Assessing the Mismatch Between Incubation and Latent Periods for Vector-Borne Diseases: The Case of Sharka.

The relative durations of the incubation period (the time between inoculation and symptom expression) and of the latent period (the time between inoculation and infectiousness of the host) are poorly documented for plant diseases. However, the extent of asynchrony between the ends of these two periods (i.e., their mismatch) can be a key determinant of the epidemic dynamics for many diseases and consequently it is of primary interest in the design of disease management strategies. In order to assess this mismatch, an experimental approach was developed and applied using sharka, a severe disease caused by Plum pox virus (PPV, genus Potyvirus, family Potyviridae) affecting trees belonging to the genus Prunus. Leaves of infected young peach trees were used individually as viral sources in aphid-mediated transmission tests carried out at different time points postinoculation in order to bracket symptom onset. By fitting a nonlinear logistic model to the obtained transmission rates, we demonstrated that the first symptoms appear on leaves 1 day before they rapidly become infectious. In addition, among symptomatic leaves, symptom intensity and transmission rate are positively correlated. These results strengthen the conclusion that, under our experimental conditions, incubation and latent periods of PPV infection are almost synchronous.

Molecular evolution and phylogeography of potato virus Y based on the CP gene.

Potato virus Y (PVY) is an important plant pathogen with a wide host range that includes, among others, potato, tobacco, tomato and pepper. The coat protein (CP) of PVY has been commonly used in phylogenetic studies for strain classification. In this study, we used a pool of 292 CP sequences from isolates collected worldwide. After detecting and removing recombinant sequences, we applied Bayesian techniques to study the influence of geography and host species in CP population structure and dynamics. Finally, we performed selection and covariation analyses to identify specific amino acids involved in adaptation. Our results show that PVY CP diversification is significantly accounted for by both geographical and host-driven adaptations. Amino acid positions detected as positively selected concentrate in the N-terminal region of the protein. Some of these selected positions may discriminate among strains, and to a much lesser extent, between potato and non-potato isolates.

Fluorescent-based techniques for viral detection, quantification, and characterization.

Fluorescent-based technologies offer opportunities for developing new assays for detection, quantification, and characterization of viral isolates. According to the intrinsic characteristics of fluorescent-based tools (high specificity, sensitivity, and reliability), such type of molecular assays makes possible investigations on original studies such as evolutionary processes (including fitness measurement of isolates), quantitative epidemiology, or the analysis of synergism and antagonism between closely related isolates. The development of these tools is very simple and requires, in complement to basic molecular knowledge such as extraction, cloning, and (RT)-PCR procedures, only the identification of short specific sequence(s) in the targeted viral genome. The Single Nucleotide Polymorphism (SNP) and the 'real-time' RT-PCR assays are proposed as fluorescent-based tools for qualitative and quantitative viral detection, respectively. Moreover, the SNaPshot technology is described as method for isolate characterization.

Analysis of population structures of viral isolates using single-strand conformation polymorphism method.

The analysis of viral populations requires the use of techniques that describe characteristics of individuals. The single-strand conformation polymorphism (SSCP) makes possible the identification of genetic differences between viral sequences and constitutes an alternative to the expensive and time-consuming cloning and sequencing strategies. Applied to small genomic regions (from 100 to 500 bases in length), SSCP patterns could describe, under appropriate experimental conditions, single nucleotide variations in the studied sequence. The different steps of a complete SSCP procedure, from sampling to pattern analysis (including nucleic acid extraction, RT-PCR amplification, double-stranded DNA quantification, polyacrylamide gel preparation, electrophoresis conditions, and staining procedures), are described using a region (500 bases) of the barley yellow dwarfvirus-PAV (BYDV-PAV, Luteovirus) genome as molecular target.

Increased expression of the E3 ubiquitin ligase RNF5 is associated with decreased survival in breast cancer.

