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<p><b>OBJECTIVE</b>To investigate Lanzhou area cases of hepatitis C virus (H-CV) infection with a 5'-non coding region (NCR) 2i genotype and core (C), envelope protein (E) and non-structural protein (NS5) 2a genotype and the relationship with therapeutic response to interferon-alpha (IFNa).</p><p><b>METHODS</b>Nine patients who received IFNa-based treatment for HCV between 2007 and 2009 at the Second People's Hospital of Gansu Province were selected for analysis.Restriction enzyme analysis was carried out for the 5'-NCR and sequencing was carried out for the other gene areas.The relationship between genetic variants and IFNaresponse was examined.</p><p><b>RESULTS</b>Of the total nine HCV cases treated with IFNa-based therapies, five of the patients achieved sustained virological response (SVR), which included two cases with type 2 genotype and three cases with no MboI restriction enzyme point of tangency (i.e.type 1b). The remaining four patients that did not achieve SVR included one case of type 2a, with a 1b and 2a mixed state, and one case with 5'-NCR 2i genotype and C area, NS5 area 2a genotype; the other two cases had 5'-NCR and C area type 1b. Of the five cases with 5'-NCR 2i genotype, all had C 2a genotype and two had C/E 2a and NS5 2a genotypes.The seven patients that showed no response to ordinary IFNa were converted to long-term IFNa plus ribavirin combination antiviral treatment; five (71.4%) of the cases showed response in HCV RNA level and the patients treated with the pegylated form showed greater response.</p><p><b>CONCLUSION</b>HCV genotyping can only provide information on the particular region of gene sequence examined, and it is important to sequence all gene regions where mutations related to antiviral drug response are located. Peg-IFNa-2a combined with ribavirin may achieve better therapeutic effect in patients infected with 2i/2a recombinant forms of HCV.</p>
Subject(s)
Humans , Angiogenesis Inhibitors , Antiviral Agents , Drug Therapy, Combination , Genetic Variation , Genotype , Hepacivirus , Genetics , Hepatitis C, Chronic , Drug Therapy , Interferon-alpha , Open Reading Frames , Polyethylene Glycols , Recombinant Proteins , Recombination, Genetic , Ribavirin , Treatment OutcomeABSTRACT
Objective A method was established for genotyping of hepatitis B virus (HBV A-D genotype),based on the PCR-restriction fragment length polymorphism (RFLP) created by Hinf Ⅰ,Ear Ⅰ,Apo Ⅰ action on an amplified segment of the S region.Methods Clinical diagnosis research.One hundred and twenty-eight HBV S sequences obtained from GeneBank were analyzed for restriction enzyme sites that would be genotype-specific.Restriction patterns following digestion with restriction enzymes Hif Ⅰ,Ear Ⅰ,Apo Ⅰ were determined to identify A-D HBV genotypes.The method was used to detect the HBV genotype of fifty severe hepatitis patients due to chronic hepatitis B in China.Then the detection results were confirmed by direct sequencing.Results The new genotyping method was established,named simple PCR-RFLP,which could identify HBV genotypes A to D.Genotypes B,C,B/C and A or D could be determined by a single step digestion with Hif Ⅰ.Eight patients of genotype A/B/C classified by single step digestion with Hif Ⅰ were conformed as genotype B variant by further digestion and direct sequencing.Extracted randomly and diluted into different concentration,three specimens were tested for genotype of HBV repeatedly and respectively.The results were all in accord with the originals,and the lowest detection limit of HBV DNA was 7 ～ 9 IU/ml.This was particularly useful in China where genotypes B and C were predominant.Twenty-three of genotype B and ten genotype C patients were classified from these fifty severe hepatitis B patients by a single step digestion with Hif Ⅰ through the simple PCR-RFLP method.The same results were also obtained by direct sequencing of PCR products (Kappa =1.00,P =0.001).The simple PCR-RFLP method was superior to direct sequencing in detecting HBV B/C polyinfection (9 cases and 0 case; x2 =18.00,P =0.001).Conclusions Both the sensitivity and repetitiveness of Simple PCR-RFLP method are satisfactory.It is superior to direct sequencing in detecting HBV B/C polyinfection,and simple,convenient.
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Objective To evaluate the performance of a DNA microarray method for detecting HBV antiviral drug-resistant mutations. Methods Two hundred and twenty four serum samples from patients with CHB were tested in parallel by DNA microarray and direct sequencing for the mutations within the HBV reverse transcriptase (rt) region, which included rtL180, rtA181, rtM204 and rtN236. Samples with discrepant results were retested by clonal sequencing. Results Complete concordance between DNA microarray and direct sequencing results was observed in 214 out of 224 samples (95. 5% ). The presence of mixed viral populations in the other 10 samples detected by DNA microarray but not by direct sequencing was confirmed by clonal analysis. The DNA microarray could detect minor viral populations which constituted 5.0%-15. 0% of the total viral load. Conclusion DNA microarray is highly consistent with direct sequencing in detecting HBV mutations conferring drug resistance and more sensitive in detecting mixed mutant and wild-type sequences than direct sequencing, which makes it a useful tool for early detection of drug resistance early.
