JVI Accepted Manuscript Posted Online 4 February 2015 J. Virol. doi:10.1128/JVI.03027-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

1

Isolation, genetic characterization and seroprevalence of Adana virus a novel

2

phlebovirus belonging to the Salehabad virus complex in Turkey

3 4

Cigdem ALKAN1,2, Sulaf ALWASSOUF1, Géraldine PIORKOWSKI,1,2 Laurence

5

BICHAUD,1,2,3 Seda TEZCAN4, Ender DINCER5, Koray ERGUNAY6, Yusuf OZBEL7,

6

Bulent ALTEN 8 , Xavier de LAMBALLERIE1,2 Rémi N. CHARREL#.1,2

7

1

, Aix Marseille Université, IRD French Institute of Research for Development,

8

EHESP French School of Public Health, EPV UMR_D 190 "Emergence des

9

Pathologies Virales", 13385, Marseille, France

10 11 12 13 14 15 16 17 18 19 20 21

2

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

France 3

Department of Parasitology, Faculty of Science, Charles University, Prague 2 128

44, Czech Republic. 4

, Mersin University Faculty of Medicine, Department of Medical Microbiology,

33343 Mersin TURKEY 5

, Mersin University, Advanced Technology Education, Research and Application

Center, 33343 Mersin TURKEY 6

, Hacettepe University Faculty of Medicine, Department of Medical Microbiology,

Virology Unit 06100 Ankara TURKEY 7 8

Ege University Medical School Department of Parasitology, Bornova, Izmir, Turkey , Faculty of Science, Department of Biology, Ecology Section, ESR Laboratories,

22

Hacettepe University, 06800, Ankara, Turkey

23 24

#, corresponding author: Rémi N. Charrel, [email protected]

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Word count of the abstract: 183 words

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Word count of the text: 4,319

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28

ABSTRACT

29

A new phlebovirus, Adana virus, was isolated from a pool of Phlebotomus sp.

30

(Diptera; Psychodidae) in the province of Adana, Mediterranean region of Turkey.

31

Genetic analysis based on complete coding genomic sequences indicated that

32

Adana virus belongs to the Salehabad virus species of the genus Phlebovirus in the

33

family Bunyaviridae. Adana virus is the third virus of the Salehabad virus species for

34

which the complete sequence has been determined. To understand the epidemiology

35

of Adana virus, a seroprevalence study using microneutralization assay was

36

performed to detect the presence of specific antibodies in human and domestic

37

animal sera collected in Adana as well as Mersin province, located 147km west of

38

Adana. The results demonstrate that (i) the virus is present in both provinces, (ii) high

39

seroprevalence rates in goats, sheep and dogs support intensive exposure to Adana

40

virus in the region, which have not been previously reported for any virus included in

41

the Salehabad serocomplex (iii) low seroprevalence rates in humans suggest that

42

Adana virus is not likely to constitute an important public health problem in exposed

43

human populations but this deserves further studies.

44 45

IMPORTANCE

46

Until recently, in the genus Phlebovirus, the Salehabad virus species consisted of two

47

viruses: Salehabad virus isolated from sandflies in Iran, and Arbia virus isolated from

48

sandflies in Italy. Here we present the isolation and complete genome

49

characterization of the Adana virus which we propose to be included in the

50

Salehabad virus species. To our knowledge, this is the first report of the isolation and

51

complete genome characterization, from sandflies in Turkey, of a Salehabad-related

52

phlebovirus with supporting seropositivity in the Mediterranean, Aegean, and Central

2

53

Anatolian region where phleboviruses have been circulating and causing outbreaks.

54

Salehabad species viruses have generally been considered to be a group of viruses

55

with little medical or veterinary interest. This view deserves to be revisited according

56

to our results which indicate a high animal infection rate of Adana virus and recent

57

evidence of human infection with Adria virus in Greece.

58 59

INTRODUCTION

60

Sandfly-borne phleboviruses (genus Phlebovirus, family Bunyaviridae) may cause

61

self-limiting febrile illness (sandfly fever) or neuro-invasive infections. The genus

62

Phlebovirus contains 9 viral species (Sandfly fever Naples, Salehabad, Rift valley

63

fever, Uukuniemi, Bujaru, Candiru, Chilibre, Frijoles, Punta Toro), and several

64

tentative species as defined in the 9th Report of the International Committee for

65

Taxonomy of Viruses (ICTV) (1). Of the 9 viral species recognized by the ICTV,

66

Sandfly fever Naples, Salehabad, Bujaru, Candiru, Chilibre, Frijoles, Punta Toro are

67

exclusively or partially vectored by sandflies. In the Old World, there are two

68

recognized species (Sandfly fever Naples [SFNV], Salehabad [SALV]) and 2 tentative

69

species (Sandfly Fever Sicilian [SFSV], Corfu [CFUV]) consisting of sandfly-borne

70

phleboviruses. In addition, many new phleboviruses have been recently isolated from

71

phlebotomine flies (Fermo, Granada, Punique) (2, 3, 4), from ticks (Heartland, Hunter

72

island group) (5, 6), or from vertebrates (Malsoor, Salanga) (7, 8). They remain to be

73

recognized by the ICTV.

