Archives of Virology https://doi.org/10.1007/s00705-018-3900-0

BRIEF REPORT

BCP/PC mutation prevalence and their association with HBV replication in HIV/HBV co‑infected patients Dongdong Li1   · Qixia Chen1 · Chuanmin Tao1 Received: 7 February 2018 / Accepted: 20 April 2018 © Springer-Verlag GmbH Austria, part of Springer Nature 2018

Abstract The high prevalence (14.3%) of HIV/HBV co-infections reported in west China makes it necessary to screen concurrent HBV infection in HIV carriers. HBV B genotype was shown to be dominant in 54 cases of HIV/HBV co-infection, accounting for 81.48% of the total. The total drug resistance rate observed was 3.70%. A1762T, G1764A and G1896A mutations were common mutations identified in the BCP/PC region. However, the prevalence of the G1896A mutation was significantly high among the HBeAg negative HIV/HBV co-infected patients, and may be associated with high HBV replication. Mutations in the PC region are related to the loss in synthesis of HBeAg and may accelerate HBV replication in HIV positive patients.

Introduction Hepatitis B virus (HBV) and human immunodeficiency virus (HIV) infections are major chronic public health concerns worldwide. Globally, about 40 million people are chronically infected with HIV, while HBV causes millions of chronic infections [26]. Our previous study showed a high prevalence of HIV/HBV (14.4%) co-infection and a low rate of effective vaccination against HBV in HIV-infected patients [30]. HIV/HBV co-infection has been associated with high morbidity and mortality [16, 23] by accelerating the course of liver diseases caused by chronic HBV infection (CHB), leading to end-stage hepatic illness and increasing the risk of hepatocellular carcinoma (HCC) [18]. Basal core promoter/ precore (BCP/PC) mutations increase the risk of HBVrelated HCC [31], and can help predict significant liver fibrosis [10, 28]. In India, the prevalence of the G1896A mutation was considerably higher among HBeAg negative HIV/ HBV co-infected individuals when compared to HBV monoinfections [20]. In addition, the 1762T/1764A mutation also occurred more frequently in HIV-infected patients in China [9]. It is known that BCP/PC mutations contribute to defects Handling Editor: Li Wu. * Chuanmin Tao [email protected] 1



Division of Clinical microbiology, Department of Laboratory Medicine, West China Hospital of Sichuan University, Chengdu 610041, Sichuan, Republic of China

or loss in synthesis of HBeAg by inhibiting translation of the protein due to either frame shift mutations or premature stop codons, or, interfering with signal peptidase cleavage. HBeAg negative patients tend to carry HBV PC mutations [7, 24]. In addition, the reduced HBeAg levels also have an important effect on HBV replication and thereby influence liver disease progression and the host response to conventional interferon-α therapy. The high prevalence of HIV/ HBV co-infection reported in west China makes it essential to screen concurrent HBV infection in HIV carriers. In HIV carriers, PC mutations related to an absence of HBeAg may accelerate HBV replication.

Materials and methods From 593 HIV positive sera or plasma confirmed by HIV western blotting (MP Dianositics, Singapore) during 2016, 85 specimens were found to be HBsAg positive (COI > 1) (Roche Diagnostics, Germany). 54 sera or plasma from those 85 cases with HBV viral loads > 103 IU/ml (Shengxiang, China), were enrolled into the HIV/HBV co-infection group. Meanwhile, 56 HBsAg positive samples (HBV viral loads > 103 IU/ml) patients were also selected as a HBV mono-infection group. We applied two nested PCR reactions (Qinke, China) to amplify the whole Reverse transcriptase (RT) gene (nt131-nt1129) and complete BCP/PC region (nt1742-nt1900) respectively using primers previously described [11, 21]. Sanger sequencing of visualized bands was performed. HBV genotyping and drug resistance results

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were obtained after submitting RT nucleotide sequences to the NCBI website (http://www.ncbi.nlm.nih.gov/proje​cts/ genot​yping​/formp​age.cgi) and Stanford’s HBV database (http://hivdb​.stanf​ord.edu/HBV/DB/cgi-bin/MutPr​evByG​ enoty​peRxH​BV.cgi).

