Accepted Manuscript

Title: The emergence of Schmallenberg virus acrossCulicoides communities and ecosystems in Europe

Author: Thomas Balenghien Nonito Pages Maria GoffredoSimon Carpenter Denis Augot Elisabeth Jacquier SandraTalavera Federica Monaco Jerome Depaquit Colette GrilletJoan Pujols Giuseppe Satta Mohamed Kasbari Marie-LaureSetier-Rio Francesca Izzo Cigdem Alkan Jean-ClaudeDelecolle Michela Quaglia Remi Charrel Andrea PolciEmmanuel Breard Valentina Federici Catherine Cetre-SossahClaire Garros

PII: S0167-5877(14)00102-0DOI: http://dx.doi.org/doi:10.1016/j.prevetmed.2014.03.007Reference: PREVET 3539

To appear in: PREVET

Received date: 23-9-2013Revised date: 24-1-2014Accepted date: 8-3-2014

Please cite this article as: Thomas BalenghienNonito PagesMaria GoffredoSimonCarpenterDenis AugotElisabeth JacquierSandra TalaveraFederica MonacoJeromeDepaquitColette GrilletJoan PujolsGiuseppe SattaMohamed KasbariMarie-LaureSetier-RioFrancesca IzzoCigdem AlkanJean-Claude DelecolleMichela QuagliaRemiCharrelAndrea PolciEmmanuel BreardValentina FedericiCatherine Cetre-SossahClaireGarros The emergence of Schmallenberg virus across Culicoides communities andecosystems in Europe (2014), http://dx.doi.org/10.1016/j.prevetmed.2014.03.007

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The emergence of Schmallenberg virus across Culicoides communities and 1

ecosystems in Europe2

Thomas Balenghien1, Nonito Pagès2, Maria Goffredo3, Simon Carpenter4, Denis Augot5, 3

Elisabeth Jacquier1, Sandra Talavera2, Federica Monaco3, Jérôme Depaquit5, Colette Grillet1, 4

Joan Pujols2, Giuseppe Satta6, Mohamed Kasbari5, Marie-Laure Setier-Rio7, Francesca Izzo6, 5

Cigdem Alkan8,9, Jean-Claude Delécolle10, Michela Quaglia6, Rémi Charrel8,9, Andrea Polci6, 6

Emmanuel Bréard11, Valentina Federici6, Catherine Cêtre-Sossah1, Claire Garros17

1 Cirad, UMR15 CMAEE, F-34398 Montpellier, France8

1 INRA, UMR1309 CMAEE, F-34398 Montpellier, France9

2 Centre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, Campus de la Universitat Autònoma de 10

Barcelona, 08193 Bellaterra (Cerdanyola del Vallès), Spain 11

3 Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise, Campo Boario, 64100 Teramo, Italy12

4 The Pirbright Institute, Ash Road, Woking, Surrey, GU24 0NF, United Kingdom13

5 Université de Reims Champagne-Ardenne, ANSES, SFR Cap Santé, EA4688 - USC « VECPAR », Reims, 14

France15

6 Istituto Zooprofilattico della Sardegna, Via Duca degli Abruzzi 8, Sardinia, Italy16

7 EID Méditerranée, 34184 Montpellier, France17

8 UMR_D 190 "Emergence des Pathologies Virales", Aix Marseille Univ, IRD French Institute of Research for 18

Development, EHESP French School of Public Health, 13005, Marseille, France19

9 IHU Mediterranee Infection, APHM Public Hospitals of Marseille, 13005 Marseille, France20

10 IPPTS, 67000 Strasbourg, France21

11 ANSES, UMR virologie, Maisons-Alfort, France22

23

Corresponding author:24

Thomas Balenghien25

Cirad TA A-15/G Campus international de Baillarguet 34 398 Montpellier Cedex 526

thomas.balenghien@cirad.fr; Phone: + 33 4 67 59 37 53 ; Fax: + 33 4 67 59 37 9527

28

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Abstract28

Schmallenberg virus (SBV), a novel arboviral pathogen, has emerged and spread across 29

Europe since 2011 inflicting congenital deformities in the offspring of infected adult 30

ruminants. Several species of Culicoides biting midges (Diptera: Ceratopogonidae) have been 31

implicated in the transmission of SBV through studies conducted in northern Europe. In this 32

study Culicoides from SBV outbreak areas of mainland France and Italy (Sardinia) were 33

screened for viral RNA. The role of both C. obsoletus and the Obsoletus complex34

(C. obsoletus and C. scoticus) in transmission of SBV were confirmed in France and SBV 35

was also discovered in a pool of C. nubeculosus for the first time, implicating this species as a 36

potential vector. While collections in Sardinia were dominated by C. imicola, only relatively 37

small quantities of SBV RNA were detected in pools of this species and conclusive evidence 38

of its potential role in transmission is required. 39

In addition to these field-based studies, infection rates in colony-derived individuals of 40

