Vector Competence and Mosquito-Arbovirus Relationships Vector Competence in the Lab Vectorial Capacity in the Fields

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vector competence and mosquito-arbovirus relationships vector competence in the lab vectorial capacity in the fields

WNV CHIKV Aedes RVFV Culex DENV ZIKV YFV Examples of scientific questions:

- What is the vector responsible of WNV transmission in my country? - What permit the persistence of dengue virus in inter-epizootic period? - Is Culex pipiens a secondary vector of WNV? - Between Aedes aegypti and Aedes albopictus, which one is the major vector of dengue virus in my country? Bad example

Entomologist Virologist Good example

Entomologist Virologist Three speakers:

Anna-Bella Failloux How to obtain vector status? the example of Zika virus

Mawlouth Diallo Experimental infection of mosquito with arbovirus in order to evaluate the vector competence

Miguel Ángel Jiménez-Clavero Mosquito-borne epornitic flaviviruses emerging in the Mediterranean How to get the status of vector? the example of Zika vectors

Anna-Bella FAILLOUX

Arboviruses and Insect Vectors Department of Virology The most important arboviruses

Dengue 1 Japan (1943)

Zika Uganda (1947) Yellow fever Nigeria (1927) Chikungunya Tanzania (1952) Transmission cycles

Non-human primates

Bridge vectors?

Arboreal canopy-dwelling Aedes spp. Anthropophilic mosquitoes Sylvatic cycle Zone of emergence Epidemic cycle The same epidemic vectors

Aedes aegypti Aedes albopictus

Ae. aegypti and Ae. albopictus Houé et al. Emerg. Microbes Infect. (2019) Aedes mosquitoes Main criteria to be a vector

1. Effective contacts between pathogen-infected host and the mosquito; the feeding behavior

2. Association in time and space of the vector and the pathogen-infected host

3. Repeated demonstration of natural infection of the vector

4. Experimental transmission of the pathogen by the vector Main criteria to be a vector

1. Effective contacts with pathogen’s host and feeding

2. Association in time and space of the vector and the pathogen infected host

3. Repeated demonstration of natural infection of the vector

4. Experimental transmission of the pathogen by the vector a Global Alliance For Zika Virus Control & Prevention (52 partners, coordination: Inserm) Mosquito sampling

Epidemic Bridge vectors vectors and enzootic transmission Brazil x x French Guyana x x Guadeloupe x Gabon x x Cambodia x x Senegal x x Suriname x x Mass screening of arboviruses

▪ Develop a high-throughput real-time PCR chip (BioMarkTM dynamic arrays)

84 viral species / more than 150 genotypes Samples

Targets

▪ Screen 17,958 mosquitoes from Africa, America, and Asia: 6 countries/territories Targeting known viruses

Number of Number of Examples of Family Genus viral species genotypes viruses targeted targeted targeted

Bunyavirus/ Bunyaviridae Orthobunyavirus/ 24 38 Rift Valley fever virus Phlébovirus

Flaviviridae Flavivirus 18 42 West Nile

Togaviridae Alphavirus 12 40 Ross River

Seadornavirus / Reoviridae 2 2 Banna Orbivirus

Mesoniviridae Mesonivirus 1 1 Nam Dinh

Rhabdoviridae Vesiculovirus 1 1 Jurona BioMarkTM Dynamic Array

Samples

- Taqman probe (FAM – BHQ1): Tm 70°C - Primers: Tm 60°C - qPCR program: Pre-incubation 95°C 5 min Amplification 95°C 10 s Targets (45 cycles) 60°C 15 s 96x96 chip → 9216 reactions 48.48 chip → 2304 reactions

Time cost: 30 min

Financial cost : 4.5 à 9.11 centimes/reaction Development of the HT system

95 Assays (primers and probe targeted viral species) and one design for E. coli (internal control)

92 reference samples and 4 negative control Number of mosquitoes Period of collections Arbovirus identified examined

French Guyana 3942 June – August 2016 ZIKV – Aedes aegypti

ZIKV – Culex Guadeloupe 2173 May – June 2016 quinquefasciatus ZIKV – Aedes aegypti Suriname 2310 March – May 2017 No virus detected

Moutailler et al. Viruses (2019) Number of mosquitoes Period of collections Arbovirus identified examined Senegal 934 August – Nov. 2017 YFV – Aedes furcifer May 2019 No virus detected Cambodia 492 Nov. 2018 YFV - Aedes scapularis YFV - Aedes taeniorhynchus YFV - Haemagogus leucocelaenus Brazil 7705 Jan. 2016 – May 2017 CHIKV - Culex erraticus CHIKV - Runchomyia humboldti Trivittatus virus - Culex nigripalpus

Moutailler et al. Viruses (2019) Main criteria to be a vector

1. Effective contact with pathogen’s host and feeding

2. Association in time and space of the vector and the pathogen infected host

3. Repeated demonstration of natural infection of the vector

4. Experimental transmission of the pathogen by the vector Vector competence

Bellone & Failloux. Front. Microbiol. (2020) May 15 2015: first cases in Brazil

➢ 1,5 million cases ➢ > 45 American countries

E gene In Brazil

100

Rio de Janeiro (Brazil) 80

June 2015 – May 2016 60

1,683 mosquitoes captured (720 ♀ + 963 ♂) 40 Day 4 pi Day 7 pi Infectionrtae(%) 20

100

40 Day 4 pi Day 7 pi 20

Ferreira-de-Brito et al. Mem Inst Oswaldo Cruz (2016) (%) rate Disseminatedinfection

