Dataset Information

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.

SUBMITTER: Wise EL

PROVIDER: S-EPMC6994106 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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

Wise Emma L EL Márquez Sully S Mellors Jack J Paz Verónica V Atkinson Barry B Gutierrez Bernardo B Zapata Sonia S Coloma Josefina J Pybus Oliver G OG Jackson Simon K SK Trueba Gabriel G Fejer Gyorgy G Logue Christopher H CH Pullan Steven T ST

PLoS neglected tropical diseases 20200121 1

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 coh ...[more]

PMID: 31961856

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Project description:Spoilage and pathogenic spore-forming bacteria are a major cause of concern for producers of dairy products. Traditional agar-based detection methods employed by the dairy industry have limitations with respect to their sensitivity and specificity. The aim of this study was to identify low-abundance sporeformers in samples of a powdered dairy product, whey powder, produced monthly over 1 year, using novel culture-independent shotgun metagenomics-based approaches. Although mesophilic sporeformers were the main target of this study, in one instance thermophilic sporeformers were also targeted using this culture-independent approach. For comparative purposes, mesophilic and thermophilic sporeformers were also tested for within the same sample using culture-based approaches. Ultimately, the approaches taken highlighted differences in the taxa identified due to treatment and isolation methods. Despite this, low levels of transient, mesophilic, and in some cases potentially pathogenic sporeformers were consistently detected in powder samples. Although the specific sporeformers changed from one month to the next, it was apparent that 3 groups of mesophilic sporeformers, namely, Bacillus cereus, Bacillus licheniformis/Bacillusparalicheniformis, and a third, more heterogeneous group containing Brevibacillus brevis, dominated across the 12 samples. Total thermophilic sporeformer taxonomy was considerably different from mesophilic taxonomy, as well as from the culturable thermophilic taxonomy, in the one sample analyzed by all four approaches. Ultimately, through the application of shotgun metagenomic sequencing to dairy powders, the potential for this technology to facilitate the detection of undesirable bacteria present in these food ingredients is highlighted.IMPORTANCE The ability of sporeformers to remain dormant in a desiccated state is of concern from a safety and spoilage perspective in dairy powder. Traditional culturing techniques are slow and provide little information without further investigation. We describe the identification of mesophilic sporeformers present in powders produced over 1 year, using novel shotgun metagenomic sequencing. This method allows detection and identification of possible pathogens and spoilage bacteria in parallel. Strain-level analysis and functional gene analysis, such as identification of toxin genes, were also performed. This approach has the potential to be of great value with respect to the detection of spore-forming bacteria and could allow a processor to make an informed decision surrounding process changes to reduce the risk of spore contamination.

Dataset Information

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

Publications

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

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