# Dengue-1 Virus Clade Replacement in Thailand Associated with Enhanced Mosquito Transmission

Louis Lambrechts, Thanyalak Fansiri, Arissara Pongsiri, Butsaya

Thaisomboonsuk, Chonticha Klungthong, Jason H Richardson, Alongkot

Ponlawat, Richard G Jarman, Thomas W Scott

► To cite this version:

Louis Lambrechts, Thanyalak Fansiri, Arissara Pongsiri, Butsaya Thaisomboonsuk, Chonticha Klungthong, et al.. Dengue-1 Virus Clade Replacement in Thailand Associated with Enhanced Mosquito Transmission. Journal of Virology, 2012, 86 (3), pp.1853-61. 10.1128/JVI.06458-11 . pasteur-00661378

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1 Dengue-1 Virus Clade Replacement in Thailand Associated

2 with Enhanced Mosquito Transmission

3

4

5 RUNNING TITLE: Role of vectors in dengue virus evolution

6

7

8 Louis Lambrechts,¹* Thanyalak Fansiri,¹,² Arissara Pongsiri,² Butsaya

9 Thaisomboonsuk,³ Chonticha Klungthong,³ Jason H. Richardson,²,ª Alongkot

10 Ponlawat,² Richard G. Jarman,³,ᵇ and Thomas W. Scott⁴,⁵

11

12

13 ¹Department of Virology, Institut Pasteur, 75252 Paris Cedex 15, France

14 ²Department of Entomology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand 10400

16 ³Department of Virology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand 10400

18 ⁴Department of Entomology, University of California, Davis, CA 95616, USA

19 ⁵Fogarty International Center, National Institutes of Health, Bethesda, MD 20892, USA

20 ªPresent address: Entomology Branch, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA

22 ᵇPresent address: Viral Disease Branch, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA
24 * Corresponding author. Mailing address: Institut Pasteur, 25 rue du Docteur Roux,

25 75724 Paris Cedex 15, France. Phone: 0033-144389127. E-mail:

26 louis.lambrechts@pasteur.fr

27

28 Abstract word count: 218. Text word count: 5,327.
29 **Abstract**

30

31 Dengue viruses (DENV) are characterized by extensive genetic diversity, which can be
32 organized in multiple, genetically distinct lineages that arise and die out on a regular basis
33 in endemic regions. A fundamental question for understanding DENV evolution is the
34 relative extent to which stochastic processes (genetic drift) and natural selection acting on
35 fitness differences among lineages contribute to lineage diversity and turnover. Here, we
36 used a set of recently collected and archived low-passage DENV-1 isolates from Thailand
37 to examine the role of mosquito vector-virus interactions in DENV evolution. By
38 comparing the ability of 23 viruses isolated on different dates between 1985 and 2009 to
39 be transmitted by a present-day *Aedes aegypti* population from Thailand, we found that a
40 major clade replacement event in the mid-1990s was associated with virus isolates
41 exhibiting increased titers in the vector's hemocoel, which is predicted to result in a
42 higher probability of transmission. This finding is consistent with the hypothesis that
43 selection for enhanced transmission by mosquitoes is a possible mechanism underlying
44 major DENV clade replacement events. There was significant variation in transmission
45 potential among isolates within each clade, indicating that in addition to vector-driven
46 selection other evolutionary forces act to maintain viral genetic diversity. We conclude
47 that occasional adaptive processes involving the mosquito vector can drive major DENV
48 lineage replacement events.

49
50 INTRODUCTION

51

52 Worldwide, dengue viruses (DENV) are the most important mosquito-borne viral

53 pathogens of humans. The four antigenically distinct DENV serotypes (DENV-1 to -4)

54 cause a broad spectrum of clinical manifestations. An estimated 50 million people

55 experience dengue illness each year, approximately 500,000 of which are associated with

56 severe, life-threatening disease (18). In addition, a significant portion of infections can be

57 inapparent and, thus, go undetected by surveillance programs (15). Despite the large

58 disease burden imposed by dengue on the human population, there is currently no

59 commercially available DENV vaccine or antiviral therapy (46). In endemic regions

60 where multiple serotypes co-circulate, DENV epidemiological dynamics are

61 characterized by complex oscillations in incidence and serotype prevalence (6, 32, 43). A

62 variety of ecological (10, 24) and immunological factors (1, 36) are thought to govern

63 these complex spatio-temporal dynamics. There is also compelling evidence for the

64 influence of virological factors in disease incidence and severity (reviewed in ref. 37).

65 DENV are single-stranded, positive-sense RNA viruses of the genus *Flavivirus* (family

66 *Flaviviridae*) with extensive genetic diversity (21). Each serotype can be divided into

67 large, genetically diverse phylogenetic clusters, which, in turn, consist of multiple,

68 distinct lineages (22). Hereafter, we use the terms clade and lineage interchangeably. In

69 the last two decades, in-depth phylogenetic analyses have significantly improved

70 understanding of DENV epidemiological and evolutionary dynamics (reviewed in refs.

71 35, 45).

72
One of the most striking features of DENV evolutionary dynamics is that viral lineages within serotypes arise and die out on a regular basis (22). This lineage turnover is detected in phylogenetic analyses in two different, non-mutually exclusive forms: (i) continuous, “ladder-like” temporal structure within clades and (ii) more dramatic, major clade replacement events. Within a clade, the majority of sub-lineages present at a specific time-point are not detected at later time-points, resulting in a ladder-like tree topology (8, 13, 26). Occasionally, an entire clade that persisted for a number of years at a given location goes extinct as an entirely new clade takes over. Such major clade replacement events have been recurrently documented at regional scales, for example DENV-3 in Sri Lanka in the late 1980s (30), DENV-4 in Puerto Rico during the 1980s-1990s (8), DENV-1 in Thailand in the mid-1990s (48), DENV-3 in Thailand in the early 1990s (47), DENV-1 in Myanmar in the late 1990s (31), DENV-2 in Vietnam in the early 2000s (43), and DENV-1 in Cambodia in the early 2000s (14). Major clade replacement events have also been reported at larger scales. For example, DENV-2 lineages from Southeast Asia displaced a native DENV-2 lineage in the Americas during the early 1990s (38). Although successive clades may be temporally non-overlapping (9), in most cases there is a transition period of co-circulation (14, 30, 31, 43, 47, 48).

