For best viewing of the website please use Mozilla Firefox or Google Chrome.
Citation: Yuqian Luo, Le Zhang, Yimin Dai, Yali Hu, Biyun Xu, Yi-Hua Zhou. Conservative Evolution of Hepatitis B Virus Precore and Core Gene During Immune Tolerant Phase in Intrafamilial Transmission [J].VIROLOGICA SINICA, 2020, 35(4) : 388-397.  http://dx.doi.org/10.1007/s12250-020-00194-6

Conservative Evolution of Hepatitis B Virus Precore and Core Gene During Immune Tolerant Phase in Intrafamilial Transmission

  • Corresponding author: Yi-Hua Zhou, zgr03summer@126.com, ORCID: 0000-0001-8880-0392
  • Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12250-020-00194-6) contains supplementary material, which is available to authorized users.
  • Received Date: 02 August 2019
    Accepted Date: 06 December 2019
    Published Date: 02 March 2020
  • Hepatitis B virus (HBV) is characterized with high mutations, which is attributed to the lack of proof-reading of the viral reverse transcriptase and host immune pressure. In this study, 31 HBV chronic carriers from 14 families were enrolled to investigate the evolution of the same original HBV sources in different hosts. Sequences of pre-C and C (pre-C/C) genes were analyzed in eight pairs of HBV-infected mothers with longitudinal sera (at an interval of 6.0–7.2 years) and their children (5.5–6.7 years old), and in 15 adults (21–78 years old) from six families with known intrafamilial HBV infection. The pre-C/C sequences had almost no change in eight mothers during 6.0–7.2 years and their children who were in immune tolerant phase. The pre-C/C sequences from the 15 adults of six families, mostly in the immune-clearance phase or the low replicative phase, showed various diversified mutations between individuals from each family. Compared to a reference stain (GQ205441) isolated nearby, the pre-C/C in individuals in immune tolerant phase showed 98.56%–99.52% homology at nucleotide level and 99.5%–100% homology at amino acid level. In contrast, multiple mutations were developed in the immune-clearance phase or the low replicative phase, affecting immune epitopes in core gene and G1896 in pre-C gene. The results indicate that the evolution of new HBV variants is not mainly resulted from the spontaneous error rate of viral reverse transcription, but from the host immune pressure.

  • 加载中
  • 10.1007s12250-020-00194-6-ESM1.pdf
    10.1007s12250-020-00194-6-ESM1.docx
    1. Abbott WG, Tsai P, Leung E, Trevarton A, Ofanoa M, Hornell J, Gane EJ, Munn SR, Rodrigo AG (2010) Associations between HLA class Ⅰ alleles and escape mutations in the hepatitis B virus core gene in New Zealand-resident Tongans. J Virol 84:621–629
        doi: 10.1128/JVI.01471-09

    2. Bertoletti A, Ferrari C, Fiaccadori F, Penna A, Margolskee R, Schlicht HJ, Fowler P, Guilhot S, Chisari FV (1991) HLA class Ⅰ-restricted human cytotoxic T cells recognize endogenously synthesized hepatitis B virus nucleocapsid antigen. Proc Natl Acad Sci USA 88:10445–10449
        doi: 10.1073/pnas.88.23.10445

    3. Bertoletti A, Chisari FV, Penna A, Guilhot S, Galati L, Missale G, Fowler P, Schlicht HJ, Vitiello A, Chesnut RC (1993) Definition of a minimal optimal cytotoxic T-cell epitope within the hepatitis B virus nucleocapsid protein. J Virol 67:2376–2380
        doi: 10.1128/JVI.67.4.2376-2380.1993

    4. Boeijen LL, Hoogeveen RC, Boonstra A, Lauer GM (2017) Hepatitis B virus infection and the immune response: the big questions. Best Pract Res Clin Gastroenterol 31:265–272

    5. Carman WF, Thursz M, Hadziyannis S, McIntyre G, Colman K, Gioustoz A, Fattovich G, Alberti A, Thomas HC (1995) Hepatitis B e antigen negative chronic active hepatitis: hepatitis B virus core mutations occur predominantly in known antigenic determinants. J Viral Hepat 2:77–84
        doi: 10.1111/j.1365-2893.1995.tb00010.x

    6. Carman WF, Boner W, Fattovich G, Colman K, Dornan ES, Thursz M, Hadziyannis S (1997) Hepatitis B virus core protein mutations are concentrated in B cell epitopes in progressive disease and in T helper cell epitopes during clinical remission. J Infect Dis 175:1093–1100
        doi: 10.1086/516447

    7. Clewley JP, Arnold C (1997) MEGALIGN. The multiple alignment module of LASERGENE. Methods Mol Biol 70:119–129

    8. Colombatto P, Barbera C, Bortolotti F, Maina AM, Moriconi F, Cavallone D, Calvo P, Oliveri F, Bonino F, Brunetto MR (2018) HBV pre-core mutant in genotype-D infected children is selected during HBeAg/anti-HBe seroconversion and leads to HBeAg negative chronic hepatitis B in adulthood. J Med Virol 90:1232–1239
        doi: 10.1002/jmv.25068

    9. Croagh CM, Lubel JS (2014) Natural history of chronic hepatitis B: phases in a complex relationship. World J Gastroenterol 20:10395–10404
        doi: 10.3748/wjg.v20.i30.10395

    10. Dandri M, Murray JM, Lutgehetmann M, Volz T, Lohse AW, Petersen J (2008) Virion half-life in chronic hepatitis B infection is strongly correlated with levels of viremia. Hepatology 48:1079–1086
        doi: 10.1002/hep.22469

