Next Article in Journal
Pre-Clade IIb Mpox Virus Exposure in Ghana: A Retrospective Serological Analysis
Previous Article in Journal
Genetic and Statistical Study of Anelloviruses and Gyroviruses in Diarrheic Cats and Their Co-Occurrence Patterns
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Viruses
Volume 17
Issue 11
10.3390/v17111414
viruses-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Detection of West Nile Virus, Usutu Virus and Insect-Specific Bunyaviruses in Culex spp. Mosquitoes, Greece, 2024

by
Katerina Tsioka
1,
Konstantina Stoikou
1,
Vasilis Antalis
2,
Elissavet Charizani
2,
Styliani Pappa
1,
Sandra Gewehr
2,
Stella Kalaitzopoulou
2,
Spiros Mourelatos
2 and
Anna Papa
1,*
1
Medical School, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
2
Ecodevelopment SA, 57010 Thessaloniki, Greece
*
Author to whom correspondence should be addressed.
Viruses 2025, 17(11), 1414; https://doi.org/10.3390/v17111414
Submission received: 15 August 2025 / Revised: 13 October 2025 / Accepted: 22 October 2025 / Published: 23 October 2025
(This article belongs to the Section Invertebrate Viruses)
Browse Figures
Versions Notes

Abstract

Greece is one of the countries in Europe most affected by West Nile virus (WNV), and since 2010, when the virus caused a large outbreak with 197 human neuroinvasive cases, outbreaks occur almost every year. Mosquito surveillance is an indirect sign of virus circulation; therefore, the purpose of the study was the molecular detection of WNV in 45,988 C. pipiens s.l. mosquitoes collected during 2024 in four Regions of Greece and the genetic characterization of the virus strains. WNV was detected in 41 of 1316 (3.12%) Culex spp. mosquito pools. Next-generation sequencing was applied to the WNV-positive samples that had a high viral load. All WNV sequences belong to Cluster B of the sub-lineage Europe WNV-2A presenting a temporal clustering. The WNV infection rates varied highly across the Regions, regional units and months, being higher in Thessaly and Central Macedonia Regions, especially in July and September. All mosquito pools were also tested for Usutu virus (USUV), and one pool was found positive, with sequence clustering into the EU-2 lineage. A subset of mosquitoes (737 pools) was tested for additional viruses, and bunya-like viruses were detected in 6 pools with sequences clustering into four distinct subclades. The prompt detection of pathogenic viruses is helpful for the design of control measures, while the detection of insect-specific viruses provides insights into viral diversity and evolution.

1. Introduction

Mosquitoes of the Culex genus are vectors of several viral pathogens, including West Nile virus (WNV) and Usutu virus (USUV). Both viruses belong to the genus Orthoflavivirus in the Flaviviridae family [1] and circulate in nature in an enzootic cycle between mosquitoes (mainly of the Culex species) as vectors and birds as amplifying reservoir hosts, while humans and other mammalian species are dead-end hosts due to low level of viremia [2,3]. Several avian species are susceptible to WNV and USUV; USUV, in particular, causes mass mortality in birds, mainly blackbirds in Europe [4,5,6].
Human WNV and USUV infections are usually asymptomatic or present as mild febrile illness, while less than 1% of the WNV infections present as neuroinvasive disease (WNND), mainly encephalitis, meningitis or flaccid paralysis, with a fatality rate of approximately 15% [7]. Neuroinvasiveness is seen less often in USUV infections; however, an increasing number of neurological cases are being reported, mainly in immunocompromised patients, with few of them with a fatal outcome [8,9,10,11,12]. It is of interest that several USUV infections have been detected in asymptomatic blood donors who tested WNV-positive in the nucleic acid amplification tests due to cross-reactivity between these two viruses [8,13].
Both WNV and USUV circulate in Europe; however, there is a big difference in the incidence and severity of the disease they cause. While WNV causes human outbreaks in southern and central Europe, the number of reported USUV infections is much lower, either because the circulation of the virus is limited, or due to the mild symptomatology of the disease, which remains unrecognized, or even due to misdiagnosis as WNV infections because of the cross-reactivity in serology. A recent study showed that in contrast to WNV, USUV cannot infect motor neurons in healthy individuals due to its restriction by the antiviral immune response, which could explain the differences in the clinical impact of these two viruses [14]. In addition, a study based on the human blood–brain barrier (BBB) model showed that USUV can replicate efficiently in BBB cells and promote immune activation, but the level of neuroinflammation is lower than that of WNV [15].
Since the geographical distribution of WNV and USUV in Europe is overlapping, there is a potential for co-infections in mosquitoes, which can influence the virus transmission. In vitro studies showed that co-infections might lead to a decreased growth of USUV in mosquitoes and of both viruses in vertebrate hosts [16]. A reduction in USUV transmission was seen in mosquitoes exposed simultaneously to both viruses compared to mosquitoes exposed only to USUV, while the infection and transmission of WNV was unaffected; in contrast, WNV transmission was significantly reduced when mosquitoes were pre-infected with USUV [17].
Both viruses are characterized by great genetic variability. Globally, nine WNV lineages have been identified (WNV-1 to WNV-9); however, most human infections are associated with WNV-1 and WNV-2. The predominant WNV lineage in recent years in Europe is WNV-2, which is divided into two sub-lineages: 2A and 2B. Specifically, sub-lineage WNV-2A emerged in Hungary around 2003–2004, and has diverged into Cluster A, which emerged in July 2006 and spread to northwest and western Europe, and Cluster B, which emerged in 2007 and spread to southern Europe, including Greece [18]. Similarly, the known USUV sequences cluster into eight lineages, Europe (EU)1 to EU5 and Africa (AF)1 to AF3. All lineages, except AF1, are present in Europe, suggesting that several virus introductions have occurred [6].
Greece is one of the most endemic countries for WNV in Europe. Specifically in 2024, it was the second-most affected country in EU/EEA after Italy [19]. The virus emerged in the country in the summer of 2010, in Central Macedonia Region, and caused a large outbreak with 197 WNND cases [20]. Since then, outbreaks occur annually, except 2016 and 2017, and up to the end of 2024, 1469 WNND cases (18% fatal) have been reported to the National Public Health Organization [21]. Molecular screening of mosquitoes for WNV infection is often used as an indirect sign of the virus circulation and as an early warning system, which facilitates the design of mosquito control measures. Previous studies in Greece showed that the WNV infection rate (IR) of Culex mosquitoes ranges from 0% to 17%, differing in years, months, regions, and regional units [22,23,24,25,26,27,28,29]. Regarding USUV, there is one report of virus detection in Culex mosquitoes in Greece [30], while in 2024, the virus was isolated from an asymptomatic blood donor (article in preparation). Therefore, the aim of the present study was the detection of WNV and USUV in Culex pipiens s.l. (hereafter C. pipiens) mosquitoes collected during 2024 and the genetic characterization of the virus strains. In addition, a generic RT-PCR, initially designed to detect phleboviruses, was applied in a subset of the mosquito collection.

2. Materials and Methods

2.1. Study Area

Mosquito collection was conducted in 250 sites in both urban and rural areas in four Regions of Greece. Specifically, mosquitoes were collected from 116 sites in Central Macedonia, 13 sites in West Macedonia, 75 sites in Thessaly, and 46 sites in West Greece. The regional units (RUs) tested per Region are shown in Table 1.
The collection sites were selected based on ecosystems favorable for mosquito breeding, such as proximity to rice fields, wetlands, and other water bodies, or in urban parks; areas where human cases had been reported in previous years were also included.

