Identification of a Novel Myxoma Virus C7-Like Host Range Factor That Enabled a Species Leap from Rabbits to Hares.

Myxoma virus (MYXV) is naturally found in rabbit Sylvilagus species and is known to cause lethal myxomatosis in European rabbits (Oryctolagus cuniculus). In 2019, an MYXV strain (MYXV strain Toledo [MYXV-Tol]) causing myxomatosis-like disease in Iberian hares (Lepus granatensis) was identified. MYXV-Tol acquired a recombinant region of ∼2.8 kb harboring several new genes, including a novel host range gene (M159) that we show to be an orthologous member of the vaccinia virus C7 host range family. Here, to test whether M159 alone has enabled MYXV to alter its host range to Iberian hares, several recombinant viruses were generated, including an MYXV-Tol ΔM159 (knockout) strain. While MYXV-Tol underwent fully productive infection in hare HN-R cells, neither the wild-type MYXV-Lau strain (lacking M159) nor vMyxTol-ΔM159 (deleted for M159) was able to infect and replicate, showing that the ability of MYXV-Tol to infect these cells and replicate depends on the presence of M159. Similar to other C7L family members, M159 was shown to be expressed as an early/late gene but was translocated into the nucleus at later time points, indicating that further studies are needed to elucidate its role in the nucleus. Finally, in rabbit cells, the M159 protein did not contribute to increased replication but was able to upregulate the replication levels of MYXV in nonpermissive and semipermissive human cancer cells, suggesting that the M159-targeted pathway is conserved across mammalian species. Altogether, these observations demonstrate that the M159 protein plays a critical role in determining the host specificity of MYXV-Tol in hare and human cells by imparting new host range functions. IMPORTANCE The coevolution of European rabbit populations and MYXV is a textbook example of an arms race between a pathogen and a host. Recently, a recombinant MYXV (MYXV-Tol) crossed the species barrier by jumping from leporid species to another species, causing lethal myxomatosis-like disease. Given the highly pathogenic nature of this new virus in hares and the incidences of other poxvirus cross-species spillovers into other animals, including humans, it is important to understand how and why MYXV-Tol was able to become virulent in a new host species. The results presented clearly demonstrate that M159 is the key factor allowing MYXV-Tol replication in hare cells by imparting new host range functions. These results have the potential to improve current knowledge about the virulence of poxviruses and provide a platform to better understand the new MYXV-Tol, rendering the virus capable of leaping into a new host species.

Determination of glyphosate exposure in the Iberian hare: A potential focal species associated to agrosystems.

Glyphosate is the most used herbicide worldwide. It is a small and highly polar pesticide whose physicochemical properties makes its analytical determination difficult. Here, a procedure based on liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) was developed for glyphosate determination in samples of gastric content from wildlife. Iberian hare (Lepus granatensis), a herbivorous mammal species, strongly associated to agrosystems was selected as model species. The procedure involves direct analysis of sample without derivatization or instead of neither further cleaning steps. The procedure was validated by inter-day accuracy and precision studies with gastric content of hare spiked with glyphosate at ecologically relevant concentrations for the species (0.1-6 µg/g), and with 1 µg/g of isotopically labelled internal standard (glyphosate-2-13C,15N). Finally, glyphosate residues in hunted animals from pesticide-treated and pesticide-free areas (n = 75 and 28, respectively), as well as from hares found dead in the field (n = 11) were analysed. The linearity of both standards in extraction solutions and procedural calibration curves with spiked samples was similar, both with determination coefficients (r2) higher than 0.99. Satisfactory recoveries in spiked samples were achieved within the range of 95% to 118% (CV ≤ 20%). The limit of detection of glyphosate in hare gastric content was 0.03 µg/g. Prevalence of glyphosate in hunted animals from pesticide-treated areas ranged between 9 and 22%, increasing to 45% in animals found dead. The glyphosate concentrations detected in the gastric content of hares ranged from 0.11 to 16 µg/g. No residues were detected in animals from pesticide-free areas. In practice, the developed methodology may be particularly useful in the context of research and other work on the exposure in wildlife of one of the most used pesticides nowadays.

Direct flow cytometry measurements reveal a fine-tuning of symbiotic cell dynamics according to the host developmental needs in aphid symbiosis.

