Chicken embryo fibroblasts (CEFs) are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus (AIV).In this study,the expression of eleven housekeeping gen...Chicken embryo fibroblasts (CEFs) are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus (AIV).In this study,the expression of eleven housekeeping genes typically used for the normalization of quantitative real-time PCR (QPCR) analysis in mammals were compared in CEFs infected with H5N1 AIV to determine the most reliable reference genes in this system.CEFs cultured from 10-day-old SPF chicken embryos were infected with 100 TCID50 of H5N1 AIV and harvested at 3,12,24 and 30 hours post-infection.The expression levels of the eleven reference genes in infected and uninfected CEFs were determined by real-time PCR.Based on expression stability and expression levels,our data suggest that the ribosomal protein L4 (RPL4) and tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ) are the best reference genes to use in the study of host cell response to H5N1 AIV infection.However,for the study of replication levels of H5N1 AIV in CEFs,the β-actin gene (ACTB) and the ribosomal protein L4 (RPL4) gene are the best references.展开更多
Inducing animal viruses to adapt to chicken embryos or chicken embryo fibroblasts(CEF) is a common method to develop attenuated live vaccines with full security.Canine distemper virus(CDV) also does this,but the mecha...Inducing animal viruses to adapt to chicken embryos or chicken embryo fibroblasts(CEF) is a common method to develop attenuated live vaccines with full security.Canine distemper virus(CDV) also does this,but the mechanisms and particular receptors remain unclear.Virus overlay protein blot assays were carried out on CEF membrane proteins,which were extracted respectively with a Mem-PER TM kit,a radioimmunoprecipitation assay buffer or a modified co-immunoprecipitation method,and revealed a common 57 kDa positive band that differed from the 42-kDa positive band in Vero cells and also from those receptors reported in lymphocytes and 293 cells,indicating a receptor diversity of CDV and the possibility of the 57-kDa protein acting as a receptor that is involved in adaptive infection of CDV Kunming strain to CEF.展开更多
RNA interference(RNAi)technology is a powerful tool for identifying gene functions.Chicken embryo fibroblast(CEF)is an ideal model for studying the interaction between avian viruses and their hosts.To establish a meth...RNA interference(RNAi)technology is a powerful tool for identifying gene functions.Chicken embryo fibroblast(CEF)is an ideal model for studying the interaction between avian viruses and their hosts.To establish a methodological platform for RNAi studies in CEF,three plasmid vectors expressing short hairpin RNAs(shRNAs)targeted against the Newcastle disease virus(NDV)NP gene were constructed.One of them,ndv1,was proven effective on blocking viral replication in CEF and chicken embryos.Four hours prior to infection with NDV,the CEF was transfected with the plasmids by Silent-fect.An unrelated shRNA sequence(HK)was used in mock transfection.The expression of a potent shRNA resulted in up to 2.3,21.1 and 9.8 fold decreases in NP gene expression at 3,6 and 9 h post infection in CEF,respectively.The ndv1 was able to completely inhibit the replication of the virus in CEF within 48 post infection.Furthermore,the pathological changes in CEF caused by NDV were delayed,and the degree of pathological changes was lighter compared with the mock transfection in the presence of ndv1.When the complex of shRNASilent-fect and NDV was co-injected into the allantoic cavity of 10-day-old embryonated eggs with 10^(5) or 10^(6) ELD50 NDV,NDV replication was decreased by 94.14% and 62.15% after 17 h,respectively.These findings suggest that the newly synthesized NP protein is critical for NDV transcription and replication and provide a basis for identifying the functions of viral genes and screening for effective siRNAs against viruses in CEF and chicken embryo by RNAi.展开更多
[Objective] To observe whether fowlpox virus （FPV） can proliferate in chicken embryo passage fibroblasts or not and then try to use chicken embryo passage fibroblasts to replace primary chicken embryo cells for FPV ...[Objective] To observe whether fowlpox virus （FPV） can proliferate in chicken embryo passage fibroblasts or not and then try to use chicken embryo passage fibroblasts to replace primary chicken embryo cells for FPV culture. [Method] Primary chicken embryo fibroblasts were prepared and subcultured. After FPV were inoculated on the 20th passage fibroblasts, cytopathy was observed. Then, the FPV culture was identified and determined quantificationally. [Result] Specific cytopathy appeared in the FPV-inoculated chicken embryo passage fibroblasts. The titer of the yielded FPV culture reached the standard for production of fowl pox vaccine. Further analysis reveals that the chorioallantoic membrane lesions were caused by FPV. [ Conclusion] FPV can reproduce in chicken embryo passage fibroblasts, and the Uter of FPV cell culture can meet the pro- duction requirements of fowl pox vaccine.展开更多
基金National "11th Five-year Plan" Scientific and Technical Supporting Programs (2006BAD06A11)
文摘Chicken embryo fibroblasts (CEFs) are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus (AIV).In this study,the expression of eleven housekeeping genes typically used for the normalization of quantitative real-time PCR (QPCR) analysis in mammals were compared in CEFs infected with H5N1 AIV to determine the most reliable reference genes in this system.CEFs cultured from 10-day-old SPF chicken embryos were infected with 100 TCID50 of H5N1 AIV and harvested at 3,12,24 and 30 hours post-infection.The expression levels of the eleven reference genes in infected and uninfected CEFs were determined by real-time PCR.Based on expression stability and expression levels,our data suggest that the ribosomal protein L4 (RPL4) and tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ) are the best reference genes to use in the study of host cell response to H5N1 AIV infection.However,for the study of replication levels of H5N1 AIV in CEFs,the β-actin gene (ACTB) and the ribosomal protein L4 (RPL4) gene are the best references.
