JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1996, p. 2623–2624 0095-1137/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 34, No. 10
Genotyping of Hepatitis C Virus Isolates from Lebanese Hemodialysis Patients by Reverse Transcription-PCR and Restriction Fragment Length Polymorphism Analysis of 59 Noncoding Region GHASSAN M. MATAR,* HANADY M. SHARARA, GEORGE E. ABDELNOUR, AND ALEX M. ABDELNOOR Department of Microbiology and Immunology, Faculty of Medicine, American University of Beirut, New York, New York 10022 Received 11 April 1996/Returned for modification 7 May 1996/Accepted 28 June 1996
We have genotyped the 5* noncoding region of hepatitis C virus in Lebanese hemodialysis patients by reverse transcription-PCR and restriction fragment length polymorphism analysis. Of 50 patients, 15 had the expected 268-bp amplicon by reverse transcription-PCR. Specificity of the amplicons was confirmed by Southern hybridization. Restriction analysis of the amplicons showed the pattern for genotype 4 (common in the Middle East). and 10 U of Moloney murine leukemia virus reverse transcriptase (Cis Bio International), was incubated at 378C for 45 min and then at 998C for 5 min. PCR was then performed according to the method of Chan et al. (3). Samples were tested in a 100-ml reaction mixture containing 10 ml of cDNA, diethyl pyrocarbonate-H2O, a 200 mM concentration of each deoxynucleoside triphosphate (Pharmacia LKB, Uppsala, Sweden), 103 PCR buffer, 0.5 mM antisense and sense PCR primers (3), and 0.5 U of Taq DNA polymerase (Pharmacia LKB). PCR conditions were set in accordance with the method used by Chan et al. (3) with few modifications: initial denaturation at 948C for 3 min followed by 40 cycles of 948C for 30 s, 558C for 35 s, and 688C for 2.5 min. A final extension step at 688C for 10 min was done at the end of the reaction. Twenty microliters of the mix was then electrophoresed on 1.5% agarose, stained with ethidium bromide, visualized under UV light, and photographed with 667 Polaroid film. To confirm the specificity of the amplicons, Southern hybridization was performed on all positive and some negative samples with a digoxigenin-labeled probe derived from the 268-bp target sequence according to the method used by Popovic et al. (6). Restriction endonuclease analysis was then used to determine the genotype(s) of HCV in our samples. The PCR products were digested with HaeIII (10 U/ml) and a combination of ScrFI (10 U/ml) and HinfI (10 U/ml) (New England Biolabs, Beverly, Mass.) according to the manufacturer’s specifications. The digested amplicons were then electrophoresed on 2.5% Nusieve agarose (FMC Bioproducts, Rockland, Maine). Gels were then stained with ethidium bromide, and bands were visualized under UV
Hepatitis C virus (HCV) is considered the major causative agent of posttransfusion non-A, non-B hepatitis throughout the world. A major characteristic of HCV infection is its tendency to establish chronic liver disease, such as cirrhosis, and eventually hepatocellular carcinoma (4). The virus has a positive-sense, single-stranded RNA genome approximately 10 kb in length. Different isolates of HCV show substantial nucleotide sequence variation distributed throughout the genome. Regions encoding the envelop proteins are the most variable, whereas the 59 noncoding region (NCR) is the most conserved (7). Because it is the most conserved with minor heterogeneity, several researchers have considered the 59 NCR the region of choice for virus detection by reverse transcription (RT)-PCR. Sequence analysis performed on isolates from different geographical areas around the world has revealed the presence of different genotypes, genotypes 1 to 6 (1). A typing scheme using restriction fragment length polymorphism analysis of the 59 NCR was able to differentiate the six major genotypes (5). The aim of this study was to detect HCV by RT-PCR of the 59 NCR and then to determine the genotype(s) of HCV present among our samples by restriction analysis of the amplicons. In a previous study blood was collected from 108 patients in various Lebanese hospitals and tested by enzyme-linked immunosorbent assay (ELISA) and Western blotting (immunoblotting). Twenty-one plasma samples positive by ELISA and Western blotting and 29 negative by ELISA were used in this study for RT-PCR testing and genotyping. Plasma was stored at 2708C, and RNA extraction was as described by Cha et al. (2) with few modifications. Briefly, 100 ml of plasma was incubated with DNase, diethyl pyrocarbonate-H2O, and proteinase K (20 mg/ml) at 568C for 1 h. tRNA (1 mg/ml) was added after incubation. RNA was then extracted with phenol and chloroform-isoamyl alcohol, precipitated with ethanol, redissolved in 20 ml of diethyl pyrocarbonate-H2O, and stored at 2208C. cDNA synthesis was carried out according to the kit instructions (CIS Bio International, Grif-sur-Yvette, France). A 20-ml reaction mixture, containing 10 ml of RNA extract, RT buffer, 5 mM deoxynucleoside triphosphate, 0.1 M dithiothreitol, 1.5 mM reverse transcriptase primer (3), 40 U of RNase inhibitor, * Corresponding author. Mailing address: Department of Microbiology and Immunology, American University of Beirut, 850 3rd Ave., New York, NY 10022. Fax: (212) 486-2867. Electronic mail address:
[email protected].
