Detection of heDatitis C virus RNA by in situ hvbridization I
J
Blight K, Trowbridge R, Rowland R, Gowans E. Detection of hepatitis C virus RNA by in situ hybridization. Liver 1992 (Spec. issue) 12: 286-289. Abstract: Persistent infection with hepatitis C virus (HCV) is associated with chronic hepatitis and cirrhosis which may eventually develop into primary hepatocellular carcinoma. The mechanism of pathogenesis is illdefined and nothing is known of the distribution, frequency or type of infected cell in the liver of HCV-infected individuals. In this study we have examined liver tissue taken at autopsy from 2 anti-HCV-positive patients by in situ hybridization for the presence of HCV RNA. Viral RNA was detected by autoradiography after hybridization with ‘251labelled riboprobes, representing approximately 35% of the HCV genome. Only a few positive cells were identified in the HCV-infected liver samples, but not in a normal liver sample. Hybridization with an unrelated probe was negative in all samples. The HCV RNA-positive cells were detected with anti-sense but not sense RNA probes, suggesting that they contained a high ratio of genomic:antigenomic RNA. The appearance and distribution of the HCV RNA-positive cells suggested that they were not hepatocytes and were more likely to be lymphocytes or macrophages.
Introduction
The genome of hepatitis C virus (HCV) isolated from chimpanzee or human carriers has been cloned recently (1, 2) and recombinant viral antigens expressed from regions of the cDNA. These are used in assays to detect antibodies to the virus (anti-HCV) (3,4). Anti-HCV antibodies were detected in a high proportion of patients with chronic non-A, non-B hepatitis (NANBH) and in a smaller proportion of patients with acute NANBH (3). A number of patients with cryptogenic cirrhosis or hepatocellular carcinoma (HCC) were also reported to be positive for anti-HCV ( 5 , 6 ) . These studies suggest that HCV is a major etiologic agent associated with NANBH, although the link with HCC is still unclear. The cloning of the HCV genome and the subsequent examination of the nucleic acid sequence suggested that HCV was unique although it has many similarities with the Pestiviruses. The genome is positive sense and contains a single open reading frame with the putative structural proteins (Core, E l , E2/NS1) encoded towards the 5’ end and the non-structural proteins (NS2-NS5) encoded towards the 3’ end. The open reading frame is flanked by two short non-coding regions (7, 8). The availability of the genome sequence has also permitted the design of oligonucleotide primers 286
Keril Blight, Rachel Trowbridge, Robert Rowland and Eric Cowans Department of Microbiology and Immunology, University of Adelaide, Division of Medical Virology and Division of Tissue Pathology, Institute of Medical and Veterinary Science, Adelaide, Australia
Key words: HCV RNA; viral pathogenesis; viral hepatitis; radiolabelled probes; autoradiography Dr E J Gowans, Division of Medical Virology, Institute of Medical and Veterinary Science, Box 14 Rundle Mall PO, Adelaide, SA 5000, Australia. Phone: 08 228 4631. Fax: 08 224 0927 Accepted for publication 7 April, 1992
and the use of reverse transcription (RT) followed by polymerase chain reaction (PCR) to detect HCV RNA (9, 10). This has not only defined a subpopulation of anti-HCV positive individuals who are viremic but also determined the appearance and patterns of viremia in naturally infected patients (1 1) and in experimentally infected chimpanzees (1 2). These studies showed that HCV RNA often appears in the serum within 1 week of infection during the acute phase and is also detectable in the serum of chronic carriers, a high proportion of whom develop liver disease. Ultrastructural changes accompanied by the appearance of an unrelated cytoplasmic antigen have been described in HCV-infected liver samples (12). Similar changes have been seen in hepatitis delta virus-infected liver samples, and it is likely that these are indirect markers of virus infection. No data are available on the distribution and cell type, which either supports HCV replication or is persistently HCV-infected, although it is highly likely that 1) the proportion of infected cells, or 2) the viral load in infected cells, or 3) both, are low as it is necessary to use PCR to detect HCV RNA in the livers of chronically-infected individuals (1 3). In this report, we have examined liver samples taken at autopsy from 2 HCV chronic carriers for the presence of HCV RNA by in situ hybridization.
