Identification Of A New Menber Of Genus Potyvirus--Thunberg Fritillary Mosaic Virus

Some plants of Thunberg fritillary grown in Ningbo and Panan, Zhejiang province show typical mosaic and mottle symptoms. A filamentous virus was purified from the infected leaves using a method described by Chen et al. (1989) with some modification. By a series of identity, it was proved to be a new species in the genus Potyvirus and it has been tentatively named to Thunberg fritillary mosaic virus (TFMV).Purified viral particles were slightly flexuous with 750nm in length and 12-13nm in diameter. Cylindrical inclusions comprising 'pinwheels' and long 'laminated aggregates', which are characteristic of the presence of a potyvirus, were observed in the virus-infected plant cells. SDS-PAGE suggested a single coat protein with molecular weight of 32 kDa. The virus was readily mechanically transmitted to its original host, but not to any of 17 other widely used plant virus indicators. There was no serology relationship between the virus and 26 species viruses from 5 different genera. Counterimmunoelectrophoresis, rocket immunoelectrophoresis and Western blot may be used as qualitative methods to detect the virus in infected leave. However, immunodiffusion and ELISA are not suitable for detection of the virus.The complete nucleotide sequences of TFMV Ningbo and Panan isolates were determined respectively. The genomes of Ningbo isolate (accession AJ851866) and Panan isolate (accession AJ885005) are all consisted of 9723 nucleotides (excluding the polyA tail) with a single large predicted ORF. Both are highly homologous with 98.1% nucleotide identity and 98.0% amino acid identity and it is sure that both are belong to the same virus species. Computer analysis indicates that 107-109 (AUG) appears to be the initiator for ORF which terminates at nucleotides 9494-9496 with an UGA codon, followed by a 224 nucleotides 3'-NTR. The predicted translation product of the ORF (polyprotein) contains 3130 amino acids with a calculated M_r of 354.4 kDa.TFMV genome possesses a typical organization characteristic of genus Potyvirus. Proteolytic cleavage sites of TFMV polyprotein are IKEF/S (P1/HC-Pro), YRVG/G (HC-Pro/P3), YIFQ/A (P3/6K1), MKFQ/S (6K1/CI), LHFQ/S (CI/6K2), YSFQ/A (6K2/NIa-VPg), LVFE/S (NIa-VPg/NIa-Pro), WNFQ/A (NIa-Pro/NIb) and FCFQ/A (NIb/CP). The deduced CP length was 269 amino acids and calculated M_r was 30.7 kDa, which was similar to that estimated by SDS-PAGE.GAP comparisons for nucleotide identity and amino acid identity was made between TFMV and each of the 39 fully-sequenced potyviruses and the phylogenetic analysis was also done. The results indicate that TFMV and LMoV were the most closely related species in the 6K1, VPg and NIb cistrons for nucleotide identity. ABLAST research for ORF amino acid sequence or ORF nucleotide sequence shown that TFMV and LMoV might be the most closely related species with 53.0% amino acid identity and 56.7% nucleotide identity. In phylogenetic analysis for ORF amino acid sequence, TFMV shows a grouping with LYSV, LMoV, BYMV and C1YVV, and there is a significant grouping relation with LYSV. Both LYSV and LMoV infect bulbous monocot plants.Phylogenetic analysis for the 3'-end coding sequence (the partial Nib and complete CP) was made between TFMV and 82 species of the genera Potyvirus and Kymovirus. The results indicate that TFMV and LyMMoV (AF399672) were the most closely related species with 69.1% nucleotide identity overall and 72.8% nucleotide identity in CP region.Species demarcation criteria for the genus Potyvirus formulated by ICTV includes CP amino acid identity (<80%) and complete nucleotide identity (<85%) (Berger et a!., 2004). Recent studies have shown that a value of 76% nucleotide identity for either the complete ORF or the CP region is the most appropriate for Potyvirus species demarcation criteria (Adams et ai, 2005). The potyvirus from Thunberg fritillary described here clearly satisfies ICTV criteria demarcation for a new Potyvirus species (<57% nucleotide identity in its complete ORF and <73% nucleotide identity in its CP cistron to other known potyviruses), and it also has much distinctive biological and serological properties.