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@#Abstract: Objective To clarify the long-term evolution of hepatitis B virus (HBV) quasi-species in HBsAg asymptomatic carriers in Long'an county, Guangxi. Methods ELISA was used to detect serological markers of HBV. Viral loads were measured by real time PCR. HBV DNA was extracted from serum by kits. The whole HBV genome was amplified using nested PCR and amplicons were sequenced by next-generation sequencing (NGS). These sequences from NGS were analyzed by the software like Mega. Results Serum samples were collected from 9 HBsAg asymptomatic carriers in Longan County，Guangxi at 4 different time points in 2004, 2007, 2013, 2019 or 2020. A total of 23 serum samples and 309 full-length gene quasi-species sequences were obtained, with an average amount of (0.18±0.07) G sequencing data for each sample. Genotype of 55.54%(5/9) the studied subjects underwent genotype conversion during the long-term evolution process of HBV quasi-species, and the genotyping results of the phylogenetic tree in the PreS/S region are in perfect agreement with the results of the whole genome analysis; recombinant B/C, I/C were found; the Sn ranged from 0 to 0.37 and the genetic diversity ranged from 0 to 0.11, respectively. A total of 21 special single nucleotide/amino acid mutations (7 in the S region, 2 in the X region, 3 in the PreC region and 9 in the BCP region) and 6 deletion mutations were detected, multiple mutations were found and no drug resistant mutations were found; 77.8%(7/9) of the HBV strains carried by the subjects in 2004 had double mutations at nt1 762(A→T) and 1 764(G→A) and a stop mutation at nt1 896(G→A); HBV mutations can be restored from the mutant type to the wild type and (or) vice versa without antiviral drug pressure, and The evolution rate of HBV genome was 2.03×10-5~3.50×10-3.Conclusion HBV genotype, recombinants, genetic complexity and diversity of HBV quasi-species can change over time during in natural infection. The transformation between HBV mutation type and wild type reduces the value of predicting clinical outcomes by genetic types and related mutations to some extent. The HBV genome evolution rate of asymptomatic carriers of HBsAg in Long'an County is very high.
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@#The Japanese encephalitis virus (JEV), a leading cause of encephalitis, exists as quasispecies in clinical isolates. Using a limiting dilution method combined with immunohistochemistry to detect viral antigens, 10 biological clones were isolated and purified from a clinical JEV isolate (CNS138/9) derived from an autopsy brain. These biological clones were tested for neurovirulence in SK-N-MC and NIE-115 neuronal cells, and a 2-week-old, footpad-infected, JE mouse model. Nine clones were found to be neurovirulent; one clone neuroattenuated. Although further studies are needed to determine genotypic differences, if any, in these clones, the limiting dilution purification and neurovirulence testing methods described herein should be useful for phenotypic studies of quasispecies of neurotropic viruses in general, and JEV and other flaviviruses in particular.
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Objective@#To analyze the genetic characteristics of whole-genome and quasispecies sequences from three hepatitis A virus (HAV) strains in China.@*Methods@#Serum samples from acute hepatitis A patients were collected and viral RNA extraction, transcription, nested PCR, sequencing and assembling were performed to gain near full-length sequences; cloning-based sequencing of the full-length VP1-2 A region was also performed.@*Results@#Genotyping showed that the nucleotide and amino acid identities among three strains on VP1-2 A junction region were both 100% and all belonged to subgenotype IA; the nucleotide and amino acid identities on whole-genome region were 99.9%-100% and 100% respectively, and shared the highest identities with AH2 strain from GenBank of 98.5% in nucleotide and 99.7% in amino acid level; no amino acid variation was found among published neutralizing antigenic sites. Within cloning sequences from each strain, the nucleotide and amino acid identities were 99.0%-100% and 98.1%-100%, while among all cloning sequences were 99.0%-100% and 97.2%-100%. The variation rate of nucleotide and amino acid in VP1-2 A junction region were both higher than that of partial VP1 region.@*Conclusions@#Sequences among three strains in VP1-2 A region were identical, the nucleotide and amino acid identities in both whole-genome region and among quasispecies sequences were relatively high to deduce that they were from the same outbreak. This study provides new insight for identification of HAV transmissions and tracing investigations.
