Assessing vector competence of mosquitoes from northeastern France to West Nile virus and Usutu virus

Jean-Philippe Martinet; Chloé Bohers; Marie Vazeille; Hubert Ferté; Laurence Mousson; Bruno Mathieu; Jérôme Depaquit; Anna-Bella Failloux

doi:10.1371/journal.pntd.0011144

Abstract

West Nile virus (WNV) and Usutu virus (USUV) are two arthropod-borne viruses that circulate in mainland France. Assessing vector competence has only been conducted so far with mosquitoes from southern France while an increasingly active circulation of WNV and USUV has been reported in the last years. The main vectors are mosquitoes of the Culex genus and the common mosquito Culex pipiens. Here, we measure the vector competence of five mosquito species (Aedes rusticus, Aedes albopictus, Anopheles plumbeus, Culex pipiens and Culiseta longiareolata) present in northeastern France. Field-collected populations were exposed to artificial infectious blood meal containing WNV or USUV and examined at different days post-infection. We show that (i) Cx. pipiens transmitted WNV and USUV, (ii) Ae. rusticus only WNV, and (iii) unexpectedly, Ae. albopictus transmitted both WNV and USUV. Less surprising, An. plumbeus was not competent for both viruses. Combined with data on distribution and population dynamics, these assessments of vector competence will help in developing a risk map and implementing appropriate prevention and control measures.

Author summary

West Nile virus (WNV) and Usutu virus (USUV) are on the rise in Europe and in France. WNV is reported in France as early as the 1960s in the Camargue and USUV more recently, in 2015 in eastern France. The re-emergence of WNV infections in the Camargue is associated with an expansion towards the North which is also favorable to maintain a viral transmission cycle. USUV frequently co-circulates with WNV sharing the same mosquito vectors. Culex pipiens, able to feed on birds and humans, is considered to be the main vector in France. Our study is the first to investigate the vector competence to WNV and USUV of five different mosquito species collected in northeastern France. We ascertain that French Cx. pipiens mosquitoes are competent to both WNV and USUV. More surprisingly, the mosquito Aedes albopictus from northeastern France was able to transmit WNV and USUV. Based on our result, we propose that surveillance of mosquitoes combined with viral detections must be implemented in northeastern France to allow early viral detection and timely intervention to prevent outbreaks of these two neurological diseases.

Citation: Martinet J-P, Bohers C, Vazeille M, Ferté H, Mousson L, Mathieu B, et al. (2023) Assessing vector competence of mosquitoes from northeastern France to West Nile virus and Usutu virus. PLoS Negl Trop Dis 17(6): e0011144. https://doi.org/10.1371/journal.pntd.0011144

Editor: Michael R. Holbrook, NIAID Integrated Research Facility, UNITED STATES

Received: February 3, 2023; Accepted: May 6, 2023; Published: June 5, 2023

Copyright: © 2023 Martinet et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: This study was funded by the ANSES APR-EST Grant N° 2020/01/129 (JPM, JD, BM, ABF) and the Laboratoire d'Excellence "Integrative Biology of Emerging Infectious Diseases" (grant n°ANR-10-LABX-62-IBEID) (ABF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Since 2007, Europe has been facing an increase in local transmission of arboviral diseases with dengue virus (DENV), chikungunya virus (CHIKV), and Zika virus (ZIKV) transmitted by the invasive species Aedes albopictus in a human-to-human transmission cycle [1]. Usutu virus (USUV) and West Nile virus (WNV) both belonging to the Japanese encephalitis (JE) serocomplex circulate primarily in an avian-mosquito cycle and are transmitted by Culex mosquitoes [2]. Mammals (human, horses) can be infected but develop a low viremia, insufficient to infect mosquitoes; they are considered as dead-end hosts [3]. WNV is regarded as the most important causative agent of viral encephalitis worldwide [4]. Among the multiple WNV genotypes described up to date, lineages 1 and 2 have been associated with outbreaks in humans [5]. On the other hand, USUV described under six lineages [6] circulates mainly among bird populations (mainly Passeriformes and Strigiformes) and human cases are rare. Both viruses are transmitted by Culex mosquitoes [7–9] which are ubiquitous in mainland France and use a wide range of bird species as amplifying hosts.

In mainland France, WNV first emerged in 1962 [10] and was first isolated in Culex modestus mosquitoes in 1964 [11]. USUV emerged in France in 2015 due to two different lineages originating from Germany and Spain [8]; it was first isolated from blackbirds (Turdus merula) and Culex spp. mosquitoes (e.g. Culex neavei, Culex pipiens, Culex perexiguus, and Culex perfuscus) [8,9]. While WNV causes outbreaks with severe neurological symptoms, only two cases of neuroinvasive USUV infections have been reported in humans [12,13]. While the distribution and population dynamics of Culex mosquitoes are well documented, data on vector competence of French mosquitoes for WNV and USUV are incomplete. We know that Culex modestus and Cx. pipiens from the Camargue are competent for WNV [14,15] and no information is available for USUV. Here, we performed a vector competence analysis of mosquitoes (Aedes rusticus, Ae. albopictus, Anopheles plumbeus, Cx. pipiens, and Culiseta longiareolata) collected in northeastern France (inside a region bounded by Paris, Reims and Strasbourg) using experimental infections with WNV and USUV.

Materials and methods

Ethics statements

Animals were housed in the Institut Pasteur animal facilities accredited by the French Ministry of Agriculture for performing experiments on live rodents. Work on animals was performed in compliance with French and European regulations on care and protection of laboratory animals (EC Directive 2010/63, French Law 2013–118, February 6th, 2013). All experiments were approved by the Ethics Committee for animal experiments of the Institut Pasteur (#89) and registered under the reference APAFIS#6573-201606l412077987 v2.

Mosquito collections

Aedes albopictus Strasbourg were sampled from June to October 2022 using 31 ovitraps; 5782 eggs were collected. Aedes rusticus, An. plumbeus, Cx. pipiens and Cu. longiareolata were collected as immature stages in 2019–2022 (Table 1). Geographical distribution of mosquito sampling sites is detailed in Fig 1. The map was generated with RStudio v1.4.1103 (in combination with ggplot2 and ggspatial packages) [16–18]. Eggs were immersed in water for hatching. Larvae and pupae were placed in pans containing 1 liter of dechlorinated water and a yeast tablet renewed every 2 days and maintained at 25±1°C. Pupae were collected in bowls placed in cages where adults emerged. Adults were fed with a 10% sucrose solution and kept at 28±1°C with a 12L:12D cycle and 80% relative humidity.

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Fig 1. Map showing the different mosquito collections sites (red triangles) in northeastern France during the period 2019–2022.

The map was generated with RStudio v1.4.1103 (in combination with ggplot2 and ggspatial packages). The data are plotted on naturalearth package v 0.1.0 shapefile belonging to the public domain (naturalearthdata.com).

https://doi.org/10.1371/journal.pntd.0011144.g001

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Table 1. Mosquito species and populations collected in 2019–21 for assessing vector competence to WNV and USUV.

https://doi.org/10.1371/journal.pntd.0011144.t001

Viral strains

The WNV strain (lineage 1a) was isolated from a horse in Camargue (France) in 2000 [10]. After 4 passages on Vero cells, the WNV stock was produced on Ae. albopictus C6/36 cells [19]. The USUV Europa 3 strain (10214) was isolated in 2015 on Vero cells from the brain of a blackbird [8]. All viral stocks were then produced on C6/36 cells and stored at -80°C until use.

Mosquito infections and processing

Batches of 60 7-10-day-old females were transferred from cages into boxes and exposed to an infectious blood meal at a titer of 10^6.7 plaque-forming unit (pfu)/mL for WNV and 10⁷ tissue culture infectious dose 50% (TCID₅₀)/mL for USUV. The infectious blood meal containing 1.4 mL of washed rabbit erythrocytes, 700 μL of viral suspension and ATP at 1 mM as a phagostimulant, was put in a capsule covered with a pork intestine as membrane. This Hemotek feeding system was maintained at 37°C. After 30 min of feeding, engorged mosquitoes were transferred in cardboard containers and supplied with 10% sucrose. Mosquitoes were maintained under controlled conditions (28±1°C, relative humidity of 80%, 12L:12D cycle) until examination. Mosquitoes were examined at different days post-infection (dpi) from 3 to 28 days depending on the number of fed mosquitoes and the mortalities observed after infection.

