Hepatitis B Virus Mutants Research Articles

HBV reactivation in allogeneic stem cell transplant recipients: Risk factors, outcome, and role of hepatitis B virus mutations.

Hepatitis B virus (HBV) reactivation (HBVr) in recipients of allogeneic hematopoetic stem cells (aHSCs) appears heterogeneously with respect to its frequency, manifestation, and outcome. The aim of this study was to present data from a large German cohort of recipients of aHSC transplantation (aHSCT), focusing on the incidence of HBVr in antibody to hepatitis B core antigen (anti‐HBc)‐positive aHSCT recipients, its clinical outcome, and the role of mutations in HBV. Between 2005 and 2015, 1,871 patients received aHSCT at University Hospital Essen. A follow‐up of at least 6 months after transplant was available in 55 patients who were anti‐HBc‐positive; clinical and virologic data were analyzed. The HBV genome was sequenced with next generation technology from serum samples of 8 patients with HBVr. Thirteen out of 55 (23.6%) patients developed HBVr at a median of 26 months after aHSCT. After initiation of antiviral treatment, complete HBV DNA suppression was achieved in 7/10 (70%) patients 1 to 40 months after HBVr. Nine of 13 patients had increased alanine aminotransferase; 3 patients had compromised coagulation and model for end‐stage liver disease scores of 18‐27, and 1 of these patients died due to liver failure 5 weeks after HBVr. As a risk factor for HBVr, we identified anti‐HBc signal to cut‐off ration (S/CO) ≥7.5 before transplantation. Complete HBV DNA suppression was achieved in 7/10 patients; therapy‐relevant mutations were found in 1 patient. In 4/8 patients, immune escape mutations were detected either as majority or minority variants. Conclusion: HBVr is common in anti‐HBc‐positive aHRCT recipients and can lead to severe hepatitis with compromised coagulation. The level of anti‐HBc S/CO before transplantation is a risk factor for HBVr. Complete virologic response under adequate antiviral treatment could not be achieved in all patients. (Hepatology Communications 2017;1:1014–1023)

Substitutions of rtL228 and/or L229 are involved in the regulation of replication and HBsAg secretion in hepatitis B virus, and do not affect susceptibility to nucleos(t)ide analogs.

Nucleos(t)ide analogs (NAs) are widely used in the treatment of hepatitis B virus (HBV) and human immunodeficiency virus (HIV). The mutation L210W of HIV‑1 reverse transcriptase (RT) is one of the six principal mutations which confer invivo resistance to zidovudine. Due to the similar 3D‑structure and high conservation between HIV‑RT and HBV‑RT, the present study aimed to clarify whether corresponding mutations in HBV may decrease its susceptibility to relevant NAs. Mutations including rtL228C/W, rtL229W and rtL228W/L229W were introduced into a HBV replication competent plasmid by fusion polymerase chain reaction. Replication capacity, HBs/e antigen (Ag) levels and susceptibility to NAs were subsequently analyzed invitro. Single or combination mutations of rtL228 and rtL229 impaired HBV replication. Decreased HBsAg secretion in the supernatant and production in the cell lysate wasobserved with single rtL229W or in combination with rtL228W, while there was no significant difference between wild‑type and mutant HBV with regard to the level of HBeAg in the supernatant and susceptibility to commonly‑used NAs. Substitution mutations of rtL228 and/or L229 in HBV did not alter the susceptibility of the virus to NAs, although replication and HBsAg secretion were affected.

HBx deletion-mutant combined with the reverse transcriptase enzyme/s* mutation is involved in HBV mediated liver cell proliferation and apoptosis

