JOURNAL OF INTERFERON AND CYTOKINE RESEARCH 21:417–422 (2001) Mary Ann Liebert, Inc.
Rapid Changes in Hepatitis C Virus Quasispecies Produced by a Single Dose of IFN-a in Chronically Infected Patients CLAUDIA FORNAI, 1 FABRIZIO MAGGI,1 FLAVIA FAVILLI, 1 MARIA LINDA VATTERONI, 1 MAURO PISTELLO,1 SANTINO MARCHI,2 PIETRO CICCOROSSI,2 GUIDO ANTONELLI, 3 and MAURO BENDINELLI 1
ABSTRACT The effects of a single dose of 3 international megaunits of interferon-a2b (IFN-a2b) on hepatitis C virus (HCV) load and quasispecies were examined 24 h after administration in 12 previously untreated, chronically infected patients. All patients had viremia loads appreciably reduced relative to baseline values, thus confirming that the viral load is rapidly affected by IFN-a2b. Five patients also exhibited changes in plasma HCV quasispecies composition that were clearly evident by single-strand conformation polymorphism, analysis, thus indicating that one dose of IFN-a2b may suffice to produce rapid perturbations in the genetic heterogeneity of circulating HCV. Prior to IFN-a2b administration, 3 patients exhibited viral quasispecies differences between plasma and peripheral blood mononuclear cells (PBMC). Interestingly, in 2 such patients, the viral quasispecies found in the 24-h plasma resembled that in the pre-IFN PBMC. The latter finding raises the possibility that in these patients, the differences in quasispecies composition between pre-IFN and postIFN plasma resulted from increased representation of lymphoid tissue-originated variants in the latter sample, possibly because of poor sensitivity to IFN-a2b of HCV replication in the lymphoid compartment.
INTRODUCTION
H
C VIRUS (HCV) is a leading cause of chronic hepatitis worldwide; 20%–40% of infected people may develop cirrhosis, and 2%–4% may ultimately develop liver cancer.(1,2) The only effective therapy is interferon-a (IFN-a), in combination or not with ribavirin, but complete recovery from infection is less than 20%.(3,4) Parameters that may help predict sustained responses to therapy—defined as absence of detectable viremia 6 months after completion of treatment—include pretreatment viral load, viral genotype, and specific amino acid positions within the viral NS5A gene, but none of these parameters, used alone or in combination, is 100% predictive. Degree of quasispecies heterogeneity, as determined in the hypervariable region 1 (HVR1) of the viral genome, has also been reported to be predictive of responsiveness to IFN-a treatment.(5) Early changes in virus-host equilibrium induced by antiviral drugs may permit identification of predictors of responsiveness EPATITIS
to therapy as well as help shed light on the natural history of viral infections. Recently, the beneficial effects of IFN-a in chronically infected patients were found to be evident very soon after initiation of treatment, in that viremia levels were significantly reduced as early as 12–24 h after the first inoculation.(6–9) Here, we investigated whether IFN-a2b treatment is followed by equally rapid modifications of HCV quasispecies. The results showed that this may indeed be the case, as in nearly half of the patients investigated, the single-strand conformation polymorphism (SSCP) patterns of the virus circulating in plasma immediately before (pre-IFN) and 24 h after the first dose of IFN-a2b (post-IFN) were consistently different. Prior to IFN-a2b administration, 3 patients exhibited viral quasispecies differences between plasma and peripheral blood mononuclear cells (PBMC). Interestingly, in 2 such patients, the viral quasispecies found in the 24-h plasma resembled that in the pre-IFN PBMC. This finding raises the possibility that differences in quasispecies composition between pre-IFN and post-IFN plasma resulted from increased representation of lym-
1 Virology
Section, Department of Biomedicine and Retrovirus Center, University of Pisa, Pisa, Italy. Medical Clinic–Gastroenterology Unit, University of Pisa, Pisa, Italy. 3 Virology Section, Department of Experimental Medicine and Pathology, University “La Sapienza,” Rome, Italy. 2I
417
418
FORNAI ET AL.
phoid tissue-originated variants in the latter sample. This may suggest that HCV replication in PBMC and possibly other lymphoid cells is more resistant to IFN-a2b than is HCV replication in hepatocytes.
the samples of each individual patient were examined simultaneously. The lower limit of sensitivity was 1000 genome copies per milliliter, and intrassay variability was #0.3 log.
