RESUMO
BackgroundIn summer 2022, SARS-CoV-2 Omicron BA.5 became dominant in Europe. In vitro studies have shown a large reduction of antibody neutralisation for this variant.AimWe aimed to investigate differences in protection from previous infection and/or vaccination against infection with Omicron BA.4/5 vs BA.2.MethodsWe employed a case-only approach including positive PCR tests from community testing between 2 May and 24 July 2022 that were tested for S gene target failure (SGTF), which distinguishes BA.4/5 from BA.2 infection. Previous infections were categorised by variant using whole genome sequencing or SGTF. We estimated by logistic regression the association of SGTF with vaccination and/or previous infection, and of SGTF of the current infection with the variant of the previous infection, adjusting for testing week, age group and sex.ResultsThe percentage of registered previous SARS-CoV-2 infections was higher among 19,836 persons infected with Omicron BA.4/5 than among 7,052 persons infected with BA.2 (31.3% vs 20.0%). Adjusting for testing week, age group and sex, the adjusted odds ratio (aOR) was 1.4 (95%â¯CI: 1.3-1.5). The distribution of vaccination status did not differ for BA.4/5 vs BA.2 infections (aORâ¯=â¯1.1 for primary and booster vaccination). Among persons with a previous infection, those currently infected with BA4/5 had a shorter interval between infections, and the previous infection was more often caused by BA.1, compared with those currently infected with BA.2 (aORâ¯=â¯1.9; 95%â¯CI: 1.5-2.6).ConclusionOur results suggest immunity induced by BA.1 is less effective against BA.4/5 infection than against BA.2 infection.
Assuntos
COVID-19 , Humanos , Países Baixos/epidemiologia , COVID-19/epidemiologia , SARS-CoV-2/genética , Europa (Continente) , Imunização SecundáriaRESUMO
The SARS-CoV-2 Omicron variant has a growth advantage over the Delta variant because of higher transmissibility, immune evasion or shorter serial interval. Using S gene target failure (SGTF) as indication for Omicron BA.1, we identified 908 SGTF and 1,621 non-SGTF serial intervals in the same period. Within households, the mean serial interval for SGTF cases was 0.2-0.6 days shorter than for non-SGTF cases. This suggests that the growth advantage of Omicron is partly due to a shorter serial interval.
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COVID-19 , SARS-CoV-2 , Humanos , Países BaixosRESUMO
Infections with the Omicron SARS-CoV-2 variant are rapidly increasing worldwide. Among 174,349 SARS-CoV-2-infected individuals (≥ 12 years), we observed an increased risk of S gene target failure, predictive of the Omicron variant, in vaccinated (odds ratio (OR): 3.6; 95% confidence interval (CI): 3.4-3.7) and previously infected individuals (OR: 4.2; 95% CI: 3.8-4.7) compared with infected naïve individuals. This suggests vaccine- or infection-induced immunity against SARS-CoV-2 infections is less effective against the Omicron than the Delta variant.
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COVID-19 , SARS-CoV-2 , Vacinas contra COVID-19 , Humanos , Países BaixosRESUMO
Insertions in the protease (PR) region of human immunodeficiency virus (HIV) represent an interesting mechanism of antiviral resistance against HIV PR inhibitors (PIs). Here, we demonstrate the improved ability of a phosphonate-containing experimental HIV PI, GS-8374, relative to that of other PIs, to effectively inhibit patient-derived recombinant HIV strains bearing PR insertions and numerous other mutations. We correlate enzyme inhibition with the catalytic activities of corresponding recombinant PRs in vitro and provide a biochemical and structural analysis of the PR-inhibitor complex.
