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Does HCV Replicate Outside The Liver and It's Significance?
 
Reported by Jules Levin
 
  Some individuals with HCV report experiencing fatigue, emotional distress, and cognitive impairment. Objective testing has found HCV can be associated with these symptoms. Some doctors believe these symptoms may be associated with causes other than HCV, such as prior alcohol or drug use, or other psychiatric disturbances; and some studies suggest HCV can enter the CSF and the brain. In HIV, it has been established that HIV can reside and replicate in the brain. Another way in which HIV or HCV may affect the brain is by disturbances caused to the immune system, disruptions in cytokine balance. Disruptions in the immune system can affect the brain and other organs. So, can HCV reside and replicate in other organs other than the liver? This question is the subject of previously reported and ongoing studies. This article reports on previous study findings and tries to put these questions in perspective.
 
Two studies reported at AASLD (Nov 2002) discuss HCV and it's possible presence in the brain suggesting implications for the presence of HCV extra-hepatically, outside the liver. Results from study in England suggests HCV replicates in the brain (abstract 185, Forton et al). Researchers at the Mayo Clinic reported finding HCV in the cerebrospinal fluid and in PBMCs of patients with HCV in serum, suggesting the PBMCs may carry HCV into the neurologic system; this study is detailed at http://www.natap.org/2002/AASLD/day15.htm
 
So, does HCV replicate and is it found outside the liver? Results are mixed from various studies conducted over recent years, and some of these studies are reported below. Apparently, this question has not been answered and whether HCV exists outside the liver is not resolved. In fact, there are enough reasons to think HCV may not reside in reservoirs outside the liver as there are to think there are reservoirs outside the liver. The first article below talks about extrahepatic manifestations of HCV. It is clear that patients with HCV experience skin, renal, hematologic, and arthritic disorders, and having these disorders is associated with having HCV. The study is one of many supporting this and it is well accepted that HCV is associated with these extrahepatic disorders. However, it is distinctly possible that this is due to HCV causing cytokine disruptions, dysregulation in the immune system, which may in turn lead to these disorders; rather thah due to HCV replicating in cells and organs outside the liver. Apparently, the studies that have found HCV in cells and organs outside the liver use lab testing methods and techniques which are not yet refined enough to be completely accepted by researchers. It appears researchers feel these test methods need further evaluation and confirmation.
 
In the end, it appears unresolved whether HCV can replicate or exists outside the liver. In addition, it may be irrelevant even if it does replicate and exist outside the liver. In HIV, reservoirs have been identified in patients who achieve and sustain undetectable HIV viral load (<50 copies/ml of HIV viral load). In addition, these patients can sustain undetectability and maintain good health for many years despite these reservoirs. It remains unanswered whether these patients will see their HIV viral load rebound due to the existence of reservoirs. So, does the existence of HCV reservoirs matter, if they do exist? Perhaps not. Studies of patients with HCV who have sustained viral responses for as long as 11 years have found no HCV in the blood or liver. Presumably, if HCV reservoirs persist these individuals would have reservoirs. Yet these patients remained undetectable. If HCV reservoirs do in fact exist, perhaps achieving sustained response eradicates HCV in these reservoirs. Perhaps patients who relapse after achieving end-of-treatment responses from IFN/RBV do so because there are reservoirs. These questions remained unanswered. But, ultimately what appears to matter is whether or not a patient can achieve and sustain a sustained viral response. If they can, studies show no HCV in the blood and liver. Of course, we do in fact need long term and larger studies to confirm that SVRs are sustainable and to evaluate the long term health outcomes for patients who sustain the SVR.
 
