Hepatitis C virus and peripheral blood mononuclear cell reservoirs Patricia Baré

The existence of hepatitis C virus (HCV) infection in extrahepatic sites has been widely demonstrated. Since peripheral blood mononuclear cells have been the most investigated, compelling evidence of an association with HCV has been shown. Different studies have revealed that HCV RNA can persist and replicate in immune cells but the relevance of its presence and persistence over time is still unknown. As the contribution of this extrahepatic reservoir could have several clinical implications in viral transmission, treatment response and disease pathogenesis, future studies are required to improve our knowledge of the extrahepatic manifestations of HCV and its possible consequences.     

Flow cytometric detection of hepatitis C virus antigens in infected peripheral blood leukocytes： Binding and entry

AIM: We designed two synthetic-core-specific peptides core 1 (C1) and core 2 (C2), and an El-specific peptide (El). We produced specific polyclonal antibodies against these peptides and used the antibodies for detection of HCV antigens on surface and within infected peripheral blood leukocytes. METHODS: Peripheral blood from a healthy individual who tested negative for HCV RNA was incubated with HCV type 4 infected serum for 1 h and 24 h at 37 ℃. Cells were stained by direct and indirect immunofluorescence and measured by flow cytometry. RESULTS: After 1 h of incubation, antibodies against C1, C2, and E1 detected HCV antigens on the surface of 27%, 26% and 73% of monocytes respectively, while 10%, 5% and 9% of lymphocytes were positive with anti-C1, anti-C2 and anti-E1 respectively. Only 1-3% of granulocytes showed positive staining with anti-C1, anti-C2 and anti E1 antibodies. After 24 h of incubation, we found no surface staining with anti-C1, anti-C2 or anti-E1. Direct immunostaining using anti-C2 could not detect intracellular HCV antigens, after 1 h of incubation with the virus, while after 24 h of incubation, 28% of infected cells showed positive staining. Only plus strand RNA was detectable intracellularly as early as 1 h after incubation, and remained detectable throughout 48 h post-infection. Interestingly, minus RNA strand could not be detected after 1 h, but became strongly detectable intracellularly after 24 h post-infection. CONCLUSION: Monocytes and lymphocytes are the preferred target cells for HCV infection in peripheral blood leukocytes. Our specific anti-core and anti-El antibodies are valuable reagents for demonstration of HCV cell cycle. Also, HCV is capable of infecting and replicating in peripheral blood mononuclear cells as confirmed by detection of minus strand HCV RNA as well as intracellular staining of core HCV antigen.     

Interleukin 2 and γ/δ T-cell receptors in peripheral blood of patients with chronic hepatitis C virus infection

Studies were conducted to determine if CD3 (T-cell) associated antigen receptors (R) such as interleukin 2 (IL-2) and γδ participate in the pathogenesis of hepatitis C virus (HCV) infection. Peripheral blood T-cells bearing IL-2R α (CD25) or β (CD122) chain, and αβ or γδ were examined using two-color flow cytometry in 92 patients with various chronic HCV infection. CD25-positive T-cells were increased with the progression of the disease; patients with hepatocellular carcinoma (HCC) had the highest percentage of CD25 + T-cells, while CD 122 + T-cells were significantly higher in asymptomatic HCV carriers with normal serum ALT levels and mild to moderate chronic hepatitis. In HCC patients, a decrease in CD25 + cells and increase in CD 122+ cells were noted after hepatic resection or percutaneous ethanol injection. Asymptomatic carriers had higher γδ T-cells than in controls, and patients with chronic liver disease. The percentage of γδ T-cells became higher during IFN treatment in responders compared with nonresponders. The absolute number of T-cells studied here showed comparable results to those expressed as percentage in each patient group. There was no correlation between serum ALT values, HCV RNA levels, and the incidence of T-cell subsets. The findings suggest that IL-2R αβ chains on T-cells and γδ T-cell are associated with the pathogenesis of chronic HCV infection.     

