Hepatitis C virus genotypes in patients with chronic hepatitis C infection in southern Iran from 2016 to 2019

Hepatitis C is a liver disease caused by the hepatitis C virus (HCV). The treatment of HCV infection has become more complicated due to various genotypes and subtypes of HCV. The treatment of HCV has made significant advances with direct-acting antivirals. However, for the choice of medicine or the combination of drugs for hepatitis C, it is imperative to detect and discriminate the crucial HCV genotypes. The main objective of this study was to determine the pattern of circulating HCV genotypes in southern Iran, from 2016 until 2019. The other aim of the study was to determine possible associations of patients’ risk factors with HCV genotypes. A total of 803 serum samples were collected in 4 years (2016–2019) from patients with HCV antibody positive results. A total of 728 serum samples were HCV-RNA positive. The prevalence of HCV genotypes was detected using the genotype-specific RT-PCR test for serum samples obtained from 615 patients. The HCV genotype 1 (G1) was the most prevalent (48.8%) genotype in the area, with G1a, G1b, and mixed G1a/b representing 38.4%, 10.1%, and 0.3%, respectively. Genotype 3a was the next most prevalent (47.2%). Mixed genotypes 1a/3a were detected in 22 (3.6%) and finally G4 was found in 3 (0.5%) patients. The other HCV genotypes were not detected in any patient. Genotype 1 (1a and 1b alone, 1a/1b and 1a/3a coinfections) is the most prevalent HCV genotype in southern Iran. HCV G1 shows a significantly higher rate in people under 40 years old.     

Cytokine expression during chronic versus occult hepatitis B virus infection in HIV co-infected individuals

Chronic hepatitis B virus infection is characterized by persistent detectable levels of hepatitis B surface antigen (HBsAg) and HBV DNA in the serum. In contrast, HBsAg is not detectable during occult HBV infection, despite the presence of HBV DNA. An altered host immune response could play a role in the development of occult HBV infection; however, potential differences in immune responses among chronic and occult HBV-infected patients have not been evaluated in vivo. In the current study, we evaluated serum levels of regulatory, apoptotic, and fibrotic/anti-fibrotic cytokines/markers as indicators of immune responses in 25 chronic and 12 occult HBV-infected patients. More than half of the patients in both chronic and occult HBV infection groups had IL-2, IL-4, IL-13, and IFN-γ levels below detectable limits. In contrast, most patients had detectable levels of IL-8, IL-10, IP-10, sFas, sFasL, and TGF-β1. Of these, only sFas was significantly different between the two groups, with lower levels observed during occult compared to chronic HBV infection (p = 0.01). As a surrogate marker of apoptotic inhibition, decreased sFas during occult HBV infection suggests that apoptosis occurs at different rates in occult compared to chronic HBV infection and therefore, may contribute to persistence of occult HBV infection.     

Soluble CD40 ligand-activated B cells from patients with chronic hepatitis B virus infection as antigen presenting cells to induce hepatitis B virus specific cytotoxic T lymphocytes

Chronic hepatitis B virus (HBV) infection is the result of an inadequate antiviral immune response to the virus. In this study, we aimed to investigate whether the soluble CD40 ligand-activated B (CD40-B) cells could present antigen and induce specific cytotoxic T lymphocytes (CTLs) in patients with chronic HBV infection. We observed that after activated by sCD40L, the expression of CD80, CD86, major histocompatibility complex (MHC) I and II molecules on the CD40-B cells was significantly increased. Cytometry and fluorescence microscopy showed that more than 41.34% CD40-B cells were loaded by the HBcAg peptide. Furthermore, after been activated and HBcAg18–27 antigen peptide pulsed, B cells obtained from patients with chronic HBV infection could induce HBcAg18–27 specific CTLs in vitro. Taken together, our results show that B cells from patients with chronic HBV infection can be activated by sCD40L and may function as antigen presenting cells and induce HBV-specific CTLs.     

Occult hepatitis B virus infection among injecting drug users in the Central-West Region of Brazil

The prevalence of occult hepatitis B virus (HBV) infection was investigated in 149 hepatitis B surface antigen (HBsAg) negative injecting drug users (IDUs) in the Central-West Region of Brazil. Of these individuals, 19 were positive for HBV DNA, resulting in an occult HBV infection prevalence of 12.7% (19/149); six of these 19 individuals had anti-HBV core and/or anti-HBV surface antibodies and 13 were negative for HBV markers. All IDUs with occult hepatitis B reported sexual and/or parenteral risk behaviours. All HBV DNA-positive samples were successfully genotyped. Genotype D was the most common (17/19), followed by genotype A (2/19). These findings reveal a high prevalence of occult HBV infection and the predominance of genotype D among IDUs in Brazil''s Central-West Region.     

