West Nile virus (WNV) is a member of the family Flaviviridae classified within the Japanese encephalitis serogroup. WNV is a significant human health problem worldwide, with nine recognized genetic lineages1–2. Lin- eage 1 and 2 of WNV genetic variants are known to pose greatest risk to human health worldwide. Before 2004, when WNV lineage 2 emerged in Hungary, only lineage 1 and 3 strains were reported from Europe3. Besides the explosive spread of WNV lineage 2 from Hungary to Austria in 2008, several outbreaks in association with WNV-2 occurred across Europe (Italy, Greece, Serbia, Croatia, etc.) affecting both human and animal life4–5. A WNV outbreak occurred in Serbia with nine fatalities in 20126, culminated in the next year as the largest human WNV outbreak in Europe to date7. The increased inci- dence of WNV infections during the consecutive out- breaks in Serbia (71 human cases in 2012 and 302 in 2013) well reflects the possible impact of mutations af- fecting pathogenicity in interaction with other factors (i.e.
environmental factors, mosquito abundance etc.) on the outcome for a human outbreak8. WNV strains differ sig- nificantly in virulence and neuroinvasiveness. Initially WNV lineage 1 strains were considered as more viru- lent, until several highly virulent and neuroinvasive vari- ants of lineage 2 WNV were detected in southern Af- rica9. The neuroinvasiveness and neurovirulence of WNV is influenced by different genetic markers, and both lin- eage 1 and 2 strains may exhibit a neuroinvasive pheno- type10. The emergence of new strains possessing the NS3249P amino acid substitution, which had been previ- ously reported to affect pathogenicity and thermotolerance could serve as a possible explanation for increased number of West Nile neuroinvasive dis- ease (WNND) cases11–12. Altogether, novel phenotypes of WNV may emerge in the future, since many different strains are co-circulating simultaneously in Europe and are evolving under various selective pressures.
In the present study, the complete polyprotein gene of six WNV strains from the Serbian outbreak of 2013 were sequenced and genetically characterized. Phyloge- netic analysis provides a potential scenario for the origin of WNV strains published in this study. Our data may indicate an increased risk for introduction of the neurovirulent NS3249P phenotype to the neighbouring countries of Serbia. In contrast with previous observa- tions13 this study confirmed that, NS3249P possessing strains were present during the Serbian outbreak in 2013.
Large-scale mosquito surveillance was conducted in Vojvodina province of Serbia during 2013. Several novel WNV strains were detected, mainly in Culex pipiens spe- cies. Mosquitoes were trapped with CDC light-traps baited with dry ice in multiple sampling events during the breeding season (May to September) of 20131. Based on the geographic location of traps, six representative virus strains were selected from Kikinda, Novi Sad and Srpski Krstur cities, respectively. Samples, analyzed in this study were collected in the month of August 2013.
Nucleotide BLAST searches were performed in or- der to find relevant reference sequences in GenBank da- tabase. PCR primers were designed using the genome sequence of strain Nea Santa-Greece-2010, lineage 2 (GenBank: HQ537483) in order to amplify four overlap- ping genomic regions. cDNA synthesis was performed with Transcriptor High Fidelity cDNA Synthesis kit (Roche, Switzerland) with random hexamer priming fol- lowing the manufacturer’s instructions. PCR assays were performed with Thermo Scientific Phusion High-Fidel- ity PCR Master Mix following the protocol provided by the manufacturer.
Next generation sequencing was performed on the mixture PCR amplicons to obtain primary sequence data for each strain14. Briefly, an Ion Torrent compatible li- brary was prepared applying the NEBNext® Fast DNA Fragmentation and Library Prep Set for Ion Torrent (New
Genomic characterization of West Nile virus strains derived from mosquito samples obtained during 2013 Serbian outbreak
Brigitta Zana
1–2, Gábor Kemenesi
1– 2, Róbert Herczeg
3, Bianka Dallos
1–2, Miklós Oldal
1–2, Szilvia Marton
4, Bosiljka Krtinic
5, Ákos Gellért
6, Krisztián Bányai
4& Ferenc Jakab
1–21Virological Research Group, János Szentágothai Research Centre, University of Pécs; 2Institute of Biology, Faculty of Sciences, University of Pécs, Pécs; 3Seqomics Ltd., Szeged; 4Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest; 5Ciklonizacija Ltd., Novi Sad, Serbia; 6Department of Applied Genomics, Agricultural Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, Martonvásár, Hungary
Key words Neuroinvasive; NS3249P mutation; phylogenetic tree; Serbia, West Nile virus
England Biolabs, USA) with the Ion Torrent Xpress barcode adapters (Life Technologies, USA). The emul- sion PCR to obtain clonally amplified fragments was car- ried out using the Ion OneTouch™ 200 Template kit (Life Technologies) on an OneTouch system (Life Technolo- gies, USA) as recommended by the manufacturer.