IL-1β and TNF-α produced by peripheral blood mononuclear cells before and during interferon therapy in patients with chronic hepatitis C

We investigated the spontaneous and phytohemagglutinin-stimulated production of interleukin-1 (IL-1) and tumor necrosis factor- (TNF-) by peripheral blood mononuclear cells in patients with chronic hepatitis C during treatment with interferon- (IFN-). Spontaneous productions of these were significantly higher in patients with chronic hepatitis C than in healthy subjects. For patients prescribed interferon, stimulated production of TNF- was significantly higher in complete responders than in partial responders, but the differences were small between the other cytokine levels and outcome of IFN treatment. Spontaneous production of these cytokines was higher in patients with genotype III with complete response than in genotype III patients with a partial response, but this was not the case in patients with genotype II. There was a negative correlation between these cytokines and histological activity index. Spontaneous production of cytokines was decreased only in complete responders after the administration of interferon. These data suggest that the elevated production of cytokines in patients with chronic hepatitis C may be due to host response to the virus, and monitoring cytokines along with alanine aminotransferase and hepatitis C virus RNA during treatment may provide more precise information of the effectiveness of therapy.     

Kinetics of phytohemaglutinin-induced IFN-γ and TNF-a expression in peripheral blood mononuclear cells from patients with chronic hepatitis B after liver transplantation

AIM:To study the association between host immunity and hepatitis B virus (HBV) recurrence after liver transplantation. METHODS:Peripheral blood mononuclear cells (PBMC) were isolated from 40 patients with hepatitis B and underwent orthotopic liver transplantation (OLT) before and 2,4,8 wk after surgery. After being cultured in vitrofor 72 h, the levels of INF-γ and TNF-a in culture supernatants were detected with ELISA. At the same time, the quantities of HBV DNA in serum and PBMCs were measured by real time PCR. RESULTS:The levels of INF-γ and TNF-a in PBMC culture supernatants decreased before and 2, 4 wk after surgery in turns (INF-γ 155.52±72.32 ng/L vs 14.76±9.88 ng/L vs 13.22±10.35 ng/L, F=6.946, P=0.027<0.05; TNF-α 80.839±46.75 ng/L vs 18.59±17.29 ng/L vs 9.758±7.96 ng/L, F=22.61, P=0.000K0.05). The levels of INF-γ and TNF-a were higher in groups with phytohemagglutinin (PHA) than in those without PHA before surgery. However, the difference disappeared following OLT. Furthermore, INF-y and TNF-a could not be detected in most patients at wk 4 and none at wk 8 after OLT. The HBV detection rate and virus load in PBMC before and 2,4 wk after surgery were fluctuated (HBV detected rate:51,4%, 13.3%, 50% respectively; HBV DNA:3.55±0.674 log(10) copies/mL vs 3.00±0.329 log(10) copies/mL vs 4.608±1.344 log(10) copies/mL, F=7.582, P=0.002<0.05). HBV DNA in serum was 4.48±1.463 log(10) copies/mL before surgery and <10~3 copies/mL after OLT except for one with 5.72xl0~6 copies/mL 4 wk after OLT who was diagnosed as HBV recurrence. The levels of INF-γ and TNF-α were lower in patients with a high HBV load than in those with a low HBV load (HBV DNA detected/undetected in PBMCs:IFN-γ 138.08±72.44 ng/L vs 164.24±72.07 ng/L, t=1.065, P=0.297>0.05, TNF-α 80.75±47.30 ng/L vs74.10±49.70 ng/L, t=0.407, P=0.686> 0.05; HBV DNA positive/negative:IFN-γ 136.77±70.04 ng/L vs 175.27±71.50 ng/L, t=1.702, P=0.097>0.05; TNF-α 75.37±43.02 ng/L vs 81.53±52.46 ng/L, t=0.402, P=0.690>0.05). CONCLUSION:The yielding of INF-γ and TNF-α from PBMCs is inhibited significantly by immunosuppressive agents following OLT with HBV load increased, indicating that the impaired immunity of host is associated with HBV recurrence after OLT.     

