Journal of Viral Hepatitis, 2015, 22, 433–440
doi:10.1111/jvh.12315
Single-nucleotide substitution of Hepatitis B virus in intrauterine infection L.-J. Wang,1 Y.-F. Xu,1 X.-X. Liu1 and Y. Chen2,3
1
Institute of Liver Disease Research, Huai’an Fourth People’s
2
Hospital, Huai’an, Jiangsu Province, China; Laboratory of Digestive Tract Tumor, Huaiyin Advanced Vocational School of Nursing, Huai’an, Jiangsu Province, China; and 3Department of Gastroenterology, Huai’an Fourth People’s Hospital, Huai’an, Jiangsu Province, China Received February 2014; accepted for publication July 2014
SUMMARY. The relationship between hepatitis B virus (HBV) gene polymorphism and intrauterine infection has not been completely illuminated. Six pairs of mother and infant from intrauterine infection group and six mothers from nonintrauterine infection group in the previous study were randomly selected and separately divided into group M (Mother group), group N (Neonate group) and group NM (Negative-mother group) in this study. We found that age, gestational weeks, HBsAg titre, HBeAg titre and HBV DNA level of mothers from group M and group NM were not significantly different. Pre-S1/S2 and S regions in HBV genome were amplified, inserted into pUC19 plasmid and sequenced. It was found that all clone sequences clustered into genotype C (AY123041) through the Genotyping tool in NCBI and phylogenetic trees. Compared with AY123041, there were 20 (11 plus 9) mutations significantly different in the three groups. Most of the mutations
INTRODUCTION Mother–infant transmission is the main pathway of hepatitis B virus transmission in China. According to the literature [1–7], combination of hepatitis B immunoglobulin (HBIG) and hepatitis B vaccine could prevent 85–95% of hepatitis B virus (HBV) infection, and antiviral therapy in late pregnancy could effectively reduce the rate of HBV mother–infant transmission. Currently, intrauterine HBV infection serves as the important mode of mother–infant transmission and has become the bottleneck for further reducing the incidence of HBV mother–infant transmission. Abbreviations: CLMIA, chemiluminescence microparticle immunoassay; CO, cut-off value; HBIG, hepatitis B immunoglobulin; HBV, hepatitis B virus; NM, negative-mother. Correspondence: Yong Chen, Laboratory of Digestive Tract Tumor, Huaiyin Advanced Vocational School of Nursing, Huai’an, Jiangsu Province, China; Department of Gastroenterology, Huai’an First People’s Hospital, Huai’an, Jiangsu Province, China. E-mail: [email protected]
© 2014 John Wiley & Sons Ltd
were synonymous in pre-S1/S2/S region, while mutations of C2990T, T3205A, A167G, C407A, A667T and A680C resulted in amino acid substitution of A90V, S162T, T47A, P127T, L213F and I218L, respectively. In addition, most of the 20 mutations caused amino acid substitution in polymerase region for the tight structure of HBV genome. The occurrence and location of mutations indicated that mutation of C2990T only existing in group NM may serve as an index for nonintrauterine infection. In contrast, the incidence of intrauterine HBV infection from mothers with mutation of T3205A was lower. Then, mutations of G403A, T670G, A673G, A167G, C407A, A667T and A680C may be closely related to intrauterine HBV infection. Keywords: hepatitis B virus, intrauterine infection, mutation, pre-S1/S2/S region, single-nucleotide substitution.
In addition, 80–90% of infants infected by HBV within 1 year after birth will develop chronic infection and are with high risk for dying from hepatocellular carcinoma [8,9], which will put huge burdens on the family and society. Some reports [10–13] showed that many factors were closely correlated with intrauterine HBV infection, including maternal viraemia, maternal immune state, placental barrier, gene heterogeneity and neonatal susceptibility. Currently, there are several different theories regarding the relationship between HBV gene heterogeneity and intrauterine infection. Cheng et al. [11] reported that HBV strains with mutations of C96T in pre-S2 region, T473C in S region and C3175T in pre-S1 region could infect foetuses easily, while Su et al. [14,15] reported that mutation of C2909T in pre-S1 region was negatively correlated with intrauterine HBV infection and believed that wild-type strains played an important role in intrauterine HBV infection. The purpose of this study was to investigate the relationship between single-nucleotide substitution and intrauterine HBV infection.
