- Email: [email protected]
Detection of hepatitis C viral sequences in non-A, non-B hepatitis
MEDICAL SCIENCE
hepatitis C viral sequences in non-A, non-B hepatitis
Detection of
The role of hepatitis C virus (HCV) in posttransfusion non-A, non-B hepatitis (NANBH) was investigated by analysing clinical samples for both HCV RNA by cDNA/polymerase chain reaction and antibodies against C100-3 by radioimmunoassay. Of fifteen chronic NANBH patients and one patient with chronic cryptogenic liver disease, ten were positive for anti-C100-3 and seven of the ten had viral sequences in their livers. However, two patients negative for anti-C100-3 also had substantial levels of HCV RNA in their livers. In acute post-transfusion NANBH (one surgical patient and two experimentally infected chimpanzees), HCV RNA was detected in the absence of anti-C100-3. In addition, infectious plasma from a seronegative patient with acute post-transfusion NANBH and a seronegative pool of plasma from a chimpanzee with chronic posttransfusion NANBH had high levels of HCV. These findings show that anti-C100-3-positive patients with chronic post-transfusion NANBH are likely to be viraemic; confirm that antibodies to C100-3 are a marker for infectivity; and suggest that the prevalence of HCV infections may be underestimated from the frequency of antibodies to C100-3 alone.
Introduction When diagnostic methods for hepatitis A and B infectionsl-3 were developed, it became clear that 60-90% of reported post-transfusion hepatitis was caused by unknown agents.1 In the United States alone, non-A, non-B hepatitis (NANBH) affects an estimated 200 000 to 300 000 people annually and causes chronic liver disease in approximately 50% of post-transfusion cases.4,5 Studies on the genome of hepatitis C virus (HCV) have shown that it is a positivestranded RNA virus.6 Although expression of HCV cDNA clones in yeast has led to the development of an effective immunodiagnostic test for antibodies to HCV in plasma and serum samples from patients with NANBH world wide,7-11 there has been no sensitive assay for HCV RNA with a potential for clinical application. Based on the nucleotide sequence of HCV cDNA clones 366 and 37b (sequence lodged with Genbank, Nov 10, 1989) a sensitive, semiquantitative cDNA/polymerase chain reaction12 (cPCR) assay to detect HCV RNA in liver and plasma or serum has ADDRESSES: Chiron Corporation, Emeryville, California (A. J. Weiner, PhD, G. Kuo, PhD, C. Lee, BA, J. Rosenblatt, BA, Q-L. Choo, PhD, M. Houghton, PhD); Hepatitis Branch, Centers for Disease Control, Atlanta, Georgia, USA (D. W. Bradley, PhD); and Division of Gastroenterology, San Giovanni Battista Molinette Hospital, Turin, Italy (F. Bonnino, MD, G. Saracco, MD). Correspondence to Dr A. J. Weiner, Chiron Corporation, 4560 Horton Street, Emeryville, CA 94608, USA.
2
been developed. We report here the results from the analysis of several clinical samples from patients and chimpanzees with respect to HCV RNA and CIOO-3 antibody content.