In situ detection of HCV RNA Material and methods Autopsy liver samples
Liver samples collected at autopsy from 2 antiHCV-positive patients and from an anti-HCVnegative patient, who died as a result of a myocardial infarct, were collected within 6 to 24 hours after death. The samples were either sliced thinly and frozen at -70°C or fixed in 10% buffered formalin for routine histologic processing. Frozen sections were fixed in 4% paraformaldehyde essentially as described (14) prior to in situ hybridization. Probe preparation and in situ hybridization
A number of subgenomic probes were employed; pRTI, a 324 bp cDNA representing the 5' noncoding region cloned into pBluescript (Stratagene) and sequenced in our laboratory. Details of this clone will be reported elsewhere (1 5). pS7/ 1-216, pU 1-4652d and pS 1-8791 were generous gifts from Dr. T. Miyamura (16) and represent the core+ envelope region, NS3 and a portion of NS5, respectively. The HCV cDNA from each of the above plasmids was subcloned into pBluescript. Limited restriction enzyme mapping was performed in order to determine the orientation of the HCV cDNA insert. The plasmids were linearized to permit transcription of insert-length sense or anti-sense RNA from either the T3- or T7-RNA promoter. 1251-labelled transcripts were produced in a standard transcription reaction (Promega Biotec) containing 50 pCi '"1-CTP (a generous gift from Amersham, Australia). The total complement of unique sequences available as probe represents approximately 35% of the HCV genome. In situ hybridization, post-hybridization washing steps and autoradiographic detection of the newly
formed hybrids were performed essentially as described previously (14). An unrelated riboprobe comprised of HBV RNA was used throughout the study. Results Liver histology
Case 1: Active irregular cirrhosis was present. The broad fibrous trabeculae separating the nodules contained lymphocytes, including aggregates; macrophages, some containing lipid droplets; and a few plasma cells. Bile ducts were present and ductural proliferation was apparent. Piecemeal necrosis was evident. Sinusoid cells showed some hypertrophy and occasional lymphocytes were apparent within the sinusoids. Mild dysplasia of hepatocyte nuclei was present in some nodules. Case 2: There was a chronic persistent hepatitis pattern present. The portal tracts were expanded and fibrotic, and contained mild to moderate numbers of lymphocytes, some macrophages and a few eosinophils. Plasma cells were sparse. The limiting plates were virtually intact. The sinusoids contained scattered lymphocytes and showed some sinusoidal lining cell hypertrophy. There was severe centrilobular congestion consistent with death from heart failure. HCV RNA in individual liver cells
Despite the different histological diagnoses, the distribution and abundance of HCV RNA-positive cells was similar in liver samples from both antiHCV-positive patients. The samples contained very few positive cells, determined as 3 4 cells per section on average, although some sections contained no positive cells. These finding were consistent over several experiments. The HCV RNA-positive cells
Fig. I. The detection of HCV RNA by in situ hybridization in liver tissues from 2 anti-HCV-positive patients, (A) Case I , (B) Case 2 and (C) an anti-HCV-negative patient.
