<i>In vitro</i> investigation of HBV clinical isolates from Chinese patients reveals that genotype C isolates possess higher infectivity than genotype B isolates

Tingting Liu; Anlei Liu; Yong Liu; Shan Cen; Quan Zhang

doi:10.1016/j.virs.2022.03.008

June 2022

Citation: Tingting Liu, Anlei Liu, Yong Liu, Shan Cen, Quan Zhang. In vitro investigation of HBV clinical isolates from Chinese patients reveals that genotype C isolates possess higher infectivity than genotype B isolates .VIROLOGICA SINICA, 2022, 37(3) : 398-407. http://dx.doi.org/10.1016/j.virs.2022.03.008

In vitro investigation of HBV clinical isolates from Chinese patients reveals that genotype C isolates possess higher infectivity than genotype B isolates

Tingting Liu ^a ,
Anlei Liu ^b ,
Yong Liu ^c ,
Shan Cen ^{d
,,} ,
Quan Zhang ^{c,e
,,}

a Department of Transfusion Medicine, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing, 210008, China;
b Department of Emergency Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing, 100730, China;
c Department of Laboratory Medicine, Nanjing Drum Tower Hospital and Jiangsu Key Laboratory for Molecular Medicine, The Affiliated Hospital of Nanjing University Medical School, Nanjing, 210008, China;

Corresponding author: Shan Cen, shancen@imb.pumc.edu.cn
Quan Zhang, huanlezq44@126.com
Received Date: 03 November 2021
Accepted Date: 15 March 2022
Available online: 18 March 2022

Abstract

Hepatitis B virus (HBV) genotype B and C are two major genotypes that are prevalent in Asia and differ in natural history and disease progression. The impact of HBV genotypes on viral replication and protein expression has been explored by the transfection of hepatoma cells with replication-competent HBV DNA, which mimics the later stages of the viral life cycle. However, the influence of HBV genotypes on the early events of viral infection remains undetermined, mainly due to the difficulties in obtaining sufficient infectious viral particles for infection assays. Here, we report that a high-titer HBV inoculum can be generated from the transient transfection-based cell model after optimizing transfection conditions and modifying the HBV-expressing construct. By performing in vitro infection assays using transiently transfected derived viruses, we found that clinical genotype C isolates possessed higher infectivity than genotype B isolates. Moreover, we identified a naturally occurring mutation sL21S in small hepatitis B surface protein, which markedly decreased the infectivity of HBV genotype C isolates, but not that of genotype B isolates. In summary, using infectious viral particles provided by the optimized transient transfection-based cell model, we have been able to investigate a wide range of HBV variants on viral infectivity, which may contribute to our understanding of the reasons for different clinical outcomes in HBV infections and the development of therapeutic drugs targeting the early stages of HBV life cycle.
- Hepatitis B virus (HBV)
- , Genotype B
- , Genotype C
- , Clinical isolates
- , Infectivity
- , sL21S

