Hepatitis C Virus (HCV) and Hepatitis B Virus (HBV
http://www.100md.com
病菌学杂志 2005年第24期
Fundación para el Estudio de las Hepatitis Virales, Madrid, Spain
Departamento de Electrónica, Automática e Informática Industrial, Universidad Politécnica de Madrid, Madrid, Spain
ABSTRACT
In this work, we have shown that hepatitis C virus (HCV) and hepatitis B virus (HBV) can coexist in the same hepatocyte using double fluorescent in situ hybridization in liver biopsy samples from patients with chronic HCV infection with occult HBV infection. Digital image analysis of hybridization signals showed that the HBV DNA levels in coinfected hepatocytes were lower than those in cells infected only with HBV. This finding supports the hypothesis of inhibition of HBV replication by HCV. Furthermore, HCV RNA levels were lower in coinfected cells than in cells infected only with HCV, suggesting that HBV may also inhibit HCV replication.
TEXT
Hepatitis B virus (HBV) belongs to the Hepadnaviridae family of animal viruses, and its genome consists of a circular partially double-stranded DNA molecule of 3.2 kb in length which contains four overlapping reading frames that code for surface proteins (HBsAg), core proteins (HBc/HBeAg), the viral polymerase, and the transcriptional transactivator X protein (HBx) (9).
Hepatitis C virus (HCV) is classified in the Hepacivirus genus of the Flaviviridae family, and its genome is a positive-stranded RNA of 9.6 kb in length that encodes a large polyprotein that undergoes proteolytic processing by cellular and viral proteinases to generate the individual viral proteins (17).
As HBV and HCV share similar transmission routes, coinfection with the two viruses is not a rare event (3, 6, 7). Clinical data obtained from chronic HBV carriers superinfected with HCV suggest that HCV may inhibit HBV replication (13, 15, 20). This hypothesis is supported by the fact that patients with chronic HCV infection frequently have a special form of HBV infection, termed occult HBV infection (2, 8, 10, 12). This is characterized by the presence of low levels of HBV DNA in serum and/or in liver in the absence of detectable HBsAg in serum. This inhibition may be mediated by the host immune response (via the induction of cytokines such as interferons) or by a direct effect of HCV proteins. In this regard, it has been shown that HCV core and NS2 proteins inhibit HBV replication and gene expression in vitro (4, 5, 19, 21, 22).
Direct interference mediated by HCV proteins can occur in vivo only if both HBV and HCV coexist in the same hepatocyte. However, it has not been demonstrated whether HBV and HCV infect the same cell in the liver of patients coinfected with the two viruses. To address this issue, we have used double fluorescent in situ hybridization to determine the presence of HBV DNA and HCV RNA in liver biopsy samples from six patients with chronic hepatitis C (anti-HCV positive, serum HCV RNA positive) and occult HBV infection (HBsAg negative, serum HBV DNA positive).
Patients underwent a liver biopsy for diagnostic purposes. After liver samples were obtained, they were divided into two portions. One fragment was processed for histological diagnosis and for in situ hybridization, and the other was embedded in RNAlater (QIAGEN, Hilden, Germany) in less than 30 seconds after being obtained and stored at –20°C. At the time of the liver biopsy, a serum sample was obtained from each patient for the determination of HBV DNA and HCV RNA concentrations. All patients gave their written informed consent to participate in the study.
As mentioned above, all patients were persistently positive for HBV DNA in serum, and at the time of the liver biopsy, the mean HBV DNA concentration in serum was 2.4 x 103 ± 1.8 x103 IU/ml as tested by the Amplicor HBV Monitor test v.2.0 (Roche Diagnostics SL, Barcelona, Spain).
In situ hybridization was performed as described previously (11, 16) except that the HBV and HCV probes were labeled with digoxigenin-11-dUTP and biotin-16-dUTP (Roche Molecular Biochemicals, Indianapolis, IN). HBV hybrids were detected with an antidigoxigenin fluorescein isothiocyanate-conjugated antibody (green fluorescence), and the HCV hybrids were detected with avidin-rhodamine (red fluorescence) (Roche Molecular Biochemicals). Slides were counterstained using 4',6'-diamidino-2-phenylindole (blue fluorescence). Signals were visualized in a Nikon Eclipse E-400 light epifluorescence microscope. Images were captured with a high-sensitivity charge-coupled device camera (Leica DFC 300FX; Leica Microsystems, Switzerland) and 24-bit red-green-blue color depth and stored as tagged image file format files.
