Usutu Virus Strains Research Articles

UK mosquitoes are competent to transmit Usutu virus at native temperatures

Usutu virus (USUV) is an emerging zoonotic virus transmitted primarily by Culex mosquitoes. Since its introduction into Europe from Africa during the late 20th century, it has caused mortality within populations of passerine birds and captive owls, and can on occasion lead to disease in humans. USUV was first detected in the UK in 2020 and has become endemic, having been detected in either birds and/or mosquitoes every subsequent year. Importantly, the vector competence of indigenous mosquitoes for the circulating UK (London) USUV strain at representative regional temperatures is still to be elucidated. This study assessed the vector competence of five field-collected mosquito species/biotypes, Culex pipiens biotype molestus, Culex pipiens biotype pipiens, Culex torrentium, Culiseta annulata and Aedes detritus for the London USUV strain, with infection rates (IR) and transmission rates (TR) evaluated between 7 and 28 days post-infection. Infection and transmission were observed in all species/biotypes aside from Ae. detritus and Cx. torrentium. For Cx. pipiens biotype molestus, transmission potential suggests these populations should be monitored further for their role in transmission to humans. Furthermore, both Cx. pipiens biotype pipiens and Cs. annulata were shown to be competent vectors at 19 °C indicating the potential for geographical spread of the virus to other UK regions.

The effect of model structure and data availability on Usutu virus dynamics at three biological scales.

Understanding the epidemiology of emerging pathogens, such as Usutu virus (USUV) infections, requires systems investigation at each scale involved in the host-virus transmission cycle, from individual bird infections, to bird-to-vector transmissions, and to USUV incidence in bird and vector populations. For new pathogens field data are sparse, and predictions can be aided by the use of laboratory-type inoculation and transmission experiments combined with dynamical mathematical modelling. In this study, we investigated the dynamics of two strains of USUV by constructing mathematical models for the within-host scale, bird-to-vector transmission scale and vector-borne epidemiological scale. We used individual within-host infectious virus data and per cent mosquito infection data to predict USUV incidence in birds and mosquitoes. We addressed the dependence of predictions on model structure, data uncertainty and experimental design. We found that uncertainty in predictions at one scale change predicted results at another scale. We proposed in silico experiments that showed that sampling every 12 hours ensures practical identifiability of the within-host scale model. At the same time, we showed that practical identifiability of the transmission scale functions can only be improved under unrealistically high sampling regimes. Instead, we proposed optimal experimental designs and suggested the types of experiments that can ensure identifiability at the transmission scale and, hence, induce robustness in predictions at the epidemiological scale.

Cellular co-infections of West Nile virus and Usutu virus influence virus growth kinetics

The mosquito-borne flaviviruses West Nile virus (WNV) and Usutu virus (USUV) pose a significant threat to the health of humans and animals. Both viruses co-circulate in numerous European countries including Germany. Due to their overlapping host and vector ranges, there is a high risk of co-infections. However, it is largely unknown if WNV and USUV interact and how this might influence their epidemiology. Therefore, in-vitro infection experiments in mammalian (Vero B4), goose (GN-R) and mosquito cell lines (C6/36, CT) were performed to investigate potential effects of co-infections in vectors and vertebrate hosts. The growth kinetics of German and other European WNV and USUV strains were determined and compared. Subsequently, simultaneous co-infections were performed with selected WNV and USUV strains. The results show that the growth of USUV was suppressed by WNV in all cell lines. This effect was independent of the virus lineage but depended on the set WNV titre. The replication of WNV also decreased in co-infection scenarios on vertebrate cells. Overall, co-infections might lead to a decreased growth of USUV in mosquitoes and of both viruses in vertebrate hosts. These interactions can strongly affect the epidemiology of USUV and WNV in areas where they co-circulate.

Experimental infections in red-legged partridges reveal differences in host competence between West Nile and Usutu virus strains from Southern Spain.

