Hepatitis C virus 5' untranslated region variability correlates with treatment outcome.

Hepatitis C virus (HCV) variability affects viral-host interactions. We analysed HCV 5'untranslated region (5'UTR) in sera and peripheral blood mononuclear cells (PBMC) from chronic hepatitis C patients undergoing antiviral treatment. We studied 139 patients treated with pegylated interferon and ribavirin. The primary endpoint was a sustained virological response (SVR) defined as negative HCV RNA level 24 weeks after the end of therapy. 5'UTR was analysed by single-strand conformational polymorphism (SSCP) and sequencing. The pretreatment SSCP pattern in serum and PBMC differed in 26 (18.7%) patients. During therapy, the SSCP pattern remained stable in 65 (60.8%) patients, number of bands declined in 16 (15.0%), and in 18 (16.8%) patients, changes were qualified as 'shift' indicating change in band positions. In univariate analysis, there was a significant (P ≤ 0.05) positive association between SVR and pretreatment serum and PBMC dissimilarities, initial viral load <10(6) IU/mL, IL-28B CC genotype of the rs12979860 single nucleotide polymorphism and change in the SSCP band pattern (either 'shift' or decline) In multivariable analysis, only low initial viral load, IL-28B genotype, and changes in the SSCP band pattern were independent factors associated with SVR. In conclusion, stability of 5'UTR correlated with infection persistence, while changes correlated with SVR.

Seronegative hepatitis C virus infection in patients with lymphoproliferative disorders.

It has been reported that hepatitis C virus (HCV) RNA may be present in serum and/or lymphoid cells in the absence of specific circulating antibodies. The current study analysed seronegative HCV infection in patients with lymphoproliferative disorders. We studied 77 anti-HCV-negative patients (45 male and 32 female, mean age 54.8 ± 14.2 years) with various lymphoproliferative disorders. HCV-RNA was detected by RT-PCR in plasma, peripheral blood mononuclear cells (PBMC) and bone marrow. Furthermore, the presence of viral nonstructural protein 3 (NS3) was determined in PBMC and bone marrow by immunostaining. HCV-RNA was detectable in at least one compartment in 27 (35.1%) patients. Viral RNA was found in bone marrow in 22 patients (28.6%), in PBMC in 13 (16.9%) and in plasma in 10 (13%) patients. In nine patients, evidence of infection was confined to the bone marrow compartment. Viral load in HCV-RNA-positive plasma ranged from 15 to 1.17 × 10(3) IU/mL. NS3 was detected in all but two HCV-RNA-positive bone marrow samples and in all but one HCV-RNA-positive PBMC samples. All 27 HCV-RNA-positive patients remained anti-HCV-negative when tested again after 6-12 months, but only four remained HCV-RNA positive. In conclusion, among patients with lymphoproliferative disorders, HCV can be present in plasma, PBMC and bone marrow despite the lack of circulating specific antibodies. Further studies are required to analyse the phenomenon of seronegative infection and to determine whether such patients are infectious.

Variability of hepatitis C virus hypervariable region 1 (HVR-1) during the early phase of pegylated interferon and ribavirin therapy.

PURPOSE: Genetic variability of hepatitis C virus (HCV) is considered to be an important factor defining viral pathogenesis, persistence and resistance to treatment. The aim of the present study was to characterize HCV genetic heterogeneity within a hypervariable region 1 (HVR-1) before and during the early period of pegylated interferon alfa (PEG-IFN-α) and ribavirin treatment in correlation with treatment outcome. MATERIAL AND METHODS: The study involved 24 patients treated with PEG-IFN-α and ribavirin whose sera were collected before (baseline) and at 7, 14, 21 28 and 56 day of treatment. HCV HVR-1 region was amplified by nested RT- PCR and subjected to SSCP (single strand conformational polymorphism) analysis. SSCP changes of HCV HVR-1 over time in each patient were compared to treatment outcome results. RESULTS: In 2/11 (18%) SVR+ and 8/13 (62%) SVR- treated patients, HVR-1 genetic changes manifested by new SSCP bands (new genetic variants) and were significantly more frequent in nonresponders (P <0.05). CONCLUSIONS: Our results indicate that HCV HVR-1 variability during the early phase of PEG-IFN-α and ribavirin therapy may be predictive of treatment outcome.

