Genotype IV Japanese encephalitis (JE) virus (GIV JEV) is the least common and most neglected genotype in JEV. We evaluated the growth and pathogenic potential of the GIV strain 19CxBa-83-Cv, which was isolated from a mosquito pool in Bali, Indonesia, in 2019, and serological analyses were also conducted. The growth ability of 19CxBa-83-Cv in Vero cells was intermediate between that of the genotype I (GI) strain Mie/41/2002 and the genotype V (GV) strain Muar, whereas 19CxBa-83-Cv and Mie/41/2002 grew faster than Muar in mouse neuroblastoma cells. The neuroinvasiveness of 19CxBa-83-Cv in mice was higher than that of Mie/41/2002 but lower than that of Muar; however, there were no significant differences in neurovirulence in mice among the three strains. The neutralizing titers of sera from 19CxBa-83-Cv- and Mie/41/2002-inoculated mice against 19CxBa-83-Cv and Mie/41/2002 were similar, whereas the titers against Muar were lower than those of the other two viruses. The neutralizing titers of JE vaccine-inoculated mouse pool serum against 19CxBa-83-Cv and Muar were significantly lower than those against Mie/41/2002. The neutralizing titers against the three viruses were similar in three out of the five serum samples from GI-infected JE patients, although the titers against Mie/41/2002 were higher than those against 19CxBa-83-Cv and Muar in the remaining two sera samples. In summary, we identified the basic characteristics of 19CxBa-83-Cv, but further studies are needed to better understand GIV JEV.

In 2022, Greece was the second most seriously affected European country in terms of the West Nile virus (WNV), after Italy. Specifically, Central Macedonia was the region with the most reported human cases (81.5%). In the present study, 30,816 female Culex pipiens sensu lato mosquitoes were collected from May to September 2022 in the seven regional units of Central Macedonia; they were then grouped into 690 pools and tested for WNV, while next-generation sequencing was applied to the samples, which showed a cycle threshold of Ct < 30 in a real-time RT-PCR test. WNV was detected in 5.9% of pools, with significant differences in the detection rate among regional units and months. It is of interest that in the Thessaloniki regional unit, where most of the human cases were observed, the virus circulation started earlier, peaked earlier, and lasted longer than in the other regional units. All sequences clustered into the Central European subclade of WNV lineage 2, and the virus strain differed from the initial Greek strain of 2010 by 0.52% and 0.27% at the nucleotide and amino acid levels, respectively. Signature substitutions were present, such as S73P and T157A in the prM and E structural proteins, respectively. The screening of mosquitoes provides useful information for virus circulation in a region with a potential for early warning, while the availability of whole-genome sequences is essential for further studies, including virus evolution.

More than 40 human cases of severe encephalitis caused by Borna disease virus 1 (BoDV-1) have been reported to German health authorities. In an endemic region in southern Germany, we conducted the seroepidemiological BoSOT study ("BoDV-1 after solid-organ transplantation") to assess whether there are undetected oligo- or asymptomatic courses of infection. A total of 216 healthy blood donors and 280 outpatients after solid organ transplantation were screened by a recombinant BoDV-1 ELISA followed by an indirect immunofluorescence assay (iIFA) as confirmatory test. For comparison, 288 serum and 258 cerebrospinal fluid (CSF) samples with a request for tick-borne encephalitis (TBE) diagnostics were analyzed for BoDV-1 infections. ELISA screening reactivity rates ranged from 3.5% to 18.6% depending on the cohort and the used ELISA antigen, but only one sample of a patient from the cohort with requested TBE diagnostics was confirmed to be positive for anti-BoDV-1-IgG by iIFA. In addition, the corresponding CSF sample of this patient with a three-week history of severe neurological disease tested positive for BoDV-1 RNA. Due to the iIFA results, all other results were interpreted as false-reactive in the ELISA screening. By linear serological epitope mapping, cross-reactions with human and bacterial proteins were identified as possible underlying mechanism for the false-reactive ELISA screening results. In conclusion, no oligo- or asymptomatic infections were detected in the studied cohorts. Serological tests based on a single recombinant BoDV-1 antigen should be interpreted with caution, and an iIFA should always be performed in addition.