287
Blight et al. were only identified with probes to detect sense (genomic) RNA; no positive cells were noted after hybridization with a probe to detect antisense RNA. The positive cells were present as isolated cells found near the edge of the sinusoid, in portal tracts in the fibrotic liver (case 2) or in fibrous trabeculae of the cirrhotic liver (case 1) and occasionally in the parenchyma. The appearance of the positive cells suggested that they were not hepatocytes (Fig. lA, B), but it was not possible to conclusively identify them. No positive cells were noted in the sample of normal liver, nor in the antiHCV-positive livers after hybridization with the HBV riboprobe (Fig. 1C). Discussion
Prior to the development of an assay to detect HCV RNA by in situ hybridization, the viral genome was detected in liver samples by RT/PCR. However, unless the RT/PCR is designed to identify antigenomic RNA present as a replicative intermediate, positive results may simply reflect serum contamination rather than active virus replication. Furthermore, the PCR provides no data relating to the distribution and numbers of virus-infected cells. In this study we were unable to convincingly detect HCV RNA in hepatocytes by in situ hybridization. However, genomic HCV RNA was detected in a small proportion of cells (not concentrated in the liver cell plates), possibly lymphocytes, macrophages, fibroblasts or endothelial cells, although we believe that they are more likely to be lymphocytes or macrophages. It is unclear if these results are a consequence of poor sensitivity associated with the in situ hybridization reaction, and experiments are currently underway to determine the sensitivity limit. Furthermore, since we were unable to detect antigenomic HCV RNA in these cells, it is possible that the genomic HCV RNA actually represents virus uptake or phagocytosis rather than viral replication. Quantitation of our assay will help to define this point, since it is likely that antigenomic HCV RNA will be 10- to 100-fold less abundant (13). On the other hand, should these cells be supporting viral replication, then they may act as a “Trojan Horse” since it is probable that lymphocytes and macrophages only inhabit the liver temporarily during normal trafficking. This may be an important factor in the pathogenesis of HCV infection. We were unable to perform the full range of controls which are desirable for in situ hybridization, due to the paucity of HCV RNA-positive cells in the liver samples examined. However, the control reactions which were performed, viz unre288
lated probe, uninfected tissue sample, suggested that the reaction was specific. Furthermore, the fact that only antisense probes generated a positive signal is additional evidence for the specifity of the reaction. Further work is in progress to examine the effect of predigestion with nucleases, and to construct a post-hybridization melt curve (14). We believe that the in situ hybridization assay is likely to make a valuable contribution to the study of HCV pathogenesis. Acknowledgements We thank Dr. Tatsuo Miyamura for the gift of HCV plasmids, Dawn Campbell for typing, Amersham Australia for the gift of ”’1-CTP and the staff of the IMVS Photographic Services. Keril Blight is a Royal Adelaide Hospital Dawes Scholar. The work is supported by a grant from the National Health and Medical Research Council of Australia, and from Abbott Diagnostic Laboratories.
References 1. CHOOQ-L, Kuo G, WEINER L R, BRADLEY D A J, OVERBY
W, HOUCHTON M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B hepatitis genome. Science 1989: 244: 359-361. 2. KATON, HIJIKATA M, OTSUYAMA Y, et al. Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis. Proc Natl Acad Sci USA 1990: 87: 95249528. 3. Kuo G, CHOOQ-L, ALTERH J, et al. An assay for circulating antibodies to a major etiologic virus of human nonA, non-B hepatitis. Science 1989: 244: 362-364. T, et al. Detection of T, SAITOI, KATAYAMA 4. MIYAMURA antibody against antigen expressed by molecular cloned hepatitis C virus cDNA: Application to diagnosis and blood screening for post-transfusion hepatitis. Proc Natl Acad Sci USA 1990: 87: 983-987. J, BARRERA J M, COSTAJ, et al. Hepatitis 5 . SANCHEZ-TAPIAS C virus infection in patients with nonalcoholic-chronic liver disease. Ann Int Med 1990: 112: 921-924. 6. WATANABE Y, HARADA S, SAITOI, MIYAMLJRA T. Prevalence of antibody against the core protein of hepatitis C virus in patients with hepatocellular carcinoma. Int J Cancer 1991 (in press). A, MORI C, FUKEI, et al. Structure and 7. TAKAMIZAWA organisation of the hepatitis C virus genome isolated from human carriers. J Virol 1991: 65: 1105-1 113. o RICHMAN K H, HANJ B, et al. Genetic organis8. C ~ o Q-L, ation and diversity of the hepatitis C virus. Proc Natl Acad Sci USA 1991: 88: 2451-2455. D W, et al. Detection of 9. WEINERA J, Kuo G, BRADLEY hepatitis C viral sequences in non-A, non-B-hepatitis. Lancet 1990: 355: 1-3. 10. GARSON J A, TEDDER R S, BRICCSM, et al. Detection of hepatitis C viral sequences in blood donations by “nested” polymerase chain reaction and prediction of infectivity. Lancet 1990: 335: 1419-1422. 11. FARCIP, ALTERH J, WONCD, et al. A long-term study of hepatitis C virus replication in non-A, non-B hepatitis. N Engl J Med 1991: 325: 98-104. 12. SHIMIZU Y, WEINER A J, ROSENBLATT J, et al. Early events in hepatitis C virus infection of chimpanzees. Proc Natl Acad Sci USA 1991: 87: 6441-6444.
