Biology subjects

Brun, A.

Publications and source records attributed to Brun, A..

11 recordsLinked to original sources

bioRxiv2026

Genome-Scale Codon Deoptimization Enables Attenuation of Rift Valley Fever Virus

Rift Valley Fever Virus (RVFV) is a mosquito-borne zoonotic pathogen responsible for severe disease in domestic and wild ungulates as well as humans, representing a major threat to livestock production and human public health. RVFV is endemic in many African countries and has the potential to spread to new geographical regions. Current vaccines have limitations in safety and efficacy, highlighting the need for strategies to develop new vaccines candidates. In this study, we explored the use of codon deoptimization (CD) as a novel attenuation approach for the development of live-attenuated vaccine (LAV) against RVFV. CD exploits the redundancy of the genetic code by replacing frequently used codons with synonymous, less-preferred codons, thereby reducing translational efficiency without altering the amino acid sequence. We recoded parts of the M and S genome segments of RVFV using the least frequently used codons in mammalian cells, ensuring complete preservation of protein functionality and immunogenicity. Using reverse genetics, we rescued a panel of recombinant (r)RVFV encoding codon-deoptimized S-segment NSs gene (rNScd), M-segment Gn/Gc genes (rMcd), or both (rMcd/NScd). These recombinant CD viruses were characterized in vitro in mammalian and insect cell lines and in vivo using wild-type and immunocompromised mice. Results demonstrated varying degrees of attenuation among the three CD rRVFV, with the one deoptimized in both viral segments, rMcd/NScd, as a promising LAV based on the safety profiles. This study provides proof of concept for the use of CD as a rational strategy to generate attenuated RVFV, for the development of next-generation vaccines against this zoonotic threat.

microbiology↗

bioRxiv2026

The route of infection shapes Rift Valley fever virus pathogenesis, humoral immune response, and horizontal transmission in sheep.

Rift Valley fever (RVF) is a zoonotic arboviral disease that causes adverse pregnancy outcomes and high mortality in domestic and wild ruminants. The disease is caused by the RVF virus (RVFV), which is transmitted by mosquitoes from several genera, mainly Aedes and Culex. However, whether ruminants can become infected by horizontal virus transmission remains unclear. In addition, how the route of RVFV inoculation may influence RVF pathogenesis and the host immune response in animals is still largely unknown. With this aim, we conducted a comparative experimental study in which young sheep were either inoculated subcutaneously (SC) or intranasally (IN) with the virulent RVFV 56/74 strain. We then evaluated disease dynamics, viremia, virus excretion, tissue damage, and the humoral immune response. We also aimed to determine whether RVFV can be transmitted from infected to in-contact animals, and to assess whether the inoculation route may influence virus excretion and the likelihood of subsequent horizontal transmission. The results showed that SC inoculated sheep had a shorter incubation period, an earlier onset of viremia, and an earlier seroconversion. In contrast, IN inoculated animals developed higher rectal temperatures, reached higher peak viremia, and developed a more robust neutralizing antibody response. They also exhibited increased concentrations of analytes indicative of moderate but more severe hepatic injury compared with the subcutaneous group, along with more pronounced histopathological damage in the central nervous system. These results demonstrate the influence of the route of inoculation on RVF pathogenesis and the host immune response. Our results also confirmed the horizontal transmission of RVFV between SC inoculated sheep and in-contact animals housed in the same room, a phenomenon not observed in the IN inoculated group. This finding underscores the influence of the inoculation route on virus transmission and the potentially significant role of horizontal transmission in RVF epidemiology and disease control. Author summaryAccording to the World Health Organization (WHO), RVFV is considered a priority pathogen due to its ability to strain animal and public health systems, especially in developing countries. RVF outbreaks have occurred across most of Africa and, since 2000, in the Arabian Peninsula. Evidence of RVFV circulation in North Africa further highlights the threat to Europe, where competent mosquito vectors are present. How the inoculation route shapes disease dynamics and hosts immunity is still largely unknown. Similarly, whether the virus can spread between infected and non-infected animals without competent vectors remains unclear. A comparative infection in which young sheep were inoculated SC or IN with the RVFV 56/74 strain showed that SC inoculated sheep had a shorter incubation period, an earlier onset of viremia, and earlier seroconversion. However, rectal temperature and peak viremia were higher in IN inoculated sheep, which also showed evidence of moderate but more severe hepatic damage, accompanied by greater central nervous system damage. Only the in-contact animals housed in the subcutaneous group became infected, demonstrating horizontal transmission. Our results show that the route of inoculation influences disease progression and that RVFV can be transmitted among sheep in the absence of mosquitoes.

immunology↗

bioRxiv2025

Incorporation of ChiA into AcMNPV occlusion bodies and its implications for oral infectivity