Assuntos
Infecções por HIV/virologia , Inibidores da Protease de HIV/química , Protease de HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Mutagênese Insercional , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Farmacorresistência Viral , Infecções por HIV/tratamento farmacológico , Protease de HIV/química , Protease de HIV/metabolismo , Inibidores da Protease de HIV/farmacologia , HIV-1/química , HIV-1/genética , Humanos , Modelos Moleculares , Organofosfonatos/análiseRESUMO
Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen that has been responsible for major nosocomial epidemics worldwide. For infection control programs, rapid and adequate detection of MRSA is of great importance. We developed a rapid and high-throughput molecular screening approach that consists of an overnight selective broth enrichment, followed by mecA, mecC, and S. aureus-specific (SA442 gene) real-time PCR assays, with subsequent confirmation using a staphylococcal cassette chromosome mec element (SCCmec)-orfX-based real-time PCR assay (GeneOhm MRSA assay) and culture. Here, the results of the screening approach over a 2-year period are presented. During this period, a total of 13,387 samples were analyzed for the presence of MRSA, 2.6% of which were reported as MRSA positive. No MRSA isolates carrying the mecC gene were detected during this study. Based on the results of the real-time PCR assays only, 95.2% of the samples could be reported as negative within 24 h. Furthermore, the performance of these real-time PCR assays was evaluated using a set of 104 assorted MRSA isolates, which demonstrated high sensitivity for both the combination of mecA and mecC with SA442 and the BD GeneOhm MRSA assay (98.1% and 97.1%, respectively). This molecular screening approach proved to be an accurate method for obtaining reliable negative results within 24 h after arrival at the laboratory and contributes to improvement of infection control programs, especially in areas with a low MRSA prevalence.
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Ensaios de Triagem em Larga Escala , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Infecções Estafilocócicas/diagnóstico , Humanos , Staphylococcus aureus Resistente à Meticilina/genética , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Fatores de TempoRESUMO
BACKGROUND: Mutations in the substrate of HIV-1 protease, especially changes in the NC/p1 cleavage site, can directly contribute to protease inhibitor (PI) resistance and also compensate for defects in viral replicative capacity (RC) due to a drug resistant protease. These NC/p1 changes are known to enhance processing of the Gag protein. To investigate the capacity of HIV-1 to modulate Gag cleavage and its consequences for PI resistance and RC, we performed a detailed enzymatic and virological analysis using a set of PI resistant NC/p1 variants (HXB2431V, HXB2436E+437T, HXB2437T and HXB2437V). RESULTS: Here, we demonstrate that single NC/p1 mutants, which displayed only a slight increase in PI resistance did not show an obvious change in RC. In contrast, the double NC/p1 mutant, which displayed a clear increase in processing efficiency and PI resistance, demonstrated a clear reduction in RC. Cleavage analysis showed that a tridecameric NC/p1 peptide representing the double NC/p1 mutant was cleaved in two specific ways instead of one.The observed decrease in RC for the double NC/p1 mutant (HXB2436E+437T) could (partially) be restored by either reversion of the 436E change or by acquisition of additional changes in the NC/p1 cleavage site at codon 435 or 438 as was revealed during in vitro evolution experiments. These changes not only restored RC but also reduced PI resistance levels. Furthermore these changes normalized Gag processing efficiency and obstructed the novel secondary cleavage site observed for the double NC/p1 mutant. CONCLUSIONS: The results of this study clearly demonstrate that HIV-1 can modulate Gag processing and thereby PI resistance. Distinct increases in Gag cleavage and PI resistance result in a reduced RC that can only be restored by amino acid changes in NC/p1 which reduce Gag processing to an optimal rate.