2.2. Mosquito Collection and Species Identification

Mosquitoes were collected by the Ecodevelopment mosquito control company using CO2-baited light traps with a constant CO2 outflow of 0.5 L/min (known as “Ecodev traps”). The traps were placed at the ground level in the morning and retrieved after approximately 24 h. The collection was conducted over a six-month period, spanning from May to October 2024, corresponding to the Culex spp. mosquito activity and abundance [31]. Each location was visited every two weeks, while additional traps were set at the locations where human or equine cases were recorded.
The mosquitoes were identified using a combination of morphological identification keys, such as arrangement of scales on the wings, the shape of the antennae, and the structure of the genitalia [32,33]. Female C. pipiens mosquitoes were transported to the laboratory on dry ice and stored at −80 °C until processing.

2.3. RNA Extraction

The mosquitoes were grouped into pools (up to 50 mosquitoes per pool), based on collection site and sampling date. The specimens were then rinsed with distilled water to remove any surface contaminants and homogenized in phosphate-buffered saline using glass beads (diameter 150–212 μm) in a TissueLyser II cell disrupter (Qiagen, Hilden, Germany) at 30 Hz for 3 min to break down the mosquito tissues and release the intracellular content. The total RNA was extracted from 200 μL supernatant of each homogenized pool using the IndiSpin Pathogen Kit (Qiagen, Hilden, Germany) and eluted in 50 µL RNase-free water following the manufacturer’s instructions.

2.4. Molecular Detection of Viruses

All mosquito pools were tested for qualitative detection of WNV and USUV. For WNV, a commercial real-time RT-PCR kit (West Nile Virus Real-TM, Sacace Biotechnologies Srl, Como, Italy) was applied, while for USUV, a conventional real-time RT-PCR with degenerate primer sets targeting conserved regions of the viral L genome segment was used [34]. The USUV-positive samples were further tested by a semi-nested RT-PCR using one forward primer (FU1) and two reverse primers (CFD3 and CFD2), which amplify a 265bp fragment of NS5 gene of flaviviruses [35]. The WNV and USUV infection rates (IRs) were estimated by dividing the number of positive pools by the total number of tested pools.
The mosquitoes of Central Macedonia were further tested using a conventional RT-nested PCR with degenerate primer sets, which was initially designed to target a region of the viral L genome segment of phleboviruses that encodes the RNA-dependent RNA polymerase (RdRp) [36].

2.5. Sanger Sequencing and Phylogenetic Analysis of USUV and Bunya-like Viruses

The PCR products of the conventional RT-PCRs were Sanger-sequenced in a 3130 ABI Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The nucleotide sequences were analyzed using the National Center for Biotechnology Information (NCBI) Basic Local Alignment Sequence Tool (BLAST) search engine (https://blast.ncbi.nlm.nih.gov/, accessed on 9 July 2025) to identify the best match.
USUV and bunya-like virus sequences were aligned with respective ones retrieved from the GenBank Database using Clustal W2, while evolutionary analyses and construction of the maximum likelihood phylogenetic trees based on the best-fitted nucleotide substitution model were conducted using MEGA version 12 [37].

2.6. Next-Generation Sequencing and Phylogenetic Analysis of West Nile Virus

The WNV-positive mosquito pools with a cycle threshold (Ct) of less than 30 in the WNV real-time RT-PCR were further processed using a PCR-based next-generation sequencing (NGS) protocol [38]. The libraries were prepared using the Ion 510 & Ion 520 & Ion 530 for Ion Chef kit for 400 base-reads and quantified using the Ion Library TaqMan Quantitation kit. Then, they were normalized to a final concentration of 35pM and sequenced on an Ion Torrent S5 platform using an Ion 520 semiconductor sequencing chip following the manufacturer’s instructions. All reagents were obtained from Life Technologies Corporation (Grand Island, NY, USA).
Raw reads were processed through the Torrent Suite Software version 5.18.1 for quality control, and the sequences were aligned using the sequence of Nea Santa-Greece-2010 strain (GenBank Accession number HQ537483) as reference. Assembly and annotation of the WNV whole-genome sequences were carried out using Geneious version 7.1.3. Reads were mapped to the refence sequence (HQ538473), with medium sensitivity/Fast, Fine-Tuning option Iterate up to 5 times, while the consensus sequences were generated with a minimum coverage of 20×, while all other parameters were used as the default values. The consensus sequences were aligned with WNV lineage 2 sequences obtained from the GenBank Database, and a maximum likelihood phylogenetic tree was generated using the best model in MEGA version 12 software [37].

2.7. Data Visualization

The locations of the mosquito traps were mapped using the ArcGIS Pro geographic information system (GIS) application, version 3.0 (Esri, Redlands, CA, USA). The sites where WNV-positive mosquito pools were collected are shown on the map in red color (Figure 1).

3. Results

A total of 45,988 C. pipiens mosquitoes were collected and grouped into 1316 pools. WNV was detected in 41 pools (IR 3.12%). Specifically, 26 positive pools were detected in Central Macedonia (3.53%), 10 in Thessaly (3.98%), and 5 in West Greece (1.74%), while all mosquitoes from West Macedonia were negative (Table 1, Figure 1). The IR varied highly across the Regions, Rus, and months. The first evidence of WNV circulation was on 15 May 2024, in Thessaly Region. Highest IRs were recorded in July (5.37%) and September (4.47%). In four RUs (Larisa, Pieria, Ilia, and Achaia), the detection of WNV in mosquitoes preceded the onset of symptoms in human cases by 15–30 days. The location and date of collection of the positive pools are seen in Table 2. In one location in Pieria RU, WNV-positive mosquitoes were detected in three consecutive months (July, August, and September).
Whole-WNV genome sequences were taken by NGS from nine positive mosquito pools, which had a Ct value of less than 30 in the real-time RT-PCR. Phylogenetic analysis showed that all sequences of the current study belong to Cluster B (previously known as southeastern clade) of the sub-lineage Europe WNV-2A (Figure 2). A temporal clustering is seen among the Greek WNV strains; the sequences of 2024 cluster in a distinct subclade which contains sequences from Greece since 2018, differing by approximately 0.5% from sequences detected in previous years in the country.
USUV was detected in one pool of mosquitoes (1/1316 pools, IR 0.07%), which were collected in September 2024 in Serres RU in Central Macedonia Region [IR for Central Macedonia 0.13% (1/737 pools)]. The result of the real-time RT-PCR was confirmed by sequencing of the product of the conventional semi-nested RT-PCR. The sequence presented 100% nucleotide identity with USUV sequences belonging to EU2 lineage (Figure 3).
The mosquitoes collected in Central Macedonia Region (737 pools) were also tested using a PCR which was originally designed to detect phleboviruses (genus phlebovirus, family Phenuiviridae). PCR products of the expected size were taken from six mosquito pools. BLAST analysis showed that the sequence of one pool shared 92% nucleotide identity with Shuangao insect virus 3, a second pool shared 87% identity with Culex bunyavirus 2, the sequences of three pools shared 83% identity with Xiang Yun bunya-arena-like virus 14, and the sequence of the sixth pool shared 73% identity with Wuhan insect virus 16. The sequences form four subclades, which are distinct from viruses of the phlebovirus genus. Specifically, GR-Z155/2024 clusters with Shuangao insect virus (unclassified virus in the Peribunyaviridae family); GR-Z221/2024, GR-Z225/2024, and GR-Z303/2024 cluster with Xiang Yun bunya-arena-like virus 14 (unclassified bunyavirus); GR-Z615/2024 clusters with Culex bunyavirus 2 (unclassified bunyavirus); and GR-Z232/2024 clusters with Wuhan insect virus 16 (unclassified RNA virus) (Figure 4). The Greek sequences were named according to the viruses of the subclade they are clustering, while a new name, Tragilos insect virus, was provisionally given to GR-232/2024, as it is distantly related to the only available virus in the subclade.