Background: One of the most common cancers worldwide is hepatocellular carcinoma (HCC). Studies have shown that Hepatitis B virus (HBV) X gene (HBx) mutations play a potentially important oncogenic role in the pathogenesis of this disease. However, the molecular mechanisms underlying the malignant transformation of liver cells following HBV infection are poorly understood. We previously identified a novel deletion mutation in HBx (HBx-d382) that is capable of transforming hepatocytes and increasing their tumorigenic capacity. As part of the afore-mentioned study, we also identified reverse transcriptase enzyme (rt)/s* double mutants in nucleoside analogue (NA)-resistant patients. This current study was conducted to determine if HBx-d382 and rt/s* mutations co-exist in HCC patients. The study also aimed to identify the role of these mutations in HCC. Methods: DNA from serum samples obtained from 152 NA-resistant patients with chronic hepatitis B (CHB) and 33 patients with HCC were sequenced to identify samples harboring both the HBx-d382 mutation and rt/s* double mutations. As part of this study, we also generated lentivirus constructs carrying different HBV mutations including rtA181T/sW172*, rtM204I/sW196*, HBx-d382, rtA181T/sW172*/HBx-d382 and rtM204I/sW196*/HBx-d382. The recombinant viral constructs were used to infect a human hepatocyte cell line (LO2). The LO2 cell line was used to evaluate the effect of the mutations on apoptosis, cellular proliferation and the rate of colony formation in the LO2 cells harboring different mutations. Results: We identified rt/s*/HBx-d382 mutations in both HBV-related HCC and NA-resistant CHB clinical samples and observed that the rate of occurrence of rt/s*/HBx-d382 mutations in HBV-related HCC is relatively high. The results of this study revealed that the rtA181T/sW172* and rtM204I/sW196* double mutants exhibited enhanced proliferation, reduced apoptosis and an increased rate of colony formation in LO2 cells. Expression of p53 and CyclinD1 were increased and the level of p21 was decreased in the rtA181T/sW172* and rtM204I/sW196* expressing LO2 cells. More importantly, rt/s* double mutations combined with the HBx-d382 mutation resulted in more severe phenotypes compared with rt/s* double mutation alone in LO2 cell lines. Conclusions: Our data suggest that rt/s*/HBx-d382 triple mutants play a crucial role in HBV-mediated HCC formation.

Heat shock proteins stimulate APOBEC-3–mediated cytidine deamination in the hepatitis B virus

Apolipoprotein B mRNA-editing enzyme catalytic subunit 3 (APOBEC-3) enzymes are cytidine deaminases that are broadly and constitutively expressed. They are often up-regulated during carcinogenesis and candidate genes for causing the major single-base substitution in cancer-associated DNA mutations. Moreover, APOBEC-3s are involved in host innate immunity against many viruses. However, how APOBEC-3 mutational activity is regulated in normal and pathological conditions remains largely unknown. Heat shock protein levels are often elevated in both carcinogenesis and viral infection and are associated with DNA mutations. Here, using mutational analyses of hepatitis B virus (HBV), we found that Hsp90 stimulates deamination activity of APOBEC-3G (A3G), A3B, and A3C during co-expression in human liver HepG2 cells. Hsp90 directly stimulated A3G deamination activity when the purified proteins were used in in vitro reactions. Hsp40, -60, and -70 also had variable stimulatory effects in the cellular assay, but not in vitro Sequencing analyses further demonstrated that Hsp90 increased both A3G cytosine mutation efficiency on HBV DNA and total HBV mutation frequency. In addition, Hsp90 shifted A3G's cytosine region selection in HBV DNA and increased A3G's 5' nucleoside preference for deoxycytidine (5'-CC). Furthermore, the Hsp90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin dose dependently inhibited A3G and A3B mutational activity on HBV viral DNA. Hsp90 knockdown by siRNA or by Hsp90 active-site mutation also decreased A3G activity. These results indicate that heat shock proteins, in particular Hsp90, stimulate APOBEC-3-mediated DNA deamination activity, suggesting a potential physiological role in carcinogenesis and viral innate immunity.

