A. L. Klepper; J. Garber; J. L. Walewski; J. A. Gutierrez; T. D. Schiano; A. D. Branch.
HCV replication has been reported in extrahepatic cells, however, ability to detect HCV RNA in these cells
often fluctuates. We tested the hypothesis that denaturation prior to RT/
Mononuclear cells from ascitic fluid (AMCs;n=5)
and PMBCs (n=1) were isolated, cultured for 1-8 wks, and RNA was Trizol
extracted. Prior to RT/
4/5 of the
These studies suggest that much of the HCV RNA human cells is in dsRNA complexes, answering a persistently vexing
question in the investigation of (extrahepatic) HCV replication: Why it is so
difficult to consistently detect HCV RNA in infected cells and human specimens?
The answer: RNA is there, it is just present in structures that evade detection
by conventional RT/
HCV Drug Pipeline – Drug Resistance
A. U. Neumann.
Current paradigm assumes that HCV has no virological
mechanism to archive mutations and that resistant strains (
A new mathematical model combining intra-cellular HCV
replication dynamics with cell infection dynamics is used. Intra-cellular RNA
(ICR) forms replication units (RU), which in turn synthesize more ICR that is
partially packaged and exported as virions.
Simulation of the ODE model shows that if
This dynamical archiving process, within a large range of
parameters, allows for establishment of relatively high level of
In contradiction to the current paradigm, resistant HCV strains can persist after the end of treatment with direct anti-virals by a process of dynamical archiving, which is first described here allowing to calculate the conditions necessary to prevent it.
HCV Drug Pipeline – Nitazoxanide
T. Schaninger; J. Hong; G. G. Luo.
Nitazoxanide is a thiazolide anti-infective with activity against a number of protozoa, bacteria, and viruses. It is FDA approved for treatment of Cryptosporidium and Giardia. Nitazoxanide inhibits replication of hepatitis C virus, hepatitis B virus, and rotavirus in vitro. Based on its broad antiviral activity, the mechanism of action is likely through cellular processes rather than specific antiviral targets. Rossignol and colleagues recently reported that the use of nitazoxanide in combination with peginterferon alfa-2a with or without ribavirin among treatment-naive hepatitis C patients infected with genotype 4 significantly improved viral response compared to the standard of care (peginterferon alfa-2a plus ribavirin). The sustained virologic response was 79%, 61%, and 50% respectively.
We tested nitazoxanide and its active metabolite, tizoxanide, in HCV cell culture. Time of addition experiments were carried out in the infectious HCV genotype 2a (JFH1) cull culture model to investigate which steps of the replicative cycle are inhibited by nitazoxanide.
Nitazoxanide and tizoxanide exhibited potent antiviral activity against subgenomic HCV 1b replicon as well as infectious HCV genotype 2a (JFH1). Nitazoxanide and tizoxanide significantly inhibited HCV infection when they were added during the infection period. However, they showed reduced activity when added post infection.
These findings indicate thiazolide affects the early stage of the HCV infection cycle. A proposed mechanism of action of nitazoxanide against HCV replication will be described and discussed.
P. Bellecave; J. Gouttenoire; M. Gajer; V. Brass; G. Koutsoudakis; H. E. Blum; M. Nassal; R. Bartenschlager; D. Moradpour.
Coinfection with hepatitis B virus (HBV) and hepatitis C virus (HCV) has been associated with severe liver disease and frequent progression to liver cirrhosis and hepatocellular carcinoma. Clinical evidence suggests reciprocal replicative suppression of the two viruses ('viral interference'). However, virtually nothing is known about molecular interactions between HBV and HCV due to the lack of appropriate model systems.
A tetracycline-regulated gene expression system was used to generate a panel of stable Huh-7 cell lines inducibly replicating HBV. These cell lines and control Huh-7 cell lines inducibly expressing the green fluorescent protein (GFP) were transfected with selectable HCV replicons or infected with cell culture-derived HCV (HCVcc).
