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A sensitive RT-PCR assay for the detection of hepatitis G virus
Abstracts /Journal
of Microbiological Methods 27 (1996) 97-107
developed during the following year a humoral anti-E2 response and in parallel turned HGV-RNA negative. Conclusions: We conclude that a humoral immune response to E2 and p&bly other HGV-epitopes is associated with loss of detectable HGV-viremia. Thus, E2-specific antibodies might serve as a useful marker for diagnosing recovery from HGV infections. 7 A sensitive virus
RT-PCR assay for the detection of hepatitis G
V. Schlueter, S. Schmolke, A.M. Engel, G. Hess and B. Ofenloch-Haehnle Boehtinger Mannheim GmbH, 82377 Penzberg, Germany
R&D
Infectious Diseases,
A novel viral agent was isolated from a patient with chronic hepatitis. !$equence analysis demonstrated the presence of mot& highly conserved among positive stranded RNA viruses, closely related to the Flauiuitidae. The homology of approx. 25% to the most related Hepatitis C Virus (HCV) demonstrated the discovery of a new virus provisionally designated Hepatitis G Virus (HGV). For a sensitive detection of this new RNA virus from human sera we have developed a RT-PCR assay in the presence of digoxigcnin-dUTP amplifying a portion of the S-non-coding region (5’-NCR) and a sequence of the putative viral NS5a gene. Following PCR, the amplified products were analyzed on a fully automated ES300 analyser using biotinylated capture probes and streptavidin-coated tubes. We could demonstrate the detection of down to 4 viral copies or 1 x lo3 genome equivalents/ml with both primer pairs. Southern blol analysis showed the specificity of the RT-PCR assays. To analyze the prevalence of HGV in high as well as low risk populations we lhave screened intravenous drug abusers (IVDA’s) and blood donors. HGV was shown to be present in 34 OUI of 122 plasmas of IVDA’s. Two of them were detected with the lNS5a primers only and one case was positive in using the S-NCR primers only, indicating the necessity to use hvo or more primer sets to optimize sensitivity. 3% of the analyzed blood donors were HGV positive with both primer sets. Further studies to determine the HGV prevalence in other populations and the clinical significance of the new virus remains to be established. In addition, work for a nucleic acid-based test to quantify HGV is in progress. 8 Hepatitis C virus genotypes in HIV+ patients coinfected with hepatitis B virus F. GalLn, M.T. Pirez-Gracia, C. Fem&dez, J.A. Gir6n
and M.A. Rodriguez-Iglesias Lab. Microbiology, Puerto Real Uniu. Hosp., School of Medicine, Plaza Fragela s/n, IJ003-Ca’diz Spain Objectives: To study the prevalence of hepatitis C virus (HCV) genotypes in HIV( +) patients coinfected with hepatitis B virus (HBV). Method: One-hundred-ninety-four patients coinfected with HCV and HBV were studied. One-hundred-six were HIV (+) and eighly-eight HIV(-) patients were used as control group. Antibodies to HCV were studied by ELISA screening (BioELISNA, Biokit, Spain) and confirmatory test (Innolia HCVIII, Innogenetics, Belgium). PCR was done
99
using the Amplicor-HCV (Roche, Switzerland). The amphlied product was genotyped by InnoLipa HCV (Innogenetits, Belgium). Conventional ELlSAs were used to determine HBV (Sorin Biomedica, Italy) and HIV (Wellcozyme HIV 1 + 2, Murex, USA) serological markers. Results: Seventy-two (67.9%) HIV( + 1 and fifty-two (59.1%) HIV(-) patients were HCV-RNA positive. Fifteen samples were not able to be genotyped. The most frequent genotypes in HIV( +) were la (45.7%) and 3a (30.5%). Other genotypes detected were lb (11.8%) and 4 (II.8 %). In HIV(-) genotypes la (37.8%), lb (37.8%) 2a/Zc (2.7%), 2b (2.7%), 3a (13.5%) and 4c/4d (5.4%) were detected. Conclusions: The distribution of HCV genotypes is very different in HIV(+) and HIV (-1 patients. The absence of genotype lb in HIV(+) can be due to epidemiological or viral facts linked to immunological response or viral dynamic. In immunocompetent individuals the avirulent genotypes could be self-controlled and not detected by PCR. 9 from amplicor products H. Petersen and B. Hennings
