- Email: [email protected]
First genetic characterization of Usutu virus from Culex pipiens mosquitoes Serbia, 2014
Infection, Genetics and Evolution 63 (2018) 58–61
Contents lists available at ScienceDirect
Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Short communication
First genetic characterization of Usutu virus from Culex pipiens mosquitoes Serbia, 2014
T
⁎
Gábor Kemenesia,b, , Dóra Buzása, Brigitta Zanaa,b, Kornélia Kurucza, Bosiljka Krtinicc, Anett Kepnerd, Fanni Földesa,b, Ferenc Jakaba,b a
Virological Research Group, Szentágothai Research Centre, University of Pécs, Pécs, Hungary Institute of Biology, Faculty of Sciences, University of Pécs, Pécs, Hungary c Ciklonizacija Ltd., Novi Sad, Serbia d PROPHYL Ltd., Mohács, Hungary b
A R T I C LE I N FO
A B S T R A C T
Keywords: Mosquito monitoring Culicidae European lineage 1 Vector
Since its first appearance in Europe, Usutu virus (USUV) diverged to several different genetic lineages. The virus was reported to date from multiple countries across Europe (Hungary, Italy, Switzerland, Spain, Germany, Czech Republic and Belgium). Considering the more frequently published impact of the virus on humans it is crucial to investigate locally circulating genetic variants and trace its evolution. We retrospectively analyzed mosquito samples from Serbia Vojvodina region, collected during 2014. In this study we report the results of the screening of 23,753 female mosquitoes (753 pools) for USUV-specific nucleic-acid. Out of the 753 pools sampled, the presence of USUV RNA was confirmed in 3 pools of Culex pipiens mosquitoes, collected in August. Based on their partial NS5 sequence, all strains were identical, therefore we adjusted one representative strain for complete genome sequencing. Based on phylogenetic analysis the Serbian USUV sequences were most closely related to the virus that emerged in Austria in 2001, in Hungary in 2005 and was circulating until 2015 in Hungary. This data presents a wider geographic distribution of this genetic variant and provides the first genetic data from this region.
USUV was first isolated in Africa in 1959 (Woodall, 1964), while the first evidence of the virus in Europe was noted in 1996 in Italy (Weissenböck et al., 2013). Since then, several European genetic lineages emerged and multiple independent introduction events were also suggested from Africa to Europe. Moreover, multiple dispersal events occurred between different European territories (Cadar et al., 2015; Cadar et al., 2017a, 2017b; Ziegler et al., 2016; Calzolari et al., 2017; Sieg et al., 2017). A recent publication described the exchange of USUV strains between Italy, Austria and Hungary; although until 2015 European lineage 1 genetic variant was described as the dominant locally circulating strain in Austria and Hungary (Bakonyi et al., 2017a). From Serbia, only serologic evidence of USUV is available, which permits any investigation on the possible origin of locally circulating genetic variants. USUV-specific antibodies were detected only in horse, wild bird and wild boar specimens in seroepidemiological studies conducted in the region so far (Lupulovic et al., 2011; Petrovic´ et al., 2013; Escribano-Romero et al., 2015). These studies denoted the Northern territory of the country (Vojvodina province) for the co-occurrence of WNV and USUV and well supported the need of surveillance
⁎
studies in order to reveal genetic origins of these strains, however mosquito-related data is still scarce from the region. In this study we first reported molecular evidence for the presence of USUV in female mosquitoes collected in 2014, Vojvodina. We also provided evidence for a wider geographic distribution of the virus in Europe. Female mosquitoes were collected as part of national mosquito control activities in Vojvodina province. A total of 23,753 adult female mosquitoes were collected from urbanized, human-inhabited areas and typical mosquito breeding sites within cities and small villages, from May to October 2014. Mosquitoes were trapped with CDC light traps baited with dry ice at 59 sampling sites belong to 9 municipalities. All collected mosquitoes were transported to the laboratory on dry ice and kept frozen at −80 °C until further processing. Each specimen was determined by species according to their taxonomic keys (Becker et al., 2003) using a stereomicroscope. Specimens were grouped by a maximum of 50 individuals per sampling event, species and collection site into pools and were processed as described previously (Kemenesi et al., 2014). These pools were subjected to USUV specific nested reverse transcription - polymerase chain reaction (RT-PCR), using degenerated
Corresponding author at: Virological Research Group, Szentágothai Research Centre, University of Pécs, Ifjúság út 20., H-7624 Pécs, Hungary. E-mail address: [email protected] (G. Kemenesi).
https://doi.org/10.1016/j.meegid.2018.05.012 Received 6 February 2018; Received in revised form 11 April 2018; Accepted 16 May 2018 Available online 17 May 2018 1567-1348/ © 2018 Elsevier B.V. All rights reserved.