Three successive transfection and selection steps allowed the establishment of Huh-7 cells inducibly replicating HBV and constitutively replicating subgenomic HCV RNA. In these cell lines, it is possible to regulate the expression of HBV proteins, HBV genome replication, and infectious viral particle formation by the concentration of tetracycline in the culture medium while HCV proteins are expressed and HCV RNA replicated constitutively. The presence or absence of replicating HBV did not influence the antiviral effect of interferon-alpha against HCV. In addition, specific inhibition of one virus did not significantly affect gene expression, replication and release of the other. Experiments using HCVcc did not reveal any significant superinfection exclusion of HCV by HBV. Finally, cell culture-adapted HCVcc isolated from long-term cultures did not display any HBV-specific adaptation.
HBV and HCV can replicate in the same cell without any significant direct interaction. Therefore, the viral interference observed in vivo in coinfected patients is likely due to innate and/or adaptive host immune responses. These findings provide new insights into the pathogenesis of HBV-HCV coinfection and may contribute in the future to its clinical management.
Epidemiology/Transmission – General
K. Goto; Y. Tanaka; T. Endo; K. Ito; A. A. Khan; M. Mizokami.
The incidence of mother-to-infant transmission is approximately 5–10% in infants born to HCV-RNA-positive mothers. This is an important route of HCV infection in childhood because HCV infection through blood transfusion has been effectively managed by blood donor screening. Many data are available for HCV evolution in adults but there is little information regarding the HCV evolutionary mechanism in mothers and their infants who got infection by vertical transmission. Our aims are to investigate the vertical transmission and characteristics of HCV evolution of mother–to-infant pairs.
Seven Japanese mother-to-infant pairs with vertical transmission of HCV were studied. The 336-bp sequences in the NS5b gene, which is an essential viral replicating enzyme encoding the viral RNA-dependent RNA polymerase, were determined from serum samples at 2-6 points (~12.0 years intervals) in each patient. The sequences were phylogenetically analyzed and the evolution of HCV was compared between mothers and their infants.
The distribution of HCV subtypes in pairs was as follows: 3 pairs with 1b; 3 pairs with 2a; and 1 pair with 2b. The sequences obtained from each pair were clustering together in a phylogenetic tree, providing evidence of vertical transmission with a 97.3-99.7% identity of sequences of infants with their respective mothers. Examining the evolution of HCV in each patient, nucleotide substitutions within the NS5b region were identified in 4 of 7 mothers (0.26-4.46 x 10^-3 substitutions/site/year). Except for one infant who showed a nucleotide substitution rate of 0.37 x 10^-3 /site/year, substitutions were not found in other 5 infants during a follow-up of ~12.0 years.
The study provides evidence of vertical transmission of HCV from HCV-RNA-positive mothers to their infants. Less evolution of HCV observed in infants may be due to low selective immunological pressure generated by the infant host, as compared to HCV evolution in adults.
HCV Drug Pipeline – Telaprevir
N. Forestier; S. Susser; M. W. Welker; U. Karey; S. Zeuzem; C. Sarrazin.
Telaprevir (TVR) is a highly selective inhibitor of the hepatitis C virus NS3/4A protease with blocking of HCV replication in patients with hepatitis C. In this study, we describe the follow up status of patients who were initially treated with telaprevir. So far, little is known, about what happens after stopping treatment with TVR.
We followed up patients with HCV who were treated in a phase
1b study for 14 days with TVR (monotherapy study, n=34) and in addition
patients who were treated in another study for 14 days with either TVR alone,
TVR+peginterferon or with placebo+peginterferon (combination study, n=20). Most
of the patients from the monotherapy study received no further treatment after
the 14 days treatment with TVR, however nearly all of the patients from the
combination study received after the initial 14 days treatment with TVR
standard of care treatment (
From the monotherapy study 5 patients were enrolled and all
patients showed TVR resistant variants during the initial treatment with TVR.
Patient 1 received no further treatment afterwards and 9 months after
Variable outcomes have been detected in patients treated with telaprevir alone or in combination with Peg-IFN for 14 days in phase 1 studies. A detailed virologic status of the patients as well as sequencing analyses results in these patients will be presented at the meeting.
HCV Treatment – Predictors of Treatment Response
P. S. Pang; P. J. Planet; J. S. Glenn.
To determine why patients infected with the various hepatitis C viral genotypes require different durations of therapy and achieve different sustained viral response rates to interferon plus ribavirin therapy.