Subjects and methods To detect HCV sequences in clinical samples, cDNA was synthesised from RNA extracted from liver biopsy or plasma samples and subjected to PCR. The cDNA synthesis and PCR used the same pair of synthetic 16-mer oligonucleotide primers derived from the two non-contiguous HCV cDNA clones 36 (5’-GCATGTCATGATGTAT-3’; anti-sense) and 37b
(5’-ACAATACGTGTGTCAC-3’; sense). Reactions were carried according to the manufacturer’s instructions (BRL cDNA kit 8085SB and Perkin Elmer Cetus PCR kit N801-0055). Thirty cycles of PCR were carried out on all samples as follows: denaturation for 1 min at 94°C, annealing of primers for 2 min at 37°C, and extension for 3 min at 72°C. PCR products were analysed on 18% alkaline agarose gels, blotted onto ’Zeta’ probe paper (BioRad), and hybridised to a phosphorus-32-labelled nicktranslated HCV cDNA insert which lies between, but not including, the cDNA/PCR primers. The blots were washed in 0.1xSSC (1 x SSC=0-15 mol/1 sodium chloride, 0-015 mol/1 sodium citrate), 01% sodium dodecyl sulphate at 68°C, and autoradiographed. All samples were assayed at least three times with reproducible results and no false-positive results in the control samples. Radioinimunoassays (RIA) to detect antibodies against C100-3 were carried out according to Kuo et al.7 In several studies, we and others have found that data obtained by RIA are equivalent to those
out
obtained with the Chiron-Ortho anti-Cl00-3 enzyme-linked immunosorbent assay. Liver biopsy samples (10 x 4 mm cylinders) were obtained from fifteen Italian patients with chronic NANBH, one patient with cryptogenic liver disease, and one patient with chronic hepatitis B (HBV) infection. All these patients were negative for hepatitis B surface antigen and e antigen and for antibody to core antigen, except the patient with chronic HBV infection who was positive for all three markers of HBV infection. Serum samples were collected on the same day as the liver samples from the fifteen chronic NANBH patients and the patient with cryptogenic liver disease. Acute NANBH plasma samples were obtained from one surgical patient known to have an HCV titre of 10 65 chimpanzee infectious doses (CID) per ml13 (gift from Dr Robert Purcell and Dr Harvey Alter, National Institutes of Health) and two experimentally infected, colony-born chimpanzees (77114,15 and 91015). Pooled chronic-phase plasma from the same two chimpanzees (HCV titres of 105 CID/ml16 [no 771] and 106 CID/ml17 [no 910]) were also analysed along with non-infectious, control plasma from four human beings and four chimpanzees.
Fig 1-ALT profiles for chimpanzee 771 (A) and 910 (B) with cPCR results (RNA) and results of RIA for anti-C100-3 (Ab). The
plasma sample collected from a surgical patient during early acute phase of post-transfusion NANBH had very high levels of HCV RNA in the cPCR assay but levels of anti-CIOO-3 were below the cutoff for positivity (results in duplicate samples 1229 and 896 cpm). The four normal control plasma samples were negative for both HCV the
sequences and anti-C100-3/
Results Nine of the fifteen patients with chronic NANBH and one patient with chronic cryptogenic liver disease were positive for anti-CIOO-3 by RIA (above cutoff of mean plus 4 SD 2222 cpm) in serum taken on the same day as the liver biopsy specimen. Seven of these patients were also positive for HCV RNA in the cPCR assay. Two patients who were negative for anti-ClOO-3 also had substantial levels of HCV RNA. The patient with chronic hepatitis B had undetectable levels of HCV RNA in the cPCR assay. The size of the PCR products observed on the autoradiogram was the same as that of the predicted amplification product based on the HCV nucleotide sequence (586 bp) lying between the 622 bp and 527 bp Mspl -digested fragments of pBR322 DNA. To verify that all the samples contained similar amounts of RNA, cPCR was carried out with =
primers designed RNA.18
to
amplify alphal-antitrypsin messenger
Fig 2-Sensitivity of HCV cPCR
assay.
Analysis of samples containing 300 CID/ml (lane 1) and 30 CID/ml (lane 2). Signal for sample containing 3 CID/ml was also clearly visible on original autoradiogram but not after photographic reproduction. Arrows indicate position of Msp1-digested pBR322 DNA (622 and 527 bp).
3
Chimpanzee 711 showed a clearly defined acute episode of post-transfusion NANBH; there was a distinct peak of alanine aminotransferase (ALT) activity 28 days after infection with two subsequent peaks of ALT activity (fig 1). Although plasma collected on day 25 did not contain detectable anti-Cl00-3 (1438 and 1472 cpm) HCV RNA was detected in this sample. HCV RNA was still detectable on day 32 (anti-Cl00-3 still undetectable [ 1116 and 1022 cpm]) but was below the experimental limit of detection on day 70 when anti-C100-3 levels were rising (5273 and 4520 cpm on day 70, 7422 and 6932 cpm on day 88). In contrast, HCV RNA was not detectable 11 or 28 days after infection in chimpanzee 910 (fig 1) but was detectable on day 67, at which time anti-Cl00-3 was still undetectable (1322 and 1045 cpm). Chimpanzee 910 was positive for anti-Cl00-3 on day 11 (4894 and 4154 cpm) owing to passive immunisation of the animal with antibodies from the plasma used to inoculate it. Plasma samples from uninfected chimpanzees were negative for both HCV RNA and
well as liver enzyme activities, varied in each case. Insight into how HCV causes chronic disease will come from further studies; it is clear that there is no simple relation between the activity of ALT, the time and magnitude of initial viraemia, and the development of chronic hepatitis. This reliable and sensitive assay for detecting HCV sequences in tissue and plasma can yield valuable information on the diagnosis of HCV infections and answer other questions, such as whether agents other than HCV are associated with NANBH and how effective are potential antiviral treatments. We thank Michelle Stampien and David Ahle, for oligonucleotide primers; Dr R. Purcell and Dr H. Alter, for human plasma samples; Dr J. Rossi, for initial technological discussions; and Michael Kiefer, for evaluating the manuscript. This study is dedicated to the memory of Martin Flacks. This study was supported by Chiron Corporation, Ortho-Diagnostic Systems Ltd, and Ciba Geigy.
