Abstract
A novel bovine astrovirus genotype species (BoAstV-CH13/NeuroS1) was recently identified in brain tissues of cattle as a plausible cause of encephalitis. The purpose of the present study was to develop and validate real time RT-PCR assays for the detection of BoAstV-CH13/NeuroS1 in brain tissues of cattle. Three different primer-probe combinations were designed based on BoAstV-CH13/NeuroS1 full-genome sequences of 11 different strains identified in cattle, and established in three distinct one-step real time RT-PCR protocols. These protocols were compared regarding their diagnostic performance using brain tissues of cattle with and without astrovirus associated encephalitis. The limit of detection (LOD) of all three assays was between 1.34 × 101 and 1.34 × 102 RNA copies, leading to an analytical sensitivity two orders of magnitude superior compared to a conventional pan-astrovirus RT-PCR protocol (LOD 1.31 × 104 RNA copies). Amplification efficiency was in the range of 97.3% to 107.5% with linearity (R2) > 0.99. The diagnostic sensitivity and specificity of the assays was determined as 100%, and all three revealed good intra- and inter-test repeatability. In conclusion, the newly developed RT-qPCRs are sensitive, specific, and reliable test formats that will facilitate BoAstV-CH13/NeuroS1 detection in routine diagnostics as well as in research settings.
Highlights
Astroviridae are non-enveloped positive-sense single-stranded RNA viruses of a broad genetic diversity causing infections in mammalian and avian species
Negative control samples consisted of medulla oblongata tissues that were tested negative for BoAstV-CH13/NeuroS1 in the pan-astrovirus RT-PCR and by next-generation RNA sequencing[17]
In vitro transcribed full-length viral RNA from cloned cDNA served for the determination of the limit of detection (LOD) of the three astrovirus RT-qPCRs
Summary
Astroviridae are non-enveloped positive-sense single-stranded RNA viruses of a broad genetic diversity causing infections in mammalian and avian species. Novel astroviruses have been found in association with encephalitis and neurological disease in mink[3], humans[4], cattle[5,6,7,8] sheep[9,10] and pigs[11,12,13] Most of these encephalitis-associated strains are phylogenetically related, belong to the so-called human-mink-ovine (HMO)-like astrovirus cluster and show less than 50% sequence similarity of structural and nonstructural viral proteins compared to the classical, gut-associated astrovirus strains[2]. Viral RNA and proteins were consistently detected in the neurons in affected brain tissues in association with pathological lesions, such as perivascular cuffs, neuronal necrosis and gliosis, which supports a plausible causal relationship of astrovirus infection and encephalitis[14,18]. As the virus has not been isolated in cell culture so far, in situ techniques are important tools to investigate and characterize pathological samples, but these procedures are rather www.nature.com/scientificreports/
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