Selection of different 5' untranslated region hepatitis C virus variants during post-transfusion and post-transplantation infection.

BACKGROUND: Hepatitis C virus (HCV) translation is initiated in a cap-independent manner by an internal ribosome entry site (IRES) located within the 5' untranslated region (5'UTR). Sequence changes in this region could affect translation efficiency and presumably viral replication. AIM: To determine translation efficiency of 5'UTR variants developing during post-transfusion hepatitis C in two immunocompetent subjects and in two immunosuppressed liver recipients with recurrent HCV. METHODS: Sequential samples were screened for 5'UTR changes by single-strand conformation polymorphism followed by cloning and sequencing whenever band pattern suggested sequence changes. 5'UTR variants were tested for IRES activity using a bicistronic dual luciferase expression plasmid transfected into HepG2 and Huh7 cell-lines. RESULTS: In the transfused patients, translation efficiency of 5'UTR variants from early post-transfusion samples was 5.1- to 13.7-fold higher than that of predominant variants found in late follow-up samples. Post-transplant variants in the other two patients had 2.6- to 5.9-fold higher translation efficiency than those present only in pretransplant samples. CONCLUSION: In the immunocompetent host there may be selection of low translation efficiency HCV variants over the course of infection. However, in immunosuppressed subjects the opposite seems to be true as low translation efficiency variants are superseded by high translation efficiency variants.

Changes in hepatitis C virus population in serum and peripheral blood mononuclear cells in chronically infected patients receiving liver graft from infected donors.

BACKGROUND: We have previously studied hepatitis C (HCV)-infected recipients of livers from HCV-infected donors and found that either the donor's strain or the recipient's strain predominate in serum. The current study was undertaken to determine whether these changes are complete and whether they are reflected in the population of virus associated with peripheral blood mononuclear cells (PBMCs). METHODS: We analyzed HCV ribonucleic acid from sequential serum and PBMC samples from 11 and 8 patients, respectively. The relatively stable NS5 region was chosen for analysis because it allowed for dependable identification of donor and recipient strains. Viral sequences were analyzed by direct sequencing and by sensitive strain-specific polymerase chain reaction assays. These assays were capable of detecting the minor sequence present at a concentration 1:104-10-7 below that of the major sequence. RESULTS: Five patients retained their original infecting strain; the donor strain was detected only transiently. In the remaining six patients, recipient strain was detected for the first few weeks, after which only the donor strain was consistently present. However, in one patient the second nondominant strain was detected from the background of the major strain on a single occasion 8 months after transplantation. All changes in serum were closely paralleled by those occurring in PBMCs. CONCLUSIONS: Viral population changes in the setting of liver transplantation from HCV-infected donors to HCV-infected recipients occur simultaneously in PBMCs and serum. The takeover of one strain by another in PBMC- and serum-derived viral populations seemed to be complete and long lasting.

Exposure of hepatitis C virus (HCV) RNA-positive recipients to HCV RNA-positive blood donors results in rapid predominance of a single donor strain and exclusion and/or suppression of the recipient strain.

We have analyzed three cases of hepatitis C virus (HCV)-infected recipients who received blood from HCV-infected donors. Two recipients were exposed to two different HCV RNA-positive donors, and one was exposed to a single donor. All parental genomes from the actual infecting units of blood and the recipients were defined, and their presence in the follow-up serum samples was determined using sensitive strain-specific assays. The strain from one of the donors was found to predominate in all recipients' serum samples collected throughout the follow-up period of 10 to 30 months. In two recipients exposed to two infected donors, the strain from the second donor was occasionally found at very low level. However, the original recipients' strains were not detected. Our observations show that HCV-infected individuals can be superinfected with different strains, and this event may lead to eradication or suppression of the original infecting strain. Furthermore, our findings demonstrate that simultaneous exposure to multiple HCV strains may result in concomitant infection by more than one strain, although a single strain could rapidly establish its dominance. The results of the present study suggest the existence of competition among infecting HCV strains which determines the ultimate outcome of multiple HCV exposure.

Exposure of hepatitis C virus-negative recipients to > or =2 infected blood donors.

