Dr Dominique Higuet

Dr Miguel Angel

Dr Jimenez Clavero

Dr Navid Dinparast Djadid

Vincent Robert

Stephanie Ghosn

Leila Tawk

Hanadi Saliba

Sandy El

Joelle Sassine

Catherine Ghanem

Najibe Abou

Marie Vazeille

Laurence Mousson

Faustine Louis

Aya Awde

Zeina Shreif

Khouloud Ghosn

Aya Nasrallah

Faycal Khodr

Mahdi Fadel

Mariana Abi Saad

Haysam Omran

Elie Hamam

Yara El Morr

Sandra Zoghby

Dr I Furthermore

Laura Kramer

Renée Zakhia

Nabil Haddad

Anna-Bella Failloux
email: bella.failloux@pasteur.fr

Keywords: West Nile Virus, Rift Valley Fever Virus, Culex pipiens, Lebanon, Vector competence, Seroprevalence. vi Virus du Nil Occidental, Virus de la fièvre de la Vallée du Rift, Culex pipiens, Liban, Compétence Vectorielle, Séroprévalence Risk assessment, Emergence, Arboviruses, Vector competence

Célestine Atyame, Fadila Amraoui, and Anubis Vega Rua for teaching me the secrets of mosquito manipulations

List of Figures

 [START_REF] Smithburn | A Neurotropic Virus Isolated from the Blood of a Native of Uganda[END_REF]. Usually, only 20% of infected individuals develop symptoms and less than 1% develops serious and potentially fatal neurological illnesses such as meningitis and encephalitis. Birds are considered the main reservoir and migratory birds are involved in the introduction of the virus into new regions [START_REF] Andreadis | The contribution of Culex pipiens complex mosquitoes to transmission and persistence of West Nile virus in North America[END_REF]. WNV infections have been reported in many tropical and temperate countries in Africa, Europe, Asia and America (CDC, 2017;[START_REF] Failloux | Surveillance of Arthropod-Borne Viruses and Their Vectors in the Mediterranean and Black Sea Regions Within the MediLabSecure Network[END_REF]. The East Mediterranean region (EMR) has long been considered endemic for WNV. Locally acquired cases have been recently reported in Israel [START_REF] Paz | Environmental drivers of West Nile fever epidemiology in Europe and Western Asia--a review[END_REF], Greece [START_REF] Papa | Emerging arboviral human diseases in Southern Europe[END_REF], and Turkey [START_REF] Kalaycioglu | Emergence of West Nile virus infections in humans in Turkey, 2010 to 2011[END_REF]. In addition, evidence of WNV circulation has been reported

from Jordan [START_REF] Batieha | Seroprevalence of West Nile, Rift Valley, and sandfly arboviruses in Hashimiah, Jordan[END_REF] and Egypt [START_REF] Soliman | Studies on West Nile virus infection in Egypt[END_REF]. It is worth noting that the introduction of WNV into the United States in 1999, which constitutes a turning point in WNV epidemiology, is thought to have originated from the EMR (Israel) following introduction from Africa [START_REF] Giladi | West Nile encephalitis in Israel, 1999: the New York connection[END_REF][START_REF] Lanciotti | Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States[END_REF].

WNV is mainly transmitted by mosquitoes of the Culex genus especially those belonging to the Culex pipiens complex [START_REF] Turell | Members of the Culex pipiens complex as vectors of viruses[END_REF]. In the Mediterranean basin, this complex is mainly represented by Cx. pipiens s.s. under its two biotypes: Cx. pipiens pipiens and Cx. pipiens molestus.

To date, no WNV cases have been reported from Lebanon. However, the country is considered at risk for WNV outbreak. Not only it is geographically part of an endemic region, but also Lebanon is located on the flyways of migratory birds. Besides, entomological studies conducted in 1999-2001 showed a widespread of Culex pipiens [START_REF] Knio | A two-year survey on mosquitoes of Lebanon[END_REF]. Additional entomological surveys conducted by our laboratory (Laboratory of Immunology and Vector-Borne Diseases, Lebanese University) during the last decade confirmed the spread of this mosquito that colonizes urban and rural habitats (unpublished data).

The abundance of Culex pipens is considered a risk factor for local transmission, not only of WNV, but of also other arboviruses. Indeed, this species is a possible vector of Rift Valley Fever Virus (RVFV). This virus is of particular interest for Lebanon and the Mediterranean countries.

It is mainly spread in the Sub-Saharan region in Africa [START_REF] Himeidan | Recent outbreaks of rift valley Fever in East Africa and the middle East[END_REF]. Recorded substantial expansion to areas close to the Mediterranean basin, such as Egypt [START_REF] Meegan | The Rift Valley fever epizootic in Egypt 1977-78[END_REF]), Mauritania (El Mamy et al., 2011), Senegal and even the Arabian Peninsula (Saudi Arabia and Yemen [START_REF] Ahmad | More deaths from Rift Valley fever in Saudi Arabia and Yemen[END_REF]) should be considered as a health threat for Mediterranean countries, including Lebanon. Intensive livestock trade between Lebanon and these countries increases the risk of RVFV introduction.

During this PhD research project, we aimed to assess the circulation of WNV in Lebanon and the capacity of local strains of Cx. pipiens to transmit WNV and RVFV under experimental conditions. Therefore, our project is divided into two sections:

The first section covers the epidemiology of WNV in Lebanon and attempts to provide evidence of WNV circulation by screening mosquito populations for WNV RNA and by detecting WNV specific antibodies among human, horses and chicken populations.

The second section of this study includes an assessment of the vector competence of local populations of Cx. pipiens against both WNV and RVFV. For this, experimental transmission assays were conducted in Biosafety Level 3 (BSL3) facilities at the Unit of Arboviruses and Insect Vectors (Institut Pasteur, Paris, France). Moreover, the difference of vector competence between both Cx. pipiens biotypes was investigated.

Part 1

Literature Review

Chapter 1

Overview on Culex pipiens mosquito vector

Culex pipiens complex mosquitoes belong to the Culicidae family, Culicinae subfamily.

They have a global distribution and are considered vectors of several medically important arboviruses such as West Nile Virus (WNV) and Rift Valley Fever Virus (RVFV).

Classification

There are more than 3500 species of mosquitoes, all belonging to the Culicidae family [START_REF] Service | Medical Entomology for Students[END_REF] that is divided into three subfamilies: Toxorhynchitinae, Anophelinae (anopheline) and Culicinae (culicine). The subfamily Culicinae comprises more than 40 genera [START_REF] Service | Medical Entomology for Students[END_REF] among which Culex and Aedes include an important number of medically important species. Culex mosquitoes are widespread in almost all the continents with more than a thousand species [START_REF] Becker | Mosquitoes and Their Control[END_REF]. Within this genus, we recognize Culex pipiens which is considered a complex or assemblage of medically important species [START_REF] Harbach | Culex pipiens: species versus species complex taxonomic history and perspective[END_REF].

Culex pipiens mosquitoes are found worldwide, but they are absent from the extreme northern parts of the temperate zones. The taxonomy of the Cx. pipiens complex is controversial and has constantly changed. Based on Harbach's last revision [START_REF] Harbach | Culex pipiens: species versus species complex taxonomic history and perspective[END_REF], the Cx. pipiens complex includes Cx. pipiens s.s., Cx. quinquefasciatus and the form pallens and possibly, Cx.

australicus [START_REF] Harbach | Culex pipiens: species versus species complex taxonomic history and perspective[END_REF][START_REF] Harbach | Culex (Culex) pipiens Linnaeus (Diptera: Culicidae): concepts, type designations, and description[END_REF][START_REF] Turell | Members of the Culex pipiens complex as vectors of viruses[END_REF]. The females of the complex members cannot be distinguished morphologically. Cx. pipiens s.s .includes two biotypes or subspecies: Cx. pipiens pipiens and Cx. pipiens molestus [START_REF] Fonseca | Emerging vectors in the Culex pipiens complex[END_REF][START_REF] Harbach | Culex pipiens: species versus species complex taxonomic history and perspective[END_REF]. [START_REF] Harbach | Culex pipiens: species versus species complex taxonomic history and perspective[END_REF] considered that the nominal forms pipiens and quinquefasciatus are phylogenetically distinct species; they do not interbreed in the region where they co-exist. They are distinct outside the areas of introgression where the form pallens is described. According to this author, the pallens form does not meet the criteria for subspecies status. Harbach did not conclude on the status of Culex australicus and considered that this taxon "may or may not be a member of the pipiens assemblage" [START_REF] Harbach | Culex pipiens: species versus species complex taxonomic history and perspective[END_REF].

Geographic distribution

A worldwide general distribution map of the Culex pipiens complex species was elaborated by Farajollahi and collaborators (2011) (Figure 1). Culex pipiens s.s. (including both biotypes and their hybrids) is present in European and Asian countries in addition to all Mediterranean countries including Lebanon [START_REF] Farajollahi | Bird biting" mosquitoes and human disease: a review of the role of Culex pipiens complex mosquitoes in epidemiology[END_REF][START_REF] Knio | A two-year survey on mosquitoes of Lebanon[END_REF]. This species has been accidentally introduced into temperate zones in North and South America, while only Cx. pipiens biotype molestus has been introduced to cities in Japan, Republic of South Korea, and Australia [START_REF] Farajollahi | Bird biting" mosquitoes and human disease: a review of the role of Culex pipiens complex mosquitoes in epidemiology[END_REF][START_REF] Fonseca | Emerging vectors in the Culex pipiens complex[END_REF]. Cx. quinquefasciatus thrives in tropical and subtropical regions including the African lowlands, Americas, Asia, and Australia as shown in the distribution map (Figure 1). A recent study conducted by Shaikevich and collaborators (2016) provided the first molecular evidence of the presence of hybrids between Cx. quinquefasciatus and Cx. pipiens in coastal regions of Southern Europe and Mediterranean region (Morocco) [START_REF] Shaikevich | Genetic diversity of Culex pipiens mosquitoes in distinct populations from Europe: contribution of Cx. quinquefasciatus in Mediterranean populations[END_REF]. In addition, they confirmed the presence of hybrids between pipiens and molestus biotypes in the study areas in Europe and around the Mediterranean Sea [START_REF] Shaikevich | Genetic diversity of Culex pipiens mosquitoes in distinct populations from Europe: contribution of Cx. quinquefasciatus in Mediterranean populations[END_REF].

External morphology of adults

Adults of Culex pipiens, as any other mosquito, have a body that is divided into three parts: a head, thorax and abdomen (Figure 2). The head has a conspicuous forward-projecting proboscis and carries a pair of segmented antennae. Mosquitoes can be sexed by examining their antennae:

individuals with feathery antennae are males while those with only short and rather inconspicuous antennal hairs are females. In addition, the palps (below the antennae) of female mosquitoes (as female Culicines) are very short while in males they are long. Arising between the palps is the single long proboscis which in females contains the piercing mouthparts, thus only female mosquitoes can bite. The thorax is ornamented with numerous scales that allow the discrimination between species. Moreover, the thorax provides an insertion site of three pairs of legs and one pair of functional wings. The abdomen of mosquitoes is composed of 10 segments, but only the eight are visible. The last two segments are reduced and form the genitalia [START_REF] Service | Medical Entomology for Students[END_REF]. The abdomen is usually covered dorsally and ventrally with brown and white scales. The dorsal parts of the abdomen are called tergites, they are important criteria for Culex species identification [START_REF] Service | Medical Entomology for Students[END_REF].

At the Culex genus level:

-a scutellum clearly trilobate in thorax (Figure 3.A)

-absence of both pre-spiracular and post-spiracular setae in thorax -sensory organs at the apex of the tarsomere 5 with an empodium and 2 pulvilli -a wing characterized by: a fringe of scales in the alula and the apex of the vein 1-A localized beyond the mcu/cuA fork (Figure 3.B)

At the species level:

-a wing with an entirely dark costal vein (Figure 3.B)

-entirely dark legs (femur, tibia and tarsomeres of the 3 pairs of legs)

-basal pale band on their tergites (Figure 3.C) Morphological discrimination between members of the Cx. pipiens complex is very difficult and sometimes not possible. Cx. pipiens pipiens and Cx. pipiens molestus can only be distinguished using molecular techniques such as Polymerase Chain Reaction (PCR) [START_REF] Bahnck | Rapid assay to identify the two genetic forms of Culex (Culex) pipiens L. (Diptera: Culicidae) and hybrid populations[END_REF][START_REF] Fonseca | Emerging vectors in the Culex pipiens complex[END_REF][START_REF] Price | Genetic divergence between populations of feral and domestic forms of a mosquito disease vector assessed by transcriptomics[END_REF].

Life cycle and biology of different stages

Culex pipiens mosquitoes, as all Culex species, go through four separate and distinct stages during their life cycle: egg, larva, pupa, and adult (Figure 4).

a. Oviposition and biology of eggs

Female Culex pipiens mosquitoes lay their eggs on the surface of fresh water or in temporarily wet areas like flood plains (Figure 5). They lay up to 300 eggs in one oviposition directly on the water surface [START_REF] Service | Medical Entomology for Students[END_REF]. Eggs are usually brown, long and cylindrical of 1mm or less in length. They are laid upright in several rows on the water surface and held together by surface tension to form an egg raft that floats on the water. These eggs cannot survive to desiccation and die when dry. Time from egg hatching to larvae stage varies from 2 to 3 days in the tropical regions, 7 to 14 days in cooler temperate areas [START_REF] Becker | Mosquitoes and Their Control[END_REF].

b. Larval biology

Larvae have a well-developed head bearing a pair of antennae and a pair of compound eyes.

The thorax is roundish and usually has long and conspicuous hairs. The last segment of the abdomen differs in shape from the other segments. It ends with two pairs of transparent sausageshaped anal papillae, which are concerned with osmoregulation [START_REF] Becker | Mosquitoes and Their Control[END_REF]. Cx. pipiens larvae must come to the water surface to breath. Atmospheric air is taken in through a pair of spiracles situated at the end of a single tube termed the respiratory siphon. Cx. pipiens larval siphon is often long and narrow, possessing always more than one pair of subventral tufts of hairs.

There are four active larval instars. They feed on yeasts, bacteria, protozoans, decaying organic matter found in the water. Larval development (time from egg hatching to pupation) depends on areas, temperatures and species, it can be 5 to 7 days, but may also last about 7 to 14 days [START_REF] Becker | Mosquitoes and Their Control[END_REF].

Larval habitats of Culex pipiens mosquitoes, as most Culex species, are ground collections of water such as pools, puddles, ditches, borrow pits and rice fields. In urban areas, this species lays eggs in man-made container-habitats such as tin cans, water receptacles, bottles and waterstorage tanks with variable amount of organic matter [START_REF] Service | Medical Entomology for Students[END_REF].

c. Pupae biology

Cx. pipiens mosquito pupae are aquatic and comma-shaped [START_REF] Service | Medical Entomology for Students[END_REF]. The head and thorax are combined to form the cephalothorax, which dorsally possesses a pair of respiratory trumpets. Pupae do not feed but spend most of their time resting at the water surface to take air [START_REF] Service | Medical Entomology for Students[END_REF]. The pupal development may take 2-3 days in tropical areas, but in cooler temperate regions, pupal period can last 9-12 days. At the end of pupal life the skin on the dorsal surface of the cephalothorax splits, and the adult mosquito struggles out [START_REF] Becker | Mosquitoes and Their Control[END_REF].

d. Adult biology and behavior

Most Cx. pipiens mosquitoes mate shortly after emergence. Sperm from a male enter the spermatheca of a female, and this usually serves to fertilize all eggs laid during her lifetime [START_REF] Becker | Mosquitoes and Their Control[END_REF]. Female mosquitoes must bite a host and take a blood-meal to obtain the necessary nutrients for the development of eggs. This is referred as anautogenous development [START_REF] Service | Medical Entomology for Students[END_REF]. However, some Cx. pipiens forms can develop the first batch of eggs without a blood-meal, and more rarely, subsequent batches. This process is called autogenous development [START_REF] Service | Medical Entomology for Students[END_REF]. The blood-meal digestion depends on temperature and varies from 2 to 3 days in hot climates, to 7-14 days in colder areas. After full digestion, the Cx. pipiens female is gravid and ready to lay eggs in suitable larval habitats [START_REF] Service | Medical Entomology for Students[END_REF]. Male Cx. pipiens mosquitoes, along with other species, cannot bite but feed on the nectar of flowers and other natural sugar secretions, along with females in order to obtain energy for flying.

Adult Cx. pipiens mosquitoes mainly disperse only a few hundred meters from their emergence sites. In Japan, a mark-release-recapture of Culex pipiens pallens showed that the maximum flight distance was estimated as 1217m with a mean distance flight that did not exceed 517m [START_REF] Tsuda | A mark-release-recapture study on dispersal and flight distance of Culex pipiens pallens in an urban area of Japan[END_REF]. In some cases, this distance is longer when the mosquitoes are windassisted. In tropical areas, adult female mosquitoes live 1-2 weeks in average whereas in temperate countries, adult life may be extended to 3-4 weeks [START_REF] Becker | Mosquitoes and Their Control[END_REF]. Species that hibernate live much longer: for instance in Europe, some fertilized female Culex pipiens survive in hibernation from season to another [START_REF] Becker | Mosquitoes and Their Control[END_REF]. Adult male mosquitoes have a shorter life span than females.

Cx. pipiens subspecies differ significantly in physiology, behavior and ecology (Table 1). Cx.

pipiens biotype pipiens is mainly present in temperate areas, ornithophilic (bird biting host preference), anautogenous and eurygamous (mating in outdoor swarms only) whereas the biotype molestus, thrives in sewers in sub-tropical regions, is mammophilic (prefers biting mammals including humans), autogenous and stenogamous (ability to mate in a confined space) [START_REF] Fonseca | Emerging vectors in the Culex pipiens complex[END_REF][START_REF] Harbach | Culex pipiens: species versus species complex taxonomic history and perspective[END_REF]. The hybrids of these two biotypes show opportunistic and different feeding behaviors. Another member of the complex, Culex quinquefasciatus is a noticeably domestic species and common in urbanized areas. Especially in towns with poor and inadequate drainage and sanitation, this mosquito breeds mainly in water polluted with organic matter [START_REF] Harbach | Culex pipiens: species versus species complex taxonomic history and perspective[END_REF].

The adult females of the Cx. pipiens complex bite humans and animals throughout the night, indoors and outdoors [START_REF] Harbach | Culex pipiens: species versus species complex taxonomic history and perspective[END_REF]. During the day, they are inactive and are often found resting in dark corners of rooms and shelters. They may also rest outdoors on vegetation and in tree holes in forested areas [START_REF] Harbach | Culex pipiens: species versus species complex taxonomic history and perspective[END_REF].

Medical importance

In several countries, members of Culex pipiens complex cause considerable nuisance, annoyance and irritation because of their troublesome bites. They are considered as main vectors, and sometimes reservoirs, of several medically important pathogens that infect humans including filarial worms, wild life pathogens such as avian malaria (Plasmodium species) and some arboviruses like Saint Louis Encephalitis Virus (SLEV), WNV, and RVFV.

Culex pipiens fatigans (now referred as Culex quinquesfasciatus) was considered a vector of Bancroftian filariasis in East Africa [START_REF] White | Studies on transmission of Bancroftian filariasis in North-Eastern Tanzania[END_REF]. This species was found infected with microfilariae of Wuchereria bancrofti in North-Eastern Tanzania between 1968 and 1970 [START_REF] White | Studies on transmission of Bancroftian filariasis in North-Eastern Tanzania[END_REF]. Culex pipiens mosquitoes were implicated as natural vectors of Dirofilariasis in Central Italy [START_REF] Cancrini | Aedes albopictus and Culex pipiens implicated as natural vectors of Dirofilaria repens in central Italy[END_REF]. In 2002-2003, mosquito collections were performed using dog-baited traps and Dirofilaria immitis and Dirofilaria repens were found in three pools of Cx.

pipiens [START_REF] Cancrini | Aedes albopictus and Culex pipiens implicated as natural vectors of Dirofilaria repens in central Italy[END_REF]. In addition, three pools of field-collected Cx. pipiens were found naturally infected with Dirofilaria immitis in Central Turkey [START_REF] Yildirim | Aedes vexans and Culex pipiens as the potential vectors of Dirofilaria immitis in Central Turkey[END_REF]. In addition, avian malaria parasites can be transmitted by Cx. pipiens species complex. Plasmodium parasites were detected in Cx. pipiens collected by bird-baited traps in 2004 in New York [START_REF] Kimura | Avian malaria parasites share congeneric mosquito vectors[END_REF].

In the United States, from 1977 to 1986, nine outbreaks of SLEV occurred [START_REF] Monath | St. Louis encephalitis: lessons from the last decade[END_REF] during which both Cx. pipiens and Cx. quinquefasciatus were found naturally infected with the virus [START_REF] Monath | St. Louis encephalitis: lessons from the last decade[END_REF]. Two strains of SLEV were isolated from overwintering

Cx. pipiens mosquitoes collected in Maryland and Pennsylvania during January and February 1977 [START_REF] Bailey | Isolation of St. Louis encephalitis virus from overwintering Culex pipiens mosquitoes[END_REF]. Moreover, a Saint Louis encephalitis epidemic in Grand Junction, Colorado, occurred in 1985. No human cases were reported, but mosquito surveillance disclosed SLEV in Culex tarsalis and Culex pipiens pipiens field collected mosquitoes [START_REF] Tasi | Entomologic studies after a St. Louis encephalitis epidemic in Grand Junction, Colorado[END_REF].

One of the predominant vectors of WNV is Cx. pipiens. Field-collected Cx. pipiens mosquitoes were found to be naturally infected with the virus in several countries in the world [START_REF] Hubálek | West Nile fever--a reemerging mosquito-borne viral disease in Europe[END_REF]. WNV was isolated from Cx. pipiens mosquito pools in Romania [START_REF] Tsai | West Nile encephalitis epidemic in southeastern Romania[END_REF], Portugal [START_REF] Almeida | Potential mosquito vectors of arboviruses in Portugal: species, distribution, abundance and West Nile infection[END_REF], Italy [START_REF] Calzolari | Evidence of simultaneous circulation of West Nile and Usutu viruses in mosquitoes sampled in Emilia-Romagna region (Italy) in 2009[END_REF], Egypt [START_REF] Soliman | Studies on West Nile virus infection in Egypt[END_REF], Israel [START_REF] Orshan | Mosquito vectors of West Nile Fever in Israel[END_REF], Greece [START_REF] Papa | West Nile virus in mosquitoes in Greece[END_REF][START_REF] Patsoula | West Nile Virus Circulation in Mosquitoes in Greece (2010-2013)[END_REF], and Turkey [START_REF] Ergunay | Arboviral surveillance of field-collected mosquitoes reveals circulation of West Nile virus lineage 1 strains in Eastern Thrace, Turkey. Vector Borne Zoonotic[END_REF]. Hubalek and Halouzka (1999) mentioned in their review that WNV was also isolated from Cx. pipiens in South Africa, Czech Republic and Bulgaria. In addition, the same authors stated that WNV was as well isolated from Cx. quinquefasciatus in India, Pakistan, and Madagascar. Moreover, a study conducted by [START_REF] Andreadis | The contribution of Culex pipiens complex mosquitoes to transmission and persistence of West Nile virus in North America[END_REF] showed that, WNV isolated from field collected Cx. quinquefasciatus mosquito pools were predominant in Southeast and Southwest United States, while Cx. pipiens was the predominant vector of WNV in northeastern United States [START_REF] Andreadis | The contribution of Culex pipiens complex mosquitoes to transmission and persistence of West Nile virus in North America[END_REF]. Furthermore, a study showed that Cx. pipiens mosquitoes were able to vertically transmit WNV [START_REF] Dohm | Experimental vertical transmission of West Nile virus by Culex pipiens (Diptera: Culicidae)[END_REF]. This type of transmission amplifies the virus and increases the transmission risk.