    11. European Association for the Study of the Liver (EASL) (2017) EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. J Hepatol 67:370–398
        doi: 10.1016/j.jhep.2017.03.021

    12. Faure-Dupuy S, Lucifora J, Durantel D (2017) Interplay between the hepatitis B virus and innate immunity: from an understanding to the development of therapeutic concepts. Viruses 9:95
        doi: 10.3390/v9050095

    13. Ferrari C, Bertoletti A, Penna A, Cavalli A, Valli A, Missale G, Pilli M, Fowler P, Giuberti T, Chisari FV et al (1991) Identification of immunodominant T cell epitopes of the hepatitis B virus nucleocapsid antigen. J Clin Invest 88:214–222
        doi: 10.1172/JCI115280

    14. Girones R, Miller RH (1989) Mutation rate of the hepadnavirus genome. Virology 170:595–597
        doi: 10.1016/0042-6822(89)90455-8

    15. Holmes EC (2008) Evolutionary history and phylogeography of human viruses. Annu Rev Microbiol 62:307–328
        doi: 10.1146/annurev.micro.62.081307.162912

    16. Hu Y, Zhang S, Luo C, Liu Q, Zhou YH (2012) Gaps in the prevention of perinatal transmission of hepatitis B virus between recommendations and routine practices in a highly endemic region: a provincial population-based study in China. BMC Infect Dis 12:221
        doi: 10.1186/1471-2334-12-221

    17. Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120
        doi: 10.1007/BF01731581

    18. Lazarevic I, Banko A, Miljanovic D, Cupic M (2019) Immune-escape hepatitis B virus mutations associated with viral reactivation upon immunosuppression. Viruses 11:778
        doi: 10.3390/v11090778

    19. Liang X, Cheng J, Sun Y, Chen X, Li T, Wang H, Jiang J, Chen X, Long H, Tang H, Yu Y, Sheng J, Chen S, Niu J, Ren H, Shi J, Dou X, Wan M, Jiang J, Xie Q, Shi G, Ning Q, Chen C, Tan D, Ma H, Sun J, Jia J, Zhuang H, Hou J (2015) Randomized, three-arm study to optimize lamivudine efficacy in hepatitis B e antigen-positive chronic hepatitis B patients. J Gastroenterol Hepatol 30:748–755
        doi: 10.1111/jgh.12835

    20. Liu J, Bi Y, Xu C, Liu L, Xu B, Chen T, Chen J, Pan M, Hu Y, Zhou YH (2015) Kinetic changes of viremia and viral antigens of hepatitis B virus during and after pregnancy. Medicine 94:e2001
        doi: 10.1097/MD.0000000000002001

    21. Luo Y, Pan M, Ning M, Chenyu X, Liu L, Chen L, Chen T, Biyun X, Yali H, Zhou YH (2019) High mutation prevalence of precore and basal core promoter in pregnant women who underwent spontaneous HBeAg seroconversion within one year postpartum. Dig Liver Dis 52:199–204

    22. Maini MK, Boni C, Lee CK, Larrubia JR, Reignat S, Ogg GS, King AS, Herberg J, Gilson R, Alisa A, Williams R, Vergani D, Naoumov NV, Ferrari C, Bertoletti A (2000) The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection. J Exp Med 191:1269–1280
        doi: 10.1084/jem.191.8.1269

    23. Murray JM, Wieland SF, Purcell RH, Chisari FV (2005) Dynamics of hepatitis B virus clearance in chimpanzees. Proc Natl Acad Sci USA 102:17780–17785
        doi: 10.1073/pnas.0508913102

    24. Murray JM, Purcell RH, Wieland SF (2006) The half-life of hepatitis B virions. Hepatology 44:1117–1121
        doi: 10.1002/hep.21364

    25. Nie H, Evans AA, London WT, Block TM, Ren XD (2012) Quantitative dynamics of hepatitis B basal core promoter and precore mutants before and after HBeAg seroconversion. J Hepatol 56:795–802
        doi: 10.1016/j.jhep.2011.11.012

    26. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    27. Salfeld J, Pfaff E, Noah M, Schaller H (1989) Antigenic determinants and functional domains in core antigen and e antigen from hepatitis B virus. J Virol 63:798–808
        doi: 10.1128/JVI.63.2.798-808.1989

    28. Sallberg M, Pushko P, Berzinsh I, Bichko V, Sillekens P, Noah M, Pumpens P, Grens E, Wahren B, Magnius LO (1993) Immunochemical structure of the carboxy-terminal part of hepatitis B e antigen: identification of internal and surface-exposed sequences. J Gen Virol 74(Pt 7):1335–1340

    29. Tamura K, Nei M, Kumar S (2004) Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci USA 101:11030–11035
        doi: 10.1073/pnas.0404206101

    30. Valaydon ZS, Locarnini SA (2017) The virological aspects of hepatitis B. Best Pract Res Clin Gastroenterol 31:257–264
        doi: 10.1016/j.bpg.2017.04.013

    31. Vanlandschoot P, Cao T, Leroux-Roels G (2003) The nucleocapsid of the hepatitis B virus: a remarkable immunogenic structure. Antiviral Res 60:67–74
        doi: 10.1016/j.antiviral.2003.08.011