The impact of mosquito-borne diseases on human and veterinary health is being exacerbated by rapid environmental changes caused mainly by changing climatic patterns and globalization. To gain insight into mosquito-borne virus circulation from two counties in eastern and southeastern Romania, we have used a combination of sampling methods in natural, urban and peri-urban sites. The presence of 37 mosquito-borne viruses in 16,827 pooled mosquitoes was analyzed using a high-throughput microfluidic real-time PCR assay. West Nile virus (WNV) was detected in 10/365 pools of Culex pipiens (n = 8), Culex modestus (n = 1) and Aedes vexans (n = 1) from both studied counties. We also report the first molecular detection of Sindbis virus (SINV) RNA in the country in one pool of Culex modestus. WNV infection was confirmed by real-time RT-PCR (10/10) and virus isolation on Vero or C6/36 cells (four samples). For the SINV-positive pool, no cytopathic effectwas observed after infection of Vero or C6/36 cells, but no amplification was obtained in conventional SINV RT-PCR. Phylogenetic analysis of WNV partial NS5 sequences revealed that WNV lineage 2 of theCentral-Southeast European clade, has a wider circulation in Romania than previously known.

Ilheus fever is a mosquito-borne, poorly known tropical disease. We aimed to report the pooled rate of exposure to the Ilheus virus (ILHV) and clinical outcomes of infection to determine the epidemiological patterns of ILHV. We conducted a meta-analysis of 37 studies (n = 17,722 individuals) from Latin America. The common clinical characteristics of ILHV infection were fever (82.3%), headache (52.9%), and myalgia (52.9%). Encephalitis complicated the course of the infection in 29.4% cases. Monotypic serological reactions detected a pooled rate of exposure of 2% to ILHV (95% CI: 1-2). Studies were mainly conducted in Brazil, with a pooled proportion of ILHV positivity of 8% (95% CI: 3-14). Males (12%) had higher rates of seropositivity than females (7%) and had high chances of ILHV infection (OR: 1.7, 95% CI: 1.2-2.5). Seropositivity increased with age, from 2% (95% CI: 2-3) among people aged 0-14 years to 8% (95% CI: 6-10) among people aged 15-64 years. Our analysis indicated a low and relatively constant burden of ILHV in Latin America. More research is needed to evaluate and innovate serological assays for ILHV to better estimate the burden and dynamics of epidemiological changes in ILHV infection in different regions.

The Usutu virus (USUV), a neurotropic mosquito-borne flavivirus discovered in 1959 in South Africa, has spread over the last twenty years across the European continent. This virus follows an enzootic cycle involving mosquitoes and birds. This caused epizootics with significant bird mortality in Europe in 2016 and 2018. It can also occasionally infect humans and other mammals, including horses and bats, which act as incidental or dead-end hosts. The zoonotic risk associated with this succession of avian epizootics in Europe deserves attention, even if, to date, human cases remain exceptional. Human infection is most often asymptomatic or responsible for mild clinical symptoms. However, human Usutu infections have also been associated with neurological disorders, such as encephalitis and meningoencephalitis. One of the major complexities of the study of USUV pathogenesis is the presence of a great diversity of lineages which could co-circulate spatiotemporally. In this review we discuss several aspects of the circulation of Usutu virus in humans in Europe, the neurological disorders associated, involved viral lineages, and the issues and questions raised by their circulation.

West Nile virus (WNV) is a mosquito-borne virus potentially causing serious illness in humans and other animals. Since 2004, several studies have highlighted the progressive spread of WNV Lineage 2 (L2) in Europe, with Italy being one of the countries with the highest number of cases of West Nile disease reported. In this paper, we give an overview of the epidemiological and genetic features characterising the spread and evolution of WNV L2 in Italy, leveraging data obtained from national surveillance activities between 2011 and 2021, including 46 newly assembled genomes that were analysed under both phylogeographic and phylodynamic frameworks. In addition, to better understand the seasonal patterns of the virus, we used a machine learning model predicting areas at high-risk of WNV spread. Our results show a progressive increase in WNV L2 in Italy, clarifying the dynamics of interregional circulation, with no significant introductions from other countries in recent years. Moreover, the predicting model identified the presence of suitable conditions for the 2022 earlier and wider spread of WNV in Italy, underlining the importance of using quantitative models for early warning detection of WNV outbreaks. Taken together, these findings can be used as a reference to develop new strategies to mitigate the impact of the pathogen on human and other animal health in endemic areas and new regions.