4. Discussion

Screening mosquitoes for WNV provides valuable information about spatial and temporal virus circulation in a region which is important for the design of prevention and control measures. The current study showed that 3.12% of the C. pipiens were WNV-positive, with the highest IRs seen in Thessaly and Central Macedonia Regions. Specifically, the IR was 3.98% in Thessaly, 3.53% in Central Macedonia, 1.74% in West Greece, and 0% in West Macedonia (Table 1). Although the geographic distribution of human cases in 2024 was almost countrywide, 70.5% (110/156) of WNND cases were reported from Thessaly, Central Macedonia, and West Greece, while no cases were reported in West Macedonia [21]. Based on a continuous phylogeographic model, it was shown that WNV-2A is attracted to areas with high crop and vegetation density, livestock cultivation and urbanization, as well as to wetlands, protected bird and habitat areas, and migratory bird flyways [18]. The largest plains with the most extensive agricultural production in Greece are in Thessaly and Central Macedonia, where the highest IRs were detected. Livestock farming is also highly concentrated in these two Regions and in West Greece. The same drivers are related also to increased transmission velocity of WNV-2A [18], which may explain the high number of clinical cases in these areas. Furthermore, major wetlands are present in Central Macedonia, while Thessaly experienced a catastrophic flood in September 2023 due to the storm Daniel, which actually did not affect the number of WNV cases in 2023 (it was already end of the WNV season), but might play a role in local ecosystem changes affecting the WNV circulation in 2024 [39,40].
The first detection of WNV in mosquitoes was in mid-May, earlier than in previous years (early to mid-June), suggesting a longer period of virus activity. The IRs in July and September were higher than in August when prolonged heat waves were observed in Greece. The hot weather during August might decrease the abundance of C. pipiens mosquitoes since extremely high temperatures reduce the abundance of mosquitoes [41].
Central Macedonia is the Region where WNV emerged in Greece in 2010; since then, it is the most entomologically studied area by our group. Investigations contacted during 2010 to 2024 in this area showed that the WNV IR of Culex spp. mosquitoes ranged from 0% to 9.6% (median 2.04%, Table 3) [22,23,25,31,42], and unpublished data}. The WNV IR in mosquitoes was 0% in 2014–2017, which coincides with the number of human WNND cases in this Region (Table 3). The discrepancy between the low IR and the highest number of human cases in 2010 could be explained by the absence of immunity in the naïve human and avian population prior to the emergence of the virus.
In a study conducted in twelve Regions of Greece during 2014–2016, WNV was detected in 1.17% (6/514) C. pipiens pools in 2014, 7.96% (9/113) pools in 2015 and 10.57% (22/208) pools in 2016 [24]. Sporadic studies in various areas in the country showed IRs ranging from 0% to 17% depending on the area, year, and number and size of the mosquito pools tested [26,27,28,29,44]. Variations in C. pipiens’ WNV positivity are also seen among European countries. As an example, WNV was detected in 12.2% (5/41) pools during 2018–2019 in Bulgaria [45], 5% (232/2337) in Emilia–Romagna and Lombardy regions (northern Italy) in 2018 [46], 2.31% (23/995) and 2.09% (20/956) in 2014 and 2015, respectively, in Serbia [47], while a study in Bucharest (Romania) showed that 21.7% (37/170) pools were positive in 2017, 17.3% (27/156) in 2018, 10.31% (23/223) in 2019, 2.88% (14/486) in 2020, 10% (21/210) in 2021, 7.77% (7/90) in 2022, and 8.06% (10/124) in 2023 [48]. Since IR depends on several parameters which differ between studies, comparison is not possible. However, it is seen that WNV mosquito screening is applied in several countries, especially the endemic ones, providing important information about the spatial and temporal circulation of the virus. In the present study, positive mosquito traps were found in one specific site for three consecutive months (July, August, and September), indicating that this site was highly affected, prompting for strengthening efforts to reduce the mosquito populations.
In four RUs (Larisa, Pieria, Ilia, and Achaia), the detection of WNV in mosquitoes preceded the human cases by 15–30 days, suggesting that sustained mosquito surveillance has the potential to serve as an early warning tool of viral activity allowing for timely intervention and application of prevention measures.
As in previous years, all WNV sequences of 2024 cluster into Cluster B of the sub-lineage WNV-2A. One exception was seen in 2018, when one sequence was found to cluster into the sub-lineage 2B [49]. Phylodymanic studies showed that specifically in 2018, three novel, independent introductions from Hungary and Bulgaria occurred in northern Greece [50]. At least 19 transmission events between Greece and other European countries occurred in the past decade, with Hungary, Serbia, and Romania being the countries with the most frequent events of virus transmission to Greece [18]. A limitation of the study was that the sequences were taken from pools of mosquitoes and not from individual mosquitoes, with a risk that more than one mosquito in the pool found to be positive. However, the pooling was performed based on the collection site and sampling date. Furthermore, the genetic distance between viruses of 2018–2024 is only 0.5% from those detected prior 2018.
Regarding USUV, the total IR was 0.07%; specifically, it was in Central Macedonia Region was 0.13% (1/737 pools) and 0% in West Macedonia, Thessaly, and West Greece. Similarly, in a previous study conducted in Greece during 2020–2023, USUV was detected only in Central Macedonia Region with IR of 1.03% (4/386 pools), while it was 0% in Thessaly (0/126 pools), suggesting a low-level virus circulation in the country [30]. Low USUV IRs in mosquitoes have been reported in several countries in southern and central Europe, mainly in C. pipiens, but also in other mosquito species, like Aedes and Anopheles [51,52,53,54,55,56,57]. The USUV-positive pool was detected in September. Similarly in Italy, USUV circulation was demonstrated in autumn (mid-October), while it was poorly detected in the summer [57]. By investigating the spatial spread in Europe, it was shown that Italy acted as main donor of USUV to neighboring countries [51]. The sequence taken from the Greek USUV-positive mosquito pool clusters with sequences of the EU2 lineage (Figure 3). Although the virus circulation in Greece is low, more studies in humans, mosquitoes, and birds are needed to gain a better insight into the USUV epidemiology in the country.
Co-infection with WNV and USUV in mosquitoes was not detected in this study. However, since both viruses are endemic in Europe, the competition between them during co-infections may reduce the vector competence of the USUV-infected mosquitoes for WNV, while the circulation of USUV in the WNV-free regions may prevent WNV transmission and spread [17]. Further field studies will show whether USUV can affect the epidemiology of WNV in Europe.
The rapidly increasing advances in genome sequencing technologies, combined with the availability of efficient bioinformatic tools, resulted in the identification of numerous novel viruses closely related to the viruses classified until recently in the family Bunyaviridae. This family included both pathogenic and insect-specific viruses (ISVs, viruses restricted to replicating only in insects). Therefore, in 2017, the International Committee on Taxonomy of Viruses (ICTV) promoted the family to order Bunyavirales [58], while in 2024, after repeated and substantial revisions, the order was promoted to class Bunyaviricetes [59]. The sequences of the present study cluster into four distinct subclades together with other bunyaviruses. Since the phylogeny was based on a small fragment of RdRp, we preferred the term “bunya-like viruses”; analysis of larger sequences will show their exact designation in virus taxonomy. Most probably, they are ISVs, which are not pathogenic for humans or animals; however, ISVs are important in understanding viral diversity and evolution, as well as their potential impact on vector competence for arboviruses [60].

