Emergence of West Nile Virus Lineage-2 in Resident Corvids in Istanbul,Turkey

Abstract

West Nile fever is a vector-borne viral disease affecting animals and humans causing significant health and economic problems globally. This study was aimed at investigating circulating West Nile virus (WNV) strains in free-ranging corvids in Istanbul, Turkey. Brain, liver, and kidney were collected from corvids (n = 34) between June 2019 and April 2020 and analyzed for the presence of WNV-specific RNA by quantitative RT-PCR. In addition, histopathologic and immunohistochemical examinations were also performed. Samples found to be positive by qRT-PCR were partially sequenced. WNV-specific RNA was detected in 8 of 34 corvids analyzed, which included 7 hooded crows (Corvus cornix) and 1 Eurasian magpie (Pica pica). Phylogenetic analysis based on partial WNV sequences from the 8 WNV-positive corvids identified in this study revealed that all sequences clustered within the WNV lineage-2; they were at least 97% homologues to WNV lineage-2 sequences from Slovakia, Italy, Czechia, Hungary, Senegal, Austria, Serbia, Greece, Bulgaria, and Germany. WNV sequences showed a divergence (87.94–94.46%) from sequences reported from Romania, Central African Republic, South Africa, Madagascar, Israel, and Cyprus, which clustered into a different clade of WNV lineage-2. Common histopathologic findings of WNV-positive corvids included lymphoplasmacytic hepatitis, myocarditis, and splenitis. The liver and heart were found to be the tissues most consistently positive for WNV-specific antigen by immunohistochemistry, followed by the kidney and brain. This study demonstrates for the first time the existence of WNV virus belonging to the genetic lineage-2 in resident corvids in Istanbul, Turkey. We hypothesize that the WNV strains circulating in Istanbul are possibly the result of a spillover event from Europe. Since WNV is a zoonotic pathogen transmitted by mosquito vectors, the emergence of WNV in Istanbul also poses a risk to humans and other susceptible animals in this densely populated city and needs to be addressed by animal and public health authorities.

Introduction

Birds are infected by a multitude of viruses (Yilmaz et al. 2001, Turan et al. 2020) and also play an important role in spreading some of these viruses to humans (Hernandez-Triana et al. 2014). West Nile virus (WNV), Usutu virus, Tembusu virus, Bagaza virus, and Israel turkey meningo-encephalitis virus are vector-borne flaviviruses that can infect mosquitoes, birds, and other vertebrates. They cause infection and disease in birds, humans, and other mammals and maintain a natural transmission cycle between birds and mosquitoes; however, humans and other mammals such as horses can be dead-end hosts (O'Guinn et al. 2013, Sudeep et al. 2013, Benzarti et al. 2019). West Nile fever caused by WNV has recently emerged in both the United States and Europe and is considered a major public health problem for the past two decades (Lanciotti et al. 1999, Calistri et al. 2010, Murray et al. 2010, Garcia-Carrasco et al. 2021).

WNV is a positive-sense single-stranded RNA virus, genus Flavivirus, in the family Flaviviridae. At present, WNV strains are separated into nine genetic lineages (Fall et al. 2017). Lineage-1 emerged in the United States in 1999 and is mostly found in the United States, Africa, Europe, Australia, and the Middle East and is subdivided into clades 1a, 1b (or Kunjin virus), and 1c (Gray and Webb 2014). Lineage-2 is present in southern Africa and Madagascar, emerged in central Europe in 2004–2005, and then spread to Austria and to Southern European countries (Savini et al. 2012, Hernandez-Triana et al. 2014, Ravagnan et al. 2015, Vilibic-Cavlek et al. 2019). Recent data indicate that WNV strains detected in Europe in humans, horses, birds, and mosquitoes mostly belong to WNV lineage-2 (Savini et al. 2013, Kurolt et al. 2014). The other lineages, such as lineages-3 and -4 circulating in Russia, lineage-5 in India, and lineage-6 in Spain, are rarely detected and seem to have evolved from separate introductions into the Northern hemisphere (Rizzoli et al. 2015, Sule et al. 2018). Lineages-7, -8, and -9 are mainly found in Africa (Fall et al. 2017).

