In situ hybridization for the detection of hepatitis C virus RNA in human liver tissue

In situ hybridization (ISH) enables visualization of specific nucleic acid in morphologically preserved cells and tissue sections. Detection of the HCV genomes in clinical specimens is useful for differential diagnosis, particularly between recurrent HCV infection and acute cellular rejection in transplant specimens. We optimized an ISH protocol that demonstrated sensitivity and specificity for detecting genomic and replicative form of HCV RNA in tissue biopsies. Digoxigenin (Dig)‐labelled sense and anti‐sense riboprobes were synthesized using a plasmid containing a fragment of the highly conserved HCV noncoding region as a template. The efficiency of the Dig‐labelled riboprobes in detecting genomic and replicative‐intermediate HCV RNA was analysed in 30 liver biopsies from patients infected or uninfected with HCV in a blinded study. A Huh7 cell line that stably replicates genome‐length HCV RNA was developed to be used as a positive control. Negative control riboprobes were used in parallel to evaluate and control for background staining. The anti‐sense probe detected HCV RNA in 20/21 specimens from HCV‐infected liver tissues obtained from patients and in 0/9 samples from patients with non‐HCV‐related liver diseases, resulting in a sensitivity and specificity of 95% and 100%, respectively. HCV genomic RNA was variably distributed in tissue sections and was located primarily in the perinuclear regions in hepatocytes. Detection of HCV RNA by our optimized ISH protocol appears to be a sensitive and specific method when processing clinical specimens. It may also be revealing when exploring the pathophysiology of HCV infection by verifying the presence of viral genetic material within heptocytes and other cellular elements of diseased liver tissue. This methodology might also evaluate the response to antiviral therapies by demonstrating the absence or alteration of genetic material in clinical specimens from successfully treated patients.     

Detection of hepatitis C virus antibodies and hepatitis C virus RNA in patients with alcoholic liver disease.

The relationship between alcoholic liver disease and hepatitis C virus was studied in 80 patients by searching for hepatitis C virus RNA with the polymerase chain reaction and by measuring hepatitis C virus antibodies. By C-100 enzyme-linked immunosorbent assay, hepatitis C virus antibodies were found in 2 of 10 patients with fibrosteatosis, 8 of 20 patients with alcoholic hepatitis, 14 of 19 patients with chronic hepatitis and 19 of 31 patients with cirrhosis. Percentages of patients with antibodies found by C-100 radioimmunoassay and by enzyme-linked immunosorbent assay based on sequence peptide 42 were lower; of the 16 patients with a low titer by C-100 enzyme-linked immunosorbent assay, 10 were negative by radioimmunoassay and 6 were negative by sequence peptide 42. By a second-generation recombinant immunoblot assay, hepatitis C virus antibodies were found in 1 of 10 patients with fibrosteatosis, 2 of 20 patients with alcoholic hepatitis, 15 of 19 patients with chronic hepatitis and 18 of 31 patients with cirrhosis. Hepatitis C virus RNA was found in 1 of 10 patients with fibrosteatosis, 3 of 20 patients with alcoholic hepatitis, 13 of 19 patients with chronic hepatitis and 20 of 31 patients with cirrhosis. Of the 37 patients with hepatitis C virus RNA, 31 had antibodies by C-100 enzyme-linked immunosorbent assay (25 patients at a high titer [cut-off index greater than 6]), and 31 had antibodies by second-generation recombinant immunoblot assay. Patients with cirrhosis and hepatitis C virus RNA had higher ALT activity than such patients without hepatitis C virus RNA (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between fatty liver, alanine aminotransferase, HBsAg and hepatitis C virus