5. Conclusions

A plethora of drivers (biological, ecological, and behavioral) play a role in the emergence and spread of mosquito-transmitted viral diseases, and WNV is a nice example for all these interactions [61]. The present study provided information about the spatial and temporal dynamic patterns of WNV in C. pipiens mosquitoes in four Regions of Greece in 2024 and showed that the highest WNV IRs were seen in two neighboring Regions, Thessaly and Central Macedonia, which are characterized by intense agricultural and pastoral activities, as well as presence of water bodies. Additionally, valuable insight into the molecular epidemiology of the disease was gained by enriching the WNV phylogeny with whole genome sequences from 2024. Regarding USUV, it was shown that the virus circulation in Greece is currently low, while the detection of insect-specific, bunya-like viruses prompts for further studies to understand better their distribution and evolution.

Author Contributions

Conceptualization, A.P.; validation, A.P.; formal analysis, A.P.; investigation, K.T., K.S., V.A., E.C., S.G., S.K. and S.P.; resources, A.P. and S.M.; data curation, A.P.; writing—original draft preparation, K.T.; writing—review and editing, A.P.; visualization, S.K. and A.P.; supervision, A.P.; project administration, A.P.; funding acquisition, A.P. and S.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Versatile Emerging Infectious Disease Observatory (VEO) project funded by the European Union Horizon 2020 Research and Innovation Program, grant number 874735 and by Ecodevelopment SA.

Data Availability Statement

The data presented in this study is available on request from the corresponding author.

Conflicts of Interest

Authors V. Antalis, E. Charizani, S. Gewehr, S. Kalaitzopoulou, and S. Mourelatos were employed by the company EcoDevelopment S.A. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received funding from the Versatile Emerging Infectious Disease Observatory (VEO) project (European Union Horizon 2020 Research and Innovation Program grant number 874735) and by EcoDevelopment S.A. The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication.