74

All members of the genus Phlebovirus have a tri-segmented, single-stranded RNA

75

genome. The L segment encodes the RNA-dependent RNA polymerase. The M

76

segment encodes the viral envelope glycoproteins (Gn and Gc, formerly G1 and G2).

77

The S segment encodes the viral nucleocapsid protein (N) and a nonstructural

3

78

protein (NSs) in an ambisense orientation (9, 10, 11). Sandfly-borne phleboviruses

79

are transmitted mainly by sandflies belonging to the genus Phlebotomus in the Old

80

World when females take a blood meal (May to October). Transovarial (vertical)

81

transmission from female to offspring (12, 13, 14, 15, 16) and venereal (horizontal)

82

transmission have been recorded from infected males to uninfected females during

83

mating (12, 17). There is no defined reservoir of sandfly-borne phleboviruses.

84

Therefore their ecological distribution and evolutionary divergence seem to be highly

85

dependent on their vectors.

86

Former seroprevalence studies indicated that Sicilian and Naples viruses are present

87

in the Mediterranean and Aegean regions of Turkey (18, 19). Extensive investigations

88

have been initiated during the last decade; especially in the regions where outbreaks

89

have occurred (Mediterranean, Aegean, and Central Anatolian regions). Circulation

90

of SFSV and a SFS-like virus (Sandfly Fever Cyprus [SFCV]) was detected

91

serologically and Sandfly fever Turkey virus (SFTV) was isolated during the

92

outbreaks (20). After the outbreaks in Kirikkale, a province 51-km from the outbreak

93

region of Ankara (21) and in Kahramanmaras, 196-km north-east of Adana (22),

94

antibodies were detected in convalescent patients. An acute hepatitis case was

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recently reported due to SFSV in Kirikkale (23). Toscana virus (TOSV) was

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serologically detected in several regions (24, 25, 26, 27, 28). Although there are

97

extensive studies on seroprevalence of phleboviruses in Turkey, virus isolation was

98

only reported for SFTV from one patient (20). The sandfly-borne viruses appear to be

99

widespread throughout the country. To understand the nature of the circulation of

100

phleboviruses in Turkey, sandfly trapping campaigns were organized in Central

101

Anatolia, in Mediterranean and in Aegean regions. Here we present the genetic and

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seroprevalence data on Adana virus (ADAV), a novel phlebovirus belonging to the

4

103

Salehabad species. ADAV was isolated from sandflies trapped in the Mediterranean

104

region of Turkey. Genetic and phylogenetic studies were performed on complete

105

genomic sequence data. Seroprevalence studies using microneutralisation assays

106

were performed in 1,000 human sera and 289 animal sera from the same region.

107 108

MATERIALS AND METHODS

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Sandfly Trapping

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Sandfly trapping campaigns were conducted from August 2012 to September 2012 in

111

Adana (Mediterranean region, Turkey) using CDC Miniature Light Traps as

112

previously reported (29). Live sandflies were pooled based on sex, trapping site and

113

trapping day, with up to 30 individuals per pool and placed in 1.5mL tubes to be

114

further stored at -80°C. No morphological identification of the captured sandflies was

115

performed prior to viral testing. The rationale for this approach was to minimize

116

manipulations to facilitate virus isolation. Adana is the the 5th-most densely-populated

117

province of Turkey with a population of 2.1 million. It is located near the Seyhan

118

River, 30-km inland from the Mediterranean Sea, in south-central Anatolia. Adana

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lies in the heart of Cukurova, a geographical, economical, cultural, and agricultural

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region that also covers the provinces of Mersin, Osmaniye, and Hatay. The region is

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agriculturally productive throughout the year.

122 123

Virus Detection

124

Pools of sandflies were ground in 600μL of Eagle minimal essential medium (EMEM)

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(supplemented with 7% fetal bovine serum, 1% Penicillin Streptomycin, and 1% L-

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Glutamine 200 mM) in the presence of a 3-mm tungsten bead using a Mixer Mill

127

MM300 (Qiagen, Courtaboeuf, France) (30). A 200-μL aliquot was used for viral

5

128

nucleic acid (NA) extraction with the BioRobot EZ1-XL Advanced (Qiagen) using the

129

Virus Extraction Mini Kit (Qiagen), and eluted in 90μL. Five μL of this solution were

130

used for RT-PCR and nested-PCR assays with primers targeting the polymerase

131

gene and the nucleoprotein gene using protocols previously described (31, 32). PCR

132

products of the expected size were column-purified (Amicon Ultra Centrifugal filters,

133

Millipore) and directly sequenced. Two real-time RT-PCR assays were designed for

134

specific detection of the newly isolated Adana virus in the polymerase (ADAV-L) and

135

nucleoprotein (ADAV-N) genes, respectively. The primers for the ADAV-L assay

136

consisted of ADAV-L-FW (CACAGATGTCTACTGAGCATGAG ), ADAV-L-REV

137

(ACTTATGAGAGGGTGAATATCTCT), and ADAV-L-Probe (6FAM-

138

TTAACTGGTCTGGATTATTCAACCC-TAMRA). The primers for the ADAV-N assay

139

consisted of ADAV-N-FW (GACCGATGATGCATCCTTGCTT), ADAV-N-REV

140

(GCGGATTGATGGTCCTTGAGAA), and ADAV-N-Probe (6FAM-

141

ATTGACAACACCCTTCCAGAGGA-TAMRA). The real-time RT-PCR was performed

142

using the Go Taq Probe 1-Step RT-qPCR (Promega) following the manufacturer’s

143

protocol with the following incubation programme on a CFX96 real-time system (Bio-

144

Rad): (i) 50°C for 15 min, (ii) 95°C for 2 min; (iii) 40 cycles consisting of 95°C for 15s

145

and 60°C for 1 min.