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Hepatitis C virus ( HCV) infection is one of the main reasons causing liver cirrhosis and hepatocellular carcinoma. The quasispecies composition and the evolution of HCV are very complicated. An in-depth analysis of the quasispecies composition of HCV is critical for elucidating the mechanisms of HCV transmission. The regions encoding the envelope glycoproteins (E1 and E2) and the nonstructural protein 2 (NS2) are hyper variable regions (HVR). Analyzing the quasispecies of HCV HVR with advanced sequen-cing and bio-information technologies would be beneficial for understanding the sources of HCV infection, the routes of transmission and the evolution of HCV. Currently, three generations of sequencing methods and some bio-information soft-wares are available for analyzing HCV HVR quasispecies. When an outbreak of HCV infection happens, using the new generation of sequencing and bio-information methods to seek the first case and the routes of transmission would provide scientific basis for the prevention and treatment of HCV in-fection.
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BACKGROUND/AIMS: To investigate pre-existing hepatitis B virus (HBV) quasispecies and the genotypic evolution of several variants. METHODS: From six patients with lamivudine (LAM) failure, serum samples at pretreatment, 6 months of LAM therapy, and virologic breakthrough were obtained. One hundred clones with HBV inserts in each patient were sequenced at each time point. Pretreatment serum samples were also analyzed from six patients who achieved good responses to LAM therapy. RESULTS: Among the six patients with LAM failure, the analysis of 100 clones from patient 1 revealed the substitutions L180M in 1% of clones and V173L in 2% of clones. Patient 2 had substitutions of L80V, W153Q, and L180M. In patient 3, mutations conferring resistance to adefovir at V84I (5%), I169L (1%), and N236H (7%) and entecavir at S202G (2%) were detected. Patient 4 had mutations at T128N (1%), I169L (1%), V173L (2%), A181V (1%), and Q215H (1%). In patient 5, M204V/I was detected in 1% and 2% of clones, respectively. L80I and V173L were also identified in patient 6. In the six patients who responded to LAM, the degree of overall quasispecies was less than those with LAM failure. CONCLUSIONS: Various HBV quasispecies associated with drug resistance existed before treatment, and the quasispecies dynamically changed through LAM therapy.
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Humans , Adenine , Clone Cells , Drug Resistance , Guanine , Hepatitis , Hepatitis B , Hepatitis B virus , Hepatitis B, Chronic , Hepatitis, Chronic , Lamivudine , Lipopolysaccharides , OrganophosphonatesABSTRACT
Objective To characterize serum hepatitis B virus(HBV)full-length genome quasispecies and to investigate its ralationship with severe exacerbation of chronic hepatitis B(CHB).Methods HBV full-length genome was amplified and cloned from four treatment naive CHB patients and four treatment naive CSHB patients.Fourteen to sixteen clones per sample were selected,sequenced and analyzed by bioinformatics software.The measurement data was compared by independent-samples t test and count data was analyzed by x2 test. Results Totally 120 HBV fulllength genome sequences were obtained.All the patients had either genotype B or C virus monoinfection.One hundred percent clones(60/60)from CSHB patients showed mutations including G1896A,A1762T/G1764A(one patient even carried A1762T/G1764A/C1766T mutations),T1753C/G and start codon mutations in preS2,preS1,which were more common than those from CHB patients(46/60,76.7%;x2=15.85,P<0.01).The quasispecies complexity and diversity were higher in CSHB patients than CHB patients within full-length genome,S,X,P genes and reverse transcriptase region,but lower within C gene at both nucleotide and amino acid levels.But the difference were not statistically significant in all regions.Conclusion The mutation frequency and quasispeeies heterogeneity in HBV genome are higher in CSHB patients than in CHB patients,which may play a role in the severe exacerbation of CHB and needs further investigation in large scale studies.
