2019 - 00 - Prudhomme & Al - 527382.full

bioRxiv preprint first posted online Jan. 26, 2019; doi: http://dx.doi.org/10.1101/527382.
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1 The native European Aedes geniculatus mosquito species
2 can transmit Chikungunya virus.

3 Jorian Prudhomme1*, Albin Fontaine2,3*§, Guillaume Lacour4, Jean-Charles Gantier5, Laure
4 Diancourt6, Enkelejda Velo7, Silva Bino7, Paul Reiter8, Aurélien Mercier8,9
1
6 UMR MIVEGEC (IRD 224 - CNRS 5290 - Université de Montpellier), Montpellier, France.
2
7 Unité de Parasitologie et Entomologie, Institut de Recherche Biomédicale des Armées, 19-21
8 Boulevard Jean Moulin, 13005 Marseille, France.

3
9 Aix Marseille Université, IRD, AP-HM, SSA, UMR Vecteurs – Infections Tropicales et
10 Méditerranéennes (VITROME), IHU - Méditerranée Infection, 19-21 bd Jean Moulin, 13385
11 Marseille, cedex 5, France.
4
12 Unité Contrôle et Adaptation des Vecteurs, Institut Pasteur de la Guyane, 23 avenue Pasteur
13 B.P. 6010, 97306 Cayenne Cedex, Guyane française.
5
14 Laboratoire des Identifications Fongiques et Entomo-parasitologiques, Mennecy, France
6
15 Institut Pasteur, Genotyping of Pathogens and Public Health, Paris, France.
7
16 Control of Infectious Diseases Department, Institute of Public Health, Str. "A. Moisiu",
17 No.80, Tirana, Albania.
8
18 Unité Insectes et Maladies Infectieuses, Institut Pasteur, Paris, France.
9
19 INSERM UMR_S 1094, Neuroépidémiologie Tropicale, Laboratoire de Parasitologie-
20 Mycologie, Faculté de Médecine, Université de Limoges, Limoges 87025, France.
21
22 * These first authors contributed equally to this article.
23 § Correspondence: Albin Fontaine, Address: Institut de Recherche Biomédicale des Armées,