The selective ubiquitination of proteins by ubiquitin E3 ligases plays an important regulatory role in control of cell differentiation, growth, and transformation and their dysregulation is often associated with pathologic outcomes, including tumorigenesis. RNF5 is an E3 ubiquitin ligase that has been implicated in motility and endoplasmic reticulum stress response. Here, we show that RNF5 expression is up-regulated in breast cancer tumors and related cell lines. Elevated expression of RNF5 was seen in breast cancer cell lines that became more sensitive to cytochalasin D- and paclitaxel-induced apoptosis following its knockdown with specific short interfering RNA. Inhibition of RNF5 expression markedly decreased cell proliferation and caused a reorganization of the actin cytoskeleton in response to stress in MCF-7 but not in p53 mutant breast cancer cells, suggesting a p53-dependent function. Significantly, high levels of RNF5 were associated with decreased survival in human breast cancer specimens. Similarly, RNF5 levels were higher in metastatic melanoma specimens and in melanoma, leukemia, ovarian, and renal tumor-derived cell lines, suggesting that increased RNF5 expression may be a common event during tumor progression. These results indicate that RNF5 is a novel regulator of breast cancer progression through its effect on actin cytoskeletal alterations, which also affect sensitivity of breast cancer cells to cytoskeletal targeting antineoplastic agents.

Yap1p, the central regulator of the S. cerevisiae oxidative stress response, is activated by allicin, a natural oxidant and defence substance of garlic.

Allicin is a thiol-reactive sulfur-containing natural product from garlic with a broad range of antimicrobial effects against prokaryotes and eukaryotes. Previous work showed that the S. cerevisiae OSI1 gene is highly induced by allicin and other thiol-reactive compounds, and in silico analysis revealed multiple Yap1p binding motifs in the OSI1 promoter sequence. An OSI1-promoter::luciferase reporter construct expressed in Wt and Δyap1 cells showed absolute Yap1p-dependence for allicin-induced OSI1-expression. A GFP: Yap1p fusion protein accumulated in the nucleus within 10min of allicin treatment and a Δyap1 mutant was highly sensitive to allicin. Yap1p regulates glutathione (GSH) metabolism genes, and Δgsh1, Δgsh2 and Δglr1 mutants showed increased sensitivity to allicin. Allicin activated the OSI1-promoter::luciferase reporter construct in Δgpx3 and Δybp1 cells, indicating that allicin activates Yap1p directly rather than via H2O2 production. A systematic series of C-to-A Yap1p exchange mutants showed that the C-term C598 and C620 residues were necessary for allicin activation. These data suggest that Yap1p is an important transcriptional regulator for the resistance of yeast cells to allicin, and that activation occurs by direct modification of C-term cysteines as shown for other electrophiles.

SNaPshot and CE-SSCP: Two Simple and Cost-Effective Methods to Reveal Genetic Variability Within a Virus Species.

The multiplex SNaPshot and the capillary electrophoresis-single-strand conformation polymorphism (CE-SSCP) procedures are here used for rapid and high-throughput description of the molecular variability of viral populations. Both approaches are based on (1) standard amplification of genomic sequence(s), (2) labeled primers or labeled single-stranded DNA, and (3) migration of fluorescent-labeled molecules in capillary electrophoresis system. The SNaPshot technology was used to describe the diversity of 20 targeted single nucleotide polymorphisms (SNPs) selected from alignment of viral genomic sequences retrieved from public database. The CE-SSCP procedure was applied to identify the polymorphisms of two small (<500 bases in length) genomic regions of viral genomes. The different steps of SNaPshot and CE-SSCP setup procedures are presented using Potato virus Y (PVY, Potyvirus) and Plum pox virus (PPV, Potyvirus) RNA viruses as molecular targets, respectively.

Metagenomics Approaches Based on Virion-Associated Nucleic Acids (VANA): An Innovative Tool for Assessing Without A Priori Viral Diversity of Plants.

This chapter describes an efficient approach that combines quality and yield extraction of viral nucleic acids from plants containing high levels of secondary metabolites and a sequence-independent amplification procedure for both the inventory of known plant viruses and the discovery of unknown ones. This approach turns out to be a useful tool for assessing the virome (the genome of all the viruses that inhabit a particular organism) of plants of interest. We here show that this approach enables the identification of a novel Potyvirus member within a single plant already known to be infected by two other Potyvirus species.

Two redox centers within Yap1 for H2O2 and thiol-reactive chemicals signaling.