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Objective To investigate the association of hepatitis B virus(HBV) S gene quasispecies with the outcome of HBV infection.Methods Serum samples were collected from three chronic HBV carriers, three chronic hepatitis B and three chronic severe hepatitis B patients.All subjects were male and with HBV genotype C.HBV S gene was amplified, and 20 clones of HBV fragment were randomly selected and sequenced from each sample.SPSS 15.0 software was adopted for analysis.Results Quasispecies complexity of HBV S gene in chronic HBV carriers and chronic hepatitis B tended lower than that of the severe chronic hepatitis B, but the difference was not of statistical significance (P＞0.05).In T cell epitope 45, 47, 85 amino acid sites of the HBV S gene, the constitution of quasispecies in the chronic hepatitis B was more complex than that of the HBV carriers (P=0.01), but compared with the severe chronic hepatitis, the difference was not significant (P=0.06).The computer model showed that both the dominant clones and the non dominant clones could effectively bind to the receptors of cytotoxic T lymphocytes.Conclusion Quasispecies in some T cell epitopes of HBV S gene may be related with the clinical outcome of hepatitis B.
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The quasispecies nature of hepatitis B and C virus (HBV, HCV) plays an important role in the pathogenesis, immune escape and drug resistance during chronic infection. Although there is still a lack of effective treatment for hepatitis C, a series of nucleoside analogs (NA) have been developed for the treatment of hepatitis B. NA resistant HBV mutants can accumulate during prolonged therapy and lead to the failure of anti-HBV therapy. Switching to other sensitive NAs can inhibit the emerged resistant mutants. Therefore, understanding the evolution of viral quasispecies under drug pressure is crucial for the establishment of antiviral strategy and the monitoring of antiviral process. Immune response and escape are complicated process, during which both host and virus factors may play their roles. Further understanding of the interaction and interrelationship between host and these viruses may lead to optimized prevention, diagnosis and treatment for chronic hepatitis.
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OBJECTIVE To investigate the quasispecies diversity and the dynamics of quasispecies evolution in patients with chronic Hepatitis C virus(HCV) infection.METHODS Changes of HCV quasispecies before and after interferon(IFN) therapy were detected by heteroduplex mobility analysis(HMA) and by automatic sequencer.RESULTS We found that no changes in viral complexity were observed with therapy,but there were marked changes in viral divergence.The proportion of major pretreatment variants was different in HVR1 in all patients.During the treatment,HCV quasispecies changed significantly.CONCLUSIONS HMA is a simple,rapid and reliable method for detecting HCV quasispecies.Responsiveness to interferon may be related to the changes in viral divergence,but not related to the numbers of viral variants.
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The human immunodeficiency viruses (HIV) exhibit tremendous genetic variability in their hosts. It is mainly due to two factors: the error-prone nature of the viral reverse transcriptase enzyme and the effects of environmental constraints such as antiviral therapy, cellular tropism, or HIV-specific immune responses. These quasispecies show fluctuation both in the overall divergence and diversity between individual sequences with different duration after primary infection. For better understand the viral quasispecies, we have performed the longitudinal genetic analysis of HIV-1 env gene V1-C5 region (1.2 kb) by two molecular cloning methods. Diversity indicated that 'single clone per PCR' value was higher than that of 'multiple clones per PCR' in subjects: 0.58-3.15 in subject 1 (P<0.05) and 0.28-2.25 in subject 2 (P<0.05). But divergence was similar in both molecular cloning methods. Phylogenetic analysis of longitudinal sequences at different sampling stages revealed the existence of different topologies individually. These data suggested that 'single clone per PCR' is more efficient than 'multiple clones per PCR' in genetic diversity analysis.
