Isolation, Identification Of Bombym Mori Infectious Flacherie Vrius In Tongxiang And Preliminary Application Of ELISA Detection Method

Infectious flacherie virus is one of four viruses infecting Silkworm Bombyx mori.It is prevalent in almost all the sericultural areas of the world,and cause severe losses to cocoons and silk produced. Infectious flacherie virus was first reported in Japan duing 1960 by Yamazaki et al.,and since then the pathogen has been vastly studied in Japan. Bombyx mori infectious flacherie virus was first obtained by ultra-speed centrifugation method by Ayuzawa in 1972.Based on nucleic acid,capsid proteins and other characteristics of the virus,it was clssified as an insect picornavirus by Kawase et al.The complete genome sequence of infectious flacherie virus was accomplished(Isawa et al.,1998), sequence comparision(2C,3C and 3D et al.) revealed that BmIFV resembled mammalian picornviruses and plant picornviruses.But based on genomic organization and phylogenetic analysis,BmIFV is a Prcorna(-like) virus .Our country is the largest silk producer in the world,so the occurrence of this virual disease may be high.But its isolation and the prevalence of BmIFV in china is not reported. Bombyx mori infectious flacherie virus was isolated and purfied in our laboratory and named BmIFV- CHN001. Two hybridoma cell lines secreting monoclonal antibodies(MAbs) against Bombyx mori infectious flacherie virus (BmIFV) were produced and two monoclonal antibodies were used in ACP-ELISA for detection of BmIFV.The presence of BmIFV in Zhejiang was investgated with ACP-ELISA.Diseased larvae with typical symptoms of flacherie in Tongxiang Zhejiang were collected and the midgut was dissected out and the midgets crushed in PBS(PH=7.2).The supernatant was treated with chloroform in order to the proteins and saturated to 40% with (NH₄)₂SO₄.The precipitate obtained by centrifugation at 10700×rpm for 30 min was suspended in PBS(PH=7.2) and then loaded on sucrose gradient of 10-40% and centrifuged at 55000×g for 2 hours. Virus was quantified by UV spectroscopy after dilution to the required concentrations.The purified virus preparation was examined by electron microscope.Electron microscopic observation of the pathogen revealed non-occluded globular particles with approximate size of 26nm. The nucleic acid of the virus was isolated and was found to be RNA,The nucleic acid reacted positive with Orcinol test and negative to Diphenylamine test.The nucleic acid of the virus was reacted with DNase I and RNase A. The nucleic acid was further confirmed to be RNA by Formaldehyde agrose gel. The virus polypeptide profiles were anayzed by SDS-PAGE on 12% gels, electrophoresis revealed the four major bands and three minor bands.This is similar to BmIFV reported but no VP4.Two hybridoma cell lines secreting monoclonal antibodies(MAbs) against Bombyx mori infectious flacherie virus (BmIFV) were preparated and identified their preliminary characterization.Two mAbs were purified from the ascites and protein concentrations were measured. MAb 4E12 contained 2.252 mg of protein/ml, 3G3 2.432mg/ml. Two mAbs provided more lines in-radial immunodiffusion with anti-isotype sera, all of them provided an anti-IgG₁ line. The titres of ascitic fluids of the two MAbs are up to 10^-6 in indirect antigen-coated plate (ACP-) ELISA. ELISA showed that Two mAbs was only strongly reacted with Bombyx mori infectious flacherie virus.No other cross-reactions were observed with Bombyx mori Densovirus( BmDNV). Western-blot analysis showed that two MAbs can react specifically with the VP1(35kD) coat protein submit of BmIFV.Two MAbs were used in ACP-ELISA for detection of BmIFV. The presence of BmIFV in Zhejiang Tongxiang was investigated and the detections showed that BmIFV are common in samples of Zhejiang.This thesis first isolated and preliminary identified Bombyx mori infectious flacherie virus (BmIFV- CHN001 ) from Zhejiang Tongxiang. Based on McAb technology, We set up ELISA for detection of BmIFV and preliminary investigated the presence of BmIFV in Zhejiang. This research will help in better researching the characterization of this virus and control its prevalence in sericulture.