The Molecular Epidemiological Investigation And Pathogenicity Research Of Avian Leukosis Virus Subgroup J In Layer Flocks

Subgroup J avian leukosis is a phymatoid infectious diseases caused by subgroup J avian leukosis virus subgroup (ALV-J) and characterized by myelocytomas and malignant tumors of other cells . Although the disease existed only in meat-type chickens when it begun to occur, field cases of ALV-J infection and tumors in layers were not found worldwide. However, subgroup J of Avian leukosis (AL) has been emerging in egg-type chickens in China, and became more and more serious in recent years. Especially, numerous cases caused by ALV-J have occurred in layer flocks in many provinces in China such as HuBei, ShanDong and Hei Long-Jiang since the beginning of 2009. And the morbidity rates and the mortality rates even reached 50% or higher, causing heavy economic losses. For better understanding the infectious stutus and the virus itself of ALV-J in layers in China, we conducted our researches followed and the results were also shown as follows.Our study firstly isolated and identified the unknown pathogen from 180 samples collecting from 40 farms of 6 provinces in China. Based on the results of ELISA, PCR amplication and immunofluorescence assay special to ALV-J, 19 ALV-J strains were isolated and identified from layer flocks.To understand the origin of ALV-J strains isolated from layer flocks in China, the gp85 gene sequences of the 19 ALV-J isolates were cloned and compared with other gp85 genes of ALV-J published. The results show that 71.3%～100% identity were shared among all isolates based on the induced amino acids of gp85 gene. Phylogenetic analysis indicated that all 19 isolates belonged to 3 branches. Among those strains, most isolates (13/19) had close relationships with the prototype ALV-J strain HPRS-103, 3 isolates highly related with the US strain ADOL-7501 while 3 isolates had an independent branch, indicating the large variation of gp85 gene of the 3 isolates.To know the sequence characteritics of ALV-J causing hemangiomas symptoms, we cloned the whole genome of three ALV-J isolates(named by SD09DP03,JL09JL3-1,HLJ09MDJ-1, respectively). The results suggested that that The full-length proviral genome sequences of JL09JL3-1, SD09DP03 and HLJ09MDJ-1 contained 7677, 7677 and 7635 nucleotides , respectively. Compared with the reference ALV-J, the three hemangiomas strains unamimously shared particular sequence of 5'LTR and 5'UTR , NFAP-1 and AIB REP1 elements were missed and a C / EBP binding site added in 5'LTR U3 area of the three strains. Moreover, all the three isolates contained a relatively conservative E element and highly conservative DR component,but completely missed rTM in the 3'UTR. Finally, gag and pol genes of the isolates were highly conserved , but env genes were relatively conservative and only 1～2 amoni acids deletions. In detail, 117S was missed in JL09JL3-1 and SD09DP03 and two amino acid deletions (61N and 116R) in HLJ09MDJ-1. Also the changes of many N-glycosylation sites in env gene may affect the viral antigen.For study the pathogenicity of current ALV-J strains in layers, 0.2 mL virus suspension containining 104..25 TCID50 virions per milliliter, was injected into the intraperitoneal per one 1-day-old SPF chick.. Since 3,5,7,9,11,13,17,21,24,28,30,32 weeks after infection, samples of cloacal swabs, anticoagulant and non-anticoagulant blood were collected to study the virus-infected stutus such as virus shedding, viremia, and specific antibody to ALV-J and clinical symptoms and pathological changes were also observed. The results showed that: (1) More than 60% chickens infected with JL09JL3-1or SD09DP03 exhibited viremia stutus after the beginning of 3 weeks. (2) The percents of cloaca detoxification of infected groups were increasing before 21 weeks , then sharply decreased during 13 to 21 weeks but more than 90% chickens remained detoxificated. (3) During the period of 3 to 21 weeks, the overall antibody positive rate rised, and after 21 weeks, the positive percent of ALV-J antibody continued to decline until the lowest lever. (4) All isolates could induce marrow tumors, the rate ranged from 6% to 10%. Moreover, Sick chicken exhibited typical myeloid cells in their liver, spleen, kidney and other features of occurred, which is consistent with the changes caused by ALV-J in meat-type chickens. In our study, newly ALV-J strains in layer flocks in China were isolated and identified, which will provide rich materials for the researches on ALV-J pathogenesis to egg-type chickens. We herein firstly reported that ALV-Js with hemangiomas symptoms contains acute transforming retroviruse-like 5'LTR and 5'UTR. And high rates of viremia and shedding in layer chickens when inoculated with ALV-J may explain the dominant stutus of ALV-J in layer flocks. To conclusion, all results may contribute to the researches on pathogenesis of ALV-J to egg-type chickens.