Cx. pipiens complex is also considered a secondary vector of RVFV. Some countries reported field-collected Cx. pipiens mosquitoes naturally infected with the virus [START_REF] Tantely | A review of mosquitoes associated with Rift Valley fever virus in Madagascar[END_REF]. RVFV was successfully isolated from Cx. pipiens mosquitoes in Egypt [START_REF] Hoogstraal | The Rift Valley fever epizootic in Egypt 1977-78[END_REF] and Sudan [START_REF] Seufi | Role of Culex and Anopheles mosquito species as potential vectors of rift valley fever virus in Sudan outbreak, 2007[END_REF]. In addition, the virus was isolated from Cx.

quinquefasciatus in Kenya [START_REF] Sang | Rift Valley fever virus epidemic in Kenya, 2006/2007: the entomologic investigations[END_REF].

Cx. pipiens was indicated as a suspected vector of Usutu virus (USUV). In Italy, USUV was detected in 54 Cx. pipiens pools [START_REF] Calzolari | Evidence of simultaneous circulation of West Nile and Usutu viruses in mosquitoes sampled in Emilia-Romagna region (Italy) in 2009[END_REF]. Moreover, sequences of this virus were found in one pool of this species in Spain in 2006 collections [START_REF] Busquets | Usutu virus sequences in Culex pipiens (Diptera: Culicidae), Spain[END_REF].

Mosquito control methods and challenges

Mosquito control manages medically important mosquito populations including Culex pipiens in order to reduce their impact on human health, economies, and pathogen transmission.

Several mosquito control measures are directed at either the immature aquatic stages of the mosquito or the adults using environmental management, physical, biological, chemical and genetic methods. In addition, personal protection measures that limit people contact with adult mosquitoes are also implemented. It is a key component in the prevention of mosquito nuisance and mosquito-borne diseases. Measures may include: simply staying indoors, wearing clothing that minimizes exposed skin, use of personal repellents on skin or clothing, and use of Insecticide treated nets (ITN) [START_REF] Becker | Mosquitoes and Their Control[END_REF][START_REF] Service | Medical Entomology for Students[END_REF].

Several environmental management concepts aiming to reduce mosquito breeding sites can be applied, but these measures differ between urban and rural areas and may sometimes be difficult to implement especially in urban areas [START_REF] Becker | Mosquitoes and Their Control[END_REF]. Habitats for many mosquito species can be diminished by monitoring and eliminating wide range of water bodies and also by, appropriately designing urban structures and sanitation to prevent formation of breeding sites.

Several physical control techniques exist for both immature aquatic mosquito stages and adults. For example, larvae and pupae can be suffocated and deprived from water tension using polystyrene beads and monomolecular surface films. Physical control techniques are attractive since they are effective against mosquitoes resistant to conventional insecticides.

Biological control is performed by the reduction of the target mosquito population, to an "acceptable" level, by the use of predators, parasites, pathogens, competitors, or toxins from microorganisms. These measures are mostly efficient against the immature aquatic stages of the mosquito, for which some vertebrate and invertebrate predators can be used successfully such as the mosquito fish (Gambusia affinis), amphibians, birds and several others [START_REF] Becker | Mosquitoes and Their Control[END_REF].

Pathogens such as fungi can also be adopted targeting larval stages of mosquitoes. These measures must be controlled in order to maintain biodiversity and seek for the minimal impact on the environment. The use of toxins such as Bacillus thuringiensis toxin (Bti), is largely adopted in many industrialized countries. This environmentally safe product remains expensive and therefore rarely used in low-income regions.

The development of chemical insecticides has led to many major advances in mosquito control. Several families of insecticides are used indoors or outdoors: chlorinated hydrocarbons (organochlorines), organophosphates (OP), carbamates, and pyrethroids, and a special class referred to as insect growth regulators (IGRs) [START_REF] Becker | Mosquitoes and Their Control[END_REF]. According to the World Health Organization Pesticide Evaluation Scheme (WHOPES), the most used organochlorine in the African region is Dichlorodiphenyltrichloroethane (DDT). In other regions, the most used insecticides are the organophosphate Malathion and the pyrethroid Permethrin. The carbamate Propoxur is also used for indoor residual spraying (IRS). There are two major groups of IGRs that differ in their mode of action. The first is the chitin synthesis inhibitors which interfere with new cuticle formation, resulting in moult disruption [START_REF] Becker | Mosquitoes and Their Control[END_REF]. The second is the juvenile hormone analogs that interfere with the metamorphic processes affecting development to the adult stage [START_REF] Becker | Mosquitoes and Their Control[END_REF].

The main constraint remains the difficulty to target the adult and larval stages in their resting or breeding sites. The auto-dissemination approach is a relatively new approach that aims to better target the adult stage. It is based on exposing wild females to artificial resting sites contaminated with pyriproxyfen. Then females can disseminate this juvenile hormone analogue to larval habitats, thus interfering with adult emergence. This approach was applied on Aedes aegypti and Aedes albopictus [START_REF] Caputo | The "auto-dissemination" approachμ a novel concept to fight Aedes albopictus in urban areas[END_REF][START_REF] Devine | Using adult mosquitoes to transfer insecticides to Aedes aegypti larval habitats[END_REF]. It was also experimentally tested on Cx. quinquefasciatus populations and provided very promising results [START_REF] Mbare | Pyriproxyfen for mosquito control: female sterilization or horizontal transfer to oviposition substrates by Anopheles gambiae sensu stricto and Culex quinquefasciatus[END_REF].

Over time, repeated uses of insecticides lead to the development of insecticide resistance, especially to organochlorines and organophosphates, becoming a problem in several mosquito populations and particularly in Culex pipiens in different countries including Lebanon (Osta et al., 2012), Iran (Salim-Abadi et al., 2016) and the USA [START_REF] Paul | Insecticide resistance in Culex pipiens from New York[END_REF]. This resistance became a limiting factor for the efficacy of control efforts.

Today, genetic control of mosquitoes relies on two main fronts. The first consists of Sterile Insect Technique (SIT) that aims to eliminate an entire population of a particular mosquito species. One example is the sterile male technique or male sterilization that consists of releasing a mass of modified male mosquitoes that mate with indigenous females to produce no fertile offspring. The second is the population replacement consisting to replace wild populations by genetically-modified mosquitoes rendered refractory to a pathogen [START_REF] Becker | Mosquitoes and Their Control[END_REF].

SIT is currently in operational use more than the population replacement technique. In Florida, in 1969, Culex quinquefasciatus populations were successfully suppressed and eliminated after the massive release of chemo-sterilized males [START_REF] Patterson | Suppression and elimination of an island population of Culex pipiens quinquefasciatus with sterile males[END_REF]. In France, in 1970, a reduction in Cx. pipiens populations was obtained after the release of hundred thousand sterile specimens following chromosomal translocation [START_REF] Laven | An experiment in genetic control of Culex pipiens in the area of Montpellier[END_REF]. More recently, in La Reunion Island, a field test was implemented to control Aedes albopictus populations; a two-fold reduction of the wild population fertility was obtained after the release of males sterilized by irradiations with Cesium-137 [START_REF] Oliva | The sterile insect technique for controlling populations of Aedes albopictus (Diptera: Culicidae) on Reunion Island: mating vigour of sterilized males[END_REF].

In addition, recent work has shown that the introduction of an over replicating strain (wMelPop) of the bacteria Wolbachia into Ae. aegypti can lead to a cytoplasmic incompatibility and inhibits the dissemination of Dengue Virus (DENV) as well as Chikungunya Virus (CHIKV) [START_REF] Moreira | A Wolbachia symbiont in Aedes aegypti limits infection with dengue, Chikungunya, and Plasmodium[END_REF]. The release of Aedes specimens infected with appropriate strains of Wolbachia was proposed to control Zika Virus (ZIKV) in Brazil [START_REF] Callaway | Rio fights Zika with biggest release yet of bacteria-infected mosquitoes[END_REF]. However, the use of Wolbachia to reduce transmission of pathogens by Culex species must be studied carefully. Indeed, a recent study demonstrated that the transmission of WNV could be improved by the presence of the Wolbachia strain wAlbB in the mosquito Culex tarsalis [START_REF] Dodson | Wolbachia enhances West Nile virus (WNV) infection in the mosquito Culex tarsalis[END_REF].

Chapter 2 West Nile Virus

West Nile Virus (WNV), an arthropod-borne virus, is the causative agent of West Nile Fever (WNF). This virus is transmitted from infected birds to humans by the bite of a broad range of mosquito species, but mainly members of the Culex genus, most importantly Culex pipiens complex species.

Transmission cycle

In an enzootic cycle, mosquitoes get infected by feeding on a bird having a sufficiently high viremia. The virus then replicates inside the mosquito before it infects another host (Figure 6).

Vertical transmission by mosquitoes provides the mechanism of virus overwintering [START_REF] Chancey | The global ecology and epidemiology of West Nile virus[END_REF]. Only females having high natural infection titers succeeded to pass WNV to their overwintering adult progeny [START_REF] Nelms | Experimental and natural vertical transmission of West Nile virus by California Culex (Diptera: Culicidae) mosquitoes[END_REF]. Migratory birds ensure the seasonal spread of the virus between countries. Thus, mosquito species feeding on these infected birds during their stopovers transmit the virus to local bird species. Transmission to people and horses is accidental; it occurs when an infected mosquito feeds on a person or a horse thereby transmitting the virus via its salivary gland secretions. People and horses are dead end hosts because they do not develop viremia high enough to infect a mosquito. Experimental infections of horses with the NY99 strain of WNV revealed low viremia levels (a maximum viral titer of 10 3 plaque forming units (PFU)/mL) of short duration and all mosquitoes that fed upon viremic horses were not able to transmit the virus [START_REF] Bunning | Experimental infection of horses with West Nile virus[END_REF]. Therefore, infected horses are unlikely to serve as important amplifying hosts for WNV in nature. In fact, infectious doses required to infect Cx.

pipiens should be greater than 10 5 PFU/mL [START_REF] Turell | Potential for New York mosquitoes to transmit West Nile virus[END_REF]. Horizontal transmission between birds is possible in cases of fecal or oral fluids contact [START_REF] Van Der Meulen | West Nile virus in the vertebrate world[END_REF]. WNV can also be transmitted between humans by blood transfusion, organ transplantation, transplacental transmission, and via breast milk. Birds are presumed to be the most important amplifying hosts of WNV. In laboratory studies, bird species of Passeriformes, Charadriiformes, Strigiformes (owls), and Falconiformes developed viremia levels sufficiently high to infect most feeding mosquitoes [START_REF] Komar | Experimental infection of North American birds with the New York 1999 strain of West Nile virus[END_REF]. Birds like house sparrows (Passer domesticus) which are common in many countries including Lebanon, develop high viremia and therefore contribute to mosquito infection without developing clinical symptoms [START_REF] Komar | Experimental infection of North American birds with the New York 1999 strain of West Nile virus[END_REF].

Geographic distribution

WNV was first isolated from a febrile woman in Uganda in 1937 [START_REF] Smithburn | A Neurotropic Virus Isolated from the Blood of a Native of Uganda[END_REF].

WNV infections have been reported in many tropical and temperate countries in Africa, Europe, Asia and America. The circulation of this virus is being continuously traced by eCDC (Figure 7).

The Middle East and North Africa (MENA) region has been long considered as a WNV-endemic area in which hundreds to thousands of human and equine cases have occurred. A series of outbreaks from the 1990s brought WNV into the spotlight. Epidemics in Algeria, Morocco, Tunisia, Italy, France, Romania, Israel, and Russia were associated with uncharacteristically severe human disease, including neurologic complications and death [START_REF] Marka | West Nile virus state of the art report of MALWEST Project[END_REF]Murgue et al., 2001a;[START_REF] Platonov | Outbreak of West Nile virus infection, Volgograd Region, Russia[END_REF][START_REF] Tsai | West Nile encephalitis epidemic in southeastern Romania[END_REF]. Recent outbreaks have affected Israel in 2000 [START_REF] Weinberger | West Nile fever outbreak, Israel, 2000: epidemiologic aspects[END_REF], Greece in 2010 [START_REF] Danis | Outbreak of West Nile virus infection in Greece, 2010[END_REF] and 2011 [START_REF] Vrioni | Laboratory and clinical characteristics of human West Nile virus infections during 2011 outbreak in southern Greece[END_REF], and Turkey in 2010 [START_REF] Kalaycioglu | Emergence of West Nile virus infections in humans in Turkey, 2010 to 2011[END_REF]. In addition, evidence of recent human WNV circulation has been reported in Libya [START_REF] Shaibi | Prevalence of IgG antibodies for the West Nile virus in human population in Tripoli, Libya[END_REF], Jordan [START_REF] Batieha | Seroprevalence of West Nile, Rift Valley, and sandfly arboviruses in Hashimiah, Jordan[END_REF],

Egypt [START_REF] Soliman | Studies on West Nile virus infection in Egypt[END_REF] and Israel [START_REF] Kopel | Surveillance of West Nile virus disease, Tel Aviv district, Israel, 2005 to 2010[END_REF]. The introduction of WNV into the United States of America (USA) in 1999 which constitutes a turning point in WNV epidemiology, is thought to have originated from Israel following introduction from Africa [START_REF] Giladi | West Nile encephalitis in Israel, 1999: the New York connection[END_REF][START_REF] Lanciotti | Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States[END_REF]. In the summer 2001, the virus was found in 10 states and WNV continued to spread until a large outbreak that occurred in 2006, with 177 deaths and 1495 cases of West Nile neurological disease (WNND) out of a total of 4260 cases (WHO, 2017). Through 2010, it was estimated that 1.1% of the United States population had been infected by WNV (WHO, 2017).

The presence of WNV was first discovered in Europe in 1958 in Albania with detection of neutralizing antibodies in human sera [START_REF] Hubálek | West Nile fever--a reemerging mosquito-borne viral disease in Europe[END_REF]. The first documented outbreak of WNV occurred in southern France in 1962-1963, causing WNND in both humans and horses (Murgue et al., 2001a). Notable outbreaks with human cases include the reemergence of WNV in France in 2000-2003, Italy in 2008[START_REF] Diamond | West Nile Encephalitis Virus Infection[END_REF], and Hungary in 2008[START_REF] Bakonyi | Explosive spread of a neuroinvasive lineage 2 West Nile virus in Central Europe, 2008/2009[END_REF][START_REF] Calistri | Epidemiology of west nile in europe and in the mediterranean basin[END_REF][START_REF] Di Sabatino | Epidemiology of West Nile disease in Europe and in the Mediterranean Basin from 2009 to 2013[END_REF][START_REF] Zeller | West Nile virus: an overview of its spread in Europe and the Mediterranean basin in contrast to its spread in the Americas[END_REF]. Recently, human WNV cases were reported in several European countries.

Epizootics in horse populations occurred in Morocco in 1996 in which 94 cases in equines (including 42 deaths) and 1 case in a human were reported [START_REF] Schuffenecker | West Nile virus in Morocco, 2003[END_REF]. During the late summer of 1998, veterinary authorities in Tuscany, Italy, received reports of neurologic disease cases among horses [START_REF] Autorino | West Nile virus epidemic in horses, Tuscany region, Italy[END_REF]. In 2000 in USA, 63 horses with neurologic and other illnesses due to WNV infection were reported from 26 counties in seven states [START_REF] Marfin | West Nile encephalitis: an emerging disease in the United States[END_REF]. Cases were reported in France in 2000, where WNND cases were confirmed in 76 horses, and 21 of them died [START_REF] Murgue | West Nile outbreak in horses in southern France, 2000: the return after 35 years[END_REF]. Recent equine outbreaks occurred in Italy in 2008 (14 horse cases), Hungary in 2008 (12 equine neuroinvasive cases) [START_REF] Angenvoort | West Nile viral infection of equids[END_REF]. Since 2010, WNV infections among horses were repeatedly reported in the Mediterranean basin and other European countries, namely Algeria [START_REF] Lafri | Seroprevalence of West Nile virus antibodies in equids in the North-East of Algeria and detection of virus circulation in 2014[END_REF], Jordan [START_REF] Abutarbush | West Nile virus infection in horses in Jordan: clinical cases, seroprevalence and risk factors[END_REF], Bulgaria, Czech and Slovak Republics [START_REF] Hubálek | West Nile Virus equine serosurvey in the Czech and Slovak republics[END_REF], Croatia, Greece, Italy, Spain [START_REF] Abad-Cobo | Serosurvey Reveals Exposure to West Nile Virus in Asymptomatic Horse Populations in Central Spain Prior to Recent Disease Foci[END_REF][START_REF] Jiménez-Clavero | West Nile virus serosurveillance in horses in Donana, Spain, 2005 to 2008[END_REF], Macedonia, Morocco, Portugal [START_REF] Barros | Serological evidence of West Nile virus circulation in Portugal[END_REF], and Romania [START_REF] Angenvoort | West Nile viral infection of equids[END_REF].

WNV has been detected in more than 150 species of wild and domestic birds worldwide. The susceptibilities of birds to WNV infection differ among bird species. Severe disease and deaths were not reported in natural WNV infections of birds until 1998, when an outbreak occurred in Israel, during which 160 of 400 domestic goslings developed fatal encephalitis [START_REF] Di Sabatino | Epidemiology of West Nile disease in Europe and in the Mediterranean Basin from 2009 to 2013[END_REF]. In 1999, during the outbreak of WNV in USA, thousands of deaths in more than 150 species of native and exotic birds were detected [START_REF] Nash | The outbreak of West Nile virus infection in the New York City area in[END_REF][START_REF] Steele | Pathology of fatal West Nile virus infections in native and exotic birds during the 1999 outbreak in New York City, New York[END_REF]. In the following years, bird infection with WNV and sometimes death were observed in several countries around the world. During the summers of 2004-2005 in Southeast Hungary, the first clinical cases were described and WNV infections in birds were detected. It corresponds to the first evidence of a neuroinvasive WNV lineage 2 circulation in continental Europe [START_REF] Erdélyi | Clinical and pathologic features of lineage 2 West Nile virus infections in birds of prey in Hungary[END_REF]. Subsequently, a similar WNV lineage 2 strain was molecularly identified and characterized from an Eurasian magpie hunted in Greece in 2010, during a WNV outbreak in humans [START_REF] Valiakos | Molecular detection and phylogenetic analysis of West Nile virus lineage 2 in sedentary wild birds (Eurasian magpie), Greece, 2010[END_REF].

In a serosurvey study conducted in Camargue France, a low WNV seroprevalencewas observed among birds in a 3-year period (2005-2007). It revealed the existence of an enzootic activity of WNV without any detectable emergence of an epizootic [START_REF] Balança | Low West Nile virus circulation in wild birds in an area of recurring outbreaks in Southern France[END_REF].

Moreover, in Germany during 2005-2009, a study discovered WNV-neutralizing antibodies in 10 species of migratory birds, but not in resident birds [START_REF] Seidowski | West Nile virus monitoring of migratory and resident birds in Germany[END_REF]. For the first time in Serbia, WNV lineage 2 RNA were detected by RT-qPCR in pooled tissue samples of investigated birds in 2012 [START_REF] Petrović | Monitoring West Nile virus (WNV) infection in wild birds in Serbia during 2012: first isolation and characterisation of WNV strains from Serbia[END_REF].

Classification and lineages distribution

West Nile Virus (WNV) belongs to the Flavivirus genus that includes the largest number of viral species compared to other genera (Pestivirus and Hepacivirus) composing the Flaviviridae family. The Flavivirus genus includes at least 53 species divided into 12 serogroups. WNV belongs to the Japanese Encephalitis Virus (JEV) group that comprises eight viral species, among which we cite, in addition to WNV, JEV, SLEV, Murray Valley Encephalitis that are frequently associated with widespread human and animal disease outbreaks [START_REF] Schweitzer | Overview of the Flaviviridae With an Emphasis on the Japanese Encephalitis Group Viruses[END_REF].

WNV is a genetically and geographically diverse virus. Up to nine distinct WNV genetic lineages with inconsistent numbering, have been proposed in the literature based on phylogenetic analyses of the complete genome or of the NS5 gene. Their genomes differ from each other by more than 20-25% and correlate well with the geographical point of isolation. Rizzoli and collaborators (2015) suggested a harmonized lineage numbering into seven lineages (Figure 8).

Lineages 1 and 2 of WNV have been associated with significant outbreaks in humans [START_REF] Chancey | The global ecology and epidemiology of West Nile virus[END_REF]. Lineage 1 is the most widespread, containing isolates found in Europe, North America, Asia, Africa and the Middle East [START_REF] Chancey | The global ecology and epidemiology of West Nile virus[END_REF][START_REF] Rizzoli | The challenge of West Nile virus in Europe: knowledge gaps and research priorities[END_REF]. This linage is further divided into different clades: WNV-1a is found mainly in Africa, Europe, North America and Asia (May et al., 2011). WNV 1-b contains the Australian Kunjin virus (May et al., 2011). Clade 1c of lineage 1, formerly considered lineage 5, includes isolates from India [START_REF] Bondre | West Nile virus isolates from India: evidence for a distinct genetic lineage[END_REF][START_REF] Rizzoli | The challenge of West Nile virus in Europe: knowledge gaps and research priorities[END_REF].

Lineage 2 strains are mainly distributed in Sub-Saharan Africa and Madagascar, and detected in humans and horses [START_REF] Venter | West Nile virus lineage 2 as a cause of zoonotic neurological disease in humans and horses in southern Africa[END_REF]. But in the last decade, Lineage 2 WNV has been introduced in Europe. Ciccozzi and collaborators (2013) suggest, based on E and NS5 gene analysis, that there were at least two introductions of this lineage to Central Europe in 1999 and to Russia in 2000 from the African continent. Since 2004, strains have been observed in Hungary from birds of prey [START_REF] Bakonyi | Lineage 1 and 2 strains of encephalitic West Nile virus, central Europe[END_REF]. In 2010, it caused outbreaks in Romania and Greece [START_REF] Valiakos | Molecular detection and phylogenetic analysis of West Nile virus lineage 2 in sedentary wild birds (Eurasian magpie), Greece, 2010[END_REF] and in 2011, it was detected for the first time in Italy [START_REF] Ciccozzi | Epidemiological history and phylogeography of West Nile virus lineage 2[END_REF].

