Isolation of Oropouche Virus from Febrile Patient, Ecuador

Wise, Emma L.; Pullan, Steven T.; Márquez, Sully; Paz, Verónica; Mosquera, Juan; Zapata, Sonia; Jackson, Simon K.; Fejér, György; Trueba, Gabriel; Logue, Christopher

doi:10.3201/eid2405.171569

Cited by 28 publications

(35 citation statements)

References 10 publications

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“…An existing S segment assay (7) failed to detect OROV RNA in an Ecuadorian clinical sample that tested positive by both metagenomic sequencing and virus isolation ( Figure 1) (6). The reverse primer (OROV R) (7) had two mismatches to the novel OROV/EC/Esmeraldas/087/2016 sequence.…”

Section: Development Of An Orov Rrt-pcrmentioning

confidence: 99%

“…Metagenomic sequencing (untargeted sequencing of all genetic material in a sample) is an attractive alternative to conventional molecular detection methods as it does not require prior knowledge of pathogen genome sequences. For example, an established OROV rRT-PCR (7) was unable to detect OROV in the plasma of an Ecuadorian febrile patient who tested positive using metagenomic sequencing and virus isolation (6). This highlights the potential for misdiagnosis when screening for pathogens that lack a large collection of reference sequences on which to base targeted amplification.…”

Section: Introductionmentioning

confidence: 99%

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

Wise

Márquez

Mellors

et al. 2019

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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 rRT-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 rRT-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.

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Section: Development Of An Orov Rrt-pcrmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Wise

Márquez

Mellors

et al. 2019

Preprint

Self Cite

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“…Six strains of OROV were isolated independently via one passage in Vero cells, from febrile patient sera sampled in 2016 in the Esmeraldas region of Ecuador. RNA was extracted and six complete genome sequences were generated using a metagenomic approach as described previously (14). Briefly, cDNA was prepared using a Sequence Independent Single Primer Amplification (SISPA) approach (27), cDNA sequencing libraries were prepared using the Nextera XT Kit (Illumina), and sequencing was performed on a MiSeq (Illumina) using 150 nucleotide paired-end runs.…”

Section: Sampling and Sequencing Of Orov Isolates From Ecuadormentioning

confidence: 99%

“…Our results show a complex and dynamic picture of the evolution of OROV in the Americas, shaped by reassortment, spatial structure, and natural selection. Our report incorporates new OROV genome sequences obtained from febrile patients in the province of Esmeraldas in northern Ecuador, representing the first direct detection and whole genome sequencing of the virus in the country (14). Previous prospective studies have suggested that OROV may have been circulating in both the coastal and Amazonian provinces of Ecuador (18,70) shown for each plot.…”

Section: Structural Analysis Of Orov Proteins and Sites Under Selectionmentioning

confidence: 99%

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The evolutionary dynamics of Oropouche Virus (OROV) in South America

Gutierrez

Wise

Pullan

et al. 2019

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The Amazon basin is host to numerous arthropod-borne viral pathogens that cause febrile disease in humans. Among these, Oropouche orthobunyavirus (OROV) is a relatively understudied member of the Peribunyavirales that causes periodic outbreaks in human populations in Brazil and other South American countries. Although several studies have described the genetic diversity of the virus, the evolutionary processes that shape the viral genome remain poorly understood. Here we present a comprehensive study of the genomic dynamics of OROV that encompasses phylogenetic analysis, evolutionary rate estimates, inference of natural selective pressures, recombination and reassortment, and structural analysis of OROV variants. Our study includes all available published sequences, as well as a set of new OROV genomes sequences obtained from patients in Ecuador, representing the first set of viral genomes from this country. Our results show that differing evolutionary processes on the three segments that encompass the viral genome lead to variable evolutionary rates and TMRCAs that could be explained by cryptic reassortment. We also present the discovery of previously unobserved putative N-linked glycosylation sites, and codons which evolve under positive selection on the viral surface proteins, and discuss the potential role of these features in the evolution of the virus through a combined phylogenetic and structural approach.

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