C. nubeculosus and field-collected C. scoticus are also examined in the laboratory. Rates of 41

infection in C. nubeculosus were low, confirming previous studies, while preliminary 42

examination of C. scoticus demonstrated that while this species can replicate SBV to a 43

potentially transmissible level, further work is required to fully define comparative44

competence between species in the region. Finally, the oral competence for SBV of two 45

abundant and widespread mosquito vector species in the laboratory is assessed. Neither Aedes 46

albopictus nor Culex pipiens were demonstrated to replicate SBV to transmissible levels and 47

appear unlikely to play a major role in transmission. Other vector competence data produced 48

from studies across Europe to date is then comprehensively reviewed and compared with that 49

generated previously for bluetongue virus. 50

Keywords51

Arbovirus; vector competence; Ceratopogonidae; Orthobunyavirus; Bunyaviridae52

53

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Introduction53

54

Schmallenberg virus (SBV) is a member of the Orthobunyavirus genus first identified in 55

North Rhine-Westphalia, Germany, during summer 2011 (Hoffmann et al., 2012). Infection 56

with SBV can lead to mild clinical signs in adult ruminants, including pyrexia, decreased milk 57

production and diarrhea (Davies et al., 2012). The primary economic impact of SBV, 58

however, lies in severe congenital malformations following transplacental transmission in 59

cattle and sheep (van den Brom et al., 2012). Initial phylogenetic studies placed SBV in the 60

Simbu serogroup, sharing a close relationship to Sathuperi and Douglas viruses and 61

secondarily to Shamonda virus and included in the same lineage than the Akabane virus 62

(Saeed et al., 2001; Goller et al., 2012). These viruses have been primarily isolated from 63

Culicoides (Doherty et al., 1972; St George et al., 1978; Lee, 1979; Cybinski, 1984; 64

Blackburn and Searle, 1985; Kurogi et al., 1987; Yanase et al., 2005) and more rarely from 65

mosquitoes (Dandawate et al., 1969; Metselaar and Robin, 1976). The recent emergence of 66

the similarly Culicoides-borne bluetongue virus (BTV) in western and northern Europe67

(Carpenter et al., 2009), therefore led to immediate suspicion that SBV was transmitted by 68

female midges from the Culicoides genus.69

70

Following detection of the SBV incursion, virus RNA was rapidly identified in field-collected 71

Culicoides from farms in the affected regions (De Regge et al., 2012; Rasmussen et al., 2012; 72

Elbers et al., 2013a; Elbers et al., 2013b; Goffredo et al., 2013; Larska et al., 2013). Taken in 73

their entirety, these studies convincingly implicated a range of widespread and abundant 74

farm-associated Culicoides species in the transmission of SBV including Culicoides75

obsoletus, Culicoides scoticus, Culicoides dewulfi and Culicoides chiopterus. A detailed study 76

of SBV replication and dissemination in the model species Culicoides sonorensis also allowed 77

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confirmation that levels of viral RNA in studies carried out in the Netherlands (Elbers et al., 78

2013b) were likely to represent transmissible infections (Veronesi et al., 2013).79

80

From 2011 to 2013, SBV has spread across a huge geographic area in Europe at a rate 81

substantially exceeding that of the BTV-8 epidemic which occurred in the same region from 82

2006 to 2010. Current distribution of SBV ranges from the Mediterranean to Scandinavian 83

regions, and from Ireland to Estonia (European Food Safety Authority, 2013). The rate of 84

seroconversion recorded in many farms was rapid (Elbers et al., 2012; Meroc et al., 2013)85

with a high proportion of ruminants apparently becoming infected in a short time frame. A 86

partial explanation for this phenomenon could be the absence of animal movement restrictions 87

that were employed for BTV but not for SBV in the vast majority of countries reporting 88

incursions. Additionally, however, it is suspected that the vector competence of Culicoides for 89

SBV may exceed rates recorded for BTV either in the number of species capable of 90

transmitting the virus or in the proportion of individuals within a species able to act as 91

vectors. This hypothesis receives support from the fact that the related Akabane virus is 92

isolated at a far higher frequency than BTV from Culicoides in Australia (St George et al., 93

1978), although comparative laboratory-based investigations of susceptibility rates in vector 94

species have not been performed. 95

96

In this study we present the first data on detection of SBV RNA in Culicoides from mainland 97

France and Italy (Sardinia), examine infection rates in colony-derived individuals of 98

Culicoides nubeculosus (Meigen) and additionally preliminary results for experimental 99

infections of field-collected individuals. A significant advantage in carrying out such 100

detections across a wide geographic range is that it enables an understanding of SBV 101

transmission across disparate ecosystems and potential vector species. This includes the 102

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examination of the role of Culicoides imicola, a major afrotropical vector of BTV with a 103

distribution that could facilitate the spread of SBV into new areas, including Asia. We also 104

examine two abundant and widespread mosquito vector species in the laboratory to assess 105

whether alternative vectors could be involved in transmission of SBV across Europe. Finally, 106

we also review vector competence data produced from studies across Europe to date 107

comprehensively and compare with that generated previously for BTV. By contrasting the 108

diverse ecosystems and potential vectors present across these countries we subsequently draw 109

conclusions regarding the vulnerability of Europe to further incursions.110

111

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Material and methods111

112

Laboratory infection of mosquitoes and Culicoides113

114

Experimental infection with SBV was carried out at the Centre de Recerca en Sanitat Animal 115