Interference of hepatitis B virus with cellular signaling

The presence of hepatitis B virus (HBV) proteins leads to changes in the cellular gene expression. As a consequence, the cellular signaling processes are influenced by the actions of HBV proteins. It has been shown that HBV nucleocapsid protein and the amino-terminal part of polymerase termed as terminal protein (TP) could inhibit interferon signaling. Further, the global gene expression profiles differ in hepatoma cells with and without HBV gene expression and replication. The expression of interferon (IFN) stimulated genes (ISGs) was differently regulated in cells with HBV replication and could be modulated by antiviral treatments. The HBV TP has been found to modulate the ISG expression and enhance the HBV replication. The modulation of the cellular signaling processes by HBV may have significant implications for pathogenesis.     

Management of individuals with chronic hepatitis B virus infection and persistent normal or mildly elevated aminotransferase levels

No consensus exists with respect to positive hepatitis B virus (HBV) DNA results and persistent normal or mildly elevated alanine aminotransferase (ALT). The aim of this study is to investigate the appropriate management and prognosis of these populations with chronic hepatitis B (CHB). A total of 235 subjects with positive HBV DNA results and persistent normal or mildly elevated ALT were enrolled in this study. Liver biopsy and liver stiffness measurements (LSM) were performed in all participants at baseline. Antiviral therapy was initiated in patients with significant hepatic inflammation (G ≥ 2) and/or fibrosis (S ≥ 2). The patients were divided into entecavir and adefovir groups based on HBV DNA load (>2000 IU/mL vs <2000 IU/mL). The liver biopsies were repeated at 72 weeks for the patients received antiviral therapy. We found that 112 subjects were hepatitis B e antigen (HBeAg) positive, and 123 subjects were negative. The corresponding median ALTs were 46 (39.5-52.5) and 48 (41.5-57.0) U/mL, respectively. G ≥ 2 and/or S ≥ 2 diseases were present in 48.8% (82/168) of the HBeAg-positive and 51.2% (86/168) of HBeAg-negative patients, respectively. In addition, 96 HBeAg-positive and 72 HBeAg-negative patients were divided into entecavir and adefovir groups. Meanwhile, liver biopsies had greater diagnostic accuracy for determining cirrhosis than LSM (0.711 vs 1.0, P < 0.0001). At the end of the study period, undetectable HBV DNA levels and normal ALT levels were observed in CHB-infected patients. Furthermore, the patients showed histologic improvement at 72 weeks compared with baseline measurements (G, 1.72 ± 1.00 vs 0.73 ± 0.88, P = 0.0002; S, 1.484 ± 0.90 vs 0.99 ± 1.13, P < 0.0001). Collectively, liver biopsy enhanced diagnostic accuracy for CHB-infected individuals with persistent normal or mildly elevated aminotransferase levels. Moreover, antiviral therapy can improve or regress the hepatic fibrosis and cirrhosis.     

Serum ficolin-2 concentrations are significantly changed in patients with hepatitis B virus infection and liver diseases

Human ficolin-2 is an important lectin complement pathway activator that is secreted from liver cells and has been implicated as an anti-infection innate immune molecule. However, the role of ficolin-2 protein and its dynamic changes over the course of and in the prognosis of chronic hepatitis B(CHB) and hepatocellular carcinoma(HCC) remain unclear. In this study, we analyzed ficolin-2 protein expression in a cohort of individuals with CHB infection, HCC and cirrhosis. A sandwich enzyme-linked immunosorbent assay(ELISA) method was used to measure serum ficolin-2 concentrations. Ficolin-2 expression in liver tissues was detected by immunohistochemical staining. Serum ficolin-2 concentrations in CHB patients were significantly higher than in healthy controls and HBV carriers. After 48 weeks of routine amelioration liver function treatment, serum ficolin-2 concentrations decreased and were positively correlated with favorable alanine aminotransferase(ALT), HBV DNA and HBe Ag-seroconversion outcomes. Interestingly, we observed much lower expression of serum and intrahepatic ficolin-2 in HCC and cirrhosis compared with healthy controls. Our findings suggest that serum and intrahepatic ficolin-2 levels may be considered one of the indicators for the response of chronic HBV infection, HCC and cirrhosis.     

