中国兽医杂志2025，Vol.61Issue(7)：56-62,7.DOI:10.20157/j.cnki.zgsyzz.2025.07.006
含尼帕病毒N基因和L基因的假病毒构建及其应用
Construction and Application of Pseudoviruses Integrating Nipah Virus N and L Genes
摘要
Abstract
Nipah virus(NiV),as a highly fatal zoonotic pathogen,has attracted widespread attention globally.To develop a rapid,sensitive detection method and a safe,stable positive control,this study inserted both the NiV N and L genes into a pseudovirus transfer plasmid(PLJM-GFP),which was co-transfected into human embryonic kidney(HEK 293T)cells with packaging plasmids.Virus stocks were collected and concentrated,and viral titers were measured at 48 and 72 hours post-transfection.The pseudovirus morphology was observed under transmission electron microscopy(TEM),and PCR amplification and sequencing were performed for identification.The packaged pseudovirus was validated with the NiV primers recommended by the World Organisation for Animal Health.Specificity and sensitivity analyses were conducted using real-time fluorescence quantitative PCR(qPCR)with four pairs of NiV detection primers from different standards and literature.Additionally,homogeneity,short-term stability,and RNase A degradation tests were performed on the constructed NiV pseudovirus.The results showed that the viral titers of the pseudovirus collected at 48 and 72 hours post-transfection were 2.72×10⁵ and 3.47×10⁴ IU/mL,respectively.Electron microscopy revealed that the pseudovirus particles were round and enveloped.Sequencing confirmed the successful insertion of the N and L gene fragments into the NiV pseudovirus nucleic acid.The four pairs of NiV detection primers exhibited good specificity and could be used for NiV detection.Among them,the primers from the ongoing national standard detection method showed higher sensitivity,with a detection limit of 100 IU/mL.The NiV pseudovirus demonstrated good homogeneity,remained stable for at least 7 days at 4℃,and was resistant to degradation by a certain amount of RNase A.This study successfully packaged a pseudovirus containing the NiV N and L genes,conducted preliminary application and stability studies,and provided material support for the prevention and control of NiV encephalitis.关键词
尼帕病毒(NiV)/假病毒构建/实时荧光定量逆转录PCR(qRT-PCR)/敏感性试验Key words
Nipah virus(NiV)/pseudovirus construction/real-time fluorescence quantitative reverse transcription PCR(qRT-PCR)/sensitivity test分类
农业科技引用本文复制引用
崔超杰,王志亮,张慧,刘春菊,魏荣,张永强,崔进,戈胜强,李金明,蔡玉梅..含尼帕病毒N基因和L基因的假病毒构建及其应用[J].中国兽医杂志,2025,61(7):56-62,7.基金项目
2025年动物疫情监测与防治 （）
"十四五"国家重点研发计划项目(2022YFD1800500) （2022YFD1800500）
世界银行"预防、准备与应对新发传染病项目"农业农村部子项目(2205-000000-04-01-465744) （2205-000000-04-01-465744）