    32. Wang HY, Chien MH, Huang HP, Chang HC, Wu CC, Chen PJ, Chang MH, Chen DS (2010) Distinct hepatitis B virus dynamics in the immunotolerant and early immunoclearance phases. J Virol 84:3454–3463
        doi: 10.1128/JVI.02164-09

    33. Wang XL, Ren JP, Wang XQ, Wang XH, Yang SF, Xiong Y (2016) Mutations in pre-core and basic core promoter regions of hepatitis B virus in chronic hepatitis B patients. World J Gastroenterol 22:3268–3274
        doi: 10.3748/wjg.v22.i11.3268

    34. Warner BG, Abbott WG, Rodrigo AG (2014) Frequency-dependent selection drives HBeAg seroconversion in chronic hepatitis B virus infection. Evol Med Public Health 2014:1–9
        doi: 10.1093/emph/eot023

    35. Xu B, Zhu D, Bi Y, Wang Y, Hu Y, Zhou YH (2017) Minimal association of alleles of human leukocyte antigen class Ⅱ gene and long-term antibody response to hepatitis B vaccine vaccinated during infancy. Vaccine 35:2457–2462
        doi: 10.1016/j.vaccine.2017.03.021

    36. Zhang ZH, Li L, Zhao XP, Glebe D, Bremer CM, Zhang ZM, Tian YJ, Wang BJ, Yang Y, Gerlich W, Roggendorf M, Li X, Lu M, Yang DL (2011) Elimination of hepatitis B virus surface antigen and appearance of neutralizing antibodies in chronically infected patients without viral clearance. J Viral Hepat 18:424–433
        doi: 10.1111/j.1365-2893.2010.01322.x

  • 加载中

Figures(2) / Tables(3)

Article Metrics

Article views(563) PDF downloads(3) Cited by()

Related
Proportional views
    通讯作者: 陈斌, bchen63@163.com
    • 1. 

      沈阳化工大学材料科学与工程学院 沈阳 110142

    1. 本站搜索
    2. 百度学术搜索
    3. 万方数据库搜索
    4. CNKI搜索

    Conservative Evolution of Hepatitis B Virus Precore and Core Gene During Immune Tolerant Phase in Intrafamilial Transmission

      Corresponding author: Yi-Hua Zhou, zgr03summer@126.com
    • 1. Department of Laboratory Medicine, Nanjing Drum Tower Hospital and Jiangsu Key Laboratory for Molecular Medicine, Nanjing University Medical School, Nanjing 210008, China
    • 2. Department of Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing 210008, China
    • 3. Department of Biostatistics, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing 210008, China
    • 4. Department of Infectious Diseases, Nanjing Drum Tower Hospital, Nanjing Medical University, Nanjing 210008, China

    Abstract: Hepatitis B virus (HBV) is characterized with high mutations, which is attributed to the lack of proof-reading of the viral reverse transcriptase and host immune pressure. In this study, 31 HBV chronic carriers from 14 families were enrolled to investigate the evolution of the same original HBV sources in different hosts. Sequences of pre-C and C (pre-C/C) genes were analyzed in eight pairs of HBV-infected mothers with longitudinal sera (at an interval of 6.0–7.2 years) and their children (5.5–6.7 years old), and in 15 adults (21–78 years old) from six families with known intrafamilial HBV infection. The pre-C/C sequences had almost no change in eight mothers during 6.0–7.2 years and their children who were in immune tolerant phase. The pre-C/C sequences from the 15 adults of six families, mostly in the immune-clearance phase or the low replicative phase, showed various diversified mutations between individuals from each family. Compared to a reference stain (GQ205441) isolated nearby, the pre-C/C in individuals in immune tolerant phase showed 98.56%–99.52% homology at nucleotide level and 99.5%–100% homology at amino acid level. In contrast, multiple mutations were developed in the immune-clearance phase or the low replicative phase, affecting immune epitopes in core gene and G1896 in pre-C gene. The results indicate that the evolution of new HBV variants is not mainly resulted from the spontaneous error rate of viral reverse transcription, but from the host immune pressure.

    • Chronic hepatitis B virus (HBV) infection is a worldwide health problem. HBV is an enveloped virus that contains a circular DNA genome, approximately 3.2 kb, including four overlapping open reading frames (S, C, P, and X genes), and replicates its genomic DNA through reverse transcription by the viral reverse transcriptase (Valaydon and Locarnini 2017). The hepatitis B core antigen (HBcAg) and hepatitis B surface antigen (HBsAg) are major targets for antiviral immunity, but the first one seems to be the most immunogenic (Vanlandschoot et al. 2003) and subjected to a wide variation of amino acid sequences during chronic HBV infection (Carman et al. 1995). Indeed, it is estimated that the mutation rate is 10-4–10-6 nucleotide substitutions/site/year in HBV pre-C/C gene, approximately 100 times higher than that of any other DNA virus (Girones and Miller 1989). Lack of proof-reading of the viral reverse transcriptase (Valaydon and Locarnini 2017) and host immune pressure (Wang et al. 2010) are reasoned to be the basis for the high mutation rate of HBV. However, investigative tools such as cell culture systems that support long-term HBV propagation are still lacking.