146 147

Virus Isolation and Electron Microscopy

148

A 50μL-volume of ground sandfly pools was inoculated onto a 12.5 cm2-flask of Vero

149

cells together with EMEM, enriched with 1% Penicilin Streptomycin, 1% L-Glutamine

150

200 mM, 1% Kanamycin, and 3% Fungizone. After incubation at room temperature

151

for 1 hr, 5mL of fresh EMEM containing 5% fetal bovine serum (FBS) were added.

152

The flasks were incubated at 37°C in 5% CO2 atmosphere and examined daily for

6

153

cytopathic effect. After detection of cytopathic effect (CPE) during passage 1, the

154

virus was passaged 4 times, and P4 was used for electron-microscopic examination.

155

Negative-stained electron-microscopic specimens were prepared using infected cell

156

supernatant mixed 1:1 with 2.5% paraformaldehyde, fixed onto formvar/carbon-

157

coated grids and stained with 2% methylamine tungstate.

158 159

Complete genome sequencing

160

The Adana virus (ADAV) passage 2 was used for complete genome characterization

161

through Next Generation Sequencing (NGS). Briefly, 140μL of cell culture

162

supernatant was incubated at 37°C for 7 hr with 30 U of benzonase (Novagen 70664-

163

3), then RNA was extracted using the Viral RNA Mini Kit (Qiagen) onto the BioRobot

164

EZ1-XL Advanced (Qiagen). Random amplification was performed using tagged

165

random primer for reverse transcription (RT) and tag-specific and random primers for

166

PCR amplification (Applied Biosystems). The PCR products were purified (Amicon

167

Ultra Centrifugal filters, Millipore) and 200ng were used for sequencing using the Ion

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PGM Sequencer (Life Technologies SAS, Saint Aubin, France). Viral sequences

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were identified from the contigs based on the best BLAST similarity against reference

170

databases. Sequence gaps were completed by PCR, using primers based on NGS

171

results, and sequenced either by Sanger sequencing or by NGS. The 5' and 3'

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extremities of each segment were sequenced using a primer including the 8-nt

173

conserved sequence as previously described (33). For the confirmation of the final

174

acquired sequences by NGS, specific primers were designed for Sanger sequencing

175

of the complete genome.

176 177

Genetic Distances and Phylogenetic Analysis

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The sequences of S, M and L segments were aligned, with homologous sequences

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of other phleboviruses retrieved from Genbank until September 2014 using the

180

CLUSTAL algorithm of the MEGA 5 software (34). Nucleotide (NT) and amino acid

181

(AA) distances were calculated with the p-distance method. Neighbor-joining analysis

182

(Kimura 2-parameter model) was done with AA sequences using MEGA version 5,

183

with 1000 bootstrap pseudoreplications. Amino acid sequences in the polymerase,

184

Gn, Gc, N and Ns proteins of all respective complete coding sequences retrieved

185

from the Genbank database were used to study the distribution of evolutionary

186

distances by pairwise comparison, as previously described (30, 35).

187 188

Microneutralisation-based seroprevalence study

189

Human and domestic animal sera were collected in Adana and Mersin provinces

190

after informed consent of the individuals and animal owners, according to the

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national regulations on the operation and procedure of animal experiments ethics

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committees (Regulation Nr.26220, Date: 09.7.2006). The study protocols were

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approved by the local ethics committees (MULEC/01.09.10 for human samples,

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AULEC/201-96-346 for animal samples), and by the Ege University Local Ethical

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Committee of Animal Experiment with the protocol number 2011-101. The virus

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microneutralisation (MN) assay, described for phleboviruses (26, 35), was adapted

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with minor modifications using the ADAV strain. Briefly, twofold serial dilutions from

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1:20 to 1:160 were prepared for each serum and a volume of 50μL of each dilution

199

was transferred into 96-well plates. A volume of 50μL containing 1000 TCID50 of virus

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was added to each well except for the controls that contained PBS. The plates were

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incubated at 37°C for one hour. Then, a 100μL suspension of Vero cells containing

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approximately 2 x105 cells/mL of EMEM medium enriched with 5% fetal bovine

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serum, 1% Penicilin Streptomycin, 1% L-Glutamine 200 mM, 1% Kanamycin, 3%

204

Fungizone, was added to each well, and incubated at 37°C in presence of 5% CO2.

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The first row of each plate contained control sera diluted 1:10 and Vero cells without

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virus. After 6 days the microplates were read under an inverted microscope, and the

207

presence (neutralization titre at 20, 40, 80 and 160) or absence (no neutralization) of

208

cytopathic effect was noted. To exclude that MN results observed with ADAV were

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not due to cross-neutralising antibodies raised against Arbia virus, all sera were

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tested in parallel with a strain of Arbia virus.

