家蠶病毒病之鑑定

Abstract

病毒的研究需要有體外增殖系統的建立，病毒種類的鑑定則需要有形態及分生資料才能克竟全功。本研究旨在鑑定家蠶 (Bombyx mori) 的病毒病之主要病原體，因此先從黑角舞蛾細胞株NTU-LY1株系找到一株高病毒 (NPV) 接受的細胞株系LY16，再利用LY16增殖令家蠶發病之某病源，以光學及電顯觀察確定引起細胞病變的病毒形態及致病現象，證實胞質內有病毒形成的內涵體及病毒形成團。利用感染的LY16培養液回飼和回注射家蠶幼蟲，感染者皆呈典型的軟化症病徵，解剖病蠶及中腸電顯切片觀察亦得到相同結果。純化的病毒粒子經負染處理及電顯觀察，病毒粒子為28 nm大小的20面體病毒，無論細胞病變或電顯病理觀察，病原體極似透翅毒蛾軟化病病毒 (PnV, Perina nuda virus) 。利用RT-PCR增幅未感染的LY16細胞之mRNA，證實LY16細胞原來是PnV之持續感染細胞株系。因此本研究的結論是 (1) LY16是PnV持續感染 (persistent infection) 的細胞株系；(2) PnV 亦可以感染黑角舞蛾細胞株及家蠶；(3) 最起初罹病之家蠶疑是在實驗室飼養時被PnV感染，抑或是罹病家蠶的病原體未能在LY16細胞內增殖。

The viral studies need to establish in vitro viral propagation, and the morphological and molecular data are necessary for the identification and classification of an unknown virus. The aim of this study was undertaken to identify the viral pathogen from moribund silkworm larvae. For in vitro propagation, we selected a high NPV susceptible cell strain, LY16, from NTU-LY1 cells. The tissue fluid extracted from the moribund silkworm larvae was used as an inoculum to infect LY16 cells. The infected LY16 cells were examined by light and electron microscopes. The viral inclusion bodies and virogenic stroma were found in the cytoplasm of the cells. The gross anatomies of the disease silkworm larvae fed or injected with the media of the infected LY16 cells showed a typical symptom of flacheries disease. The columnar cells of the midgut of the disease silkworm were main target cells of the virus and also the muscle cells surrounded the midgut were seriously infected. The purified viral particles are icosahedral in shape and 28 nm in size by negative stain. Based on cytopathic effect and electropathogenic observations, this virus is highly similar to PnV (Perina nuda virus). By RT-PCR amplification, we found that PnV exists in uninfected LY16 cells, therefore LY16 cells are persistent infection with PnV. In conclusions, (1) LY16 cells are PnV-persistent infection cells; (2) PnV can infect both LY cells and silkworm larvae; (3) and the origin pathogen of silkworm may either PnV contaminated from lab during rearing or viral pathogens of silkworm origin which can’t propagate in LY16 cells.