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Farmacorresistência Viral , Inibidores da Protease de HIV/farmacologia , Protease de HIV/metabolismo , HIV-1/fisiologia , Replicação Viral , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Substituição de Aminoácidos , Análise Mutacional de DNA , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , HIV-1/genética , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Proteólise , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genéticaAssuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Macrolídeos/farmacologia , Infecções por Mycoplasma/microbiologia , Mycoplasma genitalium/efeitos dos fármacos , Mycoplasma genitalium/genética , RNA Ribossômico 23S/genética , Doenças Bacterianas Sexualmente Transmissíveis/microbiologia , Adolescente , Adulto , Idoso , Antibacterianos/uso terapêutico , Azitromicina/uso terapêutico , Feminino , Humanos , Macrolídeos/uso terapêutico , Masculino , Pessoa de Meia-Idade , Mutação , Infecções por Mycoplasma/tratamento farmacológico , Infecções por Mycoplasma/epidemiologia , Mycoplasma genitalium/isolamento & purificação , Países Baixos/epidemiologia , Prevalência , Doenças Bacterianas Sexualmente Transmissíveis/tratamento farmacológico , Doenças Bacterianas Sexualmente Transmissíveis/epidemiologiaRESUMO
Staphylococcus argenteus is a recently described member of the Staphylococcus aureus complex (SAC) and is associated with human disease. The frequency and intensity of infections caused by S. argenteus are similar to those of Staphylococcus aureus. S. argenteus can harbor antibiotic resistance genes and a variety of virulence factors analogous to methicillin-resistant S. aureus (MRSA). The aim of our study was to analyze a collection of isolates in the Dutch national MRSA surveillance from January 2008 until March 2021 that were nontypeable by multilocus variable-number tandem-repeat analysis (MLVA). Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-ToF MS) was used for identifying the S. argenteus isolates, and whole-genome sequencing and SeqSphere were used to generate an in-house whole-genome multilocus sequence typing (wgMLST) scheme for typing the isolates. Furthermore, the presence of antibiotic resistance genes, replicons, and virulence genes was determined. Of 52,467 isolates submitted as MRSA from January 2008 until March 2021, 64 isolates (0.12%) were nontypeable with MLVA, and 54 of them were identified with mass spectrometry (MALDI-ToF MS) as S. argenteus. It appeared in retrospect that the first methicillin-resistant S. argenteus (MRSArg) was already submitted in 2008. An in-house-developed S. argenteus wgMLST scheme revealed that S. argenteus isolates clustered in 5 genomic groups which were characterized by distinct MLST types, resistomes, plasmid replicon families, and virulence factors. All but one isolate carried the staphylococcal chromosomal cassette mec (SCCmec) type IV harboring the methicillin resistance gene mecA and represent MRSArg. Most of the isolates with SCCmec subtype IVc(2B) had a trimethoprim resistance gene, dfrG, and harbored a blaZ-carrying plasmid, and most MRSArg isolates have the immune-modulating genes scn and sak. Nine of the 47 isolates carried enterotoxin-encoding genes seg, sei, sem, seo, and seu, which might be able to cause food poisoning. In some persons there was long-term persistence of MRSArg, and there were several genetically related MRSArg isolates in people living in close proximity, suggesting direct human-human transmission. IMPORTANCE We show that MRSArg has been circulating in the Netherlands since at least 2008. Although MRSArg is distinct from MRSA, it has a comparable population structure and carries similar resistance and virulence genes. The Dutch national MRSA surveillance has been expanded to include other methicillin-resistant members of the S. aureus complex, such as S. argenteus and Staphylococcus schweitzeri.
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Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Humanos , Resistência a Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus/genética , Tipagem de Sequências Multilocus , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/epidemiologia , Antibacterianos/farmacologia , Enterotoxinas , Fatores de Virulência/genéticaRESUMO
Given the emergence of the SARS-CoV-2 Omicron BA.1 and BA.2 variants and the roll-out of booster COVID-19 vaccination, evidence is needed on protection conferred by primary vaccination, booster vaccination and previous SARS-CoV-2 infection by variant. We employed a test-negative design on S-gene target failure data from community PCR testing in the Netherlands from 22 November 2021 to 31 March 2022 (n = 671,763). Previous infection, primary vaccination or both protected well against Delta infection. Protection against Omicron BA.1 infection was much lower compared to Delta. Protection was similar against Omicron BA.1 compared to BA.2 infection after previous infection, primary and booster vaccination. Higher protection was observed against all variants in individuals with both vaccination and previous infection compared with either one. Protection against all variants decreased over time since last vaccination or infection. We found that primary vaccination with current COVID-19 vaccines and previous SARS-CoV-2 infections offered low protection against Omicron BA.1 and BA.2 infection. Booster vaccination considerably increased protection against Omicron infection, but decreased rapidly after vaccination.
