Precore Region Research Articles

The Characteristic of HBV Quasispecies Is Related to Occult HBV Infection of Infants Born to Highly Viremic Mothers.

Although a combination of immunoprophylaxis and antiviral therapy can effectively prevent mother-to-child transmission (MTCT) of hepatitis B virus (HBV), a considerable number of infants born to highly viremic mothers still develop occult HBV infection (OBI). To uncover the virological factor and risk predictor for OBI in infants, we found that the diversity and complexity of maternal HBV quasispecies in the case group were lower than those in the control group. Mutations with significant differences between the two groups were most enriched in the NTCPbd and PreC regions. Genetic distance at the amino-acid level of the PreC region, especially the combination of three amino-acid mutations in the PreC region, could strongly predict the risk of OBI in infants. HBV quasispecies in OBI infants were highly complex, and the non-synonymous substitutions were mainly found in the RT and HBsAg regions. The sK47E (rtQ55R) and sP49L mutations in OBI infants might contribute to OBI through inhibiting the production of HBV DNA and HBsAg, respectively. This study found the potential virological factors and risk predictors for OBI in infants born to highly viremic mothers, which might be helpful for controlling OBI in infants.

Hepatitis B virus preCore/Core region variability in pregnant women in the Republic of Guinea

Introduction. The vertical route of hepatitis B virus (HBV) transmission is a significant problem in African countries, which is characterized by late diagnosis of the disease and high mortality. The high prevalence of hepatocellular carcinoma (HCC) in Africa may be due to variability in the HBV preCore/Core region, mutations in which contribute to disease progression. Molecular genetic characterization of strains circulating among pregnant women may reflect the overall mutational profile of the pathogen in the population.
 The objective of this study was to analyze the variability of the HBV preCore/Core region circulating among pregnant women in the Republic of Guinea.
 Materials and methods. The study material included 480 plasma samples obtained from HBV-positive pregnant women from the Republic of Guinea. For all samples, the nucleotide sequences of the preCore/Core region of the HBV genome were sequenced and analyzed.
 Results. Amino acid variability in the preCore region was determined in 211 (43.96%), and in the Core region in 473 (98.54%) patients. 12 polymorphic sites of the preCore region were identified in which amino acid substitutions occurred, including 8, 2 and 5 positions identified for genotypes E, A and D, respectively. In the Core region, 67 substitution positions were identified, including 46 in samples of genotype E, 23 in HBV genotype A and 26 in genotype D. It was shown that the distribution of substitutions in the preCore and Core regions in HBV genotypes E, A and D differs significantly with a predominance in mutations among HBV genotype E — p 0.0001. Individual characteristic mutations have been identified for each genotype. The most common clinically significant mutations in the preCore/Core region in the study group were identified, including pc-H5D (27,08%), pc-W28* (35,21%), c-E64D (33,54%), c-L116I/V/G (91,46 %), c-T146N (73,13%). The double mutation A1762T/G1764A in the basal core promoter was shown in 74 samples of HBV genotype E, which accounted for 15.42% of the total group and 16.59% of patients with HBV genotype E.
 Conclusion. The frequency of clinically significant preCore/Core mutations among pregnant women in the Republic of Guinea was determined. The data obtained reflect their prevalence in the general population and can be used to predict the progression of chronic HBV among the region's population.

Prevalence of occult hepatitis B infection among blood donors in Saint Petersburg