Electronic Supplementary Material

10.1016j.virs.2022.03.008-ESM.docx
References
1. Chan, H.L., Hui, A.Y., Wong, M.L., Tse, A.M., Hung, L.C., Wong, V.W., Sung, J.J., 2004.Genotype c hepatitis b virus infection is associated with an increased risk of hepatocellular carcinoma. Gut 53, 1494-1498.
2. Chen, C., Jia, H., Zhang, F., Qin, Y., Zong, L., Yuan, Q., Wang, Y., Xia, N., Li, J., Wen, Y., Tong, S., 2016. Functional characterization of hepatitis b virus core promoter mutants revealed transcriptional interference among co-terminal viral mrnas. J. Gen.Virol. 97, 2668-2676.
3. Chu, C.J., Hussain, M., Lok, A.S., 2002. Hepatitis b virus genotype b is associated with earlier hbeag seroconversion compared with hepatitis b virus genotype c.Gastroenterology 122, 1756-1762.
4. Fallows, D.A., Goff, S.P., 1995. Mutations in the epsilon sequences of human hepatitis b virus affect both rna encapsidation and reverse transcription. J. Virol. 69, 3067-3073.
5. Günther, S., Li, B.C., Miska, S., Krüger, D.H., Meisel, H., Will, H., 1995. A novel method for efficient amplification of whole hepatitis b virus genomes permits rapid functional analysis and reveals deletion mutants in immunosuppressed patients. J. Virol. 69, 5437-5444.
6. Glebe, D., Berting, A., Broehl, S., Naumann, H., Schuster, R., Fiedler, N., Tolle, T.K., Nitsche, S., Seifer, M., Gerlich, W.H., Schaefer, S., 2001. Optimised conditions for the production of hepatitis b virus from cell culture. Intervirology 44, 370-378.
7. Gripon, P., Diot, C., Thézé, N., Fourel, I., Loreal, O., Brechot, C., Guguen-Guillouzo, C., 1988. Hepatitis b virus infection of adult human hepatocytes cultured in the presence of dimethyl sulfoxide. J. Virol. 62, 4136-4143.
8. Gripon, P., Rumin, S., Urban, S., Le Seyec, J., Glaise, D., Cannie, I., Guyomard, C., Lucas, J., Trepo, C., Guguen-Guillouzo, C., 2002. Infection of a human hepatoma cell line by hepatitis b virus. Proc. Natl. Acad. Sci. U. S. A. 99, 15655-15660.
9. Hu, J., Lin, Y.Y., Chen, P.J., Watashi, K., Wakita, T., 2019. Cell and animal models for studying hepatitis b virus infection and drug development. Gastroenterology 156, 338-354.
10. Kim, B.K., Revill, P.A., Ahn, S.H., 2011. Hbv genotypes:relevance to natural history, pathogenesis and treatment of chronic hepatitis b. Antivir. Ther. 16, 1169-1186.
11. Ko, C., Chakraborty, A., Chou, W.M., Hasreiter, J., Wettengel, J.M., Stadler, D., Bester, R., Asen, T., Zhang, K., Wisskirchen, K., McKeating, J.A., Ryu, W.S., Protzer, U., 2018.Hepatitis b virus genome recycling and de novo secondary infection events maintain stable cccdna levels. J. Hepatol. 69, 1231-1241.
12. Kramvis, A., 2014. Genotypes and genetic variability of hepatitis b virus. Intervirology 57, 141-150.
13. Kramvis, A., Kostaki, E.G., Hatzakis, A., Paraskevis, D., 2018. Immunomodulatory function of hbeag related to short-sighted evolution, transmissibility, and clinical manifestation of hepatitis b virus. Front. Microbiol. 9, 2521.
14. Krause-Kyora, B., Susat, J., Key, F.M., 2018. Neolithic and medieval virus genomes reveal complex evolution of hepatitis b. Elife 7, e36666.
15. Lepère-Douard, C., Trotard, M., Le Seyec, J., Gripon, P., 2009. The first transmembrane domain of the hepatitis b virus large envelope protein is crucial for infectivity.J. Virol. 83, 11819-11829.
16. Li, J., Li, J., Chen, S., Yuan, Q., Zhang, J., Wu, J., Jiang, Q., Wang, Q., Xia, N.S., 2020.Naturally occurring 5' pres1 deletions markedly enhance replication and infectivity of hbv genotype b and genotype c. Gut 70, 575-584.
17. Liu, T., Sun, Q., Liu, Y., Cen, S., Zhang, Q., 2019. The mov10 helicase restricts hepatitis b virus replication by inhibiting viral reverse transcription. J. Biol. Chem. 294, 19804-19813.