Chouin-Carneiro et al. PLoS Negl Trop Dis (2016) Aedes aegypti and 100 80 Aedes albopictus 60 40

Infection rate (%) rateInfection 20

0 TUB VRB Aedes aegypti Aedes albopictus Brazil USA

100

USA (AL) 40

Dissemination efficiency (%) efficiency Dissemination TUB VRB Aedes aegypti Aedes albopictus Brazil USA

100

Brazil (AA) 60

2000 km 40

Transmission efficiency (%) efficiency Transmission 0 TUB VRB Aedes aegypti Aedes albopictus Chouin-Carneiro et al. PLoS Negl Trop Dis (2016) Brazil USA Cx. pipiens complex and ZIKV

Infection Dissemination

Cx. quinquefasciatus Cx. pipiens Cx. quinquefasciatus Cx. pipiens

100 100

80 80

60 60

40 40

20 20 Infectionrate(%)

0 0 3 7 14 21 3 7 14 21 Days post-infection (%) efficiency Dissemination Days post-infection Transmission Cx. quinquefasciatus Cx. pipiens

100

20 Transmission efficiency (%) efficiency Transmission 0 3 7 14 21 Days post-infection Credit: A. Vega-Rua (I. Pasteur) Amraoui et al. Eurosurveillance (2016) No transmission by Culex spp.

Head (Replication) Cx. quinquefasciatus Cx. pipiens

100

80 Intra-thoracic inoculation 60 40

Dissemination efficiencyDissemination 0 3 7 14 Days post-infection

Saliva (Transmission) Cx. quinquefasciatus Cx. pipiens ~2500 viral particles 100 80

20 Amraoui et al. Eurosurveillance (2016) 0

Transmission efficiency (%) efficiency Transmission 3 7 14 Days post-infection Induction of RNAi pathways? No difference in ZIKV-specific small RNAs

Non-infected ZIKV-infected

Day 3 pi

Day 7 pi

Lack of virus-derived siRNAs

Lourenço-de-Oliveira et al. J Gen Virol (2017) Field-collected populations are not able to transmit ZIKV

Fernandes et al. PLoS Negl Trop Dis (2016) Aedes albopictus in Europe

20 countries 64 departments Risk for Europe

France

French Overseas departments Guadeloupe Martinique French Guiana Jupille et al. PLoS Negl Trop Dis (2016) Ae. albopictus and other ZIKV genotypes

Corsica Montpellier

100 100

80 80

60 60 ZIKV Cambodia ZIKV Cambodia 40 ZIKV Dakar 40 ZIKV Dakar ZIKV Martinique ZIKV Martinique 20 20 Transmission rate (%) rate Transmission Transmission rate (%) rate Transmission

0 0 7 14 21 7 14 21 Days post-infection Days post-infection

Vazeille et al. Emerg. Microbes Infect. (2019) 2019: first local cases of Zika in Hyères (South of France)

Brady OJ & SI Hay. The Lancet (2019) Arboviruses and Insect Vectors

R. Bellone A. Blisnick M. Vazeille P.-S. Yen M. Hocine L. Mousson J. Alexandre C. Bohers JP Martinet G. Gabiane M. Viglietta AB. Failloux

National International Barré H. (Université de Corse) Busquets N. (IRTA, Spain) Delaunay P. (CHU Nice) Boyer S. (IP Cambodia) David JP. (LECA-CNRS, Grenoble) Calvitti M. (ENEA, Italy) De Lamballerie X. (IRD-AMU-Inserm) Chen CH. (NHRI, Taiwan) Gustave J. (ARS Guadeloupe) Delang L. (Univ. Leuven, Belgium) Lagneau C./Lambert G. (EID Med.) Gasperi G./Malacrida A. (Univ. Pavia, Italy) Leparc-Goffart I. (Irba) Grandadam M./ Calvez E. (IP Laos) Mavingui P. (PIMIT, La Réunion) Haddad N. (Univ. libanaise, Lebanon) Moutailler S. (Anses) James A. (Univ. California, USA) Paupy C./Simard F. (IRD) Kohl A./ Pondeville E. (Univ. Glasgow, UK) Vega-Rua A. (IP Guadeloupe) Lourenço-de-Oliviera R. (Instituto Oswaldo Cruz, Brazil) Yébakima A. (Cedre, Martinique) Merits A. (Univ. Tartu, Estonia) Sinkins S. (Univ. Glasgow, UK) Schnettler E. (BNI, Germany) Sousa CA. (IHMT, Portugal) Takken W., Pijman G. (Univ. Wageningen, NL) Veronesi E. (Univ. Zurich, Switzerland) Experimental infection of mosquito with arbovirus in order to evaluate the vector competence

Mawlouth Diallo Pôle de Zoologie Médicale Institut Pasteur de Dakar Transmission cycle of arboviruses

Arthopods Insects (Mosquitoes, Ticks, Sand flies, biting midges Dynamic Inter-action : Triatomes etc… The pathogen (virus, parasites…) The vertebrate host (s) Vectors The Vector(s) The environment

Pathogens Virus, Parasites Bacteria Rickettsia Mammals, Reptiles, Vertebrates Birds, Batrachians Vector competence Vector Competence : definition • Ability of a mosquito species to achieve the development cycle of a virus until been infecting. – Ability to acquire infection, – Ability to replicate the virus – Ability to transmit the virus Vector Competence : Sequential step

Step 1 : Infection from a viremic vertebrate Midgut

Pathogen

hemocoel Viremic host Vector Competence : Sequential step Unique objective of the virus/pathogens Ovaries To reach the organ governing transmission: ==> Vertical Transmission Ovaries and Salivary glands. Midgut