Identification of hepatitis B virus A1762T/G1764A double mutant strain in patients in Southern Brazil

Infection by hepatitis B virus (HBV) is a worldwide public health problem. Chronic HBV infection with high viral replication may lead to cirrhosis and/or hepatocellular carcinoma. Mutant HBV strains, such as the HBV A1762T/G1764A double mutant, have been associated with poor prognosis and higher risk of the patient for developing cirrhosis and/or hepatocellular carcinoma. This study analyzed the presence of the HBV A1762T/G1764A double mutant in patients with chronic HBV and its association with clinical parameters such as viral load, aminotransferases, and HBV antigens. A total of 49 patients with chronic hepatitis B were included in the study, and the HBV A1762T/G1764A double mutant strain was detected in four samples (8.16%) by polymerase chain reaction followed by restriction fragment length analysis (PCR-RFLP). The viral load was not significantly different between patients with or without the double mutant strain (p=0.43). On the other hand, carriers of the HBV A1762T/G1764A double mutant had higher levels of ALT (p=0.0028), while AST levels did not differ between groups (p=0.051). In this study, 75% of the samples with the HBV A1762T/G1764A double mutation were HBeAg negative and anti-HBe positive, reflecting seroconversion even though they still displayed high viral loads. Our study has shown that the HBV A1762T/G1764A double mutant strain circulates in Brazilian patients, and is associated with elevated levels of ALT and HBeAg seroconversion.

Hepatitis B genotypes and surface antigen mutants present in Pakistani blood donors.

BackgroundThe prevalence of chronic Hepatitis B Virus (HBV) infection is 2–4% in the Pakistani population, defining Pakistan as an intermediate prevalence country. In this study, hepatitis B surface antigen (HBsAg) reactive blood donations were screened using a combination of serological and molecular methods to identify immune escape HBV mutant strains and to determine the HBV genotypes and subtypes present in Pakistan.MethodsBlood donations were collected at the Armed Forces Institute of Transfusion (AFIT) located in northern Pakistan and the Hussaini Blood Bank (HBB) located in the south. From 2009 to 2013 a total of 706,575 donations were screened with 2.04% (14,409) HBsAg reactive. A total of 2055 HBsAg reactive specimens, were collected and screened using a monoclonal antibody based research assay to identify immune escape mutants followed by PCR amplification and DNA sequencing to identify the mutation present. DNA sequences obtained from 192 specimens, including mutant candidates and wild type strains, were analyzed for escape mutations, genotype, and HBsAg subtype.ResultsMutations were identified in approximately 14% of HBsAg reactive donations. Mutations at HBsAg amino acid positions 143–145 are the most common (46%) with the mutation serine 143 to leucine the most frequently occurring change (28%). While regional differences were observed, the most prevalent HBV strains are subgenotypes of D with subgenotype D1/subtype ayw2 accounting for the majority of infections; 90.2% at AFIT and 52.5% at HBB.ConclusionsThe high frequency of immune escape HBV mutants in HBV infected Pakistani blood donors highlights the need for more studies into the prevalence of escape mutants. Differences between vaccinated and unvaccinated populations, the correlation of escape mutant frequency with genotype, and impact of escape mutations in different genotype backgrounds on the performance of commercially available HBsAg assays represent avenues for further investigation.

Molecular analysis of Hepatitis B virus sub-genotypes and incidence of preS1/preS2 region mutations in HBV-infected Egyptian patients from Mansoura.

Hepatitis B virus (HBV) is one of the major causes of viral hepatitis worldwide. Despite the prevalence of HBV infection in Egypt, few studies have focused on sub-genotyping of the virus. Moreover, no studies are available regarding the mutational analysis of the preS1/preS2 region of the viral genome, or its impact on hepatocellular carcinoma (HCC) development in Egypt. In this study, we have analyzed the sub-genotypes and incidence of mutations in the preS1/preS2 region of HBV present in HBV-infected patients, from Mansoura city (located in the center of Nile Delta region of Egypt), via partial sequencing of this specific region. Moreover, we have investigated the impact of these mutations on HCC development by measuring serum alpha fetoprotein (AFP) level and abdominal ultrasound examination of the HBV-infected patients. According to our results, all samples were genotype D in which sub-genotype D1 was predominant. In addition, the results revealed mutations in the preS1/preS2 region, which could result in either immature preS1 protein or completely inhibit the translation of the preS2 protein. However, there was no incidence of HCC development in patients infected with mutated HBV in the preS1/preS2 region. In summary, for the first time our work has proved the predominance of sub-genotype D1 among HBV-infected Egyptian patients in Mansoura city, Nile Delta region, Egypt, and incidence of mutations in the preS1/preS2 region of HBV genome. This current study opens up research opportunities to discuss the impact of HBV mutations on the development of HCC in Egypt.

