Isolation And Identification Of ALV-J SSGS-1Strain And Construction Of Its Infectious Molecular Clones

Avian leukosis virus is one of a retrovirus wich are widespread in chickens. The classic avian leukemia mainly caused by A、B subgroup, little by C、D subgroup. Now it had been inhibited for purified constantly in breeding poultry. But avian leukosis virus subgroup J (ALV-J) was different from classical ALV, which was first isolated by Payne et al from chickens in1988. ALV-J mainly induces tumor, reduce production performance, increase the rate of elimination, and cause immunosuppression in chickens, so diseases associated with ALV-J have caused huge economic losses worldwide. Myelocytomatosis is the main symptom after being infected with ALV-J in meet-type chickens.In order to investigate the molecular characteristics and to construct the infectious molecular clone with molecular marker of subgroup J avian myeloid leukosis virus (ALV-J) of local strains, an isolate of subgroup J avian leukosis virus (ALV-J) with Myelocytomatosis, designed SCGS-1, was isolated from grandparent meat chickens in Sichuan province. A full-length infectious clone of ALV-J (pUC-SCGS) was constructed by cloning and combining of three fragments using PCR method from SCGS-1. SalI site was carried out on4684nt of SCGS-1by overlapping PCR to form another infectious clone and named pUC-△SCGS. The two plasmids pUC-SCGS and pUC-△SCGS were transfected into CEF, and the rescued viruses were detected by PCR, avian leukosis virus antigen test kit and indirect immunofluorescence assay (IFA).The viral genome of this isolate was sequenced as7499bp and compared with reference ALV-J strains in GenBank. The results showed that the sequence identity was ranged from95%to99%. Env gene and LTR were variated greater than pol and gag gene. The sequence identity was ranged from93%to99.5%for env gene and92%to95%for LTR, lower than97%to99%for pol gene and94%to97%for gag gene. Digestion and sequence analysis revealed that the infectious clone pUC-SCGS and pUC-△SCGS were constructed correctly. PCR, antigen test and IFA results showed the3th and4th rescued virus were positive, while the controlled CEF were negative. In conclusion, rescued virus and the virus with molecular marker of subgroup J avian myeloid leukosis virus were successfully constructed, named rSCGS-1and rASCGS-1.