Pathogen

Viremic host

hemocoel Salivary glands ==> Horizontal transmission (When the mosquito will excrete saliva during its next blood meal) Vector Competence : Sequential step Step 2. The virus pass through the peritrophic membrane The virus infect midgut epithelial cells The virus replicate into epithelial cells Peritrophic membrane Infection barrier Midgut

Ovaries

Salivary glands Hemocoel Vector Competence : Sequential step Step 3. The virus escape to midgut barrier The hemolymph disseminate the virus to other organs Step 4. The virus replicate into organs and tissues (fat body, muscles, nerves etc..) Step 5. The virus infect the salivary glands Midgut MEB : Midgut Escape Barrier

Ovaries

Hemocoel SGIB: SG Infection Barrier Salivary glands SGEB : SG Escape Barrier Vector Competence : How to proceed

Two methods of oral infection are used for experimental studies to assess vectors competence :

• Natural infection • Artificial feeding methods Context of the study

Context 1. Context 2. • YF mass immunization The YF transmission cycle proposed in East Africa programme was stopped after was likely not adapted to many contexts in west & appearance of severe adverse central Africa event among children (<10 years) Ae. africanus absent or in low density in many YF endemic Land cover sites withareas. ZIKV infected mosquitoes in the 60s Ae. simpsoni absent too in many YF endemic zones or exhibited a zoophilic tendency.

• A major epidemic reported in Senegal inVector 1965 Competence in Diourbel : Process for infection First experiments using human to demonstrate the role of Aedes aegypti as the vector Yellow fever (Carlos Finlay, 1998, Walter Reed 1900, Marchoux, 1902)

Mosquito incubation for 12 days • Virus detected in all ecological unit mainly during October • In december the unique fascias infected was the forest • Early dissemination to village of the virus during June (beginning of the rainy season)

Transmission monitoring Mosquito infection • Expose human volunteer to Expose seek human Kedougouinfected mosquito bite after 12 to mosquito bite days of incubations Limitation: ethical issues • Follow up the human volunteer Hadow et al, 1968 for symptoms occurrences Vector Competence : infection using animals

Land cover sites with ZIKV infected mosquitoes Land cover sites with ZIKVLand infectedcover sites mosquitoes with ZIKV infected mosquitoes • The principle consists of infectingLand cover sites with mosquitoes ZIKV infected mosquitoes on a viraemic animal. LandLand cover cover sitesLand sites with cover with ZIKV ZIKVsites infected infectedwith mosquitoesZIKV mosquitoes infected mosquitoes Land cover sites with ZIKV infected mosquitoes

Land cover sites with ZIKV infected mosquitoes LandLand cover cover sites sites with with ZIKV ZIKV infected infected mosquitoes mosquitoes Land cover sites with ZIKV infected mosquitoes Land cover sites with ZIKV infected mosquitoes Land cover sites with ZIKV infected mosquitoes Land cover cover sites sites Landwith withLand ZIKV cover ZIKV cover infected sitesinfected sites with mosquitoes withmosquitoes ZIKV ZIKV infected infected mosquitoes mosquitoes Land cover sites with ZIKV infected mosquitoes

Land coverLand coversites with sites ZIKV with infected ZIKV infected mosquitoes mosquitoes • Virus detectedLand in all cover ecological sites unit mainly with during ZIKV October infected mosquitoes • In december the unique fascias infected• Virus was detected the forest in all ecological unit mainly during October • Early dissemination to Virusvillage detected of the •virus In in decemberall during ecological June the unit unique (beginning mainly fascias duringof theinfected rainyOctober wasseason) the forest • Virus detected • in all ecological• Earlyunit mainly dissemination during October to village of the virus during June (beginning of the rainy season) • In december the• In unique december fascias •theVirus infected unique detected fasciaswas inthe all infected forest ecological was unitthe forestmainly during October • Early dissemination• Early to dissemination village• Inof december the tovirus village duringthe ofunique theJune virus fascias (beginning during infected June of the was (beginning rainy the season)forest of the rainy season) • Virus detected in all ecological• Virus detected unit mainly in all during ecological October unit mainly during October • Early dissemination to village of the virus during June (beginning of the rainy season) • In december the unique• Infascias december infected the uniquewas the fascias forest infected was the forest • Early dissemination to• villageEarly dissemination of• theVirus virus detected during to village in Juneall ecological of (beginning the virus unit duringofmainly the rainyJuneduring season) (beginning October of the rainy season) • Virus detected in all ecological unit mainly during October • In december the unique• In decemberfascias• Virus infected the detected unique was fascias in the all forest ecological infected was unit the mainly forest during October • Early dissemination• Virus detected •toEarly village indissemination •allIn of ecologicaldecember the virus to unitthe duringvillage unique mainly Juneof the fasciasduring (beginningvirus duringOctoberinfected of June the was (beginningrainy the forestseason) of the rainy season) • Virus detected in all ecological unit mainly• Early during dissemination October to village of the virus during June (beginning of the rainy season) • In december the uniqueVirus detected fascias infected in all ecological was the unitforest mainly during October • In december the unique• Earlyfascias dissemination infected• was to the village forest of the virus during June (beginning of the rainy season) Early dissemination to village of the virus• In during december June the (beginning unique fascias of the rainyinfected season) was the forest • • Virus detected in all ecological unit mainly during October • VirusEarly detected dissemination in all ecological to village unit of mainlythe virus during during October June (beginning of the rainy season) • In december the unique fascias• infected was the forest • In december the unique fascias infected was the forest • Virus detected• Early in all dissemination ecological unit to mainlyvillage duringof the virusOctober during June (beginning of the rainy season) • Early dissemination to village of the virus during June (beginning of the rainy season) • In december the unique fascias• Virus infected detected was inVirus allthe ecological forestdetected inunit all mainly ecological during unit October mainly during October • Early dissemination to village of the virus •during June (beginning of the rainy season) • Virus detected• inIn all december ecological the •unitIn unique decembermainly fascias during the infected Octoberunique fasciaswas the infected forest was the forest • In december the• Early unique dissemination fascias• infectedEarly to dissemination villagewas the of forest the tovirus village during of theJune virus (beginning during June of the (beginning rainy season) of the rainy season) • Early dissemination to• villageVirus detectedof the virus in duringall ecological June (beginning unit mainly of the during rainy Octoberseason) • Virus detected in all ecological• In december unit mainly the uniqueduring Octoberfascias infected was the forest • In december the unique •fasciasEarly dissemination infected was the to forest village of the virus during June (beginning of the rainy season) • Early dissemination• Virus detectedto village of in theall ecologicalvirus during unit June mainly (beginning during of October the rainy season) • In december the unique fascias infected was the forest • Early dissemination to village of the virus during June (beginning of the rainy season)