24 Département des Maladies Infectieuses, Unité Parasitologie et Entomologie, IHU -
25 Méditerranée Infection, IRD, AP-HM, SSA, UMR Vecteurs – Infections Tropicales et
26 Méditerranéennes (VITROME), 19-21 Boulevard Jean Moulin, 13385 Marseille Cedex 05, E-
27 mail: albinfont@gmail.com
28
1
bioRxiv preprint first posted online Jan. 26, 2019; doi: http://dx.doi.org/10.1101/527382. The copyright holder for this preprint
29 Abstract
30 Europe is the world’s leading tourism destination and is receiving every year travelers
31 from areas with active arbovirus transmission. There is thus a threat of mosquito-borne virus
32 emergence in Europe due to the presence of the invasive mosquito vector Aedes albopictus.
33 Little attention has been paid about the possible role of indigenous mosquito species as
34 vectors of emerging arboviruses. Here, we assessed the vector competence dynamic of
35 Ae. geniculatus, a European anthropophilic mosquito species, for chikungunya virus
36 (CHIKV) in comparison with Ae. albopictus.
37 We revealed that Ae. geniculatus was highly susceptible to CHIKV infection and could
38 transmit the virus. By specifically exploring the vector competence dynamic in both mosquito
39 species, we revealed that the cumulative distribution of CHIKV incubation period in Ae.
40 geniculatus was delayed by several days as compared to Ae. albopictus.
41 Our results strengthen the importance of considering indigenous species as potential
42 vectors for emerging arboviruses. They also revealed the importance of considering variation
43 in arbovirus dissemination or transmission dynamics in mosquitoes when performing vector
44 competence assays. We will discuss the implications of our results on a CHIKV outbreak
45 dynamic in a theoretical framework.
46
47 Sentence summary: The European mosquito Aedes geniculatus is highly susceptible to
48 CHIKV infection but disseminate and transmit the virus several days later than Ae.
49 albopictus.
50 Keywords: mosquito; Aedes geniculatus; Aedes albopictus; chikungunya; arbovirus; vector
51 competence; Albania; Europe; indigenous/autochthonous species; invasive species;
52 globalization.
2
53 Introduction
54 Chikungunya virus (CHIKV) is a mosquito-borne virus that has emerged from its
55 sylvatic habitat in Africa and is now transmitted in many urban regions world-wide
56 wherever competent vectors, primarily Aedes aegypti and Aedes albopictus, are present. Three
57 distinct lineages of CHIKV are sporadically causing outbreaks in human population [1, 2].
58 First detected in Albania and later in Italy, the Ae. albopictus species [3] is now
59 established throughout much of the Mediterranean basin and northward as far as Paris [4].
60 The ever-increasing air travel between Europe and regions with active arbovirus transmission,
61 in combination with travel times that are largely inferior to incubation period, has increased
62 the risk of arboviruses introduction into europe, as illustrated by transmission events of
63 CHIKV in several European countries [5-8]. For example, an outbreak of CHIKV in Italy in
64 2017 involved more than 420 confirmed cases [5].
65 Ae. albopictus has been implicated as the vector for all European CHIKV outbreaks
66 but an indigenous species, Aedes geniculatus (Olivier, 1791) is frequently found in ovitraps
67 used for surveillance of Ae. albopictus in outbreak areas [9]. Both are tree-hole species that
68 breed in natural containers in woodland as well as in man-made containers in the peri-urban
69 and peri-domestic environment [10, 11]. Their eggs are resistant to desiccation and can
70 overwinter in temperate areas. Both species are day-active, exophilic and feed aggressively on
71 humans and other mammals. The implication of European mosquitoes in the transmission of
72 emerging arboviruses has been poorly investigated despite evidence of transmission by other
73 Aedes species outside Europe [2]. We compared the competence of the native European
74 mosquito species Ae. geniculatus to transmit CHIKV in comparison with the invasive and
75 reference vector species for CHIKV: Ae. albopictus. Importantly, the consideration of the
76 dynamic nature of vector competence revealed that differences of vector competence between
77 mosquito species were due to a time shift in the distribution of extrinsic incubation periods
3
78 rather than differences in maximum proportion of infectious mosquitoes. The importance of
79 considering indigenous species as potential vectors for arbovirus and the implication, in a
80 theoretical framework, of our results on a CHIKV outbreak dynamic would be discussed.
81
82 Materials and Methods
83
84 Mosquito collection and identification
85 Mosquitoes from both Ae. albopictus and Ae. geniculatus species used in this study
86 originated in Tirana, the capital of Albania. Eggs were collected by ovitrap in an urban park
87 (41° 18’36” N; 19° 49’18” E) from July to August 2012. Eggs from both species were
88 hatched at the Institut Pasteur in Paris and reared under standard conditions. Adults from the
89 F0 generation were identified morphologically [12] and 500 individuals of each species
90 wereplaced per cage at 26°C±1°C with 60-70% relative humidity and a light: dark cycle of 16
91 h: 8 h. Adults were given 10% sucrose solution and females were allowed to engorge with
92 rabbit blood on a membrane feeding apparatus (Hemotek, Discovery Workshops, Lancashire,
93 United Kingdom) to obtain F1 eggs. Batches of eggs were hatched simultaneously to obtain