West Nile virus (WNV) and Usutu virus (USUV) are emerging zoonotic arboviruses sharing the same life cycle with mosquitoes as vectors and wild birds as reservoir hosts. The main objective of this study was to characterize the pathogenicity and course of infection of two viral strains (WNV/08 and USUV/09) co-circulating in Southern Spain in a natural host, the red-legged partridge (Alectoris rufa), and to compare the results with those obtained with the reference strain WNV/NY99. WNV inoculated birds were monitored for clinical and analytical parameters (viral load, viremia, and antibodies) for 15 days post-inoculation. Partridges inoculated with WNV/NY99 and WNV/08 strains showed clinical signs such as weight loss, ruffled feathers, and lethargy, which were not observed in USUV/09-inoculated individuals. Although statistically significant differences in mortality were not observed, partridges inoculated with WNV strains developed significantly higher viremia and viral loads in blood than those inoculated with USUV. In addition, the viral genome was detected in organs and feathers of WNV-inoculated partridges, while it was almost undetectable in USUV-inoculated ones. These experimental results indicate that red-legged partridges are susceptible to the assayed Spanish WNV with pathogenicity similar to that observed for the prototype WNV/NY99 strain. By contrast, the USUV/09 strain was not pathogenic for this bird species and elicited extremely low viremia levels, demonstrating that red-legged partridges are not a competent host for the transmission of this USUV strain.

Competition between two Usutu virus isolates in cell culture and in the common house mosquito Culex pipiens.

Usutu virus (USUV) is a mosquito-borne flavivirus of African origin. Over the past decades, USUV has spread through Europe causing mass die-offs among multiple bird species. The natural transmission cycle of USUV involves Culex spp. mosquitoes as vectors and birds as amplifying hosts. Next to birds and mosquitoes, USUV has also been isolated from multiple mammalian species, including humans, which are considered dead-end hosts. USUV isolates are phylogenetically classified into an African and European branch, subdivided into eight genetic lineages (Africa 1, 2, and 3 and Europe 1, 2, 3, 4, and 5 lineages). Currently, multiple African and European lineages are co-circulating in Europe. Despite increased knowledge of the epidemiology and pathogenicity of the different lineages, the effects of co-infection and transmission efficacy of the co-circulating USUV strains remain unclear. In this study, we report a comparative study between two USUV isolates as follows: a Dutch isolate (USUV-NL, Africa lineage 3) and an Italian isolate (USUV-IT, Europe lineage 2). Upon co-infection, USUV-NL was consistently outcompeted by USUV-IT in mosquito, mammalian, and avian cell lines. In mosquito cells, the fitness advantage of USUV-IT was most prominently observed in comparison to the mammalian or avian cell lines. When Culex pipiens mosquitoes were orally infected with the different isolates, no overall differences in vector competence for USUV-IT and USUV-NL were observed. However, during the in vivo co-infection assay, it was observed that USUV-NL infectivity and transmission were negatively affected by USUV-IT but not vice versa.

Simultaneous Coinfections with West Nile Virus and Usutu Virus in Culex pipiens and Aedes vexans Mosquitoes

The mosquito-borne zoonotic flaviviruses West Nile virus (WNV) and Usutu virus (USUV) are endemic in many European countries and emerged in Germany in recent years. Due to the increasing overlap of their distribution areas and their similar epidemiology, coinfections of WNV and USUV are possible. Indeed, coinfections in vertebrate hosts as a rare event have already been reported from some countries including Germany. However, it is largely unknown whether and to what extent coinfections could affect the vector competence of mosquitoes for WNV and USUV. For this purpose, the mosquito species Culex pipiens biotype pipiens, Culex pipiens biotype molestus, and Aedes vexans were orally infected in mono- and simultaneous coinfections with German strains of WNV and USUV. Mosquitoes were incubated for 14 days at 26°C, 85% relative humidity, and a 16 : 8 light-dark photocycle, before they were dissected and forced to salivate. The results showed a decrease in USUV susceptibility in Culex pipiens biotype pipiens, an increase in USUV susceptibility in Aedes vexans, and no obvious interaction between both viruses in Culex pipiens biotype molestus. Vector competence for WNV appeared to be unaffected by a simultaneous occurrence of USUV in all tested mosquito species. Coinfections with both viruses were only found in Culex mosquitoes, and cotransmission of WNV and USUV was observed in Culex pipiens biotype molestus. Overall, our results show that viral interactions between WNV and USUV vary between mosquito species, and that the interaction mainly occurs during infection and replication in the mosquito midgut. The results of this study confirm that to fully understand the interaction between WNV and USUV, studies with various mosquito species are necessary. In addition, we found that even mosquito species with a low susceptibility to both viruses, such as Ae. vexans, can play a role in their transmission in areas with cocirculation.

