Ourmia melon virus (OuMV), Epirus cherry virus (EpCV) and Cassava virus C (CsVC) are three species placed in the genus Ourmiavirus. We cloned and sequenced their RNA genomes. The sizes of the three genomic RNAs of OuMV, the type member of the genus, were 2814, 1064 and 974 nt and each had one open reading frame. RNA1 potentially encoded a 97.5 kDa protein carrying the GDD motif typical of RNA-dependent RNA polymerases (RdRps). The putative RdRps of ourmiaviruses are distantly related to known viral RdRps, with the closest similarity and phylogenetic affinity observed with fungal viruses of the genus Narnaviridae. RNA2 encoded a 31.6 kDa protein which, expressed in bacteria as a His-tag fusion protein and in plants through agroinfiltration, reacted specifically with antibodies made against tubular structures found in the cytoplasm. The ORF2 product is significantly similar to movement proteins of the genus Tombusviridae, and phylogenetic analysis supported this evolutionary relationship. The product of OuMV ORF3 is a 23.8 kDa protein. This protein was also expressed in bacteria and plants, and reacted specifically with antisera against the OuMV coat protein. The sequence of the ORF3 protein showed limited but significant similarity to capsid proteins of several plant and animal viruses, although phylogenetic analysis failed to reveal its most likely origin. Taken together, these results indicate that ourmiaviruses comprise a unique group of plant viruses that might have evolved by reassortment of genomic segments of RNA viruses infecting hosts belonging to different eukaryotic kingdoms, in particular, fungi and plants.

Isolates of Tomato spotted wilt virus (TSWV) severely and systemically infecting commercial pepper cultivars with resistance introgressed from Capsicum chinense PI152225 were found in Albenga (northwestern Italy) in July 2000. Experimentally, these resistance-breaking (RB) isolates overcame the resistance in C. chinense PI152225, but they produced infection in other hosts similarly to non-RB isolates from the same area. The RB isolates were indistinguishable from TSWV by serology and electron microscopy, and they were efficiently transmitted by Frankliniella occidentalis. Such isolates were recovered on the same farm in tomato, pepper, and artichoke 2 and 12 months later, suggesting natural spread from the resistant plants and survival. The RB isolates survived in experimental mixed infections with a non-RB isolate in susceptible pepper and C. chinense, but cross-protection in pepper acted against them. Commercial TSWV-resistant pepper but not resistant tomato cultivars from different companies were susceptible to these RB isolates after mechanical inoculation. Similar isolates were not detected among TSWV samples collected from 1993 to 2000 in the area. The management of TSWV and thrips using resistant pepper cultivars is discussed.

A functional capsid protein (CP) is essential for host plant infection and insect transmission in monopartite geminiviruses. We studied two defective genomic DNAs of tomato yellow leaf curl virus (TYLCV), Sic and SicRcv. Sic, cloned from a field-infected tomato, was not infectious, whereas SicRcv, which spontaneously originated from Sic, was infectious but not whitefly transmissible. A single amino acid change in the CP was found to be responsible for restoring infectivity. When the amino acid sequences of the CPs of Sic and SicRcv were compared with that of a closely related wild-type virus (TYLCV-Sar), differences were found in the following positions: 129 (P in Sic and SicRcv, Q in Sar), 134 (Q in Sic and Sar, H in SicRcv) and 152 (E in Sic and SicRcv, D in Sar). We constructed TYLCV-Sar variants containing the eight possible amino acid combinations in those three positions and tested them for infectivity and transmissibility. QQD, QQE, QHD, and QHE had a wild-type phenotype, whereas PHD and PHE were infectious but nontransmissible. PQD and PQE mutants were not infectious; however, they replicated and accumulated CP, but not virions, in Nicotiana benthamiana leaf discs. The Q129P replacement is a nonconservative change, which may drastically alter the secondary structure of the CP and affect its ability to form the capsid. The additional Q134H change, however, appeared to compensate for the structural modification. Sequence comparisons among whitefly-transmitted geminiviruses in terms of the CP region studied showed that combinations other than QQD are present in several cases, but never with a P129.

Big-vein is a widespread and damaging disease of lettuce, transmitted through soil by the chytrid fungus Olpidium brassicae, and generally supposed to be caused by Lettuce big-vein virus (LBVV; genus Varicosavirus). This virus is reported to have rigid rod-shaped particles, a divided double-stranded RNA genome, and one capsid protein of 48 kD, but has not been isolated or rigorously shown to cause the disease. We provide evidence that a totally different virus, here named Mirafiori lettuce virus (MiLV), is also very frequently associated with lettuce showing big-vein symptoms. MiLV was mechanically transmissible from lettuce to Chenopodium quinoa and to several other herbaceous test plants. The virus was partially purified, and an antiserum prepared, which did not react with LBVV particles in decoration tests. As reported for LBVV, MiLV was labile, soil-transmitted and had a single capsid protein of 48 kD, but the particles morphologically resembled those of ophioviruses, and like these, MiLV had a genome of three RNA segments approximately 8.5, 1.9 and 1.7 kb in size. MiLV preparations reacted strongly in Western blots and in ISEM with antiserum to Tulip mild mottle mosaic virus, an ophiovirus from Japan also apparently Olpidium-transmitted. They reacted weakly but clearly in Western blots with antiserum to Ranunculus white mottle virus, another ophiovirus. When lettuce seedlings were mechanically inoculated with crude or partially purified extracts from MiLV-infected test plants, many became systemically infected with MiLV and some developed big-vein symptoms. Such plants did not react in ELISA using an LBVV antiserum or an antiserum to tobacco stunt virus, and varicosavirus-like particles were never seen in them in the EM after negative staining. We conclude that MiLV is a hitherto undescribed virus assignable to the genus Ophiovirus. The cause or causes of lettuce big-vein disease and the properties of LBVV may need to be re-evaluated in light of our results.

BackgroundHuman papillomavirus 16 (HPV-16) L1 protein has the capacity to self-assemble into capsomers or virus-like particles (VLPs) that are highly immunogenic, allowing their use in vaccine production. Successful expression of HPV-16 L1 protein has been reported in plants, and plant-produced VLPs have been shown to be immunogenic after administration to animals.ResultsWe investigated the potential of HPV-16 L1 to act as a carrier of two foreign epitopes from Influenza A virus: (i) M2e2-24, ectodomain of the M2 protein (M2e), that is highly conserved among all influenza A isolates, or (ii) M2e2-9, a shorter version of M2e containing the N-terminal highly conserved epitope, that is common for both M1 and M2 influenza proteins. A synthetic HPV-16 L1 gene optimized with human codon usage was used as a backbone gene to design four chimeric sequences containing either the M2e2-24 or the M2e2-9 epitope in two predicted surface-exposed L1 positions. All chimeric constructs were transiently expressed in plants using the Cowpea mosaic virus-derived expression vector, pEAQ-HT. Chimeras were recognized by a panel of linear and conformation-specific anti HPV-16 L1 MAbs, and two of them also reacted with the anti-influenza MAb. Electron microscopy showed that chimeric proteins made in plants spontaneously assembled in higher order structures, such as VLPs of T = 1 or T = 7 symmetry, or capsomers.ConclusionsIn this study, we report for the first time the transient expression and the self-assembly of a chimeric HPV-16 L1 bearing the M2e influenza epitope in plants, representing also the first record of a successful expression of chimeric HPV-16 L1 carrying an epitope of a heterologous virus in plants. This study further confirms the usefulness of human papillomavirus particles as carriers of exogenous epitopes and their potential relevance for the production in plants of monovalent or multivalent vaccines.