This study analyzed 4 cases of hepatitis C virus (HCV)-naive transfusion recipients who developed hepatitis after receiving blood from >1 HCV-infected donor. One recipient was exposed to 4 donors, 2 were exposed to 3 donors, and 1 was exposed to 2 donors. For 3 recipients, the strain from 1 of the donors predominated in all follow-up samples collected for 8-40 months. For 2 recipients, the strain from the second donor was occasionally detectable with sensitive strain-specific assays. For the fourth recipient, the initially dominant strain was later supplanted by a strain from the other donor. Simultaneous exposure to multiple HCV strains may result in concomitant infection by >1 strain, although a single strain rapidly establishes its dominance. These observations are compatible with the presence of competition among infecting HCV strains that results in the dominance of 1 strain and competitive exclusion or suppression of other strains.

Detection of active hepatitis C virus and hepatitis G virus/GB virus C replication in bone marrow in human subjects.

We have analyzed the presence of hepatitis C virus (HCV) and hepatitis G virus (HGV) sequences in bone marrow and serum samples from 48 patients of a hematologic outpatient clinic. HCV RNA was detected in 18 (38%) and 15 (31%) and HGV RNA was detected in 6 (13%) and 9 (19%) of serum and bone marrow samples, respectively. In 3 patients, HGV RNA was detectable in bone marrow but not in the serum; 2 of these patients were negative for the presence of specific antibodies. Using a highly strand-specific Tth-based reverse transcriptase-polymerase chain reaction (RT-PCR), the presence of HCV RNA and HGV RNA negative strand was demonstrated in 4 and 5 bone marrow samples, respectively. Our study shows that HCV and HGV can replicate in bone marrow; in the case of HGV, analysis of serum may underestimate the true prevalence of infection. (Blood. 2000;95:3986-3989)

Late-onset renal failure after liver transplantation: role of posttransplant alcohol use.

BACKGROUND: Late-onset renal failure is being increasingly recognized as a complication in patients undergoing liver transplantation for hepatitis C virus (HCV). However, its precise incidence, predisposing risk factors, and impact on outcome after liver transplantation, have not been defined. METHODS: The development of late-onset renal failure (defined as serum creatinine persistently >2.0 mg/dl, occurring more than 6 months posttransplant) was assessed in 120 consecutive liver transplant recipients who survived at least 6 months after transplantation. Fifty-seven percent (68/120) of the patients had undergone transplantation for liver disease due to HCV. The median follow-up was 5 years. RESULTS: Late-onset renal failure developed in 28% (33/120)of the patients. Posttransplant alcohol use (P=0.0001), posttransplant diabetes (P=0.0042), and recurrent HCV hepatitis (P=0.019) were significantly associated with late onset renal failure. In multivariate analysis, alcohol use (O.R. 10.7, 95%; CI 2.4-35.9, P=0.001) and diabetes (O.R. 2.1, 95%; CI 1.1-9.9, P=.03) were independently significant predictors of late onset renal failure. When only patients transplanted for HCV were analyzed, posttransplant alcohol use (P=0.004) was the only significant independent predictor of late-onset renal failure. HCV genotype 1b, as compared with other HCV genotypes, was associated with a higher rate of late-onset renal failure in patients with HCV; 70% of the patients with genotype 1b versus 32% of those with 1a and 33% of those with 2b, developed late onset renal failure (P=0.03). At a median follow up of 5 years, mortality in patients with HCV with late-onset renal failure was 52% as compared with 2% in those without renal failure (P=.0001). CONCLUSION: Late-onset renal failure in patients with HCV portended a grave outcome. Alcohol use was an independent predictor of late-onset renal failure in patients with HCV and represents a potentially modifiable risk factor for late-onset renal failure in these patients.

Hepatitis C virus in lymphoid cells of patients coinfected with human immunodeficiency virus type 1: evidence of active replication in monocytes/macrophages and lymphocytes.

