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Journal of Clinical Microbiology, August 2009, p. 2398-2404, Vol. 47, No. 8
0095-1137/09/$08.00+0     doi:10.1128/JCM.00182-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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{triangledown}

Amy J. Lambert* and Robert S. Lanciotti

Division of Vector-Borne Infectious Diseases, National Center for Zoonotic, Vector-Borne, and Enteric Diseases, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, Fort Collins, Colorado

Received 28 January 2009/ Returned for modification 14 March 2009/ Accepted 10 June 2009

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.

* Corresponding author. Mailing address: Division of Vector-Borne Infectious Diseases, National Center for Zoonotic, Vector-Borne, and Enteric Diseases, CDC, Rampart Rd., Fort Collins, CO 80521. Phone: (970) 225-4227. Fax: (970) 494-6631. E-mail: ahk7{at}cdc.gov

{triangledown} Published ahead of print on 17 June 2009.

Journal of Clinical Microbiology, August 2009, p. 2398-2404, Vol. 47, No. 8
0095-1137/09/$08.00+0     doi:10.1128/JCM.00182-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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