| To investigate the prevalence of avian leukosis(AL)in Guangxi and the genetic variations of the avian leukosis virus(ALV),the isolation and identification of ALV in the chickens of having typical AL symptoms from different farms in Guangxi during 2014 to 2018 was performed in the study.The suspicious tumor tissue samples were collected and used for cell culture for virus isolation and the ALV in the cultures was detected by ALV group-common-antigen p27 test using the supernants.The positive samples were further identified by subgroup-specific polymerase chain reaction(PCR)and immunofluorescence assay(IFA)with the subgroup-specific monoclonal antibodies,and 10 sample of ALV-J single infection were found and the isolates were further used for full-length genome sequence analysis.The results showed that the full-length of the 10 isolates genome were 7583～7648 nt,the similarities of nucleotides and amino acids were 96.0%～98.8%and 96.8%～98.9%,respectively.The similarities between the isolates and the ALV-J reference strains were more than 91%,while with subgroups ALV-A,B,C,D,E,and K were as low as 63.1%to 86.3%.The results of genetic phylogenetic tree analysis showed that 10 isolates were far from the subgroups ALV-A,B,C,D,E and K,but were closed to the ALV-J reference strains and were in the same branch.These results also proved that the 10 isolates were all ALV-J viruses.The comparisons of the three major genes env,gag and pol revealed that the similarity of nucleotide sequence of gag and pol between the 10 isolates and the ALV-J reference strains was more than 94%,indicated that they were highly conserved,while the those of the envelope protein gene env were relatively lower at 90%～95%.Further analysis of the two structural proteins gp85 and gp37 encoded by the env gene revealed that the gp37 gene was more conserved and the similarities between the isolates and ALV-J reference strains was 92.7%-99.0%,while the those of the gp85 were relatively lower at 85.9%-96.0%.Since the sequence of gp85 determines the classification of ALV subgroups,its similarities of the 10 isolates with the ALV-J reference strains were higher,while the similarities with other subgroups of ALV were as low as 38.1%to 59.7%.The entropy analysis on gp85 showed that the main variant regions were concentrated in hrl and hr2.The analysis of LTR between the isolates and the ALV-J reference strains showed that its similarities were 87.0%～97.8%,those of the U3 region were 86.3%～98.1%,and the isolates GX17YL05,GX17NN06 and the ALV-J reference strains GX14ZS04,GX14HG01 and NHH all showed a specific continuous 11-bp-deletion within the U3 region,while another 8 isolates did not.The result of the full-length genome sequence analysis of the 10 ALV-J isolates showed that there was not significantly genetic change in Guangxi during the past five years.Two ALV-J strains GX14YYA1 and GX14YYD2 were isolated in 2014 from a bivalent Newcastle disease(ND)-infectious bronchitis(IB)vaccine and an infectious laryngotracheitis(ILT)live vaccines,respectively.In order to evaluate the pathogenicity effects of the contaminated vaccines on the Three-Yellow chickens,experimental tests followed the routine vaccination program in the field was conducted.The day-old Three-Yellow chicks in group 1 were vaccinated with the ALV-J contaminated ND-IB commercial vaccine by intranasal and eye-drop at 1-day-old for the primary vaccination and at 7-day-old repeat conducted again for the secondary vaccination,birds in group 3 were intraperitoneally injected with the ALV-J strain GX14YYD2,that was isolated and purified from the ALV-J contaminated ILT live vaccine at 1-day-old,and birds in group 4 were vaccinated with the ALV-J(GX14YYD2 strain)contaminated ILT live vaccine at 35 days,while groups 2,5 and 6 were kept as the normal non-contaminated ND-IB and ILT live vaccines’ vaccination and blank control groups,respectively.All birds were vaccinated with the commercial oil-emulsion vaccines of ND,AIV-H5 and AIV-H9 at 7-day-old,and then the body weight,viremia and the viral loads of ALV-J,antibody response titers,and cloaca virus shedding of ALV-J were measured at the ages of 1d,1W,3W,4W,5W,8W,11W,15W,21W and 25W,respectively.The results showed that the birds in groups 1 and 3 were experienced serious suppression of body-weight(p<0.05)when compared with the control groups.The normal development of the immune organs was also significantly affected by the spleen was swollen,while both the thymus and the bursa were atrophy(p<0.05),and the immune response measured by their corresponding antibody titers to the vaccinations were also significantly decreased(p<0.05).Also,birds in groups 1 and 3 showed grossly obvious blood-bud of ALV-J hemangioma-type change in the heart.While no detected viremia or clinical diseases were observed during the entire experimental period in any bird of the groups 4 and control groups 2,5 and 6.Therefore,in this study,it demonstrated that one-day and seven-day-old chickens immunization with the ALV-J contaminated live vaccine or 1-day-old intraperitoneally injected with the ALV-J isolated from commercial vaccine both can lead to chickens to be infected with ALV and caused disease.It can be found in the experiment that the vaccinations of the ALV-J contaminated commercial live vaccine at 1-day-old and 7-day-old of the commercial Three-yellow chickens could result in viral infection and clinical diseases.The results of the study demonstrated that no significantly difference of the full-length genome sequence was found in the 10 ALV-J isolates during the past 5 years.Also the animal experiment demonstrated that the ALV-J contaminated commercial live vaccines could be one of the transmission routes of ALV to the commercial chickens. |
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