Abstract Circulating bat coronaviruses present a significant pandemic threat, yet our understanding of their genetic diversity and evolutionary dynamics remains limited. Over 3 years, we sampled 1,462 bats in Cambodia’s Steung Treng province, identifying extensive and diverse coronaviruses co-circulation. Using metatranscriptomic and amplicon sequencing, we generated 33 complete sarbecovirus genomes sequences, revealing novel lineages that cluster into four distinct groups, each associated with different Rhinolophus bat species. Our analysis highlights rapid migration and recombination of sarbecovirus lineages over short distances and timescales. Of note, the receptor-binding domains of two novel viral groups exhibit high similarity to SARS-CoV-2, and pseudovirus assays confirmed the ability of this spike protein to mediate entry into cells expressing human ACE2, suggesting a potential zoonotic risk. The observed genetic diversity underscores the urgent need for continuous surveillance to identify high-risk animal-to-human interfaces and inform pandemic preparedness.

The Awassi is a native sheep breed of Lebanon with high historical and economic value. Despite its importance, its genetic background has not been characterized, leaving limited understanding of its origins, diversity, and regional connections. This study aimed to characterize the genetic diversity and population structure of Awassi sheep from Lebanon and neighboring Syria using two complementary genetic systems that trace distinct evolutionary histories: maternal mitochondrial DNA and insertional polymorphisms of endogenous Jaagsiekte sheep retroviruses. Genetic analyses were conducted on two hundred seventy-seven animals. Sequencing of a one thousand thirty-four base pair fragment of the mitochondrial cytochrome b gene identified eighty-six maternal haplotypes that clustered into four haplogroups (A, B, C, and E). Maternal haplotype diversity was high, and most individuals belonged to haplogroups A and B, indicating contributions from both western and eastern maternal lineages. Retroviral insertion profiling showed that the retroviral type carrying only endogenous Jaagsiekte sheep retrovirus element eighteen was predominant. A Mediterranean retroviral type carrying both endogenous Jaagsiekte sheep retrovirus eighteen and seven was also present, especially in coastal flocks, suggesting ancient gene flow through maritime trade. Other retroviral combinations were rare. Analysis of endogenous Jaagsiekte sheep retrovirus six revealed a high frequency of solo long terminal repeat alleles, indicating an ancient recombination event. Approximately half of the animals were heterozygous for this allele and some homozygous. No pre-integration alleles were detected, confirming fixation of endogenous Jaagsiekte sheep retrovirus six in domestic sheep. Lebanese Awassi sheep exhibit substantial genetic diversity across both maternal and retroviral systems, reflecting a complex population history shaped by multiple lineages and regional exchanges. This study provides the first comprehensive genetic characterization of this native breed and contributes to broader understanding of sheep domestication in the Middle East, while supporting evidence-based conservation of this valuable genetic resource.

Rift Valley fever virus (RVFV) is a mosquito-borne phlebovirus posing major threats to human and animal health. Despite decades of research, the determinants governing diverse clinical manifestations remain incompletely understood. This narrative review examines four central facets: i) exposure route impacts on disease outcome; ii) field strain diversity and organ-specific virulence; iii) genome reassortment driving viral evolution; and iv) organ-specific pathology including the overlooked placental/retinal infections. Differences among infection routes, viral strains, and host species complicate extrapolation from experimental models to outbreaks. Collectively, evidence indicates that RVFV pathogenesis results from interplay between infection route, viral determinants, and host factors including age and immune responses, which shape viral dissemination, tissue tropism, and organ-specific disease outcomes. Despite progress, major uncertainties remain regarding mechanisms controlling tissue tropism and field strain diversity roles in severe manifestations. Integrating field isolates, organotypic cultures, and physiologically relevant models will be essential to refine understanding and improve prevention.