Extrahepatic manifestations of hepatitis C among United States male veterans
 
Hepatitis C virus (HCV) has been associated with several extrahepatic conditions. To date, most studies assessing these associations involved small numbers of patients and lacked a control group. Using the computerized databases of the Department of Veterans Affairs, we carried out a hospital-based case-control study that examined all cases of HCV-infected patients hospitalized during 1992 to 1999 (n = 34,204) and randomly chosen control subjects without HCV (n = 136,816) matched with cases on the year of admission. The inpatient and outpatient files were searched for several disorders involving the skin (porphyria cutanea tarda [PCT], vitiligo, and lichen planus); renal (membranous glomerulonephritis [GN] and membranoproliferative glomerulonephritis); hematologic (cryoglobulin, Hodgkin's and non-Hodgkin's lymphoma [NHL]); endocrine (diabetes, thyroiditis); and rheumatologic (Sjögren's syndrome). The association between HCV and these disorders was examined in multivariate analyses that controlled for age, gender, ethnicity, and period of military service. Patients in the case group were younger in age (45 vs. 57 years), were more frequently nonwhite (39.6% vs. 26.3%), and were more frequently male (98.1% vs. 97.0%). A significantly greater proportion of HCV-infected patients had PCT, vitiligo, lichen planus, and cryoglobulinemia. There was a greater prevalence of membranoproliferative GN among patients with HCV but not membranous GN. There was no significant difference in the prevalence of thyroiditis, Sjögren's syndrome, or Hodgkin's or NHL. However, NHL became significant after age adjustment. Diabetes was more prevalent in controls than cases, but no statistically significant association was found after adjustment for age. In conclusion, we found a significant association between HCV infection and PCT, lichen planus, vitiligo, cryoglobulinemia, membranoproliferative GN, and NHL. Patients presenting with these disorders should be tested for HCV infection. (HEPATOLOGY 2002;36:1439-1445.)
 
Search for Hepatitis C Virus Extrahepatic Replication Sites in Patients With Acquired Immunodeficiency Syndrome: Specific Detection of Negative-Strand Viral RNA in Various Tissues
 
The existence of extrahepatic reservoirs of hepatitis C virus (HCV) replication remains highly controversial. We searched for the presence of HCV-RNA negative strand in various tissues from eight HCV-infected patients who died of acquired immunodeficiency syndrome (AIDS)-related complications. Negative-strand RNA was detected by a Tth-based reverse-transcriptase polymerase chain reaction (RT-PCR), which was optimized for sensitivity and strand specificity on synthetic RNA templates. This assay was capable of detecting about 102 genomic Eq molecules of the correct strand while unspecifically detecting ≥108 genomic Eq molecules of the incorrect strand. Negative-strand viral RNA was detected in all but one liver, in lymph nodes (5 cases), in pancreas (5 cases), in adrenal gland (2 cases), in thyroid (2 cases), in bone marrow (1 case), and in spleen (1 case). These data suggest a possible presence of HCV replication sites outside the liver, at least in AIDS patients. Whether these findings relate to various extrahepatic manifestations of HCV infection remains to be determined. (Hepatology 1998;28:1398-1401.)
 
The existence of extrahepatic reservoirs of hepatitis C virus (HCV) replication remains highly controversial. Several groups have reported the detection of HCV-RNA negative strand, a viral replicative intermediate, in peripheral blood mononuclear cells (PBMCs); however, the validity of these studies has been recently questioned, because it was demonstrated that reverse-transcriptase polymerase chain reaction (RT-PCR) used for the detection of HCV RNA is not strand-specific. Thus, subsequent studies using assays optimized for strand specificity either failed to demonstrate the presence of viral negative strand in PBMCs or found it to be a very rare event.
 
Even less is known about HCV replication in other extrahepatic sites, because studies using strand-specific assays are few; Landford et al. failed to detect HCV-RNA negative strand in multiple organs from a chronically infected chimpanzee, while Lerat et al. did not detect any evidence of viral replication in fresh bone marrow cells from six patients with chronic hepatitis. However, both studies have serious limitations: the former was conducted on a single ape that had a low level of HCV replication in the first place, while in the latter study, no tissues other than bone marrow were examined. Obviously, the existence of extrahepatic HCV replication sites would have broad implications for antiviral treatment and our understanding of multiple extrahepatic manifestations of HCV infection, the pathogenesis of which is currently obscure.
 
The major obstacle to a study of extrahepatic HCV replication is the availability of multiple tissues from infected individuals. Apparently, such samples could be obtained only during autopsy of patients with chronic infection. We reasoned that such an investigation could be conducted on postmortem tissues from intravenous drug addicts dying from acquired immunodeficiency syndrome (AIDS), because HCV infection in this group is almost uniform, and viral titers in human immunodeficiency virus (HIV)-infected subjects are usually elevated.
 
