Biology subjects

Pain, B.

Publications and source records attributed to Pain, B..

6 recordsLinked to original sources

bioRxiv2024

Two strains of Toscana virus show different virulence and replication capacity in mice and cell culture models

Toscana virus (TOSV), belonging to Phenuiviridae family, is circulating in most Mediterranean countries and is transmitted to humans by infected female sand flies. While most infections are asymptomatic, TOSV is considered as a leading cause of meningitis and encephalitis in humans during summer. Three TOSV genotypes (named A, B, and C) have been identified, although no virus strain belonging to lineage C has been isolated so far. To date, the relationship between TOSV genetic diversity and viral pathogenicity or replication capacity remains unknown. This study aimed to compare two TOSV strains from either lineage A (TOSV-A) or B (TOSV-B) in several cell culture and two mouse models. We showed that TOSV-A replicated more efficiently in BSR and A549 cells while TOSV-B had a replication advantage in human induced pluripotent stem cells differentiated in neural cells and LL-5 sand fly cells. In vivo, we were unable to detect any virus in the brains of immunocompetent C57BL/6JRj mice infected with either strain of TOSV. On the contrary, we showed that TOSV-A disseminated to the central nervous system of 129/Sv ifnar -/- mice unlike TOSV-B, despite higher viremia of TOSV-B and a greater dissemination of this strain in other organs. The reasons for these differences are not yet known, although we showed that the presence of TOSV neutralizing antibodies in serum was slightly delayed in TOSV-A-infected mice. Altogether, the data presented in this study provide new avenues to study TOSV-induced pathogenesis and ultimately unveil molecular viral determinants modulating TOSV replication capacity. Author SummaryToscana virus is a sand fly-borne phlebovirus with tropism for the human central nervous system (CNS) and the causal agent of aseptic meningitis and encephalitis during summer in countries surrounding the Mediterranean Sea. The pathogenesis induced by TOSV is still poorly understood as well as the correlation between TOSV genetic diversity and viral pathogenicity or replication capacity. In this study, we characterised two TOSV strains belonging to two different genetic lineages (named A and B) in several in vitro, ex vivo and in vivo models of infection. We showed that the biological properties of these two strains is strikingly different and, importantly, we identified 129/Sv ifnar -/- mice as a suitable model to study the mechanism of TOSV invasion of the CNS and viral pathogenesis. Our comparative study also highlights the importance of strengthening our efforts to identify molecular viral determinants modulating TOSV-induced pathogenesis and vector transmission, in order to better characterise the epidemiological risk posed by circulating strains.

microbiology↗

bioRxiv2024

Comparative study of two Rift Valley fever virus field strains circulating in Mauritania in 2010 and 2013 reveals the high virulence of the MRU25010-30 strain isolated from camel

Rift Valley fever (RVF) is one of the major viral arthropod-borne diseases in Africa. In recent decades, RVF virus (RVFV), the causative agent of RVF, has been responsible for multiple outbreaks in West Africa with important consequences on human and animal health. In particular, an outbreak occurred in 2010 after heavy rainfalls in the desertic region of Adrar, Mauritania. It was characterized by the appearance of severe clinical signs among dromedary camels. Another one occurred in 2013-2014 across Senegal and the southern part of Mauritania. In this study, we characterized two RVFV field strains isolated during these two outbreaks. The first strain, MRU25010-30, has been isolated in camel (2010) while the second, MRU2687-3, was isolated in goat (2013). By deep-sequencing and rapid amplification of cDNA-ends by polymerase chain reaction (RACE-PCR), we successfully sequenced the complete genome of these two RVFV strains as well as the reference laboratory strain ZH548. Phylogenetic analysis shows that the two field viruses belong to two different RVFV genetic lineages. Moreover, we show that MRU25010-30 replicates more efficiently in various in vitro cell culture models than MRU2687-3 and ZH548. In vivo, MRU25010-30 caused rapid death of BALB/c mice and proved to be more virulent than MRU2687-3, regardless of the route of inoculation (subcutaneous or intranasal). The virulence of MRU25010-30 is associated with a high viral load in the liver and serum of infected mice, while the death of mice infected with MRU2687-3 and ZH548 correlates with a high viral load in the brain. Altogether, the data presented in this study provide new avenues to unveil the molecular viral determinants that modulate RVFV virulence and replication capacity Author SummaryRift Valley fever is an arboviral zoonosis caused by Rift Valley fever virus (RVFV) belonging to the Phlebovirus genus. It poses a major risk for causing a public and animal health emergency and is a significant economic burden in many African countries. To date, our knowledge of the impact of RVFV genetic diversity on its virulence, replicative capacities and transmission by mosquitoes is limited. In this study, we fully sequenced two RVFV strains isolated in Mauritania during two distinct outbreaks (2010 and 2013) and show that they were genetically distant. Interestingly, we show that one of the strains (MRU25010-30) is able to replicate in vitro more efficiently than the other (MRU2687-3). Additionally, we show that high levels of viremia and viral load in the liver are associated with rapid death in BALB/c mice infected with MRU25010-30, whereas mice infected by MRU2687-3 tend to die later with high viral load in the brain. In conclusion, our study confirms that RVFV strains from distinct genetic lineages have different phenotypic characteristics such as virulence and replication capacity. These data provide a strong basis for further studies aimed at identifying the viral genetic determinants responsible for the observed phenotypes.

microbiology↗

bioRxiv2024

Generation of systemic rabbit chimeras via Induced Pluripotent Stem Cells Reprogrammed with KLF2, ERAS, and PRMT6