18. Qin, Y., Tang, X., Garcia, T., Hussain, M., Zhang, J., Lok, A., Wands, J., Li, J., Tong, S., 2011. Hepatitis b virus genotype c isolates with wild-type core promoter sequence replicate less efficiently than genotype b isolates but possess higher virion secretion capacity. J. Virol. 85, 10167-10177.
19. Revill, P.A., Tu, T., 2020. The evolution and clinical impact of hepatitis b virus genome diversity. Nat. Rev. Gastroenterol. Hepatol. 17, 618-634.
20. Scaglioni, P.P., Melegari, M., Wands, J.R., 1997. Posttranscriptional regulation of hepatitis b virus replication by the precore protein. J. Virol. 71, 345-353.
21. Sheets, M.D., Ogg, S.C., Wickens, M.P., 1990. Point mutations in aauaaa and the poly (a) addition site:effects on the accuracy and efficiency of cleavage and polyadenylation in vitro. Nucleic. Acids. Res. 18, 5799-5805.
22. Sozzi, V., Walsh, R., Littlejohn, M., Colledge, D., Jackson, K., Warner, N., Yuen, L., Locarnini, S.A., Revill, P.A., 2016. In vitro studies show that sequence variability contributes to marked variation in hepatitis b virus replication, protein expression, and function observed across genotypes.J. Virol. 90, 10054-10064.
23. Sugiyama, M., Tanaka, Y., Kato, T., Orito, E., Ito, K., Acharya, S.K., Gish, R.G., Kramvis, A., Shimada, T., Izumi, N., Kaito, M., Miyakawa, Y., Mizokami, M., 2006. Influence of hepatitis b virus genotypes on the intra-and extracellular expression of viral DNA and antigens. Hepatology 44, 915-924.
24. Tong, S., Revill, P., 2016. Overview of hepatitis b viral replication and genetic variability.J. Hepatol. 64, S4-s16.
25. Tu, T., Urban, S., 2018. Virus entry and its inhibition to prevent and treat hepatitis b and hepatitis d virus infections. Curr. Opin. Virol. 30, 68-79.
26. Verrier, E.R., Colpitts, C.C., Schuster, C., Zeisel, M.B., Baumert, T.F., 2016. Cell culture models for the investigation of hepatitis b and d virus infection. Viruses 8, 261.
27. Xia, Y., Carpentier, A., Cheng, X., Block, P.D., Zhao, Y., Zhang, Z., Protzer, U., Liang, T.J., 2017. Human stem cell-derived hepatocytes as a model for hepatitis b virus infection, spreading and virus-host interactions. J. Hepatol. 66, 494-503.
28. Xiang, K.H., Michailidis, E., Ding, H., Peng, Y.Q., Su, M.Z., Li, Y., Liu, X.E., Dao Thi, V.L., Wu, X.F., Schneider, W.M., Rice, C.M., Zhuang, H., Li, T., 2017. Effects of amino acid substitutions in hepatitis b virus surface protein on virion secretion, antigenicity, hbsag and viral DNA. J. Hepatol. 66, 288-296.
29. Yan, H., Zhong, G., Xu, G., He, W., Jing, Z., Gao, Z., Huang, Y., Qi, Y., Peng, B., Wang, H., Fu, L., Song, M., Chen, P., Gao, W., Ren, B., Sun, Y., Cai, T., Feng, X., Sui, J., Li, W.,2012. Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis b and d virus. Elife 1, e00049.
30. Yang, H.I., Yeh, S.H., Chen, P.J., Iloeje, U.H., Jen, C.L., Su, J., Wang, L.Y., Lu, S.N., You, S.L., Chen, D.S., Liaw, Y.F., Chen, C.J., 2008. Associations between hepatitis b virus genotype and mutants and the risk of hepatocellular carcinoma. J. Natl.Cancer Inst. 100, 1134-1143.
31. Zhang, H.W., Yin, J.H., Li, Y.T., Li, C.Z., Ren, H., Gu, C.Y., Wu, H.Y., Liang, X.S., Zhang, P., Zhao, J.F., Tan, X.J., Lu, W., Schaefer, S., Cao, G.W., 2008. Risk factors for acute hepatitis b and its progression to chronic hepatitis in shanghai, China. Gut 57, 1713-1720.
32. Zhang, Q., Chen, C.Y., Yedavalli, V.S., Jeang, K.T., 2013. Neat1 long noncoding rna and paraspeckle bodies modulate hiv-1 posttranscriptional expression. mBio 4 e00596-00512.
33. Zhang, Q., Chen, J., Pan, M., Liu, J., Liu, T., Zhou, Y.H., 2018. Comparison of replication competence of wild-type and lamivudine-resistant hepatitis b virus isolates from a chronic hepatitis b patient. Virus. Res. 255, 165-170.
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