Processing of hepatitis C virus core protein is regulated by its C-terminal sequence

Polyprotein processing of plus-strand RNA viruses is important in the regulation of gene production and replication. The core protein of hepatitis C virus (HCV), constructing the viral particle, is processed from its precursor polyprotein and observed as two forms, p23 and p21. Production of p21 by cleavage at the C-terminus of p23 is considered crucial to viral assembly and replication. In this study, this processing step was compared between clones isolated from two patients with fulminant hepatitis and from five patients with chronic hepatitis by an in vitro translation assay and cell transfection assay. The p21 core protein was predominant from the clone isolated from one of the fulminant hepatitis patient (p21 core protein production was 65.98%), while p23 was abundant with clones from five chronic hepatitis patients (p21 core protein production was 7.11+/-1.62%) and clone from another fulminant hepatitis patient (p21 core protein production was 13.36%). Investigations with chimeric and mutation-introduced constructs revealed that four amino acid residues in the C-terminus of the core region are responsible for this difference. The data suggest that core protein processing is regulated by C-terminus mutations.     

IL-33 protects murine viral fulminant hepatitis by targeting coagulation hallmark protein FGL2/fibroleukin expression

Fulminant hepatitis (FH) is characterized by rapid liver failure and high mortality. The pathogenesis of viral FH includes virus-induced immune activation, inflammation, and subsequent hepatic apoptosis and necrosis. However, the mechanisms that underlie FH progression are unclear. IL-33 is a member of the IL-1-related cytokines, considered to be an “alarmin” that participates in various diseases, but its precise role in the coagulation of FH is not very clear. In our study, we found that IL-33 is significantly elevated in mice infected with murine hepatitis virus strain 3 (MHV-3). This is accompanied by an increase in pro-coagulant fibrinogen-like protein 2 (FGL2) in the liver. Previous studies have suggested that an increase in FGL2 is diagnostic of FH and liver necrosis, and animals with no FGL2 had better survivorship during FH. Our studies showed that IL-33 administration in a MHV-3 infection promoted survival during FH, with a significant reduction in FGL2 expression and liver inflammation. In vitro IL-33 treatment abrogated MHV-3 and IFN-γ induced FGL2 expression in RAW264.7 and THP-1 cells, respectively. In conclusion, our research suggests that IL-33 protects against viral fulminant hepatitis in mice by antagonizing expression of the pro-coagulant protein FGL2.     

Acute hepatic cell necrosis experimentally produced by viral agents in rabbits.

Acute and extensive hepatic cell necrosis was produced experimentally in rabbits by means of the Shwartzman mechanism using adeno- and hepatitis B viruses. The change that occurred in the liver was quite severe, namely, areas of hemorrhagic necrosis of various sizes in gross appearance and lytic-coagulative necrosis with hemorrhage and leukocyte-mononuclear cell infiltration histologically. Thrombi formation was noted in and around the necrotic areas, and it was not unusual to see necrosis of an entire lobe. This seems to be a model, to some extent, for human fulminant hepatitis caused by hepatitis virus infection, and suggested that some nonspecific reaction such as intravascular clotting may also play an important role in causing or complicating acute, severe, and extensive necrosis of the liver in human cases. Heparin administration very effectively prevented such hepatic necrosis, which supports the view that the change we observed in the liver was really the Schwartzman reaction; further, it is reminiscent of the fact that heparin administration is sometimes effective in fulminant hepatitis treatment if given at te appropriate stage of the disease.     