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Clone Cells , Cloning, Molecular , Genes, env , Genetic Variation , HIV , HIV-1 , Polymerase Chain Reaction , RNA-Directed DNA Polymerase , TropismABSTRACT
Objective To disclose the variant rule of HIV-1 vertical transmission by studying the differences of genotypes and quasispecies in the individuals with HIV-1 vertical transmission. Methods RNA was extracted from the plasma of the individuals of vertical transmission and C2-V5 DNA segment of HIV-1 env gene was acquired by RT-PCR. Purified DNA segment was inserted into T vector and transformed into Top10 Escherichia coli. Positive clones were acquired by blue-white screening and used as models in PCR. PCR products were analyzed by conformation sensitive gel electrophoresis (CSGE). The clones of major and minor quasispecies were sequenced. Results HIV-1 quasispecies in the mother of the first group were less complex than her child, and their quasispecies nucleotide sequences were highly homologous and of HIV-1 of C subtype. HIV-1 quasispecies in the mother of the second group were more complex than her child, and their quasispecies nucleotide sequences were highly homologous and of HIV-1 of AE subtype. Conclusion HIV-1 genetic subtypes are generally not changed in vertical transmission, but HIV-1 quasispecies could be selected in vertical transmission and screened by the new environment of the new host which may both induce the changes of dominative and minor quasispecies. We also find that complexity of HIV-1 quasispecies is associated with immune status of host.
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Objective To investigate the evolution of hepatitis C virus (HCV) quasispecies in persistent and self limited infection patients. Methods Peripheral blood HCV preserved for 10 years from 8 naive individuals once infected by HCV were analyzed. HCV hypervariable region 1 (HVR1) genes were cloned and sequenced. Entropy, genetic distances, nonsynonymous mutations per nonsynonymous site (K A ), synonymous mutations per synonymous site(K S ) were evaluated for evolutionary analysis. Results The entropy values and genetic distances of HCV quasispecies were lower in self limited infection indivi duals than that of persistent infection individuals. In 3 of the 4 persistent infection individuals there exist no difference between within group and between group genetic distances in HCV HVR1. The ratios of K A /K s were higher than 1 in 7 of these 8 individuals. Conclusions The complexity and diversity degree of HCV quasispecies may affect the outcome of HCV infection. Positive Darwinian selection may be the major cause for the change of HCV quasispecies distribution in hepatitis C infection natural history.
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Objective To study the complexity of S region qusispecies in various disease stages of chronic hepatitis B virus(HBV) infection and its relation to disease activity. Methods Serum samples were obtained from 112 patients with chronic hepatitis B virus infection;22 with chronic carries(ASC),30 with chronic mild or moderate hepatitis(CH),60 with fulminant hepatitis failure(FHF). HBV qusispecies populations were separated by the single strand conformation polymorphism (SSCP) method targeted the S region and DNA sequencing analysis. Results The number of SSCP bands detected in the patients with ASC、CH and FHF was 1.45?0.13,3.70?0.22 and 5.93?0.24, respectively. There was a statistically significant difference in the number of quasispecies among various disease stages ( P
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Objective To determine the influence of viral factors and host cellular immunity on the response to interferon in the patients with chronic hepatitis C. Methods Forty patients with chronic hepatitis C were treated with interferon ?. The relationships between response to interferon a and HCV genotype, quasispecies heterogeneity, HCV RNA level and HCV specific cytotoxic T lymphocyte (HCV CTL) activity in the liver were analyzed. Results After 6 months of therapy, 21 patients had obtained end of treatment response (ETR), 10 Patients of which had obtained sustained response (SR). The other 19 patients got no response (NR). ETR rate in patients with genotype HCV1 infection (43.3%, 13/30) was significantly lower than that in patients with non HIV1 infection (80%, 8/10) [ P
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A set of specific primers was synthesized according to DNA sequence of HBV found in China, the reverse transcriptase (RT) region in polymerase gene was amplified by PCR method from the serum of 3 patients with chronic HBV infection, and then the PCR products were subcloned into pGEM Teasy vectors. 13 clones were sequenced. Sequence comparison of the selected clones was made to look for the difference. After being compared, 13 sequences of RT were found different. Besides 2 clones with long sequence deletion, the different rate of RT coding nucleic acid sequences, RT and HBsAg amino acid sequences of the 11 clones is 5 1%, 4 9% and 7 5%, respectively. Many mutation types, including point substitution and deletion mutation, were found in this region. There is quasispecies population and defective HBV genome in patients with chronic HBV infection.