It has been reported that hepatitis C virus (HCV) may be lymphotropic in the setting of human immunodeficiency virus type 1 (HIV-1) coinfection. The present study was undertaken to determine the phenotype of lymphoid cells harboring replicating HCV in HIV-1-positive subjects. By means of highly strand-specific thermostable enzyme Tth-based reverse-transcriptase polymerase chain reaction, the presence of viral RNA-negative strand was sought in different subpopulations of peripheral blood mononuclear cells from 10 HIV-positive patients. HCV RNA-negative strand was most commonly present in monocytes/macrophages (4 cases), followed by CD8+ and CD4+ lymphocytes (2 cases) and CD19+ cells (1 case). In 2 cases that were further analyzed, viral-negative strand remained detectable in monocytes/macrophages cultured for 3 weeks. Moreover, monocyte/macrophage- and serum-derived viral sequences differed in the 5' untranslated region. These findings imply that, in HIV-infected subjects, HCV may replicate in the same cells as HIV-1, which raises the possibility of direct interactions between these pathogens.

Quality of life in long-term survivors after liver transplantation: impact of recurrent viral hepatitis C virus hepatitis.

Post liver transplant recurrence of infection with hepatitis C virus (HCV) occurs in approximately 50% of patients transplanted because of HCV-related liver disease. The aim of this study was to assess long-term quality of life, psychologic distress, and coping in patients with recurrent HCV after liver transplantation in comparison to patients transplanted for other etiologies of underlying liver disease. All liver transplant recipients transplanted at a University affiliated Veterans Affairs Medical Center who had greater than 6 months follow-up were sent a questionnaire investigating quality of life (assessed by Medical Outcomes study health survey SF-36), depression (assessed by Beck Depression Inventory), total mood disturbance (assessed by Profile of Mood States scale), coping (assessed by Billing and Moos Inventory of coping with illnesses), and employment status. Lower Beck Depression Inventory score (p = 0.001), lower mood disturbance score (p = 0.0001), overall satisfaction with present work (p = 0.0001), and lesser use of avoidant coping (p = 0.06) were predictors of better quality of life in long-term survivors of liver transplantation. At a mean follow-up of 4 yr after liver transplantation, patients with histopathologically diagnosed recurrent viral HCV hepatitis had significantly lower global quality of life score (mean score of 76.4 versus 86.2, p = 0.011) and physical functioning score (mean score 20 versus 25, p = 0.015), as compared to all other patients. In summary, quality of life and physical functioning were significantly impaired in liver transplant recipients with histopathologically diagnosed recurrent HCV hepatitis, as compared to those whose HCV hepatitis had not recurred or those transplanted for other reasons.

Uneven distribution of hepatitis C virus quasispecies in tissues from subjects with end-stage liver disease: confounding effect of viral adsorption and mounting evidence for the presence of low-level extrahepatic replication.

We have found differences among the populations of hepatitis C virus sequences in serum, peripheral blood mononuclear cells (PBMCs), and various tissues in patients with chronic hepatitis C. These results are compatible with the existence of independent viral compartments in the infected host. Our results also suggest that PBMCs, and probably various tissues, can selectively adsorb viral subpopulations differing in the E2 region.

Clinical and virologic outcomes of hepatitis B and C viral coinfection after liver transplantation: effect of viral hepatitis D.

Hepatitis B (HBV) and C viral (HCV) dual-infection-associated liver disease is an uncommon indication for liver transplantation. The clinical and virologic outcomes in such patients have not been well studied. We retrospectively studied 13 patients with hepatitis B surface antigen (HBsAg) and antibody to HCV positivity who underwent orthotopic liver transplantation (OLT) and survived at least 30 days post-OLT. Antibody to hepatitis delta virus (HDV) was negative in 8 patients (group I) and positive in 5 patients (group II). Eleven of the 13 patients received standard hepatitis B immune prophylaxis, and they all remained HBsAg negative. All group I patients were HCV RNA positive after transplantation; in contrast, all group II patients were HCV RNA negative. Serum alanine aminotransferase levels were elevated in 88% (7 of 8) of the patients in group I compared with 20% (1 of 5 patients) in group II. None of the patients had graft loss from chronic rejection or recurrent hepatitis. Three patients had unsuspected hepatocellular carcinoma in the explant. We conclude that among liver transplant recipients with HBV and HCV coinfection, HDV infection is associated with the suppression of HCV replication and mild inflammatory activity after OLT.

