Masako Funakoshi

Purification and properties of the Bombyx mori nuclear polyhedrosis virus liberated from polyhedra by dissolution with silkworm gut juice

Abstract Occluded virions of the Bombyx mori nuclear polyhedrosis virus were efficiently liberated from polyhedra by dissolution with the silkworm gut juice. The liberated virions were purified by sucrose density gradient centrifugation and the bands of enveloped virions were observed in the gradients. There was no functional difference between the gut juice-liberated and the carbonate-liberated virions. Disruption of enveloped virions by the gut juice was observed, but the formation of nucleocapsids from the degradation of the occluded virions was not detected. High yields of the enveloped virions from the polyhedra dissolved by the gut juice was obtained by separating the virions through sucrose density gradient centrifugation immediately after the dissolution of the polyhedra. Many factors, e.g., rearing seasons, silkworm strains, and rearing conditions, affect the polyhedra-dissolving property of the larval gut juice.

Viral inhibitory factor produced in the hemolymph of the silkworm, Bombyx mori, infected with a nuclear polyhedrosis virus

Abstract A viral inhibitory factor (VIF) was purified from the hemolymph of silkworm larvae infected with a nuclear polyhedrosis virus of the silkworm, Bombyx mori (BmNPV). VIF inactivated BmNPV strongly at 27°C for 60 min in vitro; however, preventive and therapeutic effects by the injection of VIF into silkworm pupae were not shown. Silkworm larval hemolymph and some phospholipids showed inhibitory effect on the antiviral activity of VIF. VIF was not inactivated by trypsin and periodic acid but more than 90% of the antiviral activity was lost by heating at 60°C for 10 min. Ether extracts (VIF-E) from VIF inactivated BmNPV more strongly than VIF. The antiviral activity of VIF-E scarcely decreased by heating at 100°C for 30 min. The infrared spectrum of VIF-E showed characteristic absorption bands of carboxyl, carbonyl, and alkyl groups. In addition, its spectrum was similar to that of oleic acid. These results suggest that VIF-E is a fatty acid or a mixture of fatty acids.

A New Method for Inoculation of Poor Germinator, Nosema sp. NIS M11 (Microsporida: Nosematidae), into an Insect Cell Culture

ABSTRACT. Spores of Nosema sp. NIS M11, primed with 0.1 N KOH solution, were mixed with either physiological salt solutions or IPL‐41 medium, an insect cell culture medium, for germination. In the latter medium, only a few spores germinated, while high percentages of spore germination were obtained in physiological salt solutions, particularly in Rinaldinis solution. By using the salt solutions as inoculation media, KOH‐primed NIS M11 spores were inoculated into the Spodoptera frugiperda SF21AEII cell line. The initial infection levels were consistently higher than that obtained by using IPL‐41 medium. Among the salt solutions, Rinaldinis solution, containing KCl in place of NaCl, gave the highest percentage of initial cell infection. Increased osmolarity of salt solutions did not improve the efficiency of spore germination and infection of N. sp. NIS M11.

Effects of host cell density on cell infection level in Antheraea eucalypti (Lepidoptera: Saturniidae) cell cultures persistently infected with Nosema bombycis (Microsporida: Nosematidae)

ABSTRACT. Spores of Nosema bombycis Y9101, isolated from the beet armyworm, Spodoptera exigua, were primed with an alkaline solution and inoculated into Antheraea eucalypti cell cultures. Infected cells were subcultured every five days at three cell densities (2.5 × 103, 5.0 × 103, and 1.0 × 104 cells/cm2). A difference was observed in the spread of N. bombycis Y9101 infection between low‐density and higher‐density cultures of host cells. The host cell density did not affect the productivity of secondary infective forms of the parasite. The principal factor determining the rate of microsporidian infection in vitro was the number of host cells existing within the reach of extruded short‐coiled polar tubes from spores germinated intracellularly.

Establishment and characterization of a continuous cell line from pupal ovaries of Japanese oak silkworm Antheraea yamamai Guerin-Meneville.

SummaryPupal ovaries of the wild oak silkworm Antheraea yamamai Guerin-Meneville were cultured in MGM-448 (Modified Grace Medium-448) medium containing 10% fetal bovine serum. After the primary culture was set up in 1988, a continuous cell line was obtained in 1991, designated as NISES-Anya-0611 (Anya-0611). The population doubling time was 54 hrs. and 19 min. at 96 passages and 88 hrs. and 29 min. at 387 passages. Spindle-shaped and spherical cells coexisted in the cell group. The cell line karyotype line was typical of lepidopteran cell lines, consisting of numerous small chromosomes. The cell line was distinguished from other lepidopteran cell lines by comparing malic enzyme, phosphoglucose isomerase, phosphoglucose mutase, and isocitric dehydrogenase isozyme patterns. The cell line was highly infected to the Antheraea yamamai nuclear polyhedrosis virus (Anya NPV). The luciferase gene of recombinant Bm NPV (BmNPVP6ETL) was able to express in the cell line, too, so that luciferase recombinant products were able to be detected in the cell body and in supernatant. The Anya NPV clone group was isolated on the cell seat using plaque purification.