Development of detection methods for two important orchid viruses, Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV), and construction and characterization of an ORSV infectious cDNA clone
Date Issued
2008
Date
2008
Author(s)
Lee, Shu-Chuan
Abstract
Orchid is one of the most important commercial plants in Taiwan. Virus infection in the mass production of orchid may result in great economic loss. Detection and screening the virus infected plant will reduce the risk of mass production and increase the plant quality. Three detection methods were developed for the detection of two covalent and important orchid viruses, Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). The first one was a multiplex RT-PCR method. Specific primers were designed based on the respective viral coat protein genes. In addition, one primer pair derived from the plant mitochondrial NADH dehydrogenase gene (nad5) was used as an internal control amplified in the multiplex RT-PCR. Application of this multiplex RT-PCR could greatly reduce the cost and false negative results in the routine detection. The second method was I-ELISA detection using antisera against by purified viral capsid proteins expressed in bacteria. These antisera were then designated as home-made CymMV CP antiserum (HM-Cy) and home-made ORSV CP antiserum (HM-OR). The high specificity of HM-Cy and HM-OR were validated by immunoblotting and both of them showed low background reactivity to healthy samples, especially when compared with the commercially available anti-ORSV antibody. Furthermore, they had a higher S/H ratio (sample OD405/healthy control OD405) than commercial antibodies in tested orchids. The third method was so-called a multiplex immunocapture-reverse transcription-polymerase chain reaction (mIC-RT-PCR) which combines the advantages of ELISA and mRT-PCR. Purified HM-Cy IgG and HM-OR IgG were coated onto the polystyrene tubes to enrich viral particles from plant extracts. After heating, viral RNAs were released and followed by the multiplex RT-PCR (mRT-PCR) amplification and gel electrophoresis. The simultaneous detection of CymMV and ORSV was successfully performed by mIC-RT-PCR in single- or mixed-infection samples. The sensitivity of mIC-RT-PCR was 25 - 125 times higher than I-ELISA. These three detection methods provide more options according to different requests. To investigate the viral biological properties, the ORSV full-length cDNA clones were constructed under the driven of a T7 promoter. A full-length cDNA clone, pORSV-7, with 6611 nt containing four open reading frames showing a systemically infection, was analyzed and compared with six reported ORSV isolates. The sequence homology of different ORSV isolates was ranged from 82 to 99% in nucleotides and 94 to 100% in amino acids. The ORSV MP was somehow confusing due to its different annotation of MP ORF; however, our data suggested that the product of ORSV-7 MP ORF which only containing 840 nucleotides (279 amino acids) is sufficient for virus function. Comparisons of two full-length cDNA clones, pORSV-2 and -7, we found differences in five amino acids including one in replicase protein, two in movement protein, and two in capsid proteins, respectively. Chimeric cDNA clones were generated to investigate the viral movement determinants. All chimeric constructs could replicate in Nicotiana benthamiana protoplasts at the similar level. Inoculation tests with different combinations of MP and CP of pORSV-2 and -7 revealed a complementary interaction in ORSV long-distance movement. We further narrow down the determinants of cell-to-cell movement in Chenopodium quinoa plants to the Met97 in the MP of ORSV-7. The mutation of Thr49 to Ile49 in the MP of ORSV-2 complement the Met97 to Val97 mutant of ORSV-7 to rescue its movement. CP was dispensable for ORSV cell-to-cell movement, but is important for long-distance movement. The sufficient long-distance movement of ORSV in N. benthamiana was mapped to the Glu100 in CP of ORSV-7.
Subjects
orchid
CymMV
ORSV
virus detection
full-length infectious cDNA clone
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