Open Access

Direct evidence for an expanded circulation area of the recently identified Balkan virus (Sandfly fever Naples virus species) in several countries of the Balkan archipelago

  • Nazli Ayhan1,
  • Bulent Alten2,
  • Vladimir Ivovic3,
  • Vit Dvořák4,
  • Franjo Martinkovic5,
  • Jasmin Omeragic6,
  • Jovana Stefanovska7,
  • Dusan Petric8,
  • Slavica Vaselek8,
  • Devrim Baymak9,
  • Ozge E. Kasap2,
  • Petr Volf4 and
  • Remi N. Charrel1Email authorView ORCID ID profile
Parasites & Vectors201710:402

https://doi.org/10.1186/s13071-017-2334-y

Received: 3 March 2017

Accepted: 15 August 2017

Published: 29 August 2017

Abstract

Background

Recently, Balkan virus (BALKV, family Phenuiviridae, genus Phlebovirus) was discovered in sand flies collected in Albania and genetically characterised as a member of the Sandfly fever Naples species complex. To gain knowledge concerning the geographical area where exposure to BALKV exists, entomological surveys were conducted in 2014 and 2015, in Croatia, Bosnia and Herzegovina (BH), Kosovo, Republic of Macedonia and Serbia.

Results

A total of 2830 sand flies were trapped during 2014 and 2015 campaigns, and organised as 263 pools. BALKV RNA was detected in four pools from Croatia and in one pool from BH. Phylogenetic relationships were examined using sequences in the S and L RNA segments. Study of the diversity between BALKV sequences from Albania, Croatia and BH showed that Albanian sequences were the most divergent (9–11% [NP]) from the others and that Croatian and BH sequences were grouped (0.9–5.4% [NP]; 0.7–5% [L]). The sand fly infection rate of BALKV was 0.26% in BH and 0.27% in Croatia. Identification of the species content of pools using cox1 and cytb partial regions showed that the five BALKV positive pools contained Phlebotomus neglectus DNA; in four pools, P neglectus was the unique species, whereas P. tobbi DNA was also detected in one pool.

Conclusions

We report here (i) the first direct evidence that the Balkan virus initially described in coastal Albania has a much wider dissemination area than originally believed, (ii) two real-time RT-PCR assays that may be useful for further screening of patients presenting with fever of unknown origin that may be caused by Balkan virus infection, (iii) entomological results suggesting that Balkan virus is likely transmitted by Phlebotomus neglectus, and possibly other sand fly species of the subgenus Larroussius. So far, BALKV has been detected only in sand flies. Whether BALKV can cause disease in humans is unknown and remains to be investigated.

Keywords

Bunyaviridae Phlebovirus Arbovirus Toscana virus Meningitis Fever Sand fly Phlebotomus Phylogeny Emergence

Background

Phleboviruses (family Phenuiviridae) are arthropod-borne viruses transmitted by mosquitoes, ticks and sand flies to vertebrate hosts [1]. Several phleboviruses belong to the Sandfly fever Naples species complex (which include at least two human pathogens, namely Toscana virus causing neurological infections and Naples virus causing incapacitating febrile illness) [2]. In the Old World, sand fly-borne phleboviruses are transmitted by Phlebotomus spp. and Sergentomyia spp. and show a wide distribution in all countries of the Mediterranean basin [2], http://ecdc.europa.eu/en/healthtopics/vectors/vector-maps/Pages/VBORNET_maps_sandflies.aspx. During the last decade, several new phleboviruses were discovered in Mediterranean countries either in sand flies [39] or clinical samples [10]. Each was genetically related to any of the three following groups (based on antigenic relationships): Sandfly fever Naples species, Salehabad and Sandfly fever Sicilian/ Corfou virus group. In the Balkans, the current knowledge on circulating phleboviruses is limited. Recently, the Balkan virus (BALKV) was discovered in sand flies collected in Albania and genetically characterised as a member of the Sandfly fever Naples species complex [11]. Two specific quantitative real-time RT-PCR assays were designed to screen entomological specimens collected in the surrounding countries, i.e. Croatia, Bosnia and Herzegovina (BH), Kosovo, Republic of Macedonia (RoM), and Serbia, to gain knowledge concerning the geographical area where exposure to BALKV exists.