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Objective:To investigate the existence condition of hepatitis B surface antigen(HBsAg) termina-tion codon bias.Methods: A total of 174 reference sequences of all kinds of Hepatitis B virus(HBV) genotypes were chosen from GenBank,and compared by BioEdit.Then secondary structure of RNA was constructed and analyzed together.Results:(1) There were two types of HBsAg termination codon: TAA and TGA in 174 reference sequences.TAA was in 124 cases(71.26%);and TGA in 50 cases(28.74%).(2) There was codon bias selection in HBsAg termination codon,and it could affect the secondary structure of RNA and amino acid sequence encoding protein.Conclusion: HBsAg termination codon bias exists and may be related to RNA structure and the conservatiom of protein function in the evolutionary progress.
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Objective To analyze the relation between lamivudine-resistant mutation types and HBV genotypes.Methods 95 cases with YMDD mutation were selected from out-patient clinic and sickroom in our hospital.Restriction endonuclease MboⅠand EarⅠwere used to identify HBV genotypes of PCR product, farther a phylogenesis tree was applied to check it.Results We could divide 95 cases into two parts, 75 cases(78.95%) were HBV C genotype and 20 cases(21.05%) were B genotype, these were verified by phylogenesis tree.With regard to the types of YVDD mutation、YIDD mutation and YMDD+YVDD+YIDD mutation, there were 11 cases, 5 cases, 4 cases in genotype B, respectively, but 35 cases, 27 cases, 13 cases in genotype C.We made the cha-test to check genotypes and types of YMDD mutation, ?2= 0.856, P=0.710. There were no significant differences.Conclusions Using MboⅠand EarⅠcan genotype HBV PCR product easily and reliably. It is no statistical significance between HBV B or C genotype and the types of YMDD mutation.
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Objectives In our studies before, we reported mutation C-T at -222 of hepatitis C virus type 1b genotyped on 5′ noncoding region (5′NCR). There was no this mutation of type 1b recorded in GenBank. Since genotypiog based on 5′NCR cannot differentiate subtypes, it is necessary to investigate whether the hepatitis C virus genotype 1b strains with mutation C-T belongs to other subtypes of genotype 1. Methods 64 HCV genotype 1b samples were amplified from 5′NCR by RT-PCR.Then the products were digested by use of BamHⅠ restriction enzymes. We randomly chose 6 samples with BamHⅠ restriction site and subjected them to amplification of 5′NCR and NS5B. The sequences of 5′NCR were analyzed. Sequences of NS5B fragments from the 6 samples were respectively subjected to phylogenetic analysis with subtypes of genotype 1 and 38 complete genomes of genotype 1b from GenBank.Results The phylogenetic analysis of 6 samples and subtypes of genotype 1 indicated that the genotypes of 6 strains with BamHⅠ restriction site in 5′NCR were type 1b, instead of subtype of type 1. Tree construction with 38 complete genomes of genotype 1b showed that the 6 mutants of genotype 1b did not belong to the same branch of the tree and there were no genetic differences between the mutants and the other strains of genotype 1b.Conclusions Our research indicated that the hepatitis C virus stains of C-T in 5′NCR were mutants of type 1b. Since the 5′NCR is a highly conserved region of HCV, the mutation might have some relationship to the long-time IFN therapy.
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Objective: To develop a hepatitis C virus(HCV) reverse transcription-polymerase chain reaction (RT-PCR) assay using Uracil-DNA glycosylase (UDG) for amplicon contamination control and evaluate the temperature and UDG concentrations for anti-contamination. Methods: In this new HCV RT-PCR assay, reverse transcription, UDG anti-contamination and the first PCR were carried out at the same time. The layer candles were used to prevent the contamination in the second PCR. dU-DNA was used as quality control for UDG anti-contamination and templates to determine the sensitivity of the new HCV RT-PCR assay. HCV cDNA was detected by DNA enzyme immunoassay (DNA-EIA). Results: Complete degradation of amplicon DNA was observed on the conditions of 0.2 u UDG per reaction volume respectively at 37 ℃ and 42 ℃ for 40 min. The anti-contamination condition also could eliminate all detectible dU-DNA, including the highest concentration of amplicon DNA.The 1∶104 dilution of the HCV RNA sample containing 2.110?105copies/mL copies of RNA could be detected. Conclusion: Our results indicate that this new RT-PCR assay can control the contamination stringently and is sensitive as well.