重型肝炎血清中病毒及肿瘤坏死因子作用的研究

用酶联免疫吸附试验（ＥＬＩＳＡ）方法对４５例重型肝炎，其中急性重型肝炎３例，亚急性重型肝炎２例，慢性重型肝炎４０例，病学及血清肿瘤坏死因子（ＴＮＦ）水平检测分析。认为乙型肝炎病毒（ＨＢＶ）感染是北京地区重型肝炎的主要病原，可达９７．５％；不同肝炎病毒重叠感染是重型肝炎发生的关键，占８０％，其死亡率达５３．３％。不同肝炎病毒重叠感染及ＴＮＦ水平是影响重型肝炎预后的最重要因素。上述因素的检测分析，对认     

Genetic analysis of HAV strains in Tunisia reveals two new antigenic variants

To evaluate the genetic variability of hepatitis A virus (HAV) isolates in Tunisia, serum samples were collected from 99 patients in different Tunisian areas in 2003 containing 92 cases with acute hepatitis, five with severe acute hepatitis and two with fulminant hepatitis. The entire VP1 gene was amplified and sequenced. Sequences were then aligned and a phylogenetic analysis was performed. Additionally, the amino acid (aa) sequence of the VP1 was determined. The analysis of Tunisian HAV isolates revealed that all the isolates were sub-genotype IA with 96.4%–99.8% of identity and showed the emergence of two novel antigenic variants. The Tun31-03 antigenic variant, with a 38 aa deletion containing Met156, Val171, Leu174 and Ala176 and located between 150 and 187 aa of the VP1 protein where neutralization escape mutations, was found. The second antigenic variant, Tun36-03, was isolated from a patient with fulminant hepatitis and presented a substitution of Thr by Pro at position 10 of the VP1 protein. This amino acid is located in a peptide presenting an antigenically reactive epitope of the VP1 protein. This substitution has never been described previously.     

Detection of mutations in hepatitis B virus enhancer 2/core promoter and X protein regions in patients with fatal hepatitis B virus infection

The enhancer 2/core promoter and the X protein regions located upstream of the precore and core regions in hepatitis B virus regulate expression of core/e antigen peptides. Mutations in the precore and core regions have been reported to be associated closely with the severity of type B hepatitis, and regions regulating expression of these peptides may also be involved in severe liver damage. Mutations were examined in regions that may be related to fatal liver diseases. Nucleotide sequences and deduced amino acid sequences from 20 patients with fatal type B hepatitis (12 with fulminant hepatitis and 8 with severe exacerbation) and 10 patients with self-limited acute hepatitis were analyzed. There were 50 nucleotide alterations in the enhancer 2/core promoter region of virus from 12 patients with fulminant hepatitis (average 4.1/case), 37 alterations in 8 patients with severe exacerbation (4.6/case), and 10 mutations in 10 cases of acute hepatitis (1.0/case). The numbers of amino acid mutations in X protein were 53 in 12 cases of fulminant hepatitis (4.4/case), 27 in 8 cases of severe exacerbation (3.3/case), and 9 in 10 cases of acute hepatitis (0.9/case). In fatal cases, approximately 50% of the nucleotide mutations were located within the region spanning nucleotides 1741-1777 (14.2% of the enhancer 2/core promoter region) and 30% of the amino acid mutations in X protein were located within the region containing codons 122-132 (7.1% of X protein). In addition to mutations in the precore and core regions, mutations in the enhancer 2/core promoter and the X protein regions may be associated with the pathogenesis of fatal B hepatitis infection.     

Bcl-2 和Bax蛋白在慢性病毒性肝炎肝组织中的表达

目的研究细胞凋亡相关的Ｂｃｌ－２和Ｂａｘ蛋白在慢性病毒性肝炎（ＣＨ）肝组织中表达的情况与该病发生发展的关系。方法对３５例不同病变程度的ＣＨ肝组织用免疫组化法检测Ｂｃｌ－２和Ｂａｘ的表达及定位。结果Ｂｃ１－２和Ｂａｘ分布基本一致，轻、中度ＣＨ主要见于碎屑样坏死（ＰＮ）区中的单个核细胞（ＭＮ），邻近ＰＮ区边缘的肝细胞浆中也有强阳性表达。ＨＥ染色观察，这些Ｂｃｌ－２和Ｂａｘ阳性肝细胞多呈重度水样变性或嗜酸性变。在重度ＣＨ肝组织中Ｂｃｌ－２和Ｂａｘ阳性的ＭＮ主要分布于重度ＰＮ区和桥接坏死区，周围部分肝细胞Ｂａｘ阳性，而Ｂｃｌ－２为阴性。在慢性重型肝炎，Ｂｃｌ－２和Ｂａｘ除在亚大块肝细胞坏死区浸润的ＭＮ胞浆内表达外，残存肝细胞亦呈阳性表达。结论Ｂｃｌ－２和Ｂａｘ在肝组织中的表达可能不仅与ＣＨ的细胞凋亡有密切关系，而且与慢性重型肝炎的发生发展有关     

