The Cloning And Analysis Of The Complete Genome Sequence Of Chilli Ringspot Virus (ChiRSV)

Capsicum chinense Jacp. is a unique hot pepper species in Hainan, which is extremely sensitive to viral infection. In recent years, plant viruses had gradully became the most serious pathogen of Capsicum chinense Jacp., which significantly brought about negative impact on its economic benefit and imposed restrictions on the development of hot peppers industry of Hainan in a large scale.Chilli ringspot virus (ChiRSV) was first discovered in Vietnam in2007which has no publication of the complete genome sequence to date. There were only3’terminal sequences (about1.6kb long) from two ChiRSV Vietnam isolates (ChiRSV-VN/C8, ChiRSV-VN/C9) published in GenBank. In2009, ICTV decided to classify ChiRSV as a new species belonging to Potyvirus, Potyviridae.In the present study, we collected virus samples from infected peppers leaves in Yingzhou, Hainan, after the extraction of the total RNA, then by using RACE and genomic walking methods we successfully obtained the complete sequence of ChiRSV Yingzhou Isolate (ChiRSV-HN/Yingzhou) and submitted it to GenBank (JQ234922), which is the first publication of ChiRSV complete gemome sequence in the world. By a series of bio-informatics analysis of this sequence, we determined its evolutional position and thus paved the way of development of anti-viral genetic engineering. The detailed procedures are listed as following:1. By using3’RACE, we cloned the3’terminal sequence of ChiRSV which was1571bp in length. The result of the analysis of this sequence suggested that this region included the complete CP gene which had94%and93%identities with two Vietnam isolate in amino acids sequences, respectively. According to the criterion for the classification of Potyvirus authorized by ICTV, we concluded that this3’terminal sequence belonged to a ChiRSV genome.2. By using genomic walking method, we obtained the remaining sequence apart from the3’terminal sequence. In each "walking", the up-streaming primer was designed by the reference of the conservative sequence of Potyvirus, then the down-streaming primer was designed according the sequence obtained in last "walking". We altogether designed6pairs of primers by using which we successfully amplified6fragments with the length of1193bp,2185bp,1585bp,1426bp,871bp,2113bp, respectively.3. By using5’RACE, we cloned the74bp-long5’terminal sequence. Then by referring the overlapping regions of each amplicon, we successfully aligned the complete sequence of ChiRSV-HN/Yingzhou with the length of9615bp.4. By the analysis of the complete sequence, we found that the5’-UTR and3’-UTR were194bp and181bp in length respectively, between them, there was a large open reading frame (ORF) encoding a349.1kD polypeptide composed of3079amino acids which would be enzymatic lysis into10smaller poly peptides after translation. Pretty Interesting Potyviridae ORF (PIPO) starts at the AAA condon (No.2957-2959bp), and the most ubiquitous AUG codon is ended by the UAG termination codon (No.3137-3139bp), which encodes a6.6kD polypeptide composed of60amino acids.5. By cluster and phylogenetic analysis, we discovered that ChiRSV-HN/Yingzhou has the closest relationship with Tobacco vein banding mosaic virus, EF219408, the identities between their nucleotides and amino acids sequence were64.8%and68.7%respectively, which provide another aspect of evidence that ChiRSV belongs to Potyvirus.