Articles
Characterization of a nucleorhabdovirus from Physostegia
Article number
1193_5
Pages
29 – 38
Language
English
Abstract
In July 2014, a Physostegia plant showing severe chlorotic mottle symptoms and leaf deformation was received from Austria.
Following mechanical inoculation, the virus systemically infected several assay hosts.
Bullet-shaped particles were found in electron microscopical observations indicating the presence of rhabdoviruses.
As expected for negative strand RNA viruses, dsRNA extractions failed to extract viral replication intermediates from infected plants and RT-PCR using published sets of degenerate rhabdovirus oligonucleotide primers did not result in amplification of viral sequences.
This prompted us to use a total RNA extract as template for a next generation sequencing approach.
De Novo assembly resulted in a 13 kb contig which could be identified by BLASTn as the putative genome of a Nucleorhabdovirus, showing the highest nucleotide sequence identity to eggplant mottled dwarf virus (70.7%, EMDV). The missing extreme 5′- and 3′-ends were determined by RACE, resulting in a total genome size of 13,320 nts (GenBank accession number KX636164). Following back-inoculation to Physostegia, Koch’s postulates were fulfilled.
Based on the symptoms observed on the original host, the isolate was named Physostegia chlorotic mottle virus (PhCMoV) and is available at the DSMZ Plant Virus Collection under accession no.
PV-1182.
Following mechanical inoculation, the virus systemically infected several assay hosts.
Bullet-shaped particles were found in electron microscopical observations indicating the presence of rhabdoviruses.
As expected for negative strand RNA viruses, dsRNA extractions failed to extract viral replication intermediates from infected plants and RT-PCR using published sets of degenerate rhabdovirus oligonucleotide primers did not result in amplification of viral sequences.
This prompted us to use a total RNA extract as template for a next generation sequencing approach.
De Novo assembly resulted in a 13 kb contig which could be identified by BLASTn as the putative genome of a Nucleorhabdovirus, showing the highest nucleotide sequence identity to eggplant mottled dwarf virus (70.7%, EMDV). The missing extreme 5′- and 3′-ends were determined by RACE, resulting in a total genome size of 13,320 nts (GenBank accession number KX636164). Following back-inoculation to Physostegia, Koch’s postulates were fulfilled.
Based on the symptoms observed on the original host, the isolate was named Physostegia chlorotic mottle virus (PhCMoV) and is available at the DSMZ Plant Virus Collection under accession no.
PV-1182.
Authors
W. Menzel, K.R. Richert-Pöggeler, S. Winter, D. Knierim
Keywords
Nucleorhabdovirus, Physostegia, next generation sequencing, MiSeq, PhCMoV
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