Tick-borne encephalitis (TBE) is an emerging zoonosis that may cause long-term neurological sequelae or even death. Thus, there is a growing interest in understanding the factors of TBE pathogenesis. Viral genetic determinants may greatly affect the severity and consequences of TBE. In this study, nonstructural protein 1 (NS1) of the tick-borne encephalitis virus (TBEV) was tested as such a determinant. NS1s of three strains with similar neuroinvasiveness belonging to the European, Siberian and Far-Eastern subtypes of TBEV were studied. Transfection of mouse cells with plasmids encoding NS1 of the three TBEV subtypes led to different levels of NS1 protein accumulation in and secretion from the cells. NS1s of TBEV were able to trigger cytokine production either in isolated mouse splenocytes or in mice after delivery of NS1 encoding plasmids. The profile and dynamics of TNF-α, IL-6, IL-10 and IFN-γ differed between the strains. These results demonstrated the involvement of TBEV NS1 in triggering an immune response and indicated the diversity of NS1 as one of the genetic factors of TBEV pathogenicity.

West Nile virus (WNV) is an emerging neurotropic RNA virus and a member of the genus Flavivirus. Naturally, the virus is maintained in an enzootic cycle involving mosquitoes as vectors and birds that are the principal amplifying virus hosts. In humans, the incubation period for WNV disease ranges from 3 to 14 days, with an estimated 80% of infected persons being asymptomatic, around 19% developing a mild febrile infection and less than 1% developing neuroinvasive disease. Laboratory diagnosis of WNV infection is generally accomplished by cross-reacting serological methods or highly sensitive yet expensive molecular approaches. Therefore, current diagnostic tools hinder widespread surveillance of WNV in birds and mosquitoes that serve as viral reservoirs for infecting secondary hosts, such as humans and equines. We have developed a synthetic biology-based method for sensitive and low-cost detection of WNV. This method relies on toehold riboswitches designed to detect WNV genomic RNA as transcriptional input and process it to GFP fluorescence as translational output. Our methodology offers a non-invasive tool with reduced operating cost and high diagnostic value that can be used for field surveillance of WNV in humans as well as in bird and mosquito populations.

The intron-based stabilization approach is a very useful strategy for construction of stable flavivirus infectious clones. SA14-14-2 is a highly attenuated Japanese encephalitis (JE) live vaccine strain that has been widely used in China since 1989. To develop safe and effective recombinant vaccines with SA14-14-2 as a backbone vector, we constructed the DNA-based infectious clone pCMW-JEV of SA14-14-2 using the intron-based stabilization approach and acquired the rescued virus rDJEV, which retained the biological properties of the parental virus. Unexpectedly, a rescued virus strain with altered virulence, designated rHV-DJEV, was accidentally acquired in one of the transfection experiments. rHV-DJEV showed up to 105-fold increased neurovirulence compared with the SA14-14-2 parental strain. Genome sequencing showed that the inserted introns were still present in the genome of rHV-DJEV. Therefore, we think that the intron-based stabilization approach should be used with caution in vaccine development and direct iDNA immunization.

Mosquitoes of the genus Culex comprise important vectors of pathogenic arboviruses in our region, including West Nile and Rift Valley Fever viruses. To improve our understanding of the epidemiology and transmission dynamics of arboviruses, we need to study the behavior and ecology of their vectors. The feeding patterns of the vector mosquitoes can be very useful in determining how and where to focus control efforts. For example, determining the preferred blood hosts of the females can assist in the implementation of potentially efficacious strategies for focused control of mosquito females. Determining the plants from which both sexes derive their sugar meals can comprise the initial step towards the formulation of efficient lures for trapping mosquitoes. In the past, plant meal identification was based mainly on chemical detection of fructose and microscopical observations of cellulose particles in mosquito guts. More recent studies have utilized DNA barcoding capable of identifying plant food sources. In the current study, we identify multiple plant species from which large numbers of mosquitoes obtained their sugar meals in one experimental procedure. We employed next generation DNA sequencing to sequence the chloroplast specific plant genes atpB and rbcL.