94 females of the same physiological age for experimental infections. Larvae were reared to the
95 adult stage under the same conditions. All experiments were realized with the F1 generation.
96
97 Virus
98 The isolate CHIKV 0621 was used in this study. This strain is phylogenetically close
99 to an Italian strain with an amino acid change (A226V) in the envelope glycoprotein E1 [13-
100 15] [GenBank accession number DQ443544]. The virus had been passaged three times in
101 C6/36 cells prior to use in the experiments. Virus titration was performed by focus-forming
102 assay (FFA) as previously described [16]. The titer of the frozen virus stock was estimated as
4
103 109 focus-forming units per mL (FFU/mL). All infectious experiments were conducted in a
104 BSL-3 insectary (Institut Pasteur, Paris).
105
106 Mosquito infections
107 Females 9-10 days old were deprived of sucrose solution 24 hours before experimental
108 infection. The infectious blood-meal consisted of 1 mL of viral suspension, 2 mL of washed
109 rabbit erythrocytes supplemented with adenosine triphosphate (10 mM) as a phagostimulant.
110 Blood feeding was by an artificial feeding apparatus (Hemotek) covered with pig intestine.
111 The final virus titer in blood meal was 108 FFU/mL corresponding to the viral load
112 encountered in some patients [17, 18]. Feeders were maintained at 37°C and placed on top of
113 the mesh of a plastic box containing 60 females. After 15 minutes of feeding (to minimize the
114 effect of virus degradation in the infectious blood meal), mosquitoes were cold anesthetized
115 and sorted on ice. Fully engorged females were transferred to cardboard containers and
116 maintained with 10% sucrose in an environmental growth-cabinet set at 28°C ±1°C, 80%
117 humidity, and a 16 h: 8 h light regime.
118
119 Vector competence and virus titration
120 CHIKV infection, systemic infection and transmission was determined at 3, 5, 7, 10,
121 12, 14 and 20 days post virus exposure (DPE) for both species. Presence of CHIKV in bodies
122 indicates a midgut infection, while presence of virus in heads and saliva indicates a systemic
123 (disseminated) infection and virus transmission, respectively. Saliva collection was performed
124 using the forced salivation technique [19].
125 Virus titration was performed by visualizing infectious foci on a sub confluent culture
126 of C6/36 cells by indirect immunofluorescence using 10-fold serial sample dilutions as
127 previously described [20] with a minor modification: After fixation, cells were washed three
5
128 times in PBS and incubated for 1 hour at 37 °C with 50 μL/well of mouse ascetic fluid
129 specific to CHIKV (primary antibody) diluted 1:1000 in PBS + 1% bovine serum albumin
130 (BSA) (Interchim, Montluçon, France). This mouse ascetic fluid was made by the Centre
131 National de reference des arbovirus of the Institut Pasteur in March 2013 and provided by Pr.
132 Despres Philippe.
133
134 Statistical analysis
135 The time-dependent effect of the mosquito species on mosquito midgut infection,
136 systemic infection and virus transmission was analysed by Firth's penalized likelihood logistic
137 regression by considering each phenotype as a binary response variable. Penalized logistic
138 regression, implemented through the logistf R package [21] was used to solve problem of
139 separation that can occur in logistic regression when (i) the outcome has high prevalence, (ii)
140 when all observations have the same event status for a combination of predictors or (iii) when
141 a continuous covariate predict the outcome too perfectly. A full-factorial generalized linear
142 model that included the time post virus exposure and the mosquito species was fitted to the
143 data with a binomial error structure and a logit link function. Statistical significance of the
144 effects was assessed by an analysis of deviance.
145 Virus titres in mosquito’s bodies, heads and saliva were compared between species by
146 a Mann-Whitney-Wilcoxon rank sum test stratified on the time post-exposure as implemented
147 in the wilcox_test function from the coin R package [22]. The effect of time post exposure on
148 each quantitative phenotype for each species was assessed using a Kruskal-Wallis rank sum
149 test. Time post exposure was converted into ordered factors to implement both tests.
150 The intra-host dynamic of systemic infection was assessed by a global likelihood
151 function for each mosquito species as described in Fontaine et al, 2018 [23]. Probabilities of
152 systemic infection at each time point post virus exposure were estimated with a 3-parameter
153 logistic model. The probability of systemic infection at a given time point (t) is governed by
6
154 K: the saturation level (the maximum proportion of mosquitoes with a systemic infection), B:
155 the slope factor (the maximum value of the slope during the exponential phase of the
156 cumulative function, scaled by K) and M: the lag time (the time at which the absolute increase
157 in cumulative proportion is maximal). For easier biological interpretation, B was transformed
158 into Δt, which correspond to the time required to rise from 10% to 90% of the saturation level
159 with the formula: Δt = ln (81) / B. For each mosquito species, the subplex R function [24] was
160 used to provide the best estimates of the three parameters to maximize the global likelihood
161 function (i.e., the sum of binomial probabilities at each time point post virus exposure). This
