Comprehensive evaluation of nucleic acid amplification methods widely used for generic detection of sandfly-borne phleboviruses

Author:

Polat Ceylan12ORCID,Ayhan Nazli23ORCID,Ergünay Koray1456,Charrel Remi N.27

Affiliation:

1. Department of Medical Microbiology, Faculty of Medicine, Hacettepe University, Ankara, Turkey

2. Unité des Virus Emergents, Aix Marseille University, Marseille, France

3. National Reference Center for Arboviruses, National Institute of Health, and Medical Research (Inserm) and French Armed Forces Biomedical Research Institute (IRBA), Marseille, France

4. Walter Reed Biosystematics Unit (WRBU), Smithsonian Institution Museum Support Center, Suitland, Maryland, USA

5. One Health Branch, Walter Reed Army Institute of Research (WRAIR), Silver Spring, Maryland, USA

6. Department of Entomology, Smithsonian Institution-National Museum of Natural History (NMNH), Washington, DC, USA

7. Laboratoire des Infections Virales Aigues et Tropicales, Pole des Maladies Infectieuses, AP-HM Hopitaux Universitaires de Marseille, Marseille, France

Abstract

ABSTRACT Sandfly-borne phleboviruses (SBPs), which cause sandfly fever, aseptic meningitis, encephalitis, and meningoencephalitis, are emerging pathogens of major public health concern. Virus nucleic acid testing is essential for SBP diagnosis, especially in the early stages of infection, and for the discovery of novel SBPs. The efficacy of utilizing generic primers that target conserved nucleotide sequences for the detection of both known and novel SBPs has not been extensively evaluated. We aimed to compare and evaluate the performance of five generic primer sets, widely used to detect S- and L-segments of arthropod-borne phleboviruses and designed as singleplex ( n = 3) and nested ( n = 2) formats, including both well-known and recently characterized 15 Old World virus strains. Furthermore, we performed in silico analysis to assess the detection capabilities of these generic primer sets. The initial evaluation of previously published generic primer sets for SBP detection yielded two singleplex primer sets with the potential to be adapted for use in real-time or high-throughput detection settings. Studies are ongoing to develop and further optimize a preliminary assay and test various hosts and vectors to assess their capacity to detect known and novel viruses. IMPORTANCE Virus nucleic acid testing is the primary diagnostic method, particularly in the early stages of illness. Virus-specific or syndromic tests are widely used for this purpose. The use of generic primers has had a considerable impact on the discovery, identification, and detection of Old World sandfly-borne phleboviruses (OWSBP). The study is significant because it is the first to carry out a comparative evaluation of all published OWSBP generic primer sets.

Funder

European Commission

UK Research and Innovation

EC | Horizon 2020 Framework Programme

Publisher

American Society for Microbiology

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