FIG. 1. PCR products from hemodialysis patients. Lane 1, marker (123-bp ladder); lane 2, positive control; lane 3, no DNA added; lanes 4 to 6, DNA from hemodialysis patients negative for HCV antibodies; lanes 7 to 10, DNA from hemodialysis patients positive for HCV antibodies.
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FIG. 2. Southern hybridization of HCV amplicons with a digoxigenin-labeled probe. Lane 2, positive control; lane 3, no DNA added; lanes 4 to 6, DNA from hemodialysis patients negative for HCV antibodies; lanes 7 to 10, DNA from hemodialysis patients positive for HCV antibodies.
light, photographed, and scanned with the BioImage system (Millipore Corp., Bedford, Mass.). Our results showed that 12 of 21 patients positive by ELISA and Western blotting and 3 of 29 patients negative by ELISA and used as negative controls were positive by RT-PCR and showed the expected 268-bp sequence similar to that of the positive control. Figure 1 shows a representative sample of RT-PCR results. All amplicons, including the positive control, hybridized with the digoxigenin-labeled probe derived from the amplified sequence (Fig. 2). Our results have shown that all amplicons were digested not with HaeIII but with a combination of HinfI and ScrFI, giving the pattern for genotype 4, different from that of the positive control, which exhibited the pattern for genotype 1 (Table 1). HCV genotyping showed that the HCV in the tested samples belongs to genotype 4. This is in accordance with the finding that genotype 4 is prevalent in the Middle East (5). However, this should not be generalized to all HCV cases prevalent in Lebanon. Analysis should be done with a larger population, including hemodialysis patients and blood donors, to determine the prevalent HCV genotype in Lebanon. The genotype detected among our samples was different from that of the positive control (59 NCR inserted into the BamHI site of the
TABLE 1. Cleavage patterns of the 59 NCR of genotypes 1 and 4 with ScrFI and HinfI Amplicon source(s)
DNA fragment sizes (bp)
Genotype
HCV-positive control Clinical specimens
53, 15, 48, 9, 32, 94 53, 15, 41, 7, 9, 32, 94
1 4
pATH 10/F17R38 plasmid), which belonged to genotype 1. Genotyping is important because it provides information as to strain variation and potential association with disease severity. In addition, it is of epidemiologic value because it sheds light on whether prevalent HCV strains are similar to that endemic in the Middle East. Detection of genotypes different from genotype 4 in Lebanon may indicate importation of a foreign strain(s) that may be the cause of severe disease. As an example, genotyping analysis of HCV in South Africa identified the introduction of a new strain (genotype 5) in the population which proved to be associated with severe disease which mimics that caused by genotype 1 (8). In conclusion, a number of hemodialysis patients have ongoing infection, as determined by RT-PCR. Restriction endonuclease analysis of amplicons provided information as to HCV genotype in the tested samples. Genotyping will enable us to show the relationship between the prevalent or newly introduced genotype(s) in the community and the severity of disease and is also helpful in epidemiologic studies. We thank the American University Research Board for financial support. We thank Michael J. Beach at the Centers for Disease Control and Prevention for the provision of plasmid pATH 10/F17R38 and all those who have provided us with blood samples. REFERENCES 1. Cha, T. A., E. Beall, B. Irvine, J. Kolberg, D. Chein, G. Ruo, and M. S. Urdea. 1992. At least five related but distinct hepatitis C viral genotypes exist. Proc. Natl. Acad. Sci. USA 89:7144–7148. 2. Cha, T.-A., J. Kolberg, B. Irvine, M. Stempien, E. Beall, M. Yano, Q.-L. Choo, M. Houghton, G. Kuo, J. H. Han, and M. S. Urdea. 1991. Use of a signature nucleotide sequence of hepatitis C virus for detection of viral RNA in human serum and plasma. J. Clin. Microbiol. 29:2528–2534. 3. Chan, S. W., F. McOmish, E. C. Holmes, B. Dow, J. F. Peutherer, E. Follett, P. L. Yap, and P. Simmonds. 1992. Analysis of new hepatitis C-virus type and its phylogenetic relationship to existing variants. J. Gen. Virol. 73:1131–1141. 4. Cuthbert, J. A. 1994. Hepatitis C: progress and problems. Clin. Microbiol. Rev. 7:505–532. 5. Murphy, D., B. Willens, and G. Delage. 1994. Use of the non-coding region for the genotyping of hepatitis C-virus. J. Infect. Dis. 169:473–474. 6. Popovic, T., G. A. Bopp, Ø. Olsvik, and J. A. Kiehlbauch. 1993. Ribotyping in molecular epidemiology, p. 573–583. In D. H. Persing, T. F. Smith, F. C. Tenover, and T. J. White (ed.), Diagnostic molecular microbiology: principles and applications. American Society for Microbiology, Washington, D.C. 7. Simmonds, P., E. C. Holmes, T. A. Cha, S. W. Chan, F. McOmish, B. Irvine, E. Beall, P. L. Yap, J. Kolberg, and M. S. Urdea. 1993. Classification of hepatitis C-virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS5 region. J. Gen. Virol. 74:2391–2399. 8. Smuts, H. E. M., and J. Kannemeyer. 1995. Genotyping of hepatitis C virus in South Africa. J. Clin. Microbiol. 33:1679–1681.
