Characterization, infectious clone construction and antiserum preparation of Pitaya virus X
| Autor: | Yong-Shi Li, 李勇賜 |
|---|---|
| Rok vydání: | 2010 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 98 Pitaya (Hylocereus spp.), a climbing succulent plant with delicious fruits in the family Cactaceae, is newly cultivated in Taiwan and becoming an important tropical fruit crop. According to the literatures, Cactus virus X (CVX), a widespread potexvirus, is the first reported virus infecting pitaya plants. From the previous field survey in the Yanmingshan orchard from 2006 to 2008, we isolated and identified three potexviruses from pitaya plants. These viruses included CVX, Zygocactus virus X (ZVX), first reported in Taiwan, and Pitaya virus X (PiVX), a new species of potexvirus. Before too long we discovered that the P37 isolate of PiVX was contaminated with CVX. As a result, the original PiVX-P37 sample was used for single lesion isolations on Chenopodium amaranticolor again. Finally, a pure virus isolate was obtained and confirmed by multiplex RT-PCR, and also named as PiVX-P37. This virus isolate produced chlorotic lesions with yellow halo which turned into necrotic lesions on the inoculated leaves of C. amaranticolor at 5-7 days post inoculation. In host range test, PiVX-P37 could locally infect C. amaranticolor, Tetragonia expansa and Celosia argentea, and systemically infected C. quinoa and Hylocereus undatus. The virions of PiVX are in filamentous shape with 450-500 x 12-13 nm by transmission electron microscopy, and its capsid protein is about 24-kDa by SDS-polyacrylamide gel electrophoresis analysis. To further study the properties of PiVX-P37, the full-length cDNA clone with a 35S promoter was constructed and its biological activity was tested by inoculating plasmid DNA to C. amaranticolor and C. quinoa. Five out of seven full-length cDNA clones were infectious on both tested plants, but their infectivities were different. Three of them were completely sequenced, and the sequence identities of these clones are over 99% compared to PiVX-P37. The full-length genome of PiVX-P37 contains 6677 nucleotides without poly(A) tail and five open reading frames. The genome organization of PiVX-P37 is similar to those of other potexviruses. According to sequence comparison of potexviruses, PiVX-P37 is most homologous to Schlumbergera virus X (SVX). The nucleotide sequence identities of RdRp and CP gene between PiVX-P37 and SVX are 70% and 69%, and the amino acid sequence identities are 77% and 81%, respectively. According to the species demarcating criteria of the genus Potexvirus in the 8th ICTV report, there is no doubt that PiVX should be a new species of pitaya-infecting potexvirus. We further noticed the p35S-PiVX-1 clone with weaker infectivity containing sequences different from those of other infectious clones. To develop an effective detection tool, we expressed and purified the recombinant CP of PiVX as the antigen to generate the antiserum. This PiVX antiserum could specifically and sensitively react to PiVX in immunoblot and indirect-ELISA assays. In this study, a new pitaya-infecting potexvirus, PiVX, was purified, and the infectious cDNA clones were successfully constructed. These results provided a new material for studying the cactus-infecting potexviruses and for developing virus-based vector in the future. The anti-PiVX antiserum will be useful in research as well as in disease survey of pitaya. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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