Toscana virus (TOSV) is an emerging but neglected human pathogen currently circulating around the Mediterranean basin including North Africa. Human illness ranges from asymptomatic or mild flu-like syndromes to severe neurological diseases such as meningitis or meningoencephalitis. Despite its significant impact, understanding of TOSV transmission and epidemiology remains limited. Sand flies (Diptera: Phlebotominae), specifically Phlebotomus perniciosus and Phlebotomus perfiliewi, are believed to be the primary vectors of TOSV. However, the spread of TOSV to new geographical areas and its detection in other sand fly species suggest that additional species play a role in the circulation and transmission of this virus. This study investigated the vector competence of four sand fly species - P. tobbi, P. sergenti, P. papatasi, and Sergentomyia schwetzi - for two TOSV strains: 1500590 (TOSV A lineage) and MRS20104319501 (TOSV B lineage). Sand flies were orally challenged with TOSV via bloodmeals. None of the tested species showed susceptibility to the TOSV A strain. However, for TOSV B strain, P. tobbi demonstrated a high potential as a new vector, exhibiting high infection and dissemination rates. P. sergenti also showed some susceptibility to TOSV B, with the virus dissemination observed in all infected females. These finding suggests that P. tobbi and P. sergenti are new potential vectors for TOSV B. Given that P. tobbi and P. sergenti are the primary vectors of human leishmaniases in the Balkans, Turkey and Middle East, their susceptibility to TOSV could have significant epidemiological consequences. On the other hand, P. papatasi and S. schwetzi appeared refractory to TOSV B infection. Refractoriness of P. papatasi, a highly anthropophilic species distributed from the Mediterranean to the Middle East and India, suggests that this species does not contribute to TOSV circulation.

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.

Toscana virus (TOSV) is a pathogenic and transmissible Phlebovirus of the Bunyavirales order. To date, two principal genetic lineages (A and B) have been identified and the impact of TOSV genetic diversity on its biology is still unknown. We used a reverse genetic approach based on two TOSV strains belonging to lineage A or B (i.e., TOSV-A and TOSV-B) and displaying different in vitro replicative fitness. Our results demonstrate that the sequences of Gn and Gc glycoproteins are responsible for the differences in replicative fitness between the two TOSV strains. Moreover, our data show that TOSV-A and TOSV-B display different entry kinetics and that newly-produced virions have different infectivity. This comparative approach demonstrates that the genetic diversity of TOSV can significantly impact viral properties and highlights the need for better molecular characterisation of the genomes of circulating TOSV strains, with a particular focus on the viral Gn and Gc glycoproteins.

The Rift Valley fever virus (RVFV) is an arthropod-borne, zoonotic, hemorrhagic fever virus that can cause severe diseases both in livestock and humans. The spread of RVFV in areas previously considered as non-endemic together with the absence of licensed vaccines for use in humans and animals poses a major health and economic threat worldwide. It is therefore crucial to make major progresses in our understanding and management of this virus and its zoonosis. RVFV is considered a bioterrorism pathogen, and, thus, only a few institutes, facilities, and personnel are legally authorized to detain it and handle it. Moreover, this virus must be manipulated in a biosafety level 3 (BSL3) laboratory following strict biosafety protocols to ensure that biosecurity's highest standards are met. Only certain attenuated strains such as the MP12 strain can be handled in BSL2 laboratories, depending on the country considered. To assist researchers in working with RVFV in the safest possible conditions, this chapter presents validated methods for effective RVFV decontamination and inactivation.

While locally-acquired dengue virus (DENV) human infections occur in mainland France since 2010, data to identify the mosquito species involved and to trace the virus are frequently lacking. Supported by a local network gathering public health agencies and research laboratories, we analysed, in late summer 2023, mosquitoes from privately-owned traps within a French urban neighbourhood affected by a dengue cluster. The cluster, in Auvergne-Rhône-Alpes, comprised three cases, including two autochthonous ones. Upon return from a recent visit to the French Caribbean Islands, the third case had consulted healthcare because of dengue-compatible symptoms, but dengue had not been recognised. For the two autochthonous cases, DENV-specific antibodies in serum or a positive quantitative PCR for DENV confirmed DENV infection. The third case had anti-flavivirus IgMs. No DENV genetic sequences were obtained from affected individuals but Aedes albopictus mosquitoes trapped less than 200 m from the autochthonous cases' residence contained DENV. Genetic data from the mosquito-derived DENV linked the cluster to the 2023-2024 dengue outbreak in the French Caribbean Islands. This study highlights the importance of raising mosquito-borne disease awareness among healthcare professionals. It demonstrates Ae. albopictus as a DENV vector in mainland France and the value of private mosquito traps for entomo-virological surveillance.

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 Summary Rift 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. ### Competing Interest Statement The authors have declared no competing interest.

Rift Valley fever virus (RVFV) is an arthropod-borne virus (arbovirus) responsible for a severe zoonotic disease affecting a wide range of domestic and wild ruminants as well as humans. RVFV is endemic in many African countries and has also caused outbreaks in Madagascar and Arabian Peninsula. With regard to its wide geographical distribution, its potential to emerge in a new area, and its capability to trigger major health and economic crisis, it is essential to study and better understand several aspects of its life cycle and, in particular, its interactions with mammalian hosts and arthropod vectors. To do so, it is key for researchers to be able to amplify in vitro viral strains isolated from the field and determine accurately the viral titers of RVFV stocks. In this chapter, we present protocols that can be easily implemented to produce and titrate RVFV stocks in your laboratory.