In all six patients, hepatocytes infected by only HBV, by only HCV, or by both viruses were detected (Fig. 1). The mean percentage of infected hepatocytes was 7.9% ± 7.5%. Of these, 12% ± 11.7% were infected only by HBV, 42.2% ± 19.1% only by HCV, and 45.8% ± 18.4% by both viruses. To determine whether there was an inhibition of HBV or HCV replication in coinfected hepatocytes, the intensity of fluorescence signals corresponding to HBV DNA and HCV RNA was measured in single-infected and double-infected hepatocytes by digital image analysis. To avoid differences due to the efficiency of in situ hybridization, the intensity of fluorescence signals in single-infected hepatocytes was compared to intensity in double-infected cells within the same tissue section.
This analysis showed that the intensity of fluorescence signals corresponding to HBV DNA was 1.6 ± 0.16 times higher (range, 1.44 to 1.82) in hepatocytes infected only by HBV than in those coinfected by HBV and HCV (Fig. 2), although the differences were not significant. Similarly, the intensity of fluorescence signals corresponding to HCV RNA was 1.83 ± 0.5 times higher (range, 1.45 to 2.74) in single-infected than in double-infected cells (Fig. 2), but without statistical significance. These data suggest that there may be a mutual inhibition of the replication of the two viruses in hepatocytes coinfected by HBV and HCV. This finding is in agreement with previous reports in which inhibition of HCV replication by HBV in coinfected patients has been also suggested (14, 18, 23).
To analyze further the possible inhibition of HCV replication by HBV, serum HCV RNA concentration and levels of the antigenomic HCV RNA strand, which is the replicative intermediate of the virus (1), were tested in the six patients with chronic hepatitis C and occult HBV infection in comparison to levels in 10 patients with chronic HCV infection without HBV infection. Patients were matched for gender, age, alanine aminotransferase levels, HCV genotype, and known duration of HCV infection (Table 1). These 10 patients underwent a liver biopsy for diagnostic purposes, and liver samples were processed as described above.
The serum HCV RNA concentration (tested using the Amplicor HCV Monitor test; Roche) was significantly lower (P = 0.031) in the six patients with occult HBV infection (mean ± standard error of the mean [SEM], 2.92 x 105 ± 7.03 x 104 IU/ml) than in patients with chronic hepatitis C without HBV infection (mean ± SEM, 7.66 x 105 ± 1.28 x 105 IU/ml).
For quantitation of the antigenomic HCV RNA strand, total RNA was isolated from the liver biopsy sample fragments preserved in RNAlater. Synthesis of cDNA was performed with Tth polymerase (Applied Biosystems, Foster City, CA) using 0.5 μg of total RNA and the sense primer 5'-CTTCACGCAGAAAGCGTCTA-3'. After addition of the antisense primer 5'-CAAGCACCCTATCAGGCAGT-3', real-time PCRwas performed using the LightCycler FastStart DNA Master SYBR Green I kit (Roche Molecular Biochemicals). The reaction was run in a Light Cycler (Roche Molecular Biochemicals). The specificity of the assay was tested using synthetic genomic and antigenomic HCV RNA, and it was shown that it is capable of detecting 3.2 copies of the correct strand while detecting 107 to 108 of the incorrect strand. The amount of the antigenomic HCV RNA strand was lower in patients with occult HBV infection (mean ± SEM, 1.32 x 105 ± 4.38 x 104 copies/μg of total RNA) than in patients without HBV infection (mean ± SEM, 5.7 x 105 ± 3.1 x 105 copies/μg of total RNA), although without statistical significance. This finding is in agreement with the hypothesis of the inhibition of HCV replication by HBV in coinfected patients.
In conclusion, in this work we have demonstrated that HBV and HCV can infect the same hepatocyte in the liver of patients coinfected by the two viruses. Our results also suggest that, apart from the possible involvement of the host immune response in the inhibition of the replication of HBV and HCV, a direct mutual inhibition of the replication of the two viruses in coinfected cells may also occur.
ACKNOWLEDGMENTS
This work has been supported by grants from the Fundación de Investigaciones Biomédicas and from the Fundación para el Estudio de las Hepatitis Virales, Madrid, Spain.
