Biological And Serological Properties Of Two Orchid Viruses

Cymbidium mosaic virus (CyMV) and Odontoglossum ringspot virus (ORSV) are thetwo most prevalent and economically important viruses infecting orchids. They induce theleaf and flower of orchid greatly damaged, seriously affecting its quality and commodityvalue. In this study, antiserums specific for the two orchid viruses were prepared by differentmethods including traditional method and modern gene technique. It is very important for theorchid nurseries to control the two viruses' incidence and prevalence.Cattleya and cymbidium plants typically infected with virus were gathered from the city ofZhangzhou in Fujian. They were defined to be infected with CyMV and ORSV by ID-ELISA,electron microscopy and biological assay. The two kind of viral particles were purified fromthe infected orchid leaves. The spectrophotometric analysis showed that 6．4 mg of CyMVcould be obtained from 500 g of CyMV-infected Cattleya leaves, and 26．8mg of ORSV from100 g of ORSV-infected cymbidium leaves. Then the viral particles were used to preparespecific antiserums by conventional immunity method. The titers of antiserums wererespectively 1:512000 and 1:256000 by ID-ELISA determination.The ORFs encoding CP of CyMV and ORSV were successfully amplified by RT-PCR, andcloned into pMD18-T vector respectively, then into pET-29a(+) vector. The targeted genes weredetermined after analysis of enzymes digestion and plasmid PCR. The ORF encoding CP ofCyMV Zhangzhou isolate from Fujian is 672 bp, encoding a 23．6 KDa protein with 223 aa, andshares 98．7 % similarity with the Singapore isolate at nucleotide sequence leval, and 97．8 %at the amino acid sequence leval. The ORF encoding CP of ORSV Zhangzhou isolate fromFujian is 477 bp, encoding a 18．0 KDa protein with 158 aa, and shares 99．8 % similarity withthe Guangdong isolate in China at nucleotide sequence leval, and shares 100 % at the aminoacid sequence leval.The recombinant expression vectors were transform and induced by IPTG inEscherichia coli BL21(DE3) . The fusion proteins were purified by His-compatible column and 12 % SDS-PAGE, then were used to prepare the antiserums specific for CyMV and ORSVrespectively. High titer, sensitivity and specificity for CyMV and ORSV have been shown byID-ELISA analysis of the antiserums, both of which the titer was 1:51200, the work titer was1:1000 and 1:1600 , and the sap sensitivity was 0．39 mg/mL and 0．1 mg/mLrespectively. There were no clear serum reactions between the antiserums and 11 kind ofviruses such as Tobacco mosaic virus.With the above antiserums, the incidence of CyMV and ORSV in Phalaenopsis orchids inFujian was investigated by ID-ELISA. In the investgated nurseries 28．0 %-67．7 % of the sampleswere infected with CyMV, 10．5 %-60．0 % with ORSV, and 6．8 %-47．3 % with both of them. Ofall the 324 samples, 52．5 % were infected with CyMV, 42．6 % with ORSV, and 28．4 % withboth of them.