Two distinct nanovirus species infecting faba bean in Morocco

Abstract

Using monoclonal antibodies raised against a Faba bean necrotic yellows virus (FBNYV) isolate from Egypt and a Faba bean necrotic stunt virus (FBNSV) isolate from Ethiopia, a striking serological variability among nanovirus isolates from faba bean in Morocco was revealed. To obtain a better understanding of this nanovirus variability in Morocco, the entire genomes of two serologically contrasting isolates referred to as Mor5 and Mor23 were sequenced. The eight circular ssDNA components, each identified from Mor5- and Mor23-infected tissues and thought to form the complete nanovirus genome, ranged in size from 952 to 1,005 nt for Mor5 and from 980 to 1,004 nt for Mor23 and were structurally similar to previously described nanovirus DNAs. However, Mor5 and Mor23 differed from each other in overall nucleotide and amino acid sequences by 25 and 26%, respectively. Mor23 was most closely related to typical FBNYV isolates described earlier from Egypt and Syria, with which it shared a mean amino acid sequence identity of about 94%. On the other hand, Mor5 most closely resembled a FBNSV isolate from Ethiopia, with which it shared a mean amino acid sequence identity of approximately 89%. The serological and genetic differences observed for Mor5 and Mor23 were comparable to those observed earlier for FBNYV, FBNSV, and Milk vetch dwarf virus. Following the guidelines on nanovirus species demarcation, this suggests that Mor23 and Mor5 represent isolates of FBNYV and FBNSV, respectively. This is the first report not only on the presence of FBNSV in a country other than Ethiopia but also on the occurrence and complete genome sequences of members of two nanovirus species in the same country, thus providing evidence for faba bean crops being infected by members of two distinct nanovirus species in a restricted geographic area.

Fig. 1

Alignment of the replication origin sequences of the eight DNAs of the isolates Mor5 and Mor23 illustrating the extent of sequence conservation in the stem-loop common region (CR-SL). Inverted repeat sequences (open horizontal arrows) potentially forming a stem-loop structure are in bold. A vertical arrow indicates the ‘origin of replication’, which is the position of cleavage by the master Rep protein [44]. Conserved iteron-like sequences (a) and their respective inversions (a′) possibly acting as recognition or binding sites for the master Rep protein are boxed and indicated by horizontal solid arrows. Dots indicate gaps included to maximise alignments. The AatII site (GACGTC) conserved in many nanoviruses [17] but absent from the Mor23 sequences is shown in bold italics in the Mor5 sequences

Fig. 2

Phylogenetic trees illustrating the relationships among the nucleotide sequences of the non-coding regions of the seven non-rep DNAs of Mor5 (a) and Mor23 (b). The bar indicates the number of nt substitutions per site

Fig. 3

Phylogenetic trees illustrating the relationship between the eight DNAs of Mor5 (GQ274031-8) and Mor23 (GQ274023-30) and the corresponding DNAs of the Ethiopian isolate of Faba bean necrotic stunt virus [FBNSV-(ET)], a tentative nanovirus species [17], and virus isolates [from Australia (AU), Egypt (EG), Syria (SY), Iran (IR), and Japan (JP)] assigned to Faba bean necrotic yellows virus (FBNYV), Milk vetch dwarf virus (MDV), Subterranean clover stunt virus (SCSV), assigned species of the genus Nanovirus. Horizontal branch length are scaled (see bar) according to the number of base substitutions per site. Bootstrap values (1,000 replicates) higher than 70% are shown at nodes