Epidemiological Investigation Of ALV In Chinese Indigenous Breeds And The Research On The Biological Characteristics Of ALV-K

The avian leukemia virus belongs to the genus Alpharetrovirus of the family Retroviridae.Except to serious tumors,it can also induce several subclinical syndrome such as egg-laying decrease,immunosuppression and growth retardation in the chickens infected by ALV.ALVs were divided into ten subgroups(A-J)based on the virus and host range associated antigenicity of envelope proteins and subgroup K which was identified recently.ALVs,especially subgroup J of avian leukosis virus was firstly isolated and identified in the late 1980 s,and quickly spread worldwide and caused severe economic losses to our poultry industry,which seriously endanger the healthy development of various types of chickens such as white feather broiler chickens,egg type chickens,yellow feather broiler and inherent local breed chickens.Congenital vertical infection is an important mode of the transmission of ALV.Because the host is difficult to produce an effective immune response to the ALV,and ALV genome mutation frequency faster,no commercially available vaccine is available as so far.Due to the diversity of ALV subgroups,the complexity of infection and transmission,significant economic losses were caused in our country's chicken industry.In this study,the epidemiological investigation of ALV in different flocks of China was carried out,and the effect of cock semen on ALV transmission was systematically studied.The effects of different adjuvants and immunopotentiators on the production of ALV-J gp85 recombinant protein were studied to accelerate and assist the purification of ALV in China.The genome of the newly ALV-K isolated strains were systematically analyzed and the pathogenicity of ALV-K prototype strain JS11C1 to SPF chicken was studied.To explore the role of env gene in the pathogenicity of ALV-K,we used reverse genetic manipulation to construct the infectious clones of JS11C1 strain,and the chimeric infectious clones of env(J)-LTR(K)and env(K)-LTR(J)were constructed for the first time on this basis.1.Epidemiological investigation and quasispecies analysis of ALV in different flocks inChina.In this study,the epidemiological survey of ALV in different flocks in China was carried out,and a total of 14 ALVs were isolated and identified from 12 Chinese indigenous breeds.The analysis showed that 2 were identified as ALV-A,1 as ALV-B,8 as ALV-J,and3 as ALV-K.Among them,five strains of ALV subgroup J(ALV-J)were isolated and identified from a local chicken flock associated with serious tumor cases in Shandong in2012,and the name was SDLY1201-SDLY1205.Five gp85 genes of ALV-J were amplified and analyzed for the diversity of quasispecies of five strains.The results showed that the nuclear acid length of gp85 gene of five ALV-J isolates is 921 bp,921bp,924 bp,918bp,912 bp respectively,and amino acid homologies of different gp85 clones from the five ALV-J strains were 99.3%-100%,99.3%-100%,99.4%-100%,98.4%-100%,99.0%-100%,respectively.The proportions of dominant quasispecies were 65.0%,85.0%,85.0%,50.0%,84.2%,respectively,and homology of the gp85 among these dominant quasispecies was89.2%-92.5%.These data demonstrated the composition of the ALV-J quasispecies varied among infected individuals even within the same flock,and the dominant quasispecies continued to evolve both for their proportion and gene mutation.2.Vertical transmission of avian leukosis virus subgroup J(ALV-J)from hens infected through artificial insemination with ALV-J infected semen.Avian leukosis virus(ALV)is one of the major diseases in poultry that produces tumors,which can be both horizontally and vertically transmitted.However,the effects of ALV infection in chickens,especially roosters,during propagation on future generations is not clear for a long time.The results showed that two inseminated hens developed temporary antibody responses to ALV-J at 4–5 w,the p27 antigen was detected in cloacal swaps of six hens,and three of 26 egg albumens at 1–6 w after insemination.Moreover,no viremia was detected 6 w after insemination even when virus isolation had been conducted6 times at weekly intervals for each of the 12 females.However,ALV-J was isolated from one of their 34 progeny chicks at 1-w-old,and its gp85 had sequence identity was98.4%–99.2% of the gp85 of ALV-J isolated from semen samples of the six cocks.Ourfindings indicated that females late horizontally infected with ALV-J by artificial insemination may transmit the virus to progenies through eggs,which may cause vertical transmission.3.Cooperative effects of immune enhancer TPPPS and different adjuvants on antibody responses induced by recombinant ALV-J gp85 subunit vaccines in SPF chickens.To explore the antibody responses and protective