Identification Of A Subgroup E Avian Leukosis Virus Strain And Analysis Its Pathogenicity

Avian leukosis(AL)is a neoplastic infectious disease caused by Avian leukosis virus(ALV),which characterized by low immunity,multi-tissue tumors and reduced performance in chicken.The disease was introduced into China in the 1990s,has been affecting the development of domestic chicken industry.Although China has implemented strict ALV eradication plans in chicken flocks since 2008,it has brought great challenges to prevent and control disease in China due to the variety of chicken flocks,a large number of ALV subgroups,variation and recombination of genome,and the great difference in the impact of pathogenicity among various subgroups on chicken flocks.Therefore,the isolation and identification of ALV is of great significance to study the prevalence of the disease,the variation trend of ALV genome and population purification.In this study,the positive ALV disease materials detected by RT-PCR were treated and inoculated into primary chicken embryo fibroblast(CEF)and passable chicken embryo fibroblast cell line(DF-1)respectively,the cell supernatant was collected after continuous blind transmission to the third generation.The result of RT-PCR showed that the target band was amplified in the supernatant of CEF cells.The result of indirect immunofluorescence showed that CEF cells after inoculation appeared specific green fluorescence.The virus particles with approximately spherical shape,diameter of about80 nm and a structure of capsule and fibrin was observed by transmission electron microscopy.The morphology was basically consistent with that of ALV particles,and named HLJE2020 strain.In order to analyze the replication dynamics of the isolated strain in vitro,during different periods of ALV cultivation,the change of virus copy number was detected using the method of ALV SYBR GREEN I fluorescent quantitative PCR which was established.The results showed that the virus copy number was gradually increased after infecting with CEF cells,and reached the peak on the day of 7.In order to further analyze the passage stability of the virus,it was detected by ELISA and RT-PCR.The results showed that the virus strain could be stably passaged to the 9 th generation.In order to analyze the genomic characteristics of ALV HLJE2020 strain,the whole genome amplification,sequencing and comparative analysis of HLJE2020 strain were carried out.By comparing the whole genome sequence of this strain with the 28 ALV subgroup reference strains,the results showed that the nucleotide homology of the whole genome sequence was 83.6%-98.5%,and the nucleotide homology of the LTR gene was 58.1%-98.2%,gag gene and pol gene were relatively conserved,and the nucleotide homology between the two strains and the reference strains was more than94.5%.While env gene was significantly different from that of other reference strains,and its homology ranged from 51.6%to 97.4%,genetic evolution analysis indicated that it belongs to the same branch as subgroup E.The type of ALV subgroup was determined by gp85 gene that encode envelope protein env,further analysis of gp85 gene showed that the nucleotide homology between gp85 gene of the strain subgroup E and subgroup B of ALV were more than 90%,genetic evolution analysis showed that there was a separate branch between subgroup E and subgroup B of ALV,and the Vr2,hr1 and Vr3 regions of gp85 amino acid were similar to the subgroup B of ALV,while Vr1,hr2 and other regions were similar to the subgroup E of ALV.Combined with RDPv.4 and Sim Plot software analysis,the results showed that the gp85 gene of the isolated strain might exist the recombination phenomenon between the AF229strain of subgroup E and the SDAU09C1 strain of subgroup B.In order to clarify the pathogenicity of ALV HLJE2020 strain to chickens,this study inoculated SPF chickens intraperitoneally with HLJE2020 virus solution at a dose of 300?L 10^3.2TCID₅₀/chicken.The results showed that the weight of chickens in infection group were increased slowly,which was significantly different from that in control group(P<0.05).The development of immune organs were affected by virus in infected group,which include splenomegaly and atrophy of thymus and bursa of fabricius.Histopathological observation also showed that virus infection could cause certain damage to immune organs.The results of viremia test showed that viremia appeared in infection group at the week of 3,and maintein a relatively high level(20%)at the week of 5 and 7,and then always existed at a low level.The cloacal detoxification occurred only at the week of 5,9 and 11.The results of viral load in tissues showed that the virus could be detected in heart,liver,spleen,kidney,bursa and thymus except lung,stomach and cecum after one week infection.Combined with the dynamic changes of viral load in blood and tissues,the results showed that the viral load in chickens infected with the virus reached the peak at the week of 7,and then decreased at the week of 9,and reached the second peak at the week of11,while the first peak was higher than the second peak.In conclusion,a strain of subgroup E ALV HLJE2020 was successfully isolated in this study,and the gp85 gene in the genome of this strain might be recombined,and it was confirmed that this strain had pathogenicity to SPF chickens,which laid a foundation for the future research on variation trend and pathogenesis of ALV.