Rapid diagnostic methods for classifying avian leukosis subgroups in the field were needed for routine, large-scale screening. As a first step in method development, we inserted the avian leukosis virus subgroup A （A...Rapid diagnostic methods for classifying avian leukosis subgroups in the field were needed for routine, large-scale screening. As a first step in method development, we inserted the avian leukosis virus subgroup A （ALV-A） env gene into plasmid pcDNA3.1/Zeo （＋） and used this construct to transfect DF-1 cells. Zeocin-resistant cells were obtained after 2 weeks of zeocin selection. Then, the cells were analyzed using PCR, immunofluorescence, and Western blot for expression of the envA-encoded envelope protein after 30 serial passages. The DF-1/A cell line was completely resistant to 104 TCIDso/0.1 mL （50% tissue culture infective dose）ALV-A and was partially resistant to 10~ TCIDs0/0.1 mL ALV-A viral particles. By comparing the DF-1/A and DF-1 cell lines, an ALV-A isolate was identified using a gag-specific ELISAfor capsid protein p27. Thus, we established a DF-1/A cell line that was resistant to ALV-A infection. This cell line will be useful as a diagnostic tool.展开更多
Modified Vaccinia Ankara(MVA)is a highly promising vector for generating safe vaccine candidates against many pathogens,such as HIV-1,SARS-CoV-2,and influenza viruses.The gold standard method to titrate MVA involves v...Modified Vaccinia Ankara(MVA)is a highly promising vector for generating safe vaccine candidates against many pathogens,such as HIV-1,SARS-CoV-2,and influenza viruses.The gold standard method to titrate MVA involves visualizing MVA plaques in chicken embryo fibroblasts after immunostaining.However,this method is time-consuming and costly.In this study,we evaluated the visualization of MVA plaques formed in continuous chicken embryo fibroblasts DF-1 cells using crystal violet staining.We found that MVA titration by plaque assay using crystal violet staining in DF-1 cells yielded similar results to immunostaining,with substantially reduced time and costs.The MVA plaque assay by crystal violet staining in DF-1 cells is a reliable method with accurate results and low time and financial costs.展开更多
基金The work was founded by the National Key R&D Program of China(2016YFD0501606)the Public Industry Research Program,the Ministry of Agriculture of China(201203055)+2 种基金the Program of Science and Technology Development of Guangdong Province,China(2015A020209145)the China Meat-Type Chicken Research System(CARS-42-G09)the Modern Agriculture Talents Support Program,Ministry of Agriculture of China([2012] no.160)
文摘Rapid diagnostic methods for classifying avian leukosis subgroups in the field were needed for routine, large-scale screening. As a first step in method development, we inserted the avian leukosis virus subgroup A （ALV-A） env gene into plasmid pcDNA3.1/Zeo （＋） and used this construct to transfect DF-1 cells. Zeocin-resistant cells were obtained after 2 weeks of zeocin selection. Then, the cells were analyzed using PCR, immunofluorescence, and Western blot for expression of the envA-encoded envelope protein after 30 serial passages. The DF-1/A cell line was completely resistant to 104 TCIDso/0.1 mL （50% tissue culture infective dose）ALV-A and was partially resistant to 10~ TCIDs0/0.1 mL ALV-A viral particles. By comparing the DF-1/A and DF-1 cell lines, an ALV-A isolate was identified using a gag-specific ELISAfor capsid protein p27. Thus, we established a DF-1/A cell line that was resistant to ALV-A infection. This cell line will be useful as a diagnostic tool.
文摘Modified Vaccinia Ankara(MVA)is a highly promising vector for generating safe vaccine candidates against many pathogens,such as HIV-1,SARS-CoV-2,and influenza viruses.The gold standard method to titrate MVA involves visualizing MVA plaques in chicken embryo fibroblasts after immunostaining.However,this method is time-consuming and costly.In this study,we evaluated the visualization of MVA plaques formed in continuous chicken embryo fibroblasts DF-1 cells using crystal violet staining.We found that MVA titration by plaque assay using crystal violet staining in DF-1 cells yielded similar results to immunostaining,with substantially reduced time and costs.The MVA plaque assay by crystal violet staining in DF-1 cells is a reliable method with accurate results and low time and financial costs.