Hepatitis B virus sequencing and liver fibrosis evaluation in HIV/HBV co-infected Nigerians.

Molecular characteristics of hepatitis B virus (HBV), such as genotype and genomic mutations, may contribute to liver-related morbidity and mortality. The association of these characteristics with liver fibrosis severity in sub-Saharan Africa is uncertain. We aimed to characterise molecular HBV features in human immunodeficiency virus (HIV)/HBV co-infected Nigerians and evaluate associations between these characteristics and liver fibrosis severity before and after antiretroviral therapy (ART) initiation. HIV/HBV co-infected Nigerians underwent liver fibrosis estimation by transient elastography (TE) prior to and 36months after ART initiation. Basal core promoter/precore (BCP/PC) and preS1/preS2/S regions of HBV were sequenced from baseline plasma samples. We evaluated associations between HBV mutations and liver fibrosis severity by univariate and multivariable regression. At baseline, 94 patients underwent TE with median liver stiffness of 6.4 (IQR 4.7-8.7) kPa. Patients were predominantly infected with HBV genotype E (45/46) and HBe-antigen negative (75/94, 79.8%). We identified BCP A1762T/G1764A in 15/35 (43%), PC G1896A in 20/35 (57%), 'a' determinant mutations in 12/45 (26.7%) and preS2 deletions in 6/16 (37.5%). PreS2 mutations were associated with advanced fibrosis in multivariable analysis. At follow-up, median liver stiffness was 5.2 (IQR 4.1-6.6) kPa. No HBV molecular characteristics were associated with lack of fibrosis regression, although HIV virologic control, body mass index (BMI) and baseline CD4+ T-cell count were associated with a decline in fibrosis stage. Frequent BCP/PC and preS1/preS2/S mutations were found in ART-naïve HIV/HBV co-infected Nigerians. Median liver stiffness declined after initiation of ART, regardless of pre-ART HBV mutational pattern or virologic characteristics.

Hepatocellular carcinoma: co - evolution of hepatocytes and hepatitis B virus

Chronic hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC). In the process from chronic HBV infection to the development of HCC, there is a phenomenon of co-evolution of hepatocytes and HBV. The evolution of hepatocytes includes dedifferentiation and reverse evolution, while the evolution of HBV is mainly "telemorphosis" . Since HBV evolution occurs earlier than the development of HCC, the interaction between them is mainly reflected in the promotion of HCC evolution by HBV mutation. This article briefly summarizes the novel theory termed as cancer evolution and development and elucidates the molecular mechanism of HCC caused by HBV from the perspective of evolution.

Complete genome sequencing and clinical analysis of intrahepatic hepatitis B virus cccDNA from HCC

More than half of hepatocellular carcinomas (HCCs) are etiologically attributed to hepatitis B virus (HBV) infection, but it remains unclear whether HBV mutations are virological factors that contribute to formation of HCC or instead reflect accumulation during the progression of HBV-related disease. Rolling-cycle amplification and PCR sequencing were used to characterize covalently closed circular DNA (cccDNA) mutations in tumor tissues. Paired non-tumor tissues were used as controls. High frequencies of C1653T, T1753V, and A1762T/G1764A cccDNA mutations were observed in both tumor and non-tumor tissues. T1719G, C1329A, and T3098C mutations were related to the overall survival of HCC patients. Patients with G1719 tended to be in the high Barcelona Clinic Liver Cancer stage and had lower levels of total DNA and cccDNA per cell than patients with T1719. Additionally, invitro analysis revealed that T1719G mutation reduced viral replication efficacy. Finally, significantly higher levels of preoperative alpha-fetoprotein were observed in patients harboring the G1078T, C1653T, G1727A, C1913A, T1978C, or C3116T mutations at the cccDNA level. We speculated that HBV cccDNA mutations accumulated over the course of HBV-related disease development, and that some key mutations had prognostic value for patients with HBV-related HCC.

