Reconstitution of Authentic Nanovirus from Multiple Cloned DNAs

Abstract

We describe a new plant single-stranded DNA (ssDNA) virus, a nanovirus isolate originating from the faba bean in Ethiopia. We applied rolling circle amplification (RCA) to extensively copy the individual circular DNAs of the nanovirus genome. By sequence analyses of more than 208 individually cloned genome components, we obtained a representative sample of eight polymorphic swarms of circular DNAs, each about 1 kb in size. From these heterogeneous DNA populations after RCA, we inferred consensus sequences of the eight DNA components of the virus genome. Based on the distinctive molecular and biological properties of the virus, we propose to consider it a new species of the genus Nanovirus and to name it faba bean necrotic stunt virus (FBNSV). Selecting a representative clone of each of the eight DNAs for transfer by T-DNA plasmids of Agrobacterium tumefaciens into Vicia faba plants, we elicited the development of the typical FBNSV disease symptoms. Moreover, we showed that the virus thus produced was readily transmitted by two different aphid vector species, Aphis craccivora and Acyrthosiphon pisum. This represents the first reconstitution of a fully infectious and sustainably insect-transmissible nanovirus from its cloned DNAs and provides compelling evidence that the genome of a legume-infecting nanovirus is typically comprised of eight distinct DNA components.

Advances in Small Isometric Multicomponent ssDNA Viruses Infecting Plants

“…Eight distinct DNA components were characterised and inoculation with these cloned DNAs produced typical disease in faba bean. The progeny virus of the cloned DNAs was transmissible by its vector, Aphis craccivora and Acyrthosiphon pisium [25]. This is the first conclusive demonstration about the infective unit of Nanovirus genome.…”

“…Multicomponent ssDNA plant viruses although were identified in 1990s, many of them are known to exist for a long time as they are associated with historic plant diseases and some of them have recently emerged [25,26]. Genomics of these viruses opened up new area of studies that they are the source of promoters [19,83,88] for gene expression in plant and vectors that provide efficient expression system for studying in planta protein-protein interactions [5].…”

“…The genus Nanovirus, included three species, Faba bean necrotic yellows virus (FBNYV), Milk vetch dwarf virus (MDV) and Subterranean clover stunt virus (SCSV). Recently, two new nanoviruses, Faba bean necrotic stunt virus (FBNSV) and Pea necrotic yellow dwarf virus (PVYDV) have been reported [25,26]. The genus Babuvirus included only, Banana bunchy top virus (BBTV).…”

“…FBNYV occurs in many Arab countries of West Asia, North and East Africa and Europe (Syria, Jordan, Ethiopia, Egypt, Algeria, Morocco, Sudan, Azerbaijan, Tunisia and Spain) [20, 21, 44,48,54,[56][57][58][59]72]. FBNSV has been recently reported from Ethiopia [25] and Morocco [1], and PNYDV from Germany [26].…”

“…The first comprehensive demonstration of infectivity of the cloned DNAs was shown for FBNSV. A group of eight cloned DNA components produced typical disease symptoms as well as biologically active progeny virus that was successfully transmitted through aphid vector [25].…”