At a given dpi, surviving mosquitoes were cold anesthetized on ice. Then, legs and wings of each mosquito were removed and the proboscis was inserted into a pipette tip containing 5 μL of fetal bovine serum (FBS). After 30 min, the tip content was retrieved in 45 μL of L15 medium (Invitrogen, CA, USA). Then, the head was isolated from abdomen and thorax. These two samples (head and thorax+abdomen corresponding to body) were separately ground in 300 μL of L15 supplemented with 2% FBS (Eurobio Scientific, Les Ulis, France), and centrifuged at 10,000×g for 5 min at +4°C. Body (containing the midgut), head (possibly infected with viruses having disseminated from the midgut) were tested respectively for infection and dissemination while saliva was titrated to estimate transmission.

Vector competence indices

To measure the vector competence, we used three indices which measured the role of the two main anatomical barriers in the progression of the virus in the mosquito after the infectious blood meal: (i) infection rate (IR) corresponding to the proportion of mosquitoes with infected midgut among mosquitoes exposed to the blood meal, (ii) dissemination rate (DR) referring to the proportion of mosquitoes having succeeded in disseminating the virus inside the mosquito general cavity among mosquitoes with infected midgut, and (iii) transmission rate (TR) which measures the proportion of mosquitoes with infectious saliva among mosquitoes having disseminated the virus. DR measures the efficiency of the midgut as a barrier to the dissemination of the virus inside the hemocele; the higher DR is, the less the midgut acts as a barrier to the dissemination of the virus. In addition to DR, TR measures the efficiency of the salivary glands as a barrier to the excretion of the virus in the saliva; as DR, the higher TR is, the less the salivary glands play the role of barrier to the transmission of the virus.

Viral titration

Samples of saliva and extracts from bodies and heads of mosquitoes infected by WNV were titrated on Vero cells. Six-well plates containing confluent monolayers of Vero cells were inoculated with serial 10-fold dilutions of samples and incubated for 1 h at 37°C. Cells were then covered with an overlay consisting of DMEM (Gibco, CA, USA), 2% FBS, 1% antibiotic-antimycotic mix (Invitrogen, Gibco) and 1% agarose and incubated at 37°C. Cells were incubated 5 days. Lytic plaques were then counted after staining with a solution of safranine (0.5% in 10% formaldehyde and 20% ethanol). For mosquitoes infected by USUV, serial dilutions of saliva were inoculated on C6/36 cells in 96-well plates; each well was inoculated with 50 μL of diluted samples for one hour at 28°C and after removing the inoculum, cells were covered with 150 μL of carboxymethylcellulose (CMC) supplemented with L-15 medium. In the case of low viral load in saliva, the sample was not diluted before inoculation. After incubation at 28°C for 5 days, cells were fixed with 3.6% formaldehyde, washed and hybridized with anti-flavivirus monoclonal antibody (catalog number: MAB10216, Millipore, CA, USA), and revealed by using a fluorescent-conjugated secondary antibody (catalog number: A-11029, Life Technologies, CA, USA), with dilution factors 1:200 and 1:1000, respectively. Foci were counted under a fluorescent microscope and titers were expressed as focus forming units (ffu)/sample.

Statistical analysis

IR, DR and TR were compared using Fisher’s exact test and viral loads using Kruskal-Wallis test. Statistical analyses were conducted using the Stata software (StataCorp LP, Texas, USA). p-values < 0.05 were considered significant.

Results

Some populations of Culex pipiens are able to transmit WNV and USUV

To ascertain that Cx. pipiens mosquitoes were susceptible to WNV, we examined three populations for infection, dissemination and transmission at different dpi (Fig 2A). We analyzed a total of 153 mosquitoes: 64 from Machault, 51 from Maine, and 38 from Verzy.

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Fig 2.

Culex pipiens (Machault, Maine and Verzy) experimentally infected with WNV provided at a titer of 10^6.7 pfu/mL (A) and examined for infection (B), dissemination (C) and transmission, (D) at different days post-infection together with viral loads in body (E), head (F) and saliva (G). Infection rate refers to the proportion of mosquitoes with body infected among examined mosquitoes. Dissemination rate corresponds to mosquitoes with head infected (indicating a successful viral dissemination beyond the midgut barrier) among mosquitoes with infected body. Transmission rate indicates the proportion of mosquitoes presenting viral particles in saliva among mosquitoes with infected head. Each point represents a mosquito. The color of the point indicates the day post-infection (left panel). Horizontal bars represent the mean of the log₁₀ of viral particles in each compartment. Stars indicate statistical significance of comparisons: *p≤0.05, **0.001≤p≤0.01. The drawings were created with Microsoft PowerPoint.

https://doi.org/10.1371/journal.pntd.0011144.g002

When examining IR reflecting the success of midgut infection, we found that IRs were significantly higher at 10 dpi (77.8%, N = 27 mosquitoes examined) than at 14 dpi (51.3%, N = 37) for Machault (Fisher’s exact test: p = 0.03). For Maine, IRs were significantly different at 3 dpi (44.4%, N = 18), 7 dpi (66.7%, N = 15), and 10 dpi (22.2%, N = 18) (Fisher’s exact test: p = 0.04). And, for Verzy, IRs were low: 0% (N = 14) at 7 dpi and 8.3% (N = 24) at 14 dpi (Fig 2B). When measuring the viral load in bodies, mean values were similar at 10 dpi (10^6.7 viral particles, N = 21) and 14 dpi (10^6.6, N = 19) for Machault (Kruskal-Wallis test: p = 0.22). For Maine, mean values significantly increased over dpi (Kruskal-Wallis test: p = 0.008): 10^3.2 (N = 8) at 3 dpi, 10^4.4 (N = 10) at 7 dpi, and 10^5.2 (N = 4) at 10 dpi. For Verzy, the mean value was 10^5.3 (N = 2) at 14 dpi (Fig 2E). When measuring DR translating the success of virus dissemination into the head, we found that DRs were similar at 10 dpi (33.3%, N = 21) and 14 dpi (36.8%, N = 19) for Machault (Fisher’s exact test: p = 0.82). For Maine and Verzy, DRs were equal to 0% at all dpi examined (Fig 2C). When assessing the viral load in heads, mean values were similar at 10 dpi (10^6.2, N = 6) and 14 dpi (10^6.1, N = 7) for Machault (Kruskal-Wallis test: p = 0.67). For Maine and Verzy, no viral particles were detected (Fig 2F). When estimating TR describing the success of virus transmission, we detected that TRs were comparable between 10 dpi (14.3%, N = 7) and 14 dpi (42.9%, N = 7) for Machault (Fisher’s exact test: p = 0.24). For Maine and Verzy, TRs were equal to 0% at all dpi examined (Fig 2D). When estimating the viral load in saliva, mean values were similar at 10 dpi (10^5.3, N = 1) and 14 dpi (10^4.8, N = 3) for Machault. For Maine and Verzy, no viral particles were detected (Fig 2G).

To define whether Cx. pipiens was susceptible to USUV, we examined a total of 73 mosquitoes from Sainte-Croix at 7, 14, and 21 dpi. We found that IR was equal to 0% at 7 dpi (N = 27) and 21 dpi (N = 23) resulting in DR = 0% and TR = 0%. At 14 dpi, IR value was 4.3% (N = 23) with one mosquito hosting 10^5.1 viral particles in the body; this infected mosquito was able to disseminate (DR = 100%) and to transmit (TR = 100%) with 14 viral particles in the saliva.

Anopheles plumbeus is able to become infected but not to transmit WNV and USUV

To verify that as expected, Anopheles mosquitoes are not susceptible to WNV and USUV, we examined 46 An. plumbeus exposed to WNV and 60 to USUV.

For WNV, when examining IR, we found that IRs were comparable at 7 dpi (23.8%, N = 21), 14 dpi (31.8%, N = 22), and 21 dpi (23.5%, N = 17) (Fisher’s exact test: p = 0.79; Fig 3A). The mean values of viral load in bodies were similar at 7 dpi (10^6.6, N = 5), 14 dpi (10^6.3, N = 7) and 21 dpi (10^6.4, N = 4) (Kruskal-Wallis test: p = 0.79; Fig 3D). When measuring DR, we found that DR value was equal to 0% at 7 dpi (N = 5) and values were similar between 14 dpi (14.3%, N = 7) and 21 dpi (50%, N = 4) (Fisher’s exact test: p = 0.40; Fig 3B). The mean values of viral load in heads were similar between 14 dpi (10^5.0, N = 1) and 21 dpi (10^6.2, N = 2) (Fig 3E). When estimating TR, we found that TRs were equal to 0% (Fig 3C) and no viral particles were detected (Fig 3F).

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Fig 3.

Anopheles plumbeus (Beaumont) experimentally infected with WNV provided at a titer of 10^6.7 pfu/mL and examined for infection (A), dissemination (B) and transmission (C) at different days post-infection (7,14, and 21) together with viral loads in body (D), head (E) and saliva (F).

https://doi.org/10.1371/journal.pntd.0011144.g003

For USUV, infection of bodies was only detected at 14 dpi (IR = 4.35%, N = 23) while IR was 0% at 7 dpi (N = 23). No dissemination (DR = 0%) or transmission (TR = 0%) was detected.

