Concise Review in Mechanisms of Disease
Extrahepatic replication of HCV: Insights into clinical manifestations and biological consequences†
Potential conflict of interest: Nothing to report.
An estimated 170 million persons are infected with the hepatitis C virus (HCV) worldwide. While hepatocytes are the major site of infection, a broad clinical spectrum of extrahepatic complications and diseases are associated with chronic HCV infection, highlighting the involvement of HCV in a variety of non-hepatic pathogenic processes. There is a growing body of evidence to suggest that HCV can replicate efficiently in extrahepatic tissues and cell types, including peripheral blood mononuclear cells. Nonetheless, laboratory confirmation of HCV replication in extrahepatic sites is fraught with technical challenges, and in vitro systems to investigate extrahepatic replication of HCV are severely limited. Thus, future studies of extrahepatic replication should combine innovative in vitro assays with a prospective cohort design to maximize our understanding of this important phenomenon to the pathogenesis and treatment response rates of HCV. (HEPATOLOGY 2006;44:15–22.)
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An estimated 170 million persons are infected with the hepatitis C virus (HCV) worldwide. Infection with HCV is a major cause of chronic liver disease, with >75% of patients developing chronic hepatitis and 20% of chronically infected persons developing cirrhosis.1, 2 HCV infection is also a major cause of hepatocellular carcinoma and the primary reason for liver transplantation in the United States. While hepatocytes are the major site of viral replication, a broad clinical spectrum of extrahepatic complications and diseases are associated with chronic HCV infection,3–6 including mixed cryoglobulinemia, non-Hodgkin's lymphoma, cutaneous vasculitis, glomerulonephritis, neuropathy, and lymphoproliferative disorders to highlight just a few (Table 1). Extrahepatic manifestations are quite common among patients infected with HCV. In a large prospective cohort study, the MULTIVIRC group reported that 74% of patients had a least one clinical extrahepatic manifestation.7 The most commonly identified manifestations were mixed cryoglobulinemia (MC) (40%), arthralgia (23%), paresthesia (17%), myalgia (15%), pruritus (15%), and sicca syndrome (11%). Several of the most clinically significant extrahepatic manifestations of HCV are as follows:
Table 1. Clinical Manifestations Associated With HCV Disease
|Porphyria cutanea tarda|
Intermittent purpura of the lower extremities is the most common clinical finding and is a direct consequence of immune complex deposition in the dermal capillaries. A sensation of cutaneous burns occasionally precedes its appearance. It is usually benign and self-limited with resolution of symptoms within a week, although the vasculitic process will progress to necrotizing skin lesions with ulcerations in approximately 10% of patients. The severity of vasculitis correlates with the level of HCV viremia but not with the level of serum cryoglobulins. Skin biopsy confirms the diagnosis with the classic finding of leukocytoclastic vasculitis. Cutaneous vasculitis is the MC manifestation that responds best to antiviral therapy with pegylated interferon (PEG-IFN) and ribavirin (RBV).8, 9
The association between HCV infection and renal disease is well established. Membranoproliferative glomerulonephritis (MPGN) is the most common extrahepatic manifestation of HCV that involves the kidneys, representing 70 to 80% of HCV-associated glomerulonephritis. The prevalence of HCV infection in MPGN is 10 to 20% in the United States.10 There are two forms of HCV-associated MPGN described in the literature: cryoglobulin-related and non-cryoglobulin renal disease. Irrespective of the form, the most common presentation is proteinuria and microscopic hematuria.11 The clinical course of MPGN is variable with approximately 30% of patients having complete or partial remission, 30% with intermittent exacerbation and remission, 30% with an indolent course, and 10% progressing to chronic renal failure.12 Therapy with PEG-IFN + RBV has proven to be effective in some patients with a significant improvement in proteinuria and serum creatinine levels.13 Other therapies have achieved variable clinical response rates.14, 15
Neurologic manifestations in HCV-infected patients occur predominantly in the peripheral nervous system and may affect as many as 50% of cases.16 The prevalence of peripheral nerve involvement in patients with MC ranges between 20 and 35%, with clinical manifestations resulting from immune complex deposits within vasa nervorum of the peripheral nerves leading to vasculitis.17, 18 At least 80% with neurologic manifestations present with peripheral neuropathy in the form of a symmetrical distal polyneuropathy that involves the legs and is typically very painful, with progression to muscle weakness in some patients. The clinical course is progressive and response to antiviral therapy and plasmapheresis has been discouraging.19
Recent studies have found a high prevalence of lymphoproliferative disorders (LPD) in HCV-infected patients.20 Although there is limited data to suggest that HCV replication occurs in B lymphocytes,21–23 chronic antigenic stimulation by the virus may trigger B cell proliferation resulting in a wide spectrum of pathology ranging from minor expansion of B cell populations to an aggressive high-grade lymphoma. A high prevalence of HCV in patients with B cell non-Hodgkin's lymphoma (NHL) has been reported in some, but not all studies.24 A recent meta-analysis has also confirmed this strong positive association between HCV and the risk of NHL,20 although mechanistic studies are still lacking. In HCV-infected patients, the vast majority of NHL is low grade with predominantly extranodal involvement of organs such as the liver, spleen, salivary glands, and stomach.25 The optimal treatment of the HCV-related B-cell NHL is not clear; however, emerging data of remission of LPD after HCV eradication in some subsets of splenic and gastric mucosa-associated lymphoid tissue (MALT) lymphomas are encouraging.26
Over the last decade, epidemiological data have demonstrated a possible link between porphyria cutanea tarda, lichen planus, Sjögren's syndrome and HCV.27 More recently, HCV has been associated with fatigue28 and insulin resistance though this association is weak.29, 30 HCV has also been implicated in the pathogenesis of acquired aplastic anemia, although this hematological disorder is uncommon in HCV infected individuals and the available evidence to support a causal relationship is very limited.31, 32 A greater knowledge of HCV replication in non-hepatic organs will undoubtedly provide useful insights into the pathogenic processes by which HCV is involved in these extrahepatic manifestations.