Selection of the highly replicative and partially multidrug resistant rtS78T HBV polymerase mutation during TDF-ETV combination therapy

Patients chronically infected with the hepatitis B virus (HBV) and receiving long-term treatment with nucleoside or nucleotide analogues are at risk of selecting HBV strains with complex mutational patterns. We herein report two cases of HBV-infected patients with insufficient viral suppression, despite dual antiviral therapy with entecavir (ETV) and tenofovir (TDF). One patient died from aggressive hepatocellular carcinoma (HCC). Serum samples from the two patients at different time points were analyzed using ultra-deep pyrosequencing analysis. HBV mutations were identified and transiently transfected into hepatoma cells in vitro using replication-competent HBV vectors, and functionally analyzed. We assessed replication efficacy, resistance to antivirals and potential impact on HBV secretion (viral particles, exosomes). Sequencing analyses revealed the selection of the rtS78T HBV polymerase mutation in both cases that simultaneously creates a premature stop codon at sC69 and thereby deletes almost the entire small HBV surface protein. One of the patients had an additional 261bp deletion in the preS1/S2 region. Functional analyses of the mutations in vitro revealed that the rtS78T/sC69∗ mutation, but not the preS1/S2 deletion, significantly enhanced viral replication and conferred reduced susceptibility to ETV and TDF. The sC69∗ mutation caused truncation of HBs protein, leading to impaired detection by commercial HBsAg assay, without causing intracellular HBsAg retention or affecting HBV secretion. The rtS78T/sC69∗ HBV mutation, associated with enhanced replication and insufficient response to antiviral treatment, may favor long-term persistence of these isolates. In addition to the increased production of HBV transcripts and the sustained secretion of viral particles in the absence of antigenic domains of S protein, this HBV mutation may predispose patients to carcinogenic effects. Long-term treatment with antiviral drugs carries the risk of selecting mutations in the hepatitis B virus (HBV). We herein report two cases of patients with insufficient response to dual tenofovir and entecavir therapy. Molecular analyses identified a distinct mutation, rtS78T/sC69∗, that abolishes HBsAg detection, enhances replication, sustains exosome-mediated virion secretion and decreases susceptibility to antivirals, thereby representing a potentially high-risk mutation for HBV-infected individuals.

Baicalin benefits the anti-HBV therapy via inhibiting HBV viral RNAs

BackgroundAlthough current antiviral treatments (nucleoside analogs, NAs) for chronic hepatitis B virus (HBV) infection are effective in suppressing HBV-DNA replication, their clinical outcomes can be compromised by the increasing drug resistance and the inefficiency in promoting HBsAg/HBeAg seroconversion. ObjectivesIn this study, we will explore possible effects and mechanism of a natural product baicalin (BA) with the anti-HBV efficacy of entecavir (ETV), a first-line anti-HBV drug, in HBV-DNA, HBsAg/HBeAg seroconversion and drug-resistance. MethodsThe co-effects of BA and ETV were conducted in wild-type/NA-resistance mutant HBV cell lines and DHBV-infected duckling models. HBV-DNA/RNAs, HBsAg/HBeAg, host factors (hepatocyte nuclear factors) were explored for possible anti-HBV mechanism. Results and discussionBA could significantly enhance and reduced HBsAg and HBeAg in hepG2.2.15, a wild-type HBV cell line. Co-treatment of BA and ETV had a more dramatic effect in NA-resistant HBVrtM204V/rtLl80M transfected hepG2 cells. Our study further revealed that BA mainly inhibited the production of HBV RNAs (3.5, 2.4, 2.1kb), the templates for viral proteins and HBV-DNA synthesis. BA blocked HBV RNAs transcription possibly by down-regulating transcription and expression of HBV replication dependent hepatocyte nuclear factors (HNF1α and HNF4α). Thus, BA may benefit the anti-HBV therapy via inhibiting HBV viral RNAs.

