Identification of Divergent Isolates of Banana Mild Mosaic Virus and Development of a New Diagnostic Primer to Improve Detection

Abstract

Banana mild mosaic virus (BanMMV) (Betaflexiviridae, Quinvirinae, unassigned species) is a filamentous virus belonging to the Betaflexiviridae family. It infects Musa spp. with a very wide geographic distribution. The genome variability of plant viruses, including the members of the Betaflexiviridae family, makes their molecular detection by specific primers particularly challenging. During routine indexing of the Musa germplasm accessions, a discrepancy was observed between electron microscopy and immunocapture (IC) reverse transcription (RT) polymerase chain reaction (PCR) test results for one asymptomatic accession. Filamentous viral particles were observed while molecular tests failed to amplify any fragment. The accession underwent high-throughput sequencing and two complete genomes of BanMMV with 75.3% of identity were assembled. Based on these sequences and on the 54 coat protein sequences available from GenBank, a new forward primer, named BanMMV CP9, compatible with Poty1, an oligodT reverse primer already used in diagnostics, was designed. A retrospective analysis of 110 different germplasm accessions from diverse origins was conducted, comparing BanMMCP2 and BanMMV CP9 primers. Of these 110 accessions, 16 tested positive with both BanMMCP2 and BanMMV CP9, 3 were positive with only BanMMCP2 and 2 tested positive with only BanMMV CP9. Otherwise, 89 were negative with the two primers and free of flexuous virions. Sanger sequencing was performed from purified PCR products in order to confirm the amplification of the BanMMV sequence for the five accessions with contrasting results. It is highly recommended to use the two primers successively to improve the inclusiveness of the protocol.

First viruses infecting cockspur coral tree (Erythrina crista-galli L.): discovery of a putative novel capillovirus and a new host for the prune dwarf virus

“…Nucleic acids were resuspended in 50 microliters of sterile nuclease free water and an aliquot of 20 microliters was treated with DNase (Promega, Madison, WI, USA) according to the manufacturer's instructions. After quality checking by NanoDrop 2000c spectrophotometer (Thermo Scientific, Carlsbad, CA, USA) and Bioanalyzer (Agilent, Santa Clara, CA, USA), the RNAs from E. crista-galli was used for the sequencing library preparation as previously described (Hanafi et al 2020). HTS was performed on Illumina Nextseq 500 platform yielding a total number of 4.2 million reads (2×150 nts).…”

Detection of Banana Mild Mosaic Virus in Musa In Vitro Plants: High-Throughput Sequencing Presents Higher Diagnostic Sensitivity Than (IC)-RT-PCR and Identifies a New Betaflexiviridae Species

“…The sequencing libraries were prepared using the Ribo-Zero™ Plant Leaf Kit (Illumina Inc., San Diego, CA, USA) for ribodepletion (ribosomal RNA depletion) followed by the TruSeq Stranded Total RNA Library Prep Kit (Illumina Inc., San Diego, CA, USA) using the standard protocol as previously described [22]. The samples were sequenced on the Illumina Nextseq 500 platform with paired sequencing reads of 2 × 151 nt at the GIGA facilities of Liège University (Liège, Belgium).…”

“…De novo assembly into contigs was carried out to reconstruct the genome sequence of a potential new viral species, using the SPAdes software embedded in Geneious with default parameters and a k-mer of 55 [22]. The full genome sequence was aligned and used for phylogenetic analyses with the MEGA software package version 7.0.…”

“…The phylogenetic relationships were inferred using neighbor-joining method embedded in the same version of the MEGA software. The stability of the topology was evaluated using bootstrap (1000 replications) [22]. Accession numbers of virus sequences obtained from GenBank (http://www.ncbi.nlm.nih.gov/, accessed on 1 October 2021) have been integrated in the phylogenetic trees.…”