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建立基于克隆 DNA 的腮腺炎病毒 S79 高效反向遗传学系统。

Establishment of an efficient reverse genetic system of Mumps virus S79 from cloned DNA.

机构信息

Zhejiang University School of Medicine, Hangzhou, China.

Department of Neurology, Children's Hospital, Zhejiang University School of Medicine, Hangzhou, 310052, China.

出版信息

World J Pediatr. 2019 Oct;15(5):499-505. doi: 10.1007/s12519-019-00286-8. Epub 2019 Aug 28.

DOI:10.1007/s12519-019-00286-8
PMID:31456156
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6785654/
Abstract

BACKGROUND

Mumps is a common type of respiratory infectious disease caused by mumps virus (MuV), and can be effectively prevented by vaccination. In this study, a reverse genetic system of MuV that can facilitate the rational design of safer, more efficient mumps vaccine candidates is established.

METHODS

MuV-S79 cDNA clone was assembled into a full-length plasmid by means of the GeneArt™ High-Order Genetic Assembly System, and was rescued via reverse genetic technology. RT-PCR, sequencing, and immunofluorescence assays were used for rMuV-S79 authentication. Viral replication kinetics and in vivo experimental models were used to evaluate the replication, safety, and immunogenicity of rMuV-S79.

RESULTS

A full-length cDNA clone of MuV-S79 in the assembly process was generated by a novel plasmid assemble strategy, and a robust reverse genetic system of MuV-S79 was successfully established. The established rMuV-S79 strain could reach a high virus titer in vitro. The average viral titer of rMuV-S79 in the lung tissues was 2.68 ± 0.14 logPFU/g lung tissue, and rMuV-S79 group did not induce inflammation in the lung tissues in cotton rats. Neutralizing antibody titers induced by rMuV-S79 were high, long-lasting and could provide complete protection against MuV wild strain challenge.

CONCLUSION

We have established a robust reverse genetic system of MuV-S79 which can facilitate the optimization of mumps vaccines. rMuV-S79 rescued could reach a high virus titer and the safety was proven in vivo. It could also provide complete protection against MuV wild strain challenge.

摘要

背景

流行性腮腺炎是由腮腺炎病毒（MuV）引起的常见呼吸道传染病，可通过疫苗接种有效预防。本研究建立了 MuV 的反向遗传系统，可促进更安全、更有效的腮腺炎候选疫苗的合理设计。

方法

采用 GeneArt™ 高级遗传组装系统将 MuV-S79 cDNA 克隆组装成全长质粒，并通过反向遗传技术进行拯救。通过 RT-PCR、测序和免疫荧光检测对 rMuV-S79 进行鉴定。通过病毒复制动力学和体内实验模型评估 rMuV-S79 的复制、安全性和免疫原性。

结果

在组装过程中，采用了一种新颖的质粒组装策略生成了 MuV-S79 的全长 cDNA 克隆，并成功建立了 MuV-S79 的强大反向遗传系统。所建立的 rMuV-S79 株在体外可达到高病毒滴度。rMuV-S79 在肺组织中的平均病毒滴度为 2.68±0.14 logPFU/g 肺组织，且 rMuV-S79 组在棉鼠肺组织中未引起炎症。rMuV-S79 诱导的中和抗体滴度高、持久，并能提供针对 MuV 野生株攻击的完全保护。

结论

我们建立了 MuV-S79 的强大反向遗传系统，可促进腮腺炎疫苗的优化。拯救的 rMuV-S79 可达到高病毒滴度，并在体内得到证实安全性。它还可以提供针对 MuV 野生株攻击的完全保护。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cb48/6785654/18b5af432653/12519_2019_286_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cb48/6785654/2362ae99ef9a/12519_2019_286_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cb48/6785654/4d9435652354/12519_2019_286_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cb48/6785654/18b5af432653/12519_2019_286_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cb48/6785654/2362ae99ef9a/12519_2019_286_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cb48/6785654/4d9435652354/12519_2019_286_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cb48/6785654/18b5af432653/12519_2019_286_Fig3_HTML.jpg

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