Hepatitis E and pregnancy: current state

Hepatitis E virus (HEV) is responsible for more than 50% of acute viral hepatitis cases in endemic countries. Approximately 2 billion individuals live in hepatitis E–endemic areas and, therefore, are at risk of infection. According to World Health Organization, HEV causes about 20.1 million infections and 70 000 deaths every year. In developing countries with poor sanitation, this disease is transmitted through contaminated water and is associated with large outbreaks, affecting hundreds or thousands of people. In developed countries, autochthonous cases of HEV have been increasingly recognized in the past several years. Hepatitis E virus typically causes an acute, self‐limiting illness similar to other acute viral hepatitis, such as hepatitis A or B, with about 0.2% to 1% mortality rate in the general population. However, the course of hepatitis E in pregnancy is different than the mild self‐constraining infection described in other populations. During pregnancy, HEV infection can take a fulminant course, resulting in fulminant hepatic failure, membrane rupture, spontaneous abortions, and stillbirths. Studies from various developing countries have shown a high incidence of HEV infection in pregnancy with a significant proportion of pregnant women progressing to fulminant hepatitis with a fatality rate of up to 30%. The present review will highlight new aspects of the HEV infection and pregnancy.     

Herpesvirus saimiri has a gene specifying a homologue of the cellular membrane glycoprotein CD59.

Herpesvirus saimiri (HSV) is a T-lymphotropic tumor virus that causes fulminant lymphomas and leukemias in various New World primates other than its natural host, the squirrel monkey (Saimiri sciureus). In the course of completing the nucleotide sequence of its genome, we identified an open reading frame of 363 nucleotides, designated HVS-15, that has no detectable homology to any other viral sequences to date. HVS-15 encodes a 121-amino-acid protein which shows significant similarities to human CD59, a phosphatidyl-inositol-glycan-anchored glycoprotein involved in T-cell activation and restriction of complement-mediated lysis. The predicted HVS-15 gene product is more similar to human CD59 than to the related murine Ly-6 antigens. A nucleotide sequence identity of 64% was found between HVS-15 and the CD59 reading frame, and a 48% identity exists between the corresponding protein sequences. The comparison of the amino acid sequences revealed a number of conserved structural features such as a similar pattern of hydrophobic termini and an identical cysteine skeleton.     

Virological markers and antibody responses in fulminant viral hepatitis

Clinical profiles, serological markers, and antibody responses to antigens of hepatitis B virus (HBV) were studied in patients with fulminant viral hepatitis. Whereas hepatitis A and B were found to be uncommon causes (6.9% and 12.2%, respectively), non-A, non-B (NANB) hepatitis was found to be the most common cause of fulminant hepatitis (80.9%). As against this, the incidence of hepatitis B and NANB hepatitis was very similar in nonfulminant acute viral hepatitis in adults (41.2% and 51.9%, respectively). Pregnancy with labour was an important precipitating factor for development of fulminant hepatitis of the NANB type only; 32% of fulminant NANB hepatitis patients were pregnant women and 22.6% had a history of labour preceding hepatic coma. Only 0.8% of nonfulminant NANB hepatitis cases were pregnant women. Another major precipitating factor for the development of the fulminant form of NANB hepatitis was concomitant chronic HBV carrier state. A total of 38.6% of fulminant NANB hepatitis patients were HBV carriers, whereas only 19.2% of nonfulminant acute NANB hepatitis cases were HBV carriers. Sera of 32 chronic HBV carriers with fulminant NANB hepatitis and 10 cases of fulminant hepatitis B were tested for delta antibody, and all were nonreactive. The antibody responses of the fulminant hepatitis B patients to the antigens of HBV were found to be greater compared to those of patients with nonfulminant acute hepatitis B. Antibody responses of chronic HBV carriers with fulminant NANB hepatitis to antigens of HBV were found to be depressed in comparison with those of chronic asymptomatic carriers.     