Endosymbiotic associations constitute a driving force in the ecological and evolutionary diversification of metazoan organisms. Little is known about whether and how symbiotic cells are coordinated according to host physiology. Here, we use the nutritional symbiosis between the insect pest, Acyrthosiphon pisum, and its obligate symbiont, Buchnera aphidicola, as a model system. We have developed a novel approach for unculturable bacteria, based on flow cytometry, and used this method to estimate the absolute numbers of symbionts at key stages of aphid life. The endosymbiont population increases exponentially throughout nymphal development, showing a growing rate which has never been characterized by indirect molecular techniques. Using histology and imaging techniques, we have shown that the endosymbiont-bearing cells (bacteriocytes) increase significantly in number and size during the nymphal development, and clustering in the insect abdomen. Once adulthood is reached and the laying period has begun, the dynamics of symbiont and host cells is reversed: the number of endosymbionts decreases progressively and the bacteriocyte structure degenerates during insect aging. In summary, these results show a coordination of the cellular dynamics between bacteriocytes and primary symbionts and reveal a fine-tuning of aphid symbiotic cells to the nutritional demand imposed by the host physiology throughout development.

Stochastic fluctuations and distributed control of gene expression impact cellular memory.

Despite the stochastic noise that characterizes all cellular processes the cells are able to maintain and transmit to their daughter cells the stable level of gene expression. In order to better understand this phenomenon, we investigated the temporal dynamics of gene expression variation using a double reporter gene model. We compared cell clones with transgenes coding for highly stable mRNA and fluorescent proteins with clones expressing destabilized mRNA-s and proteins. Both types of clones displayed strong heterogeneity of reporter gene expression levels. However, cells expressing stable gene products produced daughter cells with similar level of reporter proteins, while in cell clones with short mRNA and protein half-lives the epigenetic memory of the gene expression level was completely suppressed. Computer simulations also confirmed the role of mRNA and protein stability in the conservation of constant gene expression levels over several cell generations. These data indicate that the conservation of a stable phenotype in a cellular lineage may largely depend on the slow turnover of mRNA-s and proteins.

Differential miRNA expression profiles in proliferating or differentiated keratinocytes in response to gamma irradiation.

BACKGROUND: MicroRNAs (miRNAs), a group of short non-coding RNAs that negatively regulate gene expression, have recently emerged as potential modulators of cellular response to ionizing radiations both in vitro and in vivo in various cell types and tissues. However, in epidermal cells, the involvement of the miRNA machinery in the cellular response to ionizing radiations remains to be clarified. Indeed, understanding the mechanisms of cutaneous radiosensitivity is an important issue since skin is the most exposed organ to ionizing radiations and among the most sensitive. RESULTS: We settled up an expression study of miRNAs in primary human skin keratinocytes using a microfluidic system of qPCR assay, which permits to assess the expression of almost 700 annotated miRNAs. The keratinocytes were cultured to a proliferative or a differentiated state mimicking basal or suprabasal layers of human epidermis. These cells were irradiated at 10 mGy or 6 Gy and RNA was extracted 3 hours after irradiation. We found that proliferative cells irradiated at 6 Gy display a global fall of miRNA expression whereas differentiated cells exposed to the same dose display a global increase of miRNAs expression. We identified twenty miRNAs weakly but significantly modulated after 6 Gy irradiation, whereas only 2 miRNAs were modulated after low-dose irradiation in proliferating cells. To go further into the biological meaning of this miRNA response, we over-expressed some of the responding miRNA in proliferating cells: we observed a significant decrease of cell viability 72 hours after irradiation. Functional annotation of their predicted targets revealed that G-protein related pathways might be regulated by these responding miRNAs. CONCLUSIONS: Our results reveal that human primary keratinocytes exposed to ionizing irradiation expressed a miRNA pattern strongly related to the differentiation status of irradiated cells. We also demonstrate that some miRNAs play a role in the radiation response to ensure the short-term survival of irradiated keratinocytes.

Quantifying the contribution of chromatin dynamics to stochastic gene expression reveals long, locus-dependent periods between transcriptional bursts.

BACKGROUND: A number of studies have established that stochasticity in gene expression may play an important role in many biological phenomena. This therefore calls for further investigations to identify the molecular mechanisms at stake, in order to understand and manipulate cell-to-cell variability. In this work, we explored the role played by chromatin dynamics in the regulation of stochastic gene expression in higher eukaryotic cells. RESULTS: For this purpose, we generated isogenic chicken-cell populations expressing a fluorescent reporter integrated in one copy per clone. Although the clones differed only in the genetic locus at which the reporter was inserted, they showed markedly different fluorescence distributions, revealing different levels of stochastic gene expression. Use of chromatin-modifying agents showed that direct manipulation of chromatin dynamics had a marked effect on the extent of stochastic gene expression. To better understand the molecular mechanism involved in these phenomena, we fitted these data to a two-state model describing the opening/closing process of the chromatin. We found that the differences between clones seemed to be due mainly to the duration of the closed state, and that the agents we used mainly seem to act on the opening probability. CONCLUSIONS: In this study, we report biological experiments combined with computational modeling, highlighting the importance of chromatin dynamics in stochastic gene expression. This work sheds a new light on the mechanisms of gene expression in higher eukaryotic cells, and argues in favor of relatively slow dynamics with long (hours to days) periods of quiet state.