The first phylogenetic analysis to include all available HCV genomic sequences was performed. The resultant tree was then used, in conjunction with a review of all prospective clinical trials that studied interferon and ribavirin therapy, to determine if a correlation existed between genomic age and clinical outcome. The viral loci potentially responsible for interferon treatment sensitivity differences were then screened for using ancestral protein sequence reconstruction.
A new cladogram of HCV was constructed, which, for the first time reveals the relative evolutionary ages of all the major HCV genotypes. We determined that genotype-specific clinical outcomes directly correlate with genotype age. This relationship allowed us to predict that genotypes 5 and 6, for which there currently are no published prospective trials, will likely have intermediate response rates, similar to genotype 3. Two viral loci that likely play a direct role in determining genotype-specific outcomes were also identified. Prior studies have independently identified these exact two loci as having a role in inhibiting the innate immune factor PKR.
A primary determinant of genotype-specific response rates to interferon therapy is a set of viral factors that evolved to progressively acquire an increasing ability to inhibit the innate immune response.
HCV Disease Progression – Race
M. Y. Sheikh; M. H. Bashir; A. A. Afaq; H. Sadiq; J. Choudhury.
Ethinicity can affect the natural history of chronic
hepatitis C (
The clinical data of 481 Caucasians and 472 Latinos with
Of 481 Caucasians, 58.1% were males and 41.9% females with a
mean age of 46.8 years and mean
At the time of HCV diagnosis, Latinos have advanced stages of
hepatic fibrosis as compared to Caucasians, indicating higher disease
progression rates. There is a high prevalence of steatosis in Latinos as
compared to Caucasians. Further studies are needed to evaluate various factors
affecting the natural history in Latino patients with
Latino # (%)
Total # (%)
Disease Progression – Race
A. Afendy; M. Stepanova; A. Baranova; N. Hossain; I. Younossi; A. M. Wheeler; Z. M. Younossi.
African American (AA) with CH-C have
lower response rates to pegylated interferon and Ribavirin (
To compare gene expression of Caucasians (CA) with CH-C to those of AA with CH-C.
CH-C patients who were scheduled to receive
A total of 53 CH-C patients were included in this analysis.
Of these, 42 patients were CA and 11 were AA with CH-C (Age: 47.8±5.9, 58%
male, 74% genotype 1). Overall sustained virologic response was 45% in CA and
18% in AA. Prior to treatment, AA patients had lower expression of the
following genes: MMP9 (CA/AA: 1.373),
AA CH-C patients seem to have significantly lower expression
of a number of genes involved in interferon signaling pathway, prior to and
shortly after initiation of
N. I. Rallón; M. López; V. Soriano; J. García-Samaniego; M. Romero; P. Labarga; J. González-Lahoz; J. M. Benito.
Cellular responses against HCV seem to be low in most patients with chronic hepatitis C. They may be even further impaired in HIV patients. Herein, we examine the functional profile of HCV-specific T-cells and the impact of HIV coinfection in patients with chronic hepatitis C.
HCV-specific CD4+ and CD8+ T-cell responses were examined in 30 interferon-naive patients, 10 HCV-monoinfected and 20 HIV/HCV-coinfected. The profile of cytokine production (IFN-γ, IL-2 and TNF-α) in response to 324 genotype-matched overlapping peptides spanning five HCV proteins (E2, p7, NS3, NS5a, NS5b) was measured using 5-colour flow cytometry. Differences between groups for the different immune parameters were tested using non-parametric tests.
CD4 counts and plasma HIV-RNA in coinfected patients were 390+213 cells/μl and 2.6+1.3 log copies/ml (65% of them were on HAART). Serum HCV-RNA in monoinfected and coinfected patients was 5.7+0.8 and 6.4+0.7 log IU/ml, respectively (p=0.03). Overall, 90% of monoinfected patients had genotype 1 whereas in coinfected patients 45% had HCV-1 and 55% had HCV-3.