Templated beads were enriched using an Ion OneTouch™
ES pipetting robot (Life Technologies). The 200 bp se- quencing protocol was performed on a 316 chip (Life Technologies) using the Ion Torrent PGM (Life Tech- nologies) semiconductor sequencing equipment.
Trimmed sequence reads were used for de novo assem- bly utilizing the MIRA (version 3.9.17)15. Validation with remapping was performed using the CLC Genomics Workbench v6.5.116 and the DNAStar v1217 platforms.
Basic sequence alignments and sequence manipula- tions were fulfilled with Clustal X v2.118 and GeneDoc v2.719 softwares. Multiple evolutionary models were tested on the sequence alignments with PhyML v3.020 software in order to designate the best phylogenetic model for the study. The phylogenetic trees were constructed with MEGA v5.021 software using the Maximum-Like- lihood method, based on the General Time Reversible model with Gamma distribution (GTR+G). The number of bootstraps for simulations was 1000.
A total of 10,948 nucleotides were identified from all the six WNV strains (GenBank accession Nos.:
KT757318 – KT757323), which covered the complete coding region (3434 aa) of the virus. The highest nucle- otide identity (99%) of the Serbian strains was identified with the Nea Santa-Greece-2010 strain (GenBank:
HQ537483). A total of six amino acid changes were found in different positions of the polypeptide coding region.
Several previously identified amino acid substitutions as- sociated with altered pathogenicity and virulence of WNV were also observed, such as Proline (Pro) at position NS1250P, Asparagine (Asp) at the positions NS1130N, NS1175N, NS1207N and E153/154N of the N-linked glycosylation sites22–24. More interestingly, Pro amino acid substitution at position 249 of the NS3 region (NS3249P) was also detected in all the six strains. This mutation was previously described as a potential marker of increased neuropathogenicity12. Furthermore, parallel mutations were also observed between the helicase do- main of NS3 protein and the NS4B TM protein. Although, mutations in the NSB4 TM protein were previously noted by Papa et al12 the parallelism and potential functional association with NS3249P/H loci was not mentioned be- fore. Strains with NS3249H (His) substitution showed parallel NS4B14S (Ser) and NS4B49T (Thr) mutations, while the viruses with NS3249P amino acid variant showed NS4B14G (Gly) and NS4B49A (Ala) mutations. Amino acid substitutions compared to other WNV lineage 2 strains are summarized in Tables 1 and 2.