Prodromal fever indicates a high risk of liver failure in acute hepatitis B

The role of prodromal fever in the clinical course of acute hepatitis B virus (HBV) infection is still largely unclear. This study was conducted to investigate the factors associated with prodromal fever and its role in the development of acute liver failure (ALF) in patients with acute hepatitis B (AHB). Inpatients with AHB diagnosed between January 2006 and December 2010 were evaluated and followed. Clinical manifestations, results of laboratory tests, and outcomes were compared between patients with and without prodromal fever. The diagnosis of AHB was based on the discrete onset of symptoms, jaundice, abnormal liver function tests, the detection of high-titer IgM antibody to hepatitis B core antigen (anti-HBc), and a compatible clinical history. A total of 618 AHB inpatients were identified during the study period, of whom 102 (16.5%) had prodromal fever and 41 (6.6%) developed ALF. Prodromal fever indicated more severe liver injury and was independently associated with hepatitis B e antigen (HBeAg) negativity. The occurrence of ALF was more common in febrile patients than in non-febrile patients (18.6% vs. 4.3%, p<0.001). Multivariate logistic regression showed prodromal fever and temperature >38.0°C to be independently associated with the risk of ALF, with an odds ratio (95% confidence interval) of 3.5 (1.4-8.6) and 7.1 (2.6-19.7), respectively. AHB patients with prodromal fever, which is associated with a lack of HBeAg due to HBV mutation, are at high risk of ALF. Febrile patients with AHB should be managed with particular care.

The characteristics of hepatitisB surface antigen (HBsAg)-negative hepatitisB virus (HBV) infection in Chinese blood donors: a follow-up study of donors tested negative for HBsAg and reactive for simultaneous nucleic acid testing of HBV, hepatitisC virus, and human immunodeficiency virus.

The real infection status of hepatitis B virus (HBV) of hepatitisB surface antigen (HBsAg)-negative yet nucleic acid test (NAT)-positive blood donors is difficult to clarify. Detailed follow-up study is needed for analyzing the infectivity of these blood donors. Blood donors who screened negative for HBsAg and reactive for simultaneous NAT of HBV, hepatitisC virus (HCV), and human immunodeficiency virus (HIV) were included in a follow-up epidemiologic questionnaire survey and contributed follow-up samples for further testing. The follow-up samples were tested repeatedly for the serologic markers and HBV DNA. The genotypes and sequence mutations of HBV infected by 11 HBV DNA-positive donors were analyzed through the amplification and sequencing of HBV S region. Of the 46 donors included in this study, 89.1% were infected with HBV (41/46), including one (2.2%) window period infection, three (6.5%) recovered infections, and 37 (80.4%) occult HBV infections (OBIs). The S region of HBV was successfully amplified and sequenced for seven donors, five infected with Genotype B (71.4%), one with Genotype C (14.3%), and one with Genotype D (14.3%). Mutations in the S region were detected in four donors (57.1%) CONCLUSIONS: This is the first detailed study with multiple follow-up testing of the HBV infection status among blood donors who were tested negative for HBsAg and reactive for simultaneous NAT of HBV, HCV, and HIV. Most of these donors were infected with HBV with very low viral load. Our findings indicate that it is important to improve the sensitivity of NAT so as to decrease the residual risk of transfusion-transmitted HBV infection.