The proportion of patients having a detectable CD4 or CD8 response against the five HCV proteins was high (>80%) and similar in both mono- and coinfected patients. Levels of total CD4 and CD8 responses against the five HCV proteins were also similar in both groups. The cytokine profile of HCV-specific CD4 and CD8 T-cells was dominated, in both groups, by cells producing only TNF-α. However, the contribution of single TNF-α+ cells to CD4 responses against NS3 and NS5a was significantly higher in coinfected than in monoinfected patients. In contrast, the contribution of cells producing only IL-2 into the CD4 response against the different proteins was higher in monoinfected than in coinfected patients, although differences did not reach statistical significance.
In HCV-1 patients there was a positive and significant correlation between the contribution of single TNF-α+ cells to CD4 responses against NS5a and serum HCV-RNA, after adjusting for HIV load (r=0.86, p=0.01).
The functional profile of HCV-specific T-cells is limited to a single function and dominated by TNF-α. The ability of CD4 T-cells to produce IL-2 against HCV is impaired in HIV-coinfected patients. The greater level of single TNF-α+ cells contributing to the CD4 response in coinfected patients might explain at least in part a lower control of HCV replication in this population.
Disease Progression –
A. A. Khan; Y. Tanaka; Z. Azam; Z. Abbas; F. Kurbanov; S. S. Hamid; S. Jafri; M. Mizokami.
Infection with hepatitis C virus (HCV) genotype 1b is
reportedly associated with higher incidence of hepatocellular carcinoma (
A total of 189 CLDs patients including 82 with
HCV-3a was the predominant genotype (81.4%) in the studied
cohort, followed by 3b (9.3%), 3k (2.3%), 1a (1.5%), 1c (1.5%), 1b (0.8%) and
2a (0.8%). The mean HCV RNA levels were significantly higher in
HCV-3a having earlier epidemic spread in
P. Vaghefi; G. Maurin; D. Lavillette; C. Feray; E. Dussaix; A. Roque-Afonso.
HCV genetic variability is due to the high error rate of the viral RNA polymerase and to negative and positive selection pressures. We investigated the contribution of immune selection pressure on HCV quasispecies evolution by studying the hypervariable region (HVR) in HIV co-infected subjects upon immune restoration.
Patients and methods:
Twenty eight patients were studied at 2 time points: 15
patients before and after antiretroviral treatment initiation with a mean
interval of 24.3 +/- 10.6 months and 13 non-treated patients with a mean
interval of 18.1 +/- 12 months. Quasispecies complexity and its qualitative
modifications were assessed by SSCP and by cloning and sequencing. Analysis was
performed according to CD4 cell count, HIV and HCV viral load, HCV genotype,
aminotransferases levels (
In the treated group, CD4 cell count increased and HIV viral
load decreased significantly. A trend towards increased neutralizing activity
of sera was also observed. No significant change in HCV viremia and
quasispecies complexity was noted in both groups. Complexity was higher in the
14 patients infected by HCV genotype 1, particularly at the 2nd time point
(5.57 versus 3.5), independently of the viral load. Emergence of variants and
global modification of quasispecies (emergence + disappearing) was correlated
to the initial level of
We had previously shown that qualitative variation of HVR was not related to CD4 cell count and depended on the initial complexity (Roque-Afonso, J Infect Dis 2002). Immune restoration, reflected by CD4 counts increase, appears to be associated with an increase of neutralizing antibodies titer, allowing the elimination of part of circulating variants, without any impact on HCV viremia. In association with humoral response, the cellular immunity also contributes to the quasispecies evolution as suggested by the link between liver cytolysis and variants emergence.
P. A. White; J. Grebely; J. K. Flynn; G. Matthews; M. K. Renkin; B. Yeung; W. Rawlinson; J. Kaldor; M. Hellard; A. R. Lloyd; R. A. Ffrench; G. J. Dore.
To characterise the natural history of HCV superinfection and reinfection.
ATAHC is a prospective study of the natural history and
treatment of acute/early chronic HCV. Treated subjects received
167 subjects were enrolled, 107 received treatment. Nine
cases of superinfection (n=5) and reinfection (n=4) were identified, including
7 during (n=3, all superinfection) or following treatment (n=4, all
reinfection). 8 cases had ongoing injecting reported, 5 had temporal
These findings describe a heterogeneous natural history of HCV superinfection and reinfection, consistent with chimpanzee studies. Protection from viral persistence upon reinfection can occur, but is not universal.
