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Real-time RT-PCR for Mayaro virus detection in plasma and urine.

ABSTRACT: BACKGROUND:Mayaro virus (MAYV) causes an acute febrile illness which can be difficult to differentiate from dengue or chikungunya. MAYV RNA can be detected in plasma during the first 3-5days of illness, but only a single rRT-PCR has been fully evaluated in the literature. OBJECTIVES:To develop an rRT-PCR for MAYV and evaluate assay performance using human plasma and urine samples spiked with different MAYV strains. STUDY DESIGN:A MAYV rRT-PCR targeting a region of the 5'UTR and nsp1 gene was designed from the alignment of all complete-genome MAYV sequences to be compatible with existing laboratory protocols. The assay was evaluated using human samples spiked with six MAYV strains, including strains from each of the three genotypes. RESULTS:The linear range of the MAYV rRT-PCR extended from 1.0 to 8.0 log10copies/?L, and the lower limit of 95% detection was 8.2copies/?L. No detection was observed when the MAYV rRT-PCR was tested with genomic RNA from related arboviruses. The assay demonstrated linear amplification of all 6 MAYV strains when spiked into human plasma samples as well as 2 strains spiked into urine. CONCLUSIONS:We report the design and evaluation of an rRT-PCR for MAYV. Given the concern for MAYV emergence in the Americas and the few molecular tests that have been evaluated in the literature, this assay should provide a useful diagnostic for patients with an acute febrile illness.

SUBMITTER: Waggoner JJ

PROVIDER: S-EPMC5742299 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Real-time RT-PCR for Mayaro virus detection in plasma and urine.

Waggoner Jesse J JJ Rojas Alejandra A Mohamed-Hadley Alisha A de Guillén Yvalena Arévalo YA Pinsky Benjamin A BA

Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology 20171121

<h4>Background</h4>Mayaro virus (MAYV) causes an acute febrile illness which can be difficult to differentiate from dengue or chikungunya. MAYV RNA can be detected in plasma during the first 3-5days of illness, but only a single rRT-PCR has been fully evaluated in the literature.<h4>Objectives</h4>To develop an rRT-PCR for MAYV and evaluate assay performance using human plasma and urine samples spiked with different MAYV strains.<h4>Study design</h4>A MAYV rRT-PCR targeting a region of the 5'UTR ...[more]

PMID: 29172075

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Project description:Epizootic haemorrhagic disease virus (EHDV) is an emerging arboviral pathogen of wild and domestic ruminants worldwide. It is closely related to bluetongue virus (BTV) and is transmitted by adult females of competent Culicoides vector species. The EHDV genome consists of ten linear double-stranded (ds)RNA segments, encoding five non-structural and seven structural proteins. Genome-segment reassortment contributes to a high level of genetic variation in individual virus strains, particularly in the areas where multiple and distinct virus lineages co-circulate. In spite of the relatively close relationship between BTV and EHDV herd-immunity to BTV does not appear to protect against the introduction and infection of animals by EHDV. Although EHDV can cause up to 80% morbidity in affected animals, vaccination with the homologous EHDV serotype is protective. Outer-capsid protein VP2, encoded by Seg-2, is the most variable of the EHDV proteins and determines both the specificity of reactions with neutralizing antibodies and consequently the identity of the eight EHDV serotypes. In contrast, VP6 (the viral helicase), encoded by Seg-9, is highly conserved, representing a virus species/serogroup-specific antigen. We report the development and evaluation of quantitative (q)RT-PCR assays targeting EHDV Seg-9 that can detect all EHDV strains (regardless of geographic origin/topotype/serotype), as well as type-specific assays targeting Seg-2 of the eight EHDV serotypes. The assays were evaluated using orbivirus isolates from the 'Orbivirus reference collection' (ORC) at The Pirbright Institute and were shown to be EHDV pan-reactive or type-specific. They can be used for rapid, sensitive and reliable detection and identification (typing) of EHDV RNA from infected blood, tissue samples, homogenized Culicoides, or tissue culture supernatant. None of the assays detected RNA from closely related but heterologous orbiviruses, or from uninfected host animals or cell cultures. The techniques presented could be used for both surveillance and vaccine matching (serotype identification) as part of control strategies for incursions in wild and domestic animal species.

Dataset Information

Real-time RT-PCR for Mayaro virus detection in plasma and urine.

Publications

Real-time RT-PCR for Mayaro virus detection in plasma and urine.

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