Infection, Genetics and Evolution 63 (2018) 58–61
G. Kemenesi et al.
KX601690 France 2015 73
HE599647 Germany 2011 KX601691 France 2015
80
KY263624 Belgium 2016
75
KY426755 Germany 2016
100 99
European 3
KY426759 Germany 2016
KY199558 Germany 2016 KY426760 Germany 2016 KX555629 Italy 2015
European 4
MG888044 Serbia 2014 EF206350 Hungary 2005 100
98
JQ219843 Austria 2002
European 1
78 AY453411 Austria 2001
JF266698 Italy 2009 95 100
KX555626 Italy 2010 KX555627 Italy 2010
98 99
HM569263 Italy 2009 MF991886 Austria 2017
81 76
European 2
MF063042 Austria 2016
87
MF063043 Hungary 2016 KC754956 Senegal 1993
100
KC754957 Senegal 2007 African 3
KC754955 Central African Republic 1981 KM659877 Germany 2014 100
KY128482 Netherlands 2016
KF573410 Spain 2006 AY453412 South Africa 1958
African 2
0.005
Fig. 1. The evolutionary history was inferred based on 10,302 nucleotide genome fragment by using the Maximum Likelihood method based on the Tamura-Nei model (+G). The best fit nucleotide substitution model was selected based on the Bayesian information criterion as implemented in the MEGA software. Scale bar indicates evolutionary distance, whilst branch support is indicated with bootstrap values. Group names are representing putative genetic lineages from Europe and Africa. Branch support values lower than 70 are not shown.
primers targeting the conserved NS5 gene of flaviviruses (Kuno et al., 1998). Amplicons originating from positive samples were purified with Gel/PCR DNA Fragments Extraction Kit (Geneaid Biotech, Taiwan) and bi-directionally sequenced with BigDye Terminator v1.1 Cycle Sequencing Kit according to the manufacturer's protocol on ABI Prism 310 DNA Sequencer platform (Applied Biosystems, USA). The complete genome of the Serbian USUV strain was amplified by RT-PCR method, resulting in four large genome segments. These large amplicons were subjected for nested PCR reactions, resulted in overlapping fragments of the genome. The amplicons were compiled into continuous sequence, using Geneious software (http://www.geneious.com) (Kearse et al., 2012). Phylogenetic analysis was implemented in MEGA 7 software based on 10,302 nucleotide genome fragments, containing the complete polyprotein coding sequence along with partial 3′ UTR, trimmed to the shortest sequence in the dataset. The most abundant sampled species was Culex pipiens (n = 11,099, 47% of all mosquito specimens), followed by Aedes vexans (n = 10,005, 42% of all mosquito specimens). Dominant species of the investigated area were Ochlerotatus caspius (n = 1574, 7%), Ochlerotatus sticticus (n = 559, 2.4%) and Coquillettidia richiardii (n = 332, 1.4%) as well. All other species were represented with considerably lower number (n < 100, < 0.5% of all mosquito specimens). A total of 1437 female mosquitoes (68 pools) were collected in May, n = 4252 (158 pools) in
June, n = 1512 (52 pools) in July, n = 13,683 (403 pools) in August, n = 54 (2 pools) in September and n = 2815 (70 pools) in October. Out of the 753 pools sampled, the presence of USUV RNA was confirmed in 3 pools of Culex pipiens mosquitoes, collected in August from Titel (45°12′N, 20°18′E, 1 positive out of 38 pools) and Zrejanin (45°22′N, 20°23′E, 2 positives out of the 330 pools). Cx. pipiens was frequently described as a primary vector of the virus across Europe (Ashraf et al., 2015; Fros et al., 2015; Nikolay, 2015; Cadar et al., 2017a). However, Cx. pipiens mosquitoes are originally considered with ornithophilic feeding behavior (Brugman et al., 2017), the two distinct biotypes (Cx. p. pipiens and Cx. p. molestus) show remarkable physiological and behavioral differences. While the Culex p. biotype pipiens rarely bite humans and seems to be strictly ornithophilic (i.e. bird biting host preference), females of the biotype molestus are mainly anthropophilic (Becker et al., 2012). At the same time, human host preference was identified in case of pipiens and molestus forms and their hybrids as well (Martínez-de la Puente et al., 2016). The adaptation of Cx. pipiens mosquitoes to human-altered environments led to their global distribution through dispersal via humans and, combined with their mixed feeding patterns on birds and mammals (including humans), predestine them as bridge vectors for pathogens transmitted between mammals and birds (Ashraf et al., 2015; Fros et al., 2015; Nikolay, 2015; Cadar et al., 2017a). Although we did not identify 59
Infection, Genetics and Evolution 63 (2018) 58–61
– –
–
–
–
–
–
K- >R (2656)
–
–
T->I (3257)
–
– – – –
C T
A G
T C
T C
C T
G A
G A
C T
T C
T G
G A G T T C T C T C C T
7119 (3)
7284 (3)
7425 (3)
7482 (3)
7656 (3)
NS5 7967 (2)
8751 (3)
9003 (3)
9770 (2)
9816 (3)
9819 (3) 7044 (3) NS4b 7041 (3) 6786 (3) NS4a 6636 (3) NS3 6360 (3)
The research activity of K.K. was supported by the Szentágothai Talent Program, awarded by the Szentágothai Research Centre, University of Pécs. F.J. and G.K. were supported by the TÁMOP (4.2.4.A/2-11-1-2012-0001)—National Excellence Program. G.K. was also supported by the National Talent Program (NTP-EFÖ-P-15-0001) of Human Capacities Management (EMET) and financed by the Hungarian Ministry of Human Capacities (EMMI). G.K. and F.J. was supported by the ÚNKP-17-3-III and ÚNKP-17-4-III – New Excellence Program of the Ministry of Human Capacities. A.K was supported by the ÚNKP-16-2-I – New National Excellence Program of the Ministry of Human Capacities. The project has been supported by the European Union, co-financed by the European Social Fund: Comprehensive Development for Implementing Smart Specialization Strategies at the University of Pécs (EFOP-3.6.1.-16-2016-00004), and by the University of Pécs in the frame of “Viral Pathogenesis” Talent Centre program.