HCV-genotyping
Labor Fenner, BergstraJe 14, D-20095 Hambq Objectiues: The Amplicor RT-PCR is a reliable method for the identification of HCV infections [I 1. Knowledge of the HCV genotype is of further interest. HCV isolates are divided into 6 genotypes each with 1-3 subtypes [2]. In central Europe, the most common genotype IS type 1 with subtypes la and lb, followed by genotype 3. Subtype lb seems to be more often associated with aggressive liver disease and poor response to interferon a [3]. As commercial products for HCV genotyping are very expensive, we developed an economical nested PCR (n-PCR) for genotypes 1 and 3 based on our routine HCV-PCR (Amplicor HCV, Roche). Methods: The genotype-specific primers were chosen by sequence analysis and multiple alignment of HCV 5’-non coding region (NCR) available from the EMBL database. For DNA editing and multiple alignment we used “MacMolly Tetra” (SoftGene, Bochold). Amplicons of the 5’NCR [4] derived from the Amplicor HCV routine were diluted 1:lOO prior to Amplicor detection and frozen at -20°C. Genotyping was performed in separated nested PCR reactions for type 1 and 3, respectively. For each n-PCR, 1 ~1 diluted amplicon was added to 30 ~1 prepared master-mix. Amplification was performed in a PE 9600 thermocycler with 30 cycles for 10 set at 94°C and 15 set at 67°C in microstrip reaction tubes without oil overlay. Amplicons were detected by gel electrophoresis (4% NuSieve 3: I). Results: Out of 149 examined HCV positive samples, 105 (= 70.5%) were genotype 1, 39 (= 36.2%) were genotype 3, one was genotype 1 and 3, and three were neither genotype 1, nor genotype 3. This distribution corresponds to other studies genotyping HCV patinets with chronic hepatitis in Germany [5,6]. Conclusions: Based on routine HCV PCR we established a simple and economical genotyping procedure. Since patients response to interferon cr is influenced by the HCV genotype, knowledge of the genotype will be helpful in prospecting success of therapy. At the moment we evaluate the differentiation of genotype 1 in subtypes la and Ib.
of Microbiological Methods 27 (1996) 97-107
developed during the following year a humoral anti-E2 response and in parallel turned HGV-RNA negative. Conclusions: We conclude that a humoral immune response to E2 and p&bly other HGV-epitopes is associated with loss of detectable HGV-viremia. Thus, E2-specific antibodies might serve as a useful marker for diagnosing recovery from HGV infections. 7 A sensitive virus
RT-PCR assay for the detection of hepatitis G
V. Schlueter, S. Schmolke, A.M. Engel, G. Hess and B. Ofenloch-Haehnle Boehtinger Mannheim GmbH, 82377 Penzberg, Germany
R&D
Infectious Diseases,
A novel viral agent was isolated from a patient with chronic hepatitis. !$equence analysis demonstrated the presence of mot& highly conserved among positive stranded RNA viruses, closely related to the Flauiuitidae. The homology of approx. 25% to the most related Hepatitis C Virus (HCV) demonstrated the discovery of a new virus provisionally designated Hepatitis G Virus (HGV). For a sensitive detection of this new RNA virus from human sera we have developed a RT-PCR assay in the presence of digoxigcnin-dUTP amplifying a portion of the S-non-coding region (5’-NCR) and a sequence of the putative viral NS5a gene. Following PCR, the amplified products were analyzed on a fully automated ES300 analyser using biotinylated capture probes and streptavidin-coated tubes. We could demonstrate the detection of down to 4 viral copies or 1 x lo3 genome equivalents/ml with both primer pairs. Southern blol analysis showed the specificity of the RT-PCR assays. To analyze the prevalence of HGV in high as well as low risk populations we lhave screened intravenous drug abusers (IVDA’s) and blood donors. HGV was shown to be present in 34 OUI of 122 plasmas of IVDA’s. Two of them were detected with the lNS5a primers only and one case was positive in using the S-NCR primers only, indicating the necessity to use hvo or more primer sets to optimize sensitivity. 3% of the analyzed blood donors were HGV positive with both primer sets. Further studies to determine the HGV prevalence in other populations and the clinical significance of the new virus remains to be established. In addition, work for a nucleic acid-based test to quantify HGV is in progress. 8 Hepatitis C virus genotypes in HIV+ patients coinfected with hepatitis B virus F. GalLn, M.T. Pirez-Gracia, C. Fem&dez, J.A. Gir6n
