aMPV反向遗传操作系统的建立及其重组疫苗株的构建与评价

Journal of Integrative Agriculture ›› 2025, Vol. 24 ›› Issue (5): 1972-1986.DOI: 10.1016/j.jia.2024.04.025

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aMPV反向遗传操作系统的建立及其重组疫苗株的构建与评价

收稿日期:2024-01-06 修回日期:2024-04-13 接受日期:2024-03-02 出版日期:2025-05-20 发布日期:2025-04-18

Development of an improved reverse genetics system for avian metapneumovirus (aMPV): A novel vaccine vector protects against aMPV and infectious bursal disease virus

Lingzhai Meng¹, Yuntong Chen¹, Mengmeng Yu¹, Peng Liu¹, Xiaole Qi¹, Xiaoxiao Xue¹, Ru Guo¹, Tao Zhang¹, Mingxue Hu¹, Wenrui Fan¹, Ying Wang¹, Suyan Wang¹, Yanping Zhang¹, Yongzhen Liu¹, Yulu Duan¹, Hongyu Cui¹, Yulong Gao^{1, 2#}

¹Avian Immunosuppressive Diseases Division, State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China
² National Poultry Laboratory Animal Resource Center, Harbin 150069, China

Received:2024-01-06 Revised:2024-04-13 Accepted:2024-03-02 Online:2025-05-20 Published:2025-04-18
About author:Lingzhai Meng, E-mail: menglingzhai@126.com; #Correspondence Yulong Gao, Tel: +86-451-51091691, E-mail: gaoyulong@caas.cn
Supported by:
This work was supported by the grants from the National Key Research and Development Program of China (2022YFD1800604), the China Agriculture Research System (CARS-41), and the Heilongjiang Touyan Innovation Team Program, China.

摘要/Abstract

摘要：

禽偏肺病毒（Avian metapneumovirus，aMPV）为副黏病毒科肺病毒亚科偏肺病毒属家族的成员，其主要引起火鸡鼻气管炎（Turkey rhinotracheitis，TRT）和肉鸡肿头综合征（Swollen head syndrome，SHS）。目前，B亚型aMPV是我国鸡群中主要的优势流行毒株，由于缺乏aMPV反向遗传操作技术，有关该病毒的致病与致弱机制及是否可作为病毒载体的研究相对较少。为此，本研究将B亚型aMPV弱毒株LN16-A株全长分为5个cDNA片段进行扩增，并在基因组的3′端和5′端分别添加了T7启动子和丁型肝炎核酶序列，构建了全长cDNA感染性克隆质粒pOKLN16-A。将pOKLN16-A与4个辅助质粒pCAGGS-N、pCAGGS-P、pCAGGS-M21和pCAGGS-L共转染至表达T7 RNA聚合酶的BSR-T7/5细胞中，拯救出了病毒，成功建立了基于T7 RNA聚合酶的aMPV反向遗传操作系统。为进一步探究aMPV作为疫苗载体的潜力，利用反向遗传操作技术将增强型绿色荧光蛋白基因（Enhanced green fluorescent protein，EGFP）插入aMPV基因组的不同位点，并比较了其表达水平，结果显示，EGFP在B亚型aMPV的G和L基因之间的表达水平显著高于另外两个插入位点（前导基因和N基因之间及替换SH基因），因此确定外源基因表达的最佳插入位点为G和L基因之间。为进一步验证该插入位点的可用性，以鸡传染性法氏囊病病毒超强毒株（Very virulent infection bursal disease virus，vvIBDV）为模式病毒，利用反向遗传操作技术在该位点插入了其保护性抗原VP2基因，成功拯救了稳定表达vvIBDV VP2蛋白的重组B亚型aMPV，命名为rLN16A-vvVP2株。将rLN16A-vvVP2株以5000 TCID₅₀/只的剂量免疫SPF鸡，免疫3周后使用B亚型aMPV LN16-F4强毒株及vvIBDV HLJ0504强毒株进行攻毒。结果显示，单次免疫rLN16A-vvVP2株可同时诱导机体产生针对B亚型aMPV及vvIBDV两种病毒的中和抗体，免疫3周后的中和抗体效价分别为8.7及8.2 log2。此外，单次免疫rLN16A-vvVP2株对B亚型aMPV强毒及vvIBDV强毒的攻毒保护率均为100%，并能有效预防vvIBDV攻击后引起的法氏囊损伤。本研究成功建立了B亚型aMPV的反向遗传操作系统并鉴定了外源基因表达的最佳插入位点，首次评价了B亚型aMPV作为疫苗载体的潜力，研究结果为进一步研究aMPV的致病机制和安全有效的新型载体疫苗提供了技术支撑。

Abstract:

Avian metapneumovirus (aMPV), a paramyxovirus, causes acute respiratory diseases in turkeys and swollen head syndrome in chickens. This study established a reverse genetics system for aMPV subtype B LN16-A strain based on T7 RNA polymerase. Full-length cDNA of the LN16-A strain was constructed by assembling 5 cDNA fragments between the T7 promoter and hepatitis delta virus ribozyme. Transfection of this plasmid, along with the supporting plasmids encoding the N, P, M2-1, and L proteins of LN16-A into BSR-T7/5 cells, resulted in the recovery of aMPV subtype B. To identify an effective insertion site, the enhanced green fluorescent protein (EGFP) gene was inserted into different sites of the LN16-A genome to generate recombinant LN16-As. The results showed that the expression levels of EGFP at the site between the G and L genes of LN16-A were significantly higher than those at the other two sites (between the leader and N genes or replacing the SH gene). To verify the availability of the site between G and L for foreign gene expression, the VP2 gene of very virulent infectious bursal disease virus (vvIBDV) was inserted into this site, and recombinant LN16-A (rLN16A-vvVP2) was successfully rescued. Single immunization of specific-pathogen-free chickens with rLN16A-vvVP2 induced high levels of neutralizing antibodies and provided 100% protection against the virulent aMPV subtype B and vvIBDV. Establishing a reverse genetics system here provides an important foundation for understanding aMPV pathogenesis and developing novel vector vaccines.

Key words: avian metapneumovirus , reverse genetics system , insertion site , protection , vector vaccines

Lingzhai Meng, Yuntong Chen, Mengmeng Yu, Peng Liu, Xiaole Qi, Xiaoxiao Xue, Ru Guo, Tao Zhang, Mingxue Hu, Wenrui Fan, Ying Wang, Suyan Wang, Yanping Zhang, Yongzhen Liu, Yulu Duan, Hongyu Cui, Yulong Gao. aMPV反向遗传操作系统的建立及其重组疫苗株的构建与评价[J]. Journal of Integrative Agriculture, 2025, 24(5): 1972-1986.

Lingzhai Meng, Yuntong Chen, Mengmeng Yu, Peng Liu, Xiaole Qi, Xiaoxiao Xue, Ru Guo, Tao Zhang, Mingxue Hu, Wenrui Fan, Ying Wang, Suyan Wang, Yanping Zhang, Yongzhen Liu, Yulu Duan, Hongyu Cui, Yulong Gao. Development of an improved reverse genetics system for avian metapneumovirus (aMPV): A novel vaccine vector protects against aMPV and infectious bursal disease virus[J]. Journal of Integrative Agriculture, 2025, 24(5): 1972-1986.

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