Understanding the causes of DENV lineage birth-and-death has important implications for dengue epidemiology and control because it is often associated with changes in disease incidence and severity (16, 17, 30, 31, 38, 40). Elucidating the mechanisms underlying lineage turnover helps to retrospectively understand spatio-temporal patterns of dengue incidence and to make predictions about the risk of future epidemics (43). It
also has implications for vaccine design because DENV lineages may differ in their
antigenic properties (39, 44).

98

Despite their epidemiological significance, the evolutionary forces underlying major
clade replacement events are especially unclear. In particular, the extent to which clade
replacement events result from the random sampling of viral variants (genetic drift) due
to the stochastic nature of DENV transmission and/or from differences in fitness among
variants (i.e., adaptive evolution) is an unresolved question. Analyses of DENV gene
sequence data have shown that clade replacement events are sometimes associated with
signals of positive natural selection, such as an increase in the rate of nonsynonymous
substitutions (7), but not always (26, 47). Some phylogenetic studies have concluded that
major clade replacements were primarily due to stochastic events (31, 47), whereas others
found potentially causal differences in viral fitness between lineages, such as higher
viremia level in the human host (43) or enhanced infectivity to mosquito vectors (3, 5,
19).

111

Here, we addressed the question whether DENV evolution could be driven by adaptation
to mosquito vectors using a set of 23 low-passage DENV-1 isolates from Thailand
collected during the period 1985-2009. The 23 isolates were recovered from human
serum as part of the routine dengue diagnostic and surveillance services performed at the
Armed Forces Research Institute of Medical Sciences (AFRIMS) in Bangkok. We
compared the ability of the 23 isolates to be transmitted by *Aedes aegypti*, the principal
DENV vector species, in experimental *in vivo* vector competence assays. We
hypothesized that if viruses are positively selected for enhanced mosquito transmission, a relative increase in transmission potential should be observed for viruses collected over advancing years. All experiments were carried out during 2009-2010 using the first laboratory-reared generation of mosquitoes derived from a wild *Ae. aegypti* population collected in Kamphaeng Phet, Thailand. Although we cannot be sure that these mosquitoes exactly reproduce the vector-virus interactions that occurred at different dates in the past, they are the most relevant vector population of reference available to test our hypothesis. We estimated virus transmissibility with two distinct, complementary vector competence indices: the proportion of *Ae. aegypti* females that developed a DENV infection that disseminated from their midgut into their hemocoel, a requirement for virus transmission by a mosquito, and the infectious titer of disseminated virus (27).

130
131 MATERIALS AND METHODS

132

133 **Mosquitoes.** Wild *Ae. aegypti* immatures (larvae and pupae) were collected from a

134 variety of artificial containers in several households in Nhong Pling, Kon Tee, Nakorn

135 Choom, Na Bo Kham, and Thep Na Korn sub-districts, Muang district, Kamphaeng Phet

136 Province, Thailand during July 2009 (experiment 1) and March 2010 (experiment 2). In a

137 preliminary experiment, immatures were collected from containers in villages of

138 Ladkrabang district, Bangkok, and Muang district, Kamphaeng Phet Province, in January

139 2009. F₀ adults were allowed to emerge in the laboratory, mate randomly, and feed on

140 defibrinated sheep blood (National Laboratory Animal Center, Mahidol University,

141 Bangkok, Thailand) through a membrane feeding system. F₁ eggs, which were collected

142 and stored on dry pieces of paper towel and maintained under high humidity, were

143 hatched synchronously by placing them under low pressure for 30 min. Larvae were

144 reared in 24 x 34 x 9 cm plastic trays filled with 2.0 l of dechlorinated tap water at a

145 density of approximately 200 first instars per tray and fed a standard diet of

146 approximately 1.0 g of fish food pellets (C.P. Hi Pro®, Perfect Companion Group Co.

147 Ltd, Bangkok, Thailand) per tray. After emergence, F₁ adults were housed in plastic 30 x

148 30 x 30 cm cages (Megaview Science Education Service Co. Ltd, Taichung, Taiwan)

149 with permanent access to 10% sucrose. They were maintained under standard insectary

150 conditions at 28±1°C, 80% humidity, and with a 12:12 hour light:dark cycle.

151

152 **Virus isolates.** Viruses were originally isolated and archived as seed stocks from serum

153 samples collected during routine surveillance for diagnostic public health testing at
154 AFRIMS from clinically ill dengue patients attending Kamphaeng Phet Provincial Hospital in Kamphaeng Phet (experiment 1) and Queen Sirikit National Institute of Child Health in Bangkok (experiment 2). Virus isolation and identification was performed as previously described (25). Each isolate underwent 3-4 passages in cell culture (Table 1), which, according to standard procedures in our AFRIMS laboratory, is the minimum required to obtain a viral titer sufficiently high to infect mosquitoes orally using an artificial blood meal.

