RESUMO
Higher accessibility and decreasing costs of next generation sequencing (NGS), availability of commercial kits, and development of dedicated analysis pipelines, have allowed an increasing number of laboratories to adopt this technology for HIV drug resistance (HIVDR) genotyping. Conventional HIVDR genotyping is traditionally carried out using population-based Sanger sequencing, which has a limited capacity for reliable detection of variants present at intra-host frequencies below a threshold of approximately 20%. NGS has the potential to improve sensitivity and quantitatively identify low-abundance variants, improving efficiency and lowering costs. However, some challenges exist for the standardization and quality assurance of NGS-based HIVDR genotyping. In this paper, we highlight considerations of these challenges as related to laboratory, clinical, and implementation of NGS for HIV drug resistance testing. Several sources of variation and bias occur in each step of the general NGS workflow, i.e., starting material, sample type, PCR amplification, library preparation method, instrument and sequencing chemistry-inherent errors, and data analysis options and limitations. Additionally, adoption of NGS-based HIVDR genotyping, especially for clinical care, poses pressing challenges, especially for resource-poor settings, including infrastructure and equipment requirements and cost, logistic and supply chains, instrument service availability, personnel training, validated laboratory protocols, and standardized analysis outputs. The establishment of external quality assessment programs may help to address some of these challenges and is needed to proceed with NGS-based HIVDR genotyping adoption.
Assuntos
Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/genética , Sequenciamento de Nucleotídeos em Larga Escala , Animais , Avaliação Pré-Clínica de Medicamentos , Genótipo , Infecções por HIV/tratamento farmacológico , HIV-1/isolamento & purificação , HIV-1/fisiologia , HumanosRESUMO
Sequence analysis can be used to evaluate transmission networks. We have used retrospective samples to examine two HIV-1 transmission networks established by contact tracing. Regions of the HIV-1 region representing segments of gag and env were amplified by RT-PCR from frozen plasma samples and the sequence of each PCR product was determined. Within one of the networks (composed of 38 subjects) we found only a subset of the tested sequence clusters was consistent with the reported epidemiological linkage. Of 15 presumed transmission events where sequence data were available, 9 could be rejected either by subtype mismatch or by phylogenetic tests. In the other network (composed of 89 subjects) we were able to assess sequences for 26 presumed transmission events, 18 of which were rejected based on subtype discordance. Long lags in time between the time of transmission and the time of sequence sampling (ranging from 2 to 18 years) may limit the sensitivity for the detection of sequence linkage. Also, superinfection and incomplete epidemiological information are other factors that will limit the concordance of phylogenetic reconstruction and reported epidemiological linkage.
Assuntos
Busca de Comunicante/métodos , Transmissão de Doença Infecciosa , Infecções por HIV/transmissão , HIV-1/genética , Filogenia , RNA Viral/classificação , Análise por Conglomerados , Cuba/epidemiologia , Evolução Molecular , Genes env/genética , Genes gag/genética , Infecções por HIV/epidemiologia , Humanos , Epidemiologia Molecular , Dados de Sequência Molecular , Estudos RetrospectivosRESUMO
Venezuelan equine encephalitis virus replicon particles (VRP) were engineered to express different forms of SIV Gag to compare expression in vitro, formation of intra- and extracellular structures and induction of humoral and cellular immunity in mice. The three forms examined were full-length myristylated SIV Gag (Gagmyr+), full-length Gag lacking the myristylation signal (Gagmyr-) or a truncated form of Gagmyr- comprising only the matrix and capsid domains (MA/CA). Comparison of VRP-infected primary mouse embryo fibroblasts, mouse L929 cells and primate Vero cells showed comparable expression levels for each protein, as well as extracellular virus-like particles (VRP-Gagmyr+) and distinctive cytoplasmic aggregates (VRP-Gagmyr-) with each cell type. VRP were used to immunize BALB/c mice, and immune responses were compared using an interferon (IFN)-gamma ELISPOT assay and a serum antibody ELISA. Although all three VRP generated similar levels of IFN-gamma-producing cells at 1 week post-boost, at 10 weeks post-boost the MA/CA-VRP-induced response was maintained at a significantly higher level relative to that induced by Gagmyr+-VRP. Antibody responses to MA/CA-VRP and Gagmyr+-VRP were not significantly different.
