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In recent decades, increases in temperature and tropical rainfall have facilitated the spread of mosquito species into temperate zones. Mosquitoes are vectors for many viruses, including West Nile virus (WNV) and dengue virus (DENV), and pose a serious threat to public health. This review covers most of the current knowledge on the mosquito species associated with the transmission of WNV and DENV and their geographical distribution and discusses the main vertebrate hosts involved in the cycles of WNV or DENV. It also describes virological and pathogenic aspects of WNV or DENV infection, including emerging concepts linking WNV and DENV to the reproductive system. Furthermore, it provides an epidemiological analysis of the human cases of WNV and DENV reported in Europe, from 1 January 2018 to 31 December 2023, with a particular focus on Italy. The first autochthonous cases of DENV infection, with the most likely vector being Aedes albopictus, have been observed in several European countries in recent years, with a high incidence in Italy in 2023. The lack of treatments and effective vaccines is a serious challenge. Currently, the primary strategy to prevent the spread of WNV and DENV infections in humans remains to limit the spread of mosquitoes.

The aim of this study was to analyze the seroprevalence of West Nile virus (WNV) among equids in Bulgaria, confirm the results of a competitive ELISA versus the virus neutralization test (VNT) and investigate some predisposing factors for WNV seropositivity. A total of 378 serum samples from 15 provinces in northern and southern Bulgaria were tested. The samples originated from 314 horses and 64 donkeys, 135 males and 243 females, aged from 1 to 30 years. IgG and IgM antibodies against WNV protein E were detected by ELISA. ELISA-positive samples were additionally tested via VNT for WNV and Usutu virus. Thirty-five samples were WNV-positive by ELISA (9.26% [CI = 6.45–12.88]), of which 15 were confirmed by VNT; hence, the seroprevalence was 3.97% (CI = 2.22–6.55). No virus-neutralizing antibodies to Usutu virus were detected among the 35 WNV-ELISA-positive equids in Bulgaria. When compared with VNT, ELISA showed 100.0% sensitivity and 94.5% specificity. A statistical analysis showed that the risk factors associated with WNV seropositivity were the region (p < 0.0001), altitude of the locality (p < 0.0001), type of housing (p < 0.0001) and breed (p = 0.0365). The results of the study demonstrate, albeit indirectly, that WNV circulates among equids in northern and southern Bulgaria, indicating that they could be suitable sentinel animals for predicting human cases and determining the risk in these areas or regions of the country.

The West Nile Virus (WNV), a member of the family Flaviviridae, is an emerging mosquito-borne flavivirus causing potentially severe infections in humans and animals involving the central nervous system (CNS). Due to its emerging tendency, WNV now occurs in many areas where other flaviviruses are co-occurring. Cross-reactive antibodies with flavivirus infections or vaccination (e.g., tick-borne encephalitis virus (TBEV), Usutu virus (USUV), yellow fever virus (YFV), dengue virus (DENV), Japanese encephalitis virus (JEV)) therefore remain a major challenge in diagnosing flavivirus infections. Virus neutralization tests are considered as reference tests for the detection of specific flavivirus antibodies, but are elaborate, time-consuming and need biosafety level 3 facilities. A simple and straightforward assay for the differentiation and detection of specific WNV IgG antibodies for the routine laboratory is urgently needed. In this study, we compared two commercially available enzyme-linked immunosorbent assays (anti-IgG WNV ELISA and anti-NS1-IgG WNV), a commercially available indirect immunofluorescence assay, and a newly developed in-house ELISA for the detection of WNV-NS1-IgG antibodies. All four tests were compared to an in-house NT to determine both the sensitivity and specificity of the four test systems. None of the assays could match the specificity of the NT, although the two NS1-IgG based ELISAs were very close to the specificity of the NT at 97.3% and 94.6%. The in-house WNV-NS1-IgG ELISA had the best performance regarding sensitivity and specificity. The specificities of the ELISA assays and the indirect immunofluorescence assays could not meet the necessary specificity and/or sensitivity.

