The relationship between hepatitis C virus (HCV) genotypes and antibody ... non-A, non-B hepatitis patients and asymptomatic HCV-infected blood donors.
Vol. 32, No. 1
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1994, p. 211-212
0095-1137/94/$04.00+0 Copyright © 1994, American Society for Microbiology
Serological Responses to Different Genotypes of Hepatitis C Virus in France C. ALONSO,1 D. QU,1 J. P. LAMELIN,1 S. DE SANJOSE,2 L. VITVITSKI,1 J. LI,1t F. V. LAMBERT,1 M. L. CORTEY,1 AND C. TRPOl*
BERBY,'
Unite de Recherche sur les Hepatites, le SIDA et les Retrovirus Humains, Institut National de la Sante et de la Recherche Medicale, 1 and Centre International de Recherche sur le Cancer,2 Lyon, France Received 23 June 1993/Returned for modification 19 August 1993/Accepted 19 October 1993 The relationship between hepatitis C virus (HCV) genotypes and antibody status was studied in 104 chronic non-A, non-B hepatitis patients and asymptomatic HCV-infected blood donors. On the basis of amplification of the nonstructral protein 3 (NS3) coding region by PCR and hybridization with speific probes, 55 and 42 patients were identified as being infected with tpe I and ype II, respectively, according to the classification by H. Okamoto, K. Kua"i, S. Okada, K. Yamamoto, H. Lizuka, T. Tanaka, S. Fukuda, F. Tsudaand, and S. Mishiro (Virology 188:331-341, 1992). All samples were tested for antibodies to 5.1.1, C-100, C-33, and C-22 proteins by a second-generation recombinant immunoblot assay. Among 97 patients with known HCV genotypes, 31 of 42 patients inected with type H and 24 of 55 infected with type I had antibodies against all four antigens (P < 0.01). In the type H-infected group, more patients had detectable antibodies to 5.11, C-33, and C-22 proteins than in the type I group (P < 0.05). No difference was found in the serological response to C-100 between the two groups.
ing (5'-NC) region. For genotyping of samples, HCV RNA extracted from 200 ,ul of serum and reverse transcribed into cDNA in the presence of external antisense primer as previously described (6). HCV cDNA was amplified on part of the nonstructural protein 3 (NS3) region by nested PCR, with two sets of oligonucleotide primers synthesized according to prototype of HCV type I (3). The external primers were sense, 5'-AGACTGTAATACGTGTGTCACC-3' (nt 4357 to 4378), and antisense, 5'-AGCTCATACCAAGCAC AGCC-3' (nt 4571 to 4590); internal primers were sense, 5'-GACAGTCGAC-TTCAGCCTTGA-3' (nt 4381 to 4401), and antisense, 5'-TCATAGCACTCACAGAGGAC-3' (nt 4547 to 4566). PCR was carried out in a DNA thermal cycler (Perkin-Elmer Cetus) for 35 cycles with external primers and for another 35 cycles with internal primers (95°C for 1 mi, 37TC for 1 min, and 72°C for 2 min). The genotypes of the NS3-PCR product (186 bp) were identified by Southern blot hybridization. The probes were prepared by purification of an Fl- or F2-derived insert from a pUC 18 vector and labelled with [a-32P]dCTP by a DNA 3'-end-labelling kit (Boehringer Mannheim). Filters were hybridized with an Fl-specific probe. The hybridization and washing conditions were as previously described (6). The filters were then boiled in 0.5% sodium dodecyl sulfate for 10 min or more to remove the Fl probe and rehybridized with the F2 probe under the same conditions. The purified Fl and F2 fragments were used as controls. One hundred of the 104 samples (96.2%) were positive by NS3-PCR. Fifty-five samples (52.9%) hybridized with the Fl probe only (identified as type I), and 42 (40.4%) hybridized with the F2 probe only (identified as type II); three samples (2.9%) hybridized with both probes. Among the 12 blood donors, 6 and 1 were identified as being infected with type II and type I, respectively. This suggested that in France, blood donors are predominantly infected with type II, as are their Japanese counterparts (9). The three samples (one from a blood donor and two from chronic hepatitis patients) hybridized with both probes may be due either to the presence of both HCV strains or to the amplified fragments showing distances similar to those of Fl and F2 isolates (12). The four