Mosquito vectors of eastern equine encephalitis virus (EEEV) and West Nile virus (WNV) in the USA reside within broad multi-species assemblages that vary in spatial and temporal composition, relative abundances and vector competence. These variations impact the risk of pathogen transmission and the operational management of these species by local public health vector control districts. However, most models of mosquito vector dynamics focus on single species and do not account for co-occurrence probabilities between mosquito species pairs across environmental gradients. In this investigation, we use for the first time conditional Markov Random Fields (CRF) to evaluate spatial co-occurrence patterns between host-seeking mosquito vectors of EEEV and WNV around sampling sites in Manatee County, Florida. Specifically, we aimed to: (i) quantify correlations between mosquito vector species and other mosquito species; (ii) quantify correlations between mosquito vectors and landscape and climate variables; and (iii) investigate whether the strength of correlations between species pairs are conditional on landscape or climate variables. We hypothesized that either mosquito species pairs co-occur in patterns driven by the landscape and/or climate variables, or these vector species pairs are unconditionally dependent on each other regardless of the environmental variables. Our results indicated that landscape and bioclimatic covariates did not substantially improve the overall model performance and that the log abundances of the majority of WNV and EEEV vector species were positively dependent on other vector and non-vector mosquito species, unconditionally. Only five individual mosquito vectors were weakly dependent on environmental variables with one exception, Culiseta melanura, the primary vector for EEEV, which showed a strong correlation with woody wetland, precipitation seasonality and average temperature of driest quarter. Our analyses showed that majority of the studied mosquito species' abundance and distribution are insignificantly better predicted by the biotic correlations than by environmental variables. Additionally, these mosquito vector species may be habitat generalists, as indicated by the unconditional correlation matrices between species pairs, which could have confounded our analysis, but also indicated that the approach could be operationalized to leverage species co-occurrences as indicators of vector abundances in unsampled areas, or under scenarios where environmental variables are not informative.

The paper deals with a West Nile virus (WNv) model, in which the nonlocal diffusion characterizes the long-range movement of birds and mosquitoes, the free boundaries describe their spreading fronts, and the seasonal succession accounts for the effect of the warm and cold seasons. The well-posedness of the mathematical model is established, and its long-term dynamical behaviours, which depend upon the generalized eigenvalues of the corresponding linearized differential operator, are investigated. For both spatially independent and nonlocal WNv models with seasonal successions, the generalized eigenvalues are studied and applied to determine whether the spreading or vanishing occurs. Our results extend those for the case with nonlocal diffusion but no free boundary and those for the case with free boundary but local diffusion, respectively. The generalized eigenvalues reveal that there exists positive correlation between the duration of the warm season and the risk of infection. Moreover, the initial infection length, the initial infection scale and the spreading ability to new areas all play important roles for the long time behaviors of the time dependent solutions.

Rift Valley fever (RVF) is a zoonotic disease caused by Rift Valley fever virus (RVFV), an emerging arbovirus within the Phenuiviridae family of Bunyavirales that has potential to cause severe diseases in both humans and livestock. It increases the incidence of abortion or foetal malformation in ruminants and leads to clinical manifestations like encephalitis or haemorrhagic fever in humans. Upon virus invasion, the innate immune system from the cell or the organism is activated to produce interferon (IFN) and prevent virus proliferation. Meanwhile, RVFV initiates countermeasures to limit antiviral responses at transcriptional and protein levels. RVFV nonstructural proteins (NSs) are the key virulent factors that not only perform immune evasion but also impact the cell replication cycle and has cytopathic effects. In this review, we summarize the innate immunity host cells employ depending on IFN signal transduction pathways, as well as the immune evasion mechanisms developed by RVFV primarily with the inhibitory activity of NSs protein. Clarifying the arms race between host innate immunity and RVFV immune evasion provides new avenues for drug target screening and offers possible solutions to current and future epidemics.

The increasing trend of mosquito-borne pathogens demands more accurate global estimations of infection and transmission risks between mosquitoes. Here, we systematically review field and laboratory studies to assess the natural field infection and experimental laboratory transmission risk in Culex mosquitoes. We studied four worldwide flaviviruses: West Nile, Usutu, Japanese encephalitis, and St. Louis encephalitis, belonging to the Japanese encephalitis Serocomplex (JES). The PRISMA statement was carried out for both approaches. The Transmission-Infection Risk of the diverse mosquito species for the different viruses was estimated through seven variables. We considered 130 and 95 articles for field and experimental approach, respectively. We identified 30 species naturally infected, and 23 species capable to transmit some of the four flaviviruses. For the JES, the highest Transmission-Infection Risk estimate was recorded in Culex quinquefasciatus (North America). The maximum Infection-Transmission Risk values for West Nile was Culex restuans, for Usutu it was Culex pipiens (Europe), for St. Louis encephalitis Culex quinquefasciatus (North America), and for Japanese encephalitis Culex gelidus (Oceania). We conclude that on a worldwide scale, a combination of field and experimental data offers a better way of understanding natural infection and transmission risks between mosquito populations.

