A New Multiplex Real-Time RT-PCR for Simultaneous Detection and Differentiation of Avian Bornaviruses

Brigitte Sigrist,Jessica Geers,Sarah Albini,Dennis Rubbenstroth,Nina Wolfrum

doi:10.3390/v13071358

Brigitte Sigrist, Jessica Geers + Show 3 more

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https://doi.org/10.3390/v13071358

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Abstract

Avian bornaviruses were first described in 2008 as the causative agents of proventricular dilatation disease (PDD) in parrots and their relatives (Psittaciformes). To date, 15 genetically highly diverse avian bornaviruses covering at least five viral species have been discovered in different bird orders. Currently, the primary diagnostic tool is the detection of viral RNA by conventional or real-time RT-PCR (rRT-PCR). One of the drawbacks of this is the usage of either specific assays, allowing the detection of one particular virus, or of assays with a broad detection spectrum, which, however, do not allow for the simultaneous specification of the detected virus. To facilitate the simultaneous detection and specification of avian bornaviruses, a multiplex real-time RT-PCR assay was developed. Whole-genome sequences of various bornaviruses were aligned. Primers were designed to recognize conserved regions within the overlapping X/P gene and probes were selected to detect virus species-specific regions within the target region. The optimization of the assay resulted in the sensitive and specific detection of bornaviruses of Psittaciformes, Passeriformes, and aquatic birds. Finally, the new rRT-PCR was successfully employed to detect avian bornaviruses in field samples from various avian species. This assay will serve as powerful tool in epidemiological studies and will improve avian bornavirus detection.

Highlights

For the new multiplex real-time RT-PCR (rRT-PCR), X gene primers were designed to detect a broad range of avian bornaviruses
RT-PCR, which might be beneficial for diagnostic facilities without access to a real-time cycler
A conventional RT-PCR using these new primers was shown to detect the RNAs of ten available orthobornaviruses with varying sensitivity

Summary

Introduction

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Bornaviruses (family Bornaviridae) are enveloped viruses containing a non-segmented single-stranded RNA genome of negative polarity [1,2]. Until 2008, Borna disease virus 1 and 2 (BoDV-1 and -2), causing neurologic diseases in humans, horses, sheep, and other domestic mammals, were the only known members of the family. In 2008, the first avian bornaviruses were discovered in Psittaciformes and shown to be the causative agents of proventricular dilatation disease (PDD) in pet parrots.

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