- Open Access
Experimental studies on comparison of the vector competence of four Italian Culex pipiens populations for West Nile virus
- Claudia Fortuna†1Email author,
- Maria Elena Remoli†1,
- Marco Di Luca2,
- Francesco Severini2,
- Luciano Toma2,
- Eleonora Benedetti1,
- Paola Bucci1,
- Fabrizio Montarsi3,
- Giada Minelli4,
- Daniela Boccolini2,
- Roberto Romi2 and
- Maria Grazia Ciufolini1
© Fortuna et al. 2015
- Received: 27 April 2015
- Accepted: 1 September 2015
- Published: 17 September 2015
West Nile virus (WNV) is a vector-borne disease responsible for causing epidemics in many areas of the world. The virus is maintained in nature by an enzootic bird-mosquito-bird cycle and occasionally transmitted to other hosts, such as equines and humans. Culex species, in particular the ubiquitous species Culex pipiens is thought to play a major vector role both in enzootic and epizootic maintenance and transmission of WNV. Introduced in Europe in recent years, since 2008 WNV has been stably circulating mainly in the Northeastern regions of Italy, although sporadic equine and/or human cases, as well as WNV infected Cx. pipiens pools, have been recorded in other Italian areas. The scope of our study was to evaluate the potential competence of some Italian populations of Cx. pipiens to transmit WNV and to assess their ability for vertical transmission of the virus. For this purpose four Italian populations, from different areas, were experimentally infected.
After the infectious blood meal, fed females were monitored for 32 days to determine the length of viral extrinsic incubation period. WNV titre of infected mosquitoes was evaluated both by quantitative Real Time PCR and viral titration by Plaque Forming Units/ml (PFU/mL) in VERO cells. Potential Infection, Dissemination, Transmission rates (IR, DR, TR) were assessed by detection of the virus in body, legs plus wings and saliva of the fed females, respectively.
All tested populations were susceptible to the WNV infection. The viral presence in legs and wings demonstrated the ability of WNV to disseminate in the mosquitoes. Viral RNA was detected in the saliva of tested populations. No significant differences in TR values were observed among the four studied populations. The offspring of the Cx. pipiens infected females were WNV negative.
Our study addressed an important issue in the knowledge on the complex WNV-vector relationships in Italy, indicating that all Italian Cx. pipiens populations tested exhibited vector competence for WNV. Further studies should be performed in order to better clarify the role of other factors (vector density, climatic conditions, reservoir presence etc.) in order to predict where and when WNV outbreaks could occur.
- Vector competence
- West Nile virus
- Cx. pipiens
West Nile virus (WNV, family Flaviviridae, genus Flavivirus) is an emerging zoonotic arthropod-borne virus (arbovirus) widely distributed throughout the world and with considerable impact on veterinary and human health [1, 2]. The virus is maintained and amplified in nature within an enzootic transmission cycle among birds and ornithophilic mosquitoes . The virus was first isolated in 1937 in Uganda, then sporadic cases and epidemic outbreaks were reported in other African countries and in the Middle-East, mainly involving horses and humans . In 1999 an important WNV outbreak occurred in New York City, while in Europe the virus was only reported up to the end of the 1990’s [5–7]. The largest human outbreaks occurred in Romania in 1996 and Russia in 1999 [8, 9]. Concerning other countries in the EU area, cases of WNV encephalitis were initially recorded in 2000 on horses in Southern France  and seropositivity for the WNV was reported for horses, humans and birds in Portugal [11, 12] and Spain [13, 14]. In Spain, the first clinical cases of WNV were reported on horses and humans in 2010 . Since 2003 evidence of WNV circulation among birds has been reported also in the UK . Nevertheless, in Austria, Germany and Czech Republic increasing WNV antibody titres were highlighted between 2009–2010 . In the last years, WNV caused large human epidemics in the Balkan area including Greece and Romania [18, 19]. In Italy, the first WNV outbreak occurred in Tuscany in 1998 involving horses only . No further outbreaks were reported until late summer of 2008 when human cases of WNV disease were reported in the regions of Emilia Romagna, Lombardia and Veneto (North-Eastern Italy). These regions are now considered endemic for this virus. From 2008–2012 WNV foci were also reported in other Italian regions (Sardinia, Sicily, Calabria, Basilicata and Marche), with a total of 73 human cases of confirmed neuro-invasive disease [21–25]. More recently, from June to November 2013, 40 cases of neuro-invasive disease have been reported in Italy with a mortality rate of 17.5 % . Entomological and epidemiological surveillance have highlighted the circulation in Italy of both lineage 1 and 2 [25, 27].