PCR amplification and SSCP analysis MATERIALS AND METHODS
HVR1 quasispecies analysis by SSCP and its validation have been described.(12) Briefly, viral RNA was reverse-transcribed and then amplified with a set of nested primers covering the E2/NS1 region to obtain a 307-bp fragment spanning nucleotides 1284–1590 of the viral genome.(13) The primers used were: outer sense 59-CGCATGGCGTGGGACATGATG-3 9 outer antisense 59-GGTGTGGAGGGAGTCATTGCAGTT-3 9, inner sense 59-GCTTGGGATATGATGATGAACTGGTC-3 9, inner antisense 59-TGCCACCTGCCATTGGTGTT-3 9. Thermal cycling conditions for both sets of primers were exactly as described.(12) The PCR products were extracted from agarose gel and then purified using a commercial gel extraction kit (Qiagen, Chatsworth, CA) according to the manufacturer’s instructions. Purified products were amplified by asymmetric PCR, using the same conditions as for the second-round PCR, except that only the inner sense primer was used. Single-stranded DNA amplicons were electrophoresed in 10% nondenaturing polyacrylamide gels containing 0.5 3 TBE, 1.5% N,N9-methylenbisacrylamide, and 5% glycerol. Electrophoresis was carried out on the Dcode Universal Mutation Detection System apparatus (Bio-Rad, Hercules, CA) at 23°C with 40 mA for 4–5 h. Finally, the gel was visualized using silver staining. Electrophoretic DNA mobility was identified using a GDS 7500 Gel Documentation System (UVP, Cambridge, England), and SSCP band intensity was analysed using the GelBase Pro software program (UVP). Under the optimized conditions used, the technique discriminated variants differing by a single nucleotide and representing approximately 5% of the entire viral population.(14) The following measures were implemented to minimize the risk of artifactual polymorphisms(15,16): (1) for each sample, the analysis was performed twice, starting from two independent RNA extractions, and when the quasispecies compositions of
Patients and samples The study group consisted of 12 randomly selected, treatment-naive, chronically infected patients for whom the decision has been made to start IFN-a therapy (3 international megaunits of rIFN-a2b [Intron A, Schering-Plough Corp., Madison, NJ] administered subcutaneously [s.c] 3 times per week, for a total of 24 weeks). Active HCV infection was determined by positive serology (third-generation tests) (Ortho, Milan, Italy), positive testing for HCV by RT-PCR in the 59-untranslated region (59-UTR),(10) and abnormal serum alanine aminotransferase (ALT) levels. HCV genotyping was carried out by a commercial assay (LIPA II) (Innogenetics, Zwijndrecht, Belgium). The plasma samples examined for HCV load and quasispecies were obtained, with the patients’ written consent, immediately before the first dose of IFN-a2b (pre-IFN) and 24 h thereafter (post-IFN). PBMC were obtained from the time 0 bleeding by Ficoll-Hypaque density-gradient centrifugation, repeatedly washed with abundant sterile phosphate-buffered saline (PBS), incubated at 37°C with 0.5% trypsin for 20 min, followed by 0.5 mg/ml RNAse for 30 min, and finally washed again twice. This treatment has been shown to effectively remove the HCV absorbed onto the surface of PBMC,(11) and, accordingly, the last wash was HCV negative when tested by RT-PCR. All samples were stored at 280°C until use.
HCV RNA quantification Viral RNA plasma levels were measured with Monitor (Roche Diagnostic, Basel, Switzerland) according to the manufacturer’s instruction. All samples were tested in duplicate, and TABLE 1.