Effects of lamivudine on the hepatitis B virus specific CD8+ cytotoxic T lymphocyte responses via peptide-MHC tetrameric complexes assay

Lamivudine, an oral nucleoside analogue, inhibitshepatitis B virus (HBV) replication. It has been shown tobe able to restore T cell responsiveness and to induce atype 1 T helper cell (Th 1) immunity in chronic HBVpatients. To further examine the effects of lamivudineon cytotoxic T lmphocyte (CTL) responses, two HBVantigenic peptide-HLA-A2 tetrameric complexes containingpeptides derived from HBV core protein (residues 18-27;FLPSDFFPSV) and polymerase (residue 551-559; YMDDVVLGA)were constructed. These two tetramers were used toserially determine the frequency of HBV antigen-specificCD8⁺ T cells before and during the treatment oflamivudine. The specificity of these tetramers wasconfirmed by (a) nonstaining of CD8⁺ T cells fromHLA-A2-negative HBV patients, (b) having variablefrequency data in the different teteramer measurement,and (c) showing peptide-specific CTL activity in thesorted tetramer-stanining CD8⁺T cells. Lowfrequency of HBV-specific CTLs was measured for bothtetramers before lamivudine treatment. However, thenumber of CD8⁺ T cells specific for HBV core 18-27increased significantly during lamivudine treatment. Incontrast, relatively lower frequency of HBV pol 551-559specific CD8⁺ T cells was persistently measuredafter lamivudine treatment. These results indicated thatthe lamivudine treatment could enhance HBV specific CTLresponses.     

Effects of lamivudine on the hepatitis B virus specific CD8+ cytotoxic T lymphocyte responses via peptide-MHC tetrameric complexes assay

Summary Lamivudine, an oral nucleoside analogue, inhibits hepatitis B virus (HBV) replication. It has been shown to be able to restore T cell responsiveness and to induce a type 1 T helper cell (Th 1) immunity in chronic HBV patients. To further examine the effects of lamivudine on cytotoxic T Imphocyte (CTL), responses, two HBV antigenic peptide-HLA-A2 tetrameric complexes containing peptides derived from HBV core protein (residues 18–27; FLPSDFFPSV) and polymerase (residue 551–559; YMDDVVLGA) were constructed. These two tetramers were used to serially determine the frequency of HBV antigen-specific CD8⁺ T cells before and during the treatment of lamivudine. The specificity of these tetramers was confirmed by (a) nonstaining of CD8⁺ T cells from HLA-A2-negative HBV patients, (b) having variable frequency data in the different teteramer measurement, and (c) showing peptide-specific CTL activity in the sorted tetramer-stanining CD8⁺T cells. Low frequency of HBV-specific CTLs was measured for both tetramers before lamivudine treatment. However, the number of CD8⁺ T cells specific for HBV core 18–27 increased significantly during lamivudine treatment. In contrast, relatively lower frequency of HBV pol 551–559 specific CD8⁺T cells was persistently measured after lamivudine treatment. These results indicated that the lamivudine treatment could enhance HBV specific CTL responses.     

Expression and characterization of hepatitis C virus core protein fused to hepatitis B virus core antigen

Recombinant plasmids were constructed by fusing the gene fragments encoding the full-length (1-191aa) and the truncated (1-40aa and 1-69aa) HCV core proteins (HCc) respectively to the core gene of HBV at the position of amino acid 144 and expressed in E. coli. The products were analyzed by ELISA, Western blotting as well as the immunization of the mice. The results showed that those fusion proteins (B144C191, B144C69, B144C40) possessed the dual antigenicity and immunogenicity of both hepatitis B virus core antigen (HBcAg) and hepatitis C virus core protein (HCc). Analysis by electron microscopy and CsCl density gradient ultra-centrifugation revealed that similar to the HBcAg itself, all fusion proteins were able to form particles. Comparison of the antigenicity and immunogenicity of those fusion proteins showed that the length of HCc gene fused to HBeAg had no much effect on the antigenicity and immunogenicity of HBcAg, however, B144C69 and B144C40 induced higher titres antibodies against HCc than B14d     

Increased serum oxysterol concentrations in patients with chronic hepatitis C virus infection