162 method accounts for differences in sample size when estimating parameters values.
163
164 Results
165 A total of 150 Ae. geniculatus and 141 Ae. albopictus engorged females were
166 analysed, considering the mosquito mortality during the experiment. High midgut infection
167 prevalences (>85%) were obtained for both species from the first time point after virus
168 exposure. With 100% midgut infection among the exposed mosquitoes, Ae. geniculatus was
169 highly susceptible to CHIKV infection (Supplementary figure 1). Midgut infection
170 prevalences were not influenced by the time post exposure (analysis of deviance, p-value =
171 0.0659) and a significantly higher midgut infection prevalence was observed in Ae.
172 geniculatus vs Ae. albopictus(analysis of deviance, p-value = 0.0438).
173 CHIKV titres in bodies were significantly higher in Ae. geniculatus vs Ae. albopictus
174 (stratified Mann-Whitney-Wilcoxon rank sum test, p-value < 2.2e-16), with approximately
175 one log 10 difference between values average across time points (5.1 vs 4.2 log 10 FFU/mL
176 for Ae. geniculatus and Ae. albopictus, respectively). Virus titres in bodies were significantly
177 different across time points post virus exposure in both species (Kruskal-Wallis rank sum test,
178 p-value = 0.0002402 and 2.984e-15 for Ae. geniculatus and Ae. albopictus, respectively).
7
179 Systemic infection, as measured by the head infection prevalence, was significantly
180 influenced by the time post exposure (analysis of deviance, p-value = 5.43e-13) and the
181 mosquito species (analysis of deviance, p-value = 4.10e-15) but the time post exposure effect
182 was not significantly different across the mosquito species according to logistic regression
183 (analysis of deviance, interaction term, p-value= 0.6). The intra-host dynamic of systemic
184 CHIKV infection was quantified in both mosquito species by fitting a 3 parameters logistic
185 model that assumes a sigmoidal distribution of the cumulative proportion of mosquitoes with
186 a systemic infection over time. Both species presented a saturation level (K, or maximum
187 proportion of infected mosquitoes with a systemic infection) equal to 100% (Figure 1).
188 However, this saturation level was later for Ae. geniculatus than for Ae. albopictus. Indeed, an
189 estimated 14.6 days were needed for Ae. geniculatus to go from 10% to 90% of the saturation
190 level whereas 100% of the saturation level was reached in less than one day for Ae.
191 albopictus. The lag time M, which can represent a proxy for the extrinsic incubation period-50
192 (EIP-50—the time required for the systemic infection prevalence to reach 50% of the
193 saturation level)—was 2.7 days in Ae. albopictus mosquitoes compared to 7.6 days in Ae.
194 geniculatus (Figure 1).
195 CHIKV titres in heads were significantly lower in Ae. geniculatus vs Ae. albopictus
196 (stratified Mann-Whitney-Wilcoxon rank sum test, p-value = 3.3e-4), with approximately 1.3
197 log 10 difference between values average across time points (2.5 vs 3.8 log 10 FFU/mL for Ae.
198 geniculatus and Ae. albopictus, respectively). Virus titres in heads were also significantly
199 different across time points post virus exposure in both species (Kruskal-Wallis rank sum test,
200 p-value = 2.621e-07 and 1.781e-10 for Ae. geniculatus and Ae. albopictus, respectively).
201 Virus transmission prevalences were measured by assessing the presence of infectious
202 virus in mosquito saliva. Virus transmission prevalences were significantly different between
203 mosquito species (analysis of deviance, p-value = 0.0038) and the effect of time post-
8
204 exposure varied significantly between mosquito species (analysis of deviance, interaction
205 term, p-value = 3.05e-4). It was not possible to model the cumulative virus transmission
206 prevalence over time for the Ae. albopictus species because of a lack of saturation level.
207 Proportion of virus in saliva clearly collapsed from day 5 post virus exposure. The same
208 phenomenon could be observed for Ae. geniculatus. However, the 3-parameter model could
209 nonetheless fit the data, giving the following estimates: 70% of saturation level, an EIP-50 of
210 8 days and a rising time of 6.2 days (Supplementary figure 2).
211 CHIKV titres in saliva were significantly higher in Ae. geniculatus vs Ae. albopictus
212 (stratified Mann-Whitney-Wilcoxon rank sum test, p-value = 1.8e-3), with 0.24 log 10
213 difference between values average across time points (0.51 vs 0.27 log 10 FFU/mL for Ae.
214 geniculatus and Ae. albopictus, respectively). Titres in heads were significantly different
215 across time points after virus exposure in mosquitoes from the Ae. geniculatus species only
216 (Kruskal-Wallis rank sum test, p-value = 4e-03 and 0.14 for Ae. geniculatus and Ae.
217 albopictus, respectively). Virus titres in saliva were clearly increasing over time in
218 mosquitoes from the Ae. geniculatus species.
219
220 Discussion
221 Chikungunya virus (CHIK) is one of four arboviruses withdengue virus (DENV), Zika
222 virus (ZIKV) and yellow fever virus (YFV) that can be sustained in a human-vector-human
223 transmission cycle. All four originated in wild primates but can cycle in the urban
224 environment, transmitted by peridomestic mosquitoes. Intercontinental travel and trade were
225 historically involved in the transmission of these viruses world-wide, sometimes involving
226 recruitment of new local vectors [25, 26]. Thus, the YFV, originating in Africa, was