Table 1. Previously identified amino acid substitutions for pathogenicity and virulence of WNV compared to different WNV lineage 2 strains
Strain Country GenBank No. Year Protein, amino acid position
E NS1 NS3 NS4B
154 130 175 207 250 249 14 23 32 49
Nea Santa-Greece-2010 Greece HQ537483 2010 Asn Asn Asn Asn Pro Pro Gly Thr Asn Ala
Greece/2013/Xanthi_3 Greece KJ883345 2013 Asn Asn Asn Asn Pro Pro Gly Thr Asn Ala
Greece/2013/Thessaloniki_4 Greece KJ883346 2013 Asn Asn Asn Asn Pro Pro Gly Thr Asn Ala
Italy-2012 Italy JX878386 2012 – – – – – Pro – – – –
Serbia/2013/Vojvodina-1 Serbia KT7573718 2013 Asn Asn Asn Asn Pro Pro Gly Thr Asn Ala Serbia/2013/Vojvodina-2 Serbia KT7573719 2013 Asn Asn Asn Asn Pro Pro Gly Thr Asn Ala Serbia/2013/Vojvodina-3 Serbia KT7573720 2013 Asn Asn Asn Asn Pro Pro Gly Thr Ser Ala Serbia/2013/Vojvodina-4 Serbia KT7573721 2013 Asn Asn Asn Asn Pro Pro Gly Thr Asn Ala Serbia/2013/Vojvodina-5 Serbia KT7573722 2013 Asn Asn Asn Asn Pro Pro Gly Thr Asn Ala Serbia/2013/Vojvodina-6 Serbia KT7573723 2013 Asn Asn Asn Asn Pro Pro Gly Thr Asn Ala
– DRC HM147824 1958 Asn Asn Asn Asn Pro His Ser Ala Ser Thr
SA93/01 South Africa EF429198 2001 Asn Asn Asn Asn Pro His Ser Ala Ser Thr
Goshawk-Hungary/04 Hungary DQ116961 2004 Asn Asn Asn Asn Pro His Ser Thr Asn Thr
Austria/2008_gh Austria KF179640 2008 Asn Asn Asn Asn Pro His Ser Thr Asn Thr
Novi Sad-2010 Serbia KC496016 2010 Asn Asn Asn Asn Pro His Ser Thr Asn Thr
Cz 13-502 Czech Republic KM203863 2013 Asn Asn Asn Asn Pro His Ser Thr Asn Thr
Cz 13-329 Czech Republic KM203861 2013 Asn Asn Asn Asn Pro His Ser Thr Asn Thr
Italy/2013/Rovigo/32.1 Italy KF588365 2013 Asn Asn Asn Asn Pro His Ser Thr Asn Thr
Parallel mutations between NS3249H/P and NS4B14 and NS4B49 observed in the current study are emphasized in bold; Asn—Asparagines;
Pro—Proline; His—Histidine; Gly—Glycine; Ser—Serine; Thr—Threonine; Ala—Alanine; E—Envelope; NS—Nonstructural protein.
Phylogenetic and sequence analysis of the complete coding sequence revealed that the 2013 Serbian strains belonged to the group of WNV genetic lineage 2. As the phylogenetic tree clearly shows, the Serbian WNV strains formed a single monophyletic group which unambigu- ously clusters with other Greek strains from 2010 and 2013, containing NS3249P mutation (Fig. 1a). Due to the lack of WNV sequences with NS3249P mutation from Italy, only a single partial NS3 sequence was available, which serves as an evidence for the presence of NS3249P strains in the country (Table 1; Fig. 1b). This observa- tion along with phylogenetic- and molecular character- ization suggests multiple introductions of different WNV strains from other European territories. Based on com- plete sequence coding and partial NS3 phylogenetic
analysis, a notable aggregation of NS3249P possessor strains in a separate clade from other European strains can be observed.
Mosquito surveillance, vector competence studies, human or animal case reports and serosurveillance ac- tivities are all important pillars for understanding the risk of WNV infections in Europe and to predict scenarios for future outbreaks. In addition, examining locally cir- culating WNV strains for the early detection of those emerging mutations or amino acid alterations which may affect neurovirulence or pathogenicity is essential for evaluating the possible impact of forthcoming outbreaks.
The complete coding region of six WNV strains for the presence of amino acid alterations was examined in this study, which had been described as putative viru-
Fig. 1:Phylogenetic analysis of West Nile virus lineage 2 strains detected in Serbia, 2013. Phylogenetic trees were constructed based on the complete coding region of the virus (a); and on a partial region (743 nt) of the NS3 gene (b). Strains possessing the NS3249P mutation are marked with black triangle. Strains identified in this study are emphasized in bold.
