| Citrus tristeza virus (CTV) is the causal agent of citrus tristeza disease, which has extensively affected the production of citrus. CTV is a member of genus Closterovirus in family Closteroviridae. The virus has a genomic RNA of approximately19.3kb, which ranges the largest genome among the currently known plant RNA viruses. At present, the genomes of27CTV isolates have been completely sequenced. It is known that CTV is highly variable in molecular constitution and recombination between different genotypes occurs frequently in the virus, which contributes to the complex of the population structure of CTV. In nature, CTV is mainly spread by aphids, among which Toxoptera citricidus is the most effective vector. Our previous study indicated that CTV isolate S45(CTV-S45) contains multiple molecular variants and obtained it partial genomic sequences. To understand the population structure and molecular characteristics of CTV-S45, this study was taken to analyze the genomic sequences of CTV-S45and its molecular variant constitution post the transmission by citrus brown aphid. Results are illustrated as follows:1. Eight sets of primers targeting to six fragments in the regions6-5340nt at the5’ end and9516-15965nt at proximal3’end of CTV genome were designed on the basis of sequence information of the virus obtained previously. RT-PCR was employed to amplify those six fragments, and3-11clones of each amplicon were sequenced. Sequence analysis showed that all those fragments had molecular variation and consisted of two to six molecular variants. The fragments U (6-725nt)、X (9516-11236nt) and Z (13426-15965nt) contained sequence variants having the highest similarities of97.8-99.0%to that of CTV mild strain T30and severe strain NZRB-G90, respectively. The fragment X was highly variable, and six sequence variants were identified in the fragment. Sequence alignments revealed the presence of recombinants in fragments X and W.2. Sequences obtained in this study and a previous study were compared and assembly by using DNAMAN, A full genome sequence of19256nt (S45-R) and two partial genome sequences of9690nt (S45-R1,1-9690nt) and5120nt (S45-R2,10846-15965nt) were obtained. Sequence analyzes showed that the nucleotide sequence of S45-R was80.0%-90.9%identical to the genomic sequences of27CTV isolates available in GenBank, and had the highest similarity to the mild strain T30. However, its CP gene had the highest similarity of97.5%to the isolate NZRB-G90, and its P23had the signature amino acid sits of T30, indicating that S45-R was a recombinant between divergent variants. S45-R1and S45-R2had the highest similarity of98.2%and98.1%to isolates T30and NZRB-G90, respectively.3. CTV-S45was transmitted onto sweet orange seedlings by brown citrus aphids. After three months post their transmission, RT-PCR results confirmed that three seedlings (marked as TV1, TV12and TV13) of sweet orange were positive to the CTV isolate. The fragments U, X and Z and CP gene were amplified by RT-PCR and sequenced. Results showed that nt similarities intro-and inter-subisolates transmitted by aphid were over99%, and all those sequences had similarities of98.5-99.8%to S45-R. Those results indicated that brown citrus aphids selectively transmitted the CTV variant and the full genomic sequences S45-R determined in this study was from the aphid-transmitted subisolate. |
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