227 introduced to the Americas during the slave trade,where it entered new transmission cycles
228 involving autochthonous arboreal species, such as Haemagogus janthinomys and mosquito
9
229 from the Sabethes genus, and non-human primates in primary tropical rain forest. The virus
230 then regularly propagated from the jungle into urban transmission cycles [27].
231 Europe is the world’s leading tourism destination with 671 million arrivals (50% of all
232 international tourists) in 2017, a number that continues to increase. [28]. Travellers with
233 arbovirus infections [29-35], including CHIKV [36] have initiated autochthonous
234 transmission [5, 6] vectored by the invasive mosquito Ae. albopictus. At the same time,
235 Europe harbours native mosquito species potentially able to transmit emerging arboviruses.
236 Yet, only two studies have assessed the potential for CHIKV infection in six indigenous
237 species from the south of France and Italy (Culex pipiens, Aedes caspius, Aedes detritus,
238 Aedes vexans, Aedes vittatus, and Anopheles maculipennis). The four Aedes species were
239 susceptible to virus infection, but the transmission potential of infectious viral particles in the
240 mosquito saliva was not investigated. These insects represent a small portion of all European
241 anthropophilic mosquitoes.
242 Our study has revealed the ability of the European mosquito Ae. geniculatus to
243 transmit CHIKV experimentally. Aedes geniculatus was revealed to be highly susceptible to
244 CHIKV infection. Midgut infection by an arbovirus is achieved very rapidly after exposure to
245 the infectious blood meal when infectious virus particles are still in contact with the midgut
246 wall cells. Virus dissemination in secondary tissues can only occur in mosquitoes with
247 infected midgut and is, in contrast to the infection phenotype, a dynamic process scaled in a
248 day unit that can have epidemiological significance. Variation in DENV dissemination
249 dynamics in mosquitoes was reported to significantly affects the risk and magnitude of
250 dengue outbreaks [23]. We revealed that both species were completely able to disseminate
251 CHIKV in their secondary tissues. However, this 100% saturation level of infected
252 mosquitoes with a disseminated infection was reached in Ae. albopictus 15 days earlier than
253 Ae. geniculatus. The estimated virus dissemination lag time, that can be considered as a proxy
10
254 for the EIP-50 (extrinsic incubation period in days until 50% of maximum infectiousness),
255 was nearly 8 days for Ae. geniculatus whereas is was less than 3 days for Ae. albopictus.
256 Combined with vector longevity, EIP is the most powerful contributor to vectorial capacity
257 according to the Ross-MacDonald equation [37]. Vectorial capacity defines the number of
258 secondary infections expected to occur from the introduction of a single infection in a naive
259 population [38]. The longer it would take for a virus to disseminate from the infected midgut
260 to the saliva, the fewer opportunities the mosquito would have to transmit the virus to a
261 human host before its death. This theory can be thwarted if Ae. geniculatus mosquito’s life
262 span is equal or longer than Ae. albopictus one [39]. Ae. geniculatus might then be involved
263 in autochthonous CHIKV transmissions in Europe in complementation with or in absence of
264 Ae. albopictus. In addition, possibility of rapid virus adaptation to new vectors was already
265 observed [40, 41]. CHIKV evolution toward restricted EIP duration in this autochthonous
266 species would greatly increase the epidemic potential of this vector-virus couple.
267 The biology of Ae. geniculatus is poorly studied [10]. From what is known, the species
268 can bite aggressively both humans and animals mainly during daylight hours [10, 57] (and
269 personal observation). Its larvae are commonly found in mature trees’ holes [42]. Adults often
270 coexist with Ae. albopictus in Europe but population densities of Ae. geniculatus generally
271 didn’t reached those of Ae. albopictus in peri-urban areas [64]. More data are needed to fully
272 characterise the level of contact with human hosts. As an anecdote, we captured Ae.
273 geniculatus on Jim Morrison grave, one of the most visited graves by tourists of all
274 nationalities in Pere Lachaise cemetery, in the center of Paris, where millions of people come
275 every year, strengthening the threat for this species in local virus transmission. Its flight
276 dispersion is not considered superior than Ae. albopictus. Consequently, this species would
277 not carry viruses outside of the area treated in the frame of the vector control intervention
278 [59]. Arbovirus vertical transmission is a rare event, and its occurrence hazard is correlated
11
279 with vector density and the surveillance of virus. If higher CHIKV vertical transmission rates
280 are achieved in Ae. geniculatus species, vertical transmission of CHIKV in overwintering
281 Aedes mosquitoes might contribute to the maintenance of this virus during winter.
282 Populations of Ae. geniculatus are generally monovoltine (ie. one generation per year), and
283 their diapause termination is asynchronous [10], enabling a reintroduction of the virus later in
284 the following year when the population of the primary vector Ae. albopictus is already high. It
285 seems therefore necessary to improve the knowledge on this species, including longevity,
286 flight and host-seeking behaviour, diapause and virus overwintering to fully assess its