Little is known about the molecular underpinnings of pluripotent stem cells (PSCs) ability to colonize the epiblast of preimplantation embryos and generate chimeras. In our study, using rabbit PSCs as a model system, we conducted unbiased screening of a cDNA library that encodes a panel of 36 pluripotency factors. From this screening, we identified KLF2, ERAS and PRMT6, whose overexpression confers the ability for self-renewal in a KOSR/FGF2-free culture medium supplemented with LIF, activin A, PKC and WNT inhibitors. The reprogrammed cells acquired transcriptomic and epigenetic features of naive pluripotency, including the reactivation of the 2nd X-chromosome. Leveraging these PSC lines, we determined the transcriptomic signature of embryonic colonization-competence, demonstrating transcriptional repression of genes involved in MAPK, WNT, HIPPO, and EPH signaling pathways, alongside the activation of genes involved in amino-acid metabolism, NF-kB signaling, and p53 pathway. Remarkably, a subset of reprogrammed cells, expressing CD75 at a high level, gained the ability to produce chimeric fetuses with a high contribution from PSCs in all lineages.

cell biology↗

bioRxiv2023

Cell specification and functional interactions in the pig blastocyst inferred from single cell transcriptomics

The early embryonic development of the pig comprises a long in utero pre- and peri-implantation development, which dramatically differs from mouse and human. During this pro-tracted peri-implantation period, an intimate dialogue between the embryo and the uterus is established through a complex series of paracrine signals. It leads to concomitant drastic changes in the embryonic morphology and uterine receptivity to implantation. From day 7 after fertilization, the spherical blastocyst elongates as ovoid, tubular and filamentous wich transforms from a 0.5-1 mm diameter sphere to a 1000 mm long filamentous blastocyst at day 16. At the same time, the inner cell mass moves up to the trophoblast as the Raubers layer disappears and evolves as an embryonic disc that is directly exposed to molecules present in the uterine fluids. These drastic changes occurring before implantation are driven and coordinated by interactions between embryonic cells and maternal tissues. To better understand the biology of pig embryos before implantation, we generated a large dataset of single-cell RNAseq at different embryonic stages (early and hatched blastocyst, spheroid and ovoid conceptus) and proteomic datasets from corresponding uterine fluids. These data were cleaned, filtered and represent a total of 34,888 cells. We first characterised the embryonic and extra-embryonic cell populations and their evolution, and identified population-specific markers of the three main populations (epiblast, trophectoderm and hypoblast). Our analysis also confirmed known functions and predicted new biological functions associated with these cell populations.We then inferred gene regulatory networks working on modules of gene regulation (regulon) and selected those that were specifically active in each embryonic population. We confirmed the relevance of the identified regulons by a meta-analysis of two other scRNAseq datasets (porcine and human preimplantation embryos). We then linked these regulons to signalling pathways and biological processes. Our results confirm the molecular specificity and functionality of the three main cell populations and identify novel stage-specific subpopulations. In particular, we discovered two previously unknown subpopulations of the trophectoderm, one characterised by the expression of LRP2, which could represent a subpopulation of progenitor cells, and the other, expressing many pro-apoptotic markers, which could correspond to the cells of the Raubers layer. We also provide new insights into the biology of these populations, their reciprocal functional interactions and the molecular dialogue established with the maternal uterine environment.

developmental biology↗

bioRxiv2019

Reprogrammed Pteropus Bat Stem Cells Present Distinct Immune Signature And Are Highly Permissive For Henipaviruses

Bats are unique among mammals due to the ability of powered flight and exceptional longevity. They are also asymptomatic hosts for numerous viruses, including recently emerged zoonotic Henipaviruses Nipah and Hendra, which are highly pathogenic for humans and other mammals. Better understanding of how bats control viral infection requires development of relevant permissive cellular experimental models. By applying a somatic reprogramming protocol to Pteropus bat primary cells, using a novel combination of ESRRB, CDX2, and c-MYC transcription factors, we generated bat reprogrammed cells exhibiting stem cell-like characteristics and a neural stem cell-like molecular signature. These cells present a unique interferon-stimulated transcriptomic signature and both produce and respond to interferon type-I, highlighting differences between stem cells from bats and other mammals. In contrast to primary bat cells, these reprogrammed cells are highly susceptible to infection by Henipavirus, thereby enabling isolation of new bat viruses, study of virus-bat interactions, and better understanding of bat biology. Summary sentenceSomatic reprogramming provides new bat stem cells with unique immune properties and original viral permissivness

cell biology↗

bioRxiv2018

Three-dimensional culture of chicken primordial germ cells in chemically defined media containing the functional polymer FP003

Scalable production of avian suspension cell exhibits a valuable potential on therapeutic application by producing recombinant protein and as the substrate for virus growth. This study sought to establish a system with chemically defined components for three-dimensional (3D) culture of chicken primordial germ cells (cPGCs), a pluripotent avian cell type. cPGCs were cultured in medium supplemented with the functional polymer FP003. Viscoelasticity was low in this medium, and cPGCs did not sediment,and consequently their expansion was improved. The total number of cPGCs increased by 17-fold after 1week of culture in 3D-FAot medium, an aseric chemically defined medium containing FP003, indicating that this medium enhances the expansion of cPGCs. Moreover, cPGC cell lines stably expressed the germline-specific reporter VASA:tdTOMATO, as well as other markers of cPGCs, for more than 1 month upon culture in 3D-FAot medium, indicating that the characteristics of these cells are maintained. cPGCs harboring both PGK:EGFP and VASA:tdTOMATO robustly expressed both fluorescent proteins upon culture in 3D-FAot, suggesting that this approach is perspective for recombinant protein production. In summary,this novel 3D culture system can be used to efficiently expand cPGCs in suspension without mechanical stirring or loss of cellular properties. This system provides a platform for large-scale culture ofcPGCs in industry.

cell biology↗