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 rains 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, was isolated from a camel (2010) while the second, MRU2687-3, was isolated from a goat (2013). By deep-sequencing and rapid amplification of cDNA-ends by polymerase chain reaction, we successfully sequenced the complete genome of these two RVFV strains as well as the reference laboratory strain ZH548. Phylogenetic analysis showed that the two field viruses belong to two different RVFV genetic lineages. Moreover, we showed 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 correlated 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.

In this study, we identified and assembled a strain of American nodavirus (ANV) in the Phlebotomus papatasi-derived PP9ad cell line. This strain most closely resembles Flock House virus and ANV identified in the Drosophila melanogaster S2/S2R cell line. Through small RNA sequencing and analysis, we demonstrate that ANV replication in PP9ad cells is primarily targeted by the exogenous small interfering RNA (exo-siRNA) pathway, with minimal engagement from the PIWI-interacting RNA (piRNA) pathway. In mosquitoes such as Aedes and Culex, the PIWI pathway is expanded and specialised, which actively limits virus replication. This is unlike in Drosophila spp., where the piRNA pathway does not restrict viral replication. In Lutzomyia sandflies (family Psychodidae), close relatives of Phlebotomus species and Drosophila, there appears to be an absence of virus-derived piRNAs. To investigate whether this absence is due to a lack of PIWI pathway proteins, we analysed the piRNA and siRNA diversity and repertoire in PP9ad cells. Previous assemblies of P. papatasi genome (Ppap_1.0) have revealed a patchy repertoire of the siRNA and piRNA pathways. Our analysis of the updated P. papatasi genome (Ppap_2.1) has shown no PIWI protein expansion in sandflies. We found that both siRNA and piRNA pathways are transcriptionally active in PP9ad cells, with genomic mapping of small RNAs generating typical piRNA signatures. Our results suggest that the piRNA pathway may not respond to virus replication in these cells, but an antiviral response is mounted via the exo-siRNA pathway.

Toscana virus (TOSV) ( Bunyavirales , Phenuiviridae , Phlebovirus , Toscana phlebovirus ) and other related human pathogenic arboviruses are transmitted by phlebotomine sand flies. TOSV has been reported in nations bordering the Mediterranean Sea among other regions. Infection can result in febrile illness as well as meningitis and encephalitis. Understanding vector-arbovirus interactions is crucial to improving our knowledge of how arboviruses spread, and in this context, immune responses that control viral replication play a significant role. Extensive research has been conducted on mosquito vector immunity against arboviruses, with RNA interference (RNAi) and specifically the exogenous siRNA (exo-siRNA) pathway playing a critical role. However, the antiviral immunity of phlebotomine sand flies is less well understood. Here we were able to show that the exo-siRNA pathway is active in a Phlebotomus papatasi -derived cell line. Following TOSV infection, distinctive 21 nucleotide virus-derived small interfering RNAs (vsiRNAs) were detected. We also identified the exo-siRNA effector Ago2 in this cell line, and silencing its expression rendered the exo-siRNA pathway largely inactive. Thus, our data show that this pathway is active as an antiviral response against a sand fly transmitted bunyavirus, TOSV.

Rift Valley Fever virus (RVFV) and Toscana virus (TOSV) are two pathogenic arthropod-borne viruses responsible for zoonotic infections in both humans and animals; as such, they represent a growing threat to public and veterinary health. Interferon-induced transmembrane (IFITM) proteins are broad inhibitors of a large panel of viruses belonging to various families and genera. However, little is known on the interplay between RVFV, TOSV, and the IFITM proteins derived from their naturally infected host species. In this study, we investigated the ability of human, bovine, and camel IFITMs to restrict RVFV and TOSV infection. Our results indicated that TOSV was extremely sensitive to inhibition by all the animal IFITMs tested, while RVFV was inhibited by human IFITM-2 and IFITM-3, but not IFITM-1, and exhibited a more heterogeneous resistance phenotype towards the individual bovine and camel IFITMs tested. Overall, our findings shed some light on the complex and differential interplay between two zoonotic viruses and IFITMs from their naturally infected animal species.