Gene Nucleotide position (codon position) MG888044 EF206350 JQ219843 AY453411 Amino Acid mutation (position)
– F - > L (54)
T- >A (702)
–
–
L->I (1240)
–
–
–
–
–
–
– – – –
C T A G T C T C C T C T A C T C T C G A T C C T
660 (3) 507 (3) 160 (1)
Nucleotide position (codon position) MG888044 EF206350 JQ219843 AY453411 Amino Acid mutation (position)
PreM Core protein C
T C
5898 (3) 5880 (3) 5598 (3) 5376 (3) 4965 (3) 4563 (3) 3718 (1) 2104 (1)
2259 (3)
2517 (3)
NS3 NS2b NS2a NS1 Envelope
C T G A T C
Acknowledgments
6024 (3) 2010 (3) 1473 (3)
members of the Cx. pipiens complex to biotype level, mosquitoes of the molestus form are more frequently found in urban than in natural areas (Martínez-de la Puente et al., 2016) and biotype molestus was identified already in Novi Sad, Serbia (Becker et al., 2012). Regarding the close geographic location, same vector species and the 100% identity of partial NS5 sequences, we subjected one positive pool for longer genomic fragment amplification and used it as a reference strain from the region. The complete coding region of the virus was determined along with a partial 3′ UTR fragment (Accession number: MG888044) with a putative polyprotein encoding open reading frame of 3434 amino acid. Representative members of known putative genetic lineages were selected (Africa 2 and 3; Europe 1–4) to infer presumed evolutionary relationships to known genetic variants. Africa 1 genetic variant (KC754958) was excluded from the analysis for a better resolution of the tree. Based on phylogeny results the Serbian USUV strain from 2014 was unambiguously clustered to European genetic lineage 1 clade which was originally emerged in Austria, 2001 (Fig. 1). Unique mutation on both nucleotide and amino acid level compared to the most closely related sequences from the European genetic lineage 1 are summarized in Table 1. Functional analyses are needed to decipher the possible role of these mutations on the pathogenicity of this strain. The presence of this lineage in Serbia indicates a geographic expansion to southward territories in the Balkanian peninsula (Fig. 2) and urges the establishment of surveillance system in neighboring countries as well, especially where serologic evidence of the virus was already published (e.g. Croatia) (Barbic et al., 2013; Vilibic-Cavlek et al., 2014; Santini et al., 2015). It is not clear if the serologic evidence of USUV in the Southern part of the Balkans in Greece indicates the circulation of the same genetic variant, or it is an evidence for the presence of another genetic lineage of USUV (Chaintoutis et al., 2014). Intensified surveillance efforts are needed to clarify this. The implementation of such a program could serve to clarify if this genetic lineage reached a wide distribution on Balkan Peninsula or other genetic variants are co-circulating. Based on several evidences of human infection with USUV (Bakonyi et al., 2017b; Percivalle et al., 2017; Grottola et al., 2017; Cadar et al., 2017b), it is necessary to expand the surveillance efforts to human samples and note the presence of the virus in the region for public health authorities and blood transfusion services. This work represents the first evidence for the geographic expansion of European lineage 1 to southern territories in the Balkanian peninsula and provides the first genetic data of USUV in the region. However it is necessary to further investigate locally circulating strains to clarify if any possible strain exchange as published recently from Hungary and Austria (Bakonyi et al., 2017a).
Gene
Table 1 Unique mutations of the study sequence compared to the most closely related sequences from the European genetic lineage 1 with available complete ORF coding sequence (EF206350 – Hungary 2005; JQ219843 – Austria 2002; AY453411 – Austria 2001). Amino acid mutations and their positions within the amino acid sequence of the complete open reading frame are indicated with bold letters.