Results and discussion The high prevalence of hepatitis B virus (HBV) in China, together with the poor outcomes of HBV/HIV co-infection [18, 19], make it critical to determine HBV prevalence in HIV-infected patients. The prevalence of HBV/HIV coinfection varies in countries or regions [1, 2, 5, 14, 17, 27, 32]. In this study, 85 of 593 HIV samples (14.3%) were HBsAg positive, similar to the HIV/HBV prevalence seen in Eastern China (14.5%) [27]. Sharma A et al. recommended to screen HIV patients routinely for concurrent HBV infection [22]. Oliveira et al reported that OBI prevalence was 3.8% in HIV positive patients, highlighting the role of HBV DNA as a diagnostic separate from serological markers [15]. Firstly, the genotype distribution for B, C, and C/D recombinant was 81.48%, 14.82% and 3.70% in the HIV/ HBV co-infection group, and 80.36%, 17.86% and 1.78% in the HBV mono-infection group (Table 1). These genotype distribution differences were not statistically different (P > 0.05), which is similar to previous reports in the same geographical origin [25, 34]. Secondly, two of the 54 HIV positive patients (3.79%) had two mutations at

Table 1  Demographic data of patients involved in the present study

established drug resistance positions (rtA181S, rtV173A) (Table 1). The rate is lower than the incidence of spontaneous hepatitis B virus resistance mutations (5.73%) [33], suggesting the HBV/HIV co-infection group in our study was under no selective pressure. HBV BCP/PC variants may predict the progression of liver disease or fibrosis, and correlate with an increased risk of HCC [10, 28]. Thirdly, 97 out of the 110 HBV BCP/PC sequences were successfully obtained. G1896A, A1762T/ G1764A, and G1899A were the main BCP/PC mutations identified in this study (Table 2). The prevalence of the A1762T/G1764A mutation was lower in the HBV/HIV co-infection group (35.2%) when compared to the HBV mono-infection group (63.6%) (P < 0.05), which was in accordance with previous studies [3, 20]. However, there is a contrary report that the prevalence of A1762T/G1764A mutation in HBV/HIV coinfection group (52.5%) was significantly higher than that in the HBV mono-infected group [9]. Higher HBeAg positive prevalences in the HBV mono-infection group might lead to an overall decrease in the frequency of the BCP mutations which are more often associated with a HBeAg negative status [20]. However, the BCP A1762T/G1764A double mutation was more prevalent in the HBV monoinfection group than in the HBV/HIV co-infection group (P < 0.05), although its prevalence was not independent of HBeAg status (P > 0.05) (Table 2). Finally, as expected, the PC (A1846T, G1862T/A, and G1896A) mutations were more prevalent in the HBeAg-negative subjects than in the HBeAg-positive subjects (P < 0.05). G1896A

Gender (male/female) Age, median (IQR) HBeAg+, n (%) HBeAb+ n (%) ALT, IU/L, median (IQR) AST, IU/L, median (IQR) B C D C/D recombinant Mutations at established drug resistance positions  lg (HBV DNA) IU/ml, median (IQR)   lg (HBV DNA) HBeAg+   lg (HBV DNA) HbeAg-

HIV/HBV coinfection (n = 54)

HBV mono-infection (n = 56)

P

46/8 44 (36-57) 18 (33.33%) 36 (66.7%) 34 (13-70) 59 (42-103) 44 (81.48%) 8 (14.82%) 0 2 (3.70%) 2 (3.7%) 6.8 (5.1,7.8) 7.1 (5.8,> 8.0) 5.84 (4.06,7.3)

44/12 41 (31-49) 30 (53.6%) 26 (46.44%) 54 (28-86) 41 (33-76)) 45 (80.36%) 10 (17.86%) 1 (1.78%) 0 5 (8.93%) 4.6 (3.6,6.0) 6.5 (4.8,7.9) 4.0 (3.3,4.9)

0.37 0.08 0.32 0.032 < 0.01 < 0.01 0.88

< 0.01 < 0.01

Chi-square test, student’s t test and Kruskal-Wallis test were used for statistical analysis in SPSS software (Version 16.0, SPSS Inc., USA)

