximum specific growth rate observed in this culture medium as well (0.0074 h -1 ). Higher production of OrNV was observed in IPL-41 and TC-100 (4.1 9 10 7 TCID 50 /ml) than in cultures infected in Sf-900 III (2.0 9 10 7 TCID 50 /ml) and Sf-900 II (1.4 9 10 7 TCID 50 /ml). At the end of the growth period, glucose was completely consumed in cultures grown in TC-100, while remained in excess in the other three culture media. The cell line produced lactate and ammonia to very low levels in the TC-100 culture medium which is a promising aspect for its cultivation at large-scale.

Visualizing Nudivirus Assembly and Egress

“…Strategies to overcome slow growth kinetics of the DSIR-HA-1179 cell line and inconsistencies in productivity using traditional approaches of virus production were previously reported ( 5 , 20 , 21 ). By monitoring of virus titer in insect cells infected with OrNV at a multiplicity of infection of five, we observed an increase of virus production up to day six postinoculation.…”

“…Viruses make extensive use of this mechanism to exit the cell (49). Considering that only uncoated nucleocapsids enter into the nucleoplasm for replication (5,19), it could be speculated that the formation of MMVs and their association with both NR and NE might be crucial for OrNV transport into the cytoplasm. Importantly, the shedding of both free-floating and vesicle-enclosed virions from infected cells (Fig.…”

“…For long-term storage, 1 × 10 7 DSIR-HA-1179 cells were resuspended in freezing medium (TC-100, 10% FBS, 10% dimethyl sulfoxide), allowed to freeze gradually using a Mr. Frosty freezing container (Nalgene) and later transferred to liquid nitrogen. Confluent monolayers of attached DSIR-HA-1179 cells at day 10 of culture were treated with TrypLE Express as previously described by Pushparajan et al ( 19 ) to dissociate the cell monolayer into a single cell suspension. The cell suspension was counted on a hemocytometer.…”

“…The fact that beetle population is drastically reduced after infection made OrNV a widely used biocontrol agent, which helps the recovery of badly damaged coconut and oil palm trees in Southeast Asia and the Pacific Islands ( 16 ). Establishment of DSIR-HA-1179, a susceptible and permissive host cell line that was isolated from the black beetle Heteronychus arator , has proven to be an important step toward producing a higher yield of viable OrNV ( 17 – 19 ). Although the timeline for virus entry, replication, and budding has been previously described ( 5 ), the mechanism behind viral replication and assembly is still unclear.…”

Integrated Multi-Omic Characterization of the Detachment Process of Adherent Vero Cells with Animal-Based and Animal-Origin-Free Enzymes

“…In recent years, as an alternative to traditional animal-based enzymes, animal-origin-free enzymes have been gradually applied [ 23 , 24 , 25 ]; they can cleave peptide bonds at the C-terminus of lysine and arginine, and can directly replace animal-based enzymes. Animal-origin-free enzymes are stable at room temperature and have mild effects on the cells.…”