The aim of this study was to assess the prevalence of occult hepatitis B infection among blood donors in St. Petersburg, as well as to characterize the identified virus isolates. The study material was represented by 2800 blood plasma samples collected in 2019 from blood donors living in St. Petersburg. The ELISA study for HBV marker rate consisted of HBsAg, anti-HBs IgG, anti-HBcore IgG. HBV DNA was analyzed by nested PCR with real-time hybridization-fluorescence detection on three targets allowing to determine virus DNA at low viral load, including HBsAg-negative chronic hepatitis B. Hepatitis B serological markers were detected in 69.43% of those surveyed, HBsAg was found in 0.43% of individuals, and all of which donated blood first time. A significant excess of the anti-HBcore IgG antibodies occurrence among primary donors (15.1%) compared with repeated/regular donors (7.48%) was shown. The prevalence of virus DNA in the group was 3.14%, including 2.71% of cases in HBsAg-negative CHB. Based on phylogenetic analysis of 88 isolates, HBV subgenotypes were determined in the following order: D1 and D2, 40.91% each, D3 and A2, 9.09% each. While determining the serological subtype in detected isolates, the serotype ayw3 (52.27%) vs ayw2 (46.59%) and adw2 (10.23%) prevailed. Drug resistance mutations, including compensatory ones, were detected in six examined patients (6.82%). In all genotype D isolates, multiple amino acid substitutions were identified in the RT, SHB, MHB, LHB, and Core regions; mutations in the preCore region were detected in 21.59% samples. In the MHR of the HBV genotype D genome, twenty-six positions were identified in which amino acid substitutions occurred, and all isolates showed modifications at positions 113, 114, 131, 134, 159, 161, 168, in 76 — at position 122, in 68 — at position 127, in 36 — at position 118, in 24 — at position 128. In HBV A2 isolates, mutations T113S, S143T, Y161F were identified. Nine isolates in the preCore region showed a polymorphism including a stop codon W28*W; in five isolates the W28S substitution was shown in the same position, and the W28*S variant was found in one more sample. The high incidence of HBsAg-negative CHB cases among blood donors, as well as the predominance of HBV isolates that simultaneously carry mutations resulting in diagnostic failure of HBsAg tests and prophylactic failure of immunoglobulin or vaccines and virus reactivation, mutations that contribute to disease progression obviously pose a threat to health and require to be further examined.

Core Promoter and Pre-Core Variants of the Hepatitis B Virus (HBV) Are Frequent in Chronic Hepatitis B HBeAg-Negative Patients Infected by Genotypes A and D.

In Brazil, hepatitis B virus endemicity is low, moderate, or high in some areas, such as Espírito Santo State in the southeast region. In this study, we intend to characterize the basal core promoter (BCP) and pre-core region (PC) variants and their association with clinical/epidemiological disease patterns in patients infected with genotypes A and D. The study included 116 chronic hepatitis B patients from Espírito Santo State, Southeast Brazil, infected with genotypes A and D. Basal core promoter (BCP) and pre-core mutations were analyzed in these patients. The frequency of BCP and PC mutations was compared with age, HBeAg status, HBV genotype and subgenotype, HBV-DNA level, clinical classification, and transmission route. HBeAg-negative status was found in 101 (87.1%) patients: 87 (75.0%) were infected with genotype A (A1 = 85; A2 = 2) and 29 (25.0%) were infected with genotype D (D3 = 24; D4 = 3; D2 = 2). BCP + PC variants altogether were more frequent (48.1%) in genotype D than in genotype A strains (6.0%) (p < 0.001). When this evaluation was performed considering the cases that presented only the A1762T and/or G1764A (BCP) mutations, it was observed that the frequency was higher in genotype A (67.5%) compared to genotype D (7.4%) (p < 0.001). On the other hand, considering the samples with mutations only in positions G1896A and/or G1899A (PC), the frequency was higher in genotype D (75.8%) than in genotype A (6.9%) (p < 0.001). Interestingly, HBV DNA was lower than 2000 IU/mL especially when both BCP/PC mutations were present (p < 0.001) or when only PC mutations were detected (p = 0.047), reinforcing their role in viral replication.

Detection of Mutations in Precore and Basal Core Region in HBeAg Negative Chronic Hepatitis B Patients from Jaipur

Detection of Mutations in Precore and Basal Core Region in HBeAg Negative Chronic Hepatitis B Patients from Jaipur

HBV precore G1896A mutation promotes growth of hepatocellular carcinoma cells by activating ERK/MAPK pathway

Chronic hepatitis B virus (HBV) infection is one of the leading causes of hepatocellular carcinoma (HCC). The HBV genome is prone to mutate and several variants are closely related to the malignant transformation of liver disease. G1896A mutation (G to A mutation at nucleotide 1896) is one of the most frequently observed mutations in the precore region of HBV, which prevents HBeAg expression and is strongly associated with HCC. However, the mechanisms by which this mutation causes HCC are unclear. Here, we explored the function and molecular mechanisms of the G1896A mutation during HBV-associated HCC. G1896A mutation remarkably enhanced the HBV replication in vitro. Moreover, it increased tumor formation and inhibited apoptosis of hepatoma cells, and decreased the sensitivity of HCC to sorafenib. Mechanistically, the G1896A mutation could activate ERK/MAPK pathway to enhanced sorafenib resistance in HCC cells and augmented cell survival and growth. Collectively, our study demonstrates for the first time that the G1896A mutation has a dual regulatory role in exacerbating HCC severity and sheds some light on the treatment of G1896A mutation-associated HCC patients.

