Kobuvirus comprises 6 officially recognized species,namely Aichivirus A-F,and can be further divided into 20 genotypes through VP1 gene phylogenetic analysis(https://ictv.global/report/chapter/picornaviridae/picornavi...Kobuvirus comprises 6 officially recognized species,namely Aichivirus A-F,and can be further divided into 20 genotypes through VP1 gene phylogenetic analysis(https://ictv.global/report/chapter/picornaviridae/picornaviridae/kobuvirus).Aichivirus A in human,Aichivirus B in bovine,Aichivirus C in porcine and caprine,Aichivirus D in yak have been proved to be associated with diarrhea(Chen Y S et al.2013;Yang et al.2015;Zhu et al.2016;Zhai et al.2017;Wang et al.2020;Abi et al.2022;Yan et al.2023).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
基金sponsored by the Southwest Minzu University Double World-Class ProjectChina(XM2023014)+3 种基金the Sichuan Veterinary Medicine and Drug Innovation Group of China Agricultural Research System(SCCXTD-2020-18)the Key Laboratory of Veterinary Medicine of Universities of Sichuan Province of Chinathe Fundamental Research Funds for the Central UniversitiesSouthwest Minzu University China(ZYN2023043)。
文摘Kobuvirus comprises 6 officially recognized species,namely Aichivirus A-F,and can be further divided into 20 genotypes through VP1 gene phylogenetic analysis(https://ictv.global/report/chapter/picornaviridae/picornaviridae/kobuvirus).Aichivirus A in human,Aichivirus B in bovine,Aichivirus C in porcine and caprine,Aichivirus D in yak have been proved to be associated with diarrhea(Chen Y S et al.2013;Yang et al.2015;Zhu et al.2016;Zhai et al.2017;Wang et al.2020;Abi et al.2022;Yan et al.2023).
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.