Mosquitoes species belonging to the genus Culex are considered important vectors for WNV. In particular Culex pipiens is thought to play a major role both in enzootic and epizootic cycles [28–31]. This species comprises two distinct forms, pipiens and molestus, that differ in several behavioural and ecological characteristics affecting their vector competence . When in sympatry, molestus and pipiens forms may interbreed generating hybrids that with their intermediate features may act as WNV-bridge vectors for WNV [33, 34].
Currently most of our knowledge of ecology, dynamics and genetic composition of vector populations, come from North America [35, 36]. Conversely, in Europe the distribution and circulation dynamics of WNV are not completely clear. WNV outbreaks occur nearly every year but in different and often widely separated regions. A number of studies have proposed that this pattern is the result of infected migratory birds arriving from Africa and seeding the virus in different areas .
The complexity of WNV eco-epidemiologic characteristics, involving different vectors and host species, has probably contributed to the recent increase in the circulation of this virus, that has become the most widespread mosquito-borne flavivirus in recent years . Recently several laboratory studies have been carried out on the potential vector competence of Cx. pipiens by experimental infections [35, 38, 39]. Vector competence is a measure of the intrinsic ability of an arthropod to become infected, to support the development or replication of a pathogen, and to transmit it to a vertebrate host.
The aim of this study was to evaluate the vector competence and to determine the potential role in WNV transmission of Cx. pipiens mosquitoes collected in different areas of Italy. For this purpose the susceptibility to WNV of Cx. pipiens laboratory colonies and field populations was assessed by experimental infections using membrane feeding technique. In addition the progeny from both the first (FGC) and the second (SGC) gonotrophic cycle of the tested populations was analysed in order to assess the possible virus overwintering and amplification maintenance by vertical transmission.
No specific permissions are required for the field activities which do not involve endangered or protected species. The field sites are not privately-owned or protected properties. The Istituto Superiore di Sanità (ISS) is the National Health Institute of Italy and represents the technical-scientific arm of the Ministry of Health (MoH). All our operative procedures and scientific protocols are previously authorized by the MoH. In this frame, it is involved in vector control activities which authorize it to operate without any specific permission of access to breeding sites and mosquito collections. Moreover the activities reported in the paper were carried out within the 7th Framework Program: European West Nile collaborative research project (Grant agreement n. 261391), approved by European and Italian scientific Committees. This study was carried out in accordance with the recommendations in the Animal Experimentation protocol. All mosquitoes used in our experiments were sacrificed by freezing at the end of the study. Rabbits and quails, used in this study, were not sacrificed. They were from the animal facility of the ISS, where they were maintained according to the Italian and European rules on Laboratory animal care. Blood was collected from the ear vein of the rabbit according to the European legislation for the care and the use of laboratory animals. This is not a part of veterinary care routine but only a blood sample for the infection experiments. At the time, when experiments were performed, the use of laboratory animals in Italy was regulated by legislative decree no. 116/92, which implemented the European Directive 86/609/EEC on laboratory animal protection. In accordance with this legislation the presence and approval of an Ethical Committee is not required; however local welfare veterinarians had the same functions as IACUCs. In particular, at ISS, the veterinarians working for the Service for Biotechnology and Animal Welfare performed the functions of local IACUC; they approved animal research protocols and they verified that the guidelines of legislative decree no. 116/92 on animal welfare were strictly and constantly implemented. All efforts were made to minimize animal suffering. Pig intestine epithelium, used for the experimental infections, is a commercial fresh product, very common in Italy (used for making sausages); it can be found in every butchery or supermarket. We just rinsed it with a saline solution and cut a square portion before use. According to European regulations, manipulations of pathogens belonging to the group 3 (WNV) were carried out in biosafety level 3 (BSL3) facilities. The viral stocks, obtained from human specimens after several passages on Vero cells, are used routinely for research studies. Our laboratory, as National Reference Laboratory for Arboviruses, ISS, routinely analyses human specimens for Arbovirus diagnosis, within the National epidemiologic surveillance activity “Surveillance of human cases of vector-borne diseases with particular reference to Chikungunya, Dengue, and West Nile Disease-2013” (Ministry of Health, DGPRE 0013699-P-14 June 2013 I.4.c.a.9/2011/24). All patient data is anonymized and a written informed consent has been obtained from all participants and/or parents of participants.