Towards experimental manipulation of stochasticity in gene expression.

For decades, most of molecular biology was driven by the "central dogma" in which the phenotype is defined by the genotype following a fully deterministic point of view. However, during the last 10 years, a wealth of studies has demonstrated that a given genotype can generate multiple phenotypes in identical environmental conditions, mainly because of the inherently probabilistic nature of the transcription process. It has also been shown that cells can tune this variability at the molecular level. Although previously described as a useless "noise", stochastic gene expression has now been shown by many authors to be an essential part of diverse biological processes. Chromatin dynamics having a central role in higher eukaryotes, we decided to investigate its involvement in the generation and control of stochasticity in gene expression (SGE). Our experiments reveal that the chromatin environment of a gene plays an important role in regulating SGE. Indeed, we find that histone acetylation and DNA methylation significantly affect SGE, suggesting that cells are able to adjust the variability of the expression of their genes through modification of chromatin marks. Given that the alteration of chromatin marks is itself subject to the expression of chromatin modifiers, our results shed light on a complex circular causality with on the one hand, the effect of gene expression on chromatin and on the other hand, the influence of the local chromatin environment of a gene on the dynamics of its expression.

Multimodal dynamic response of the Buchnera aphidicola pLeu plasmid to variations in leucine demand of its host, the pea aphid Acyrthosiphon pisum.

Aphids, important agricultural pests, can grow and reproduce thanks to their intimate symbiosis with the γ-proteobacterium Buchnera aphidicola that furnishes them with essential amino acids lacking in their phloem sap diet. To study how B. aphidicola, with its reduced genome containing very few transcriptional regulators, responds to variations in the metabolic requirements of its host, we concentrated on the leucine metabolic pathway. We show that leucine is a limiting factor for aphid growth and it displays a stimulatory feeding effect. Our metabolic analyses demonstrate that symbiotic aphids are able to respond to leucine starvation or excess by modulating the neosynthesis of this amino acid. At a molecular level, this response involves an early important transcriptional regulation (after 12 h of treatment) followed by a moderate change in the pLeu plasmid copy number. Both responses are no longer apparent after 7 days of treatment. These experimental data are discussed in the light of a re-annotation of the pLeu plasmid regulatory elements. Taken together, our data show that the response of B. aphidicola to the leucine demand of its host is multimodal and dynamically regulated, providing new insights concerning the genetic regulation capabilities of this bacterium in relation to its symbiotic functions.

Systemic analysis of the symbiotic function of Buchnera aphidicola, the primary endosymbiont of the pea aphid Acyrthosiphon pisum.

Buchnera aphidicola is the primary obligate intracellular symbiont of most aphid species. B. aphidicola and aphids have been evolving in parallel since their association started, about 150 Myr ago. Both partners have lost their autonomy, and aphid diversification has been confined to smaller ecological niches by this co-evolution. B. aphidicola has undergone major genomic and biochemical changes as a result of adapting to intracellular life. Several genomes of B. aphidicola from different aphid species have been sequenced in the last decade, making it possible to carry out analyses and comparative studies using system-level in silico methods. This review attempts to provide a systemic description of the symbiotic function of aphid endosymbionts, particularly of B. aphidicola from the pea aphid Acyrthosiphon pisum, by analyzing their structural genomic properties, as well as their genetic and metabolic networks.

A combination of transposable elements and magnetic cell sorting provides a very efficient transgenesis system for chicken primary erythroid progenitors.

BACKGROUND: Stable transgenesis is an undeniable key to understanding any genetic system. Retrovirus-based insertional strategies, which feature several technical challenges when they are used, are often limited to one particular species, and even sometimes to a particular cell type as the infection depends on certain cellular receptors. A universal-like system, which would allow both stable transgene expression independent of the cell type and an efficient sorting of transfected cells, is required when handling cellular models that are incompatible with retroviral strategies. RESULTS: We report here on the combination of a stable insertional transgenesis technique, based on the Tol2 transposon system together with the magnetic cell sorting (MACS) technique, which allows specific selection of cells carrying the transgene in an efficient, reliable and rapid way. CONCLUSION: This new Tol2/MACS system leads to stable expression in a culture of primary chicken erythroid cells highly enriched in cells expressing the transgene of interest. This system could be used in a wide variety of vertebrate species.

