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1.
Virol J ; 19(1): 114, 2022 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-35765099

RESUMO

BACKGROUND: Chronic infection with hepatitis B virus (HBV) has been proved highly associated with the development of hepatocellular carcinoma (HCC). AIMS: The purpose of the study is to investigate the association between HBV preS region quasispecies and HCC development, as well as to develop HCC diagnosis model using HBV preS region quasispecies. METHODS: A total of 104 chronic hepatitis B (CHB) patients and 117 HBV-related HCC patients were enrolled. HBV preS region was sequenced using next generation sequencing (NGS) and the nucleotide entropy was calculated for quasispecies evaluation. Sparse logistic regression (SLR) was used to predict HCC development and prediction performances were evaluated using receiver operating characteristic curves. RESULTS: Entropy of HBV preS1, preS2 regions and several nucleotide points showed significant divergence between CHB and HCC patients. Using SLR, the classification of HCC/CHB groups achieved a mean area under the receiver operating characteristic curve (AUC) of 0.883 in the training data and 0.795 in the test data. The prediction model was also validated by a completely independent dataset from Hong Kong. The 10 selected nucleotide positions showed significantly different entropy between CHB and HCC patients. The HBV quasispecies also classified three clinical parameters, including HBeAg, HBVDNA, and Alkaline phosphatase (ALP) with the AUC value greater than 0.6 in the test data. CONCLUSIONS: Using NGS and SLR, the association between HBV preS region nucleotide entropy and HCC development was validated in our study and this could promote the understanding of HCC progression mechanism.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Humanos , Modelos Logísticos , Nucleotídeos , Quase-Espécies
2.
Virus Res ; 315: 198786, 2022 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-35429618

RESUMO

Studies are needed to better understand the genomic evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study aimed to describe viral quasispecies population of upper and lower respiratory tract by next-generation sequencing in patients admitted to intensive care unit. A deep sequencing of the S gene of SARS-CoV-2 from 109 clinical specimens, sampled from the upper respiratory tract (URT) and lower respiratory tract (LRT) of 77 patients was performed. A higher incidence of non-synonymous mutations and indels was observed in the LRT among minority variants. This might be explained by the ability of the virus to invade cells without interacting with ACE2 (e.g. exploiting macrophage phagocytosis). Minority variants are highly concentrated around the gene portion encoding for the Spike cleavage site, with a higher incidence in the URT; four mutations are highly recurring among samples and were found associated with the URT. Interestingly, 55.8% of minority variants detected in this locus were T>G and G>T transversions. Results from this study evidenced the presence of selective pressure and suggest that an evolutionary process is still ongoing in one of the crucial sites of spike protein associated with the spillover to humans.


Assuntos
COVID-19 , SARS-CoV-2 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Quase-Espécies , Sistema Respiratório , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
3.
Acta Virol ; 66(1): 18-26, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35380862

RESUMO

Human immunodeficiency virus (HIV)/hepatitis B virus (HBV) co-infection accelerates the progression of HBV-related liver diseases. HBV basic core promoter (BCP)/pre-core (preC) gene mutations may be one of the most important risk factors. In this study, a total of 230 patients were recruited, and 199 patients whose HBV BCP/preC gene were successfully amplified and sequenced, including 99 HIV/HBV co-infected and 100 HBV mono-infected patients. Next-generation sequencing was used for detection of BCP/preC mutations which were then compared in patients with different HBV genotypes and different HBeAg statuses, and 1% and 20% cutoff values were defined to evaluate the mutations. HBV quasispecies diversity was also compared in HIV/HBV co-infected and HBV mono-infected patients. Among the patients infected with HBV genotype C and HBeAg-negative status, the frequency of A1762T/G1764A double mutations was significantly lower in HIV/HBV co-infected patients than in HBV mono-infected patients (53.3% vs. 100.0%, P = 0.008) regardless of the 1% or 20% cutoff value level. However, A1762T/G1764A double mutations did not differ in the other groups (P >0.05). Viral quasispecies diversity was lower in HIV/HBV co-infected patients than in HBV mono-infected patients (P Keywords: human immunodeficiency virus, hepatitis B virus; mutations; viral quasispecies; next-generation sequencing.