and M.A. Rodriguez-Iglesias Lab. Microbiology, Puerto Real Uniu. Hosp., School of Medicine, Plaza Fragela s/n, IJ003-Ca’diz Spain Objectives: To study the prevalence of hepatitis C virus (HCV) genotypes in HIV( +) patients coinfected with hepatitis B virus (HBV). Method: One-hundred-ninety-four patients coinfected with HCV and HBV were studied. One-hundred-six were HIV (+) and eighly-eight HIV(-) patients were used as control group. Antibodies to HCV were studied by ELISA screening (BioELISNA, Biokit, Spain) and confirmatory test (Innolia HCVIII, Innogenetics, Belgium). PCR was done
99
using the Amplicor-HCV (Roche, Switzerland). The amphlied product was genotyped by InnoLipa HCV (Innogenetits, Belgium). Conventional ELlSAs were used to determine HBV (Sorin Biomedica, Italy) and HIV (Wellcozyme HIV 1 + 2, Murex, USA) serological markers. Results: Seventy-two (67.9%) HIV( + 1 and fifty-two (59.1%) HIV(-) patients were HCV-RNA positive. Fifteen samples were not able to be genotyped. The most frequent genotypes in HIV( +) were la (45.7%) and 3a (30.5%). Other genotypes detected were lb (11.8%) and 4 (II.8 %). In HIV(-) genotypes la (37.8%), lb (37.8%) 2a/Zc (2.7%), 2b (2.7%), 3a (13.5%) and 4c/4d (5.4%) were detected. Conclusions: The distribution of HCV genotypes is very different in HIV(+) and HIV (-1 patients. The absence of genotype lb in HIV(+) can be due to epidemiological or viral facts linked to immunological response or viral dynamic. In immunocompetent individuals the avirulent genotypes could be self-controlled and not detected by PCR. 9 from amplicor products H. Petersen and B. Hennings
HCV-genotyping
Labor Fenner, BergstraJe 14, D-20095 Hambq Objectiues: The Amplicor RT-PCR is a reliable method for the identification of HCV infections [I 1. Knowledge of the HCV genotype is of further interest. HCV isolates are divided into 6 genotypes each with 1-3 subtypes [2]. In central Europe, the most common genotype IS type 1 with subtypes la and lb, followed by genotype 3. Subtype lb seems to be more often associated with aggressive liver disease and poor response to interferon a [3]. As commercial products for HCV genotyping are very expensive, we developed an economical nested PCR (n-PCR) for genotypes 1 and 3 based on our routine HCV-PCR (Amplicor HCV, Roche). Methods: The genotype-specific primers were chosen by sequence analysis and multiple alignment of HCV 5’-non coding region (NCR) available from the EMBL database. For DNA editing and multiple alignment we used “MacMolly Tetra” (SoftGene, Bochold). Amplicons of the 5’NCR [4] derived from the Amplicor HCV routine were diluted 1:lOO prior to Amplicor detection and frozen at -20°C. Genotyping was performed in separated nested PCR reactions for type 1 and 3, respectively. For each n-PCR, 1 ~1 diluted amplicon was added to 30 ~1 prepared master-mix. Amplification was performed in a PE 9600 thermocycler with 30 cycles for 10 set at 94°C and 15 set at 67°C in microstrip reaction tubes without oil overlay. Amplicons were detected by gel electrophoresis (4% NuSieve 3: I). Results: Out of 149 examined HCV positive samples, 105 (= 70.5%) were genotype 1, 39 (= 36.2%) were genotype 3, one was genotype 1 and 3, and three were neither genotype 1, nor genotype 3. This distribution corresponds to other studies genotyping HCV patinets with chronic hepatitis in Germany [5,6]. Conclusions: Based on routine HCV PCR we established a simple and economical genotyping procedure. Since patients response to interferon cr is influenced by the HCV genotype, knowledge of the genotype will be helpful in prospecting success of therapy. At the moment we evaluate the differentiation of genotype 1 in subtypes la and Ib.