…..

Hamster Guinea pig Mouse Chiken Primates Prerequisites • The animal should be susceptible to infection with the virus to be tested (free of antibodies against the virus to be tested). • The approach require a precise knowledge of the period and duration of the viraemia (presence of the virus in the bloodstream) • The animal is inoculated several hours/days before the exposition of the arthropods to be tested for feeding. Vector Competence : infection using animals

…..

Hamster Guinea pig Mouse Chiken Primates Prerequisites • Animal ethic and species protection • Lack of animal facilities / infrastructures (ABSL2 or ABSL 3) to handle for each virus the appropriate vertebrate host (Biosecurity and biosafety) Matériel et méthodes

Matériel et méthodesSystème artificiel d’infection par voie orale: Vector CompetenceSystème artificiel : Infection d’infection par Process voie orale:

Oral Infection ofØ mosquitoesLa méthode du coton using imbibéØ artificial Laet celle méthode des gouttes feeding de sangdu method virémiquecoton imbibéhave et celle des gouttes de sang virémique been developed to (Gublerovercomeet al., 1976) issues concerning(Gubler et the al., use 1976) of animal :

(Mattingly, 1947)

(Gubler 1976)

Placer sur le moustiquaireCotton recouvrant pledget le pot d’élevage/stick une support à trois gouttes system de sans virémique ou un coton imprégné de sang virémique.

Placer sur le moustiquaire recouvrant le pot d’élevage une à trois gouttes de sans virémique ou un coton imprégné de sang virémique. (Rutledge et al, 1964) (Hemotek Ltd, UK) (Luo, 2014) 16 Finality: Feeding mosquito through a membrane and maintaining the blood at 37°C METHODOLOGIE (7/8) L’infection expérimentaleInfectionMETHODOLOGIE expérimentale (7/8) Elevage Expériences d’exposition artificielle des moustiques au repas de sang Expériences d’exposition artificielle des moustiques au repas de sang Vector Competencev Procédure : Infection expérimentale process Colonisation des v Procédure expérimentale • 1/3 Hématies lavées moustiques pour What do we need for this infection? 5 minutes 3 l’élevage 5 minutes 3 3 Serum • 1/3 Suspension10 minutes virale 3 Stades : Remove after 10 minutes 15 minutes 3 centrifugation • Adulte 15Tempsminutesd’exposition3 • Œuf Temps d’exposition • 1/3 (SVF,ATP20 minutes à 0,005M,3 20 minutes 3 Blood (washed Membrane • Larve Parafilm paper Baudruche 25 minutes 3 erhythrocytesto avoid 25 minutes 3 • Nymphe •potential29-30 presenceC of Chiken skin Mouse shaven skin Sucrose)30 minutes 3 Prélèvementantibodies & lavage in the serum) du sang etc. 30 minutes 3 Mosquito Virus Sang jour du Prélèvement : Expérience 1 • Génération : F1 • Geographic origin Sang•jourGeographicdu Prélèvement origin : Expérience 1 • Period of collection • Host origin (vertebrateSystèmeSang conservé de Rutledgeà J+1 : Expérience 2 • Age (3-5 days) Sang conservé/invertebrateà J+1 : Expérience 2 Sang conservé à J+2 : Expérience 3 • âges : 3-5 jours • Feeding background after Sang• conservéStock preparationà J+2 : (cellExpérience lines, 3 emergence (sucrose 10% only) mice, Mosquito inoculation) • Generation (F 1 lab generation • Stock aliquoted to avoid • sucrose : 10% • 27C more appropriate to be close to the defrosting/refreezing12 field population) 12• 80% HR • Titer of the stock • Starved for 24-72h (water provide • Passage level • affamées : 48h to avoid weakness)• 12/12 h jour/nuit• Genotype • Up to 50 female per Cardboard

7 Conditions standards Système HemotekTM

10 Infection expérimentale

• 1/3 Hématies lavées

• 1/3 Suspension virale

• 1/3 (SVF,ATP à 0,005M, Vector Competence : Process for infection Sucrose) FedPrélèvement individuals sorted & lavage du sang METHODOLOGIEWhat (5/8) do we need for this infection: on chill table and put Mixed blood meal into Cardboard Système de Rutledge • volume blood (1/2 or 1/3) Expériences d’exposition artificielle des moustiques au• virusrepas (1/2 de or 1/3)sang • ATP 510-3M. v Principe expérimental du Système de Rutledge• Sucrose 10% (optional)

