Biological Characteristic And Genomic Organization Of Grapevine Fanleaf Virus Hangzhou Isolate

1. A virus isolate with isometric particles, about 28-30 nm in diameter wascollected from grapevine (Vitis vinifera L. cv. Hongfushi) with fanleaf-like symptoms in the experimental orchard of Zhejiang University. In addition to typically intact particles, "empty" shell particles, which were penetrated by phosphotungstate, were found. On Chenopodium amaranticolor, the virus induced chlorotic local lesions or systemic mottle, and the symptoms usually faded as the plants aged. Whereas, in C. quinoa it caused deformation and necrosis on young leaves in addition to the above symptoms, and the symptoms maintained longer than on C. amaranticolor. The virus caused chlorotic local lesions on inoculated bean (Phaseolus vulgaris cv. Bountiful) leaves, and mottle, vein clearing and distortion on systemically leaves. The virus did not infected cucumber (Cucumis sativus) and several species of tobacco (Nicotiana spp.). Positive serological reaction of this isolate with antisera of Grape vine fanleaf virus (GFLV) from German and France was verified by analysis of ELISA and immun-electron respectively.SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) showed that the protein extracted from the virus particles was comprised of single polypeptide with molecular weight of 54kDa. Formaldehyde-denatured gel electrophoresis indicated that the genome of this isolate contained two RNA molecules with length of 7.3kb and 3.7kb respectively. A fragment with about 1.5kb cDNA was amplified by RT-PCR with the specific primers corresponding to coat protein gene of Grapevine fanleaf virus strain F13 (GFLV-F13). Thus, it was concluded that the Hangzhou isolate we obtained from grapevine is an isolate of GFLV, named GFLV-H (Hangzhou isolate).2. About 30 rhizosphere soil samples were collected from Xiaoshan Botanical Garden of Chinese Academy of Sciences (Beijing), Experimental Orchard in Shandong Agricultural University (Taian) and Experimental Orchards in Zhejiang University (Hangzhou) where the GFLV were reported. Nematode isolation and identification were carried out, and Xiphinema insigne Loos andX. brevicolle lordel lo & Da Costa were found to be the major species belonging to Xiphinema genus. Viral transmission test didn't demonstrate that X. insigae was capable of transmiting GFLV-H. Since the disease has not been spread in field. We suggested that the natural nematode vector for virus transmission wasn't existed in China.3. A pair of specific primer based on the sequences of coat protein and movement protein genes of GFLV-F13 was synthesized. A fragment about 2.5kb cDNA was amplified by RT-PCR, and the PCR product was cloned into pGEM-T easy vector, and transformed into E. coli. The sequence analysis of the clones showed that cloned cDNA composed of 2559 nucleotide residues, encoded 852 amino acids. The homology of ammo acid sequences between CPs of reported 7 GFLV isolates were over 90%.Alignment of GFLV-H CP animo acid with the corresponding region of 9 species Nepovirus indicated that the identity of GFLV-H CP amino acid sequence with that of 9nepoviruses CP was less than 30% additional to it was 69% common with that ofArabis mosaic virus (ArMV). MP animo acid sequence of GFLV-H was 38.5% identical to that o f Tomato ringspot virus (TomRSV), but less than 30% homology to other 4 nepoviruses. Amino acid sequence comparisons revealed a proline residue existed within a hydrophobic domain (P motif, upstream of GFLV-H CP). The motif is conserved between the putative MPs of nepoviruse and como-, capilo- and caulimoviruses. MPs of TomRSV and two comoviruses, CPMV and Red clover mottle virus (RCMV) have been shown to be structural components of tubule structures that are required for cell-to cell movement of theses virus.4. The ultrastructural alteration of C. amaranticolor and C. quinoa infected with GFLV-H was studied by transmission electron microscope. Isometric virus particles were found in the cytoplasm of infected parenchyma cells. The membrane-walled tubules contained virus particles often aggregated. Virus part...