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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) Ⅰ 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 Ⅱ 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 Ⅲ 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 Ⅳ 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 Ⅴ 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 Ⅵ 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 Ⅶ 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 Ⅷ 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 Ⅸ 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 Ⅺ 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 Ⅻ 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.
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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).
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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).
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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.
Study Population
Detection of Serological Markers for HBV Infection
HBV Genotyping and Analysis of Pre-C/C Mutations
Statistical Analysis
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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).
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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 Ⅰ 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) Ⅱ 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) Ⅲ 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) Ⅳ 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) Ⅴ 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) Ⅵ 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) Ⅶ 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) Ⅷ 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 Ⅸ 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) Ⅹ 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) Ⅺ 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) Ⅻ 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.
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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).
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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.