The Effect Of Duck Circovirus Rep Protein N-glycosylation Site Mutations On Proliferation And Pathogenicity

Duck circovirus (DuCV) is a member of circovirus, mainly against duck immune system to produce immune suppression, their genome comprised of multiple ORFs, the ORF1 encoding Rep protein (Replication protein), and the protein may be associated with viral Replication. Glycosylation plays an important role in protein translation and regulation of protein function. In this study, we explore the effects of Rep protein on viral proliferation and pathogenicity by DuCV Rep protein N-glycosylation site mutation.1. Construction of DuCV Rep protein N-glycosylation site mutant strainThe double-copy clones have been constructed (has been introduced viral Replication independent regulatory region restriction sites Xho I) as a template, the overlap PCR method was used to construct the Rep protein N-glycosylation sites (sixth and eighteen amino acids of Rep protein) mutation strains W-6 and W-18, and by the immune ducklings to test the effect of rescue. The results showed that the DuCV target fragment was not amplified in the sera of the 1d and 2d after inoculation; but at the 4d after inoculation, the DuCV target fragment was amplified in the serum, which showed that our PCR amplification products are not directly derived on the mutant DNA. Confirmed by sequencing that the viral DNA extracted from the serum containing the modified Xho I enzyme cleavage site, it can be seen that we build the virus mutation strains has been in the duck body ring success and proliferation.2. The effect of DuCV Rep protein N-glycosylation site on the proliferationFor analysis the effect of DuCV Rep protein N-glycosylation site mutation on the proliferation, the 3 weeks old ducks were divided for 4 groups, and then infected with mutant strain W-6 (group ?), W-18 (group ?) and infectious clone strains pIC-2DuCV (positive control group) and PBS (negative control group). In 7d,14d, 21d,28d,35d,42d after infection, the thymus, spleen, bursa and other tissues were collected for fluorescence quantitative PCR and immunohistochemical detection. Fluorescence quantitative PCR results showed that compared with the positive control group, the content of group ? virus DNA was relatively large in the others, a copy of which the highest number for 1049 copies/mg. In the bursa of Fabricius virus DNA content was the lowest, the copy number was the highest 103.2 copies/mg; the group ? and the positive control group phase ratio content similar, virus DNA content in tissue specimen is relatively more organization for liver and the copy number were the highest for 106.3copies/mg (group ?) and 107.0copies/mg (positive control group), and with low content of tissue is Fabricius, the copy number and the highest values were 104.2 copies/mg (group ?) and 104'5 copies/mg (positive control group). Immunohistochemistry results showed that after being infected, the average optical density (AOD) value of positive cells in group ? is relatively higher in the liver, the highest value between 0.1774±0.00076 and 0.1679±0.01469; in group?, the top positive cells of AOD values in the organization is the liver and bursa of Fabricius, the highest value in the 0.2697±0.05571 and 0.2339±0.02187. And by difference analysis showed that the AOD value was no significant difference between group ? and the control group. Based on the results, we can clearly seen that DuCV Rep protein of the six amino acids mutated can weaken its replication in vivo, and DuCV Rep protein 18 amino acids mutated activity have no significant influence on its replication. The effects of mutation on the replication of DuCV Rep protein N-glycosylation site were studied, and provide a theoretical basis for the further study on DuCV.3. The effect of DuCV Rep protein N-glycosylation site on the pathogenicityFor analysis of viral pathogenicity of N- glycosylation site mutations of DuCV Rep protein, the mutation strain W-6, W-18, infectious clone strains pIC-2DuCV and PBS were used to infect the 3weekS old ducks, and collected the thymus, spleen, bursa and other tissues at 7d,14d,21d,28d,35d,42d after infection for histopathological observation. The results showed that after being Infected, necropsy found that the experimental group ?, ? and positive control group showed the thymus powder on the bleeding point, the liver has a small amount of bleeding and the tissue lesions of each groups are not obvious. By HE staining shows the main immune organs such as the thymus, spleen, etc in the experimental groups and positive control group are different degree of pathological damage, the mainly for capillary hyperemia; lymphocyte gap widened; lymphocyte nuclear condensation and nuclear fragmentation phenomenon. The pathological results showed that the ducks pathogenicity of DuCV Rep protein N-glycosylation site mutation on the pathogenicity of no obvious effect, which provided a theoretical basis for further research.