Javier Romero

Genome diversity and evidence of recombination and reassortment in nanoviruses from Europe

The recent identification of a new nanovirus, pea necrotic yellow dwarf virus, from pea in Germany prompted us to survey wild and cultivated legumes for nanovirus infections in several European countries. This led to the identification of two new nanoviruses: black medic leaf roll virus (BMLRV) and pea yellow stunt virus (PYSV), each considered a putative new species. The complete genomes of a PYSV isolate from Austria and three BMLRV isolates from Austria, Azerbaijan and Sweden were sequenced. In addition, the genomes of five isolates of faba bean necrotic yellows virus (FBNYV) from Azerbaijan and Spain and those of four faba bean necrotic stunt virus (FBNSV) isolates from Azerbaijan were completely sequenced, leading to the first identification of FBNSV occurring in Europe. Sequence analyses uncovered evolutionary relationships, extensive reassortment and potential remnants of mixed nanovirus infections, as well as intra- and intercomponent recombination events within the nanovirus genomes. In some virus isolates, diverse types of the same genome component (paralogues) were observed, a type of genome complexity not described previously for any member of the family Nanoviridae. Moreover, infectious and aphid-transmissible nanoviruses from cloned genomic DNAs of FBNYV and BMLRV were reconstituted that, for the first time, allow experimental reassortments for studying the genome functions and evolution of these nanoviruses.

Effect of the Host and Temperature on the Formation of Defective RNAs Associated with Broad bean mottle virus Infection

ABSTRACT Previously, we demonstrated that Broad bean mottle virus (BBMV), a member of the genus Bromovirus, could accumulate RNA 2-derived defective interfering (DI) RNAs during infection. In this work, we study how host and environmental factors affect the accumulation of DI RNAs. Serial passages of BBMV through selected plant species reveal that, with low-multiplicity inocula, some systemic hosts (Vicia faba, Nicotiana clevelandii, and N. tabacum cv. Samsum) support DI RNA accumulation after the first passage cycle but other hosts (Phaseolus vulgaris, Pisum sativum, and Glycine max) do not. However, several passages with the high-multiplicity inocula can generate DI RNAs in pea plants. Local lesion hosts (Chenopodium quinoa, C. amaranticolor, and C. murale) remain free of the DI RNA components. The size of the de novo-formed DI RNAs depends on the host and on environmental conditions. For instance, broad bean plants cultivated in a greenhouse or in a growth chamber at 20 degrees C accumulated DI RNAs of 2.4 or 1.9 kb in size, respectively. A reverse trend was observed in pea plants. Lower temperatures greatly facilitated the formation of DI RNAs in broad bean and pea hosts after the first passage. The importance of these findings for the studies on DI RNAs are discussed.

Use of a defective RNA of broad bean mottle bromovirus for stable gene expression in legumes

A viral vector that expressed foreign genes was engineered using a cDNA clone of broad bean mottle bromovirus (BBMV) defective interfering (DI) RNA. The expression vector required an active ORF to ensure efficient accumulation and replication in the host plants. After mechanical inoculation with BBMV RNAs, expression of the green fluorescent protein (GFP) reporter was driven by DI RNA constructs during consecutive passages through broad bean plants. Our data prove that BBMV DI RNAs, whose yield is similar to the virus genomic components, are useful as gene vectors after mechanical inoculation on legume plants.