High Prevalence of Occult Hepatitis B Virus Infection among Iranian Hemodialysis Patients.

Considering the potential risks associated with occult hepatitis B virus (HBV) infection, this study was designed to investigate the magnitude and genotypic pattern of occult HBV infection among hemodialysis patients. All patients on regular hemodialysis attending the dialysis centers located in southern Iran and 277 nonhemodialysis controls were invited to participate in this study. Serum samples were tested for detection of hepatitis B core antibody (HBcAb) and hepatitis B surface antigen (HBsAg) by competitive enzyme immunoassay and sandwich ELISA, respectively. The molecular evaluation of HBV infection was conducted by two nested polymerase chain reaction (PCR) assays, targeting S, X, and precore regions of HBV genome, and sequencing by Sanger dideoxy sequencing technology. Moreover, HBV viremic samples were tested for hepatitis C virus (HCV) coinfection by HCV Ab ELISA and seminested reverse transcriptase PCR. Of 279 hemodialysis patients, five (1.8%) were positive for HBsAg, 66 (23.7%) were positive for HBcAb, and 32 (11.5%) had HBV viremia with HBV genotype D, sub-genotype D3 and subtype ayw2. Moreover, 90.6% of the hemodialysis patients with HBV viremia had occult HBV infection. Hemodialysis patients (11.5%) had significantly higher prevalence of HBV viremia than nonhemodialysis controls (1.08%; P = 0.0001). The prevalence of HBV viremia among hemodialysis patients was not statistically associated with duration of hemodialysis, age and gender distribution. In contrast, HBV viremia was significantly associated with place of residency and ethnicity, with residents of Dashtestan and Arab having had significantly higher prevalence of HBV viremia compared with the residents of other cities and Fars patients. Notably, 27.6% and 6.9% of hemodialysis patients infected with occult HBV infection were also positive for anti-HCV antibodies and HCV viremia, respectively. High prevalence of occult HBV infection was detected in hemodialysis patients, whereas 62% of patients with occult HBV infection were negative for HBcAb. Therefore, screening of all hemodialysis patients by sensitive molecular tests, regardless of the pattern of HBV serological markers, is recommended to increase the diagnosis rate of HBV infection.

Different mutation rates in the basal core promoter and precore regions between hepatitis B virus subgenotype F1b clusters.

Hepatitis B virus (HBV) is the main etiological agent of hepatocellular carcinoma (HCC) worldwide. It has been classified into nine genotypes and several subgenotypes, with uneven global distribution. There is growing evidence that the viral genotype influences the course and outcome of chronic hepatitis B infection. Two evolutionarily different clusters of the subgenotype F1b, called basal and cosmopolitan, have been described. The two clusters have constrained geographical distribution, with the particular feature that the basal cluster is present in regions of high HCC incidence, while the Cosmopolitan cluster is found in regions of low HCC incidence. The BCP/pC region was sequenced in 68 cases chronically infected with the F1b subgenotype to determine if there was a differential pattern of pathogenic-associated mutations between both clusters. Twenty-two of the 68 cases belonged to the subgenotype F1b basal cluster and 46 to the cosmopolitan cluster. Among the HBeAg-negative patients the A1762T/G1764A and G1896A mutations were more frequently found in the basal samples (85.7 and 92.9%) compared to the cosmopolitan ones (50 and 18.2%). Interestingly, no HBeAg loss-associated mutations were observed in 7.1 and 36.4% of the basal and cosmopolitan cases, respectively. The different rate of mutations associated with a more severe course of chronic hepatitis in the basal cluster would support the difference in the HCC incidence rate in the geographical regions where the basal cluster is restricted.

HBV with precore and basal core promoter mutations exhibits a high replication phenotype and causes ER stress-mediated cell death in humanized liver chimeric mice.