Arbovirus emergence and epidemic potential, as approximated by the vectorial capacity formula, depends on host and vector parameters, including the vector’s intrinsic ability to replicate then transmit the pathogen known as vector competence. Vector competence is a complex, time-dependent, quantitative phenotype influenced by biotic and abiotic factors. A combination of experimental and modelling approaches is required to assess arbovirus intra-vector dynamics and estimate epidemic potential. In this study, we measured infection, dissemination, and transmission dynamics of chikungunya virus (CHIKV) in a field-derived Aedes albopictus population (Lyon, France) after oral exposure to a range of virus doses spanning human viraemia. Statistical modelling indicates rapid and efficient CHIKV progression in the vector mainly due to an absence of a dissemination barrier, with 100% of the infected mosquitoes ultimately exhibiting a disseminated infection, regardless of the virus dose. Transmission rate data revealed a time-dependent, but overall weak, transmission barrier, with individuals transmitting as soon as 2 days post-exposure (dpe) and >50% infectious mosquitoes at 6 dpe for the highest dose. Based on these experimental intra-vector dynamics data, epidemiological simulations conducted with an agent-based model showed that even at low mosquito biting rates, CHIKV could trigger outbreaks locally. Together, this reveals the epidemic potential of CHIKV upon transmission by Aedes albopictus in mainland France.

Rift Valley fever virus (RVFV) is a pathogenic arthropod-borne virus that can cause serious illness in both ruminants and humans. The virus can be transmitted by an arthropod bite or contact with contaminated fluids or tissues. Two live-attenuated veterinary vaccines-the Smithburn (SB) and Clone 13 (Cl.13)-are currently used during epizootic events in Africa. However, their residual pathogenicity (i.e., SB) or potential of reversion (i.e., Cl.13) causes important adverse effects, strongly limiting their use in the field. In this study, we infected immunocompetent mice with SB or Cl.13 by a subcutaneous or an intranasal inoculation. Interestingly, we found that, unlike the subcutaneous infection, the intranasal inoculation led to a high mortality rate. In addition, we detected high titers and viral N antigen levels in the brain of both the SB- and Cl.13-infected mice. Overall, we unveil a clear correlation between the pathogenicity and the route of administration of both SB and Cl.13, with the intranasal inoculation leading to a stronger neurovirulence and higher mortality rate than the subcutaneous infection.

Arbovirus emergence and epidemic potential, as approximated by the vectorial capacity formula, depends on several host and vector parameters including vector intrinsic ability to transmit the pathogen. Such ability, called vector competence is influenced by biotic (e.g. virus and vector genotype) and abiotic (e.g. temperature). Vector competence is often evaluated as a qualitative phenotype although it is a multistep, time-dependent, quantitative phenotype. Combination of experimental and modelling approaches can i) capture intra-vector dynamics of arboviral infection and ii) use data to estimate arbovirus epidemic potential. Here, we measured individual Aedes albopictus (Lyon, France) mosquitoes infection, dissemination, and transmission rate upon oral exposure to chikungunya virus (CHIKV, La Reunion Island isolate) at eleven time-points from day 2 to day 20 post-exposure (dpe) for a range of CHIKV infectious doses spanning human viremia. Statistical modelisation indicates an explosive CHIKV intra-vector dynamics mainly due to an absence of dissemination barrier with 100% of the infected mosquitoes ultimately exhibiting a disseminated infection regardless of the viral dose. Transmission rate data revealed a time and dose-dependent but overall weak transmission barrier with individuals transmitting as soon as 2 dpe and >50% infectious mosquitoes at 6 dpe for the highest dose. Epidemiological simulations conducted with an Agent-Based Model based on experimental intra-vector dynamics data showed that even at low mosquito biting rates, CHIKV triggers explosive outbreaks. Together, this reveals the high epidemic potential of this CHIKV isolate with this French metropolitan population of Aedes albopictus.

Toscana virus (TOSV) is a Phlebovirus in the Phenuiviridae family, order Bunyavirales, found in the countries surrounding the Mediterranean. TOSV is an important cause of seasonal acute meningitis and encephalitis within its range. Here, we determined the full sequence of the TOSV strain 1500590, a lineage A virus obtained from an infected patient (Marseille, 2007) and used this in combination with other sequence information to construct functional cDNA plasmids encoding the viral L, M, and S antigenomic sequences under the control of the T7 RNA promoter to recover recombinant viruses. Importantly, resequencing identified two single nucleotide changes to a TOSV reference genome, which, when corrected, restored functionality to the polymerase L and made it possible to recover infectious recombinant TOSV (rTOSV) from cDNA, as well as establish a minigenome system. Using reverse genetics, we produced an NSs-deletant rTOSV and also obtained viruses expressing reporter genes instead of NSs. The availability of such a system assists investigating questions that require genetic manipulation of the viral genome, such as investigations into replication and tropism, and beyond these fundamental aspects, also the development of novel vaccine design strategies.