Study On Molecular Characterization Of Two Filamentous Viruses Associated With Grapevine Rugose Wood Complex

Grapevine virus A (GVA) and Grapevine virus B (GVB) are agents of grapevine stem-grooving and cork bark disease respectively, which are component of Grapevine Rugose wood complex (RW). Although these viruses have been reported in China for about 2 decades, until date a few of researches has been done on Chinese isolates. In this study, investigations were carried out on GVA and GVB infection in three distant areas: Zhengzhou Henan province; Wuhan Hubei province and Liaoning province. In addition, Group identification on GVA Chinese isolates was also done using two sets of group-specific primers. GVA genetic variability and population structure were analyzed based on its coat protein (CP) gene utilizing single-strand conformation polymorphisms (SSCP). In addition, the characterization of GVB Chinese isolates was studied. The main results obtained are as follows:1. With the use of Triple antibody sandwich ELISA (TAS-ELISA) and reverse transcription-polymerase chain reaction (RT-PCR), forty and six samples were detected positive to GVA and GVB respectively. The detection rates of GVA in three areas were much higher in accordance with previous results. Especially for samples collected in 2008 in Zhengzhou, the detection rate was 82.8%, and was much higher than 2002. Vectors' transmissions maybe charge to be a core cause of this effect. The detection rate of GVB was comperatively lower to that of GVA, the highest rate was 25% in Liaoning Samples.2. Group identification of 40 isolates showed that all the isolates obtained belonged to groupâ… orâ…¡, no isolate belonged to groupâ…¢. CP genes were cloned from 14 GVA Chinese isolates with the size of 597 nt. SSCP profiles based on clones could be divided into three categories according to the number of predominant haplotype. Clones representing different haplotypes of each isolate were sequenced. Genetic distance revealed that seven isolates contained highly divergent variants, which showed the mixed infection and the heterogeneous population of GVA. Phylogenetic analysis revealed that there were three main clusters represented by groupâ… ,â…¡andâ…¢. Groupâ… was divided into two subgroups named IA and IB. All the variants obtained were clustered into groupâ… ; moreover, the majority of them belonged to subgroup IA.3. Isolate AF showed a unique population structure. Among seven mixed infection isolates, four isolates contained variants of two subgroups. Variants of subgroup IA were predominant in three isolates, but only for AF, frequency of variants of subgroup IB was slightly higher than that of subgroup IA.4.3'terminal region containing ORF5 and 3'Un-translated region (3'UTR) of GVB was cloned from two isolates LCLA and QMWH. Nucleotide sequences analysis revealed a higher homology in 3'UTR than that of ORF5. Phylogenetic analysis showed that these two isolates clustered together.5. CP genes of GVB were cloned from isolates LCLA, TR and WHBJX. Homology of nucleotide and predicted amino acid sequences of isolates TR and WHBJX were 98.5%. Phylogenetic analysis showed a close relationship between them, while isolate LCLA was clustered with Sichuan isolate SL10 and Japanese isolate. Comparison of the predicted 197 aa sequence of the CP of TR/WHBJX and other isolates of GVA revealed five amino acid alterations, but only at the position 56, the neutral amino acids-glutamine was substituted by the strong basic arginine (R) in TR and WHBJX which changed net charge, which might be associated with the pathogencity of virus.