G. Kemenesi et al.
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Infection, Genetics and Evolution 63 (2018) 58–61
G. Kemenesi et al.
Fig. 2. Places of mosquito collection in Vojvodina province, Serbia. Black dots represent positive sampling sites for Usutu virus RNA. Mosquito numbers involved from each sampling points are shown along with the number of tested mosquito pools from each location. Positive pool numbers are indicated in brackets.
Conflicts of interest
West Nile virus transmission potential by local Culex pipiens mosquitoes in NorthWestern Europe. One Health. 1, 31–36. Grottola, A., Marcacci, M., Tagliazucchi, S., Gennari, W., Di Gennaro, A., Orsini, M., Monaco, F., Marchegiano, P., Marini, V., Meacci, M., Rumpianesi, F., Lorusso, A., Pecorari, M., Savini, G., 2017. Usutu virus infections in humans: a retrospective analysis in the municipality of Modena, Italy. Clin. Microbiol. Infect. 23, 33–37. Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., Buxton, S., Cooper, A., Markowitz, S., Duran, C., Thierer, T., Ashton, B., Mentjies, P., Drummond, A., 2012. Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28, 1647–1649. Kemenesi, G., Krtinić, B., Milankov, V., Kutas, A., Dallos, B., Oldal, M., Somogyi, N., Németh, V., Bányai, K., Jakab, F., 2014. West Nile virus surveillance in mosquitoes, April to October 2013, Vojvodina province, Serbia: implications for the 2014 season. Euro. Surveill. 19, 20779. Kuno, G., Chang, G.J.J., Tsuchiya, K.R., Karabatsos, N., Cropp, C.B., 1998. Phylogeny of the genus Flavivirus. J. Virol. 72, 73–83. Lupulovic, D., Martin-Acebes, M.A., Lazic, S., Alonso-Padilla, J., Blazquez, A.B., Escribano-Romero, E., Petrovic´, T., Saiz, J.C., 2011. First serological evidence of West Nile virus activity in horses in Serbia. Vector Borne Zoonotic Dis. 11, 1303–1305. Martínez-de la Puente, J., Ferraguti, M., Ruiz, S., Roiz, D., Soriguer, R.C., Figuerola, J., 2016. Culex pipiens forms and urbanization: effects on blood feeding sources and transmission of avian Plasmodium. Malar. J. 15, 589. Nikolay, B., 2015. A review of West Nile and Usutu virus co-circulation in Europe: how much do transmission cycles overlap? Trans. Royal Soc. Trop. Med. Hyg 109, 609–618. Percivalle, E., Sassera, D., Rovida, F., Isernia, P., Fabbi, M., Baldanti, F., Marone, P., 2017. Usutu virus antibodies in blood donors and healthy forestry Workers in the Lombardy Region, northern Italy. Vector Borne Zoonotic Dis. 17, 658–661. Petrovic´, T., Blazquez, A., Lupulovic, D., Lazic, G., Escribano-Romero, E., Fabijan, D., Kapetanov, M., Lazic, S., Saiz, J., 2013. Monitoring West Nile virus (WNV) infection in wild birds in Serbia during 2012: first isolation and characterisation of WNV strains from Serbia. Euro. Surveill. (44), 20622. Santini, M., Vilibic-Cavlek, T., Barsic, B., Barbic, L., Savic, V., Stevanovic, V., Listes, E., Di Gennaro, A., Savini, G., 2015. First cases of human Usutu virus neuroinvasive infection in Croatia, August-September 2013: clinical and laboratory features. J. Neurovirol. 21, 92–97. Sieg, M., Schmidt, V., Ziegler, U., Keller, M., Höper, D., Heenemann, K., Rückner, A., Nieper, H., Muluneh, A., Groschup, M.H., Vahlenkamp, T.W., 2017. Outbreak and Cocirculation of three different Usutu virus strains in eastern Germany. Vector-Borne and Zoonotic Diseases. 17, 662–664. Vilibic-Cavlek, T., Kaic, B., Barbic, L., Pem-Novosel, I., Slavic-Vrzic, V., Lesnikar, V., Kurecic-Filipovic, S., Babic-Erceg, A., Listes, E., Stevanovic, V., Gjenero-Margan, I., Savini, G., 2014. First evidence of simultaneous occurrence of West Nile virus and Usutu virus neuroinvasive disease in humans in Croatia during the 2013 outbreak. Infection 42, 689–695. Weissenböck, H., Bakonyi, T., Rossi, G., Mani, P., Nowotny, N., 2013. Usutu virus, Italy, 1996. Emerg. Infect. Dis. 19, 274–277. Woodall, J.P., 1964. The viruses isolated from arthropods at the East African Virus Research Institute in the 26 years ending December. Proc. E. Afr. Acad. II, 141–146. Ziegler, U., Fast, C., Eiden, M., Bock, S., Schulze, C., Hoeper, D., Ochs, A., Schlieben, P., Keller, M., Zielke, D.E., Luehken, R., Cadar, D., Walther, D., Schmidt-Chanasit, J., Groschup, M.H., 2016. Evidence for an independent third Usutu virus introduction into Germany. Vet. Microbiol. 192, 60–66.