161

162 **Oral challenge.** Two sets of two-day-old confluent cultures of *Aedes albopictus* cells (C6/36, ATCC #CRL-1660TM) in 25 cm² flasks (approximately 10⁷ cells/flask) were inoculated with 1.0 ml of stock virus per flask and incubated at 35°C under 5% CO₂. A similar titer can be reached by growing virus in C6/36 cells at 35°C for 4-6 days instead of the usual 7-10 days at 28°C. In experiment 1, supernatant was harvested at 5 and 6 days post-inoculation to prepare the infectious blood meal of experimental blocks 1 and 2, respectively. In experiment 2, supernatant was harvested at 4 days post-inoculation to prepare the infectious blood meal of both experimental blocks. The protocol difference between experiments 1 and 2 was due to logistical constraints. The artificial blood meal consisted of 1:1 mix of defibrinated sheep blood (National Laboratory Animal Center, Mahidol University, Bangkok, Thailand) and virus suspension. Three- to seven-day-old *Ae. aegypti* F₁ females deprived of sucrose and water for 24 hours were offered an infectious blood meal for 30 min through pieces of desalted porcine intestine stretched over water-jacketed glass feeders maintained at 37°C. Samples of the blood meal were saved for subsequent titration by plaque assay. After blood feeding, mosquitoes were
177 briefly sedated with CO₂ from dry ice and fully engorged females were transferred to
178 clean paper cups. Unfed or partially fed females were discarded. Engorged females were
179 maintained under standard insectary conditions and provided cotton soaked with 10%
180 sucrose *ad libitum*.

181

182 **Vector competence.** The ability of DENV isolates to be transmitted by *Ae. aegypti* was
183 assessed at 7 and 14 days post blood meal (pbm) with two vector competence
184 phenotypes: (i) the proportion of mosquitoes that developed a disseminated infection and
185 (ii) the infectious titer of disseminated virus. These two complementary indices of vector
186 competence represent two successive, non-overlapping aspects of the infection process in
187 mosquitoes that lead to their ability to transmit virus, namely that virus disseminates
188 beyond the midgut and the magnitude of infectious titer in the hemocoel. As such, we
189 analyzed them separately. The two periods of extrinsic incubation (7 and 14 days pbm)
190 were chosen to represent early and late phases of dissemination kinetics (28).

191 Relationship between the infectious titer of disseminated virus and the potential for
192 transmission *in vitro* was established in a preliminary experiment (see below). Upon
193 harvest, the head of each female was cut off and placed individually in 1.0 ml of
194 mosquito diluent (MD), consisting of RPMI 1640 medium with 10% heat-inactivated
195 FBS with 100 units/ml penicillin and 100 µg/ml streptomycin. Bodies were kept
196 separately in 1.0 ml of MD. Samples were stored at -70°C before processing. Body and
197 head samples were quickly thawed in a water bath at 35±2°C and homogenized in a
198 mixer mill (Qiagen) at 24 cycles/sec for 2 min. Infected bodies were screened by
199 serotype-specific RT-PCR (experiment 1) or plaque assay (experiment 2). In experiment
200 1, total RNA was extracted from 140 µl of body homogenates using QIAamp viral RNA
201 mini kit (Qiagen) according to the manufacturer's instructions. RT-PCR was performed
202 with 5 µl of extracted RNA following a standard protocol (29) with the following
203 modifications: (i) 1X PCR buffer II supplied with Amplitaq DNA Polymerase (Applied
204 Biosystems) was used instead of the original buffer (50 mM KCl, 10 mM Tris pH 8.5 and
205 0.01 mM gelatin) in both the first round RT-PCR and the second round PCR (nested
206 PCR); (ii) the first round RT-PCR reaction contained Avian myeloblastosis virus reverse
207 transcriptase (Promega) instead of rav-2 recombinant reverse transcriptase; (iii) the 1:50
208 dilution of the first round RT-PCR product was used as the template in the nested PCR;
209 (iv) the nested PCR reaction contained 12.5 pmol of each primer instead of 50 pmol; and
210 (v) the number of the nested PCR cycles was increased from 20 cycles to 25 cycles (29).
211 Plaque assay was performed in rhesus monkey kidney cells (LLC-MK₂, ATCC #CCL-
212 7TM) as described previously (42). Briefly, the homogenized samples were passed
213 individually through a 0.22 µm syringe filter unit and 1:2, 1:10 and 1:100 dilutions were
214 prepared in MD. The samples were placed in an ice bath and 100 µl/well were inoculated
215 into a monolayer of LLC-MK₂ cells in 24-well plates. The virus was adsorbed for 1 hour
216 at room temperature (20-28°C) on a rocker platform. The inoculum was removed and 0.5
217 ml/well of a first overlay of medium was added. The cells were incubated for 5 days at
218 35±1°C in a 5±0.5% CO₂ incubator. The cells were stained with a second overlay of
219 medium containing 4% neutral red (Sigma). Plaques were counted and plaque forming
220 units (PFU)/ml were calculated. Head samples of infected bodies were titrated by plaque
221 assay in LLC-MK₂ cells. Mosquitoes whose bodies were negative by RT-PCR
222 (experiment 1) or plaque assay (experiment 2) were considered uninfected and their
223 heads were not processed further.

224

225 **In vitro transmission.** A preliminary experiment was carried out to define the
226 relationship between the titer of disseminated virus and the potential for *in vitro* virus
227 transmission. This experiment involved two F₁ populations of *Ae. aegypti* sampled during
228 January-February 2009 in Bangkok and Kamphaeng Phet, respectively. Both populations
229 were experimentally exposed to two 2009 DENV-1 isolates from Bangkok (ID#
230 00076/09 and 00088/09) and two 2009 DENV-1 isolates from Kamphaeng Phet (ID#
231 30015/09 and 30025/09). The relationship between the titer of disseminated virus and *in
232 vitro* transmission success was evaluated both at 8 and 14 days pbm. Mosquitoes were
233 anesthetized with triethylamine (Sigma-Aldrich), and their legs were removed
234 individually and placed into 1.0 ml of MD. Saliva samples were collected using a forced
235 salivation technique (2). Briefly, mosquito mouthparts were inserted for 15 min into a
236 glass microcapillary tube filled with approximately 10 µl of FBS. After 15 min, the
237 contents of the microcapillary tube were placed into 0.3 ml of MD and snap-frozen in dry
238 ice. The remainder of the mosquito bodies were placed individually into 1.0 ml of MD,
239 and all samples were stored at -70°C before processing. Body and leg samples were
240 quickly thawed in a water bath at 35±2°C and homogenized in a mixer mill. Infected
241 bodies were screened by serotype-specific RT-PCR as described above. The titer of
242 disseminated virus in the legs of mosquitoes whose body was positive by RT-PCR was
243 determined by plaque assay in LLC-MK₂ cells as described above. Mosquitoes whose
244 bodies were negative by RT-PCR were considered uninfected and their legs were not
245 processed further. The presence of virus in the saliva samples from mosquitoes with a disseminated infection was tested by plaque assay in LLC-MK₂ cells.