A community health survey of 923 residents aged 30 years or more was performed in Putai Township of Taiwan. To elucidate the relationships between hepatitis C virus (HCV) and surrogate tests for non-A, non-B hepatitis in hyperendemic areas of hepatitis B virus (HBV) serum levels of alanine aminotransferase (ALT), triglycerides, cholesterol, hepatitis B surface antigen (HBsAg) and antibody to HCV (anti-HCV) were examined. Glucose tolerance tests and the history of diabetes treatment were used to define the diabetes status. Fatty liver was diagnosed by sonography. The prevalence of anti-HCV was 2.6% (95% confidence interval, 1.6-3.6%). Elevated ALT and fatty liver were significantly associated with anti-HCV in univariate analysis. Anti-HCV was not an associated factor for fatty liver after adjusting for serum triglycerides and cholesterol, sex, body mass index and diabetes status through multiple logistic regression. However elevated ALT was still associated with anti-HCV after adjusting for serum triglycerides, sex, body mass index, HBsAg and age through multiple linear regression. The anti-HCV prevalence was similar between HBsAg-positive and negative subjects. Aggregation of HCV infection was found among spouses. It was concluded that elevated ALT and intimate contact with HCV carriers might be associated factors for HCV infection, and that HBV infection and fatty liver were not related to HCV infection in Taiwan.     

HIV and hepatitis C virus RNA in seronegative organ and tissue donors

The objective of our study was to determine whether nucleic acid testing could detect HIV RNA or hepatitis C virus (HCV) RNA in a large series of seronegative organ and tissue donors, and whether this technique should be routinely used to improve viral safety of grafts. We studied 2236 organ donors, 636 tissue donors, and 177 cornea donors. We identified five HCV RNA-positive donors in 2119 HCV-seronegative organ donors, and one HCV RNA-positive donor in 631 HCV-seronegative tissue donors. No HIV-seronegative, HIV RNA-positive donor was identified. Our data suggest that routine nucleic acid testing of organ and tissue donors might increase viral safety in transplantation.     

丙型肝炎肝组织和血清HCV RNA含量及其关系的研究

目的 了解丙型肝炎肝组织和血清中丙型肝炎病毒（ＨＣＶ）ＲＮＡ的含量及其相互关系。方法 采用荧光定量聚合酶链反应（ＰＣＲ）检测２４例慢性丙型肝炎患者肝组织和血清ＨＣＶＲＮＡ含量。结果 肝组织中ＨＣＶＲＮＡ含量（１０＾８至１０＾１２拷贝／克）与血清病毒含量（１０＾５至１０＾９．２拷贝／毫升）呈显著正相关（Ｐ〈０．０１）,每克感染肝组织ＨＣＶＲＮＡ含量高于每毫升血清的１０＾２至１０＾４拷贝。慢性活性肝炎     

Relationship between gastric localization of hepatitis C virus and mucosa-associated lymphoid tissue in Helicobacter pylori infection

BACKGROUND: Hepatitis C virus (HCV) has been localized in several extra-hepatic sites. Recent evidence suggests that the stomach can harbour HCV. We therefore evaluated the prevalence of gastric localization of HCV and its possible relationship with the chronic inflammatory response to Helicobacter pylori infection. METHODS: Sixty patients with HCV infection (group A) and 60 subjects without HCV infection (control group), who underwent upper endoscopy for dyspeptic symptoms, were consecutively enrolled. Biopsy specimens of gastric mucosa obtained from each patient were assessed for H. pylori and chronic inflammatory infiltrates (classified as mild, moderate or marked). Furthermore, polymerase chain reaction (PCR) analyses were performed on the gastric biopsies to detect HCV and immunoglobulin heavy-chain (IgH) gene rearrangements of mucosal B cells. RESULTS: In group A, 24 of 36 patients with H. pylori infection and 6 of 24 without H. pylori hosted HCV in their stomach (P = 0.0017). In these subjects, the presence of both HCV in the gastric mucosa and H. pylori was significantly associated with marked or moderate inflammatory infiltrates. Oligoclonal IgH gene rearrangements were detected in three group A patients who harboured both H. pylori and HCV in their stomach. In the control group, PCR analyses failed to find HCV in the gastric mucosa, and polyclonal patterns were detected in all individuals. CONCLUSIONS: HCV is frequently localized in the stomach and is associated with the chronic lymphocytic inflammatory response to H. pylori. H. pylori and HCV, when both present, may favour the selection of clonal B cells.     