Role of transforming growth factor beta 1 on hepatic regeneration and apoptosis in liver diseases.

AIMS--To investigate the effects of transforming growth factor beta 1 (TGF-beta 1) on regeneration and induction of apoptosis of liver cell and bile duct in various liver diseases. METHODS--Formalin fixed paraffin wax sections of 18 liver tissue samples were obtained by needle biopsy, surgery, or necropsy; these included six liver cirrhosis, three obstructive jaundice; five fulminant hepatitis, one subacute hepatitis, and three normal liver. Expression of TGF-beta 1, apoptosis related Le(y) antigen, Fas antigen, a receptor for tumour necrosis factor, and biotin nick end labelling with terminal deoxynucleotidyl transferase mediated dUTP (TUNEL) for locating DNA fragmentation, was investigated histochemically. RESULTS--TGF-beta 1 was expressed in areas of atypical bile duct proliferation, where bile duct continuously proliferated from liver cells. In occlusive jaundice and fulminant hepatitis, TUNEL was positive in nuclei and cytoplasm of metaplastic cells which formed incomplete bile ducts, and these cells appeared to extend from TGF-beta 1 expressing liver cells. Fas antigen was found only on the cell membrane of proliferated bile duct in fulminant hepatitis, which differed from TGF-beta 1 and TUNEL positive areas. Le(y) antigen was expressed in liver cell and bile duct at the areas with atypical bile duct proliferation, but its coexpression with TUNEL was rare. CONCLUSIONS--TGF-beta 1 plays a role in the arrest of liver cell regeneration and atypical bile duct proliferation, and in areas of rapidly progressing atypical bile duct proliferation, such as in fulminant hepatitis or bile retention. Apoptosis appears to be induced by TGF-beta 1. This phenomenon may account for the inadequate hepatic regeneration that occurs with liver disease.     

Expression of a new woodchuck IFN-alpha gene by a helper-dependent adenoviral vector in woodchuck hepatitis virus-infected primary hepatocytes.

Recombinant interferon-alpha (rIFN-alpha) is currently used in the treatment of viral hepatitis either alone or in combination with small molecules. However, this treatment is not very efficacious, and more effective protocols are needed. To this end, we have explored the woodchuck hepatitis system, validated as an infection model for vaccination and antiviral studies against human hepatitis B virus (HBV) infection. The lack of a woodchuck IFN-alpha (WoIFN-alpha) homolog has prevented study of viral inhibition, which may be instrumental in understanding the IFN-alpha-induced antiviral pathways responsible for HBV clearance in humans. We have, therefore, cloned two WoIFN-alpha homologs from the woodchuck genome, which showed high similarity to the human IFN-alpha (HuIFN-alpha) gene at both nucleotide and amino acid levels. WoIFN-alpha showed a species-specific activity without any efficacy on human or mouse cells. In agreement with this antiviral activity, induction of Mx protein was observed in woodchuck cells only on WoIFN-alpha treatment. The antiviral efficacy of a WoIFN-alpha gene transfer was explored using a helper-dependent adenoviral (Ad) vector (HD-WoIFN) as a delivery vehicle. This treatment resulted in the reduction of woodchuck hepatitis viral proteins in primary hepatocytes from chronically woodchuck hepatitis virus (WHV)-infected woodchucks.     

