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家蚕来源的一株单链RNA病毒的分离和特性研究

Vootla.Shyam Kumar  
【摘要】:Infectious flacherie virus of Silkworm Bombyx mori is prevalent in almost all the sericultural areas of the world, and is reported to cause severe losses to cocoon crops due to the death of the larvae, or losses due to decrease in the quality of cocoons and silk produced. Infectious flacherie virus was first reported in Japan during 1960 by Yamazaki et al, and since then the pathogen has been vastly studied in Japan, accomplishing the whole genome sequencing of the virus. India is the second largest silk producer in the world after China, and Karnataka state is the leading silk producing state in India, where sericulture is practiced at large scale and the occurrence of diseases is also high. Crop loss of about 47.9% has been reported from northern districts of Karnataka due infectious flacherie (Savanurmath et al, 1994) Northern Districts of Karnataka are non-traditional areas, where the farmers have taken up sericulture as a source of remunerative agricultural crop and thus the farmers are not well trained and expertise in diagnosing the diseases infecting the silkworms in these areas. As the silkworms are infected by various kinds of pathogens, including bacterial, protozoan, fungal and viral and as such the viral pathogens cannot be distinguished and diagnosed very easily, the proper knowledge of the pathogen causing the crop losses is very necessary before stepping up any control measures. Diseased silkworm larvae with typical symptoms of flacherie like initially decrease in consumption of mulberry leaves, and ununiform and delayed moulting, and variation in size of the larvae with in the same batch, The main symptoms of the diseased larvae were empty foregut, diarrhea and vomiting of brownish fluid, were collected from farmers of Northern districts of Karnataka. Initial diagnosis could not identify the pathogen under a microscope, and thus the pathogen was considered to be a virus. Such diseased larvae with symptoms were collected and the midgut was dissected out and the midguts crushed and treated with 0.2 M Acetic acid pH 4 for 15 minutes and neutralized to pH 7, and the extract was fed to the disease free silkworm larvae. Disease free layings of the popular hybrid silkworm race Feng 1x54A was selected and reared in the laboratory following the standard rearing procedures and using healthy mulberry leaves. The crude viral extract was fed to such silkworms by smearing the virus on the leaves and fed to the silkworm. The infected silkworms showed the same symptoms described above from third day post infection. It was also observed that the secondary bacterial infection to the diseased larvae resulted in enhanced larval death. The pathogen was isolated from the infected midgut tissues of silkworms. Silkworm midguts were homogenized in 50 mM Tris-HCl buffer, pH 7.2 and centrifuged at 4600 rpm for 10 min. The supernatant was treated with chloroform 5 times followed by centrifugation 3000 xg for 10 min. The aqueous phase was saturated to 40% with (NH4)2SO4. The precipitate obtained by centrifugation at 12000xg for 30 min was suspended in 50 mM Tris-HCl buffer with 100 mM EDTA pH7.2. The suspension was dialyzed overnight with 50 mM Tris-HCl buffer pH7.2 with 100 mM EDTA, and then centrifuged at 12000xg for 20 min. Virus was pelleted by centrifugation at 65,100xg for 2 h. The dissolved peilet was loaded on 30% sucrose and centrifuged at 125,000xg for 5 h. the sediment was dissolved in 0.05 M Tris-HCl buffer pH 7.2.Virus was quantified by UV spectroscopy after dilution to the required concentrations, 0.1% NaN3 was added before storing to prevent bacterial contamination. The UV absorbance spectroscopy of the pathogen showed a clear nucleoprotein pattern A240/260 =1.86 / 2.22, and at A 260/280 =2.22 / 4.51. The purified virus preparation was examined by electron microscope. Electron microscopic observation of the pathogen revealed non-occluded globular particles with approximate size of 25? run, while virus particles reported earlier from the same region were 28? run. Even though the size of the virus particl


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