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Objective: To investigate the infection state of hepatitis C virus genotype 6a in China.Methods: Three(95,126,150)HCV genotype 6a serum samples were identified by digesting 5′NCR with compound enzyme method.Then,HCV 5′NCR and NS5B fragments were amplified from these samples by RT-PCR assay and sequenced.The phylogenetic trees of the samples were analyzed and compared with 24 HCV complete gene sequences from GenBank.Results: The sequencing reports on 5′NCR showed "CA" bases in 3 serum samples(95,126,150) were inserted into-145 site,and the sequences of 3 serum samples had the highest homology with sequence Y12083(0.934,0.930,and 0.926,respectively).The results of the phylogenetic trees suggested these 3 serum samples belonged to HCV genotype 6a.The sequencing reports on NS5B showed the 3 serum samples also had the highest homology with HC-J4(0.934,0.930,and 0.926,respectively),and the results of the phylogenetic trees suggested these 3 serum samples belonged to HCV genotype 1b.To exclude the influence of amplification efficiency of primers,NS5B fragments were amplified by HCV genotype 6a specific primers and no amplification products appeared.Conclusion: There are different results of HCV genotype by analyzing 5′NCR and NS5B in 3 samples infected with HCV genotype 6a.It may be related with gene recombination.It suggests HCV genotype should be analyzed on more than two regions.
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Objective To investigate the genetic heterogeneity of 5’-NCR of HCV genotype 1b.Methods We selected 147 HBV genotype 1b serum samples. HCV 5’-NCR fragments were amplified from these samples by use of RT-PCR assay and sequenced after using restriction endonuclease Mbo Ⅰ and BamH Ⅰ. We analyzed the phylogenetic trees of the samples and compared them with 40 isolates of HCV genotype 1b.Results The sequencing reports indicated the isolates of HCV genotype 1b recognized by BamHⅠat position 117 by a substitution (C-T) at position 120 in HCV genotype 1b viruses in China. Of 147 samples, 17 (11.56%) samples for one BamHⅠrecognization position, 26 (17.69%) for neither BamH Ⅰ recognization position nor Mbo Ⅰ,6 samples (4.08%) for two Mbo I recognization position, 18(12.24%)for one MboⅠrecognization position.Conclusions Of 147 HBV genotype 1b serum samples, 54.42% for one Mbo I recognization position, whether this have relationship with the response to IFN therapy provide the framwork for future detailed investigation of HCV antviral therapy. There is specific BamH I recognization position in isolates of HCV genotype 1b in China as compared to the other 40 HCV 1b isolates. The role of this specific mutation needs to be further researched.
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Objective To develop a rapid specific method to detect HBV gene mutation by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Methords The patients of YMDD and YIDD mutation were detected by DNA sequencing.Results the DNA sequencing demonstrated that not only YMDD and YIDD/YVDD mutation, but LLLL,LMLL,DLHD,DMHD,LLAQ and LMAQ mutation were also detected in different hepatitis B patients. The results of PCR-RFLP indicated that there were different restricted sites in the distinct mutation.Conclusion The results above indicated that all these mutants exist in the initiator codon of ATG(Met). The mutation of LMLL DMHD LMAQ was associated with Lamivordin treatment. Whether mutation of Met links with the second replicating of HBV needs to be further studied.
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To explore the destructive effect of residual uracil DNA glycosylase on dUTP incorporation to the cDNA PCR products,PAGE and DNA-EIA hybridization technique were applied to detect the efficiency of anti-contamination and the rate of DNA hybridization.In this study,it was found that:⑴Good effect could be achieved with the reaction of 2u of uracil DNA glycosylase(UDG)for 20min at 37℃.⑵UDG could be inactivated at 94℃ for 10min.The hybridization rate of control group of PCR products was 94 18% at room temperature for 65 hours.The hybridization rates of test groups at-20℃ for 65 hours were 87 69%,77 24%,76 83%,respectively.And the inactivation times of anti-contamination groups at 94℃ were“0”min,“10”min and “20”min,respectively;at room temperature for 65 hours,these hybridization rates were 74 77%,72 50%,70 29%,respectively.There were significant differences between control and rest groups(P0 05).Our study suggested that the hybridization rate of test groups kept at-20℃ decreased 19 41%,after 65 hours,the rate decreased 27 45%,but in control group only decreased 5 82% for 65h at room temperature.So that,we conclude that the residual UDP can cause above results.
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Objective To explore the effect of residual UDG on all dU-DNA PCR products. Methods The hybridization percentage of dU-DNA PCR products, which were stored at 37℃ for 20 hours as well as at -20℃,4℃,room temperature and 37℃,respectively, were detected by employing microplate hybridization technique. The effect of inactivating the products at 94 ℃ for 10 minutes on the activity of Taq polymerase was also analysed. Results Compared with the control, A value decreased by 13.281% and 20.557%, respectively after adding 0.2u and 10u UDG (P