Lineage 3 contains strains circulating in certain Culex and Aedes mosquito species in southern Moravia, Czech Republic; and it is not known to be pathogenic to mammals [START_REF] Bakonyi | Lineage 1 and 2 strains of encephalitic West Nile virus, central Europe[END_REF]. Lineage 4 comprises three clades of viruses as well: clade 4awasdetectedin a tick specimen from the Caucasus (May et al., 2011). Clade 4b, previously noted as lineages 6 and 7, contains a number of isolates from Culex pipiens mosquitoes captured in Spain in 2006 [START_REF] Vazquez | Putative new lineage of west nile virus, Spain[END_REF]. And, Clade 4c, previously classified as lineage 9, comprises viral strains isolated from Uranotaenia mosquitoes in Austria [START_REF] Rizzoli | The challenge of West Nile virus in Europe: knowledge gaps and research priorities[END_REF]. A re-classification of Sarawak Kunjin virus as lineage 6 has been proposed as this strain is different from other Kunjin viruses (Lineage 1-b). Lineages 6 (called Koutango virus) and 7 comprise isolates of the virus from Senegal [START_REF] Chancey | The global ecology and epidemiology of West Nile virus[END_REF][START_REF] Rizzoli | The challenge of West Nile virus in Europe: knowledge gaps and research priorities[END_REF].

Virus description and genome structure

WNV virion is a 50 nm icosahedral particle, enveloped by a lipid bilayer (Figure 9). It has a single stranded positive-sense RNA genome of approximately 11 kb [START_REF] Knipe | Fields Virology[END_REF].

The virion presents a host-derived lipid membrane surrounding a nucleocapsid core [START_REF] Knipe | Fields Virology[END_REF]. The envelope, derived from the host cell membrane, is composed of two virally derived major proteins: protein E (association with the outer viral envelope) and protein M. The protein E is a viral hemagglutinin and the primary target of the host immune response while the protein M acts as a stabilizing factor to protect the protein E as it is transported from the infected cell during replication [START_REF] Schweitzer | Overview of the Flaviviridae With an Emphasis on the Japanese Encephalitis Group Viruses[END_REF]. The genome contains 10 genes flanked by 5' and 3' non coding regions (NCR) without a polyadenylation tail at the 3' end [START_REF] Knipe | Fields Virology[END_REF]. WNV genome has a single open reading frame (ORF) and encodes one large polyprotein that is co-and post-translationally processed and cleaved into 3 structural proteins that are encoded in the 5' end of the genome, and 7 non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) proteins that are encoded in the 3' end [START_REF] Knipe | Fields Virology[END_REF].

Structural proteins of WNV include: the nucleocapsid protein (C); a precursor glycoprotein (prM) that is cleaved during virus maturation to yield the transmembrane protein (M); and the major spike glycoprotein of the envelope (E) [START_REF] Knipe | Fields Virology[END_REF]. The nucleocapsid is composed of C protein, which associates with the RNA genome and mediates viral assembly [START_REF] Knipe | Fields Virology[END_REF]. Heterodimers of prM and E protein become embedded in the lipid bilayer of the virus during assembly and are exposed on the virion surface until cleavage during the viral maturation process [START_REF] Knipe | Fields Virology[END_REF]. The E protein mediates binding of the receptor on the cell surface for viral entry. It also helps fusion with the membrane of the host cell and is considered the major target for neutralizing antibodies [START_REF] Knipe | Fields Virology[END_REF].

All seven non-structural proteins appear to be directly or indirectly involved in viral RNA synthesis. NS1 plays an early role in regulation of viral RNA replication [START_REF] Knipe | Fields Virology[END_REF]. NS2A, NS2B, NS4A, and NS4B are small, hydrophobic proteins that do not have conserved motifs characteristic of known enzymes. NS3 has multiple enzymatic functions, serving as a viral serine protease which cleaves the other non-structural proteins from the viral polyprotein, in association with NS2B; and an RNA helicase in association with NS4A [START_REF] Knipe | Fields Virology[END_REF]. NS5 is the C-terminal protein of the viral polyprotein and is the largest and most conserved of the mature flavivirus proteins. It is necessary for viral replication, containing RNA-dependent RNA polymerase activity in the C-terminal region and methyltransferase activity in the N-terminal region [START_REF] Knipe | Fields Virology[END_REF].

The presence of the envelope makes WNV, like other Flaviviruses, susceptible to organic solvents. Moreover, these viruses are easily inactivated by heat and by common disinfectants [START_REF] Knipe | Fields Virology[END_REF][START_REF] Maclachlan | Fenner's Veterinary Virology[END_REF].

Replication

WNV replicates well and cause cytopathic changes in many cell cultures such as Vero cell line (African green monkey kidney), and mosquito cell line (C6/36). Cellular attachment appears to be mediated by ligands on the E glycoprotein(s), although cellular receptors have not been unambiguously identified (Figure 10). After attachment, the viruses enter cells via receptormediated endocytosis. In the endosome, E proteins fuse the viral membrane with the endosomal vesicle membrane then release the nucleocapsid into the cytoplasm (Figure 10A). The genomic RNA is released from the capsid by an unknown mechanism and is translated (Figure 10B). The polyprotein is co-and post-translationally cleaved at multiple sites to generate the mature viral proteins (Figure 10C). The viral RNA dependent RNA polymerase (from the NS5 protein) copies complementary negative polarity strands from the positive polarity genomic RNA template (Figure 10D). The newly formed negative strand RNAs serve as templates for the synthesis of new genomic RNAs (Figure 10E). Virion assembly occurs in association with rough endoplasmic reticulum membranes (Figure 10F). Intracellular immature virions containing heterodimers of E and prM accumulate in vesicles and are then transported through the host secretory pathway where E and prM glycans are modified (Figure 10G). A portion of prM on immature virions is cleaved in the trans-Golgi network and at the end, virions are transported to the plasma membrane in vesicles and released by exocytosis (Figure 10H) [START_REF] Diamond | West Nile Encephalitis Virus Infection[END_REF].

Pathogenesis

The majority of WNV infections in humans are asymptomatic. Approximately, 20 to 30% of infected individuals develop flu-like clinical manifestations as an acute febrile illness accompanied by headache, polyarthropathy, rash and lymphadenopathy [START_REF] Knipe | Fields Virology[END_REF]. But WNV is also able to cause a neuroinvasive disease (WNND) in a limited subpopulation of individuals (approximately 1 in 150). The clinical features of severe WNV infection vary and include muscle weakness, cognitive impairment, tremors, and a poliomyelitislike flaccid paralysis. In some cases, infections can lead to encephalitis, causing long-term disability and rarely leading to death [START_REF] Knipe | Fields Virology[END_REF].

Horses, just like humans, are dead-end hosts, meaning that while they become infected, they do not spread the infection. Horses infected with WNV mostly show no symptoms or rarely acquire mild febrile illness. However, neuroinvasive (encephalomyelitis) forms are also reported.

In these cases, the disease shows reserved prognosis and 30-40% of horses would die [START_REF] Maclachlan | Fenner's Veterinary Virology[END_REF]. Horses affected by West Nile Encephalitis (WNE) are usually euthanized. Many horses that recover from WNE have been reported to have some residual neurological deficits, such as gait abnormalities and a change in their usual temperament or behavior. The signs and course of the disease are highly variable since not all infected horses react similarly. Horses may become abnormally sensitive to sights, sounds, or touch. They may present signs of discomfort or anxiety, low-grade fever, or malaise. Neurologic signs can include muzzle twitching, impaired vision, trembling, lack of coordination, and muscle contractions. The most severe cases evolve to paralysis of the hind legs, terminal convulsion and death [START_REF] Maclachlan | Fenner's Veterinary Virology[END_REF].

In Europe, Africa, Middle East and Asia, mortality in birds associated with WNV infection is rare. In contrast, the virus is highly pathogenic for birds in USA [START_REF] Maclachlan | Fenner's Veterinary Virology[END_REF].

Infection may result in a very high viremia titer and widespread necrosis, hemorrhage, and inflammation in several organs including heart, lungs, spleen, liver, kidney, and nervous system.

These severe infections might lead to death [START_REF] Maclachlan | Fenner's Veterinary Virology[END_REF]. In Serbia, evidence of WNV circulation (neutralizing antibodies or viral RNA) was reported in several bird species such as Northern Goshawks (Accipiter gentilis), White-tailed Eagles (Haliaeetus albicilla),

Legged Gull (Larus michahelis), Hooded Crow (Corvus cornix), Bearded Parrot-bill (Panarus biramicus), and Common Pheasant (Phasianus colchicus) [START_REF] Petrović | Monitoring West Nile virus (WNV) infection in wild birds in Serbia during 2012: first isolation and characterisation of WNV strains from Serbia[END_REF]. In Southern

France and Southern Spain, House sparrow (Passer domesticus) and Magpie (Corvidae family) birds were found infected with WNV [START_REF] Balança | Low West Nile virus circulation in wild birds in an area of recurring outbreaks in Southern France[END_REF][START_REF] Ferraguti | West Nile virus-neutralizing antibodies in wild birds from southern Spain[END_REF]. In USA,

Corvid species appear to be susceptible for WNV infection. Following the large outbreak in 1999, crows in addition to flamingos were found highly infected with this virus [START_REF] Steele | Pathology of fatal West Nile virus infections in native and exotic birds during the 1999 outbreak in New York City, New York[END_REF].

Diagnosis

The clinical signs of WNV infection among humans and horses are characteristic but not [START_REF] Rizzoli | The challenge of West Nile virus in Europe: knowledge gaps and research priorities[END_REF].

To date, the most consistent manner to verify WNV infection is serology [START_REF] Rossi | West Nile virus[END_REF]. et al., 2015). But the problem of PCR remains in certain protocols to miss some circulating WNV lineages, therefore a multiplex PCR is advised in order to avoid such issues [START_REF] Rizzoli | The challenge of West Nile virus in Europe: knowledge gaps and research priorities[END_REF].

The presence of reactive lymphocytes or monocytes in CSF samples is indicative of WNV neurologic infection. In patients with WNV neuroinvasion, more than 40% of cells in the CSF are neutrophils. Furthermore, Magnetic Resonance (MR) imaging suggests abnormalities in the brain and meninges of WNV infected patients presenting a central nervous system disease [START_REF] Rossi | West Nile virus[END_REF].

Treatment and vaccine development

Treatment for WNV infections is mainly supportive. No specific antiviral therapy is available. Management is focused on pain control and inflammation. There are no licensed human WNV vaccines, but there are multiple equine vaccines. The development of safe and effective vaccines against WNV remains as a high priority. The only preventive methods to limit the spread of WNV are based on controlling the vector population and on vaccinating horse populations in endemic countries. Vector control is performed using several techniques such as personal prevention, the use of insecticides, impregnated bed nets and the implementation of some challenging genetic control protocols. The main enzootic vectors belong to the Aedes genus. However, several Culex species, including

Cx. pipiens are considered secondary vectors and contribute to the transmission of the virus.

Transmission cycle

RVFV circulates following two types of cycles: an enzootic cycle that occurs during the average rainfall season and an epizootic-epidemic cycle that occurs during the heavy rainfall season (Figure 11). During the enzootic cycle, the virus is maintained through transovarial transmission between the female mosquito belonging to the Aedes genus and her eggs and occasionally, through amplification in a vertebrate host such as African buffaloes. During the epizootic-epidemic cycle, the virus is over-amplified with massive hatching of Aedes eggs (including those RVFV-infected) following an abnormally high rainfall or floods. Obtained infected adults will infect susceptible livestock triggering an epizootic phase. A very high level of viremia is maintained for 3 to 5 days in infected sheep and cattle, allowing many more mosquitoes to become infected when feeding on infected animals. These mosquitoes may be transported for long distances in low level wind or air currents, which may lead to the rapid spread of the virus from region to region. Humans can become infected following the bites of Aedes (as a primary vector) or Culex (as a secondary vector) mosquitoes but the majority of human cases are thought to result from handling infected fluids and tissues: blood, secretions or excretions of infected animals, notably after abortion via contaminated placentae [START_REF] Maclachlan | Fenner's Veterinary Virology[END_REF].

Geographic distribution

RVFV was first identified in an outbreak of abortions and deaths in wool sheep and illness in humans that occurred in the Rift Valley of Kenya after heavy rainfalls in 1930-1931 [START_REF] Daubney | Enzootic hepatitis or rift valley fever. An undescribed virus disease of sheep cattle and man from east africa[END_REF].

Since then, outbreaks have been reported in the African region extending from sub-Sahara [START_REF] Himeidan | Recent outbreaks of rift valley Fever in East Africa and the middle East[END_REF] up to Mauritania (El Mamy et al., 2011) (Figure 12). In Egypt in 1977, RVF was introduced via infected livestock trade along the Nile irrigation system and caused a large outbreak among both animals and humans resulting in over 600 human deaths [START_REF] Meegan | The Rift Valley fever epizootic in Egypt 1977-78[END_REF]. Another example of RVF spillover into human populations occurred in West Africa in 1987, and was linked to construction of a dam on the Senegal river [START_REF] Guillaud | Prevalence of antibodies against Rift Valley fever virus in sheep and goats in Senegal[END_REF]. The project caused flooding in the lower Senegal River area, altering ecological conditions and interactions between animals and humans. As a result, a large RVF outbreak occurred in animals and humans. In 1997-1998, a major outbreak occurred in Kenya, Somalia and Tanzania following El Niño event and extensive flooding [START_REF] Woods | An outbreak of Rift Valley fever in Northeastern Kenya, 1997-98[END_REF]. Following infected livestock trade from the horn of Africa, RVF spread in September 2000 to Saudi Arabia and Yemen [START_REF] Ahmad | More deaths from Rift Valley fever in Saudi Arabia and Yemen[END_REF][START_REF] Madani | Rift Valley fever epidemic in Saudi Arabia: epidemiological, clinical, and laboratory characteristics[END_REF], marking the first reported occurrence of the disease outside the African continent and raising concerns that it could extend to other parts of Asia and Europe. In October and November 2010, an outbreak of RVF was reported in the northern part of Mauritania [START_REF] El Mamy | Unexpected Rift Valley fever outbreak, northern Mauritania[END_REF]. Cases in animals and humans seemed to occur shortly after intense and unusual rainfalls [START_REF] Faye | Reemergence of Rift Valley fever, Mauritania, 2010[END_REF]. Severe clinical signs and high mortality was described among camel. At least, 63 human cases were noted including 13 deaths [START_REF] Faye | Reemergence of Rift Valley fever, Mauritania, 2010[END_REF]. RVFV strains were isolated from humans and also from Cx. antennatus mosquitoes [START_REF] Faye | Reemergence of Rift Valley fever, Mauritania, 2010[END_REF]. In 2012, a large but unspecified number of ruminants were affected, together with more than 34 human cases, including 17 deaths, from six regions in Mauritania. Most of the human cases had a history of contacts with animals or sick animals [START_REF] Sow | Rift Valley fever outbreak, southern Mauritania, 2012[END_REF]. Then, from September 2013, domestic ruminants and humans from Senegal were tested positive for RVFV by RT-PCR in several regions [START_REF] Sow | Widespread Rift Valley Fever Emergence in Senegal in 2013-2014[END_REF]. The outbreak coincided with preparations for the annual Muslim celebration [START_REF] Sow | Widespread Rift Valley Fever Emergence in Senegal in 2013-2014[END_REF]. From September to November 2015, 31 hospitalized patients who presented severe clinical forms of RVF disease were subsequently tested positive for the virus. At least, eight people died [START_REF] Boushab | Severe Human Illness Caused by Rift Valley Fever Virus in Mauritania[END_REF].

Most recently, in 2016 in Uganda, CDC localized an outbreak of three confirmed and two probable human cases. In addition, in August 2016, health authorities in Niger reported cases of febrile illness, including hemorrhagic symptoms, in humans, as well as sickness and abortions in ruminants. Subsequent diagnostic testing confirmed 17 RVF human cases and 33 deaths (CDC, 2017).

Classification and lineages distribution

RVFV belongs to the Bunyaviridae family, Phlebovirus genus comprising 70 viruses that can be divided in 2 groups: the sandfly fever virus group and the Uukuniemi-like virus group [START_REF] Elliott | Emerging phleboviruses[END_REF]. There are important differences between these groups; notably that the Uukuniemi-like viruses do not encode a nonstructural protein and they are all transmitted by ticks. The sandfly fever group are transmitted by dipterans (phlebotomines and mosquitoes). This group contains important human and animal pathogens, including RVFV and the sandfly fever viruses [START_REF] Maclachlan | Fenner's Veterinary Virology[END_REF].

A number of studies on the genetic diversity of RVFV have been published over the past years revealing an increasing number of lineages. These studies showed that the genetic diversity of RVFV across all lineages is limited, with maximum pair-wise differences for the S segment and M segments of 4% and 5.4% for the nucleotide sequence and 1% and 2.8% for the amino acid sequence, respectively.

Extensive analysis of the complete genomes (S, M, and L segments) of 33 diverse RVFV, via high-throughput technique, showed that this virus can be separated into 7 distinct genetic lineages (A to G) [START_REF] Bird | Complete genome analysis of 33 ecologically and biologically diverse Rift Valley fever virus strains reveals widespread virus movement and low genetic diversity due to recent common ancestry[END_REF]. RVFV strains with diverse geographic origins can be found in each lineage, which is indicative of widespread dispersal and movement of the virus genotypes throughout Africa. consisted of isolates having a common root node deriving from strains isolated from the same mosquito species [START_REF] Grobbelaar | Molecular epidemiology of Rift Valley fever virus[END_REF].

A more recent study compared sequences from the three mini-genomic segments of viral strains from 18 countries across Africa plus Saudi Arabia. In this study, 11 RVFV lineages (A-K) were identified [START_REF] Samy | Phylogeography of Rift Valley Fever Virus in Africa and the Arabian Peninsula[END_REF]. The differences between this phylogeny and the previous ones could be the result of a larger dataset.

Virus description and genome structure

RVFV virions, as the majority of Bunyaviruses, are spherical, approximately 100 nm in diameter, and are composed of a lipid envelope with glycoprotein spikes (Figure 13). Inside it, there are three circular ribonucleoprotein (RNP) complexes comprised of individual genome RNA segments associated with the viral nucleoprotein [START_REF] Maclachlan | Fenner's Veterinary Virology[END_REF][START_REF] Mansfield | Rift Valley fever virus: A review of diagnosis and vaccination, and implications for emergence in Europe[END_REF]. The genome of RVFV as all viruses belonging to the Phlebovirus group, is approximately 12 kb and consists of three segments of negative-sense (or ambisense), single-stranded RNA, designated large (L), medium (M), and small (S) (Figure 13). The RNA segments differ in size:

L RNA segment is of 6.4 kb, the M RNA segment is of 3.2 kb, and the S RNA segment is of 1.7

kb [START_REF] Maclachlan | Fenner's Veterinary Virology[END_REF]. The L RNA encodes a single large protein L, the RNAdependent RNA polymerase (transcriptase). The M RNA encodes a polyprotein that is processed to form two glycoproteins (Gn and Gc) and a non-structural protein (NSm). The S RNA, an ambisense segment (single-stranded RNA containing both positive-sense and negative-sense sequences), encodes the nucleocapsid (N) protein and a non-structural (NSs) proteins [START_REF] Maclachlan | Fenner's Veterinary Virology[END_REF]. The N and NSs proteins of RVFV are each translated from a separate subgenomic mRNA. The non-structural NSs protein interferes with the innate host-cell antiviral response via inhibition of the cell signaling molecules leading to global suppression of the type I interferon response [START_REF] Maclachlan | Fenner's Veterinary Virology[END_REF][START_REF] Mansfield | Rift Valley fever virus: A review of diagnosis and vaccination, and implications for emergence in Europe[END_REF].

RVFV, like other Phleboviruses, is sensitive to heat and acid conditions, and is easily inactivated by detergents, lipid solvents, and common disinfectants [START_REF] Maclachlan | Fenner's Veterinary Virology[END_REF].

Replication

RVFV, like most Bunyaviruses, replicates in Vero (African green monkey) cells, BHK-21

(baby hamster kidney) cells, and C6/36 mosquito (Aedes albopictus) cells.

Viral entry into its host cell occurs by receptor-mediated endocytosis; all subsequent steps take place in the cytoplasm. After penetration and uncoating, the virion RNA polymerase (transcriptase) is activated and RNA is transcribed to viral mRNAs from each of the three virion RNAs (the S, M and L segments) [START_REF] Maclachlan | Fenner's Veterinary Virology[END_REF]. These proteins mature by budding into smooth-surfaced vesicles in or near the Golgi apparatus of the host cell to be eventually released.

Pathogenesis

Infected people remain mainly asymptomatic; otherwise, the disease begins after a very short incubation period (2 to 6 days) with mild fever, severe headache, chills, "back-breaking" myalgia, diarrhea, vomiting. Usually the clinical disease lasts 4 to 6 days, followed by a prolonged convalescence and complete recovery. In 1% to 2% of affected individuals, the disease can become more severe including fulminant hepatitis, encephalitis, retinitis, blindness, or a hemorrhagic syndrome. Among severely affected persons, the case fatality ratio is approximately 10% to 20% [START_REF] Madani | Rift Valley fever epidemic in Saudi Arabia: epidemiological, clinical, and laboratory characteristics[END_REF].

Affected cattle, water buffaloes, sheep and goats often remain asymptomatic; but fever lasting 24 to 96 hours, dull coat, lacrimation, nasal discharge, excessive salivation, anorexia, weakness, bloody diarrhea, low milk yield, and high abortion rates in pregnant cows are common symptoms. In ruminant livestock, mortality rate reaches 100% in neonates and 10 to 20% in adults [START_REF] Bird | Rift Valley fever virus[END_REF]FAO, 2017).

Diagnosis

RVF epidemics should always be strongly suspected when there is a sudden onset of large numbers of abortions in sheep, goats, cattle or camels and deaths in lambs, or calves [START_REF] Maclachlan | Fenner's Veterinary Virology[END_REF]. Rapid diagnosis aiming for virus detection is achieved using RT-PCR assays, a very sensitive technique able to detect approximately 5 to 10 RNA copies/sample.

Consequently, it allows for the detection of infection during the early phase of the disease. This technique is easily adapted to high-throughput use during epizootics and epidemics but can detect viral nucleic acids only during the viremic period, which is relatively transient.

Moreover, the presence of the virus can be confirmed by virus isolation in cell culture such as Vero E6 and BHK-21 cells. The virus is rapidly cytopathic and forms plaques [START_REF] Maclachlan | Fenner's Veterinary Virology[END_REF]. In this case, nucleic acid sequencing is used to prove the identity of isolates.

Virus culture requires high safety level setting and usually conducted in reference laboratories.

Serologic diagnosis of acute infection is commonly used to seek for the presence of immunoglobulin (IgM) capture on sera samples by enzyme immunoassays, neutralization or hemagglutination-inhibition assays on sera samples from surviving animals [START_REF] Maclachlan | Fenner's Veterinary Virology[END_REF].However, the duration of detectable anti-RVF IgM antibody in these animals is limited(approximately 60 to 90 days) [START_REF] Morvan | Duration of immunoglobulin M antibodies against Rift Valley fever virus in cattle after natural infection[END_REF]. Tests for detection of total RVF virus-specific immunoglobulins (IgM and IgG) in cattle, sheep, and African buffaloes became commercially available [START_REF] Paweska | An inhibition enzymelinked immunosorbent assay for the detection of antibody to Rift Valley fever virus in humans, domestic and wild ruminants[END_REF]. Although this assay is unable to differentiate acute and convalescent phases of infection, it may be useful in emergency situations. The Virus Neutralization Assay is the golden standard assay that is recommended by the World Organization for Animal Health (OIE) for international animal trade. But, this technique can only be performed in adequate biosecurity facilities.