In the current study, we searched for the presence of HCV-RNA negative strand in various tissues from eight HCV-positive drug addicts who died of AIDS-related complications. To avoid mispriming events during RT-PCR, cDNA synthesis was conducted at a high temperature with the thermostable enzyme, Tth. We have recently used such a Tth-based RT-PCR for the identification of hepatitis G replication sites. The sensitivity and specificity of our assay was determined using synthetic RNA templates.
 
We studied tissue samples from eight HIV-1-infected drug addicts who died from AIDS-related complications between March and June 1997. All eight patients were anti-HCV-positive and hepatitis G virus (HGV) RNA-negative in serum; their CD4 count at the time of admission was below 200 cells per cubic millimeter. Tissue samples were obtained during routine autopsy conducted within 48 hours of death and stored at -80°C until analysis. The study was conducted in accordance with institutional IRB requirements.
 
Samples of the following tissues were collected postmortem from each patient: liver, bone marrow, mediastinal lymph node, pancreas, thyroid, adrenal gland, kidney, lung, skeletal muscle, spleen, and spinal cord. Serum samples were drawn within the last week of the patient's life; however, in two cases (patients 1 and 5), they were collected a few hours before death, and in one subject (patient 4), serum was collected at death. In addition, in four cases (patients 2, 3, 6, and 7), an additional serum sample was collected at autopsy.
 
We studied tissue samples from eight HIV-1-infected drug addicts who died from AIDS-related complications between March and June 1997. All eight patients were anti-HCV-positive and hepatitis G virus (HGV) RNA-negative in serum; their CD4 count at the time of admission was below 200 cells per cubic millimeter. Tissue samples were obtained during routine autopsy conducted within 48 hours of death and stored at -80°C until analysis. The study was conducted in accordance with institutional IRB requirements.
 
Samples of the following tissues were collected postmortem from each patient: liver, bone marrow, mediastinal lymph node, pancreas, thyroid, adrenal gland, kidney, lung, skeletal muscle, spleen, and spinal cord. Serum samples were drawn within the last week of the patient's life; however, in two cases (patients 1 and 5), they were collected a few hours before death, and in one subject (patient 4), serum was collected at death. In addition, in four cases (patients 2, 3, 6, and 7), an additional serum sample was collected at autopsy.
 
RESULTS
 
All analyzed samples were positive for the presence of HCV RNA when tested with the MMLV RT-based assay, although the actual titer varied widely from tissue to tissue, being the highest in the liver, and at least two logs lower at other sites.
 
Using a Tth-based strand-specific assay, the presence of HCV-RNA negative strand was documented in all but one liver sample in titers that were one to two logs lower than the titers of the positive strand. In two patients (patients 7 and 8), all extrahepatic tissue samples were negative, while in the remaining six patients, HCV-RNA negative strand was detected in at least one tissue. RNA negative strand was most commonly present in lymph node and pancreas tissue (five patients), followed by adrenal gland tissue (two patients), thyroid (two patients), bone marrow (one patient), and spleen (one patient). All serum samples, including those collected at autopsy, tested negative for the presence of HCV-RNA negative strand. These results were confirmed in two independent experiments using two separate extraction procedures.
 
The present work is currently the only extensive study on extrahepatic HCV replication sites in humans. Because the tissue samples were collected at the time of autopsy, some RNA might have been degraded, and low-level replication at various organs could have been missed; however, the presence of HCV-RNA negative strand was commonly demonstrated in lymph nodes and pancreas, and occasionally in adrenal gland, bone marrow, thyroid tissue, and spleen. These results cannot be simply explained by contamination with liver-derived HCV-RNA negative strand, because the latter was not detected in any of pre- or postmortem serum samples. Although limited to AIDS patients, our study provides evidence that HCV is not strictly hepatotropic.
 
However, we cannot exclude the possibility of low-level viral replication at other sites as well. Because RNA negative strands seem to be present at a level that is one to two logs lower than the level of the positive strand, in some instances, replication could be below the sensitivity level of our Tth-based strand-specific assay.
 