In situ detection of HCV RNA 13. FONGT-L, SHINDO M, FEINSTONE S M, HOOFNAGLE J H, Dr BISCEGLIE A M. Detection of replicative intermediates of hepatitis C viral RNA in the liver and serum of patients with chronic hepatitis C. J Clin Invest 1991: 88: 1058-1060. 14. GOWANSE J, JILBERT A R, BURRELL C J. Detection of specific DNA and RNA sequences in tissues and cells by in situ hybridization. In: SYMONSR H, ed. Nucleic acid probes. Boca Raton: CRC Press. 1989, 139-158. 15. TROWBRIDGE R, BEARDM, BLIGHTK, BEARDSLEY A,
E. PCR analysis of anti-HCV positive sera using GOWANS diverse primer combinations. (in preparation). 16. TAKEUCHI K, KUBO Y, BOONMAR S, et at. The putative nucleocapsid and envelope protein genes of hepatitis C virus determined by comparison of nucleotide sequences of two isolates derived from an experimentally infected chimpanzee and healthy human carriers. J Gen Virol 1990: 71: 3027-3033.
289
J
Blight K, Trowbridge R, Rowland R, Gowans E. Detection of hepatitis C virus RNA by in situ hybridization. Liver 1992 (Spec. issue) 12: 286-289. Abstract: Persistent infection with hepatitis C virus (HCV) is associated with chronic hepatitis and cirrhosis which may eventually develop into primary hepatocellular carcinoma. The mechanism of pathogenesis is illdefined and nothing is known of the distribution, frequency or type of infected cell in the liver of HCV-infected individuals. In this study we have examined liver tissue taken at autopsy from 2 anti-HCV-positive patients by in situ hybridization for the presence of HCV RNA. Viral RNA was detected by autoradiography after hybridization with ‘251labelled riboprobes, representing approximately 35% of the HCV genome. Only a few positive cells were identified in the HCV-infected liver samples, but not in a normal liver sample. Hybridization with an unrelated probe was negative in all samples. The HCV RNA-positive cells were detected with anti-sense but not sense RNA probes, suggesting that they contained a high ratio of genomic:antigenomic RNA. The appearance and distribution of the HCV RNA-positive cells suggested that they were not hepatocytes and were more likely to be lymphocytes or macrophages.
