RESUMO
Two one-step real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for the detection of Zika virus (ZIKV) were developed, based on two different primer design approaches: (1) open source, based on a combination of sequence diversity clustering (phylogeny and principal component analysis) and LAVA algorithm, using 45 whole genome ZIKV sequences retrieved from the National Center for Biotechnology Information (NCBI) database; (2) standard software for LAMP primer design (Primer Explorer V4), using 59 sequences of the ZIKV 3' UTR. The assays were firstly evaluated with External Quality Assessment panels from INSTAND e.V. (Germany) and EVD-LabNet (The Netherlands) including 4 and 12 unknown samples, respectively, and secondly, with 9 human, mosquito, and monkey ZIKV isolates from Africa (Senegal, Ivory Coast, and Uganda) and America (Brazil). The limit of detection as determined by probit analysis was 181 molecules for both RT-LAMP assays, and 100% reproducibility in the assays was obtained for 103 molecules (4/8 repetitions were positive for 102 molecules). Both assays were specific, amplifying only ZIKV RNA and not cross-detecting other arboviruses included in this study.
Assuntos
Algoritmos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Software , Infecção por Zika virus/diagnóstico , Zika virus/genética , África , Animais , Brasil , Células Cultivadas , Culicidae/virologia , Alemanha , Haplorrinos , Humanos , Ensaio de Proficiência Laboratorial , Limite de Detecção , Técnicas de Diagnóstico Molecular/normas , Países Baixos , Técnicas de Amplificação de Ácido Nucleico/normas , RNA Viral/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Zika virus/isolamento & purificação , Infecção por Zika virus/veterináriaRESUMO
Molecular detection of Zika virus (ZIKV) is a key element of outbreak management. Multiple PCR and isothermal ZIKV assays targeting different ZIKV sequences have been published. In this study, we compared a qRT-PCR, 2 RT-LAMP assays (based on different primer design approaches), and an RT-RPA for the detection of African and Asian/American lineages of ZIKV isolates from human, mosquito, and monkey. Results showed that RT-LAMP detected 100% of samples with a time threshold (Tt) of 18.01 ± 11.71 min while qRT-PCR detected 88.88% of samples with a Tt of 58.30 ± 16.58 min and RT-RPA 50% of samples with a Tt of 3.70 ± 0.44 min.
Assuntos
Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Infecção por Zika virus/diagnóstico , Zika virus/genética , Animais , Brasil , Células Cultivadas , Côte d'Ivoire , Culicidae , Haplorrinos , Humanos , Recombinases/metabolismo , Reprodutibilidade dos Testes , Senegal , Uganda , Zika virus/isolamento & purificação , Infecção por Zika virus/virologiaRESUMO
During 2015-2016, Cape Verde, an island nation off the coast of West Africa, experienced a Zika virus (ZIKV) outbreak involving 7,580 suspected Zika cases and 18 microcephaly cases. Analysis of the complete genomes of 3 ZIKV isolates from the outbreak indicated the strain was of the Asian (not African) lineage. The Cape Verde ZIKV sequences formed a distinct monophylogenetic group and possessed 1-2 (T659A, I756V) unique amino acid changes in the envelope protein. Phylogeographic and serologic evidence support earlier introduction of this lineage into Cape Verde, possibly from northeast Brazil, between June 2014 and August 2015, suggesting cryptic circulation of the virus before the initial wave of cases were detected in October 2015. These findings underscore the utility of genomic-scale epidemiology for outbreak investigations.
Assuntos
Microcefalia , Infecção por Zika virus , Zika virus , África Ocidental , Brasil/epidemiologia , Cabo Verde , Surtos de Doenças , Genômica , Humanos , Microcefalia/epidemiologia , Zika virus/genética , Infecção por Zika virus/epidemiologiaRESUMO
The differential diagnosis of dengue virus (DENV) and yellow fever virus (YFV) infections in endemic areas is complicated by nonspecific early clinical manifestations. In this study, we describe an internally controlled, multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) for the detection of DENV and YFV. The DENV-YFV assay demonstrated specific detection and had a dynamic range of 2.0-8.0 log10 copies/µL of eluate for each DENV serotype and YFV. Clinical performance was similar to a published pan-DENV assay: 48/48 acute-phase samples from dengue cases were detected in both assays. For YFV detection, mock samples were prepared with nine geographically diverse YFV isolates over a range of concentrations. The DENV-YFV assay detected 62/65 replicates, whereas 54/65 were detected using a reference YFV rRT-PCR. Given the reemergence of DENV and YFV in areas around the world, the DENV-YFV assay should be a useful tool to narrow the differential diagnosis and provide early case detection.
Assuntos
Vírus da Dengue/isolamento & purificação , Dengue/diagnóstico , Febre Amarela/diagnóstico , Vírus da Febre Amarela/isolamento & purificação , Dengue/virologia , Vírus da Dengue/genética , Diagnóstico Precoce , Humanos , Reação em Cadeia da Polimerase Multiplex , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Febre Amarela/virologia , Vírus da Febre Amarela/genéticaRESUMO
Rift Valley Fever virus (RVFV) is a member of Bunyaviridae family that causes a febrile disease affecting mainly ruminants and occasionally humans in Africa, with symptoms that range from mid to severe. RVFV has a tri-segmented ssRNA genome that permits reassortment and could generate more virulent strains. In this study, we reveal the importance of reassortment for RVFV evolution using viral gene genealogy inference and phylodynamics. We uncovered seven events of reassortment that originated RVFV lineages with discordant origins among segments. Moreover, we also found that despite similar selection regimens, the three segments have distinct evolutionary dynamics; the longer segment L evolves at a significant lower rate. Episodes of discordance between population size estimates per segment also coincided with reassortment dating. Our results show that RVFV segments are decoupled enough to have distinct demographic histories and to evolve under different molecular rates.