Detection of Extrahepatic Replication
Because positive-sense HCV RNA resides within individual virions, the mere detection of positive-sense HCV RNA in cell preparations does not definitively prove replication. Thus, the most important indicator of HCV genomic replication is the production of negative-strand HCV RNA, as sustained viral replication within a given cellular environment can only be maintained through the constant production of replicative intermediate molecules.33 A predominance of positive-strand over negative-strand HCV RNA occurs during intrahepatic replication of HCV34–36; however, the various factors that control this asymmetry are not well described, and this phenomenon has not been adequately characterized in non-hepatic cell types.
While traditional HCV “viral load” assays quantify HCV RNA in the serum, they are unable to distinguish positive- from negative-strand HCV RNA and, thus have limited utility in assessing extrahepatic replication. Nonetheless, highly sensitive detection methods are continuously being developed and should facilitate a more accurate assessment of the true extent of HCV infection/replication in a variety of cell types and tissues.37, 38 To date, extrahepatic replication of HCV has been explored using a plethora of cell culture and molecular biological assays. The use of “strand-specific” reverse transcriptase polymerase chain reaction (RT-PCR) to detect negative-strand HCV RNA is often cited as evidence for viral replication. However, due to the potential for contamination and the lack of strand-specificity, “standard” RT-PCR assays should not be used to characterize extrahepatic replication of HCV.33 A new approach has been to enhance the strand specificity of RT-PCR through the use of recombinant Tth enzyme, which has independent reverse transcriptase and polymerase activities.39 Because of the 10,000-fold ability of Tth to discriminate between specific strands, it is ideal for the specific detection of negative-strand HCV RNA and has quickly replaced several of the previous, less-specific PCR-based strategies. Real-time, strand-specific PCR assays utilizing Tth are also now available.35, 40, 41
Recently, the development of self-replicating HCV RNA replicons has revolutionized the study of HCV;42 however, these systems are restricted to a single, highly permissive hepatocyte-derived cell line.43, 44 Only within the last year have HCV systems that support infectious HCV production been reported,45–48 although they do not support HCV replication in non-hepatocyte-derived cells. Consequently, additional systems to study sustained viral replication in multiple hepatocyte- and non-hepatocyte-derived cells are critically needed to advance the molecular characterization of extrahepatic replication of HCV and its role in the progression of the disease.
Several complementary strategies to investigate extrahepatic replication of HCV include DNA-based expression systems,41, 49–53 lymphocyte cell lines that constitutively express HCV,21, 23, 54 peripheral blood mononuclear cell (PBMC) cultures from HCV-infected persons,21, 55, 56 direct viral infection of extrahepatic cell types with HCV-infected sera,35, 57–60in situ hybridization and immunohistochemistry,38, 61–64 and laser capture microdissection.37, 65 The relevance of each of these strategies to extrahepatic replication of HCV is not currently known and can only be determined through extensive validation and cross-comparison amongst all available methodologies.