WNV is mainly transmitted by Culex mosquitoes but can also be transmitted by direct contact with tissues or bodily fluids of viremic animals and humans, vertically in utero, and iatrogenically by blood transfusions or organ transplants. Birds are the main reservoir and amplifying hosts for WNV, whereas horses, humans, and most other mammals are considered to be dead-end hosts (Chancey et al. 2015). WNV infection has been reported in a wide range of avian species (Steele et al. 2000, Erdelyi et al. 2007). In birds, infections are generally subclinical but can be fatal in highly susceptible bird species, such as corvids and birds of prey (Eidson et al. 2001, Komar et al. 2003, Wünschmann et al. 2004, Bakonyi et al. 2013).

WNV is an important pathogen for avians, equines, and humans and has been reported in many countries (David and Abraham 2016). After the initial discovery of WNV in the blood of a febrile woman in Uganda in 1937 (Smithburn et al. 1940), the virus was identified for the first time in avian species in 1953 in Egypt in hooded crows (Corvus cornix) and rock pigeons (Columba livia) (Work et al. 1953). Between 1997 and 2000, WNV outbreaks were reported in the migratory white stork (Ciconia ciconia) and domesticated geese (Anser anser domesticus) in Israel (Malkinson et al. 2002) and associated with high mortality in 342 avian species in the United States (Murray et al. 2010, Centers for Disease Control and Prevention 2018). WNV emerged in the United States in 1999, about 27,000 horses and 48,183 people were infected resulting in 2163 deaths in humans (California Department of Food and Agriculture 2018, Centers for Disease Control and Prevention 2018). In addition, WNV caused sporadic infections and neurological illness in humans and horses in Europe and Eastern Mediterranean countries, including Turkey and Iran (David and Abraham 2016, Benzarti et al. 2019, Eybpoosh et al. 2019).

WNV has been present in the Mediterranean Basin since the 1950s and in other parts of Europe since the 1960s, however, causing only sporadic outbreaks (Michel et al. 2019). During the past two decades, there has been an emergence of the virus in Europe, including Turkey. Human and equine cases were reported in several European countries, mainly in Hungary, Greece, Austria, Czech Republic, and Italy (Papa et al. 2011, Wodak et al. 2011, Bakonyi et al. 2013, Popovic et al. 2013, Barzon et al. 2015, Chancey et al. 2015). In 2018, WNV cases increased compared to 2017 by 7.2-fold; a total of 2083 human WNV cases and 285 WNV outbreaks among equids were recorded in European Union (EU) member states with the largest number detected in Italy, Serbia, and Greece as well as neighboring countries, including Turkey (Ozkul et al. 2013, Haussig et al. 2018, Riccardo et al. 2018, European Center for Disease Prevention and Control 2019a, Michel et al. 2019, Yilmaz et al. 2019, Garcia-Carrasco et al. 2021).

In Turkey, human and equine cases of WNV have been previously reported, including the presence of WNV in local mosquito species (Ozkul et al. 2013, Ergunay et al. 2015). Following the first introduction of WNV lineage-2 into Europe in 2004, it was found that both short- and long-distance migratory birds and resident birds are responsible for the spread of WNV from Africa, as well as for the transmission and maintenance of WNV in Europe and Eastern Europe (Bakonyi et al. 2006, Valiakos et al. 2012). Migratory birds play a key role in the transboundary and transcontinental spread of a multitude of viruses via their major flyways (Rappole et al. 2003, Hubalek et al. 2004, Michel et al. 2019). Therefore, the prevention and control of WNV depend on effective biosurveillance in reservoir host species, which includes monitoring of birds, Culex mosquitoes, and susceptible mammals including humans (Chancey et al. 2015, Romeo et al. 2018, Michel et al. 2019). Corvids such as crows, blue jays, and magpies are particularly susceptible to the virus and are often used as indicator species for WNV biosurveillance (Eidson et al. 2001).

Despite previous investigations on WNV epidemiology in the Eastern Mediterranean Region, there are still many unknown aspects regarding the circulation and distribution of WNV in Turkey and the driving factors behind its emergence in animals and humans. Therefore, this study was undertaken to investigate the presence of WNV in birds in Istanbul, Turkey, which borders the EU. Up to date, there has not been a comprehensive study demonstrating molecular, histopathologic, and immunohistochemical features of WNV infection in birds in Turkey.