The Yap1 transcription factor regulates yeast responses to H2O2 and to several unrelated chemicals and metals. Activation by H2O2 involves Yap1 Cys303-Cys598 intra-molecular disulfide bond formation directed by the H2O2 sensor Orp1/Gpx3. We show here that the electrophile N-ethylmaleimide activates Yap1 by covalent modification of Yap1 C-terminal Cys598, Cys620, and Cys629, in an Orp1 and Yap1-oxidation-independent way, thus establishing an alternate and distinct mode of Yap1 activation. We also show that menadione, a superoxide anion generator and a highly reactive electrophile, operates both modes of Yap1 activation. Further, the Yap1 C-terminal domain reactivity towards other electrophiles (4-hydroxynonenal, iodoacetamide) and metals (cadmium, selenium) suggests a common mechanism for sensing thiol reactive chemicals, involving thiol chemical modification. We propose that Yap1 has two distinct molecular redox centers, one triggered by ROS (hydroperoxides and the superoxide anion) and the other by chemicals with thiol reactivity (electrophiles and divalent heavy metals cations). These data indicate that yeast cells cannot sense these compounds through the same molecular devices, albeit they are all electrophilic.

Yap8p activation in Saccharomyces cerevisiae under arsenic conditions.

Yap8p, a member of the Saccharomyces cerevisiae Yap family, is activated in response to arsenic. Both the mechanisms by which this activation takes place and its regulation have not yet been identified. In this report, we show that Yap8p is not activated at the transcriptional level but, rather, its nuclear transport is actively regulated and dependent on the exportin chromosome region maintenance protein. In addition, it is shown that Cys(132), Cys(137)and Cys(274) are essential for Yap8p localization and transactivation function both of which are required for its biological activity.

Assessment of SNaPshot and single step RT-qPCR methods for discriminating Potato virus Y (PVY) subgroups.

Potato virus Y (PVY) is the most important virus infecting potato (Solanum tuberosum), causing potato tuber necrotic ringspot disease (PTNRD), with a great impact on seed potato production. Numerous PVY strain groups with different pathogenicity and economical impact are distributed worldwide. Tools for accurate and reliable detection and discrimination of PVY strain groups are therefore essential for successful disease management. Two state of the art characterization tools based on detecting molecular markers - RT-qPCR (Kogovsek et al., 2008) and SNaPshot (Rolland et al., 2008) - were assessed for their ability to assign PVY accurately to the correct group. The results were validated by bioassay, ELISA and in silico sequence analysis. The spectrum of PVY strain groups distinguished by SNaPshot is broader than that by RT-qPCR. However, the latter was more reliable in discriminating the PVY(NTN) group members, known for their ability to induce PTNRD on selected potato cultivars. The difference in discrimination precision was due to different molecular markers being targeted by RT-qPCR and SNaPshot. Both tools use genotypic markers for detecting PVY(NTN) strain groups. Future development, however, should be focused on identifying the genomic determinants of the tuber necrosis property. Until then, the RT-qPCR and SNaPshot methods remain the most powerful diagnostic tools for detecting the PVY subgroup isolates found in Europe.

Phylogeography and molecular evolution of potato virus Y.

Potato virus Y (PVY) is an important plant pathogen, whose host range includes economically important crops such as potato, tobacco, tomato, and pepper. PVY presents three main strains (PVY(O), PVY(N) and PVY(C)) and several recombinant forms. PVY has a worldwide distribution, yet the mechanisms that promote and maintain its population structure and genetic diversity are still unclear. In this study, we used a pool of 77 complete PVY genomes from isolates collected worldwide. After removing the effect of recombination in our data set, we used bayesian techniques to study the influence of geography and host species in both PVY population structure and dynamics. We have also performed selection and covariation analyses to identify evolutionarily relevant amino acid residues. Our results show that both geographic and host-driven adaptations explain PVY diversification. Furthermore, purifying selection is the main force driving PVY evolution, although some indications of positive selection accounted for the diversification of the different strains. Interestingly, the analysis of P3N-PIPO, a recently described gene in potyviruses, seems to show a variable length among the isolates analyzed, and this variability is explained, in part, by host-driven adaptation.

Sulfiredoxin protects mice from lipopolysaccharide-induced endotoxic shock.