      Dictated by the host immunity, the natural course of chronic HBV infection may undergo five phases: the immune tolerant phase (HBeAg-positive chronic infection), the immune-clearance phase (HBeAg-positive chronic hepatitis), the low replicative phase (HBeAg-negative chronic infection), the immune reactivation phase (HBeAgnegative chronic hepatitis), and, rarely, the recovery (HBsAg-negative phase) (EASL 2017). Patients with mother-to-infant transmission of HBV often remain in the immune tolerant phase for decades. These patients have very weak specific T cell responses, as the HBV DNA levels are high and constant, alanine transaminase (ALT) values are normal, and there is minimal liver inflammation (EASL 2017). Later they may enter into the immuneclearance phase, during which the host immunity is, for unknown reason, awakened to cause notable liver inflammation. The immune-clearance phase may then transit into the low replicative phase, during which seroconversion to positive anti-HBe sometimes occurs, HBV DNA remains at very low levels, and ALT return to normal; alternatively some patients enter into the reactivation phase with higher ALT and return of viremia.

      The key to tackle HBV infection seems to lie on the interaction between the host immunity and the virus adaptation. Given the same source of infection, mother-to-infant or intrafamilial transmission of HBV provides a useful model to characterize the virus evolution pattern under different host immune background. In the present study, to shed light on the interaction between host immunity and HBV evolution, we performed longitudinal analysis of HBV pre-C/C sequences within family members infected with the same original pathogen source.

    • The subjects enrolled in this study contained two groups of individuals with ongoing HBV infection. Group one included eight pairs of relatively young mothers (aged 28–37 years) and their children (aged 5.5–6.7 years) (Hu et al. 2012). During a follow-up period of 6.0–7.2 years, these mothers' health conditions were generally well, with normal liver function and normal ultrasound B liver scanning image, and they were positive for both HBsAg and HBeAg and had constantly high HBV DNA levels. Thus, they were in HBeAg-positive chronic infection phase of infection. The longitudinal blood samples from these mothers, collected at an interval of 6.0–7.2 years (Hu et al. 2012), were kept at - 20 ℃. Their eight children received hepatitis B immunoglobulin and/or three doses of hepatitis B vaccine on a standard 0-, 1-, and 6-month schedule after birth, but they were infected with HBV; the infection was defined in three children before 3 years age (Table 1: Ⅰb, Ⅴb, and ⅩⅢb) and in five others at 5.6–6.7 years age. These children were considered be perinatally infected with HBV as a consequence of immunoprophylaxis failure. The blood samples of these children were only retained at the age of 5.5–6.7 years.

      Family Patient Relation Sex Age (Years) HBsAg (IU/mL) HBeAg (S/CO) HBV DNA (IU/mL)
      Ia Mother F 30.7 128640 2299.3 1.42×108
      37.0 256360 2928.0 5.50×107
      Ⅰb Daughter F 5.9 70720 2055.2 1.14×108
      Ⅱa Mother F 21.4 52890 2607.8 9.04×106
      28.0 29124 2119.4 3.00×106
      Ⅱb Daughter F 6.1 57550 1719.4 8.99×106
      Ⅲa Mother F 25.5 3448 3270.6 5.91×106
      32.3 8752 1885.7 5.29×106
      Ⅲb Daughter F 6.4 11130 1876.5 5.97×106
      Ⅳa Mother F 25.8 149280 2297.9 1.06×107
      33.0 139260 2794.2 1.08×107
      Ⅳb Son M 6.7 133200 2178.9 2.32×107
      Ⅴa Mother F 25.6 36758 1820.9 2.39×106
      32.7 94220 2612.9 3.67×107
      Ⅴb Son M 6.7 31676 1662.9 4.11×107
      Ⅵa Mother F 22.5 32580 1542.3 5.99×106
      28.5 81490 1829.3 2.63×107
      Ⅵb Daughter F 5.6 60600 2279.6 1.27×108
      Ⅶa Mother F 27.4 37688 1133.0 5.68×106
      34.3 60530 1510.8 1.27×107
      Ⅶb Daughter F 6.5 69900 1693.3 2.14×107
      Ⅷa Mother F 24.2 84270 1655.5 2.16×107
      30.2 27038 1759.0 2.60×107
      Ⅷb Son M 5.5 70942 1489.8 2.13×107
      All patients were negative for IgM antibody against hepatitis B core antigen, and had normal levels of ALT. They were in the phase 1, the HBeAg-positive chronic infection phase, previously known as the immune tolerant phase of HBV infection.

      Table 1.  Virological characteristics in group one consisting of younger mothers and their children.

      Group two contained 15 individuals from six families, including six index patients (aged 40–78 years), and nine patients (aged 21–53 years) who were assumed to have acquired the infection in their early childhood from the index, except in family ⅩⅣ who were two spouses, and the husband was assumed to have acquired the infection from his wife as he was negative for HBV before he got married.

      Of the above 31 patients, 20 (64.5%) were women. All the patients had no co-infection of HIV or hepatitis C virus, and had not been treated with antiviral agents. The demographic data of these patients are presented in Tables 1 and 2.