HCV RNA LOADS BEFORE
AND
AFTER
A
SINGLE DOSE
OF
IFNa2b
IN
12 STUDY PATIENTS
HCV RNA (log copies/ml)b Patienta 121 151 173 213 640 982 2888 6081 6583 4931 5011 6886 Mean 6 SD a Some
HCV genotype
Pre-IFN
Post-IFN
% reduction
1
5.94 6.39 5.73 6.50 5.53 5.76 4.72 5.28 4.36 5.54 6.45 5.70 5.66 6 0.65
5.76 5.81 5.11 5.79 ,3c 000 4.66 3.28 4.68 2.95 3.30 6.09 4.83 4.75 6 1.12
340 740 760 810 .99 920 960 750 960 990 560 860 80 6 20
2 3
of these data were included in ref. 9. and post-IFN HCV RNA loads were significantly different at p 5 0.007. c A value of 3 logs was arbitrarily used for calculations. b Pre-IFN
419
IFN AND HCV QUASISPECIES
Statistical analysis A two-sample paired Student’s t-test was used to compare means. A p value of ,0.05 was considered significant.
RESULTS AND DISCUSSION
FIG. 1. Graphic representation of the HCV SSCP patterns obtained from plasma samples collected immediately before (PreIFN) and 24 h after the first dose of IFN-a2b (post-IFN). Three representative patients of the five who exhibited appreciable intersample pattern modifications are shown. Dotted and solid lines indicate bands present in both samples or in only one sample, respectively. Bands were considered distinct when they differed in electrophoretic mobility by at least 5 pixels. Bar length indicates the intensity of gel silver staining.
the two replicas were different, analysis was repeated at least a third time from a new RNA extract, (2) PCR reactions were performed after the amount of viral RNA template used in each amplification was adjusted to contain approximately the same number of viral genomes, and (3) HCV variants found in one replica only were considered artifacts and eliminated from subsequent computations.
Table 1 shows the results of the HCV load determinations performed on the pre-IFN and post-IFN plasma samples of the 12 therapy-naive patients enrolled in the study. At 24 h postIFN, all patients had viremia loads appreciably reduced relative to the corresponding pre-IFN samples, although the extent of reduction varied considerably in individual patients. Of note, in 10 patients, viremia reduction was greater than the intraassay variability exhibited by the quantification method used in our laboratory, thus demonstrating that the observed reduction is real. The average viremia reduction was 80%, which is somewhat higher than that observed by Lam et al.(6) in patients given the same dose of IFN-a2b. No correlation was observed between extent of viremia reduction and baseline viremia loads (Table 1) and ALT levels (data not shown). Thus, these findings were in accordance with previous data.(6–9) HCV exists in infected hosts as a population of related genetic variants known as quasispecies,(17,18) which appears to be important for viral persistent.(19) To investigate whether the rapid reduction of HCV viremia induced by a single dose of IFN-a2b was accompanied by changes in the quasispecies of the infecting virus, all the pre-IFN and post-IFN plasma samples were examined by HVR1 SSCP analysis. HVR1 is a highly variable segment in the E2/NS1 gene of HCV. Elevated tolerance for amino acid substitution and sensitivity to the selective pressure exerted by the host’s immune system make this region particularly suitable for quasispecies heterogeneity studies.(20–22) As is usually observed,(23,24) HCV was found to be present in the form of quasispecies in all the pre-IFN samples, with numbers of genetic variants ranging between 3 and 10 in individual patients. Following IFN-a2b administration, 7 patients showed SSCP patterns essentially identical to the pre-IFN patterns (data not shown). In contrast, the other 5 patients showed clearly evident changes in the SSCP patterns (Fig. 1). The changes were disappearance of bands (4 patients), appearance of novel bands (4 patients), and substitution of the master sequence (2 patients). Previous studies on the effects of IFN-a on HCV quasi-
TABLE 2. CHANGES IN PLASMA HCV QUASISPECIES COMPOSITION AFTER A SINGLE DOSE OF IFNa2b IN RELATION QUASISPECIES COMPLEXITY PRIOR TO TREATMENT AND RESPONSE TO TREATMENT AT 24 H AND 6 MONTHS
TO
Post-IFN quasispecies composition
Pre-IFN quasispecies complexitya
Post-IFN viremia log reduction
Sustained response to treatmentb
Varied Unvaried
5.8 6 2.9c,d 5.6 6 3.60
0.95 6 0.79c,d 1.2 6 0.700
3/5 (60%) 4/7 (57%)
a Number
of SSCP bands. with plasma viremia below detectability at 6 months after initiation of treatment. c Mean 6 SD. d Varied group not significantly different from unvaried group (p . 0.05). b Patients