S. C. Gordon; D. Moonka; K. a. Brown; M. Y. Huang; S. E. Anteau; L. Lamerato.
Chronic HCV mediates the development of
We used administrative data from a large integrated health
care system to explore the incidence of RCC among HCV infected persons. Adults
(over 18 yrs) testing HCV+ between 1997 and 2006 were compared to a control
cohort testing anti-HCV negative (HCV-) during the same time period; HBsAg and
HIV positive patients were excluded. Data from the system’s registry identified
all RCC pts diagnosed between 1997 and April 2008.
The cohort consisted of 74,570 pts with 12.6% HCV+ (n=9,401)
and 65,169 HCV negative. Males and African Americans (AA) had a higher
prevalence of HCV (15.4% males vs. 9.8% females, p<0.001 and 16.4% AA vs.
9.8% non-AA, p<0.001). The mean age of HCV+ patients was older than the HCV-
(52 vs. 48, p<0.001). There were 163 RCC patients (0.25/100) in the
unaffected vs. 35 (0.37/100) in the HCV+ group. The unadjusted OR for RCC was
1.49 (CI: 1.03, 1.83, p=0.03). The mean age at RCC diagnosis was much younger
in HCV+ individuals (52 vs. 63, p<0.001). When adjusting for age, race and
gender the overall OR for HCV among RCC patients was 1.2 (CI: 0.83, 1.74,
p=0.34). However, at particular risk were males less than age of 50 at HCV
diagnosis, where the adjusted OR for RCC was 4.8 (CI: 2.48, 9.17, p<0.001).
As expected, the risk of
Chronic HCV is associated with a higher incidence of primary renal cell carcinoma than non-infected persons, and this risk is particularly high, with nearly 5-fold increase, among younger males. Potential limitations include unmeasured risk factors and a referral bias at a tertiary medical center; larger studies are required to confirm these findings.
R. Narita; M. Hiura; S. Abe; Y. Kihara; A. Tabaru; M. Otsuki.
Background and Aims:
Recent epidemiological studies have suggested that hepatitis
C (HCV) infection is associated with an increased risk of development of type 2
diabetes mellitus (DM). Elucidation of the relationship between chronic HCV
We enrolled a total of 202
There was a significant correlation between [AUC]0-180 min of glucose or insulin and HOMA-IR. Pearson’s correlations coefficient (r) and the P values for these correlations were r = 0.334 and 0.562, and P<0.0001 and <0.001 respectively. Matsuda index [104/√(mean insulin after OGTT x mean glucose after OGTT x fasting glucose x fasting insulin)] strongly correlated with HOMA-IR (r = 0.855, P<0.0001). Glucose 0-30[AUC] x insulin 0-30[AUC], which reflects an index of hepatic resistance to insulin, had a significant but much weaker correlation with HOMA-IR (r = 0.534, P<0.0001).
Although HOMA-IR is an useful index
of IR, determination of hepatic insulin resistance using of HOMA-IR is not
Disease Progression –
S. Seronello; C. Ito; T. Wakita; J. Choi.
Hepatitis C virus (HCV) causes severe alteration of the host
redox status, and ethanol is a well-known cofactor in the hepatopathogenesis
induced by HCV. Ethanol metabolism generates reactive oxygen species (
In this study, we use JFH1 strain that produces infectious
virus particles in Huh7 cells to demonstrate that ethanol, at subtoxic and
physiologically relevant concentrations, elevates HCV RNA level in the context
of the complete lifecycle of HCV. Ethanol also increases HCV RNA in the
subgenomic JFH1-Luc (genotype 2a) and Con1-Neo (genotype 1b) RNA-transfected
cells, suggesting that the HCV RNA genome replication is affected, independent
of the genotype. Acetaldehyde likewise elevates HCV RNA at physiological
concentrations. In contrast, decreasing intracellular glutathione (
Therefore, ethanol is likely to enhance HCV replication
through acetaldehyde rather than
Y. Eguchi; T. Mizuta; E. Ishibashi; T. Eguchi; A. Matsunobu; N. Oza; S. Nakashita; Y. Kitajima; H. Takahashi; Y. Kawaguchi; R. Iwakiri; I. Ozaki; N. Ono; K. Fujimoto.