anti-Cl00-3.
REFERENCES
An infectious, high-titre pool of 17 plasma samples from chimpanzee 910 during chronic infection contained substantial levels of viral RNA but levels of anti-Cl00-3 were below the cutoff for positivity (1204 and 907 cpm). The sensitivity of the cPCR assay was determined by analysing ten-fold serial dilutions of a plasma pool of known titre. Approximately 300 CID, 30 CID, and 3 CID were easily detected in a 15 h exposure of the autoradiogram (fig 2). The higher titre dilutions were detectable within 2 h. Since the average titre of HCV in infected patients is believed to be between 102 and 104 CID/ml of plasma, the cPCR assay should be clinically useful.
Discussion We have shown a strong correlation between the presence of circulating antibodies to C100-3 and the presence ofHCV sequences in the livers of patients with chronic NANBH. Our study also provides molecular evidence for the chronic carrier condition caused by chronic NANB infections19 and further evidence that anti-ClOO-3 is a marker for infectivity.’ Two patients with chronic NANBH had HCV RNA in the liver but no anti-Cl 00-3 detected in the plasma. Similarly, a high-titre pool of infectious plasma from a chronically infected chimpanzee lacked anti-ClOO-3 but had high levels of HCV RNA. Although some patients with chronic post-transfusion NANBH are clearly negative for anti-Cl00-3, it remains to be seen whether other HCV antigens allow serological identification of these samples as well. This study, however, suggests that estimated numbers of HCV infections may increase when both direct and indirect tests for HCV are used in epidemiological studies. The high percentage of Italian patients positive for HCV RNA in the cPCR assay suggests that the predominant strain of HCV in Italy is similar to that found in the USA. cPCR and RIA studies on samples from the early acute period of NANBH showed that virus is usually present during the major peak of liver enzyme activities in both human beings and chimpanzees before the appearance of circulating IgG anti-Cl00-3, as might be expected for a typical immunological response to viral antigens. Interestingly, the infection progressed to chronic liver disease in the patient and the two chimpanzees even though the patterns and amounts of viral RNA and anti-Cl00-3, as
HJ, Purcell RH, Holland PV, Feinstone SM, Morrow AG, Moritsugu Y. Clinical and serological analysis of transfusion-associated hepatitis. Lancet 1975; ii: 838-41. 2. Feinstone SM, Kapikian AZ, Purcell RH, et al. Transfusion-associated hepatitis not due to hepatitis type A or B. N Engl J Med 1975; 292: 1. Alter
767-70. 3. Knodell RG, Conrad ME, Dienstag JL, et al. Etiological spectrum of transfusion hepatitis. Gastroenterology 1975; 69: 1278-85.
post
Dienstag JL, Alter HJ. Non-A, non-B hepatitis; evolving epidemiologic and clinical perspective. Semin Liver Dis 1986; 6: 67-81. 5. Alter HJ. Post transfusion hepatitis: clinical features, risk and donor testing. In: Infection, immunity and blood transfusion. New York: Alan R Liss, 1985: 47-61. 6. Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from a blood-home non-A, non-B viral hepatitis genome. Science 1989; 244: 359-62. 7. Kuo G, Choo QL, Alter HJ, et al. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 1989;
4.
244: 362-64.
8. Van Der Poel CL, Reesink HW, Lelie PN, et al. antibodies and non-A, non-B post-transfusion Netherlands. Lancet 1989; ii: 297-98.