In a recent study, we used a similar group of patients to search for possible replication sites of HGV.11 In striking contrast to HCV infection, in which high titers of negative-strand viral RNA were almost uniformly detected in liver tissue, HGV-RNA negative strand was rarely present in liver samples. However, it was detected in bone marrow and spleen, but not in any of the other analyzed tissues. Thus, although the specific cellular site of replication within the positive tissues was not identified, HGV is likely to have a different tissue tropism than HCV.
 
HCV has been suggested to be a lymphotropic virus; though its replication is rarely if ever detected in PBMCs from human subjects, it has been reported to infect lymphocyte cell lines in vitro,12 and some recent studies on chimpanzees suggest the existence of strains with particular affinity for lymphocytes. The common presence of severe lymphopenia prevented the collection of PBMCs in our patients. However, it is the prevailing opinion that in AIDS patients, the study of lymph nodes is far superior to the study of PBMCs.14 In the current investigation, we found the presence of negative-strand viral RNA in lymph nodes from 5 of 8 patients, but it is unclear which cells were infected. Nevertheless, because lymph nodes in AIDS are depleted of dendritic and T cells, it is likely that HCV replicated in the cells of the monocyte/macrophage lineage or even in B cells. The latter possibility is particularly intriguing, because chronic HCV infection was credibly associated with B-cell lymphoproliferative disorders such as mixed cryoglobulinemia and B-cell non-Hodgkin's lymphoma. Interestingly, it was recently shown that HCV core protein has broad trans-acting properties, including the activation of some known oncogenes.
 
In our previous study,6 we did not detect HCV-RNA negative strand in PBMCs from HIV-negative patients with chronic hepatitis C, which is in agreement with the results published by two other groups. However, considering the expected low level of viral replication at extrahepatic sites and the fact that strand-specific assays are relatively insensitive—in several studies, they were found to be at least one log less sensitive than standard RT-PCR4-7—replication could have been below the limit of detection. Thus, detection of HCV-RNA negative strand in lymphoid tissue in AIDS patients could be related to enhanced viral replication in the presence of severe immunodeficiency. Alternatively, HCV replication could be more efficient in activated cells, and/or weakened immune pressure against HCV could lead to the development of lymphotropic viral strains. However, it should be emphasized that the types and ratios of cells in the lymph nodes of AIDS patients are not representative of the typical PBMC composition in HIV-negative subjects.
 
Equally intriguing is the finding of HCV-replicative intermediates in thyroid and pancreas. The correlation between various forms of thyroid dysfunction and HCV infection is long known and was initially attributed solely to the effects of treatment with interferon. However, it was soon realized that thyroid abnormalities are commonly present in patients even before the actual treatment is started. A similar association was recently reported between HCV infection and the development of diabetes mellitus. Nevertheless, it remains unclear what cell lineage supports replication at extrahepatic sites. Interestingly, thyroid, pancreas, and adrenal glands were reported to be commonly involved in disseminated cytomegalovirus infection in AIDS patients.
 
There remains the important question of whether the ratio of infected cells in the extrahepatic tissues would be high enough to sustain infection and to be of clinical relevance....However, the actual number of infected cells at the extrahepatic sites could be higher because part of the viral RNA could have degraded between the time of the patient's death and autopsy.
 
In summary, using highly strand-specific Tth-based RT-PCR, we detected the common presence of negative-strand HCV RNA in lymph nodes and pancreas, and occasionally in thyroid, adrenal gland, spleen, and bone marrow from HCV-infected patients with AIDS. Because the study was conducted on severely ill and immunocompromised patients, the significance of our findings for HIV-negative patients is still uncertain. However, they could possibly relate to the various extrahepatic manifestations of HCV infection.
 