REFERENCES
Bartenschlager R., and V. Lohmann. 2000. Replication of the hepatitis C virus. J. Gen. Virol. 81:1631-1648.
Cacciola, I., T. Pollicino, G. Squadrito, G. Cerenzia, M. E. Orlando, and G. Raimondo. 1999. Occult hepatitis B virus infection in patients with chronic hepatitis C liver disease. N. Engl. J. Med. 341:22-26.
Chen, D. S., G. C. Kuo, J. L. Sung, M. Y. Lai, J. C. Sheu, P. J. Chen, P. M. Yang, H. M. Hsu, M. H. Chang, C. J. Chen, L. C. Hahn, Q. L. Choo, T. H. Wang, and M. Houghton. 1990. Hepatitis C virus infection in an area hyperendemic for hepatitis B and chronic liver disease: the Taiwan experience. J. Infect. Dis. 162:817-822.
Chen, S. Y., C. F. Kao, C. M. Chen, C. M. Shih, M. J. Hsu, C. H. Chao, S. H. Wang, L. R. You, and Y. H. Lee. 2003. Mechanisms for inhibition of hepatitis B virus gene expression and replication by hepatitis C virus core protein. J. Biol. Chem. 278:591-607.
Dumoulin, F. L., A. von dem Bussche, J. Li, L. Khamzina, J. R. Wands, T. Sauerbruch, and U. Spengler. 2003. Hepatitis C virus NS2 protein inhibits gene expression from different cellular and viral promoters in hepatic and nonhepatic cell lines. Virology 305:260-266.
Fattovich, G., A. Tagger, L. Brollo, G. Giustina, P. Pontisso, G. Realdi, A. Alberti, and A. Ruol. 1991. Hepatitis C virus infection in chronic hepatitis B virus carriers. J. Infect. Dis. 163:400-402.
Fong, T. L., A. M. Di Bisceglie, J. G. Waggoner, S. M. Banks, and J. H. Hoofnagle. 1991. The significance of antibody to hepatitis C virus in patients with chronic hepatitis B. Hepatology 14:64-67.
Fukuda, R., N. Ishimura, M. Niigaki, S. Hamamoto, S. Satoh, S. Tanaka, Y. Kushiyama, Y. Uchida, S. Ihihara, S. Akagi, M. Watanabe, and Y. Kinoshita. 1999. Serologically silent hepatitis B virus coinfection in patients with hepatitis C virus-associated chronic liver disease: clinical and virological significance. J. Med. Virol. 58:201-207.
Ganem, D., and H. E. Varmus. 1987. The molecular biology of the hepatitis B viruses. Annu. Rev. Biochem. 56:651-693.
Gonzalez, S., S. Navas, A. Madejon, J. Bartolome, I. Castillo, G. Moraleda, J. Martin, E. Marriott, M. Herrero, and V. Carreno. 1995. Hepatitis B and D genomes in hepatitis B surface antigen negative patients with chronic hepatitis C. J. Med. Virol. 45:168-173.
Gosalvez, J., E. Rodriguez-I?igo, J. L. Ramiro-Diaz, J. Bartolome, J. F. Tomas, H. Oliva, and V. Carreno. 1998. Relative quantification and mapping of hepatitis C virus by in situ hybridization and digital image analysis. Hepatology 27:1428-1434.
Koike, K., M. Kobayashi, M. Gondo, I. Hayashi, T. Osuga, and S. Takada. 1998. Hepatitis B virus DNA is frequently found in liver biopsy samples from hepatitis C virus-infected chronic hepatitis patients. J. Med. Virol. 54:249-255.
Liaw, Y. F., S. M. Lin, I. S. Sheen, and C. M. Chu. 1991. Acute hepatitis C virus superinfection followed by spontaneous HBeAg seroconversion and HBsAg elimination. Infection 19:250-251.
Ohkawa, K., N. Hayashi, N. Yuki, H. Hagiwara, M. Kato, K. Yamamoto, H. Eguchi, H. Fusamoto, M. Masuzawa, and T. Kamada. 1994. Hepatitis C virus antibody and hepatitis C virus replication in chronic hepatitis B patients. J. Hepatol. 21:509-514.
Pontisso, P., M. Gerotto, L. Benvegnu, L. Chemello, and A. Alberti. 1998. Coinfection by hepatitis B virus and hepatitis C virus. Antivir. Ther. 3(Suppl. 3):137-142.