The authors declare that there are no conflicts of interest. References Ashraf, U., Ye, J., Ruan, X., Wan, S., Zhu, B., Cao, S., 2015. Usutu Virus: An emerging Flavivirus in Europe. Viruses. 7, 219–238. Bakonyi, T., Erdélyi, K., Brunthaler, R., Dán, Á., Weissenböck, H., Nowotny, N., 2017a. Usutu virus, Austria and Hungary, 2010–2016. Emerg. Microb. & Infect.(10), e85. Bakonyi, T., Jungbauer, C., Aberle, S.W., Kolodziejek, J., Dimmel, K., Stiasny, K., Allerberger, F., Nowotny, N., 2017b. Usutu virus infections among blood donors, Austria, July and August 2017 – raising awareness for diagnostic challenges. Euro. Surveill. (41), 17–00644. Barbic, L., Vilibic-Cavlek, T., Listes, E., Stevanovic, V., Gjenero-Margan, I., LjubinSternak, S., Pem-Novosel, I., Listes, I., Mlinaric-Galinovic, G., Di Gennaro, A., Savini, G., 2013. Demonstration of Usutu virus antibodies in horses, Croatia. Vector Borne Zoonotic Dis. (10), 772–774. Becker, N., Petrić, D., Zgomba, M., Boase, C., Madon, M., Dahl, C., et al., 2003. Mosquitoes and their Control. Vol. 498 Kluwer Academic/Plenum Publisher, New York. Becker, N., Jöst, A., Weitzel, T., 2012. The Culex pipiens complex in Europe. J. Am. Mosq. Control Assoc. 28, 53–67. Brugman, V.A., Hernández-Triana, L.M., England, M.E., Medlock, J.M., Mertens, P.P.C., Logan, J.G., Wilson, A.J., Fooks, A.R., Johnson, N., Carpenter, S., 2017. Blood-feeding patterns of native mosquitoes and insights into their potential role as pathogen vectors in the Thames estuary region of the United Kingdom. Parasit. Vectors 10, 163. Cadar, D., Bosch, S., Jöst, H., Börstler, J., Garigliany, M.M., Becker, N., Schmidt-Chanasit, J., 2015. Putative lineage of novel African Usutu virus, Central Europe. Emerg. Infect. Dis. 21, 1647–1650. Cadar, D., Lühken, R., van der Jeugd, H., Garigliany, M., Ziegler, U., Keller, M., Lahoreau, J., Lachmann, L., Becker, N., Kik, M., Oude Munnink, B.B., Bosch, S., Tannich, E., Linden, A., Schmidt, V., Koopmans, M.P., Rijks, J., Desmecht, D., Groschup, M.H., Reusken, C., Schmidt-Chanasit, J., 2017a. Widespread activity of multiple lineages of Usutu virus, western Europe, 2016. Euro. Surveill. 22, 30452. Cadar, D., Maier, P., Müller, S., Kress, J., Chudy, M., Bialonski, A., Schlaphof, A., Jansen, S., Jöst, H., Tannich, E., Runkel, S., Hitzler, W.E., Hutschenreuter, G., Wessiepe, M., Schmidt-Chanasit, J., 2017b. Blood donor screening for West Nile virus (WNV) revealed acute Usutu virus (USUV) infection, Germany, September 2016. Euro. Surveill. 22, 30501. Calzolari, M., Chiapponi, C., Bonilauri, P., Lelli, D., Baioni, L., Barbieri, I., Lavazza, A., Pongolini, S., Dottori, M., Moreno, A., 2017. Co-circulation of two Usutu virus strains in northern Italy between 2009 and 2014. Infect. Genet. Evol. 51, 255–262. Chaintoutis, S.C., Dovas, C.I., Papanastassopoulou, M., Gewehr, S., Danis, K., Beck, C., Lecollinet, S., Antalis, V., Kalaitzopoulou, S., Panagiotopoulos, T., Mourelatos, S., Zientara, S., Papadopoulos, O., 2014. Evaluation of a West Nile virus surveillance and early warning system in Greece, based on domestic pigeons. Comp. Immunol. Microbiol. Infect. Dis. 37, 131–141. Escribano-Romero, E., Lupulovic´, D., Merino-Ramos, T., Blázquez, A.B., Lazic´, G., Lazic´, S., Saiz, J.C., Petrovic, T., 2015. West Nile virus serosurveillance in pigs, wild boars, and roe deer in Serbia. Vet. Microbiol. 176, 365–369. Fros, J.J., Miesen, P., Vogels, C.B., Gaibani, P., Sambri, V., Martina, B.E., Koenraadt, C.J., van Rij, R.P., Vlak, J.M., Takken, W., Pijlman, G.P., 2015. Comparative Usutu and
61
Contents lists available at ScienceDirect
Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Short communication
First genetic characterization of Usutu virus from Culex pipiens mosquitoes Serbia, 2014
T
⁎
Gábor Kemenesia,b, , Dóra Buzása, Brigitta Zanaa,b, Kornélia Kurucza, Bosiljka Krtinicc, Anett Kepnerd, Fanni Földesa,b, Ferenc Jakaba,b a
Virological Research Group, Szentágothai Research Centre, University of Pécs, Pécs, Hungary Institute of Biology, Faculty of Sciences, University of Pécs, Pécs, Hungary c Ciklonizacija Ltd., Novi Sad, Serbia d PROPHYL Ltd., Mohács, Hungary b
A R T I C LE I N FO
A B S T R A C T
Keywords: Mosquito monitoring Culicidae European lineage 1 Vector