211 212

Genotyping of sandflies in the virus-positive pool

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To attempt identification of the sandfly species present in the Adana virus positive

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pool, PCR was performed using 3-μL of nucleic acid extract of the pool to amplify the

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cytochrome c oxidase I (COI) gene, frequently used for biological barcoding (37). The

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PCR products were processed and sequenced through NGS as described above.

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NGS reads were compared with available sequences in Genbank using the CLC

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Genomic Workbench 6.5.

219 220

RESULTS

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Sandfly trapping and virus detection

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A total of 7,731 (3,524 females and 4,207 males) sandflies were collected in August

223

and September 2012 from six villages (Fig. 1) located within the district of Adana

224

province (Mediterranean Turkey). They were organized into 380 pools (including 179

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female and 201 male pools). The number of sandflies and pools originating from

226

individual villages are shown in Table 1. Pool #195 that consisted of 20 males

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trapped in Damyeri village (3650733357 North and 4140570 East, altitude: 194m)

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was positive with primers N-phlebo1S and 1R (32). The resulting 505-nt sequence in

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the polymerase gene was most closely related with the Salehabad virus (GenBank

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accession no: JX472403) sequence (86% and 77% identity at the AA and NT levels,

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respectively). Using the two real-time RT-PCR assays specifically designed to detect

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ADAV, only the pool #195 was found to be positive (Ct values < 26). Four-fold

233

dilutions of the pool #195 were tested and found positive until 1:4,096 dilution with Ct

234

values ranging from 36.2 to 38.2 for the 1:4,096 dilution. This is a convincing

235

argument for excellent sensitivity of these ADAV specific real-time RT-PCR tests.

236 237

Virus Isolation and Electron Microscopy

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Vero cells that were inoculated with pool #195 showed a clear cytopathic effect after

239

4 days. Material corresponding to passage 3 was used for mass production, and

240

subsequent freeze-drying; these vials have been included in the collection of the

241

European Virus Archive (www.european-virus-archive.com) where they are publicly

242

available for academic research. The morphology of the virus was shown by electron-

243

microscopic examination (Fig. 2). EM micrographs showed spherical or pleomorphic

244

structures, with a size of 80-120 nm in diameter, and surface projections (5-10 nm

245

long) that evenly covered the virions, and were compatible with images observed for

246

other members of the Bunyaviridae family.

247 248

Complete genome sequencing

249

The reads obtained through using next generation sequencing were processed by

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CLC Genomics Workbench 7.0.4. Reads, of minimum length 30 nucleotides, were

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trimmed using CLC Genomic Workbench 6.5, with a minimum of 99% quality per

252

base and mapped to reference sequences (Arbia Virus, GenBank accession no:

10

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JX472400, JX472401, and JX472402 for the L, M and S segments, respectively).

254

Parameters were set such that each accepted read had to map to the reference

255

sequence for at least 50% of its length, with a minimum of 80% identity to the

256

reference. The complete genome of Adana virus consists of 6,405 nts, 4,229 nts and

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1,758 nts for the L, M and S segment, respectively (GenBank acc. no KJ939330,

258

KJ939331, and KJ939332). The polymerase gene encodes a 6,288-nt long ORF

259

(2,096 AA), whereas the glycoprotein gene encodes a 4,005-nt long ORF (1,335 AA).

260

The small segment encodes a 744-nt and an 819-nt long ORFs which are translated

261

to a nucleocapsid protein (248 AA) and a nonstructural protein (273 AA),

262

respectively. Sequences obtained using NGS were confirmed by direct sequencing

263

performed on overlapping PCR products using Sanger sequencing.

264 265

Genetic distances

266

Pairwise distances of the nucleotide and amino acid sequences among ADAV and

267

viruses in the Salehabad virus complex as well as other phleboviruses are shown in

268

Table 2. Amino acid pairwise distances between ADAV and other Salehabad

269

complex viruses were ≥ 21.4% (N), ≥ 25.3% (NS), ≥ 26.1 % (Gn), ≥ 15.4 % (Gc) and

270

≥ 11.5% (L), whereas compared with other Old World phlebovirus species they were

271

≥ 45.4% (N), 71.4% (NS), 57.7 % (Gn), 51.1 % (Gc) and 40.5% (L).

272

To determine if it was possible to distinguish the species using quantitative genetic

273

data, the distribution of amino acid genetic distance was studied independently for

274

each of the genes (L, Gn, Gc, N, and NS) (Supplementary Table 1) using only the

275

complete sequences in the Genbank database . For each of the 9 species

276

recognized by the ICTV, interspecies cut-off values and the highest distance

277

observed between ADAV and other members of the Salehabad species were

11

278

indicated on the histograms. The highest amino acid distances observed between

279

ADAV and Salehabad species for the L, Gn, Gc, N and NS genes are 15.3%, 35.6%,

280

28.3%, 21.8%, and 32.2%, respectively. When compared gene by gene, these

281

distances are consistently lower than the lowest distances observed between ADAV

282

and phleboviruses other than Salehabad species which are shown in Supplementary

283

Table 1 as 40.0%, 58.1%, 50.6%, 44.4%, and 70.8% for the L, Gn, Gc, N and NS

284

genes, respectively. The lowest interspecific distances detected for the L, Gn, Gc, N

285

and NS genes as 40.0%, 46.2%, 33.6%, 35.8%, and 54.8%, respectively among

286

phlebovirus species groups were higher than the lowest distances observed between

287

ADAV and Salehabad species when compared gene by gene (species groups

288

defined by the ICTV (1). They are indicated in different colours in Supplementary

289

Table 1). This suggests that ADAV may be included in the Salehabad species group.

