Viruses express proteins with silencing suppression activity to counteract the RNA silencing-mediated defense response of the host. In the family Closteroviridae, examples of multiple-component RNA silencing suppression systems have been reported. To ascertain if this is a general strategy in this group of viruses, we have explored the bipartite genome of Tomato chlorosis virus (ToCV, genus Crinivirus). We have identified the RNA1-encoded p22 protein as an effective silencing suppressor by using a Agrobacterium co-infiltration assay. p22 suppressed local RNA silencing induced either by sense RNA or dsRNA very efficiently, but did not interfere with short or long-distance systemic spread of silencing. We have also demonstrated by using the heterologous vector PVX the silencing suppression activity of the RNA-2 encoded coat protein (CP) and minor coat protein (CPm). In this study, we demonstrate an even greater complexity of silencing suppressor activity for a plant virus, and for the first time we show the presence of RNA silencing suppressor genes encoded by both genomic RNA molecules of a bipartite genome in the complex family Closteroviridae.

SummaryThe small size of plant viral genomes, the ease with which they can be manipulated, and the simplicity of the infection process is making the viral vectors an attractive alternative to the transgenic systems for the expression of foreign proteins in plants. One use of these virus expression systems is for vaccine production. There are two basic types of viral system that have been developed for the production of immunogenic peptides and proteins in plants: epitope presentation and polypeptide expression systems. In this review, we discuss advances made in this field.

INTRODUCTIONThe first plant viruses to be developed as expression vectors in the early 1980s were those with DNA genomes (for reviews, see 42, 56). This was due to the fact that, at the time, only the DNA genomes could be manipulated and the technology for creating infectious cDNA copies of viruses with RNA genomes did not exist. However, the vast majority of plant viruses have genomes that consist of one or more strands of positive-sense RNA. These viruses infect a wide range of hosts and some can reach extremely high titres. Following the construction of the first full-length cDNA clones shown to be infectious (1), the past 25 years has seen a large number of RNA viruses developed as vectors for the expression of foreign sequences and other uses, such as gene silencing (10, 31,42, 55, 56,70).Many proteins have been successfully expressed with virus vectors and significant progress in vector design has been driven by the demands of this application. Generally developed with the expression of fluorescent marker proteins, RNA virus-based vectors have become a highly effective means of producing recombinant heterologous protein in plant tissue within a short time frame (28, 35,44,67). In addition to their use as vectors for the production of heterologous polypeptides and as gene silencing vectors, plant RNA viruses have also provided a source of particles for various applications. Virus capsids provide nano-scale particles with consistent size and shape, which can be exploited for a number of chemical and biological applications (72). For example, a number of systems make use of the repetitive geometry of plant virus capsids to present multiple copies of antigenic sequences, to increase their potential as a source of novel vaccines (10,39, 58). Also, both wild type and genetically modified capsids of plant viruses are also being used as biotemplates for novel materials in nanotechnology (88). 3Cowpea mosaic virus (CPMV) has borne witness to most of the aforementioned biotechnological uses of RNA viruses. The year 2009 marks the 50 th anniversary of the first description of CPMV as a pathogen of cowpeas (Vigna unguiculata) in West Africa (13). As a result of its ease of propagation, high yield and the stability of the viral particles, CPMV rapidly became an object of intense scientific research. Early studies revealed the bipartite nature of the viral genome (7,78), the structural similarities between CPMV and the animal picornaviruses (87) and the mechanism of gene expression (polyprotein processing; 53).Subsequent work resulted in the determination of the nucleotide sequences of both genomic segments (81, 43), a realisation of the genetic similarities between CPMV and picornaviruses (21), an atomic resolution structure of the virus particles (32, 33), and the creation of infectious cDNA clones (16, 25,84). A crucial step for the development of practical CPMVbased expression systems was the creation of vectors that could be inoculated by agroinfiltration (38), and this approach is now the method of choice for intr...