The study of virus structures by X-ray free-electron lasers (XFELs) has attracted increased attention in recent decades. Such experiments are based on the collection of 2D diffraction patterns measured at the detector following the application of femtosecond X-ray pulses to biological samples. To prepare an experiment at the European XFEL, the diffraction data for the tick-borne encephalitis virus (TBEV) was simulated with different parameters and the optimal values were identified. Following the necessary steps of a well established data-processing pipeline, the structure of TBEV was obtained. In the structure determination presented, a priori knowledge of the simulated virus orientations was used. The efficiency of the proposed pipeline was demonstrated.

Tick-borne encephalitis virus (TBEV) is a major cause of neurological infections in many regions of central, eastern and northern Europe and northern Asia. In approximately 15% of cases, TBEV infections lead to the development of severe encephalitis or meningitis. The main route of TBEV transmission is tick bites; however, ingestion of dairy products from infected animals (goats, cattle and sheep) is also a frequent cause of the disease. Therefore, vaccination of livestock in virus endemic regions could also contribute to the decrease in TBEV infection among humans. Although few vaccines against TBEV based on inactivated viruses are available for humans, due to high costs, vaccination is not mandatory in most of the affected countries. Moreover, there is still no vaccine for veterinary use. Here, we present a characterization and immunogenicity study of a new potential TBEV vaccine based on virus-like particles (VLPs) produced in Leishmania tarentolae cells. VLPs, which mimic native viral particles but do not contain genetic material, show good immunogenic potential. For the first time, we showed that the protozoan L. tarentolae expression system can be successfully used for the production of TBEV virus-like particles with highly efficient production. We confirmed that TBEV recombinant structural proteins (prM/M and E) from VLPs are highly recognized by neutralizing antibodies in in vitro analyses. Therefore, VLPs in combination with AddaVax adjuvant were used in immunization studies in a mouse model. VLPs proved to be highly immunogenic and induced the production of high levels of neutralizing antibodies. In a challenge experiment, immunization with VLPs provided full protection from lethal TBE in mice. Thus, we suggest that Leishmania-derived VLPs may be a good candidate for a safe alternative human vaccine with high efficiency of production. Moreover, this potential vaccine candidate may constitute a low-cost candidate for veterinary use.

West Nile virus (WNV) and Usutu virus (USUV), two antigenically related flaviviruses co-circulating in Europe, can cause severe neurological disease in animals and humans. The immune response against USUV and WNV and their immunopathogenesis are still poorly investigated. Here we present results upon sequential infections of adult immunocompetent CD-1 and BALB/c mice primed with two different doses (high dose, HD or low dose, LD) of an USUV isolate and challenged with HD or LD of three different WNV isolates. CD-1 and BALB/c LD USUV-primed mice, regardless of the dose, are largely protected from lethal WNV challenges despite showing no detectable neutralizing antibodies. Furthermore, mice immunized with a chimeric virus harboring the E protein of USUV within the WNV backbone (WNVE-USUV) are protected against a lethal challenge with WNV. We believe these findings could contribute to understanding the dynamics of the interaction during sequential infection of these two flaviviruses.

Tick-borne encephalitis (TBE) is a potentially fatal disease common in much of Europe and Asia. There is no specific therapy for the treatment of TBE patients. However, several efforts are being made to develop small molecules that specifically interfere with the life cycle of TBE virus. In particular, recently various nucleoside analogues that can inhibit the viral replicase, such as the RNA-dependent RNA polymerase or viral methyltransferases, have been explored. In addition, human or chimeric (i.e., structural chimeras that combine mouse variable domains with human constant domains) monoclonal antibodies with promising potential for post-exposure prophylaxis or early therapy have been developed. This review summarizes the latest directions and experimental approaches that may be used to combat TBE in humans.

We collected >40,000 mosquitoes from 5 provinces in South Africa during 2011-2018 and screened for zoonotic flaviviruses. We detected West Nile virus in mosquitoes from conservation and periurban sites and potential new mosquito vectors; Banzi virus was rare. Our results suggest flavivirus transmission risks are increasing in South Africa.