Oxidative stress and dysregulated cholesterol metabolism are characteristic features of chronic hepatitis C virus infection (CHC). Therefore, we analyzed serum oxysterol profiles in CHC patients and examined the significance of oxysterols in CHC. The concentrations of 7α-hydroxycholesterol, 4β-hydroxycholesterol and 25-hydroxycholesterol as determined by LC–ESI–MS/MS were significantly elevated by +236%, +29% and +44%, respectively, in CHC patients compared with controls. Moreover, the elevated levels were significantly decreased by anti-viral therapy using PEGylated-interferon and ribavirin for 3 months. In contrast, 24S-hydroxycholesterol, 27-hydroxycholesterol and 7α-hydroxy-4-cholesten-3-one concentrations were not affected by CHC or anti-viral treatment. These results suggest that some oxysterols that are elevated in CHC are produced by cholesterol autoxidation due to oxidative stress or inflammation in the liver. Oxysterols may represent novel targets for the inhibition of disease progression and the prevention of hepatocarcinogenesis in CHC patients.     

Mechanism of inhibiting type I interferon induction by hepatitis B virus X protein

Hepatitis B virus (HBV) is regarded as a stealth virus, invading and replicating efficiently in human liver undetected by host innate antiviral immunity. Here, we show that type I interferon (IFN) induction but not its downstream signaling is blocked by HBV replication in HepG2.2.15 cells. This effect may be partially due to HBV X protein (HBx), which impairs IFNβ promoter activation by both Sendai virus (SeV) and components implicated in signaling by viral sensors. As a deubiquitinating enzyme (DUB), HBx cleaves Lys63-linked polyubiquitin chains from many proteins except TANK-binding kinase 1 (TBK1). It binds and deconjugates retinoic acid-inducible gene I (RIG I) and TNF receptor-associated factor 3 (TRAF3), causing their dissociation from the downstream adaptor CARDIF or TBK1 kinase. In addition to RIG I and TRAF3, HBx also interacts with CARDIF, TRIF, NEMO, TBK1, inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase epsilon (IKKi) and interferon regulatory factor 3 (IRF3). Our data indicate that multiple points of signaling pathways can be targeted by HBx to negatively regulate production of type I IFN.     

Genotype and subtype analyses of hepatitis B virus (HBV) and possible co-infection of HBV and hepatitis C virus (HCV) or hepatitis D virus (HDV) in blood donors, patients with chronic liver disease and patients on hemodialysis in Surabaya, Indonesia

Four subtypes (adw, adr, ayw, and ayr ) and eight genotypes (A to H) of the hepatitis B virus (HBV) have been identified. They appear to be associated with particular geographic distribution, ethnicity, and possibly clinical outcomes. In this study, hepatitis B surface antigen (HBsAg) subtyping and HBV genotyping were carried out on sera obtained from HBsAg-positive HBV carriers, including healthy blood donors; patients with acute hepatitis, chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma; and patients on hemodialysis all located in Surabaya, Indonesia. We report here that all HBV isolates tested in Surabaya belonged to genotype B, with more than 90% of them being classified into subtype adw. Our results also revealed that prevalence of hepatitis C virus (HCV) co-infection among HBV carriers in Surabaya was approximately 10% for healthy blood donors and patients with chronic liver disease, and approximately 60% for patients on maintenance hemodialysis. Interestingly, HBsAg titers were lower in HBV carriers with HCV co-infection than in those without HCV co-infection. We also found that prevalence of hepatitis D virus (HDV) co-infection was < 0.5% among HBV carriers in Surabaya.     