Culex pipiens population features
Before starting the experimental infections, the four Cx. pipiens populations were tested to exclude the presence of WN, Chikungunya and Dengue viruses. A representative number of specimens (more than 100) of each populations was analysed as pools of 20 individuals by using both quantitative Real Time PCR (qRT-PCR), and isolation on VERO cells.
WNV strain Ma V3, belonging to lineage 1, isolated on VERO cells from the cerebrospinal fluid (CSF) of a patient from Sardinia outbreak in 2011  was used for the experimental infections. The WNV stocks were obtained by propagation on VERO cells and then stored at - 80 °C in aliquots until use. The viral titre of the stocks was 8.43 log10 Plaque Forming Units/mL (PFU/mL).
To standardize the experimental infection technique, different conditions in the feeding system (membrane system, blood, humidity and room temperature) were tested, using Frascati as reference population, the most adapted to laboratory conditions. The experimental conditions showing the highest efficiency in mosquito feeding were selected and used for all infection experiments.
Infection experiments of the four populations were performed, at the same time, in BSL3 cabinet at room temperature of 28 °C and about 70 % RH. The infectious blood meal was composed of 2/3 rabbit blood with EDTA (Ethylenediaminetetraacetic acid) and 1/3 viral seed, with a final concentration of 7.97 log10 PFU/mL.
Female mosquitoes, 8–12 days old, were allowed to feed for 60 min through a pig intestine membrane covering the base of a glass feeder containing the blood-virus mixture, maintained at 37 °C by a warm water circulation system.
After the infectious blood meal, engorged females were selected, transferred to cages and maintained with 10 % sucrose in a climatic chamber at 28 ± 1 °C and 70 % RH.
In order to determine the length of viral extrinsic incubation period, fed females were monitored for 32 days after the infected blood meal. Collections of fed females were carried out at 0–6-9–14-21–28-32 days post-infection (p.i.) and at each time point 5–10 specimens were dissected to examine the viral content in body, legs plus wings and saliva. After chilling, wings and legs were removed from each mosquito to allow for saliva collection. For saliva collection the proboscis was inserted into a quartz capillary filled with 5 μl of Fetal Bovine Serum (FBS) for saliva collection. The mosquitoes were induced to salivate applying on the thorax 2 μl of 1 % pilocarpine in Phosphate buffered saline (PBS) . After 30 min, medium containing the saliva was collected into 500 μl of mosquito diluent (MD) consisting of PBS, 20 % heat-inactivated FBS, 1 % penicillin/streptomycin/amphotericin B mix (Invitrogen, GIBCO). Body, legs plus wings and saliva were stored at -80 °C until processing.
Cx. pipiens offspring
In order to detect a potential vertical transmission of the WNV, a sample of potentially infected females were allowed to lay eggs. Larvae from the first gonotrophic cycle (FGC) were reared up to adulthood in the climatic chamber. Samples of adults (separated by sex) were collected from the early (4th day p.i.) and late (8th - 10th day p.i.) ovipositions. A second uninfected blood meal was provided to the remaining females at 14th day p.i. and offspring from the second gonotrophic cycle (SGC) were also reared and the adult mosquitoes collected. All samples from FGC and SGC were stored at - 80 °C and processed as pools of 5–20 specimens.
Real time PCR and viral titration
WNV titre of infected mosquitoes was evaluated both by qRT-PCR and titration by PFU/mL on VERO cells.
For each mosquito, body and legs plus wings were homogenized separately, suspended in 1 mL of MD and centrifuged at 3000 × g for 30 min. The two supernatants obtained and the saliva were used for RNA extraction by using the QIAamp viral RNA kit (Qiagen Inc., Valencia, CA, USA) and for VERO cells inoculation. The qRT-PCR was performed by using WNV TaqMan primers and probe . Briefly, 7 μl of RNA was combined with 20 pmol of each primer and 4 pmol of the FAM- and TAMRA-labelled probe in a 20 μl total reaction volume by using the RNA Virus Master Roche (Roche Diagnostics, Basel, CH). The samples obtained were amplified in an CFX96 Touch™ Real-Time PCR Detection (Bio-Rad), with the following cycling times and temperatures: 1 cycle of retro transcription at 50 °C for 10 min, 1 cycle at 94 °C for 3 min and 45 cycles of 95 °C for 1 s, 60 °C for 20 s (acquisition mode: single), and 72 °C for 1 s (Ramp Rate: 2 °C/s).