Advancement in proteomics methods for interrogating biological samples has helped identify disease biomarkers for early diagnostics and unravel underlying molecular mechanisms of disease. Herein, we examined the serum proteomes of 23 study participants presenting with one of two common arthropod-borne infections: Lyme disease (LD), an extracellular bacterial infection or West Nile virus infection (WNV), an intracellular viral infection. The LC/MS based serum proteomes of samples collected at the time of diagnosis and during convalescence were assessed using a depletion-based high-throughput shotgun proteomics (dHSP) pipeline as well as a non-depleting blotting-based low-throughput platform (MStern). The LC/MS integrated analyses identified host proteome responses in the acute and recovery phases shared by LD and WNV infections, as well as differentially abundant proteins that were unique to each infection. Notably, we also detected proteins that distinguished localized from disseminated LD and asymptomatic from symptomatic WNV infection. The proteins detected in both diseases with the dHSP pipeline identified unique and overlapping proteins detected with the non-depleting MStern platform, supporting the utility of both detection methods. Machine learning confirmed the use of the serum proteome to distinguish the infection from healthy control sera but could not develop discriminatory models between LD and WNV at current sample numbers. Our study is the first to compare the serum proteomes in two arthropod-borne infections and highlights the similarities in host responses even though the pathogens and the vectors themselves are different.

The blood brain barrier (BBB) is a multicellular microenvironment that plays an important role in regulating bidirectional transport to and from the central nervous system (CNS). Infections by many acutely infectious viruses such as alphaviruses and flaviviruses are known to impact the integrity of the endothelial lining of the BBB. Infection by Venezuelan Equine Encephalitis Virus (VEEV) through the aerosol route causes significant damage to the integrity of the BBB, which contributes to long-term neurological sequelae. An effective therapeutic intervention strategy should ideally not only control viral load in the host, but also prevent and/or reverse deleterious events at the BBB. Two dimensional monocultures, including trans-well models that use endothelial cells, do not recapitulate the intricate multicellular environment of the BBB. Complex in vitro organ-on-a-chip models (OOC) provide a great opportunity to introduce human-like experimental models to understand the mechanistic underpinnings of the disease state and evaluate the effectiveness of therapeutic candidates in a highly relevant manner. Here we demonstrate the utility of a neurovascular unit (NVU) in analyzing the dynamics of infection and proinflammatory response following VEEV infection and therapeutic effectiveness of omaveloxolone to preserve BBB integrity and decrease viral and inflammatory load.

West Nile virus (WNV) is a zoonotic flavivirus transmitted by mosquitoes as a biological vector. Because of its biting behavior, the widespread snow-melt mosquito Aedes punctor could be a potential bridge vector for WNV to humans and nonhuman mammals. However, little is known on its role in transmission of WNV. The aim of this study was to determine the vector competence of German Ae. punctor for WNV lineages 1 and 2. Field-collected larvae and pupae were reared to adults and offered infectious blood containing either an Italian WNV lineage 1 or a German WNV lineage 2 strain via cotton stick feeding. Engorged females were incubated for 14/15 or 21 days at 18 °C. After incubation; surviving mosquitoes were dissected and forced to salivate. Mosquito bodies with abdomens, thoraces and heads, legs plus wings and saliva samples were investigated for WNV RNA by RT-qPCR. Altogether, 2/70 (2.86%) and 5/85 (5.88%) mosquito bodies were found infected with WNV lineage 1 or 2, respectively. In two mosquitoes, viral RNA was also detected in legs and wings. No saliva sample contained viral RNA. Based on these results, we conclude that Ae. punctor does not play an important role in WNV transmission in Germany.

West Nile virus (WNV) is a virus of the Japanese encephalitis antigenic complex and belongs to the family Flaviviridae of the genus flavivirus. The virus can cause infection in humans which in most cases is asymptomatic, however symptomatic cases exist and the disease can be severe causing encephalitis and meningoencephalitis. The virus is maintained in an enzootic cycle involving mosquitoes and birds, humans and other mammals such as horses can be accidental hosts. A mosquito-based arbovirus surveillance system and the sentinel syndromic surveillance network (4S) have been in place since 1988 and 2015 respectively, to better understand the transmission dynamics of arboviruses including WNV in Senegal. Arthropod and human samples have been collected from the field and analysed at Institut Pasteur de Dakar using different methods including RT-PCR, ELISA, plaque reduction neutralization test and viral isolation. RT-PCR positive samples have been analysed by Next Generation Sequencing. From 2012 to 2021, 7912 samples have been analysed and WNV positive cases have been detected, 20 human cases (19 IgM and 1 RT-PCR positive cases) and 41 mosquito pools. Phylogenetic analyzes of the sequences of complete genomes obtained showed the circulation of lineage 1a, with all these recent strains from Senegal identical to each other and very close to strains isolated from horse in France in 2015, Italy and Spain. Our data showed lineage 1a endemicity in Senegal as previously described, with circulation of WNV in humans and mosquitoes. Phylogenetic analyzes carried out with the genome sequences obtained also revealed exchanges of WNV strains between Europe and Senegal which could be possible via migratory birds. The surveillance systems that have enabled the detection of WNV in humans and arthropods should be extended to animals in a one-health approach to better prepare for global health threats.