37C Conditions standards Système HemotekTM

Water Stream Water Stream - Water stream to maintain outlet Incubation entrance the blood meal at 37°C 10 - Exposition (30-60mn) • Mosquito feed with sucrose 10% only Maintain mosquitoes at Cone receiving • viremic blood 27±1°C and 70-80%HR in an incubator Gorgeur en verre

Ø Gorgeurs en verre connectés entre eux et à un bain polystat

10 Handling exposed mosquito – assess transmission

Saliva collection using capillary tube containing Dissection and saliva collection PBS ; Immersion oil, FBS etc… after N days post exposure to blood meal /incubation (days 14 or every days) Saliva

Legs and wings Whole body

Virus detection using cell line ; molecular tools Data analysis

• Infection rates: number of positive bodies/total number of engorged mosquitoes incubated and tested • Disseminated infection rate : number of mosquitoes with positive wings-legs/ total number of engorged mosquitoes incubated and tested • Transmission rate : number of mosquitoes with infected saliva/ total number of engorged mosquitoes incubated and tested

• Rates are calculated for each species and each days post exposure to blood meal. • The rates obtained are compared using Chi2 or Fisher’s exact test American continent than the ZIKV Asian lineage

(5) Virus adaptation to different mosquito species appears an important evolutionary force for CHIKV evolution (La Reunion in 2005 of the A226V amino acid substitution in the E1 envelope glycoprotein its role in DENVs evolution is still controversial

(6) Limited data are available on co-infections with different viruses or serotypes/genotypes of one viral species.Some data from Senegal

Susceptibility of Aedes aegypti to Zika Virus

Tissue Culture Infectious Dose50 Assay eclipse phase typically associated with low virus midgut titer can be Viral titers in this study were determined with a tissue culture seen on day 1 pe, with only one of the midgut showing detectable infectious dose50 assay, an endpoint dilution technique, using Vero ZIKV. Virus titers in day 2 pe were higher than that observed for cells as described by Higgs et al. [40]. Briefly, 100 mL of 10-fold day 1 pe, mirroring the results obtained on percentage of midguts serial dilutions of each sample were titrated (in duplicate) in 96-well infected (Figure 1). These suggest that midgut ZIKV titer observed microtititer plates and incubated with Vero cells at 37uC and 5% during day 2 pe was most probably due to virus replication in the midgut rather than to the remaining amount of blood observed in CO2. At the end of day-7 incubation, the cells were examined microscopically for ZIKV-induced cytopathic effect (CPE). A well is some of the mosquitoes. A significant increase (P,0.026) in mean scored positive if any CPE is observed compared to the uninfected viral titers was observed between days 3 pe (3.9 Log10 TCID50/ mL) and day 5 pe (5.6 Log TCID /mL). From day 6 pe control cells. All virus titers were expressed as Log10 TCID50/mL. 10 50 onwards, mean viral titers showed a decreasing trend from Statistical analysis fluctuated between 5.4 Log10 TCID50/mL and 5.9 Log10 TCID /mL but the differences observed were not statistically Proportion infected was calculated by dividing the number of 50 significant (P$0.91). infected midguts (or salivary glands) by the total number of miguts ZIKV titers in the salivary glands increased from day 4 pe (or salivary glands) sampled. To compare viral titers at different time onwards (Figure 3). Although the difference in mean viral titers points, raw data was subjected to a normality test using SPSS Ver 18 from day 5 pe (2.7 Log TCID /mL) to day 7 pe (3.7 Log (IBM, USA). Data that passed the normality test were analyzed by 10 50 10 TCID /mL) was not significant (P = 0.68), the mean viral load analysis of variance using the above mentioned software. 50 increased significantly (P,0.001) by day 10 pe (6.4 Log10 TCID /mL), achieving the highest mean viral load of .8.0 Vector competence Aedes from ResultsSenegal and ZIKAV 50 Log10 TCID50/mL by day 14 pe. Oral susceptibility of Ae. aegypti to ZIKV Presence or absence of blood in the midgut was verified during Discussion dissection under a Stereoscope (Olympus, USA). By Day 3, when Recent unprecedented spread of chikungunya virus (CHIKV) in blood had been completely digested, seven (87.5%) of the analyzed many parts of the world, with millions of people affected, mosquitoes were positive for ZIKV (Figure 1). From day 6 pe exemplifies how arboviruses can adapt and affect human health onwards, all midguts were positive for ZIKV except for one of the on a global scale [31]. Singapore’s vulnerability to emerging and mosquitoes that was negative for the virus at day 7-pe. re-emerging arboviruses is accentuated by the country’s location as The presence of viable ZIKV in the salivary glands (n = 1) was a popular tourist and business hub, high dependency on migrant first observed on day 4 pe (Figure 1) and 62% of mosquitoes workers, tropical climate, dense population, and the presence of sampled on day 5-pe showed detectable virus in the salivary potential mosquito vectors. An outbreak of chikungunya in glands. ZIKV was observed in salivary glands of all infected Singapore during the 2008–09 period attests the country’s mosquitoes sampled at days 10 and 14 pe. vulnerability to mosquito-borne diseases [31,41]. The outbreaks of ZIKV on Yap Island and the worldwide spread of CHIKV have ZIKV midgut and salivary gland titers shown the propensity of arboviruses to spread outside their known Figure 2 presents ZIKV midgut titers at different days pe. geographical range and their potential to cause large-scale Although remaining blood meal in midgut was not removed, an epidemics. Diagne et al. BMC Infect Diseases, 2015 15:492

