hNTCP‑expressing primary pig hepatocytes are a valuable tool for investigating hepatitis B virus infection and antiviral drugs

Zhou,Ming; Qin,Bo; Deng,Xue‑Song; Zeng,Xiao‑Li; Lu,Ying; Huang,Zi‑Gang; Wu,Chun‑Chen; Mou,Li‑Sha

doi:10.3892/mmr.2019.10628

hNTCP‑expressing primary pig hepatocytes are a valuable tool for investigating hepatitis B virus infection and antiviral drugs

Authors:
- Ming Zhou
- Bo Qin
- Xue‑Song Deng
- Xiao‑Li Zeng
- Ying Lu
- Zi‑Gang Huang
- Chun‑Chen Wu
- Li‑Sha Mou
View Affiliations

Affiliations: Shenzhen Xenotransplantation Research and Development Center, Institute of Translational Medicine, Health Science Center, Shenzhen University School of Medicine, Shenzhen Second People's Hospital, The First Afﬁliated Hospital of Shenzhen University, Shenzhen, Guangdong 518035, P.R. China, Clinical Laboratory Center, Shaoxing People's Hospital, Shaoxing Hospital of Zhejiang University, Shaoxing, Zhejiang 312000, P.R. China, Department of Hepatobiliary Surgery, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen, Guangdong 518035, P.R. China, Department of Internal Medicine, The Second People's Hospital of Futian District, Shenzhen, Guangdong 518049, P.R. China, Liver‑Biotechnology (Shenzhen) Co., Ltd., Shenzhen, Guangdong 518110, P.R. China, Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071, P.R. China
Published online on: August 29, 2019 https://doi.org/10.3892/mmr.2019.10628
Pages: 3820-3828
Copyright: © Zhou et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Primary human hepatocytes (PHHs) are the ‘gold standard’ for investigating hepatitis B virus (HBV) infection and antiviral drugs. However, poor availability, variation between batches and ethical issues regarding PHHs limit their applications. The discovery of human sodium taurocholate co‑transporting polypeptide (hNTCP) as a functional HBV receptor has enabled the development of a surrogate model to supplement the use of PHHs. In the present study, the evolutionary distance of seven species was assessed based on single‑copy homologous genes. Based on the evolutionary distance and availability, PHHs and primary rabbit hepatocytes (PRHs) were isolated and infected with hNTCP‑recombinant lentivirus, and susceptibility to HBV infection in the two cell types was tested and compared. In addition, HBV infection efficiency of hNTCP‑expressing PPHs with pooled HBV‑positive serum and purified particles was determined. The potential use of HBV‑infected hNTCP‑expressing PPHs for drug screening was assessed. The results demonstrated that pigs and rabbits are closer to humans in the divergence tree compared with mice and rats, indicating that pigs and rabbits were more likely to facilitate the HBV post‑entry lifecycle. Following hNTCP complementation and HBV infection, PPHs and Huh7D human hepatocellular carcinoma cells, but not PRHs, exhibited increased hepatitis B surface antigen and hepatitis B e‑antigen secretion, covalently closed circular DNA formation and infectious particle secretion. hNTCP‑expressing PPHs were susceptible to infection with HBV particles purified from pooled HBV‑positive sera, but were poisoned by raw HBV‑positive sera. The use of HBV‑infected hNTCP‑expressing PPHs for viral entry inhibitor screening was revealed to be applicable and reproducible. In conclusion, hNTCP‑expressing PPHs may be valuable tool for investigating HBV infection and antiviral drugs.