(CReSA) using lines of Culex pipiens Linnaeus and Aedes albopictus Skuse that had been 116

maintained for two and four years, respectively. In addition, a line of C. nubeculosus was also 117

infected at CReSA which had originated from that originally established at The Pirbright 118

Institute. All studies using both intrathoracic inoculation and artificial membrane-based 119

blood-feeding were conducted at the CReSA Biosafety level 3 facilities.120

121

For intrathoracic (IT) inoculation, females of each species (of ages 2 to 4 days) were122

anesthetised using carbon dioxide and placed under a stereomicroscope. A volume of 0.2 to 123

0.4µl (Culicoides) and 1 to 2 µl (mosquitoes) of a 4.38 TCID50/ml SBV viral suspension, 124

produced on a baby hamster kidney (BHK-21) cell line was inoculated intrathoracically into 125

each individual using a glass micro-needle. Insertion of the needle was made between the 126

epimeron and episterum using a manual micro-injector (Sutter instruments, California, USA).127

Inoculated mosquitoes and midges were placed in groups of 15 to 20 individuals inside 128

cardboard and plastic primary containers for Culicoides and mosquitoes respectively. Females 129

were fed with 5% sucrose ad libitum and maintained at 24±2 ºC and 80% RH, with a 14:10 130

(light:dark) photoperiod for eight to nine days prior to processing. Females of the same age 131

range were also exposed to oral infection using an artificial membrane system fitted with a 132

one day old chick skin membrane (Hemotek, UK). This study used a 1:1 mixture of bovine 133

blood and 4.38 TCID50/ml SBV suspension, to obtain a final concentration of 2.17 134

TCID50/ml, that during the trial was heated to 38ºC during a 45-60 minute exposure. Fully 135

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engorged females of all three species were subsequently selected and placed inside primary 136

containers for an extrinsic incubation period of 10 days using same maintenance conditions as137

for IT inoculations.138

139

Experimental infection trials with SBV were also carried out with field collected Culicoides140

to provide preliminary estimates of their vector competence for SBV at the University 141

Champagne-Ardenne Biosafety level 2 facilities. Culicoides were collected in a farm located 142

in north-eastern France (Louvois: 49°06’06”N, 4°07’00”E) using light/suction traps (UV 143

CDC trap, John W. Hock Compagny, Gainesvile, FL, USA) or after emergence from manure144

or the margins of ponds. A total of 60 females were allowed to feed on 10 ml of sucrose 145

solution containing a final concentration of 103 TCID50/ml SBV (produced on Vero3 cell line) 146

using a cotton pledglet for four hours. Females were fed with 10% sucrose ad libitum and 147

maintained at 25±2 ºC and 80% RH with a 15:9 (light:dark) photoperiod for four to eight days 148

prior to processing. Identification of orally exposed Culicoides was confirmed by cytochrome 149

oxydase I sequencing, as barcode sequence reference (Hebert and Gregory, 2005).150

151

Screening of field-collected Culicoides for SBV presence152

153

Collection sites for detection of SBV transmission in Culicoides were selected in temperate154

Europe (north-eastern France) and in the Mediterranean island of Sardinia (Italy) (Figure 1).155

In France, wide-scale monitoring of the activity of Culicoides populations was already in 156

place prior to the SBV outbreak and was subsequently carried out from 2009 to 2012 across 157

both mainland areas and Corsica (Balenghien et al., 2011; Venail et al., 2012). This consisted 158

of approximately 160 light-suction UV traps (either one or two based in each department), run 159

at weekly intervals during mid-February to April and during November and December and at 160

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monthly intervals for the rest of the year (Balenghien et al., 2011; Venail et al., 2012). SBV 161

detection was targeted at traps located in departments where SBV surveillance had 162

highlighted significant incidence in 2011, i.e. > 1.5% as defined by the French national 163

surveillance program (Dominguez et al., 2013). In total, Culicoides collected from 53 traps in 164

28 distinct departments were selected (including 6 traps selected prior to the final results of 165

the SBV surveillance which subsequently were found to have an incidence comprised 166

between 0 and 0.5%). Screening for SBV RNA was conducted on Culicoides collected from 167

the 3rd to the 6th October 2011 as late summer/autumn represented the likely time of 168

transmission of the virus described in other studies.169

170

In Italy, clinical outbreaks of SBV infection were reported in Sardinia from October to 171

December 2012, characterised by abortions that occurred in small ruminants held at farms in 172

Sassari, Ogliastra and Cagliari provinces (Figure 1). Following the confirmation of SBV, an 173

entomological survey was performed in the island. Between the 30th October and 4th 174

