To compare the oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus in Chinese patients with chronic hepatitis B. METHODS： Mixture of samples with different genotypes and clinical se...To compare the oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus in Chinese patients with chronic hepatitis B. METHODS： Mixture of samples with different genotypes and clinical serum samples from 126 chronic hepatitis B patients was tested for hepatitis B virus genotypes by oligonucleotide chip, real-time PCR and sequencing of PCR products, respectively. Clinical performances, time required and costs of the three assays were evaluated. RESULTS： Oligonucleotide chips and real-time PCR detected 1% and 0.1% genotypes, respectively, in mixed samples. Of the 126 clinical samples from patients with chronic hepatitis B, genotype B was detected in 41 （33%）, 41 （33%） and 45 （36%） samples, and genotype C in 76 （60%）, 76 （60%） and 81 （64%） samples, by oligonucleotide chip, real-time PCR and sequencing, respectively. Oligonucleotide chip and real-time PCR detected mixed genotypes B and C in 9 samples. Real- time PCR was the rapidest and cheapest among the three assays. CONCLUSION： Oligonucleotide chip and real-time PCR are able to detect mixed genotypes, while sequencing only detects the dominant genotype in clinical samples.展开更多
AIM To detect infection rate of GBV-C/HGV inhepatitis C patients,to determine the methodsof higher sensitivity and the primers of higherefficiency for GBV-C/HGV RNA detection and tostudy the dominant subtype and mutat...AIM To detect infection rate of GBV-C/HGV inhepatitis C patients,to determine the methodsof higher sensitivity and the primers of higherefficiency for GBV-C/HGV RNA detection and tostudy the dominant subtype and mutation ofGBV-C/HGV.METHODS Quantitative RT-PCR for detectionpf HCV RNA concentration in serum samples,RT-nested PCR with two sets of primers fordetection of GBV-C RNA,RT-PCR ELISA with twosets of primers for detection of HGV RNA,nucleotide sequence and putative amino acidsequence analysis.RESULTS The positive rates of GBV-C RNA atthe 5’-NCR and NS3 region in 211 serums amplesfrom the patients with HCV infection were 31.8%and 22.8% respectively.The positive rates ofHGV RNA at the 5’-NCR and NS5 region in thesame samples were 47.9% and 31.8%respectively.The total positive rate of GBV-C/HGV RNA was as high as 55.5%.HCV copynumbers in the patients without GBV-C/ HGVcoinfection were statistically higher than that inthe patients with GBV-C/ HGV coinfection(P【0.01).Frequent mutation of nucleotideresidue was present in the amplificationproducts.Frameshift mutation was found in twosamples with GBV-C NS3 region nucleotidesequences.All nucleotide sequences fromamplification products showed higher homologyto HGV genome than to GBV-C genome even though part of the sequences were amplifiedwith GBV-C primers.CONCLUSION A high frequency of GBV-C/ HGV coinfection existed in the hepatitis C patients. RT-PCR ELISA was more sensitive than RT-nested PCR for detection of GBV-C/ HGV RNA. The primers derived from the 5 -NCR was more efficient than those derived from the NS3 and NS5 regions. A reverse relationship was found to exist between HCV RNA concentration and GBV-C/ HGV infection frequency. HGV was the dominant subtype of the virus in the local area. The major mutations of GBV-C/ HGV genomes were random mutation of nucleotide residue.展开更多
AIM To determine the variability/conservation of the domain of hepatitis B virus(HBV) pre S1 region that interacts with sodium-taurocholate cotransporting polypeptide(hereafter, NTCP-interacting domain) and the preval...AIM To determine the variability/conservation of the domain of hepatitis B virus(HBV) pre S1 region that interacts with sodium-taurocholate cotransporting polypeptide(hereafter, NTCP-interacting domain) and the prevalence of the rs2296651 polymorphism(S267 F, NTCP variant) in a Spanish population. METHODS Serum samples from 246 individuals were included and divided into 3 groups: patients with chronic HBV infection(CHB)(n = 41, 73% Caucasians), patients with resolved HBV infection(n = 100, 100% Caucasians) and an HBV-uninfected control group(n = 105, 100% Caucasians). Variability/conservation of the amino acid(aa) sequences of the NTCPinteracting domain,(aa 2-48 in viral genotype D) and a highly conserved pre S1 domain associated with virion morphogenesis(aa 92-103 in viral genotype D) were analyzed by next-generation sequencing and compared in 18 CHB patients with viremia > 4 log IU/mL. The rs2296651 polymorphism was determined in all individuals in all 3 groups using an in-house real-time PCR melting curve analysis.RESULTS The HBV pre S1 NTCP-interacting domain showed a high degree of conservation among the examined viral genomes especially between aa 9 and 21(in the genotype D consensus sequence). As compared with the virion morphogenesis domain, the NTCPinteracting domain had a smaller proportion of HBV genotype-unrelated changes comprising > 1% of the quasispecies(25.5% vs 31.8%), but a larger proportion of genotype-associated viral polymorphisms(34% vs 27.3%), according to consensus sequences from Gen Bank patterns of HBV genotypes A to H. Variation/conservation in both domains depended on viral genotype, with genotype C being the most highly conserved and genotype E the most variable(limited finding, only 2 genotype E included). Of note, proline residues were highly conserved in both domains, and serine residues showed changes only to threonine or tyrosine in the virion morphogenesis domain. The rs2296651 polymorphism was not detected in any participant.CONCLUSION In our CHB population, the NTCP-interacting domain was highly conserved, particularly the proline residues and essential amino acids related with the NTCP interaction, and the prevalence of rs2296651 was low/null.展开更多
文摘To compare the oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus in Chinese patients with chronic hepatitis B. METHODS： Mixture of samples with different genotypes and clinical serum samples from 126 chronic hepatitis B patients was tested for hepatitis B virus genotypes by oligonucleotide chip, real-time PCR and sequencing of PCR products, respectively. Clinical performances, time required and costs of the three assays were evaluated. RESULTS： Oligonucleotide chips and real-time PCR detected 1% and 0.1% genotypes, respectively, in mixed samples. Of the 126 clinical samples from patients with chronic hepatitis B, genotype B was detected in 41 （33%）, 41 （33%） and 45 （36%） samples, and genotype C in 76 （60%）, 76 （60%） and 81 （64%） samples, by oligonucleotide chip, real-time PCR and sequencing, respectively. Oligonucleotide chip and real-time PCR detected mixed genotypes B and C in 9 samples. Real- time PCR was the rapidest and cheapest among the three assays. CONCLUSION： Oligonucleotide chip and real-time PCR are able to detect mixed genotypes, while sequencing only detects the dominant genotype in clinical samples.