Aedes rusticus is able to transmit WNV but not USUV

To search for secondary vectors of WNV and USUV beside the main vector Cx. pipiens, we examined 30 Ae. rusticus mosquitoes from Berru exposed to WNV and 51 mosquitoes from Berru and 45 from Verzy exposed to USUV. For WNV, we found that at 7 dpi, IR was 50% (N = 30), DR of 33.33% (N = 15), and TR of 40% (N = 5) (Fig 4A). The mean numbers of viral particles were 10^7.05 viral particles in bodies (N = 15), 10^6.6 in heads (N = 5), and 37 in saliva (N = 2) (Fig 4B). For USUV, infection (IR = 0%), dissemination (DR = 0%), and transmission (TR = 0%) were not detected at 4, 7, and 10 dpi for Berru and Verzy populations.

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Fig 4.

Aedes rusticus (Berru) experimentally infected with WNV provided at a titer of 10^6.7 pfu/mL and examined at 7 days post-infection for infection, dissemination and transmission (A), and viral loads in body, head and saliva (B).

https://doi.org/10.1371/journal.pntd.0011144.g004

Aedes albopictus transmits WNV and to a lesser extent, USUV

To see if this invasive mosquito could transmit Culex-borne viruses such as WNV and USUV, we examined 156 Ae. albopictus mosquitoes exposed to WNV and 150 to USUV.

For WNV, when examining IR, we found that IRs were similar at 3 dpi (20.8%, N = 24), 7 dpi (25%, N = 24), 10 dpi (29.2%, N = 24), 14 dpi (29.2%, N = 24), 17 dpi (54.2%, N = 24), 21 dpi (41.7%, N = 24), and 28 dpi (8.3%, N = 12) (Fisher’s exact test: p = 0.07; Fig 5A). Mean values of viral load in bodies were comparable at 3 dpi (10^4.07, N = 5), 7 dpi (10^5.6, N = 6), 10 dpi (10^7.2, N = 7), 14 dpi (10^7.8, N = 7), 17 dpi (10^7.5, N = 13), 21 dpi (10^8.0, N = 10), and 28 dpi (10^7.3, N = 1) (Kruskal-Wallis test: p = 0.22; Fig 5D). When measuring DR, we found that DR was equal to 0% (N = 5) at 3 dpi, increased to 33.33% (N = 6) at 7 dpi, 100% (N = 7) at 10 dpi, 100% (N = 7) at 14 dpi, and dropped to 61.5% (N = 13) at 17 dpi et 80% (N = 10) at 21 dpi (Fisher’s exact test: p = 0.001; Fig 5B). Mean values of viral load in heads were similar at 7 dpi (10^4.5, N = 2), 10 dpi (10^6.5, N = 7), 14 dpi (10^7.3, N = 7), 17 dpi (10^5.9, N = 8), 21 dpi (10^5.3, N = 8), and 28 dpi (10^5.8, N = 1) (Kruskal-Wallis test: p = 0.65; Fig 5E). When estimating TR, we found that TRs were equal to 0% (N = 2) at 7 dpi, 42.8% (N = 7) at 10 dpi, 100% (N = 7) at 14dpi, 62.5% (N = 8) at 17 dpi, 75% (N = 8) at 21 dpi, and 100% (N = 1) at 28 dpi (Fisher’s exact test: p = 0.072; Fig 5C). Mean values of viral load in saliva were similar at 10 dpi (10^2.9, N = 3), 14 dpi (10^2.3, N = 7), 17 dpi (10^3.3, N = 5), 21 dpi (10^3.3, N = 6), and 28 dpi (10^1.5, N = 1) (Kruskal-Wallis test: p = 0.52; Fig 5F).

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Fig 5.

Aedes albopictus (Strasbourg) experimentally infected with WNV provided at a titer of 10^6.7 pfu/mL and examined for infection (A), dissemination (B) and transmission (C) at different days post-infection (3,7,10,14,17,21, and 28) together with viral loads in body (D), head (E) and saliva (F). Stars indicate statistical significance of comparisons: **0.001≤p≤0.01.

https://doi.org/10.1371/journal.pntd.0011144.g005

For USUV, we only examined the transmission. TRs were equal to 4.2% (N = 24) at 10 dpi, 29.2% (N = 24) at 14 dpi, 12.5% (N = 24) at 17 dpi, 16.7% (N = 24) at 21 dpi, and 16.7% (N = 6) at 28 dpi (Fisher’s exact test: p = 0.01). Mean values of viral load in saliva were 10^0.8 (N = 1) at 10 dpi, 10^2.2 (N = 7) at 14 dpi, 10^2.4 (N = 3) at 17 dpi, 10^2.0 (N = 4) at 21 dpi, and 10^3.2 (N = 1) at 28 dpi (Kruskal-Wallis test: p = 0.26).

Culiseta longiareolata is not able to transmit USUV

Due to the difficulty in feeding Cs. longiareolata mosquitoes in BSL-3 conditions and keeping them alive, only 8 individuals were examined at 10 dpi. None of them were able to transmit USUV.

Discussion

We show that some field-collected mosquitoes from northeastern France are competent vectors of WNV and USUV; WNV was transmitted by Cx. pipiens, Ae. rusticus, and Ae. albopictus and USUV was transmitted by Cx. pipiens and Ae. albopictus.

Aedes albopictus is an invasive mosquito species introduced in mainland France in 1999 [20] and is now established in 67 out of 96 departments [21]. It is a known vector of multiple arboviruses such as DENV or CHIKV [22]. French populations of Ae. albopictus are experimentally competent to DENV, CHIKV and ZIKV [23,24]. We show that Ae. albopictus from Strasbourg was able to transmit WNV from 10 days post-infection. Variations of DR values are likely due to the sampling bias. This length of the extrinsic incubation period (EIP) is close to the value estimated for Italian mosquitoes (i.e. 9–14 days; [25]). We also found that Ae. albopictus was able to transmit USUV from 10 days post-infection with 10^0.8 viral particles detected in saliva of one mosquito. Combined with the detection of USUV in field-collected Ae. albopictus [26,27], our results on vector competence for USUV, are in favor of a role of Ae. albopictus in the transmission cycle of USUV [28,29]. As Ae. albopictus is spreading in Northern France, surveillance should be reinforced as it could coincide with the expansion of WNV.

Aedes rusticus is ubiquitous in northern France and present in 12 countries of western Europe [30]. The species mainly present in forested environments in close contacts with avian populations is a human biting mosquito, active from April to August (Martinet, personal communication). We show that viral transmission occurs at 7 dpi and certainly before, indicating a high potential of Ae. rusticus to behave as competent WNV vector on the field. This species is the fourth referenced Aedes species of northeast Europe to be competent to WNV beside Ae. caspius, Ae. detritus and Ae. japonicus [14,31,32]. However, no WNV-infected mosquitoes were detected on the field [33].

Anopheles plumbeus was one of the historical malaria vectors in northern Europe [34]. This mosquito is present from late spring to the end of September, and females feed mostly on mammals [35]. This species had been described as competent for the WNV lineage 2 [36]. We found that An. plumbeus became infected but was not able to transmit WNV lineage 1a. More studies are required to clarify the vector status of An. plumbeus as this species is actively colonizing anthropic biotopes [37]. Screenings performed on An. plumbeus in Germany in 2007–2008 showed no evidence of WNV-infected specimens [38].

Culex pipiens is ubiquitous in mainland France and populations used in this study were collected in different biotopes. Culex pipiens Machault from the department of Ardennes was collected in a rural area, in proximity of human habitations and domestic animals. The Verzy population from the department of Marne was sampled in a sylvatic environment often visited by hikers. The Sainte-Croix population in the department of Lorraine was collected in a zoological park at a short distance from Strix nebulosa enclosure and Maine population from Paris was collected in an urban environment. We provide some of the first vector competence data on Culex populations from northern France in addition to data for mosquitoes from southern France [14,15]. Populations from West Europe (Germany, The Netherlands, Switzerland, United-Kingdom) were experimentally capable to transmit WNV [39–42]. Furthermore, circulation of WNV in Germany has been reported in 2018 [43]. With low DRs and TRs, we found that Machault population was able to transmit WNV. Maine and Verzy demonstrated a midgut escape barrier, highlighting variations of vector competence depending on the mosquito population in addition to the virus genotype [44]. We determined that Machault population was composed of the two biotypes, Cx. pipiens pipiens and Cx. pipiens molestus, and hybrids pipiens/molestus (S1 Table). The two forms have distinct host preferences (Cx. pipiens pipiens biting mainly birds and Cx. pipiens molestus, mainly mammals, especially humans) [45]. Further attention is needed regarding the genetic or epigenetic factors that can cause variation of transmission for hybrids populations.