420 species were carried out on plasma samples collected after several weeks of treatment and revealed clearly visible alterations of this parameter.(22,25) Taken together with previous findings demonstrating that in the absence of therapy, the quasispecies of HCV shows no abrupt changes but evolves slowly over prolonged periods of time,(12,22,24) the results show that in a subpopulation of hepatitis C patients, a single dose of IFN-a2b suffices to produce marked and rapid perturbations in the infecting viral quasispecies. To the best of our knowledge, this is the first study investigating the behavior of HCV quasispecies as early as 1 day after initiation of IFN-a2b therapy. The occurrence of detectable quasispecies changes was independent of baseline ALT level (data not shown), viral load (Table 1), quasispecies complexity, and also of the extent of post-IFN viremia reduction and clinical or virologic response to treatment at 6 months (Table 2). Because a time interval of 24 h appeared too short for the formation and massive emergence in plasma of novel viral variants, we speculated that the prompt quasispecies changes might result from differences in the efficacy of IFN-a2b on HCV replication in different body compartments. HCV quasispecies composition may vary depending on the body district where it is analyzed, and this is believed to reflect the fact that the infection is at least partially compartimentalized, and hence the virus undergoes partially independent evolutions in different cell types or body tissues.(12,14,16,26–30) In particular, previous studies by us and other groups have shown that although most viral variants found in the plasma of patients with chronic HCV infection are detected in concomitant liver biopsy specimens as well, some are found in the concomitant PBMC but not in the liver, suggesting that they are produced in lymphoid cells.(14,29,43) To investigate whether plasma HCV variants were affected in a similar fashion by a single dose of IFN-a2b independently of their presence or absence in the PBMC, we compared the HCV quasispecies in the pre-IFN plasma and PBMC samples of 3 of the patients with that in the post-IFN plasma samples. As illustrated by Figure 2, in 2 such patients (6583 and 6886), the HCV quasispecies of pre-IFN plasma and PBMC were quite divergent (100% and 80%, respectively), and, interestingly, in each of these 2 patients, the quasispecies of postIFN plasma was similar to that in the pre-IFN PBMC, as shown by nearly superimposable SSCP patterns. This indicates that most of the IFN-a2b-induced viral load reduction observed in these patients was at the expense of the HCV variants, presumably of liver origin, which were not represented in the PBMC before treatment. The third patient (5011), as well as 2 patients with invariant quasispecies examined for control (2888 and 6081), exhibited pre-IFN plasma and PBMC quasispecies too similar between themselves to permit insights on the aspect investigated (Fig. 2). Lam et al.(6) and Neumann et al.(8) applied mathematical modeling techniques to the decay of HCV viremia elicited by IFN-a and suggested that the major initial effect of IFN is blockage of viral production or release rather than inhibition of de novo infection of susceptible cells or increased viral clearance. Substantiating earlier clues inferred from the study of liver transplanted patients,(32,33) the same authors also provided evidence that the rapid viremia decline observed after IFN-a administration is possible because the half-life of virions in plasma is only a few hours. Thus, by showing that 1 day after a single
FORNAI ET AL. dose of IFN-a2b, 2 patients had circulating in their plasma HCV with quasispecies features similar to those of the virus that was present within the PBMC prior to treatment, our results suggest the possibility that at least in some patients HCV replication in the lymphoid compartment is particularly resistant to IFN-a treatment. This might stem from the characteristics of these host cells or from the fact that HCV variants dwelling in lymphoid tissues are less sensitive to the cytokine. In either case, characterization of PBMC-associated HCV might be more informative than characterization of plasma HCV in regard to long-
FIG. 2. SSCP patterns of HCV in the pre-IFN plasma and PBMC and in the post-IFN plasma of 3 patients who exhibited quasispecies changes between the pre-IFN and post-IFN plasma samples (5011, 6583, 6886) and of 2 patients with invariant quasispecies (2888, 6081).
421
IFN AND HCV QUASISPECIES term outcome of therapy. Further investigations are warranted to elucidate these aspects.