      Family No. Patient No. Relation Sex Age (Years) HBsAg (IU/mL) HBeAg (S/CO) anti-HBe (S/CO) HBV DNA (IU/mL) ALT (U/L) Phasea
      Ⅸa Index F 78 92 0.4 0.2 5.51×106 969.8 2
      Ⅸb Daughter F 53 1803 - + 2.40×102 12.6 3
      Ⅸc Son M 45 97005 2214.9 - 1.59×108 34.5 1
      X Xa Index F 71 254 - + 2.86×106 237.1 4
      Xb Son M 40 168 - - 1.32×102 16.9 3
      Ⅺa Index F 63 244.11 1.58 1.72 Undetectable 78.2 2
      Ⅺb Son M 41 983 6.9 1.75 1.05×106 44.8 2
      Ⅺc Son M 38 827 - + 3.28×102 17.9 3
      Ⅻa Indexb F 46 - - + Undetectable 22.4 5
      Ⅻb Daughter F 23 10182 - + 3.40×102 24.6 3
      Ⅻc Son M 21 4678 490.4 16.02 1.26×106 550.3 2
      ⅩⅢ ⅩⅢa Index M 46 5725 - + 1.51×102 40.3 3
      ⅩⅢb Son M 22 5202 - + 1.43×102 19.5 3
      ⅩⅣ ⅩⅣa Index F 40 155 0.6 + 1.94×103 71.8 2
      ⅩⅣb Husband M 40 0.8 - - 1.25×102 14.7 3
      All patients were positive for total antibody against hepatitis B core antigen.
      aPhases 1–5 refer to HBeAg-positive chronic infection, HBeAg-positive chronic hepatitis, HBeAg-negative chronic infection, HBeAg-negative chronic hepatitis, and HBsAg-negative phase, respectively.
      bThis patient had history of chronic HBV infection and did not receive antiviral therapy, but showed negative HBsAg and positive anti-HBs at the enrollment.

      Table 2.  Virological characteristics in group two consisting of elderly parents and adult offspring.

    • Serum samples were tested for HBsAg, antibody against HBsAg (anti-HBs), HBeAg, anti-HBe, and anti-HBc using enzyme-linked immunosorbent assay kits (Huakang Biotech, Shenzhen, China). Quantification of serum HBsAg and HBeAg was performed by a microparticle enzyme immunoassay (Architect System, Abbott, North Chicago, IL, USA), as previously reported (Liu et al. 2015). HBeAg levels were presented as the ratio of relative light units of the samples to negative controls (S/CO). Quantification of HBV DNA was performed by a fluorescent real-time PCR assay (Shenyou Biotechnology, Shanghai, China) as described before (Liu et al. 2015).

    • Serum DNA was extracted from 200 μL serum by phenol/ chloroform extraction method, and dissolved in 20 μL Tris–EDTA buffer as reported previously (Xu et al. 2017). The pre-C/C regions were amplified by nested PCR using primers as listed in Supplementary Table S1. The first round PCR was carried out using primers C1 and C2. The second round was performed using C3 and C4 primers. To prevent cross contamination, each step was performed in separate areas with dedicated equipment, and always included negative controls.

      The purified PCR products were directly sequenced on an ABI Prism 3730xl sequencer (Applied Biosystems, Hitachi, Tokyo, Japan) after reaction with BigDye Terminator v3.1 (Applied Biosystems, Foster, CA, USA). When mixed signals (multiple peaks) were seen in the chromatograms of sequencing results, the PCR products were subcloned using pUCm-T vector (Sangon Biotech, Shanghai, China).

      HBV genotypes were determined by phylogenetic analysis as previously described (Luo et al. 2019) based on the pre-C/C sequence. Sequences were also aligned with reference stains, including one (GQ205441) isolated in nearby city Hefei in eastern China (Zhang et al. 2011), one (KR013798) in Guangzhou in southern China (Liang et al. 2015), one (KU519422) in Tibet in western China, and one (LC170476) in Japanese patients. Multiple sequence alignments were performed using Clustal W method. Phylogenetic trees were constructed using neighboringjoining methods (Saitou and Nei 1987) with pairwise distances being estimated by Kimura's two-parameter method (Kimura 1980). The evolutionary distances were computed using the maximum composite likelihood method (Tamura et al. 2004). These analyses were done automatically using MegAlign software program (Clewley and Arnold 1997).

    • Statistical analysis was performed with SPSS software (SPSS Standard v. 17.0, Chicago, IL). Unpaired t test was used to determine the significance in mutation rates between two groups with 95% confidence intervals (CI). All tests were two-sided; P < 0.05 was considered as a significant difference.

    • As shown in Table 1, the serum levels of HBsAg, HBeAg, and HBV DNA in each of eight mothers in group one had no significant changes during 6.0–7.2 years (from the second trimester of pregnancy to 5.5–6.7 years postpartum). The HBV DNA levels were constantly higher than 1.0 × 106 IU/mL among all eight mothers. Meanwhile, the levels of HBsAg, HBeAg, and HBV DNA in each child were also comparably high. In addition, these patients had normal ALT levels (data not shown). Thus, all patients in group one were in the HBeAg-positive chronic infection phase, also known as the immune tolerant phase according to 2017 European association for the study of the liver (EASL) guidelines (EASL 2017).

      Table 2 shows the virological characteristics in patients in group two at a single time point. Of the six index patients, three were over 60 years old and three others were over 40 years old. Most of the patients were in natural phases 2–4 based on the EASL guideline (EASL 2017). Noticeably, seven patients underwent spontaneous HBeAg seroconversion, two had seroclearance of HBeAg without development of anti-HBe, and five showed coexistence of HBeAg and anti-HBe. Additionally, one index patient (Ⅻa) spontaneously cleared HBsAg and developed antiHBs with undetectable HBV DNA and normal ALT value, indicative of recovery from HBV infection, thus falling into the HBsAg-negative phase (EASL 2017).