Assuntos
Coinfecção , Infecções por HIV , Hepatite B Crônica , Coinfecção/genética , DNA Viral/genética , Genótipo , Infecções por HIV/complicações , Vírus da Hepatite B/genética , Hepatite B Crônica/complicações , Hepatite B Crônica/genética , Humanos , Mutação , Regiões Promotoras Genéticas , Quase-Espécies
4.
J Math Biol ; 84(4): 29, 2022 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-35254508

RESUMO

We propose a modified mathematical model of the quasispecies type to analyze an unstable tumor progression evolution. In our study, we consider a heterogeneous population with different individuals, generated by the accumulation of successive mutations. Our model's main feature is that it allows for variable growth rates for each subpopulation and takes into account mutations from nonconsecutive types of mutants. Bifurcations and linear stability of the steady states are analyzed. We focus on two equilibria; one of them implies the coexistence of anomalous growth and genetically unstable cells. The other one yields the dominance of the anomalous growth population and the extinction of the malignant cells. However, linear stability analysis of the second equilibrium is inconclusive and suggests a suitable environment for the study of periodic therapy. This is carried out by introducing a small perturbation modeling the effect of a periodic medical treatment. As a result, a Zero-Hopf periodic orbit is identified, showing a cyclic behavior among the populations, with a strong dominance of the parental anomalous growth cell population.


Assuntos
Modelos Biológicos , Quase-Espécies , Humanos , Modelos Teóricos
5.
Viruses ; 14(2)2022 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-35215809

RESUMO

The hepatitis delta virus (HDV) genome has an autocatalytic region called the ribozyme, which is essential for viral replication. The aim of this study was to use next-generation sequencing (NGS) to analyze the ribozyme quasispecies (QS) in order to study its evolution and identify highly conserved regions potentially suitable for a gene-silencing strategy. HDV RNA was extracted from 2 longitudinal samples of chronic HDV patients and the ribozyme (nucleotide, nt 688-771) was analyzed using NGS. QS conservation, variability and genetic distance were analyzed. Mutations were identified by aligning sequences with their specific genotype consensus. The main relevant mutations were tested in vitro. The ribozyme was conserved overall, with a hyper-conserved region between nt 715-745. No difference in QS was observed over time. The most variable region was between nt 739-769. Thirteen mutations were observed, with three showing a higher frequency: T23C, T69C and C64 deletion. This last strongly reduced HDV replication by more than 1 log in vitro. HDV Ribozyme QS was generally highly conserved and was maintained during follow-up. The most conserved portion may be a valuable target for a gene-silencing strategy. The presence of the C64 deletion may strongly impair viral replication, as it is a potential mechanism of viral persistence.


Assuntos
Vírus Delta da Hepatite/genética , RNA Catalítico/genética , Sequência Conservada , Genótipo , Hepatite D Crônica/virologia , Vírus Delta da Hepatite/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Quase-Espécies , RNA Viral/genética , Análise de Sequência de RNA , Replicação Viral/genética
6.
Virol J ; 19(1): 4, 2022 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-34991646

RESUMO

INTRODUCTION: Transplantation among HIV positive patients may be a valuable therapeutic intervention. This study involves an HIV D+/R+ kidney-liver transplantation, where PBMC-associated HIV quasispecies were analyzed in donor and transplant recipients (TR) prior to transplantation and thereafter, together with standard viral monitoring. METHODS: The donor was a 54 year of age HIV infected woman: kidney and liver recipients were two HIV infected men, aged 49 and 61. HIV quasispecies in PBMC was analyzed by ultra-deep sequencing of V3 env region. During TR follow-up, plasma HIV-1 RNA, HIV-1 DNA in PBMC, analysis of proviral integration sites and drug-resistance genotyping were performed. Other virological and immunological monitoring included CMV and EBV DNA quantification in blood and CD4 T cell counts. RESULTS: Donor and TR were all ART-HIV suppressed at transplantation. Thereafter, TR maintained a nearly suppressed HIV-1 viremia, but HIV-1 RNA blips and the increase of proviral integration sites in PBMC attested some residual HIV replication. A transient peak in HIV-1 DNA occurred in the liver recipient. No major changes of drug-resistance genotype were detected after transplantation. CMV and EBV transient reactivations were observed only in the kidney recipient, but did not require specific treatment. CD4 counts remained stable. No intermixed quasispecies between donor and TR was observed at transplantation or thereafter. Despite signs of viral evolution in TR, HIV genetic heterogeneity did not increase over the course of the months of follow up. CONCLUSIONS: No evidence of HIV superinfection was observed in the donor nor in the recipients. The immunosuppressive treatment administrated to TR did not result in clinical relevant viral reactivations.