Hepatitis C virus (HCV), the major etiological agent in non-A, non-B hepatitis, has been characterized recently (1, 5). The HCV genome is a positive single-stranded RNA. Its genetic organization is similar to that of flaviviruses and pestiviruses and shows significant heterogeneity (3, 7, 13). On the basis of the comparative analysis of both complete and partial nucleotide (nt) sequences of most isolates published up to 1992, Okamoto et al. classified HCV into four genotypes: types I, II, III, and IV (8). In France, two predominant HCV genotypes, Fl and F2, corresponding to types I and II, respectively, have been reported (6). HCV infection can be diagnosed by serological methods and by PCR. Antibody response in acute and chronic HCV hepatitis is variable, and no particular pattern associated with clinical stage, viral replication, or remission of hepatitis has been found (2, 11). One explanation of this variability of the serological response could be the existence of several strains of HCV differing in their immunogenicity and therefore in the host immune response. The severity of liver disease and the response to interferon therapy vary between patients infected with different HCV genotypes (10, 12, 14). The influence of the different genotypes on the serological profile of HCV infection is not yet clear. In the present study, the correlation between HCV genotype and the serological response to HCV antigens was investigated in patients infected with HCV. We studied 92 patients with chronic non-A, non-B hepatitis referred to our Liver Unit between November 1989 and December 1992 and 12 asymptomatic HCV-infected blood donors with serological HCV positivity from the Blood Transfusion Center of Lyon. All of them were anti-HCV positive by enzyme immunoassay and HCV RNA positive by PCR amplification using the primers from the 5' noncod-
was
* Corresponding author. Mailing address: Hepatitis Research Unit, Institut National de la Sante et de la Recherche Medicale U271, 151 Cours A. Thomas, 69424 Lyon Cedex 03, France. Phone: 72 33 06 91. t Present address: Molecular Hepatology Laboratory, Charlestown, MA 02129.
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J. CLIN. MICROBIOL.
TABLE 1. Patterns of RIBA-2 antibodies in 97 patients infected with HCV type I or II RIBA reactivity to: 5.1.1 -
No. of patients with HCV
C-100
C-33
C-22
Type I
Type II
_ + -
_ + + + + +
-
7 24 3 1 8 1 1 6 2 2
1 31 4 0 2 0 0 3 0 1
+ + + + -
+ + + -
-
-
+ + + + + +
+
-
-
+
samples from blood donors which were 5'-NC-PCR positive but NS3-PCR negative could be due to the presence of other different genotypes. All samples studied were tested for antibodies to 5.1.1, C-100, C-33, and C-22 proteins by the second-generation
recombinant immunoblot assay (RIBA-2; Ortho Diagnostics). A sample was considered to be positive when it reacted with at least two HCV-specific bands of an intensity superior to the low-normal control level of the human immunoglobulin G band. The four recombinant proteins characteristic of RIBA-2, deduced from HCV type I, correspond to relatively well-conserved sequences within the different HCV genotypes, which may yield a broadly reactive diagnostic test (4). Table 1 shows the serological response to each of the four HCV antigens in 97 patients infected with known genotypes. Thirty-one of 42 samples (73.8%) in the type II-infected group, compared with 24 of 55 samples (43.6%) in the type I-infected group, were positive for the four proteins. The difference between the two groups was statistically significant (P = 0.003). Seven type I-infected patients but only one type II-infected patient had no detectable antibodies to any of the four antigens (P = 0.067). Of four samples that failed to be amplified from blood donors, three reacted with all four antigens in the RIBA-2 and the fourth reacted with only the C-100 protein. Samples from the other eight blood donors, who were identified as type I or/and type II, had complete serological patterns. Then, the antibody to each antigen was analyzed (Table 2). In the type I-infected group, 52.7, 80.0, and 81.8% of the patients had antibody to 5.1.1, C-33, and C-22, respectively, while these proportions were 83.8, 95.2, and 97.6%, respectively, among patients infected with type II (P < 0.05). No statistical difference was found in the serological response to C-100 between the two groups. Our results indicate that type 1I-infected patients have a broader serological response, especially anti-5.1.1, anti-C33, TABLE 2. Analysis of reactivity to individual recombinant proteins in patients infected with HCV type I or II No. (%) of patients with detectable
antibody specificity HCV type II
Protein
pa
HCV type I
5.1.1 C-100 C-33 C-22
29 34 44 45
(52.7) (61.8) (80.0) (81.8)
a By the x2 test. NS, not significant.