290 291

Phylogenetic Analysis

292

ADAV belongs to the cluster that comprises viruses belonging to the Salehabad

293

species, regardless of the viral gene used for analysis. The monophyly of the 3

294

viruses (SALV, ARBV, and ADAV) is supported with bootstrap values ≥ 99% for the 4

295

ORFs (Fig. 3). In phylogenetic analysis (Fig. 3), the major nodes enable identification

296

of the virus species, and confirm previously reported topologies (30, 33, 38, 39). For

297

comparison, we also performed Maximum likelihood analysis which showed the

298

same phlylogenetic relationships for all the gene segments (data not shown).

299 300

Microneutralisation-based (MN) seroprevalence study

301

Detailed results are presented in Table 3 and Fig 1. A total of 124 dog sera were

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collected from the Adana region, of which 17 (13.7%) contained neutralising

12

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antibodies against ADAV. These 124 sera consisted of 2 batches of 35 and 89 sera,

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respectively. Detailed information (village, sex, age) and the nature of dog usage

305

(hunting, guard, sheep dog, pet, and village dog) were available for the 89-sera batch

306

only. There was no correlation between these parameters and the presence /

307

absence of neutralising antibodies against ADAV. They were all negative when

308

tested with Arbia virus.

309

A total of 1,000 human sera were collected from individuals living in the Mersin

310

region, as well as 51, 48, and 66 sera from goats, sheep and dogs, respectively. Of

311

the 1,000 human sera, only 7 had neutralising antibodies against ADAV (0.7%). In

312

contrast, 39 of 165 (23.6%) animal sera collected in Mersin were positive. All, except

313

one human serum, were negative when tested with Arbia virus.

314 315

Genotyping of sandflies in the virus-positive pool

316

The analysis of NGS reads indicated that pool #195 contained P. tobbi (675 reads),

317

P. perfiliewi (65 reads), and P. papatasi (58 reads) corresponding to cytochrome c

318

gene.

319 320

DISCUSSION

321

The first evidence for the presence of sandfly-borne phleboviruses in Turkey was

322

reported in 1976 in a neutralisation-based seroprevalence study (19). Recently,

323

widespread circulation of these viruses was revealed via seroprevalence studies,

324

clinical case reports, and a series of human cases (20, 21, 22, 23, 24, 25, 27, 28).

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Sandfly fever occurs commonly amongst local populations in three regions of Turkey

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(Mediterranean, Aegean, and Central Anatolia) as recorded in several outbreaks

327

reported since 2004 (20, 21, 22). The presence of Sandfly fever Turkey virus (SFTV)

13

328

and Toscana virus (TOSV) was established in Turkey through virus isolation and

329

molecular detection, respectively (20, 24, 25, 26, 27, 28). However, most field-based

330

studies that combined entomological and virological aspects to understand the

331

distribution of phleboviruses and their vectors have been inadequately conducted in

332

the past. One noticeable exception was a study which identified P. major sensu lato

333

as a vector of SFTV in Central Anatolia although the virus was not isolated from

334

sandflies. At the outset of this study, STFV was the only phlebovirus isolated in

335

Turkey (20).

336

In this study, from 7,731 sandflies organised in 380 pools, we isolated a novel

337

phlebovirus, tentatively named Adana virus (ADAV) from the eponymous name of the

338

studied province (Figure 1). The complete sequence of ADAV consists of 3 segments

339

of 6,405, 4,229 and 1,758 nucleotides for the L, M and S segment, respectively.

340

Genetic and phylogenetic analyses showed that the SALV-ARBV-ADAV cluster is

341

supported by high bootstrap values (≥ 99%) regardless of the gene segment used for

342

the analysis. As previously reported by Palacios et al (2013) for other Old World

343

sandfly-borne phleboviruses, the consistent grouping of ADAV together with viruses

344

belonging to the Salehabad species may exclude the mechanism of recombination in

345

the generation of ADAV.

346

According to ICTV Salehabad virus consists of two viruses: Salehabad virus isolated

347

in 1959 from sandflies in Iran (40), and Arbia virus isolated in 1988 from sandflies in

348

Italy (41).