Baculoviruses are insect-specific viruses primarily employed in biological pest control and recombinant protein expression. As biopesticides, they offer numerous advantages over chemical pesticides and hold significant potential for application in integrated pest management (IPM). However, their slower action compared to conventional synthetic strategies has limited their widespread adoption. This study aimed to investigate the impact of incorporating a viral protein, AcChiA chitinase, which disrupts the gut peritrophic membrane of susceptible insects, into AcMNPV occlusion bodies in trans. This was achieved through the development of an insect cell line that facilitates the passive incorporation of this protein. The presence of ChiA was detected in dissolved occlusion bodies (OBs) preparations from infected transgenic lines, in a method-dependent manner, with the stronger signals observed in chimeric polyhedra. It is hypothesized that ChiA in OBs will increase peritrophic membrane permeability and facilitate primary infection. In bioassays, OBs produced in ChiA-expressing lines were associated with higher larval mortality than OBs produced in Sf9, consistent with a ChiA-mediated effect. Notably, the addition of ChiA proteins from stably transformed insect cell lines improved infection rates, particularly in recombinant baculoviruses, which exhibited a significant reduction in occlusion-derived virion (ODV) occlusion. This underscores the critical role of these proteins in the oral infection process. Consequently, it was possible to incorporate the ChiA protein from stably transformed insect cell lines into wild-type and recombinant baculovirus occlusion bodies, paving the way for potential applications in IPM with AcMNPV.

bioengineering↗

bioRxiv2025

Generation and characterization of an ovine cell line derived from peripheral blood and its potential use to study livestock and zoonotic viral infections

BackgroundMesenchymal stromal cells (MSCs) are a population of undifferentiated non-hematopoietic fibroblast-like cells isolated from several tissues with multipotent differentiation capacity in vitro. This study focused on the establishment and characterization of a mesenchymal stromal cell line derived from ovine peripheral blood mononuclear cells (PBMCs). MethodsMSC were isolated from ovine blood and used to develop a cell line. The characterization of the cell line to confirm its mesenchymal origin was carried out by different assays. First at all, cells were characterized by flow cytometry (FACS) using monoclonal antibodies specific for MSC and hematopoietic non-stromal cell surface antigens. Real-time quantitative PCR (RT-qPCR) was performed using gene primer pairs specific for hematopoietic and mesenchymal cell surface markers. In addition, we assayed the potential of the cell line to differentiate in vitro to other lineages such as osteoblasts and neurons and confirmed that by specific staining with Alizarin Red (AR) and RT-qPCR and immunofluorescence (IFA). Finally, we assayed the susceptibility of the cell line to various livestock, animal and human viruses. ResultsFACS and RT-qPCR analyses revealed that the ovine cell line expressed mesenchymal markers and was negative for hematopoietic markers assayed. MSC were able to differentiate to osteoblasts and neurons, these results were confirmed by quantitative analyses of AR staining and RT-qPCR and IFA assays. The cell line showed susceptibility to infection with all viruses assayed confirmed by IFA asssays. ConclusionsIn this work, we used isolated MSCs from peripheral blood to establish a new ovine cell line. The characterization and the osteogenic and neuronal differentiation carried out confirmed that the cell line had a mesenchymal stromal origin. In fact, the new established ovine cell line was permissive to all viruses studied in this work. The new sheep cell line could be a useful tool for isolating and characterizing viruses and studying virus-host interactions.

cell biology↗

bioRxiv2025

The hyper-attenuated RVFV 40Fp8 strain can be safely administered to pregnant ewes to protect them from a virulent challenge

In the present study, we evaluated the immunogenicity, safety and protective efficacy of the attenuated RVFV-40Fp8 strain in natural hosts (non-pregnant ewes) and in a highly susceptible host infection model such as pregnant ewes in the first third of pregnancy. Our results confirm the immunogenicity of 40Fp8 administration in non-pregnant and pregnant ewes, as well as the absence of foetal damage even after a high-dose vaccination regime in pregnant ewes. In addition, the ewes and their foetuses were protected against a virulent RVFV-56/74 strain challenge, as shown by comparative histopathological evaluation of tissue samples from vaccinated and non-vaccinated pregnant ewes. These results confirm the potential use of 40Fp8 as a RVF live-attenuated vaccine candidate and pave the way for further clinical developments.

microbiology↗

bioRxiv2024

Novel replication-competent reporter-expressing Rift Valley fever viruses for molecular studies

