云南省新分离蚊浓核病毒的分子特征研究

Molecular Characterization of Genome of Mosquito Densovirus Newly Isolated in Yunnan Province

对云南省新分离的14株浓核病毒进行基因序列测定和分析,以了解浓核病毒基因组结构及特性.用浓核病毒基因组扩增引物（DNV3F,DNV3R）,RT-PCR扩增片段,PCR产物直接测序,拼接后获得基因序列.通过Clustal X（1.8）、DNAStar、GeneDoc （3.2） 等生物学软件进行核苷酸序列及氨基酸序列分析和病毒的系统进化分析.结果表明,2007年7～8月,在云南省西部中缅边境地区采获蚊虫4属29种54 673只,分离到14株浓核病毒,其中来自三带喙库蚊Culex tritaeniorhynchus 10株,中华按蚊Anopheles sinensis、伪杂鳞库蚊Culex pseudovishnui、环带库蚊Culex annulus和致倦库蚊Culex pipiens quinquefasciatus各1株.新分离株与我国其他省市的分离株核苷酸同源性很高,在99.9%～100%之间;氨基酸同源性在99.7%～100%之间.基于NS1和NS2基因进行进化分析显示,云南新分离的14株浓核病毒与以往中国分离株进化关系最接近.

To provide information for the genomic structure of mosquito densovirus （ DNV ）, nucleotide of DNVs new strain isolated in Yunnan was sequenced and analyzed. Primers （DNV3F, DNV3R） were used for genome amplification. RT-PCR was performed to amplify the fragments, which were sequenced directly and all the nucleotides were connected to acquire the genome. The computer software was used to analyze the nucleic acid data, deduce the amino acid sequence and phylogeny including ClustalX （1.8）, DNAStar, GeneDoc （3.2）. The 14 DNVs were isolated from Culex tritaeniorhynchus （10 isolates）, Anopheles sinensis （1 isolate）, Cx. pseudovishnui （1 isolate）, Cx. annulus （1 isolate） and Cx. pipiens quinquefasciatus （ 1 isolate） of Yunnan, China. The results revealed that the new isolates were with high homology in nucleotide sequence （99.9% to 100% ）, and in amino acid sequence （99.7% to 100% ）, to the strains isolated from other provinces. Analysis based on the NS1 and NS2 genome sequences showed that the 14 new isolates of densovirus from Yunnan were with the closest homology to those formerly isolated from China.