慢性乙型肝炎病毒感染自然史中HBsAg和HBsAb共存血清学模式分析

目的分析慢性乙肝病毒感染者HBsAg和HBsAb共存模式中血清学指标、HBV-DNA和肝酶等指标与自然病程的关系,探讨其临床意义。方法回顾性分析2016年重庆医科大学附属第一医院HBsAg和HBsAb双阳性患者的血清学指标、HBV-DNA和ALT、GGT检测结果,并对其感染的自然病程进行分析。结果 2016年该院HBsAg和HBsAb双阳性患者共520例,占全部HBV感染者的2.80%,占总送检标本数的0.42%。可分期的184例双阳性患者中,免疫耐受期47例(25.54%),免疫清除期17例(9.24%),低复制期108例(58.70%),再活动期12例(6.52%),HBsAg、HBsAg/HBsAb比值、HBV-DNA、ALT和GGT水平差异均有统计学意义(P<0.05),低复制期患者HBsAg/HBsAb比值均低于其他患者(P<0.05)。不同分期患者HBsAb、年龄和性别比较差异无统计学意义(P>0.05),且HBsAb水平均较低。284例资料完整HBsAg和HBsAb共存病例中HBV-DNA阳性136例,占47.89%。HBsAg浓度与HBV-DNA载量成正相关(r=0.295,P<0.05),HBsAb浓度与HBV-DNA载量之间没有显著相关性(r=0.04,P>0.05)。结论 HBsAg和HBsAb共存患者并不少见,与性别无关,可发生在各个年龄阶段,以低复制期患者为最多。HBsAg和HBsAb共存患者中HBsAb多以低浓度形式存在,且浓度与自然病程无关。HBsAb的出现并非代表患者体内病毒复制停止,在诊断及治疗HBsAg和HBsAb共存模式的乙肝病毒感染者时仍需结合HBV-DNA载量来判断感染状态。     

Mutation analyses of integrated HBV genome in hepatitis B patients

Little has been learnt in the last 30 years about detection of HBV genome as well as its mutation analysis between hepatitis B fathers （HBF） and their children. In this study, we used nest polymerase chain reaction （PCR）, fluorescence in situ hybridization （FISH）, and DNA sequencing analysis, to examine the integrated HBV genome in paraffin-embedded testis tissues, which were taken as samples from HBE and in peripheral blood mononuclear cells （PBMC） from 74 cases of HBFs and their children who were born after their fathers＇ HBV infection （caHBF）. We found that HBV DNA existed in testis tissues, mainly in the basilar parts of the seminiferous tubules, and also in PBMC of HBE It was also documented that there were point mutations of poly-loci, insertions and deletions of nucleotides in integrated HBV genomes, and the types of gene mutations in the HBFs were similar to those in caHBE This study addresses the major types of gene mutations in integrated HBV genome in human patients and also presents reliable evidence of possible genetic transmission of hepatitis B.     

乙型肝炎病毒B2和C2亚型中SSRs的比较分析

乙型肝炎病毒(hepatitis B virus,HBV)属于嗜肝DNA病毒科(hepadnaviridae),它分布在世界各地并严重危害人类的健康。本文从NCBI的GenBank中下载了106条B2亚型和130条C2亚型的基因组全长序列,以下载的数据为材料,分析简单重复序列(simple sequence repeats,SSRs)在B2和C2亚型基因组序列中的分布情况。结果显示,简单重复序列的重复次数比较少;二型简单重复序列在研究的五种简单重复序列类型中占绝对优势;这可能与B2和C2亚型基因组序列有较高的突变率有关。同时还发现最普遍的SSRs、序列间SSRs的平均相对丰度和平均相对密度在B2和C2亚型中分布情况相似。     

HBsAg定量检测在慢性乙型肝炎自然病程中的意义

目的 研究慢性乙型肝炎病毒(HBV)感染自然病程中HBsAg水平变化情况以及和HBV DNA的关系.方法 将183名HBV感染者按感染的不同阶段分为免疫耐受期(IT)42例,免疫清除期(IC)46例,非/低水平复制期(LR)57例,和再活动期(HBeAg阴性肝炎组ENH)38例,回顾性分析每个阶段血清中HBsAg和HBV DNA水平以及两者之间的关系.结果 HBsAg含量分别为IT期最高[(3.81±0.46) Ig IU/mL],其次为IC期[(3.41±0.77) Ig IU/mL],最低是LR期[(2.87±0.61)Ig0.61IU/mL],每期之间HBsAg含量差异有统计学意义(P＜0.05),ENH组HBsAg含量又有所升高[(3.31 ±0.78)Ig IU/mL].IT期HBsAg与HBV DNA无相关(r=0.231,P＞0.05),IC期HBsAg与HBV DNA呈正相关(r=0.612,P＜0.05),ENH期两者无相关(r=0.189,P＞0.05);不同时期HBsAg含量与ALT均无相关性.结论 HBV感染的不同阶段HBsAg水平有差异,IT期最高,进入IC期后开始下降,在LR期降至最低,ENH期再度升高,HBsAg与HBV DNA仅在IC期有相关性,血清HBsAg的表达与不同的免疫背景有关.