Retrovirus Component Integrate Within The Genomes Of Fowlpox Virus And Construction Of Recombinant Fowlpox Virus Expressing GFP

To clarify the integration mechanism of Reticuloendotheliosis virus (REV) into the genome of FowlPox virus (FPV), some different vaccines and wild- type isolation strains of FPV were detected and analysized by Polymerase Chain Reaction (PCR) with severa pair primers and homologous recombination based on the plasmid containing the Green Fluencescence Protein (GFP). This article has a important intention for the natural recombination of different RNA virus and DNA virus.1 The integration of REV sequence into the geneome of different FPV vaccinesThe different FPV vaccines were passaged on the SPF embryo and chicken fibroblast cells and detected by indirect immuno-fluencescence assay (IFA) with monoclonal antibody of REV. According to the REV integration site in the genome of FPV, we designed a pair of primers which its forward primer located on the 201 open reading frame gene (ORF201) and reverse primer on the 203 gene (ORF203). After amplifivation from these vaccines, some PCR products of different sizes were founded ranging from 400 bp,700 bp,1400bp,1700bp,2000bp,2500bp,5000bp and 8000bp. The 1700 bp and 2000 bp products were then cloned, sequenced and compared with other FPV and REV strains by BLAST software in the NCBI website. The results of comparison show that different FPV vaccines have different REV LTR integration sequence of 191bp and 504bp. To my interest, the integration site and the size REV sequence were the same as other FPV strains identified by American and Austria experts. Furthermore, these FPV vaccines share the high homology of 98%-99% with other FPV strains and 83.6%-99.6% with other REV strains from NCBI.2 The full -length REV genome sequence integrated wild FPV of Anqiu and Pingdu strainsWe choose two wild FPV strains of Anqiu and Pingdu which appeared positive results in the IFA as templates to amplify the full-length REV genome sequence integrated in the FPV strains. Some pairs of primers were synthesized according to REV sequences published. The amplified products were sequenced and compared showed that the REV genome sequences in the Anqiu strain and Pingdu strain have almost the same size except 8 bp difference and lacking of 3 basepair. The full length of Anqiu REV Genome was 7941 bp, which located in the ORF201 of FPV from the the beginning of 30th bp and ended in the 213th bp in the REV LTR. The comparison showed Anqiu REV genome had a high homology of 97%-99% with other strains of DQ387450.1(APC-566),AF246698.2,AY842951.1(HA9901),DQ003591.1(SNV). The phylogenetic tree indicated that thse wild FPV strains of Anqiu and Pingdu maybe the prominent in China.3 The artificial recombinant FPVIn previous study we have testified the FPV strain 282E4 have integrated REV LTR between its ORF 201 and 203. So we selected the Enhanced green fluorescent protein as reporter gene and p7.5 plasimd fragment as promter to construct the recombinant FPV. Afer transfection with this plasmid,some green plaques were appeared 48 hours later under UV light. This recombinant FPV including GFP were purified by a serious of dilution on 96-cell plates. This study provides the foundation to learn more mechanism of REV different integration sequences and the pathology of FPV.