247

248 **Sequencing and phylogenetic analysis.** Viral RNA extracted by QIAamp viral RNA mini kit was converted to cDNA using Transcription High Fidelity kit (Roche) and random hexamer oligonucleotides according to the manufacturer's instructions. Sequencing of *E* genes and complete genomes was performed following previously described methods (48). The DNA fragments of the *E* gene and the overlapping DNA fragments covering the entire DENV-1 genome were amplified using Ampliq DNA Polymerase (Applied Biosystems) and purified using QIAquick PCR purification kit and QIAquick gel extraction kit (Qiagen). Purified DNA fragments were used as templates in cycle sequencing reactions using the DYEnamic ET dye terminator sequencing kit (GE Healthcare) according to the manufacturer's instructions. The sequencing primers were described previously (48). The sequencing products were purified by standard ethanol precipitation before sequencing in a MegaBACE 500 automated DNA sequencer (GE Healthcare). The overlapping sequences obtained from forward and reverse primers were combined for analysis and edited by using the Sequencher software (Gene Code Corporation). Phylogenetic trees were constructed with PAUP* version 4.0 (41) using a maximum likelihood (ML) method. ML trees included the new sequences generated in this study and 17 "background" DENV-1 sequences from GenBank. The best model of nucleotide substitution was chosen with jModelTest (33) based on the lowest akaike information criterion (AIC) value (34). The TIM2+Γ and GTR+Γ models of nucleotide substitution were applied to construct the trees for *E* gene and complete genome
sequences, respectively. Reliability of particular phylogenetic groupings was calculated using bootstrap resampling analysis with 1,000 replicate neighbor-joining (NJ) trees under the ML substitution models. All sequences have been submitted to GenBank and assigned accession numbers JN638322-JN638344.

272

**Data analysis.** All statistical analyses of vector competence data included the confounding effects of the experiment and the experimental block. The study was run in two separate experiments that involved two different sets of DENV isolates and used a population of *Ae. aegypti* that was sampled at two different times. In both experiments, the same batch of mosquitoes was exposed twice to the same set of DENV isolates on two successive days (i.e., two experimental blocks). In the two blocks, viruses came from the same passage in cell culture but were harvested separately (see above). The effect of the block was thus nested within each experiment and accounted for the difference in titer between the two harvests, the effect of the one-day difference in mosquito age, and the effect of any uncontrolled differences between the two days. The isolate effect was nested within each experiment and clade because each isolate belonged to only one phylogenetic clade and each experiment involved a different set of isolates. Blood meal titers were log-transformed (to ensure normal distribution of the residuals) and compared using a multifactorial analysis of variance (MANOVA) accounting for the effects of clade, experiment, block and their interactions. The proportion of mosquitoes with a disseminated infection was analyzed with a nominal logistic regression that included the effects of blood meal titer, experiment, experimental block, clade, isolate and their interactions. The titer of disseminated virus was log-transformed (to ensure normal
291 distribution of the residuals) and analyzed with a MANOVA that included the effects of

292 blood meal titer, experiment, experimental block, clade, isolate and their interactions.

293 Differences were considered statistically significant at *P*<0.05. Analyses were performed

294 with the software JMP version 5.1.2.

295
296 **RESULTS**

297

298 **Estimation of transmission potential.** Transmission potential was defined as the
299 presence of infectious virus in the mosquito's saliva. In a preliminary experiment, a total
300 of 322 individual *Ae. aegypti* females that developed a disseminated infection following
301 oral challenge were tested for virus titer in their legs and virus presence in their saliva
302 collected *in vitro*. The data included two F₁ *Ae. aegypti* populations (sampled in Bangkok
303 and Kamphaeng Phet, respectively), four DENV-1 isolates (two from Bangkok and two
304 from Kamphaeng Phet), and two extrinsic incubation periods (8 and 14 days pbm). Leg
305 titers ranged from 1 x 10¹ to 2.3 x 10⁴ plaque forming units per ml (PFU/ml) and
306 infectious virus was detected in 7.5% of saliva samples. The probability of detecting
307 infectious virus in saliva was positively correlated with the infectious titer of
308 disseminated virus (Fig. 1). Logistic regression showed that the leg titer was a highly
309 significant predictor of virus detection in saliva (df=1; L-R χ²=15.48; P<0.0001). In
310 particular, no virus was detected in any of the saliva samples from mosquitoes with a leg
311 titer < 1 x 10³ PFU/ml. For the rest of the study, the infectious titer of disseminated virus
312 was used as an estimate of transmission potential. Because the number of mosquito legs
313 may vary among individuals (they can lose them over time), the head titer was used
314 instead of the leg titer. Previous data showed that the leg titer and the head titer are
315 strongly correlated in individual *Ae. aegypti* (27).

