Paul J. Iwai

Propagation of multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata in larvae of Trichoplusia ni

Abstract The multicapsid nuclear polyhedrosis virus (OpMNPV) of Orgyia pseudotsugata (Lepidoptera: Lymantriidae) was adapted by serial passage to a substitute host, Trichoplusia ni (Lepidoptera: Noctuidae). During the first five virus passages, we found only localized lesions in fat body and epidermis of infected T. ni larvae. Disseminated lesions were observed in the sixth passage. Systemic fat body, tracheal, and epidermal lesions typical of nuclear polyhedrosis appeared in the seventh passage. The adapted virus (OpMNPV-Tn) maintained the high virulence of OpMNPV for larvae of the natural host, O. pseudotsugata. After one reverse passage in O. pseudotsugata, the virulence of OpMNPV-Tn for the natural host increased to tenfold that of technical-grade OpMNPV. The reverse-passaged virus had the highest activity in O. pseudotsugata thus far observed in our laboratory. The DNA profile of OpMNPV by five restriction endonucleases was maintained upon passage in T. ni. Because T. ni has many advantages over the natural host for propagation of OpMNPV, we performed several small-scale virus production trials and computed average virus yields. Presently, the average yield of OpMNPV per T. ni larva is lower than expected for larvae of this size. The substitute host, however, offers a continuous and abundant supply of nondiapausing eggs and has a very brief life cycle. For large-scale production, these advantages outweigh the reduced virus yield.

Thermal inactivation characteristics of two strains of nucleopolyhedrosis virus (Baculovirus subgroup A) pathogenic for Orgyia pseudotsugata

Abstract Two nucleopolyhedrosis viruses of the Douglas-fir tussock moth, Orgyia pseudotsugata , one with a single nucleocapsid per envelope (SV) and one with multiple nucleocapsids per envelope (BV), are inactivated by a first-order reaction at 55° and 60°C. BV is the more thermostable of the two viruses: At both test temperatures, it has a lower inactivation rate than SV. BV is also the more virulent of the two viruses, with respect to acute course of the disease and severity of the histological lesions. The greater thermostability of BV and the acute course of the disease caused by this pathogen support the choice of BV as the virus most suitable for industrial production and field use.

Serum neutralization of nucleopolyhedrosis viruses (Baculovirus subgroup A) pathogenic for Orgyia pseudotsugata

The preparation of antisera to intracellular nonoccluded virions and an in vivo neutralization test procedure (constant serum and virus in dilutions) are described. Results of homologous neutralization tests showed that rabbit antisera to two multicapsid viruses pathogenic for Orgyia pseudotsugata had higher neutralization indices than antiserum to a unicapsid Baculovrus from O. pseudotsugata. Based on reciprocal tests, the three viruses are antigenically distinguishable. Blood serum of rats which had been exposed by inhalation to 25 projected acre doses of a technical-grade Baculovirus preparation demonstrated no viral neutralizing activity. Since the neutralization test used in this study does not require availability of susceptible cell lines and is sensitive and accurate, it could find application in quality control programs and in field monitoring of Baculovirus strains.

A cytoplasmic polyhedrosis of Hemerocampa pseudotsugata

Abstract Histochemical and histological studies of midguts of larvae of the Douglas-fir tussock moth, Hemerocampa pseudotsugata , infected with cytoplasmic-polyhedrosis virus showed that the viral inclusion bodies formed in apicobasally oriented rows in goblet and columnar cells of the epithelium. Electron micrographs of ultrathin sections of infected cells in the late stages of the disease revealed that the cytoplasmic polyhedra, were surrounded by high numbers of virus particles, adjacent to the surface of the inclusion bodies. Biological assay gave an ld 50 estimate of 104 polyhedra/g larval weight. In our samples, the mean volume of the inclusion bodies was 7.7 μ 3 and the mean diameter of the virus particles was 56 mμ.

Heterogeneous buoyant density in batches of viral nucleopolyhedra

Abstract Nucleopolyhedron preparations of the bundle virus strain, obtained from large volumes of tissue homogenates of Hemerocampa pseudotsugata larvae, contain populations of inclusion bodies which differ with regard to their buoyant density in isopycnic sucrose gradients. The heavier polyhedra contain higher numbers of virus rods per polyhedron as well as higher numbers of rods per virus bundle. Polyhedra of the single-rod virus strain occlude larger virus rods and band at a higher density than bundle virus polyhedra with similar numbers of virus rods. This fact favors the hypothesis that the single-rod and the bundle virus strains are distinct entities. This study includes a determination of the sedimentation constants of two polyhedron fractions, a mathematical simulation of the sedimentation behavior of nucleopolyhedra in isopycnic sucrose gradients, and an empirical analysis of the sedimentation of a polyhedron fraction. These data are intended to provide further criteria for improving the purification of polyhedral inclusion bodies in Andersons zonal rotors.