Outcome of liver transplantation in hepatitis C virus-infected patients who received hepatitis C virus-infected grafts.

BACKGROUND & AIMS: The present organ shortage has brought into question the suitability of hepatitis C virus (HCV)-positive grafts. This study reviewed the outcome of such transplantations in our institution. METHODS: Twenty-three HCV-positive patients who underwent orthotopic liver transplantation (OLT) for end-stage liver disease with HCV-positive grafts in 1992-1995 were studied. Only patients who survived more than 30 days were included in the analysis. Control group included 169 patients who underwent transplantation for HCV-related cirrhosis and received HCV-negative organs. RESULTS: Patients who received HCV-infected organs had a cumulative survival rate of 89% and 72% at 1 and 5 years, respectively, vs. 88% and 73% for the control group (NS). There was no difference in graft survival, incidence of cirrhosis, mean hepatitis activity index score, fibrosis, or mean activity of serum transaminases. There was a trend toward lower incidence of recurrent hepatitis C in the study group compared with control (21% vs. 23% at 1 year and 47% vs. 64% at 5 years; NS). Patients in whom the donor strain became predominant after transplantation had significantly longer disease-free survival than patients who retained their own HCV strain (P < 0.003). CONCLUSIONS: HCV-infected livers transplanted into HCV-infected recipients do not appear to convey a worse outcome in the initial years after OLT than HCV-negative grafts.

Analysis of hepatitis G virus/GB virus C quasispecies and replication sites in human subjects.

Although the hepatitis G virus is unlikely to be a primary hepatotropic virus, its replication sites remain unclear. Using highly strand-specific Tth-based reverse transcriptase PCR we searched for the presence of the viral RNA negative strand in various autopsy tissues in two patients who died of end-stage liver disease. In addition, amplified viral sequences were compared in the 5' untranslated and the putative capsid regions by the single-strand conformation polymorphism (SSCP). Negative strand HGV RNA was detected in bone marrow and spleen from both patients and in lymph node tissue from one. All amplified sequences from a given patient were identical when compared by SSCP and direct sequencing. This lack of difference in the composition of quasispecies recovered from various tissues suggests the presence of a single, common viral compartment in the infected host.

Differences between hepatitis C virus 5' untranslated region quasispecies in serum and liver.

It is unclear whether the sequence populations of hepatitis C virus (HCV) quasispecies in the liver and in serum are different, as a variety of studies on this subject provide conflicting results. In the current study, the populations of HCV 5' untranslated region (5' UTR) sequences in paired serum and liver samples from six patients with chronic hepatitis were analysed. Liver-derived, negative-strand viral RNA was amplified with a highly strand-specific Tth-based assay, and extensive measures, including accounting for template copy number, were undertaken to lower the risk of sporadic artefactual polymorphism. Amplified sequences were compared by single-strand conformation polymorphism analysis and by direct sequencing of identified differences. In four patients, liver samples were found to contain variants within the quasispecies which were not found in serum or negative-strand viral RNA, while in the remaining two patients, low virus titre prevented a reliable quasispecies analysis. These results suggest the presence in the same individual of HCV variants differing in the 5' UTR and possibly replicating with different kinetics.

Hepatitis C virus in peripheral blood mononuclear cells from a chronically infected patient receiving liver graft from infected donor.

BACKGROUND: In hepatitis C virus (HCV)-positive patients receiving HCV-positive liver allografts either the donor or recipient strain overtakes the other strain. Whether these changes are reflected in peripheral blood mononuclear cell (PBMC)-associated virus is unknown. METHODS: We analyzed by single-strand conformation polymorphism and sequencing HCV RNA from serum and PBMCs from a liver transplant recipient whose indigenous strain was replaced by the donor strain. RESULTS: Only the recipient strain was detectable in serum and PBMCs 3 and 5 days after transplantation; at day 7 and 8, a mixture of both was present in the PBMCs, but only recipient strain was detectable in serum. This coincided with the peak presence of donor DNA in recipient PBMCs. From day 14 on, HCV sequences in serum and PBMCs were indistinguishable. CONCLUSIONS: Overtake phenomenon in the setting of liver transplantation from infected donors to infected recipients is manifested in PBMCs. Cells released from infected graft carry donor HCV strain.