Rodriguez-Inigo, E., L. Mariscal, J. Bartolome, I. Castillo, C. Navacerrada, N. Ortiz-Movilla, M. Pardo, and V. Carreno. 2003. Distribution of hepatitis B virus in the liver of chronic hepatitis C patients with occult hepatitis B virus infection. J. Med. Virol. 70:571-580.
Rosenberg, S. 2001. Recent advances in the molecular biology of hepatitis C virus. J. Mol. Biol. 313:451-464.
Sagnelli, E., N. Coppola, V. Messina, D. Di Caprio, C. Marrocco, A. Marotta, M. Onofrio, C. Scolastico, and P. Filippini. 2002. HBV superinfection in hepatitis C virus chronic carriers, viral interaction, and clinical course. Hepatology 36:1285-1291.
Schuttler, C. G., N. Fiedler, K. Schmidt, R. Repp, W. H. Gerlich, and S. Schaefer. 2002. Suppression of hepatitis B virus enhancer 1 and 2 by hepatitis C virus core protein. J. Hepatol. 37:855-862.
Sheen, I. S., Y. F. Liaw, C. M. Chu, and C. C. Pao. 1992. Role of hepatitis C virus infection in spontaneous hepatitis B surface antigen clearance during chronic hepatitis B virus infection. J. Infect. Dis. 165:831-834.
Shih, C. M., S. J. Lo, T. Miyamura, S. Y. Chen, and Y. H. Lee. 1993. Suppression of hepatitis B virus expression and replication by hepatitis C virus core protein in HuH-7 cells. J. Virol. 67:5823-5832.
Shih, C. M., C. M. Chen, S. Y. Chen, and Y. H. Lee. 1995. Modulation of the trans-suppression activity of hepatitis C virus core protein by phosphorylation. J. Virol. 69:1160-1171.
Zarski, J. P., B. Bohn, A. Bastie, J. M. Pawlotsky, M. Baud, F. Bost-Bezeaux, J. Tran van Nhieu, J. M. Seigneurin, C. Buffet, and D. Dhumeaux. 1998. Characteristics of patients with dual infection by hepatitis B and C viruses. J. Hepatol. 28:27-33.(E. Rodríguez-í?igo, J. Ba)
Departamento de Electrónica, Automática e Informática Industrial, Universidad Politécnica de Madrid, Madrid, Spain
ABSTRACT
In this work, we have shown that hepatitis C virus (HCV) and hepatitis B virus (HBV) can coexist in the same hepatocyte using double fluorescent in situ hybridization in liver biopsy samples from patients with chronic HCV infection with occult HBV infection. Digital image analysis of hybridization signals showed that the HBV DNA levels in coinfected hepatocytes were lower than those in cells infected only with HBV. This finding supports the hypothesis of inhibition of HBV replication by HCV. Furthermore, HCV RNA levels were lower in coinfected cells than in cells infected only with HCV, suggesting that HBV may also inhibit HCV replication.
TEXT
Hepatitis B virus (HBV) belongs to the Hepadnaviridae family of animal viruses, and its genome consists of a circular partially double-stranded DNA molecule of 3.2 kb in length which contains four overlapping reading frames that code for surface proteins (HBsAg), core proteins (HBc/HBeAg), the viral polymerase, and the transcriptional transactivator X protein (HBx) (9).
Hepatitis C virus (HCV) is classified in the Hepacivirus genus of the Flaviviridae family, and its genome is a positive-stranded RNA of 9.6 kb in length that encodes a large polyprotein that undergoes proteolytic processing by cellular and viral proteinases to generate the individual viral proteins (17).
As HBV and HCV share similar transmission routes, coinfection with the two viruses is not a rare event (3, 6, 7). Clinical data obtained from chronic HBV carriers superinfected with HCV suggest that HCV may inhibit HBV replication (13, 15, 20). This hypothesis is supported by the fact that patients with chronic HCV infection frequently have a special form of HBV infection, termed occult HBV infection (2, 8, 10, 12). This is characterized by the presence of low levels of HBV DNA in serum and/or in liver in the absence of detectable HBsAg in serum. This inhibition may be mediated by the host immune response (via the induction of cytokines such as interferons) or by a direct effect of HCV proteins. In this regard, it has been shown that HCV core and NS2 proteins inhibit HBV replication and gene expression in vitro (4, 5, 19, 21, 22).