One Cx. pipiens Sainte-Croix was able to transmit USUV at 14 days post-infection with 14 viral particles detected in the saliva. Our result is on the line with the vector competence described for local populations of Cx. pipiens in Germany and Switzerland [41,42]. It is also correlated with the different episodes of USUV infections of Strix nebulosa and Strix aluca in zoological parks in the last years [46]. Therefore, monitoring of mosquito populations in zoological parks housing susceptible bird species could be of help to prevent USUV circulation.

Culiseta longiareolata is an ornithophilic species widely distributed in the southern Paleartic region [47]. Recently, it has been spreading to western Europe including several countries bordering France such as Belgium and Germany [47,48]. This species is considered a vector of blood parasites in birds and is not likely to feed on humans [35]. Due to the limited number of mosquitoes examined, we cannot strongly conclude that Cs. longiareolata from Reims was able to transmit USUV. Further experiments with increased sample size and more timepoints are needed, especially if the EIP for this species is longer than 10 days.

WNV-competent birds mainly belong to the Passeriformes and Charadriiformes [7]. West-Nile virus introduction in mainland France is done by migrating birds following main migratory routes. These routes crosses France from southwest to northeast [49]. The viraemia in birds does not last long enough (4-5days) to cover the duration of the migration (15–20 days from sub-Saharan Africa to Europe). Therefore, the probability that WNV-infected bird arrives in Europe directly from sub-Saharan Africa is low [50] and secondary spots of viral contaminations are suspected in resting sites for migrating birds, which are numerous in northeastern France.

WNV and USUV share the same hosts in their transmission cycle: birds as amplifying hosts and Culex mosquitoes as vectors. An increasing circulation of USUV would have important implications on WNV as it would affect human health and potentially convoluting diagnostics. USUV may have a strong cross-neutralizing potential towards WNV; the two closely related flaviviruses of the JEV serocomplex cannot persist in the same ecological niche due to cross-protective avian herd immunity [51]. Furthermore, Cx. pipiens mosquitoes appear to be a major vector for both WNV and USUV in Europe. This species feeds on birds and humans which then, can act as a bridge vector for spillovers to humans and horses [52]. Co-infections of Cx. pipiens with USUV and WNV show that WNV outcompetes USUV in mosquitoes. Therefore, the chance of concomitant USUV and WNV transmission via a single mosquito bite is low [53].

Differences of climate could also be considered in the disparity of WNV circulation in the country between north and south of France. Warm Mediterranean climate in the south could enhance virus circulation by: (i) contributing to increased mosquito densities and intensify contacts with bird hosts, and (ii) shortening the EIP allowing WNV to be transmitted earlier in the south than in the north. In the context of global change, warmer summers and autumns can decrease the temperature differential between North and South of France, expanding the geographical area and the period of viral contamination. Climate modelling studies suggest an increase of infection probability by WNV in Northern France by the end of the century [54].

In conclusion, we report two new putative vectors for WNV in northeastern France (Ae. albopictus and Ae. rusticus) aside from its known vector Cx. pipiens. WNV and USUV transmission overlaps sharing the same hosts and highlighting the importance of further studies on the interactions between the two viruses within vertebrate hosts and vector populations.

Supporting information

S1 Table. Molecular assay based on indels in the flanking region of a microsatellite locus CQ11 to distinguish the two forms of Culex pipiens, pipiens and molestus.

The method is described in [45]. One mosquito leg was placed in a tube containing the PCR mix composed of two antisense primers at a final concentration of 0.15 μM, the sense primer at 0.25 μM, buffer (1X), dNTPs at 250 μM, MgCl2 at 1.5 mM, BSA (Bovine serum Albumin) at 0.135μg/μL, one unit of Taq polymerase and 5 μL of the DNA extract. The primers used were: pipCQ11R 5’-CATGTTGAGCTTCGGTGAA-3’, molCQ11R 5’-CCCTCCAGTAAGGTATCAAC-3’ and CQ11F2 5’-GATCCTAGCAAGCGAGAAC-3’. The amplification program started with 15 min at 94°C, 35 cycles of 94°C for 30s, 54°C for 30s and 72°C for 40s and finally a 5 min elongation phase at 72°C. PCR products were separated by electrophoresis on a 2% agarose gel.

https://doi.org/10.1371/journal.pntd.0011144.s001

(PDF)

Acknowledgments

The authors are grateful to Eva Nast and Laure Augendre for field collection of mosquitoes, and André Yébakima and Pierre Manuellan for collecting Culex mosquitoes in Paris 14^th district. We warmly thank Dr Jennifer Lahoreau for sampling at Parc de Sainte-Croix zoological park. We also thank Benjamin Dupuis for helping in genotyping Culex pipiens mosquitoes.