Expression of a hepatitis B virus pre-S2 deletion mutant in the liver results in hepatomegaly and hepatocellular carcinoma in mice.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer and has a poor prognosis and a low survival rate; its incidence is on the rise. Hepatitis B virus (HBV) infection is one of the main causes of HCC. A high prevalence of pre-S deletions of HBV surface antigen, which encompass T-cell and/or B-cell epitopes, is found in HBV carriers; antiviral therapy and viral immune escape may cause and select for these HBV mutants. In particular, the presence of pre-S2 deletion mutants is an important risk factor associated with cirrhosis and HCC. We generated Alb-preΔS2 transgenic mice that express a naturally occurring pre-S2 mutant protein containing a 33-nucleotide deletion (preΔS2); the aim was to investigate its effect on hepatocarcinogenesis. After 30 months of follow-up, the liver pathology of the mice fell into four groups: G1, chronic inflammation solely; G2, chronic inflammation and fibrosis; G3, inflammation, fibrosis, and hepatomegaly accompanied by rectal prolapse (4-12%); and G4, hepatomegaly and spontaneous HCC (12-15%). Striking degeneration of the endoplasmic reticulum (ER) was present in the mouse livers at an early stage (4 months old). At 8 months, overt ER stress and the Atf6 pathway of the unfolded protein response (UPR) were induced; at the same time, metabolic pathways associated with mevalonate and cholesterol biogenesis, involving the peroxisomes and the ER, were disturbed. At 20 months and older, the protein kinase RNA-like endoplasmic reticulum kinase (PERK) pathway of the UPR was induced and the Hippo transducer Yap was activated. Together, these ultrastructural aberrations and metabolic disturbance all seem to contribute to the molecular pathogenesis and hepatocarcinogenesis present in the Alb-preΔS2 mice. These findings may contribute to the development of therapies for the liver disorders and HCC associated with pre-S2 deletion mutations among HBV carriers. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Serological Investigation against Recombinant Hepatitis B Virus Surface Mutants in Vaccinated Healthcare Workers and HBV Chronic Carriers with Anti-HBs

Aims: Hepatitis B virus (HBV) infection is still a worldwide public health problem despite regular vaccination programs around the world. This study was performed to investigate the serological pattern of vaccinated subjects against recombinant repeatedly isolates of hepatitis B virus surface mutants, and evaluate the characteristics of HBsAg positive patients having HBs antibodies concurrently. Methods: Fifty vaccinated health care staffs participated in this study accompanied with five patients simultaneously carry HBsAg and anti-HBs antibody. For all the samples, the anti-HBs Enzyme-linked immunosorbent assay was performed for detecting antibody against thirteen recombinant mutants. Findings: The results showed that all vaccinated individuals had clinically sufficient protective antibody values (greater than 10 MIU/ml) with the mean value of 165 mIU/ml ± 29 mIU/ml. whereas, only 30% of individuals showed the complete protective neutralizing immune response to all specific HBsAg recombinant mutants. The lack of responsiveness mainly observed in three of HBV mutants; D144A (70%), Q129R (48.5%) and G145R (ad) (30%) respectively. The HBV chronic carriers with anti-HBs showed having the Q129R, G145R (ay), D144A, T126 N, T126s and G145 (ad) escaped mutants. Conclusion: The lack of responsiveness to some specific HBV mutants in vaccinated subjects, point out the potential risk of exposure to hepatitis B virus mutants.

Mutations in Hepatitis-B X-Gene Region: Chronic Hepatitis-B versus Cirrhosis

Specific mutations in Hepatitis-B Virus (HBV) genome would proceed the development of chronic hepatitis B to more serious consequences like cirrhosis and end-stage liver disease. This study was designed to detect deletion and insertion mutational patterns in the X-gene region in a population of chronic HBV and related cirrhosis patients. Sixty eight chronic HBV patients and 34 HBV-related cirrhotics were recruited from the eligible cases (N=50) referred to the academic hospitals of Gorgan city, Northeast of Iran, between Jan 2011 to Dec 2013. The HBx region was amplified by semi-nested PCR using serum samples and analyzed by sequencing. Our findings showed deletions and insertions in the C-terminal of HBx of the cirrhotic group and 8 bp found in two chronic HBV cases (2.9%). We detected 15 types of deletions in cirrhotic cases such as 1762-1768, 1763-1770, 1769-1773 and T1771/A1775. We found that the frequencies of deletion and insertion mutations in C-terminal of X-gene were more seen in cirrhotic patients comparing to chronic HBV cases in our area of study.

Hepatitis B Virus-Associated Hepatocellular Carcinoma.

