构建并鉴定鸭甲型肝炎病毒 1 型 DNA 载体疫苗的改进型感染性克隆。
Construction and characterization of an improved DNA-launched infectious clone of duck hepatitis a virus type 1.
机构信息
Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Taian, Shandong, 271018, China.
Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Taian, Shandong, 271018, China.
出版信息
Virol J. 2017 Nov 3;14(1):212. doi: 10.1186/s12985-017-0883-5.
BACKGROUND
DNA-launched infectious system is a useful tool with high rescue efficiency that allows the introduction of mutations in specific positions to investigate the function of an individual viral element. Rescued virus particles could be harvested by directly transfecting the DNA-launched recombinant plasmid to the host cells, which will reduce labor and experimental cost by skipping the in vitro transcription assay.
METHODS
A total of four overlapping fragments covering the entire viral genome were amplified and then were assembled into a transformation vector based on pIRES2-EGFP to establish the DNA-launched infectious system of duck hepatitis A virus type 1 (DHAV-1), named pIR-DHAV-1. Reverse transcription polymerase chain reaction (RT-PCR) detection, quantitative real-time polymerase chain reaction (qRT-PCR), western blotting assay and indirect immunofluorescence (IFA) were conducted for rescued virus identification. A total of 4.0 μg of recombinant plasmid of pIR-DHAV-1 and in vitro transcribed product of 4.0 μg of RNA-launched infectious clone named pR-DHAV-1 were transfected into BHK-21 cells to analyze the rescue efficiency. Following that, tissue tropism of rescued virus (rDHAV-1) and parental virus (pDHAV-1) were assayed for virulence testing in 1-day-old ducklings.
RESULTS
Rescued virus particles carry the designed genetic marker which could be harvested by directly transfecting pIR-DHAV-1 to BHK-21 cells. The qRT-PCR and western blotting results indicated that rDHAV-1 shared similar growth characteristics with pDHAV-1. Furthermore, DNA-launched infectious system possessed much higher rescue efficiency assay compared to RNA-launched infectious system. The mutation at position 3042 from T to C has no impact on viral replication and tissue tropism. From 1 h post infection (hpi) to 48 hpi, the viral RNA copies of rDHAV-1 in liver were the highest among the six tested tissues (with an exception of thymus at 6 hpi), while the viral RNA copy numbers in heart and kidney were alternately the lowest.
CONCLUSION
We have constructed a genetically stable and highly pathogenic DNA-launched infectious clone, from which the rescued virus could be harvested by direct transfection with recombinant plasmids. rDHAV-1 shared similar growth characteristics and tissue tropism with pDHAV-1. The DNA-launched infectious system of DHAV-1 possessed higher rescue efficiency compared to the traditional RNA-launched infectious system.
背景
DNA 启动的感染性系统是一种高效的工具，可实现特定位置的突变引入，从而研究单个病毒元件的功能。通过直接将 DNA 启动的重组质粒转染到宿主细胞中，可以回收病毒颗粒，从而避免体外转录测定，从而降低劳动和实验成本。
方法
总共扩增了覆盖整个病毒基因组的四个重叠片段，然后将其组装到基于 pIRES2-EGFP 的转化载体中，建立了鸭甲型肝炎病毒 1 型（DHAV-1）的 DNA 启动的感染性系统，命名为 pIR-DHAV-1。通过逆转录聚合酶链反应（RT-PCR）检测、定量实时聚合酶链反应（qRT-PCR）、Western blot 分析和间接免疫荧光（IFA）鉴定回收病毒。将 4.0μg 的重组质粒 pIR-DHAV-1 和 4.0μg 的 RNA 启动的感染性克隆 pR-DHAV-1 的体外转录产物转染到 BHK-21 细胞中，分析拯救效率。然后，在 1 日龄雏鸭中进行病毒毒力试验，检测回收病毒（rDHAV-1）和亲本病毒（pDHAV-1）的组织嗜性。
结果
回收的病毒颗粒带有设计的遗传标记，可以通过直接转染 pIR-DHAV-1 到 BHK-21 细胞中进行收获。qRT-PCR 和 Western blot 结果表明，rDHAV-1 与 pDHAV-1 具有相似的生长特征。此外，与 RNA 启动的感染性系统相比，DNA 启动的感染性系统具有更高的拯救效率。位置 3042 从 T 到 C 的突变对病毒复制和组织嗜性没有影响。从 1 小时感染后（hpi）到 48 hpi，rDHAV-1 在肝脏中的病毒 RNA 拷贝数在六个检测组织中最高（胸腺在 6 hpi 时除外），而心脏和肾脏中的病毒 RNA 拷贝数则交替最低。
结论
我们构建了一种遗传稳定且高致病性的 DNA 启动的感染性克隆，可通过直接转染重组质粒回收病毒。rDHAV-1 与 pDHAV-1 具有相似的生长特征和组织嗜性。与传统的 RNA 启动的感染性系统相比，DHAV-1 的 DNA 启动的感染性系统具有更高的拯救效率。
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