References

  1. International Committee on Taxonomy of Viruses. ICTV: Family: Flaviviridae. Genus: Orthoflavivirus. 2025. Available online: https://ictv.global/report/chapter/flaviviridae/flaviviridae/orthoflavivirus (accessed on 2 May 2025).
  2. Martina, B.E.; Barzon, L.; Pijlman, G.P.; de la Fuente, J.; Rizzoli, A.; Wammes, L.J.; Takken, W.; van Rij, R.P.; Papa, A. Human to human transmission of arthropod-borne pathogens. Curr. Opin. Virol. 2017, 22, 13–21. [Google Scholar] [CrossRef]
  3. Engler, O.; Savini, G.; Papa, A.; Figuerola, J.; Groschup, M.H.; Kampen, H.; Medlock, J.; Vaux, A.; Wilson, A.J.; Werner, D.; et al. European surveillance for West Nile virus in mosquito populations. Int. J. Environ. Res. Public Health 2013, 10, 4869–4895. [Google Scholar] [CrossRef]
  4. de Bruin, E.; van Irsel, J.; Chandler, F.; Kohl, R.; van de Voorde, T.; van der Linden, A.; de Boer, F.; Koopmans, M.; van der Jeugd, H.; Reusken, C. Usutu Virus Antibody Dynamics in Naturally Infected Blackbirds, the Netherlands, 2016–2018. Emerg. Infect. Dis. 2025, 31, 1244–1246. [Google Scholar] [CrossRef]
  5. Weissenbock, H.; Bakonyi, T.; Rossi, G.; Mani, P.; Nowotny, N. Usutu virus, Italy, 1996. Emerg. Infect. Dis. 2013, 19, 274–277. [Google Scholar] [CrossRef]
  6. Cadar, D.; Luhken, R.; van der Jeugd, H.; Garigliany, M.; Ziegler, U.; Keller, M.; Lahoreau, J.; Lachmann, L.; Becker, N.; Kik, M.; et al. Widespread activity of multiple lineages of Usutu virus, western Europe, 2016. Eurosurveillance 2017, 22, 30452. [Google Scholar] [CrossRef]
  7. Sejvar, J.J. Clinical manifestations and outcomes of West Nile virus infection. Viruses 2014, 6, 606–623. [Google Scholar] [CrossRef]
  8. Angeloni, G.; Bertola, M.; Lazzaro, E.; Morini, M.; Masi, G.; Sinigaglia, A.; Trevisan, M.; Gossner, C.M.; Haussig, J.M.; Bakonyi, T.; et al. Epidemiology, surveillance and diagnosis of Usutu virus infection in the EU/EEA, 2012 to 2021. Eurosurveillance 2023, 28, 2200929. [Google Scholar] [CrossRef]
  9. Cadar, D.; Simonin, Y. Human Usutu Virus Infections in Europe: A New Risk on Horizon? Viruses 2022, 15, 77. [Google Scholar] [CrossRef]
  10. Pecorari, M.; Longo, G.; Gennari, W.; Grottola, A.; Sabbatini, A.; Tagliazucchi, S.; Savini, G.; Monaco, F.; Simone, M.; Lelli, R.; et al. First human case of Usutu virus neuroinvasive infection, Italy, August-September 2009. Eurosurveillance 2009, 14, 15–16. [Google Scholar] [CrossRef]
  11. Simonin, Y.; Sillam, O.; Carles, M.J.; Gutierrez, S.; Gil, P.; Constant, O.; Martin, M.F.; Girard, G.; Van de Perre, P.; Salinas, S.; et al. Human Usutu Virus Infection with Atypical Neurologic Presentation, Montpellier, France, 2016. Emerg. Infect. Dis. 2018, 24, 875–878. [Google Scholar] [CrossRef]
  12. Szabo, B.G.; Nagy, A.; Nagy, O.; Koroknai, A.; Csonka, N.; Korozs, D.; Jeszenszky, K.; Hardi, A.; Deezsi-Magyar, N.; Sztikler, J.; et al. First documented case of a fatal autochthonous Usutu virus infection in an immunocompromised patient in Hungary: A clinical-virological report and implications from the literature. Virol. J. 2025, 22, 261. [Google Scholar] [CrossRef]
  13. Cadar, D.; Maier, P.; Muller, S.; Kress, J.; Chudy, M.; Bialonski, A.; Schlaphof, A.; Jansen, S.; Jost, H.; Tannich, E.; et al. Blood donor screening for West Nile virus (WNV) revealed acute Usutu virus (USUV) infection, Germany, September 2016. Eurosurveillance 2017, 22, 30501. [Google Scholar] [CrossRef]
  14. Marshall, E.M.; Bauer, L.; Nelemans, T.; Sooksawasdi Na Ayudhya, S.; Benavides, F.; Lanko, K.; de Vrij, F.M.S.; Kushner, S.A.; Koopmans, M.; van Riel, D.; et al. Differential susceptibility of human motor neurons to infection with Usutu and West Nile virus. J. Neuroinflammation 2024, 21, 236. [Google Scholar] [CrossRef]
  15. Constant, O.; Maarifi, G.; Barthelemy, J.; Martin, M.F.; Tinto, B.; Savini, G.; Van de Perre, P.; Nisole, S.; Simonin, Y.; Salinas, S. Differential effects of Usutu and West Nile viruses on neuroinflammation, immune cell recruitment and blood-brain barrier integrity. Emerg. Microbes Infect. 2023, 12, 2156815. [Google Scholar] [CrossRef]
  16. Korsten, C.; Reemtsma, H.; Ziegler, U.; Fischer, S.; Tews, B.A.; Groschup, M.H.; Silaghi, C.; Vasic, A.; Holicki, C.M. Cellular co-infections of West Nile virus and Usutu virus influence virus growth kinetics. Virol. J. 2023, 20, 234. [Google Scholar] [CrossRef]
  17. Wang, H.; Abbo, S.R.; Visser, T.M.; Westenberg, M.; Geertsema, C.; Fros, J.J.; Koenraadt, C.J.M.; Pijlman, G.P. Competition between Usutu virus and West Nile virus during simultaneous and sequential infection of Culex pipiens mosquitoes. Emerg. Microbes Infect. 2020, 9, 2642–2652. [Google Scholar] [CrossRef]
  18. Lu, L.; Zhang, F.; Oude Munnink, B.B.; Munger, E.; Sikkema, R.S.; Pappa, S.; Tsioka, K.; Sinigaglia, A.; Dal Molin, E.; Shih, B.B.; et al. West Nile virus spread in Europe: Phylogeographic pattern analysis and key drivers. PLoS Pathog. 2024, 20, e1011880. [Google Scholar] [CrossRef]
  19. European Centre for Disease Prevention and Control. Historical Data by Year—West Nile Virus Seasonal Surveillance: West Nile Virus Infections in 2024. Available online: https://www.ecdc.europa.eu/en/west-nile-fever/surveillance-and-disease-data/historical (accessed on 9 July 2025).
  20. Papa, A.; Danis, K.; Baka, A.; Bakas, A.; Dougas, G.; Lytras, T.; Theocharopoulos, G.; Chrysagis, D.; Vassiliadou, E.; Kamaria, F.; et al. Ongoing outbreak of West Nile virus infections in humans in Greece, July–August 2010. Eurosurveillance 2010, 15, 19644. [Google Scholar] [CrossRef]
  21. National Organisation of Public Health. West Nile Virus. 2025. Available online: https://eody.gov.gr/en/disease/west-nile-virus/ (accessed on 9 October 2025).
  22. Papa, A.; Xanthopoulou, K.; Tsioka, A.; Kalaitzopoulou, S.; Mourelatos, S. West Nile virus in mosquitoes in Greece. Parasitol. Res. 2013, 112, 1551–1555. [Google Scholar] [CrossRef]
  23. Papa, A.; Papadopoulou, E.; Kalaitzopoulou, S.; Tsioka, K.; Mourelatos, S. Detection of West Nile virus and insect-specific flavivirus RNA in Culex mosquitoes, central Macedonia, Greece. Trans. R. Soc. Trop. Med. Hyg. 2014, 108, 555–559. [Google Scholar] [CrossRef]
  24. Patsoula, E.; Beleri, S.; Tegos, N.; Mkrtsian, R.; Vakali, A.; Pervanidou, D. Entomological Data and Detection of West Nile Virus in Mosquitoes in Greece (2014–2016), Before Disease Re-Emergence in 2017. Vector-Borne Zoonotic Dis. 2020, 20, 60–70. [Google Scholar] [CrossRef]
  25. Papa, A.; Gewehr, S.; Tsioka, K.; Kalaitzopoulou, S.; Pappa, S.; Mourelatos, S. Detection of flaviviruses and alphaviruses in mosquitoes in Central Macedonia, Greece, 2018. Acta Trop. 2020, 202, 105278. [Google Scholar] [CrossRef]
  26. Papa, A.; Tsioka, K.; Gewehr, S.; Kalaitzopoulou, S.; Pappa, S.; Mourelatos, S. West Nile virus lineage 2 in Culex mosquitoes in Thessaly, Greece, 2019. Acta Trop. 2020, 208, 105514. [Google Scholar] [CrossRef] [PubMed]
  27. Mavridis, K.; Fotakis, E.A.; Kioulos, I.; Mpellou, S.; Konstantas, S.; Varela, E.; Gewehr, S.; Diamantopoulos, V.; Vontas, J. Detection of West Nile Virus—Lineage 2 in Culex pipiens mosquitoes, associated with disease outbreak in Greece, 2017. Acta Trop. 2018, 182, 64–68. [Google Scholar] [CrossRef] [PubMed]
  28. Vakali, A.; Beleri, S.; Tegos, N.; Fytrou, A.; Mpimpa, A.; Sergentanis, T.N.; Pervanidou, D.; Patsoula, E. Entomological Surveillance Activities in Regions in Greece: Data on Mosquito Species Abundance and West Nile Virus Detection in Culex pipiens Pools (2019–2020). Trop. Med. Infect. Dis. 2022, 8, 1. [Google Scholar] [CrossRef] [PubMed]
  29. Sofia, M.; Giannakopoulos, A.; Giantsis, I.A.; Touloudi, A.; Birtsas, P.; Papageorgiou, K.; Athanasakopoulou, Z.; Chatzopoulos, D.C.; Vrioni, G.; Galamatis, D.; et al. West Nile Virus Occurrence and Ecological Niche Modeling in Wild Bird Species and Mosquito Vectors: An Active Surveillance Program in the Peloponnese Region of Greece. Microorganisms 2022, 10, 1328. [Google Scholar] [CrossRef]
  30. Panagopoulou, A.; Tegos, N.; Beleri, S.; Mpimpa, A.; Balatsos, G.; Michaelakis, A.; Hadjichristodoulou, C.; Patsoula, E. Molecular detection of Usutu virus in pools of Culex pipiens mosquitoes in Greece. Acta Trop. 2024, 258, 107330. [Google Scholar] [CrossRef]
  31. Tsioka, K.; Gewehr, S.; Kalaitzopoulou, S.; Pappa, S.; Stoikou, K.; Mourelatos, S.; Papa, A. Detection and molecular characterization of West Nile virus in Culex pipiens mosquitoes in Central Macedonia, Greece, 2019–2021. Acta Trop. 2022, 230, 106391. [Google Scholar] [CrossRef]
  32. Becker, N. Mosquitoes and Their Control, 2nd ed.; Springer: Heidelberg, Germany, 2010. [Google Scholar]
  33. Darsie, R.F., Jr.; Samanidou-Voyadjoglou, A. Keys for the identification of the mosquitoes of Greece. J. Am. Mosq. Control Assoc. 1997, 13, 247–254. [Google Scholar]
  34. Del Amo, J.; Sotelo, E.; Fernandez-Pinero, J.; Gallardo, C.; Llorente, F.; Aguero, M.; Jimenez-Clavero, M.A. A novel quantitative multiplex real-time RT-PCR for the simultaneous detection and differentiation of West Nile virus lineages 1 and 2, and of Usutu virus. J. Virol. Methods 2013, 189, 321–327. [Google Scholar] [CrossRef]