Assuntos
Infecções por HIV , Transplante de Fígado , Humanos , Rim , Leucócitos Mononucleares , Fígado , Quase-Espécies
7.
Clin Microbiol Infect ; 28(1): 139.e5-139.e8, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34537363

RESUMO

OBJECTIVES: To evaluate the impact of neutralizing monoclonal antibody (mAb) treatment and to determine whether the selective pressure of mAbs could facilitate the proliferation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with spike protein mutations that might attenuate mAb effectiveness. PATIENTS AND METHODS: We evaluated the impact of mAbs on the nasopharyngeal (NP) viral load and virus quasispecies of mAb-treated patients using single-molecule real-time sequencing. The mAbs used were: Bamlanivimab alone (four patients), Bamlanivimab/Etesevimab (23 patients) and Casirivimab/Imdevimab (five patients). RESULTS: The NP SARS-CoV-2 viral load of mAb-treated patients decreased from 8.2 log10 copies/mL before administration to 4.3 log10 copies/mL 7 days after administration. Five immunocompromised patients given Bamlanivimab/Etesevimab were found to have mAb activity-reducing spike mutations. Two patients harboured SARS-CoV-2 variants with a Q493R spike mutation 7 days after administration, as did a third patient 14 days after administration. The fourth patient harboured a variant with a Q493K spike mutation 7 days post-treatment, and the fifth patient had a variant with a E484K spike mutation on day 21. The emergence of the spike mutation was accompanied by stabilization or rebound of the NP viral load in three of five patients. CONCLUSION: Two-mAb therapy can drive the selection of resistant SARS-CoV-2 variants in immunocompromised patients. Patients given mAbs should be closely monitored and measures to limit virus spread should be reinforced.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos Imunológicos , COVID-19 , Evolução Molecular , SARS-CoV-2/genética , Carga Viral , Anticorpos Monoclonais Humanizados , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais , Antineoplásicos Imunológicos/uso terapêutico , COVID-19/terapia , Humanos , Mutação , Quase-Espécies , Seleção Genética
8.
World J Gastroenterol ; 27(41): 7144-7158, 2021 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-34887634

RESUMO

BACKGROUND: Different forms of pregenomic and other hepatitis B virus (HBV) RNA have been detected in patients' sera. These circulating HBV-RNAs may be useful for monitoring covalently closed circular DNA activity, and predicting hepatitis B e-antigen seroconversion or viral rebound after nucleos(t)ide analog cessation. Data on serum HBV-RNA quasispecies, however, is scarce. It is therefore important to develop methodologies to thoroughly analyze this quasispecies, ensuring the elimination of any residual HBV-DNA. Studying circulating HBV-RNA quasispecies may facilitate achieving functional cure of HBV infection. AIM: To establish a next-generation sequencing (NGS) methodology for analyzing serum HBV-RNA and comparing it with DNA quasispecies. METHODS: Thirteen untreated chronic hepatitis B patients, showing different HBV-genotypes and degrees of severity of liver disease were enrolled in the study and a serum sample with HBV-DNA > 5 Log10 IU/mL and HBV-RNA > 4 Log10 copies/mL was taken from each patient. HBV-RNA was treated with DNAse I to remove any residual DNA, and the region between nucleotides (nt) 1255-1611 was amplified using a 3-nested polymerase chain reaction protocol, and analyzed with NGS. Variability/conservation and complexity was compared between HBV-DNA and RNA quasispecies. RESULTS: No HBV-DNA contamination was detected in cDNA samples from HBV-RNA quasispecies. HBV quasispecies complexity showed heterogeneous behavior among patients. The Rare Haplotype Load at 1% was greater in DNA than in RNA quasispecies, with no statistically significant differences (P = 0.1641). Regarding conservation, information content was equal in RNA and DNA quasispecies in most nt positions [218/357 (61.06%)]. In 102 of the remaining 139 (73.38%), HBV-RNA showed slightly higher variability. Sliding window analysis identified 4 hyper-conserved sequence fragments in each quasispecies, 3 of them coincided between the 2 quasispecies: nts 1258-1286, 1545-1573 and 1575-1604. The 2 hyper-variable sequence fragments also coincided: nts 1311-1344 and 1461-1485. Sequences between nts 1519-1543 and 1559-1587 were only hyper-conserved in HBV-DNA and RNA, respectively. CONCLUSION: Our methodology allowed analyzing HBV-RNA quasispecies complexity and conservation without interference from HBV-DNA. Thanks to this, we have been able to compare both quasispecies in the present study.


