Direct broad-range detection of alphaviruses in mosquito extracts

Abstract

Members of the genus Alphavirus are a diverse group of principally mosquito-borne RNA viruses. There are at least 29 species and many more subtypes of alphaviruses and some are considered potential bioweapons. We have developed a multi-locus RT-PCR followed by electrospray ionization mass spectrometry (RT-PCR/ESI-MS) assay that uses the amplicon base compositions to detect and identify alphaviruses. A small set of primer pairs targeting conserved sites in the alphavirus RNA genome were used to amplify a panel of 36 virus isolates representing characterized Old World and New World alphaviruses. Base compositions from the resulting amplicons could be used to unambiguously determine the species or subtype of 35 of the 36 isolates. The assay detected, without culture, Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), and mixtures of both in pools consisting of laboratory-infected and -uninfected mosquitoes. Further, the assay was used to detect alphaviruses in naturally occurring mosquito vectors collected from locations in South America and Asia. Mosquito pools collected near Iquitos, Peru, were found to contain an alphavirus with a very distinct signature. Subsequent sequence analysis confirmed that the virus was a member of the Mucambo virus species (subtype IIID in the VEEV complex). The assay we have developed provides a rapid, accurate, and high-throughput assay for surveillance of alphaviruses.

Detection and Identification of Ehrlichia Species in Blood by Use of PCR and Electrospray Ionization Mass Spectrometry

“…First, a small set of PCRs can be used to detect and distinguish among a wider range of organisms (15,29). PCR/ESI-MS can also be used to detect mixtures of bacteria or viruses in a single sample (14,17,30). Ticks carry numerous pathogens, including coinfections with different pathogens (7,32).…”

Outbreak of gastroenteritis caused byYersinia pestisin Afghanistan

“…All bacterial culture attempts from patients' blood and camel tissue were negative or only yielded contaminants, and few samples were available for testing ; the key to identifying the causative agent in this outbreak was the use of the Ibis T5000 PCR/ESI-MS assay. This diagnostic tool has previously been shown to detect a broad range of bacterial pathogens [16,17] and several viruses [18][19][20]. The inclusion of certain key gene targets in the Ibis T5000 biodefence assay allows it to detect and identify a wide range of pathogens in potentially complex clinical or environmental samples with little or no background effect.…”

Development of multiplex suspension microarrays for the detection of alphaviruses and flaviviruses of public health significance

“…World members (Eshoo et al, 2007). They can be classified into eight antigenic complexes and share a minimum amino acid sequence identity of approximately 40% in the structural proteins and 60% in the non-structural proteins (Fauquet et al, 2005).…”

“…The primers published by Pfeffer et al (1997) have been the most widely adopted and have been utilised in numerous studies including a duplex RT-PCR reaction for the detection of Brazilian alphaviruses and flaviviruses (de Morais Bronzoni et al, 2005), a macroarray for the simultaneous detection of alphaviruses and orthopox viruses (Fitzgibbon & Sagripanti, 2006) and an RT-PCR-enzyme linked immuno-assay for the detection of alphaviruses in clinical samples and mosquito pools . Broad-range alphavirus primers that target the NS1 region of genome have also proven to be successful in the detection of a variety of alphaviruses in mosquito extracts (Eshoo et al, 2007). Therefore, genus-specific alphavirus primers targeting the NS1 conserved region have proven very useful in a clinical and surveillance capacity.…”

“…RT-PCR has been used to detect both flaviviruses (Pyke et al, 2004 and alphaviruses in mosquito pools (Eshoo et al, 2007). These studies demonstrate that molecular assays have the appropriate sensitivity to detect the viral titres produced in infected mosquitoes, even in pooled samples.…”