Isolation And Genomic Sequence Analysis Of Deformed Wing Virus

Deformed wing virus(DWV) is a pathogen of causing residual wing bee disease, can infect various developmental stages of the bees, the hazard in the pupa stage is not obvious, but there will be incomplete wings after emergence and rapid death; when the adult bees were infected, the symptoms were wings malformations, abdominal shrinking and fading. Although the latent infection bees were no obvious clinical symptoms but its life will be shortened. Because DWV is closely related to Varroa mite, so, there is a lot of research about them. Recent studies show that DWV is one of the main virus that triggered "colony collapse disorder"(Colony collapse disorder, CCD). DWV has become an important reason for the abnormal disappearance of honeybee colonies in popularity worldwide. DWV produces a 30 nm icosahedral particle consisting of a single, positive strand RNA genome and belong to the iflaviruses.In order to obtain DWV, we have isolated and cultured the suspected DWV samples in healthy bee larvae, due to propagation characteristics DWV invisible, we do not found deformities and other typical symptoms, but by transmission electron microscopy, we found the DWV virus particle. According to the GenBank published bees residual wing disease(DWV) genomic sequence(GenBank: NC004830 and AY292384), in which the conservative district designed a pair of specific primers by RT-PCR detection methods identified after the initial judge get to a DWV. After obtaining the genes were cloned and sequenced by Blast program on GenBank for comparison, the United States found that the residual strain of bees on the wing virus sequences highly homologous in GenBank, the homology of 96%. This study is further evidence of the success of a bee isolated remnant wing virus.According to the bee deformed wing disease(DWV) genomic sequence(GenBank: NC004830 and AY292384) published in GenBank, using TaqMan fluorescent probe technique principle, for residues conserved sequence of DWV designed a pair of specific primers and a probe to establish a rapid detection method,a fluorescent PCR method detect DWV. The method can detect acute bee paralysis virus, chronic bee paralysis virus, Sacbrood Virus and black queen residual virus and no cross-reaction units, meanwhile, this method can detect low levels of virus of positive plasmids about 1.0 × 102 copies/μL.The method has high sensitivity, reproducibility and stability.The coefficient of variation less than 1.6%. The method is rapid, sensitive, specific and reproducible, suitable for rapid quarantine DWV of bees and bee products.In order to know the molecular structure and molecular biological characteristics of DWV, we had sequenced the virus genome. According to deformed wing virus genome sequences published on GenBank(GenBank: NC004830 and AY292384), we had designed 11 pairs of primers, utilizing primer walking method, take virus total RNA extracted from samples as template, we had achieved 11 fragments, after cloning, sequencing and software stitching, ultimately get the genome sequence. Sequence analysis showed that the deformed wing virus genome sequence was consist of 9826 nucleotides, and encoding 2375 amino acids. Through the analysis of relevant molecular biology software for the measured sequences found that it has a high homology with Korea-1(JX878304) published on GenBank about 96.6%. Through comparative analysis of the evolutionary tree we found that the virus is related to Korea1(JX878304)and Korea2(JX878305) on genetic relationship.