Serological evidence of circulation of West Nile virus in equids in Algerian eastern drylands and its epidemiological risk factors

In order to determine the prevalence of equine infectious anemia virus (EIAV), Usutu virus (USUV), and West Nile virus (WNV) in eastern Algerian drylands, 340 sera from distinct equids have been collected from 2015 to 2017. Serological analysis for the presence of antibodies against EIAV and flaviviruses was performed using commercially available ELISAs. Sera detected positive, doubtful, or negative close to the doubtful threshold in flavivirus ELISA were tested by the virus neutralization test (VNT), using WNV and USUV strains. The prevalence of WNV antibodies with ELISA was 11.47% (39/340) against 13.53% (46/340) by WNV VNT. EIAV antibodies were not detected in any samples. WNV seroprevalence varies with species, breed and location of horses. Only, one equid was positive for both WNV and USUV neutralizing antibodies. This is the first screening on equids sera of EIAV and USUV in Algeria. This study indicate that WNV and possibly USUV have circulated/are circulating in the Algerian equine population, unlike EIAV does not seem to be present.

West Nile Virus Vaccination Protects against Usutu Virus Disease in Mice.

West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne flaviviruses that can cause neuroinvasive disease in humans. WNV and USUV circulate in both Africa and Europe and are closely related. Due to antigenic similarity, WNV-specific antibodies and USUV-specific antibodies have the potential to bind heterologous viruses; however, it is unclear whether this interaction may offer protection against infection. To investigate how prior WNV exposure would influence USUV infection, we used an attenuated WNV vaccine that contains the surface proteins of WNV in the backbone of a dengue virus 2 vaccine strain and protects against WNV disease. We hypothesized that vaccination with this attenuated WNV vaccine would protect against USUV infection. Neutralizing responses against WNV and USUV were measured in vitro using sera following vaccination. Sera from vaccinated CD-1 and Ifnar1−/− mice cross-neutralized with WNV and USUV. All mice were then subsequently challenged with an African or European USUV strain. In CD-1 mice, there was no difference in USUV titers between vaccinated and mock-vaccinated mice. However, in the Ifnar1−/− model, vaccinated mice had significantly higher survival rates and significantly lower USUV viremia compared to mock-vaccinated mice. Our results indicate that exposure to an attenuated form of WNV protects against severe USUV disease in mice and elicits a neutralizing response to both WNV and USUV. Future studies will investigate the immune mechanisms responsible for the protection against USUV infection induced by WNV vaccination, providing critical insight that will be essential for USUV and WNV vaccine development.

Modeling the dynamics of Usutu virus infection in birds

Usutu virus is an emerging zoonotic flavivirus causing high avian mortality rates and occasional severe neurological disorders in humans. Several virus strains are co-circulating and the differences in their characteristics and avian pathogenesis levels are still unknown. In this study, we use within–host mathematical models to characterize the mechanisms responsible for virus expansion and clearance in juvenile chickens challenged with four Usutu virus strains. We find heterogeneity between the virus strains, with the time between cell infection and viral production varying between 16 h and 23 h, the infected cell lifespan varying between 48 min and 9.5 h, and the basic reproductive number R0 varying between 12.05 and 19.49. The strains with high basic reproductive number have short infected cell lifespan, indicative of immune responses. The virus strains with low basic reproductive number have lower viral peaks and longer lasting viremia, due to lower infection rates and high infected cell lifespan. We discuss how the host and virus heterogeneities may differently impact the public health threat presented by these virus strains.

Structure of Usutu virus SAAR-1776 displays fusion loop asymmetry

Usutu virus (USUV) is an emerging arbovirus in Europe that has been increasingly identified in asymptomatic humans and donated blood samples and is a cause of increased incidents of neuroinvasive human disease. Treatment or prevention options for USUV disease are currently nonexistent, the result of a lack of understanding of the fundamental elements of USUV pathogenesis. Here, we report two structures of the mature USUV virus, determined at a resolution of 2.4 Å, using single-particle cryogenic electron microscopy. Mature USUV is an icosahedral shell of 180 copies of envelope (E) and membrane (M) proteins arranged in the classic herringbone pattern. However, unlike previous reports of flavivirus structures, we observe virus subpopulations and differences in the fusion loop disulfide bond. Presence of a second, unique E glycosylation site could elucidate host interactions, contributing to the broad USUV tissue tropism. The structures provide a basis for exploring USUV interactions with glycosaminoglycans and lectins, the role of the RGD motif as a receptor, and the inability of West Nile virus therapeutic antibody E16 to neutralize the mature USUV strain SAAR-1776. Finally, we identify three lipid binding sites and predict key residues that likely participate in virus stability and flexibility during membrane fusion. Our findings provide a framework for the development of USUV therapeutics and expand the current knowledge base of flavivirus biology.