Peroxiredoxins constitute a major family of cysteine-based peroxide-scavenging enzymes. They carry an intriguing redox switch by undergoing substrate-mediated inactivation via overoxidation of their catalytic cysteine to the sulfinic acid form that is reverted by reduction catalyzed by the sulfinic acid reductase sulfiredoxin (Srx). The biological significance of such inactivation is not understood, nor is the function of Srx1. To address this question, we generated a mouse line with a null deletion of the Srx1-encoding Srxn1 gene. We show here that Srxn1(-/-) mice are perfectly viable and do not suffer from any apparent defects under laboratory conditions, but have an abnormal response to lipopolysaccharide that manifests by increased mortality during endotoxic shock. Microarray-based mRNA profiles show that although the response of Srxn1(-/-) mice to lipopolysaccharide is typical, spanning all spectrum and all pathways of innate immunity, it is delayed by several hours and remains intense when the response of Srxn1(+/+) mice has already dissipated. These data indicate that Srx1 activity protects mice from the lethality of endotoxic shock, adding this enzyme to other host factors, as NRF2 and peroxiredoxin 2, which by regulating cellular reactive oxygen species levels act as important modifiers in the pathogenesis of sepsis.

Complementations and exclusions between mutated versions of a potato virus Y genotype during mixed infections of Nicotiana hosts.

Understanding the processes that have led to the recent prevalence of necrotic genotypes in PVY populations is an important challenge for research programs studying this virus. Non-necrotic PVY(O)-139, necrotic PVY(N)-605 and point mutated versions of PVY(N)-605 (PVY(KRED), PVY(KR) and PVY(ED)), were used in mixtures to inoculate two Nicotiana hosts which express (N. tabacum cv. Xanthi) or not (N. clevelandii) necrosis symptoms in response to infection by PVY(N) group members. The comparison during serial passage experiments of proportions of PVY genotypes produced in mixed infected plants with those of the inocula was used to describe: (i) complementation between PVY(KR) and PVY(N) and between PVY(KRED) and PVY(O) genotypes; (ii) exclusion of the PVY(KRED) genotype, previously described as fitter, during mixed infections in the presence of one of the less fit PVY(N), PVY(ED) and PVY(KR) genotypes and (iii) the prevalence of the non-necrotic PVY(KR) genotype in the presence of PVY(N) parental sequence. These results indicate that the role of both A/G(2213) and A/C(2271) nucleotides in the fitness of PVY genotypes depends on other genetic information in the viral genome that has not yet been identified. Moreover, the collected data indicate that mutation of the nucleotide 2213 in the PVY(N)-605 sequence could lead to the prevalence, both in N. tabacum cv. Xanthi and in N. clevelandii, of the non-necrotic PVY(KR) genotype.

A single-stranded conformational polymorphism (SSCP)-derived quantitative variable to monitor the virulence of a Barley yellow dwarf virus-PAV (BYDV-PAV) isolate during adaptation to the TC14 resistant wheat line.

A standardized single-stranded conformational polymorphism (SSCP) procedure is proposed as an alternative to the time-consuming biological characterization of Barley yellow dwarf virus-PAV (BYDV-PAV) isolates. Using this procedure, six of 21 overlapping regions used to scan the viral genome gave patterns specific to '4E' (avirulent) or '4T' ('4E'-derived virulent) isolates. The calibration of samples and integration of SSCP patterns corresponding to the nucleotide region 1482-2023 allowed the estimation of P(T) values that reflect the proportions of a '4T'-specific band. Analysis of the biological (area under the pathogen progress curve) and molecular (P(T)) data suggested a positive linear relation between these variables. Moreover, sequence analysis of the nucleotide region 1482-2023 highlighted the presence of a nucleotide polymorphism (C/A(1835)) which can be considered as a candidate for virus-host interactions linked to the monitored virulence. According to these parameters, P(T) values associated with '4E'- and '4T'-derived populations show that: (i) long-term infection of a BYDV-PAV isolate on the 'TC14' resistant host leads to the fixation of virulent individuals in viral populations; and (ii) the introduction of susceptible hosts in successive 'TC14' infections results in the maintenance of low virulence of the populations. Thus, the presented study demonstrates that SSCP is a useful tool for monitoring viral populations during the host adaptation process. The described impact of host alternation provides new opportunities for the use of the 'TC14' resistance source in BYDV-resistant breeding programmes. This study is part of the global effort made by the scientific community to propose sustainable alternatives to the chemical control of this viral disease.

Regulation of endoplasmic reticulum-associated degradation by RNF5-dependent ubiquitination of JNK-associated membrane protein (JAMP).