Anti-hepatitis C hepatitis in the
9. Esteban JI, Esteban R, Viladomiu I, et al. among risk groups in Spain. Lancet 1989;
Hepatitis C virus antibodies ii: 294-96. 10. Kuhnl P, Seidl S, Stangel W, Beyer J, Sibrowski W, Flik J. Antibody to hepatitis C virus in German blood donors. Lancet 1989; ii: 324. 11. Roggendorf M, Deinhardt F, Rasshofer R, et al. Antibodies to hepatitis C virus. Lancet 1989; ii: 324-25. 12. Saiki RK, Scarf S, Faloona F, et al. Enzymatic amplification of &bgr;-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 1985; 230: 1350-54. SM, Alter HJ, Dienes HP, et al. Non-A, non-B hepatitis in chimpanzees and marmosets. J Infect Dis 1981; 144: 588-98.
13. Feinstone
DW, Cook EH, Maynard JE, et al. Experimental infection of chimpanzees with antihemophilic (factor VIII) materials: recovery of virus like particles associated with non-A, non-B hepatitis. J Med Viral 1979; 3: 253-69. 15. Bradley DW, Maynard JE, Krawczynski KZ, et al. Non-A, non-B hepatitis in chimpanzees infected with a factor VIII agent: evidence of persistent hepatic disease. In: Szmuness W, Alter HJ, Maynard JE, eds. Viral hepatitis. Philadelphia: Franklin Institute Press, 1981: 14. Bradley
319-29. 16. Bradley DW. The agents of 1985; 10: 307—19. 17.
18.
non-A, non-B viral hepatitis. J Virol Methods
Bradley DW, Maynard JE, Etiology and natural history of posttransfusion and enterically-transmitted non-A, non-B hepatitis. Semin Liver Dis 1986; 6: 56-66. Rosenberg SR, Barr PJ, Narjarian RC, Hallewell RA. Synthesis in yeast of a functional oxidation-resistant mutant of human &agr;l-anti-trypsin. Nature 1984; 312: 77-80.
HJ, Holland PV, Purcell RH, Popper H. Transmissible agent in non-A, non-B hepatitis. Lancet 1978; i: 459-66.
19. Alter
hepatitis C viral sequences in non-A, non-B hepatitis
Detection of
The role of hepatitis C virus (HCV) in posttransfusion non-A, non-B hepatitis (NANBH) was investigated by analysing clinical samples for both HCV RNA by cDNA/polymerase chain reaction and antibodies against C100-3 by radioimmunoassay. Of fifteen chronic NANBH patients and one patient with chronic cryptogenic liver disease, ten were positive for anti-C100-3 and seven of the ten had viral sequences in their livers. However, two patients negative for anti-C100-3 also had substantial levels of HCV RNA in their livers. In acute post-transfusion NANBH (one surgical patient and two experimentally infected chimpanzees), HCV RNA was detected in the absence of anti-C100-3. In addition, infectious plasma from a seronegative patient with acute post-transfusion NANBH and a seronegative pool of plasma from a chimpanzee with chronic posttransfusion NANBH had high levels of HCV. These findings show that anti-C100-3-positive patients with chronic post-transfusion NANBH are likely to be viraemic; confirm that antibodies to C100-3 are a marker for infectivity; and suggest that the prevalence of HCV infections may be underestimated from the frequency of antibodies to C100-3 alone.
Introduction When diagnostic methods for hepatitis A and B infectionsl-3 were developed, it became clear that 60-90% of reported post-transfusion hepatitis was caused by unknown agents.1 In the United States alone, non-A, non-B hepatitis (NANBH) affects an estimated 200 000 to 300 000 people annually and causes chronic liver disease in approximately 50% of post-transfusion cases.4,5 Studies on the genome of hepatitis C virus (HCV) have shown that it is a positivestranded RNA virus.6 Although expression of HCV cDNA clones in yeast has led to the development of an effective immunodiagnostic test for antibodies to HCV in plasma and serum samples from patients with NANBH world wide,7-11 there has been no sensitive assay for HCV RNA with a potential for clinical application. Based on the nucleotide sequence of HCV cDNA clones 366 and 37b (sequence lodged with Genbank, Nov 10, 1989) a sensitive, semiquantitative cDNA/polymerase chain reaction12 (cPCR) assay to detect HCV RNA in liver and plasma or serum has ADDRESSES: Chiron Corporation, Emeryville, California (A. J. Weiner, PhD, G. Kuo, PhD, C. Lee, BA, J. Rosenblatt, BA, Q-L. Choo, PhD, M. Houghton, PhD); Hepatitis Branch, Centers for Disease Control, Atlanta, Georgia, USA (D. W. Bradley, PhD); and Division of Gastroenterology, San Giovanni Battista Molinette Hospital, Turin, Italy (F. Bonnino, MD, G. Saracco, MD). Correspondence to Dr A. J. Weiner, Chiron Corporation, 4560 Horton Street, Emeryville, CA 94608, USA.
