Oropouche virus cases identified in Ecuador using an optimised qRT-PCR informed by metagenomic sequencing

Abstract

Oropouche virus (OROV) is responsible for outbreaks of Oropouche fever in parts of South America. We recently identified and isolated OROV from a febrile Ecuadorian patient, however, a previously published qRT-PCR assay did not detect OROV in the patient sample. A primer mismatch to the Ecuadorian OROV lineage was identified from metagenomic sequencing data. We report the optimisation of an qRT-PCR assay for the Ecuadorian OROV lineage, which subsequently identified a further five cases in a cohort of 196 febrile patients. We isolated OROV via cell culture and developed an algorithmically-designed primer set for whole-genome amplification of the virus. Metagenomic sequencing of the patient samples provided OROV genome coverage ranging from 68-99%. The additional cases formed a single phylogenetic cluster together with the initial case. OROV should be considered as a differential diagnosis for Ecuadorian patients with febrile illness to avoid mis-diagnosis with other circulating pathogens.

Fig 1. Sensitivity analysis of three reverse primers used in the qRT-PCR assay.

Cq values are shown for ten-fold serial dilutions of OROV RNA, either from the prototype strain (GenBank KP026181.1), or the Ecuadorian strain OROV/EC/Esmeraldas/087/2016 (GenBank MF926352.1). Three separate experiments were performed, from which the means (plotted as data points) and standard deviations (indicated by error bars) were calculated. Reverse primer sequences (5’–3’) are given in the embedded table.

Fig 2. Cq values from 49 pathogen positive or potentially positive plasma samples, from a cohort of 196 screened by qRT-PCR.

Samples with a Cq value of 35–39.9 should be considered potentially positive because (except for the OROV sample) confirmatory tests were not performed. Lepto = Leptospira spp.

Fig 3. OROV genome coverage generated from six OROV-positive patient plasma samples, using a metagenomic sequencing approach.

Reads were mapped to reference sequences OROV/EC/Esmeraldas/087/2016 (GenBank accessions MF926352.1- MF926354.1). Panel A: OROV genome segment (S, M, L) coverage (%). qRT-PCR Cq value is given at the top of each plot. Coverage is defined as a minimum of 5 reads at any given position. Panel B: OROV genome coverage shown as the number of reads at each genomic position. Plots are separated by genome segment (S, M, L). Red dashed line indicates 5x coverage.

Fig 4. Genome coverage of two OROV strains (Ecuadorian strain OROV/EC/Esmeraldas/087/2016 Genbank MF926352.1—MF926354.1, and prototype strain Genbank KP026179.1—KP026181.1), sequenced using a MinION (Oxford Nanopore), from multiplex tiling RT-PCR amplicons.

Plots are separated by genome segment (S, M, L). Red dashed line indicates 20x coverage.