Rice yellow mottle virus is a suitable amplicon vector for an efficient production of an anti-leishmianiasis vaccine in Nicotiana benthamiana leaves.

BACKGROUND: Since the 2000's, plants have been used as bioreactors for the transient production of molecules of interest such as vaccines. To improve protein yield, "amplicon" vectors based on plant viruses are used. These viral constructs, engineered to carry the gene of interest replicate strongly once introduced into the plant cell, allowing significant accumulation of the protein. Here, we evaluated the suitability of the monocot-infecting RNA virus Rice yellow mottle virus (RYMV) as an amplicon vector. The promastigote surface antigen (PSA) of the protozoan Leishmania was considered as a protein of interest due to its vaccine properties against canine leishmaniasis. RESULTS: Since P1 (ORF1) and CP (ORF3) proteins are not strictly necessary for viral replication, ORF1 was deleted and the PSA gene was substituted to ORF3 in the RYMV-based vector. We evaluated its expression in the best described plant bioreactor system, Nicotiana benthamiana which, unlike rice, allows transient transformation by Agrobacterium. Despite not being its natural host, we demonstrated a low level of RYMV-based vector replication in N. benthamiana leaves. Under optimized ratio, we showed that the P19 silencing suppressor in combination with the missing viral CP ORF significantly enhanced RYMV amplicon replication in N. benthamiana. Under these optimized CP/P19 conditions, we showed that the RYMV amplicon replicated autonomously in the infiltrated N. benthamiana cells, but was unable to move out of the infiltrated zones. Finally, we showed that when the RYMV amplicon was expressed under the optimized conditions we set up, it allowed enhanced PSA protein accumulation in N. benthamiana compared to the PSA coding sequence driven by the 35S promoter without amplicon background. CONCLUSION: This work demonstrates that a non-dicot-infecting virus can be used as an amplicon vector for the efficient production of proteins of interest such as PSA in N. benthamiana leaves.

First Report of Rice stripe necrosis virus Infecting Rice in Burkina Faso.

Rice stripe necrosis virus (RSNV) was first described in 1977 as a new virus infecting rice in Cote d'Ivoire (3) and was subsequently observed in Liberia, Nigeria, and Sierra Leone (2). RSNV is a soil-borne virus transmitted by the fungus Polymyxa graminis (1) and belongs to the genus Benyvirus (4). During a survey carried out in April of 2013, severe symptoms characterized by seedling death, severe plant malformation, and foliar striping were observed on rice plants in an experimental field of INERA at Banfora located in western Burkina Faso. Disease incidence in the field was estimated to be 80 ± 5%. The symptoms of disease were successfully transmitted to the susceptible rice (Oryza sativa) cultivar IR64 by soil transmission experiments (1). RSNV was detected by ELISA using a polyclonal antiserum (1), kindly provided by Dr. Denis Fargette, IRD, Montpellier, France. Total nucleic acid was extracted with TRIzol reagent (Invitrogen) from IR64 and field infected samples. The presence of the virus was confirmed by RT-PCR using primers 5'-CATCTTGTCGAGATGAG-3' and 5'-GCGTTGTCTTTATCAGTG-3' for specific sequences flanking the RNA2 CP gene. The RT-PCR product was directly sequenced and the sequence was deposited in GenBank (Accession No. LK023710). Sequence analysis showed that the CP gene of the RSNV isolate from Burkina Faso shared the highest nucleotide sequence identity (97.6%) with the known RSNV CP gene sequence from the Colombian isolate (EU099845) available in GenBank, confirming the presence of RSNV in the rice crops in Burkina Faso. To our knowledge, this is the first confirmed report of RSNV in Burkina Faso. Further studies are needed to determine its incidence and spread in the country. Detection of RSNV in Burkina Faso signals the urgent need for adoption of appropriate measures to restrict the spread and impact of this virus within Africa. References: (1) C. Fauquet and J. C. Thouvenel. Proc. Acad. Sci. Ser. D 296:575, 1983. (2) C. Fauquet et al. Develop. Appl. Biol. 2:71, 1988. (3) D. Louvel and J.-M. Bidaux. Agronomie Tropicale 32:257, 1977. (4) I. Lozano and F. Morales. Eur. J. Plant Pathol. 124:673, 2009.

Recombination, selection and clock-like evolution of Rice yellow mottle virus.

The clock-like diversification of Rice yellow mottle virus (RYMV), a widespread RNA plant virus that infects rice in Africa, was tested following a three-step approach with (i) an exhaustive search of recombinants, (ii) a comprehensive assessment of the selective constraints over lineages, and (iii) a stepwise series of tests of the molecular clock hypothesis. The first evidence of recombination in RYMV was found in East Africa, in the region most favorable to co-infection. RYMV evolved under a pronounced purifying selection, but the selection pressure did vary among lineages. There was no phylogenetic evidence of transient deleterious mutations. ORF2b, which codes for the polymerase and is the most constrained ORF, tends to diversify clock-like. With the other ORFs and the full genome, the departure from the strict clock model was limited. This likely reflects the dominant conservative selection pressure and the clock-like fixation of synonymous mutations.

Rice yellow mottle virus, an RNA plant virus, evolves as rapidly as most RNA animal viruses.

The rate of evolution of an RNA plant virus has never been estimated using temporally spaced sequence data, by contrast to the information available on an increasing range of animal viruses. Accordingly, the evolution rate of Rice yellow mottle virus (RYMV) was calculated from sequences of the coat protein gene of isolates collected from rice over a 40-year period in different parts of Africa. The evolution rate of RYMV was estimated by pairwise distance linear regression on five phylogeographically defined groups comprising a total of 135 isolates. It was further assessed from 253 isolates collected all over Africa by Bayesian coalescent methods under strict and relaxed molecular clock models and under constant size and skyline population genetic models. Consistent estimates of the evolution rate between 4 x 10(-4) and 8 x 10(-4) nucleotides (nt)/site/year were obtained whatever method and model were applied. The synonymous evolution rate was between 8 x 10(-4) and 11 x 10(-4) nt/site/year. The overall and synonymous evolution rates of RYMV were within the range of the rates of 50 RNA animal viruses, below the average but above the distribution median. Experimentally, in host change studies, substitutions accumulated at an even higher rate. The results show that an RNA plant virus such as RYMV evolves as rapidly as most RNA animal viruses. Knowledge of the molecular clock of plant viruses provides methods for testing a wide range of biological hypotheses.