View Figures

View References

1	Beasley RP: Rocks along the road to the control of HBV and HCC. Ann Epidemiol. 19:231–234. 2009. View Article : Google Scholar : PubMed/NCBI
2	Sheahan T, Jones CT and Ploss A: Advances and challenges in studying hepatitis C virus in its native environment. Expert Rev Gastroenterol Hepatol. 4:541–550. 2010. View Article : Google Scholar : PubMed/NCBI
3	Weizsäcker FV and Roggendorf M: Models of viral hepatitisBasel; New York: Karger; 2005
4	Yan H, Zhong G, Xu G, He W, Jing Z, Gao Z, Huang Y, Qi Y, Peng B, Wang H, et al: Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. Elife. 1:e000492012. View Article : Google Scholar : PubMed/NCBI
5	He W, Ren B, Mao F, Jing Z, Li Y, Liu Y, Peng B, Yan H, Qi Y, Sun Y, et al: Hepatitis D virus infection of mice expressing human sodium taurocholate co-transporting polypeptide. PLoS Pathog. 11:e10048402015. View Article : Google Scholar : PubMed/NCBI
6	Ni Y, Lempp FA, Mehrle S, Nkongolo S, Kaufman C, Fälth M, Stindt J, Königer C, Nassal M, Kubitz R, et al: Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes. Gastroenterology. 146:1070–1083. 2014. View Article : Google Scholar : PubMed/NCBI
7	Yuan W, Wu JY, Zhao YZ, Li J, Li JB, Li ZH and Li CS: Comparison of early sequential hypothermia and delayed hypothermia on neurological function after resuscitation in a swine model. Am J Emerg Med. 35:1645–1652. 2017. View Article : Google Scholar : PubMed/NCBI
8	Zhou M, Zhao F, Li J, Cheng Z, Tian X, Zhi X, Huang Y and Hu K: Long-term maintenance of human fetal hepatocytes and prolonged susceptibility to HBV infection by co-culture with non-parenchymal cells. J Virol Methods. 195:185–193. 2014. View Article : Google Scholar : PubMed/NCBI
9	Zhou M, Huang Y, Cheng Z, Zhao F, Li J, Zhi X, Tian X, Sun W and Hu K: Revival, characterization, and hepatitis B virus infection of cryopreserved human fetal hepatocytes. J Virol Methods. 207:29–37. 2014. View Article : Google Scholar : PubMed/NCBI
10	Zhou M, Zhao K, Yao Y, Yuan Y, Pei R, Wang Y, Chen J, Hu X, Zhou Y, Chen X and Wu C: Productive HBV infection of well-differentiated, hNTCP-expressing human hepatoma-derived (Huh7) cells. Virol Sin. 32:465–475. 2017. View Article : Google Scholar : PubMed/NCBI
11	Ladner SK, Otto MJ, Barker CS, Zaifert K, Wang GH, Guo JT, Seeger C and King RW: Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: A novel system for screening potential inhibitors of HBV replication. Antimicrob Agents Chemother. 41:1715–1720. 1997. View Article : Google Scholar : PubMed/NCBI
12	Köck J, Nassal M, MacNelly S, Baumert TF, Blum HE and von Weizsäcker F: Efficient infection of primary tupaia hepatocytes with purified human and woolly monkey hepatitis B virus. J Virol. 75:5084–5089. 2001. View Article : Google Scholar : PubMed/NCBI
13	Köck J, Rösler C, Zhang JJ, Blum HE, Nassal M and Thoma C: Generation of covalently closed circular DNA of hepatitis B viruses via intracellular recycling is regulated in a virus specific manner. PLoS Pathog. 6:e10010822010. View Article : Google Scholar : PubMed/NCBI
14	Glebe D, Aliakbari M, Krass P, Knoop EV, Valerius KP and Gerlich WH: Pre-s1 antigen-dependent infection of Tupaia hepatocyte cultures with human hepatitis B virus. J Virol. 77:9511–9521. 2003. View Article : Google Scholar : PubMed/NCBI
15	Doitsh G and Shaul Y: A long HBV transcript encoding pX is inefficiently exported from the nucleus. Virology. 309:339–349. 2003. View Article : Google Scholar : PubMed/NCBI
16	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
17	Nassal M: The arginine-rich domain of the hepatitis B virus core protein is required for pregenome encapsidation and productive viral positive-strand DNA synthesis but not for virus assembly. J Virol. 66:4107–4116. 1992.PubMed/NCBI
18	Fushan AA, Turanov AA, Lee SG, Kim EB, Lobanov AV, Yim SH, Buffenstein R, Lee SR, Chang KT, Rhee H, et al: Gene expression defines natural changes in mammalian lifespan. Aging Cell. 14:352–365. 2015. View Article : Google Scholar : PubMed/NCBI
19	Drexler JF, Geipel A, König A, Corman VM, van Riel D, Leijten LM, Bremer CM, Rasche A, Cottontail VM, Maganga GD, et al: Bats carry pathogenic hepadnaviruses antigenically related to hepatitis B virus and capable of infecting human hepatocytes. Proc Natl Acad Sci USA. 110:16151–16156. 2013. View Article : Google Scholar : PubMed/NCBI
20	Vieira YR, dos Santos DR, Portilho MM, Velloso CE, Arissawa M, Villar LM, Pinto MA and de Paula VS: Hepadnavirus detected in bile and liver samples from domestic pigs of commercial abattoirs. BMC Microbiol. 14:3152014. View Article : Google Scholar : PubMed/NCBI
21	Li W, She R, Liu L, You H and Yin J: Prevalence of a virus similar to human hepatitis B virus in swine. Virol J. 7:602010. View Article : Google Scholar : PubMed/NCBI
22	Tang H, Zhou S, Zhao L, Wang J, Lei B and Cao Z: Primary serologic investigation of HBV-like virus existence in domestic animals and fowls. Hua Xi Yi Ke Da Xue Xue Bao. 21:181–184. 1990.(In Chinese). PubMed/NCBI
23	Ye X, Zhou M, He Y, Wan Y, Bai W, Tao S, Ren Y, Zhang X, Xu J, Liu J, et al: Efficient inhibition of hepatitis B virus infection by a preS1-binding peptide. Sci Rep. 6:293912016. View Article : Google Scholar : PubMed/NCBI
24	Glebe D, Urban S, Knoop EV, Cag N, Krass P, Grün S, Bulavaite A, Sasnauskas K and Gerlich WH: Mapping of the hepatitis B virus attachment site by use of infection-inhibiting preS1 lipopeptides and tupaia hepatocytes. Gastroenterology. 129:234–245. 2005. View Article : Google Scholar : PubMed/NCBI
25	Doong SL, Tsai CH, Schinazi RF, Liotta DC and Cheng YC: Inhibition of the replication of hepatitis B virus in vitro by 2′,3′-dideoxy-3′-thiacytidine and related analogues. Proc Natl Acad Sci USA. 88:8495–8499. 1991. View Article : Google Scholar : PubMed/NCBI
26	Joziasse DH and Oriol R: Xenotransplantation: The importance of the Galalpha1,3Gal epitope in hyperacute vascular rejection. Biochim Biophys Acta. 1455:403–418. 1999. View Article : Google Scholar : PubMed/NCBI
27	Lempp FA, Wiedtke E, Qu B, Roques P, Chemin I, Vondran FWR, Le Grand R, Grimm D and Urban S: Sodium taurocholate cotransporting polypeptide is the limiting host factor of Hepatitis B Virus infection in macaque and pig hepatocytes. Hepatology. 66:703–716. 2017. View Article : Google Scholar : PubMed/NCBI