ACKNOWLEDGMENTS This work was supported in part by grants from the Ministero della Sanità and Ministero della Università e Ricerca Scientifica, Rome, Italy.
REFERENCES 1. ALTER, M.J., MARGOLIS, H.S., KRAWCZYNSKI, K., JUDSON, F.N., MARES, A., ALEXANDER, W.J., HU, P.Y., MILLER, J.K., GERBER, M.A., and SAMPLINER, R.E. (1992). The natural history of community-acquired hepatitis C in the United States. The Sentinel Counties Chronic non-A, non-B Hepatitis Study Team. N. Engl. J. Med. 327, 1899–1905. 2. BROWN, J.L. (1998). Efficacy of combined interferon and ribavirin for treatment of hepatitis C. Lancet 35, 178–180. 3. POYNARD, T., LEROY, V., COHARD, M., THEVENOT, T., MATHURIN, P., OPOLON, P., and ZARSKI, J.P. (1996). Metaanalysis of interferon randomized trials in the treatment of viral hepatitis C: effects of dose and duration. Hepatology 24, 778–789. 4. HOOFNAGLE, J.H., and DI BISCEGLIE, A.M. (1997). The treatment of chronic viral hepatitis. N. Engl. J. Med. 336, 347–356. 5. BENDINELLI, M., VATTERONI, M.L., MAGGI, F., and PISTELLO, M. (1999). Hepatitis C virus. Biology, pathogenesis, epidemiology, clinical description, and diagnosis. In: S. Specter (ed.) Viral Hepatitis: Diagnosis, Therapy, and Prevention. Totowa, NJ: Humana Press, pp. 4–127. 6. LAM, N.P., NEUMANN, A.U., GRETCH, D.R., WILEY, T.E., PERELSON, A.S., and LAYDEN, T.J. (1997). Dose-dependent acute clearance of hepatitis C genotype 1 virus with interferon alpha. Hepatology 26, 226–231. 7. YASUI, K., OKANOUE, T., MURAKAMI, Y., ITOH, Y., MINAMI, M., SAKAMOTO, S., SAKAMOTO, M., and NISHIOJI, K. (1998). Dynamics of hepatitis C viremia following interferona administration. J. Infect Dis. 177, 1475–1479. 8. NEUMANN, A.U., LAM, N.P., DAHARI, H., GRETCH, D.R., WILEY, T.E., LAYDEN, T.J., and PERELSON, A.S. (1998). Hepatitis C viral dynamics in vivo and the antiviral efficacy of interferon-a therapy. Science 282, 103–107. 9. ANTONELLI, G., RIVA, E., MAGGI, F., VATTERONI, M.L., and SIMEONI, E. (1999). Influence of hepatitis C virus (HCV) genotype, HCV RNA load, and alanine aminotransferase level on reduction of HCV RNA after a single administration of interferona. J. Infect. Dis. 180, 1411–1412. 10. VATTERONI, M.L., PISTELLO, M., MAGGI, F., CECCONI, N., PANICUCCI, F., and BENDINELLI, M. (1994). Hepatitis C virus serological and polymerase chain reactions in human immunodeficiency virus-positive and negative patients. Clin. Diagn. Virol. 2, 7–16. 11. WILLEMS, M., PEERLINCK, K., MOSHAGE, H., DELEU, I., VAN DEN EYNDE, C., VERMYLEN, J., and YAP, S.H. (1994). Hepatitis C virus-RNAs in plasma and in peripheral blood mononuclear cells of hemophiliacs with chronic hepatitis C: evidence for viral replication in peripheral blood mononuclear cells. J. Med. Virol. 42, 272–278. 12. MAGGI, F., FORNAI, C., VATTERONI, M.L., GIORGI, M., MORRICA, A., PISTELLO, M., CAMMAROTA, G., MARCHI, S., CICCOROSSI, P., BIONDA, A., and BENDINELLI, M. (1997). Differences in hepatitis C virus quasispecies composition
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