Aubry et al, 2018 ; bioRxivJun. 10, 2018; doi: http://dx.doi.org/10.1101/342741Figure 1. Midguts and salivary glands infectionLi et al rates 2012 of ZIKV Plos in NTDAe. aegypti at different days post-infectious bloodmeal. Eight mosquitoes were sampled per day. doi:10.1371/journal.pntd.0001792.g001 • Susceptibility to ZIKV for all species ; Low dissemination rate . • Ae. aegypti significantly less susceptible than non-AfricanPLOS Neglected Ae. Tropical aegypti Diseases | www.plosntds.orgacross all ZIKV3 August 2012 | Volume 6 | Issue 8 | e1792 strains tested. Some data. Aedes aegypti from Senegal and YFV

Dickson et al, 2014 PLoS Negl Trop Dis 8(10): Some data. Aedes from Senegal and CHIKV 100

50 5dpi • Results revealed significant difference of Infection, 0 Dissemination and Transmission rates depending to the 100 origin of the population 50 10dpi

0 ArD30237 • No transmission with Ae. aegypti from Dakar whatever 100 the virus strains and the population was not infected 50 15dpi animal strain (CS13-288) 0

100 100 RAtes(%)

50 5dpi 50 5dpi 0 0 Legend 100 100

288 Ae. aegypti CV 50 10dpi - 50 10dpi Ae. aegypti DK HD180738 0 CS13 0 100 100 Ae. aegypti KDG Ae. vittatus KDG 50 15dpi 50 15dpi

0 0

Diagne et al, AJTMH (2014) • Viruses used: HD180738 isolated from human ; CS13-288 isolated from bats (Scotophillus sp.) ; ArD30237 isolated from mosquitoes (Ae. neoafricanus) • NB: None of the CHIKV strains analyzed in this study had a valine subsititution at position 226 Some data. Aedes aegypti from Senegal and DENV

Diallo et al, 2008 TRSTMH

Sylla et al, 20O9 PLoS NTD 3(4) Aaf had significantly lower vector competence Low suceptibility of all Ae. aegypti pop (Kedougou less susceptible) than Aaa. Aaa. 73.9% DIR ; 6.8% MIB rate of, 19.3%, MEB rate of 19.3%. Aaf = DIR = 34.2%, MIB rate = 7.4%, and MEB rate = 58.4%.

• Competence dependent to virus & vector strain • Sylvatic popupaltion with high DIR

Dickson et al, Plos NTD 2014) Gaye et al, 2019 PLoS NTD 13(2) Ndiaye et al. Parasites & Vectors (2016) 9:94 Page 6 of 9 Some data. Aedes aegypti from Senegal and RVFV

Ae. vexans Cx. quinquefasciatus Cx. poicilipes 100

25 infection

0 N = 30 30 76 9 25 25 50 10 40 30 76 18 N = 30 30 79 20 30 30 70 18 40 50 100 66 N = 25 25 36 6 42 48 68 10 30 30 52 8 100

50 Rate (%) 25 Dissemination 0 N = 23 19 57 5 9 8 25 3 34 24 54 13 N = 2 1 7 1 7 7 19 5 9 8 21 11 N = na na 3 na 11 10 28 2 11 14 22 na 100 Legend 75 5 dpe dpe : Days post exposure

50 10 dpe N : Sample size 15 dpe na : Not applicable 25 20 dpe : 95% confidence intervals Transmission 0 N = na na 6 na na 2 4 1 5 8 20 2 N = na na 2 na na na na na na 1 2 2 N = na na na na 2 1 1 na 1 3 9 na

AnD 133719 ArD 141967 SH 172805 AnD 133719 ArD 141967 SH 172805 AnD 133719 ArD 141967 SH 172805 Virus strains Fig. 3 RVFV infection, dissemination and transmission rates throughout incubation periods as indicated for Ae. vexans, Cx. quinquefasciatus and Cx. poicilipes• Culexthat wereless orally exposed susceptible to infectious blood to mealRVFV containing infection 106-108 PFU/ml than virus suspension Aedes with RVFV strains SH72805, ArD14196 or AnD133719.• The N: animal number of specimens strains tested. is dpe: less day postinfectious exposure • SH 172805 (human/East African lineage) the unique strain transmitted among the viral strains revealed that at 10 dpe, the the dissemination rates remain similar irrespective of the SH172805 strain was more infectious than the dpe (P ≥ 0.1). This species did not disseminate the ArD141967 strain (P = 0.016), and at 15 dpe, the infection ArD141967 strain, despite the high viral titres of 5.5108 rate of the AnD133719 strain was significantly lower than PFU/ml detected in the blood meal post-feeding. The both the ArD141967 (P = 0.00049) and SH172805 strain observation was similar for Cx. poicilipes, which did not (P = 0.0002). disseminate the animal strain (AnD133719). Based on a pairwise analysis of infection rates between For all mosquito species tested, only the SH172805 strain the species, Ae. vexans was generally more susceptible to was transmitted (i.e. present in saliva) from 10 dpe and infection than Cx. quinquefasciatus. The unique excep- thereafter for Ae. vexans,15dpeforCx. poicilipes and only tion was the infection obtained with the ArD141967 after 20 dpe for Cx. quinquefasciatus.Thetransmission strain at 15 dpe, when the infection rate of Ae. vexans was rates ranged from 13.–33.3 % for Ae. vexans and was significantly higher than that of Cx. quinquefasciatus 11.1 % for Cx. poicilipes and 50 % for Cx. quinquefasciatus, (P = 0.001). Indeed using the same strain of virus, a com- but the differences between the three species and the dpe parable rate of infection was found for Ae. vexans and Cx. were not significant (P ≥ 0.05). quinquefasciatus at 5, 10, and 20 dpe (P ≥ 0.3). Independent of the virus strain used, dissemination Discussion rates were relatively low for the three species of mosqui- Our study showed that the three mosquito species were toes ranging from 10.5–37 % in Ae. vexans, from 9.5– susceptible to infection by the different RVFV strains 28.6 % in Cx. quinquefasciatus and from 3–40.9 % in with Ae. vexans mosquitoes being more susceptible than Cx. poicilipes. Dissemination was recorded relatively Cx. poicilipes or Cx. quinquefasciatus. The highest infec- quickly in Ae. vexans at 5 dpe for the SH172805 strain tion rates were obtained with Ae. vexans, which was also but was delayed to 10, and 15 dpe, for the ArD141967 the only species that disseminated all the virus strains and AnD133719 strains, respectively. With the exception and transmitted the SH172805 strains at different dpe. of the significant difference observed at 15 dpe between Despite a lower viral titre (≈ 106 PFU/ml) in the blood the AnD133719 and SH172805 strains (P = 0.001), the meal post-feeding, the rates of infection, dissemination dissemination rates were comparable for all virus strains and transmission rates obtained were as high as those and incubation periods (P ≥ 0.07). In Cx. quinquefasciatus, obtained in US populations of Ae. vexans that were Vector competence. Global observation Overall, significant variation was observed for infection, dissemination or transmission rates.