349

The highest amino acid distances observed between ADAV and Salehabad species

350

for the L, Gn, Gc, N and NS genes are 15.3%, 35.6%, 28.3%, 21.8%, and 32.2%,

351

respectively (Table 2). These distances are consistently lower than the lowest

352

distances observed between ADAV and non-Salehabad phleboviruses (40.0%,

14

353

58.1%, 50.6%, 44.4%, and 70.8% for the L, Gn, Gc, N and NS genes, respectively)

354

(Supplementary Table 1). Thus, genetic data indicate that ADAV belongs to the

355

Salehabad virus species. This is also supported by the fact that the lowest

356

interspecific distances among ICTV recognized species (1) (40.0%, 46.2%, 33.6%,

357

35.8%, and 54.8% for the L, Gn, Gc, N and NS genes, respectively) are higher than

358

the highest distances observed between ADAV and Salehabad viruses.

359

Recently, molecular data (although not confirmed by virus isolation) support the

360

existence of other viruses in the Salehabad virus group: (i) sequences of Adria virus

361

were reported from sandflies in Albania (42); (ii) one case of meningitis was attributed

362

to Adria virus in a Greek patient with no history of travelling abroad (43); (iii) in north

363

western Turkey (Eastern Thrace), sequences related to but clearly distinct from

364

Salehabad, Arbia, ADAV or Adria viruses were detected in sandflies (44).

365

For many years, the lack of genetic data for most phleboviruses has dictated that the

366

species are defined by their serological relationships, and are distinguishable by four-

367

fold differences in two-way neutralization tests (1). We could not perform these tests

368

due to the lack of ADAV hyperimmune antisera. In a previous study, amino acid

369

pairwise distances of Gc and L were deemed suitable for delineating species of the

370

Phlebovirus genus. Cut off values for intraspecies distances were 31% (L) were observed at interspecies

372

level (30). The increased number of complete sequences available for phleboviruses

373

has drastically modified the picture and specific studies are needed to revisit the

374

possible utilisation of genetic distance for taxonomy (33, 39, 45).

375

The high rates of neutralising antibodies in domestic animal sera (13.7% for dogs in

376

the Adana region; 6.1%, 35.3% and 35.4% for dogs, goats and sheep respectively in

377

Mersin region) demonstrate that ADAV is present and circulates actively in these

15

378

contiguous regions of Mediterranean Turkey. We considered the possibility that

379

antigenic cross-reactivity with SFTV or TOSV might have biased our results.

380

However, the following points contradict this argument: (i) neutralization assay is the

381

most specific and discriminative technique for seroprevalence studies (36), (ii) we

382

employed a stringent microneutralisation assay by using 1000TCID50 of virus (for

383

both ADAV and Arbia virus), i.e. a dose that is 10 times higher than that used in other

384

studies (25, 46), (iii) none of the 289 animal sera possessed neutralising antibodies

385

against Arbia virus. These results constitute compelling evidence that the positive

386

sera contained antibodies truly elicited against ADAV and not another virus of the

387

Salehabad virus complex.

388

Salehabad species viruses were long considered a group of viruses with no medical

389

or veterinary interest. This view deserves to be revisited according to our results and

390

to recent evidence of human infection with Adria virus in Greece (43).

391

In this study, we found that 0.7% of the human sera from people living in Mersin

392

(147km far from Adana) had neutralising antibodies against ADAV. This very low

393

prevalence suggests that local populations are either not exposed to ADAV, or that

394

ADAV is poorly or not replicating in humans. Since in Adana region local populations

395

commonly live in the vicinity of domestic animals, human exposure to ADAV is likely

396

to be equivalent to that of domestic animals. Therefore, we favor the second

397

hypothesis. The 0.7% seroprevalence rate may relate to repeated exposure to virus

398

antigen through significant and repetitive contact with the virus. Similar results were

399

recently observed in Tunisia with Punique virus, where seroprevalence rates in

400

humans were 0.4% despite frequent detection in sandflies and high seroprevalence

401

in dogs (4, 36, 47). The low seroprevalence in humans suggest that ADAV is not

402

likely to present an important public health importance in exposed human

16

403

populations. However, further studies must be conducted to investigate its capacity to

404

cause febrile illness, neuroinvasive infections or other clinical manifestations in

405

humans.

406

Sandflies are present in almost all regions of Turkey due to favorable climatic and

407

ecological conditions such as temperate, humidity, compatible microhabitat and

408

social dynamics. In the study region, the most abundant species is P. tobbi (49%),

409

followed by Larroussius spp. (26%), P. papatasi (8%), S. dentata (6%), and P. p.

410

transcaucasicus (9%), P. major s. l. (1%) and P. sergenti (1%) (48). Our results

411

showing that pool #195 contained P. tobbi, P. perfiliewi and P.papatasi are consistent

412

with previously established species distribution in the Adana region (48). The region

413

is also a well-known focus of cutaneous leishmaniasis due to Leishmania infantum

414

transmitted by P. tobbi which feeds on cattle (70%) and humans (10%) according to

415

blood-meal identification (49, 50, 51). It is therefore likely that ADAV is transmitted by

416

Larroussius sandflies, most probably P. tobbi. However, further studies using

417

individual sandflies are required for indisputable identification of the vector of ADAV.

418

ADAV rates of infection in sandflies (0.01%) are lower than rates reported with other

419

phleboviruses in other countries. This rate was calculated using two RT-nested PCR

420

assays that are commonly used in such studies (31, 32). It was confirmed by using

421

two real-time RT-PCR assays specifically designed for ADAV. Firstly, although lower

422

than in other studies, the ADAV infection rate is in the same order of magnitude as

423

that of Toscana virus in Tunisia (0.03%) and in Spain (0.05%) (52, 53). Secondly, this

424

study is the first one to calculate a rate of infection for a phlebovirus belonging to the

425

Salehabad virus species.

