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Marine Biotechnology

, Volume 21, Issue 2, pp 186–195 | Cite as

Screening for Antiviral Medaka Haploid Embryonic Stem Cells by Genome Wide Mutagenesis

  • Wanwan Zhang
  • Peng Jia
  • Wei Liu
  • Kuntong JiaEmail author
  • Meisheng YiEmail author
Original Article

Abstract

Nervous necrosis virus (NNV), one of the most prevalent fish pathogens, has caused significant losses in both yield and economy to the aquaculture. Host factors involved in NNV infection remain to be identified due to the lack of ideal model for the study of NNV and host interaction. Haploid stem cells have proven to be ideal materials in genetic screens. Here, we generated a cell line HX1G1 (simply named G1) with the activity against red-spotted grouper nervous necrosis virus (RGNNV) by N-ethyl-N-nitrosourea (ENU)-mediated whole genome random mutagenesis from the haploid embryonic stem cell HX1a, a cell clone from haploid cell line HX1 that we previously derived from the medaka fish. G1 cells retained the characteristics of haploidy and pluripotency as indicated by the EBs differentiation ability after genetic mutagenesis. Compared with HX1a cells, no typical cytopathic effects were observed, and the expression of RNA-dependent RNA polymerase (RDRP) was significantly reduced in G1 cells post RGNNV infection, indicating the enhanced anti-RGNNV activity of G1. Furthermore, we demonstrated that RGNNV entry into G1 cells was partially inhibited, and this inhibition might be relevant to the induced mutation of heat shock cognate protein 70 (HSC70) which was decisive for NNV entry. Interestingly, G1 cells were to some extent permissive to RGNNV infection, but RGNNV was spontaneously cleared in G1 cells during serial passage. In addition, we also found that the expression levels of interferon (IFN)-related genes were higher in G1 cells than those in HX1a cells, suggesting that viral clearance might be associated with the elevated expression of IFN-related genes in G1 cells.

Keywords

Haploid embryonic stem cells Red-spotted grouper nervous necrosis virus N-ethyl-N-nitrosourea Antiviral activity 

Notes

Funding Information

This work was supported by the Natural Science Foundation of Guangdong Province (2015A030308012), the National Natural Science Foundation of China (31502195, 31602191, 31771587), the Zhuhai Scholar Professor Program (2015), Fundamental Research Funds for the Central Universities (17lgpy61), the Science and Technology Planning Project of Guangdong Province (2017A030303010), and the Pearl River S&T Nova Program of Guangzhou (201806010047).

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflicts of interest.

Supplementary material

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Fig. S1

Alkaline phosphatase staining of medaka liver (a) and brain cells (b). (PNG 1772 kb)

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High definition image (TIF 896 kb)
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Fig. S2

Expression of clathrin in HX1a and G1 cells post RGNNV infection. (PNG 164 kb)

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High definition image (TIF 92 kb)
10126_2018_9870_Fig10_ESM.png (781 kb)
Fig. S3

Alignment of HX1a-HSC70 and G1-HSC70. Nucleotide sequences of HSC70 in HX1a and G1 cells. The different nucleotides were highlighted in blue. The predicted amino acid sequence of HX1a-HSC70 is indicated below the DNA sequence, G1-HSC70 mutations on the protein were highlighted in red. The stop codons were indicated by asterisks. The red triangle indicated an A insertion (at position 1861 bp) in HSC70 gene of G1 cells resulted in the reading frame shift mutation. (PNG 781 kb)

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High definition image (TIF 2284 kb)
10126_2018_9870_MOESM4_ESM.xlsx (17 kb)
ESM 1 (XLSX 17 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.School of Marine SciencesSun Yat-sen UniversityGuangdongChina
  2. 2.Guangdong Provincial Key Laboratory of Marine Resources and Coastal EngineeringGuangzhouChina
  3. 3.Zhuhai Key Laboratory of Marine Bioresources and EnvironmentGuangdongChina

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