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L.-J. Wang et al.
MATERIALS AND METHODS Subjects and blood collection With informed consent, six pairs of mother and neonate from intrauterine infection group and six mothers from nonintrauterine infection group in our previous study [10] were randomly recruited in this project. Neonates whose cord blood was positive for both HBV DNA and hepatitis B surface antigen (HBsAg) were defined as a Neonate group (group N) and their mothers were classified as a Mother group (group M). Six mothers whose infants were negative with HBV DNA or HBsAg in cord blood were defined as the Negative-mother group (group NM). After approval of the hospital’s ethics committee, venous blood of mothers from group M and group NM and cord blood from group N were collected. In order to minimize the risk of contamination, we used the following steps to draw cord blood: firstly, both ends of the umbilical cord were clamped; then blood on the surface of the umbilical cord was removed by rinsing with the normal saline; the part for blood collection was wiped for three times with 75% ethanol; at last, the cord blood was drawn out with a syringe. All infants from group N were given 200 IU of hepatitis B immunoglobulin (HBIG) in the first 24 h after birth and 10 lg of hepatitis B vaccine at 0, 1 and 6 months.
Detection of HBsAg and HBeAg HBsAg and HBeAg were detected by chemiluminescence microparticle immunoassay (CLMIA). All kits and the automated immunoassay analyser (ARCHITECT i1000SR) were from Abbott Laboratories (Abbott Park, IL, USA). Samples were defined as positive, when their chemiluminescence signals were greater than or equal to the cut-off value. The reference range of HBsAg is
doi:10.1111/jvh.12315
Single-nucleotide substitution of Hepatitis B virus in intrauterine infection L.-J. Wang,1 Y.-F. Xu,1 X.-X. Liu1 and Y. Chen2,3
1
Institute of Liver Disease Research, Huai’an Fourth People’s
2
Hospital, Huai’an, Jiangsu Province, China; Laboratory of Digestive Tract Tumor, Huaiyin Advanced Vocational School of Nursing, Huai’an, Jiangsu Province, China; and 3Department of Gastroenterology, Huai’an Fourth People’s Hospital, Huai’an, Jiangsu Province, China Received February 2014; accepted for publication July 2014
SUMMARY. The relationship between hepatitis B virus (HBV) gene polymorphism and intrauterine infection has not been completely illuminated. Six pairs of mother and infant from intrauterine infection group and six mothers from nonintrauterine infection group in the previous study were randomly selected and separately divided into group M (Mother group), group N (Neonate group) and group NM (Negative-mother group) in this study. We found that age, gestational weeks, HBsAg titre, HBeAg titre and HBV DNA level of mothers from group M and group NM were not significantly different. Pre-S1/S2 and S regions in HBV genome were amplified, inserted into pUC19 plasmid and sequenced. It was found that all clone sequences clustered into genotype C (AY123041) through the Genotyping tool in NCBI and phylogenetic trees. Compared with AY123041, there were 20 (11 plus 9) mutations significantly different in the three groups. Most of the mutations
INTRODUCTION Mother–infant transmission is the main pathway of hepatitis B virus transmission in China. According to the literature [1–7], combination of hepatitis B immunoglobulin (HBIG) and hepatitis B vaccine could prevent 85–95% of hepatitis B virus (HBV) infection, and antiviral therapy in late pregnancy could effectively reduce the rate of HBV mother–infant transmission. Currently, intrauterine HBV infection serves as the important mode of mother–infant transmission and has become the bottleneck for further reducing the incidence of HBV mother–infant transmission. Abbreviations: CLMIA, chemiluminescence microparticle immunoassay; CO, cut-off value; HBIG, hepatitis B immunoglobulin; HBV, hepatitis B virus; NM, negative-mother. Correspondence: Yong Chen, Laboratory of Digestive Tract Tumor, Huaiyin Advanced Vocational School of Nursing, Huai’an, Jiangsu Province, China; Department of Gastroenterology, Huai’an First People’s Hospital, Huai’an, Jiangsu Province, China. E-mail: [email protected]
