Establishment Of An Indirect Immunofluorescence Assay For Detection Of Avian Reticuloendotheliosis Virus

Reticuloendotheliosis (RE) is a set of pathological syndrome characterized by the hyperplasia of reticular cells in chickens, turkeys, ducks, geese and other poultries, which is caused by REV. It is a great damage to the poultry industry because it can not only lead to poor weight gain and feed efficiency but also result in reduced egg production and egg quality. It can also lead to tumors of poultries. REV can spread by vertical and horizontal transmission. It can cause a wide range of diffusion by vaccine contamination. The infection of REV is common in China. It is necessary to establish a method to accurately detect REV of live attenuated vaccines and clinical samples.The p30gene which codes group antigens was amplified by PCR. Then the p30gene was cloned into vector pGEX-6P-1and pET-32a (+) for prokaryotic expression, and the49kD recombination GST-p30protein and43.4kD recombination His-p30protein were obtained in this study. The recombinant proteins were purified by High Affinity GST Resin and High Affinity NI-NTA Resin. The purified recombinant protein GST-p30was immunized with BALB/c mice as antigen for its spleen cells, and then the spleen cells were fused with SP2/0. And the purified recombinant protein His-p30was chosen as detection antigen for screening positive clones, eight hybridoma cells that could stably secrete monoclonal antibodies (MAbs) anti-p30antigen were produced in all through subcloning three or four times, which were named as1A10,2G1,4G2,4D9,2-4D9,8G3,9F2and12F2. The eight MAbs had the ELISA titers ranged from1:6,400to1:204,800. The subclass of MAbs4D9and4G2were IgG3, and the other six MAbs were IgGl. Western-blotting analysis showed that the MAbs including2G1,4G2,4D9,2-4D9,8G3,9F2and12F2could be recognized by recombination His-p30protein specifically. The results of indirect immunofluorescence assay (IFA) approved that the MAbs consisted of1A10,4G2,4D9,2-4D9,8G3,9F2and12F2could have a special reaction with DF1cells infected with REV. And according to the research, an IFA method for REV detection was established with the purified MAb4D9, which had a high sensitivity and specificity against REV p30antigen. The results showed that this IFA method had a good sensitivity for REV detection, and furthermore had a100%agreement with PCR method. The minimum amount of REV detection of this method was1TCID50when infected with DF1cells just after four days. In addition, DF1cells infected with12.5TCID50REV could be detected after24hours. The IFA method also had a good specificity for REV detection, and without reactivity with avian leukosis virus (ALV) and marek’s disease virus (MDV).In addition,420samples consisted of120live attenuated vaccine samples and300diseased-chickens samples were detected by this established IFA method for REV detection, which were collected from chicken flocks in4provinces and Beijing between2011and2014. One strain of REV was detected from120live attenuated vaccine samples. Another two strains of REV were isolated from these diseased-chickens samples. gp90gene of REV isolates were amplified for phylogenetic analysis. The analysis revealed that these REV isolates classified as subtype I were all closed to the strain REV-A, REV-T and HA9901on the genetic relationship.