2
been developed. We report here the results from the analysis of several clinical samples from patients and chimpanzees with respect to HCV RNA and CIOO-3 antibody content.
Subjects and methods To detect HCV sequences in clinical samples, cDNA was synthesised from RNA extracted from liver biopsy or plasma samples and subjected to PCR. The cDNA synthesis and PCR used the same pair of synthetic 16-mer oligonucleotide primers derived from the two non-contiguous HCV cDNA clones 36 (5’-GCATGTCATGATGTAT-3’; anti-sense) and 37b
(5’-ACAATACGTGTGTCAC-3’; sense). Reactions were carried according to the manufacturer’s instructions (BRL cDNA kit 8085SB and Perkin Elmer Cetus PCR kit N801-0055). Thirty cycles of PCR were carried out on all samples as follows: denaturation for 1 min at 94°C, annealing of primers for 2 min at 37°C, and extension for 3 min at 72°C. PCR products were analysed on 18% alkaline agarose gels, blotted onto ’Zeta’ probe paper (BioRad), and hybridised to a phosphorus-32-labelled nicktranslated HCV cDNA insert which lies between, but not including, the cDNA/PCR primers. The blots were washed in 0.1xSSC (1 x SSC=0-15 mol/1 sodium chloride, 0-015 mol/1 sodium citrate), 01% sodium dodecyl sulphate at 68°C, and autoradiographed. All samples were assayed at least three times with reproducible results and no false-positive results in the control samples. Radioinimunoassays (RIA) to detect antibodies against C100-3 were carried out according to Kuo et al.7 In several studies, we and others have found that data obtained by RIA are equivalent to those
out
obtained with the Chiron-Ortho anti-Cl00-3 enzyme-linked immunosorbent assay. Liver biopsy samples (10 x 4 mm cylinders) were obtained from fifteen Italian patients with chronic NANBH, one patient with cryptogenic liver disease, and one patient with chronic hepatitis B (HBV) infection. All these patients were negative for hepatitis B surface antigen and e antigen and for antibody to core antigen, except the patient with chronic HBV infection who was positive for all three markers of HBV infection. Serum samples were collected on the same day as the liver samples from the fifteen chronic NANBH patients and the patient with cryptogenic liver disease. Acute NANBH plasma samples were obtained from one surgical patient known to have an HCV titre of 10 65 chimpanzee infectious doses (CID) per ml13 (gift from Dr Robert Purcell and Dr Harvey Alter, National Institutes of Health) and two experimentally infected, colony-born chimpanzees (77114,15 and 91015). Pooled chronic-phase plasma from the same two chimpanzees (HCV titres of 105 CID/ml16 [no 771] and 106 CID/ml17 [no 910]) were also analysed along with non-infectious, control plasma from four human beings and four chimpanzees.
Fig 1-ALT profiles for chimpanzee 771 (A) and 910 (B) with cPCR results (RNA) and results of RIA for anti-C100-3 (Ab). The
plasma sample collected from a surgical patient during early acute phase of post-transfusion NANBH had very high levels of HCV RNA in the cPCR assay but levels of anti-CIOO-3 were below the cutoff for positivity (results in duplicate samples 1229 and 896 cpm). The four normal control plasma samples were negative for both HCV the
sequences and anti-C100-3/
Results Nine of the fifteen patients with chronic NANBH and one patient with chronic cryptogenic liver disease were positive for anti-CIOO-3 by RIA (above cutoff of mean plus 4 SD 2222 cpm) in serum taken on the same day as the liver biopsy specimen. Seven of these patients were also positive for HCV RNA in the cPCR assay. Two patients who were negative for anti-ClOO-3 also had substantial levels of HCV RNA. The patient with chronic hepatitis B had undetectable levels of HCV RNA in the cPCR assay. The size of the PCR products observed on the autoradiogram was the same as that of the predicted amplification product based on the HCV nucleotide sequence (586 bp) lying between the 622 bp and 527 bp Mspl -digested fragments of pBR322 DNA. To verify that all the samples contained similar amounts of RNA, cPCR was carried out with =
primers designed RNA.18
to
amplify alphal-antitrypsin messenger
Fig 2-Sensitivity of HCV cPCR
assay.
