Construction And Rescuing Of Avian Reticuloendotheliosis Virus Expressing Exogenous Gene And Its Characteristics

Avian reticuloendotheliosis virus(REV)belongs to Avian C retroviruses in retroviral family.This disease caused by REV is characterized by syndromes of the proliferation in reticular endothelial cells.It includes acute reticulosis,short syndrome and other chronic tumor proliferative disease in tissue.Retroviruses have been extensively reported as vectors for expressing and delivering exogenous gene.However,REV has not been used as a vector for expressing foreign gene.In this study,the enhanced green fluorescence protein(EGFP)gene was first inserted into the N-terminus of the viral Env gene and REV expressing the EGFP was rescued.And then,a HA fragment with epitopes from H9 influenza virus was inserted into the N-terminus of the viral Env,and REV expressing the HA epitope was rescued and characterized.1.Construction and Rescuing of Avian Reticuloendotheliosis Virus Expressing EGFPIn order to evaluate whether REV can be as viral vector for expressing exogenous gene and identify which site can be inserted for the foreign gene,in this study,the EGFP gene was first inserted into the N-terminus of the viral Env gene in the REV infectious clone through ExnaseTM ? Cloning technique,and the positive recombinant was named as rREV-EGFP-Env.The REV expressing EGFP-Env was rescued through the transfection of rREV-EGFP-Env in DF1 cells and verified under fluorescence microscopy.Our data demonstrated that the N-terminus of the viral Env gene in the REV genome could be as an inserting site for eapressing foreign gene and showed the possibility for developing REV vector used for the expression of foreign genes.Additionally,the REV expressing EGFP-Env could not only be applied to track the replication of REV,but also provide a powerful tool for further investigate the pathogenesis and tissue tropism of REV.2.Construction and Rescuing of Avian Reticuloendotheliosis Virus Expressing partial antigenic epitope of HA from H9 Influenza virus and its characterizationIn order to study on the possibility of REV as a viral vaccine vector to express foreign genes,a HA fragment with epitopes from H9 influenza virus was inserted into the N-terminus of the viral Env gene and the positive recombinant was named as rREV-HAep-Env.The REV expressing HAep-Env was rescued through the transfection of rREV-HAep-Env in DF1 cells and verified by PCR,IFA and Western blot,designated as REV-HA.The growth curves in DF1 cells showed that the replication ability of REV-HA was similar to that of the wild REV.The infection study in chickens showed that the bodyweight changes in chickens infected with REV and REV-HA were very similar within seven weeks post infection.The Antibody against REV could be detected both in REV and REV-HA groups at 2-week post infection,and the level of the antibody reached its peak at 3-week post infection.The chickens infected with REV and REV-HA were challenged with H9 influenza virus at 7-week post infection,.At day 2 post challenge,the viral shedding in treachle of chickens infected with REV-HA was significantly lower than that from REV group and PBS group.All these data demonstrated that REV-HA had similar biological characteristics with REV,indicating that REV could be as a potential viral vaccine vector for expressing the foreign gene.