Dataset Information

Differentiation of Capripox Viruses by Nanopore Sequencing.

ABSTRACT: The genus capripoxvirus (CaPV), family Poxviridae, includes three virus species: goatpox virus (GPV), sheeppox virus (SPV) and lumpy skin disease virus (LSDV). CaPV causes disease outbreaks with consequent economic losses in Africa and the Middle East. LSDV has recently spread to Southeast Europe. As CaPVs share 96-97% genetic similarity along the length of the entire genome and are difficult to distinguish using serological assays, simple, reliable and fast methods for diagnosis and species differentiation are crucial in cases of disease outbreak. The present study aimed to develop a field-applicable CaPV differentiation method. Nanopore technology was used for whole genome sequencing. A local database of complete CaPV genomes and partial sequences of three genes (RPO30, P32 and GPCR) was established for offline Basic Local Alignment Search Tool (BLAST). Specificities of 98.04% in whole genome and 97.86% in RPO30 gene runs were obtained among the three virus species, while other databases were less specific. The total run time was shortened to approximately 2 h. Functionality of the developed procedure was proved by samples with high host background sequences. Reliable differentiation options for the quality and capacity of hardware, and sample quality of suspected cases, were derived from these findings. The whole workflow can be performed rapidly with a mobile suitcase laboratory and mini-computer, allowing application at the point-of-need with limited resource settings.

SUBMITTER: Eltom KH

PROVIDER: S-EPMC8067513 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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Differentiation of Capripox Viruses by Nanopore Sequencing.

Eltom Kamal H KH Althoff Anna Christina AC Hansen Sören S Böhlken-Fascher Susanne S Yousif Ausama A El-Sheikh Hussein A HA ElWakeel Ahmed A AA Elgamal Mahmoud A MA Mossa Hadeer M HM Aboul-Soud Emad A EA Wolff Janika J Korthase Christian C Hoffmann Bernd B Adam Nabawia M NM Abdelaziz Sanaa A SA Shalaby Mohamed A MA Abd El Wahed Ahmed A

Vaccines 20210406 4

The genus capripoxvirus (CaPV), family <i>Poxviridae</i>, includes three virus species: goatpox virus (GPV), sheeppox virus (SPV) and lumpy skin disease virus (LSDV). CaPV causes disease outbreaks with consequent economic losses in Africa and the Middle East. LSDV has recently spread to Southeast Europe. As CaPVs share 96-97% genetic similarity along the length of the entire genome and are difficult to distinguish using serological assays, simple, reliable and fast methods for diagnosis and spec ...[more]

PMID: 33917413

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Project description:High-throughput sequencing (HTS) has become an important tool for plant virus detection and discovery. Nanopore sequencing has been rapidly developing in the recent years and offers new possibilities for fast diagnostic applications of HTS. With this in mind, a study was completed, comparing the most established HTS platform (MiSeq benchtop sequencer-Illumina), with the MinION sequencer (Oxford Nanopore Technologies) for the detection of plant viruses and viroids. Method comparisons were performed on five selected samples, containing two viroids, which were sequenced using nanopore technology for the first time and 11 plant viruses with different genome organizations. For all samples, sequencing libraries for the MiSeq were prepared from ribosomal RNA-depleted total RNA (rRNA-depleted totRNA) and for MinION sequencing, direct RNA sequencing of totRNA was used. Moreover, for one of the samples, which contained five different plant viruses and a viroid, three additional variations of sample preparation for MinION sequencing were also used: direct RNA sequencing of rRNA-depleted totRNA, cDNA-PCR sequencing of totRNA, and cDNA-PCR sequencing of rRNA-depleted totRNA. Whilst direct RNA sequencing of total RNA was the quickest of the tested approaches, it was also the least sensitive: using this approach, we failed to detect only one virus that was present in a sample at an extremely low titer. All other MinION sequencing approaches showed improved performance with outcomes similar to Illumina sequencing, with cDNA-PCR sequencing of rRNA-depleted totRNA showing the best performance amongst tested nanopore MinION sequencing approaches. Moreover, when enough sequencing data were generated, high-quality consensus viral genome sequences could be reconstructed from MinION sequencing data, with high identity to the ones generated from Illumina data. The results of this study implicate that, when an appropriate sample and library preparation are selected, nanopore MinION sequencing could be used for the detection of plant viruses and viroids with similar performance as Illumina sequencing. Taken as a balance of practicality and performance, this suggests that MinION sequencing may be an ideal tool for fast and affordable virus diagnostics.

Dataset Information

Differentiation of Capripox Viruses by Nanopore Sequencing.

Publications

Differentiation of Capripox Viruses by Nanopore Sequencing.

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