Analysis of samples containing 300 CID/ml (lane 1) and 30 CID/ml (lane 2). Signal for sample containing 3 CID/ml was also clearly visible on original autoradiogram but not after photographic reproduction. Arrows indicate position of Msp1-digested pBR322 DNA (622 and 527 bp).
3
Chimpanzee 711 showed a clearly defined acute episode of post-transfusion NANBH; there was a distinct peak of alanine aminotransferase (ALT) activity 28 days after infection with two subsequent peaks of ALT activity (fig 1). Although plasma collected on day 25 did not contain detectable anti-Cl00-3 (1438 and 1472 cpm) HCV RNA was detected in this sample. HCV RNA was still detectable on day 32 (anti-Cl00-3 still undetectable [ 1116 and 1022 cpm]) but was below the experimental limit of detection on day 70 when anti-C100-3 levels were rising (5273 and 4520 cpm on day 70, 7422 and 6932 cpm on day 88). In contrast, HCV RNA was not detectable 11 or 28 days after infection in chimpanzee 910 (fig 1) but was detectable on day 67, at which time anti-Cl00-3 was still undetectable (1322 and 1045 cpm). Chimpanzee 910 was positive for anti-Cl00-3 on day 11 (4894 and 4154 cpm) owing to passive immunisation of the animal with antibodies from the plasma used to inoculate it. Plasma samples from uninfected chimpanzees were negative for both HCV RNA and
well as liver enzyme activities, varied in each case. Insight into how HCV causes chronic disease will come from further studies; it is clear that there is no simple relation between the activity of ALT, the time and magnitude of initial viraemia, and the development of chronic hepatitis. This reliable and sensitive assay for detecting HCV sequences in tissue and plasma can yield valuable information on the diagnosis of HCV infections and answer other questions, such as whether agents other than HCV are associated with NANBH and how effective are potential antiviral treatments. We thank Michelle Stampien and David Ahle, for oligonucleotide primers; Dr R. Purcell and Dr H. Alter, for human plasma samples; Dr J. Rossi, for initial technological discussions; and Michael Kiefer, for evaluating the manuscript. This study is dedicated to the memory of Martin Flacks. This study was supported by Chiron Corporation, Ortho-Diagnostic Systems Ltd, and Ciba Geigy.
anti-Cl00-3.
REFERENCES
An infectious, high-titre pool of 17 plasma samples from chimpanzee 910 during chronic infection contained substantial levels of viral RNA but levels of anti-Cl00-3 were below the cutoff for positivity (1204 and 907 cpm). The sensitivity of the cPCR assay was determined by analysing ten-fold serial dilutions of a plasma pool of known titre. Approximately 300 CID, 30 CID, and 3 CID were easily detected in a 15 h exposure of the autoradiogram (fig 2). The higher titre dilutions were detectable within 2 h. Since the average titre of HCV in infected patients is believed to be between 102 and 104 CID/ml of plasma, the cPCR assay should be clinically useful.