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic disease that causes severe disease in both domestic and wild ungulates and humans, making it a significant threat to livestock and public health. The RVFV genome consists of three single-stranded, negative-sense RNA segments differing in size: Small (S), Medium (M) and Large (L). Segment S encodes the virus nucleoprotein N and the virulence-associated factor non-structural (NSs) protein in opposite orientations, separated by an intergenic region (IGR). To overcome the current need of using secondary techniques to detect the presence of RVFV in infected cells, we used T7-driven polymerase plasmid-based reverse genetics to generate replication-competent recombinant (r)RVFV expressing Nanoluciferase (Nluc) or Venus fluorescent proteins. These reporter genes were used as valid surrogates to track the presence of RVFV in mammalian and insect cells. Notably, we explored the genome plasticity of RVFV and compared four different strategies by modifying the viral segment S in order to introduce the reporter gene foreign sequences. The reporter-expressing rRVFV were stable and able to replicate in cultured mammalian and insect cells, although to a lesser extent than the recombinant wild-type (WT) counterpart. Moreover, rRVFV expressing reporter genes were validated to identify neutralizing antibodies or compounds with antiviral activity. In vivo, all mice infected with the reporter-expressing rRVFV displayed an attenuated phenotype, although at different levels. These rRVFV expressing reporter genes provide a novel approach to better understand the biology and pathogenesis of RVFV, and represent an excellent biotechnological tool for developing new therapeutics against RVFV infections. IMPORTANCERift Valley fever virus (RVFV) is a mosquito-borne virus and zoonotic agent threat that can be deadly to domestic or wild ungulates, and humans. In this work, we used reverse genetics approaches to explore the genome plasticity of RVFV by generating a set of recombinant rRVFV that express fluorescent or luminescent proteins to track viral infection. All the generated reporter-expressing rRVFV were able to propagate in mammalian or insect cells, and in a mouse model of infection. Our studies may contribute to advances in research on RVFV and other bunyaviruses and pave the way for the development of novel vaccines and the identification of new antivirals for the prophylactic and therapeutic treatment, respectively, of RVFV infections.

microbiology↗

bioRxiv2024

The Rift Valley fever (RVF) vaccine candidate 40Fp8 shows an extreme attenuation in IFNARKO mice following intranasal inoculation

Rift Valley fever (RVF) is an important zoonotic viral disease affecting several species of domestic and wild ruminants, causing major economic losses and dozens of human deaths in various geographical areas of Africa, where it is endemic. Although it is not present in Europe, there is a risk of its introduction and spread linked to globalisation and climate change. At present, the only measure that could help to prevent the disease is vaccination of flocks in areas at risk of RVF. Available live attenuated vaccines are an effective means of controlling the disease, but their use is often questioned due to residual virulence, particularly in susceptible hosts such as pregnant sheep. On the other hand, no vaccine is currently licensed for use in humans. The development of safe and effective vaccines is therefore a major area of research. In previous studies, we selected under selective mutagenic pressure a highly attenuated RVFV 56/74 virus variant called 40Fp8. This virus showed an extremely attenuated phenotype in both wild-type and immunodeficient A129 (IFNARKO) mice, yet was still able to induce protective immunity after a single inoculation, thus supporting its use as a safe, live attenuated vaccine. To further investigate its safety, in this work we have analysed the attenuation level of 40Fp8 in immunosuppressed mice (A129) when administered by the intranasal route, and compared it with other attenuated RVF viruses that are the basis of vaccines in use or in development. Our results show that 40Fp8 has a much higher attenuated level than these other viruses and confirm its potential as a candidate for safe RVF vaccine development. Author SummaryVaccines that use attenuated viruses are highly effective in terms of providing protection, duration, breadth, and quality of the immune response. However, they may pose a risk to immunosuppressed individuals or in certain situations, such as during pregnancy. It is therefore important to analyze the residual virulence of attenuated vaccines to ensure their safety. In this work, we analysed the safety of the RVF virus vaccine variant 40Fp8 developed in our laboratory. We studied its effect on immunodeficient mice and used a more aggressive route of administration such as the intranasal delivery route. To assess the degree of attenuation of 40Fp8, we compared it with other attenuated prototype RVF vaccines. The results clearly demonstrate that 40Fp8 is highly attenuated, and its residual virulence level is extremely low compared to other vaccine viruses tested.

pathology↗

bioRxiv2023

Rapid genome functional annotation pipeline anchored to the House sparrow (Passer domesticus, Linnaeus 1758) genome reannotation

House sparrow (Passer domesticus) is an important avian model for both laboratory and field-based studies of evolutionary genetics, development, neurobiology, physiology, behavior, and ecology. The current annotation of the P. domesticus genome is available at Ensembl Rapid Release site, which currently only focuses on gene set building. Here, we provide the first functional reannotation of the P. domesticus genome based on enrichment with intestinal Illumina RNA-Seq libraries. This revised annotation describes 38592 transcripts, compared to 23574 currently for Ensembl, and 14717 predicted protein-coding genes, with 96.4% complete Passeriformes lineage BUSCOs. A key improvement in this revised annotation is the definition of untranslated region (UTR) sequences, with 82.7% and 93.8% of transcripts containing 5 and 3 UTRs, respectively. Our reannotation highlights the benefits to genome annotation improvement when additional specific RNA-Seq data is available for analysis and rapid data throughput (>200 Mb h-1) is used.

