Consensus amplification and novel multiplex sequencing method for S segment species identification of 47 viruses of the Orthobunyavirus, Phlebovirus, and Nairovirus genera of the family Bunyaviridae

  • Lambert A
  • Lanciotti R
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Abstract

A reverse {transcription-PCR} {(RT-PCR)} assay was designed, according to previously determined and newly derived genetic data, to target S genomic segments of 47 viruses, including 29 arthropod-borne human pathogens, of the family Bunyaviridae. The analytical sensitivity of the presented assay was evaluated through its application to {RNAs} extracted from quantitated dilutions of bunyaviruses of interest. Additionally, the assay's analytical specificity was determined through the evaluation of {RNAs} extracted from selected bunyaviruses and other representative arthropod-borne viruses isolated from a diverse group of host species and temporal and geographic origins. After {RT-PCR} amplification, {DNAs} amplified from bunyaviruses of interest were subjected to a novel multiplex sequencing method to confirm bunyavirus positivity and provide preliminary, species-level S segment identification. It is our goal that this assay will be used as a tool for identification and characterization of emergent arthropod-borne bunyavirus isolates of medical import as well as related viruses of the family Bunyaviridae that have not been associated with human illness.

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Authors

  • Amy J. Lambert

  • Robert S. Lanciotti

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