287 epidemic potential. At last, temperature is strongly influencing vector competence. It can be
288 of interest to compare vector competence for CHIKV between both mosquito species at lower
289 temperatures.
290 Our study has several limitations. First, our estimates of virus dissemination dynamic
291 can be biased. They rely on the modelisation of the measured cumulative proportion of
292 mosquitoes with a disseminated infection over time. Concerning Ae. geniculatus, the
293 modelized proportion is not null at the time of mosquito exposure to the virus as it should be.
294 The number and repartition of time points and the proportions accuracy (that depend on
295 sample size at each time point) are influential in determining accurate parameter estimates of
296 the virus dissemination dynamic. This method can nonetheless provide realistic estimates and
297 offer the possibility to consider the dynamic nature of vector competence. Importantly, this
298 allows to disentangle the incubation period effect from the maximum proportion of systemic
299 virus infection. Then, the transmission dynamic estimates must be considered with caution
300 because the method to detect viruses in mosquito saliva do not distinguish true negatives from
301 negatives resulting from mosquito that did not expectorate saliva. This can explain decrease
302 of transmission rates for Ae. albopictus. Alternatively, this decrease could be explained by the
303 fact that infected mosquitoes died before non-infected mosquitoes or that oldest mosquitoes
12
304 begin to clear the infection through immune function [43] or by the natural death of virions. It
305 can however be concluded that both species can transmit infectious CHIKV particles as soon
306 as 3 and 7 days post exposure for Ae. albopictus and Ae. geniculatus, respectively.
307 CHIKV is in expansion throughout the world and Europe is not spared. Since the
308 CHIKV outbreak in Northern Italy in 2007, and more recently the isolated cases of
309 chikungunya and dengue recorded in France and Croatia from 2010 to 2013 [30, 44], it has
310 been acknowledged that Europe is vulnerable to local transmission of “tropical” arboviruses.
311 Epidemics risk is in connection with the steady increase of imported cases of Aedes-bornes
312 viruses as well as with the European expansion of Ae. albopictus [45]. So far, studies on the
313 epidemiology of the arboviral diseases chikungunya and dengue in European countries have
314 focused on the invasive "Asian tiger mosquito" without considering the potential role for
315 indigenous vector species. These results show the importance of considering European
316 indigenous species to assess overall risk of arbovirus transmission in Europe.
317 Assessing the vector competence of the different European mosquito species, but also
318 other regions around the world potentially exposed to arboviral outbreak, for other
319 arboviruses or other strains, will help to anticipate patterns of transmission of arbovirus as
320 CHIKV and DENV and the relative contribution of different vector species to virus’s
321 amplification and persistence (transmission and reservoir) in these areas. Moreover, we
322 recommend to monitor on the field all mosquito species, whether native or invasive, during
323 surveillance programs on future outbreaks, to further characterize the autochthonous mosquito
324 fauna that can be potentially involved in arbovirus transmission.
325
326 Acknowledgments
327 We thank Pr. Despres Philippe for providing technical assistance. This study was funded by
328 EU grant FP7-261504 EDENext (http://www.edenext.eu). The contents of this publication are
13
329 the sole responsibility of the authors and do not necessarily reflect the views of the European
330 Commission.
331
332
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Tables
438 Table 1: Body infection rate (BIR), systemic infection rate (SIR) and
439 transmission rate (TR) at different days post-infection of Ae. geniculatus and Ae.
440 albopictus exposed to CHIKV. Mosquitoes were contained in cardboard boxes after virus
441 exposure. For all surviving mosquitoes per box and time point, we measured three parameters
442 describing vector competence: (i) mosquito infection as measured by the presence of viral
443 infectious particles in the body (abdomen and thorax): BIR, (ii) virus dissemination as
444 measured by the presence of viral infectious particles in mosquito head: SIR, and (iii)
445 transmission efficiency as measured by the number of mosquitoes with viral infectious
446 particles in their saliva: TR.
Ae. geniculatus Ae. albopictus
Day pi BIRa SIRb TRc BIRa SIRb TRc
3 100 (20) 15 (20) 0 (3) 100 (20) 90 (20) 27.8 (18)
5 100 (20) 20 (20) 0 (4) 95 (20) 100 (19) 63.1 (19)
7 100 (18) 72.2 (18) 30.8 (13) 100 (18) 100 (18) 38.9 (18)
10 100 (18) 72.2 (18) 38.5 (13) 100 (18) 100 (18) 27.8 (18)
12 100 (18) 72.2 (18) 84.6 (13) 100 (17) 100 (17) 23.5 (17)
14 100 (19) 78.9 (19) 73.3 (15) 93.3 (15) 100 (14) 28.6 (14)
20 100 (21) 100 (21) 61.9 (21) 87.5 (16) 100 (14) 14.3 (14)
a
Percent of mosquitoes with infected body among all exposed mosquitoes.
b
Percent of mosquitoes with infected head among body infected mosquitoes.
17
c
Percent of mosquitoes with infectious saliva among mosquitoes with a systemic infection
(mosquitoes that disseminate the virus beyond the midgut barrier).
The number of mosquitoes analysed is displayed in brackets.
18
Figures
CHIKV systemic infection Ae. albopictus Ae. geniculatus