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BCP/PC mutations in HIV/HBV co-infections Table 2  BCP/PC mutation analysis in relation to HIV and HBeAg status

T1753C/G T1754G A1762T G1764A C1766T T1768A G1862T/A A1846T G1896A G1899A

HIV+eAg+ n = 18

HIV-eAg+ n = 22

HIV+eAgn = 36

HIV-eAgn = 22

P

1 (5.6%) 0 8 (44.4%) 9 (50.0%) 1 (5.6%) 1 (5.6%) 0 (0%) 2 (9.1%) 6 (33.3%) 2 (11.1%)

4 (18.2%) 0 12 (54.5%) 12 (54.5%) 0 1 (3.3%) 5 (16.7%) 2 (9.1%) 2 (9.1%) 7 (31.8%)

4 (11.1%) 5 (13.9%) 11 (30.6%) 12 (33.3%) 1 (2.8%) 2 (5.6%) 7 (19.4%) 17 (47.2%) 26 (72.2%) 8 (22.2%)

2 (9.1%) 1 (4.5%) 16 (72.7%) 16 (72.7%) 1 (3.8%) 1 (3.8%) 11 (42.3%) 8 (36.4%) 3 (13.6%) 13 (22.4%)

0.72 n/a 0.017 0.033 n/a n/a 0.000 0.004 0.000 0.005

n/a: not applied

and A1846T were more prevalent in the HBV/HIV coinfection group than in the HBV mono-infected group (P < 0.05), and G1862T/A was less prevalent in the HBV/ HIV co-infection group than in the HBV mono-infected group (P < 0.05). The frequency of G1896A and A1846T mutation was noticeably high among the HBeAg negative HIV individuals. The HBV viral loads in the BCP+/PCsubjects were much lower than those in the other three groups (Fig 1) (P < 0.05), and the HBV viral loads in the HBeAg negative controls were also much lower than those in the other three groups (P < 0.05) (Table 1). BCP mutations can result in lower levels of viral release via an epigenetic mechanism [8]. HBV DNA levels were significantly higher in patients with the PC mutations and

HBeAg negative status [6]. There is an opposite association between the PC mutation and viral loads before and after HBeAg seroconversion in patients with HBV infection [6]. It is inferred that the PC mutations may accelerate HBV replication regardless of the HBeAg status. Castelain, S, et al., demonstrated that a high serum HBV DNA level was also associated with PC mutations [4]. This may be the reason why HBV DNA levels were significantly higher in co-infected patients. Wang et al, showed that PC mutations do not cause increased DNA copies, but associate with damage to liver function [24]. A1762T/G1764A, A1846T and G1896A mutations have a higher possibility to develop HBV-ACLF in CHB patients [12, 29]. Nikolopoulos, G. K et al., showed that HBV-DNA levels predict

Fig. 1  BCP/PC mutations in relation to HBV replication in two groups. The A1762T/ G1764A dual mutation is represented as the BCP mutation, and the G1896A mutation is represented as G1896A.

The HBV viral loads in BCP+/PC- subjects was the lowest, both in cases (P > 0.05) and in controls (P < 0.05). *represents P < 0.05

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overall mortality in HIV positive patients [13]. Here, PC mutations may be associated with poor outcomes in the setting of HIV positive patients. In conclusion, high prevalence of HIV/HBV co-infection makes it essential to screen HIV patients routinely for concurrent HBV infection [22]. However, the prevalence of G1896A was markedly high among HBeAg negative HIV patients, and may promote HBV replication. Acknowledgements  We acknowledge all the colleagues in our laboratory for their assistance with routine testing. We also thank Dr. Shu Feng and Mrs Mengna Zou for reviewing the English and the grammar in the manuscript.

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Funding  The present study was supported by: the National Natural Science Foundation of China (#81301400/H1901).

Compliance with ethical standards  Ethic statements  The Ethics Committee of West China Hospital of Sichuan University approved this study. All procedures performed in studies involving human participants were in accordance with 1964 Helsinki declaration and its later amendments. Conflict of interest  The authors declare that they have no competing interests

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