Diversity of West Nile and Usutu virus strains in mosquitoes at an international airport in Austria.

Increased globalization and international transportation have resulted in the inadvertent introduction of exotic mosquitoes and new mosquito‐borne diseases. International airports are among the possible points of entry for mosquitoes and their pathogens. We established a mosquito and mosquito‐borne diseases monitoring programme at the largest international airport in Austria and report the results for the first two years, 2018 and 2019. This included weekly monitoring and sampling of adult mosquitoes, and screening them for the presence of viral nucleic acids by standard molecular diagnostic techniques. Additionally, we surveyed the avian community at the airport, as birds are potentially amplifying hosts. In 2018, West Nile virus (WNV) was detected in 14 pools and Usutu virus (USUV) was detected in another 14 pools of mosquitoes (minimum infection rate [MIR] of 6.8 for each virus). Of these 28 pools, 26 consisted of female Culex pipiens/torrentium, and two contained male Culex sp. mosquitoes. Cx. pipiens/torrentium mosquitoes were the most frequently captured mosquito species at the airport. The detected WNV strains belonged to five sub‐clusters within the sub‐lineage 2d‐1, and all detected USUV strains were grouped to at least seven sub‐clusters among the cluster Europe 2; all strains were previously shown to be endemic in Austria. In 2019, all mosquito pools were negative for any viral nucleic acids tested. Our study suggests that airports may serve as foci of arbovirus activity, particularly during epidemic years, and should be considered when designing mosquito control and arbovirus monitoring programmes.

Co-infections: Simultaneous detections of West Nile virus and Usutu virus in birds from Germany.

The emergence of West Nile virus (WNV) and Usutu virus (USUV) in Europe resulted in significant outbreaks leading to avifauna mortality and human infections. Both viruses have overlapping geographical, host and vector ranges, and are often co-circulating in Europe. In Germany, a nationwide bird surveillance network was established to monitor these zoonotic arthropod-borne viruses in migratory and resident birds. In this framework, co-infections with WNV and USUV were detected in six dead birds collected in 2018 and 2019. Genomic sequencing and phylogenetic analyses classified the detected WNV strains as lineage 2 and the USUV strains as lineages Africa 2 (n=2), Africa 3 (n=3) and Europe 2 (n=1). Preliminary attempts to co-propagate both viruses in vitro failed. However, we successfully cultivated WNV from two animals. Further evidence for WNV-USUV co-infection was obtained by sampling live birds in four zoological gardens with confirmed WNV cases. Three snowy owls had high neutralizing antibody titres against both WNV and USUV, of which two were also positive for USUV-RNA. In conclusion, further reports of co-infections in animals as well as in humans are expected in the future, particularly in areas where both viruses are present in the vector population.

Development and characterization of infectious clones of two strains of Usutu virus

Usutu virus (USUV; genus Flavivirus; family Flaviviridae) is a mosquito-borne, positive-sense RNA virus that is currently causing significant die-offs in numerous bird species throughout Europe and has caused infections in humans. Currently, there are no molecular clones for USUV, hence, hindering studies on the pathogenesis and transmission of USUV. Here, we demonstrate the development and characterization of infectious clones for two modern strains of USUV isolated from Europe and Africa. We show that the infectious clone-derived viruses replicated similarly to the parental strains in mammalian and insect cells. Additionally, we observed similar levels of replication and disease in two mouse models. These clones will aid the study of USUV infection, transmission, diagnostics, and vaccines.

In Vitro and In Vivo Models to Study the Zoonotic Mosquito-Borne Usutu Virus.