Treatment and vaccine development

There is no specific treatment for RVF. The most widely tested antiviral agent is the nucleoside analogue ribavirin along with its closely related chemical derivatives. However, the limited penetration of ribavirin across the blood-brain barrier may limit the effectiveness of this treatment in preventing delayed-onset neurologic disease.

To date, there is no licensed vaccines against RVF available to immunize humans but various strains for livestock and ruminants are licensed and commercially produced such as the live-attenuated Smithburn vaccine, Formalin-Inactivated vaccine, and Clone 13 (a form of freeze dried live attenuated virus) [START_REF] Alhaj | Safety and Efficacy Profile of Commercial Veterinary Vaccines against Rift Valley Fever: A Review Study[END_REF]. Although, the first two vaccines are widely used in control, both of them were accompanied by significant concerns. The first one requires multiple doses for protection, causes abortion in pregnant ewes, and has teratogenic effects; and the second has a risk of causing abortion and fetal malformation in pregnant animals especially in European breeds [START_REF] Botros | Adverse response of non-indigenous cattle of European breeds to live attenuated Smithburn Rift Valley fever vaccine[END_REF]. For Clone13 vaccine, it is based on an attenuated strain which contains a large deletion in the main virulence factor, the NSs gene [START_REF] Muller | Characterization of clone 13, a naturally attenuated avirulent isolate of Rift Valley fever virus, which is altered in the small segment[END_REF].

This strain was shown to be highly immunogenic and experimentally safe. However, intranasal inoculation with this attenuated virus has been shown to cause severe neurological disease in mice [START_REF] Dodd | Rift valley Fever virus encephalitis is associated with an ineffective systemic immune response and activated T cell infiltration into the CNS in an immunocompetent mouse model[END_REF], implying that NSs-deleted viruses may still retain some virulence.

Moreover, the use of this vaccine during the first trimester of gestation remains controversial and was associated with malformations and stillbirths [START_REF] Alhaj | Safety and Efficacy Profile of Commercial Veterinary Vaccines against Rift Valley Fever: A Review Study[END_REF].

A newly developed live-attenuated MP-12 vaccine in USA is in clinical trials. It has been developed for both animal and humans but was conditionally licensed for animal vaccination [START_REF] Ikegami | Rift Valley fever vaccines: an overview of the safety and efficacy of the liveattenuated MP-12 vaccine candidate[END_REF]. The MP-12 vaccine was developed from the pathogenic ZH548 strain, it is well attenuated, and a single dose induces neutralizing antibodies in ruminants [START_REF] Ikegami | Rift Valley fever vaccines: an overview of the safety and efficacy of the liveattenuated MP-12 vaccine candidate[END_REF]. This attenuated viral strain contains mutations in the three viral gene segments [START_REF] Ikegami | Rift Valley fever vaccines: an overview of the safety and efficacy of the liveattenuated MP-12 vaccine candidate[END_REF][START_REF] Mansfield | Rift Valley fever virus: A review of diagnosis and vaccination, and implications for emergence in Europe[END_REF].Vaccination trials results have been encouraging with minimal teratogenic effects seen in ruminants and suitable immunogenicity in non-human primates [START_REF] Ikegami | Rift Valley fever vaccines: an overview of the safety and efficacy of the liveattenuated MP-12 vaccine candidate[END_REF].

Control is based primarily on livestock vaccination, but vector control via the use of larvicides and insecticides is also applied during outbreaks to kill mosquitoes. In addition, the management the livestock export during RVF epizootic periods is also mandatory.

Chapter 4

Vector-virus interactions

Arboviruses are dependent on intricate relationships with their arthropod vectors and vertebrate hosts, and these relationships are further shaped by environmental conditions.

Vector Capacity and vector competence

The inter-relationships between viruses, vectors and vertebrate hosts are complex and dynamic and shaped by several biotic (viral strain, vector genetics, host susceptibility) and abiotic (temperature, rainfall, human land use) factors [START_REF] Kramer | Complexity of virus-vector interactions[END_REF].

The Vector Capacity (VC) is the measure of a mosquito population's capacity to transmit an infectious agent to a new susceptible population [START_REF] Kramer | Complexity of virus-vector interactions[END_REF]. This capacity integrates biotic and abiotic factors and is quantified by the following formula as reviewed by [START_REF] Kramer | Complexity of virus-vector interactions[END_REF]:

VC = [ma 2 (I*T) p n ]/-ln(p) in which:

 m is the vector density in relation to the host  a is the probability that a vector feeds on a host in one day  p is the probability that a vector survives one day  n is the duration of the extrinsic incubation period (EIP) in days  1/-ln(p) is the duration of the vector's life in days after surviving the EIP  I is the infection rate and T is the transmission rate, so I*T represents the vector competence.

This equation shows that the vector capacity implicitly depends on both, extrinsic factors such as the availability of the vertebrate host, its immune status, temperature and rainfall, the abundance of vectors; and intrinsic factors like vector survivorship, trophic preferences, the blood meal frequency and vector competence.

Vector competence is one aspect of vector capacity. It defines the ability of the mosquito to become infected, replicate and transmit the virus to a host following an infectious blood meal [START_REF] Kramer | Complexity of virus-vector interactions[END_REF]. Vector competence is affected also by both extrinsic and intrinsic factors.

Intrinsic factors include genetic characteristics of both the virus and the vector origins which determines the vector's receptivity to the pathogen, and extrinsic factors such as the viral dose ingested by the vector and the incubation temperature of vectors after ingestion of an infected blood meal [START_REF] Hardy | Intrinsic factors affecting vector competence of mosquitoes for arboviruses[END_REF].

The study of the receptivity of mosquitoes to different arboviruses has shown that the virusvector relationship could be very specific. Different species, subspecies or even populations of mosquitoes do not have the same vector competence to the same virus. For instance, Culex pipiens mosquitoes, collected from different areas in the Maghreb region, showed diverse viral transmission capacities when infected with WNV [START_REF] Amraoui | Culex pipiens, an experimental efficient vector of West Nile and Rift Valley fever viruses in the Maghreb region[END_REF]. Another study showed that Culex pipiens biotypes (molestus, pipiens and their hybrid) do not show similar WNV transmission rates in the same conditions [START_REF] Vogels | Vector competence of northern European Culex pipiens biotypes and hybrids for West Nile virus is differentially affected by temperature[END_REF]. In the other way around, vector competence of one mosquito species is different when tested with different viruses or different lineages of the same virus. For example, Culex quinquefasciatus (of the Culex pipiens complex)

from Florida had different transmission rates with diverse WNV isolates [START_REF] Richards | Vector competence of Culex pipiens quinquefasciatus (Diptera: Culicidae) for West Nile virus isolates from Florida[END_REF].

These observations show that viral and vector genotypes may affect vector competence, but most importantly, the interaction between these two factors shapes the characteristics of viral infection, dissemination and transmission.

As already mentioned, vector competence is influenced, not just by intrinsic factors, but also by extrinsic factors such as incubation temperature. In fact, several studies have shown that a higher temperature leads to an increase in the rate of infection and a decrease in the extrinsic incubation period (EIP). This has been demonstrated for DENV [START_REF] Watts | Effect of temperature on the vector efficiency of Aedes aegypti for dengue 2 virus[END_REF], the RVFV [START_REF] Brubaker | Effect of environmental temperature on the susceptibility of Culex pipiens (Diptera: Culicidae) to Rift Valley fever virus[END_REF], WNV [START_REF] Dohm | Effect of incubation at overwintering temperatures on the replication of West Nile Virus in New York Culex pipiens (Diptera: Culicidae)[END_REF] and CHIKV [START_REF] Mourya | Effect of temperature stress on immature stages and susceptibility of Aedes aegypti mosquitoes to chikungunya virus[END_REF].

Experimental observations allow us to notice that the vector competence, in particular viral transmission, depends on complex interactions between the three factors: mosquito genotype, viral genotype and temperature. It has been demonstrated that some populations of Ae.

albopictus better transmit some strains of CHIKV at 20°C than at 28°C, whereas this is not the case for other populations of Ae. albopictus infected with the same viral strains [START_REF] Zouache | Three-way interactions between mosquito population, viral strain and temperature underlying chikungunya virus transmission potential[END_REF]. This three-way interaction between mosquito population, virus strain and temperature, is known as genotype-by-genotype-by-environment (G×G×E) interactions [START_REF] Zouache | Three-way interactions between mosquito population, viral strain and temperature underlying chikungunya virus transmission potential[END_REF].

Arbovirus transmission by mosquito vectors: mechanism and barriers

The transmission of an arbovirus by the mosquito vector proceeds in several steps as shown in figure 14 [START_REF] Franz | Tissue Barriers to Arbovirus Infection in Mosquitoes[END_REF][START_REF] Hardy | Intrinsic factors affecting vector competence of mosquitoes for arboviruses[END_REF][START_REF] Mellor | Replication of arboviruses in insect vectors[END_REF]. For the transmission to occur the vector should first be attracted and feed on a viremic host. The ingested virus within the blood meal must then rapidly penetrate into the epithelial cells of the intestine before it is altered by the digestive enzymes. Due to specific recognition by interaction of a viral protein with a receptor present on the surface of the digestive epithelium, the virus penetrates into the epithelial cell by endocytosis and replicates there (Figure 14, steps 1 and 2). After replication, newly formed virions must cross the basal lamina which supports the digestive epithelium and then be released into the general cavity of the insect or hemocoel where circulates the hemolymph (arthropod circulatory fluid) (Figure 14, step 3). Once the virus has reached the circulatory system, it can be disseminated in various tissues and organs such as muscle tissues, nervous tissues, fatty body, reproductive organs and salivary glands (Figure 14, step 4). The dissemination to the salivary glands allows the excretion of the virus in the saliva and its potential transmission during a subsequent blood meal (Figure 14, step 5). Thus, the mosquito's gastrointestinal tract and salivary glands appear to be the two main physical barriers to the transmission of the virus by a mosquito [START_REF] Franz | Tissue Barriers to Arbovirus Infection in Mosquitoes[END_REF]. It is largely known that the mosquito defense system is unable to clear arboviral infections but can effectively limit viral dissemination to a tolerable level (by the mosquito). This requires a complex interaction between the mosquito immune system and viruses which makes mosquitoes an ideal vector for virus transmission without being seriously affected.

The mosquito gut is the first-line barrier to limit viral dissemination. Studies conducted on

Ae. aegypti suggest the existence of several pathways to restrict infections by arboviruses, including Flaviviruses: the RNA interference (RNAi) pathway, the Toll pathway, and the JAK-STAT pathway. Moreover, the gut microbial flora is also considered an important factor to control the midgut viral infection [START_REF] Cheng | Mosquito Defense Strategies against Viral Infection[END_REF].

In the hemocoel, the virus is immersed in a nutrient-rich environment that contains immune cells (innate cellular responses) and humoral response factors produced by hemocytes pericardial cells and fatty substances. Although mosquito hemocytes are permissive for the replication of arboviruses, humoral immunity in the hemolymph playsan important role in limiting systemic dissemination of these arboviruses [START_REF] Xiao | Complement-related proteins control the flavivirus infection of Aedes aegypti by inducing antimicrobial peptides[END_REF]. The humoral components refer to soluble molecules in hemolymph such as pattern recognition receptors, antimicrobial peptides, and components of the phenoloxidase (PO) cascade that leads to the formation of melanin [START_REF] Cheng | Mosquito Defense Strategies against Viral Infection[END_REF][START_REF] Hillyer | Mosquito immunity[END_REF]. The role of melanization in eliminating bacterial and parasitic infections in invertebrates is relatively well elucidated. However, the antiviral role of melanization is poorly understood.

The salivary glands are apparently permissive for arboviral replication [START_REF] Vega Rua | Émergence du virus chikungunya en Amérique et en Europe[END_REF].

Nevertheless, recent studies revealed that, at this level, immune reactions that limit viral replication do exist (reviewed in [START_REF] Cheng | Mosquito Defense Strategies against Viral Infection[END_REF]. The salivary peptide cecropin (CEC) like (an antibacterial peptide) has been suggested to have a potent anti-DENV and anti-CHIKV activity. This antiviral activity was validated in an in vitro cell culture system [START_REF] Luplertlop | Induction of a peptide with activity against a broad spectrum of pathogens in the Aedes aegypti salivary gland, following Infection with Dengue Virus[END_REF] and reviewed in [START_REF] Cheng | Mosquito Defense Strategies against Viral Infection[END_REF]). The virus present in the salivary glands ensures horizontal transmission to a new vertebrate host by the saliva emitted by infected females [START_REF] Hardy | Intrinsic factors affecting vector competence of mosquitoes for arboviruses[END_REF].

In some cases, the virus can disseminate to mosquito's reproductive organs. The presence of the virus in the ovaries and eggs can promote its vertical transmission (vertical transmission:

from infected females to eggs). This rare phenomenon allows some arboviruses to persist in the absence of receptive hosts and during dry or cold periods, or during the absence or low density of mosquitoes that adversely affects horizontal transmission [START_REF] Dohm | Effect of incubation at overwintering temperatures on the replication of West Nile Virus in New York Culex pipiens (Diptera: Culicidae)[END_REF][START_REF] Dohm | Experimental vertical transmission of West Nile virus by Culex pipiens (Diptera: Culicidae)[END_REF][START_REF] Lequime | Vertical transmission of arboviruses in mosquitoes: a historical perspective[END_REF].

Viral infection is generally persistent in females. Therefore, once infected, the mosquito will be able to transmit the virus throughout its life. The time between the ingestion of the infectious meal and the time the mosquito becomes capable of transmitting the virus present in the saliva corresponds to the extrinsic incubation period (EIP) [START_REF] Hardy | Intrinsic factors affecting vector competence of mosquitoes for arboviruses[END_REF]. EIP varies according to the dose of virus infected in the mosquito, the viral genotype, the vector genotype, and external factors such as larval nutrition and temperature [START_REF] Hardy | Intrinsic factors affecting vector competence of mosquitoes for arboviruses[END_REF]. For effective transmission of a pathogen, the EIP must be shorter than the life of the vector. Thus, EIP is a key parameter of viral transmission.

Vector competence assessment

As previously noted, the determination of vector competence is essential for evaluating the potential for transmission of arboviruses by vectors. The vector competence is unique and characteristic of each mosquito-virus combination. Indeed, the evaluation of vector competence makes it possible to predict the epidemiological risks posed by the introduction of an arbovirus in a certain environment where the populations of local mosquitoes can present a good receptivity to the viral infection [START_REF] Hardy | Intrinsic factors affecting vector competence of mosquitoes for arboviruses[END_REF][START_REF] Kramer | Dissecting vectorial capacity for mosquito-borne viruses[END_REF]. This helps to identify localities in need of strengthening epidemiological surveillance (control and isolation of imported cases).

The evaluation of vector competence is carried out in the laboratory using experimental To estimate the rate and efficiency of transmission, mosquito saliva should be recovered by forced salivation technique [START_REF] Dubrulle | Chikungunya virus and Aedes mosquitoes: saliva is infectious as soon as two days after oral infection[END_REF]. Briefly, after removing the legs and wings of the female mosquito, the proboscis is inserted gently into a tip containing Fetal Bovine Serum (FBS) and the mosquito is left to salivate for 30-45 minutes (Figure 15). The infectious status and the number of infectious viral particles in collected saliva are determined by viral titration on insect (C6/36) or mammalian (Vero) cells. -Morphologically identified using specific keys.

-Screened for WNV RNA by molecular analysis (RNA extraction and RT-PCR) on identified mosquito pools.

 Collecting blood samples from human residents, horses and chicken in different areas in the country. The collected sera were then analyzed by serological techniques as follows:

-Detection of IgG or IgY antibodies against WNV by in-house ELISA.

-Confirmation of ELISA positive sera by PRNT and estimating the circulation rate of the virus among each population.

-Observation of potential seroconversion in sentinel chickens in order to assess potential current WNV circulation. This was performed by detecting specific IgY level increase in these sentinels during the transmission season.

The second part of this study aimed to assess the vector competence of local populations of Cx. pipiens against both WNV and RVFV. For this, the following activities were performed:

 Culex pipiens egg rafts were collected.

 Mosquitoes were reared and maintained in BSL3 facilities.

 Females of Cx. pipiens were artificially fed with blood infected with WNV or RVFV.

 Estimation of viral infection, dissemination and transmission rates at different days after experimental infection with each virus.

 The vector competence of both Cx. pipiens biotypes was compared in order to detect any genetic influence on the virus transmission.

Introduction

WNF is an arboviral infection caused by a flavivirus that is transmitted from infected birds to humans by the bite of a broad range of mosquito species, but mainly members of the Culex genus. WNV infects mostly birds which constitute the main reservoir. Humans, just like horses, are "dead-end" hosts, meaning that they might be infected but do not spread the virus. The infection is usually asymptomatic in humans. WNV infections have been reported in many tropical and temperate countries in Africa, Europe, Asia, America and the Middle East. Recent outbreaks have affected Palestine in 2000, Greece in 2010 and 2011, and Turkey in 2010. In addition, evidence of virus circulation has been reported from Jordan and Egypt.

In Lebanon, human and animal cases caused by WNV have never yet been reported.

However, we suspect the existence of this disease in the country. The introduction of this virus is likely to occur through the migratory birds that usually cross Lebanon during the migration season. Besides, Culex pipiens, the primary vector, is widespread in the country. 17).

The fourth region is formed by the Anti Lebanon Mountains that form the natural eastern border with Syria. The range's highest peak is Mount Hermon at 2814m. Collections were performed using two types of traps: CDC (Centers for Disease Control) light trap (Florida, USA), fitted or not with a CO 2 source (Figure 19A), and BG-sentinel traps (Biogents ®) using Bg-lure attractant containing lactic acid, ammonia and fatty acids, substances usually found on the human skin (Figure 19B). Traps were placed outdoor (mainly houses backyards) and in animal shelters (cattle and horses) from sunset till the next morning.

Screening for vector infection

Total RNA extraction followed by standard Reverse Transcription Polymerase Chain Reaction (RT-PCR) was performed on mosquito pools using WNV specific primers targeting the NS5 viral gene.

a. RNA extraction RNA stabilizing solution was removed from the tubes, in which mosquitoes were pooled, before any manipulation. Total RNA was extracted using the NucleoSpin RNA isolation kit (Macherey-Nagel, Germany). Following the manufacturer's protocol, a first step of homogenization of the tissues was done by using the kit's Lysis Solution/2-Mercaptoethanol mixture that is required to fully inactivate RNases. Two sterile stainless steel beads (5mm diameter, Qiagen, USA) were added to each tube and tissues were grinded and crushed by vortex until no visible debris was observed (around two minutes). Note that after homogenization, two centrifugations were performed on supernatants of homogenates in order to get rid of mosquito debris that may block the filter/column during the RNA extraction process. After homogenization, proteinase K (Qiagen, USA) was added to the homogenate and incubated at 55°C for 10 min to effectively release RNA from fibrous tissues. RNA extraction process continued as per the manufacturer's protocol. RNA was collected and stored at -80°C. A dosage of the RNA products was performed using the NanoDrop Lite Spectrophotometer (Thermo Scientific, USA). This quantification was used to determine the volume of RNA extract needed for the reverse transcription (RT) step.

b. Reverse transcription

Subsequent to RNA dosage, the GeneAmp Gold RNA PCR Reagent Kit (Applied Biosystems, USA) was used to obtain a cDNA and perform a PCR. A Two-Step RNA PCR was done in order to keep the cDNA for any further studies. As per the manufacturer's protocol, in the Reverse Transcription (RT) step, 5µL RNA samples or templates of the mosquitoes' RNA pools, the positive control RNA (pAW109 RNA) provided by the kit and the negative control or blank were transcribed. This reverse transcription reaction was performed using the GeneAmp PCR System 9700, Applied Biosystems, Thermal Cycler (USA). Then the cDNA products were conserved at -20°C.

c. PCR amplification and viral characterization

The PCR mix was prepared using the Thermo Scientific (USA) PCR master mix kit. Specific selected WNV desalted primers (Integrated DNA Technologies IDT, USA) WNV NS5 WN 9794 and WNV NS5 WN 9483 were used to screen the pools for WNV RNA [START_REF] Kauffman | Detection Protocols for West Nile Virus in Mosquitoes, Birds, and Nonhuman Mammals[END_REF][START_REF] Lanciotti | Rapid detection of west nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan reverse transcriptase-PCR assay[END_REF]. These primers amplify a 326 base pairs (bp) fragment within the Non Structural protein (NS5) gene which encodes for the viral RNA-dependent RNA polymerase [START_REF] Lanciotti | Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States[END_REF]. This polymerase is the most highly conserved protein of WNV (and of Flaviviruses in general) [START_REF] Brinton | The molecular biology of West Nile Virus: a new invader of the western hemisphere[END_REF]. As a consequence of the conserved nature of the region, chosen primers should readily bind to any potential strain of WNV. The sequences of the used primers are shown in table 2. In addition, Primer DM151 and Primer DM152, upstream and downstream respectively, were used to amplify the positive control gene (pAW109 RNA) for the reverse transcriptase reaction. DNA purification was performed using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Germany). According to the manufacturer's protocol, the band of interest was cut from the gel and prepared in order to bind DNA on a silica membrane within the spin column. The eluted DNA was ready for immediate use or stored at -20°C. Purified DNA samples were submitted to sequencing using the same PCR primers. The obtained sequences were identified after their alignment with sequences of different strains published in GenBank.

Blood samples collection

The serological study was conducted on three types of hosts: humans, horses and chickens.

Blood samples were collected in 5mL gel tubes. Human blood samples were all collected by certified laboratory technicians whereas horse and chicken blood samples, they were withdrawn by veterinarians. Most of the collected samples belonged to horses but some were from ponies; the horses were of diverse age, breed and gender.

Blood was obtained from the jugular vein (Figure 20). Horses were checked for microchips using a microchip scanner. ID numbers, horse's name, age, gender, breed and medical history were registered, when available. None of the horses or ponies being tested has been vaccinated against WNV. Some farms and clubs employed pest control measurements on the horses and in the facility.

c. Chicken blood collection

In order to estimate the seroprevalence of WNV, blood samples were collected from egglaying hens that were raised in gardens of private houses in different villages of the Bekaa Plain.

Blood collection was performed between September and December 2015. Samples were collected from 15 different locations in West Bekaa, Zahle, and Baalbek districts (Figure 18).

Moreover, and in order to assess the current circulation of WNV in the Bekaa area, a seroconversion study was conducted. Therefore, two groups of ten sentinel chickens each were placed in Younine (Baalbek) and El Marj (West Bekaa) villages (Figure 18). In each locality, five white and five red chickens (Leghorn) were marked with wing nodes made by ribbons of five different colors. These chickens were entrusted to a villager in each locality who placed them in his private garden (outdoor) during the study period. Blood samples were withdrawn from each of these chickens: one in September 2015 (coinciding with the beginning of bird migration period), one in October 2015 and the last one in December 2015. The aim of this study is to detect the appearance of specific antibodies against WNV in these chickens and to monitor their evolution over time. All Chicken blood samples were collected from the alary vein (Figure 21).

Sera screening by ELISA

After blood collection, all samples of different origins were brought to the laboratory and centrifuged at 4000g for 10 min in order to obtain the sera. Aliquots from each serum were prepared under sterile conditions and stored at -20°C for further use.