Liver cancer is the fifth most common cancer worldwide in men and the ninth in women. It is also the second most common cause of cancer mortality. Hepatocellular carcinoma (HCC) is the most common type of liver cancer. About 350 million people globally are chronically infected with HBV. Chronic hepatitis B virus (HBV) infection accounts for at least 50% cases of HCC worldwide. Other non-HBV factors may increase HCC risk among persons with chronic HBV infection. Both indirect and direct mechanisms are involved in HCC oncogenesis by HBV. HCC-promoting HBV factors include long-lasting infection, high levels of HBV replication, HBV genotype, HBV integration, specific HBV mutants, and HBV-encoded oncoproteins (e.g., HBx and truncated preS2/S proteins). Recurrent liver inflammation caused by host immune responses during chronic HBV infection can lead to liver fibrosis and cirrhosis and accelerate hepatocyte turnover rate and promote accumulation of mutations. Major breakthroughs have been achieved in the prevention of HBV-associated HCC with HBV vaccines and antiviral therapies.

Characteristics of escape mutations from occult hepatitis B virus infected patients with hematological malignancies in South Egypt.

AIMTo investigate the prevalence and virological characteristics of occult hepatitis B virus (HBV) infections in patients with hematological malignancies in South Egypt.METHODSSerum samples were collected from 165 patients with hematological malignancies to monitor titers of HBV DNA, hepatitis B surface antigen (HBsAg), and antibodies to HBV core (anti-HBc) and surface antigens. Serum samples negative for HBsAg and positive for anti-HBc were subjected to nucleic acid extraction and HBV DNA detection by real-time polymerase chain reaction. DNA sequences spanning the S region were analyzed in cases with occult HBV infection. In vitro comparative study of constructed 1.24-fold wild type and S protein mutant HBV genotype D clones was further performed.RESULTSHBV DNA was detected in 23 (42.6%) of 54 patients with hematological malignancies who were HBsAg negative, but anti-HBc positive, suggesting the presence of occult HBV infection. The complete HBV genome was retrieved from 6 occult HBV patients, and P120T and S143L were detected in 3 and 2 cases, respectively. Site directed mutagenesis was done to produce 1.24-fold genotype D clones with amino acid mutations T120 and L143. The in vitro analyses revealed that a lower level of extracellular HBsAg was detected by chemiluminescence enzyme immunoassay (CLEIA) with the clone containing T120 mutation, compared with the wild type or the clone with S143L mutation despite the similar levels of extracellular and intracellular HBsAg detected by Western blot. Southern blot experiments showed that the levels of intracellular HBV DNA were not different between these clones.CONCLUSIONOccult HBV infection is common in patients with hematological malignancies and associated with P120T and S143L mutations. 120T mutation impairs the detection of HBsAg by CLEIA.

Clinical features and viral quasispecies characteristics associated with infection by the hepatitis B virus G145R immune escape mutant

Coexistence of the hepatitis B surface antigen (HBsAg) and hepatitis B surface antibody (anti-HBs) is an uncommon phenomenon, and the underlying mechanisms remain largely unknown. Amino-acid (aa) substitution from glycine to arginine at aa 145 (G145R), in the major hydrophilic region, has been reported in patients with HBsAg and anti-HBs coexistence. However, there is limited knowledge about the clinical features and viral quasispecies characteristics associated with G145R mutant hepatitis B virus (HBV) infection. We herein describe the dynamic changes in the serological and virological markers in a case of hepatitis B with coexisting HBsAg and anti-HBs, caused by a G145R immune escape mutant (genotype C). Entecavir was administered during the 4th week after admission. Alanine aminotransferase peaked in the 16th week, while both the HBsAg and HBeAg declined rapidly. HBsAg clearance and hepatitis B e antigen (HBeAg)/hepatitis B e antibody (anti-HBe) seroconversion were achieved in the 36th week, and then entecavir was withdrawn. A follow-up of 96 weeks showed that HBV DNA remained undetectable and that anti-HBs was maintained above 100 mIU/mL. The quasispecies characteristics of the G145R mutant HBV were investigated via ultra-deep sequencing. The complexity and genetic distance of the S and RT regions were much higher in the 8th week than at baseline or in the 4th week. Moreover, the frequencies of mutations (L173P, Q181R and A184V) in cytotoxic T lymphocyte epitopes increased before entecavir treatment. These findings extend understanding of the evolution of HBV under host immune pressure and of the clinical outcomes of affected patients.Emerging Microbes & Infections (2017) 6, e15; doi:10.1038/emi.2017.2; published online 22 March 2017