December 2012, a total of 26 Culicoides collections were performed on 15 farms in seven 175

municipalities of Sardinia, located in the provinces of Carbonia-Iglesias (San Giovanni 176

Suergiu and Sant’Anna Arresi), Cagliari (Muravera), Ogliastra (Barisardo, Girasole and 177

Tertenia) and Sassari (Mores).178

179

In both France and Italy, Culicoides were stored in 70% ethanol and subsequently categorized 180

according to their physiological status (nulliparous, parous and blood engorged). All 181

Culicoides were identified morphologically using a stereomicroscope (Delécolle, 1985) and182

females of C. obsoletus and C. scoticus were grouped as the Obsoletus complex. In each site183

in France, up to 50 unengorged females of the Obsoletus complex were pooled (maximum 1184

pool) and additional pools of up to 50 unengorged females of other species (maximum 3185

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pools) were assayed for SBV presence. In each site in Italy, parous and engorged females 186

were assayed separately.187

188

Detection of SBV genome189

190

In experimental infections conducted in Spain and in field-collected Culicoides screenings 191

conducted in France, viral RNA was extracted using NucleoSpin RNA Virus (Macherey 192

Nagel, Germany) following the manufacturer’s instructions. In Italy, Culicoides nucleic acids 193

were extracted and purified using a BioSprint 96 One-For-All Vet Kit procedure based on the 194

BioSprint 96 instrument (Qiagen, California, USA). In experimental infections conducted in 195

France, RNA was extracted using EZ1 virus mini kit v2.0 (Qiagen, California, USA) 196

following the manufacturer’s instructions. RNA was extracted individually from entire insects 197

for both inoculated and orally exposed females in Spain or France, and for females from the 198

Obsoletus complex collected in France and in Italy, and from pools of maximum 50 entire 199

midges for all other species collected in the field. The S gene of the Schmallenberg virus was 200

transcribed and amplified according to the one step real-time RT-PCR protocol published by 201

Hoffman et al. (2012). Following extraction, SBV RNA was quantified using the one step 202

real-time reverse transcription PCR (RT-PCR) following the protocol developed by Friedrich 203

Loeffler Institut (FLI) targeting the S3 genomic fragment (Bilk et al., 2012), except for Cirad 204

where RT-PCR was performed according to Taq Vet Schmallenberg virus S gene 50 kit 205

(Laboratoire Service International, LSI, France). Assessments of infection status were made 206

using Ct values generated from the samples as a semi-quantitative measure of viral RNA 207

quantity. In France, pools screened for SBV presence and with a 37<Ct<45 values were tested 208

twice and stated positive when both tests gave the same 37<Ct <45 values with appropriate 209

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shape curves. Positive females of the Obsoletus complex were identified to species level using 210

molecular species identification assay (Nolan et al., 2007).211

212

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Results212

213

Experimental infections using laboratory colonies214

215

Intrathoracic inoculations216

Presence of SBV RNA was detected by real-time RT-PCR in a subsample of the inoculated 217

mosquitoes and midges on the day of inoculation (Table 1): the mean Ct values were 218

equivalent between the mosquito species assessed (28.7±0.5 [confidence interval (CI) 28.3-219

29.2] for Ae. albopictus and 28.8±0.4 [CI 28.4-29.2] for Cx. pipiens) but higher in 220

C. nubeculosus (34.1±1.5 [CI 33.1-35.1]), due to a smaller inoculation volume used. The 221

presence of SBV RNA was detected after 8 to 9 days post-inoculation (dpi) in all 222

Ae. albopictus, in 69.7% of Cx. pipiens females and in 99.2 % of C. nubeculosus females 223

(Table 1). The mean Ct values decreased in positive females between the inoculation day and 224

8 to 9 dpi by 8.0 Ct in Ae. albopictus (20.7±5.1 [CI 19.7-21.7]), by 5.4 in Cx. pipiens225

(23.4±4.2 [CI 22.6-24.3]) and by 10.0 in C. nubeculosus (24.2±4.9 [CI 23.3-25.0]) (Figure 2).226

227

Oral exposure228

Presence of SBV RNA was detected by real time RT-PCR in a subsample of the orally 229

exposed mosquitoes and Culicoides on the day of exposure (Table 1): the mean Ct values 230

were similar between mosquitoes and Culicoides (29.0±1.1 [CI 28.2-29.7] for Ae. albopictus, 231

28.8±0.1 [CI 28.6-29.0] for Cx. pipiens and 30.0±2.1 [CI 28.9-31.2] for C. nubeculosus) 232

despite a smaller blood-meal in Culicoides. At 10 days post-exposure, SBV RNA was 233

detected in 1.3% of Ae. albopictus (2/149), in none of the 27 blood-fed Cx. pipiens females 234

and in 3.9% of the blood-fed C. nubeculosus (4/102). The infection rate was significantly235

greater for C. nubeculosus (95% binomial confidence interval of 1.08 to 9.73%) than for236