35 33 40 41
(83.8) (78.6) (95.2) (97.6)
0.0016 NS 0.0291 0.015
and anti-C22, than those infected with the type I. The different serological responses may be due to differences in virus replication capacities or in the immune response to viral antigens or to the presence of different epitopes in both strains. Further investigations are needed to determine the significance of these findings in HCV infection. We thank J. C. Chevre from the Blood Transfusion Center of
Lyon for giving us access to blood donor samples. This study was supported by a clinical research grant from the
French
Ministry of Health.
REFERENCES 1. Alter, H. J., R. H. Purcell, J. W. Shih, J. C. Melpolder, M. Houghton, Q.-L. Choo, and G. Kuo. 1989. Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis. N. Engl. J. Med. 321:1494-1500. 2. Bradley, D. W. 1992. Virology, molecular biology, and serology of hepatitis C virus. Transfus. Med. Rev. 2:93-102. 3. Choo, Q.-L., K. H. Richman, J. H. Han, B. Berger, C. Lee, C. Dong, C. Gallegos D. Colt, A. Medina-Selby, D. J. Barr, A. J. Weiner, D. W. Bradley, G. Kuo, and M. Houghton. 1991. Genetic organization and diversity of the hepatitis C virus. Proc. Natl. Acad. Sci. USA 88:2451-2455. 4. Houghton, M., A. Weiner, J. Han, G. Kuo, and Q. Choo. 1991. Molecular biology of the hepatitis C viruses: implications for diagnosis, development and control of viral disease. Hepatology 14:381-388. 5. Koo, G., Q.-L. Choo, H. J. Alter, C. L. Gitnick, A. G. Redeker, R. H. Purcell, T. Miyamura, J. L. Dienstag, M. J. Alter, C. E. Stevens, G. E. Tegtmeier, F. Bonino, M. Colombo, W. S. Lee, K. Berger, J. R. Shuster, L. R. Overby, D. W. Bradley, and M. Houghton. 1989. An assay for circulating antibodies to a major aetiologic virus of a human non-A, non-B hepatitis. Science 244:362-364. 6. Li, J.-S., S.-P. Tong, L. Vitvitsky, D. Lepot, and C. Trepo. 1991. Two French genotypes of hepatitis C virus: homology of the predominant genotype with the prototype American strain. Gene 105:165-172. 7. Miller, L H., and R. H. Purcell. 1990. Hepatitis C virus shares amino acid sequence similarity with pestivirus and flavivirus as well as members of two plant virus super groups. Proc. Natl. Acad. Sci. USA 87:2057-2061. 8. Okamoto, H., K. Kurai, S. Okada, K. Yamamoto, H. Lizuka, T. Tanaka, S. Fukuda, F. Tsudaand, and S. Mishiro. 1992. Fulllength sequence of a hepatitis C virus genome having poor homology to reported isolates: comparative study of four distinct genotypes. Virology 188:331-341. 9. Okamoto, H., Y. Sujuama, S. Okada, K. Kurai, Y. Akahane, Y. Sugay, T. Tanaka, K. Sato, F. Tsuda, Y. Mjakawaand, and M. Mayumi. 1992. Typing hepatitis C virus by polymerase chain reaction with type-specific primers: application to clinical surveys and tracing infectious sources. J. Gen. Virol. 73:673-679. 10. Pozzato, G., M. Moretti, F. Franzin, L. S. Croce, C. Tiribelli, T. Masayu, S. Kaneko, M. Unoura, and K. Kobayashi. 1991. Severity of liver disease with different hepatitis C viral clones. Lancet 338:509. 11. Puoti, M., A. Zonaro, A. Kavaggi, M. G. Marin, F. Castelnuovo, and E. Cariani. 1992. Hepatitis C virus RNA antibody response in the clinical course of acute hepatitis C virus infection. Hepatology 16:877-881. 12. Qu, D., J. Li, L. Vitvitsky, S. Mechal, F. Berby, S. Tong, F. Bailly, Q. Wang, and C. Trepo. Submitted for publication. 13. Takamizawa, A., C. Mori, I. Fuke, S. Manabe, S. Murakami S, J. Fujita, E. Onisni, T. Andoh, I. Yoshida, and H. Okayama. 1991. Structure and organization of the hepatitis C virus genome isolated from human carriers. J. Virol. 65:1105-1113. 14. Yoshioka, K., K. Shinichi, T. Waldta, T. Ishikawa T., Y. Itoh, M. Takayanagy, Y. Higashi, M. Shibata, and T. Morishima. 1992. Detection of hepatitis C virus by polymerase chain reaction and response to interferon therapy: relationship to genotypes of hepatitis C virus. Hepatology 16:293-299.