316

317 **Phylogenetic relationships between DENV isolates.** All but two of the 23 DENV-1
318 isolates were passaged four times in cell culture (Table 1) prior to their use in
experimental mosquito infections; two were passaged three times. The study was run in two separate experiments involving two different sets of virus isolates (11 in the first experiment and 12 in the second) and *Ae. aegypti* mosquitoes sampled at two different times. Phylogenetic analysis based on the complete DENV *E* gene sequence indicated that the virus isolates could be divided into two major phylogenetic clades within the genotype I of DENV-1 (Fig. 2A). The first clade included 8 isolates sampled between 1985 and 1995; the second clade included 15 isolates sampled between 1992 and 2009.
Hereafter, the first and second clades are termed “early clade” and “new clade”, respectively, in reference to the chronological order of their detection. Complete genome sequences obtained for a selection of 9 isolates sampled between 1986 and 1997 (6 from the early clade and 3 from the new clade) confirmed the phylogenetic relationships inferred using the *E* gene sequence (Fig. 2B).

331

**Variation in transmission potential among DENV isolates.** Vector competence assays included a total of 738 individual *Ae. aegypti* females. Transmission potential was characterized based on 7-44 (mean 32) individuals per isolate. Infectious titer of artificial blood meals used in vector competence assays varied among isolates (Fig. 3), ranging from 3.5 x 10⁴ PFU/ml to 3.0 x 10⁶ PFU/ml. Blood meal titers were significantly higher in the second experiment (SS=3.251; df=1; *F*=19.63; *P*<0.0001), but they were not significantly different between blocks within experiments (SS=0.574; df=2; *F*=1.734; *P*=0.191). The higher blood meal titers in experiment 2 may have resulted from the different harvest date post-inoculation in cell culture (day 4) compared to experiment 1 (day 5-6). Importantly, blood meal titers did not differ between clades (SS=0.439; df=1;
342 F=2.649; P=0.112) (Fig. 2). In the early clade, blood meal titers ranged from 4.5 x 10⁴ PFU/ml to 3.0 x 10⁶ PFU/ml (mean 7.0 x 10⁵ PFU/ml). In the new clade, blood meal titers ranged from 3.5 x 10⁴ PFU/ml and 2.4 x 10⁶ PFU/ml (mean 8.2 x 10⁵ PFU/ml).
343
344
345
346 Overall, about two thirds of mosquitoes were infected (65.6% at 7 days pbm and 69.6% at 14 days pbm). Nearly all infected females developed disseminated infections within the first two weeks pbm (52.9% at 7 days pbm and 94.6% at 14 days pbm). The percentage of females with a disseminated infection (including uninfected individuals) varied significantly among isolates at both 7 and 14 days pbm (Table 2). It ranged from 0 to 81% at 7 days pbm (mean 32%) and from 20 to 100% (mean 64%) at 14 days pbm. The percentage of females with a disseminated infection was not significantly influenced by the blood meal titer, but it was significantly higher in the first than in the second experiment at 14 days pbm (Table 2). The higher level of dissemination in the second experiment may have been due to either the different geographical origin of viruses (Bangkok vs. Kamphaeng Phet) or any uncontrolled, environmental difference between the two experiments. There was no significant difference between the early and new clade in the mean percentage of females with a disseminated infection at either 7 or 14 days pbm (Table 2). Dissemination tended to increase as a function of the year of virus isolation in the early clade whereas it tended to decrease as a function of the year of virus isolation in the new clade, both at 7 days pbm (Fig. 4A) and 14 days pbm (Fig. 4B).
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362 These trends may have been a consequence of variation in blood meal titers, which showed similar patterns (Fig. 3).
363
364
Among females with a disseminated infection, head titers ranged from 1.0 x 10¹ to 2.3 x 10⁴ PFU/ml at 7 days pbm (N=128, mean 5.4 x 10² PFU/ml, median 3.0 x 10¹ PFU/ml), and from 1.0 x 10¹ to 2.9 x 10⁴ PFU/ml at 14 days pbm (N=243, mean 3.4 x 10³ PFU/ml, median 1.5 x 10³ PFU/ml). The titer of disseminated virus was not significantly influenced by the blood meal titer; it differed significantly among isolates at 14 days pbm (Table 3). The mean head titer of the new clade was significantly higher than that of the early clade at 14 days pbm (Table 3; Fig. 5). At 7 days pbm, the new clade had a higher mean head titer than the early clade, but the difference was marginally insignificant (Table 3; Fig. 5). The statistical power at 7 days pbm, however, was reduced compared to 14 days pbm because the number of mosquitoes with a disseminated infection was half as large. At 7 days pbm the head titer tended to increase slightly as a function of the year of virus isolation in the new clade, whereas it did not vary temporally in the early clade (Fig. 4C). At 14 days pbm, the head titer tended to increase as a function of the year of virus isolation in the early clade, whereas it did not vary temporally in the new clade (Fig. 4D).

379

380

381
382 DISCUSSION

383

384 By comparing the ability of 23 DENV-1 isolates from Thailand spanning a 24-year

385 period to infect and be transmitted by *Ae. aegypti*, we found that a major clade

386 replacement event in the mid-1990s was associated with a higher transmission potential

387 of the isolates belonging to the new clade. Higher transmissibility was mainly due to a

388 higher infectious titer of virus in the vector's hemocoel, which is predicted to result in a

389 higher probability of transmission. This finding supports the hypothesis that major clade

390 replacement events can be driven by natural selection and emphasizes the potentially

391 important role of vector-virus interactions in DENV evolution.