Souza-Neto et al 2019 General conclusions of a review of literature on infection, dissemination and transmission rates of arboviruses by Ae. aegypti mosquitoes

• Lack of uniformity in the procedures to test for vector competence • Cases of complete refractoriness to arboviral infection are rare • Complete susceptibility to infection has been detected for some Ae. aegypti populations for DENVs, ZIKV and CHIKV • complete susceptibility was not observed for any population tested for YFV • Initial infection dose of virus positively correlates with infection rate. • Virus adaptation to different mosquito species appears an important evolutionary force for CHIKV (La Reunion in 2005 of the A226V amino acid substitution in the E1 envelope glycoprotein) • Virus adaptation role in DENVs evolution is still controversial • Limited data are available on co-infections with different viruses or serotypes/genotypes of one viral species. Keys Factors that influence Vector competence. Intrinsic factors • Midgut Infection barrier (MIB) • Midgut Escape barrier (MEB) • Fat body playing a role in the immune response • Salivary Gland Infection barrier (SGIB) • Salivary Gland Infection barrier (SGEB) • The gut microbiota of the mosquito including Wolbachia endosymbiont regulate virus replication as an inhibitor or to enhance • The mosquito virome (Insect specific viruses) that may enhance or alliterate vector competence. • Immune response in insects against arboviruses (RNAi Responses) Keys Factors that influence Vector competence. Extrinsic factors • Extrinsic Incubation period (EIP) • Temperature. Increased temperature enhance vector competence • Viral dose / Titer • Virus passage history • Genotype of the virus • Mosquito colonization history Thank for your attention Mosquito-borne epornitic flaviviruses emerging in the Mediterranean

Miguel Ángel Jiménez-Clavero Animal Health Research Center (CISA), INIA-CSIC, Madrid, Spain

On-line workshop “Vector competence & mosquito-arbovirus relationships” 7th July 2021 Arboviruses, cycles and reservoirs

SPILLOVER FROM ENZOOTIC CYCLE - Most zoonotic arboviruses hold enzootic cycles that can spillover and lead to peri-domestic (urban) infections. Their vertebrate reservoirs are wildlife species, from which they depend upon for transmission (e.g.: WNV, USUV, TBEV). HUMAN AMPLIFICATION - Some arboviruses have AMPLIFICATION IN adapted to humans as DOMESTIC ANIMALS - their vertebrate Some zoonotic reservoirs, and hold a arboviruses have “urban cycle” so they do adapted to domestic not depend on an animal animals, which act as reservoir for transmission, reservoirs to complete i.e. they are no longer their transmission zoonotic. (e.g.: DENV, (“epizootic”) cycle (e.g. YFV, CHIKV, ZIKV). JEV, RVFV, CCHFV).

From: Weaver S.C. (2006) Evolutionary Influences in Arboviral Disease.. Current Topics in Microbiology and Immunology, 299:285–314 Springer, Berlin, Heidelberg. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7120121/pdf/978-3-540-26397-5_Chapter_10.pdf The flavivirus genus within the Flaviviridae family

- The Flavivirus genus comprises 53 viral species with global distribution - Many of them are human and/or animal pathogens of concern: - Dengue virus - Yellow fever virus - West Nile virus - Japanese encephalitis virus - Tick-borne encephalitis virus - Zika virus

- Common features: - Arthropod-borne viruses (=arboviruses) - Many of them are zoonotic (need an animal reservoir host to complete their cycles) - Severe clinical outcomes: neurological (encephalitis) or hemorrhagic syndromes The flaviviruses evolve firstly to adapt to different Primate reservoir transmission vectors Non-neurotropic Hemorrhagic disease

Bird reservoir Neurotropic Encephalitis

In most cases Beck et al., 2013 associated to rodents Flaviviruses are emerging worldwide Main mosquito-borne epornitic flaviviruses emerging in the Mediterranean region Flavivirus Serocomplex Main vector Main host West Nile virus (WNV) Japanese encephalitis Culex sp Avian Usutu virus (USUV) Japanese encephalitis Culex sp Avian Bagaza virus (BAGV) Ntaya Culex sp Avian