Mutations within the precore (PC) and basal core promoter (BCP) regions of the HBV genome are associated with fulminant hepatitis and HBV reactivation. These mutations may enhance viral replication, but little is known about whether they directly induce damage to the liver. We investigated mechanisms of direct cytopathic effects induced by the infection with PC/BCP mutants in the absence of immune response in vitro and in vivo . Mice with humanized livers and hepatocytes derived from humanized mice were infected with either wild-type or mutant-type PC/BCP HBV, and the HBV replication and human hepatocyte damage were evaluated. HBV proliferated vigorously in mice with PC/BCP-mutant infection, and the severe loss of human hepatocytes with a slight human ALT elevation subsequently occurred only in PC/BCP mutant mice. In PC/BCP mutant infection, the accumulation of HBsAg in humanized livers colocalized with the endoplasmic reticulum, leading to apoptosis through unfolded protein response in HBV-infected hepatocytes. RNA-sequencing revealed the molecular characteristics of the phenotype of PC/BCP mutant infection in a humanized mouse model. Reduced ALT elevation and higher HBV DNA levels in this model are consistent with characteristics of HBV reactivation, indicating that the hepatocyte damage in this model might mimic HBV reactivation followed by hepatocyte damage under immunosuppressive conditions. PC and BCP mutations were associated with enhanced viral replication and cell death induced by ER stress using HBV infection models. These mutations might be associated with liver damage in patients with fulminant hepatitis or HBV reactivation.

Hepatitis B infection among β-thalassemia major patients in Bushehr province of southern Iran

ABSTRACT This study was performed to determine the prevalence, genotype distribution and risk factors of hepatitis B virus (HBV) infection among β-thalassemia patients. ELISA was used to detect HBsAg and HBcAb. Molecular evaluation of HBV infection was performed by nested PCR, targeting S, X and pre-C regions of the genome, and sequencing. Of 126 thalassemia patients, 4 cases (3.17%) were positive for HBsAg, 23 cases (18.25%) were positive for HBcAb, and 6 cases (4.76%) had HBV viremia with genotype D, sub-genotype D3 and subtype ayw2. HBV prevalence among thalassemia patients was not statistically associated with gender distribution, place of residency, marital status and frequency of blood transfusion. HBsAg seroprevalence was significantly higher in Afghan immigrants and patients with ALT levels of 41–80 IU/L. The prevalence of HBV viremia was significantly higher among thalassemia patients aged >20 years compared to the patients aged <20 years. Moreover, 1.59% of thalassemia patients had seropositive occult HBV infection, which was positive for HBV-DNA and HBcAb but negative for HBsAg. Considering the relatively high prevalence of occult HBV infection among thalassemia patients, there is a possibility of their contamination through donated blood. Therefore, screening of donated blood based on detection of HBsAg cannot abolish HBV transmission through blood transfusion.

Whole genome analysis of hepatitis B virus before and during long-term therapy in chronic infected patients: Molecular characterization, impact on treatment and liver disease progression.

Hepatitis B virus (HBV) infection remains a serious public health concern worldwide despite the availability of an efficient vaccine and the major improvements in antiviral treatments. The aim of the present study is to analyze the mutational profile of the HBV whole genome in ETV non-responder chronic HBV patients, in order to investigate antiviral drug resistance, immune escape, and liver disease progression to Liver Cirrhosis (LC) or Hepatocellular Carcinoma (HCC). Blood samples were collected from five chronic hepatitis B patients. For each patient, two plasma samples were collected, before and during the treatment. Whole genome sequencing was performed using Sanger technology. Phylogenetic analysis comparing the studied sequences with reference ones was used for genotyping. The mutational profile was analyzed by comparison with the reference sequence M32138. Genotyping showed that the studied strains belong to subgenotypes D1, D7, and D8. The mutational analysis showed high genetic variability. In the RT region of the polymerase gene, 28 amino acid (aa) mutations were detected. The most significant mutations were the pattern rtL180M + rtS202G + rtM204V, which confer treatment resistance. In the S gene, 35 mutations were detected namely sP120T, sT126S, sG130R, sY134F, sS193L, sI195M, and sL216stop were previously described to lead to vaccine, immunotherapy, and/or diagnosis escape. In the C gene, 34 mutations were found. In particular, cG1764A, cC1766G/T, cT1768A, and cC1773T in the BCP; cG1896A and cG1899A in the precore region and cT12S, cE64D, cA80T, and cP130Q in the core region were associated with disease progression to LC and/or HCC. Other mutations were associated with viral replication increase including cT1753V, cG1764A/T, cC1766G/T, cT1768A, and cC1788G in the BCP as well as cG1896A and cG1899A in the precore region. In the X gene, 30 aa substitutions were detected, of which substitutions xT36D, xP46S, xA47T, xI88F, xA102V, xI127T, xK130M, xV131I, and xF132Y were previously described to lead to LC and/or HCC disease progression. In conclusion, our results show high genetic variability in the long-term treatment of chronic HBV patients causing several effects. This could contribute to guiding national efforts to optimize relevant HBV treatment management in order to achieve the global hepatitis elimination goal by 2030.