  35. Kuno, G.; Chang, G.J.; Tsuchiya, K.R.; Karabatsos, N.; Cropp, C.B. Phylogeny of the genus Flavivirus. J. Virol. 1998, 72, 73–83. [Google Scholar] [CrossRef] [PubMed]
  36. Matsuno, K.; Weisend, C.; Kajihara, M.; Matysiak, C.; Williamson, B.N.; Simuunza, M.; Mweene, A.S.; Takada, A.; Tesh, R.B.; Ebihara, H. Comprehensive molecular detection of tick-borne phleboviruses leads to the retrospective identification of taxonomically unassigned bunyaviruses and the discovery of a novel member of the genus phlebovirus. J. Virol. 2015, 89, 594–604. [Google Scholar] [CrossRef] [PubMed]
  37. Kumar, S.; Stecher, G.; Suleski, M.; Sanderford, M.; Sharma, S.; Tamura, K. MEGA12: Molecular Evolutionary Genetic Analysis Version 12 for Adaptive and Green Computing. Mol. Biol. Evol. 2024, 41, msae263. [Google Scholar] [CrossRef] [PubMed]
  38. Pappa, S.; Chaintoutis, S.C.; Dovas, C.I.; Papa, A. PCR-based next-generation West Nile virus sequencing protocols. Mol. Cell. Probes 2021, 60, 101774. [Google Scholar] [CrossRef]
  39. Mourelatos, S.; Charizani, E.; Kalaitzopoulou, S.; Tseni, X.; Lazos, N.; Tsioka, K.; Papa, A.; Dafka, S.; Rocklov, J.; Gewehr, S. Extreme flood and WNV transmission in Thessaly, Greece, 2023. Sci. Rep. 2025, 15, 22433. [Google Scholar] [CrossRef]
  40. Mavroulis, S.; Mavrouli, M.; Lekkas, E.; Tsakris, A. Impact of the September 2023 Storm Daniel and Subsequent Flooding in Thessaly (Greece) on the Natural and Built Environment and on Infectious Disease Emergence. Environments 2024, 11, 163. [Google Scholar] [CrossRef]
  41. Lim, A.Y.; Cheong, H.K.; Chung, Y.; Sim, K.; Kim, J.H. Mosquito abundance in relation to extremely high temperatures in urban and rural areas of Incheon Metropolitan City, South Korea from 2015 to 2020: An observational study. Parasit. Vectors 2021, 14, 559. [Google Scholar] [CrossRef]
  42. Tsioka, K.; Gewehr, S.; Pappa, S.; Kalaitzopoulou, S.; Stoikou, K.; Mourelatos, S.; Papa, A. West Nile Virus in Culex Mosquitoes in Central Macedonia, Greece, 2022. Viruses 2023, 15, 224. [Google Scholar] [CrossRef]
  43. National Public Health Organisation. Annual Epidemiological Data: West Nile Virus Infection. 2024. Available online: https://eody.gov.gr/en/epidemiological-statistical-data/annual-epidemiological-data/ (accessed on 9 October 2025).
  44. Papa, A.; Tsioka, K.; Gewehr, S.; Kalaitzopouou, S.; Pervanidou, D.; Vakali, A.; Kefaloudi, C.; Pappa, S.; Louka, X.; Mourelatos, S. West Nile fever upsurge in a Greek regional unit, 2020. Acta Trop. 2021, 221, 106010. [Google Scholar] [CrossRef]
  45. Christova, I.; Papa, A.; Trifonova, I.; Panayotova, E.; Pappa, S.; Mikov, O. West Nile virus lineage 2 in humans and mosquitoes in Bulgaria, 2018–2019. J. Clin. Virol. 2020, 127, 104365. [Google Scholar] [CrossRef]
  46. Calzolari, M.; Angelini, P.; Bolzoni, L.; Bonilauri, P.; Cagarelli, R.; Canziani, S.; Cereda, D.; Cerioli, M.P.; Chiari, M.; Galletti, G.; et al. Enhanced West Nile Virus Circulation in the Emilia-Romagna and Lombardy Regions (Northern Italy) in 2018 Detected by Entomological Surveillance. Front. Vet. Sci. 2020, 7, 243. [Google Scholar] [CrossRef] [PubMed]
  47. Petrovic, T.; Sekler, M.; Petric, D.; Lazic, S.; Debeljak, Z.; Vidanovic, D.; Ignjatovic Cupina, A.; Lazic, G.; Lupulovic, D.; Kolarevic, M.; et al. Methodology and results of integrated WNV surveillance programmes in Serbia. PLoS ONE 2018, 13, e0195439. [Google Scholar] [CrossRef] [PubMed]
  48. Dinu, S.; Stancu, I.G.; Cotar, A.I.; Ceianu, C.S.; Pintilie, G.V.; Karpathakis, I.; Falcuta, E.; Csutak, O.; Prioteasa, F.L. Continuous and Dynamic Circulation of West Nile Virus in Mosquito Populations in Bucharest Area, Romania, 2017–2023. Microorganisms 2024, 12, 2080. [Google Scholar] [CrossRef]
  49. Papa, A.; Papadopoulou, E.; Chatzixanthouliou, C.; Glouftsios, P.; Pappa, S.; Pervanidou, D.; Georgiou, L. Emergence of West Nile virus lineage 2 belonging to the Eastern European subclade, Greece. Arch. Virol. 2019, 164, 1673–1675. [Google Scholar] [CrossRef]
  50. Chaintoutis, S.C.; Papa, A.; Pervanidou, D.; Dovas, C.I. Evolutionary dynamics of lineage 2 West Nile virus in Europe, 2004–2018: Phylogeny, selection pressure and phylogeography. Mol. Phylogenet. Evol. 2019, 141, 106617. [Google Scholar] [CrossRef]
  51. Zecchin, B.; Fusaro, A.; Milani, A.; Schivo, A.; Ravagnan, S.; Ormelli, S.; Mavian, C.; Michelutti, A.; Toniolo, F.; Barzon, L.; et al. The central role of Italy in the spatial spread of USUTU virus in Europe. Virus Evol. 2021, 7, veab048. [Google Scholar] [CrossRef] [PubMed]
  52. Constant, O.; Gil, P.; Barthelemy, J.; Bollore, K.; Foulongne, V.; Desmetz, C.; Leblond, A.; Desjardins, I.; Pradier, S.; Joulie, A.; et al. One Health surveillance of West Nile and Usutu viruses: A repeated cross-sectional study exploring seroprevalence and endemicity in Southern France, 2016 to 2020. Eurosurveillance 2022, 27, 2200068. [Google Scholar] [CrossRef]
  53. Honig, V.; Palus, M.; Kaspar, T.; Zemanova, M.; Majerova, K.; Hofmannova, L.; Papezik, P.; Sikutova, S.; Rettich, F.; Hubalek, Z.; et al. Multiple Lineages of Usutu Virus (Flaviviridae, Flavivirus) in Blackbirds (Turdus merula) and Mosquitoes (Culex pipiens, Cx. modestus) in the Czech Republic (2016–2019). Microorganisms 2019, 7, 568. [Google Scholar] [CrossRef]
  54. Camp, J.V.; Kolodziejek, J.; Nowotny, N. Targeted surveillance reveals native and invasive mosquito species infected with Usutu virus. Parasit. Vectors 2019, 12, 46. [Google Scholar] [CrossRef]
  55. Klobucar, A.; Savic, V.; Curman Posavec, M.; Petrinic, S.; Kuhar, U.; Toplak, I.; Madic, J.; Vilibic-Cavlek, T. Screening of Mosquitoes for West Nile Virus and Usutu Virus in Croatia, 2015–2020. Trop. Med. Infect. Dis. 2021, 6, 45. [Google Scholar] [CrossRef]
  56. Cabanova, V.; Ticha, E.; Bradbury, R.S.; Zubrikova, D.; Valentova, D.; Chovancova, G.; Gresakova, L.; Vichova, B.; Sikutova, S.; Csank, T.; et al. Mosquito surveillance of West Nile and Usutu viruses in four territorial units of Slovakia and description of a confirmed autochthonous human case of West Nile fever, 2018 to 2019. Eurosurveillance 2021, 26, 2000063. [Google Scholar] [CrossRef]
  57. Gobbo, F.; Chiarello, G.; Sgubin, S.; Toniolo, F.; Gradoni, F.; Danca, L.I.; Carlin, S.; Capello, K.; De Conti, G.; Bortolami, A.; et al. Integrated One Health Surveillance of West Nile Virus and Usutu Virus in the Veneto Region, Northeastern Italy, from 2022 to 2023. Pathogens 2025, 14, 227. [Google Scholar] [CrossRef]
  58. Adams, M.J.; Lefkowitz, E.J.; King, A.M.Q.; Harrach, B.; Harrison, R.L.; Knowles, N.J.; Kropinski, A.M.; Krupovic, M.; Kuhn, J.H.; Mushegian, A.R.; et al. Changes to taxonomy and the International Code of Virus Classification and Nomenclature ratified by the International Committee on Taxonomy of Viruses (2017). Arch. Virol. 2017, 162, 2505–2538. [Google Scholar] [CrossRef]
  59. Kuhn, J.H.; Brown, K.; Adkins, S.; de la Torre, J.C.; Digiaro, M.; Ergunay, K.; Firth, A.E.; Hughes, H.R.; Junglen, S.; Lambert, A.J.; et al. Promotion of order Bunyavirales to class Bunyaviricetes to accommodate a rapidly increasing number of related polyploviricotine viruses. J. Virol. 2024, 98, e0106924. [Google Scholar] [CrossRef] [PubMed]
  60. Carvalho, V.L.; Long, M.T. Insect-Specific Viruses: An overview and their relationship to arboviruses of concern to humans and animals. Virology 2021, 557, 34–43. [Google Scholar] [CrossRef] [PubMed]
  61. Horigan, V.; Kelly, L.; Papa, A.; Koopmans, M.P.G.; Sikkema, R.S.; Koren, L.G.H.; Snary, E.L. Assessing the quality of data for drivers of disease emergence. Rev. Sci. Tech. 2023, 42, 90–102. [Google Scholar] [CrossRef] [PubMed]
Viruses 17 01414 g001
Figure 1. Map of Greece with the sites where WNV-positive and -negative C. pipiens mosquitoes were trapped in four Regions of Greece from May to October 2024.
Figure 1. Map of Greece with the sites where WNV-positive and -negative C. pipiens mosquitoes were trapped in four Regions of Greece from May to October 2024.
Viruses 17 01414 g001
Viruses 17 01414 g002
Figure 2. Maximum Likelihood phylogenetic tree based on whole-genome consensus sequences encoding the West Nile virus polyprotein (10,302 nucleotides). The percentage of replicate trees in which the associated taxa clustered together (100 replicates) is shown below the branches; only values > 75% are shown. The evolutionary rate differences among sites were modeled using a discrete Gamma distribution across 5 categories (+G, parameter = 1.2424), with 52.88% of sites deemed evolutionarily invariant (+I). The sequences of the present study are marked with a black circle. Sequences from Greece are shown as accession number, regional unit, and year of detection; sequences from other countries are shown in capital letters as accession number, country, and year of detection.