Assuntos
Vírus da Encefalite Equina Venezuelana/genética , Produtos do Gene gag/imunologia , Vetores Genéticos/genética , Vírus da Imunodeficiência Símia/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Embrião de Mamíferos/citologia , Ensaio de Imunoadsorção Enzimática , Feminino , Fibroblastos , Produtos do Gene gag/química , Antígenos H-2/imunologia , Interferon gama/biossíntese , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos , Microscopia Eletrônica de Transmissão , Modelos Animais , Gravidez , Replicon/genética , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Células Vero , Vacinas Virais/genéticaRESUMO
VEE replicon particles (VRP), non-propagating vaccine vectors derived from Venezuelan equine encephalitis virus (VEE), were engineered to express immunogens from the cloned isolate SIVsmH-4, combined in a vaccine cocktail and inoculated subcutaneously to immunize rhesus macaques. The virulent, uncloned challenge stock, SIVsmE660, represented a type of heterologous challenge and the intrarectal challenge modeled infection across a mucosal surface. Prechallenge neutralizing antibodies against SIVsmH-4 were induced in all vaccinates, and a prechallenge cellular immune response could be detected in one of six. Post-challenge, virus loads were reduced at the peak, at set point and at termination (41 weeks post-challenge), although these differences did not reach statistical significance. Significantly elevated levels of CD4+ T cells were observed post-challenge. A strong correlation was noted between a net increase in CD4+ T cell count and lowered virus load at set point.
Assuntos
Vírus da Encefalite Equina Venezuelana/genética , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Contagem de Linfócito CD4 , Modelos Animais de Doenças , Vetores Genéticos , Imunidade Celular , Injeções Subcutâneas , Macaca mulatta , Testes de Neutralização , Replicon/genética , Vacinas contra a SAIDS/administração & dosagem , Vacinas contra a SAIDS/genética , Linfócitos T Citotóxicos/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Carga Viral , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
Se demostró la eficacia de la enzima transcriptasa inversa SensiScript para obtener material genético útil en la secuenciación de ácido nucleico de VIH-1, a partir de sueros colectados entre 1989 y 1998 y conservados a temperatura subóptima. Con el empleo de la enzima SensiScript se obtuvo amplificación del ARN del VIH-1 en 86,5 % de las muestras estudiadas, comparado con 20 % al utilizar la enzima AMV-RT. En 13,5 % de las muestras no se obtuvo amplificación con ninguna de las 2 enzimas empleadas.
The efficiency of SensiScript reverse transciptase to obtain useful genetic material in the sequencing of the nucleic acid from HIV-1, starting from sera collected between 1989 and 1998 and kept at suboptimal temperatures, was proved. On using the SensiScript enzyme it was obtained an amplification of the RNA of the HIV-1 in 86.5 % of the studied samples, compared with 20 % on using the AMV-RT enzyme . No amplification was obtained in 13.5 % of the studied samples with any of the 2 enzymes used.
Assuntos
Humanos , HIV-1 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Viral/isolamento & purificação , Sangue/virologia , Criopreservação , Fatores de TempoRESUMO
The efficiency of SensiScript reverse transciptase to obtain useful genetic material in the sequencing of the nucleic acid from HIV-1, starting from sera collected between 1989 and 1998 and kept at suboptimal temperatures, was proved. On using the SensiScript enzyme it was obtained an amplification of the RNA of the HIV-1 in 86.5 % of the studied samples, compared with 20 % on using the AMV-RT enzyme . No amplification was obtained in 13.5 % of the studied samples with any of the 2 enzymes used.