Molecular evaluation of hepatitis B virus infection and predominant mutations of pre-core, basal core promoter and S regions in an Iranian population with type 2 diabetes mellitus: a case–control study

BackgroundThis study was designed to evaluate the prevalence, genotypic patterns, and predominant mutations of hepatitis B virus (HBV) infection among diabetic patients.MethodsSerum samples were obtained from 733 patients with type 2 diabetes mellitus and 782 non-diabetic controls. The presence of HBsAg and HBcAb was determined by ELISA. Nested PCR, targeting S and pre-core regions of the HBV genome, followed by sequencing was carried out to determine HBV genotypes and predominant mutations in the S, basal core promoter (BCP), and pre-core regions of the HBV genome.ResultsOf 733 diabetic patients, 94 cases (12.82%) were positive for HBcAb, 28 cases (3.82%) were positive for HBsAg, and 19 cases (2.59%) had HBV-DNA with genotype D, sub-genotype D1/D3 and subtype ayw2. An occult HBV infection was found in one of the HBV DNA-positive samples, which was positive for HBcAb but negative for HBsAg. P120T/G145R, G1896A/G1899A, and A1762T/G1764T were the most frequent point substitution mutations detected in the S, pre-core, and BCP regions of the HBV genome, respectively. P120T and G145R mutations were associated with low levels or undetectable levels of HBsAg in serum. Therefore, routine tests based on HBsAg detection cannot detect HBsAg-negative infected patients.ConclusionsRelatively high prevalence of HBV infection was found in diabetic patients, while all of the HBV-infected patients were unaware of their infection. Therefore, screening for HBV infection should be included in the management program of diabetes for timely diagnosis and treatment of infected but asymptomatic patients.

High-Frequency Notable HBV Mutations Identified in Blood Donors With Occult Hepatitis B Infection From Heyuan City of Southern China.

BackgroundAll Chinese blood centers have implemented mini pool (MP) HBV nucleic acid testing (NAT) together with HBsAg ELISA in routine donor screening since 2015. The prevalence of occult hepatitis B virus infection (OBI) in donors from different regions varies, and the molecular characterization of the HBV DNA and clinical outcomes of these OBIs remain largely unexplored.MethodsBlood donations from Heyuan city in Southern China were screened by HBsAg ELISA and HBV MP8 NAT. Donations with HBsAg-/HBV DNA+ were collected for this study. Molecular characterizations of HBV DNAs were further analyzed by various DNA amplification assays including quantitative PCR (qPCR) and nested PCR, amplifying the basic core and pre-core promoter regions (BCP/PC). The HBsAg (S) region from HBV DNA was isolated by high-volume nucleic acid extraction. Notable mutations were identified by comparison to the HBV reference sequences. The clinical outcomes of the donors with OBIs were further followed for nearly 3 years.ResultsSeventy OBIs from 44,592 donations (0.15%) that we identified and reported previously were enrolled for this current study. HBV sequences were obtained from 44/70 OBIs, and genotyping analysis showed that 42/44 (95.2%) OBIs were genotype B, and 2/44 (4.8%) were genotype C. Interestingly, mutation analysis revealed that various mutations including M133L/T, F134L, P142L, V168A, R169H, S174N, L175S, and V177A of HBV DNA affecting HBsAg detection were observed in genotype B OBIs. Two notable mutations, T47K and L53S, were identified in genotype C OBIs. Follow-up studies showed that 3/31 (9.7%) OBIs converted to HBsAg+ as chronic infections while 1/31 (3.2%) HBV DNA was undetectable (classified as recovery) and 27/31 (87.1%) remained as OBIs.ConclusionVarious notable mutations affecting HBsAg detection were observed in blood donors with OBIs in Heyuan city of Southern China. Follow-up studies showed that most OBIs remained as OBIs with fluctuating or low viral loads. Higher sensitive HBV ID NAT is recommended for donor screening to further reduce the transmission risk of OBIs.

Review on hepatitis B virus precore/core promoter mutations and their correlation with genotypes and liver disease severity.