Figure 2. Maximum Likelihood phylogenetic tree based on whole-genome consensus sequences encoding the West Nile virus polyprotein (10,302 nucleotides). The percentage of replicate trees in which the associated taxa clustered together (100 replicates) is shown below the branches; only values > 75% are shown. The evolutionary rate differences among sites were modeled using a discrete Gamma distribution across 5 categories (+G, parameter = 1.2424), with 52.88% of sites deemed evolutionarily invariant (+I). The sequences of the present study are marked with a black circle. Sequences from Greece are shown as accession number, regional unit, and year of detection; sequences from other countries are shown in capital letters as accession number, country, and year of detection.
Viruses 17 01414 g002
Viruses 17 01414 g003
Figure 3. Maximum Likelihood phylogenetic tree based on a 212-bp fragment of USUV polyprotein gene. The tree was constructed using the Kimura-2 parameter model. The percentage of replicate trees in which the associated taxa clustered together (100 replicates) is shown below the branches; only values > 75% are shown. The sequence of the present study is marked with a black circle. Sequences are shown as accession number, country, and year of detection.
Figure 3. Maximum Likelihood phylogenetic tree based on a 212-bp fragment of USUV polyprotein gene. The tree was constructed using the Kimura-2 parameter model. The percentage of replicate trees in which the associated taxa clustered together (100 replicates) is shown below the branches; only values > 75% are shown. The sequence of the present study is marked with a black circle. Sequences are shown as accession number, country, and year of detection.
Viruses 17 01414 g003
Viruses 17 01414 g004
Figure 4. Maximum Likelihood phylogenetic tree based on 160 amino acid sequences of the viral-encoded RNA polymerase of bunyaviruses. The percentage of replicate trees in which the associated taxa clustered together (100 replicates) is shown below the branches; only values >75% are shown. The evolutionary rate differences among sites were modeled using a discrete Gamma distribution across 5 categories (+G, parameter = 1.2600), with 8.02% of sites deemed evolutionarily invariant (+I). The sequences of the present study are marked with a black circle. Sequences are shown as accession number, virus, country and year of detection; sequences from other countries are shown in capital letters as accession number, country, and year of detection.
Figure 4. Maximum Likelihood phylogenetic tree based on 160 amino acid sequences of the viral-encoded RNA polymerase of bunyaviruses. The percentage of replicate trees in which the associated taxa clustered together (100 replicates) is shown below the branches; only values >75% are shown. The evolutionary rate differences among sites were modeled using a discrete Gamma distribution across 5 categories (+G, parameter = 1.2600), with 8.02% of sites deemed evolutionarily invariant (+I). The sequences of the present study are marked with a black circle. Sequences are shown as accession number, virus, country and year of detection; sequences from other countries are shown in capital letters as accession number, country, and year of detection.
Viruses 17 01414 g004
Table 1. Detection of West Nile virus in Culex pipiens s.l. mosquitoes per month and regional unit in the Regions of Central Macedonia (CM), West Macedonia (WM), Thessaly (TH), and West Greece (WG), 2024. P: number of C. pipiens s.l. pools; M: number of C. pipiens s.l.
Table 1. Detection of West Nile virus in Culex pipiens s.l. mosquitoes per month and regional unit in the Regions of Central Macedonia (CM), West Macedonia (WM), Thessaly (TH), and West Greece (WG), 2024. P: number of C. pipiens s.l. pools; M: number of C. pipiens s.l.
Regional UnitMayJuneJulyAugustSeptemberOctoberTotal
MPPos P (%)MPPos P (%)MPPos P (%)MPPos P (%)MPPos P (%)MPPos P (%)MPPos P (%)
Imathia23790 (0)572170 (0)830221 (4.55)602161 (6.25)880170 (0)000 (0)3121812 (2.47)
Thessaloniki1383430 (0)1944620 (0)3162977 (7.22)2180671 (1.49)2564694 (5.80)000 (0)11,23333812 (3.55)
Kilkis12050 (0)301100 (0)357130 (0)19491 (11.11)240110 (0)000 (0)1212481 (2.08)
Pella25160 (0)41690 (0)710130 (0)968140 (0)948152 (13.33)000 (0)3293572 (3.51)
Pieria10350 (0)18590 (0)498192 (10.53)620162 (12.50)1160191 (5.26)000 (0)2566685 (7.35)
Serres216120 (0)383130 (0)869210 (0)526162 (12.50)577172 (11.76)000 (0)2571794 (5.06)
Chalkidiki20290 (0)315110 (0)539160 (0)582140 (0)717160 (0)000 (0)2355660 (0)
Subtotal CM2512890 (0)41161310 (0)696520110 (4.98)56721527 (4.61)70861649 (5.49)000 (0)26,35173726 (3.53)
Grevena000 (0)1410 (0)1110 (0)000 (0)000 (0)000 (0)2520 (0)
Kastoria000 (0)1010 (0)2720 (0)1010 (0)1410 (0)000 (0)6150 (0)
Kozani5020 (0)5310 (0)5330 (0)5030 (0)7040 (0)000 (0)276130 (0)
Florina000 (0)2720 (0)28170 (0)33570 (0)12350 (0)000 (0)766210 (0)
Subtotal WM5020 (0)10450 (0)372130 (0)395110 (0)207100 (0)000 (0)1128410 (0)
Karditsa8060 (0)23080 (0)14160 (0)5010 (0)5010 (0)000 (0)551220 (0)
Larisa1107272 (7.41)1046240 (0)3704595 (8.47)1951410 (0)1765332 (6.06)8620 (0)96591869 (4.84)
Magnisia12430 (0)4520 (0)8770 (0)12950 (0)330121 (8.33)15470 (0)869361 (2.78)
Sporades000 (0)000 (0)8130 (0)2620 (0)1920 (0)000 (0)12670 (0)
Subtotal TH1311362 (5.56)1321340 (0)4013755 (6.67)2156490 (0)2164483 (6.25)24090 (0)11,20525110 (3.98)
Aitoloak/nia371130 (0)339130 (0)145110 (0)78100 (0)221180 (0)12590 (0)1279740 (0)
Axaia446160 (0)482140 (0)1251393 (7.69)458280 (0)877341 (2.94)663200 (0)41771514 (2.65)
Ileia26560 (0)17660 (0)549151 (6.67)297100 (0)355170 (0)20680 (0)1848621 (1.61)
Subtotal WG1082350 (0)997330 (0)1945654 (6.15)833480 (0)1453691 (1.45)994370 (0)73042875 (1.74)
Total49551622 (1.23)65382030 (0)13,29535419 (5.37)90562607 (2.69)10,91029113 (4.47)1234460 (0)45,988131641 (3.12)
Table 2. Location and collection date of the WNV-positive mosquito pools detected in the present study.
Table 2. Location and collection date of the WNV-positive mosquito pools detected in the present study.
Pool IDCollection DateRegional UnitRegion
Z32417 July 2024ImathiaCentral Macedonia
Z49712 August 2024ImathiaCentral Macedonia
Z46407 August 2024KilkisCentral Macedonia
Z61802 September 2024PellaCentral Macedonia
A30516 September 2024PellaCentral Macedonia
A16815 July 2024PieriaCentral Macedonia
A21729 July 2024Pieria *Central Macedonia
A25109 August 2024Pieria *Central Macedonia
A26009 August 2024PieriaCentral Macedonia
A31109 September 2024Pieria *Central Macedonia
Z468-A07 August 2024SerresCentral Macedonia
Z468-B07 August 2024SerresCentral Macedonia
Z61304 September 2024SerresCentral Macedonia
Z64304 September 2024SerresCentral Macedonia
Z33215 July 2024ThessalonikiCentral Macedonia
Z35515 July 2024ThessalonikiCentral Macedonia
Z37024 July 2024ThessalonikiCentral Macedonia
Z37324 July 2024ThessalonikiCentral Macedonia
Z36624 July 2024ThessalonikiCentral Macedonia
Z41731 July 2024ThessalonikiCentral Macedonia
Z44031 July 2024ThessalonikiCentral Macedonia
A31628 August 2024ThessalonikiCentral Macedonia
Z62704 September 2024ThessalonikiCentral Macedonia
Z63904 September 2024ThessalonikiCentral Macedonia
Z61104 September 2024ThessalonikiCentral Macedonia
Z66811 September 2024ThessalonikiCentral Macedonia
A1115 May 2024LarissaThessaly
A1615 May 2024LarissaThessaly
A11903 July 2024LarissaThessaly
A15305 July 2024LarissaThessaly
A18117 July 2024LarissaThessaly
Z38024 July 2024LarissaThessaly
A18324 July 2024LarissaThessaly
Z62304 September 2024LarissaThessaly
A31504 September 2024LarissaThessaly
A32711 September 2024MagnesiaThessaly
Z252-A01 July 2024AchaiaWest Greece
Z252-B01 July 2024AchaiaWest Greece
Z252-C01 July 2024AchaiaWest Greece
Z61704 September 2024AchaiaWest Greece
A15103 September 2024IliaWest Greece
* Entries with an asterisk are referred to one specific location where WNV positive pools were serially detected.
Table 3. Infection rates (IRs) of mosquito pools tested by our group in Central Macedonia (CM) Region, Greece, 2010–2023. The number of human WNND cases from Central Macedonia reported to the National Public Health Organization are also shown [43].
Table 3. Infection rates (IRs) of mosquito pools tested by our group in Central Macedonia (CM) Region, Greece, 2010–2023. The number of human WNND cases from Central Macedonia reported to the National Public Health Organization are also shown [43].
YearPositive Pools/Pools Tested (IR)ReferenceHuman WNND Cases in CM
20103/224 (1.34)[22]186
20112/53 (3.77)[22]23
20120/100 (0) 15
20139/295 (3.1)[23]13
20140/207 (0) 0
20150/438 (0) 0
20160/62 (0) 0
20170/55 (0) 0
201810/229 (4.4)[25]78
20195/346 (1.5)[31]37
202013/362 (3.6)[31]76
202137/391 (9.6)[31]31
202241/ 690 (5.9)[42]148
202319/736 (2.58) 55
202426/737 (3.53)current study60
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Tsioka, K.; Stoikou, K.; Antalis, V.; Charizani, E.; Pappa, S.; Gewehr, S.; Kalaitzopoulou, S.; Mourelatos, S.; Papa, A. Detection of West Nile Virus, Usutu Virus and Insect-Specific Bunyaviruses in Culex spp. Mosquitoes, Greece, 2024. Viruses 2025, 17, 1414. https://doi.org/10.3390/v17111414

