RESUMO
Quantification of hepatitis C virus (HCV)-RNA in serum or plasma samples is an essential parameter in HCV diagnostics. Here, the NeuMoDx™Molecular System (Qiagen) was tested for the most common HCV genotypes and compared to the cobas c6800 system (Roche). HCV-RNA from 131 plasma/serum samples from chronically infected patients was determined in parallel on the NeuMoDx and c6800 systems. Linearity was analysed using the four most common HCV genotypes (1-4) in our cohort. The coefficient of variation (CV) within (intra-assay) and between (inter-assay) runs was calculated based on HCV-RNA concentration. Quantitative HCV-RNA results were highly correlated on both test systems (R2 =â¯0.7947; yâ¯=â¯0.94â¯xâ¯+â¯0.37). On average, the NeuMoDx and c6800 HCV RNA levels showed a mean difference of only 0.05 log10 IU/mL but with a broad distribution (±1.2 2â¯xâ¯SD). The NeuMoDx demonstrated very good linearity across all HCV genotypes tested at concentrations between 1.7 and 6.2 log10 IU/mL (R2 range: 0.9257-0.9991) with the highest mean coefficient of determination for genotype 1 (R2 =â¯0.9909). The mean intra- and inter-assay CV for both serum and plasma samples was <5â¯%. The NeuMoDx HCV-RNA Assay demonstrates high subtype-independent comparability, linearity, and reproducibility for the quantification of HCV-RNA in serum and plasma samples from chronically infected patients.
Assuntos
Genótipo , Hepacivirus , RNA Viral , Carga Viral , Humanos , Hepacivirus/genética , Hepacivirus/isolamento & purificação , RNA Viral/sangue , RNA Viral/genética , Carga Viral/métodos , Reprodutibilidade dos Testes , Hepatite C Crônica/virologia , Hepatite C Crônica/diagnóstico , Hepatite C Crônica/sangue , Sensibilidade e Especificidade , Hepatite C/diagnóstico , Hepatite C/virologia , Hepatite C/sangue , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Kit de Reagentes para Diagnóstico/normasRESUMO
We provide follow-up data on the humoral immune response after COVID-19 vaccinations of two distinct cohorts aged below 60 and over 80 years to screen for age-related differences in the longevity and magnitude of the induction of the antibody responses post booster-vaccinations. While anti-SARS-CoV-2 spike-specific IgG and neutralization capacity waned rapidly after the initial vaccination schedule, additional boosters highly benefitted the humoral immune responses especially in the elderly cohort, including the neutralization of Omikron variants. Thus, adjusted COVID-19 booster vaccination schedules are an appropriate tool to overcome limitations in the success of vaccinations.
RESUMO
Immune evasion of pathogens can modify the course of infection and impact viral persistence and pathology. Here, using different strains of the lymphocytic choriomeningitis virus (LCMV) model system, we show that slower propagation results in limited type I interferon (IFN-I) production and viral persistence. Specifically, cells infected with LCMV-Docile exhibited reduced viral replication when compared to LCMV-WE and as a consequence, infection with LCMV-Docile resulted in reduced activation of bone marrow derived dendritic cells (BMDCs) and IFN-I production in vitro in comparison with LCMV-WE. In vivo, we observed a reduction of IFN-I, T cell exhaustion and viral persistence following infection of LCMV-Docile but not LCMV-WE. Mechanistically, block of intracellular protein transport uncovered reduced propagation of LCMV-Docile when compared to LCMV-WE. This reduced propagation was critical in blunting the activation of the innate and adaptive immune system. When mice were simultaneously infected with LCMV-Docile and LCMV-WE, immune function was restored and IFN-I production, T cell effector functions as well as viral loads were similar to that of mice infected with LCMV-WE alone. Taken together, this study suggests that reduced viral propagation can result in immune evasion and viral persistence.