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COVID-19 , Vacinas Virais , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Humanos , SARS-CoV-2 , VacinaçãoRESUMO
BACKGROUND: Maturation inhibitors are an experimental class of antiretrovirals that inhibit Human Immunodeficiency Virus (HIV) particle maturation, the structural rearrangement required to form infectious virus particles. This rearrangement is triggered by the ordered cleavage of the precursor Gag polyproteins into their functional counterparts by the viral enzyme protease. In contrast to protease inhibitors, maturation inhibitors impede particle maturation by targeting the substrate of protease (Gag) instead of the protease enzyme itself. Direct cross-resistance between protease and maturation inhibitors may seem unlikely, but the co-evolution of protease and its substrate, Gag, during protease inhibitor therapy, could potentially affect future maturation inhibitor therapy. Previous studies showed that there might also be an effect of protease inhibitor resistance mutations on the development of maturation inhibitor resistance, but the exact mechanism remains unclear. We used wild-type and protease inhibitor resistant viruses to determine the impact of protease inhibitor resistance mutations on the development of maturation inhibitor resistance. RESULTS: Our resistance selection studies demonstrated that the resistance profiles for the maturation inhibitor bevirimat are more diverse for viruses with a mutated protease compared to viruses with a wild-type protease. Viral replication did not appear to be a major factor during emergence of bevirimat resistance. In all in vitro selections, one of four mutations was selected: Gag V362I, A364V, S368N or V370A. The impact of these mutations on maturation inhibitor resistance and viral replication was analyzed in different protease backgrounds. The data suggest that the protease background affects development of HIV-1 resistance to bevirimat and the replication profiles of bevirimat-selected HIV-1. The protease-dependent bevirimat resistance and replication levels can be explained by differences in CA/p2 cleavage processing by the different proteases. CONCLUSIONS: These findings highlight the complicated interactions between the viral protease and its substrate. By providing a better understanding of these interactions, we aim to help guide the development of second generation maturation inhibitors.
Assuntos
Farmacorresistência Viral , Inibidores da Protease de HIV/farmacologia , Protease de HIV/genética , HIV-1/fisiologia , Mutação , Succinatos/farmacologia , Triterpenos/farmacologia , Montagem de Vírus/efeitos dos fármacos , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Linhagem Celular , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Protease de HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Replicação Viral/efeitos dos fármacos , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
While the selection of amino acid insertions in human immunodeficiency virus (HIV) reverse transcriptase (RT) is a known mechanism of resistance against RT inhibitors, very few reports on the selection of insertions in the protease (PR) coding region have been published. It is still unclear whether these insertions impact protease inhibitor (PI) resistance and/or viral replication capacity. We show that the prevalence of insertions, especially between amino acids 30 to 41 of HIV type 1 (HIV-1) PR, has increased in recent years. We identified amino acid insertions at positions 33 and 35 of the PR of HIV-1-infected patients who had undergone prolonged treatment with PIs, and we characterized the contribution of these insertions to viral resistance. We prepared the corresponding mutated, recombinant PR variants with or without insertions at positions 33 and 35 and characterized them in terms of enzyme kinetics and crystal structures. We also engineered the corresponding recombinant viruses and analyzed the PR susceptibility and replication capacity by recombinant virus assay. Both in vitro methods confirmed that the amino acid insertions at positions 33 and 35 contribute to the viral resistance to most of the tested PIs. The structural analysis revealed local structural rearrangements in the flap region and in the substrate binding pockets. The enlargement of the PR substrate binding site together with impaired flap dynamics could account for the weaker inhibitor binding by the insertion mutants. Amino acid insertions in the vicinity of the binding cleft therefore represent a novel mechanism of HIV resistance development.