© 2014 John Wiley & Sons Ltd
were synonymous in pre-S1/S2/S region, while mutations of C2990T, T3205A, A167G, C407A, A667T and A680C resulted in amino acid substitution of A90V, S162T, T47A, P127T, L213F and I218L, respectively. In addition, most of the 20 mutations caused amino acid substitution in polymerase region for the tight structure of HBV genome. The occurrence and location of mutations indicated that mutation of C2990T only existing in group NM may serve as an index for nonintrauterine infection. In contrast, the incidence of intrauterine HBV infection from mothers with mutation of T3205A was lower. Then, mutations of G403A, T670G, A673G, A167G, C407A, A667T and A680C may be closely related to intrauterine HBV infection. Keywords: hepatitis B virus, intrauterine infection, mutation, pre-S1/S2/S region, single-nucleotide substitution.
In addition, 80–90% of infants infected by HBV within 1 year after birth will develop chronic infection and are with high risk for dying from hepatocellular carcinoma [8,9], which will put huge burdens on the family and society. Some reports [10–13] showed that many factors were closely correlated with intrauterine HBV infection, including maternal viraemia, maternal immune state, placental barrier, gene heterogeneity and neonatal susceptibility. Currently, there are several different theories regarding the relationship between HBV gene heterogeneity and intrauterine infection. Cheng et al. [11] reported that HBV strains with mutations of C96T in pre-S2 region, T473C in S region and C3175T in pre-S1 region could infect foetuses easily, while Su et al. [14,15] reported that mutation of C2909T in pre-S1 region was negatively correlated with intrauterine HBV infection and believed that wild-type strains played an important role in intrauterine HBV infection. The purpose of this study was to investigate the relationship between single-nucleotide substitution and intrauterine HBV infection.
434
L.-J. Wang et al.
MATERIALS AND METHODS Subjects and blood collection With informed consent, six pairs of mother and neonate from intrauterine infection group and six mothers from nonintrauterine infection group in our previous study [10] were randomly recruited in this project. Neonates whose cord blood was positive for both HBV DNA and hepatitis B surface antigen (HBsAg) were defined as a Neonate group (group N) and their mothers were classified as a Mother group (group M). Six mothers whose infants were negative with HBV DNA or HBsAg in cord blood were defined as the Negative-mother group (group NM). After approval of the hospital’s ethics committee, venous blood of mothers from group M and group NM and cord blood from group N were collected. In order to minimize the risk of contamination, we used the following steps to draw cord blood: firstly, both ends of the umbilical cord were clamped; then blood on the surface of the umbilical cord was removed by rinsing with the normal saline; the part for blood collection was wiped for three times with 75% ethanol; at last, the cord blood was drawn out with a syringe. All infants from group N were given 200 IU of hepatitis B immunoglobulin (HBIG) in the first 24 h after birth and 10 lg of hepatitis B vaccine at 0, 1 and 6 months.
Detection of HBsAg and HBeAg HBsAg and HBeAg were detected by chemiluminescence microparticle immunoassay (CLMIA). All kits and the automated immunoassay analyser (ARCHITECT i1000SR) were from Abbott Laboratories (Abbott Park, IL, USA). Samples were defined as positive, when their chemiluminescence signals were greater than or equal to the cut-off value. The reference range of HBsAg is