392

393 The major clade replacement event revealed by the phylogenetic analysis of the 23

394 isolates of our study was described previously (48). In their study, Zhang *et al.* (48)

395 observed that the DENV-1 clade replacement was associated with a decline in DENV-1

396 prevalence and a concomitant rise of DENV-4 in Thailand. They speculated that clade

397 replacement events might result from differential susceptibility to cross-reactive immune

398 responses in the human host. Here, we provide a non-mutually exclusive, alternative

399 explanation. Our data suggest that this clade replacement event may have been driven, at

400 least in part, by adaptation to mosquito vectors. Even though it occurred in a context of

401 declining DENV-1 incidence, the new clade may have outcompeted the early clade

402 through enhanced mosquito transmission. Isolates in the new clade had a higher

403 dissemination titer, which is expected to result in higher viral fitness because the
404 probability of transmission by a mosquito is positively correlated with the quantity of
405 virus that circulates in the insect's hemocoel.

406

407 Our estimate of DENV transmission potential relied on an experiment demonstrating a
408 positive correlation between the titer of disseminated virus and the probability of virus
detection in the mosquito's saliva (Fig. 1). Virus presence in the saliva was determined
409 using an established *in vitro* method (2) that likely underestimates transmission
410 probability because it may fail to detect small, but transmissible amounts of virus. Indeed,
411 infectious virus was only detected in the saliva of 7.5% of females with a disseminated
412 infection. Our analysis showed, however, that the probability to detect infectious virus in
413 saliva samples was positively correlated with the infectious titer of disseminated virus,
414 across two different *Ae. aegypti* populations and four different DENV-1 isolates. In
415 particular, there was a lower threshold titer below which none of the mosquitoes with a
416 disseminated infection had a positive saliva sample. Although the actual threshold may be
417 lower than we estimate due to the relatively low sensitivity of the method, our data
418 established that the titer of disseminated virus is a strong predictor of transmission
419 probability in this system.
420

421

422 It is worth noting that we did not consider other entomological parameters underlying
423 DENV transmission such as vector survival or biting rate, which could potentially vary
424 among viruses. Future studies will determine whether variation of these traits contribute
425 to fitness differences among viruses. Another limitation of the study was that adaptation
426 to cell culture could have occurred during the 3-4 passages of virus amplification prior to
427 their use in experimental infections of mosquitoes. The selection regime, however, was
428 the same for all isolates, thus comparisons among vector competence phenotypes
429 remained meaningful. The mechanisms underlying the phenotypic differences we
430 observed between the two clades remain unknown. Authors of a recent study suggested
431 that variation in vector competence among DENV-2 lineages does not depend on
432 mosquito midgut binding affinity, but rather on replication ability (12).

433

434 Investigators who preceded us and studied DENV-2 and DENV-3 indicated that vector-
435 driven selection could play an important role in DENV evolution. By comparing the
436 ability of three DENV-3 isolates of a native lineage and three DENV-3 isolates of an
437 invasive lineage to be transmitted by *Ae. aegypti*, Hanley *et al*. (19) showed that a clade
438 replacement event in Sri Lanka during the 1980s was associated with enhanced mosquito
439 transmission potential for the invasive lineage. Enhanced transmissibility was associated
440 with a higher body titer and an increased proportion of *Ae. aegypti* females with a
441 disseminated infection (19). At a larger geographic scale, Rico-Hesse and others
442 suggested in a series of studies that displacement of a native DENV-2 lineage in the
443 Americas by Southeast Asian DENV-2 lineages during the early 1990s was driven in part
444 by adaptation to mosquitoes (3-5, 11). In both the DENV-3 and DENV-2 cases, invasive
445 lineages were associated with more severe disease in humans, which highlights the
446 critical need to more fully explore the connection between DENV evolutionary biology
447 and dengue epidemiology.

448
Our study helps to understand the respective contributions of stochastic and adaptive processes in DENV evolution. Frequent introductions of new lineages (gene flow) are known to maintain substantial DENV genetic diversity even at a very local scale (23). In contrast, elimination of deleterious mutations (purifying selection) strongly limits *in situ* evolution of lineages (23). Purifying selection is likely the dominant evolutionary force in DENV evolution (20), and nucleotide fixation events are due primarily to genetic drift (13, 20, 26). Although stochastic processes are undoubtedly important in DENV evolution, the present study indicates that more dramatic major clade replacement events may have an adaptive basis (43). That extinction of the early DENV-1 clade was concomitant with rise of the new clade, with an overlapping period in the early 1990s (48), was suggestive of a fitness difference between the two clades. The relatively modest average fitness difference between the two clades (a 0.5-log difference in head titer) may explain why they co-circulated for at least four years before the early clade went extinct. It is worth noting that we also observed significant variation in vector competence indices within each clade (Fig. 4), indicating that other evolutionary forces than vector-driven selection promote DENV genetic diversity. In particular, the temporal pattern within each clade did not provide a clear indication that adaptation to mosquitoes occurs continuously through time (Fig. 4). We speculate that the temporal trends observed within clades resulted from uncontrolled variation in experimental blood meal titers (Fig. 3), but this merits further investigation.

Although other selective forces may be important drivers of DENV evolution, especially those occurring in the human host that we did not examine, we provide evidence for the
472 possible role of adaptation to mosquito vectors in a major DENV-1 clade replacement

473 event in Thailand in the early 1990s. Our results reinforce the idea that vector-virus

474 interactions can play a critical role in DENV epidemiological and evolutionary dynamics,

475 and suggest that the role of vector-mediated selection be investigated in other instances of

476 clade replacement.