Kinetics of phytohemaglutinin-induced IFN-γ and TNF-α expression in peripheral blood mononuclear cells from patients with chronic hepatitis B after liver transplantation

AIM: To study the association between host immunity and hepatitis B virus (HBV) recurrence after liver transplantation. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from 40 patients with hepatitis B and underwent orthotopic liver transplantation (OLT) before and 2, 4, 8 wk after surgery. After being cultured in vitro for 72 h, the levels of INF-gamma and TNF-alpha in culture supernatants were detected with ELISA. At the same time, the quantities of HBV DNA in serum and PBMCs were measured by real time PCR. RESULTS: The levels of INF-gamma and TNF-alpha in PBMC culture supernatants decreased before and 2, 4 wk after surgery in turns (INF-gamma 155.52+/-72.32 ng/L vs 14.76+/-9.88 ng/L vs 13.22+/-10.35 ng/L, F = 6.946, P = 0.027 < 0.05; TNF-alpha 80.839+/-46.75 ng/L vs 18.59+/-17.29 ng/L vs 9.758+/-7.96 ng/L, F = 22.61, P = 0.0001 < 0.05). The levels of INF-gamma and TNF-alpha were higher in groups with phytohemagglutinin (PHA) than in those without PHA before surgery. However, the difference disappeared following OLT. Furthermore, INF-gamma and TNF-alpha could not be detected in most patients at wk 4 and none at wk 8 after OLT. The HBV detection rate and virus load in PBMC before and 2, 4 wk after surgery were fluctuated (HBV detected rate: 51.4%, 13.3%, 50% respectively; HBV DNA: 3.55+/-0.674 log10 copies/mL vs 3.00+/-0.329 log10 copies/mL vs 4.608+/-1.344 log10 copies/mL, F = 7.582, P = 0.002 < 0.05). HBV DNA in serum was 4.48+/-1.463 log10 copies/mL before surgery and <10(3) copies/mL after OLT except for one with 5.72 x 10(6) copies/mL 4 wk after OLT who was diagnosed as HBV recurrence. The levels of INF-gamma and TNF-alpha were lower in patients with a high HBV load than in those with a low HBV load (HBV DNA detected/undetected in PBMCs: IFN-gamma 138.08+/-72.44 ng/L vs 164.24+/-72.07 ng/L, t = 1.065, P = 0.297 > 0.05, TNF-alpha 80.75+/-47.30 ng/L vs 74.10+/-49.70 ng/L, t = 0.407, P = 0.686 > 0.05; HBV DNA positive/negative: IFN-gamma 136.77+/-70.04 ng/L vs 175.27+/-71.50 ng/L, t = 1.702, P = 0.097 > 0.05; TNF-alpha 75.37+/-43.02 ng/L vs 81.53+/-52.46 ng/L, t = 0.402, P = 0.690 > 0.05). CONCLUSION: The yielding of INF-gamma and TNF-alpha from PBMCs is inhibited significantly by immunosuppressive agents following OLT with HBV load increased, indicating that the impaired immunity of host is associated with HBV recurrence after OLT.     

Effect of in vitro interferon-beta administration on hepatitis C virus in peripheral blood mononuclear cells as a predictive marker of clinical response to interferon treatment for chronic hepatitis C

AIM:To test whether in vitro incubation of peripheral bloodmononuclear cells (PBMC) with interferon (IFN) couldefficiently decrease hepatitis C virus-RNA (HCV-RNA) amountand to analyze whether this effect was associated with clinicalresponse to IFN.METHODS:Twenty-seven patients with histologically provenchronic hepatitis C were given intravenous administrationof 6 million units (MU) IFN-β daily for 6 weeks followed bythree times weekly for 20 weeks.PBMC collected beforeIFN therapy were incubated with IFN-β and HCV-RNA inPMBC was semi-quantitatively determined.RESULTS:Twenty-five patients completed IFN therapy.Eight patients (32%) had sustained loss of serum HCV-RNAwith normal serum ALT levels after IFN therapy (completeresponders).HCV-RNA in PBMC was detected in all patients,whereas it was not detected in PBMC from healthy subjects.In vitro administration of IFN-β decreased the amount ofHCV-RNA in PMBC in 18 patients (72%).Eight of thesepatients obtained complete response.On the other hand,none of the patients whose HCV-RNA in PBMC did notdecrease by IFN-β was complete responders.Multiple logisticregression analysis revealed that the decrease of HCV-RNAamount in PBMC by IFN-β was the only independent predictorfor complete response (P<0.05).CONCLUSION:The effect of in vitro IFN-β on HCV in PBMCreflects clinical response and would be taken into accountas a predictive marker of IFN therapy for chronic hepatitis C.     