Evidence for extrahepatic Replication of HCV
HCV binds several cell surface receptors,66 although cell tropism and the specific host determinants required for HCV genome replication are not well characterized. Thus, it is difficult to determine a priori which cell types will support HCV replication, as indicated by the synthesis and accumulation of HCV negative-strand RNAs. Despite these limitations, extrahepatic replication of HCV has been documented extensively in vivo. For instance, negative-strand HCV RNA has been identified in a variety of non-hepatic tissues (Table 2), including the pancreas, thyroid, bone marrow, adrenal gland, spleen, lymph node, cervicovaginal fluid, and brain.29, 67–72
Table 2. Detection of Negative-Strand HCV RNA in Extrahepatic Tissues
|Tth-based assays|| || |
|Chronic HCV||Not detected in spleen, pancreas, kidney, muscle, lymph node, or bone marrow tissues from infected chimpanzees||109|
|HIV/AIDS||Pancreas and thyroid||67|
|HIV/AIDS||Bone marrow, lymph node, pancreas, thyroid, adrenal gland, and spleen; Not detected in kidney, lung, muscle, or spinal cord tissues||68|
|HIV/AIDS||Lymph node, pancreas, and adrenal gland; Not detected in bone marrow, thyroid, muscle, kidney, skin, or lung tissues||69|
|Chronic HCV||Peripheral blood dendritic cells||79|
|Outpatient hematology patients||Bone marrow||70|
|Chronic HCV||Not detected in leukocytes||110|
|Virologic responders||Lymphocytes and macrophages||80|
|Chronic HCV*||Granulocytes, macrophages, and B lymphocytes; Not detected in T lymphocytes or thrombocytes||81|
Initial studies similarly reported extrahepatic replication of HCV in PBMCs (Table 3).58, 73–77 However, as the detection assays were not able to adequately differentiate between positive- and negative-strand HCV RNAs, theses results should be interpreted with extreme caution. Subsequent studies have now convincingly demonstrated negative-strand HCV RNA - albeit in a minority of PBMCs tested - particularly among immunosuppressed patients, such as HIV/AIDS patients68, 69, 71 and liver transplant recipients.63, 78 There is also a growing body of evidence to suggest that HCV replication may occur within peripheral dendritic cells, granulocytes, B lymphocytes, and monocytes/macrophages.79–81 While some of these data should be viewed cautiously, as a variety of techniques were utilized and a limited number of samples were analyzed, they are intriguing in light of the extrahepatic manifestations of HCV reviewed earlier.
Table 3. Detection of Negative-Strand HCV RNA in PBMCs
|Tth-based assays|| || || |
| ||Chronic HCV||0 of 10 PBMCs (human), 0 of 5 PBMCs (chimpanzee), 5 of 5 livers||109|
| ||Chronic HCV||0 of 27 PBMCs||111|
| ||HIV/AIDS||0 of 5 PBMCs, 3 of 3 livers||67|
| ||HIV/AIDS||2 of 5 PBMCs, 2 of 2 livers||69|
| ||Chronic HCV||0 of 4 PBMCs, 2 of 3 livers||93|
| ||Chronic HCV||0 of 16 PBMCs||79|
| ||HIV/AIDS||5 of 14 PBMCs||71|
| ||Virologic responders||1 of 17 PBMCs||80|
| ||Hemodialysis patients||5 of 45 PBMCs||98|
| ||Virologic responders||9 f 12 PBMCs, 8 of 10 livers||102|
| ||Chronic HCV and/or HIV/AIDS||17 of 45 PBMCs||40|
| ||Occult HCV||11 of 18 PBMCs||101|
|Not Tth-based assays|| || || |
|Taq-based||Chronic hepatitis HCV/HBV||98 of 154 with HCV mono-infection, 14 of 54 with HBV/HCV co-infection||112|
|Tagged RT-PCR||HCV chronic||2 of 26 PBMCs, 16 of 20 livers||113|
|Taq-based||Chronic HCV||1 of 46 PBMCs||114|
|Taq-based||Chronic HCV||15 of 22 PBMCs, 11 of 25 livers||100|
|Taq-based||Chronic HCV||13 of 20 PBMCs||99|
|CAP assay||Chronic HCV||3 of 11 PBMCs||81|
|Taq-based||Chronic HCV||33 of 106 PBMCs||97|
|Enzyme not described||Acute and chronic HCV||14 of 35 chronics, 1 of 19 acutes||103|
|Tagged primer||Chronic HCV||1 of 10 PBMCs (dendritic cell subpopulation)||115|
Pioneering studies by Shimizu et al. demonstrated that HCV infection of lymphocytes occurs both in vitro and in vivo.82–84 Interestingly, sequence analysis has subsequently revealed that certain viral variants may be selected for growth in extrahepatic cell types, implying that HCV diversity directly impacts cell tropism.85, 86 Furthermore, several studies have described a non-random distribution of HCV sequences in hepatic and extrahepatic compartments,72, 87–96 leading to the conclusion that the presence of tissue-specific sequences is compatible with independent viral replication in extrahepatic sites. It has also been speculated that HCV variants within distinct compartments may differ in their sensitivity to interferon, although this hypothesis has not been formally tested. Viral sequence evolution after HCV therapy cessation could also suggest emergence from a protected compartment, although this would require extensive characterization of viruses before and after treatment in multiple tissue/cellular compartments. The potential correlation between extrahepatic replication and HCV genotype has also been examined,81, 97 with at least one study suggesting that negative-strand HCV RNA may be more frequently detected in the PBMCs from HCV genotype 1-infected persons.81 Nonetheless, these data will require rigorous validation in larger clinical populations and additional follow-up data to determine if certain HCV genotypes are more frequently associated with the development of specific extrahepatic manifestations.