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Ae. albopictus (0.16 to 4.76%): p = 0.049 under bionomial assumptions. It was not possible to 237

distinguish the infection rates between Cx. pipiens (0 to 12.77%) and the two other species 238

due to too few mosquitoes being tested (N = 27). The Ct values were similar ten days post-239

infection than the day of exposure for the two positive Ae. albopictus (29.53 and 31.25 versus 240

29.0), whereas they were much lower for 3 of the 4 positive C. nubeculosus (21.67, 24.21 and 241

25.3 versus 30.0) strongly suggesting SBV replication.242

243

Culicoides scoticus was the dominant species of field-collected females exposed orally to 244

SBV (45 of the 60 individuals). For this species, mean Ct values were 32.2 at day 0, 18.5 at 245

day 4, 33.8 at day 5 and 29.0 at day 8 (Table 2) suggesting SBV replication in some 246

C. scoticus individuals. Variation in Ct values may be due to differences in the quantity of 247

ingested blood and in individual susceptibility within C. scoticus population, and was 248

amplified by the limited number of individuals.249

It was not possible to interpret results for other species due to too few individuals. Single viral 250

RNA identification in Forcipomyia or C. newsteadi individuals, especially with a high Ct251

value, was not sufficient to incriminate these species as potential vectors without further 252

investigations.253

254

Screening of field-collected individuals for SBV presence255

256

In France, a total of 224,870 Culicoides, belonging to at least 20 species, were collected in the 257

studied period. The species diversity was dominated by C. dewulfi (58.4% of the total catch), 258

the Obsoletus complex (35.9%) and C. chiopterus (4.5%). The parity rates (No. parous 259

females/No. females) of C. dewulfi and of the Obsoletus complex were about 32.0% and 40%. 260

In Italy, a total of 53,531 Culicoides were collected (Table 3). Culicoides imicola was the 261

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dominant species in these collections, (74.5% of the total catch), followed by Culicoides262

newsteadi Austen (21.7%), species of the Obsoletus complex (0.8%), Culicoides pulicaris263

(Linneaus) (0.4%) and Culicoides punctatus (Meigen) (0.01%) (Table 3). The parity rate of 264

C. imicola was approximately 55.0%.265

266

For collections made in France 5,135 females were tested for SBV RNA presence. Ten pools 267

of females from the Obsoletus complex contained detectable quantities of SBV RNA and all 268

females processed individually that tested positive were identified as C. obsoletus. Pools of 269

the Obsoletus complex had an overall minimum infection rate (MIR) of 0.58%, with270

C. chiopterus giving an MIR of 0.16%, C. pulicaris possessing an overall MIR of 0.37% and 271

of C. nubeculosus giving an overall MIR of 2.33% (Table 4). Excluding sites where SBV was 272

not recovered increased the MIR to approximately 4% for the Obsoletus complex and 273

C. nubeculosus and around 1% for C. chiopterus and C. pulicaris (Table 4). For Italian 274

collections, 27,837 parous females (727 pools) and 340 engorged females (32 pools) were 275

tested for SBV presence. Detectable SBV RNA was identified in three pools of C. imicola of 276

which two were pools of 50 pigmented females and one was a pool of 33 engorged females. 277

All these pools were collected at San Giovanni Suergiu (Carbonia-Iglesias province) leading 278

to a MIR = 0.04% within the pigmented population of C. imicola in this site (Table 5).279

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Discussion280

281

This study, together with previous work in the Netherlands (Elbers et al., 2013b), Belgium282

(De Regge et al., 2012) and Italy (Goffredo et al., 2013) confirmed the role of C. obsoletus as 283

a highly probable vector of SBV in northern Europe. This species is among the most abundant 284

livestock-associated species in the region (Meiswinkel et al., 2008; Carpenter et al., 2009; 285

Venail et al., 2012) and its apparently ubiquitous distribution on farms across the Palaearctic 286

and Nearctic may facilitate spread of SBV to new regions. In addition, C. nubeculosus was 287

implicated for the first time as a potential vector in France, although quantities of SBV RNA 288

detected were equivocal in defining the level of dissemination that had occurred (Veronesi et 289

al., 2013). Studies of vector competence for SBV in colony lines of this species, both in the 290

current study and in previous studies in the UK (Veronesi et al., 2013), have indicated 291

extremely low rates of competence of approximately 3%. It is important to note, however, 292

that such infection rates have been demonstrated to vary with vector population for other 293

Culicoides-borne arboviruses (Tabachnick, 1996). Preliminary evidence was also provided 294

that C. scoticus is able to replicate SBV to transmissible levels, albeit using a technique 295

(pledglet feeding with sugar) that is likely to result in virus being transported to the crop 296

rather than the gut (Jennings and Mellor, 1988).297

298

Studies of C. imicola in Sardinia (Italy) failed to convincingly implicate this species in 299

transmission through detection of SBV RNA, despite its well documented role in transmission 300

of other Culicoides-borne arboviruses (Mellor et al., 2009b) and association with BTV 301

outbreaks in Italy (Goffredo et al., 2003; Goffredo et al., 2004). Indeed, all numerous pools of 302