(a) (b)
Table 2. Unique amino acid substitution changes in the Serbian lineage 2 WNV strains compared to Neo Santa-Greece-2010 virus
Strain GenBank No. Protein, amino acid position
NS1 NS2A NS4A NS5
77 109 166 155 61 650
Serbia/2013/Vojvodina-1 KT7573718 Thr Glu Met Val → Ala Ser Thr
Serbia/2013/Vojvodina-2 KT7573719 Thr Glu Met Val →Ala Ser Thr
Serbia/2013/Vojvodina-3 KT7573720 Thr →Ala Glu → Gly Met Val Ser → Gly Thr → Ala Serbia/2013/Vojvodina-4 KT7573721 Thr → Ala Glu → Gly Met → Val Val Ser Thr
Serbia/2013/Vojvodina-5 KT7573722 Thr →Ala Glu → Gly Met Val Ser Thr
Serbia/2013/Vojvodina-6 KT7573723 Thr →Ala Glu → Gly Met Val Ser Thr
Changes are emphasized in bold; Thr—Threonine; Ala—Alanine; Glu—Glutamic acid; Gly—Glycine; Met—Methionine; Val—Valine; Ser—
Serine.
lence factors in previous studies. Asparagine changes were observed in all the examined lineage 2 sequences at the 3 N-linked glycosylation sites of NS1 (NS1130N, NS1175N, NS1207N) and E (E153/154N) proteins. Amino acid changes might be important due to the special func- tions of these proteins. NS1 has co-factor activity of the viral replicase, while22–23 E protein is responsible for the attachment to the cell surface, membrane fusion and vi- ral assembly23. In a study, E protein also proved to be a molecular determinant (E153/154N) of neuroinvasiveness in case of a WNV NY99 strain (GenBank: HQ596519)25. In this study, NS3249P substitution was detected at NS3 genomic region in all the Serbian strains analyzed12.
The NS3 protein of most WNV lineage 2 strains contains His at the amino acid position 249, which is also characteristic of most neuroinvasive lineage 1 strains12. In previous experimental studies, NS3249P sub- stitution of NS3 protein has been reported to be associ- ated with increased viraemia and mortality among Ameri- can crows (Corvus brachyrhynchos) which might have had an influence on the increased virus transmission rates11–12, 23. Furthermore, NS3249P mutants with in- creased viraemia and elevated thermotolerance had been demonstrated to show greater pathogenicity11. This im- portant locus was only detectable in a single lineage 2 Italian sequence, in several Greek strains and the present Serbian lineage 2 viruses (Table 1). Introduction of the new WNV strains possessing NS3249P substitution in Serbia could serve as an explanation for the increased number of WNND cases during 2012 and 2013 outbreaks in the country6–7.
Additionally, the study revealed parallel mutations between NS3 protein helicase domain and NS4B TM protein (NS3249H with NSB14S, and NS3249P with NS4B14G), which might influence the putative protein- protein interaction between the two viral proteins.
Phylogenetic relationships with other European strains suggest multiple introduction events to WNV strains in Serbia as mentioned before26–27. Serbian WNV strains from mosquito samples of 2013 were clustered with Greek strains from 2010 and 2013. This suggests the possible origin of these strains from Greece, which explains the presence of NS3249P substitution in the Serbian samples. Based on the phylogenetic analysis, a possible WNV introduction from Greece to Italy in 2012 is also assumable based on a partial NS3 sequence from Italy (JX878386), which also contains the NS3249P alter- ation like the Greek and Serbian samples. Previously de- tected WNV strains from Serbia (2010 and 2012) unam- biguously clustered with other European WNV strains with the absence of the NS3249P alteration. The presence
of NS3249P mutant WNV strains in Serbia might explain the increased number of WNND cases in the outbreak of 2013, and warn for possible more serious outbreaks in the affected areas in future.
In summary, this study analyzed the complete cod- ing region of six WNV strains, detected in mosquito samples during the outbreak of 2013, Serbia. The NS3249P amino acid substitution was observed in all the strains, which has been previously described as a potential marker of increased neuropathogenicity. Additionally, parallel mutations between NS3 protein helicase domain and NS4B TM protein were described, which might influ- ence the protein-protein interaction, and thereby RNA binding of NS3, which might facilitate viral replication.
These results may support future surveillance activities by shedding light on the importance of examining muta- tions possibly affecting neuropathogenicity. This may lead to more precise predictions on the possible scenario of future outbreaks.
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Correspondence to: Dr Habil Ferenc Jakab, Virological Research Group, Jáinos Szentágothai Research Centre, University of Pécs, Ifjúság útja 20, H-7624, Pécs, Hungary.
E-mail: [email protected]
Received: 12 April 2016 Accepted in revised form: 5 September 2016