genomics↗

bioRxiv2022

Identification of small molecules with virus growth enhancement properties

The novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused the pandemic disease known as coronavirus disease 2019 (COVID-19). COVID-19 vaccines were developed at record speed and were authorized approximately a year after the original outbreak. This fast response saved the lives of countless individuals and reduced the disease burden of many more. The experience has served as a reminder of the necessity to implement solid vaccine development platforms and fast production pipelines. Manufacturing vaccines for enveloped viruses, including some SARS-CoV-2 vaccines, often relies on the production of large quantities of viruses in vitro. Thus, speeding up or increasing virus production would expedite vaccine development. With this objective in mind, we established a high throughput screening (HTS) to identify small molecules that enhance or speed up host-virus membrane fusion. Among the HTS hits, we identified that ethynylestradiol augments SARS-CoV-2 fusion activity in both the absence and presence of TMPRSS2. Furthermore, we confirmed that ethynylestradiol can boost the growth of not only SARS-CoV-2 but also Influenza A virus in vitro. A small molecule with these characteristics could be implemented to improve vaccines production. ImportanceThe (COVID-19) pandemic had a tremendous impact on our healthcare systems and the global economy. The rapid development of effective vaccines saved the lives of countless individuals and reduced the disease burden of many more. Intending to increase vaccine production, we developed and performed a high-throughput screening (HTS) to identify small molecules that enhance viral and cellular membrane fusion. Among the HTS hits, we confirmed that Ethynylestradiol can boost the growth of SARS-CoV-2 and Influenza A virus in vitro.

microbiology↗

bioRxiv2020

A hyper-attenuated variant of Rift Valley fever virus (RVFV) generated by a mutagenic drug (favipiravir) unveils potential virulence markers

SUMMARY/ABSTRACTIn a previous work, we showed that favipiravir, a promising drug with antiviral activity against a number of RNA viruses, led to the extinction of RVFV from infected cell cultures. Nevertheless, certain drug concentrations allowed the recovery of a virus variant showing increased resistance to favipiravir. In this work, we characterized this novel resistant variant both at genomic and phenotypic level in vitro and in vivo. Interestingly, the resistant virus displayed reduced growth rates in insect cells and was highly attenuated but still immunogenic in vivo. Some amino acid substitutions were identified in the viral RNA-dependent RNA-polymerase (RdRp) gene and in the encoded IFN antagonist NSs gene, in catalytic core motifs and nuclear localization associated positions respectively. These data may help to characterize novel potential virulence markers, offering additional strategies for further safety improvements of RVF live attenuated vaccine candidates. SIGNIFICANCE STATEMENTLive attenuated virus vaccines usually provide long lasting immune responses upon administration. These vaccines are not recommended for use in immune compromised hosts, due to the presence of uncontrolled residual virulence. Cell culture virus propagation in the presence of mutagenic drugs often results in weakened virus lacking virulence as well as limited spreading capabilities. Here, we have characterized a mutagen-induced RVFV variant (40F-p8) that is not virulent in a highly sensitive mouse strain lacking antiviral response. The observed lack of virulence correlates with the presence of specific mutations along key residues in the viral genome, unveiling potential virulence determinants. Thus, 40F-p8 constitutes the basis for a novel RVFV vaccine strain with additional safety features.

microbiology↗

bioRxiv2019

Lethal mutagenesis of Rift Valley fever virus induced by favipiravir

Rift Valley fever virus (RVFV) is an emerging, mosquito-borne, zoonotic pathogen with recurrent outbreaks paying a considerable toll of human deaths in many African countries, for which no effective treatment is available. In cell culture studies and with laboratory animal models, the nucleoside analogue favipiravir (T-705) has demonstrated great potential for the treatment of several seasonal, chronic and emerging RNA virus infections of humans, suggesting applicability to control some viral outbreaks. Treatment with favipiravir was shown to reduce the infectivity of Rift Valley fever virus both in cell cultures and in experimental animal models, but the mechanism of this protective effect is not understood. In this work we show that favipiravir at concentrations well below the toxicity threshold estimated for cells is able to extinguish RVFV from infected cell cultures. Nucleotide sequence analysis has documented RVFV mutagenesis associated with virus extinction, with a significant increase in G to A and C to U transition frequencies, and a decrease of specific infectivity, hallmarks of lethal mutagenesis.

microbiology↗