All collected sera were tested for the presence of specific antibodies against WNV: IgG antibodies (in human and equine) and IgY antibodies (in chicken). The technique used was an inhouse indirect Enzyme Linked Immunosorbent Assay (ELISA) based on the protocol performed at the Laboratory of Arboviruses (Slingerlands, New York State Department of Health, USA). A series of optimization tests were done to determine Coating Antigens' concentrations, secondary antibody dilutions, and buffers to be used in the laboratory conditions.

The coating antigens consisted of a normal slurry antigen (also called negative antigen) and another positive WNV antigen. The first is composed of the lysate of uninfected Vero cell culture. The latter consisted of the lysate of WNV (New York 1999 strain) cultured on Vero cell.

Both antigens in addition the required human and horse positive and negative controls were generously provided by Dr. Laura Kramer's Laboratory. Chicken positive and negative controls were kindly provided by Dr. Sylvie Lecollinet from Maison Alford, Paris, France. All used controls belonged to sera confirmed positive or negative against WNV by PRNT.

The applied ELISA protocol was similar for human, equine and chicken sera. Briefly, 50µL

of antigen diluted at 1:200 in fresh coating buffer (Appendix E: Coating buffer preparation) were applied to each 96 wells of Nunc-Immuno MicroWell ELISA plates (Sigma, USA). The normal slurry antigen was dispensed in the wells of the 1 st , 4 th , 7 th and 10 th columns while the positive antigen was distributed in the wells of the remaining columns of the plate (Appendix F: plate plan). The antigens were allowed to bind during an overnight incubation at 4°C.

The following day, the coat solution was discarded, the plate was washed three times with Phosphate Buffered Saline (1×PBS) 0.05% Tween 20 (PBS-T) (Appendix E: 10XPBS buffer preparation). 100 L blocking buffer (5% skimmed milk in PBS-T) was added, and the plates were left in a humid chamber at 37°C for 1 hour. Following incubation, blocking solution was discarded and plates were washed as above. 50 L of each serum sample, diluted 1μ200 in PBS-T-BA buffer (1×PBS, 0.05% Tween 20, 0.5% Bovine Serum Albumin (BSA)), were dispensed to one negative and two positive antigen-containing wells. Plates were then returned to a humid chamber and incubated at 37°C for 1 h.

Afterward, plates were removed, washed as above, and 50 L of goat anti-human IgG (H+L)

Horseradish Peroxidase (HRP) secondary antibody (Southern Biotech, USA), diluted 1:4000 in PBS-T-BA buffer, were added to each well. For horse and chicken sera screening, goat antiequine IgG (H+L) HRP (Southern Biotech, USA) or goat anti-chicken IgY (H+L) HRP (Southern Biotech, USA) were respectively used at the same dilution. After incubation and washing as above, potentially formed immune complexes were revealed by the addition of 50 L of 3,3',5,5'-Tetramethylbenzidine (TMB) liquid substrate (Eurobio, France) for 10 min. The reactions were stopped with 50 L of 1μ20 HCl, and the optical density (OD) of each well was read at 450 nm ( read = 450 nm and reference = 4λ2 nm) using ELISA reader Multiskan FC (Thermo Scientific, USA). It is worth noting that in each plate, in addition to the control sera samples, blank (wells without serum and secondary antibodies) and secondary antibodies (wells without serum) controls were designated. For each serum, a ratio of OD obtained on the positive antigen (ODP) over the OD obtained on the negative antigen (ODN) was calculated. We considered a human serum as positive for WNV when the value of ODP/ODN was greater than or equal to 2. Whereas for horse and chicken sera samples, we considered suspected positive all sera showing an ODP/ODN ratio greater than 1.2. These ratio cut-off values were chosen according to the number of tested sera samples, in a way not to miss any potential positive sample.

Some validation criteria were taken into consideration to validate observed results in each plate. These criteria depended mainly on the OD values of the positive and negative control sera, the secondary antibody and blank controls.

Confirmation by PRNT

The plaque-reduction neutralization test (PRNT) is considered the gold standard procedure to confirm arboviral infection especially when other serological techniques, such as ELISA and Immunofuluorescence Assay (IFA), show important cross reactivity between closely related circulating viruses (in our case, other flaviviruses). PRNT is used to determine the titer of neutralizing antibodies for a virus in a serum sample. The assay is time consuming and laborious.

For this reason, it is mainly performed in reference laboratories and in high security level settings.

All sera that turned to be positive for WNV by ELISA (at the fixed threshold), were tested by PRNT for confirmation at the Laboratory of Arboviruses (Wadsworth Center, New York State Department of Health, Slingerlands, New York, USA) as part of a scientific collaboration project with our laboratory (Laboratory of Immunology and Vector-Borne Diseases, Faculty of Public Health, Lebanese University). Here, we take this opportunity to thank Dr. Laura Kramer, head of the American partner laboratory, for her generous collaboration and precious advices that guided our project.

Fresh Vero cell suspension (2.0×10 5 cells/mL) is prepared and dispensed (3 mL/well) in 6 well plates. Plates are then incubated at 37 o C and 5% CO 2 . Test sera were serially diluted from 1:5 through 1:160 in BA-1 diluent (Medium-199H, 0.05M Tris pH 7.6, 1% bovine serum albumin, 0.35g/L sodium bicarbonate, 100 units/mL penicillin, 100 µg/ mL streptomycin, and 1 µg/mL fungizone) in a 96-well plate (100 µL/well). The WNV (strain NY-1999) was diluted to obtain 200 PFU/100µL. Afterward, the viral suspension was distributed on the plate wells and mixed with the existing 100µL of diluted sera. The plate was then incubated overnight at 4°C.

The following day, 100µL of each serum-virus mixture was added onto confluent monolayers of Vero cells (in the 6 well plates) and allowed to adsorb at 37 o C for 1 h, after which an agarose overlay (3 mL/well) was added and incubation was continued. When virus plaques became visible, a second overlay of nutrient-agar (3 mL/well) containing Neutral Red (1mL of Neutral Red/100 mL of overlay) was added. Neutral red stains viable cells leaving white/cleared plaques where virus is present. The plates are then returned to the incubators.

In order to determine the neutralizing titer, a virus control (also called back-titration) was prepared. It consists of duplicate virus suspensions containing 200, 20, and 2 PFU/0.1 mL mixed with an equal volume of BA-1 diluent to contain 100, 10 and 1 PFU/0.1 mL.

Plaques were counted on the 3rd (WNV) day after the viral inoculation. The highest dilution of serum neutralizing 90% of the inoculum as determined by back-titration (control) was considered the neutralizing titer. WNV positive and negative control sera were used in this test.

Statistical analysis

All human serological results were statistically analyzed in terms of frequencies and percentages. For each qualitative variable, seropositivity against WNV, human participant gender, place of residence and presence of domestic animals, frequencies and percentages were calculated. The chi-square test was used to compare the frequencies and percentages between groups of each qualitative variable. Fisher's exact test was used when indicated. Statistical

Package for the Social Sciences (SPSS) software version 16.0 was used for the analysis of obtained data.

Results

Description of mosquito collections

Adult mosquito trapping in West Bekaa (Jebb Jennine, Qaraoun, Ein El Jawzi and El Marj villages), over the period of 6 days, yielded 2934 adult mosquitoes with a total of 78 trap-days.

Identification of these specimens showed that 91.65% of collected mosquitoes were Culex pipiens. The remaining specimens (8.35%) belonged to four other species: Culex martini, Culex perexiguus, Culiseta longiareolata and Orthopodomyia pulcripalpis (Table 3).

Adult mosquito trapping in Aakar, North Lebanon, in three different locations, using CDC light traps and BG sentinel traps, over the period of 15 days, yielded 3912 adult mosquitoes with a total of 88 trap-days. Identification of these specimens showed that 83.90% of collected mosquitoes were Culex pipiens. In addition, other species were found in much lower percentages such as, Culex martini, Culex perexiguus and Culiseta longiareolata (Table 4).

Molecular screening of mosquitoes for WNV

Collected mosquito specimens were separated into pools according to collection site, species identity and gender. Mosquitoes collected from West Bekaa were divided into 66 pools, each contained up to 50 mosquitoes. Fifty seven of them were composed of females Cx. pipiens as shown in Appendix G. Similarly, mosquitoes collected in Aakar were divided into 141 pools.

Each pool contained up to 25 mosquitoes; 119 of these pools were composed of females Cx.

pipiens (Appendix G). Following DNA extraction, each pool was screened for the presence of WNV gene by a 2-steps RT-PCR. Screening results showed that all tested pools were negative for WNV (Figure 22).

Serological study of human population a. Description of participants

For this study, we analyzed 803 serum samples of consenting outpatients attending laboratory facilities of three hospitals in West Bekaa: 168 from Tal Chiha Hospital in Zahle, from Bekaa Hospital in Taanayel-Chtoura and 134 from Farhat Hospital in Kamed El Lawz. In addition, in Northern Lebanon, a total of 288 sera were collected from consenting participants recruited equally in the UNRWA dispensary at Nahr El Bared Camp, Akkar and in the laboratory of the military clinic of El Kobbeh Army Barrack, Tripoli (Table 5). Almost two third of the participants were females (63.34%) (Table 5). The age range of the population was between 9 and 85 years with an average age of 40.7, a median of 39, and a high representation of participants aging between 21 and 30 years old (Figure 23). Their occupations varied from indoor and outdoor types of jobs, to soldiers or students (Figure 24). Moreover, female participants (72.21%) were either house wives or unemployed; and 97 male participants (24.25%) were either unemployed or retired. 143 male participants had outdoor jobs such as in agriculture, farms, construction or drivers or several other occupations. 104 male participants had indoor or desk jobs in banks, administration of companies, or hospital staff or other occupations.

The remaining participants were soldiers and students (Figure 24). Sera analysis using an in-house ELISA technique was performed on all collected samples, and those showing an OD ratio above 2 were considered as suspected positive. Results revealed that 74 individuals (9.22%, N=803) from Bekaa region and 2 participants (0.69%, N=288) from North area were seropositive against WNV (Figures 25 and 26). This brings the seroprevalence rate for both regions to 6.97% (76 sera out of 1091). In order to confirm seropositivity after ELISA, PRNT was performed on suspected sera (OD ratio ≥2). Nevertheless, for the North area, and since the number of suspected positive samples is limited (only 2), we included in the seroneutralization test those sera having OD ratio ≥ 1.7 in order to avoid missing any potential positive specimen (Figure 26). Results showed that only 11 samples from the Bekaa region had neutralizing antibodies at a titer of 40 or higher whereas no sera from North Lebanon had WNV specific neutralizing antibodies at the fixed titer. This brings the seroprevalence rate of WNV to 1.37% in the Bekaa area and 0% in North Lebanon area (1.01% in both regions together). It is worth noting that three and four samples, from Bekaa and North regions respectively, had neutralizing antibodies at a titer of 20 or below (Table 6).

b. Seroprevalence results

ELISA testing showed that 10 sera samples had an OD ratio above 1.2 (Figure 30). Thus the seroprevalence rate is 4.95 % for the fixed threshold. PRNT was performed on these samples. It revealed that only four had neutralizing antibodies for WNV at a titer of 40 or higher (Table 7).

Thus, the seroprevalence rate dropped to 1.98%. Only one horse had specific antibodies at titer Similarly, the 20 chickens (10 white and 10 red) used for the seroconversion study part were of Leghorn breed.

b. Seroprevalence results

ELISA screening of chickens for WNV specific IgY did not reveal any clear positive serum.

In fact, the OD ratios observed were all less than 1.2, except one serum that had an OD ratio of 1.26 (Figure 32). Two chicken sera (those having the highest OD ratio value) were further tested by PRNT against WNV and both turned to be negative. Thus, in the present study, the observed seroprevalence rate against WNV among chicken is null.

c. Seroconversion results

As for the temporal analysis of chicken sera by ELISA technique, results did not show any onset or increase of WNV specific antibody levels in any of the selected individuals in either locations. The observed OD ratios remained comparable in the three samples taken for each chicken (Figure 33). These results show that during the study period and in the Bekaa area, there was no detectable circulation of WNV and therefore, the tested chickens were not exposed to infectious bites of the mosquito vector.

Statistical analysis

Among serology results of human sample populations, no statistical significant differences were observed between seropositivity against WNV and any factor for human (sex, profession…).

Discussion and Conclusion

Lebanon is considered non-endemic and non-enzootic for WNV. To date, no WNF cases were officially reported. Our study brings a strong evidence of its occurrence in the country. The detection of relatively high titer of neutralizing antibodies IgG (≥320) in participants who never travelled out of the country confirms a local exposure to this virus. Moreover, the fact that all seropositive human cases (n=11) originated from the Bekaa area suggests that this region is the primary introduction site of WNV in the country. This area is considered a stopover zone for migratory birds. It harbors several water plans (Aammik swamps and Qaraoun lake) and important agriculture activities, which make it suitable for the introduction and local spread of the WNV. The detection of confirmed positive sera (by PRNT) among horses is an additional evidence of WNV circulation as all seropositive horses were of local breed with no record of cross-border transfer. However, their movement within the country did not allow us to conclude on the exposure area to WNV.

The absence of WNV specific antibodies (IgY) in the poultry study population from the Bekaa and the absence of seroconversion in sentinel chickens suggest that there is no active current circulation of WNV in this region. Apparently, the circulation of WNV in Lebanon, when it occurs, seems to happen at low rate, which explains why human and animal cases have not yet been reported in the country.

In a previous study, conducted by Gallian and collaborators (2010), among 639 Lebanese blood donors, six were WNV seropositive by ELISA among which only 3 (0.47%) were confirmed positive by PRNT at titers higher than 1/10. Seropositive participants lived in South Lebanon, Beirut, and the Bekaa however the authors did not specify whether these participants had any travel history or not [START_REF] Gallian | Seroprevalence of West Nile virus in blood donors at Hôtel Dieu de France, Beirut, Lebanon[END_REF].

The observed seroprevalence rate in our study, although relatively low, is comparable to what is usually reported from geographically close endemic countries such as in Greece where the seroprevalence rate was 1.02% following confirmation by PRNT [START_REF] Papa | West Nile virus--neutralizing antibodies in humans in Greece[END_REF]. In the neighboring Turkey, the WNV seroprevalence rate, as also confirmed by PRNT, have been reported to be as low as 0.8% in blood donors [START_REF] Ayturan | Investigation of West Nile virus seroprevalence in Hacettepe University Hospital blood donors and confirmation of the positive results by plaque reduction neutralization test[END_REF], and as high as 17% in Mardin province (near the Syrian border) among human population following the appearance suspected encephalitis cases [START_REF] Karakoç | West nile virus infection in the Mesopotamia region, Syria border of Turkey[END_REF]. In another study conducted in the Manisa province on the Aegean Sea, West of Turkey, 4.3% of tested sera of healthy rural residents were seropositive following ELISA screening and confirmation by IIFA [START_REF] Gazi | Seroprevalence of West Nile virus, Crimean-Congo hemorrhagic fever virus, Francisella tularensis and Borrelia burgdorferi in rural population of Manisa, western Turkey[END_REF]. Similarly, in Egypt, WNV neutralizing antibodies (by PRNT) were found in humans and the rate varied from 1% (in Northern Sinai) to 35% (in Upper Egypt) [START_REF] Soliman | Studies on West Nile virus infection in Egypt[END_REF]. In Jordan, that, like Lebanon, is considered non-endemic for WNV, the seroprevalence rate was estimated at 8% in care center population following screening by ELISA [START_REF] Batieha | Seroprevalence of West Nile, Rift Valley, and sandfly arboviruses in Hashimiah, Jordan[END_REF].

In Israel, a country at the Southern border of Lebanon, WNV has been responsible for several outbreaks. In the year 2000, hundreds of cases were reported from this country, mainly from the central and northern regions, close to Lebanon [START_REF] Weinberger | West Nile fever outbreak, Israel, 2000: epidemiologic aspects[END_REF]. A cross-sectional study, involving more than 3100 healthy individuals recruited between 2011 and 2014, showed that 11% of subjects had specific antibodies IgG for WNV following screening by ELISA and confirmation by PRNT. This study revealed that the Coastal Plain, the Inland Plain and the Great Rift Valley, which is a geographic extension of the Bekaa valley, had the highest seroprevalence of WNV antibodies [START_REF] Bassal | The seroprevalence of West Nile Virus in Israel: A nationwide cross sectional study[END_REF]. These observations suggest that WNV has a lower prevalence in Lebanon than in Israel.

The serosurvey among horses revealed a 4.95% seropositive rate for WNV by ELISA which dropped to 1.98% after PRNT. All confirmed positive horses (n=4) came from different geographical locations (Tripoli, Aakar and Nabatiyeh). These results represent the first record of equine exposure to WNV in Lebanon. The observed seroprevalence rate is considered low compared to that observed in neighboring or distant endemic countries. In Turkey, following the WNV outbreak in 2011, the seroprevalence rate among horses was 21% after confirmation by PRNT [START_REF] Ozkul | Concurrent occurrence of human and equine West Nile virus infections in Central Anatolia, Turkey: the first evidence for circulation of lineage 1 viruses[END_REF]. In South of France, following an outbreak among horses in 2000, a serosurvey conducted among equines revealed a seroprevalence rate of 8.5% by ELISA [START_REF] Durand | West Nile virus outbreak in horses, southern France, 2000: results of a serosurvey[END_REF]. Moreover, in Ukraine, a similar study was conducted in 2013 following limited WNV outbreak among humans in 2011 and 2012 and showed a seroprevalence rate of 13.5% by PRNT [START_REF] Ziegler | West nile virus antibody prevalence in horses of Ukraine[END_REF]. Nevertheless, the seroprevalence observed in our study is comparable to what was reported from Iran where the endemicity of WNV is considered relatively low among both human and horses. Following screening by ELISA technique, WNV seroprevalence rate among horses (N=315) was 2.8% [START_REF] Chinikar | Seroprevalence of West Nile virus in Iran[END_REF]. Interestingly, in Jordan and Algeria, the seroprevalence rates among horses using ELISA technique were 24.9% and 26.8% respectively [START_REF] Abutarbush | West Nile virus infection in horses in Jordan: clinical cases, seroprevalence and risk factors[END_REF][START_REF] Lafri | Seroprevalence of West Nile virus antibodies in equids in the North-East of Algeria and detection of virus circulation in 2014[END_REF]. In the absence of confirmation by PRNT, these high rates should be carefully interpreted since they can reflect the circulation of not only WNV, but also other Flaviviruses.

The ELISA technique is considered a screening method to test a relatively large number of sera. This technique is very sensitive but can lead to the detection of false positive sera due to the cross-reaction with other Flaviviruses (such as Dengue Virus, Zika virus, Usutu virus, or other viruses) that might be circulating in the region without being noticed. Previous studies in Lebanon confirmed arbovirus circulation in [1945][1946], in which an epidemic of Dengue fever was reported from Beirut region and affected more than 100,000 individuals [START_REF] Hitti | A Report on the Recent Epidemic of Dengue in Beirut, Lebanon, and some of its Complications[END_REF]. Another serological survey conducted during 1962-1963 showed a seroprevalence for DENV of 61.9% using hemagglutination-inhibition test [START_REF] Garabedian | Serologic evidence of arbovirus infection in Lebanon[END_REF]. Besides, the tiger mosquito (Aedes albopictus) is well spread in Lebanon [START_REF] Haddad | Presence of Aedes albopictus in Lebanon and Syria[END_REF]. Lebanese strains of this mosquito appear to be able to transmit Dengue virus under experimental conditions [START_REF] Haddad | Aedes albopictus in Lebanon, a potential risk of arboviruses outbreak[END_REF]. Moreover, the tiger mosquito is a possible vector of Zika virus [START_REF] Wong | Aedes (Stegomyia) albopictus (Skuse): a potential vector of Zika virus in Singapore[END_REF] 2017). Some of these species of wild and domestic birds were seropositive against WNV in endemic countries such as Israel [START_REF] Di Sabatino | Epidemiology of West Nile disease in Europe and in the Mediterranean Basin from 2009 to 2013[END_REF], Greece [START_REF] Valiakos | Molecular detection and phylogenetic analysis of West Nile virus lineage 2 in sedentary wild birds (Eurasian magpie), Greece, 2010[END_REF], Hungary [START_REF] Erdélyi | Clinical and pathologic features of lineage 2 West Nile virus infections in birds of prey in Hungary[END_REF], and USA [START_REF] Nash | The outbreak of West Nile virus infection in the New York City area in[END_REF][START_REF] Steele | Pathology of fatal West Nile virus infections in native and exotic birds during the 1999 outbreak in New York City, New York[END_REF]. Moreover, in Germany, during 2005-2009, a study detected WNV neutralizing antibodies in 10 species of migratory birds, but not in resident birds [START_REF] Seidowski | West Nile virus monitoring of migratory and resident birds in Germany[END_REF]. Migratory birds are very difficult to monitor. It was proven that chickens are very sensitive to WNV infection but they usually do not develop clinical symptoms. Besides, they develop relatively low viremia that is insufficient to infect mosquito vectors [START_REF] Langevin | Experimental infection of chickens as candidate sentinels for West Nile virus[END_REF]. Nevertheless, following infections, these birds develop detectable neutralizing antibodies. Consequently, chickens are largely used during WNV surveillance programs as sentinel animals to detect the circulation of the virus. In order to assess WNV circulation, we screened Leghorn chickens for viral exposure.

These chickens have a life span of 1 to 6 years. Therefore, their serological monitoring is useful to detect relatively recent WNV infection. In our study, we were not able to detect WNV exposure. To our knowledge, no other studies attempted to assess the circulation of WNV neither in domestic nor in migratory birds in Lebanon.

Our entomological surveys in Bekaa and Akkar revealed high presence of Culex pipiens, the known primary vector of WNV. Moreover, another potential vector, Culex perexiguus, was also present but in limited numbers. These findings are in concordance with previous studies [START_REF] Knio | A two-year survey on mosquitoes of Lebanon[END_REF] and entomological surveys conducted by our laboratory (unpublished data). Molecular analysis results revealed that none of the mosquito pools was found naturally infected with WNV. This is not very surprising if we consider the low circulation of the virus in the country as suggested by the observed low seroprevalence rates among human and horses. Therefore, a much higher number of mosquitoes should have been screened in order to increase the chances of virus detection in the potential vectors.