Relationship between genotypes of hepatitis C virus and histopathological manifestations in chronic hepatitis C patients

OBJECTIVE: The aim of this study was to assess the relationship between HCV genotype and histological liver injury. DESIGN: Prospective study on a cohort of patients with biopsy proven chronic hepatitis C. SETTING: University medical centre. PARTICIPANTS: Enrolled were 324 consecutive patients (male 197, median age 52 years, range 19-68; chronic hepatitis, 224; cirrhosis, 100). METHODS: HCV genotype was determined by the INNO LiPA assay and HCV RNA levels by the bDNA assay. The histological features were scored according to the histology activity index. RESULTS: The distribution of HCV genotypes was 1a, 4.6%; 1b, 52.4%; 2a/c, 27%; 3a, 8%; 4, 2%; mixed, 6%. Serum HCV RNA levels were similar for all genotypes. There was no difference in the distribution of HCV genotypes between patients with chronic hepatitis and those with cirrhosis. Patients with genotype 1b and those with type 2a/c showed a similar prevalence of cases of cirrhosis (33% versus 31%, respectively). In addition, in a subgroup of 102 patients with an established date of infection, the progression to cirrhosis occurred with a similar length of time for HCV type 1b and 2a/c (median 16 versus 15 years, respectively). Patients with HCV genotype 2a/c or mixed genotype showed a higher histology activity index than those with type 1b (P< 0.01), whereas there was no difference in the fibrosis score for the different genotypes. Patients with genotype 3a showed a significantly higher prevalence of steatosis compared to those infected with other genotypes. Alanine aminotransferase (ALT) values were higher in patients with HCV type 2a/c, 3a and mixed genotype than those with type 1 (P < 0.002). CONCLUSIONS: The data indicate that there is no association between a particular HCV genotype and the progression to cirrhosis, and that specific genotypes are associated with distinct histopathological and biochemical manifestations although none of them is correlated with an increase of the fibrosis stage.     

Detection of hepatitis C virus (HCV) RNA sequences in liver tissue by in situ hybridization.

In situ hybridization was used to identify the cell types infected by hepatitis C virus (HCV) in the liver. Using an antisense HCV-RNA probe from the 5' non-coding region, HCV-RNAs molecules were detected in liver sections of 4/11 patients with chronic hepatitis C. These 4 positive subjects were also infected by HIV. HCV-RNA-positive strands were detected in scattered hepatocytes as well as in cells identified as mononuclear cells within the inflammatory infiltrates. HCV-RNA negative strands, likely replicative intermediates, were also detected in these cells. This study therefore indicates that replication of HCV may occur in both hepatocytes and mononuclear liver cells.     

Detection and partial sequencing of hepatitis C virus RNA in the liver.

To detect hepatitis C virus RNA, total RNA was extracted from liver tissue, reverse transcribed to complementary DNA, and amplified by polymerase chain reaction. The reaction products were analyzed by ethidium bromide staining in acrylamide gel and hybridization with a radiolabeled probe. Hepatitis C virus RNA was thereby detected in 17 of 27 (63%) liver tissue specimens obtained from patients with non-A, non-B chronic liver diseases. Of these 27 patients, viral RNA was detected in 12 of 17 (71%) liver tissues from anti-hepatitis C virus-positive patients and in 5 of 10 (50%) liver tissues from anti-hepatitis C virus-negative patients. Direct sequencing of amplified complementary DNA (35 nucleotides) of the 17 RNA-positive samples showed only 66% to 77% homology to the reported hepatitis C virus complementary DNA sequence. These results indicate that the majority of anti-hepatitis C virus-positive patients are currently infected with hepatitis C virus, and some of the anti-hepatitis C virus-negative patients with non-A, non-B hepatitis are harboring hepatitis C virus in the liver. Detection of hepatitis C virus RNA appears to provide a useful indicator in the study of hepatitis C virus infection.     