In this work, we presented data from a two-year study of flavi-, flavi-like, and phenuiviruses circulation in the population of ixodid ticks in the Chelyabinsk region. We isolated three tick-borne encephalitis virus (TBEV) strains from I. persulcatus, which was not detected in the ticks of the genus Dermacentor. The virus prevalence ranged from 0.66% to 2.28%. The Yanggou tick virus (YGTV) is widespread in steppe and forest-steppe zones and is mainly associated with ticks of the genus Dermacentor. We isolated 26 strains from D. reticulatus, D. marginatus, and I. persulcatus ticks in the HAE/CTVM8 tick cell line. The virus prevalence ranged from 1.58% to 4.18% in D. reticulatus, ranged from 0.78% to 3.93% in D. marginatus, and was 0.66% in I. persulcatus. There was combined focus of TBEV and YGTV in the territory of the Chelyabinsk region. The Alongshan virus (ALSV) was found to be associated with I. persulcatus ticks and is spread in forest zone. We detected 12 amplicons and isolated 7 strains of ALSV in tick cells. The virus prevalence ranged from 1.13% to 6.00%. The phlebovirus Gomselga and unclassified phenuivirus Stavropol were associated with I. persulcatus and D. reticulatus ticks, respectively. Virus prevalence of the unclassified phenuivirus Stavropol in the Chelyabinsk region is lower than that in neighbouring regions.

The Japanese encephalitis virus (JEV) is the most common cause of neurodegenerative disease in Southeast Asia and the Western Pacific region; approximately 1.15 billion people are at risk, and thousands suffer from permanent neurological disorders across Asian countries, with 10-15 thousand people dying each year. JEV crosses the blood-brain barrier (BBB) and forms a complex with receptors on the surface of neurons. GRP78, Src, TLR7, caveolin-1, and dopamine receptor D2 are involved in JEV binding and entry into the neurons, and these receptors also play a role in carcinogenic activity in cells. JEV binds to GRP78, a member of the HSP70 overexpressed on malignant cells to enter neurons, indicating a higher chance of JEV infection in cancer patients. However, JEV enters human brain microvascular endothelial cells via an endocytic pathway mediated by caveolae and the ezrin protein and also targets dopamine-rich areas for infection of the midbrain via altering dopamine levels. In addition, JEV complexed with CLEC5A receptor of macrophage cells is involved in the breakdown of the BBB and central nervous system (CNS) inflammation. CLEC5A-mediated infection is also responsible for the influx of cytokines into the CNS. In this review, we discuss the neuronal and macrophage surface receptors involved in neuronal death.

The tick-borne encephalitis virus (TBEV) causes a most important viral life-threatening illness transmitted by ticks. The interactions between the virus and ticks are largely unexplored, indicating a lack of experimental tools and systematic studies. One such tool is recombinant reporter TBEV, offering antibody-free visualization to facilitate studies of transmission and interactions between a tick vector and a virus. In this paper, we utilized a recently developed recombinant TBEV expressing the reporter gene mCherry to study its fitness in various tick-derived in vitro cell cultures and live unfed nymphal Ixodes ricinus ticks. The reporter virus was successfully replicated in tick cell lines and live ticks as confirmed by the plaque assay and the mCherry-specific polymerase chain reaction (PCR). Although a strong mCherry signal determined by fluorescence microscopy was detected in several tick cell lines, the fluorescence of the reporter was not observed in the live ticks, corroborated also by immunoblotting. Our data indicate that the mCherry reporter TBEV might be an excellent tool for studying TBEV-tick interactions using a tick in vitro model. However, physiological attributes of a live tick, likely contributing to the inactivity of the reporter, warrant further development of reporter-tagged viruses to study TBEV in ticks in vivo.