C. imicola collected in farms reporting abortions tested negative for SBV. On the contrary, 303

relatively small quantities of SBV RNA were detected in pools of C. imicola in Iglesias 304

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province where no case was reported (Figure 1). The fact that C. imicola dominated the 305

Culicoides fauna in Sardinia and especially at outbreak sites where only very limited numbers 306

of the Obsoletus complex were present, however, indicates its probable involvement in 307

transmission of SBV. An absence of pools of C. imicola containing significant quantities of 308

SBV RNA may have been due to the time of sampling. Samples were made in farms 309

experiencing SBV reports few months after the probable infection date. On the contrary, SBV 310

RNA was detected in C. imicola populations late in the season when animal infection may be 311

unapparent. Thus further screening of field-collected Culicoides within the distribution of this 312

species would be desirable.313

314

Results from IT inoculation of mosquitoes strongly suggested that SBV can replicate in 315

individuals when introduced directly into the haemocoel, bypassing mid-gut barriers to 316

arbovirus dissemination (Mellor, 2000). Oral infection, however, did not result in Ct values 317

indicative of full SBV dissemination in either mosquito species. While these results should be 318

interpreted with caution as they utilised inbred colony lines, this study provides preliminary 319

evidence that the Culicidae may not play a substantial role in transmission of SBV in the 320

field. Previously it was demonstrated that Culicoides were efficient experimental vectors for 321

the closely related Akabane virus (Jennings and Mellor, 1989), whereas attempts to 322

demonstrate replication of this virus in mosquitoes have so far proved unsuccessful (Kay et 323

al., 1975). Systematic studies to characterise biting rates of mosquito species on livestock in 324

Europe, however, would be useful in understanding the role of this group to potentially325

transmit pathogens such as SBV.326

327

The spread of SBV has been noted to have been quicker and its transmission more intense in328

comparison to BTV and this has been hypothesised to be due to Culicoides populations being 329

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more susceptible to infection (Elbers et al., 2013b). A review of studies conducted to date 330

found equivocal support for this hypothesis, as expected from the fact that the proportion of 331

Culicoides exposed to viraemic hosts within screened populations is unknown and in general 332

the numbers of individuals and sites investigated in initial studies were low (De Regge et al., 333

2012; Rasmussen et al., 2012; Elbers et al., 2013b). The most straightforward way to assess 334

the true competence of populations is to carry out infection studies of field collected 335

Culicoides in the laboratory using either viraemic hosts or artificial means of feeding, as 336

conducted for BTV (Jennings and Mellor, 1988; Carpenter et al., 2006; Carpenter et al., 337

2008). As the timing of animal based experiments in biosecure containment with population 338

peaks in Culicoides is logistically challenging, pledglet-based blood feeding methods are most 339

commonly employed to assess infection rates. Membrane-based methods, such as those 340

employed to feed colony Culicoides and mosquitoes in this study currently result in extremely 341

poor rates of feeding in field collected Culicoides from northern Europe (Jennings and Mellor, 342

1988; Venter et al., 2005). As it is known that pledglet feeding significantly underestimates 343

the proportion of competent Culicoides in a population (Venter et al., 2005) it is therefore 344

vital that standard membrane-based techniques are developed for northern European species.345

346

A key observation during the SBV outbreak is the rapid spread of the virus into new 347

ecosystems in Europe which are primarily dominated by different multitudes of Culicoides348

species. This phenomenon has been observed previously during large-scale outbreaks of BTV 349

in Europe, initially in the case of BTV-9, which was able to move from areas dominated by 350

C. imicola into the Balkans, where the Culicoides fauna was dominated by Palaearctic species 351

(Mellor and Wittmann, 2002). In later outbreaks, BTV-8 was introduced by an as yet 352

undefined route into northern Europe and then caused disease outbreaks in southern Europe 353

on farms dominated by C. imicola. Similarly, BTV-1 moved from C. imicola dominated 354

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farms in Spain to areas dominated by the Obsoletus complex and C. dewulfi in France 355

(Carpenter et al., 2009; Mellor et al., 2009a; Mellor et al., 2009b). The ease by which these 356

arboviruses have moved between disparate ecosystems implies that any barriers presented by 357

the process of adaptation of Culicoides-borne arboviruses to novel vector groups are at best 358

only partially restrictive between these two primary vector groups. In a period of significant 359

globalisation of trade and environmental change this has significant consequences for the 360

worldwide spread of such pathogens. Conducting studies to understand factors limiting the 361

current distribution of arboviruses is therefore likely to be useful in determining the risk that 362

emerging pathogens pose to other regions.363

364

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Acknowledgements: 364

The study was mainly funded by the Directorate General for Health and Consumers (DG-365

SANCO), European Commission (2012/349/UE). This study was partially funded by EU 366

grant FP7-261504 EDENext and is catalogued by the EDENext Steering Committee as 367

EDENext175 (http://www.edenext.eu). The contents of this publication are the sole 368

responsibility of the authors and do not necessarily reflect the views of the European 369