Direct interference mediated by HCV proteins can occur in vivo only if both HBV and HCV coexist in the same hepatocyte. However, it has not been demonstrated whether HBV and HCV infect the same cell in the liver of patients coinfected with the two viruses. To address this issue, we have used double fluorescent in situ hybridization to determine the presence of HBV DNA and HCV RNA in liver biopsy samples from six patients with chronic hepatitis C (anti-HCV positive, serum HCV RNA positive) and occult HBV infection (HBsAg negative, serum HBV DNA positive).
Patients underwent a liver biopsy for diagnostic purposes. After liver samples were obtained, they were divided into two portions. One fragment was processed for histological diagnosis and for in situ hybridization, and the other was embedded in RNAlater (QIAGEN, Hilden, Germany) in less than 30 seconds after being obtained and stored at –20°C. At the time of the liver biopsy, a serum sample was obtained from each patient for the determination of HBV DNA and HCV RNA concentrations. All patients gave their written informed consent to participate in the study.
As mentioned above, all patients were persistently positive for HBV DNA in serum, and at the time of the liver biopsy, the mean HBV DNA concentration in serum was 2.4 x 103 ± 1.8 x103 IU/ml as tested by the Amplicor HBV Monitor test v.2.0 (Roche Diagnostics SL, Barcelona, Spain).
In situ hybridization was performed as described previously (11, 16) except that the HBV and HCV probes were labeled with digoxigenin-11-dUTP and biotin-16-dUTP (Roche Molecular Biochemicals, Indianapolis, IN). HBV hybrids were detected with an antidigoxigenin fluorescein isothiocyanate-conjugated antibody (green fluorescence), and the HCV hybrids were detected with avidin-rhodamine (red fluorescence) (Roche Molecular Biochemicals). Slides were counterstained using 4',6'-diamidino-2-phenylindole (blue fluorescence). Signals were visualized in a Nikon Eclipse E-400 light epifluorescence microscope. Images were captured with a high-sensitivity charge-coupled device camera (Leica DFC 300FX; Leica Microsystems, Switzerland) and 24-bit red-green-blue color depth and stored as tagged image file format files.
In all six patients, hepatocytes infected by only HBV, by only HCV, or by both viruses were detected (Fig. 1). The mean percentage of infected hepatocytes was 7.9% ± 7.5%. Of these, 12% ± 11.7% were infected only by HBV, 42.2% ± 19.1% only by HCV, and 45.8% ± 18.4% by both viruses. To determine whether there was an inhibition of HBV or HCV replication in coinfected hepatocytes, the intensity of fluorescence signals corresponding to HBV DNA and HCV RNA was measured in single-infected and double-infected hepatocytes by digital image analysis. To avoid differences due to the efficiency of in situ hybridization, the intensity of fluorescence signals in single-infected hepatocytes was compared to intensity in double-infected cells within the same tissue section.
This analysis showed that the intensity of fluorescence signals corresponding to HBV DNA was 1.6 ± 0.16 times higher (range, 1.44 to 1.82) in hepatocytes infected only by HBV than in those coinfected by HBV and HCV (Fig. 2), although the differences were not significant. Similarly, the intensity of fluorescence signals corresponding to HCV RNA was 1.83 ± 0.5 times higher (range, 1.45 to 2.74) in single-infected than in double-infected cells (Fig. 2), but without statistical significance. These data suggest that there may be a mutual inhibition of the replication of the two viruses in hepatocytes coinfected by HBV and HCV. This finding is in agreement with previous reports in which inhibition of HCV replication by HBV in coinfected patients has been also suggested (14, 18, 23).
To analyze further the possible inhibition of HCV replication by HBV, serum HCV RNA concentration and levels of the antigenomic HCV RNA strand, which is the replicative intermediate of the virus (1), were tested in the six patients with chronic hepatitis C and occult HBV infection in comparison to levels in 10 patients with chronic HCV infection without HBV infection. Patients were matched for gender, age, alanine aminotransferase levels, HCV genotype, and known duration of HCV infection (Table 1). These 10 patients underwent a liver biopsy for diagnostic purposes, and liver samples were processed as described above.