Emerging infectious diseases have inflicted a significant health and socioeconomic burden upon the global population and governments worldwide. West Nile virus, a zoonotic, mosquito-borne flavivirus, was originally isolated in 1937 from a febrile patient in the West Nile Province of Uganda. It remained confined mainly to Africa, the Middle East, and parts of Europe and Australia until 1999, circulating in an enzootic mosquito-bird transmission cycle. Since the beginning of the 21st century, a new, neurotropic, more virulent strain was isolated from human outbreaks initially occurring in North America and later expanding to South and South-eastern Europe. Since 2010, when the first epidemic was recorded in Greece, annual incidence has fluctuated significantly. A variety of environmental, biological and socioeconomic factors have been globally addressed as potential regulators of the anticipated intensity of the annual incidence rate; circulation within the zoonotic reservoirs, recruitment and adaptation of new potent arthropod vectors, average winter and summer temperatures, precipitation during the early summer months, and socioeconomic factors, such as the emergence and progression of urbanization and the development of densely populated areas in association with insufficient health policy measures. This paper presents a review of the biological and socioenvironmental factors influencing the dynamics of the epidemics of West Nile virus (WNV) cases in Greece, one of the highest-ranked European countries in terms of annual incidence. To date, WNV remains an unpredictable opponent as is also the case with other emerging infectious diseases, forcing the National Health systems to develop response strategies, control the number of infections, and shorten the duration of the epidemics, thus minimizing the impact on human and material resources.

Usutu virus (USUV) and West Nile virus (WNV) are known to cause diseases and mortalities in bird populations. Since 2010/2011, USUV has circulated in Germany and spread nationwide, while WNV was only introduced into East Germany in 2018. The zoological garden investigated is located in Northern Germany, where USUV infections in wild birds have been detected for several years. In this longitudinal study conducted over a four-year period, zoo birds were sampled biannually and screened for molecular and serological evidence of USUV and WNV. USUV genomes were detected in eight of the sampled birds and whole-genome sequences revealed the circulation of USUV lineages Europe 3 and Africa 3. Of the eight birds infected with USUV during the study period, four died after the infection, while four survived without displaying clinical signs. Furthermore, in a few of the birds, a USUV (re-)infection was confirmed on a serological level with three birds producing USUV-neutralizing antibodies (nAbs) over a period of four years. Nonetheless, in two birds sampled throughout this longitudinal study, neither a USUV nor a WNV infection was evident. In 2022, WNV nAbs were detected for the first time in a juvenile zoo bird, indicating the introduction of the virus into this region.

Costa Rica harbors several flaviviruses, including Dengue (DENV), Zika (ZIKV), West Nile virus (WNV), and Saint Louis encephalitis virus (SLEV). While DENV and ZIKV are hyperendemic, previous research indicates restricted circulation of SLEV and WNV in animals. SLEV and WNV seroprevalence and high transmission areas have not yet been measured. To determine the extents of putative WNV and SLEV circulation, we sampled peri-domestic and domestic animals, humans, and mosquitoes in rural households located in two DENV and ZIKV hyperendemic regions during the rainy and dry seasons of 2017–2018 and conducted plaque reduction neutralization test assay for serology (PRNT) and RT-PCR for virus detection. In Cuajiniquil, serological evidence of WNV and SLEV was found in equines, humans, chickens, and wild birds. Additionally, five seroconversion events were recorded for WNV (2 equines), SLEV (1 human), and DENV-1 (2 humans). In Talamanca, WNV was not found, but serological evidence of SLEV circulation was recorded in equines, humans, and wild birds. Even though no active viral infection was detected, the seroconversion events recorded here indicate recent circulation of SLEV and WNV in these two regions. This study thus provides clear-cut evidence for WNV and SLEV presence in these areas, and therefore, they should be considered in arboviruses differential diagnostics and future infection prevention campaigns.

The annual movements of migratory birds can contribute to the spread of African ticks and tick-borne pathogens of potential public health concern across Europe. The aim of the study was to investigate their role in the possible introduction of African ticks and tick-borne pathogens into European countries during spring migration. A total of 2344 ticks were collected during three spring seasons from 1079 birds captured on three Italian stop-over islands during their northbound migration. Once identified, each tick was tested by RT-PCR for the presence of Crimean-Congo hemorrhagic fever (CCHFV), West Nile (WNV), and Usutu (USUV) viruses. Moreover, carcasses of birds found dead were collected and tested for the possible presence of WNV and USUV. Results confirmed a higher contribution of trans-Saharan migrants compared to intra-Palearctic ones and the prevalence of African tick species in the sample. CCHFV was detected for the second time in Italy in a Hyalomma rufipes, and WNV was found in two ticks of the same genus, all carried by trans-Saharan birds. WNV lineage 1 was also found in the organs of a Garden warbler. These results confirm the role of migratory birds in carrying African ticks, as well as viruses of zoonotic importance, from Africa into Europe.