AMA Style

Tsioka K, Stoikou K, Antalis V, Charizani E, Pappa S, Gewehr S, Kalaitzopoulou S, Mourelatos S, Papa A. Detection of West Nile Virus, Usutu Virus and Insect-Specific Bunyaviruses in Culex spp. Mosquitoes, Greece, 2024. Viruses. 2025; 17(11):1414. https://doi.org/10.3390/v17111414

Chicago/Turabian Style

Tsioka, Katerina, Konstantina Stoikou, Vasilis Antalis, Elissavet Charizani, Styliani Pappa, Sandra Gewehr, Stella Kalaitzopoulou, Spiros Mourelatos, and Anna Papa. 2025. "Detection of West Nile Virus, Usutu Virus and Insect-Specific Bunyaviruses in Culex spp. Mosquitoes, Greece, 2024" Viruses 17, no. 11: 1414. https://doi.org/10.3390/v17111414

APA Style

Tsioka, K., Stoikou, K., Antalis, V., Charizani, E., Pappa, S., Gewehr, S., Kalaitzopoulou, S., Mourelatos, S., & Papa, A. (2025). Detection of West Nile Virus, Usutu Virus and Insect-Specific Bunyaviruses in Culex spp. Mosquitoes, Greece, 2024. Viruses, 17(11), 1414. https://doi.org/10.3390/v17111414

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop