Skip to main content
SpringerLink
Log in

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

  • Original Article
  • Published:
Marine Biotechnology Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Aydin Y, Chatterjee A, Chandra PK, Chava S, Chen W, Tandon A, Dash A, Chedid M, Moehlen MW, Regenstein F, Balart LA, Cohen A, Lu H, Wu T, Dash S (2017) Interferon-alpha-induced hepatitis C virus clearance restores p53 tumor suppressor more than direct-acting antivirals. Hepatol Commun 1:256–269

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Béjar J, Hong Y, Schartl M (2003) Mitf expression is sufficient to direct differentiation of medaka blastula derived stem cells to melanocytes. Development 130:6545–6553

    Article  CAS  PubMed  Google Scholar 

  • Bull KR, Rimmer AJ, Siggs OM, Miosge LA, Roots CM, Enders A, Bertram EM, Crockford TL, Whittle B, Potter PK, Simon MM, Mallon AM, Brown SDM, Beutler B, Goodnow CC, Lunter G, Cornall RJ (2013) Unlocking the bottleneck in forward genetics using whole-genome sequencing and identity by descent to isolate causative mutations. PLoS Genet 9:e1003219

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Caignard G, Eva MM, van Bruggen R, Eveleigh R, Bourque G, Malo D, Gros P, Vidal SM (2014) Mouse ENU mutagenesis to enderstand immunity to infection: methods, selected examples, and perspectives. Genes 5:887–925

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chang JS, Chi SC (2015) GHSC70 is involved in the cellular entry of nervous necrosis virus. J Virol 89:61–70

    Article  CAS  Google Scholar 

  • Chisada S, Okamoto H, Taniguchi Y, Kimori Y, Toyoda A, Sakaki Y, Takeda S, Yoshiura Y (2011) Myostatin-deficient medaka exhibit a double-muscling phenotype with hyperplasia and hypertrophy, which occur sequentially during post-hatch development. Dev Biol 359:82–94

    Article  CAS  PubMed  Google Scholar 

  • Choe J, Lee SM, Lim S, Choi SH, Kim N, Do K-H, Seo JB (2017) Doubling time of thymic epithelial tumours on CT: correlation with histological subtype. Eur Radiol 27:4030–4036

    Article  PubMed  Google Scholar 

  • Costa JZ, Thompson KD (2016) Understanding the interaction between Betanodavirus and its host for the development of prophylactic measures for viral encephalopathy and retinopathy. Fish Shellfish Immunol 53:35–49

    Article  CAS  PubMed  Google Scholar 

  • Dong C, Shuang F, Weng S, He J (2014) Cloning of a new fibroblast cell line from an early primary culture from mandarin fish (Siniperca chuatsi) fry for efficient proliferation of megalocytiviruses. Cytotechnology 66:883–890

    Article  CAS  PubMed  Google Scholar 

  • Hai T, Guo W, Yao J, Cao C, Luo A, Qi M, Wang X, Wang X, Huang J, Zhang Y, Zhang H, Wang D, Shang H, Hong Q, Zhang R, Jia Q, Zheng Q, Qin G, Li Y, Zhang T, Jin W, Chen ZY, Wang H, Zhou Q, Meng A, Wei H, Yang S, Zhao J (2017) Creation of miniature pig model of human Waardenburg syndrome type 2A by ENU mutagenesis. Hum Genet 136:1463–1475

    Article  CAS  PubMed  Google Scholar 

  • Hoebe K, Beutler B (2005) Unraveling innate immunity using large scale N-ethyl-N-nitrosourea mutagenesis. Tissue Antigens 65:395–401

    Article  CAS  PubMed  Google Scholar 

  • Hong Y, Winkler C, Schartl M (1996) Pluripotency and differentiation of embryonic stem cell lines from the medakafish (Oryzias latipes). Mech Dev 60:33–44

    Article  CAS  PubMed  Google Scholar 

  • Hong Y, Chen S, Gui J, Schartl M (2004) Retention of the developmental pluripotency in medaka embryonic stem cells after gene transfer and long-term drug selection for gene targeting in fish. Transgenic Res 13:41–50

    Article  CAS  PubMed  Google Scholar 

  • Huang X, Huang Y, Cai J, Wei S, Ouyang Z, Qin Q (2013) Molecular cloning, expression and functional analysis of ISG15 in orange-spotted grouper, Epinephelus coioides. Fish Shellfish Immunol 34:1094–1102

    Article  CAS  PubMed  Google Scholar 

  • Huang R, Zhu G, Zhang J, Lai Y, Xu Y, He J, Xie J (2017) Betanodavirus-like particles enter host cells via clathrin-mediated endocytosis in a cholesterol-, pH- and cytoskeleton-dependent manner. Vet Res 48:8–24

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Janiak M, Caraballo Cortes K, Demkow U, Radkowski M (2018) Spontaneous elimination of hepatitis C virus infection. Adv Exp Med Biol 1039:45–54