During these past years, several studies have provided serological evidence regarding the circulation of West Nile virus (WNV) in Brazil. Despite some reports, much is still unknown regarding the genomic diversity and transmission dynamics of this virus in the country. Recently, genomic monitoring activities in horses revealed the circulation of WNV in several Brazilian regions. These findings on the paucity of genomic data reinforce the need for prompt investigation of WNV infection in horses, which may precede human cases of encephalitis in Brazil. Thus, in this study, we retrospectively screened 54 suspicious WNV samples collected between 2017 and 2020 from the spinal cord and brain of horses with encephalitis and generated three new WNV genomes from the Ceará and Bahia states, located in the northeastern region of Brazil. The Bayesian reconstruction revealed that at least two independent introduction events occurred in Brazil. The first introduction event appears to be likely related to the North American outbreak, and was estimated to have occurred in March 2013.The second introduction event appears to have occurred in September 2017 and appears to be likely related to the South American outbreak. Together, our results reinforce the importance of increasing the priority of WNV genomic monitoring in equines with encephalitis in order to track the dispersion of this emerging pathogen through the country.

In Italy, West Nile virus (WNV) appeared for the first time in the Tuscany region in 1998. After 10 years of absence, it re-appeared in the areas surrounding the Po River delta, affecting eight provinces in three regions. Thereafter, WNV epidemics caused by genetically divergent isolates have been documented every year in the country. Since 2018, only WNV Lineage 2 has been reported in the Italian territory. In October 2020, WNV Lineage 1 (WNV-L1) re-emerged in Italy, in the Campania region. This is the first occurrence of WNV-L1 detection in the Italian territory since 2017. WNV was detected in the internal organs of a goshawk (Accipiter gentilis) and a kestrel (Falco tinnunculus). The RNA extracted in the goshawk tissue samples was sequenced, and a Bayesian phylogenetic analysis was performed by a maximum-likelihood tree. Genome analysis, conducted on the goshawk WNV complete genome sequence, indicates that the strain belongs to the WNV-L1 Western-Mediterranean (WMed) cluster. Moreover, a close phylogenetic similarity is observed between the goshawk strain, the 2008–2011 group of Italian sequences, and European strains belonging to the Wmed cluster. Our results evidence the possibility of both a new re-introduction or unnoticed silent circulation in Italy, and the strong importance of keeping the WNV surveillance system in the Italian territory active.

West Nile virus (WNV) is a flavivirus transmitted by mosquitoes. Birds are the reservoir for the virus; humans, horses and other mammals are dead-end hosts. Infections caused by WNV in humans can vary from asymptomatic infections to West Nile fever (WNF) or West Nile neuroinvasive disease (WNND). In 1995, a serosurvey was performed in Slovenia on forest workers, and WNV specific IgG antibodies were confirmed in 6.8% of the screened samples, indicating that WNV is circulating in Slovenia. No human disease cases were detected in Slovenia until 2013, when the first case of WNV infection was confirmed in a retrospective study in a 79-year old man with meningitis. In 2018, three patients with WNND were confirmed by laboratory tests, with detection of IgM antibodies in the cerebrospinal fluid of the patients. In one of the patients, WNV RNA was detected in the urine sample. In 2017, 2018 and 2019, a mosquito study was performed in Slovenia. Mosquitoes were sampled on 14 control locations and 35 additional locations in 2019. No WNV was detected in mosquitoes in 2017 and 2019, but we confirmed the virus in a pool of Culex sp. mosquitoes in 2018. The virus was successfully isolated, and complete genome sequence was acquired. The whole genome of the WNV was also sequenced from the patient’s urine sample. The whole genome sequences of the WNV virus detected in Slovenian patient and mosquito indicate the virus most likely spread from the north, because of the geographic proximity and because the sequences cluster with the Austrian and Hungarian sequences. A sentinel study was performed on dog sera samples, and we were able to confirm IgG antibodies in 1.8% and 4.3% of the samples in 2017 and 2018, respectively. Though Slovenia is not a highly endemic country for WNV, we have established that the virus circulates in Slovenia.

The arthropod-borne flaviviruses are important human pathogens, and a deeper understanding of the virus–host cell interaction is required to identify cellular targets that can be used as therapeutic candidates. It is well reported that the flaviviruses hijack several cellular functions, such as exosome-mediated cell communication during infection, which is modulated by the delivery of the exosomal cargo of pro- or antiviral molecules to the receiving host cells. Therefore, to study the role of exosomes during flavivirus infections is essential, not only to understand its relevance in virus–host interaction, but also to identify molecular factors that may contribute to the development of new strategies to block these viral infections. This review explores the implications of exosomes in flavivirus dissemination and transmission from the vector to human host cells, as well as their involvement in the host immune response. The hypothesis about exosomes as a transplacental infection route of ZIKV and the paradox effect or the dual role of exosomes released during flavivirus infection are also discussed here. Although several studies have been performed in order to identify and characterize cellular and viral molecules released in exosomes, it is not clear how all of these components participate in viral pathogenesis. Further studies will determine the balance between protective and harmful exosomes secreted by flavivirus infected cells, the characteristics and components that distinguish them both, and how they could be a factor that determines the infection outcome.