Assuntos
Ácidos Nucleicos Livres , Hepatite B Crônica , Hepatite B , Antivirais/uso terapêutico , Estudos Transversais , DNA Viral/genética , DNA Viral/uso terapêutico , Hepatite B/tratamento farmacológico , Vírus da Hepatite B/genética , Hepatite B Crônica/diagnóstico , Hepatite B Crônica/tratamento farmacológico , Humanos , Quase-Espécies , RNA
9.
Front Immunol ; 12: 775461, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34899733

RESUMO

Background: This study aimed to explore the molecular mechanism of the coexistence of hepatitis B surface antigen (HBsAg) and hepatitis B surface antibody (HBsAb) serological pattern via intensive characterization of HBV s gene in both chronic hepatitis B (CHB) and hepatocellular carcinoma (HCC) patients. Method: A total of 73 HBsAg+/HBsAb+ patients (CHB = 36, HCC = 37) and 96 HBsAg+/HBsAb- patients (CHB = 47, HCC = 49) were enrolled from 13 medical centers in China. The sequence features were elaborated based on the combination of next-generation sequencing (NGS) and multidimensional bioinformatics analysis. Results: The 16 high-frequency missense mutations, changes of stop codon mutation, clustering, and random forest models based on quasispecies features demonstrated the significant discrepancy power between HBsAg+/HBsAb+ and HBsAg+/HBsAb- in CHB and HCC, respectively. The immunogenicity for cytotoxic T lymphocyte (CTL) epitope Se and antigenicity for the major hydrophilic region (MHR) were both reduced in HBsAg+/HBsAb+ patients (CTL Se: p < 0.0001; MHR: p = 0.0216). Different mutation patterns were observed between HBsAg+/HBsAb+ patients with CHB and with HCC. Especially, mutations in antigenic epitopes, such as I126S in CHB and I126T in HCC, could impact the conformational structure and alter the antigenicity/immunogenicity of HBsAg. Conclusion: Based on NGS and bioinformatics analysis, this study indicates for the first time that point mutations and quasispecies diversities of HBV s gene could alter the MHR antigenicity and CTL Se immunogenicity and could contribute to the concurrent HBsAg+/HBsAb+ with different features in HCC and CHB. Our findings might renew the understanding of this special serological profile and benefit the clinical management in HBV-related diseases.


Assuntos
Carcinoma Hepatocelular/virologia , Anticorpos Anti-Hepatite B/sangue , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Neoplasias Hepáticas/virologia , Mutação Puntual , Quase-Espécies/genética , Adulto , Idoso , Biomarcadores/sangue , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/imunologia , China , Biologia Computacional , Feminino , Genótipo , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Hepatite B Crônica/sangue , Hepatite B Crônica/imunologia , Humanos , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/imunologia , Masculino , Pessoa de Meia-Idade
10.
Microbiol Spectr ; 9(3): e0145921, 2021 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-34756074

RESUMO

RNA viruses replicate as complex mutant spectra termed viral quasispecies. The frequency of each individual genome in a mutant spectrum depends on its rate of generation and its relative fitness in the replicating population ensemble. The advent of deep sequencing methodologies allows for the first-time quantification of haplotype abundances within mutant spectra. There is no information on the haplotype profile of the resident genomes and how the landscape evolves when a virus replicates in a controlled cell culture environment. Here, we report the construction of intramutant spectrum haplotype landscapes of three amplicons of the NS5A-NS5B coding region of hepatitis C virus (HCV). Two-dimensional (2D) neural networks were constructed for 44 related HCV populations derived from a common clonal ancestor that was passaged up to 210 times in human hepatoma Huh-7.5 cells in the absence of external selective pressures. The haplotype profiles consisted of an extended dense basal platform, from which a lower number of protruding higher peaks emerged. As HCV increased its adaptation to the cells, the number of haplotype peaks within each mutant spectrum expanded, and their distribution shifted in the 2D network. The results show that extensive HCV replication in a monotonous cell culture environment does not limit HCV exploration of sequence space through haplotype peak movements. The landscapes reflect dynamic variation in the intramutant spectrum haplotype profile and may serve as a reference to interpret the modifications produced by external selective pressures or to compare with the landscapes of mutant spectra in complex in vivo environments. IMPORTANCE The study provides for the first time the haplotype profile and its variation in the course of virus adaptation to a cell culture environment in the absence of external selective constraints. The deep sequencing-based self-organized maps document a two-layer haplotype distribution with an ample basal platform and a lower number of protruding peaks. The results suggest an inferred intramutant spectrum fitness landscape structure that offers potential benefits for virus resilience to mutational inputs.