Of 350 million people worldwide are chronically infected with hepatitis B virus (HBV) and are at risk of developing cirrhosis and hepatocellular carcinoma (HCC) later in life. HBV is the most diverse DNA virus, and its genome is composed of four open reading frames: Presurface antigen/surface antigen gene (preS/S), precore/core gene (preC/C), polymerase gene (P), and the X gene (X). HBV produces quasispecies naturally or in response to antiviral agents because of the absence of proofreading activity amid reverse transcription and a high replication rate. The virus has 10 genotypes (A to J) with different geographical distributions. There are various HBV mutations in the HBV genome, including preC/C mutations, preS/S mutations, P gene mutations, and X gene mutations. The core promoter region plays a vital part in the replication, morphogenesis and pathogenesis of the virus. The precore region also plays a crucial role in viral replication. Both core promoter and precore mutations rescue the virus from host immune surveillance and result in the formation of mutated strains that may have altered pathogenicity. preC/C mutations are associated with liver disease progression. Precore mutations stop hepatitis B e antigen (HBeAg) production and basal core promoter mutations downregulate HBeAg production. Mutations in the basal core promoter are also associated with increased HBV replication and an increased incidence of advanced liver diseases such as cirrhosis and HCC. The emergence of antiviral-resistant mutations is the main reason for treatment failure. This review focuses mainly on preC/C promoter mutations and their correlation with genotypes and liver disease severity. Thorough perception and knowledge of HBV genetic variety and mutants could be vital to discover techniques for the prognosis and control of HBV infection.

Prevalence, genotype distribution and mutations of hepatitis B virus and the associated risk factors among pregnant women residing in the northern shores of Persian Gulf, Iran.

Considering perinatal transmission and the high rate of chronic hepatitis B virus (HBV) infection in infants, diagnosis of HBV infection during pregnancy and timely interventions are of great importance. Therefore, this study was performed to investigate the prevalence and genotype distribution of HBV infection and the associated risk factors among pregnant women in the northern shores of the Persian Gulf, South of Iran. Serum samples of 1425 pregnant women were tested for the presence of HBsAg and HBcAb by ELISA (HBsAg one-Version ULTRA and HBc Ab ELISA kits, DIA.PRO, Milan, Italy). The seropositive samples were tested for the presence of HBV DNA by nested PCR, targeting S, X, pre-core (pre-C), and basal core promoter (BCP) regions of the HBV genome. The amplified fragments were sequenced by Sanger dideoxy sequencing technology to evaluate the genotype distribution and mutations of HBV infection by using the MEGA 7 software. The HBV seropositive pregnant women were tested for HCV and HIV coinfections by ELISA (HCV Ab and HIV Ab/Ag ELISA kits, DIA.PRO, Milan, Italy). Of 1425 participants, 15 pregnant women (1.05%, 95% CI: 0.64%-1.73%) were positive for HBsAg, 41 women (2.88%, 95% CI: 2.10%-3.88%) were positive for HBcAb, and 5 women (0.35%, 95% CI: 0.15% -0.82%) had HBV viremia with genotype D, sub-genotype D3 and subtype ayw2. One of the viremic samples was positive for HBcAb but negative for HBsAg, which is indicative of an occult HBV infection. HBsAg seroprevalence was higher among pregnant women aged 20 to 29 years, women in the third trimester of pregnancy, residents of Khormuj city, Afghan immigrants, illiterate women, and pregnant women with a history of tattoo and HBV vaccination. The highest rate of HBcAb seroprevalence was observed in residents of Borazjan city, Turk ethnicity, the age group >39 years, and those women with more parities and a history of abortion. Nevertheless, HBV seroprevalence among pregnant women was not statistically associated with these variables. In contrast, HBcAb seropositivity was significantly associated with the history of tattoo (P = 0.018). According to mutations analyses, seven amino acid substitutions in the HBsAg, one point mutation in the pre-C region, and five points mutations in the BCP region were detected. Besides, the BCP mutations caused amino acid substitutions in the X protein. Of note, the conversion of Ala → Val at amino acid 168 (A168V) and Thr → Pro at amino acid 127 (T127P) were detected in HBsAg of the occult HBV strain. These results indicate a relatively low prevalence of HBV infection among pregnant women in the South of Iran, while tattooing is a risk factor for exposure to HBV infection. Moreover, all of the HBV-positive pregnant women were asymptomatic and unaware of their infection. Therefore, routine screening for HBV markers during pregnancy, appropriate treatment of HBV-infected women, and HBV vaccination are recommended to decrease mother-to-child transmission of HBV.