Methods

Sand flies were collected in the field in 2014; 10 stations in Kosovo and 8 stations in Serbia, in 2015; 5 stations in Croatia, 6 stations in BH, 5 stations in RoM, 1 station in Montenegro and 1 station in Serbia (Table 1) using a previously described method [11]. Traps were placed near animals with the consent of the owners. BALKV RNA was detected using 2 SYBR Green real-time RT-PCR specific assays targeting the polymerase gene (BALKV-L-F; 5′-CTD ATY AGY TGC TGC TAC AAT G-3′, BALKV-L-R; 5′-CCA TAA CCA AGA TAY TCA T-3′) and the nucleoprotein gene (BALKV-S-F; 5′-AGA GTR TCT GCA GCC TTT GTT CC-3′, BALKV-S-R; 5′-CAG CTA TCT CAT TAG GYT GT-3′). The cycling program consisted of 50 °C for 30 min and 95 °C for 15 min, followed by 40 cycles at 94 °C for 15 s, 60 °C for 30 s, and 72 °C for 45 s, with a final melting curve step at 95 °C for 1 min, 60 °C 30 s and 95 °C for 30 s. Melting curves for positives were at 75 °C for the polymerase assay and 79.5 °C for the nucleoprotein test.
Table 1

Trapping campaigns and geographical information of the Balkan virus positive pools

Trapping region

Number of collected sand flies

No. of pools (positive pools_gender)

Female

Male

Mix/unknown

 

2014: Kosovo

 Vermice

12

16

4

6

 Zhur

10

4

2

6

 Landrovice

11

14

2

6

 Krusha E. Vogel

0

0

2

1

 Junik

28

27

3

7

 Donji Livoc

1

0

0

1

 Cernice

2

0

0

1

 Nishor

2

4

0

2

 Studencan

18

11

2

6

 Semetiste

50

43

2

12

Total

134

119

17

48

2014: Serbia

 Aleksinac / Kraljevo

8

8

0

2

 Brest

3

3

0

2

 Arbanasce

8

0

0

1

 Prugovac

5

5

0

2

 Subotinac

0

1

1

2

 Mozgovo

0

1

0

1

 Bovan

2

0

0

1

 Jugbogdanovac

8

0

0

2

Total

34

18

1

13

2015: Bosnia and Herzegovina

 Sovici

105

77

0

11//(#B1_male)

 Mikanjici

22

18

0

4

 Zakovo

10

3

0

2

 Grab

47

0

0

5

 Stolac

55

44

0

7

 Tuli

0

5

0

1

Total

239

147

0

19

2015: Croatia

 Duba

176

129

30

18/(#C13_male)

 Jesenice

81

0

25

6

 Gorna Ljuta

22

18

2

4

 Zvekovica

12

9

0

3

 Vidonje

490

55

404

47/(#C41, #C50, #C51_females)

Total

781

211

461

78

2015: Montenegro

 Ozrinici

21

14

2

4

Total

21

14

2

4

2015 Romania

 Mokrino

48

91

3

26

 Kezhovica

85

10

42

15

 Dedeli

25

7

1

10

 Suvo Grlo

274

0

0

30

 Furka

11

4

1

4

Total

443

112

47

85

2015: Serbia

 Krasava

19

9

1

16

Total

19

9

1

16

Grand total

1671

630

529

263

Phylogenetic relationships were reconstructed using sequences of the S and L RNA segments. Positive samples were PCR-amplified targeting a portion of the polymerase [12] and the nucleoprotein genes [13, 14] (two systems producing overlapping sequences which were concatenated before analysis). Sand fly species identification within positive pools was performed using as previously described cytochrome c oxidase subunit 1 (cox1) and cytochrome b (cytb) barcoding gene regions followed by NGS sequencing of the corresponding PCR products [11]. A 50 μl-volume of BALKV positive pools was inoculated onto Vero cells for attempting virus isolation [7, 9].