Assuntos
Adaptação Fisiológica/genética , Genoma Viral/genética , Haplótipos/genética , Hepacivirus/genética , RNA Polimerase Dependente de RNA/genética , Proteínas não Estruturais Virais/genética , Substituição de Aminoácidos/genética , Linhagem Celular Tumoral , Mapeamento Cromossômico , Evolução Molecular , Hepacivirus/crescimento & desenvolvimento , Hepatite C/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação/genética , Quase-Espécies/genética , RNA Viral/genética , Replicação Viral
11.
Viruses ; 13(11)2021 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-34834980

RESUMO

Enterovirus, like the majority of RNA viruses, evolves to survive the changeable environments by a variety of strategies. Here, we showed that HY12 virus evolved to alter its characteristics and pathogenicity by employing a non-synonymous mutation. Analyses of 5'UTR, VP1 and VP2 gene sequences revealed the existence of HY12 virus in an array of mutants defined as quasispecies. The determination of diversity and complexity showed that the mutation rate and complexity of HY12 virus quasispecies increased, while the proportion of HY12 VP1 and VP2 consensus (master) sequences decreased with increasing passages. Synonymous mutation and non-synonymous mutation analysis displayed a positive selection for HY12 quasispecies evolution. A comparison of HY12 virus in different passages demonstrated that HY12 virus altered its characteristic, phenotype, and pathogenicity via non-synonymous mutation. These findings revealed the evolution pattern for HY12 virus, and the alteration of HY12 virus characteristics and pathogenicity by mutation.


Assuntos
Enterovirus/genética , Evolução Molecular , Quase-Espécies , Regiões 5' não Traduzidas , Animais , Antígenos Virais/genética , Proteínas do Capsídeo/genética , Chlorocebus aethiops , Enterovirus/fisiologia , Infecções por Enterovirus/virologia , Mutação , Alinhamento de Sequência , Células Vero , Virulência
12.
Genes (Basel) ; 12(11)2021 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-34828337

RESUMO

Cirrhosis derived from chronic hepatitis C virus (HCV) infection is still a common indication for liver transplantation (LT). Reinfection of the engrafted liver is universal in patients with detectable viral RNA at the time of transplant and causes fast progression of cirrhosis (within 5 years) in around one-third of these patients. To prevent damage to the liver graft, effective direct-acting antiviral (DAA) therapy is required as soon as possible. However, because of post-LT clinical instability, it is difficult to determine the optimal time to start DAAs with a low risk of complications. Evaluate changes in quasispecies complexity following LT and seek a predictive index of fast liver damage progression to determine the timing of DAA initiation. HCV genomes isolated from pre-LT and 15-day post-LT serum samples of ten patients, who underwent orthotopic LT, were quantified and sequenced using a next-generation sequencing platform. Sequence alignments, phylogenetic trees, quasispecies complexity measures, biostatistics analyses, adjusted R2 values, and analysis of variance (ANOVA) were carried out. Three different patterns of reinfection were observed (viral bottlenecking, conserved pre-LT population, and mixed populations), suggesting that bottlenecking or homogenization of the viral population is not a generalized effect after liver graft reinfection. None of the quasispecies complexity measures predicted the future degree of liver damage. Higher and more uniform viral load (VL) values were observed in all pre-LT samples, but values were more dispersed in post-LT samples. However, VL increased significantly from the pre-LT to 15-day post-LT samples in patients with advanced fibrosis at 1-year post-LT, suggesting that a VL increase on day 15 may be a predictor of fast liver fibrosis progression. HCV kinetics after LT differ between patients and are not fibrosis-dependent. Higher VL at day 15 post-LT versus pre-LT samples may predict fast liver fibrosis progression.


Assuntos
Hepacivirus/classificação , Hepatite C Crônica/terapia , Cirrose Hepática/virologia , Transplante de Fígado/efeitos adversos , Antivirais/farmacologia , Antivirais/uso terapêutico , Progressão da Doença , Hepacivirus/genética , Hepacivirus/isolamento & purificação , Humanos , Cirrose Hepática/prevenção & controle , Filogenia , Quase-Espécies , Tempo para o Tratamento
13.
Virol J ; 18(1): 236, 2021 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-34844612