between liver, peripheral blood mononuclear cells and plasma. J. Gen. Virol. 78, 1521–1525. LEE, J.H., STRIPF, T., ROTH, W.K., and ZEUZEM, S. (1997). Non-Isotopic detection of hepatitis C virus quasispecies by single strand conformation polymorphism. J. Med. Virol. 53, 245–251. MAGGI, F., FORNAI, C., MORRICA, A., VATTERONI M.L., GIORGI, M., MARCHI, S., CICCOROSSI, P., BENDINELLI, M., and PISTELLO, M. (1999). Divergent evolution of hepatitis C virus in liver and peripheral blood mononuclear cells of infected patients. J. Med. Virol. 57, 57–63. SMITH, D.B., McALLISTER, J., CASINO, C., and SIMMONDS, P. (1997). Virus “quasispecies”: making a mountain out of a molehill? J. Gen. Virol. 78, 1511–1519. JANG, S.J., WANG, L.F., RADKOWSKI, M., RAKELA, J., and LASKUS, T. (1999). Differences between hepatitis C cirus 59 untranslated region quasispecies in serum and liver. J. Gen. Virol. 80, 711–716. MARTELL, M., ESTEBAN, J.I., GENESCA, J., WEINER, A., ESTEBAN, R., GUARDIA, J., and GOMEZ, J. (1992). Hepatitis C virus (HCV) circulates as a population of different but closely related genomes: quasispecies nature of HCV genome distribution. J. Virol. 66, 3225–3229. WILSON, J.J., POLYAK, S.J., DAY, T.D., and GRETCH, D.R. (1995). Characterization of simple and complex hepatitis C virus quasispecies by heteroduplex gel shift analysis: correlation with nucleotide sequencing. J. Gen. Virol. 76, 1763–1771. RAY, S.C., WANG, Y.M., LAEYENDECKER, O., TICEHURST, J.R., VILLANO, S.A., and THOMAS, D.L. (1999). Acute hepatitis C virus structural gene sequences as predictors of persistent viremia: hypervariable region 1 as a decoy. J. Virol. 73, 2938–2946. WEINER, A.J., GEYSEN, H.M., CHRISTOPHERSON, C., HALL, J.E., MASON, T.J., SARACCO, G., BONINO, F., CRAWFORD, K., MARION, C.D., CRAWFORD, K.A., BRUNETTO, M., BARR, P.J., MIYAMURA, T., McHUTCHINSON, J., and HOUGHTON, M. (1992). Evidence for immune selection of hepatitis C (HCV) putative virus envelope glycoprotein variants: potential role in chronic HCV infections. Proc. Natl. Acad. Sci. USA 89, 3468–3472. FARCI, P., ALTER, H.J., WONG, D.C., MILLER, R.H., GOVINDARAJAN, S., ENGLE, R., SHAPIRO, M., and PURCELL, R.H. (1994). Prevention of hepatitis C virus infection in chimpanzees after antibody-mediated in vitro neutralization. Proc. Natl. Acad. Sci. USA 91, 7792–7796. PAWLOTSKY, J.M., GERMANIDIS, G., FRAINAIS, P.O., BOUVIER, M., SOULIER, A., PELLERIN, M., and DHUMEAUX, D. (1999). Evolution of the hepatitis C virus second envelope protein hypervariable region in chronically infected patients receiving alpha interferon therapy. J. Virol. 73, 6490–6499. PAWLOTSKY, J.M., PELLERIN, M., BOUVIER, M., ROUDOTTHORAVAL, F., GERMANIDIS, G., BASTIE, A., DARTHUY, F., RÉMIRÉ, J., SOUSSY, C.J., and DHUMEAUX, D. (1998). Genetic complexity of the hypervariable region 1 (HVR1) of hepatitis C virus (HCV): influence on the characteristics of the infection and responses to interferon alfa therapy in patients with chronic hepatitis C. J. Med. Virol. 54, 256–264. POLYAK, S.J., McARDLE, S., LIU, S.L., SULLIVAN, D.G., CHUNG, M., HOFGÄRTNER, W.T., CARITHERS, R.L., JR., McMAHON, B.J., MULLINS, J.I., COREY, L., and GRETCH, D.R. (1998). Evolution of hepatitis C virus quasispecies in hypervariable region 1 and the putative interferon sensitivity-determining region during interferon therapy and natural infection. J. Virol. 72, 4288–4296. GEROTTO, M., SULLIVAN, D.G., POLYAK, S.J., CHEMELLO, L., CAVALLETTO, L., PONTISSO, P., ALBERTI, A., and GRETCH, D.R. (1999). Effect of retreatment with interferon alone or interferon plus ribavirin on hepatitis C virus quasispecies di-
422
26.