Assuntos
Farmacorresistência Viral , Protease de HIV/química , Protease de HIV/genética , HIV-1/enzimologia , Mutagênese Insercional , Inibidores da Transcriptase Reversa/química , Sequência de Aminoácidos , Fármacos Anti-HIV/uso terapêutico , Sítios de Ligação , Catálise , Linhagem Celular , Sequência Consenso , Protease de HIV/isolamento & purificação , Protease de HIV/metabolismo , HIV-1/genética , HIV-1/fisiologia , Humanos , Rim/citologia , Cinética , Modelos Químicos , Dados de Sequência Molecular , Ligação Proteica , RNA Viral/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Replicação Viral , Difração de Raios XRESUMO
The C-type lectin dendritic cell (DC)-specific intercellular adhesion molecule grabbing non-integrin (DC-SIGN; CD209) facilitates binding and internalization of several viruses, including HIV-1, on DCs, but the underlying mechanism for being such an efficient phagocytic pathogen-recognition receptor is poorly understood. By high resolution electron microscopy, we demonstrate a direct relation between DC-SIGN function as viral receptor and its microlocalization on the plasma membrane. During development of human monocyte-derived DCs, DC-SIGN becomes organized in well-defined microdomains, with an average diameter of 200 nm. Biochemical experiments and confocal microscopy indicate that DC-SIGN microdomains reside within lipid rafts. Finally, we show that the organization of DC-SIGN in microdomains on the plasma membrane is important for binding and internalization of virus particles, suggesting that these multimolecular assemblies of DC-SIGN act as a docking site for pathogens like HIV-1 to invade the host.
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Moléculas de Adesão Celular/metabolismo , Membrana Celular/metabolismo , Células Dendríticas/metabolismo , Lectinas Tipo C/metabolismo , Infecções por Vírus de RNA/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Virais/metabolismo , Moléculas de Adesão Celular/imunologia , Moléculas de Adesão Celular/ultraestrutura , Membrana Celular/ultraestrutura , Membrana Celular/virologia , Células Cultivadas , Células Dendríticas/ultraestrutura , Células Dendríticas/virologia , Endocitose/fisiologia , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , HIV-1/patogenicidade , HIV-1/fisiologia , Humanos , Imuno-Histoquímica , Lectinas Tipo C/imunologia , Lectinas Tipo C/ultraestrutura , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/ultraestrutura , Microscopia Eletrônica , Monócitos/metabolismo , Monócitos/ultraestrutura , Monócitos/virologia , Estrutura Terciária de Proteína/fisiologia , Infecções por Vírus de RNA/imunologia , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/ultraestrutura , Receptores Virais/imunologia , Receptores Virais/ultraestruturaRESUMO
BACKGROUND: HIV protease inhibitor (PI) therapy results in the rapid selection of drug resistant viral variants harbouring one or two substitutions in the viral protease. To combat PI resistance development, two approaches have been developed. The first is to increase the level of PI in the plasma of the patient, and the second is to develop novel PI with high potency against the known PI-resistant HIV protease variants. Both approaches share the requirement for a considerable increase in the number of protease mutations to lead to clinical resistance, thereby increasing the genetic barrier. We investigated whether HIV could yet again find a way to become less susceptible to these novel inhibitors. METHODS AND FINDINGS: We have performed in vitro selection experiments using a novel PI with an increased genetic barrier (RO033-4649) and demonstrated selection of three viruses 4- to 8-fold resistant to all PI compared to wild type. These PI-resistant viruses did not have a single substitution in the viral protease. Full genomic sequencing revealed the presence of NC/p1 cleavage site substitutions in the viral Gag polyprotein (K436E and/or I437T/V) in all three resistant viruses. These changes, when introduced in a reference strain, conferred PI resistance. The mechanism leading to PI resistance is enhancement of the processing efficiency of the altered substrate by wild-type protease. Analysis of genotypic and phenotypic resistance profiles of 28,000 clinical isolates demonstrated the presence of these NC/p1 cleavage site mutations in some clinical samples (codon 431 substitutions in 13%, codon 436 substitutions in 8%, and codon 437 substitutions in 10%). Moreover, these cleavage site substitutions were highly significantly associated with reduced susceptibility to PI in clinical isolates lacking primary protease mutations. Furthermore, we used data from a clinical trial (NARVAL, ANRS 088) to demonstrate that these NC/p1 cleavage site changes are associated with virological failure during PI therapy. CONCLUSIONS: HIV can use an alternative mechanism to become resistant to PI by changing the substrate instead of the protease. Further studies are required to determine to what extent cleavage site mutations may explain virological failure during PI therapy.
