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

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.

FIG. 1.

Targeted regions of the presented assay shown within S segment viral cRNA N and nonstructural (NSs) ORF coding strategies of arthropod-borne genera of the family Bunyaviridae. N, nucleocapsid; nts, nucleotides.

FIG. 2.

Gel image of fragments amplified by the presented consensus assay. Fragments range in size from approximately 210 to 400 bp. Fragments from the amplification of La Crosse (lane 1), Wyeomyia (lane 2), Bunyamwera (lane 3), Oropouche (lane 4), Punta Toro (lane 5), and Dugbe (lane 6) viruses are represented. Five microliters of each DNA was loaded onto a 2% agarose gel containing an ethidium bromide stain.