Assuntos
Infecções por Arenaviridae/virologia , Células Dendríticas/virologia , Vírus da Coriomeningite Linfocítica/imunologia , Receptor de Interferon alfa e beta/fisiologia , Linfócitos T/virologia , Replicação Viral , Animais , Infecções por Arenaviridae/imunologia , Infecções por Arenaviridae/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has led to the development of various vaccines. Real-life data on immune responses elicited in the most vulnerable group of vaccinees older than age 80 years old are still underrepresented despite the prioritization of the elderly in vaccination campaigns. METHODS: We conducted a cohort study with 2 age groups, young vaccinees below the age of 60 years and elderly vaccinees over the age of 80 years, to compare their antibody responses to the first and second dose of the BNT162b2 coronavirus disease 2019 vaccination. RESULTS: Although the majority of participants in both groups produced specific immunoglobulin G antibody titers against SARS-CoV-2 spike protein, titers were significantly lower in elderly participants. Although the increment of antibody levels after the second immunization was higher in elderly participants, the absolute mean titer of this group remained lower than the <60 years of age group. After the second vaccination, 31.3% of the elderly had no detectable neutralizing antibodies in contrast to the younger group, in which only 2.2% had no detectable neutralizing antibodies. CONCLUSIONS: Our data showed differences between the antibody responses raised after the first and second BNT162b2 vaccination, in particular lower frequencies of neutralizing antibodies in the elderly group. This suggests that this population needs to be closely monitored and may require earlier revaccination and/or an increased vaccine dose to ensure stronger long-lasting immunity and protection against infection.
Assuntos
Vacina BNT162 , COVID-19 , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vacina BNT162/imunologia , COVID-19/prevenção & controle , Estudos de Coortes , Feminino , Humanos , Imunidade , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Glicoproteína da Espícula de Coronavírus/imunologia , VacinaçãoRESUMO
The underlying molecular mechanism and their general effect on the replication capacity of HIV 1 drug-resistance-associated mutations is often poorly understood. To elucidate the effect of two such mutations located in a region with a high density of spicing regulatory elements on the HIV-1-splicing outcome, bioinformatic predictions were combined with transfection and infection experiments. Results show that the previously described R263K drug-resistance-associated integrase mutation has additionally a severe effect on the ESE2b splicing regulatory element (SRE) in exon 2b, which causes loss of SD2b recognition. This was confirmed by an R263R silent mutation with a similar predicted effect on the exon 2b SRE. In contrast, a V260I mutation and its silent counterpart with a lower effect on ESS2b did not exhibit any differences in the splicing pattern. Since HIV-1 highly relies on a balanced splicing reaction, changes in the splicing outcome can contribute to changes in viral replication and might add to the effect of escape mutations toward antiviral drugs. Thus, a classification of mutations purely addressing proteins is insufficient.
Assuntos
Farmacorresistência Viral/genética , Éxons/genética , HIV-1/genética , Mutação/genética , Splicing de RNA/genética , RNA Mensageiro/genética , Linhagem Celular , Farmacorresistência Viral/efeitos dos fármacos , Éxons/efeitos dos fármacos , Células HEK293 , Integrase de HIV/genética , Inibidores de Integrase de HIV/farmacologia , HIV-1/efeitos dos fármacos , Células HeLa , Humanos , Mutação/efeitos dos fármacos , Sítios de Splice de RNA/efeitos dos fármacos , Sítios de Splice de RNA/genética , Splicing de RNA/efeitos dos fármacos , Sequências Reguladoras de Ácido Nucleico/genética , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
Most human pathogenic mutations in 5' splice sites affect the canonical GT in positions +1 and +2, leading to noncanonical dinucleotides. On the other hand, noncanonical dinucleotides are observed under physiological conditions in â¼1% of all human 5'ss. It is therefore a challenging task to understand the pathogenic mutation mechanisms underlying the conditions under which noncanonical 5'ss are used. In this work, we systematically examined noncanonical 5' splice site selection, both experimentally using splicing competition reporters and by analyzing a large RNA-seq data set of 54 fibroblast samples from 27 subjects containing a total of 2.4 billion gapped reads covering 269,375 exon junctions. From both approaches, we consistently derived a noncanonical 5'ss usage ranking GC > TT > AT > GA > GG > CT. In our competition splicing reporter assay, noncanonical splicing was strictly dependent on the presence of upstream or downstream splicing regulatory elements (SREs), and changes in SREs could be compensated by variation of U1 snRNA complementarity in the competing 5'ss. In particular, we could confirm splicing at different positions (i.e., -1, +1, +5) of a splice site for all noncanonical dinucleotides "weaker" than GC. In our comprehensive RNA-seq data set analysis, noncanonical 5'ss were preferentially detected in weakly used exon junctions of highly expressed genes. Among high-confidence splice sites, they were 10-fold overrepresented in clusters with a neighboring, more frequently used 5'ss. Conversely, these more frequently used neighbors contained only the dinucleotides GT, GC, and TT, in accordance with the above ranking.