Hepatitis G virus infection in patients with chronic non-A-E hepatitis

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Testing for hepatitis C virus sequences in peripheral blood mononuclear cells of patients with chronic hepatitis C in the absence of serum hepatitis C virus RNA

Hepatitis C virus (HCV) is able to replicate in peripheral blood mononuclear cells (PBMC) of HCV-infected patients. Few data are available on PBMC testing for HCV RNA in serum HCV RNA negative patients, positive for anti-HCV and with histological evidence of chronic hepatitis. Twenty such patients were studied; of these, 11 were tested during interferon α (IFN) treatment, at the time of serum HCV RNA clearance and ALT normalisation: only one was found to be positive for HCV sequences in PBMC. Within 3 months of IFN withdrawal all 11 patients relapsed with high ALT and recurrence of serum HCV RNA. Of nine serum HCV RNA negative patients with chronic hepatitis C who were not receiving IFN when tested (four untreated patients and five patients who had already completed IFN schedule), PBMC HCV RNA was detected in four. Evidence of active HCV replication (presence of the minus strand genome) in PBMC was also observed in two cases. Thus, five of the 20 patients without detectable serum HCV RNA turned out to be carriers of HCV sequences in PBMC. These data indicate that: 1. PBMC are an extrahepatic replication site of HCV; this is true also in the absence of serum HCV RNA; 2. the role of PBMC as a “viral reservoir” after IFN-induced serum HCV RNA clearance is questioned; 3. the absence of both serum and PBMC HCV RNA in patients under IFN is not predictive of sustained viral loss; 4. testing for PBMC viral sequences might enhance the chances of detecting HCV infection.     

慢性乙型肝炎患者外周血单个核细胞内HBV DNA存在的意义

目的探讨外周血单个核细胞（PBMC）内HBVDNA与HBV标志物的关系及在慢性肝病发展过程中的作用．方法应用RCR技术结合斑点杂交技术测定血清HBVDNA及PBMC内HBVDNA血清学标志物采用ELISA法结果在HBsAg＋，HBeAg十组48例中，血清与PBMC内HBVDNA阳性检出率分别为93．8％和83．3％；在HBsAg＋，抗-HBe 组41例中，其阳性检出率分别为48．8％和58．5％.两组间存在显著性差异（P＜0．01）在其他各组中血清及PBMC内HBVDNA检出率均较抵有6例呈单纯PBMC内HBVDNA阳性.在不同类型慢性乙肝患者中，CSH和CMoH组PBMC-HBVDNA检出率分别为78．6％和61．8％，与CMiH组（25．0％）、ASC组（14．3％）比较，存在非常显著性差异（P＜0．01），呈随病情加重而检出率增高的趋势结论PBMC清除HBVDNA较血液缓慢，在病毒持续感染及造成肝细胞损伤过程中可能起一定作用．     

外周血单核细胞中丙型肝炎病毒RNA正负链检测的临床意义

目的通过对外周血单核细胞（ＰＢＭＣ）中ＨＣＶＲＮＡ正负链的检测,来探讨其与丙型肝炎慢性化及干扰素治疗的关系．方法慢性丙型肝炎患者４０例,其中干扰素治疗者１０例,分离血清及ＰＢＭＣ．异硫氰酸胍－酚－氯仿抽提法提取ＨＣＶＲＮＡ,应用逆转录－巢式ＰＣＲ技术检测ＨＣＶＲＮＡ正负链．结果血清正链ＨＣＶＲＮＡ阳性率为６７５％,但负链均为阴性,ＰＢＭＣ中正链ＨＣＶＲＮＡ阳性率为５７５％,负链的阳性率为３５０％．其中３例患者血清中正链ＨＣＶＲＮＡ为阴性,而ＰＢＭＣ中为阳性．１０例干扰素治疗者在治疗结束时血清正链ＨＣＶＲＮＡ６０％转阴,ＰＢＭＣ中负链ＨＣＶＲＮＡ８０％转阴,而正链仅３７５％转阴．结论ＨＣＶ能在ＰＢＭＣ中存在和复制,这可能是导致丙型肝炎易发生慢性化的原因之一．ＰＢＭＣ中ＨＣＶＲＮＡ正负链的检测对于临床判断干扰素的疗效及预后有重要意义     

