Introduction

West Nile virus (WNV), a member of the genus Flavivirus, family Flaviviridae, is considered one of the most important endemic arboviruses in Hungary. WNV has a single-stranded positive-sense RNA genome of approximately 11 kilobases in length and encodes a single polyprotein [1]. Since the first documented human neuroinvasive infections in 2004, West Nile fever (WNF) and the more severe manifestation of the infection, West Nile neuroinvasive disease (WNND) have been regularly diagnosed in Hungary with the highest peak of reported cases in 2018 [2]. The National Reference Laboratory (NRL) for Viral Zoonoses of the National Center for Public Health (NCPH), Hungary is exclusively responsible for the laboratory diagnosis of human infections, across the entire territory of the country. The Department of Epidemiological and Vaccination Surveillance of the NCPH collects, evaluates, and reports epidemiological and microbiological data directly to The European Surveillance System (TESSy) operated by the European Centre for Disease Prevention and Control (ECDC) [2]. For laboratory case confirmation the European Union's case definition criteria are applied: isolation of WNV from blood or cerebrospinal fluid (CSF), detection of WNV nucleic acid in blood or CSF, detection of WNV specific IgM antibodies in CSF, presence of WNV IgM antibodies in high titer and detection of WNV IgG and confirmation by neutralization [3]. In addition, nucleic acid detection in urine samples is also part of the routine laboratory confirmatory protocol [2]. Molecular typing of the WNV RNA by Sanger-method or by next-generation whole genome sequencing (NGS) is also an important element of the surveillance activities, as data regarding the circulating lineages are regularly reported to the ECDC. To date, at least eight genetic variants of WNV have been identified, with huge differences in pathogenicity potential. Among the genetic variants (lineages), lineage 1 and 2 pose the largest health risk to humans and animals worldwide [4]. Until the first emergence of WNV lineage 2 in Hungary in 2004, only lineage 1 and 3 WNV strains had been reported across Europe [5]. Shortly after its first emergence, lineage 2 strains began to spread rapidly in the region, characterized by a high degree of genetic diversification. During the last decade, WNV lineage 2 strains were responsible for several outbreaks in European countries (e.g. Austria, Italy, Greece, Romania, etc.) in both human and animal populations [6–7]. Furthermore, the largest European WNV epidemic in 2018 was also caused by lineage 2 strains [8–9]. The different clades and clusters of lineage 2 in Europe show geographic clustering due to the independent introduction from Africa via migratory birds and the local genetic evolution of viral strains [6]. The wide diversity of WNV strains requires continuous monitoring and accurate molecular typing of genomic sequences. This study aimed to sequence or re-sequence WNV strains between 2015 and 2022 to obtain a detailed phylogenetic analysis of currently circulating variants in Hungary. The geographical clustering of the sequences and the cumulative WNV incidence data were also compared.

Methods

A detailed description of the epidemiological surveillance and data collection, as well as the laboratory diagnostic algorithm, was previously published elsewhere [2].

Results

Summary of the epidemiological data

Between 2004 and 2022, altogether 499 human WNF and WNND cases were diagnosed and reported by the NCPH, Hungary (Fig. 1), including 483 autochthonous and 16 imported infections.

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The annual number of laboratory-diagnosed WNF and WNND cases in Hungary, between 2004 and 2022

Citation: Acta Microbiologica et Immunologica Hungarica 70, 2; 10.1556/030.2023.02040

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To date, the highest peak in case numbers was detected in 2018 (225), during the largest WNV epidemic in Europe, while in 2020 (3) and 2021 (8), a significant decrease could be observed in the number of reported WNF and WNND cases (Fig. 1). This trend coincides with the COVID-19 pandemic, which posed a significant burden on the healthcare system, resulting in a reduced number of samples sent to the reference laboratory of the NCPH for diagnosis of zoonotic infections. The clinical and laboratory diagnostics of infectious agents other than SARS-CoV-2, including WNV remained underrepresented during this period, probably as an indirect effect of the pandemic.