Usutu virus (USUV), a mosquito-borne zoonotic flavivirus discovered in South Africa in 1959, has spread to many European countries over the last 20 years. The virus is currently a major concern for animal health due to its expanding host range and the growing number of avian mass mortality events. Although human infections with USUV are often asymptomatic, they are occasionally accompanied by neurological complications reminiscent of those due to West Nile virus (another flavivirus closely related to USUV). Whilst USUV actually appears less threatening than some other emergent arboviruses, the lessons learned from Chikungunya, Dengue, and Zika viruses during the past few years should not be ignored. Further, it would not be surprising if, with time, USUV disperses further eastwards towards Asia and possibly westwards to the Americas, which may result in more pathogenic USUV strains to humans and/or animals. These observations, inviting the scientific community to be more vigilant about the spread and genetic evolution of USUV, have prompted the use of experimental systems to understand USUV pathogenesis and to boost the development of vaccines and antivirals. This review is the first to provide comprehensive coverage of existing in vitro and in vivo models for USUV infection and to discuss their contribution in advancing data concerning this neurotropic virus. We believe that this paper is a helpful tool for scientists to identify gaps in the knowledge about USUV and to design their future experiments to study the virus.

Structural and antigenic investigation of Usutu virus envelope protein domain III

The mosquito-borne flavivirus Usutu virus (USUV) has recently emerged in birds and humans in Europe. Symptoms of a USUV infection resemble those of West Nile virus (WNV); further, the close antigenic relationship of domain III (DIII) of the USUV and WNV envelope (E) proteins has prevented the development of a reliable serological test to distinguish USUV from WNV. To begin to address this deficiency, we identified ten different sequence groups of DIII from 253 complete and 80 partial USUV genome sequences. We solved the DIII structures of four groups, including that of the outlying CAR-1969 strain, which shows an atypical DIII structure. Structural comparisons of the USUV DIII groups and the DIII of WNV bound to the neutralizing antibody E16 revealed why the E16 failed to neutralize all USUV strains tested except for USUV CAR-1969. The analyses allowed predictions to be made to engineer an antibody specific for USUV CAR-1969.

Development and characterization of specific anti-Usutu virus chicken-derived single chain variable fragment antibodies.

Usutu virus belongs to the Japanese encephalitis serogroup within the Flaviviridae family. Mammals may become incidental hosts after the bite of an infected mosquito while birds act as the main reservoir. Human cases have become more common recently and elicit various outcomes ranging from asymptomatic to severe illness including encephalitis. Problematically, antisera against Usutu virus cross‐react with other flaviviruses such as the co‐circulating West Nile virus. As an approach to generate Usutu virus‐specific antibodies, we immunized chickens with purified Usutu virus envelope protein domain III, isolated the spleen mRNA and generated an scFv phage display library. The most potent binders for Usutu virus domain III were selected via biopanning and their affinity to domain III was examined using SPR. Four scFvs bound the domain III of Usutu virus in the nanomolar region; two bound the protein over 40 times more strongly than West Nile virus domain III. We further characterized these scFv antibodies for suitability in standard laboratory tests such as western blots, ELISA, and neutralization tests. Four specific and one cross‐reactive antibody performed well in western blots with domain III and the full‐length envelope protein of Usutu virus and West Nile virus. All antibodies bound in virus ELISA assays to Usutu virus strain Vienna‐2001. However, none of the antibodies neutralized either Usutu virus or West Nile virus. These antibody candidates could be crucial in future diagnostic tests to distinguish Usutu virus from other flaviviruses and might even offer virus neutralization after a conversion to Fab or IgG.

Epidemiology of Usutu Virus: The European Scenario.

Usutu virus (USUV) is an emerging arbovirus isolated in 1959 (Usutu River, Swaziland). Previously restricted to sub-Saharan Africa, the virus was introduced in Europe in 1996. While the USUV has received little attention in Africa, the virus emergence has prompted numerous studies with robust epidemiological surveillance programs in Europe. The natural transmission cycle of USUV involves mosquitoes (vectors) and birds (amplifying hosts) with humans and other mammals considered incidental (“dead-end”) hosts. In Africa, the virus was isolated in mosquitoes, rodents and birds and serologically detected in horses and dogs. In Europe, USUV was detected in bats, whereas antibodies were found in different animal species (horses, dogs, squirrels, wild boar, deer and lizards). While bird mortalities were not reported in Africa, in Europe USUV was shown to be highly pathogenic for several bird species, especially blackbirds (Turdus merula) and great gray owls (Strix nebulosa). Furthermore, neurotropism of USUV for humans was reported for the first time in both immunocompromised and immunocompetent patients. Epizootics and genetic diversity of USUV in different bird species as well as detection of the virus in mosquitoes suggest repeated USUV introductions into Europe with endemization in some countries. The zoonotic potential of USUV has been reported in a growing number of human cases. Clinical cases of neuroinvasive disease and USUV fever, as well as seroconversion in blood donors were reported in Europe since 2009. While most USUV strains detected in humans, birds and mosquitoes belong to European USUV lineages, several reports indicate the presence of African lineages as well. Since spreading trends of USUV are likely to continue, continuous multidisciplinary interventions (“One Health” concept) should be conducted for monitoring and prevention of this emerging arboviral infection.

