Development of a Rhinovirus Inoculum Using a Reverse Genetics Approach.
Development of a Rhinovirus Inoculum Using a Reverse Genetics Approach.
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DOI：
10.1093/infdis/jiy629
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发表时间：
2019-06-19
期刊：
影响因子：
--
通讯作者：
Crisafi G
中科院分区：
文献类型：
--
作者：
Gern JE;Lee WM;Swenson CA;Nakagome K;Lee I;Wolff M;Grindle K;Sigelman S;Liggett SB;Togias A;Evans M;Denlinger L;Gangnon R;Bochkov YA;Crisafi G
实验接种是研究普通感冒和哮喘的重要工具。从鼻分泌物生产鼻病毒（RV）接种物需要对病毒供体进行长时间观察，以排除外来病原体。我们使用反向遗传学生产了RV-A16接种物，并确定了在血清阴性志愿者中引起中度感冒所需的剂量。克隆来自先前接种物的RV-A16的共有序列，并使用反向遗传学技术产生接种物病毒。在安全性测试后，志愿者接种RV-A16（n = 26）或安慰剂（n = 10），记录杰克逊感冒评分，并测试鼻分泌物中RV-A16核糖核酸的脱落。反向遗传学过程产生的感染性病毒被特异性抗血清中和，突变率与传统的病毒生长技术相似。1000中位组织培养感染剂量（TCID 50）剂量在大多数个体中产生中度感冒，其效果与先前测试的常规RV-A16接种物相似。反向遗传学技术产生了一种RV-A16接种物，可以在血清阴性志愿者中引起临床感冒，它们也可以作为实验室使用的稳定病毒来源。重组生产程序消除了从鼻分泌物中获得种子病毒的需要，从而排除了通过该途径引入外源性病原体。反向遗传学技术被用于开发一种新的鼻病毒接种物，该接种物在实验接种的志愿者中诱导呼吸道疾病。这种接种病毒是克隆的，而不是从人鼻分泌物中培养的，因此降低了外来病原体污染的风险。
Experimental inoculation is an important tool for common cold and asthma research. Producing rhinovirus (RV) inocula from nasal secretions has required prolonged observation of the virus donor to exclude extraneous pathogens. We produced a RV-A16 inoculum using reverse genetics and determined the dose necessary to cause moderate colds in seronegative volunteers. The consensus sequence of RV-A16 from a previous inoculum was cloned, and inoculum virus was produced using reverse genetics techniques. After safety testing, volunteers were inoculated with either RV-A16 (n = 26) or placebo (n = 10), Jackson cold scores were recorded, and nasal secretions were tested for shedding of RV-A16 ribonucleic acid. The reverse genetics process produced infectious virus that was neutralized by specific antisera and had a mutation rate similar to conventional virus growth techniques. The 1000 median tissue culture infectious dose (TCID50) dose produced moderate colds in most individuals with effects similar to that of a previously tested conventional RV-A16 inoculum. Reverse genetics techniques produced a RV-A16 inoculum that can cause clinical colds in seronegative volunteers, and they also serve as a stable source of virus for laboratory use. The recombinant production procedures eliminate the need to derive seed virus from nasal secretions, thus precluding introduction of extraneous pathogens through this route. Reverse genetics technology was used to develop a new rhinovirus inoculum, which induced respiratory illness in experimentally inoculated volunteers. This inoculum virus was cloned instead of being grown from human nasal secretions, therefore reducing risks of contamination with extraneous pathogens.
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15.9
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