Clearance of misfolded proteins by endoplasmic reticulum (ER)-associated degradation (ERAD) requires concerted activity of chaperones, adaptor proteins, ubiquitin ligases, and proteasomes. RNF5 is a ubiquitin ligase anchored to the ER membrane implicated in ERAD via ubiquitination of misfolded proteins. Among RNF5-associated proteins is JNK-associated membrane protein (JAMP), a 7-transmembrane protein located within the ER membrane that facilitates degradation of misfolded proteins through recruitment of proteasomes and ERAD regulatory components. Here we demonstrate that RNF5 associates with JAMP in the ER membrane. This association results in Ubc13-dependent RNF5-mediated noncanonical ubiquitination of JAMP. This ubiquitination does not alter JAMP stability but rather inhibits its association with Rpt5 and p97. Consequently, clearance of misfolded proteins, such as CFTRDelta508 and T cell receptor alpha, is less efficient, resulting in their greater accumulation. Significantly, the RNF5 effect on JAMP is seen prior to and after ER stress response, thereby highlighting a novel mechanism to limit ERAD and proteasome assembly at the ER, to the actual ER stress response.

JAMP optimizes ERAD to protect cells from unfolded proteins.

Clearance of misfolded proteins from the ER is central for maintenance of cellular homeostasis. This process requires coordinated recognition, ER-cytosol translocation, and finally ubiquitination-dependent proteasomal degradation. Here, we identify an ER resident seven-transmembrane protein (JAMP) that links ER chaperones, channel proteins, ubiquitin ligases, and 26S proteasome subunits, thereby optimizing degradation of misfolded proteins. Elevated JAMP expression promotes localization of proteasomes at the ER, with a concomitant effect on degradation of specific ER-resident misfolded proteins, whereas inhibiting JAMP promotes the opposite response. Correspondingly, a jamp-1 deleted Caenorhabditis elegans strain exhibits hypersensitivity to ER stress and increased UPR. Using biochemical and genetic approaches, we identify JAMP as important component for coordinated clearance of misfolded proteins from the ER.

The ER-bound RING finger protein 5 (RNF5/RMA1) causes degenerative myopathy in transgenic mice and is deregulated in inclusion body myositis.

Growing evidence supports the importance of ubiquitin ligases in the pathogenesis of muscular disorders, although underlying mechanisms remain largely elusive. Here we show that the expression of RNF5 (aka RMA1), an ER-anchored RING finger E3 ligase implicated in muscle organization and in recognition and processing of malfolded proteins, is elevated and mislocalized to cytoplasmic aggregates in biopsies from patients suffering from sporadic-Inclusion Body Myositis (sIBM). Consistent with these findings, an animal model for hereditary IBM (hIBM), but not their control littermates, revealed deregulated expression of RNF5. Further studies for the role of RNF5 in the pathogenesis of s-IBM and more generally in muscle physiology were performed using RNF5 transgenic and KO animals. Transgenic mice carrying inducible expression of RNF5, under control of beta-actin or muscle specific promoter, exhibit an early onset of muscle wasting, muscle degeneration and extensive fiber regeneration. Prolonged expression of RNF5 in the muscle also results in the formation of fibers containing congophilic material, blue-rimmed vacuoles and inclusion bodies. These phenotypes were associated with altered expression and activity of ER chaperones, characteristic of myodegenerative diseases such as s-IBM. Conversely, muscle regeneration and induction of ER stress markers were delayed in RNF5 KO mice subjected to cardiotoxin treatment. While supporting a role for RNF5 Tg mice as model for s-IBM, our study also establishes the importance of RNF5 in muscle physiology and its deregulation in ER stress associated muscular disorders.

Infectivity of Cryptosporidium hominis and Cryptosporidium parvum genotype 2 isolates in immunosuppressed Mongolian gerbils.

One-month-old dexamethasone-immunosuppressed Mongolian gerbils were challenged with 1 oocyst to 2 x 10(5) oocysts from two isolates genotyped as Cryptosporidium hominis and C. parvum (genotype 2), respectively. A similar dose-dependent gut infection was obtained, and the initial genotype maintained for 21 to 22 days. The data suggest that immunosuppressed gerbils provide a reliable rodent model of persistent C. hominis infection.