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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This manuscript describes a detailed protocol for Hepatitis B virus (HBV) infection in novel engineered 293T cells (293T-NE-3NRs, expressing human NTCP, HNF4α, RXRα and PPARα) and traditional hepatic cells (HepG2-NE, expressing human NTCP).

Abstract

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV models are limited to hepatoma cell lines (such as HepG2 and Huh7) overexpressing a functional HBV receptor, sodium taurocholate co-transporting polypeptide (NTCP). Here, we used 293T-NE-3NRs (293T overexpressing human NTCP, HNF4α, RXRα and PPARα) and HepG2-NE (HepG2 overexpressing NTCP) as model cell lines. HBV infection in these cell lines was performed either by using concentrated HBV virus particles from HepG2.2.15 or co-culturing HepG2.2.15 with the target cell lines. HBcAg immunofluorescence for HBcAg was performed to confirm HBV infection. The two methods presented here will help us study HBV infection in non-hepatic cell lines.  

Introduction

Hepatitis B affects the lives of more than 2 billion people and is one of the major threats to public health. Approximately 257 million people are chronically infected with hepatitis B virus (HBV) worldwide, causing a big burden to the society1. Hepatocytes are not the only cells infected by HBV and other cells in non-hepatic tissue, such as kidney and testis, are also infected by this virus2,3. Currently, HBV infection cell models are limited to human hepatocytes with only few non-hepatic cell line models. This hampers the study of HBV infection and HBV-related pathology of non-hepatic....

Protocol

Culture, collection of supernatants from HepG2.2.15 cells and HBV infection should be performed in biosafety level II (P2) or biosafety level III (P3) laboratory according to the biosafety guidance in the country. Laboratory safety practices should be followed to ensure the safety of laboratory personnel, and all should be vaccinated and detected HBsAb positive before performing HBV experiments. Observe the state of the cells at every step before proceeding to the next step. Human serum samples were used in accordance wi.......

Representative Results

We constructed pSIN-NTCP-EGFP plasmid expressing NTCP and EGFP fusion and with puromycin resistance. The plasmid was transfected into HepG2 and 293T cells to construct stable cell lines HepG2-NE and 293T-NE expressing NTCP and EGFP. Plasmids (pSIN-HNF4α, pSIN-RXRα, pLV-PPARα-puro-flag) with puromycin resistance and expression were transfected into 293T-NE cells to construct a stable cell line expressing 4 host genes9. The expression of NTCP-EGFP can be observed by green fluorescence.......

Discussion

Here, we introduce protocols for HBV infection in non-hepatic 293T-NE-3NRs and hepatoma-based HepG2-NE cells. 293T-NE-3NRs were suitable for HBV infection at both high and low GEq. The following critical steps need to be taken into consideration while using our protocol. The cell status is an important factor for a successful infection. The infection medium must be changed timely after the initial period of HBV infection. 293T-NE-3NRs cells are typically fragile following infection with high viral titers. Therefore, thes.......

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 81870432 and 81570567 to X.L.Z.), (No. 81571994 to P.N.S.), Research Fund for International Young Scientists (No. 81950410640 to W.I.); The Li Ka Shing Shantou University Foundation (No. L1111 2008 to P.N.S.). We would like to thank Prof. Stanley Lin from Shantou University Medical College for useful advice.

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Materials

NameCompanyCatalog NumberComments
0.45μm membrane filterMillex-HVSLHU033RBFilter for HepG 2.2.15 supernatant
293T-NELaboratory construction——Cell model for HBV infection
293T-NE-3NRsLaboratory construction——Cell model for HBV infection
594 labeled goat against rabbit IgGZSGB-BIOZF-0516For immunofluorescence assay,second antibody
6-well plateBIOFILTCP010006For co-culture
Amicon Ultra 15 mlMilliporeUFC910008For concentration of HepG 2.2.15 supernatant
BSABeyotimeST023For immunofluorescence assay
Cyclosporin ASangon biotech59865-13-3inhibitor of HBV infection
DAPIBeyotimeC1006For nuclear staining
Diagnostic kit for Quantification of Hepatitis B Virus DNA(PCR-Fluorescence Probing)DAAN GENE7265-2013For HBV DNA detection
DMEMHyClongSH30243.01For culture medium
DMSOSigma-AldrichD5879For improvement of infection efficiency
Fetal bovine serum(FBS)CLARK BioscienceFB25015For culture medium
Fluorescence microscopeZEISSAxio observer Z1For immunofluorescence assay
HepG2-NELaboratory construction——Cell model for HBV infection
HBcAg antibodyZSGB-BIOZA-0121For immunofluorescence assay, primary antibody
PBSZSGB-BIOZLI-9062For cell wash
PEG8000MerckP8260For infection medium
Penicillin-Streptomycin-GlutamineThermo Fisher10378016For culture medium
TranswellCORNING3412For co-culture
Tween 20sigma-AldrichWXBB7485VFor PBST
VirkonDouban6971728840012Viruside

References

  1. World Health Organization. Global hepatitis report, 2017. World Health Organization. , (2017).
  2. Yoffe, B., Burns, D. K., Bhatt, H. S., Combes, B. Extrahepatic hepatitis B virus DNA sequences in patients with acute hepatitis B infection. ....

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Hepatitis B VirusHBVNon hepatic Cells293T NE 3NRIn Vitro ModelHepG2 2 15Cell CultureViral ReplicationExtrahepatic SyndromesCell Line EngineeringBiosafetyViral SupernatantVirus ConcentrationReal time PCR

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