基金supported by a grant from Yunnan Provincial Education Board(08C0070)a grant from Yunnan Provincial Program for Introducing High-level Scientists (2009CI125)
文摘Inducing animal viruses to adapt to chicken embryos or chicken embryo fibroblasts(CEF) is a common method to develop attenuated live vaccines with full security.Canine distemper virus(CDV) also does this,but the mechanisms and particular receptors remain unclear.Virus overlay protein blot assays were carried out on CEF membrane proteins,which were extracted respectively with a Mem-PER TM kit,a radioimmunoprecipitation assay buffer or a modified co-immunoprecipitation method,and revealed a common 57 kDa positive band that differed from the 42-kDa positive band in Vero cells and also from those receptors reported in lymphocytes and 293 cells,indicating a receptor diversity of CDV and the possibility of the 57-kDa protein acting as a receptor that is involved in adaptive infection of CDV Kunming strain to CEF.
基金The study was supported by the National Science and Technology Foundation during the 10th Five-Year Plan Period(2004BA519A58)Applied Basic Research Program of Sichuan Province(05JY029-007-5).
文摘RNA interference(RNAi)technology is a powerful tool for identifying gene functions.Chicken embryo fibroblast(CEF)is an ideal model for studying the interaction between avian viruses and their hosts.To establish a methodological platform for RNAi studies in CEF,three plasmid vectors expressing short hairpin RNAs(shRNAs)targeted against the Newcastle disease virus(NDV)NP gene were constructed.One of them,ndv1,was proven effective on blocking viral replication in CEF and chicken embryos.Four hours prior to infection with NDV,the CEF was transfected with the plasmids by Silent-fect.An unrelated shRNA sequence(HK)was used in mock transfection.The expression of a potent shRNA resulted in up to 2.3,21.1 and 9.8 fold decreases in NP gene expression at 3,6 and 9 h post infection in CEF,respectively.The ndv1 was able to completely inhibit the replication of the virus in CEF within 48 post infection.Furthermore,the pathological changes in CEF caused by NDV were delayed,and the degree of pathological changes was lighter compared with the mock transfection in the presence of ndv1.When the complex of shRNASilent-fect and NDV was co-injected into the allantoic cavity of 10-day-old embryonated eggs with 10^(5) or 10^(6) ELD50 NDV,NDV replication was decreased by 94.14% and 62.15% after 17 h,respectively.These findings suggest that the newly synthesized NP protein is critical for NDV transcription and replication and provide a basis for identifying the functions of viral genes and screening for effective siRNAs against viruses in CEF and chicken embryo by RNAi.
基金Liaoning Agricultural College for providing test site and fund for Doctors of Liaoning Medical College
文摘[Objective] To observe whether fowlpox virus （FPV） can proliferate in chicken embryo passage fibroblasts or not and then try to use chicken embryo passage fibroblasts to replace primary chicken embryo cells for FPV culture. [Method] Primary chicken embryo fibroblasts were prepared and subcultured. After FPV were inoculated on the 20th passage fibroblasts, cytopathy was observed. Then, the FPV culture was identified and determined quantificationally. [Result] Specific cytopathy appeared in the FPV-inoculated chicken embryo passage fibroblasts. The titer of the yielded FPV culture reached the standard for production of fowl pox vaccine. Further analysis reveals that the chorioallantoic membrane lesions were caused by FPV. [ Conclusion] FPV can reproduce in chicken embryo passage fibroblasts, and the Uter of FPV cell culture can meet the pro- duction requirements of fowl pox vaccine.
