Isolation, Identification And Analysis Of The Complete Genome Sequence Of Black Queen Cell Virus China-JL1

In nature, honey bees are the most important pollinators. In recent years the epidemic disease on bees caused huge losses. Black Queen Cell Virus(BQCV) is a kind of pathogen and it was first reported in 1955. It mainly infects bee larvae and pupae, making the bodies dark and black, causing massive dead. More seriously, it will make the wall of the cell black. BQCV is a seasonal epidemic and often in unapparent infection, it can be spread by means of horizontal and vertical transmission of proliferation. In recent years, the research show that Nosema can not only as a medium organisms play an important role in BQCV transmission but also make the bees carried Nosema and then infected BQCV died soon. BQCV infected with host ranges widely, not only can infect a variety of bee species,but also infect spiders, centipedes and other arthropods. It can co-infected with the other honeybee viruses.The honeybee pupae were collected from Jilin apiaries, RNA was extracted as template for amplification. Based on the complete genome sequence of Black queen cell virus(BQCV) published on GenBank, we design 10 pairs of primers, using reverse transcription- polymerase chain reaction(RT-PCR)to amplify genes. We obtain the first complete genome sequence of BQCV isolates in China, named BQCV-JL1 strain. The BQCV-JL1 strain is consist of 8358 nucleotides, compared with the complete genome sequence of the other six BQCV strains published on GenBank, the homology is between 86 % and 93 %. The nucleotides positions of the ORF 1 of BQCV-JL1 strain is between 546 and 4676(4131 nt), and the ORF 2 of BQCV-JL1 strain is located between nt positions 5750 and 8203, between the two ORFs of BQCV-JL1 strain, there is a short ORF, called ORF 3, between nucleotides4891 and 5433(543 nt). The nucleotides positions of the first gene functional domain of BQCV-JL1 strain is between 546 and 5429, it’s contain both ORF 1 and ORF 3.There is an internal ribosome entry site(IRES) located before ORF 2, its last three bases are CCU(5642 nt~5644 nt),as an initiation codon facilitating the translation of ORF 2. The second gene functional domain of BQCV-JL1 strain is located between nt positions 5642 and 8203. The BQCV-JL1 strain shared high sequence identity(93 %)with the Hungary10 genotype. When we analyse the nucleotide sequences and amino acid sequences, we find that BQCV-JL1 strain showed close genetic relationshipswith the South Korea strain, that is to say, BQCV-JL1 strain and South Korea strain might have migrated from European countries. BQCV-JL1 strain exists differences on dividing the nucleotides positions of ORF with the other 6 strains, because of the gene mutation.In this paper, we isolate and identify BQCV from honeybee samples. We establish quantitative PCR method for detecting it. We also using DNAStar software for analyzing the complete genome sequence. We establish in vitro cultivate system for the proliferation of BQCV.