426

Despite studies searching for phleboviruses in sandflies in Turkey using the same

427

molecular tools, ADAV was not previously identified (29, 44). Firstly, this is the only

17

428

study screening field-caught sandflies for the presence of phleboviruses in Adana

429

and Mersin. Secondly, a possible reason for these observations in a cross-sectional

430

surveillance effort is the typically-limited activity range of sandflies (54). Thirdly,

431

similar findings were observed in Central Anatolia and Eastern Thrace regions where

432

novel strains seem to be confined to relatively few sampling locations in rural areas

433

(29, 44). Collecting sandflies over longer periods may help to understand the

434

circulation of Adana virus in the same region and also in the neighbouring city Mersin

435

where seropositivities were detected.

436

Our discovery of ADAV, together with recent data (44), demonstrate that the

437

Salehabad virus species have a much greater genetic diversity and may exhibit a

438

much wider geographical distribution than initially believed. Future studies are

439

required to address these points and to confirm whether or not specific members of

440

the Salehabad virus species cause human or animal disease.

441 442

Acknowledgements

443

We would like to thank Ozge Erisoz Kasap, Asli Belen Saglam and Mehmet Karakus

444

for their contributions during the sandfly collection campaigns and Karine Barthelemy

445

for excellent technical assistance in the Sanger and next generation sequencing. We

446

are very grateful to Prof. Ernest Gould for proofreading and editing the final

447

manuscript. The research leading to these results has received funding from the

448

European Union’s Seventh Framework Programme for research, technological

449

development and demonstration under Grant Agreement numbers 261504-EDENext-

450

FP7 (http://www.edenext.eu) and FP7 CAPACITIES project GA no228292-EVA

451

(European Virus Archive, http://www.european-virus-archive.com/). This article is

18

452

catalogued by the EDENext Steering Committee as EDENext-number pending. The

453

work of RNC was done under the frame of EurNegVec COST Action TD1303.

454 455

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655

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656 657

FIGURE LEGENDS

658

FIG. 1. Geographic representation of the results.

659

FIG. 2. Negative staining electron microscopy of Vero cell culture supernatant

660

medium at day five post infection with the Adana virus.

661

FIG. 3. Phylogenetic analysis of the phlebovirus amino acid sequences. (a) L protein,

662

(b) Gn protein, (c) Gc protein, (d), Nucleocapsid protein, (e) Non-structural protein.

663

The recognized species by ICTV were indicated in bold and italics and the tentative

664

species were indicated in bold. The GenBank accession numbers of all the

665

phleboviruses included in the analysis can be found in the Supplementary table.

666 667

TABLE FOOTNOTES

668

Table 1. . Distribution of sandfly specimens and pools according to the sampling

669

locations in Adana, Mediterranean region of Turkey.

670

Table 2. Estimates (%) of evolutionary divergence between sequences of the A. L, B.

671

Gn C. Gc D. N and E. Ns genes of the selected phleboviruses and the Adana virus.

672

*Partial sequences. The upper-right matrix represents pairwise distances between

673

amino acids alignments. The lower-left matrix represents pairwise distances between

674

nucleotides alignments. Genbank accession numbers are in the following order: L

27

675

segment; KJ939330, JX472400, JX472403, HM043726, HM566144, HM566174,

676

NC_015412, EF095551, KF297909, HM566172, JF939846, EU725771, NC_006319.

677

Gn and Gc: KJ939331, JX472401, JX472404, HM566143, HM566173, NC_015411,

678

AY129740, KF297907, HM566171, JF939847, EU725772, EU003177. S segment:

679

KJ939332, JX472402, JX472405, HM566145, HM566175, NC_015413, EF201827,

680

KF297914, EF201829, JF939848, EU725773, NC_006318.

681

Table 3. Distribution of Adana virus neutralising antibodies according to sampling

682

locations and species.

683 684

28

A.

Adana Virus-KJ939330

100

Arbia Virus-JX472400 100

Salehabad Virus-JX472403

100

Arumowot Virus-HM566144

50

Odrenisrou Virus-HM566174

100 39

Aguacate virus group species

100

58

Rift Valley Fever Virus- DQ375430 Karimabad Virus-KF297909

59

Gabek Forest Virus-KF297903

100 99

Sandfly Fever Turkey Virus- GQ847513 Candiru virus group species

100

100

Saint Floris Virus-JF920136 Gordil Virus-KF297900 100

100 99

Sandfly Fever Naples Virus Sequences Granada Virus-GU135606

100 100

100

Massilia Virus-EU725771 Punique Virus-JF920133 Sandfly Fever Naples Virus--JF920139

95

53

Tehran Virus-JF939846 100

Toscana Virus Sequences

100

Uukuniemi virus group species

100

100

Bhanja Virus-JQ956376 Palma Virus-JQ956379 Heartland Virus-JX005847

100 100

0.05

Severe Fever Thrombocytopenia Syndrome Virus-HQ141604

B.