RESUMO

BACKGROUND: Previous works have observed that younger infants with chronic hepatitis B virus (HBV) infection are more responsive to antiviral treatment. However, the underlying mechanism remains unclear. In this study, the dynamic changes of HBV quasispecies in infants with immunoprophylaxis failure were investigated to provide virological explanations for clinical management on infantile antiviral therapy. METHODS: Thirteen 7-month-old infants with immunoprophylaxis failure and their mothers were enrolled from a prospective cohort, and 8 of them were followed up to 3 years old. The sequences of HBV quasispecies were analyzed by the full-length genome clone-based sequencing, and compared among mothers and their infants at different ages. RESULTS: The results revealed that the complexity, mutation frequency and genetic distance of HBV quasispecies decreased significantly at full-length, partial open reading frames and regulatory regions of HBV genome at nucleotide level in 7-month-old infants comparing with their mothers, whereas increased significantly to near the maternal level when infants grew up to 3 years old. Furthermore, similar changes were also found in Core, PreS2, RT and P regions of HBV genome at amino acid level, especially for potential NAs-resistant mutants in RT region and immune-escape mutants in Core and PreS2 regions. CONCLUSIONS: This study uncovered the evolution of HBV quasispecies in infancy after mother-to-child transmission, which may provide the virological evidence for explaning that younger children are more responsive to antiviral therapy.


Assuntos
Hepatite B Crônica , Hepatite B , Criança , Estudos de Coortes , DNA Viral , Feminino , Hepatite B/prevenção & controle , Vírus da Hepatite B/genética , Hepatite B Crônica/tratamento farmacológico , Humanos , Lactente , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Estudos Prospectivos , Quase-Espécies
14.
Vet Microbiol ; 263: 109252, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34673357

RESUMO

Previous studies resulted in the isolation of a low-virulence plaque-purified variant from the third passage (P3) in BHK-21 cells of a Tembusu virus (TMUV) isolate, suggesting the presence of viral quasispecies in the P3 culture. To confirm this notion, the fourth passage virus (P4) was prepared by infecting BHK-21 cells with P3 for isolation of more variants. We isolated 10 plaque-purified viruses. Comparative genome sequence analysis identified six of the 10 viruses as genetically different variants, which harbored a total of eight amino acid differences in the envelope, NS1, NS3, and NS5 proteins. When tested in a 2-day-old Pekin duck model, P4 caused 80 % mortality, belonging to a high-virulence TMUV strain. Out of the six genetically different variants, two presented high-virulence, one exhibited moderate-virulence, and three displayed low-virulence, causing 60 %-70 %, 40 %, and 10 % mortalities, respectively. These results demonstrate that P4 contains at least three groups of variants with distinct virulence phenotypes. Analysis of links between the eight residues and virulence of the six variants identified NS1 protein residue 183 and NS5 protein residues 275 and/or 287 as novel determinants of TMUV virulence. The analysis also provided a new clue for future studies on the molecular basis of TMUV virulence in terms of genetic interaction of different proteins. Overall, our study provides direct evidence to suggest that TMUV exists in in vitro culture of a virulent isolate as a quasispecies, which may enhance our understanding of molecular mechanism of TMUV virulence.


Assuntos
Infecções por Flavivirus , Flavivirus , Doenças das Aves Domésticas , Animais , Linhagem Celular , Patos , Flavivirus/patogenicidade , Infecções por Flavivirus/veterinária , Infecções por Flavivirus/virologia , Fenótipo , Doenças das Aves Domésticas/virologia , Quase-Espécies , Virulência
15.
Antiviral Res ; 195: 105178, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34509461

RESUMO

BACKGROUND: Telbivudine (LdT) - a synthetic thymidine ß-L-nucleoside analogue (NA) - is an effective inhibitor for hepatitis B virus (HBV) replication. The quasispecies spectra in the reverse transcriptase (RT) region of the HBV genome and their dynamic changes associated with LdT treatment remains largely unknown. METHODS: We prospectively recruited a total of 21 treatment-naive patients with chronic HBV infection and collected sequential serum samples at five time points (baseline, weeks 1, 3, 12, and 24 after LdT treatment). The HBV RT region was amplified and shotgun-sequenced by the Ion Torrent Personal Genome Machine (PGM)® system. We reconstructed full-length haplotypes of the RT region using an integrated bioinformatics framework, including de novo contig assembly and full-length haplotype reconstruction. In addition, we investigated the quasispecies' dynamic changes and evolution history and characterized potential NAs resistant mutations over the treatment course. RESULTS: Viral quasispecies differed obviously between patients with complete (n = 8) and incomplete/no response (n = 13) at 12 weeks after LdT treatment. A reduced dN/dS ratio in quasispecies demonstrated a selective constraint resulting from antiviral therapy. The temporal clustering of sequential quasispecies showed different patterns along with a 24-week observation, although its statistic did not differ significantly. Several patients harboring pre-existing resistant mutations showed different clinical responses, while NAs resistant mutations were rare within a short-term treatment. CONCLUSION: A complete profile of quasispecies reconstructed from in-depth shotgun sequencing may has important implications for enhancing clinical decision in adjusting antiviral therapy timely.