Correlation of serum HCV RNA and alanine aminotransferase levels in chronic hepatitis C patients during treatment with ribavirin

to evaluate the effect of ribavirin on serum hepatitis C virus (HCV) RNA and alanine aminotransferase (ALT) levels, 22 patients with chronic HCV infection were treated with oral ribavirin 1200 mg daily in three divided doses for 4 weeks. At the end of 4 weeks treatment, the serum ALT decreased in all but one patient and became normal in three individuals. The mean pretreatment serum ALT was reduced significantly from 193 ± 45 i.u./L to 95 ± 16 i.u./L after 4 weeks therapy (P= 0.009). However, 8 weeks after cessation of treatment, the serum ALT rose to a mean value of 154 ± 21 i.u./L. The mean pretreatment serum HCV RNA was not significantly decreased at the end of 4 weeks treatment (7.0 × 10⁵vs 4.1 × 10⁵ copies/mL, P > 0.05). However, serum HCV RNA levels were decreased in 12 and increased in 10 patients at the end of 4 weeks therapy. Eight weeks after cessation of therapy, the serum HCV RNA of 22 patients rose to a mean value of 4.9 ± 10⁵ copies/mL. Six patients who continued to have elevated serum ALT and positive HCV RNA after the initial 4 weeks treatment received oral ribavirin at the same dosage for an additional 24 weeks. The serum ALT again decreased in all six patients during therapy, but rose to pretreatment values by 8 weeks after cessation of the treatment. In addition, no significant changes were noted in the mean serum HCV RNA levels during and after 24 weeks of ribavirin therapy. Our results indicate that oral ribavirin only transiently lowered serum ALT values and did not efficiently suppress HCV synthesis in patients with chronic hepatitits C infection.     

Quantitative assessment of hepatitis C virus RNA in peripheral blood mononuclear cells during therapy with interferon-α2a

A significant number of patients with hepatitis C (HCV) treated with interferon (IFN) will initially clear their serum of HCV RNA, but will then have recurrence of viraemia either during or after therapy. One proposed mechanism for relapse is that HCV may persist in peripheral blood mononuclear cells (PBMCs) and that the PBMCs serve as a 'viral reservoir' that is resistant to IFN. To address this hypothesis, we performed serial, quantitative polymerase chain reaction (PCR) of HCV RNA in serum and PBMCs from 26 consecutive patients treated with IFN-α2a. Of the 26 patients, 11 (42%) did not clear virus from their serum during therapy and were termed non-responders. Five patients (19%) had sustained clearance of virus from serum and were termed complete responders. The remaining 10 patients (39%) initially eliminated HCV RNA from their serum, but had relapse of viraemia. They were termed partial responders. In all 10 partial responders HCV RNA was undetectable in PBMCs at the same time that it was undetectable in serum. When virus recurred in serum, it was preceded by or occurred at the same time as the return of virus in PBMCs. The results of our study indicate that PBMCs did not serve as an IFN-resistant 'viral reservoir' during therapy. Partial responders who transiently cleared virus from serum also cleared virus from PBMCs and the presence or titre of HCV RNA in PBMCs at the initiation of therapy did not predict response to therapy.     

Hepatitis C virus RNA genome in plasma of patients with non-A,non-B hepatitis

Recently, the assay system of anti-hepatitis C virus antibody (HCV-Ab) was developed. However, there is no clinically useful method to detect hepatitis C virus (HCV) itself. The authors recently developed a method to detect the HCV-RNA genome in plasma using polymerase chain reaction (PCR). In the present study, the specificity of this assay in detecting HCV infection was investigated. Freshly obtained 1 ml plasma specimens from 100 patients with various liver diseases and from 11 control subjects were studied. In patients with non-A, non-B (NANB) hepatitis-related liver diseases, HCV-RNA was detected in 2 out of 7 cases of acute hepatitis, in 29 out of 31 cases of chronic hepatitis, in 17 out of 21 cases of cirrhosis and in 2 out of 6 cases of hepatocellular carcinoma. On the other hand, no HCV-RNA was detected in 15 cases of various types of alcoholic liver diseases, in 12 cases of hepatitis B related liver diseases, and in 11 controls. HCV-RNA was detected in 2 of 6 drinkers with chronic hepatitis. The prevalence of HCV-RNA was not closely related to a history of blood transfusions. These results suggest that our method for HCV-RNA is specific for HCV infection and HCV infection is the likely etiology of most chronic NANB hepatitis cases. The clinical usefulness of our method is illustrated by the fact that we were able to study 100 patients and needed only 1 ml plasma per HCV-RNA assay.     