Materials and Methods

History and collection of tissue specimens

This study consisted of 34 corvids (22 hooded crows and 12 Eurasian magpies) found sick or dead in the environment and submitted to the Department of Wild Animal Diseases and Ecology (DWADE) of Istanbul University-Cerrahpasa between June 2019 and April 2020 by the public. Sick birds that died at the rehabilitation center of the DWADE shortly after submission (n = 19) and the dead birds (n = 15) were taken to the Department of Pathology (Veterinary Faculty of Istanbul University-Cerrahpasa) for postmortem examination.

At necropsy, carcasses were weighed, the gross findings including age and sex were recorded. Birds were aged by plumage as juvenile (first year) or adult (after first year) (Emlen 1936). Samples of the brain, heart, lungs, trachea, liver, kidneys, spleen, proventriculus, ventriculus, intestines, and skeletal muscle were fixed in 10% buffered formalin for 24 h, embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin for histopathologic examination.

A set of brain, liver, and kidney tissues from each bird were frozen at −80°C for WNV testing by quantitative RT-PCR. Brain samples were analyzed separately from each bird, whereas liver and kidney samples were pooled. Brain samples of the birds number 1, 2, 3, and 6 could not be analyzed with qRT-PCR; this is summarized in Supplementary Table S1.

RNA extraction

For the extraction of viral RNA from tissue samples, the RNeasy RNA extraction kit was used as described by the manufacturer (Cat. No.: 74106; Qiagen). The amount of RNA in the extracted material was measured using a NanoDrop spectrophotometer (NanoDrop 1000c; Thermo Scientific, Waltham, MA).

qRT-PCR for the detection of WNV-RNA

Reverse transcription and generation of complementary DNA (cDNA) were performed by using High-Capacity cDNA Reverse Transcription Kit (Cat. No.: 4368814; Applied Biosystems) as described by the manufacturer. After completion of the reverse transcription reaction, 30 μL of nuclease-free water was added to each cDNA sample. Primers and probes used in this study were derived from a previously published study (Linke et al. 2007) (Supplementary Table S2). The methods and amplification conditions for the qRT-PCR to detect WNV-RNA in tissue samples were the same as described previously (Linke et al. 2007). qRT-PCR were performed for the test samples in a 25 μL PCR by using positive (provided by Dr. Robert S. Lanciotti, Centers for Disease Control and Prevention, USA) and negative controls, and amplification was performed in a Stratagene MX3005P qPCR instrument. An optimized PCR mix consisted of 2 μL (7.5 μM) of each forward and reverse primer, 1 μL (2.5 μM) probe, 12.5 μL Maxima Hot Start PCR Master Mix (K1052; Thermo Scientific), 0.5 μL MgCl₂, 5 μL of cDNA, and 2 μL nuclease-free water.

Sequencing and phylogenetic analysis

Samples found to be positive by qRT-PCR were also analyzed by conventional RT-PCR. For this purpose, conventional RT-PCR was performed for the amplification of WNV-RNA using the primers previously published by Lanciotti et al. (2000) (Supplementary Table S2). Briefly, an optimized 50 μL RT-PCR mixture consisted of 7 μL of RNA and 20 pmol of each primer, 25 μL Platinum™ SuperFi™ PCR Master Mix (12358010; Invitrogen), and negative and positive controls (provided by Dr. Robert S. Lanciotti, Centers for Disease Control and Prevention) (Bilgin et al. 2020). Amplification was performed in a Stratagene MX3005P PCR machine by following the instructions included in the Platinum SuperFi PCR Master Mix kit. Products were separated by gel electrophoresis using a 1.5% agarose gel. Products of 408bp length were sequenced, and phylogenetic analyses were performed as described previously (Bilgin et al. 2020).

Alignments of the partial sequences of the nucleocapsid and pre-membrane protein genes of WNV were made using the Molecular Evolutionary Genetics Analysis-7 software. Phylogenetic analyses were carried out using the criterion of neighbor-joining trees based on genetic distance model by Tamura et al. (2004). The reference sequences from the GenBank for WNV classification were used to reconstruct the topology of the partial sequences of the nucleocapsid and pre-membrane protein genes of WNV in this study. The partial sequences of the nucleocapsid and pre-membrane protein genes of WNV obtained in this study were submitted to the GenBank.

