Courot, O. | Search bioRxiv

bioRxiv2026

Early-warning surveillance of West Nile and Usutu viruses in water and mosquito excreta using digital PCR

1.West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne pathogens maintained in bird-mosquito cycles and increasingly cause human and equine disease in temperate regions. As most infections are asymptomatic, surveillance based on clinical and veterinary reports provides delayed signals for vector control and equine vaccination strategies. We implemented, in 2024 and 2025, an operational environmental surveillance strategy combining molecular xenomonitoring of adult mosquito excreta with water sampling from wetlands and mosquito breeding sites, coupled to a standardized multiplex reverse transcription digital PCR workflow. Viral detection was performed using a multiplex digital PCR assay with characterized limits of blank, detection, and quantification, and low-level positives were confirmed by Sanger sequencing. This multi-matrix approach detected WNV RNA in southern France as early as 1 July 2024, 40 days before the first human case and 67 days before the first equine alert, and on 9 April 2025, a full 19 weeks ahead of the first human case that year. USUV RNA was similarly detected from 5 July 2024 and 22 April 2025, providing actionable early warnings to guide targeted vector control and preparedness. Across both years, WNV and USUV were detected 29 and 8 times among 396 samples in 2024, and 90 and 14 times among 815 samples in 2025, revealing previously unrecognized, cryptic virus circulation. These results demonstrate that integrating environmental surveillance provides a sensitive, proactive framework for the early detection of emerging Culex-borne arboviruses, offering precious lead time for public one-health strategies.

ecology↗

bioRxiv2024

First application of digital-PCR in oenology for the specific detection of intact cells of Brettanomyces bruxellensis in the winemaking process

Wine is a complex matrix resulting from a fermentation process carried out by specific microbial communities. These communities can be in competition and the development of some microorganisms, as the yeast Brettanomyces bruxellensis, can impact the fermentation process and lead to organoleptic alterations of wine. To manage this risk, microbiological diagnostic methods as microscopic observations, qPCR or flow cytometry are already used in oenology, but remain either not specific enough, or tedious. In this context, IAGE (Ingenierie et Analyses en Genetique Environnementale) has developed the first digital-PCR system enabling the detection and quantification of B. bruxellensis during the whole winemaking process. Furthermore, wine DNA extraction was optimized to enable a representative and sensitive analysis of B. bruxellensis intact cells, as well as an easy-to-implement protocol to cope with the increasing number of samples to analyze. The IAGE workflow for B. bruxellensis quantification has been proven to be successful when analyzing naturally-contaminated samples during the different steps of the winemaking process and offers a robust method to oenologists for appropriate treatments and risk management in wine cellars. HighlightsO_LIDevelopment of a dPCR method led to a highly-specific analysis of B. bruxellensis intact cells in different steps of the winemaking process. C_LIO_LIDNA extraction method has been optimized to be robust across various types of wine with varying concentrations of inhibitors, as well as throughout different stages of the wine making process. C_LIO_LIThe complete process was proven successful in analyzing a large number of naturally-contaminated samples, giving results in less than 48 hours. C_LI

microbiology↗