Recent high incidence of fulminant hepatitis in Samara, Russia: molecular analysis of prevailing hepatitis B and D virus strains

Until 1991, the Russian city of Samara was largely isolated from other parts of Russia and the rest of the world. Very recently, Samara has seen an alarming increase in the incidence of hepatitis. The proportion of fulminant cases is unusually high. We wanted to assess the roles of hepatitis B virus (HBV) and hepatitis D virus (HDV) in acute viral hepatitis in this region by analyzing the prevailing strains of both and by determining their genotypes and possible origin. Serum samples were screened for different serological markers and by PCR followed by direct sequencing. Of the 94 HBV-positive samples (80% of which were acute infections), 37 (39%) were also HDV positive. Sixty-seven percent of the patients had anti-HCV antibodies. Twenty-five percent of all patients in the study had fulminant hepatitis. Statistically significant sex differences were found among fulminant cases. For HBV, the core promoter sequences of 62 strains were determined and all but one were found to be of genotype D. None of these had any deletions. Only one strain, from a patient with fulminant fatal hepatitis, showed multiple mutations. The pre-S2 region sequences of 31 HBV strains were also compared. Phylogenetically, these fell into two distinct groups within genotype D, suggesting different origins. For HDV, part of the region encoding the delta-antigen was sequenced from four strains. All proved to be of genotype I and were similar to Far Eastern and Eastern European strains. The contribution of intravenous drug use to the sharp increase in viral hepatitis in this unique setting is discussed.     

Morphologic and immunohistochemical features of fulminant delta hepatitis

Recent studies have indicated that some cases of fulminant hepatitis that were ostensibly type B were actually acute viral hepatitis, types B and delta, or persistent hepatitis, type B with superimposed acute delta infection. Histologic characterization of the various types has not yet been undertaken. Within ten to 37 days of the onset of initial symptoms, liver tissues from a total of 16 patients who had acute delta hepatitis with fulminant clinical courses were examined. Tissues were from biopsies in nine cases and from autopsies in seven. Based on the serologic absence of IgM hepatitis B core antibody, chronic forms of hepatitis B with acute delta infection were diagnosed in five of the 16 patients. The underlying liver disease in three of these five patients was identifiable as chronic active hepatitis with fibrosis, and that in the others was apparently persistent hepatitis B. Coded liver tissues from these seven autopsies were evaluated without the knowledge of serologic diagnosis for qualitative and quantitative light microscopic features, together with autopsy liver tissues from five patients with fulminant B hepatitis without serologic evidence of delta infection and five patients with fulminant non-A, non-B hepatitis. No specific features that would allow discrimination among these three types of hepatitis were found.     

Analysis of the full-length genome of genotype 4 hepatitis E virus isolates from patients with fulminant or acute self-limited hepatitis E

It was suggested that hepatitis E virus (HEV) genotype 4 is associated more closely with the severity of hepatitis E than genotype 3, although the virological basis remains unknown. The aim of this study was to examine whether genomic differences among genotype 4 HEVs are responsible for the development of fulminant hepatitis. Full-length sequences of genotype 4 HEVs from three patients with fulminant hepatitis and six patients with acute self-limited hepatitis were determined. The sequences were analyzed with those of 13 genotype 4 HEV isolates whose entire nucleotide sequence is known. Analysis of 22 full-length sequences (fulminant hepatitis, 5; acute hepatitis, 17) revealed that C at nt 1816 and U at nt 3148 (U3148), both of which do not change the amino acid sequences, were significantly associated with fulminant hepatitis (P = 0.0489, respectively). When partial nucleotide sequences containing nt 1816 or nt 3148 were determined in 16 additional HEV isolates of genotype 4, a closer association between U3148 and fulminant hepatitis (P = 0.0018) was observed. The comparison of 86 HEV isolates of all four genotypes showed that U3148 had a stronger association with fulminant hepatitis than other nucleotides at nt 3148 (P = 0.0006). Patients infected with HEV with U3148 had a significantly lower value of the lowest prothrombin activity (P = 0.0293). Nt 3148 is located within the RNA helicase domain, and 22-nt sequence including nt 3148 was well conserved among all genotypes. A silent substitution of U3148 in HEV may be associated with the development of fulminant hepatitis. Further studies are needed to clarify the underlying mechanism.     