Results

In 2014 a total of 270 and 53 sand flies were collected from Kosovo and Serbia, respectively. In 2015, 1453, 386, 37, 602 and 29 sand flies were trapped in Croatia, BH, Montenegro, RoM and Serbia, respectively (Table 1). BALKV RNA was detected in 4 pools from Croatia (3 collected in Vidonje [C41, C50, C51 at 42.98244N, 17.64294E (240 m)], 1 in Duba [C13 at 42.60032N, 18.33946E (475 m)]) and in 1 pool from BH in Sovici (B1 at 43.408240N, 17.329175E, 283 m) (Table 1, Fig. 1).
Fig. 1

Geographical distribution of the sand fly trapping stations. Black circles denote trapping stations. Red circles denote trapping stations in which at least one pool was found to contain Balkan virus RNA

Although not quantitative, the low Ct values observed with the polymerase gene (Ct range 19.9–24.4) and the nucleoprotein gene (Ct range 19.8–32.8) SYBR Green real-time RT-PCR was indicative of high viral load in the positive pools. Phylogeny was reconstructed by using sequences in the S and L RNA segments that were 572 nt (Fig. 2) and 525 nt long, respectively (Fig. 3). Identical groupings were observed using both markers. BALKV formed a homogenous cluster with common ancestor supported by a high bootstrap value. BALKV was included in the subgroup I of the Sandfly fever Naples species complex together with SFNV, Tehran, Zerdali and Fermo viruses.
Fig. 2

Phylogeny of the Balkan virus and closely related phleboviruses using partial nucleotide sequences of the nucleoprotein gene (572 nt). Neighbor-joining analysis (Kimura 2-parameter model) was performed using MEGA6, with 1000 bootstrap replicates

Fig. 3

Phylogeny of the Balkan virus and closely related phleboviruses using partial nucleotide sequences of the polymerase gene (525 nt). Neighbor-joining analysis (Kimura 2-parameter model) was performed using MEGA6, with 1000 bootstrap replicates

For pool B1, failure to obtain a positive PCR with Nphlebo primers led us to sequence the 136 bp SYBR Green RT-qPCR product for genetic and phylogenetic analysis. Study of the diversity between BALKV sequences from Albania, Croatia and BH showed that (i) Albanian sequences were the most divergent (9–11% [NP]) from the others, and (ii) that Croatian and BH sequences were grouped (0.9–5.4% [NP]; 0.7–5% [L]) (GenBank: KY662276–KY662287).

Identification of the sand fly species contained in the BALKV-positive pools detected Phlebotomus neglectus sequences in all five pools; P. neglectus was the unique species in four pools, whereas P. tobbi DNA was present in 1 pool from Croatia (Table 2).
Table 2

Details of the Balkan virus positive pools with sandfly species identification using cytochrome b and cox1 sequences

Trapping locality

Pool code

Sand fly species

Gene

Reads

No. of sand flies

Gender

Collection date

Altitude (m)

Bosnia and Herzegovina

 Sovici

B1

P. neglectus

cytb

1427

27

male

06/07/2015

283

  

cox1

4257

   

Croatia

 Duba

C13

P. tobbi

cytb

1211

20

male

13/07/2015

475

  

cox1

546

   
 

P. neglectus

cytb

967

   
  

cox1

7351

   

 Vidonje

C41

P. neglectus

cytb

950

20

female

16/07/2015

240

  

cox1

8182

   

 Vidonje

C50

P. neglectus

cytb

1834

20

female (bf)

16/07/2015

240

  

cox1

5302

   

 Vidonje

C51

P. neglectus

cytb

3143

20

female (bf)

16/07/2015

240

  

cox1

22,867

   

Discussion

The Balkan Peninsula is the region where sand fly fever was first described at the end of the nineteenth century in BH [15, 16]. Subsequent studies provided direct and indirect evidence for the presence of viruses belonging to the SFNV in BH [1721]. In Croatia, antibodies against SFNV were found in human populations, with highest rates (up to 53.9%) observed on islands and in coastal regions [18, 2227]. BALKV belongs to the Sandfly fever Naples species complex where it is most closely to Fermo, SFNV YU 8–76, Zerdali and Tehran viruses isolated in Italy, Serbia, Turkey and Iran which are grouped in the subgroup I [6, 9, 19, 28] (Figs. 2, 3). BALKV was first detected from two sand fly pools from Albania, Kruje region [11]. Here, we demonstrated that BALKV has a much larger circulation area that seems to be confined to the Adriatic coast of the Balkan Peninsula. This merits further confirmation through similar studies conducted north and south of the current study area (Fig. 1).