References

1. Cochet A, Calba C, Jourdain F, Grard G, Durand GA, Guinard A, et al. Autochthonous dengue in mainland France, 2022: geographical extension and incidence increase. Eurosurveillance. 2022 Nov 3;27(44):2200818. pmid:36330819
- View Article
- PubMed/NCBI
- Google Scholar
2. Nikolay B. A review of West Nile and Usutu virus co-circulation in Europe: how much do transmission cycles overlap? Trans R Soc Trop Med Hyg. 2015 Oct;109(10):609–18. pmid:26286946
- View Article
- PubMed/NCBI
- Google Scholar
3. Rudolf I, Betášová L, Blažejová H, Venclíková K, Straková P, Šebesta O, et al. West Nile virus in overwintering mosquitoes, central Europe. Parasit Vectors. 2017 Dec;10(1):452. pmid:28969685
- View Article
- PubMed/NCBI
- Google Scholar
4. Chancey C, Grinev A, Volkova E, Rios M. The Global Ecology and Epidemiology of West Nile Virus. BioMed Res Int. 2015;2015:1–20. pmid:25866777
- View Article
- PubMed/NCBI
- Google Scholar
5. Bakonyi T, Ivanics É, Erdélyi K, Ursu K, Ferenczi E, Weissenböck H, et al. Lineage 1 and 2 Strains of Encephalitic West Nile Virus, Central Europe. Emerg Infect Dis. 2006 Apr;12(4):618–23. pmid:16704810
- View Article
- PubMed/NCBI
- Google Scholar
6. Engel D, Jöst H, Wink M, Börstler J, Bosch S, Garigliany MM, et al. Reconstruction of the Evolutionary History and Dispersal of Usutu Virus, a Neglected Emerging Arbovirus in Europe and Africa. Meng XJ, editor. mBio. 2016 Mar 2;7(1):e01938–15. pmid:26838717
- View Article
- PubMed/NCBI
- Google Scholar
7. Pérez-Ramírez E, Llorente F, Jiménez-Clavero MÁ. Experimental Infections of Wild Birds with West Nile Virus. Viruses. 2014 Feb;6(2):752–81. pmid:24531334
- View Article
- PubMed/NCBI
- Google Scholar
8. Lecollinet S, Blanchard Y, Manson C, Lowenski S, Laloy E, Quenault H, et al. Dual Emergence of Usutu Virus in Common Blackbirds, Eastern France, 2015. Emerg Infect Dis. 2016;Volume 22(12):2225–7. pmid:27869608
- View Article
- PubMed/NCBI
- Google Scholar
9. Eiden M, Gil P, Ziegler U, Rakotoarivony I, Marie A, Frances B, et al. Emergence of two Usutu virus lineages in Culex pipiens mosquitoes in the Camargue, France, 2015. Infect Genet Evol. 2018 Jul;61:151–4.
- View Article
- Google Scholar
10. Murgue B, Murri S, Triki H, Deubel V, Zeller HG. West Nile in the Mediterranean Basin: 1950–2000. Ann N Y Acad Sci. 2006 Jan 25;951(1):117–26.
- View Article
- Google Scholar
11. Hannoun C, Panthier R, Mouchet J, Eouzan JP. [ISOLATION IN FRANCE OF THE WEST NILE VIRUS FROM PATIENTS AND FROM THE VECTOR CULEX MODESTUS FICALBI]. Comptes Rendus Hebd Seances Acad Sci. 1964 Nov 30;259:4170–2.
- View Article
- Google Scholar
12. Simonin Y, Sillam O, Carles MJ, Gutierrez S, Gil P, Constant O, et al. Human Usutu Virus Infection with Atypical Neurologic Presentation, Montpellier, France, 2016. Emerg Infect Dis. 2018 May;24(5):875–8. pmid:29664365
- View Article
- PubMed/NCBI
- Google Scholar
13. Communiqué de presse—Confirmation d’une infection autochtone à virus Usutu (secteurs des Landes et de Gironde) [Internet]. [cited 2023 Jan 11]. Available from: https://www.nouvelle-aquitaine.ars.sante.fr/communique-de-presse-confirmation-dune-infection-autochtone-virus-usutu-secteurs-des-landes-et-de.
14. Balenghien T, Vazeille M, Grandadam M, Schaffner F, Zeller H, Reiter P, et al. Vector Competence of Some French Culex and Aedes Mosquitoes for West Nile Virus. Vector-Borne Zoonotic Dis. 2008 Oct;8(5):589–96. pmid:18447623
- View Article
- PubMed/NCBI
- Google Scholar
15. Balenghien T, Vazeille M, Reiter P, Schaffner F, Zeller H, Bicout DJ. Evidence of laboratory vecto competence of Culex modestus for West Nile virus. J Am Mosq Control Assoc. 2007 Jun;23(2):233–6.
- View Article
- Google Scholar
16. RStudio Team. RStudio: Integrated Development Environment for R [Internet]. Boston, MA: RStudio, PBC; 2021. Available from: http://www.rstudio.com/.
17. Wickham H. ggplot2: Elegant Graphics for Data Analysis [Internet]. Springer-Verlag New York; 2016. Available from: https://ggplot2.tidyverse.org.
18. Dunnington D. ggspatial: Spatial Data Framework for ggplot2 [Internet]. 2022. Available from: https://paleolimbot.github.io/ggspatial/, https://github.com/paleolimbot/ggspatial.
- View Article
- Google Scholar
19. Igarashi A. Isolation of a Singh’s Aedes albopictus Cell Clone Sensitive to Dengue and Chikungunya Viruses. J Gen Virol. 1978 Sep 1;40(3):531–44.
- View Article
- Google Scholar
20. Schaffner F, Karch S. Première observation d’Aedes albopictus (Skuse, 1894) en France métropolitaine. Comptes Rendus Académie Sci—Ser III—Sci Vie. 2000 Apr;323(4):373–5.
- View Article
- Google Scholar
21. Aedes albopictus—current known distribution: March 2022 [Internet]. European Centre for Disease Prevention and Control. 2022 [cited 2023 Jan 13]. Available from: https://www.ecdc.europa.eu/en/publications-data/aedes-albopictus-current-known-distribution-march-2022
- View Article
- Google Scholar
22. Paupy C, Delatte H, Bagny L, Corbel V, Fontenille D. Aedes albopictus, an arbovirus vector: From the darkness to the light. Microbes Infect. 2009 Dec;11(14–15):1177–85.
- View Article
- Google Scholar
23. Jupille H, Seixas G, Mousson L, Sousa CA, Failloux AB. Zika Virus, a New Threat for Europe? Pimenta PF, editor. PLoS Negl Trop Dis. 2016 Aug 9;10(8):e0004901. pmid:27505002
- View Article
- PubMed/NCBI
- Google Scholar
24. Vega-Rua A, Zouache K, Caro V, Diancourt L, Delaunay P, Grandadam M, et al. High Efficiency of Temperate Aedes albopictus to Transmit Chikungunya and Dengue Viruses in the Southeast of France. Moreira LA, editor. PLoS ONE. 2013 Mar 18;8(3):e59716.
- View Article
- Google Scholar
25. Fortuna C, Remoli ME, Severini F, Di Luca M, Toma L, Fois F, et al. Evaluation of vector competence for West Nile virus in Italian Stegomyia albopicta (= Aedes albopictus) mosquitoes. Med Vet Entomol. 2015;29(4):430–3.
- View Article
- Google Scholar
26. Calzolari M, Gaibani P, Bellini R, Defilippo F, Pierro A, Albieri A, et al. Mosquito, Bird and Human Surveillance of West Nile and Usutu Viruses in Emilia-Romagna Region (Italy) in 2010. Coffey LL, editor. PLoS ONE. 2012 May 30;7(5):e38058. pmid:22666446
- View Article
- PubMed/NCBI
- Google Scholar
27. Calzolari M, Bonilauri P, Bellini R, Albieri A, Defilippo F, Tamba M, et al. Usutu Virus Persistence and West Nile Virus Inactivity in the Emilia-Romagna Region (Italy) in 2011. Coffey LL, editor. PLoS ONE. 2013 May 7;8(5):e63978. pmid:23667694
- View Article
- PubMed/NCBI
- Google Scholar
28. Calzolari M, Bonilauri P, Bellini R, Albieri A, Defilippo F, Maioli G, et al. Evidence of Simultaneous Circulation of West Nile and Usutu Viruses in Mosquitoes Sampled in Emilia-Romagna Region (Italy) in 2009. Baylis M, editor. PLoS ONE. 2010 Dec 15;5(12):e14324. pmid:21179462
- View Article
- PubMed/NCBI
- Google Scholar
29. Tamba M, Bonilauri P, Bellini R, Calzolari M, Albieri A, Sambri V, et al. Detection of Usutu Virus Within a West Nile Virus Surveillance Program in Northern Italy. Vector-Borne Zoonotic Dis. 2011 May;11(5):551–7. pmid:20849275
- View Article
- PubMed/NCBI
- Google Scholar
30. Robert V, Günay F, Le Goff G, Boussès P, Sulesco T, Khalin A, et al. Distribution chart for Euro-Mediterranean mosquitoes (western Palaearctic region). J Eur Mosq Control Assoc. 2019;37:1–28.
- View Article
- Google Scholar
31. Veronesi E, Paslaru A, Silaghi C, Tobler K, Glavinic U, Torgerson P, et al. Experimental evaluation of infection, dissemination, and transmission rates for two West Nile virus strains in European Aedes japonicus under a fluctuating temperature regime. Parasitol Res. 2018 Jun;117(6):1925–32. pmid:29705877
- View Article
- PubMed/NCBI
- Google Scholar
32. Blagrove MSC, Sherlock K, Chapman GE, Impoinvil DE, McCall PJ, Medlock JM, et al. Evaluation of the vector competence of a native UK mosquito Ochlerotatus detritus (Aedes detritus) for dengue, chikungunya and West Nile viruses. Parasit Vectors. 2016 Dec;9(1):452. pmid:27527700
- View Article
- PubMed/NCBI
- Google Scholar
33. Martinet JP, Ferté H, Failloux AB, Schaffner F, Depaquit J. Mosquitoes of North-Western Europe as Potential Vectors of Arboviruses: A Review. Viruses. 2019 Nov 14;11(11):1059. pmid:31739553
- View Article
- PubMed/NCBI
- Google Scholar
34. Kruger A, Rech A, Su XZ, Tannich E. Two cases of autochthonous Plasmodium falciparum malaria in Germany with evidence for local transmission by indigenous Anopheles plumbeus. Trop Med Int Health. 2001 Dec;6(12):983–5.
- View Article
- Google Scholar
35. Becker N, editor. Mosquitoes and their control. 2nd ed. Heidelberg: Springer; 2010. 577 p.
36. Vermeil CL, Reeb E. Survival and transmission of West Nile virus by various arthropods. Bull Société Pathol Exot. 1960;53(2):273–9.
- View Article
- Google Scholar
37. Dekoninck W, Hendrickx F, Van Bortel W, Versteirt V, Coosemans M, Damiens D, et al. Human-Induced Expanded Distribution of Anopheles plumbeus, Experimental Vector of West Nile Virus and a Potential Vector of Human Malaria in Belgium. J Med Entomol. 2011 Jul 1;48(4):924–8. pmid:21845955
- View Article
- PubMed/NCBI
- Google Scholar
38. Timmermann U, Becker N. Mosquito-borne West Nile virus (WNV) surveillance in the Upper Rhine Valley, Germany. J Vector Ecol J Soc Vector Ecol. 2010 Jun;35(1):140–3. pmid:20618659
- View Article
- PubMed/NCBI
- Google Scholar
39. Fros JJ, Geertsema C, Vogels CB, Roosjen PP, Failloux AB, Vlak JM, et al. West Nile Virus: High Transmission Rate in North-Western European Mosquitoes Indicates Its Epidemic Potential and Warrants Increased Surveillance. Liang S, editor. PLoS Negl Trop Dis. 2015 Jul 30;9(7):e0003956. pmid:26225555
- View Article
- PubMed/NCBI
- Google Scholar
40. Vogels CBF, Göertz GP, Pijlman GP, Koenraadt CJM. Vector competence of northern and southern European Culex pipiens pipiens mosquitoes for West Nile virus across a gradient of temperatures: West Nile virus transmission by mosquitoes. Med Vet Entomol. 2017 Dec;31(4):358–64.
- View Article
- Google Scholar
41. Wagner S, Mathis A, Schönenberger AC, Becker S, Schmidt-Chanasit J, Silaghi C, et al. Vector competence of field populations of the mosquito species Aedes japonicus japonicus and Culex pipiens from Switzerland for two West Nile virus strains: Ae. japonicus WNV vector competence. Med Vet Entomol. 2018 Mar;32(1):121–4.
- View Article
- Google Scholar
42. Leggewie M, Badusche M, Rudolf M, Jansen S, Börstler J, Krumkamp R, et al. Culex pipiens and Culex torrentium populations from Central Europe are susceptible to West Nile virus infection. One Health. 2016 Dec;2:88–94.
- View Article
- Google Scholar
43. Ziegler U, Lühken R, Keller M, Cadar D, van der Grinten E, Michel F, et al. West Nile virus epizootic in Germany, 2018. Antiviral Res. 2019 Feb;162:39–43. pmid:30550796
- View Article
- PubMed/NCBI
- Google Scholar
44. Lambrechts L, Chevillon C, Albright RG, Thaisomboonsuk B, Richardson JH, Jarman RG, et al. Genetic specificity and potential for local adaptation between dengue viruses and mosquito vectors. BMC Evol Biol. 2009;9(1):160. pmid:19589156
- View Article
- PubMed/NCBI
- Google Scholar
45. Bahnck CM, Fonseca DM. Rapid assay to identify the two genetic forms of Culex (Culex) pipiens L.(Diptera: Culicidae) and hybrid populations. Am J Trop Med Hyg. 2006;75(2):251–5. pmid:16896127
- View Article
- PubMed/NCBI
- Google Scholar
46. Beck C, Gonzalez G, Eraud C, Decors A, Desvaux S. BILAN DE LA CIRCULATION DU VIRUS USUTU EN FRANCE AU 27 AOUT 2018 [Internet]. ESA; 2018 Sep [cited 2023 Jan 12] p. 5. Available from: https://www.plateforme-esa.fr/sites/default/files/2021-11/USUTU%20bilan%20270818_v4.pdf.
47. Deblauwe I, Ibáñez-Justicia A, De Wolf K, Smitz N, Schneider A, Stroo A, et al. First Detections of Culiseta longiareolata (Diptera: Culicidae) in Belgium and the Netherlands. Wilkerson R editor. J Med Entomol. 2021 Nov 9;58(6):2524–32. pmid:34313772
- View Article
- PubMed/NCBI
- Google Scholar
48. Becker N, Hoffmann D. First record of Culiseta longiareolata (Macquart) for Germany. Eur Mosq Bull. 2011 Jan 1;29.
- View Article
- Google Scholar
49. Jourdain E, Toussaint Y, Leblond A, Bicout DJ, Sabatier P, Gauthier-Clerc M. Bird species potentially involved in introduction, amplification, and spread of West Nile virus in a Mediterranean wetland, the Camargue (Southern France). Vector Borne Zoonotic Dis Larchmt N. 2007;7(1):15–33.
- View Article
- Google Scholar
50. Pradier S, Lecollinet S, Leblond A. West Nile virus epidemiology and factors triggering change in its distribution in Europe: -EN- -FR- L’épidémiologie du virus West Nile et les facteurs favorisant les changements de sa distribution en Europe -ES- Epidemiología del virus West Nile y factores desencadenantes de cambios en su distribución europea. Rev Sci Tech OIE. 2012 Dec 1;31(3):829–44.
- View Article
- Google Scholar
51. Escribano-Romero E, Jiménez de Oya N, Camacho MC, Blázquez AB, Martín-Acebes MA, Risalde MA, et al. Previous Usutu Virus Exposure Partially Protects Magpies (Pica pica) against West Nile Virus Disease But Does Not Prevent Horizontal Transmission. Viruses. 2021 Jul 20;13(7):1409. pmid:34372622
- View Article
- PubMed/NCBI
- Google Scholar
52. Rizzoli A, Jiménez-Clavero MA, Barzon L, Cordioli P, Figuerola J, Koraka P, et al. The challenge of West Nile virus in Europe: knowledge gaps and research priorities. Eurosurveillance [Internet]. 2015 May 21 [cited 2023 Jan 16];20(20). Available from: https://www.eurosurveillance.org/content/10.2807/1560-7917.es2015.20.20.21135 pmid:26027485
- View Article
- PubMed/NCBI
- Google Scholar
53. Wang H, Abbo SR, Visser TM, Westenberg M, Geertsema C, Fros JJ, et al. Competition between Usutu virus and West Nile virus during simultaneous and sequential infection of Culex pipiens mosquitoes. Emerg Microbes Infect. 2020 Jan 1;9(1):2642–52.
- View Article
- Google Scholar
54. Semenza JC, Tran A, Espinosa L, Sudre B, Domanovic D, Paz S. Climate change projections of West Nile virus infections in Europe: implications for blood safety practices. Environ Health. 2016 Dec;15(S1):S28. pmid:26961903
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Cochet A, Calba C, Jourdain F, Grard G, Durand GA, Guinard A, et al. Autochthonous dengue in mainland France, 2022: geographical extension and incidence increase. Eurosurveillance. 2022 Nov 3;27(44):2200818. pmid:36330819
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Nikolay B. A review of West Nile and Usutu virus co-circulation in Europe: how much do transmission cycles overlap? Trans R Soc Trop Med Hyg. 2015 Oct;109(10):609–18. pmid:26286946
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Rudolf I, Betášová L, Blažejová H, Venclíková K, Straková P, Šebesta O, et al. West Nile virus in overwintering mosquitoes, central Europe. Parasit Vectors. 2017 Dec;10(1):452. pmid:28969685
View Article
PubMed/NCBI
Google Scholar