HSV-1 establishes life-long latency that can result in clinical relapses or in asymptomatic virus shedding. Although virtually all adults have been exposed to HSV-1, the clinical course varies remarkably. Genetic host variability could be related to this clinical diversity. In this study, we analyzed the contribution of gene families in chromosomes 1, 6, 12, and 19, which encode key regulators of the innate and adaptive immunity, in a cohort of 302 individuals. Class I and class II alleles of the HLA system, the copy-number variation of NK cell receptor genes (KIR and NKG2C), the combinations of killer cell Ig-like receptor and their HLA ligands, and CD16A and CD32A allotypes of variable affinity for IgG subclasses were all studied. Although no major susceptibility locus for HSV-1 was identified, our results show that the risk of suffering clinical HSV-1 infection is modified by MHC class I allotypes (B*18, C*15, and the group of alleles encoding A19), the high-affinity receptor/ligand pair KIR2DL2/HLA-C1, and the CD16A-158V/F dimorphism. Conversely, HLA class II and CD32A polymorphisms and NKG2C deletion did not seem to influence the clinical course of herpetic infection. Collectively, these findings support an important role in host defense against herpetic infection for several polymorphic genes implicated in adaptive immunity and in surveillance of its subversion. They confirm the crucial role of cytotoxic cells (CTL and NK) and the contribution of genetic diversity to the clinical course of HSV-1 infection.

Hop stunt viroid (HSVd) is able to infect a number of herbaceous and woody hosts, such as grapevine, Citrus or Prunus plants. Previous phylogenetic analyses have suggested the existence of three major groups of HSVd isolates (plum-type, hop-type and citrus-type). The fact that these groups often contain isolates from only a limited number of isolation hosts prompted the suggestion that group-discriminating sequence variations could, in fact, represent host-specific sequence determinants which may facilitate or be required for replication in a given host. In an effort to further understand the relationships between HSVd and its different hosts, HSVd variants from eight naturally infected Prunus sources, including apricot, peach and Japanese plum have been cloned and sequenced. In total, ten

The avirulence determinant triggering the resistance conferred by the tomato gene Sw-5 against Tomato spotted wilt virus (TSWV) is still unresolved. Sequence comparison showed two substitutions (C118Y and T120N) in the movement protein NSm present only in TSWV resistance-breaking (RB) isolates. In this work, transient expression of NSm of three TSWV isolates [RB1 (T120N), RB2 (C118Y) and non-resistance-breaking (NRB)] in Nicotiana benthamiana expressing Sw-5 showed a hypersensitive response (HR) only with NRB. Exchange of the movement protein of Alfalfa mosaic virus (AMV) with NSm supported cell-to-cell and systemic transport of the chimeric AMV RNAs into N. tabacum with or without Sw-5, except for the constructs with NBR when Sw-5 was expressed, although RB2 showed reduced cell-to-cell transport. Mutational analysis revealed that N120 was sufficient to avoid the HR, but the substitution V130I was required for systemic transport. Finally, co-inoculation of RB and NRB AMV chimeric constructs showed different prevalence of RB or NBR depending on the presence or absence of Sw-5. These results indicate that NSm is the avirulence determinant for Sw-5 resistance, and mutations C118Y and T120N are responsible for resistance breakdown and have a fitness penalty in the context of the heterologous AMV system.

Infected tomato plants are stunted or dwarfed, with leaflets rolled upwards and inwards; young leaves are slightly chlorotic; in recently infected plants, fruits might not be produced or, if produced, are small and unmarketable. In common bean, some TYLCVs produce the bean leaf crumple disease, with thickening, epinasty, crumpling, blade reduction and upward curling of leaves, as well as abnormal shoot proliferation and internode reduction; the very small leaves result in a bushy appearance.

Mycoviruses from plant pathogens can induce hypovirulence (reduced virulence) in their host fungi and have gained considerable attention as potential biocontrol tools. An increasing number of mycoviruses that induce fungal hypovirulence, from a wide variety of taxonomic groups, are currently being reported. Successful application of these viruses in disease management is greatly dependent on their ability to spread in the natural populations of the pathogen. Mycoviruses generally lack extracellular routes of transmission. Hyphal anastomosis is the main route of horizontal mycovirus transmission to other isolates, and conidia of vertical transmission to the progeny. Transmission efficiencies are influenced by both the fungal host and the infecting virus. Interestingly, artificial transfection methods have shown that potential biocontrol mycoviruses often have the ability to infect a variety of fungi. This expands their possible use to the control of pathogens others than those where they were identified. Mycovirus research is also focused on gaining insights into their complex molecular biology and the molecular bases of fungus−virus interactions. This knowledge could be exploited to manipulate the mycovirus and/or the host and generate combinations with enhanced properties in biological control. Finally, when exploring the use of mycoviruses in field conditions, the pathogen life style and the characteristics of the disease and crops affected will deeply impact the specific challenges to overcome, and the development of biocontrol formulations and delivery methods.