Hepatitis C virus negative strand RNA is not detected in peripheral blood mononuclear cells and viral sequences are identical to those in serum: a case against extrahepatic replication
 
Peripheral blood mononuclear cells (PBMCs) from 27 hepatitis C virus (HCV)-infected patients were analysed for the presence of HCV negative strand RNA with strand-specific Tth-based RT-PCR. No negative strand RNA was detected in any sample, and positive strand HCV sequences amplified from PBMCs were identical to those found in serum. These findings suggest that HCV does not replicate in PBMCs, and the presence of HCV sequences at this site is compatible with passive virus adsorption and/or contamination by circulating virus. J Gen Virol 1997 Nov;78 ( Pt 11):2747-50
 
Specific detection of hepatitis C virus minus strand RNA in hematopoietic cells
 
The presence of hepatitis C virus (HCV) negative strand RNA in extrahepatic compartments based on PCR detection assays has been suggested in many reports with a very heterologous detection rate (from 0 to 100%). In this study, we have analyzed the presence of HCV negative strand in hepatic (liver biopsies, n = 20) and extrahepatic (sera, n = 32; PBMC, n = 26 and fresh bone marrow cells, n = 8) compartments from infected patients with three different reverse transcriptase (RT)-PCR-based assays using primers located in the 5' noncoding region, with or without a tag selected to display different viral loads (10(5)-3 x 10(7) genomic equivalent/ml or gram) and viral genotypes (n = 5). Using synthetic as well as biological templates, we could document extensive artifactual detection of negative strand RNA, due to self priming and mispriming events, even either 5' noncoding region primer pair was used, whereas both artifacts were dramatically reduced (mispriming) or eliminated (selfpriming) using CAP-based RT-PCR assay. Mispriming artifacts were directly correlated to the titer of positive strand RNA present in the sample. Using the CAP-PCR assay, the presence of HCV negative strand RNA was found in 75% of livers (16:20) and only 8% of PBMC, independent of the genotype involved, but could not be documented in sera (0:32) and fresh bone marrow cells (0:6). These findings suggest that caution regarding the type of RT-PCR assay used and the level of HCV positive strand RNA present in the biological sample analyzed has to be taken to avoid false identification of viral reservoirs. The findings suggest that hematopoietic peripheral cells can support HCV replication, although in a very limited number of carriers. J Clin Invest 1996 Feb 1;97(3):845-51
 
EDITORIAL in Hepatology by Negro and Levrero
 
Although HCV infection of hepatocytes has been demon-strated convincingly by in situ hybridization and immunohis-tochemistry, the localization of HCV in both peripheral blood mononuclear cells (PBMC) and bone marrow cells has repeatedly been questioned, although similar techniques, as well as the strand-specific reverse-transcriptase polymerase chain reaction (RT-PCR) amplification, have been used...
 
...The presence of the replicative intermediate of HCV-negative strand RNA in PBMC and in vitro infected lymphoblastoid cells was reported soon after the discovery of HCV; however, the strand specificity of these early RT-PCR procedures was questioned in subsequent work based on the
 
...Suitably modified assays resulted in a more highly specific detection of the negative-strand RNA; however, increased specificity may have adversely affected sensitivity, possibly explaining why different authors who studied the HCV tropism for cells of the hematopoietic lineage reported conflicting results. In fact, one likely explanation may be that HCV replicates in PBMC but only at very low levels, which makes its detection difficult and inconsistent. Furthermore, another explanation suggests that extrahepatic sites may contribute little to the total plasma level of HCV; that is an idea that seems to be confirmed by indirect data on serum HCV RNA kinetics in the liver transplantation model. It should be noted that in this same model, some authors have made claims that the reinfection of the grafted liver can take place only via the existence of an extrahepatic reservoir of HCV replication….. (editorial note: and this post-transplant reinfection appears to occur only in patients who were not able to achieve SVR, suggesting that once an SVR is achieved extrahepatic HCV reservoirs, if there are any, are eradicated or are irrelevant).
 
...The infection of PBMC by HCV has also been reported using in situ hybridization, with conflicting results. Oddly enough, the proportion of infected cells does not correlate with the sensitivity of the assay. However, if the RT-PCR data are to be believed, the proportion of PBMC infected by HCV must be very low…. Two in situ hybridization studies provided an estimate of 1 to 3 cells per 10,000 34 and 0.15% to 4%, respectively. A more sensitive in situ PCR analysis, using fluorescent primers, increased the percentage up to a maximum of 8.1% of the total number of cells. Also mononuclear cells infiltrating the liver have reportedly been infected by HCV, but these data are contradicted by most other groups who have applied similar techniques...
 