Cloning of Tabby, the murine homolog of the human EDA gene: evidence for a membrane-associated protein with a short collagenous domain

X-Linked hypohidrotic ectodermal dysplasia (XLHED) is a human congenital disorder resulting in abnormal tooth, hair and sweat gland development. A candidate gene for the disorder has been cloned, but the function and full size of its putative protein product is unclear. We have identified a candidate cDNA for the mouse Tabby gene (Ta), which, based on phenotype and syntenic mapping, is postulated to represent the analogous murine disorder. Mutations have been identified in three different Ta alleles and Northern analysis indicates that the gene is expressed at increasing levels during embryogenesis (11-17 days p.c.), the period when affected structures develop. The putative protein product encoded by exon 1 is highly homologous (87% identical) to the predicted EDA protein product (135 amino acids), including the presence of a single transmembrane domain. However, the murine cDNA also encodes an additional 246 amino acids, which contains a short collagenous domain (Gly-X-Y)19. This predicted structure is similar to a number of membrane-associated proteins with either single or multiple collagenous domains in their extracellular C-terminal regions. Since mutations can only be identified in 10-15% of families with XLHED, it is likely that additional homologous exons exist for the human EDA gene. Hybridization of YACs from the EDA region with the Ta cDNA support this hypothesis. The predicted extracellular collagenous domain of this membrane protein may play a key role in epithelial-mesenchymal interactions, defects of which are thought to underlie the Ta/XLHED phenotype.      

Mutations in the precore region of hepatitis B virus DNA in patients with fulminant and severe hepatitis

BACKGROUND. The presence of the hepatitis B e antigen (HBeAg) in serum is known to be a marker of a high degree of viral infectivity. However, fulminant hepatitis may occur in persons who are negative for HBeAg. A single point mutation has been reported to produce a stop codon in the precore region of hepatitis B virus DNA and prevent the formation of the precore protein required to make HBeAg. To determine whether a precore-mutant virus is causally related to severe liver injury, we analyzed the entire precore region in viral strains isolated from patients with fatal cases and uncomplicated cases of hepatitis B. METHODS. Serum was obtained from 9 patients with fatal hepatitis B (5 with fulminant and 4 with severe exacerbations of chronic hepatitis) and 10 patients with acute, self-limited hepatitis B. Serum samples from a sex partner implicated as the source of the virus in one case of fulminant hepatitis were also studied. The 87 nucleotides in the precore region of the hepatitis B virus were amplified by the polymerase chain reaction and then directly sequenced. RESULTS. Of the nine patients with fatal hepatitis, seven had retrievable hepatitis B DNA. In all seven there was a point mutation from G to A at nucleotide 1896 of the precore region, converting tryptophan (TGG) to a stop codon (TAG). In contrast, this mutation was not found in the 10 patients with acute, self-limited hepatitis B. The hepatitis B DNA from the implicated source contained a sequence with the stop-codon mutation that was identical to the sequence in her partner, who had fulminant hepatitis. CONCLUSIONS. The presence of a mutant viral strain is associated with and may be involved in the pathogenesis of fulminant hepatitis B and severe exacerbations of chronic hepatitis B.     

Influences on hepatitis B virus replication by a naturally occurring mutation in the core gene

Little is known about specific naturally occurring mutations of hepatitis B virus (HBV) and underlying mechanisms of their association with fulminant hepatitis. A HBV clone isolated from a patient with fulminant hepatitis was analyzed, and the features of the particular mutations observed around furin cleavage site in core region (A2339G/G2345A) were assessed using an in vitro replication model. The clone belonged to genotype B with precore stop codon mutation (G1896A). Replication efficiency of 1.24-fold HBV genome in Huh-7 cells was increased in the presence of A2339G. Further in vitro studies using furin inhibitor indicated that the effect of the mutation was probably associated with accumulation of the full-length core protein without cleavage by furin-like protease, suggesting that a processing of the core protein might play an important role in regulation of viral replication. In conclusion, the A2339G mutation was considered as one of the viral factors involved in high replication efficiency.