Overall, the cumulative incidence (laboratory-diagnosed WNF and WNND cases per 100,000 inhabitants) was 4.88 in the average population between 2004 and 2022. Considering the geographical distribution of the cumulative incidence rates at NUTS 3 (Nomenclature of Territorial Units for Statistics level 3) Hajdú-Bihar county (12.70) proved to be the most affected area, followed by Békés (11.06), Csongrád-Csanád (10.40), Jász-Nagykun-Szolnok (9.01) and Fejér (8.04) counties. The lowest incidence rate was measured in Zala county (0.35). Overall, the Eastern and Southeastern parts of the country are highly endemic for WNV, while in the Western counties, moderate or low incidence rates were observed. This tendency is also supported by seroprevalence data, obtained from a nationwide WNV serosurvey of blood donors in Hungary, in 2019 [11]. Table 1 summarizes the geographic distribution of the total number of cases and the cumulative incidence rates at the NUTS 3 level in Hungary.

Table 1.

Geographic distribution of the cumulative number of WNV cases and the cumulative incidence rates between 2004 and 2022, at the NUTS 3 level of Hungary. (CI: Confidence interval, WNV: West Nile virus) Incidence rates are marked with the same color scale as in Fig. 3

^aAccording to the Hungarian Central Statistical Office, regional population data are available until 2021.

Phylogenetic analysis

Between 2015 and 2018 altogether 15 virus isolates were available for sequencing. The number of WNV PCR-positive clinical specimens was more limited, as between 2018 and 2022 only 10 samples were suitable for direct whole genome sequencing. Considering the high potential of the adaptation of the viruses to the cell cultures and the permanent change in the nucleotide sequence of the viral RNA resulting from virus replication, only virus isolates with passage 0 (n = 13 isolates) or passage 1 (n = 2 isolates) were used for the NGS. Horizontal coverage of the consensus whole genomes compared to the reference genome was proved to be 99.2–99.8% in 24 samples, and 95.1% in one urine sample (GenBank accession number: OK239667). The depth of genome coverage varied between 2,782 and 13,597 (mean: 7,660). All relevant data are summarized in the supplementary document. Lineage 2 WNV was identified in all samples, according to our previous results based on the Sanger sequencing method, although whole genome sequencing enabled a more detailed analysis of the circulating virus strains of the country [8]. According to the maximum likelihood phylogenetic tree (Fig. 2), lineage 2 WNV strains form two main clades, the Central European (or Hungarian) clade and the Eastern European (or Russian) clade. Only one Hungarian sample (isolated in the Eastern part of the country in 2017) belongs to the Eastern European clade (OK239669; Fig. 2: highlighted in blue). The nucleotide sequence of this strain differs in only 35 nucleotides (99% nucleotide sequence identity) from a Romanian sequence (KJ934710). Most of the samples (n = 24) belong to the Central European clade, however, this clade seems to be substantially diverse (Fig. 2). Within the Central European clade, three main clusters can be further separated into the Central European, the Southwest European, and the Balkan clusters (Fig. 2). Hungarian samples collected between 2015 and 2018 form a separate monophyletic group in the phylogenetic tree (highlighted in yellowish green in Fig. 2). This monophyletic group is further defined as the Hungarian cluster within the Central European (Hungarian) clade. Furthermore, other sequences of the Central European cluster can be divided into smaller groups. A WNV PCR-positive urine sample from 2014 (KT359349; Fig. 2: highlighted in dark green) shows high (>99%) nucleotide sequence similarity with a Serbian WNV strain (KC496016 Novi Sad, Serbia; 2010). KT359349 was obtained from an imported WNV case, as the patient traveled to Serbia during the incubation period. Sequence analysis of this WNV strain has already been described in our previously published study [12].

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Maximum likelihood phylogenetic tree of the lineage 2 WNV sequences. (Accession numbers and names of the Hungarian samples are written in red.) Sequence data are defined as follows: GenBank accession number, the geographical location where the virus was isolated or detected, and the year of isolation or detection

Citation: Acta Microbiologica et Immunologica Hungarica 70, 2; 10.1556/030.2023.02040

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When considering the geographical distribution of the WNV strains, the location of WNV exposure has been taken into account. In one case (OK129334) the location of the WNV exposure could not be precisely determined (only at NUTS 3 level), therefore this sample was omitted from the map. Figure 3 represents the geographical distribution of the WNV sequences, as well as the geographical distribution of the cumulative incidence between 2004 and 2022. Our sequence data represent mainly the Central, Eastern, and Southeast parts of Hungary, as lower incidence rates were measured in the Western part of the country, resulting in the lack of cases and PCR-positive samples from this region (Fig. 3). Based on our data, a characteristic geographical distribution of WNV strains, that belong to different clusters can be established. Virus strains of the Balkan cluster (Fig. 3, symbolized by green asterisks) are located in the Eastern and Southeast parts of Hungary, while Central European WNV strains are distributed in the Central and Southern counties (Fig. 3). The virus strain that belongs to the Eastern European (Russian) clade originated from an East Central county of Hungary, namely Jász-Nagykun-Szolnok (Fig. 3, marked with a blue asterisk). This strain was isolated in 2017 from a human urine sample of an autochthonous case of WNV infection.