Quantification of WNV in RNA samples was determined via qRT-PCR with specifications described previously to estimate plaque forming unit equivalents (PFUeq) [41, 42]. Crossing points values were compared to standard curve based on data acquired from 10-fold serial dilutions of virus stocks with estimated concentration by titration on VERO cells and expressed as log10 PFU/mL .
The viral titration was performed on six-well plates containing confluent monolayers of VERO cells infected with serial 10-fold dilutions of body and legs plus wings supernatants and of saliva. Cells were incubated at 37 °C for five days under an overlay consisting of Dulbecco’s MEM (DMEM), 2 % FBS, 1 % antibiotic-antimycotic mix (Invitrogen, Gibco) and 2 % tragacant gum (Sigma Aldrich). The plaques were counted after staining them with a solution of crystal violet (0.2 % in 10 % formaldehyde and 20 % ethanol).
Vector competence indexes
The vector competence of all mosquito populations was assessed by calculating the Infection Rate (IR), Dissemination Rate (DR) and Transmission Rate (TR). Mosquito bodies were analysed in order to evaluate the IR, calculated as the number of WNV positive bodies with respect to the total number of fed females. Legs plus wings were tested to assess the DR, calculated as the number of the specimens with WNV positive legs plus wings out of the number of specimens with WNV positive bodies. The saliva of the potentially infected females was processed to assess the TR, defined as the number of WNV positive saliva out of the number of specimens with WNV positive bodies. The potential vector competence was also expressed as Population Transmission Rate (PTR) calculated as the number of specimens with WNV positive saliva with respect to the total number of fed mosquitoes [42, 43].
Pearson’s Chi-squared test with Yates’ continuity correction was used to determine significant differences (p < 0.05) in IR, DR, TR and PTR among the four Cx. pipiens populations, and the Kruskall-Wallis test to compare the viral means titres. Statistical analyses were performed using the Stata®13 software (StartCorp LP, Texas, USA).
WNV replication dynamics in analysed mosquito populations
The trend of the mean viral titres, calculated by plaque titration, was very similar to that obtained by qRT-PCR (Fig. 2b). Indeed the mean WNV titre in the bodies increased progressively reaching a value of 6.02 ± 0.26 log10 PFU/mL at the 32nd day p.i. The presence of the virus in legs plus wings and saliva was also detected starting from day 14th p.i. with a titre of 3.06 and 1.02 log10 PFU/mL, respectively. The viral titres increased in these two compartments up to 32nd day p.i. with values of 4.43 ± 0.46 log10 PFU/mL in the legs plus wings and 3.45 ± 0.17 log10 PFU/mL in the saliva (Fig. 2b).
Our results highlighted that in all Cx. pipiens populations, WNV was able to infect mosquitoes and replicate over time, disseminating in wings and legs and reaching the salivary glands.
WNV vector competence analysis
Vector competence indexes for WNV of four Italian populations of Cx. pipiens
Chi 2 Test
p = 0.037
p = 0.001
(legs plus wings WNV+/bodies WNV+)
p = 0.915
(saliva WNV+/bodies WNV+)
p = 0.758
In order to detect a possible vertical transmission of WNV in mosquitoes, a total of 377 adult specimens from both the FGC and SGC of the four Cx. pipiens populations were analysed in pools. No evidence of vertical transmission was detected, with all 245 male and 132 female progeny testing negative for WNV.
Although WNV has been steadily circulating since 2008 in some regions of Northeastern Italy, and sporadically in other areas of the Country, up to now, no study of vector competence on Cx. pipiens Italian populations has been carried out. According to the literature, Cx. pipiens is the most common mosquito in many European countries and is also considered to be the primary vector for WNV [21, 28, 31].