Clinical Implications of Extrahepatic Replication
To date, prospective studies of extrahepatic replication are missing from the available literature. Nonetheless, retrospective studies suggest several potential associations between extrahepatic replication of HCV and important clinical outcomes. For example, several studies have found that HCV RNA may be present in PBMCs but not the corresponding serum or liver.98–101 Thus, current detection systems may underestimate the true extent of replication. Moreover, HCV RNA, including replicative intermediate forms, can persist at very low levels in peripheral lymphoid cells for many years after apparently complete spontaneous or treatment-induced resolution of chronic HCV,102 strongly suggesting that sub-populations within PBMCs may represent true reservoirs of HCV replication. Interestingly, patients with detectable negative-strand HCV RNA in PBMCs had lower interferon (IFN) sustained response rates compared to those without detectable negative-strand HCV RNA in PBMCs.103 Thus, it is provocative to speculate that low-level replication of HCV in PBMCs may lead to reactivation of HCV after termination of therapy and/or predict response to therapy.80, 99, 100, 102–105
As reviewed elsewhere, HCV is likely involved in several neurologic syndromes.16 While central nervous system (CNS) involvement is less frequent, the detection of negative-strand HCV RNA in the CNS29 suggests a potential link between HCV and these extrahepatic pathologies. However, the underlying biological mechanisms have not been elucidated. Similarly, the detection of HCV RNA in cervical lymphocytes106 and HCV compartmentalization within cervicovaginal fluid72 suggests that this compartment may contribute to the sexual transmission of HCV. Nonetheless, few studies have examined the temporal relationship between HCV RNA levels in extrahepatic compartments and the development of specific extrahepatic complications, although successful treatment of HCV is frequently associated with resolution of several extrahepatic complications of HCV infection.8, 9, 13, 16, 26
The proposed pathogenic processes by which HCV may result in the extrahepatic manifestations described above have been described in detail elsewhere.4–6, 107 Importantly, multiple immunologic, virologic, and environmental factors are likely involved. For instance, given that many extrahepatic manifestations are lymphoproliferative disorders and/or autoimmune disorders, it is tempting to speculate that the lymphotropism of HCV may be an important contributing factor. Moreover, it has long been hypothesized that chronic antigen stimulation by HCV activates B cells and may ultimately result in cryoglobulinemia and non-Hodgkin lymphoma.5 Furthermore, specific genomic regions, such as the 5′UTR and HVR-1, have also been identified that may impact cell tropism,85, 86 thereby influencing the extent of extrahepatic replication. There is also evidence to suggest that the HCV core protein can regulate various cellular oncogenes and viral promoters that regulate transformation and/or carcinogenesis.108
Extrahepatic Replication: the Research Road Ahead
Until recently, the existence of extrahepatic reservoirs of HCV has been somewhat controversial due to conflicting findings in vivo. A number of current reports now suggest that extrahepatic replication of HCV does indeed occur and that it may have profound effects on HCV treatment and disease pathogenesis. Nevertheless, a focused research agenda is still lacking with several critical research areas remaining to be explored.
While epidemiological studies provide important data regarding possible associations, it is essential that the link between HCV replication and development of extrahepatic diseases be examined at a mechanistic level to understand the precise pathogenic processes that underlie these clinical outcomes. This is a large field of research that includes characterization of the pathogenic mechanisms by which HCV can lead to the development of lymphomas, the mechanisms that link HCV to insulin resistance and diabetes, and the association of HCV with autoimmune-mediated thyroid diseases. Additional prospective studies could further define the true extent of extrahepatic replication of HCV and the subsequent development of extrahepatic manifestations by measuring multiple virological, immunological, and clinical parameters in large, well-characterized cohorts. In the absence of robust culture systems that permit detailed examinations of HCV replication in extrahepatic cell types, such studies are also absolutely critical to demonstrate a causal relationship between viral replication in specific extrahepatic compartments and the subsequent development of certain extrahepatic manifestations of HCV. Moreover, several groups have suggested that extrahepatic replication is associated with treatment outcome, although this has not been formally tested in large clinical cohorts receiving HCV therapy. In such cohorts, the association of baseline detection of negative-strand HCV RNA, as well as a more rigorous examination of HCV viral variants present in extrahepatic compartments, should be examined as possible predictors of treatment failure. Finally, additional in vitro replication systems that allow direct comparison of HCV replication among a variety of cell types of hepatic and extrahepatic origin will be absolutely essential to elucidate the complex pathogenic processes by which extrahepatic HCV occurs and results in a myriad of disease manifestations.