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Geographical distribution of the Hungarian WNV strains and the geographic distribution of the cumulative incidence rate of WNF and WNND cases between 2004 and 2022. (Geographical location of strains belonging to different clusters is marked with the same colors as clusters in Fig. 2.)

Citation: Acta Microbiologica et Immunologica Hungarica 70, 2; 10.1556/030.2023.02040

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Discussion

The main limitation of our retrospective study was the available number of suitable PCR-positive human samples and virus isolates. Besides environmental and ecological factors, the number of diagnosed WNV cases also depends on the number of samples received by the NRL for Viral Zoonoses for analysis. During the COVID-19 pandemic, the number of tested samples for WNV decreased, thereby limiting the number of PCR-positive samples and sequencing between 2020 and 2021. The time period between sampling and symptom onset, sample storage, and shipment conditions also affect the integrity of viral RNA and the detection of viral nucleic acid [13]. As human WNV infections can be characterized by a low level of viremia, in many cases WNV load is insufficient for virus isolation on cell cultures or for the amplification of the whole viral genome. Anticoagulant-treated whole blood and urine samples are the most suitable sample types for WNV nucleic acid detection [14–15]. In our study, whole genome amplification was successful from nine urine samples, and one whole blood (real-time PCR Ct values for each sample are available in the supplementary document). To date, according to our results, urine proved to be the most suitable sample type for virus isolation, and it may be more suitable for whole genome amplification and sequencing as well. However, a further comparative examination of more clinical specimens is needed.

Prior to 2004 only lineage 1 WNV strains were detected in Europe, while in 2004 WNV lineage 2 was first identified outside of Africa [7]. The virus was isolated from the brain of a goshawk (Accipiter gentilis) in Hungary [7]. After its first detection, an explosive spread of lineage 2 strains could be observed across Europe, resulting in local epidemics in Greece, Italy, and Austria. Furthermore, lineage 2 WNV strains have already been detected in France, Spain, and the northern parts of Europe, such as the Czech Republic, Germany, and the Netherlands [6, 16]. Following its geographical clustering, Southwest, Balkan, and Central European clusters could be distinguished within the Central European (Hungarian) clade (Fig. 2).

Among the sequenced strains between 2015 and 2022, only one Hungarian WNV strain belongs to the Eastern European (Russian) clade (OK239669; Fig. 2). Regardless of the appearance of Hungarian WNV lineage 2 in 2004, another lineage 2 strain with African origin (GenBank accession number: FJ425721.1) was recorded in Volgograd; Southern Russia, in 2007 [17]. Subsequently, in 2010, similar lineage 2 strains caused a large WNND epidemic in Romania and since then, these strains became endemic in Romania and Russia, forming the Eastern European (Russian) clade of WNV lineage 2 [17]. OK239669 has 99.68% nucleotide sequence homology with a WNV strain detected in a Hyalomma marginatum marginatum tick collected from a song thrush (Turdus philomelos), in the Danube delta; Eastern Romania in 2013 (GenBank Accession number: KJ934710; Fig. 2) [17]. Phylogenetically closely related WNV strains were recorded in Israel, Iran, and Italy [18–19]. In addition, during the 2018 epidemic, Eastern European lineage 2 WNV was detected in a 72-year-old female WNND patient in Greece, however, the other WNV sequences from 2018 were similar to strains found in previous years [18]. Overall, based on our current knowledge, the detection of Eastern European lineage 2 WNV sequences is uncommon in Central Europe.

