Sequence Analysis Of Genomic Coding Region Of Chronic Bee Paralysis Virus Ch1 And The Establishment Of Semi-nested RT-PCR Detection Method

ObjectiveThe CBPV(Ch1 strain of CBPV)was separated and purified from suspicious infected chronic bee paralysis virus(Chronic bee paralysis virus,CBPV)and then identified by RT-PCR.Thegene coding region of Ch1 strain was cloned,and then the gene sequenceof coding region was analysised with DNAStarsoftware.On this basis,we established a semi-nested PCR testing methodof CBPV for clinical test.MethodsDifferential centrifugation method was used toisolate and identify the C h1 strain.Primers were designed referring to the French region CBPV isol ates Fr1(Gen Bank No.EU122229,EU122230).Thegene coding region of C h1 strain was cloned,sequenced,spliced and thecompared with reference str ain by RT-PCR.The Ch1 strain coding region of nucleotide sequenceand deduced amino acid sequence were obtainedand analysed.Referring to re ports,the use of DNAStar software analysis inferredstructuralproteins SP1,SP2,and RNA-dependent RNA polymerase(RNA-dependent RNA polymera se,Rd Rp),homology comparison of SP1 and SP2 protein gene,phylogenetic analysis Rd Rp as a arget genes.On thisb-asis,three specificprimers were designed,asemi-nested RT-PCR detectionmethod was established,Optimiz ed reaction conditions,and its sensitivity,sp-ecificity were detect ed for clinical application.Resu LtsThe Ch1 strain of CBPV was separated,purified and identified from suspiciousinfected sample.Sequence analysis revealed that,CBPV coding region of the gene from the Ch1 RNA1 and RNA2 of two parts,a total length of3467 nucleotides RNA1 genes encoding three reading frames,ORF3 main encoding Rd Rp;RNA2 full length c DNA nucleotide 2305,4 encoding reading frame encoding structural proteins which ORF2 SP2,ORF3 encoding structural proteins SP1.Homology comparisos showed that,Ch1 strain RNA1 nucleotide sequence and the French isolates Fr 1 and Fr 2 homology were 91.7% and96.1%;Ch1 strain RNA2 nucleotide sequence and the French isolates Fr 1 and Fr 2 isolates nucleotide sequence homology 91.6% and 95.5%,where Ch1 strain and isolates nucleotide sequences SP2 Fr 1 and Fr 2 nucleotide sequence homology were 91.7% and 95.4%,while the highest and Uruguay SP1 isolates nucleotide sequence homology up 96.9%,with the French isolates Fr10 nucleotide sequence homology to a minimum of 93.2%,Ch1 Rd Rp nucleotide sequence and the nucleotide sequence of France with isolates Fr2 borne up to 96.5%,with the French isolates Fr9,Spain isolates Sp1 nucleotide sequence homology to a minimum of 91.5%.As the target genes were Phylogenetic analysis showed that,Ch1 Strain and Fr2(Gen Bank No.EU122231)located in the same phylogenetic tree based on the branch Rd Rp,high affinity.On this basis,a formula established by RT-PCR method,the minimum detection limit of 10-3pg,bee health,acute bee paralysis virus(Acute paralysis virus,AB-PV),the bee larvae cystic disease virus(Chinese sacbrood bee virus,C-SBV),black queen station virus(black queen cell virus,BQCV),bee residual wing virus(Deformed wing virus,DWV)c DNA was no cross reaction,detected in 4 samples were positive in 20 clinical samples samples can be used in the clinical testing CBPV.Conclusions1 ? The Ch1 strain of CBPV was isolated,homology comparison of its nucleotide sequence and deduced amino acid sequence was made,and phylogenetic tree was constructed with Rd Rp as a target gene.2?A semi-nested PCR detection was established,which has high sensitivity and specificity for detection of clinical samples and preliminary resu Lts show that this method can be used for clinical test of CBPV.