To our knowledge, BALKV is the first phlebovirus to be genetically identified in BH. Assuming that each positive pool contained one infected sand fly only, the sand fly infection rate of BALKV is 0.26% in BH and 0.27% in Croatia; which is higher than Zerdali virus (0.035%) and similar to Fermo virus (0.20%) [6, 9]. Identification of the species content of pools using cox1 and cytb showed that P. neglectus is the only species to be found in all BALKV RNA positive pools; indicating that this species might be the vector of BALKV. Interestingly, P. neglectus belongs to subgenus Larroussius, similar to P. tobbi which seems to be a typical vector for Zerdali virus, another member of the Sandfly fever Naples species [9]. Together, these data support the hypothesis that Larroussius sand flies are typical vectors of the members of this virus group.

Conclusions

We report here (i) the first direct evidence that Balkan virus initially described in Coastal Albania has a much wider dissemination area than originally believed, (ii) two real-time RT-PCR assays that may be useful for further screening of patients presenting with fever of unknown origin that may be caused by Balkan virus infection, (iii) entomologic results suggesting that Balkan virus is likely transmitted by Phlebotomus neglectus, and possibly other sand fly species of the subgenus Larroussius. So far, BALKV has been detected only in sand flies. Whether BALKV can cause disease in humans is unknown and remains to be investigated.

Abbreviations

BALKV: 

Balkan virus

BH: 

Bosnia and Herzegovina

L: 

Large RNA segment

NGS: 

Next generation sequencing

NP: 

Nucleoprotein

RoM: 

Republic of Macedonia

RT-PCR: 

Reverse transcriptase polymerase chain reaction

S: 

Small RNA segment

Declarations

Acknowledgements

The authors wish to thank Karine Almani for excellent technical assistance. The work of RNC was done under the frame of EurNegVec (TD1303) COST Action. NA is a PhD student supported by a grant from Fondation Mediterranee Infection.

Funding

This work was supported by funds received from (i) VectorNet, a European network for sharing data on the geographic distribution of arthropod vectors, transmitting human and animal disease agents (Contract OC/EFSA/ AHAW/2013/02-FWC1) funded by the European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (ECDC) (http://ecdc.europa.eu/en/healthtopics/vectors/VectorNet/Pages/VectorNet.aspx), (ii) the European Virus Archive goes Global (EVAg) project in the European Union’s Horizon 2020 research and innovation programme under grant agreement No 653316 (http://global.european-virus-archive.com/). Nazli Ayhan is a PhD student supported by a grant from Fondation Mediterranee Infection. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Availability of data and materials

Sequences generated in this study are available in the GenBank database under the accession numbers KY662276–KY662287.

Authors’ contributions

NA participated in field work, performed PCR and sequencing, and wrote the original MS; BA, VI, FM, JO, JS, DP, DB and PV organized and participated in the field work; VD set-up of PCR-based NGS identification of sand flies; SV participated to field work, performed PCR-based NGS identification; RNC analysed results, and coordinated the lab work. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Traps were placed near animals with the consent of the owners.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Authors’ Affiliations

(1)
UMR “Emergence des Pathologies Viralesˮ (EPV: Aix-Marseille Univ – IRD 190 – Inserm 1207 – EHESP – IHU Méditerranée Infection)
(2)
Faculty of Science, Department of Biology, Ecology Division, VERG Labs, Hacettepe University
(3)
University of Primorska, FAMNIT
(4)
Faculty of Science, Department of Parasitology, Charles University
(5)
Faculty of Veterinary Medicine, Department of Parasitology and Parasitic Diseases with Clinics, University of Zagreb
(6)
Department of Parasitology, Veterinary Faculty of Sarajevo
(7)
Department of Parasitology and Parasitic Diseases, Faculty of Veterinary Medicine, Ss. Cyril and Methodius University
(8)
Faculty of Agriculture, Laboratory for Medical and Veterinary Entomology, University of Novi Sad
(9)
National Institute of Public Health

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© The Author(s). 2017

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