Immunohistochemistry

Paraffin blocks of the eight birds, which were positive for WNV qRT-PCR, including brain, heart, kidney, and liver tissues were submitted to the California Animal Health and Food Safety Laboratory System (Davis, CA) for immunohistochemistry.

A mouse monoclonal antibody directed against an epitope of the E protein was used (diluted 1:300, 45 min, room temperature, clone 7H2; VRL, Gaithersburg, MD). This antibody was proved that it does not have cross-reactivity for viruses closely related to WNV, such as Saint Louis encephalitis virus and Japanese encephalitis virus. The immunohistochemistry (IHC) protocol was performed as previously described (Wünschmann et al. 2004) with the exception of using citrate buffer for antigen retrieval.

The amount of antigen expression was graded subjectively as negative (−), mild (+) representing occasional scattered immunoreactivity, moderate (++) when moderate numbers of cells were stained, and marked (+++) when there was a generalized frequent immunostaining of cells (Supplementary Table S1).

Results

Detection of WNV-RNA in corvids

The presence of WNV-specific RNA was observed in 8 (Nos. 1–8) of 34 birds. The C_T values of these samples ranged from 18.67 to 36.65, whereas a C_T value of 18.20 was obtained with the positive WNV RNA control. WNV-specific RNA was detected in the brain (C_T values: 23.99 and 26.07) and kidney–liver pool (C_T values: 28.96 and 30.66) of the birds Nos. 4 and 5, where the birds Nos. 1, 2, 3, 6, 7, and 8 were positive in the kidney–liver pool only (C_T values: 18.67–36.65). There was no positive RT-PCR signal in brain, kidney, and liver tissue samples taken from the remaining 26 corvids and in the negative controls (Supplementary Table S1).

Sequencing and phylogenetic analysis

A 408 bp PCR product on gel electrophoresis was seen after the WNV conventional RT-PCR for all eight WNV RNA-positive samples. The PCR amplicons from positive corvid samples were sequenced, and the sequences were submitted to the GenBank (accession numbers: MT521982, MT521983, MT521984, MT521985, MT521986, MT521987, MT521988, MT521989).

Phylogenetic analyses based on partial sequences of the WNV pre-membrane gene revealed that all sequences clustered in WNV lineage-2 were 98.57% similar to WNV lineage-2 sequences from Austria, Serbia, Greece, Bulgaria, and Germany (GenBank accession numbers: MF984344, MF984345, KT757318, KT757323, MN652880, KU206781, and MH910045; Fig. 1). High homology was also seen between the Turkish corvid WNV sequences and sequences from Slovakia (98.1%), Italy (98.1%), Czechia (98.1%), Hungary (97.63%), and Senegal (97.09%) (GenBank accession numbers: MH244511, KF647250, KM203861, DQ116961, DQ318019, respectively) (Fig. 1). In contrast, WNV sequences obtained in this study showed some degree of divergence from sequences reported from Romania (94.46%), Central African Republic (94.46%), Madagascar (94.13%), Israel (93.48%), South Africa (90.22%), and Cyprus (87.94%) (GenBank accession numbers: KJ934710, DQ318020, HM147822, HM147823, AY688948, and GQ903680) and clustered into a different clade within WNV lineage-2 (Fig. 1).

FIG. 1.

ML phylogenetic tree constructed based on partial sequences of the nucleocapsid and pre-membrane protein genes of WNV. The classification has been performed using the reference sequences submitted to the GenBank. Circular dots indicate the WNV detected in this study. ML, maximum likelihood; WNV, West Nile virus.

Clinical signs and postmortem findings including histopathology and immunohistochemistry

Five (Nos.1–5) of the eight birds found positive for WNV RNA by qRT-PCR, showed neurological signs including tremors, head tilt, torticollis, and inability to fly, and the animals died within 2 days after submission. The other three birds (Nos. 6–8) were found dead. Five (Nos. 1–5) birds were admitted to the DWADE in July, two (Nos. 6, 7) birds in August, and one (No. 8) bird in September 2019. Seven corvids were determined adults (Nos. 1, 2, 4–8) and one was a subadult (No. 3). Birds 3, 5, and 8 were females (37.5%, 3/8), and birds 1, 2, 4, 6, and 7 were males (62.5%, 5/8) (Supplementary Table S1).