WNV outbreaks in humans, equids and birds in 2020 WNV outbreaks in humans 2011-2020 Main mosquito-borne epornitic flaviviruses emerging in the Mediterranean region Flavivirus Serocomplex Main vector Main host West Nile virus (WNV) Japanese encephalitis Culex sp Avian Usutu virus (USUV) Japanese encephalitis Culex sp Avian Bagaza virus (BAGV)* Ntaya Culex sp Avian *Also known as “Israel turkey meningoencephalomyelitis virus (ITV)” USUV BAGV/ITV West Nile virus as a model of an EMERGING epizootic, epornitic and zoonotic flavivirus

Transovarial The virus: 7-8 genetic lineages, many sub- transmission? lineages, a lot of clades, clusters... All equivalent? Other vectors Direct (e.g. ticks)? Many other susceptible contact? Knowledge gaps stillvertebrates remain (mammals, frogs, aligators). around WNV transmission,All “dead-end hosts”? introduction, spread and Persistence? Oral transmission? maintainanceCross-immunity?in circulationTrasfusions, transplants, Birds: >300 transplacent., Mosquitos: >60 maternal milk.. susceptible carrier species. Introduction species. All competent & spread All reservoir vectors? Spillover hosts? Overwintering WNV has a complex eco-epidemiology

Every transmission setting has different conditions: WNV adapts to many different settings WNV is a human and animal pathogen

Impact on human health Impact on animal health

BIRDS - Highly variable susceptibility, depending on the bird species and viral strain. - CORVIDS and RAPTORS: very susceptible, neurological symptoms and high mortality.

HORSES - 80% of cases: asymptomatic - 10-20% mild signs - 1-10%: neurological signs (ataxia, paralysis, fasciculations…) No available vaccine - Mortality: 20-40% of clinical cases. - Vaccine Europe, 2010-20: WNV re-emergence

Human cases Year Europe USA. (*) (#) 2010 926 1021 2011 340 712 2012 935 5674 2013 785 2469 2014 210 2205 2015 301 2175 2016 477 2038 Lineage 2 2017 288 2097 L3 L4a L4b 2018 2083 2647 Lineage 1 2019 425 958 L4c 2020 336 540

(*) ECDC (#) CDC Changes in Europe in the last 10 years: WNV REMAINS AND SPREADS. WNV surveillance

ACTIVE PASSIVE

• Sentinel birds • Diseased or dead birds (essential role of wildlife • Sampling of healthy birds during rehabilitation centers) ringing or other capture activities ORAL SWABS and FEATHERS (PCR) ORAL SWABS, ORGANS and FEATHERS (PCR) SERUM (Serology) SERUM (Serology)

• Sentinel horses sampled • Horses with neurological signs periodically SERUM (Serology- IgM) CEREBROSPINAL FLUID (PCR) SERUM (Serology)

• Mosquito traps periodically sampled during transmission season (May-October)

MOSQUITO POOLS (PCR) Adapted to each transmission setting WNV surveillance

EARLY WARNING Cost of control outbreak Exposure Infection Exposure in humans in vectors in animals Clinical signs in Clinical humans signs in animals Humans seek medical care

Modified from The World Bank, 2012 The economics of One Health Usutu & Bagaza viruses

• Less knowledge available on them as compared to WNV • In general, less pathogenic for humans/mammals • Highly pathogenic for certain bird species • Bagaza: vaccine for turkeys • IMPORTANCE: their co-circulation with WNV may have important effects on: • Diagnosis (cross-reactions) • Epidemiology (transmission, outbreak frequency, spillover, etc) • Clinical outcome (co-infections, cross-protection, superinfection exclusion, ADE risk…). Flavivirus cross-protection studies in bird hosts

Cross- BAGV USUV WNV prot.

1st 2nd YES

1st 2nd YES Flavivirus interactions in vectors

WNV USUV (…)

? Transcontinental spread of flaviviruses

• It is a widely known phenomenon: • Phylogenetic relationships support the existence of flavivirus flows between Africa and Europe. • Introductions of WNV (L1 and USUV L2), USUV and BAGV in Europe, BAGV WNV presumably from Africa, have been ascertained. BAGV • Other known transcontinental WNV flavivirus introductions are, e.g.: USUV • WNV in North America in 1999, presumably from Israel. • Barkedji virus in Israel. • Japanese encephalitis virus in Australia from Indonesia. Transcontinental spread of flaviviruses

• Migratory birds are often pointed out as the source of these introductions. • BUT: • Bird migrations not always “guilty” (e.g. WNV in NY, 1999). • Short-lived viremia (few days) do not allow for long virus “jumps”. • SEASONALITY: winter migration more likely to bring virus-loaded birds from Europe to Africa. • In Europe, WNV has demonstrated capacity to establish enzootic cycles (no need for new introductions). • These introduction events are possible but probably infrequent. Main take-home messages • Many flaviviruses around the world are spreading their geographic ranges. • Among them, mosquito-borne epornitic flaviviruses are becoming important threats for animal and human health in large parts of the world. • West Nile virus is probably the most important mosquito- borne epornitic flavivirus and represents a paradigm for this spread. • Other mosquito-borne epornitic flaviviruses recently emerging in the Mediterranean region are Usutu and Bagaza virus. • Important gaps still remain regarding transmission, ecology, epidemiology and diagnosis of these viruses. • As different flaviviruses sharing hosts and vectors simultaneously spread in the same territory, interactions between them may occur. • The outcomes of these interactions are still unpredictable. Thank you very much for your attention!!

CISA: Animal Health Research Centre A high biosecurity facility Transboundary and emerging diseases group

• Miguel Ángel Jiménez Clavero • Group leader