Study On Pathogenicity Of LTR Mutant Of Subgroup K Avian Leukosis Virus

Avian leukosis?AL?is a class of transmissible neoplastic diseases caused by avian leukosis virus?ALV?causing malignant or benign tumors.According to the host range,envelope protein specificity,etc.,ALVs were divided into A-J 10 subgroups.In 2012,a new ALV subgroup,the K subgroup?ALV-K?,was firstly isolated from some local chickens.In recent years,the virus has gradually increased.The ALV-K replication ability is so weak that it could be easily missed in the past eradiction tests,but its specific pathogenic mechanism is not yet clear.In our previous study,three strains of ALV-K?GDFX0601,GDFX0602 and GDFX0603?were isolated from chicken in southern China and their gene sequence analysis revealed that the similarity of the nucleotide sequence between the LTR sequences of the three strains of the ALV-K isolate and the E-subgroup ALV was as high as98.5%,while it was comparable to other subgroup reference strains is only 65%.Based on the luciferase expression activity of different subgroups of ALV 5'LTR sequence,it was found that the activation activity of ALV-K?GDFX0601?5'LTR sequence was significantly lower than other subgroups,but its LTR to the replicate ability and the influence on pathogenicity of ALV-K was not well understood.In order to analyze the effect of LTR sequence on the pathogenicity of ALV-K,the ALV-K isolate GDFX0601 was selected as the parental virus for infectious clone and virus rescue,and both ends LTR of the pathogenic ALV-J?CHN06?were selected to replace the corresponding LTR of ALV-K?GDFX0601?to obtain ALV-K LTR mutant strain HN-GDFX0601.Using GDFX0601 proviral DNA as a template,the target fragment was amplified in two segments and then cloned into the pBluescriptII KS?+?vector to obtain the recombinant plasmid pB-GDFX0601.The ALV-K LTR mutant recombinant plasmid pB-HN-GDFX0601 was successfully constructed by using the overlapping PCR method.After the recombinant plasmids were transfected with DF-1 cells,the cell supernatant ALV p27antigen ELISA test results showed that the S/P value was 0.8 and 1.25?S/P value?0.2 was positive?,demonstrating that the ALV-K?GDFX0601?clone strain rGDFX0601 and ALV-K LTR mutant viruses HN-GDFX0601 were successfully rescued.The rescued virus was infected to DF-1 cells and passaged blindly for 3 generations then tested by ELISA,RT-PCR,IFA and Western Blot.ELISA results showed that all infection groups were positive and RT-PCR method could detecte ALV-K gp85 and LTR,IFA detection results showed that the infected group showed green fluorescence,yet non-infected control group had no green fluorescence signal,Western Blot using ALV-p27 antibody as primary antibody showed that there was a clear protein band in the infected group but no band in the control group,indicating that rGDFX0601 and HN-GDFX0601 were successfully rescued and had biological activity.ALV-K clonal strain rGDFX0601 and ALV-K LTR mutant strain HN-GDFX0601 were inoculated into 24-well cell plates in DF-1 cells,and the parental strains ALV-K?GDFX0601?and ALV-J?CHN06?were used as a control,the cell supernatants from 1 to 7days after infection were used for ELISA detection and the replication kinetic curve was drawn.The results showed that the ALV-K clonal strain rGDFX0601 was almost identical to the parental strain ALV-K?GDFX0601?in terms of the replication ability.The replication capacity of the ALV-K LTR mutant strain HN-GDFX0601 was significantly higher than that of ALV-K clone strain rGDFX0601?P<0.05?,but significantly lower than that of ALV-J?CHN06?.That means mutation of LTR can increase the replicate ability of ALV-K?GDFX0601?in DF-1 cells.To further clarify the effect of LTR on the pathogenicity of ALV-K,150 SPF chicken of one-day-old were randomly divided into 5 groups.Four of them were injected with10³TCID₅₀ of rGDFX0601,HN-GDFX0601,ALV-K?GDFX0601?and ALV-J?CHN06?respectively.The group 5was injected with equal volume of DMEM medium as a blank control group.By comparing the effects of different viral infections on body weight gain and immune organs of SPF chicken,dynamic changes of viremia and cloaca shedding,and expression of IL-6 and IFN-?in the serum of late-infection chickens to evaluate the role of LTR on ALV-K pathogenicity.The results showed that LTR mutation did not increase the effect of ALV-K on weight gain and immune organs of SPF chickens.After LTR mutation cloaca shedding only had positive cloaca shedding during the 6th and 7th weeks,while rGDFX0601 strain had positive cloaca shedding except the 2nd and 7th weeks.However,after the LTR mutation,the virus viremia was more positive than before the mutation.Serum cytokine assay results showed that LTR mutants infected IFN-?levels at 35dpi were1.68-fold higher than cloned strains,while IL-6 levels were 1.17-fold lower than cloned strains,but both IL-6 and IFN-?levels are lower than before the mutation at 56 days post infecton.The results show that LTR replacement from ALV-J does not increase the pathogenicity of ALV-K in SPF chickens.This study provides a scientific basis for further elucidating the molecular mechanisms underlying the pathogenicity of ALV-K.