Partial Purification of Kuchijirosho Causative Agent by Sodium lotalamate Density Gradient Centrifugation

Abstract

ABSTRACT--To purify the kuchijirosho causative agent derived from brains of kuchijirosho-affected tiger pufferTakifugu rubripes, the method of density gradient centrifu gation using iodinated contrast medium, Angio-Conray (solium iotalamata solution), was used. The extracts of kuchijirosho-affected brains were loaded on 3-55% gradient of Angio-Conray, and centrifuged at 280,000•~g for 2 h.The lethal activity to grass puffer Takifugu niphobles was found in a fraction with a density around 1.096 g/cm3. The infectivity of the extracts of kuchijirosho-affected brains was lost by treatment with ether, UV-irradiation, propiolacton or proteinase K.

Proliferation of kuchijirosho causative agent in a fugu‐derived cell line

“…The density of KCA derived from fugu brain was distributed around 1.1 g mL −1 on Angio‐Conray (sodium iotalamate) density gradient centrifugation (Miyadai et al. 2004).…”

“…The density of KCA produced from Fugu eye cells was similar to that from kuchijirosho‐affected fugu brain as previously reported (Miyadai et al. 2004).…”

“…The density of KCA derived from fugu brain was distributed around 1.1 g mL )1 on Angio-Conray (sodium iotalamate) density gradient centrifugation (Miyadai et al 2004). To compare the density of fugu eye cell-derived KCA with that of brainderived KCA, the former was subjected to density gradient centrifugation and each fraction injected into five fugu.…”

“…Ex. 1-5: extract of kuchijirosho-affected brains (Miyadai et al 2004), Ex. 6 and 7: media from inoculated fugu eye cells (7 and 3 days postinfection, respectively).…”

“…Injection of an extract of kuchijirosho-affected brain into several species of fugu, red sea bream, Pagrus major (Temminck & Schlegel), and black rockfish, Sebastes schlegeli Hilgendorf, caused kuchijirosho in these fish (Miyadai, Kitamura, Uwaoku & Tahara 2001) and the brains of experimentally infected fish other than fugu also transmitted the condition, which indicates that KCA propagates in the brain. KCA derived from kuchijirosho-affected fugu brain was fractionated using sodium iotalamate density gradient centrifugation (Miyadai, Hashimoto, Hashimoto, Watari, Ohtani & Tahara 2004); however, a quantity of viral particles sufficient for electron microscopic observation was not obtained.…”