Sequencing And Sequence Analysis Of The Genome Of Honeybee Sacbrood Virus

Honeybees is a very important economic resource insects, it can create a very substantial economic value for human. But, it also often violated by a variety of diseases, and thus hinders the development of beekeeping. Honeybee sacbrood is a very serious hazards disease, it is a contagious disease of honeybees, and it is caused by sacbrood virus proliferation in the larval stage, pupa stage and adult bee stage. The larva infected sacbrood virus made it cannot pupate, and the ecdysial fluid gathered in the epidermis to form a "capsule" shape. Honey bee sacbrood virus (SBV) is kinds of picornavirus, a small positive strand RNA virus, and the genome include 8832 nucleotides.According to the genome sequences of honeybee sacbrood virus (Genbank:AF092924) published on genbank, design a pair of specific primers, and detect the highly suspected samples with the method of RT-PCR. The results we amplified target band, then cloned and sequenced the PCR products, and found high identity between SBV of GUANGDONG by blast program of genbank. It proved that the sample infected the sacbrood virus.To further understand the molecular structure and the pathogenesis of the virus, we determined the genome sequence of the virus. According to the genome sequences of honeybee sacbrood virus (CSB V-GD (AF469603), CSBV-LN (HM237361.1), SBV-UK (AF092924)) published on genbank, design ten pairs of amplifiable primers. Using primer walking sequencing methods and total RNA extracted from the sample as the template, amplified by 10 overlapping coverage of the genome fragments, after cloning, sequencing and software stitching, we obtained the genomic sequence. The sequencing results showed that the genome of the sacbrood virus include 8519 nucleotides, encoding 2839 amino acids. Through the relevant molecular biology sequence analysis software, found posted on the Internet CSBV-LN (HM237361.1) has a high homology, reaching 98.8%. By comparison of the phylogenetic tree analysis found that the genetic relationship between the virus and CSBV-LN (HM237361.1) recent. Otherwise, nucleotide and deduced amino acid sequence analysis showed that the nucleotide sequence missing part of base at 2296-2366, resulting in the deduced amino acid sequence missing part of amino acids at 766-775 and 786-789. This part of the base deletion affect on molecular biological characteristics and pathogenesis need further study,we did not use the RACE method determined genome sequence, so only got a partial sequence of the genome, because of relatively large variability of the virus, it is also a great reference for researching on molecular biology of the virus.