A Novel Pan-FlavivirusDetection and Identification Assay Based on RT-qPCR and Microarray

Abstract

The genus Flavivirus includes arthropod-borne viruses responsible for a large number of infections in humans and economically important animals. While RT-PCR protocols for specific detection of most Flavivirus species are available, there has been also a demand for a broad-range Flavivirus assay covering all members of the genus. It is particularly challenging to balance specificity at genus level with equal sensitivity towards each target species. In the present study, a novel assay combining a SYBR Green-based RT-qPCR with a low-density DNA microarray has been developed. Validation experiments confirmed that the RT-qPCR exhibited roughly equal sensitivity of detection and quantification for all flaviviruses tested. These PCR products are subjected to hybridization on a microarray carrying 84 different oligonucleotide probes that represent all known Flavivirus species. This assay has been used as a screening and confirmation tool for Flavivirus presence in laboratory and field samples, and it performed successfully in international External Quality Assessment of NAT studies. Twenty-six Flavivirus strains were tested with the assay, showing equivalent or superior characteristics compared with the original or even with species-specific RT-PCRs. As an example, test results on West Nile virus detection in a panel of 340 mosquito pool samples from Greece are presented.

Tick-Borne Flaviviruses, with a Focus on Powassan Virus

“…High-density panviral and panmicrobial DNA probe-based solid-phase microarrays are known to target hundreds to thousands of viral pathogens or a mixture of viral, bacterial, and fungal pathogens and parasites of vertebrates, respectively. Some of these assays were reported to include POWV-specific capture DNA probes (251)(252)(253)(254). Also, a more recent advancement in panvirus detection methods is the combination of viral metagenomics with high-throughput next-generation sequencing (NGS).…”

A Duplex Quantitative Real-Time Reverse Transcription-PCR for Simultaneous Detection and Differentiation of Flaviviruses of the Japanese Encephalitis and Ntaya Serocomplexes in Birds

“…Although some pan-flavivirus PCR methods have been described so far, most are focused on public health application or entomological surveillance (27)(28)(29)(30)(31)(32)(33), while none of them has been developed for birds. This is significant in the current epidemiological situation, where the recent and potential emergence of bird-pathogenic flaviviruses in different territories poses a complex challenge for the diagnostic laboratories and for the veterinary authorities.…”

“…However, the new dRRT-PCR has demonstrated to be a specific and highly sensitive tool, capable of detecting the wide range of JE and Ntaya flaviviral species analyzed, and posing a performance similar to the RRT-PCR methods used as reference (34,35). On the other hand, most PCR methods described for flaviviruses detection have been verified with human or mosquito samples, when available (28,(31)(32)(33). In this study, WNV, USUV and BAGV positive samples from a variety of wild bird species were available for the evaluation of the dRRT-PCR.…”

“…As the number of flaviviruses circulating in given geographic areas (such as those already mentioned) grows, molecular diagnosis of flaviviral infections relies more and more on generic RT-PCR approaches, which may be particularly advantageous in bird disease diagnostics and surveillance. However, panflavivirus PCR methods described so far are focused essentially on public health application or entomological surveillance (27)(28)(29)(30)(31)(32)(33), and no PCR-based system is currently available for avian monitoring. Most important bird-pathogenic flaviviruses belong to the above-mentioned JE and Ntaya serocomplexes.…”

Le virus Usutu : la menace fantôme

“…Cette surveillance inclut désormais un volet vétérinaire et un volet humain avec une sensibilisation SYNTHÈSE REVUES Les auteurs dé clarent n'avoir aucun lien d'inté rê t concernant les donné es publié es dans cet article.RÉFÉRENCESLes techniques d'amplification d'ARN du WNV, utilisées lors de la qualification des dons de sang pour le risque WNV (type Cobas WNV, Roche), permettent également de détecter le génome viral d'USUV[42,51]. De nombreuses techniques de RT-PCR ont été décrites et certaines approches de PCR ont été développées afin d'être spécifiques des séquences d'USUV[38,52] ou d'être, au contraire, consensuelles de la plupart des flavivirus en ciblant une région conservée de 260 paires de bases du gène NS5, commun à ces virus[53][54][55] ; le typage du virus peut alors être réalisé secondairement par séquençage ou hybridation[54]. Cette approche « panflavivirus » est certes plus fastidieuse, mais elle présente un double avantage.…”