Assuntos
Farmacorresistência Viral/genética , Inibidores da Protease de HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/genética , Mutação , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Linhagem Celular , Códon/genética , Mutação da Fase de Leitura , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Genoma Viral , Protease de HIV/genética , Protease de HIV/metabolismo , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , Humanos , Immunoblotting , Dados de Sequência Molecular , RNA Viral/genética , Ritonavir/farmacologia , Especificidade por Substrato , TransfecçãoRESUMO
BACKGROUND: HIV-HBV-coinfected individuals who need to be treated only for their HBV infection have limited therapeutic options, since most approved anti-HBV agents have a risk of selecting for drug-resistant HIV mutants. In vivo data are inconclusive as to whether telbivudine (LdT) may exert antiviral effects against HIV. Thus, we investigated in further detail the antiviral activity and the biochemical properties of LdT against HIV-1. METHODS: To investigate the activity of LdT against HIV-1 in humans we analysed viral dynamics and genotypic and phenotypic resistance development in two HIV-HBV-coinfected individuals with no prior antiviral exposure. To investigate the activity of LdT against HIV-1 in vitro, LdT susceptibility for HIV-1 wild-type strains as well as drug-resistant strains was determined. Furthermore, we studied whether the 5'-triphosphate form of LdT (LdT-TP) can act as a substrate for wild-type HIV-1 RT. RESULTS: In the two patients studied, LdT treatment did not result in a significant decline of HIV-1 RNA load nor in selection of genotypic or phenotypic resistance in HIV-1 RT. In vitro virological analyses demonstrated that LdT had no activity (50% effective concentration >100 µM) against wild type HIV and drug-resistant variants. Biochemical analyses demonstrated that LdT-TP is not incorporated by wild-type HIV-1 RT. CONCLUSIONS: Based on the in vivo and in vitro evidence obtained in this study, we conclude that LdT has no anti-HIV-1 activity and is currently the only selective anti-HBV agent among the five FDA-approved nucleoside/nucleotide analogues for treatment of HBV infections in HIV-infected individuals.
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Antivirais/farmacologia , Farmacorresistência Viral , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Hepatite B Crônica/tratamento farmacológico , Nucleosídeos/farmacologia , Pirimidinonas/farmacologia , Adulto , Antivirais/uso terapêutico , Contagem de Linfócito CD4 , Linhagem Celular , Coinfecção , DNA Viral/sangue , Genótipo , Células HEK293 , Infecções por HIV/complicações , Infecções por HIV/virologia , HIV-1/fisiologia , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/complicações , Hepatite B Crônica/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Nucleosídeos/sangue , Nucleosídeos/uso terapêutico , Fenótipo , Pirimidinonas/sangue , Pirimidinonas/uso terapêutico , RNA Viral/sangue , Telbivudina , Timidina/análogos & derivados , Carga ViralRESUMO
HIV protease plays a crucial role in the viral life cycle and is essential for the generation of mature infectious virus particles. Detailed knowledge of the structure of HIV protease and its substrate has led to the design of specific HIV protease inhibitors. Unfortunately, resistance to all protease inhibitors (PIs) has been observed and the genetic basis of resistance has been well documented over the past 15 years. The arrival of the early PIs was a pivotal moment in the development of antiretroviral therapy. They made possible the dual class triple combination therapy that became known as HAART. However, the clinical utility of the first generation of PIs was limited by low bioavailability and high pill burdens, which ultimately reduced adherence and limited long-term viral inhibition. When therapy failure occurred multiple protease resistance mutations were observed, often resulting in broad class resistance. To combat PI-resistance development, second-generation approaches have been developed. The first advance was to increase the level of existing PIs in the plasma by boosting with ritonavir. The second was to develop novel PIs with high potency against the known PI-resistant HIV protease variants. Both approaches increased the number of protease mutations required for clinical resistance, thereby raising the genetic barrier. This review provides an overview of the history of protease inhibitor therapy, its current status and future perspectives. It forms part of a special issue of Antiviral Research marking the 25th anniversary of antiretroviral drug discovery and development, vol. 85, issue 1, 2010.