Assuntos
Regulação da Expressão Gênica , Genes Reporter , Estudo de Associação Genômica Ampla , Sítios de Splice de RNA , Splicing de RNA , Adolescente , Adulto , Idoso , Processamento Alternativo , Sequência de Bases , Linhagem Celular , Elementos Facilitadores Genéticos , Éxons , Feminino , Estudo de Associação Genômica Ampla/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência de RNA , Adulto JovemRESUMO
The HIV-1 accessory protein Vif is essential for viral replication by counteracting the host restriction factor APOBEC3G (A3G), and balanced levels of both proteins are required for efficient viral replication. Noncoding exons 2/2b contain the Vif start codon between their alternatively used splice donors 2 and 2b (D2 and D2b). For vif mRNA, intron 1 must be removed while intron 2 must be retained. Thus, splice acceptor 1 (A1) must be activated by U1 snRNP binding to either D2 or D2b, while splicing at D2 or D2b must be prevented. Here, we unravel the complex interactions between previously known and novel components of the splicing regulatory network regulating HIV-1 exon 2/2b inclusion in viral mRNAs. In particular, using RNA pulldown experiments and mass spectrometry analysis, we found members of the heterogeneous nuclear ribonucleoparticle (hnRNP) A/B family binding to a novel splicing regulatory element (SRE), the exonic splicing silencer ESS2b, and the splicing regulatory proteins Tra2/SRSF10 binding to the nearby exonic splicing enhancer ESE2b. Using a minigene reporter, we performed bioinformatics HEXplorer-guided mutational analysis to narrow down SRE motifs affecting splice site selection between D2 and D2b. Eventually, the impacts of these SREs on the viral splicing pattern and protein expression were exhaustively analyzed in viral particle production and replication experiments. Masking of these protein binding sites by use of locked nucleic acids (LNAs) impaired Vif expression and viral replication.IMPORTANCE Based on our results, we propose a model in which a dense network of SREs regulates vif mRNA and protein expression, crucial to maintain viral replication within host cells with varying A3G levels and at different stages of infection. This regulation is maintained by several serine/arginine-rich splicing factors (SRSF) and hnRNPs binding to those elements. Targeting this cluster of SREs with LNAs may lead to the development of novel effective therapeutic strategies.
Assuntos
HIV-1/genética , Sítios de Splice de RNA , Fatores de Processamento de RNA/análise , RNA Viral/genética , Sequências Reguladoras de Ácido Ribonucleico , Produtos do Gene vif do Vírus da Imunodeficiência Humana/genética , Sítios de Ligação , Linhagem Celular , Centrifugação , Análise Mutacional de DNA , Éxons , Humanos , Espectrometria de Massas , RNA Viral/metabolismoRESUMO
A critical step in exon definition is the recognition of a proper splice donor (5Îss) by the 5' end of U1 snRNA. In the selection of appropriate 5Îss, cis-acting splicing regulatory elements (SREs) are indispensable. As a model for 5Îss recognition, we investigated cryptic 5Îss selection within the human fibrinogen Bß-chain gene (FGB) exon 7, where we identified several exonic SREs that simultaneously acted on up- and downstream cryptic 5Îss. In the FGB exon 7 model system, 5Îss selection iteratively proceeded along an alternating sequence of U1 snRNA binding sites and interleaved SREs which in principle supported different 3' exon ends. Like in a relay race, SREs either suppressed a potential 5Îss and passed the splicing baton on or splicing actually occurred. From RNA-Seq data, we systematically selected 19 genes containing exons with silent U1 snRNA binding sites competing with nearby highly used 5Îss. Extensive SRE analysis by different algorithms found authentic 5Îss significantly more supported by SREs than silent U1 snRNA binding sites, indicating that our concept may permit generalization to a model for 5Îss selection and 3' exon end definition.