Introduction
The genome of hepatitis C virus (HCV) isolated from chimpanzee or human carriers has been cloned recently (1, 2) and recombinant viral antigens expressed from regions of the cDNA. These are used in assays to detect antibodies to the virus (anti-HCV) (3,4). Anti-HCV antibodies were detected in a high proportion of patients with chronic non-A, non-B hepatitis (NANBH) and in a smaller proportion of patients with acute NANBH (3). A number of patients with cryptogenic cirrhosis or hepatocellular carcinoma (HCC) were also reported to be positive for anti-HCV ( 5 , 6 ) . These studies suggest that HCV is a major etiologic agent associated with NANBH, although the link with HCC is still unclear. The cloning of the HCV genome and the subsequent examination of the nucleic acid sequence suggested that HCV was unique although it has many similarities with the Pestiviruses. The genome is positive sense and contains a single open reading frame with the putative structural proteins (Core, E l , E2/NS1) encoded towards the 5’ end and the non-structural proteins (NS2-NS5) encoded towards the 3’ end. The open reading frame is flanked by two short non-coding regions (7, 8). The availability of the genome sequence has also permitted the design of oligonucleotide primers 286
Keril Blight, Rachel Trowbridge, Robert Rowland and Eric Cowans Department of Microbiology and Immunology, University of Adelaide, Division of Medical Virology and Division of Tissue Pathology, Institute of Medical and Veterinary Science, Adelaide, Australia
Key words: HCV RNA; viral pathogenesis; viral hepatitis; radiolabelled probes; autoradiography Dr E J Gowans, Division of Medical Virology, Institute of Medical and Veterinary Science, Box 14 Rundle Mall PO, Adelaide, SA 5000, Australia. Phone: 08 228 4631. Fax: 08 224 0927 Accepted for publication 7 April, 1992
and the use of reverse transcription (RT) followed by polymerase chain reaction (PCR) to detect HCV RNA (9, 10). This has not only defined a subpopulation of anti-HCV positive individuals who are viremic but also determined the appearance and patterns of viremia in naturally infected patients (1 1) and in experimentally infected chimpanzees (1 2). These studies showed that HCV RNA often appears in the serum within 1 week of infection during the acute phase and is also detectable in the serum of chronic carriers, a high proportion of whom develop liver disease. Ultrastructural changes accompanied by the appearance of an unrelated cytoplasmic antigen have been described in HCV-infected liver samples (12). Similar changes have been seen in hepatitis delta virus-infected liver samples, and it is likely that these are indirect markers of virus infection. No data are available on the distribution and cell type, which either supports HCV replication or is persistently HCV-infected, although it is highly likely that 1) the proportion of infected cells, or 2) the viral load in infected cells, or 3) both, are low as it is necessary to use PCR to detect HCV RNA in the livers of chronically-infected individuals (1 3). In this report, we have examined liver samples taken at autopsy from 2 HCV chronic carriers for the presence of HCV RNA by in situ hybridization.
In situ detection of HCV RNA Material and methods Autopsy liver samples
Liver samples collected at autopsy from 2 antiHCV-positive patients and from an anti-HCVnegative patient, who died as a result of a myocardial infarct, were collected within 6 to 24 hours after death. The samples were either sliced thinly and frozen at -70°C or fixed in 10% buffered formalin for routine histologic processing. Frozen sections were fixed in 4% paraformaldehyde essentially as described (14) prior to in situ hybridization. Probe preparation and in situ hybridization
A number of subgenomic probes were employed; pRTI, a 324 bp cDNA representing the 5' noncoding region cloned into pBluescript (Stratagene) and sequenced in our laboratory. Details of this clone will be reported elsewhere (1 5). pS7/ 1-216, pU 1-4652d and pS 1-8791 were generous gifts from Dr. T. Miyamura (16) and represent the core+ envelope region, NS3 and a portion of NS5, respectively. The HCV cDNA from each of the above plasmids was subcloned into pBluescript. Limited restriction enzyme mapping was performed in order to determine the orientation of the HCV cDNA insert. The plasmids were linearized to permit transcription of insert-length sense or anti-sense RNA from either the T3- or T7-RNA promoter. 1251-labelled transcripts were produced in a standard transcription reaction (Promega Biotec) containing 50 pCi '"1-CTP (a generous gift from Amersham, Australia). The total complement of unique sequences available as probe represents approximately 35% of the HCV genome. In situ hybridization, post-hybridization washing steps and autoradiographic detection of the newly
formed hybrids were performed essentially as described previously (14). An unrelated riboprobe comprised of HBV RNA was used throughout the study. Results Liver histology