[10] View Article

[11] PubMed/NCBI

[12] Google Scholar

[ref4] 4. Chancey C, Grinev A, Volkova E, Rios M. The Global Ecology and Epidemiology of West Nile Virus. BioMed Res Int. 2015;2015:1–20. pmid:25866777
View Article
PubMed/NCBI
Google Scholar

[14] View Article

[15] PubMed/NCBI

[16] Google Scholar

[ref5] 5. Bakonyi T, Ivanics É, Erdélyi K, Ursu K, Ferenczi E, Weissenböck H, et al. Lineage 1 and 2 Strains of Encephalitic West Nile Virus, Central Europe. Emerg Infect Dis. 2006 Apr;12(4):618–23. pmid:16704810
View Article
PubMed/NCBI
Google Scholar

[18] View Article

[19] PubMed/NCBI

[20] Google Scholar

[ref6] 6. Engel D, Jöst H, Wink M, Börstler J, Bosch S, Garigliany MM, et al. Reconstruction of the Evolutionary History and Dispersal of Usutu Virus, a Neglected Emerging Arbovirus in Europe and Africa. Meng XJ, editor. mBio. 2016 Mar 2;7(1):e01938–15. pmid:26838717
View Article
PubMed/NCBI
Google Scholar

[22] View Article

[23] PubMed/NCBI

[24] Google Scholar

[ref7] 7. Pérez-Ramírez E, Llorente F, Jiménez-Clavero MÁ. Experimental Infections of Wild Birds with West Nile Virus. Viruses. 2014 Feb;6(2):752–81. pmid:24531334
View Article
PubMed/NCBI
Google Scholar

[26] View Article

[27] PubMed/NCBI

[28] Google Scholar

[ref8] 8. Lecollinet S, Blanchard Y, Manson C, Lowenski S, Laloy E, Quenault H, et al. Dual Emergence of Usutu Virus in Common Blackbirds, Eastern France, 2015. Emerg Infect Dis. 2016;Volume 22(12):2225–7. pmid:27869608
View Article
PubMed/NCBI
Google Scholar

[30] View Article

[31] PubMed/NCBI

[32] Google Scholar

[ref9] 9. Eiden M, Gil P, Ziegler U, Rakotoarivony I, Marie A, Frances B, et al. Emergence of two Usutu virus lineages in Culex pipiens mosquitoes in the Camargue, France, 2015. Infect Genet Evol. 2018 Jul;61:151–4.
View Article
Google Scholar

[34] View Article

[35] Google Scholar

[ref10] 10. Murgue B, Murri S, Triki H, Deubel V, Zeller HG. West Nile in the Mediterranean Basin: 1950–2000. Ann N Y Acad Sci. 2006 Jan 25;951(1):117–26.
View Article
Google Scholar

[37] View Article

[38] Google Scholar

[ref11] 11. Hannoun C, Panthier R, Mouchet J, Eouzan JP. [ISOLATION IN FRANCE OF THE WEST NILE VIRUS FROM PATIENTS AND FROM THE VECTOR CULEX MODESTUS FICALBI]. Comptes Rendus Hebd Seances Acad Sci. 1964 Nov 30;259:4170–2.
View Article
Google Scholar

[40] View Article

[41] Google Scholar

[ref12] 12. Simonin Y, Sillam O, Carles MJ, Gutierrez S, Gil P, Constant O, et al. Human Usutu Virus Infection with Atypical Neurologic Presentation, Montpellier, France, 2016. Emerg Infect Dis. 2018 May;24(5):875–8. pmid:29664365
View Article
PubMed/NCBI
Google Scholar

[43] View Article

[44] PubMed/NCBI

[45] Google Scholar

[ref13] 13. Communiqué de presse—Confirmation d’une infection autochtone à virus Usutu (secteurs des Landes et de Gironde) [Internet]. [cited 2023 Jan 11]. Available from: https://www.nouvelle-aquitaine.ars.sante.fr/communique-de-presse-confirmation-dune-infection-autochtone-virus-usutu-secteurs-des-landes-et-de.