Reactivation of Occult Hepatitis B Virus Infection During Long-Term Entecavir Antiviral Therapy.

Up to now, it has not been clear whether occult hepatitis B virus (HBV) infection (OBI) can be treated with antiviral therapy whether OBI can develop drug resistance gene mutation or not. We report a middle-aged female patient with OBI who showed HBV reactivation (HBVr) during more than 3 years of intermittent entecavir (ETV) antiviral therapy: seropositive HBV surface antigen (HBsAg), increased e antigen (HBeAg), and repeatedly elevated serum HBV DNA. Genotype analysis showed that the patient was infected with HBV type B. Genetic sequencing of HBV showed the mutants of S143T, D144G, and G145R in the S gene region, and the mutant of site 1896 in the pre-Core region coexisted with the wild type (G1896A/G). No mutation was found in other HBV gene segments. Drug resistance gene analysis found RtL229W mutant, resistant to lamivudine but sensitive to ETV and other nucleoside analogs. This case of OBI provides us with the following clinical experiences: Firstly, it is necessary to detect HBV genotype, mutation, and drug-resistant genes at the initial diagnosis, which can be helpful for reasonable treatment. Secondly, identifying the risk factors and mechanisms associated with HBVr could help quantify the risk of HBVr and manage the clinical consequences. Thirdly, the OBI patients with hepatitis B e antigen-positive, HBV DNA > 1 × 103 IU/ml should be recommended regular and continuous antiviral therapy as soon as possible to prevent the occurrence of hepatocirrhosis and hepatocellular carcinoma (HCC).

Precore/core mutations of hepatitis B virus genotype D arising in different states of infection.

Aim of the studyPrecore/core variations and liver disease progression have been suggested. In this study, we aimed to determine the frequency of precore/core mutations in hepatitis B virus (HBV)-infected patients at various clinical stages.Material and methodsIn total, 73 HBV-infected patients including 26 inactive carriers (IC), 20 chronic active (CA), and 27 patients with liver cirrhosis/hepatocellular carcinoma (C/HCC) were randomly selected. The HBV DNA was extracted from the sera and subjected to nested PCR for amplification of precore/core region. The PCR product was then sequenced by the Sanger method.ResultsThe stop codon of W28*(G1896A) was determined as the most prevalent mutation (55%) of the precore region. The comparison of groups also demonstrated that core substitutions at residues of S21, E40 and I105 (< 0.05) correlated with the development of the inactive carrier state. Furthermore, the total substitutions in Th epitopes (117-131) were significantly higher in the C/HCC group than the IC and CA groups (p = 0.001).ConclusionsOur results indicated a high frequency of W28* mutation in HBV studied patients. Moreover, variations including S21, E40 and I105 and R151 that were mapped onto cellular epitopes might be related to inactive state development.

Immunogenicity and Antiviral Response of Therapeutic Hepatitis B Vaccination in a Mouse Model of HBeAg-Negative, Persistent HBV Infection.

During the natural course of chronic hepatitis B virus (HBV) infection, the hepatitis B e antigen (HBeAg) is typically lost, while the direct transmission of HBeAg-negative HBV may result in fulminant hepatitis B. While the induction of HBV-specific immune responses by therapeutic vaccination is a promising, novel treatment option for chronic hepatitis B, it remains unclear whether a loss of HBeAg may influence its efficacy or tolerability. We therefore generated an adeno-associated virus (AAV)-vector that carries a 1.3-fold overlength HBV genome with a typical stop-codon mutation in the pre-core region and initiates the replication of HBeAg(−) HBV in mouse livers. Infection of C57BL/6 mice established persistent HBeAg(−) HBV-replication without any detectable anti-HBV immunity or liver damage. HBV-carrier mice were immunized with TherVacB, a therapeutic hepatitis B vaccine that uses a particulate HBV S and a core protein for prime vaccination, and a modified vaccinia Ankara (MVA) for boost vaccination. The TherVacB immunization of HBeAg(+) and HBeAg(−) HBV carrier mice resulted in the effective induction of HBV-specific antibodies and the loss of HBsAg but only mild liver damage. Intrahepatic, HBV-specific CD8 T cells induced in HBeAg(−) mice expressed more IFNγ but showed similar cytolytic activity. This indicates that the loss of HBeAg improves the performance of therapeutic vaccination by enhancing non-cytolytic effector functions.