Commission. The French authors would like to thank the national veterinary services, the 370

farmers and the technicians for collecting the biting midges; Xavier Allène, Ignace 371

Rakotoarivony, Jonathan Lhoir (Cirad), Bethsabée Scheid (EID-Med) and Delphine Delécolle 372

(IPPTS) for identifying the Culicoides samples.373

374

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508509510

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Table 1. Experimental infections of mosquito and Culicoides laboratory colonies with Schmallenberg virus

Species Aedes albopictus Culex pipiens Culicoides nubeculosus

Days post-inoculation 0 8-9 0 8-9 0 8-9

No. inoculated/No. tested 5 51 4 89 9 130

No. positive (%) 5 (100.0) 51 (100.0) 4 (100.0) 62 (69.7) 9 (100.0) 129 (99.2)

Intrathoracic

inoculation

Mean Ct value [min-max] 28.7 [28.0-29.2] 20.0 [16.3-40.6] 28.8 [28.3-29.2] 23.4 [18.2-37.8] 34.1 [32.2-36.5] 24.2 [15.2-38.6]

Days post-exposure 0 10 0 10 0 10

No. exposed 8 149 2 27 12 102

No. positive (%) 8 (100.0) 2 (1.3) 2 (100.0) 0 12 (100.0) 4 (3.9)

Oral exposure

Mean Ct value [min-max] 29.0 [28.0-30.8] 30.4 [29.5-31.3] 28.8 [28.7-28.9] 30.0 [26.9-33.8] 27.5 [21.7-38.7]

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Table 2. Experimental infections (orally) of field-collected midges with Schmallenberg virus

Species No. exposed Days post-exposure No. positive Mean Ct value [min-max]

C. scoticus 45 0 4 32.2 [31.4-34.2]

4 1 18.5

5 3 33.8 [33.1-34.2]

8 2 29.0 [28.9-29.1]

Forcipomyia sp. 6 4 1 31.5

C. obsoletus 1 0 1 33.9

C. newsteadi 3 8 1 35.2

Other species 5 - 0

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Table 3. Culicoides collected in Sardinia (Italy) from the 30th October to the 5th December 2012 and assessed for the presence of Schmallenberg

virus

Province Ogliastra Sassari Cagliari Carbonia-IglesiasSpecies

Municipality (No.

collections)

Barisardo (7) Girasole (1) Tertenia (5) Mores (4) Muravera (1) San Giovanni Suergiu (3)

Sant'Anna Arresi (5)

Total

C. imicola Total No. midges 19,576 11 769 3,778 99 15,589 44 39,866 (74.47%)

No. PFa (No. pools) 10,909 (221) 7 (1) 603 (14) 395 (10) 50 (1) 10,132 (204) 30 (5) 22,126 (456)

No. EFa (No. pools) 75 (3) 4 (1) 13 (3) 1 (1) 106 (4) 199 (12)

C. newsteadi Total No. midges 695 3 108 9,248 151 1,326 103 11,634 (21.73%)

No. PF (No. pools) 303 (10) 45 (4) 4,299 (87) 107 (3) 684 (15) 65 (5) 5,503 (124)

No. EF (No. pools) 12 (2) 2 (2) 104 (5) 2 (1) 8 (1) 1 (1) 129 (12)

Obsoletus complex Total No. midges 48 0 81 137 7 130 9 421 (0.77%)

No. PF 7 41 59 6 14 4 131

No. EF 4 4 2 10

C. pulicaris Total No. midges 81 3 74 33 1 0 0 192 (0.36%)

No. PF (No. pools) 25 (3) 2 (1) 28 (4) 16 (4) 1 (1) 72 (13)

No. EF (No. pools) 2 (2) 2 (2)

C. punctatus Total No. midges 1 0 0 0 0 0 5 6 (0.01%)

No. PF (No. pools) 1 (1) 4 (2) 5 (3)

Other species Total No. midges 740 5 74 149 5 228 220 1,421 (2.65%)

Total Total No. midges 21,141 22 1,106 13,345 263 17,273 381 53,531

No. PF (No. pools) 11,245 (242) 9 (2) 717 (63) 4,769 (160) 164 (11) 10,830 (233) 103 (16) 27,837 (727)

No. EF (No. pools) 87 (5) 10 (5) 123 (13) 3 (2) 116 (6) 1 (1) 340 (32)

a PF: parous females; EF: blood-fed females

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Table 4. Retrospective detection of Schmallenberg virus from Culicoides collected in France in October 2011

Species No. Culicoides (pools)

tested

No. positive pools Mean Ct value [min-max] Overall minimum infection

rate (MIR)

Mean MIR in positive sites

[min-max]

Obsoletus complexa 1 734 (347) 10 32.9 [23.4-38.2]a 0.58%a 4.14% [2.00-6.00]