477
478 ACKNOWLEDGEMENTS

479

480 We acknowledge the invaluable contributions of the clinical, laboratory, and

481 entomological personnel of AFRIMS and Kamphaeng Phet AFRIMS Virology Research

482 Unit (KAVRU). This study was primarily supported by grant R01 GM-083224 from the

483 National Institutes of Health. L.L. was supported by grant ANR-09-RPDOC-007-01 from

484 the French Agence Nationale de la Recherche. T.W.S. received support from the

485 Research and Policy for Infectious Disease Dynamics program of the Science and

486 Technology Directorate, Department of Homeland Security, and Fogarty International

487 Center, National Institutes of Health. We are grateful to Robert Gibbons, Timothy Endy

488 and Brian Evans for their continuous support. We thank Eddie Holmes, Robert Gibbons

489 and four anonymous reviewers for helpful comments on a previous version of the

490 manuscript. The opinions or assertions contained herein are the private views of the

491 authors and are not to be construed as reflecting the official views of the United States

492 Army, Royal Thai Army, or the United States Department of Defense.

493

494 L.L. conceived and coordinated the study, designed the research, analyzed the data and

495 wrote the manuscript. T.F. and A.P. performed the field collection of mosquitoes and

496 vector competence assays. B.T. supervised the isolation, amplification and titration of

497 viruses. C.K. supervised the molecular detection and sequencing of viruses, and

498 performed the phylogenetic analyses. J.H.R. and A.P. coordinated the field collection of

499 mosquitoes and vector competence assays. R.G.J. coordinated the collection of virus

500 isolates, virological assays, and phylogenetic analyses. T.W.S. conceived and coordinated
501 the study, and helped to design the research and write the manuscript. All authors read

502 and approved the final manuscript.

503
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<table><caption>**Table 1.** DENV-1 isolates used in experimental mosquito infections. For each isolate, the year of virus isolation, location of origin, number of passages in cell culture, and phylogenetic group (clade) are indicated. KPPH: Kamphaeng Phet Provincial Hospital; QSNICH: Queen Sirikit National Institute for Child Health (Bangkok); TS: *Toxorhynchites splendens* mosquitoes; C6/36: *Aedes albopictus* cells.</caption><thead><tr><th>Experiment</th><th>Isolation year</th><th>Isolate ID</th><th>Source</th><th>Passage History</th><th>Clade</th></tr></thead><tbody><tr><td rowspan="10">1</td><td>1986</td><td>KD86-035</td><td>KPPH</td><td>TS-1, C6/36-3</td><td>Early</td></tr><tr><td>1990</td><td>KD90-157</td><td>KPPH</td><td>TS-1, C6/36-2</td><td>Early</td></tr><tr><td>1992</td><td>KD92-080</td><td>KPPH</td><td>TS-1, C6/36-2</td><td>Early</td></tr><tr><td>1995</td><td>30399/95</td><td>KPPH</td><td>C6/36-4</td><td>New</td></tr><tr><td>1997</td><td>30231/97</td><td>KPPH</td><td>C6/36-4</td><td>New</td></tr><tr><td>2000</td><td>30529/00</td><td>KPPH</td><td>C6/36-4</td><td>New</td></tr><tr><td>2003</td><td>30247/03</td><td>KPPH</td><td>C6/36-4</td><td>New</td></tr><tr><td>2005</td><td>30230/05</td><td>KPPH</td><td>C6/36-4</td><td>New</td></tr><tr><td>2006</td><td>30118/06</td><td>KPPH</td><td>C6/36-4</td><td>New</td></tr><tr><td>2009</td><td>30015/09</td><td>KPPH</td><td>C6/36-4</td><td>New</td></tr><tr><td>2009</td><td>30025/09</td><td>KPPH</td><td>C6/36-4</td><td>New</td></tr><tr><td rowspan="5">2</td><td>1985</td><td>D85-372</td><td>QSNICH</td><td>TS-1, C6/36-3</td><td>Early</td></tr><tr><td>1986</td><td>D86-412</td><td>QSNICH</td><td>TS-1, C6/36-3</td><td>Early</td></tr><tr><td>1987</td><td>D87-116</td><td>QSNICH</td><td>TS-1, C6/36-3</td><td>Early</td></tr><tr><td>1990</td><td>D90-1197</td><td>QSNICH</td><td>TS-1, C6/36-3</td><td>Early</td></tr><tr><td>1992</td><td>03881/92</td><td>QSNICH</td><td>TS-1, C6/36-3</td><td>New</td></tr></tbody></table>
<table><tr><td></td><td>1995</td><td>00407/95</td><td>QSNICH</td><td>TS-1, C6/36-3</td><td>Early</td></tr><tr><td></td><td>1997</td><td>00616/97</td><td>QSNICH</td><td>TS-1, C6/36-3</td><td>New</td></tr><tr><td></td><td>2000</td><td>04805/00</td><td>QSNICH</td><td>TS-1, C6/36-3</td><td>New</td></tr><tr><td></td><td>2003</td><td>01417/03</td><td>QSNICH</td><td>TS-1, C6/36-3</td><td>New</td></tr><tr><td></td><td>2005</td><td>00442/05</td><td>QSNICH</td><td>TS-1, C6/36-3</td><td>New</td></tr><tr><td></td><td>2007</td><td>02128/07</td><td>QSNICH</td><td>C6/36-4</td><td>New</td></tr><tr><td></td><td>2009</td><td>00132/09</td><td>QSNICH</td><td>C6/36-4</td><td>New</td></tr></table>

671

672
Table 2. Logistic regression analysis of dissemination prevalence 7 and 14 days post-blood meal among mosquitoes experimentally exposed to different DENV-1 isolates.

df: degrees of freedom; L-R: likelihood ratio. \*P\*<0.05; \*\*P\*<0.01.