...The presence of HCV has been evaluated in several other extrahepatic compartments. Analogous to other flaviviruses, HCV has been sought in the bone marrow. HCV RNA of both strands and HCV antigens have been detected by nonradioisotopic in situ hybridization and immunofluores-cence, respectively, in a low percentage of bone marrow mononuclear cells derived from 54% of patients with chronic hepatitis C. RT-PCR-based studies seemed to confirm the presence of HCV replicative forms in the bone marrow, especially in the presence of cryoglobulinemia. However, using a more strand-specific RT-PCR, Lerat et al. Questioned these findings, as did others looking for HCV-negative strand in bone marrow and other extrahepatic organs (PBMC, spleen, muscle, lymph nodes, pancreas, and kidney) taken from a chimpanzee at autopsy. Data which suggest that the presence of HCV in bone marrow preparations should also be interpreted with caution owing to the varying degree of contamination with peripheral blood cells. In situ staining of HCV has been demonstrated in both salivary gland epithelium and keratinocytes at the site of cryoglobulinemia-associated vasculitis. Again, however, contradictory data have been reported: immunohistochemistry has failed to stain non-structural HCV proteins in the skin of cryoglobulinemic, chronic hepatitis C patients, and a careful RT-PCR study has suggested the absence of HCV RNA in salivary gland epithelial cells. Finally, the presence of HCV in the brain has only been anecdotally reported and awaits confirmation...
 
....In light of these conflicting reports, new data concerning the extrahepatic replication of HCV is expected to elicit skepticism….The paper by Bronowicki et al……presents new evidence favoring the persistent replication of HCV in hematopoietic cells. The authors used the elegant model of mice with severe combined immunodeficiency. These severe combined immunodeficiency mice lack both humoral and cellular immunity caused ……Although this model will be used infrequently to study the pathobiology of HCV, it has provided convincing evidence for HCV replication in hematopoietic cells…..
 
...HCV may directly affect the function of immune cells by interfering with their capability to eliminate HCV-infected hepatocytes. HCV-infected lymphocytes may be crippled in their ability either to undergo activation in response to proper stimulation or to exert effector functions. These considerations remain purely speculative without specific studies, which have been hampered by the lack of a reproducible in vitro infection system and by the lack of an easy ex vivo access to adequate samples of infected lymphoid cells. Alternatively, HCV-infected cells, either lymphoid or epithelial cells, may become resistant to the induction of programmed cell death...
 
...The functional implications of this finding await clarification. Defects in the control of immune system homeostasis by apoptosis are important for the pathogenesis of both autoimmune and lymphoproliferative disorders. Indeed, malignancies of the hematopoietic lineage have been associated with HCV infection, especially non-Hodgkin's lymphoma; one study suggests that other extrahepatic malignancies may be more frequent among HCV-infected patients than in the general population…..HCV may directly transform infected cells, including cells of lymphoid origin…..B-cell non-Hodgkin's lymphoma is often associated with HCV infection, and a primary extranodal localization (espe-cially liver, stomach, and salivary glands) is more frequently observed in HCV-positive than in HCV-negative indi-viduals. However, it is interesting to note that the non-Hodgkin's lymphomatous tissue does not seem to support HCV replication. Moreover, the weight of evidence of HCV infection with hematopoietic malignancies is epidemiologic and does not provide insight into the mechanism of this association. The paper by Bronowicki et al. does not resolve that issue...
 
...this syndrome share the same cross-idiotype. Thus, the mechanisms leading to the development of a fully malignant form of B-cell non-Hodgkin's lymphoma may involve local factors, including cytokine secretion, or factors independent of HCV, such as Helicobacter pylori. The presence of a clear geographical heterogeneity in the prevalence of HCV- positive non-Hodgkin lymphomas indicates that other genetic and environmental cofactors must be sought to explain the pathogenesis of these lymphoproliferative disor-ders. In the absence of clear evidence that the transformed lymphocytes are infected by HCV, the pathogenesis of HCV-associated lymphomas is uncertain. Despite the potential ability of the virus to affect crucial regulatory mechanisms in the cell, its role seems to be that of an infectious cofactor in a multi-step process which relies on the accumulation of several as yet unidentified genetic alterations.
 
 
 
 
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