Assuntos
Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/tratamento farmacológico , Quase-Espécies/efeitos dos fármacos , DNA Polimerase Dirigida por RNA/efeitos dos fármacos , Telbivudina/uso terapêutico , Adulto , Antivirais/farmacologia , Farmacorresistência Viral , Feminino , Vírus da Hepatite B/classificação , Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Humanos , Masculino , Mutação/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico , Estudos Prospectivos , Quase-Espécies/genética , DNA Polimerase Dirigida por RNA/genética , Adulto Jovem
16.
Annu Rev Virol ; 8(1): 51-72, 2021 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-34586874

RESUMO

Viral quasispecies are dynamic distributions of nonidentical but closely related mutant and recombinant viral genomes subjected to a continuous process of genetic variation, competition, and selection that may act as a unit of selection. The quasispecies concept owes its theoretical origins to a model for the origin of life as a collection of mutant RNA replicators. Independently, experimental evidence for the quasispecies concept was obtained from sampling of bacteriophage clones, which revealed that the viral populations consisted of many mutant genomes whose frequency varied with time of replication. Similar findings were made in animal and plant RNA viruses. Quasispecies became a theoretical framework to understand viral population dynamics and adaptability. The evidence came at a time when mutations were considered rare events in genetics, a perception that was to change dramatically in subsequent decades. Indeed, viral quasispecies was the conceptual forefront of a remarkable degree of biological diversity, now evident for cell populations and organisms, not only for viruses. Quasispecies dynamics unveiled complexities in the behavior of viral populations,with consequences for disease mechanisms and control strategies. This review addresses the origin of the quasispecies concept, its major implications on both viral evolution and antiviral strategies, and current and future prospects.


Assuntos
Vírus de RNA , Vírus , Animais , Antivirais , Genoma Viral , Quase-Espécies/genética , Vírus de RNA/genética , Vírus/genética
17.
Microbiol Spectr ; 9(1): e0026121, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34346744

RESUMO

The dynamics of quasispecies afford RNA viruses a great fitness on cell tropism and host range. To study the quasispecies features and the intra-host evolution of SARS-CoV-2, we collected nine confirmed patients and sequenced the haplotypes of spike gene using a single-molecule real-time platform. Fourteen samples were extracted from sputum, nasopharyngeal swabs, or stool, which in total produced 283,655 high-quality circular consensus sequences. We observed a stable quasispecies structure that one master mutant (mean abundance ∼0.70), followed by numerous minor mutants (mean abundance ∼1.21 × 10-3). Under high selective pressure, minor mutants may obtain a fitness advantage and become the master ones. The later predominant substitution D614G existed in the minor mutants of more than one early patient. An epidemic variant had a possibility to be independently originated from multiple hosts. The mutant spectrums covered ∼85% amino acid variations of public genomes (GISAID; frequency ≥ 0.1) and likely provided an advantage mutation pool for the current/future epidemic variants. Notably, 32 of 35 collected antibody escape substitutions were preexistent in the early quasispecies. Virus populations in different tissues/organs revealed potentially independent replications. The quasispecies complexity of sputum samples was significantly lower than that of nasopharyngeal swabs (P = 0.02). Evolution analysis revealed that three continuous S2 domains (HR1, CH, and CD) had undergone a positive selection. Cell fusion-related domains may play a crucial role in adapting to the intrahost immune system. Our findings suggested that future epidemiologic investigations and clinical interventions should consider the quasispecies information that has missed by routine single consensus genome. IMPORTANCE RNA virus population in a host does not consist of a consensus single haplotype but rather an ensemble of related sequences termed quasispecies. The dynamics of quasispecies afford SARS-CoV-2 a great ability on genetic fitness during intrahost evolution. The process is likely achieved by changing the genetic characteristics of key functional genes, such as the spike glycoprotein. Previous studies have applied the next-generation sequencing (NGS) technology to evaluate the quasispecies of SARS-CoV-2, and results indicated a low genetic diversity of the spike gene. However, the NGS platform cannot directly obtain the full haplotypes without assembling, and it is also difficult to predict the extremely low-frequency variations. Therefore, we introduced a single-molecule real-time technology to directly obtain the haplotypes of the RNA population and further study the quasispecies features and intrahost evolution of the spike gene.