Case 1: Active irregular cirrhosis was present. The broad fibrous trabeculae separating the nodules contained lymphocytes, including aggregates; macrophages, some containing lipid droplets; and a few plasma cells. Bile ducts were present and ductural proliferation was apparent. Piecemeal necrosis was evident. Sinusoid cells showed some hypertrophy and occasional lymphocytes were apparent within the sinusoids. Mild dysplasia of hepatocyte nuclei was present in some nodules. Case 2: There was a chronic persistent hepatitis pattern present. The portal tracts were expanded and fibrotic, and contained mild to moderate numbers of lymphocytes, some macrophages and a few eosinophils. Plasma cells were sparse. The limiting plates were virtually intact. The sinusoids contained scattered lymphocytes and showed some sinusoidal lining cell hypertrophy. There was severe centrilobular congestion consistent with death from heart failure. HCV RNA in individual liver cells
Despite the different histological diagnoses, the distribution and abundance of HCV RNA-positive cells was similar in liver samples from both antiHCV-positive patients. The samples contained very few positive cells, determined as 3 4 cells per section on average, although some sections contained no positive cells. These finding were consistent over several experiments. The HCV RNA-positive cells
Fig. I. The detection of HCV RNA by in situ hybridization in liver tissues from 2 anti-HCV-positive patients, (A) Case I , (B) Case 2 and (C) an anti-HCV-negative patient.
287
Blight et al. were only identified with probes to detect sense (genomic) RNA; no positive cells were noted after hybridization with a probe to detect antisense RNA. The positive cells were present as isolated cells found near the edge of the sinusoid, in portal tracts in the fibrotic liver (case 2) or in fibrous trabeculae of the cirrhotic liver (case 1) and occasionally in the parenchyma. The appearance of the positive cells suggested that they were not hepatocytes (Fig. lA, B), but it was not possible to conclusively identify them. No positive cells were noted in the sample of normal liver, nor in the antiHCV-positive livers after hybridization with the HBV riboprobe (Fig. 1C). Discussion
Prior to the development of an assay to detect HCV RNA by in situ hybridization, the viral genome was detected in liver samples by RT/PCR. However, unless the RT/PCR is designed to identify antigenomic RNA present as a replicative intermediate, positive results may simply reflect serum contamination rather than active virus replication. Furthermore, the PCR provides no data relating to the distribution and numbers of virus-infected cells. In this study we were unable to convincingly detect HCV RNA in hepatocytes by in situ hybridization. However, genomic HCV RNA was detected in a small proportion of cells (not concentrated in the liver cell plates), possibly lymphocytes, macrophages, fibroblasts or endothelial cells, although we believe that they are more likely to be lymphocytes or macrophages. It is unclear if these results are a consequence of poor sensitivity associated with the in situ hybridization reaction, and experiments are currently underway to determine the sensitivity limit. Furthermore, since we were unable to detect antigenomic HCV RNA in these cells, it is possible that the genomic HCV RNA actually represents virus uptake or phagocytosis rather than viral replication. Quantitation of our assay will help to define this point, since it is likely that antigenomic HCV RNA will be 10- to 100-fold less abundant (13). On the other hand, should these cells be supporting viral replication, then they may act as a “Trojan Horse” since it is probable that lymphocytes and macrophages only inhabit the liver temporarily during normal trafficking. This may be an important factor in the pathogenesis of HCV infection. We were unable to perform the full range of controls which are desirable for in situ hybridization, due to the paucity of HCV RNA-positive cells in the liver samples examined. However, the control reactions which were performed, viz unre288
lated probe, uninfected tissue sample, suggested that the reaction was specific. Furthermore, the fact that only antisense probes generated a positive signal is additional evidence for the specifity of the reaction. Further work is in progress to examine the effect of predigestion with nucleases, and to construct a post-hybridization melt curve (14). We believe that the in situ hybridization assay is likely to make a valuable contribution to the study of HCV pathogenesis. Acknowledgements We thank Dr. Tatsuo Miyamura for the gift of HCV plasmids, Dawn Campbell for typing, Amersham Australia for the gift of ”’1-CTP and the staff of the IMVS Photographic Services. Keril Blight is a Royal Adelaide Hospital Dawes Scholar. The work is supported by a grant from the National Health and Medical Research Council of Australia, and from Abbott Diagnostic Laboratories.