[ref14] 14. Balenghien T, Vazeille M, Grandadam M, Schaffner F, Zeller H, Reiter P, et al. Vector Competence of Some French Culex and Aedes Mosquitoes for West Nile Virus. Vector-Borne Zoonotic Dis. 2008 Oct;8(5):589–96. pmid:18447623
View Article
PubMed/NCBI
Google Scholar

[48] View Article

[49] PubMed/NCBI

[50] Google Scholar

[ref15] 15. Balenghien T, Vazeille M, Reiter P, Schaffner F, Zeller H, Bicout DJ. Evidence of laboratory vecto competence of Culex modestus for West Nile virus. J Am Mosq Control Assoc. 2007 Jun;23(2):233–6.
View Article
Google Scholar

[52] View Article

[53] Google Scholar

[ref16] 16. RStudio Team. RStudio: Integrated Development Environment for R [Internet]. Boston, MA: RStudio, PBC; 2021. Available from: http://www.rstudio.com/.

[ref17] 17. Wickham H. ggplot2: Elegant Graphics for Data Analysis [Internet]. Springer-Verlag New York; 2016. Available from: https://ggplot2.tidyverse.org.

[ref18] 18. Dunnington D. ggspatial: Spatial Data Framework for ggplot2 [Internet]. 2022. Available from: https://paleolimbot.github.io/ggspatial/, https://github.com/paleolimbot/ggspatial.
View Article
Google Scholar

[57] View Article

[58] Google Scholar

[ref19] 19. Igarashi A. Isolation of a Singh’s Aedes albopictus Cell Clone Sensitive to Dengue and Chikungunya Viruses. J Gen Virol. 1978 Sep 1;40(3):531–44.
View Article
Google Scholar

[60] View Article

[61] Google Scholar

[ref20] 20. Schaffner F, Karch S. Première observation d’Aedes albopictus (Skuse, 1894) en France métropolitaine. Comptes Rendus Académie Sci—Ser III—Sci Vie. 2000 Apr;323(4):373–5.
View Article
Google Scholar

[63] View Article

[64] Google Scholar

[ref21] 21. Aedes albopictus—current known distribution: March 2022 [Internet]. European Centre for Disease Prevention and Control. 2022 [cited 2023 Jan 13]. Available from: https://www.ecdc.europa.eu/en/publications-data/aedes-albopictus-current-known-distribution-march-2022
View Article
Google Scholar

[66] View Article

[67] Google Scholar

[ref22] 22. Paupy C, Delatte H, Bagny L, Corbel V, Fontenille D. Aedes albopictus, an arbovirus vector: From the darkness to the light. Microbes Infect. 2009 Dec;11(14–15):1177–85.
View Article
Google Scholar

[69] View Article

[70] Google Scholar

[ref23] 23. Jupille H, Seixas G, Mousson L, Sousa CA, Failloux AB. Zika Virus, a New Threat for Europe? Pimenta PF, editor. PLoS Negl Trop Dis. 2016 Aug 9;10(8):e0004901. pmid:27505002
View Article
PubMed/NCBI
Google Scholar

[72] View Article

[73] PubMed/NCBI

[74] Google Scholar

[ref24] 24. Vega-Rua A, Zouache K, Caro V, Diancourt L, Delaunay P, Grandadam M, et al. High Efficiency of Temperate Aedes albopictus to Transmit Chikungunya and Dengue Viruses in the Southeast of France. Moreira LA, editor. PLoS ONE. 2013 Mar 18;8(3):e59716.
View Article
Google Scholar

[76] View Article

[77] Google Scholar

[ref25] 25. Fortuna C, Remoli ME, Severini F, Di Luca M, Toma L, Fois F, et al. Evaluation of vector competence for West Nile virus in Italian Stegomyia albopicta (= Aedes albopictus) mosquitoes. Med Vet Entomol. 2015;29(4):430–3.
View Article
Google Scholar

[79] View Article

[80] Google Scholar

[ref26] 26. Calzolari M, Gaibani P, Bellini R, Defilippo F, Pierro A, Albieri A, et al. Mosquito, Bird and Human Surveillance of West Nile and Usutu Viruses in Emilia-Romagna Region (Italy) in 2010. Coffey LL, editor. PLoS ONE. 2012 May 30;7(5):e38058. pmid:22666446
View Article
PubMed/NCBI
Google Scholar

[82] View Article

[83] PubMed/NCBI

[84] Google Scholar

[ref27] 27. Calzolari M, Bonilauri P, Bellini R, Albieri A, Defilippo F, Tamba M, et al. Usutu Virus Persistence and West Nile Virus Inactivity in the Emilia-Romagna Region (Italy) in 2011. Coffey LL, editor. PLoS ONE. 2013 May 7;8(5):e63978. pmid:23667694
View Article
PubMed/NCBI
Google Scholar

[86] View Article

[87] PubMed/NCBI

[88] Google Scholar

[ref28] 28. Calzolari M, Bonilauri P, Bellini R, Albieri A, Defilippo F, Maioli G, et al. Evidence of Simultaneous Circulation of West Nile and Usutu Viruses in Mosquitoes Sampled in Emilia-Romagna Region (Italy) in 2009. Baylis M, editor. PLoS ONE. 2010 Dec 15;5(12):e14324. pmid:21179462
View Article
PubMed/NCBI
Google Scholar

[90] View Article

[91] PubMed/NCBI

[92] Google Scholar

[ref29] 29. Tamba M, Bonilauri P, Bellini R, Calzolari M, Albieri A, Sambri V, et al. Detection of Usutu Virus Within a West Nile Virus Surveillance Program in Northern Italy. Vector-Borne Zoonotic Dis. 2011 May;11(5):551–7. pmid:20849275
View Article
PubMed/NCBI
Google Scholar

[94] View Article

[95] PubMed/NCBI

[96] Google Scholar

[ref30] 30. Robert V, Günay F, Le Goff G, Boussès P, Sulesco T, Khalin A, et al. Distribution chart for Euro-Mediterranean mosquitoes (western Palaearctic region). J Eur Mosq Control Assoc. 2019;37:1–28.
View Article
Google Scholar

[98] View Article

[99] Google Scholar

[ref31] 31. Veronesi E, Paslaru A, Silaghi C, Tobler K, Glavinic U, Torgerson P, et al. Experimental evaluation of infection, dissemination, and transmission rates for two West Nile virus strains in European Aedes japonicus under a fluctuating temperature regime. Parasitol Res. 2018 Jun;117(6):1925–32. pmid:29705877
View Article
PubMed/NCBI
Google Scholar

[101] View Article

[102] PubMed/NCBI

[103] Google Scholar

[ref32] 32. Blagrove MSC, Sherlock K, Chapman GE, Impoinvil DE, McCall PJ, Medlock JM, et al. Evaluation of the vector competence of a native UK mosquito Ochlerotatus detritus (Aedes detritus) for dengue, chikungunya and West Nile viruses. Parasit Vectors. 2016 Dec;9(1):452. pmid:27527700
View Article
PubMed/NCBI
Google Scholar

[105] View Article

[106] PubMed/NCBI

[107] Google Scholar

[ref33] 33. Martinet JP, Ferté H, Failloux AB, Schaffner F, Depaquit J. Mosquitoes of North-Western Europe as Potential Vectors of Arboviruses: A Review. Viruses. 2019 Nov 14;11(11):1059. pmid:31739553
View Article
PubMed/NCBI
Google Scholar

[109] View Article

[110] PubMed/NCBI

[111] Google Scholar

[ref34] 34. Kruger A, Rech A, Su XZ, Tannich E. Two cases of autochthonous Plasmodium falciparum malaria in Germany with evidence for local transmission by indigenous Anopheles plumbeus. Trop Med Int Health. 2001 Dec;6(12):983–5.
View Article
Google Scholar

[113] View Article

[114] Google Scholar

[ref35] 35. Becker N, editor. Mosquitoes and their control. 2nd ed. Heidelberg: Springer; 2010. 577 p.