Assuntos
Epidemias , Mutação , Quase-Espécies , SARS-CoV-2/classificação , SARS-CoV-2/genética , Adulto , Idoso , Sequência de Bases , COVID-19/virologia , Criança , Feminino , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Glicoproteína da Espícula de Coronavírus/genética
18.
Viruses ; 13(8)2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34452416

RESUMO

Vaccination is an effective method for the prevention of influenza virus infection. Many manufacturers use embryonated chicken eggs (ECE) for the propagation of vaccine strains. However, the adaptation of viral strains during subsequent passages can lead to additional virus evolution and lower effectiveness of the resulting vaccines. In our study, we analyzed the distribution of single nucleotide variants (SNVs) of equine influenza virus (EIV) during passaging in ECE. Viral RNA from passage 0 (nasal swabs), passage 2 and 5 was sequenced using next generation technology. In total, 50 SNVs with an occurrence frequency above 2% were observed, 29 of which resulted in amino acid changes. The highest variability was found in passage 2, with the most variable segment being IV encoding hemagglutinin (HA). Three variants, HA (W222G), PB2 (A377E) and PA (R531K), had clearly increased frequency with the subsequent passages, becoming dominant. None of the five nonsynonymous HA variants directly affected the major antigenic sites; however, S227P was previously reported to influence the antigenicity of EIV. Our results suggest that although host-specific adaptation was observed in low passages of EIV in ECE, it should not pose a significant risk to influenza vaccine efficacy.


Assuntos
Ovos/virologia , Vírus da Influenza A Subtipo H3N8/genética , Vírus da Influenza A Subtipo H3N8/fisiologia , Polimorfismo de Nucleotídeo Único , Quase-Espécies/genética , RNA Viral/genética , Adaptação Fisiológica/genética , Animais , Galinhas/imunologia , Cavalos/virologia , Filogenia , Análise de Sequência de DNA , Inoculações Seriadas
19.
J Gen Virol ; 102(7)2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34292864

RESUMO

Hepatitis B virus surface antigen (HBsAg) encoded by the S gene is highly expressed during the replication cycle of hepatitis B virus (HBV). However, the frequent usage of tryptophan in HBsAg, which leads to a high cost of biosynthesis, is inconsistent with the high expression level of this protein. Tryptophan-truncated mutation of HBsAg, that is, a tryptophan to stop codon mutation resulting in truncated HBsAg, might help to maintain its high expression with lower biosynthetic cost. We aimed to investigate the prevalence of tryptophan-truncated S quasispecies in treatment-naïve patients with chronic hepatitis B (CHB) by applying CirSeq as well as a site-by-site algorithm developed by us to identify variants at extremely low frequencies in the carboxyl terminus of HBsAg. A total of 730 mutations were identified in 27 patients with CHB, varying from seven to 56 mutations per sample. The number of synonymous mutations was much higher than that of nonsynonymous mutations in the reverse transcriptase (RT) coding region and vice versa in the S coding region, implying that the evolutionary constraints on the RT and S genes might be different. We showed that 25 (92.6 %) of 27 patients had at least one S-truncated mutation, most of which were derived from tryptophan, indicating a high prevalence of tryptophan-truncated S mutations in treatment-naïve patients with CHB. In terms of the RT gene, 21 (77.8 %) patients had pre-existing drug-resistant mutations, while no truncated mutations were detected. Our findings that tryptophan-truncated S quasispecies and drug-resistant RT mutants were highly prevalent in treatment-naïve patients with CHB provide new insights into the composition of the HBV population, which might help optimize the treatment and management of patients with CHB.


Assuntos
Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Mutação , Triptofano/genética , Adolescente , Adulto , Algoritmos , Motivos de Aminoácidos , Antivirais/farmacologia , Antivirais/uso terapêutico , Códon , Farmacorresistência Viral , Evolução Molecular , Feminino , Genes Virais , Antígenos de Superfície da Hepatite B/química , Vírus da Hepatite B/química , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/tratamento farmacológico , Humanos , Masculino , Pessoa de Meia-Idade , Quase-Espécies , DNA Polimerase Dirigida por RNA/genética , Análise de Sequência de DNA , Adulto Jovem
20.
Curr Opin Virol ; 49: 157-163, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34153841

RESUMO

The genetic diversity of virus populations within their hosts is known to influence disease progression, treatment outcome, drug resistance, cell tropism, and transmission risk, and the study of dynamic changes of genetic heterogeneity can provide insights into the evolution of viruses. Several measures to quantify within-host genetic diversity capturing different aspects of diversity patterns in a sample or population are used, based on incidence, relative frequencies, pairwise distances, or phylogenetic trees. Here, we review and compare several of these measures.


Assuntos
Variação Genética , Viroses/virologia , Vírus/genética , Genoma Viral , Haplótipos , Humanos , Mutação , Filogenia , Quase-Espécies
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