A high-throughput, polymerase-targeted RT-PCR for broad detection of mammalian filoviruses.

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  • Additional Information
    • Source:
      Publisher: ASM Press Country of Publication: United States NLM ID: 101634614 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 2165-0497 (Electronic) Linking ISSN: 21650497 NLM ISO Abbreviation: Microbiol Spectr Subsets: MEDLINE
    • Publication Information:
      Original Publication: Washington, DC : ASM Press, 2013-
    • Subject Terms:
      Filoviridae*/genetics ; Filoviridae*/isolation & purification ; Filoviridae*/classification ; Filoviridae Infections*/virology ; Reverse Transcriptase Polymerase Chain Reaction*/methods; Humans ; Animals ; RNA, Viral/genetics ; Sensitivity and Specificity ; High-Throughput Nucleotide Sequencing/methods ; Mammals/virology ; High-Throughput Screening Assays/methods ; Viral Proteins/genetics
    • Abstract:
      Filoviruses are some of the most lethal viruses in the modern world, and increasing numbers of filovirus species and genera have been discovered in recent years. Despite the potential severity of filovirus outbreaks in the human population, comparably few sensitive pan-filovirus RT-PCR assays have been described that might facilitate early detection and prevention. Here, we present a new pan-filovirus RT-PCR assay targeting the L polymerase gene for detection of all known mammalian filoviruses. We demonstrate the detection of 10 synthetic filovirus RNA templates with analytical sensitivity ranging from 178 to 3,354 copies/mL, without cross-reactivity on 10 non-filoviral human viral species. We verified assay performance on 10 inactivated filovirus isolates, yielding initial sensitivities of 0.012-44.17 TCID 50 /mL. We coupled this broadly reactive RT-PCR with a deep sequencing workflow that is amenable to high-throughput pooling to maximize detection and discovery potential. In summary, this pan-filovirus RT-PCR assay targets the most conserved filovirus gene, offers the widest breadth of coverage to date, and may help in the detection and discovery of novel filoviruses.IMPORTANCEFiloviruses remain some of the most mysterious viruses known to the world, with extremely high lethality rates and significant pandemic potential. Yet comparably few filovirus species and genera have been discovered to date and questions surround the definitive host species for zoonotic infections. Here, we describe a novel broadly reactive RT-PCR assay targeting the conserved L polymerase gene for high-throughput screening for filoviruses in a variety of clinical and environmental specimens. We demonstrate the assay can detect all known mammalian filoviruses and determine the sensitivity and specificity of the assay on synthetic RNA sequences, inactivated filovirus isolates, and non-filoviral species.
      Competing Interests: The authors declare no conflict of interest.
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    • Grant Information:
      U19 AI171403 United States AI NIAID NIH HHS; 1U19AI171403-01 HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)
    • Contributed Indexing:
      Keywords: RT-PCR; broad range PCR; filovirus; pan-filovirus; zoonosis
    • Accession Number:
      0 (RNA, Viral)
      0 (Viral Proteins)
    • Publication Date:
      Date Created: 20240724 Date Completed: 20240903 Latest Revision: 20240905
    • Publication Date:
      20240905
    • Accession Number:
      PMC11370238
    • Accession Number:
      10.1128/spectrum.01010-24
    • Accession Number:
      39046245