Possible association between serum GB virus C RNA level and disease activity in fulminant hepatitis type G.

BACKGROUND/AIMS: Whether GB virus C causes serious liver diseases remains controversial. The aim of the present study was to determine whether there is an etiological relationship between GB virus C and fulminant hepatitis. METHODS: The level of GB virus C RNA in the sera of three patients with fulminant hepatitis was quantitatively determined using the newly developed real-time detection polymerase chain reaction method, which is based on Taq Man chemistry. The NS 3 region of the viral genome isolated from the sera was sequenced at several time points to confirm whether the same virus was responsible for fulminant hepatitis during the patients' clinical courses. RESULTS: The sensitivity of the PCR was comparable to that of nested PCR and a linear relationship between RNA copy number and threshold cycle was observed for 10(1) and 10(6) RNA copies/ml (r = 0.99). The serum level of GB virus C RNA closely paralleled that of ALT in all patients. Sequence analysis of the NS3 region isolated from the patients' sera revealed that the same GB virus C strain infected the patients during their entire clinical courses, despite plasma exchange therapy. CONCLUSIONS: These observations suggest that GB virus C may be etiologically associated with fulminant hepatic failure, and is not merely an inactive bystander introduced by therapeutic plasma exchange.     

慢性丙型肝炎患者HCV基因型与甲状腺功能的关系分析

目的初步探讨慢性丙型肝炎(CHC)2a、1b基因型与甲状腺激素水平及甲状腺自身抗体的关系。方法收集2013年10月-2014年12月在西安交通大学医学院第二附属医院就诊及住院的CHC患者196例,采用RT-PCR法对患者进行HCV基因型的检测,使用化学发光免疫分析法检测甲状腺激素水平及甲状腺自身抗体水平,并对基因型与甲状腺功能的关系进行分析。计量资料组间比较采用t检验,相关危险因素采用Logistic回归分析。结果 196例丙型肝炎患者,HCV-2a型占57.7%(113例),HCV-1b型占42.3%(83例)。对HCV-2a型、HCV-1b型患者甲状腺激素水平、甲状腺自身抗体水平比较,差异均无统计学意义(P值均0.05);其中92例HCV患者使用干扰素治疗,甲状腺激素水平、甲状腺自身抗体水平在HCV-2a型、HCV-1b型中差异均无统计学意义(P值均0.05)。多元Logistic回归分析显示,基因分型不是甲状腺自身抗体异常的危险因素(比值比为2.012,P0.05)。结论 CHC基因分型与甲状腺功能无明显内在联系,基因分型对甲状腺功能无明显影响。     

Hepatitis C virus RNA quantitation in hepatic veins and peripheral blood in patients with liver cirrhosis: evidence for low level intrahepatic hepatitis C virus replication in advanced liver disease