    • Comparison of pre-C/C gene (624 bp) sequences recovered during the second trimester and 5.5–6.7 years postpartum revealed that pre-C/C gene had no mutation in seven women, and one single nucleotide substitution in one woman (ⅩⅢa) (Table 3). The sequences recovered from their children were identical to the sequences in their mothers during pregnancy. Moreover, among four reference strains (GQ205441, KR013798, KU519422, LC170476) that were isolated in China or Japan, the strain (GQ205441) in a neighboring city is evolutionarily closest to the sequences in group one (Fig. 1A). The variation rate of nucleotides between GQ205441 and the sequences in group one was only 0.48%–1.44%, and none leads to an amino acid variation (Table 3).

      Family Patient Relation Sex Age (Years) Compared to indexa Compared to GQ205441
      nt (%) aa (%) nt (%) aa (%)
      Group one
      Ⅰa Index F 30.7 - - 3 (0.48) 0 (0.0)
      37.0 0 (0.0) 0 (0.0) 3 (0.48) 0 (0.0)
      Ⅰb Daughter F 5.9 0 (0.0) 0 (0.0) 3 (0.48) 0 (0.0)
      Ⅱa Index F 21.4 - - 5 (0.8) 0 (0.0)
      28.0 0 (0.0) 0 (0.0) 5 (0.8) 0 (0.0)
      Ⅱb Daughter F 6.1 0 (0.0) 0 (0.0) 5 (0.8) 0 (0.0)
      Ⅲa Index F 25.5 - - 4 (0.64) 0 (0.0)
      32.3 0 (0.0) 0 (0.0) 4 (0.64) 0 (0.0)
      Ⅲb Daughter F 6.4 0 (0.0) 0 (0.0) 4 (0.64) 0 (0.0)
      Ⅳa Index F 25.8 - - 4 (0.64) 0 (0.0)
      33.0 0 (0.0) 0 (0.0) 4 (0.64) 0 (0.0)
      Ⅳb Son M 6.7 0 (0.0) 0 (0.0) 4 (0.64) 0 (0.0)
      Ⅴa Index F 25.6 - - 5 (0.8) 0 (0.0)
      32.7 0 (0.0) 0 (0.0) 5 (0.8) 0 (0.0)
      Ⅴb Son M 6.7 0 (0.0) 0 (0.0) 5 (0.8) 0 (0.0)
      Ⅵa Index F 22.5 - - 8 (1.28) 0 (0.0)
      28.5 0 (0.0) 0 (0.0) 8 (1.28) 0 (0.0)
      Ⅵb Daughter F 5.6 0 (0.0) 0 (0.0) 8 (1.28) 0 (0.0)
      Ⅶa Index F 27.4 - - 4 (0.64) 0 (0.0)
      34.3 0 (0.0) 0 (0.0) 4 (0.64) 0 (0.0)
      Ⅶb Daughter F 6.5 0 (0.0) 0 (0.0) 4 (0.64) 0 (0.0)
      Ⅷa Index F 24.2 - - 9 (1.44) 0 (0.0)
      30.2 1 (0.001) 0 (0.0) 8 (1.28) 0 (0.0)
      Ⅷb Son M 5.5 0 (0.0) 0 (0.0) 9 (1.44) 0 (0.0)
      Group two
      Ⅸa Index F 78 - - 23 (3.69) 15 (7.2)
      Ⅸb Daughter F 53 35 (5.61) 28 (13.46) 24 (3.85) 17 (8.2)
      Ⅸc Son M 45 22 (3.53) 14 (6.73) 6 (0.96) 1 (0.5)
      Ⅹa Index F 71 - - 9 (1.44) 5 (2.4)
      Ⅹb Son M 40 14 (2.24) 10 (4.80) 13 (2.08) 7 (3.4)
      Ⅺa Index F 63 Undetectable
      Ⅺb Son M 41 - - 6 (0.96) 4 (1.9)
      Ⅺc Son M 38 19 (3.04) 11 (5.29) 23 (3.69) 15 (7.2)
      Ⅻa Index F 46 Undetectable
      Ⅻb Daughter F 23 - - 14 (2.24) 10 (4.8)
      Ⅻc Son M 21 20 (3.21) 15 (7.21) 13 (2.08) 8 (3.8)
      ⅩⅢ ⅩⅢa Index M 46 - - 24 (3.85) 14 (6.7)
      ⅩⅢb Son M 22 27 (4.33) 19 (9.13) 7 (1.12) 7 (3.4)
      ⅩⅣ ⅩⅣa Index F 40 - - 15 (2.4) 10 (4.8)
      ⅩⅣb Husband M 40 16 (2.56) 12 (5.76) 13 (2.08) 7 (3.4)
      aPre-C/C sequences were compared to the sequences recovered from the index patients during the second trimester in families Ⅰ–Ⅷ, and to the sequences recovered from the index patients or other infected family members in families Ⅸ–ⅩⅣ.

      Table 3.  Nucleotide and amino acid variations in pre-C/C gene among family members or compared to GQ205441.

      Figure 1.  Phylogenetic analysis of pre-C/C region of HBV. Phylogenetic tree was constructed based on the pre-C/ C sequences (624 bp) from samples in families Ⅰ–Ⅷ in group one (A) and families Ⅸ– ⅩⅣ in group two (B). The sequences GQ205441 (Heifei, China), KR013798 (Guangzhou, China), KU519422 (Tibet, China), LC170476 (Tokyo, Japan) (all genotype C) retrieved from GenBank were used as references.