    Article  PubMed  Google Scholar 

  • Jia P, Jia KT, Yi MS (2015) Complete genome sequence of a fish nervous necrosis virus isolated from sea perch (Lateolabrax japonicus) in China. Genome Announc 3:e00048–e00015

    Article  PubMed  PubMed Central  Google Scholar 

  • Le Y, Li Y, Jin Y, Jia P, Jia K, Yi M (2017) Establishment and characterization of a brain cell line from sea perch, Lateolabrax japonicus. In Vitro Cell Dev Biol Anim 53:834–840

    Article  CAS  PubMed  Google Scholar 

  • Liu W, Hsu CH, Hong YR, Wu SC, Wang CH, Wu YM, Chao CB, Lin CS (2005) Early endocytosis pathways in SSN-1 cells infected by dragon grouper nervous necrosis virus. J Gen Virol 86:2553–256

  • Liu Q, Wang Y, Lin F, Zhang L, Li Y, Ge R, Hong Y (2015) Gene transfer and genome-wide insertional mutagenesis by retroviral transduction in fish stem cells. PLoS One 10:e0127961

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Long H, Sun L (2014) CsIFIT1, an interferon-induced protein with tetratricopeptide repeat, inhibits viral infection in tongue sole (Cynoglossus semilaevis). Fish Shellfish Immunol 41:231–237

    Article  CAS  PubMed  Google Scholar 

  • Poynter SJ, DeWitte-Orr SJ (2016) Fish interferon-stimulated genes: the antiviral effectors. Dev Comp Immunol 65:218–225

    Article  CAS  PubMed  Google Scholar 

  • Raaben M, Jae LT, Herbert AS, Kuehne AI, Stubbs SH, Chou YY, Blomen VA, Kirchhausen T, Dye JM, Brummelkamp TR, Whelan SP (2017) NRP2 and CD63 are host factors for lujo virus cell entry. Cell Host Microbe 22:688–696

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Riblett AM, Blomen VA, Jae LT, Altamura LA, Doms RW, Brummelkamp TR, Wojcechowskyj JA (2015) A haploid genetic screen identifies heparan sulfate proteoglycans supporting rift valley fever virus infection. J Virol 90:1414–1423

    Article  CAS  PubMed  Google Scholar 

  • Wang D, Manali D, Wang T, Bhat N, Hong N, Li Z, Wang L, Yan Y, Liu R, Hong Y (2011) Identification of pluripotency genes in the fish medaka. Int J Biol Sci 7:440–451

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yang Y, Wang X, Liu Y, Fu Q, Tian C, Wu C, Shi H, Yuan Z, Tan S, Liu S, Gao D, Dunham R, Liu Z (2018) Transcriptome analysis reveals enrichment of genes associated with auditory system in swimbladder of channel catfish. Comp Biochem Physiol Part D Genomics Proteomics 27:30–39

    Article  CAS  PubMed  Google Scholar 

  • Yi M, Hong N, Hong Y (2009) Generation of medaka fish haploid embryonic stem cells. Science 326:430–433

    Article  CAS  PubMed  Google Scholar 

  • Yi M, Hong N, Hong Y (2010) Derivation and characterization of haploid embryonic stem cell cultures in medaka fish. Nat Protoc 5:1418–1430

    Article  CAS  PubMed  Google Scholar 

  • Yilmaz A, Peretz M, Sagi I, Benvenisty N (2016) Haploid human embryonic stem cells: half the genome, double the value. Cell Stem Cell 19:569–572

    Article  CAS  PubMed  Google Scholar 

  • Yuan Y, Huang X, Zhang L, Zhu Y, Huang Y, Qin Q, Hong Y (2013) Medaka haploid embryonic stem cells are susceptible to Singapore grouper iridovirus as well as to other viruses of aquaculture fish species. J Gen Virol 94:2352–2359

    Article  CAS  PubMed  Google Scholar 

Download references

Funding

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).

Author information

Authors and Affiliations

  1. School of Marine Sciences, Sun Yat-sen University, Guangdong, China

    Wanwan Zhang, Peng Jia, Wei Liu, Kuntong Jia & Meisheng Yi

  2. Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, Guangzhou, China

    Wanwan Zhang, Peng Jia, Wei Liu, Kuntong Jia & Meisheng Yi

  3. Zhuhai Key Laboratory of Marine Bioresources and Environment, Guangdong, China

    Wanwan Zhang, Peng Jia, Wei Liu, Kuntong Jia & Meisheng Yi

Authors
  1. Wanwan Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Peng Jia
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Wei Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kuntong Jia
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Meisheng Yi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Kuntong Jia or Meisheng Yi.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflicts of interest.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Fig. S1

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

High definition image (TIF 896 kb)

Fig. S2

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

High definition image (TIF 92 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)

High definition image (TIF 2284 kb)

ESM 1

(XLSX 17 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, W., Jia, P., Liu, W. et al. Screening for Antiviral Medaka Haploid Embryonic Stem Cells by Genome Wide Mutagenesis. Mar Biotechnol 21, 186–195 (2019). https://doi.org/10.1007/s10126-018-09870-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10126-018-09870-x

Keywords

Search

Navigation