BACKGROUND: Very few data exist concerning the level of hepatitis C virus replication within the cirrhotic liver and its relationship to disease severity and progression. AIMS: To quantitate hepatitis C virus RNA in hepatic vein blood and peripheral blood in patients with cirrhosis, to evaluate the correlation of hepatitis C virus levels in paired blood samples, and to compare the results with clinical features. PATIENTS: A series of 25 patients with hepatitis C virus-related liver cirrhosis undergoing hepatic vein catheterization were studied: 11 belonged to Child Pugh class A, 8 to class B and 6 to class C. RESULTS: Hepatitis C virus RNA levels did not differ between hepatic vein blood and peripheral blood (p = 0.26), despite a trend towards higher peripheral hepatitis C virus RNA levels. Hepatitis C virus RNA levels did not differ between patients with genotype 1b and non-1b either in hepatic veins or peripheral blood. Hepatitis C virus loads varied according to the severity of cirrhosis. The patients with more severe liver disease had significantly lower RNA titres than those with less advanced cirrhosis, both in hepatic veins (p = 0.002) and peripheral blood (p = 0.004). No differences in hepatitis C virus load were observed between patients in Child Pugh classes B and C. CONCLUSIONS: The present data show that in patients with cirrhosis hepatitis C virus RNA concentrations do not differ between hepatic blood and peripheral blood and, furthermore, confirm that hepatitis C virus replication is reduced in patients with advanced cirrhosis, compared with patients with less severe liver disease. These findings might indicate that patients with liver cirrhosis maintain an efficient intrahepatic hepatitis C virus replication even in end-stage disease, although hepatitis C virus viraemia decreases according to the severity of liver disease.     

Detection of hepatitis C virus RNA in the liver by in situ hybridization

ABSTRACT— To examine HCV infection histologically, we attempted nonradioactive in situ hybridization of HCV-RNA in the liver. We amplified cDNA probe (360 base pairs) by PCR using the primers deduced from the core region of the HCV genome. The probe was labelled with digoxigenin by PCR and used for in situ hybridization on paraformaldehyde-fixed frozen liver sections. The hybrids were visualized immunohistochemically with alkaline-phosphatase-conjugated anti-digoxigenin and alkaline-phosphatase substrates. HCV-RNA-cDNA hybrids were detected in 21 of 24 patients with positive serum HCV markers, whereas there were no positive signals in the liver of 12 cases without HCV infection. The signal intensity of HCV-RNA-cDNA hybrids was abolished after RNase treatment. Various other specificity experiments also verified specific hybridization of HCV-RNA-cDNA. HCV-RNA was visualized in liver cells and most of them were regarded as hepatocytes from their characteristic features. The infected hepatocytes were frequently associated with mononuclear cell infiltration. Hepatocytes positive for HCV-RNA were sometimes binuclear and distributed in various patterns among cases tested. The present in situ hybridization of HCV RNA is highly sensitive and specific and the results suggest the host immune response to HCV-infected cells.     

Translation of hepatitis C virus RNA

The 341-nucleotide 5' non-translated region is the most conserved part of the hepatitis C virus (HCV) genome. It contains a highly structured internal ribosomal entry site (IRES) that mediates cap-independent initiation of translation of the viral polyprotein by a mechanism that is unprecedented in eukaryotes. The first step in translation initiation is assembly of eukaryotic initiation factor (eIF) 3, eIF2, GTP, initiator tRNA and a 40S ribosomal subunit into a 43S preinitiation complex. The HCV IRES recruits this complex and directs its precise attachment at the initiation codon to form a 48S complex in a process that does not involve eIFs 4A, 4B or 4F. The IRES contains sites that bind independently with the eIF3 and 40S subunit components of 43S complexes, and structural determinants that ensure the correct spatial orientation of these binding sites so that the 48S complex assembles precisely at the initiation codon.     

Translation of hepatitis C virus RNA

The 341-nucleotide 5' non-translated region is the most conserved part of the hepatitis C virus (HCV) genome. It contains a highly structured internal ribosomal entry site (IRES) that mediates cap-independent initiation of translation of the viral polyprotein by a mechanism that is unprecedented in eukaryotes. The first step in translation initiation is assembly of eukaryotic initiation factor (eIF) 3, eIF2, GTP, initiator tRNA and a 40S ribosomal subunit into a 43S preinitiation complex. The HCV IRES recruits this complex and directs its precise attachment at the initiation codon to form a 48S complex in a process that does not involve eIFs 4A, 4B or 4F. The IRES contains sites that bind independently with the eIF3 and 40S subunit components of 43S complexes, and structural determinants that ensure the correct spatial orientation of these binding sites so that the 48S complex assembles precisely at the initiation codon.