% of mosquitoes with a
_ _ _ _ _ _ _
1.00 _
_ _ _
_ Sample
_ _ _ _
_ size
0.75 _ _
_
14
_ 16
0.50 _ _ _ _
18
_
20
0.25
Lag time: 2.7 days Lag time: 7.6 days
0.00
Δt: 0.52 days _
_
Δt: 14.6 days
6
Mean CHIKV titer in heads
(log FFU/mL)
0
0 5 10 15 20 25 0 5 10 15 20 25
Days post-exposure
Figure 1: Systemic infection kinetic for Ae. albopictus and Ae. geniculatus species
infected with CHIKV. The upper panel shows the cumulative prevalence of systemic
infection over time post CHIKV exposure. Data points represent the observed prevalence at
each time point with their size being proportional to the sample size. Dashes represent the
95% confidence interval of the prevalence. The fitted values obtained with a 3-parameter
logistic model are represented for each species by a coloured line. The lag time and rising
time estimates are represented for each species. The lower panel shows CHIKV titres in
mosquito heads measured at each time points after oral exposure to the virus.
19
Ae. albopictus Ae. geniculatus

_ _ _ _ _ _ _ _ _ _ _ _ _
1.0 _
% of mosquitoes with a Sample

CHIKV infection 0.9 size
15
_ _ _ _ 16
0.8 _ _ _ _ _
_
17
_
_
18
19
0.7 20
_
21
_
0.6
6
in bodies (log FFU/mL)
Mean CHIKV titer
0
0 5 10 15 20 25 0 5 10 15 20 25
Days post-exposure
Supplementary figure 1: Midgut infection for Ae. albopictus and Ae. geniculatus species
infected with CHIKV. The upper panel shows the percentage of midgut infection over time
post CHIKV exposure. Data points represent the observed prevalence at each time point with
their size being proportional to the sample size. Dashes represent the 95% confidence interval
of the prevalence. The lower panel shows CHIKV titres in mosquito bodies measured at each
time points after oral exposure to the virus.
20
Ae. albopictus Ae. geniculatus

1.00 _
_
with CHIKV in saliva

% of mosquitoes _ _
Sample
0.75 _
size
_
_
_ _
_ 5
_ _ _
0.50 _ 10
_ _
_ _ 15
20
Saturation lev.: 70%
0.25 EIP-50: 8 days
Δt: 6.2 days
_
_
_ _ _ _
_
_
_ _
0.00
Mean CHIKV titer in saliva
3
(log FFU/mL)
0
0 5 10 15 20 25 0 5 10 15 20 25
Days post-exposure
Supplementary figure 2: Saliva infection for Ae. albopictus and Ae. geniculatus species
exposed to CHIKV. The upper panel shows the percentage of saliva infection over time post
CHIKV exposure. Data points represent the observed prevalence at each time point with their
size being proportional to the sample size. Dashes represent the 95% confidence interval of
the prevalence. EIP-50 and rising time estimates are represented for Ae. geniculatus species.
The lower panel shows CHIKV titres in mosquito saliva measured at each time points after
oral exposure to the virus.
21
Mean CHIKV titer in heads % of mosquitoes with a
(log FFU/mL) CHIKV systemic infection
0.00
0.25
0.50
0.75
1.00
6
0
_
5
_
_
Ae. albopictus
10
_
_
_
Δt: 0.52 days
Lag time: 2.7 days
_
15
20
_
Days post-exposure
25
0
_
5
_ _
Ae. geniculatus
10
_
_
_
Δt: 14.6 days
Lag time: 7.6 days
_
15
20
_
25
Sample
size
20
18
16
14
Mean CHIKV titer % of mosquitoes with a
in bodies (log FFU/mL) CHIKV infection
0.6
0.7
0.8
0.9
1.0
6
_
5
_
_
_
Ae. albopictus
10
_
_
_
_
_
15
20
_
Days post-exposure
25
0
_
5
_
_
_
Ae. geniculatus
10
_
_
_
_
_
15
20
_
25
Sample
size
21
20
19
18
17
16
15
Mean CHIKV titer in saliva % of mosquitoes
(log FFU/mL) with CHIKV in saliva
0.00
0.25
0.50
0.75
1.00
3
_
5
_
_
Ae. albopictus
10
_
_
_
_
_
15
20
_
Days post-exposure
25
0
_
5
_
_
Ae. geniculatus
10
_
_
_
_
_
15
Δt: 6.2 days