In the EMR, WNV was naturally isolated from different Culex species in endemic countries in which the seroprevalence rates among human and horses were much higher than those observed in the present study. In Turkey, after an outbreak in 2012, WNV was isolated from 1.9% of collected mosquito pools [START_REF] Ergunay | Arboviral surveillance of field-collected mosquitoes reveals circulation of West Nile virus lineage 1 strains in Eastern Thrace, Turkey. Vector Borne Zoonotic[END_REF]. Whereas in Israel, analysis of 277186 mosquitoes collected during 15 years (1985)(1986)(1987)(1988)(1989)(1990)(1991)(1992)(1993)(1994)(1995)(1996)(1997)(1998)(1999)(2000), allowed the detection of WNV lineages 1 and 2 in 336 mosquito pools [START_REF] Lustig | Mosquito Surveillance for 15 Years Reveals High Genetic Diversity Among West Nile Viruses in Israel[END_REF]. The virus was isolated from 2.39% and 9.22% of Cx. pipiens and Cx. perexiguus pools respectively [START_REF] Lustig | Mosquito Surveillance for 15 Years Reveals High Genetic Diversity Among West Nile Viruses in Israel[END_REF]. Lustig and collaborators' observations in 2016 highlighted the role of this latter species in the spread of the virus. This mosquito was previously associated with WNV infection. Following molecular screening of almost 430,000 mosquitoes collected during eight consecutive years (2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007), the majority of WNV infections were found in the three most common species: Cx. pipiens, Cx. perexiguus, and Ae. caspius. The WNV infection rate of Cx. perexiguus (2.6%) was significantly higher than those of the first and the third species (0.5% and 0.8% respectively) [START_REF] Orshan | Mosquito vectors of West Nile Fever in Israel[END_REF]. In the same study, the authors noticed that in the year 2004, when a sharp decline of the number of human cases occurred, only Cx. perexiguus was found infected. In Spain, Munoz and collaborators (2012) analyzed the feeding pattern of different potential WNV vectors belonging to the Culex genus. They noticed that the feeding behavior of Cx. perexiguus is in favor of WNV transmission from bird to bird and from bird to horse, this mosquito species being then the primary candidate for enzootic (bird-to-bird) transmission and for epizootic transmission to horses [START_REF] Muñoz | Feeding patterns of potential West Nile virus vectors in south-west Spain[END_REF]. The authors noticed that Culex pipiens is also an enzootic vector, but has only a low risk for epizootic transmission to humans. In Lebanon, the ecology of both vectors should be further investigated in order to better understand the pattern of transmission of WNV in the country.

In conclusion, during this study, we were able to confirm that WNV circulates in Lebanon.

This circulation was more obviously detected in the Bekaa region where the seroprevalence rate for humans was 1.37%. Besides, and for the first time, we detected seropositive horses in the country (1.98%). We assume that the exposure to WNV is not recent as no seroconversion in sentinel chickens and no WNV particles in mosquito populations were detected. Culex pipiens,

Introduction

West Nile virus (WNV) and Rift Valley fever virus (RVFV) are two important emerging mosquito-borne zoonotic agents transmitted by Culex pipiens, a complex of sibling species that includes Cx. pipiens s.s., Cx. quinquefasciatus and possibly Cx. australicus [START_REF] Harbach | Culex pipiens: species versus species complex taxonomic history and perspective[END_REF][START_REF] Turell | Members of the Culex pipiens complex as vectors of viruses[END_REF]. Cx. pipiens s.s. includes two biotypes or subspecies: Cx. pipiens pipiens and Cx.

pipiens molestus [START_REF] Fonseca | Emerging vectors in the Culex pipiens complex[END_REF][START_REF] Harbach | Culex pipiens: species versus species complex taxonomic history and perspective[END_REF]. The first biotype is primarily a birdfeeding mosquito present in temperate areas while Cx. pipiens biotype molestus feeds on mammals (mainly human) and thrives in sewers in temperate and sub-tropical regions [START_REF] Bahnck | Rapid assay to identify the two genetic forms of Culex (Culex) pipiens L. (Diptera: Culicidae) and hybrid populations[END_REF][START_REF] Fonseca | Emerging vectors in the Culex pipiens complex[END_REF]. Because morphological identification of these biotypes is not possible, they can only be distinguished using molecular techniques [START_REF] Bahnck | Rapid assay to identify the two genetic forms of Culex (Culex) pipiens L. (Diptera: Culicidae) and hybrid populations[END_REF][START_REF] Fonseca | Emerging vectors in the Culex pipiens complex[END_REF][START_REF] Price | Genetic divergence between populations of feral and domestic forms of a mosquito disease vector assessed by transcriptomics[END_REF].

WNV is a member of the Flavivirusgenus (Flaviviridae family) and was first isolated in Uganda in 1937 [START_REF] Smithburn | A Neurotropic Virus Isolated from the Blood of a Native of Uganda[END_REF]. Usually, only 20% of infected individuals develop symptoms and less than 1% of infected people develop serious and potentially fatal neurological illnesses such as meningitis and encephalitis. Birds are considered the main reservoir of the virus and Cx. pipiens is recognized as the primary enzootic vector [START_REF] Andreadis | The contribution of Culex pipiens complex mosquitoes to transmission and persistence of West Nile virus in North America[END_REF]. WNV infections have been reported in many tropical and temperate countries in Africa, Europe, Asia and America. The Middle East and North Africa (MENA) region has been long considered as a WNV-endemic area [START_REF] Marka | West Nile virus state of the art report of MALWEST Project[END_REF]Murgue et al., 2001a). Locally acquired cases have been recently reported in Israel [START_REF] Paz | Environmental drivers of West Nile fever epidemiology in Europe and Western Asia--a review[END_REF], Greece [START_REF] Papa | Emerging arboviral human diseases in Southern Europe[END_REF], Turkey [START_REF] Failloux | Surveillance of Arthropod-Borne Viruses and Their Vectors in the Mediterranean and Black Sea Regions Within the MediLabSecure Network[END_REF], and Italy [START_REF] Calistri | Epidemiology of west nile in europe and in the mediterranean basin[END_REF]. In addition, evidence of WNV circulation has been reported in Jordan [START_REF] Batieha | Seroprevalence of West Nile, Rift Valley, and sandfly arboviruses in Hashimiah, Jordan[END_REF] and Egypt [START_REF] Soliman | Studies on West Nile virus infection in Egypt[END_REF]. The introduction of WNV into the United States in 1999, which constitutes a turning point in WNV epidemiology, is thought to have originated from Israel following introduction from Africa [START_REF] Giladi | West Nile encephalitis in Israel, 1999: the New York connection[END_REF][START_REF] Lanciotti | Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States[END_REF].

RVFV belongs to Phlebovirus genus (Bunyaviridae family). It was first identified in Kenya in 1931 [START_REF] Daubney | Enzootic hepatitis or rift valley fever. An undescribed virus disease of sheep cattle and man from east africa[END_REF]. This virus usually affects livestock and causes abortion. The main enzootic vectors belong to the Aedes genus [START_REF] Tantely | A review of mosquitoes associated with Rift Valley fever virus in Madagascar[END_REF]. However, several Culex species, including Cx. pipiens are considered secondary vectors and contribute to the transmission of WNV to humans [START_REF] Tantely | A review of mosquitoes associated with Rift Valley fever virus in Madagascar[END_REF]. Infected people can be asymptomatic or develop a mild febrile disease. In less than 10% of cases, people may develop more severe symptoms such as encephalitis and hemorrhagic fever. RVFV was responsible for numerous outbreaks among animals and humans in Sub-Saharan Africa [START_REF] Himeidan | Recent outbreaks of rift valley Fever in East Africa and the middle East[END_REF] up to Mauritania (El Mamy et al., 2011) but also in Egypt [START_REF] Meegan | The Rift Valley fever epizootic in Egypt 1977-78[END_REF]. In the Middle East, epizootics were reported in Saudi Arabia and Yemen [START_REF] Ahmad | More deaths from Rift Valley fever in Saudi Arabia and Yemen[END_REF].

In Lebanon, Cx. pipiens is a predominant mosquito species besides another vector of arboviruses, Aedes albopictus [START_REF] Haddad | Presence of Aedes albopictus in Lebanon and Syria[END_REF][START_REF] Knio | A two-year survey on mosquitoes of Lebanon[END_REF]. Cx. pipiens colonizes urban and rural habitats whereas Ae. albopictus is mostly present in the densely populated coastal fringe. Local Ae. albopictus are competent to transmit Chikungunya virus and to a lesser extent, Dengue virus [START_REF] Haddad | Aedes albopictus in Lebanon, a potential risk of arboviruses outbreak[END_REF]. The vector competence of local populations of Cx.

pipiens to transmit WNV and RVFV has never been evaluated. Diseases caused by these two viruses have never been reported in Lebanon. Nevertheless, a serological study conducted in a main hospital in the capital city of Beirut, confirmed the presence of neutralizing WNV antibodies in blood donors [START_REF] Gallian | Seroprevalence of West Nile virus in blood donors at Hôtel Dieu de France, Beirut, Lebanon[END_REF]. In fact, Lebanon is situated in a WNV-endemic area and located on the flyways of migratory birds with potential introduction of the virus into the country. Moreover, Lebanon is geographically close to Yemen and Saudi Arabia, regions where RVFV had circulated actively. Intensive livestock trade between Lebanon and these countries increases the risk of RVFV introduction.

Here, we assess the vector competence of local populations of Cx. pipiens towards WNV and RVFV. For each Cx. pipiens biotype, we estimate viral infection, dissemination and transmission at different days after experimental infections.

Material and Methods

Mosquito collections

Culex egg rafts were sampled in June 2015 in Bab Mareh, in the West Bekaa district, a subhumid, agricultural area in east of Lebanon with large stagnant water systems (Fig. 34). Egg rafts were collected on the water surface in an artificial basin and placed individually in a tube containing 30mL of water collected from the breeding site.

Viruses and blood meal preparation

Two viruses were used in this study: WNV lineage 1 strain isolated from a horse in Camargue (France) in 2000 [START_REF] Murgue | West Nile outbreak in horses in southern France, 2000: the return after 35 years[END_REF] and an avirulent RVFV strain Clone 13 isolated from a human case in Bangui (Central African Republic) in 1974 [START_REF] Muller | Characterization of clone 13, a naturally attenuated avirulent isolate of Rift Valley fever virus, which is altered in the small segment[END_REF]. After passages on Vero (E6) cells (ATCC cell lines), both viruses were produced on C6/36 mosquito cells. Viral stocks were stored at -80°C until use. The infectious blood meal was composed of a viral suspension (1:3) diluted in washed rabbit erythrocytes (New Zealand White rabbit, Charles

River) collected at the day of mosquitoes infection. A phagostimulant (ATP) was added at a final concentration of 5mM. Virus titer in the blood meal was 1.6×10 8 plaque forming units (PFU)/mL for WNV and 1.33×10 7 PFU/mL for RVFV.

Artificial feeding of female mosquitoes

The susceptibility of Lebanese Cx. pipiens mosquitoes to WNV and RVFV was tested on F0 and F1 generation respectively. Ten-to-twelve day-old female Cx. pipiens mosquitoes were left to starve for 48 h in Biosafety Level 3 (BSL3) insectary at 28±1°C with 80% relative humidity and a 16h:8h photoperiod. Females were then allowed to feed for one hour through a chicken skin membrane (obtained from a commercially purchased chicken) covering the base of a capsule of the feeding system (Hemotek) containing the blood-virus mixture maintained at 37°C. Fully engorged females were sorted, then transferred in cardboard containers and maintained with 10% sucrose at 28±1°C until examination.

The capacity of the WNV and RVFV to cross the salivary glands barrier was evaluated by determining the transmission efficiency (TE) which corresponds to the proportion of female mosquitoes that secrete infectious saliva among tested specimens. The number of infectious particles within collected saliva samples was estimated on Vero cell culture and expressed as PFU/saliva. Briefly, 20µL of each saliva were diluted in 280µL DMEM 2% FBS. The total volume was inoculated on a monolayer of Vero cells (8.10 5 cells/well) in six-well plates. Cells were incubated at 37°C for 6days under an overlay consisting of DMEM, 2% FBS, 1%

antibiotic-antimycotic mix and 1% agarose. The lytic plaques were counted after staining with a crystal violet solution.

Statistical Analysis

Proportions (IR, DE and TE) were compared using Fisher's exact test and sample distributions (number of viral particles) with the Kruskal-Wallis test. Statistical analyses were conducted using the Stata software (StataCorp LP, Texas, and USA). P-values <0.05 were considered significant.

Results

Artificial feeding

Collected egg rafts were hatched in laboratory conditions and provided 480 adult females of Culex pipiens (F0 generation); of those only 174 (36.25%) had successfully fed on a WNVinfected blood. A batch of 600 F1 female mosquitoes was used for the RVFV infection assay. Of those, only 91 (15.16%) had successfully fed on infected blood. challenged with WNV at a titer of 1.6×10 8 PFU/mL and RVFV at titer of 1.33×10 7 PFU/mL using an artificial feeding system (Hemotek). At 3, 7, 14, and 19 dpi, homogenates of mosquito bodies and heads, and saliva collected from females were tested for the presence of virus on Vero cells to estimate infection rate, dissemination efficiency, and transmission efficiency, respectively. In brackets, the number of tested mosquitoes. Error bars show the confidence interval (95%).

Vector competence of Cx. pipiens biotypes

Mosquitoes previously examined were then analyzed according to their biotype. When infected with WNV, the three biotypes presented similar IR, DE and TE at each dpi (Fisher's exact testμ p >0.05) (Fig. 37A-C). Cx. pipiens molestus started to transmit WNV from 3 dpi while pipiens biotype from 7 dpi and hybrids from 14 dpi. When infected with RVFV, IR, DE and TE were slightly similar at each dpi whatever the biotype (Fisher's exact testμ p > 0.05) (Fig. 38A-C).

RVFV was only detected in saliva of Cx. pipiens biotype pipiensfrom 19 dpi. No virus was detected in saliva of molestus and hybrid biotypes. For these results, it is important to consider the low number of individuals analyzed. When comparing the amount of virus expectorated by the two Cx. pipiens biotypes and their hybrids, they delivered more than 500 viral particles from day 3 for molestus biotype, day 7 for pipiens biotype and day 14 for hybrids (Fig. 39A). Only one Cx. pipiens biotype pipiens delivered 42 particles of RVFV in saliva at 19 dpi (Fig. 39B). interactions between mosquito and virus genotypes; these interactions promoting adaptation of viral lineages to specific mosquito vector genotypes influence the outcome of transmission [START_REF] Fansiri | Genetic mapping of specific interactions between Aedes aegypti mosquitoes and dengue viruses[END_REF]. In addition, when viral dose increases in blood meals, transmission efficiency also increases suggesting that hosts presenting a high viremia may infect more mosquitoes [START_REF] Martin | Phylogeography of West Nile virus: from the cradle of evolution in Africa to Eurasia, Australia, and the Americas[END_REF]. Animals susceptible to RVFV can develop very high viremia, higher than 10 10.1 MIPLD50 (mouse intraperitoneal 50% lethal dose/mL) in lambs [START_REF] Easterday | The pathogenesis of Rift Valley fever in lambs[END_REF]. Then, at higher titers of blood meal, RVFV may infect more mosquitoes.

Our study showed that different biotypes of Cx. pipiens exhibit different vector competence to WNV and RVFV. Cx. pipiens biotype molestus transmits earlier WNV than pipiens and hybrid biotypes. Therefore, Cx. pipiens biotype molestus has the shortest EIP under our experimental conditions. Mosquitoes of the molestus biotype persist all year long (not able to diapause during winter), are autogeneous (lay the first batch of eggs without taking a bloodmeal) and stenogamous (mate in confined spaces) mainly in underground areas in cities [START_REF] Harbach | Culex (Culex) pipiens Linnaeus (Diptera: Culicidae): concepts, type designations, and description[END_REF]. Mosquitoes of this biotype feed mainly on mammals and then can potentially transmit pathogens such as WNV to humans. It can be suggested that the molestus biotype predominant in urban settings could intervene in an inter-human transmission of WNV.

It has been shown that transmission of WNV by molestus was not affected by temperature [START_REF] Vogels | Vector competence of northern European Culex pipiens biotypes and hybrids for West Nile virus is differentially affected by temperature[END_REF]; the colonization of hypogenous sites (where temperatures are more stable) may lead to select genetically differentiated mosquito populations more adapted to this environment with presumably effects on their phenotypic characteristics including vector competence for arboviruses.

With RVFV, the biotype pipiens was alone capable of transmitting the virus at 19 dpi.

While the infection was efficient from 3 dpi for the three biotypes, only Cx. pipiens biotype pipiens was able to disseminate the virus at 19 dpi. This result indicates that the midgut plays a significant role of barrier to the release of the virus into the mosquito general cavity. Moreover, the RVFV strain Clone 13 which is a naturally attenuated virus characterized by a deletion of 70% of the NSs gene encoding for a virulence factor [START_REF] Billecocq | NSs protein of Rift Valley fever virus blocks interferon production by inhibiting host gene transcription[END_REF][START_REF] May | TFIIH transcription factor, a target for the Rift Valley hemorrhagic fever virus[END_REF] replicates differently in mosquitoes. Viral dissemination was higher when mosquitoes were exposed to a virulent RVFV such as ZH548 [START_REF] Moutailler | Potential vectors of Rift Valley fever virus in the Mediterranean region[END_REF]. In our study, only one Cx.

pipiens biotype pipiens was able to transmit the virus at 19 dpi with 42 viral particles detected in saliva. This viral load was more than 14 times lower than the dose detected in Cx. pipiens mosquitoes from the Maghreb region (i.e. 620 viral particles) [START_REF] Amraoui | Culex pipiens, an experimental efficient vector of West Nile and Rift Valley fever viruses in the Maghreb region[END_REF], but probably enough to infect hosts, animals and humans.

In conclusion, the predominant Cx. pipiens mosquito in Lebanon is susceptible to both viruses, WNV and RVFV. The biotype molestus can transmit WNV earlier than pipiens and hybrid biotypes whereas the biotype pipiens acts as the sole efficient experimental vector of RVFV, i.e. capable of transmitting at late days post-infection. As Lebanon is located in a region

where WNV and RVFV can be potentially introduced (respectively through migratory birds and animal trades), local health authorities should establish an active surveillance to detect any new human cases in addition to reinforce the entomological surveillance allowing an early viral detection in field-collected mosquitoes.

Chapter 8

General Discussion and Perspectives

WNV and RVFV are mosquito-borne viruses. Their introduction to a given country usually occurs following the introduction of viremic hosts. The vector capacity of the local vectors is the determinant factor that would allow the transmission of these viruses in the country. This capacity does not depend only on the receptivity of the vector to the virus but also on several extrinsic factors such as attractiveness to the host and vector density. Culex pipiens is a potential vector for both viral species. In the Middle East, high human population density and humaninduced habitat changes creating potential mosquito breeding sites favor the presence of this mosquito [START_REF] Conley | Modeling the distribution of the West Nile and Rift Valley Fever vector Culex pipiens in arid and semi-arid regions of the Middle East and North Africa[END_REF].

The transmission of WNV occurs in Lebanon as we were able to demonstrate the exposure of local hosts (humans and horses) to this virus. The Bekaa plane is the probable introduction region of the virus. Besides, Culex pipiens of this region showed to be receptive to WNV. The biotype molestus which is known to feed on mammals including humans and also on birds [START_REF] Harbach | Culex pipiens: species versus species complex taxonomic history and perspective[END_REF], showed a shorter EIP than the pipiens biotype under our experimental conditions. Therefore, molestus biotype is highly competent to ensure transmission of WNV to humans. The observed low endemicity of this virus, compared to what is usually observed in the neighboring Israel, that is located on the same flyway of migratory birds as Lebanon, is intriguing. The climatic conditions of the Bekaa plane, which is a plateau of 800-900 m of altitude, during the bird migration season, could explain this difference. Indeed, during spring (northward migration) and fall (southward migration), the local temperatures in the Bekaa valley are not high enough and consequently, mosquito population densities are not very important.

WNV does not seem to be the only flavivirus circulating in Lebanon as shown in the important cross-reactivity with WNV antigens by the In-House ELISA testing. Several avian flaviviruses, occurring in Africa, are likely to occur in the EMR, including Lebanon, through bird migration as one of their major routes from Africa passing through the Middle East (Figure 17).

Bagaza virus (BAGV), which was recently considered a synonymous of Israel Turkey Menigoencephalitis virus [START_REF] Fernández-Pinero | Bagaza virus and Israel turkey meningoencephalomyelitis virus are a single virus species[END_REF], is one of these avian flaviviruses that can potentially occur in the EMR. This virus affects mainly birds; however, specific antibodies were detected in patients suffering from encephalitis in India [START_REF] Bondre | Genetic characterization of Bagaza virus (BAGV) isolated in India and evidence of anti-BAGV antibodies in sera collected from encephalitis patients[END_REF]. Usutu Virus (USUV) is another avian flavivirus, with more zoonotic potential, that can be spread to the EMR.

This virus was described in South Africa and was introduced to Europe where it caused sudden death in birds and affected several immune-compromised persons in Italy during the year 2009 [START_REF] Becker | Epizootic emergence of Usutu virus in wild and captive birds in Germany[END_REF][START_REF] Savini | Usutu virus in Italy: an emergence or a silent infection?[END_REF]. Both BAGV and USUV can be transmitted by Cx.

pipiens [START_REF] Braverman | Detection of Israel turkey meningo-encephalitis virus from mosquito (Diptera: Culicidae) and Culicoides (Diptera: Ceratopogonidae) species and its survival in Culex pipiens and Phlebotomus papatasi (Diptera: Phlebotomidae)[END_REF][START_REF] Calzolari | Evidence of simultaneous circulation of West Nile and Usutu viruses in mosquitoes sampled in Emilia-Romagna region (Italy) in 2009[END_REF].

Non-Avian Flaviviruses could also circulate in Lebanon. Dengue virus was historically spread in the EMR. Since the elimination of its vector, Ae. aegypti at that time, the country is considered Dengue free. Nevertheless, the currently established Ae. albopictus can ensure the transmission of this disease [START_REF] Haddad | Aedes albopictus in Lebanon, a potential risk of arboviruses outbreak[END_REF] as it occurred in some Mediterranean countries [START_REF] Gjenero-Margan | Autochthonous dengue fever in Croatia, August-September 2010[END_REF][START_REF] La Ruche | First two autochthonous dengue virus infections in metropolitan France, September 2010[END_REF][START_REF] Marchand | Autochthonous case of dengue in France, October 2013[END_REF]. In addition, Zika virus can be also transmitted by Ae. albopictus as suggested by field and laboratory based observations [START_REF] Chouin-Carneiro | Differential Susceptibilities of Aedes aegypti and Aedes albopictus from the Americas to Zika Virus[END_REF][START_REF] Grard | Zika virus in Gabon (Central Africa)--2007: a new threat from Aedes albopictus?[END_REF][START_REF] Wong | Aedes (Stegomyia) albopictus (Skuse): a potential vector of Zika virus in Singapore[END_REF]. The involvement of Cx. pipiens in the transmission of this virus is unlikely [START_REF] Amraoui | Culex mosquitoes are experimentally unable to transmit Zika virus[END_REF].

The potential introduction of RVFV to Lebanon depends on the livestock trade with countries in Africa and even in the Arabian Peninsula, where the virus occurs. Culex pipiens from Lebanon, did not seem to be very competent to transmit RVFV. However, the exposure of this dominant mosquito to a highly viremic animal could lead to an efficient transmission of the virus. Moreover, the possibility of this virus to be locally transmitted by other mosquitoes, especially those of the Aedes genus, should be considered. Based on field observation, Aedes albopictus, is the most dominant species of this genus in Lebanon [START_REF] Haddad | Aedes albopictus in Lebanon, a potential risk of arboviruses outbreak[END_REF]. The role of this species in transmitting RVFV was suggested by Turell and collaborators (1988). In a vector competence study conducted on Ae. albopictus strains from Texas (USA), 15% of the orally infected mosquitoes, with a disseminated infection, transmitted the virus. This rate is similar to what was observed in Aedes mcintoshi that has been implicated in RVFV transmission in Africa [START_REF] Turell | Vector competence of a Houston, Texas strain of Aedes albopictus for Rift Valley fever virus[END_REF]. The vector competence of the Lebanese tiger mosquito toward this virus needs to be assessed.