27.
28.
29.
30.
31.
FORNAI ET AL. versification in nonresponder patients with chronic hepatitis C. J. Virol. 73, 7241–7247. CABOT, B., ESTEBAN, J.I., MARTELL, M., GENESCÀ, J., VARGAS, V., ESTEBAN, R., GUARDIA, J., and GOMEZ, J. (1997). Structure of replicating hepatitis C virus (HCV) quasispecies in the liver may not be reflected by analysis of circulating HCV virions. J. Virol. 71, 1732–1734. NAVAS, S., MARTIN, J., QUIROGA, J.A., CASTILLO, I., and CARRENO, V. (1998). Genetic diversity and tissue compartmentalization of the hepatitis C virus genome in blood mononuclear cells, liver, and serum from chronic hepatis C patients. J. Virol. 72, 1640–1646. HSU, C.W., YEH, C.T., CHEN, P.G.C., and LIAW, Y.F. (1999). Replication of hepatitis C virus in ascitic mononuclear cells with development of distinct viral quasispecies. J. Infect. Dis. 180, 992–1000. OKUDA, M., HINO, K., KORENAGA, M., YAMAGUCHI, Y., KATOH, Y., and OKITA, K. (1999). Differences in hypervariable region 1 quasispecies of hepatitis C virus in human serum, peripheral blood mononuclear cells, and liver. Hepatology 29, 217–222. LASKUS, T., RADKOWSKI, M., PIASEK, A., NOWICKI, M., HORBAN, A., CIANCIARA, J., and RAKELA, J. (2000). Hepatitis C virus in lymphoid cells of patients coinfected with human immunodeficiency virus type 1: evidence of active replication in monocytes/macrophages and lymphocytes. J. Infect. Dis. 181, 442–448. SHIMIZU, Y.K., IGARASHI, H., KANEMATU, T., FUJIWARA, K., WONG, D.C., PURCELL, R.H., and YOSHIKURA, H. (1997).
Sequence analysis of the hepatitis C virus genome recovered from serum, liver, and peripheral blood mononuclear cells of infected chimpanzees. J. Virol. 71, 5769–5773. 32. GRETCH, D.R., BACCHI, C.E., COREY, L., DELA ROSA, C., LESNIEWSKI, R.R., KOWDLEY, K., GOWN, A., FRANK, I., PERKINS, J.D., and CARITHERS, R.L., Jr. (1995). Persistent hepatitis C virus infection after liver transplantation: clinical and virological features. Hepatology 22, 1–9. 33. FUKUMOTO, T., BERG, T., KU, Y., BECHSTEIN, W.O., KNOOP, M., LEMMENS, H.P., LOBECK, H., HOPF, U., and NEUHASU, P. (1996). Viral dynamics of hepatitis C early after orthotopic liver transplantation: evidence for rapid turnover of serum virions. Hepatology 24, 1351–1354.
Address reprint requests to: Dr. Mauro Bendinelli Department of Biomedicine University of Pisa Via San Zeno 37 I-56127 Pisa Italy Tel: 39-050-553-562 Fax: 39-050-559-455 E-mail:
[email protected] Received 15 January 2001/Accepted 16 February 2001
This article has been cited by: 1. Xiao-Ling Liao , Hao Ren , Ping Zhao , Shi-Ying Zhu , Jie Cao , Zhi-Hui Chen , Lan-Juan Zhao , Wei Pan , Mark Alan Feitelson , Zhong-Tian Qui . 2007. Efficient Induction of Mouse Immune Responses to Hepatitis C Virus by Viral Core Protein-Carrying Attenuated Salmonella typhimuriumEfficient Induction of Mouse Immune Responses to Hepatitis C Virus by Viral Core Protein-Carrying Attenuated Salmonella typhimurium. Viral Immunology 20:3, 469-478. [Abstract] [PDF] [PDF Plus]