EIP-50: 8 days
Saturation lev.: 70%
20
_
25
Sample
size
20
15
10
5

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bioRxiv preprint first posted online Jan. 26, 2019; doi: http://dx.doi.org/10.1101/527382.

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1 The native European Aedes geniculatus mosquito species

2 can transmit Chikungunya virus.

4 Diancourt6, Enkelejda Velo7, Silva Bino7, Paul Reiter8, Aurélien Mercier8,9

8 Boulevard Jean Moulin, 13005 Marseille, France.

23 § Correspondence: Albin Fontaine, Address: Institut de Recherche Biomédicale des Armées,

34 vectors of emerging arboviruses. Here, we assessed the vector competence dynamic of

35 Ae. geniculatus, a European anthropophilic mosquito species, for chikungunya virus

36 (CHIKV) in comparison with Ae. albopictus.

40 geniculatus was delayed by several days as compared to Ae. albopictus.

41 Our results strengthen the importance of considering indigenous species as potential

43 in arbovirus dissemination or transmission dynamics in mosquitoes when performing vector

45 dynamic in a theoretical framework.

47 Sentence summary: The European mosquito Aedes geniculatus is highly susceptible to

50 Keywords: mosquito; Aedes geniculatus; Aedes albopictus; chikungunya; arbovirus; vector

51 competence; Albania; Europe; indigenous/autochthonous species; invasive species;

62 the risk of arboviruses introduction into europe, as illustrated by transmission events of

64 2017 involved more than 420 confirmed cases [5].

68 breed in natural containers in woodland as well as in man-made containers in the peri-urban

78 rather than differences in maximum proportion of infectious mosquitoes. The importance of

80 theoretical framework, of our results on a CHIKV outbreak dynamic would be discussed.

82 Materials and Methods

84 Mosquito collection and identification

92 rabbit blood on a membrane feeding apparatus (Hemotek, Discovery Workshops, Lancashire,

104 BSL-3 insectary (Institut Pasteur, Paris).

106 Mosquito infections

108 infection. The infectious blood-meal consisted of 1 mL of viral suspension, 2 mL of washed

117 humidity, and a 16 h: 8 h light regime.

119 Vector competence and virus titration

124 using the forced salivation technique [19].

132 Despres Philippe.

134 Statistical analysis

144 effects was assessed by an analysis of deviance.

172 geniculatus vs Ae. albopictus(analysis of deviance, p-value = 0.0438).

194 geniculatus (Figure 1).

218 mosquitoes from the Ae. geniculatus species.

241 anthropophilic mosquitoes.

263 in autochthonous CHIKV transmissions in Europe in complementation with or in absence of

316 indigenous species to assess overall risk of arbovirus transmission in Europe.

324 fauna that can be potentially involved in arbovirus transmission.

334 1. Pulmanausahakul, R., et al., Chikungunya in Southeast Asia: understanding the

446 particles in their saliva: TR.

Ae. geniculatus Ae. albopictus

Day pi BIRa SIRb TRc BIRa SIRb TRc

3 100 (20) 15 (20) 0 (3) 100 (20) 90 (20) 27.8 (18)

5 100 (20) 20 (20) 0 (4) 95 (20) 100 (19) 63.1 (19)

(mosquitoes that disseminate the virus beyond the midgut barrier).

The number of mosquitoes analysed is displayed in brackets.

CHIKV systemic infection Ae. albopictus Ae. geniculatus

Ae. albopictus Ae. geniculatus

% of mosquitoes with a Sample

time points after oral exposure to the virus.

Ae. albopictus Ae. geniculatus

with CHIKV in saliva

oral exposure to the virus.

Δt: 6.2 days

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