Hepatitis C virus RNA and long-term response to recombinant interferon-α2b in patients with chronic hepatitis C

Summary. The effectiveness of recombinant interferon-α2b (rIFN-α2b) in eradicating hepatitis C virus (HCV) RNA from serum has not been completely assessed. We studied 39 patients with compensated chronic hepatitis C diagnosed by liver biopsy and positive HCV RNA measured by polymerase chain reaction (PCR). Group I consisted of 26 patients treated with 3 MU of rIFN-α2b for 6 months; group II, 13 control patients observed for six months; and group III, 12 out of 13 patients from group n who subsequently received 5 MU of rIFN-α2b for 6 months. In group I, 11 out of 23 (47.8%) patients who completed treatment had an immediate response and five (21.7%) had a sustained response to therapy six months after treatment. No response was observed in patients from group II. In group III, 7 out of 12 (58.3%) patients who completed treatment had an immediate response and none had a sustained response. Considering all patients who completed rIFN-α2b treatment, HCV RNA remained positive at the end of therapy in three of five sustained responders (60%), six of 13 patients who relapsed (46.1%), and in all non-responders (100%). HCV RNA was positive 6 months after therapy in four (80%), 13 (100%). and 17 (100%) patients respectively. All patients with a sustained response had normal aminotransferase levels 18 months after therapy. We conclude that in chronic hepatitis C rIFN-α2b causes a significant immediate response but this is not sustained, only 2.8% of treated patients had a sustained loss of HCV RNA. Normal aminotransferase persist in the long term, despite persistence of HCV RNA.     

实时荧光定量RT-PCR检测血清和单个核细胞中HCV RNA的意义

目的探讨实时荧光定量聚合酶链反应(FQ-RT-PCR)检测血清和外周血单个核细胞(PBMCs)中HCV RNA含量及其临床价值。方法采集可疑丙型病毒性肝炎病人血,用FQ-RT-PCR分别检测血清和PBMCs中HCV RNA含量。血清或PBMCS HCV RNA阳性者都视为HCV RNA阳性。结果检测可疑患者180例,HCV RNA阳性106例。其中血清HCV RNA阳性84例,占79.25%;PBMCs HCV RNA阳性63例,占59.43%。HCV RNA阳性比率血清高于PBMCs(χ2=9.78,P<0.01)。单独血清HCV RNA阳性43例,占40.57%;单独PBMCs HCV RNA阳性22例,占20.75%;血清和PBMCs中HCV RNA同时阳性41例,占38.68%。血清和PBMCs HCV RNA含量差异无显著性。结论同时检测血清和PBMCs HCV RNA可提高HCV感染诊断的阳性率,检测PBMCs HCV RNA对抗病毒治疗的疗效评价及治疗时间有重要意义。     

Hepatitis C virus RNA quantification in right and left lobes of the liver in patients with chronic hepatitis C

Summary. Quantification of hepatitis C virus RNA in liver tissue is likely to be useful in the study of the natural history, pathogenesis, progression and treatment of hepatitis C virus-associated liver disease. Quantitative measurements of hepatitis C virus RNA in liver biopsy samples using the branched DNA (bDNA) signal amplification assay were carried out. The aims of this study were threefold: first, to assess the level of hepatitis C virus RNA in biopsy samples from the right and left lobes of the liver; second, to evaluate the correlation between hepatitis C virus RNA levels in serum and liver; and third, to investigate the relationship between serum and liver hepatitis C virus RNA levels and the severity of hepatic histology in non-cirrhotic patients with chronic hepatitis C. There was a strong correlation ( r = 0.92, P < 0.01) between hepatitis C virus RNA levels in the right and left lobes of the liver as well as a strong correlation between hepatitis C virus RNA levels in liver and serum ( r = 0.82, P < 0.01). However, there was no significant correlation between the severity of hepatic histology and levels of hepatitis C virus RNA in serum and liver among patients with chronic active hepatitis classified according to Knodell's hepatic activity index (KI). Our results indicate that hepatitis C virus RNA quantification from a single liver biopsy is representative of both lobes in patients with chronic hepatitis, and suggest that serum hepatitis C virus RNA levels are a meaningful reflection of hepatitis C virus RNA levels in the liver.