effects induced by subgroup J avian leukosis virus(ALV-J)gp85 protein vaccine plus different adjuvants(CpG and white oil adjuvant YF01)combined with the immune enhancer Taishan Pinus massoniana pollen polysaccharide(TPPPS),we immunised SPF chickens with the recombinant ALV-J gp85 protein,along with different adjuvants and immune enhancer,which protected the chickens by inducing different levels of protective antibodies.Results showed that a single injection of gp85 recombinant protein could only produce low-titre antibodies that were maintained over a short time in few chickens.When combined with YF01 or CpG adjuvants,the recombinant protein could induce high-titre antibodies in most of the immunised chickens.Moreover,when the immune enhancer TPPPS was used with the two adjuvants,it further elevated the antibody levels for a longer duration.The eggs from four groups with the highest levels of ALV-J antibodies were collected,hatched,and examined for maternal antibodies.The protection by the maternal antibodies against ALV-J infection in the TPPPS-immunised group was higher than that in the group without TPPPS,which was consistent with the observations in the parents.This study shows that the immune enhancer TPPPS,combined with YF01 or CpG adjuvants,can enhance the immunogenicity of gp85 recombinant proteins,and provide a better immuno-protection.It provides a powerful experimental basis for the development of ALV-J subunit vaccine.Efficient subunit vaccine development will also accelerate the process of purification of ALV-J.4.Genome analysis and pathogenicity of ALV-K.In this study,three ALV-K strains were isolated and identified from Chinese indigenous breeds,and genome analysis was carried out.The results showed that the three ALV-Kstrains in this study and the previously published ALV-K strains were located in a separate branch,and they were not in the same branch as the ALVs of other known subgroups in the flock.Among them,the three ALV-K strains SDAUAK-11,SDAUAK-12 and SDAUAK-13 have the highest homology with the GD14 LZ strain,GDFX0601 strain,which were isolated from Guangdong province and JS11C1 strain,which was isolated from Jiangsu province,respectively.Their homology with other ALV-K strains was 90.8%-97.7%,90.8%-97.7%,91.2%-97.3%,respectively.To investigate the pathogenicity of JS11C1 strain,a suspected new subgroup(subgroup K)of avian leukosis virus(ALV-K)isolated from Chinese local breed in 2011,specific-pathogen-free(SPF)leghorn chickens were infected with JS11C1 by yolk sac inoculation,intravenous inoculation and intraperitoneal inoculation respectively.Several indices such as dynamics of viremia and antibody in infected chickens,suppression of body weight,and influence on immune responses to vaccination were observed.The results demonstrated that,inoculation of JS11C1 could induce the persistent viremia,suppress the body weight and reduce the immune response to NDV and AIV-H9 vaccination in infected chickens by yolk sac inoculation or intravenous inoculation.In addition,it only could induce a low incidence of lymphocytoma in infected chickens.Overall,the transmission efficiency and pathogenicity of JS11C1 were lower than those belonging to other subgroups.This study paved the foundation for further research on the pathogenic mechanism and prevention of ALV-K.5.The study on the pathogenicity of env gene in ALV-K.The differences in the ALV genome of the different subgroups are mainly concentrated in the env gene,which binds to cell-specific receptors and determines the characteristics of viral tropism and pathogenicity.In this study,the infectious cloning plasmids of ALV-K prototype strain JS11C1 and the chimeric infectious cloning plasmid of env(J)-LTR(K)and env(K)-LTR(J)were constructed by routine molecular biology techniques such as PCR and restriction enzyme digestion.The above four infectious cloning plasmids were transfected into DF-1 cells,respectively,and four rescue viruses were saved.And the replication ability of four viruses rescued by infectious clones on DF-1 cells was compared.The resultsshowed that the rescue virus rSDAU1005 had the strongest replication ability on DF-1 cells,followed by renv(J)-LTR(K),rJS11C1,renv(K)-LTR(J).In addition,this study also compared the pathogenicity of four rescue viruses in SPF chickens through the yolk sac inoculation to explore the role of ALV-K env gene in its pathogenicity.The results showed that four rescue viruses had a pathogenic effect on SPF chickens,rSDAU1005 can cause the most serious growth retardation and immunosuppression.Compared with the two chimeric virus groups,renv(J)-LTR(K)can cause more severe growth retardation and immunosuppression than renv(K)-LTR(J).This study is the basis for the study of ALV-K pathogenic molecular basis and its comprehensive prevention and control.