RVF epidemics are more and more frequent in Africa (CDC, 2017) and in some countries of the Middle East, probably in relation with climatic changes and intensified livestock trade [START_REF] Chevalier | Rift Valley fever--a threat for Europe? Euro Surveill[END_REF]. Therefore, the probability of spread of RVF outside the historically known endemic area remains a possibility especially to regions with large population of livestock. Therefore, control measures especially on imported animals should be reinforced.

Current and previous entomological observations confirmed the dominance of two important vectors: Cx. pipiens and Ae. albopictus species. Both species are competent vectors to several arboviruses that are likely to be introduced to Lebanon as the result of globalization and climate change. Consequently, vector control programs that have ceased since 1975, at the beginning of the Lebanese war, should resume in order to prevent a potential local spread of these pathogens.

Our study gave evidence, for the first time, of WNV circulation in human and horses in Lebanon. Although the circulation seems to appear at a low pace, we observed a 1.01% and 1.98% seroprevalence rates against WNV among human and horses respectively (after PRNT).

In addition, experimental infections of Cx. pipiens from Lebanon showed that these local mosquitoes are competent vectors of WNV and to a lower extent, RVFV. In this study, we were able to identify molestus and pipiens biotypes and their hybrid in the country. Both biotypes were competent to transmit WNV, while the molestus biotype had a shorter EIP and therefore can ensure faster transmission of this virus.

In this project we were able to describe important elements related to the epidemiology of WNV in Lebanon, however we failed to characterize the identity of the virus certainly because of the observed low endemicity. Nevertheless, this study highlighted the potential circulation of other flaviviruses in the country. Therefore, and in order to gather more epidemiological information, we suggest to perform the following studies:

 Repeat serosurveillance study on larger numbers of chickens and use more sentinel birds in order to pick up potential future circulation of WNV.

 Carry out additional entomological studies and screen by molecular tools greater numbers of mosquitoes for WNV or even for flaviviruses using generic primers.

 Conduct molecular screening for flaviviruses on cerebrospinal fluids of patients with aseptic meningitis in order to detect neuroinvasive flaviviruses.

 Conduct serological and entomological studies in new geographical areas such as in the South that border with the endemic Israel.

 Establish a surveillance system to conduct regular monitoring of horses to detect any potential exposure to WNV.

ix 2 .

 2 Molecular screening of mosquitoes for WNV .............................................................. 3. Serological study of human population........................................................................ a. Description of participants ....................................................................................... b. Seroprevalence results ............................................................................................. 4. Serological study of horse population .......................................................................... a. Description of horse sample population ................................................................... b. Seroprevalence results ............................................................................................. 5. Serological study of chicken population ...................................................................... a. Description of the chicken study population ............................................................ b. Seroprevalence results ............................................................................................. c. Seroconversion results ............................................................................................. 6. Statistical analysis ....................................................................................................... Discussion and Conclusion .................................................................................................... Chapter 7 Experimental transmission of West Nile Virus and Rift Valley Fever Virus by Culex pipiens from Lebanon .......................................................................................................... Introduction .............................................................................................................. Material and Methods ............................................................................................... Mosquito collections ............................................................................................. Mosquito preparation for vector competence study ................................................ Viruses and blood meal preparation ....................................................................... Artificial feeding of female mosquitoes ................................................................. Saliva collection .................................................................................................... Viral detection in mosquitoes ................................................................................ Statistical Analysis ................................................................................................ Results ...................................................................................................................... Artificial feeding ................................................................................................... Viral infection ....................................................................................................... Viral dissemination ................................................................................................ Viral transmission.................................................................................................. Intensity of transmission ........................................................................................ Vector competence of Cx. pipiens biotypes ........................................................

Figure 1 .Figure 2 .Figure 3 . 11 Figure 4 . 12 Figure 5 .Figure 6 .Figure 7 .Figure 11 .Figure 12 .Figure 16 .Figure 17 .Figure 18 . 71 Figure 21 .Figure 27 .

 123114125671112161718712127 Figure 1. Global distribution of Culex pipiens complex mosquitoes .............................................8 Figure 2. Culex pipiens anatomy diagram ....................................................................................9 Figure 3. Culex pipiens morphological characteristics. .............................................................. 11 Figure 4. Culex pipiens life cycle .............................................................................................. 12 Figure 5. Female Culex pipiens laying eggs ............................................................................... 13 Figure 6. WNV transmission cycle ............................................................................................ 23 Figure 7. Past and present WNV transmission activities in the Euro-Mediterranean region ........ 25 Figure 8. Phylogenetic tree of WNV lineages based on complete genome sequences ................. 28 Figure 9. WNV genome organization and virion composition ................................................... 30 Figure 10. The replication cycle of WNV. ................................................................................. 33 Figure 11. RVFV transmission cycle ......................................................................................... 40 Figure 12. RVFV geographic distribution .................................................................................. 41 Figure 13. Schematic diagram of RVFV structure ..................................................................... 45 Figure 14. Steps of virus dissemination in the mosquito vector .................................................. 53 Figure 15. Forced salivation technique ...................................................................................... 57 Figure 16. Lebanon landscape map............................................................................................ 62 Figure 17. Flyways of migratory birds ....................................................................................... 64 Figure 18. Collection sites of mosquitoes, humans, horses and chicken blood samples .............. 65 Figure 19. A. CDC light trap. B. BG sentinel trap...................................................................... 66 Figure 20. Blood withdrawal from the jugular vein of a horse ................................................... 71 Figure 21. Blood withdrawal from the alary vein of a chicken. .................................................. 72 Figure 22. Gel electrophoresis picture of PCR products ............................................................. 81 Figure 23. Bar chart representing the age distribution of the study participants. ......................... 83 Figure 24. Bar chart showing the profession distribution of the study participants. .................... 83 Figure 25. Scatter graph showing OD ratio obtained by ELISA on human sera samples collected from Bekaa region ..................................................................................................................... 84 Figure 26. Scatter graph showing OD ratio obtained by ELISA on human sera samples collected from North Lebanon .................................................................................................................. 85

Figure 28 .

 28 Figure 28. Pie chart showing the horse sample population breed distribution. ............................

Figure 29 .

 29 Figure 29. Bar chart representing the age distribution of the horse population ...........................

Figure 30 .

 30 Figure 30. Scatter graph showing OD ratio obtained by ELISA on horse sera samples ..............

Figure 31 .

 31 Figure 31. Pie chart representing the chicken sample population distribution by district ............

Figure 32 .

 32 Figure 32. Scatter graph showing OD ratio obtained by ELISA on chicken sera samples ...........

Figure 33 .

 33 Figure 33. Bar chart showing OD ratios of chicken samples of the seroconversion study. ..........

Figure 34 .

 34 Figure 34. Map showing the geographic location of Lebanon East of the Mediterranean basin Figure 35. Infection (A), Dissemination (B) and Transmission (C) of Culex pipiens at different days after ingestion of an infectious blood meal containing WNV or RVFV............................

Figure 36 .

 36 Figure 36. Mean titer of infectious viral particles secreted in saliva of Culex pipiens at different days after ingestion of an infectious blood meal containing WNV or RVFV............................

Figure 37 .

 37 Figure 37. Comparison between Culex pipiens biotypes challenged with WNV: infection rate (A), dissemination efficiency (B) and transmission efficiency (C) ...........................................

Figure 38 .

 38 Figure 38. Comparison between Culex pipiens biotypes challenged with RVFV: infection rate (A), dissemination efficiency (B) and transmission efficiency (C) ...........................................

Figure 39 .

 39 Figure 39. Mean number of viral particles of WNV (A) and RVFV (B) in saliva of the biotypes of Cx. pipiens examined at different days after ingestion of an infectious blood meal ..............

Figure 1 .

 1 Figure 1. Global distribution of Culex pipiens complex mosquitoes (Farajollahi et al., 2011).

Figure 2 .

 2 Figure 2. Culex pipiens anatomy diagram (Credit: Callista Images).

Figure 3 .

 3 Figure 3. Culex pipiens morphological characteristics: A. Trilobate scutellum. B. Wing characteristics: fringe of scales in the alula (left arrow), dark costal vein (right arrow), apex of the vein 1-A (middle red arrow) beyond the mcu/cuA fork (blue arrow). C. Pale basal band on tergites (The mosquitoes of the Mediterranean Africa, IRD, France, CD-ROM, 1999).

Figure 4 .

 4 Figure 4. Culex pipiens life cycle (AMCA, 2017).

Figure 5 .

 5 Figure 5. Female Culex pipiens laying eggs. Photograph by Sean McCann (University of Florida).

Figure 6 .

 6 Figure 6. WNV transmission cycle (Chancey et al., 2015).

Figure 7 .

 7 Figure 7. Past and present WNV transmission activities in the Euro-Mediterranean region (eCDC, 2017).

Figure 8 .

 8 Figure 8. Phylogenetic tree of WNV lineages based on complete genome sequences (Rizzoli et al., 2015).

Figure 9 .

 9 Figure 9. WNV genome organization and virion composition: (a) the viral genome is represented with one Open Reading Frame that includes 10 genes: 3 encode for structural proteins and 7 for nonstructural proteins. (b) Structure of WNV virion showing membrane and capsid proteins enveloping the RNA (Chancey et al., 2015).

Figure 10 .

 10 Figure 10. The replication cycle of WNV. The virus attaches to cell membrane, enters the host cell and releases the RNA that is translated and copied (A-C). The viral RNA negative and positive polarity are copied using viral RNA polymerase (D-E). The newly formed virions assemble with the endoplasmic reticulum membrane (F) then they are cleaved by the Golgi network (G). Finally virions are transported by vesicles and secreted by exocytosis (H) (Diamond, 2009).

  pathognomonic. Routine clinical laboratory analysis do not distinguish WNV infection from many other viral infections. Thus the diagnosis must be confirmed by further laboratory tests based on the detection of WNV RNA in blood and cerebrospinal fluid (CSF) or virus isolation in cell culture from serum samples (direct diagnosis) or the detection of a specific immune response against the virus (indirect diagnosis)

Figure 11 .

 11 Figure 11. RVFV transmission cycle. An enzootic (sylvatic) cycle occurs during the average rainfall season in which the virus is maintained through transovarial transmission and through amplification in a vertebrate host. An epizootic-epidemic (domestic) cycle occurs during the heavy rainfall season in which the virus is over-amplified with massive hatching of mosquito eggs (FAO, 2017).

Figure 12 .

 12 Figure 12. RVFV geographic distribution (CDC, 2017).

  Grobbelaar and collaborators (2011) analyzed five derived strains and 198 isolates of RVFV that have circulated over 67 year period. It showed that despite low genetic diversity, these isolates resolved into 15 lineages (A-O): Six lineages (B, D, F, J, O and I) contained isolates from South Africa, six other lineages (A, C, E, G, H and N) contained clusters of isolates associated with outbreaks in individual countries or regions and three lineages (K, L, and M)

Figure 13 .

 13 Figure 13. Schematic diagram of RVFV structure. The virion is enveloped by glycoproteins and comprises three segments (S, M and L) of RNA encoding for structural and nonstructural proteins (Pepin et al., 2010).

Figure 14 .

 14 Figure 14. Steps of virus dissemination in the mosquito vector: 1. Ingestion of an infectious blood meal. 2. Viral replication and passage in the midgut cells. 3. Virions pass in the hemocoel. 4. The virus infects secondary organs such as salivary glands. 5. The virus is secreted in saliva and other hosts may be infected. Adapted from AIV, IP.



  infections. Mosquitoes of the species and population of interest are raised under controlled conditions of nutrients, temperature, humidity and photoperiod. Females are starved 48 hours before being fed on an infectious mixture containing fresh rabbit blood and a viral suspension of known titer is prepared. Fully engorged females are kept for analysis. The viral dynamic within these females is monitored at different days during the post-infection incubation period (pi) in order to assess the vector competence. In this perspective, the following parameters are usually evaluated at all kinetic points:  Midgut infection rate: proportion of mosquito females with infected midgut among tested ones (alive and engorged at Day 0)  Disseminated infection rate: proportion of females with infected head or legs or wings among females with infected midgut. Dissemination efficiency: proportion of mosquito females with infected head or legs or wings among tested ones. Transmission rate: proportion of females with infectious saliva among females able to disseminate the virus beyond the midgut barrier (with infected head or legs or wings). Transmission efficiency: proportion of mosquito females with infectious saliva among tested ones.Thus, the disseminated infection rate and the dissemination efficiency allow the evaluation of the ability of the virus to cross the midgut barrier of the mosquito and to disseminate to other internal organs/tissues. While the transmission rate and the transmission efficiency indicate the capacity of the virus to cross the ultimate barrier of the salivary glands to be released into the saliva of the mosquito. Therefore, transmission rate and efficiency are the main parameters for assessing the epidemic risk associated with the transmission of a given virus by local mosquitoes.

Figure 15 .

 15 Figure 15. Forced salivation technique. After removing the legs and wings of the female mosquito, the proboscis is inserted gently into a tip containing Fetal Bovine Serum.

Figure 18 .

 18 Figure 18. Collection sites of mosquitoes, humans, horses and chicken blood samples.

a.

  Human blood collection Two human populations were included in this seroprevalence study. The first population consisted of consenting donors from the Bekaa region, an area characterized by the presence of large water systems. All involved participants were outpatients attending the laboratories of three hospitals in the study area in order to conduct routine blood analysis: Tal Chiha Hospital in Zahle (June 2013), Bekaa Hospital in Taanayel, Chtoura (June-September 2013) and Farhat Hospital in Kamed El Lawz (July and August 2013) (Figure 18). The sera of the second population were taken from a serum bank and belong to patients that were involved in a previous serosurvey on Sand fly fever virus. All patients were recruited in September 2012 from outpatients attending two laboratory facilities in North Lebanon district: the laboratory of health dispensary of the United Nations Relief and Works Agency (UNRWA) inside Nahr El Bared Camp (NBC) for Palestinian refugees in Akkar and the laboratory of the military clinic in Al Kobbeh, Tripoli, North Lebanon (Figure 18). All participants have signed a consent form and filled a questionnaire in Arabic language (Appendices A and B). Translated versions of both forms appear in Appendices C and D. The consent form notified each participant about the objectives of the study, the requirements and the confidentiality of results. The questionnaire aimed to collect information about the participant's socio-economical factors (gender, place of residence, work nature, summer fever history, travel history…). Only patients below λ and above 85 years old were excluded from this study.b. Horse blood collectionEquine sera samples for the seroprevalence study part were collected from June to October 2015 from farms and riding clubs in rural areas of the following districts in Lebanon: Tripoli, Akkar, Baalbeck, Hermel, Zahle, West Bekaa, Nabatiye, Saida and Jezzine districts (Figure18).

Figure 20 .

 20 Figure 20. Blood withdrawal from the jugular vein of a horse (The Courier Journal, 2015).

Figure 21 .

 21 Figure 21. Blood withdrawal from the alary vein of a chicken.

Figure 22 .

 22 Figure 22. Gel electrophoresis picture of PCR products. Wells from left to right represent: DNA ladder, reverse transcription positive control band on 308 bp, reverse transcription negative control, WNV positive control band of 326 bp, PCR negative control, pools # 1 to 5 all negative for WNV.

Figure 23 .

 23 Figure 23. Bar chart representing the age distribution of the study participants.

Figure 24 .

 24 Figure 24. Bar chart showing the profession distribution of the study participants.

Figure 25 .Figure 26 .

 2526 Figure 25. Scatter graph showing OD ratio obtained by ELISA on human sera samples collected from Bekaa region. The red line represents the fixed threshold (OD ratio = 2).

  of horse sample population According to the Lebanese ministry of agriculture, the estimated number of horses in Lebanon is around 4000 horses. In this study, 202 horse samples were collected from 16 different farms/stables in eight districts in Lebanon (Figure27). Seven of them were ponies and the others belonged to different breeds such as Arabian, Thoroughbred, Dutch, Belgian Draft, Turkish, and the local breed called Alef which was the most represented breed in the study population (144 samples, 70.59%) (Figure28). More than two thirds of the horse samples were females (140 samples, 69.31%). The age of included horses ranged from 4 months to 22 years with an average of 8.74 years (Figure29).

Figure 27 .Figure 28 .

 2728 Figure 27. Pie chart showing the horse population distribution by district.

Figure 29 .

 29 Figure 29. Bar chart representing the age distribution of the horse population.

  This serum was considered negative as it is below the fixed threshold. All positive horses were of Alef breed with no cross border movement history. They were recruited from farms in Tripoli, Aakar and Nabatiyeh districts.

Figure 30 .

 30 Figure 30. Scatter graph showing OD ratio obtained by ELISA on horse sera samples. The red line represents the fixed threshold (OD ratio = 1.2).

  of the chicken study populationIn order to estimate the seroprevalence rate among chicken population, 163 chicken sera were sampled between September and December 2015 from 15 different locations. These chickens are of Leghorn breed and belonged to house poultries of residents and farmers in the Bekaa valley in West Bekaa, Zahle, and Baalbek districts. The majority (106 chickens, 65%) of the collected chicken originated from Zahle district (Figure31).

Figure 31 .

 31 Figure 31. Pie chart representing the chicken sample population distribution by district.

Figure 32 .

 32 Figure 32. Scatter graph showing OD ratio obtained by ELISA on chicken sera samples. The red line represents the fixed threshold (OD ratio = 1.2).

Figure 33 .

 33 Figure 33. Bar chart showing OD ratios of chicken samples of the seroconversion study.

Figure 35 .

 35 Figure 35. Infection (A), Dissemination (B) and Transmission (C) of Culex pipiens at different days after ingestion of an infectious blood meal containing WNV or RVFV. Culex pipiens F0/F1 mosquitoes were orallychallenged with WNV at a titer of 1.6×10 8 PFU/mL and RVFV at titer of 1.33×10 7 PFU/mL using an artificial feeding system (Hemotek). At 3, 7, 14, and 19 dpi, homogenates of mosquito bodies and heads, and saliva collected from females were tested for the presence of virus on Vero cells to estimate infection rate, dissemination efficiency, and transmission efficiency, respectively. In brackets, the number of tested mosquitoes. Error bars show the confidence interval (95%).

Figure 36 .

 36 Figure 36. Mean titer of infectious viral particles secreted in saliva of Culex pipiens at different days after ingestion of an infectious blood meal containing WNV or RVFV. The number of infectious particles in saliva was estimated by inoculation of collected saliva on Vero cells and expressed as pfu/saliva. In brackets, the number of tested mosquitoes. Error bars refer to the standard error.

Figure 37 .

 37 Figure 37. Comparison between Culex pipiens biotypes challenged with WNV: infection rate (A), dissemination efficiency (B) and transmission efficiency (C). Mosquito legs or wings were used to amplify the CQ11 gene ofCx. pipiens, a diagnostic gene to distinguish the two Cx. pipiens biotypes and their hybrids. IR, DE and TR were calculated for each biotype. In brackets, the number of tested mosquitoes.

Figure 38 .

 38 Figure 38. Comparison between Culex pipiens biotypes challenged with RVFV: infection rate (A), dissemination efficiency (B) and transmission efficiency (C). Mosquito legs or wings were used to amplify the CQ11 gene of Cx. pipiens, a diagnostic gene to distinguish the two Cx. pipiens biotypes and their hybrids. IR, DE and TR were calculated for each biotype. In brackets, the number of tested mosquitoes.

Figure 39 .

 39 Figure 39. Mean number of viral particles of WNV (A) and RVFV (B) in saliva of the biotypes of Cx. pipiens examined at different days after ingestion of an infectious blood meal provided. Blood meals were provided at a titer of 1.6×10 8 PFU/mL for WNV and 1.33×10 7 PFU/mL for RVFV. The number of infectious particles in saliva was estimated by inoculation of collected saliva on Vero cells and expressed as PFU/saliva. In brackets, the number of tested mosquitoes. Error bars refer to the standard error.

  ....

	IgY s.s.	Immunoglobulin Y sensu stricto
	IRS SIT	Indoor Residual Spraying Sterile Insect Technique
	ITN SLEV	Insecticide treated nets St. Louis Encephalitis Virus
	JEV SPNL	Japanese Encephalitis Virus Society for the Protection of Nature in Lebanon
	°C MENA SPSS	Degree Celsius Middle East and North Africa Statistical Package for the Social Sciences
	Ae. MR TBE	Aedes Magnetic Resonance Tris Borate EDTA
	BAGV BHK NBC TMB NCR UNRWA	Bagaza Virus Nahr El Bared Camp 3,3',5,5'-Tetramethylbenzidine Baby Hamster Kidney Non Coding Region United Nations Relief and Works Agency
	bp NS USA	base pairs Nonstructural United States of America
	BSA NY USUV	Bovine Serum Albumin New York Usutu Virus
	Bti OD VC	Bacillus thuringiensis toxin Optical Density Vector Capacity
	CDC ODN WHO	Centers for Disease Control and Prevention Optical Density on Negative antigen World Health Organization
	cDNA ODP WHOPES	Copy Deoxyribonucleic acid Optical Density on Positive antigen World Health Organization Pesticide Evaluation Scheme
	CEC OIE WNE	Cecropin World Organization for Animal Health West Nile Encephalitis
	CHIKV OP WNF	Chikungunya Virus Organophosphate West Nile Fever
	CSF ORF WNND	Cerebrospinal Fluid Open Reading Frame West Nile Neurological Disease
	Cx. PBS WNV	Culex Phosphate Buffered Saline West Nile Virus
	DDT PBS-T YFV	Dichlorodiphenyltrichloroethane Phosphate Buffered Saline Tween Yellow Fever Virus
	DENV PBS-T-BA ZIKV	Dengue Virus Phosphate Buffered Saline Tween Bovine Albumin Zika Virus
	dpi PCR	days post infection Polymerase Chain Reaction
	eCDC PFU	European Centre for Disease Prevention and Control Plaque Forming Units
	EIP pi	Extrinsic Incubation Period post infection
	ELISA PO	Enzyme Linked Immunosorbent Assay Phenoloxidase
	EMR PRNT	East Mediterranean Region Plaque Reduction Neutralization Test
	FAO RNA	Food and Agriculture Organization of the United Nations Ribonucleic Acid
	FBS RNAi	Fetal Bovine Serum Ribonucleic Acid interference
	FDA RNP	Food and Drug Administration Ribonucleoprotein
	HRP RT-PCR	Horseradish Peroxidase Reverse Transcription Polymerase Chain Reaction
	IFA RVF	Immunofuluorescence Assay Rift Valley Fever
	IgG RVFV	Immunoglobulin G Rift Valley Fever Virus
	IgM s.l.	Immunoglobulin M sensu lato
	IGR	Insect Growth Regulator
		x xii xiii xiv

Table 1 .

 1 Behavioral and physiological traits associated with three nominal forms of the pipiens assemblage ................................................................................................................................ 16

Table 2 .

 2 WNV NS5 primer sequences ....................................................................................... 69

Table 3 .

 3 Species composition and description of collected mosquitoes in localities of West Bekaa during August 2014. .................................................................................................................. 79

Table 4 .

 4 Species composition and description of collected mosquitoes in localities of North Lebanon during September 2014. .............................................................................................. 80

Table 5 .

 5 Description of human sample population. .................................................................... 82

Table 6 .

 6 Summary of human serology results ............................................................................ 86

Table 7 .