The serum HCV RNA concentration (tested using the Amplicor HCV Monitor test; Roche) was significantly lower (P = 0.031) in the six patients with occult HBV infection (mean ± standard error of the mean [SEM], 2.92 x 105 ± 7.03 x 104 IU/ml) than in patients with chronic hepatitis C without HBV infection (mean ± SEM, 7.66 x 105 ± 1.28 x 105 IU/ml).
For quantitation of the antigenomic HCV RNA strand, total RNA was isolated from the liver biopsy sample fragments preserved in RNAlater. Synthesis of cDNA was performed with Tth polymerase (Applied Biosystems, Foster City, CA) using 0.5 μg of total RNA and the sense primer 5'-CTTCACGCAGAAAGCGTCTA-3'. After addition of the antisense primer 5'-CAAGCACCCTATCAGGCAGT-3', real-time PCRwas performed using the LightCycler FastStart DNA Master SYBR Green I kit (Roche Molecular Biochemicals). The reaction was run in a Light Cycler (Roche Molecular Biochemicals). The specificity of the assay was tested using synthetic genomic and antigenomic HCV RNA, and it was shown that it is capable of detecting 3.2 copies of the correct strand while detecting 107 to 108 of the incorrect strand. The amount of the antigenomic HCV RNA strand was lower in patients with occult HBV infection (mean ± SEM, 1.32 x 105 ± 4.38 x 104 copies/μg of total RNA) than in patients without HBV infection (mean ± SEM, 5.7 x 105 ± 3.1 x 105 copies/μg of total RNA), although without statistical significance. This finding is in agreement with the hypothesis of the inhibition of HCV replication by HBV in coinfected patients.
In conclusion, in this work we have demonstrated that HBV and HCV can infect the same hepatocyte in the liver of patients coinfected by the two viruses. Our results also suggest that, apart from the possible involvement of the host immune response in the inhibition of the replication of HBV and HCV, a direct mutual inhibition of the replication of the two viruses in coinfected cells may also occur.
ACKNOWLEDGMENTS
This work has been supported by grants from the Fundación de Investigaciones Biomédicas and from the Fundación para el Estudio de las Hepatitis Virales, Madrid, Spain.
REFERENCES
Bartenschlager R., and V. Lohmann. 2000. Replication of the hepatitis C virus. J. Gen. Virol. 81:1631-1648.
Cacciola, I., T. Pollicino, G. Squadrito, G. Cerenzia, M. E. Orlando, and G. Raimondo. 1999. Occult hepatitis B virus infection in patients with chronic hepatitis C liver disease. N. Engl. J. Med. 341:22-26.
Chen, D. S., G. C. Kuo, J. L. Sung, M. Y. Lai, J. C. Sheu, P. J. Chen, P. M. Yang, H. M. Hsu, M. H. Chang, C. J. Chen, L. C. Hahn, Q. L. Choo, T. H. Wang, and M. Houghton. 1990. Hepatitis C virus infection in an area hyperendemic for hepatitis B and chronic liver disease: the Taiwan experience. J. Infect. Dis. 162:817-822.
Chen, S. Y., C. F. Kao, C. M. Chen, C. M. Shih, M. J. Hsu, C. H. Chao, S. H. Wang, L. R. You, and Y. H. Lee. 2003. Mechanisms for inhibition of hepatitis B virus gene expression and replication by hepatitis C virus core protein. J. Biol. Chem. 278:591-607.
Dumoulin, F. L., A. von dem Bussche, J. Li, L. Khamzina, J. R. Wands, T. Sauerbruch, and U. Spengler. 2003. Hepatitis C virus NS2 protein inhibits gene expression from different cellular and viral promoters in hepatic and nonhepatic cell lines. Virology 305:260-266.
Fattovich, G., A. Tagger, L. Brollo, G. Giustina, P. Pontisso, G. Realdi, A. Alberti, and A. Ruol. 1991. Hepatitis C virus infection in chronic hepatitis B virus carriers. J. Infect. Dis. 163:400-402.
Fong, T. L., A. M. Di Bisceglie, J. G. Waggoner, S. M. Banks, and J. H. Hoofnagle. 1991. The significance of antibody to hepatitis C virus in patients with chronic hepatitis B. Hepatology 14:64-67.
Fukuda, R., N. Ishimura, M. Niigaki, S. Hamamoto, S. Satoh, S. Tanaka, Y. Kushiyama, Y. Uchida, S. Ihihara, S. Akagi, M. Watanabe, and Y. Kinoshita. 1999. Serologically silent hepatitis B virus coinfection in patients with hepatitis C virus-associated chronic liver disease: clinical and virological significance. J. Med. Virol. 58:201-207.
