In vitro culture of Lambdina fiscellaria lugubrosa nucleopolyhedrovirus in heterologous cell lines

Abstract

Infections of two heterologous insect cell lines derived from Malacosoma disstria (Md108) and Choristoneura fumiferana (Cf70) by the Lambdina fiscellaria lugubrosa nucleopolyhedrovirus (LafiNPV-W) were characterized. Cytopathic effects characteristic of LafiNPV-W infection, including rounding of cells, nuclear hypertrophy, and occlusion body (OB) production, were observed in both cell lines. Budded virus titers were slightly higher in Md108 cells than Cf70 cells (5.8 x 10(7) versus 3.1 x 10(7) TCID(50) units mL(-1)). Viral replication kinetics and cytopathic effects induced by LafiNPV-W infection were very similar in both cell lines. Actin rearrangements and redistribution of heterochromatin and euchromatin were observed within 24 h post-inoculation (hpi), and large quantities of nucleocapsids and virions were observed by electron microscopy at 48 hpi in both cell lines. Cf70 cultures produced OBs with numerous embedded virions, while OBs in Md108 cultures contained few virions or were empty with nucleocapsids packed in the nucleoplasm between OBs. In bioassays against second instar L. fiscellaria lugubrosa, OBs derived from LafiNPV-W-infected Md108 cells induced significantly lower levels of mortality than OBs derived from LafiNPV-W-infected Cf70 cells or from infected L. fiscellaria fiscellaria larvae.

Kinetic characterization of the group II helicoverpa armigera nucleopolyhedrovirus propagated in suspension cell cultures: Implications for development of a biopesticides production process

“…Such a comparison has not been made previously because of the scarcity of suspension‐derived kinetics for baculoviruses other than AcMNPV. Simple BV kinetic studies have been reported for a wide variety of both Groups I and II NPVs,11–16 but the majority of these were conducted in kinetically inefficient stationary cultures, which have been shown to support poorer infection outcomes when compared with suspension cultures6, 53–55 and in which PCDs were rarely monitored to establish cell‐specific BV titers.…”

“…However, there is a paucity of well‐characterized suspension culture baculovirus kinetics data in the literature, except for the well‐established recombinant Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV, Group I NPV),5, 6 which benefits from a wide variety of model proposals 5, 7–10. Simple kinetic studies have been published for a large number of other baculoviruses, but in general the data were inadequate for accurate estimation of cell‐specific virus infection rates and were often obtained from poorly defined stationary cultures 11–16. Hence, there is a need for well‐characterized suspension culture kinetic studies for a wider variety of baculoviruses to enable the scale‐up of in vitro baculovirus biopesticide production processes.…”

Canadian contributions to forest insect pathology and to the use of pathogens in forest pest management

“…The availability of cloning techniques in the 1980s lead to the construction of physical DNA maps for a number of forest insect nucleopolyhedroviruses (Arif et al 1984, 1985; Arif 1986), and characterisation of genes and genomic regions coding for specific polypeptides (Arella et al 1988; Barrett et al 1995; Bah et al 1997; Echeverry et al 1997; Li et al 1997a, 1997b). Critical to these achievements was the parallel development of methods for establishing and culturing permissive insect cell lines (Wyatt 1956; Sohi and Cunningham 1972; Sohi et al 1984; Sohi 1995; Whittome-Waygood et al 2009) as systems for virus purification and gene expression (Arif and Pavlik 2013).…”

“…The availability of cloning techniques in the 1980s lead to the construction of physical DNA maps for a number of forest insect nucleopolyhedroviruses (Arif et al 1984(Arif et al , 1985Arif 1986), and characterisation of genes and genomic regions coding for specific polypeptides (Arella et al 1988;Barrett et al 1995;Bah et al 1997;Echeverry et al 1997;Li et al 1997aLi et al , 1997b. Critical to these achievements was the parallel development of methods for establishing and culturing permissive insect cell lines (Wyatt 1956;Sohi and Cunningham 1972;Sohi et al 1984;Sohi 1995;Whittome-Waygood et al 2009) as systems for virus purification and gene expression (Arif and Pavlik 2013). The advent of ever more efficient and less expensive sequencing technologies early in the new century facilitated in-depth elucidation of phylogenetic relationships based on gene sequence homologies (Jakubowska et al 2007;Graham et al 2008;Zhang et al 2010), and eventually sequencing of entire genomes.…”