C. obsoletus 8 34.4 [28.3-38.2]a

C. dewulfi 1 729 (47) 0

C. chiopterus 1 224 (40) 2 32.0 [30.6-33.4] 0.16% 1.17% [1.12-1.22]

C. pulicaris 271 (27) 1 38.3 0.37% 1.25%

C. newsteadi 65 (12) 0

C. nubeculosus 43 (7) 1 28.8 2.33% 3.70%

C. lupicaris 24 (9) 0

C. punctatus 17 (7) 0

C. festivipennis 9 (3) 0

C. clastrieri 8 (1) 0

C. circumscriptus 4 (2) 0

C. fascipennis 2 (1) 0

C. alazanicus 1 (1) 0

C. cataneii/gejgelensis 1 (1) 0

C. parroti 1 (1) 0

C. puncticollis 1 (1) 0

C. subfasciipennis 1 (1) 0

a Eight of the 10 positive individuals from the Obsoletus complex were identified as C. obsoletus; it was not possible to identify the two others at the species level. The Ct values associated with the Obsoletus

complex individuals correspond to assay on single individuals (compared to pools for the other species) leading to minimum infection rates being the effective infection rate sampled populations of the Obsoletus

complex.

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Table 5. Detection of Schmallenberg virus from Culicoides collected at San Giovanni Suergiu, Carbonia-Iglesias province, in Sardinia (Italy),

the 7th November 2012

Species No. midges collected Physiological status No. midges (pools)

tested

No. positive pools Mean Ct value [min-

max]

Minimum infection rate

C. imicola 7,704 Parous 5,050 (101) 2 36 [34-38] 0.04%

Engorged 66 (2) 1 33

C. newsteadi 846 Parous 500 (10) 0

Engorged 8 (1) 0

Obsoletus complex 27 Parous 6 (6) 0

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Table 6. Published reports of Schmallenberg virus detection from field-collected Culicoides in Belgium, Denmark, Netherlands, Italy and Poland

using detection assays from Hoffmann et al. (2012) and Bilk et al. (2012)

Country Period Pool constitutiona Species No. midges

(pools) tested

No. positive

pools

Mean Ct value

[min-max]

Minimum

infection rate

Reference

Belgium August to October 2011 25 heads (PF) Obsoletus complex 688 (34) 5 33.9 [30.7-36.0] 0.73% (De Regge et al., 2012)

C. obsoletus 283 (32) 3 35.9 [34.9-36.5] 1.06%

C. scoticus 240 (27) 0

C. dewulfi 181 (20) 2 35.2 [32.2-38.1] 1.10%

C. chiopterus 227 (23) 1 28.7 0.44%

C. pulicaris 89 (11) 1 37.9 1.12%

Denmark October 2011 5 entire females Obsoletus group 91 2 26.0 [25.0-27.6] 2.20% (Rasmussen et al.,

2012)

Netherlands August to September 2011 10 heads (NF or PF) Obsoletus complex 2,300 (230) 12 24.6 [19.6-36.0] 0.52% (Elbers et al., 2013b)

C. obsoletus 1 24.6

C. scoticus 10 25.0 [19.6-36.0]

C. dewulfi 1,300 (130) 0

C. chiopterus 1,440 (144) 2 31.6 [27.9-35.4] 0.14%

C. punctatus 1,050 (105) 0

May to September 2012 Obsoletus complex 2,100 (42) 2 36.3 [35.0-37.7] 0.10% (Elbers et al., 2013a)50 entire females (PF

or GF) C. dewulfi 1,300 (26) 0

C. chiopterus 1,050 (21) 0

C. punctatus 1,550 (31) 0

C. pulicaris 500 (10) 0

Italy June 2011 to June 2012 < 50 entire females

(PF or GF)

Obsoletus complex 5,146 6 28.7 [26.0-33.0] 0.12% (Goffredo et al., 2013)

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C. pulicaris 29 (17) 0

C. punctatus 28 (14) 0

C. dewulfi 1 (1) 0

Nubeculosus complex 296 (34) 0

C. flavipulicaris 1 (1) 0

September to November 2011 Obsoletus complex 1,104 5 29.0 [26.0-33.0] 0.45%

May 2012 Obsoletus complex 769 1 27.0 0.13%

C. obsoletus 1 27.0

Poland Obsoletus complex ~ 3,600 (181) 28 ~ 29.8 [17.5-39.4] 0.78% (Larska et al., 2013)September/October 2011 and

April to October 2012

~ 20 entire females

(NP, PF or GF)b C. punctatus ~ 2,100 (108) 6 ~ 31.4 [23.9-37.2] 0.29%

a PF: parous females; NF: nulliparous females; GF: gravid femalesb The number of Culicoides per pools was not given precisely, it ranged from 9 to 60 (meanly 20). Blood-fed females were also tested in this study, but we did not report the results here

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Figure captions12

Figure 1. Localisation of traps and Schmallenberg virus reports in France and Sardinia (Italy)34

Figure 2. Observed Ct values for Schmallenberg virus in mosquitoes and Culicoides 8 to 9 5days after intrathoracic inoculation6

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Figure1

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Figure2