Phylogenetic analysis revealed that virus isolates from the 2018 WNV epidemic belonged to different clusters within the Central European clade (Fig. 2). The fact that these samples do not form a single monophyletic group suggests that the 2018 European outbreak was rather associated with the impact of certain environmental factors, than with the emergence of a novel genetic variant. These results are consistent with our previously published study, based on partial genomic sequence analysis [8]. Since 2003, no lineage 1 WNV has been detected in Hungary, neither in humans nor in animals, although, at the same time, a high degree of genetic heterogeneity of lineage 2 strains has been observed. In conclusion, our phylogenetic analysis demonstrates the co-circulation of different WNV lineage 2 clusters in Hungary with characteristic geographical distribution. However, any possible differences in the virulence of these strains are currently not clear and require further investigations, such as plaque size assays or in vivo studies of pathogenicity in vertebrate hosts. The geographical distribution of different genetic clusters of WNV lineage 2 might be related to the bird migration routes and breeding sites. In Hungary, there are many important bird areas (IBA), that provide favorable habitats for one or more bird species, including sites for breeding, wintering, or routes of migration. According to the Hungarian Bird Watching Association's website (https://www.hungarianbirdwatching.com/), there are 54 IBAs, covering 1.4 million hectares (representing 15.1% of the territory of Hungary). Some of the most important birding hotspots are localized in the Eastern and Southeastern parts of the country, such as Jász-Nagykun-Szolnok and Hajdú-Bihar counties, which are characterized by high WNV incidence rates (Table 1 and Fig. 3). It can be hypothesized, that besides the genetic evolution of the local strains in a given geographic area, the diversity of WNV in Central Europe is associated with the independent introduction and continuous fluctuation of viral variants between Africa and Europe, triggered by migratory species.

Our study is limited to the sequencing of WNV PCR-positive human samples or virus isolates derived from human clinical specimens, as the NRL for Viral Zoonoses of the NCPH, Hungary is responsible for the microbiological diagnosis of human infections. In Hungary, a passive equine and avian surveillance system exists and animal cases (e.g. equine encephalomyelitis due to WNV) are notified by the Veterinary Diagnostic Directorate of the National Food Chain Safety Office and regularly reported to the European Animal Disease Notification System (ADNS) of the European Commission. A country-wide mosquito surveillance system is still not in operation in Hungary, however real-time monitoring of native and invasive mosquito species, based on citizen science and local trapping is being developed within the One Health concept. In light of the 2018 WNV outbreak in Europe, the European Centre for Disease Prevention and Control anticipates the need for multi-country outbreak investigations through sequence-based typing. Further findings in different European countries also strengthen the need for such an approach: In 2019 a death of a little grebe (Tachybaptus ruficollis subsp. ruficollis) in Italy was associated with WNV lineage 2 infection [20]. Furthermore, in January 2022, WNV lineage 2 was also detected in a female goshawk rescued in the region of Umbria [20]. WNV circulation has never been reported in this territory before 2019 [20]. The westward spread of lineage 2 WNV was also evidenced, as WNV lineage 2 was molecularly detected in 2017, for the first time in Catalonia, Spain, away from the lineage 1-affected areas of the country [21]. In 2020, WNV lineage 2, belonging to the Southwest European cluster within the Central European (Hungarian) clade was detected again in northern goshawks with clinical signs compatible with WNV infection, highlighting the spread of WNV lineage 2 from the central regions of Europe to the west [21]. Due to the emerging number of human and animal cases and the increasing geographical spread of lineage 2 strains, WNV should be kept under extensive surveillance, including whole genome sequencing of viral variants, following the One-Health approach. Based on previous findings, it seems that Hungary has an essential role in the dissemination of WNV strains in Europe, thus it is important to enhance the collection of whole genome sequencing data from this region of Europe. An increase in the number of tested human clinical samples and diagnosed cases may help to obtain even more data regarding the circulating virus variants. Besides whole blood, the collection of urine samples can highly support virus isolation, viral RNA detection, and molecular typing.

Funding sources

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Ethical statement

Ethical approval and special permission was not needed for this study as it was performed as part of the routine diagnostic and surveillance activities.

Data availability

All relevant data are within the manuscript and its Supporting Information files.

Conflict of interest

None declared.

Authors' contributions

AN, MT conceptualized the study, AH, AN, NM, and CSN performed laboratory experiments, AH, AN interpreted the laboratory results and performed bioinformatics analysis, JH helped in NGS data analysis and reviewed the results, EM provided epidemiological data and prepared Fig. 3, ON, AK, MT reviewed the manuscript.

Supplementary material

Supplementary data to this article can be found online at https://doi.org/10.1556/030.2023.02040.