In this study, we evaluated the susceptibility for WNV infection, dissemination, and transmission of four Cx. pipiens populations originating from North, Central and South/Island areas of Italy, as representative of the whole Country. To our knowledge, this is the first description of the time course study of WNV growth in Italian populations of Cx. pipiens. All of the mosquito populations tested were susceptible to infection and the viral growth titres were not significantly different (Figs. 2 and 3). Cx. pipiens mosquitoes, both wild (Legnaro and Zafferana) and reared in Insectarium for several generations (Frascati F11 and Caffarella F7), showed a low barrier to midgut infection and WNV was able to escape from the midgut to legs and wings (cumulative IR and DR ≥50 %). IR and DR values of the wild Legnaro population (Table 1), coming from an Italian WNV endemic area, were significantly higher compared to the other populations (p = 0.037 and p = 0.001, respectively). The cumulative TR of the tested populations showed values ranging from 37–47 % (differences not statistically significant, p = 0.915), suggesting the capacity of WNV to reach the salivary glands in all four populations. In addition, the viral titres in the saliva, estimated as genome equivalents, showed no remarkable differences among the four populations, exhibiting values similar to those observed in the literature (2–4 log10 PFU/mL) and thought sufficient to successfully infect a potential host [39, 44, 45]. In accordance with the cumulative TR values, the PTRs obtained, measuring the real potential susceptibility of a mosquito population to be infected, confirmed the comparable vector competence of the four Italian Cx. pipiens populations (values ranging from 21–33 %, p = 0.758).
Generally, the genetic origin (i.e. the degree of anthropophily) and local environmental components (i.e. climate, host seeking, accessibility to humans) represent factors influencing the real efficiency of a vector species (vectorial capacity) [46–48]. The experimental infections, performed under controlled conditions, eliminate or normalize the influence of the extrinsic factors above mentioned. On these bases, eventual differences in the vector competence among mosquito populations of the same species should be reasonably attributed mainly to intrinsic factors. Actually, the question whether the efficiency of a mosquito species to be a good vector may depend on a genetic trait only, is still the object of discussion. Different studies led to hypothesize that the molestus form, or even the hybrid ones, may be more competent to transmit WNV [33, 34]. In our study, we assumed a pipiens characterization for both the Northern and Southern populations due to the absence of autogenous females. On the contrary, the molestus component was certainly present in the other two populations from Central Italy, as displayed already from the first generation with the autogenous ovipositions. Indeed, our results demonstrated that the Cx. pipiens populations, even if potentially different in their genetic traits, showed similar capacity to be infected and potentiality to transmit the virus. In addition although different levels of susceptibility to WNV infection have been documented in Cx. pipiens populations collected from different areas of California [46, 47], our populations, collected in different areas of Italy, showed a similar vector competence. Despite these results, recurring outbreaks occur in Northeastern Italy but occasionally also in other areas of the Country. These differences in the actual circulation of WNV in Italy seem to depend on a complex of extrinsic factors rather than simply on different intrinsic traits within Cx. pipiens populations. Recently Mulatti et al. highlighted how the mosquito density, in combination with key environmental factors, is relevant for a robust prediction of Cx. pipiens population expansion and WNV transmission risk . Therefore, the assessment of the genetic characterization of the Italian Cx. pipiens populations tested, and the role of the local environmental factors, is in progress in order to better understand of the vectorial capacity of this species.
The analysis of the adults of the FGC and SGC offspring of the four populations didn’t show any evidence of WNV transmission to progeny. However, even if the number of offspring analysed in our study may be considered low (245 males and 132 females), our findings seem to suggest the hypothesis that the vertical transmission would represent a sporadic event not strictly needed for the survival of WNV in the temperate areas of Europe . In addition, very low rates of vertical transmission have been reported also in some WNV experimental infections for North American Cx. pipiens populations [50, 51]. Moreover, even if Monaco et al., on the basis of epidemiological studies in North East areas of Italy, strongly suggested the possibility of the WNV overwintering , up to now no vertical transmission has been demonstrated in mosquitoes field collected in Italy.
The results of our study indicate that Cx. pipiens Italian populations exhibited a good vector competence for Italian WNV lineage 1 strain, addressing an important issue in the complex WNV-vector relationships. Insights on vector competence, distribution, seasonal density, and natural feeding patterns of Cx. pipiens can help to improve WNV surveillance in Italy, since current knowledge does not allow yet to predict where and when WNV outbreaks will occur.
The authors thank Andrea Martinelli and Gioia Bongiorno, Simone Martini, Fabrizio Piccari, Marco Fermetti and Giulietta Venturi for their fundamental contribution to this work and Judith Sutcliffe [BA (Hons) Eng. Lang and Lit] for the linguistic revision. This work was funded by the 7th Framework Program: European West Nile collaborative research project (Grant agreement n. 261391).
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