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Terapia Antirretroviral de Alta Atividade/métodos , Infecções por HIV/tratamento farmacológico , Inibidores da Protease de HIV/farmacologia , Inibidores da Protease de HIV/uso terapêutico , HIV/efeitos dos fármacos , Farmacorresistência Viral , Inibidores da Protease de HIV/história , História do Século XX , História do Século XXI , HumanosRESUMO
BACKGROUND: Norovirus, Rotavirus group A, Astrovirus, Sapovirus and Adenovirus serotypes 40 and 41, are common causes of gastroenteritis. Conventional diagnosis of these causative agents is based on antigen detection and electron microscopy. OBJECTIVE: To improve the diagnostic possibilities for viral gastroenteritis, two internally controlled multiplex real-time PCRs have been developed. STUDY DESIGN: Individual real-time PCRs were developed and optimized for the specific detection of Norovirus genogroup I, Norovirus genogroup II, Rotavirus group A, Astrovirus, Adenovirus group F and Sapovirus. Subsequently, the PCRs were combined to two multiplex PCR reactions. The multiplex assays were clinically evaluated using 239 fecal samples submitted to our laboratory over a 1-year period for the routine detection of Rotavirus and/or Adenovirus antigens using the Vikia(®) Rota/Adeno test (bioMérieux, Boxtel, The Netherlands). RESULTS: In general, the multiplex real-time PCR assays showed comparable sensitivity and specificity to the individual assays. A retrospective clinical evaluation showed increased pathogen detection in samples from 14% using conventional methods to 45% using PCR. Subsequently, the assay was implemented as a routine diagnostic tool. From September 2007 up to December 2009, 486 positive results were obtained in 1570 samples (31%) analyzed. Norovirus genogroup II was found the most frequently (61.1%), followed by Adenovirus (9.9%), Rotavirus (9.3%), Astrovirus (6.0%), Norovirus genogroup I (3.3%) and Sapovirus (0.4%). CONCLUSIONS: Two internally controlled multiplex real-time PCR assays for the simultaneous detection of Astrovirus, Adenovirus group F, Rotavirus, Norovirus genogroups I and II and Sapovirus have shown significant improvement in the diagnosis of viral gastroenteritis.
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Gastroenterite/virologia , Reação em Cadeia da Polimerase/métodos , Virologia/métodos , Viroses/diagnóstico , Adenoviridae/isolamento & purificação , Pré-Escolar , Fezes/virologia , Humanos , Lactente , Mamastrovirus , Norovirus/isolamento & purificação , Reação em Cadeia da Polimerase/normas , Padrões de Referência , Rotavirus/isolamento & purificação , Sapovirus/isolamento & purificação , Sensibilidade e Especificidade , Viroses/virologiaRESUMO
PURPOSE OF REVIEW: Several alternative mechanisms that cause protease inhibitor resistance have been proposed. A summary of the proposed mechanisms and the status regarding their clinical relevance is given. RECENT FINDINGS: At this moment only changes in the cleavage sites of protease (either alone or in the background of protease mutations) have been associated with phenotypic changes in IC50 and virological failure. SUMMARY: Further studies are necessary to unravel the mechanism, the clinical relevance and potential effect of transmission of these cleavage site changes.
