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

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.

Development of a dipstick immunoassay to detect nucleopolyhedroviruses in Douglas-fir tussock moth larvae.

In this paper, we describe the development of a novel field detection system for the identification of Orgyia pseudotsugata nucleopolyhedrovirus (OpNPV) and OpNPV infections in Douglas-fir tussock moth (O. pseudotsugata) (DFTM) larvae, utilizing antibodies in a dipstick immunoassay. The dipstick method is sensitive to a minimum of 10ng of extracted virus protein, or approximately 1070 virus occlusion bodies, and is sufficiently sensitive to detect OpNPV infections in DFTM prior to mortality. Additionally, the method can be used to unambiguously detect virus in infected larvae without purification of the test sample. This research provides a novel tool for on-site assessment of the incidence of OpNPV in field populations of DFTM, and has the potential to improve the biological control of the DFTM by facilitating on-site pest management decisions.

Development and evaluation of methods to detect nucleopolyhedroviruses in larvae of the Douglas-fir tussock moth, Orgyia pseudotsugata (McDunnough).

Various molecular methods are used to detect pathogenic microorganisms and viruses within their hosts, but these methods are rarely validated by direct comparison. Southern hybridization, enzyme-linked immunosorbent assay (ELISA), and a novel DNA extraction/PCR assay were used to detect Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) in Douglas-fir tussock moth larvae. PCR was more sensitive than Southern hybridization and ELISA at detecting semipurified virus. ELISA, however, was the most accurate method for detecting virus within larvae, given that Southern hybridization and PCR produced false-negative results (31% and 2.5%, respectively). ELISA may be preferable in some applications because virus infections can be quantified (r(2) = 0.995). These results may be applicable to both applied and academic research that seeks to accurately identify the incidence of viruses and microorganisms that regulate insect populations.