The Study on the Effect of NS2A-C60A Site Mutation of Japanese Encephalitis Virus on Its Biological Characteristics

doi:10.13523/j.cb.2005059

China Biotechnology

2020, Vol. 40

Issue (9): 1-10 DOI: 10.13523/j.cb.2005059

The Study on the Effect of NS2A-C60A Site Mutation of Japanese Encephalitis Virus on Its Biological Characteristics

GUO Guang-chao¹,ZHOU Yu-yong¹,CAO San-jie^1,^2,^3,^**(

),WU Yao-min¹,WU Rui¹,ZHAO Qin¹,WEN Xin-tian¹,HUANG Xiao-bo¹,WEN Yi-ping¹

1 Research Center for Swine Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China
2 National Teaching and Experiment Center of Animal, Sichuan Agricultural University, Chengdu 611130, China
3 Sichuan Science-observation Experimental Station of Veterinary Drugs and Veterinary Diagnostic Technology, sMinistry of Agriculture, Chengdu 611130, China

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Abstract

Objective: In order to explore the effect of the NS2A-C60A gene mutation in the attenuation process on the biological characteristics of Japanese encephalitis virus. Method: First of all, the target research site NS2A-C60A that may be related to the expression of NS1' and virulence weakening was determined by sequencing and comparison of the genomes of the JEV attenuated strain and the original strain, prediction of the secondary structure of the 5'-terminal sequence of NS2A gene, and changes in NS1' protein expression; then, the NS2A-C60A single point mutation virus strain rJEV-C60A was rescued by using reverse genetic site-directed mutation technology; Finally, the influences on the biological characteristics for JEV were studyed by the observation of plaque morphology, growth curve, dual luciferase analysis, WB, inflammatory factor detection and animal experiments. Results: The research found for the first time that the passage attenuated of type Ⅰ JEV would lead to a significant decrease in NS1' protein expression and the related site NS2A-C60A, and successfully rescued the NS2A-C60A single point mutant strain rJEV-C60A. Plaque and growth curve results show that the NS2A-C60A mutation have no effect on the growth characteristics of JEV, but dual luciferase analysis and WB results reveal that NS2A-C60A mutation can significantly reduce the expression of viral NS1' protein. The results of inflammatory factors suggeset that the NS2A-C60A site mutation can slightly hinder the JEV suppression of inflammatory factors expression, but animal experiments show that the NS2A-C60A point mutation virus has similar neurovirulence to the original strain, indicating that the site mutation is not a key site that affects the virulence of JEV. Conclusion: The novel mutation NS2A-C60A can significantly reduce the expression of the JEV NS1' protein, but it has no significant effects on its other biological characteristics, like proliferation, induced inflammation and neurovirulence.

Key words： Japanese encephalitis virus Passage attenuation NS2A-C60A point mutation NS1' protein Biological characteristics

Received: 27 May 2020 Published: 12 October 2020

ZTFLH:

Q819

Corresponding Authors: San-jie CAO E-mail: veterinary226@163.com

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	Guang-chao GUO
	Yu-yong ZHOU
	San-jie CAO
	Yao-min WU
	Rui WU
	Qin ZHAO
	Xin-tian WEN
	Xiao-bo HUANG
	Yi-ping WEN

Cite this article:

GUO Guang-chao,ZHOU Yu-yong,CAO San-jie,WU Yao-min,WU Rui,ZHAO Qin,WEN Xin-tian,HUANG Xiao-bo,WEN Yi-ping. The Study on the Effect of NS2A-C60A Site Mutation of Japanese Encephalitis Virus on Its Biological Characteristics. China Biotechnology, 2020, 40(9): 1-10.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2005059 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I9/1

Fig.1 The map of the target research site NS2A-C60A determined (a) F1 and F120 generation NS1' protein WB (b) NS2A gene sequence alignment (c) NS2A gene secondary structure prediction

Fig.2 The map of the target research site NS2A-C60A determined (a) F1 and F120 generation NS1' protein WB (b) NS2A gene sequence alignment (c) NS2A gene secondary structure prediction

Fig.3 Plaque morphology of the original strain rJEV-WT and the single point mutant strain rJEV-C60A rJEV-WT: Original strain; rJEV-C60A: NS2A-C60A point mutation strain(rescued)

Fig. 4 Growth curve detection results of the original strain rJEV-WT and the single point mutant strain rJEV-C60A

Fig.5 The effect of NS2A-C60A point mutation on dual luciferase activity *P<0.05

Fig.6 WB detection results of NS1 'protein expression of the original strain rJEV-WT and the single point mutant strain rJEV-C60A *P<0.05

Fig.7 The detection results of inflammatory factors TNF-α, CCL-2, IL-6 and IFN-beta by using qRT-PCR ns:P>0.05

Fig.8 Neurovirulence test results of the original strain rJEV-WT and the point mutant strain rJEV-C60A


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