Discussion We have shown a strong correlation between the presence of circulating antibodies to C100-3 and the presence ofHCV sequences in the livers of patients with chronic NANBH. Our study also provides molecular evidence for the chronic carrier condition caused by chronic NANB infections19 and further evidence that anti-ClOO-3 is a marker for infectivity.’ Two patients with chronic NANBH had HCV RNA in the liver but no anti-Cl 00-3 detected in the plasma. Similarly, a high-titre pool of infectious plasma from a chronically infected chimpanzee lacked anti-ClOO-3 but had high levels of HCV RNA. Although some patients with chronic post-transfusion NANBH are clearly negative for anti-Cl00-3, it remains to be seen whether other HCV antigens allow serological identification of these samples as well. This study, however, suggests that estimated numbers of HCV infections may increase when both direct and indirect tests for HCV are used in epidemiological studies. The high percentage of Italian patients positive for HCV RNA in the cPCR assay suggests that the predominant strain of HCV in Italy is similar to that found in the USA. cPCR and RIA studies on samples from the early acute period of NANBH showed that virus is usually present during the major peak of liver enzyme activities in both human beings and chimpanzees before the appearance of circulating IgG anti-Cl00-3, as might be expected for a typical immunological response to viral antigens. Interestingly, the infection progressed to chronic liver disease in the patient and the two chimpanzees even though the patterns and amounts of viral RNA and anti-Cl00-3, as
HJ, Purcell RH, Holland PV, Feinstone SM, Morrow AG, Moritsugu Y. Clinical and serological analysis of transfusion-associated hepatitis. Lancet 1975; ii: 838-41. 2. Feinstone SM, Kapikian AZ, Purcell RH, et al. Transfusion-associated hepatitis not due to hepatitis type A or B. N Engl J Med 1975; 292: 1. Alter
767-70. 3. Knodell RG, Conrad ME, Dienstag JL, et al. Etiological spectrum of transfusion hepatitis. Gastroenterology 1975; 69: 1278-85.
post
Dienstag JL, Alter HJ. Non-A, non-B hepatitis; evolving epidemiologic and clinical perspective. Semin Liver Dis 1986; 6: 67-81. 5. Alter HJ. Post transfusion hepatitis: clinical features, risk and donor testing. In: Infection, immunity and blood transfusion. New York: Alan R Liss, 1985: 47-61. 6. Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from a blood-home non-A, non-B viral hepatitis genome. Science 1989; 244: 359-62. 7. Kuo G, Choo QL, Alter HJ, et al. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 1989;
4.
244: 362-64.
8. Van Der Poel CL, Reesink HW, Lelie PN, et al. antibodies and non-A, non-B post-transfusion Netherlands. Lancet 1989; ii: 297-98.
Anti-hepatitis C hepatitis in the
9. Esteban JI, Esteban R, Viladomiu I, et al. among risk groups in Spain. Lancet 1989;
Hepatitis C virus antibodies ii: 294-96. 10. Kuhnl P, Seidl S, Stangel W, Beyer J, Sibrowski W, Flik J. Antibody to hepatitis C virus in German blood donors. Lancet 1989; ii: 324. 11. Roggendorf M, Deinhardt F, Rasshofer R, et al. Antibodies to hepatitis C virus. Lancet 1989; ii: 324-25. 12. Saiki RK, Scarf S, Faloona F, et al. Enzymatic amplification of &bgr;-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 1985; 230: 1350-54. SM, Alter HJ, Dienes HP, et al. Non-A, non-B hepatitis in chimpanzees and marmosets. J Infect Dis 1981; 144: 588-98.
13. Feinstone
DW, Cook EH, Maynard JE, et al. Experimental infection of chimpanzees with antihemophilic (factor VIII) materials: recovery of virus like particles associated with non-A, non-B hepatitis. J Med Viral 1979; 3: 253-69. 15. Bradley DW, Maynard JE, Krawczynski KZ, et al. Non-A, non-B hepatitis in chimpanzees infected with a factor VIII agent: evidence of persistent hepatic disease. In: Szmuness W, Alter HJ, Maynard JE, eds. Viral hepatitis. Philadelphia: Franklin Institute Press, 1981: 14. Bradley
319-29. 16. Bradley DW. The agents of 1985; 10: 307—19. 17.
18.
non-A, non-B viral hepatitis. J Virol Methods
Bradley DW, Maynard JE, Etiology and natural history of posttransfusion and enterically-transmitted non-A, non-B hepatitis. Semin Liver Dis 1986; 6: 56-66. Rosenberg SR, Barr PJ, Narjarian RC, Hallewell RA. Synthesis in yeast of a functional oxidation-resistant mutant of human &agr;l-anti-trypsin. Nature 1984; 312: 77-80.
HJ, Holland PV, Purcell RH, Popper H. Transmissible agent in non-A, non-B hepatitis. Lancet 1978; i: 459-66.
19. Alter