• Journal of Sericultural and Entomological Science
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• v.25 no.2
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• pp.1-31
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• 1984

Flacherie, as one of the most prevalent silkworm diseases, causes severe economic damage to sericultural industry and its pathogens have been proved to be flacherie virus (FV) and densonucleosis virus (DNV). Multiplications of the viruses in the larvae of the silkworm, Bombyx mori, were studied by the sucrose density gradient centrifugation and electron microscopy. The quantitative and qualitative changes of nucleic acids and proteins were investigated from the midgut and hemolymph in the silkworm larvae infected separately with FV and DNV. The histopathological changes of epithelial cells of infected midgut also were examined by an electron microscope. 1. Purified fractions of FV or DNV in a sucrose density gradient centrifugation yielded one homogenous and sharp peak without a shoulder, suggesting no heterogenous materials in the preparation. Electron microscopy also revealed that FV and DNV were spherical particles, 27nm and 21nm in diameter, respectively. 2. Silkworm larvae showed a decrease in body weight on the 6th day and in midgut weight on the 3rd day after inoculation with FV or DNV. 3. DNA content was higher in the midgut when infected with FV or DNV, but the hemolymph of the infected larvae showed no difference during first 6 days after inoculation, after which DNA concentration declined rapidly. 4. RNA synthesis of silkworm larvae infected separately with FV and DNV was stimulated in the midgut, but RNA content was reduced in the hemolymph at the early stage of virus multiplication. At the late stage of virus multiplication, however, it was extremely reduced in both midgut and hemolymph. 5. The concentration of protein in the midgut and hemolymph of silkworm larvae infected separately with FV and DNV showed no difference from that of the healthy larvae at the early stage of virus multiplication, but it was significantly reduced at the late stage of virus multiplication. 6. There was no difference in the electrophoretic patterns of RNAs extracted from the midgut of healthy or virus-infected larvae. 7. The electrophoresis of proteins extracted from the midgut infected with FV or DNV, when carried out on the 1st and 5th day after virus inoculation, showed no difference from that of the healthy larvae. But, there was an additional band with medium motility in the proteins on the 8th day after virus inoculation, while a band with low mobility shown in the proteins of healthy larvae disappeared in the infected larvae. However, a band with high mobility in the healthy larvae was separated into two fractions in the infected larvae. 8. The electrophoretic pattern of hemolymph proteins of the silkworm larvae infected separately with FV and DNV was similar to that of the healthy larvae, but the concentration of hemolymph proteins in the infected larvae was lower than that of the healthy larvae at the late stage. 9. Two types of inclusion bodies were shown by the double staining of pyronin-methyl green in the columnar cell of the midgut on the 8th day after FV inoculation. 10. Electron microscopy of the infected midgut revealed that the 'cytoplasmic wall' of the goblet cell thickened on the 5th day after FV inoculation and several types of the cytopathogenic structures, such as virus$.$specific vesicles, virus particles, linear structures, tubular structures, and high electron-dense matrices were observed in the cytoplasm of the goblet cell. The virus particles were also observed in the microvilli and the structures similar to spherical virus particles were observed around the virus-specific vesicles, suggesting the virus assembly in the cytoplasm. 11. Fluorescence micrograph of the infected midgut stained with acridine orange showed that the nucleus, the site of DNV multiplication in the columnar cell, enlarged on the 5th day after virus inoculation. 12. Electron microscopic examination of DNV infected midgut revealed that the nucleolus of the columnar cell was broken into granules and those granules dispersed into apical region of the nucleus on the 5th day after virus inoculation. On the 8th day after inoculation, it was also observed that the nucleus of the columnar cell was full with the high electron-dense virogenic stroma which were similar to virus particles. These facts suggest that the virogenic stroma were the sites of virus assembly in the process of DNV multiplication.