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

Abstract

Billions of people are potentially at risk from mosquito-borne arboviruses belonging to the genera Alphavirus and Flavivirus, representing a serious, global, public health concern. Some of the medically important alphaviruses include chikungunya virus (CHIKV), Barmah Forest virus (BFV) and Ross River virus (RRV) while medically important flaviviruses include dengue viruses (DENV), Japanese encephalitis virus (JEV), Murray Valley encephalitis virus (MVEV), West Nile virus (WNV), and yellow fever virus (YFV). Factors that may be contributing to the emergence and re-emergence of these viruses include ineffective vector control, increasing global travel, urbanisation and expanding host ranges. The clinical signs associated with alphaviral and flaviviral disease can be non-specific, thus necessitating a laboratory diagnosis.Rapid diagnostic tests have been identified as an area of critical importance for alphavirus and flavivirus research, as they are essential tools for both the clinical treatment of patients and public health surveillance. Effective arbovirus surveillance requires a multi-faceted approach combining surveillance for fevers of unknown origin, particularly in travellers, as well as animal and entomological surveillance. Early detection of clinical cases, followed by the implementation of control measures such as vaccination, mosquito-control and raising public awareness, can be effective in limiting outbreaks. Conversely, the failure of effective surveillance can have a huge economic and public health impact, even in developed countries. The introduction, spread and establishment of WNV in the USA demonstrates the need for effective surveillance systems for both endemic and exotic diseases, and the high costs associated with a failure to rapidly implement effective public health measures.Multiplex suspension microarrays, using Luminex technology, have been reported to be costefficient, and easy to design and perform. Most importantly, their proven ability to detect multiple viruses in a single sample has suggested an important role for surveillance work. For these reasons, a suspension microarray for the detection of alphaviruses and flaviviruses was developed as a useful, rapid, diagnostic test for both clinical diagnosis and public health surveillance. Consensus primers were used for the amplification of alphaviruses and flaviviruses and virus-specific probes were either adapted from previously validated TaqMan assays or designed to enable a specific diagnosis. The multiplex test was validated against a panel of reference viruses, clinical samples where available, and individual virus-positive mosquitoes or mosquito pools. The standardised assay conditions that were developed will facilitate the multiplexing of all assays in future.The multiplex suspension microarrays developed have been shown to be sensitive and virusspecific. The alphavirus suspension microarray could successfully detect Ross River virus (RRV), Barmah Forest virus (BFV), Chikungunya virus (CHIKV) and Sindbis virus (SINV). The encephalitic flavivirus suspension microarray which could initially detect four different encephalitic flaviviruses was expanded to an assay that was capable of detecting 12 different flaviviruses through the incorporation of 14 probes. This was accomplished without a loss in sensitivity in the assay from multiplexing. The flavivirus suspension microarray detected the dengue viruses (DENV-1-4), Kokobera virus (KOKV), Sepik virus (SEPV), Stratford virus (STRATV) and yellow fever virus (YFV), as well as the encephalitic viruses, Japanese encephalitis virus (JEV), the Kunjin strain of West Nile virus (KUNV), Murray Valley encephalitis virus (MVEV) and West Nile virus (WNV). These viruses are either important global pathogens or endemic flaviviruses of public health interest in Australia. Some cross-reactivity was observed between closely related viruses; however use of the highest signal allowed the differentiation of the correct virus species. The suspension microarrays were found to be rapid, sensitive, specific and cost-effective.It is anticipated that these cost-effective diagnostic tests may enable adoption of improved surveillance programs and the routine screening of samples against a wider panel of viruses. We also report for the first time that this technology can be useful for detecting virus in mosquito pools. This has important implications for cost-effective vector-surveillance programs. We also successfully explored the potential to use an amplification strategy that would allow the same PCR product to be used in both suspension microarray and chip microarray assays, potentially allowing epidemiological analysis to occur rapidly in-house. A novel synthetic amplification control was also incorporated in the protocol, as an additional quality control measure. In conclusion, we report a rapid, multiplex suspension microarray which has the potential to be a useful test as a screening tool for clinical diagnosis, and improve surveillance and diagnosis of endemic and exotic alphaviruses and flaviviruses in Australia.