<table><thead><tr><th rowspan="2">Source</th><th rowspan="2">df</th><th colspan="2">Dissemination 7<br>days pbm</th><th colspan="2">Dissemination 14<br>days pbm</th></tr><tr><th>L-R χ²</th><th>P-value</th><th>L-R χ²</th><th>P-value</th></tr></thead><tbody><tr><td>Blood meal titer</td><td>1</td><td>1.451</td><td>0.228</td><td>0.749</td><td>0.387</td></tr><tr><td>Experiment</td><td>1</td><td>1.058</td><td>0.304</td><td>11.45</td><td>0.001**</td></tr><tr><td>Clade</td><td>1</td><td>0.877</td><td>0.349</td><td>3.619</td><td>0.057</td></tr><tr><td>Experiment*Clade</td><td>1</td><td>0.443</td><td>0.506</td><td>0.461</td><td>0.497</td></tr><tr><td>Block [within Experiment]</td><td>2</td><td>1.604</td><td>0.448</td><td>11.34</td><td>0.003**</td></tr><tr><td>Block*Clade [within Experiment]</td><td>2</td><td>0.080</td><td>0.961</td><td>4.236</td><td>0.120</td></tr><tr><td>Isolate [within Experiment,<br>Clade]</td><td>19</td><td>34.82</td><td>0.015*</td><td>46.21</td><td>0.001**</td></tr></tbody></table>
Table 3. Analysis of variance of head titers at 7 and 14 days post-blood meal among mosquitoes experimentally exposed to different DENV-1 isolates. Titers were log-transformed to satisfy statistical assumptions. The analysis is performed on individual females with a disseminated infection (i.e., excluding those that did not develop a disseminated infection). df: degrees of freedom; SS: sum of squares. \*P\*<0.05; \*\*P\*<0.01.

<table><thead><tr><th rowspan="2">Source</th><th colspan="4">Head titer 7 days pbm</th><th colspan="4">Head titer 14 days pbm</th></tr><tr><th>df</th><th>SS</th><th>F ratio</th><th>P-value</th><th>df</th><th>SS</th><th>F ratio</th><th>P-value</th></tr></thead><tbody><tr><td>Blood meal titer</td><td>1</td><td>2.005</td><td>4.086</td><td>0.046*</td><td>1</td><td>0.027</td><td>0.048</td><td>0.828</td></tr><tr><td>Experiment</td><td>1</td><td>2.651</td><td>5.403</td><td>0.022*</td><td>1</td><td>0.353</td><td>0.633</td><td>0.427</td></tr><tr><td>Clade</td><td>1</td><td>1.898</td><td>3.869</td><td>0.052</td><td>1</td><td>5.494</td><td>9.838</td><td>0.002**</td></tr><tr><td>Experiment*Clade</td><td>1</td><td>0.235</td><td>0.479</td><td>0.491</td><td>1</td><td>0.007</td><td>0.013</td><td>0.908</td></tr><tr><td>Block [within Experiment]</td><td>2</td><td>0.787</td><td>0.802</td><td>0.451</td><td>2</td><td>0.483</td><td>0.433</td><td>0.649</td></tr><tr><td>Block*Clade [within Experiment]</td><td>2</td><td>3.215</td><td>3.276</td><td>0.042*</td><td>2</td><td>3.153</td><td>2.823</td><td>0.062</td></tr><tr><td>Isolate [within Experiment, Clade]</td><td>19</td><td>15.60</td><td>1.673</td><td>0.054</td><td>19</td><td>26.76</td><td>2.522</td><td>0.001**</td></tr><tr><td>Error</td><td>100</td><td>49.06</td><td></td><td></td><td>215</td><td>120.1</td><td></td><td></td></tr></tbody></table>
687 FIGURE LEGENDS

688

689 **Figure 1.** Mosquito transmission potential strongly correlates with infectious titer of disseminated DENV-1. The percentage of female *Ae. aegypti* tested positive for infectious virus in saliva samples collected *in vitro* is shown as a function of infectious virus titers in their legs at either 8 or 14 days post-blood meal. Bars represent individuals with a leg liter > the lower value of the corresponding interval on the x-axis. Data shown is pooled from vector competence assays involving two mosquito populations exposed to four different DENV-1 isolates (see Materials and Methods for details). Dotted, vertical lines indicate the 95% confidence intervals of percentages. Sample sizes are indicated above the bars.

698

699 **Figure 2.** Phylogenetic relationships among DENV-1 isolates. Maximum likelihood trees based on *E* gene (A) or complete genome (B) sequences. Sequences from GenBank are in black font and sequences generated in this study are in color. Blue and red fonts indicate isolates used in experiment 1 and 2, respectively. All horizontal branch lengths are drawn to scale, with bootstrap support values shown next to relevant nodes.

704

705 **Figure 3.** Variation of artificial DENV-1 blood meal titers in vector competence assays. Infectious titers of the artificial blood meals measured by plaque assay are shown for each isolate as a function of their date of isolation and phylogenetic group. Each isolate was tested twice in two experimental blocks (for details see Materials and Methods). Lines represents linear *y*=*f*(*x*) regressions to give a graphical indication of
temporal trends. Lower lines correspond to experimental block 1; upper lines correspond to experimental block 2.

712

**Figure 4. Variation in vector competence indices among DENV-1 isolates.** In the upper panels, the percentage of females with a disseminated infection is indicated at 7 days post-blood meal (pbm) (A) and 14 days pbm (B) as a function of the year of virus isolation and phylogenetic group (clade). In the lower panels, mean infectious titer in heads of females with a disseminated infection is indicated for each isolate at 7 days pbm (C) and 14 days pbm (D) as a function of virus isolation year and phylogenetic group. Each isolate is represented by the mean of two experimental blocks (for details see Materials and Methods). Lines represents linear y=f(x) regressions to give a graphical indication of temporal trends.

722

**Figure 5. DENV-1 isolates in the new clade disseminate at higher titers in orally challenged Ae. aegypti.** Bars show the mean head titer of isolates belonging to the early and new clades at 7 and 14 days post-blood meal. Error bars indicate the standard errors of the means. Statistical significance of difference between clades is given by *P*-values derived from a complete multifactorial analysis of variance accounting for the effects of infectious dose (blood meal titer), experiment, block, clade and isolates; the analysis only includes mosquitoes with a disseminated infection.
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A

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B

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Blood meal titer (log PFU/ml)
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