West Nile virus (WNV) is a positive-sense RNA arbovirus responsible for recent outbreaks of severe neurological disease within the US and Europe. Large-scale analyses of antiviral compounds that inhibit virus replication have been limited due to the lack of an adequate WN reporter virus. Previous attempts to insert a reporter into the 3’ untranslated region of WNV generated unstable viruses, suggesting that this region does not accommodate additional nucleotides. Here, we engineered two WNV infectious clones containing insertions at the Capsid (C)/Capsid Anchor (CA) junction of the viral polyprotein. Recombinant viruses containing a TAT(1-67) or Gaussia Luciferase (GLuc) gene at this location were successfully recovered. However, rapid loss of most, if not all, of the reporter sequence occurred for both viruses, indicating that the reporter viruses were not stable. While the GLuc viruses predominantly reverted back to wild-type WNV length, the TAT viruses retained up to 75 additional nucleotides of the reporter sequence. These additional nucleotides were stable over at least five passages and did not significantly alter WNV fitness. Thus, the C/CA junction of WNV can tolerate additional nucleotides, though insertions are subject to certain constraints.

Flaviviruses are the most medically relevant group of arboviruses causing a wide range of diseases in humans and are associated with high mortality and morbidity, as such posing a major health concern. Viruses belonging to this family can be endemic (e.g., dengue virus), but can also cause fulminant outbreaks (e.g., West Nile virus, Japanese encephalitis virus and Zika virus). Intense research efforts in the past decades uncovered shared fundamental strategies used by flaviviruses to successfully replicate in their respective hosts. However, the distinct features contributing to the specific host and tissue tropism as well as the pathological outcomes unique to each individual flavivirus are still largely elusive. The profound footprint of individual viruses on their respective hosts can be investigated using novel technologies in the field of proteomics that have rapidly developed over the last decade. An unprecedented sensitivity and throughput of mass spectrometers, combined with the development of new sample preparation and bioinformatics analysis methods, have made the systematic investigation of virus−host interactions possible. Furthermore, the ability to assess dynamic alterations in protein abundances, protein turnover rates and post-translational modifications occurring in infected cells now offer the unique possibility to unravel complex viral perturbations induced in the infected host. In this review, we discuss the most recent contributions of mass spectrometry−based proteomic approaches in flavivirus biology with a special focus on Zika virus, and their basic and translational potential and implications in understanding and characterizing host responses to arboviral infections.

The Usutu virus (USUV) is a flavivirus that is drawing increasing attention because of its potential for emergence. First isolated in Africa, it was introduced into Europe where it caused significant outbreaks in birds, such as in Austria in 2001. Since then, its geographical distribution has rapidly expanded, with increased circulation, especially in the last few years. Similar to West Nile virus (WNV), the USUV enzootic transmission cycle involves Culex mosquitoes as vectors, and birds as amplifying reservoir hosts, with humans and other mammals likely being dead-end hosts. A similarity in the ecology of these two viruses, which co-circulate in several European countries, highlights USUV’s potential to become an important human pathogen. While USUV has had a severe impact on the blackbird population, the number of human cases remains low, with most infections being asymptomatic. However, some rare cases of neurological disease have been described, both in healthy and immuno-compromised patients. Here, we will discuss the transmission dynamics and the current state of USUV circulation in Europe.

Flaviviruses cause numerous pathologies in humans across a broad clinical spectrum with potentially severe clinical manifestations, including hemorrhagic and neurological disorders. Among human flaviviruses, some viral proteins show high conservation and are good candidates as targets for drug design. From an epidemiological point of view, flaviviruses cause more than 400 million cases of infection worldwide each year. In particular, the Yellow Fever, dengue, West Nile, and Zika viruses have high morbidity and mortality-about an estimated 20,000 deaths per year. As they depend on human vectors, they have expanded their geographical range in recent years due to altered climatic and social conditions. Despite these epidemiological and clinical premises, there are limited antiviral treatments for these infections. In this review, we describe the major compounds that are currently under evaluation for the treatment of flavivirus infections and the challenges faced during clinical trials, outlining their mechanisms of action in order to present an overview of ongoing studies. According to our review, the absence of approved antivirals for flaviviruses led to in vitro and in vivo experiments aimed at identifying compounds that can interfere with one or more viral cycle steps. Still, the currently unavailability of approved antivirals poses a significant public health issue.