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PURPOSE OF REVIEW: This review focuses on the evolution of protease inhibitor resistance and replication capacity in the presence and absence of protease inhibitor pressure. RECENT FINDINGS: Classically, HIV escapes through mutations in the protease itself causing a decrease in affinity to the inhibitor, leading to resistance. These changes also affect the binding of the enzyme to the natural substrate, and as a consequence cause a decrease in replication capacity of the virus. Continuous replication of these viruses may result in the acquisition of compensatory changes, which will fixate the drug-resistant variant in the viral population. Furthermore, novel treatment strategies have been developed to combat the development of classic protease inhibitor resistance. Using these strategies, the development of resistance in the viral protease is blocked because single or double mutations do not confer significant resistance. Alternative protease inhibitor resistance pathways are described, which enable the virus to escape these novel strategies. SUMMARY: Suboptimal protease inhibitor pressure clearly results in the selection of mutations conferring resistance and in the acquisition of mutations compensating the initial reduction in viral replicative capacity. The major implications of the selection of these compensatory changes on evolution in the absence of protease inhibitor pressure are discussed.
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OBJECTIVE: To investigate the mechanism explaining the persistence of human immunodeficiency virus (HIV) type 1 variants with multiple protease inhibitor (PI)-resistance mutations in the absence of PI therapy. METHODS: Longitudinal genotypic analyses were performed on sequential samples obtained from 2 HIV-1-infected patients who had stopped PI therapy for 4 years. Replication capacity (RC) was determined using recombinant viruses. Subsequently, the effect that changing individual protease mutations back to wild type has on RC was analyzed. RESULTS: We observed prolonged persistence (up to 4 years) of viruses with multiple protease mutations after PI therapy was stopped, despite the fact that the RC of the viruses was severely reduced. Forcing the virus to evolve toward wild type by changing individual protease mutations to wild type was unsuccessful, because all variants displayed a decreased RC in comparison with that of their predecessors. CONCLUSIONS: We propose compensatory fixation as a mechanism for the in vivo persistence of variants with multiple PI-resistance mutations in the absence of PI therapy. Viruses with multiple PI mutations have (partially) compensated for the initial loss in RC. Therefore, reversion of a single mutation causes a (further) reduction in RC and, as a consequence, the route to wild type is blocked.
Assuntos
Evolução Molecular , Infecções por HIV/virologia , Inibidores da Protease de HIV/uso terapêutico , Protease de HIV/genética , HIV-1/fisiologia , Sequência de Aminoácidos , Linhagem Celular Tumoral , Farmacorresistência Viral Múltipla/genética , Variação Genética , Infecções por HIV/tratamento farmacológico , Inibidores da Protease de HIV/farmacologia , HIV-1/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , Mutação , Inibidores da Transcriptase Reversa/uso terapêutico , Alinhamento de Sequência , Replicação Viral , Suspensão de TratamentoRESUMO
Little is known about the factors which drive the evolution of protease inhibitor-resistant human immunodeficiency virus type-1 in the absence of drugs. To examine if viral replicative capacity (RC) is an important determinant, we performed in vitro evolution experiments in the absence of drugs with a unique panel of 6 drug-resistant human immunodeficiency virus type-1 recombinant protease variants with a range of different RC. The experiments revealed that an increase in viral RC was indeed an important determinant of evolution. Initial protease inhibitor-resistant viruses with only a few protease mutations and a lowered RC evolved into viruses with an increased RC, either by reversion of primary resistance mutations or by the acquisition of compensatory mutations. For these viruses with a lowered RC, higher fitness peaks are most likely available in the sequence space. Evolution of these viruses in the absence of drugs will therefore drive them to new fitness peaks. In contrast, viruses with an RC comparable to wild type or even higher than wild type did not show any evolution. In the case of these viruses, it is not so likely that higher fitness peaks are present within the sequence space, and therefore, these variants will persist in the absence of drug pressure.