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Infectious diseases are characterized as their pathogens.So the detection of pathogen is very important for the diagnosis of infectious diseases.Molecular diagnosis based on polymerase chain reaction (PCR) is playing an important role in the diagnosis,therapy,and prevention for infectious diseases.The concept of quasispecies must be put into consideration for quasispecies are universal for almost every pathogen and important for the diagnosis.Resistance is emerging along with anti-microbial therapy and has comprehensive impaet for treatment of infectious diseases.Diagnosis of resistance will be helpful for seleetion of right drug,and modification of therapy protocol,and reduction of resistance.Genotyping technique is meaningful for individualized therapy for patients with infectious diseases.
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Objective To investigate the the quasispecies character of 3′ untranlated region(3′ UTR) in hepatitis C virus(HCV) by analysing the nucleotide sequence polymorphism and mutation features in 3′ UTR region.Methods Patients infected with genotype 1b HCV were identified by reverse transcription-nested polymerase chain reaction(RT-PCR) and restriction fragment length polymorphism(RFLP) assay.Fragments of the cDNA of 3′ UTR were amplified using semi-nested RT-PCR,and subjected to cloning.The 12-15 clones that contained HCV 3′UTR gene fragments amplified from each patients were sequenced.Results The full-length sequence of 1b genotype HCV 3′UTR in cDNA were obtained.The 3′UTR region consists of four elements: the 5′ region,the poly(U),poly(U/C) and 98-base region.The nucleotide sequence diversity ranged from 0.2%～2.1% and the mutation points were almost distributed in the the 5′region and poly(U/C).Conclusions The HCV has complex quasispecies character in 3′ UTR.
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Interferon-alpha (IFN-alpha) has been used to treat hepatitis C virus (HCV)-induced hepatitis, but it has been effective in only about half of the treated patients, with recurrence appearing in the other half. As a consequence of the possible complications associated with IFN-alpha and the high cost of treatment, it has become extremely important to select the proper patients for IFN-alpha treatment. In our previous study, we found that the quasispecies in the hypervariable region (HVR) 1 of HCV were various and that a new quasispecies can appear in non-responders and/or lead to deterioration in the patients' condition. The preliminary data we obtained in the process of our previous research led us to believe that the quasispecies of HVR 1 has something to do with the effect of IFN-alpha. Thus, in this investigation, we tried to determine the predictive factors of IFN-alpha therapy. Thirty patients with HCV infection were treated with IFN-alpha. Among them, 15 patients recovered after six months IFN-alpha treatment, but the remaining 15 patients showed no response after six months IFN-alpha treatment. We cloned HVR 1 DNA by reverse transcription-polymerase chain reaction (RT-PCR) and examined the quasispecies of HVR 1. As the quasispecies of HVR 1 in non-responders varied more than in the complete remission group, we concluded that the sequence variation in HVR 1 of HCV can be used to predict the effect of IFN-alpha.
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Adult , Aged , Female , Humans , Male , Amino Acid Sequence , Genotype , Hepatitis C/virology , Hepatitis C/drug therapy , Hepacivirus/classification , Interferon-alpha/therapeutic use , Middle Aged , Molecular Sequence Data , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/bloodABSTRACT
Primers were synthesized according to DNA sequence of HBV found in China, and the reverse transcriptase (RT) region in polymerase gene, HBV preC/C region and the whole genome were amplified by PCR method from the serum of 8 patients with chronic HBV infection. Then the PCR products were ligated into pGEM Teasy vectors. 27 clones were sequenced. Deduced amino acid sequences were compared, and the results showed that mutations were widely distributed in structural and non structural viral proteins. The substitution mutations occur in clones isolated from specific patients, which led to a characteristic mutation in the quasispecies group. The results indicated that the diversity of HBV gene might play an important role in the pathogenesis of chronic HBV infection.
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Mutations of hepatitis B virus (HBV) viral genomes are normal biological events and result in the heterogeneity, such as coexistence of viral genotypes and serotypes in infected individuals('quasispecies'). The HBV DNA heterogeneity is ubiquitous all chronic HBV infected patients, and it involves each DNA fragment of both structural and regulatory genes. The process of heterogeneity is continuing,with important biological and clinical implications, playing an important role inthe pathogenesis, drug resistance, and even hepato carcinogenesis in hepatitis patients. The introduction of the conception of quasispecies may contribute to a revolutionary re evaluation of the mutations of HBV DNA.