Adana Virus-KJ939331

100 100

Arbia Virus-JX472401 Salehabad Virus-JX472404

51

Arumowot Virus-HM566143 Odrenisrou Virus-HM566173

100

72

Rift Valley Fever Virus--DQ380193 Karimabad Virus-KF297910

100 46 98

Gabek Forest Virus-KF297904 Sandfly Fever Turkey Virus-NC_015411

100

100

36

Aguacate virus group species Punta Toro Virus-DQ363407

38 100 66

Candiru virus group species Saint Floris Virus-JF920137

92

Gordil Virus-KF297901 Massilia Virus-EU725772

100 92

100

Punique Virus-JF920134 Granada Virus-GU135607 100

Sandfly Naples Virus Sequences

93

Sandfly Fever Naples Virus -JF920140

100

68

Tehran Virus-JF939847 79

Toscana virus sequences

100

100

Uukuniemi virus group species 100

Bhanja Virus-JQ956377 Palma Virus-JQ956380 Heartland Virus-JX005845

100 100

0.1

Severe Fever Thrombocytopenia Syndrome Virus-JQ684872

C.

Adana Virus-KJ939331

100 100

Arbia Virus-JX472401 Salehabad Virus-JX472404

96

Arumowot Virus-HM566143 Odrenisrou Virus-HM566173

100

Rift Valley Fever Virus- DQ380193

83

Karimabad Virus-KF297910 100

Gabek Forest Virus-KF297904

100

32

100

Aguacate virus group species Punta Toro Virus-DQ363407

60

8935

Echarte Virus-HM119411 100

Candiru virus group species

99

Sandfly Fever Turkey Virus-NC_015411 94

Gordil Virus-KF297901 Saint Floris Virus-JF920137 100

100 85

Sandfly Fever Naples Virus Sequences Tehran Virus-JF939847

90

Sandfly Fever Naples Virus-JF920140

100

Massilia Virus-EU725772

100

98 58

Punique Virus-JF920134 Granada Virus-GU135607

60

Toscana Virus Sequences

100

Uukuniemi virus group species

100 100

Bhanja Virus-JQ956377 Palma Virus-JQ956380 Heartland Virus-JX005845

100 100

0.1

Severe Fever Thrombocytopenia Syndrome Virus-JQ684872

D. Adana Virus-KJ939332

99

Salehabad Virus-JX472405 25

Arbia Virus-JX472402

99

Odrenisrou Virus-HM566175 Arumowot Virus-HM566145

99

Rift Valley Fever Virus- DQ380157

56 10

Aguacate virus group species

99

Corfou Virus-EF201821 85

Sandfly Fever Sicilian Virus-EF201827

99

6

Sandfly Fever Cyprus Virus-GU119908

99 90

Sandfly Fever Turkey Virus-NC_015411

Karimabad Virus-KF297914

8

Gabek Forest Virus-KF297905

87

Punta Toro Virus-EF201835

97

Buenaventura Virus-EF201839 62 15

Candiru virus group species

99

99

Frijoles virus group species

82 99

Salobo virus group species Gordil Virus-KF297902

43

Saint Floris Virus-JF920138 99

Tehran Virus-JF939848

58

Sandfly Fever Naples Virus- EF201831

51 99 99 63

Granada Virus-GU135608 Massilia Virus-EU725773

Punique Virus-GQ165520 99

31

Sandfly Fever Naples Virus Sequences

66 99

Toscana Virus Sequences 99

Bhanja Virus-JQ956378 Palma Virus-JQ956381 Heartland Virus-JX005843

99 99

Severe Fever with Thrombocytopenia Syndrome Virus-HQ141606 Uukuniemi virus group species

99

0.05

E.

Adana Virus-KJ939332S

87 100

Salehabad Virus-JX472405 Arbia Virus-JX472402

100

Odrenisrou Virus-HM566175 Arumowot Virus-HM566145

100 100

Aguacate virus group species

89

BeAn578142 Virus-EF201815 53

99

Frijoles virus group species

100 100

Gabek Forest Virus-KF297905 34

Karimabad Virus-KF297914

100

Corfou Virus-EF201821 Sandfly Fever Sicilian Virus-EF201827

100

Sandfly Fever Sicilian Virus-AJ811547

100 84

82 87

99

Sandfly Fever Turkey Virus-NC_015411 Sandfly Fever Cyprus Virus-GU119908 Rift Valley Fever Virus- DQ380157 Buenaventura Virus-EF201839 Punta Toro Virus-EF201835

99

42 100

Candiru virus group species

100

Saint Floris Virus-JF920138

97

Gordil Virus-KF297902 100 100

100

Massilia Virus-EU725773 Granada Virus-GU135608 Punique Virus-FJ848987

100

Sandfly Fever Naples Sequences

95 98

Sandfly Fever Naples Virus-EF201831

53

Tehran Virus-JF939848 89

Toscana Virus Sequences

100

100

Uukiniemi virus group species 100

Bhanja Virus-JQ956378 Palma Virus-JQ956381 Heartland Virus-JX005843

92 100

0.1

Severe Fever with Thrombocytopenia Syndrome Virus-HQ141606

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