Impact of host developmental age on the transcriptome of the symbiotic bacterium Buchnera aphidicola in the pea aphid (Acyrthosiphon pisum).

Of the 617 genes from Buchnera aphidicola, the obligate bacterial symbiont of the pea aphid, 23% were differentially expressed in embryos compared to adults. Genes involved in flagellar apparatus and riboflavin synthesis exhibited particularly robust upregulation in embryos, suggesting functional differences between the symbiosis in the adult and embryo insect.

Conservation of the links between gene transcription and chromosomal organization in the highly reduced genome of Buchnera aphidicola.

BACKGROUND: Genomic studies on bacteria have clearly shown the existence of chromosomal organization as regards, for example, to gene localization, order and orientation. Moreover, transcriptomic analyses have demonstrated that, in free-living bacteria, gene transcription levels and chromosomal organization are mutually influenced. We have explored the possible conservation of relationships between mRNA abundances and chromosomal organization in the highly reduced genome of Buchnera aphidicola, the primary endosymbiont of the aphids, and a close relative to Escherichia coli. RESULTS: Using an oligonucleotide-based microarray, we normalized the transcriptomic data by genomic DNA signals in order to have access to inter-gene comparison data. Our analysis showed that mRNA abundances, gene organization (operon) and gene essentiality are correlated in Buchnera (i.e., the most expressed genes are essential genes organized in operons) whereas no link between mRNA abundances and gene strand bias was found. The effect of Buchnera genome evolution on gene expression levels has also been analysed in order to assess the constraints imposed by the obligate symbiosis with aphids, underlining the importance of some gene sets for the survival of the two partners. Finally, our results show the existence of spatial periodic transcriptional patterns in the genome of Buchnera. CONCLUSION: Despite an important reduction in its genome size and an apparent decay of its capacity for regulating transcription, this work reveals a significant correlation between mRNA abundances and chromosomal organization of the aphid-symbiont Buchnera.

Different levels of transcriptional regulation due to trophic constraints in the reduced genome of Buchnera aphidicola APS.

Symbiotic associations involving intracellular microorganisms and animals are widespread, especially for species feeding on poor or unbalanced diets. Buchnera aphidicola, the obligate intracellular bacterium associated with most aphid species, provides its hosts with essential amino acids (EAAs), nutrients in short supply in the plant phloem sap. The Buchnera genome has undergone severe reductions during intracellular evolution. Genes for EAA biosynthesis are conserved, but most of the transcriptional regulatory elements are lost. This work addresses two main questions: is transcription in Buchnera (i) regulated and (ii) scaled to aphid EAA demand? Two microarray experiments were designed for profiling the gene expression in Buchnera. The first one was characterized by a specific depletion of tyrosine and phenylalanine in the aphid diet, and the second experiment combined a global diminution of EAAs in the aphid diet with a sucrose concentration increase to manipulate the aphid growth rate. Aphid biological performance and budget analysis (the balance between EAAs provided by the diet and those synthesized by Buchnera) were performed to quantify the nutritional demand from the aphids toward their symbiotic bacteria. Despite the absence of known regulatory elements, a significant transcriptional regulation was observed at different levels of organization in the Buchnera genome: between genes, within putative transcription units, and within specific metabolic pathways. However, unambiguous evidence for transcriptional changes underpinning the scaling of EAA biosynthesis to aphid demand was not obtained. The phenotypic relevance of the transcriptional response from the reduced genome of Buchnera is addressed.

Codon usage bias and tRNA over-expression in Buchnera aphidicola after aromatic amino acid nutritional stress on its host Acyrthosiphon pisum.

Codon usage bias and relative abundances of tRNA isoacceptors were analysed in the obligate intracellular symbiotic bacterium, Buchnera aphidicola from the aphid Acyrthosiphon pisum, using a dedicated 35mer oligonucleotide microarray. Buchnera is archetypal of organisms living with minimal metabolic requirements and presents a reduced genome with high-evolutionary rate. Codonusage in Buchnera has been overcome by the high mutational bias towards AT bases. However, several lines of evidence for codon usage selection are given here. A significant correlation was found between tRNA relative abundances and codon composition of Buchnera genes. A significant codon usage bias was found for the choice of rare codons in Buchnera: C-ending codons are preferred in highly expressed genes, whereas G-ending codons are avoided. This bias is not explained by GC skew in the bacteria and might correspond to a selection for perfect matching between codon-anticodon pairs for some essential amino acids in Buchnera proteins. Nutritional stress applied to the aphid host induced a significant overexpression of most of the tRNA isoacceptors in bacteria. Although, molecular regulation of the tRNA operons in Buchnera was not investigated, a correlation between relative expression levels and organization in transcription unit was found in the genome of Buchnera.