Construction Of Infectious Clone Of Subgroup J Avian Leukosis Virus NX0101 Strain And Its Biological Characterization

Avian leucosis virus Subgroup J (ALV-J) is a retrovirus which infects meat-type chickens, ALV-J was first reported as a new subgroup of ALV by Payne in 1991.It induced avian myelcytomatosis (ML) in broiler.ALV-J was isolated and identified in 1999 from two broiler breeder farms in China.ALV-J was highly virable virus, whether immune selective pressures exiting or not, could evolute into different quasispecies.that is to say,virus isolated from field was composed by different strains in genome.So seeking for one-oriented virus was important for virus research. Reverse genetic manipulation was a newly developed technique in study of RNA virus.Infectious clonal virus could rescued from molecular clone.so far, the molecular clone of ALV-J HPRS-103 was acquired in the world.According to provirus form in the virul life circle, 3 fragments of provirus cDNA of avian leukosis virus(ALV-J) strain NX0101 were amplified from the genomic DNA of ALV-J infected cells by using PCR,and then combined in the right direction and sequences into recombinant plasmid pALV-J-NX, containing the whole genome of NX0101.After transfection of chicken embryo fibroblast (CEF) cells with plasmid pALV-J-NX DNA, the rescued virus was identified in CEF by indirect fluorescence antibody test with ALV-J specific monoclonal antibody JE9. The rescued virus could replicate in CEF at a titer of 10^5.16/mL. Through cross neutralization test,the results showed that there is 100% cross antigenicity between NX0101 strain and rNX0101 strain.In chicken experiment, Chickens were injected at day 1 of incubation with ALV-J NX0101 strain or rNX0101 strain, similar significant growth suppression compared with Negative control chickens was found, atrophy in the bursa and thymus was detected, at the same time , indexes of immune organs to body weight showed there are a similar tendency to a lower response in NX0101 infected and rNX0101 infected birds. In addition, antibody reaction to Newcastle disease virus vaccines was inhibited, hile compared to the control birds (P<0.05). otherwise NX0101 or rNX0101 infection gave no influence on reaction to IBDV vaccination in birds.The chicken experiment also demonstrated that the rescued virus was still able to induce tumors in commercial meat-type broilers.In order to perform more effective assey for diagnosis of naturally ALV-J infected from flocks, PCR^ IFAn PAP asseys were used to detect the virus in tumor tissue section.