    • HBV DNA were undetectable in the serum samples from two index patients in group two, presumably due to the extremely low viral load. In 13 other patients in group two, pre-C/C sequences varied significantly among the patients in each family. Compared to those in the index patients or between other family members, the nucleotide sequences and amino acid residues in pre-C/C region showed 2.24%–5.61% and 4.8%–13.46% differences respectively (Table 3), significantly higher than those observed in group one (P < 0.001). When compared to GQ205441, the nucleotide sequences and amino acid residues in patient Ⅸc, who was in phase 1, had 0.96% and 0.5% difference respectively, similar to those observed in group one; whereas the nucleotide sequences and amino acid residues in the other 12 patients had 1.12%–3.68% and 1.9%–8.2% difference respectively, significantly higher than those observed in group one (P < 0.001). Phylogenetic analysis showed highly close evolutionary relationship between each mother (both during pregnancy and at 5.5–6.7 years postpartum) and her child in all families in group one (Fig. 1A), but relatively distant relationship among members in each family in group two (Fig. 1B).

    • When compared with GQ205441, a total of 106 different point substitutions were detected in pre-C/C gene in this study. No deletions or insertions were found within pre-C/ C gene. Of them, 75 nucleotide substitutions, including six double mutations and two triple mutations, lead to 64 missense mutations and one nonsense mutation (i.c. G1896A). Sixty-one amino acid variations occurred in core protein (Fig. 2), the majority (72%) of which spread out within previously reported epitopes for T cells, cytotoxic T lymphocytes (CTLs), or B cells (Salfeld et al. 1989; Bertoletti et al. 1991, 1993; Ferrari et al. 1991; Sallberg et al. 1993; Carman et al. 1997). The other four non-synonymous mutations occurred in pre-C gene, among which the most prevalent one is the nonsense mutation G1896A, found in five HBeAg-negative patients (Ⅺc, Ⅸb, Ⅹa, Ⅹb, ⅩⅣb) and three (Ⅸa, Ⅺb, ⅩⅣa) who was undergoing seroconversion.

      Figure 2.  All sixty-one missense mutations found in core protein in this study. Missense mutations were found in amino acids (aa) of core protein in each sample from families Ⅸ–ⅩⅣ, the majority of which spread out known immune epitopes for T cells, CTLs, or B cells.

    • In the present study, we investigated the evolution of preC/C gene sequences in 31 patients from 14 families with known perinatal/intrafamilial HBV infection. We found that in the immune tolerant phase, pre-C/C sequences remained almost unchanged during 6.0–7.2 years in both the spreaders and the infected children, and had high homology to the sequences of HBV strain GQ205441 isolated from a nearby city (Zhang et al. 2011). By contrast, the intrafamilial transmitted HBV had gained diversified mutations in pre-C/C gene among the family members mostly in immune-clearance and low replicative phases. These results indicate that HBV pre-C/C gene is highly conservative during the immune tolerant phase, and mutations emerge around the time of the immune-clearance phase that is often coincided with decrease or loss of HBeAg. Seemingly, it may be the host immune pressure that decidedly drives the genesis of HBV mutations.

      It has long been considered that the viral reverse transcriptase of HBV has an error-prone nature, and HBV thus evolves with a high mutation rate similar to that of RNA viruses (Holmes 2008). The estimated half-life of circulating HBV varies, from 2.5 to 46 min (Dandri et al. 2008) to 4–24 h (Murray et al. 2005, 2006). In the present study, HBV DNA levels in the mothers from group one during the second trimester were comparable to those at 5.5–6.7 years postpartum (Table 1). To maintain such constant circulating viral loads, the circulating HBV should be replenished at least by 50% per day based on an estimated half-life of 24 h. By simple calculation, the HBV in the paired mother–child should have noticeable mutations during the observation period of 6.0–7.2 years (Girones and Miller 1989). However, of eight women, seven did not have any substitution, and one had only one nucleotide substitution yet nonsense mutation at 6.0–7.2 years follow-up (Table 3). Moreover, the pre-C/C gene in the eight children had identical sequences to their mothers (Table 3, Fig. 1A). These results indicate that in the immune tolerant phase, even the error-prone nature of HBV replication was hardly able to introduce detectable mutation into pre-C/C gene.

      Noticeably, compared with the sequence of HBV GQ205441, which was isolated in a nearby city in China, the pre-C/C sequences from individuals in immune tolerant phase (group one) had high (98.56%–99.52%) homology at nucleotide level and complete homology (100%) at amino acid level (Table 3). The patient (Ⅸc) in immune tolerant phase in group two also had high homology at nucleotide and amino acid levels with GQ205441 (Table 3). The high homology was unlikely caused by cross-contamination, as GQ205441 was isolated in a different institute, and the sequences among all the index patients were indeed diversified (Fig. 1). These findings suggest that a predominant HBV isolate is circulating in this region, and its pre-C/C gene has maintained highly conservative in immune tolerant phase even among different individuals. These results also imply that with little host immune pressure in play, proof-reading-deficient viral reverse transcriptase rarely causes detectable replication error.

      On the other hand, the pre-C/C gene in the subjects who were in non-immune tolerant phases (group two) showed diversified sequences (Table 3, Fig. 1B), despite that the family members most likely acquired the same HBV sources from the index patients. Of the 15 patients, one was in the immune tolerant phase with high viral load over 1.0 × 108, and 14 others were in other phases among which 10 had significantly reduced HBV DNA levels of ~ 1.0 × 10-4 IU/mL (Table 2). Thus, it is probable that most patients in group two had developed specific immune pressure against HBV, at least during a certain period. Therefore, the pre-C/C gene mutations were likely resulted from the long-term immune pressure as reported previously (Wang et al. 2010).