Usutu Virus Infection of Embryonated Chicken Eggs and a Chicken Embryo-Derived Primary Cell Line.

Usutu virus (USUV) is a mosquito-borne flavivirus, closely related to the West Nile virus (WNV). Similar to WNV, USUV may cause infections in humans, with occasional, but sometimes severe, neurological complications. Further, USUV can be highly pathogenic in wild and captive birds and its circulation in Europe has given rise to substantial avian death. Adequate study models of this virus are still lacking but are critically needed to understand its pathogenesis and virulence spectrum. The chicken embryo is a low-cost, easy-to-manipulate and ethically acceptable model that closely reflects mammalian fetal development and allows immune response investigations, drug screening, and high-throughput virus production for vaccine development. While former studies suggested that this model was refractory to USUV infection, we unexpectedly found that high doses of four phylogenetically distinct USUV strains caused embryonic lethality. By employing immunohistochemistry and quantitative reverse transcriptase-polymerase chain reaction, we demonstrated that USUV was widely distributed in embryonic tissues, including the brain, retina, and feather follicles. We then successfully developed a primary cell line from the chorioallantoic membrane that was permissive to the virus without the need for viral adaptation. We believe the future use of these models would foster a significant understanding of USUV-induced neuropathogenesis and immune response and allow the future development of drugs and vaccines against USUV.

Multiple Lineages of Usutu Virus (Flaviviridae, Flavivirus) in Blackbirds (Turdus merula) and Mosquitoes (Culex pipiens, Cx. modestus) in the Czech Republic (2016-2019).

Usutu virus (USUV) is a flavivirus (Flaviviridae: Flavivirus) of an African origin transmitted among its natural hosts (diverse species of birds) by mosquitoes. The virus was introduced multiple times to Europe where it caused mortality of blackbirds (Turdus merula) and certain other susceptible species of birds. In this study, we report detection of USUV RNA in blackbirds, Culex pipiens and Cx. modestus mosquitoes in the Czech Republic, and isolation of 10 new Czech USUV strains from carcasses of blackbirds in cell culture. Multiple lineages (Europe 1, 2 and Africa 3) of USUV were found in blackbirds and mosquitoes in the southeastern part of the country. A single USUV lineage (Europe 3) was found in Prague and was likely associated with increased mortalities in the local blackbird population seen in this area in 2018. USUV genomic RNA (lineage Europe 2) was detected in a pool of Cx. pipiens mosquitoes from South Bohemia (southern part of the country), where no major mortality of birds has been reported so far, and no flavivirus RNA has been found in randomly sampled cadavers of blackbirds. The obtained data contributes to our knowledge about USUV genetic variability, distribution and spread in Central Europe.

Impact of genetic diversity on biological characteristics of Usutu virus strains in Africa

Usutu virus (USUV) previously restricted to Africa where it caused mild infections, emerged in 2001 in Europe and caused more severe infections among birds and humans with neurological forms, suggesting an adaptation and increasing virulence. This evolution suggests the need to better understand USUV transmission patterns for assessing risks and to develop control strategies. Phylogenetic analysis conducted in Africa showed low genetic diversity of African USUV strains except for one human and the USUV subtype (USUVsub) strains, which exhibited a deletion in the 3′UTR and nucleotide substitutions throughout the genome. Here we analyzed their viral replication in vitro in mosquito and mammalian cells, and vector competence of Culex quinquefasciatus, compared to a reference strain. Growth kinetics of the different strains showed comparable replication rates however variations in replication and translation efficiency were observed. Vector competence analysis showed that all strains were able to infect Culex quinquefasciatus the main peridomestic Culex species in Africa, with detection of USUV viral genomes and infectious particles. Dissemination and transmission were observed only for USUVsub, but infectious particles were not detected in Culex quinquefasciatus saliva. Our findings suggest that genetic variability can affect USUV in vitro replication in a cell type-dependent manner and in vivo in mosquitoes. In addition, the results show that Culex quinquefasciatus is not competent for the USUV strains analyzed here and also suggest an aborted transmission process for the USUVsub, which requires further investigations.