A Universal RT-PCR Detection And Identification Methods To Potyvirus

The Potyvirus genus in Potyviridae is the largest RNA virus genus with146definitive species and32tentative species. The genus harbors some most important and destructive plant pathogens. The universal RT-PCR detection methods for Potyvirus have been reported since1990s. Due to the rapid expansion of the genus and the sequences within the genus in the last decade, the conserved motifs used for designing primers for potyvirus detection are no longer conserved, leading to failures in the detection of some potyviruses.In order to develop a rapid, sensitive, and universal RT-PCR detection method, some species of potyviruses which have significant sequence differences among them were used for searching the conserved motifs.A pair of degenerate primers was designed in the middle of NIb and end of CP. The primers were tested in a touchdown RT-PCR detection method. The universality and sensitivity of the method were validated by its detection of8known potyviruses from13species of common plants, and no non-specific reactions were observed in the tests.Using the method,15suspected virus-infected plant samples were tested. The result showed5positive samples. DNA fragments amplified by RT-PCR were sequenced using the Sanger method and the sequences were analyzed. The results showed that:1. The potyvirus in the wild tomato collected from Chengmai is Wild tomato mosaic virus (WTMV). It shares the highest sequence homology with WTMV Laichau isolate from Vietnam, having91.1%and93.3%similarity on nucleotide and amino level respectively; the potyvirus in the wild tomato collected from Guizhou is Tobacco veinal binding mottle virus (TVbMV) as it shares the highest sequence homology with TVbMV YN9.1isolate from Yunnan, having99%similarity at both the nucleotide and the amino acid levels. This is the first report of TVbMV natural infection of wild tomato.2. The potyviruses found in datura are two new strains of Chilli ringspot virus (ChiRSV), sharing the highest sequence homology with ChiRSV HN/14isolate from Capsicum chinense, having80%,83%similarity at the nucleotide level and88%,91%similarity at the amino acid level, respectively. Using a pair of primers annealing to the NIb region and the3’UTR of ChiRSV, the3’terminal sequences of ChiRSV infecting datura were cloned and sequenced. A deletion of three nucleotides was found at the end of the CP3’-terminal, resulting in the early translation termination of the CP protein by two amino acids. A recombination event was detected during the construction of the phylogenetic tree and confirmed by RDP analysis. The ChiRSVRec strain which is more relative to ChiRSV HN/14is a recombinant formed between the datura strain (ChiRSVD) and a strain (ChiRSVc) isolated from C. chinense. This is the first report of ChiRSV recombination.3. The datura sample was also co-infected by another potyvirus, Chilli veinal mottle virus (ChiVMV). By using genome-walking and RACE, an8067-bp (polyA end was not included) partial genomic sequence exhibiting typical Potyvirus signatures was obtained. Sequence analyses of the CP gene and partial genomic sequence showed that the ChiVMV isolate from datura is a novel strain distinct from other ChiVMV isolates.4. The potyvirus detected in jasmine from Haikou is Jasmine virus T (JaVT). By using genome-walking and RACE, a8749bp (PolyA end was not included) partial genomic sequence was obtained. Sequence analysis of the CP gene showed that the isolate shares the highest homology with JaVT Taiwan isolate, having91%and96%similarity at the nucleotide and the amino levels, respectively. The phylogenetic analysis showed that JaVT is most closely related to Scallion mosaic virus and Narcissus late season yellows virus.5. The potyvirus in Catharanthus spp. is a novel virus, tentative named Catharanthus veinal mottle virus (CaVMV). By using genome-walking and RACE, a9243bp (PolyA end was not included) nearly complete genomic sequence was acquired. The sequence shares a typical Potyvirus genome organization, but the virus movement motif CCC was replaced by ISC. The virus is distinct from other species, according to CP gene phylogenetic analysis, the closest relative is Cocksfoot streak virus sharing52.98%similarity; according to phylogenetic analysis of nearly complete genome, the closest relative is Papaya leaf distortion mosaic virus sharing50.34%similarity.