References 1. CHOOQ-L, Kuo G, WEINER L R, BRADLEY D A J, OVERBY
W, HOUCHTON M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B hepatitis genome. Science 1989: 244: 359-361. 2. KATON, HIJIKATA M, OTSUYAMA Y, et al. Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis. Proc Natl Acad Sci USA 1990: 87: 95249528. 3. Kuo G, CHOOQ-L, ALTERH J, et al. An assay for circulating antibodies to a major etiologic virus of human nonA, non-B hepatitis. Science 1989: 244: 362-364. T, et al. Detection of T, SAITOI, KATAYAMA 4. MIYAMURA antibody against antigen expressed by molecular cloned hepatitis C virus cDNA: Application to diagnosis and blood screening for post-transfusion hepatitis. Proc Natl Acad Sci USA 1990: 87: 983-987. J, BARRERA J M, COSTAJ, et al. Hepatitis 5 . SANCHEZ-TAPIAS C virus infection in patients with nonalcoholic-chronic liver disease. Ann Int Med 1990: 112: 921-924. 6. WATANABE Y, HARADA S, SAITOI, MIYAMLJRA T. Prevalence of antibody against the core protein of hepatitis C virus in patients with hepatocellular carcinoma. Int J Cancer 1991 (in press). A, MORI C, FUKEI, et al. Structure and 7. TAKAMIZAWA organisation of the hepatitis C virus genome isolated from human carriers. J Virol 1991: 65: 1105-1 113. o RICHMAN K H, HANJ B, et al. Genetic organis8. C ~ o Q-L, ation and diversity of the hepatitis C virus. Proc Natl Acad Sci USA 1991: 88: 2451-2455. D W, et al. Detection of 9. WEINERA J, Kuo G, BRADLEY hepatitis C viral sequences in non-A, non-B-hepatitis. Lancet 1990: 355: 1-3. 10. GARSON J A, TEDDER R S, BRICCSM, et al. Detection of hepatitis C viral sequences in blood donations by “nested” polymerase chain reaction and prediction of infectivity. Lancet 1990: 335: 1419-1422. 11. FARCIP, ALTERH J, WONCD, et al. A long-term study of hepatitis C virus replication in non-A, non-B hepatitis. N Engl J Med 1991: 325: 98-104. 12. SHIMIZU Y, WEINER A J, ROSENBLATT J, et al. Early events in hepatitis C virus infection of chimpanzees. Proc Natl Acad Sci USA 1991: 87: 6441-6444.
In situ detection of HCV RNA 13. FONGT-L, SHINDO M, FEINSTONE S M, HOOFNAGLE J H, Dr BISCEGLIE A M. Detection of replicative intermediates of hepatitis C viral RNA in the liver and serum of patients with chronic hepatitis C. J Clin Invest 1991: 88: 1058-1060. 14. GOWANSE J, JILBERT A R, BURRELL C J. Detection of specific DNA and RNA sequences in tissues and cells by in situ hybridization. In: SYMONSR H, ed. Nucleic acid probes. Boca Raton: CRC Press. 1989, 139-158. 15. TROWBRIDGE R, BEARDM, BLIGHTK, BEARDSLEY A,
E. PCR analysis of anti-HCV positive sera using GOWANS diverse primer combinations. (in preparation). 16. TAKEUCHI K, KUBO Y, BOONMAR S, et at. The putative nucleocapsid and envelope protein genes of hepatitis C virus determined by comparison of nucleotide sequences of two isolates derived from an experimentally infected chimpanzee and healthy human carriers. J Gen Virol 1990: 71: 3027-3033.
289