Ganem, D., and H. E. Varmus. 1987. The molecular biology of the hepatitis B viruses. Annu. Rev. Biochem. 56:651-693.
Gonzalez, S., S. Navas, A. Madejon, J. Bartolome, I. Castillo, G. Moraleda, J. Martin, E. Marriott, M. Herrero, and V. Carreno. 1995. Hepatitis B and D genomes in hepatitis B surface antigen negative patients with chronic hepatitis C. J. Med. Virol. 45:168-173.
Gosalvez, J., E. Rodriguez-I?igo, J. L. Ramiro-Diaz, J. Bartolome, J. F. Tomas, H. Oliva, and V. Carreno. 1998. Relative quantification and mapping of hepatitis C virus by in situ hybridization and digital image analysis. Hepatology 27:1428-1434.
Koike, K., M. Kobayashi, M. Gondo, I. Hayashi, T. Osuga, and S. Takada. 1998. Hepatitis B virus DNA is frequently found in liver biopsy samples from hepatitis C virus-infected chronic hepatitis patients. J. Med. Virol. 54:249-255.
Liaw, Y. F., S. M. Lin, I. S. Sheen, and C. M. Chu. 1991. Acute hepatitis C virus superinfection followed by spontaneous HBeAg seroconversion and HBsAg elimination. Infection 19:250-251.
Ohkawa, K., N. Hayashi, N. Yuki, H. Hagiwara, M. Kato, K. Yamamoto, H. Eguchi, H. Fusamoto, M. Masuzawa, and T. Kamada. 1994. Hepatitis C virus antibody and hepatitis C virus replication in chronic hepatitis B patients. J. Hepatol. 21:509-514.
Pontisso, P., M. Gerotto, L. Benvegnu, L. Chemello, and A. Alberti. 1998. Coinfection by hepatitis B virus and hepatitis C virus. Antivir. Ther. 3(Suppl. 3):137-142.
Rodriguez-Inigo, E., L. Mariscal, J. Bartolome, I. Castillo, C. Navacerrada, N. Ortiz-Movilla, M. Pardo, and V. Carreno. 2003. Distribution of hepatitis B virus in the liver of chronic hepatitis C patients with occult hepatitis B virus infection. J. Med. Virol. 70:571-580.
Rosenberg, S. 2001. Recent advances in the molecular biology of hepatitis C virus. J. Mol. Biol. 313:451-464.
Sagnelli, E., N. Coppola, V. Messina, D. Di Caprio, C. Marrocco, A. Marotta, M. Onofrio, C. Scolastico, and P. Filippini. 2002. HBV superinfection in hepatitis C virus chronic carriers, viral interaction, and clinical course. Hepatology 36:1285-1291.
Schuttler, C. G., N. Fiedler, K. Schmidt, R. Repp, W. H. Gerlich, and S. Schaefer. 2002. Suppression of hepatitis B virus enhancer 1 and 2 by hepatitis C virus core protein. J. Hepatol. 37:855-862.
Sheen, I. S., Y. F. Liaw, C. M. Chu, and C. C. Pao. 1992. Role of hepatitis C virus infection in spontaneous hepatitis B surface antigen clearance during chronic hepatitis B virus infection. J. Infect. Dis. 165:831-834.
Shih, C. M., S. J. Lo, T. Miyamura, S. Y. Chen, and Y. H. Lee. 1993. Suppression of hepatitis B virus expression and replication by hepatitis C virus core protein in HuH-7 cells. J. Virol. 67:5823-5832.
Shih, C. M., C. M. Chen, S. Y. Chen, and Y. H. Lee. 1995. Modulation of the trans-suppression activity of hepatitis C virus core protein by phosphorylation. J. Virol. 69:1160-1171.
Zarski, J. P., B. Bohn, A. Bastie, J. M. Pawlotsky, M. Baud, F. Bost-Bezeaux, J. Tran van Nhieu, J. M. Seigneurin, C. Buffet, and D. Dhumeaux. 1998. Characteristics of patients with dual infection by hepatitis B and C viruses. J. Hepatol. 28:27-33.(E. Rodríguez-í?igo, J. Ba)