High Frequency Occult Hepatitis B Virus Infection Detected in Non-Resolved Donations Suggests the Requirement of Anti-HBc Test in Blood Donors in Southern China.

BackgroundMost Chinese Blood Centers adopted mini pool (MP) nucleic acid testing (NAT) for HBV screening due to high cost of Individual donation (ID) NAT, and different proportions of MP-reactive but ID-non-reactive donations (MP+/ID−, defined as non-resolved donations) have been observed during daily donor screening process. Some of these non-resolved donations are occult HBV infections (OBIs), which pose potential risk of HBV transmission if they are not deferred. This study is aimed to further analyze these non-resolved donations.MethodsThe non-resolved plasma samples were further analyzed by serological tests and various HBV DNA amplification assays including quantitative PCR (qPCR) and nested PCR amplifying the basic core and pre-core promoter regions (BCP/PC; 295 base pairs) and HBsAg (S) region (496 base pairs). Molecular characterizations of HBV DNA+ non-resolved samples were determined by sequencing analysis.ResultsOf 17,226 MPs from 103,356 seronegative blood donations, 98 MPs were detected reactive for HBV. Fifty-six out of these 98 (57.1%) reactive MPs were resolved as HBV DNA+, but the remaining 42 pools (42.9%, 252 donations) were left non-resolved with a high rate (53.2%) of anti-HBc+. Surprisingly, among 42 non-resolved MPs, 17 contained one donation identified as OBIs by alternative NAT assays. Sequence analysis on HBV DNAs extracted from these OBI donations showed some key mutations in the S region that may lead to failure in HBsAg detection and vaccine escape.ConclusionA total of 53.2% of the non-resolved donations were anti-HBc+, and OBIs were identified in 40.5% of these non-resolved pools. Therefore, non-resolved donations with anti-HBc+ might pose potential risk for HBV transmission. Our present analysis indicates that anti-HBc testing in non-resolved donations should be used to identify OBIs in order to further increase blood safety in China.

Relationship between C genotype HBV mutation and intrauterine transmission

Objective: To analyze the virus genome mutation of mothers with C genotype HBV and explore its relationship with HBV intrauterine transmission. Methods: A total of 399 mothers carrying HBV and their newborns hospitalized in the obstetrics department of the Third People's Hospital of Taiyuan from 2011 to 2013 were selected. Necessary information about mothers and children was obtained through a questionnaire survey and medical records. HBV DNA and HBV serological markers were detected by quantitative fluorescence PCR and electrochemiluminescence. Within 24 hours after birth and before active/passive immunization, those with positive HBsAg and/or HBV DNA in femoral venous blood were determined as HBV intrauterine transmission. According to the requirements of cloning and sequencing, mothers' HBV DNA load should be ≥106 IU/ml. Among 54 cases of HBV intrauterine transmission, 22 pairs of mothers and their newborns meeting the requirements of cloning and sequencing were used as the intrauterine transmission group. The same number of mothers and their newborns without intrauterine transmission was selected as the random seed method's control group. After PCR amplification of HBV DNA, gene cloning, and sequencing, the gene mutation analysis of mothers with C genotype HBV was performed. Results: Among the 44 samples, 39 (88.63%, 39/44) were genotype C, 2 were genotype B, and 3 were mixed genotype B, and C. A total of 406 clone beads from 42 mothers with C genotype HBV were analyzed for gene mutation, including 204 in the intrauterine transmission group and 202 in the control group. The base substitution mutation rate of PreS1, S, C, and P regions in the HBV intrauterine transmission group were significantly lower than those in the control group (χ2 ranged from 8.67 to 40.73, P<0.05). The mutation rate of base deletion in PreC and X regions in the HBV intrauterine transmission group was lower than that in the control group (χ2 values were 17.82 and 34.78, P<0.001). Two clones in the X region had 31 bp insertion mutations between nt1644 and nt1645, and two clones had 27 bp insertion mutations between nt1649 and nt1650, all of which took place in the control group. Conclusions: The base substitution mutations in the PreS1, S, C, and P segments of the HBV genome in mothers with C genotype HBV were associated with the occurrence of intrauterine transmission of HBV. Deletion mutations in the PreC region, insertion and deletion mutations in the X region may reduce intrauterine transmission risk.