The Construction And Applicationof DF-1/B And DF-1/K Cell Line

Avian leukosis?AL?is one of the main viral tumor diseases caused by avian leukosis virus?ALV?via vertical and horizontal transmission,whose clinical symptoms are multiple tumors of organ or tissue,immunosuppression,performance descends,the increase of death and culling rate,and caused great harm to poultry industry.There were A,B,J and K subgroups were frequently reported in C hina over the past 20 years.Subgroups A and B ALV in Chinese indigenous chickenshave caused great economic losses,while they are not easily distinguishable.It is necessary to establish a specificdifferention method for ALV-A and ALV-B.Notably,a novel ALV subgroup named ALV-K was recently isolated from Chinese local chickens and is different from subgroups A to E and J.ALV-K might have existed in native C hinese chickens for a long time.At present,ALV-K diagnostic methods mainly rely on virus isolation followed by envelope gene sequence comparisons,and there is a lack of other diagnostic methods.Although there are some unique chicken lines in United States that the primary cells produced can resistant to some specific subgroup ALV infection.The rareness of these chicken lines,the absence of permanent passage of primary cells and inconvenience to take along has limited the cells to apply.In the 1990s,the ADO L laboratory in the United States has established a special cell line?DF-1/J?based on DF-1cell line,which was resistant to ALV-J infection,and has its application in fields.A DF-1/A cell lines that specifically restricted ALV-A infection had been established in our laboratory.Therefore,construction cell line?ALV-B and ALV-K?can enrich the diagnostic toolbox,which will promote the eradication and control of AL infection in C hina..In this study,the env gene from ALV-B was inserted into pcDNA3.1/Zeo?+?eukaryotic expression vector to construct pc DNA-env-B,and then the recombinant plasmid pcDNA-env-B was transfected into DF-1 cell line.The transfected cells were scree ned by Zeocin for 21 days.After that the cells were continuously passaged 60 times with Zeocin.In order to detect the genetic stability of env gene in cell line,env gene was detected by bothordinary PCR and real-time PCR.The ALV-B env protein was further examined by IFA,Western blot and immune electron microscopy.The results showed that cell line,named as DF-1/B,can stably express ALV-B envprotein and the immunogold particles were observed in the cell membrane.The DF-1/B cell line isresistant to ALV-B infection in vitro at a viral dose of 1×10⁴TCID₅₀,but had no significant inhibitory effect on ALV-J replication.In this study,the env gene from ALV-K was also inserted into pc DNA3.1/Zeo?+?eukaryotic expression vector to construct pc DNA-env-K,and then the recombinant plasmid pcDNA-env-K was transfected into DF-1 cell line.The transfected cells were screened by Zeocin for 21 days.After that the cells were continuously passaged 60times with Zeocin.In order to detect the genetic stability of env gene in cell line,env gene was detected byboth ordinary PCR and real-time PCR.The ALV-K env protein was further examined by IFA,Western blot and immune electron microscopy.The results showed that cell line,named as DF-1/K,can stably express ALV-K envproteinand the immunogold particles were observed in the cell membrane.The DF-1/K cell line was resistant to ALV-K infection in vitro at a viral dose of 1×10⁴TCID₅₀,but had no significant inhibitory effect on either ALV-A,ALV-B or ALV-Jreplication.In order to verify the antiviral activity of resistant cell lines,another fourALV-K field isolates were inoculated into DF-1 and DF-1/K,respectively.DF-1/K cell line,based on ELISA detection of p27,were found all resistant to those4 ALV-K isolates infection.In order to evaluate the effect of DF-1/B and DF-1/K in clinical differential diagnosis,108 clinical plasma samples were inoculated into DF-1,DF-1/A,DF-1/B and DF-1/K.The results showed that ALV p27 S/P of one sample in DF-1/B was significantly lo wer than those in DF-1,DF-1/A and DF-1/K,suggesting the sample could contain ALV-B,which was confirmed by env gene sequencing.The ALV p27 S/P values of DF-1/Koftwo samples were significantly lower thanthose in DF-1,DF-1/A and DF-1/B,suggesting that the two samples may contain ALV-K,which was confirmed by env gene sequencing.The results indicate DF-1/B and DF-1/K have the ability to differentiate subgroups of ALV in clinical diagnosis.The construction and application of DF-1/B and DF-1/K have enriched the identification toolbox of different subgroup ALV,which can promote the epidemiological investigation and purification of avian leukosis in China,especially in the AL control and eradication of yellow feather breeder chickens.These cell lines provide a good tool for further study of the molecular mechanism of the interaction between ALV and its host cells.