[ref36] 36. Vermeil CL, Reeb E. Survival and transmission of West Nile virus by various arthropods. Bull Société Pathol Exot. 1960;53(2):273–9.
View Article
Google Scholar

[117] View Article

[118] Google Scholar

[ref37] 37. Dekoninck W, Hendrickx F, Van Bortel W, Versteirt V, Coosemans M, Damiens D, et al. Human-Induced Expanded Distribution of Anopheles plumbeus, Experimental Vector of West Nile Virus and a Potential Vector of Human Malaria in Belgium. J Med Entomol. 2011 Jul 1;48(4):924–8. pmid:21845955
View Article
PubMed/NCBI
Google Scholar

[120] View Article

[121] PubMed/NCBI

[122] Google Scholar

[ref38] 38. Timmermann U, Becker N. Mosquito-borne West Nile virus (WNV) surveillance in the Upper Rhine Valley, Germany. J Vector Ecol J Soc Vector Ecol. 2010 Jun;35(1):140–3. pmid:20618659
View Article
PubMed/NCBI
Google Scholar

[124] View Article

[125] PubMed/NCBI

[126] Google Scholar

[ref39] 39. Fros JJ, Geertsema C, Vogels CB, Roosjen PP, Failloux AB, Vlak JM, et al. West Nile Virus: High Transmission Rate in North-Western European Mosquitoes Indicates Its Epidemic Potential and Warrants Increased Surveillance. Liang S, editor. PLoS Negl Trop Dis. 2015 Jul 30;9(7):e0003956. pmid:26225555
View Article
PubMed/NCBI
Google Scholar

[128] View Article

[129] PubMed/NCBI

[130] Google Scholar

[ref40] 40. Vogels CBF, Göertz GP, Pijlman GP, Koenraadt CJM. Vector competence of northern and southern European Culex pipiens pipiens mosquitoes for West Nile virus across a gradient of temperatures: West Nile virus transmission by mosquitoes. Med Vet Entomol. 2017 Dec;31(4):358–64.
View Article
Google Scholar

[132] View Article

[133] Google Scholar

[ref41] 41. Wagner S, Mathis A, Schönenberger AC, Becker S, Schmidt-Chanasit J, Silaghi C, et al. Vector competence of field populations of the mosquito species Aedes japonicus japonicus and Culex pipiens from Switzerland for two West Nile virus strains: Ae. japonicus WNV vector competence. Med Vet Entomol. 2018 Mar;32(1):121–4.
View Article
Google Scholar

[135] View Article

[136] Google Scholar

[ref42] 42. Leggewie M, Badusche M, Rudolf M, Jansen S, Börstler J, Krumkamp R, et al. Culex pipiens and Culex torrentium populations from Central Europe are susceptible to West Nile virus infection. One Health. 2016 Dec;2:88–94.
View Article
Google Scholar

[138] View Article

[139] Google Scholar

[ref43] 43. Ziegler U, Lühken R, Keller M, Cadar D, van der Grinten E, Michel F, et al. West Nile virus epizootic in Germany, 2018. Antiviral Res. 2019 Feb;162:39–43. pmid:30550796
View Article
PubMed/NCBI
Google Scholar

[141] View Article

[142] PubMed/NCBI

[143] Google Scholar

[ref44] 44. Lambrechts L, Chevillon C, Albright RG, Thaisomboonsuk B, Richardson JH, Jarman RG, et al. Genetic specificity and potential for local adaptation between dengue viruses and mosquito vectors. BMC Evol Biol. 2009;9(1):160. pmid:19589156
View Article
PubMed/NCBI
Google Scholar

[145] View Article

[146] PubMed/NCBI

[147] Google Scholar

[ref45] 45. Bahnck CM, Fonseca DM. Rapid assay to identify the two genetic forms of Culex (Culex) pipiens L.(Diptera: Culicidae) and hybrid populations. Am J Trop Med Hyg. 2006;75(2):251–5. pmid:16896127
View Article
PubMed/NCBI
Google Scholar

[149] View Article

[150] PubMed/NCBI

[151] Google Scholar

[ref46] 46. Beck C, Gonzalez G, Eraud C, Decors A, Desvaux S. BILAN DE LA CIRCULATION DU VIRUS USUTU EN FRANCE AU 27 AOUT 2018 [Internet]. ESA; 2018 Sep [cited 2023 Jan 12] p. 5. Available from: https://www.plateforme-esa.fr/sites/default/files/2021-11/USUTU%20bilan%20270818_v4.pdf.

[ref47] 47. Deblauwe I, Ibáñez-Justicia A, De Wolf K, Smitz N, Schneider A, Stroo A, et al. First Detections of Culiseta longiareolata (Diptera: Culicidae) in Belgium and the Netherlands. Wilkerson R editor. J Med Entomol. 2021 Nov 9;58(6):2524–32. pmid:34313772
View Article
PubMed/NCBI
Google Scholar

[154] View Article

[155] PubMed/NCBI

[156] Google Scholar

[ref48] 48. Becker N, Hoffmann D. First record of Culiseta longiareolata (Macquart) for Germany. Eur Mosq Bull. 2011 Jan 1;29.
View Article
Google Scholar

[158] View Article

[159] Google Scholar

[ref49] 49. Jourdain E, Toussaint Y, Leblond A, Bicout DJ, Sabatier P, Gauthier-Clerc M. Bird species potentially involved in introduction, amplification, and spread of West Nile virus in a Mediterranean wetland, the Camargue (Southern France). Vector Borne Zoonotic Dis Larchmt N. 2007;7(1):15–33.
View Article
Google Scholar

[161] View Article

[162] Google Scholar

[ref50] 50. Pradier S, Lecollinet S, Leblond A. West Nile virus epidemiology and factors triggering change in its distribution in Europe: -EN- -FR- L’épidémiologie du virus West Nile et les facteurs favorisant les changements de sa distribution en Europe -ES- Epidemiología del virus West Nile y factores desencadenantes de cambios en su distribución europea. Rev Sci Tech OIE. 2012 Dec 1;31(3):829–44.
View Article
Google Scholar

[164] View Article

[165] Google Scholar

[ref51] 51. Escribano-Romero E, Jiménez de Oya N, Camacho MC, Blázquez AB, Martín-Acebes MA, Risalde MA, et al. Previous Usutu Virus Exposure Partially Protects Magpies (Pica pica) against West Nile Virus Disease But Does Not Prevent Horizontal Transmission. Viruses. 2021 Jul 20;13(7):1409. pmid:34372622
View Article
PubMed/NCBI
Google Scholar

[167] View Article

[168] PubMed/NCBI

[169] Google Scholar

[ref52] 52. Rizzoli A, Jiménez-Clavero MA, Barzon L, Cordioli P, Figuerola J, Koraka P, et al. The challenge of West Nile virus in Europe: knowledge gaps and research priorities. Eurosurveillance [Internet]. 2015 May 21 [cited 2023 Jan 16];20(20). Available from: https://www.eurosurveillance.org/content/10.2807/1560-7917.es2015.20.20.21135 pmid:26027485
View Article
PubMed/NCBI
Google Scholar

[171] View Article

[172] PubMed/NCBI

[173] Google Scholar

[ref53] 53. Wang H, Abbo SR, Visser TM, Westenberg M, Geertsema C, Fros JJ, et al. Competition between Usutu virus and West Nile virus during simultaneous and sequential infection of Culex pipiens mosquitoes. Emerg Microbes Infect. 2020 Jan 1;9(1):2642–52.
View Article
Google Scholar

[175] View Article

[176] Google Scholar

[ref54] 54. Semenza JC, Tran A, Espinosa L, Sudre B, Domanovic D, Paz S. Climate change projections of West Nile virus infections in Europe: implications for blood safety practices. Environ Health. 2016 Dec;15(S1):S28. pmid:26961903
View Article
PubMed/NCBI
Google Scholar

[178] View Article

[179] PubMed/NCBI

[180] Google Scholar

Figures

Abstract

Author summary

Introduction

Materials and methods

Ethics statements

Mosquito collections

Viral strains

Mosquito infections and processing

Vector competence indices

Viral titration

Statistical analysis

Results

Some populations of Culex pipiens are able to transmit WNV and USUV

Anopheles plumbeus is able to become infected but not to transmit WNV and USUV

Aedes rusticus is able to transmit WNV but not USUV

Aedes albopictus transmits WNV and to a lesser extent, USUV

Culiseta longiareolata is not able to transmit USUV

Discussion

Supporting information

S1 Table. Molecular assay based on indels in the flanking region of a microsatellite locus CQ11 to distinguish the two forms of Culex pipiens, pipiens and molestus.

Acknowledgments

References