 7 Summary of horse serology results .............................................................................. 89 West Nile virus (WNV) is a mosquito-borne zoonotic agent member of the Flavivirus genus (Flaviviridae family). It was first isolated in Uganda in 1937

  This manuscript is divided in two parts Litterature Review and Empirical Studies. The first part is composed of four chapters. Chapter 1 is an overview of the mosquito vector Culex pipiens, its biology and medical importance. In this chapter, we present classical and advanced methods to control mosquito populations and discuss the related challenges. Chapters 2 and 3 describe West Nile Virus and Rift Valley Fever Virus, their epidemiology, diagnosis and pathogenesis. Chapter 4 discusses the knowledge related to the vector-virus interactions. Chapter 5 specifies the study objectives of the present research project. The second part presents the work

performed during this thesis project. It is composed of three chapters. In chapter 6, we assess the circulation of WNV in Lebanon by conducting entomological and seroprevalence surveys. In chapter 7, we assess the vector competence of Lebanese strains of Culex pipiens against West Nile Virus and Rift Valley Fever Virus. Chapter 8 presents the general discussion and conclusion related to the epidemiology of WNV in the country.

Table 1 . Behavioral and physiological traits associated with three nominal forms of the pipiens assemblage (Harbach, 2012).

 1

	Trait	pipiens	molestus	Cx. quinquefasciatus
	Host feeding	ornithophilic,	anthropophilic,	anthropophilic,
		zoophilic	mammophilic,	mammophilic,
			ornithophilic	ornithophilic
	Feeding location	exophagic	endo-/ exophagic	endo-/ exophagic
	Resting location	exophilic	endo-/ exophilic	endo-/ exophilic
	Egg development anautogenous	autogenous	anautogenous
	Mating space	eurygamous	stenogamous	stenogamous
	Diapause	heterodynamous	homodynamous	homodynamous
	Larval habitat	clean water normally	clean / polluted water	clean / polluted /
		above ground	below ground	brackish water above
				ground

  other Flaviviruses. Thus, Plaque reduction neutralization test (PRNT) is the gold standard method used for further confirmation of WNV infection[START_REF] Rossi | West Nile virus[END_REF].Moreover, virology tests can directly confirm the presence of the virus in patient serum or CSF. The virus is first amplified within permissive cells (such as Vero cells) and then the viral identity is confirmed by molecular methods. Nevertheless, molecular tools can be directly employed to confirm the presence of virus (or its RNA). Different quantitative reverse transcription polymerase chain reaction (RTqPCR) protocols have been developed to amplify

	Serological tests are performed on serum or cerebrospinal fluid (CSF) of acute or convalescent
	cases to determine the WNV specific antibody profile by ELISA. In order to detect recent
	infections, the test involves immunoglobulin M antibodies IgM-specific ELISA in which serum
	is collected within 8 to 21 days after the appearance of clinical symptoms. This test provides
	strong evidence of recent WNV infection. For seroprevalence studies, immunoglobulin G (IgG)

antibodies are seeked out by ELISA. One disadvantage of ELISA test is cross-reactivity with infections caused by minimal amounts of WNV RNA. But, viraemia is short-lived in dead-end hosts, so detection of viral genomes becomes increasingly challenging over time. Recent studies demonstrated that WNV RNA can be detected in urine much longer (up to 20 days after the onset of symptoms) and at higher concentrations than in serum or CSF in individuals infected with WNND (Rizzoli

  It is the most advanced vaccine in development, and currently, it has progressed beyond the second part of clinical phase II trial[START_REF] De Filette | Recent progress in West Nile virus diagnosis and vaccination[END_REF]. Although the live vaccine candidates induced high humoral and cell-mediated immune responses similar to that obtained with wild-type WNV, vaccination has raised some safety concerns in susceptible individuals[START_REF] De Filette | Recent progress in West Nile virus diagnosis and vaccination[END_REF]. Long-term immunogenicity, safety and cost effectiveness are the most important problems in current development of vaccines against WNV and potential candidates for use in humans.

	recombinant live virus, chimeric virus (ChimeriVax-WN), DNA, and recombinant subunit vaccines (De Filette et al., 2012). ChimeriVax-West Nile vaccine (Acambis, Sanofi-Pasteur) utilizes the attenuated Yellow Fever Virus (YFV) vaccine strain to build a live chimeric virus that consists of the prM and E proteins of WNV in the context of the YFV capsid and non-Rift Valley Fever Virus structural proteins. Chapter 3 Rift Valley Fever Virus (RVFV) is a mosquito-borne virus that affects humans and livestock.

Three vaccine formulations are currently available for prevention of WNV in equids: two are inactivated vaccines and one is a non-replicating live canary pox recombinant vector vaccine

[START_REF] De Filette | Recent progress in West Nile virus diagnosis and vaccination[END_REF]

. The first licensed vaccine contains a formalin-inactivated whole WNV. This vaccine is currently commercialized in USA under the trade name West Nile-Innovator®.

Another killed virus vaccine (Vetera® WNV vaccine) was developed and licensed. A third commercialized WNV vaccine in USA for horses is Recombitek® Equine West Nile Virus Vaccine which is a chimeric recombinant canary poxvirus vaccine. It expresses the prM and E genes derived from a 1999 New York isolate of WNV

[START_REF] De Filette | Recent progress in West Nile virus diagnosis and vaccination[END_REF]

. Administration, dosage and vaccination schedule differ between these three vaccines.

Although no approval from FDA (Food and Drug Administration) was obtained, vaccines for human use exist and currently, there are a number of ongoing clinical trials. There are several strategies being pursued for WNV vaccine development for humans including inactivated,

  During this outbreak, 516 cases with 87 deaths of RVF were reported by the Ministry of Health of Saudi Arabia while the Ministry of Public Health in Yemen reported 1087 suspected cases including 121 deaths[START_REF] Ahmad | More deaths from Rift Valley fever in Saudi Arabia and Yemen[END_REF] CDC, 2017;[START_REF] Madani | Rift Valley fever epidemic in Saudi Arabia: epidemiological, clinical, and laboratory characteristics[END_REF] WHO, 2017). The African continent remains the most affected. Outbreaks continue to be reported from different countries. In 2003 in Egypt, there were 148 cases including 27 deaths of RVF reported by the Ministry of Health of Egypt(CDC, 2017; WHO, 2017). Several other outbreaks occurred in

	Kenya (2006, 2007), Tanzania (2007), Somalia (2007), Sudan (2007), Madagascar (2008, 2009),
	and Republic of South Africa (2010) (CDC, 2017; WHO, 2017).

  This species is established in the coastal zone in addition to inland locations where the stagnant water systems are scattered. During this research project, we assessed the circulation of WNV in Lebanon by screening mosquito populations for this virus and by detecting specific antibodies among human, horse and chicken populations. agricultural life is limited to the north and the south of the country in which Citrus trees are planted. During the summer, and specially July and August, the weather is hot and steamy, and humidity can reach 90%, with temperatures over 30°C. The rain falls are mainly from December to March with relatively cold weather. The second region is formed by the Mount Lebanon chain, running parallel to the coastline, and peaking at the northern part at 3083m. It has a typically Alpine climate with important snowfall and temperatures below zero on the mountains during winter.The third region is the Bekaa plain, which is considered part of the Great Rift Valley that extents until South East Africa. It is an inland plain, situated 1000m above sea level. The northern part of this plain is arid with an average rainfall. The Bekaa Valley is shielded from the influence of the sea by the Lebanon Mountains. Therefore, the Bekaa is drier than the rest of the

country with considerably less precipitation not exceeding 100 mm in its upper part (Hermel district) and a wider variation in daily and yearly temperatures. Summers are hot and dry, while winters are cold and frosty. It is a fertile and farming region in which two rivers originate: the Orontes River in the Northern district of Hermel and the Litani River that crosses the West Bekaa region. Moreover, the Bekaa region is relatively rich in stagnant water plans such as the Qaraoun artificial lake and Aammik natural swamps which provide a favorable environment for the breeding and proliferation of mosquitoes. Besides, this region is considered an important stopover for migratory birds (Figure

Table 2 . WNV NS5 primer sequences (Kauffman et al., 2016; Lanciotti et al., 2000).

 2

	Description	Sequence (5'-3')	Size bp
	WN 9483 WN 9794	CAC-CTA-CGC-CCT-AAA-CAC-TTT-CAC-C GGA-ACC-TGC-TGC-CAA-TCA-TAC-CAT-C	326
	Primers were used at a final concentration of 0.5µM in a reaction volume of 50µL PCR
	mixture. 3µL cDNA was used for amplification. After optimization, PCR cycling parameters
	were as follows: Enzyme activation at 95°C for 10 min, PCR (denaturation, annealing,
	extension) with 35 cycles at 94°C for 45 s, 56°C for 45 s, 72°C for 1 min, PCR final step
	(elongation) at 72°C for 10 min. This amplification reaction was performed using the GeneAmp

PCR System 9700, Applied Biosystems, Thermal Cycler (USA). WNV cDNA positive control was generously provided by Dr. Laura Kramer (Laboratory of Arboviruses, Slingerlands, New York, USA).

The obtained PCR products were run on a 1.5% agarose gel electrophoresis suspended in Tris Borate EDTA (1×TBE) running buffer (Appendix E: 10×TBE buffer Preparation). DNA fragments were colored by Gel Red Nucleic acid gel stain (Biotium, USA). Potential WNV amplicons are expected to appear at 326 bp position.

Table 3 . Species composition and description of collected mosquitoes in localities of West Bekaa during August 2014.

 3

	Place	Coordinates LAT LONG	Altitude (m)	# of collection days	Trap type	Trap-days	Culex pipiens F M	Culex perexiguus F M	Culex martini F M	Culiseta longiareolata F M	Orthopodomyia pulcripalpis F M	Incompl ete
	Jebb Jenni ne	33°37' 58" N	35°46' 55" E	870	3	BG sentinel CDC	8 19	27 360	58 33	7		2 18		3 1	3 2		2 2	4 37
	El Marj	33°45' 42" N	35°53' 10" E	870	2	BG sentinel CDC	8 19	119 1042	256 11	2		5	2	3 4	5 1		1 4	11 53
	Qarao un	33°34' 10" N	35°43' 10" E	900	2	BG sentinel CDC	6 13	37 658	73 9	1		3 2		4 2	2 6		1	7 41
	Deir Ein El Jawzi	33°35' 49" N	35°41' 29" E	1000	1	BG sentinel CDC	2 3	1 5								2	2
				Total			78	2249 440	10	0	30	2	17	19	2	12	153
			Total of specimens (%)			2689 (91.65%)	10 (0.34%)	32 (1.09%)	36 (1.23%)	14 (0.48%)	153 (5.21%)
				Grand total									2934
	LAT: Latitude, LONG: Longitude, #: number, F: female, M: male

Table 4 . Species composition and description of collected mosquitoes in localities of North Lebanon during September 2014.

 4

	Place	Coordinates LAT LONG	Altitude (m)	# of collection days	Trap type	Trap-days	Culex pipiens F M	Culex perexiguus F M	Culex martini F M	Culiseta longiareolata Incomplete F M
	Darine	34°36' 15" N	36°06' 25" E	74	1	BG sentinel	2	22	11				6
	Klayaat	34°35' 10" N	35°59' 35" E	5	2	CDC	12	1				1
	El Bahsa Bebnine	34°30' 90" N	35°59' 25" E	70	12	CDC	74	2748 500	82		8	12	521
				Total			88	2771 511	82	0	9	0	12	0	527
			Total of specimens (%)			3282 (83.90%)	82 (2.10%)	9 (0.23%)	12 (0.31%)	527 (13.47%)
				Grand total								3912

LAT: Latitude, LONG: Longitude, #: number, F: female, M: male

Table 5 . Description of human sample population.

 5

	Area	Location	Sample population (N) Females Males Median Age
		Bekaa Hospital	501	298	203	36
	Bekaa	Tel Chiha Hospital	168	97	71	49
		Farhat Hospital	134	89	45	31
		Bekaa total	803	484	319	39
	Akkar Nahr El Bared Camp	144	106	38	37.5
	Tripoli El Kobbeh Barrack	144	101	43	40.5
		North total	288	207	81	40
	Cumulative total	1091	691	400	39

Table 7 . Summary of horse serology results. Samples included in this table have PRNT titer ≥ 10.

 7

	Serum #	ELISA OD ratio	PRNT titer result	Breed Age Gender	Location
	83	1.371	160	Alef	7	Male	Tripoli
	128	1.511	160	Alef	7	Female	Aakar
	183	1.246	10	Alef	20	Female	Nabatiyeh
	199	1.238	40	Alef	10	Female	Nabatiyeh
	202	1.701	320	Alef	7	Female	Nabatiyeh

  . The exposure of Lebanese population to this virus was never assessed. Thus, and in order to increase the specificity of sera screening, all ELISA positive samples were confirmed by PRNT, a clearly more specific technique than other serological methods. It is worth noting that 65 sera samples had high OD ratio by ELISA but did not have neutralizing antibodies against WNV. This observation suggests the circulation of other Flaviviruses which should be identified.According to the Society for the Protection of Nature in Lebanon (SPNL) and Bird Life Lebanon, there are more than 300 bird species in the country. Migratory birds in Lebanon include Cranes, Pelicans, Common Buzzard, Lesser Spotted Eagle, and White Storks (SPNL,

Acknowledgments..................................................................................................... Chapter 8 General Discussion and Perspectives ................................................................... List of References ................................................................................................................... Appendix A: Informed Consent Form in Arabic ......................................................................

Acknowledgments

We are grateful Anubis Vega-Rua for technical advice.

I would like to thank my supervisor in France Anna Bella Failloux for allowing me to pass

Survey of West Nile Virus in Lebanon

Material and Methods

Geographical description of Lebanon

Lebanon is a mountainous country located on the eastern side of the Mediterranean Sea. It is characterized by a typical Mediterranean climate. The landscape of the country is characterized by parallel alternation of plains and high lands creating four longitudinal geographic regions (Figure 16). The first region is a narrow coastal strip that becomes relatively broader at the Northern part of the country near Tripoli. This region is heavily urbanized and contains the major cities. The

b. Mosquito identification

Mosquito specimens were identified under a stereomicroscope and placed in tubes containing RNA stabilizing solution (MCLAB, USA). The morphological identification was performed using a key for Mediterranean mosquitoes [START_REF] Dubose | Identification keys to the adult and larval mosquitoes of the Mediterranean Area[END_REF] in addition to the following digital identification keysμ "The Mosquitoes of Europe" (IRD, 2001) and "The mosquitoes of the Mediterranean Africa" (IRD, 1999). Identified mosquitoes were pooled according to collection location, gender and species. Each pool contained up to 25 specimens.

Mosquito pools were then preserved at -80°C for subsequent molecular analysis. Ten (5 females and 5 males) of the confirmed seropositive samples had more than 65 years old. All of them were either unemployed or housewives. One male seropositive participant is 31 years old and works indoor in a private company. When analyzing travel history of seropositive participants, only four of them had travelled to countries endemic to WNV (Egypt, USA and Canada). The remaining seropositive participants, either had never travelled out of Lebanon ( 2patients) or had travelled to Syria or to other countries non-endemic for WNF (Table 6). These observations constitute a strong evidence of local circulation of WNV and local exposure of human population to this virus.

the most abundant mosquito species, is the suspected vector. Besides, Cx. perexiguus, a known WNV vector in the East Mediterranean Region, was also present but in limited numbers. The feeding behavior of both mosquitoes should be analyzed in order to determine their implication in the transmission cycle. We recommend conducting similar surveys for several consecutive years in order to detect potential introduction of WNV to Lebanon.

Chapter 7 Experimental transmission of West Nile Virus and Rift Valley Fever Virus by Culex pipiens from Lebanon

In this chapter, we present the submitted article to Plos Neglected Tropical Diseases journal.

Peer reviewing is under process.

Experimental transmission of West Nile Virus and Rift Valley Fever Virus by

Mosquito preparation for vector competence study

Collected egg rafts were shipped to the Laboratory of Arboviruses and Insect Vectors (AIV) at the Institut Pasteur, Paris. They were reared until the adult stage. Adults emerging from each raft were morphologically identified and only Cx. pipiens species were retained for this study.

Saliva collection

Around 20 female mosquitoes were tested at 3, 7, 14 and 19 days post-infection (dpi). For each mosquito, saliva was collected using the forced salivation technique [START_REF] Dubrulle | Chikungunya virus and Aedes mosquitoes: saliva is infectious as soon as two days after oral infection[END_REF].

Briefly, mosquitoes were chilled, their legs and wings removed and the proboscis was inserted into 20µL tip filled with 5µL of Fetal Bovine Serum (FBS). After 45 min, medium containing the saliva was expelled into 0.2mL tube containing 45µL of Dulbecco's MEM (DMEM)

medium. Collected saliva and the remaining mosquito bodies were conserved at -80°C for further analysis.

Viral detection in mosquitoes

In order to assess the ability of both viruses to invade and cross the midgut barrier, the infection rate (IR) and the dissemination efficiency (DE) were determined. IR reflects the proportion of female mosquitoes with infected bodies (thorax and abdomen including the midgut) among tested specimens while DE is the proportion of female mosquitoes with infected head (detection of the virus having succeeded to reach the mosquito general cavity) among tested ones. Thus, heads and bodies were separated and ground each in 300µL DMEM supplemented with 3% FBS.

After centrifugation, the supernatant of each homogenate was conserved at -80°C. Then, 20µL of each sample were diluted in 180µL DMEM supplemented with 2% FBS and distributed in serial dilutions from 10 -1 to 10 -3 in duplicates on Vero cell monolayers (3.10 5 cells/well) in 96-well plates. After incubation at 37°C for 6 days, inoculum was removed and the cells were fixed and stained using a crystal violet solution (0.2% in 10% formaldehyde and 20% ethanol). After washing, the presence or absence of cytopathic effect was noted.

Viral infection

Infection rate (IR) for each virus was estimated by determining the number of infected bodies (abdomen and thorax) among all engorged female mosquitoes examined at each dpi (3, 7, 14 and 19) (Fig. 35A). For WNV, IRs were very high (94.7-100%) from 3 to19 dpi. For RVFV, IRs were much lower: at 3 dpi, the IR was 44% ±12.05 (mean±SE) and increased gradually to reach 65% (±10.9) at 14 dpi and 64.3 (±13.3) at 19 dpi.

Viral dissemination

The detection of viral particles in mosquito heads allowed estimating the ability of the virus to disseminate from the midgut to internal organs. For WNV, dissemination efficiency (DE) increased from 31.6% (3 dpi) to 94.7% (19 dpi) (Fig. 35B). For RVFV, virus was only detected at 19 dpi with a DE of 21.4% (±10.9) (Fig. 35B).

Viral transmission

The ability of mosquitoes to transmit the virus was measured by detecting viral particles in saliva expectorated by mosquitoes. With WNV, transmission efficiencies were much higher than with RVFV (Fig. 35C). At 3 dpi, 10.5% (±7.0) of mosquitoes had WNV particles detected in their saliva. TE increased gradually to reach 86.4% (±7.3) at 14 dpi and slightly dropped to 68.4% (±10.9) at 19 dpi. For RVFV, viral particles were only detected in saliva at 19 dpi with a TE of 7.1% (±6.9) (Fig. 35C).

Intensity of transmission

Mosquitoes were able to deliver an average of 550 (±450) PFU/saliva at 3 dpi with WNV, which increased to reach1004 (±442) PFU/saliva at 7 dpi (Fig. 36). Despite a decrease at 14 dpi, the viral load remained high at 19 dpi with 1028 (±405) PFU/saliva. For RVFV, only one female had infectious particles in saliva at 19 dpi with a viral load of 42 PFU (Fig. 36).

Discussion

Culex pipiens is the most widely distributed mosquito species in Lebanon and is suspected to transmit WNV and RVFV in several countries [START_REF] Andreadis | The contribution of Culex pipiens complex mosquitoes to transmission and persistence of West Nile virus in North America[END_REF][START_REF] Tantely | A review of mosquitoes associated with Rift Valley fever virus in Madagascar[END_REF]. Using experimental infections, we showed that Cx. pipiens populations collected from West Bekaa, Lebanon were susceptible to infection by these two viruses and ensured efficient transmission of WNV and to a lesser extent, RVFV.

When exposed to an infectious blood-meal containing WNV, Cx. pipiens was capable to ensure viral infection, dissemination and transmission starting from 3 dpi. Most mosquitoes exposed to the infectious blood-meal were infected as IRs reached 100% at the 4 dpi examined [START_REF]What is your work type?[END_REF]7,14 dpi. This suggests a significant role of the midgut and the salivary glands as respective barriers to the release of viruses into the body cavity and their excretion in saliva [START_REF] Kramer | Dynamics of flavivirus infection in mosquitoes[END_REF].

In this manner, Cx. pipiens was less susceptible to RVFV than to WNV.

Overall, Cx. pipiens can transmit experimentally both viruses but the time interval between the ingestion of the viremic blood-meal and the ability to transmit the virus termed the extrinsic incubation period (EIP) was 3 days for WNV and 19 days for RVFV. Cx. pipiens from Tunisia showed similar EIP of 3 days with WNV and a much shorter EIP of 3 days with RVFV [START_REF] Amraoui | Culex pipiens, an experimental efficient vector of West Nile and Rift Valley fever viruses in the Maghreb region[END_REF] underlining the significant role of mosquito genotype in specific  Study blood preferences and feeding behaviors of Culex pipiens and other Culex species (such as Cx. perexiguus) in order to understand their potential involvement in WNV transmission cycle.

Finally and based on all the above observations, we invite local authorities to start an integrative surveillance system that include entomological monitoring and control programs in addition to human and animal serological screening programs in order to detect the introduction and prevent the spread of potential arboviruses.

Appendix C: Informed Consent Form in English

Dear Participant, The research team of the Laboratory of Immunology at the Lebanese University, in collaboration with the University of New York, is conducting a study to evaluate the immune status of Lebanese population against West Nile Virus. This virus is transmitted from infected birds to humans by the bite of a mosquito. In most of the cases, the infection remains asymptomatic. Otherwise, it causes acute febrile illness, and rarely leads to encephalitis, causing long term disability or even death.

The main objective of this study is to search for the presence of specific antibodies in human which is indicative of previous exposure to the virus. The obtained results will be submitted to the Lebanese Health authorities to take necessary preventive measures.

In order to participate in this study, you are kindly requested to:

fill out the attached questionnaire that will be used for statistical and epidemiological analysis.

donate 5 ml of blood for laboratory analysis.

We would like to inform you that:

-The laboratory tests are free of charge. Their costs will be covered by a research grant.

-All results and provided personal information will remain secret and be treated in complete anonymity. The blood analysis results of each donor can be provided to him upon his request.

-The blood sera will be solely used for the purpose of this current study.

Preparation of 1L of 10X TBE Buffer

The following salts were dissolved in 800ml deionized water: 108g of Tris Base (Sigma Aldrich) and 55g of Boric Acid (Amresco). Then 40ml of prepared 0.5M EDTA (Scharlou) (pH=8) was added to the mixture and the volume was completed to 1L with deionized water.

Preparation of 5L of 10X PBS Buffer

The following salts were dissolved and stirred in 5L deionized water until all the salts were well dissolved: 340g of NaCl (Sigma Aldrich), 139.5g of Na 2 HPO 4 , 7H