      The underlying mechanism that drives HBV evolution during its chronic infection has intrigued microbiologists for long (Croagh and Lubel 2014; Warner et al. 2014; Boeijen et al. 2017; Faure-Dupuy et al. 2017; Lazarevic et al. 2019). A quasi-species theory has been proposed to explain the interplay between the host immune pressure and the HBV genomic diversity (Warner et al. 2014). When there is only low immune pressure (such as in the immune tolerant phase), high viral load is maintained, whereas a low selective pressure exists that leads to only few emerging adaptive mutants. Positive selection pressure occurs (such as in the immune-clearance phase) when host immunity that curtails HBV replication forces the selection of virions that contain escape mutations in the immune epitopes they recognize. Mutations under positive selection can be identified in viral subcloning by finding a high rate of non-synonymous mutations in the genes that encode the immune epitopes. An increased frequency of positively selected mutations has been shown in the pre-C/C gene of the HBV from HBeAg-negative patients (Abbott et al. 2010). In this study, samples from 9 out of 13 patients in group two required subcloning to determine the correct sequences due to mixed trace signals in the chromatogram after direct sequencing (data not shown). For each sample, a consensus sequence was determined and used. Whereas samples from group one showed rival diversity to much less extent that only one sample needed subcloning and showed variants with only synonymous mutations in pre-C/C gene. In consistent, a previous longitudinal study spanning 4–14 years that followed-up 18 patients who were treatment naı ¨ve showed that the viral diversity may stay very low and stable for many years during immune tolerant phase, followed by an increase in the viral diversity within 0–3 years around the time of HBeAg seroconversion (Nie et al. 2012). To reveal more detailed viral mutational spectrums in patients, next generation sequencing (NGS) may be required in further in-depth analysis. Indeed, NGS technologies are able to assess the mutational frequency per site and have revolutionized the way to study diversity of viral population.

      HBV is a non-cytopathic virus, and the infection itself does not damage hepatocytes. Liver damage arises from cytolytic effects of the immune system, mainly the CTLs which attempt to clear HBV by killing the infected cells (Maini et al. 2000). The magnitude of such immune response has been noted to determine the course of the infection and clinical outcomes (Boeijen et al. 2017). Vigorous immune attack against infection is evidenced by elevated ALT (which are released from injured or killed hepatocytes) with suppressed viral replication. While normal ALT and active viral replication are signs for host immunity tolerant to HBV infection. This is the rationale behind the phase classification of HBV infection. Although we did not directly examine the CTL immunity in these patients, subcloning results revealed variants in group two harboring a dozen of different amino acid changes spread out immune epitopes in the core protein, including those for CD8+ T cells, CD4+ T cells, and B cells, co-exist in the serum. Clustering mutations occurred in known immunological target regions may reflect a role of the immune response for selection. On the other hand, no conclusive evidence has so far demonstrated that the accumulation of pre-C/C mutations could influence the outcomes of liver diseases.

      There are several limitations in this study. First, the blood samples in the eight children in group one were available only at 5.5–6.7 years age, leaving us impossible to observe the evolution of viral sequences in these children. However, the full sequence homology between the children and their mothers indicated that the sequences in children did not undergo mutation. Second, the samples in all individuals in group two were cross-sectional. Thus, we could not longitudinally observe the evolution of the same original HBV during the different phases of infection. Third, we cannot exclude potential mutations in regions other than pre-C/C. In fact, pre-C/C encodes HBeAg and HBcAg, two major targets for CTLs- and B cells-mediated antiviral immunity. HBV e and c antigens are prone to a wide variation of amino acid sequences during chronic HBV infection (Wang et al. 2016; Colombatto et al. 2018; Luo et al. 2019). The pre-C/C is arguably the most common site for mutations in HBV (Wang et al. 2016; Luo et al. 2019). Mutations occurred to pre-C/C should reflect how conservative or radical the virus had evolved so far. Fourth, the number of study subjects was relatively small. Nevertheless, these limitations did not taint the finding that pre-C/C is highly conservative for 6.0–7.2 years in the immune tolerant phase regardless of individuals.

      In summary, HBV pre-C/C gene in perinatally HBV-infected patients is highly conservative during the immune tolerant phase irrespective of different individuals, and undergoes diversified mutations around the time of ongoing immune-clearance phase. These results indicate that the emergence of viral variants is mainly driven by the host immune pressure. Mutations occurred in pre-C/C gene may mark the switch of immune tolerant to immune reactive phase in the natural course of HBV infection.

    • We thank Ms. Zhenhua Feng (Nanjing Drum Tower Hospital, Nanjing 210008, China) for performing sequencing of HBV pre-C/C gene. This study was supported by the National Natural Science Foundation of China (81672002), the Science and Technology Department of Jiangsu Province (BK20161105), and the Jiangsu Provincial Department of Health (H201537), China.

    • YHZ designed the study and critically revised the manuscript; YL and LZ performed the experiments, analyzed data and wrote the manuscript; YD and YH followed the patients, collected and analyzed the clinical data; BX participated in the design and performed the statistical analysis. All authors read and approved the final manuscript.

    • The authors declare no conflict of interests.

    • This study was approved by the institutional review boards of Nanjing Drum Tower Hospital, essentially following the ethical guidelines of the Declaration of Helsinki. Before sample collection, written informed consent was obtained from all the patients or their guardians for which identifying information is included in this article.

    Figure (2)  Table (3) Reference (36) Relative (20)

    目录

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return