Since its first appearance in Europe, Usutu virus (USUV) diverged to several different genetic lineages. The virus was reported to date from multiple countries across Europe (Hungary, Italy, Switzerland, Spain, Germany, Czech Republic and Belgium). Considering the more frequently published impact of the virus on humans it is crucial to investigate locally circulating genetic variants and trace its evolution. We retrospectively analyzed mosquito samples from Serbia Vojvodina region, collected during 2014. In this study we report the results of the screening of 23,753 female mosquitoes (753 pools) for USUV-specific nucleic-acid. Out of the 753 pools sampled, the presence of USUV RNA was confirmed in 3 pools of Culex pipiens mosquitoes, collected in August. Based on their partial NS5 sequence, all strains were identical, therefore we adjusted one representative strain for complete genome sequencing. Based on phylogenetic analysis the Serbian USUV sequences were most closely related to the virus that emerged in Austria in 2001, in Hungary in 2005 and was circulating until 2015 in Hungary. This data presents a wider geographic distribution of this genetic variant and provides the first genetic data from this region.
USUV was first isolated in Africa in 1959 (Woodall, 1964), while the first evidence of the virus in Europe was noted in 1996 in Italy (Weissenböck et al., 2013). Since then, several European genetic lineages emerged and multiple independent introduction events were also suggested from Africa to Europe. Moreover, multiple dispersal events occurred between different European territories (Cadar et al., 2015; Cadar et al., 2017a, 2017b; Ziegler et al., 2016; Calzolari et al., 2017; Sieg et al., 2017). A recent publication described the exchange of USUV strains between Italy, Austria and Hungary; although until 2015 European lineage 1 genetic variant was described as the dominant locally circulating strain in Austria and Hungary (Bakonyi et al., 2017a). From Serbia, only serologic evidence of USUV is available, which permits any investigation on the possible origin of locally circulating genetic variants. USUV-specific antibodies were detected only in horse, wild bird and wild boar specimens in seroepidemiological studies conducted in the region so far (Lupulovic et al., 2011; Petrovic´ et al., 2013; Escribano-Romero et al., 2015). These studies denoted the Northern territory of the country (Vojvodina province) for the co-occurrence of WNV and USUV and well supported the need of surveillance
⁎
studies in order to reveal genetic origins of these strains, however mosquito-related data is still scarce from the region. In this study we first reported molecular evidence for the presence of USUV in female mosquitoes collected in 2014, Vojvodina. We also provided evidence for a wider geographic distribution of the virus in Europe. Female mosquitoes were collected as part of national mosquito control activities in Vojvodina province. A total of 23,753 adult female mosquitoes were collected from urbanized, human-inhabited areas and typical mosquito breeding sites within cities and small villages, from May to October 2014. Mosquitoes were trapped with CDC light traps baited with dry ice at 59 sampling sites belong to 9 municipalities. All collected mosquitoes were transported to the laboratory on dry ice and kept frozen at −80 °C until further processing. Each specimen was determined by species according to their taxonomic keys (Becker et al., 2003) using a stereomicroscope. Specimens were grouped by a maximum of 50 individuals per sampling event, species and collection site into pools and were processed as described previously (Kemenesi et al., 2014). These pools were subjected to USUV specific nested reverse transcription - polymerase chain reaction (RT-PCR), using degenerated
Corresponding author at: Virological Research Group, Szentágothai Research Centre, University of Pécs, Ifjúság út 20., H-7624 Pécs, Hungary. E-mail address: [email protected] (G. Kemenesi).
https://doi.org/10.1016/j.meegid.2018.05.012 Received 6 February 2018; Received in revised form 11 April 2018; Accepted 16 May 2018 Available online 17 May 2018 1567-1348/ © 2018 Elsevier B.V. All rights reserved.