文摘AIM To detect infection rate of GBV-C/HGV inhepatitis C patients,to determine the methodsof higher sensitivity and the primers of higherefficiency for GBV-C/HGV RNA detection and tostudy the dominant subtype and mutation ofGBV-C/HGV.METHODS Quantitative RT-PCR for detectionpf HCV RNA concentration in serum samples,RT-nested PCR with two sets of primers fordetection of GBV-C RNA,RT-PCR ELISA with twosets of primers for detection of HGV RNA,nucleotide sequence and putative amino acidsequence analysis.RESULTS The positive rates of GBV-C RNA atthe 5’-NCR and NS3 region in 211 serums amplesfrom the patients with HCV infection were 31.8%and 22.8% respectively.The positive rates ofHGV RNA at the 5’-NCR and NS5 region in thesame samples were 47.9% and 31.8%respectively.The total positive rate of GBV-C/HGV RNA was as high as 55.5%.HCV copynumbers in the patients without GBV-C/ HGVcoinfection were statistically higher than that inthe patients with GBV-C/ HGV coinfection(P【0.01).Frequent mutation of nucleotideresidue was present in the amplificationproducts.Frameshift mutation was found in twosamples with GBV-C NS3 region nucleotidesequences.All nucleotide sequences fromamplification products showed higher homologyto HGV genome than to GBV-C genome even though part of the sequences were amplifiedwith GBV-C primers.CONCLUSION A high frequency of GBV-C/ HGV coinfection existed in the hepatitis C patients. RT-PCR ELISA was more sensitive than RT-nested PCR for detection of GBV-C/ HGV RNA. The primers derived from the 5 -NCR was more efficient than those derived from the NS3 and NS5 regions. A reverse relationship was found to exist between HCV RNA concentration and GBV-C/ HGV infection frequency. HGV was the dominant subtype of the virus in the local area. The major mutations of GBV-C/ HGV genomes were random mutation of nucleotide residue.
基金Supported by Instituto de Salud Carlos Ⅲ,No.PI14/01416 and No.PI15/00856cofinanced by the European Regional Development Fund(ERDF)the Gilead Fellowship Program,No.GLD14-00296
文摘AIM To determine the variability/conservation of the domain of hepatitis B virus(HBV) pre S1 region that interacts with sodium-taurocholate cotransporting polypeptide(hereafter, NTCP-interacting domain) and the prevalence of the rs2296651 polymorphism(S267 F, NTCP variant) in a Spanish population. METHODS Serum samples from 246 individuals were included and divided into 3 groups: patients with chronic HBV infection(CHB)(n = 41, 73% Caucasians), patients with resolved HBV infection(n = 100, 100% Caucasians) and an HBV-uninfected control group(n = 105, 100% Caucasians). Variability/conservation of the amino acid(aa) sequences of the NTCPinteracting domain,(aa 2-48 in viral genotype D) and a highly conserved pre S1 domain associated with virion morphogenesis(aa 92-103 in viral genotype D) were analyzed by next-generation sequencing and compared in 18 CHB patients with viremia > 4 log IU/mL. The rs2296651 polymorphism was determined in all individuals in all 3 groups using an in-house real-time PCR melting curve analysis.RESULTS The HBV pre S1 NTCP-interacting domain showed a high degree of conservation among the examined viral genomes especially between aa 9 and 21(in the genotype D consensus sequence). As compared with the virion morphogenesis domain, the NTCPinteracting domain had a smaller proportion of HBV genotype-unrelated changes comprising > 1% of the quasispecies(25.5% vs 31.8%), but a larger proportion of genotype-associated viral polymorphisms(34% vs 27.3%), according to consensus sequences from Gen Bank patterns of HBV genotypes A to H. Variation/conservation in both domains depended on viral genotype, with genotype C being the most highly conserved and genotype E the most variable(limited finding, only 2 genotype E included). Of note, proline residues were highly conserved in both domains, and serine residues showed changes only to threonine or tyrosine in the virion morphogenesis domain. The rs2296651 polymorphism was not detected in any participant.CONCLUSION In our CHB population, the NTCP-interacting domain was highly conserved, particularly the proline residues and essential amino acids related with the NTCP interaction, and the prevalence of rs2296651 was low/null.
