RESUMO
Rabies is a generally fatal encephalitis caused by a negative-sense single-stranded RNA lyssavirus transmitted to humans mainly from dog bite. Despite the recommendation by WHO and OIE to use the direct immunofluorescence test as standard method, molecular diagnostic assays like reverse transcription quantitative polymerase chain reaction (RT-qPCR) are increasing as a confirmatory method. However, both technologies are inaccessible in resource-limited settings. Moreover, the available point-of-need molecular assay is of poor detection limit for African strains. Herein, we developed a reverse transcription recombinase polymerase amplification (RT-RPA) assay as potential point-of-need diagnostic tool for rapid detection of various strains of rabies virus including locally isolated African strains. The sensitivity and specificity of the method was evaluated using a molecular RNA standard and different Rabies-related viruses belonging to the Rhabdoviridea family, respectively. The RABV-RPA performances were evaluated on isolates representative of the existing diversity and viral dilutions spiked in non-neural clinical specimen. The results were compared with RT-qPCR as a gold standard. The RABV-RPA detected down to 4 RNA molecules per reaction in 95% of the cases in less than 10 min. The RABV-RPA assay is highly specific as various RABV isolates were identified, but no amplification was observed for other member of the Rhabdoviridea family. The sample background did not affect the performance of the RABV-RPA as down to 11 RNA molecules were identified, which is similar to the RT-qPCR results. Our developed assay is suitable for use in low-resource settings as a promising alternative tool for ante-mortem rabies diagnosis in humans for facilitating timely control decisions.
Assuntos
Testes Imediatos/organização & administração , RNA Viral/genética , Vírus da Raiva/genética , Raiva/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Rhabdoviridae/genética , África/epidemiologia , Animais , Primers do DNA/síntese química , Primers do DNA/metabolismo , Cães , Corantes Fluorescentes/química , Humanos , Limite de Detecção , Testes Imediatos/economia , Raiva/epidemiologia , Raiva/transmissão , Raiva/virologia , Vírus da Raiva/isolamento & purificação , Recombinases/genética , Recombinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/economia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Rhabdoviridae/classificaçãoRESUMO
Host-parasite coevolution is considered to be an important factor in maintaining genetic variation in resistance to pathogens. Drosophila melanogaster is naturally infected by the sigma virus, a vertically transmitted and host-specific pathogen. In fly populations, there is a large amount of genetic variation in the transmission rate from parent to offspring, much of which is caused by major-effect resistance polymorphisms. We have found that there are similarly high levels of genetic variation in the rate of paternal transmission among 95 different isolates of the virus as in the host. However, when we examined a transmission-blocking gene in the host, we found that it was effective across virus isolates. Therefore, the high levels of genetic variation observed in this system do not appear to be maintained because of coevolution resulting from interactions between this host gene and parasite genes.
Assuntos
Drosophila melanogaster/virologia , Variação Genética , Interações Hospedeiro-Patógeno/genética , Locos de Características Quantitativas , Rhabdoviridae/genética , Animais , Primers do DNA/genética , Feminino , Modelos Lineares , Masculino , Cadeias de Markov , Método de Monte CarloRESUMO
The genomic RNA of the fish rhabdovirus infectious hematopoietic necrosis virus (IHNV) contains a small NV gene which is not present in any other rhabdoviruses characterized to date. We have constructed a plasmid which carries a full length cDNA copy of the IHNV NV gene between 2 RNA polymerase promoters such that plus-sense and minus-sense RNA transcripts of the IHNV NV gene can be synthesized. These were used to develop an RNase protection assay which was capable of detecting as little as 1-5 ng of NV mRNA or genomic RNA. RNA from tissue culture cells infected with 3 different strains of IHNV showed distinct, diagnostic cleavage patterns reflecting sequence variation in their NV genes.