Infection, Genetics and Evolution 63 (2018) 58–61
G. Kemenesi et al.
KX601690 France 2015 73
HE599647 Germany 2011 KX601691 France 2015
80
KY263624 Belgium 2016
75
KY426755 Germany 2016
100 99
European 3
KY426759 Germany 2016
KY199558 Germany 2016 KY426760 Germany 2016 KX555629 Italy 2015
European 4
MG888044 Serbia 2014 EF206350 Hungary 2005 100
98
JQ219843 Austria 2002
European 1
78 AY453411 Austria 2001
JF266698 Italy 2009 95 100
KX555626 Italy 2010 KX555627 Italy 2010
98 99
HM569263 Italy 2009 MF991886 Austria 2017
81 76
European 2
MF063042 Austria 2016
87
MF063043 Hungary 2016 KC754956 Senegal 1993
100
KC754957 Senegal 2007 African 3
KC754955 Central African Republic 1981 KM659877 Germany 2014 100
KY128482 Netherlands 2016
KF573410 Spain 2006 AY453412 South Africa 1958
African 2
0.005
Fig. 1. The evolutionary history was inferred based on 10,302 nucleotide genome fragment by using the Maximum Likelihood method based on the Tamura-Nei model (+G). The best fit nucleotide substitution model was selected based on the Bayesian information criterion as implemented in the MEGA software. Scale bar indicates evolutionary distance, whilst branch support is indicated with bootstrap values. Group names are representing putative genetic lineages from Europe and Africa. Branch support values lower than 70 are not shown.
primers targeting the conserved NS5 gene of flaviviruses (Kuno et al., 1998). Amplicons originating from positive samples were purified with Gel/PCR DNA Fragments Extraction Kit (Geneaid Biotech, Taiwan) and bi-directionally sequenced with BigDye Terminator v1.1 Cycle Sequencing Kit according to the manufacturer's protocol on ABI Prism 310 DNA Sequencer platform (Applied Biosystems, USA). The complete genome of the Serbian USUV strain was amplified by RT-PCR method, resulting in four large genome segments. These large amplicons were subjected for nested PCR reactions, resulted in overlapping fragments of the genome. The amplicons were compiled into continuous sequence, using Geneious software (http://www.geneious.com) (Kearse et al., 2012). Phylogenetic analysis was implemented in MEGA 7 software based on 10,302 nucleotide genome fragments, containing the complete polyprotein coding sequence along with partial 3′ UTR, trimmed to the shortest sequence in the dataset. The most abundant sampled species was Culex pipiens (n = 11,099, 47% of all mosquito specimens), followed by Aedes vexans (n = 10,005, 42% of all mosquito specimens). Dominant species of the investigated area were Ochlerotatus caspius (n = 1574, 7%), Ochlerotatus sticticus (n = 559, 2.4%) and Coquillettidia richiardii (n = 332, 1.4%) as well. All other species were represented with considerably lower number (n < 100, < 0.5% of all mosquito specimens). A total of 1437 female mosquitoes (68 pools) were collected in May, n = 4252 (158 pools) in
June, n = 1512 (52 pools) in July, n = 13,683 (403 pools) in August, n = 54 (2 pools) in September and n = 2815 (70 pools) in October. Out of the 753 pools sampled, the presence of USUV RNA was confirmed in 3 pools of Culex pipiens mosquitoes, collected in August from Titel (45°12′N, 20°18′E, 1 positive out of 38 pools) and Zrejanin (45°22′N, 20°23′E, 2 positives out of the 330 pools). Cx. pipiens was frequently described as a primary vector of the virus across Europe (Ashraf et al., 2015; Fros et al., 2015; Nikolay, 2015; Cadar et al., 2017a). However, Cx. pipiens mosquitoes are originally considered with ornithophilic feeding behavior (Brugman et al., 2017), the two distinct biotypes (Cx. p. pipiens and Cx. p. molestus) show remarkable physiological and behavioral differences. While the Culex p. biotype pipiens rarely bite humans and seems to be strictly ornithophilic (i.e. bird biting host preference), females of the biotype molestus are mainly anthropophilic (Becker et al., 2012). At the same time, human host preference was identified in case of pipiens and molestus forms and their hybrids as well (Martínez-de la Puente et al., 2016). The adaptation of Cx. pipiens mosquitoes to human-altered environments led to their global distribution through dispersal via humans and, combined with their mixed feeding patterns on birds and mammals (including humans), predestine them as bridge vectors for pathogens transmitted between mammals and birds (Ashraf et al., 2015; Fros et al., 2015; Nikolay, 2015; Cadar et al., 2017a). Although we did not identify 59
Infection, Genetics and Evolution 63 (2018) 58–61
– –
–
–
–
–
–
K- >R (2656)
–
–
T->I (3257)
–
– – – –
C T
A G
T C
T C
C T
G A
G A
C T
T C
T G
G A G T T C T C T C C T
7119 (3)
7284 (3)
7425 (3)
7482 (3)
7656 (3)
NS5 7967 (2)
8751 (3)
9003 (3)
9770 (2)
9816 (3)
9819 (3) 7044 (3) NS4b 7041 (3) 6786 (3) NS4a 6636 (3) NS3 6360 (3)
The research activity of K.K. was supported by the Szentágothai Talent Program, awarded by the Szentágothai Research Centre, University of Pécs. F.J. and G.K. were supported by the TÁMOP (4.2.4.A/2-11-1-2012-0001)—National Excellence Program. G.K. was also supported by the National Talent Program (NTP-EFÖ-P-15-0001) of Human Capacities Management (EMET) and financed by the Hungarian Ministry of Human Capacities (EMMI). G.K. and F.J. was supported by the ÚNKP-17-3-III and ÚNKP-17-4-III – New Excellence Program of the Ministry of Human Capacities. A.K was supported by the ÚNKP-16-2-I – New National Excellence Program of the Ministry of Human Capacities. The project has been supported by the European Union, co-financed by the European Social Fund: Comprehensive Development for Implementing Smart Specialization Strategies at the University of Pécs (EFOP-3.6.1.-16-2016-00004), and by the University of Pécs in the frame of “Viral Pathogenesis” Talent Centre program.