Recently, an association between hepatitis C virus (HCV) infection and insulin resistance (IR) has been noted. Although visceral fat accumulation is closely related to IR in subjects with metabolic syndrome and nonalcoholic fatty liver disease (NAFLD), the possible effect of HCV on visceral fat accumulation remains unclear.
To evaluate the relationship between IR and visceral fat accumulation in HCV-infected patients and then compared the incidence of IR between HCV-infected patients and NAFLD patients with or without visceral fat accumulation.
A total of 87 HCV-infected patients in comparison with 125 sex- and age-matched patients with NAFLD were recruited. In order to exclude a confounding effect of advanced hepatic fibrosis on IR, the patients were limited to HCV patients with fibrosis stages 1 and 2 (n=87; 39 males and 48 females) and platelet counts of >15 x 104 /mm3. The patients were included if they fulfilled the following criteria: absence of diabetes mellitus; absence of markers of hepatitis B virus; no evidence of autoimmune liver disease or alcoholic liver disease (>20 g of alcohol per day). We evaluated the degree of visceral fat area (VFA; cm2) at the umbilical level was measured by computed tomography and divided into two grades: no visceral obesity, VFA<100; visceral obesity, VFA≥100. IR was evaluated by homeostasis model assessment of IR (HOMA-IR) and the quantitative insulin sensitivity check index (QUICKI). Pancreatic beta-cell function was evaluated by homeostasis model assessment of beta-cell function (HOMA-beta). Serum soluble TNF-receptors 1 and 2 and adiponectin were measured.
Body mass index, waist circumference, VFA, total cholesterol, triglyceride and fasting plasma glucose were significantly higher in NAFLD patients than in HCV-infected patients, whereas aminotransferase and HOMA-beta were significantly higher in HCV-infected patients than in NAFLD patients. HOMA-IR and QUICKI was correlated with visceral fat accumulation, and higher in HCV patients than in NAFLD patients with visceral obesity. HOMA-beta was higher in HCV patients than in NAFLD patients for each VFA grade. There was no difference between the levels of adiponectin in HCV-infected patients and NAFLD patients with visceral obesity. Serum soluble TNF-receptor 1 and 2 were higher in HCV patients than in NAFLD patients with visceral obesity.
These results indicate that HCV infection is a risk factor for development of IR, particularly in patients with visceral obesity. The progression of IR may be enhanced by an increase in TNF activity in HCV-infected patients with visceral obesity.
Microsomal triglyceride transfer protein (
É. F. Siqueira; C. P. Oliveira; M. Corrêa-Giannella; J. Stefano; A. Cavaleiro; M. Z. Fortes; M. C. Muniz; K. A. Silva; F. S. Silva; L. B. Pereira; F. J. Carrilho.
Background & Aims:
Chronic hepatitis C (
Eight six untreated patients with HCV RNA and liver biopsy
Our results indicate that the presence of G allele of
Disease Progression – HCC
C. Braconi; F. Meng; N. Huang; T. Suzuki; C. Taccioli; C. Croce; T. Patel.
Compared to controls, HCV-positive Hep-394 cells had a lower
IC50 to sorafenib (1.0 +/- 0.1 vs 3.5+/- 0.8 μΜ) or doxorubicin (0.8
+/- 0.1 vs 1.7 +/- 0.1 μΜ). Compared to controls, HCV positive cells
had significant alterations in expression of selected miRNA, with 10 miRNA
(3.1% of total) > 2-fold down-regulated and 27 miRNA (8.4% of total) >
2-fold up-regulated (p<0.01). Of these, miR-193b and miR-29b were prominently
increased in expression (by 3.6 and 1.8-fold respectively), whereas miR-206 and
miR-627 were decreased (by 5.0 and 3.0-fold respectively) in HCV-positive
Summary and Conclusions:
Cellular expression of HCV proteins increases sensitivity to
sorafenib by miRNA-dependent modulation of Mcl-1 and cellular apoptosis. We
conclude that enhancing the miRNA responses involved in response to
chemotherapy may be useful for individualized therapy in