Molecular characterization and diagnosis of a new emerging Cucurbit chlorotic yellows virus in Taiwan

[[abstract]]Viruses of the family Closteroviridae have large positive-sense single-stranded RNA genomes encapsidated in long flexuous virions and are divided into three genera, Closterovirus, Ampelovirus and Crinivirus. Different from the other two genera, Crinivirus has bipartite particles of 650-900 � 12 nm comprising two genome segments, denoted RNA1 and RNA2, and is transmitted by whitefly. Criniviruses infect several important crops worldwide, causing symptoms of interveinal chlorosis, yellowing and brittleness of leaves accompanied by severe yield losses. In April 2009, typical crinivirus-induced symptoms were observed on melon (Cucumis melo L.) plants in Yunlin County, Taiwan. The degenerate primers designed for Closterovirus and Crinivirus were used in nested reverse transcription-polymerase chain reaction (RT-PCR) to amplify a 0.5 kb DNA fragment from the symptomatic melon tissues. Subsequently, the amplified DNA fragment was determined to share 100% identity with the reported sequence of Cucurbit chlorotic yellows virus (CCYV), a new Crinivirus species found in Japan. In this study, an isolate of crinivirus denoted as Crinivirus-TW was obtained from a plant of Nicotiana benthamiana inoculated by whitefly transmission. Furthermore, the complete nucleotide (nt) sequence of Crinivirus-TW RNA2 was determined as 8,041 nt in length containing eight open reading frames (ORFs), P5, Hsp70h, P6, P60, P9, CP, CPm and P26. Sequence analyses revealed that the RNA2 sequence of Crinivirus-TW shares 99% nt identity with that of the original Japan isolate of CCYV. Thus, Crinivirus-TW was identified as an isolate of CCYV, renamed CCYV-TW. Phylogenetic analyses of the Hsp70h, P60, CP, CPm and P26 among Crinivirus species revealed that CCYV-TW is closely related to Lettuce chlorosis virus (LCV), Bean yellow disorder virus (BnYDV) and Cucurbit yellow stunting disorder virus (CYSDV). The CP of CCYV-TW expressed by the bacterial pET expression system was used as an immunogen for production of monoclonal antibodies (MAbs). The MAbs prepared were successfully used to detect CCYV in diseased plants by western blotting and to examine the accumulation of virus in phloem cells by tissue blot immunoassay (TBIA). Moreover, primer pair Crini-hsp70h-f/Crini-hsp70-r, designed from the Hsp70h gene, coupled with primer pair mt-F2/mtR1, designed from the plant mitochondrial NADH dehydrogenase (nad5) gene, used in multiplex one-step RT-PCR was developed as an effective detection method and applied in field survey. A number of 253 diseased melon samples collected from central Taiwan during February to June in 2010 were tested by multiplex one-step RT-PCR. An increased incidence of 3.8% to 100% was noticed. Our results indicated that CCYV has become an important threat for the production of cucurbits in Taiwan, and it should be majorly concerned by government