Gene Nucleotide position (codon position) MG888044 EF206350 JQ219843 AY453411 Amino Acid mutation (position)
– F - > L (54)
T- >A (702)
–
–
L->I (1240)
–
–
–
–
–
–
– – – –
C T A G T C T C C T C T A C T C T C G A T C C T
660 (3) 507 (3) 160 (1)
Nucleotide position (codon position) MG888044 EF206350 JQ219843 AY453411 Amino Acid mutation (position)
PreM Core protein C
T C
5898 (3) 5880 (3) 5598 (3) 5376 (3) 4965 (3) 4563 (3) 3718 (1) 2104 (1)
2259 (3)
2517 (3)
NS3 NS2b NS2a NS1 Envelope
C T G A T C
Acknowledgments
6024 (3) 2010 (3) 1473 (3)
members of the Cx. pipiens complex to biotype level, mosquitoes of the molestus form are more frequently found in urban than in natural areas (Martínez-de la Puente et al., 2016) and biotype molestus was identified already in Novi Sad, Serbia (Becker et al., 2012). Regarding the close geographic location, same vector species and the 100% identity of partial NS5 sequences, we subjected one positive pool for longer genomic fragment amplification and used it as a reference strain from the region. The complete coding region of the virus was determined along with a partial 3′ UTR fragment (Accession number: MG888044) with a putative polyprotein encoding open reading frame of 3434 amino acid. Representative members of known putative genetic lineages were selected (Africa 2 and 3; Europe 1–4) to infer presumed evolutionary relationships to known genetic variants. Africa 1 genetic variant (KC754958) was excluded from the analysis for a better resolution of the tree. Based on phylogeny results the Serbian USUV strain from 2014 was unambiguously clustered to European genetic lineage 1 clade which was originally emerged in Austria, 2001 (Fig. 1). Unique mutation on both nucleotide and amino acid level compared to the most closely related sequences from the European genetic lineage 1 are summarized in Table 1. Functional analyses are needed to decipher the possible role of these mutations on the pathogenicity of this strain. The presence of this lineage in Serbia indicates a geographic expansion to southward territories in the Balkanian peninsula (Fig. 2) and urges the establishment of surveillance system in neighboring countries as well, especially where serologic evidence of the virus was already published (e.g. Croatia) (Barbic et al., 2013; Vilibic-Cavlek et al., 2014; Santini et al., 2015). It is not clear if the serologic evidence of USUV in the Southern part of the Balkans in Greece indicates the circulation of the same genetic variant, or it is an evidence for the presence of another genetic lineage of USUV (Chaintoutis et al., 2014). Intensified surveillance efforts are needed to clarify this. The implementation of such a program could serve to clarify if this genetic lineage reached a wide distribution on Balkan Peninsula or other genetic variants are co-circulating. Based on several evidences of human infection with USUV (Bakonyi et al., 2017b; Percivalle et al., 2017; Grottola et al., 2017; Cadar et al., 2017b), it is necessary to expand the surveillance efforts to human samples and note the presence of the virus in the region for public health authorities and blood transfusion services. This work represents the first evidence for the geographic expansion of European lineage 1 to southern territories in the Balkanian peninsula and provides the first genetic data of USUV in the region. However it is necessary to further investigate locally circulating strains to clarify if any possible strain exchange as published recently from Hungary and Austria (Bakonyi et al., 2017a).
Gene
Table 1 Unique mutations of the study sequence compared to the most closely related sequences from the European genetic lineage 1 with available complete ORF coding sequence (EF206350 – Hungary 2005; JQ219843 – Austria 2002; AY453411 – Austria 2001). Amino acid mutations and their positions within the amino acid sequence of the complete open reading frame are indicated with bold letters.
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Fig. 2. Places of mosquito collection in Vojvodina province, Serbia. Black dots represent positive sampling sites for Usutu virus RNA. Mosquito numbers involved from each sampling points are shown along with the number of tested mosquito pools from each location. Positive pool numbers are indicated in brackets.
Conflicts of interest
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