Gross pathology findings included poor body condition (3/8, Nos. 3–5), hemorrhage in the calvaria (4/8, Nos. 1, 3, 7, 8), diffusely darkened liver, kidneys, and spleen (6/8, Nos. 1–5, 7), splenomegaly (4/8, Nos. 1, 3–5), multifocal myocardial pallor (1/8, No. 7), and small foci of hemorrhage and thickening in the mucosa of small intestinal wall (2/8, Nos. 3–6).

The most consistent histopathologic lesions were present in the liver, spleen, and heart, as shown in Supplementary Table S1. Mild-to-severe, periportal to random lymphoplasmacytic hepatitis was present in five birds (Nos. 1–3, 7, 8; Supplementary Fig. S1). Severe hemorrhage (3/8, Nos. 1, 3, 6) and diffuse coagulative necrosis (2/8; Nos. 7, 8) were other prominent hepatic lesions. Moderate-to-severe lymphoplasmacytic and histiocytic splenitis with diffuse coagulative necrosis was detected in three birds (Nos. 1, 3, 8). Myocardial lesions were mild lymphoplasmacytic myocarditis (3/8; Nos. 1, 4, 7; Supplementary Fig. S2) and multifocal hemorrhages (1/8; No. 8).

Gross and histopathologic examinations of WNV-negative corvids (n = 26) showed that the birds died from pneumonia (n = 11), traumatic injuries (n = 7), and unknown causes (n = 8).

Immunohistochemistry was performed on the brain, heart, and liver of all eight birds positive for WNV RNA by qRT-PCR (Nos. 1–8) and on kidneys of seven WNV-positive animals (Nos. 1, 3–8; not available from No. 2). The heart and liver were the most consistently immunoreactive tissues, followed by the kidneys (Supplementary Table S1). Immunoreactivity in the heart of all eight birds was observed in the major artery and coronary artery walls, in the smooth muscle cells and the adventitia (Supplementary Fig. S3). Cardiomyocyte staining was extensive in two birds (Nos. 7–8). The livers of WNV-positive animals had moderate to marked generalized staining, predominantly in Kupffer cells (Supplementary Fig. S4).

Discussion

Bird migration plays an important role in the spread of the avian-borne viruses from Africa to Europe, from Europe to Africa, and to other continents (Rappole et al. 2000, Sule et al. 2018). The Middle East represents an important transit zone for bird migration between Africa and Eurasia, and biosurveillance in this region provides valuable information on circulation of WNV and other bird-associated viruses (Lustig et al. 2017a, 2017b, Salama et al. 2019). This study was performed to investigate the presence of WNV in free-ranging corvids that were either sick or were found dead in Istanbul, Turkey.

In the present study, WNV was detected in 8 of 34 deceased wild corvids by qRT-PCR. Histopathologic and immunohistochemical analyses indicated the presence of WNV infection in various organs of PCR-positive birds. Gross findings included diffusely darkened liver, spleen, and kidneys and splenomegaly; these findings are consistent with previously reported gross lesions of WNV infection in birds (Steele et al. 2000, Komar et al. 2003). Histopathologic hallmarks of the present study were lymphoplasmacytic hepatitis detected in five birds (5/8; 62.5%), lymphoplasmacytic splenitis with diffuse coagulative necrosis in three birds (3/8; 37.5%), and a mild encephalitis in one bird (1/8; 12.5%), consistent with previous findings of WNV infections in corvids (Steele et al. 2000, Panella et al. 2001, Wünschmann et al. 2004, Gibbs et al. 2005). WNV infection in corvids does not produce florid histological lesions, particularly in the brain (Weingartl et al. 2004, Wünschmann et al. 2004, Gibbs et al. 2005) likely due to the acute to peracute nature of the disease.

Immunohistochemistry for WNV-specific antigens in the liver, heart, kidney, and brain demonstrated strong and prominent staining in the liver of all eight WNV-positive corvids. Corresponding with previous studies (Steele et al. 2000, Fitzgerald et al. 2003, Weingartl et al. 2004, Ellis et al. 2005), the heart also proved to be consistently immunoreactive in all eight birds, although less intense when compared with the liver. Brain tissue revealed mostly mild immunoreactivity in six of eight birds, confirming that IHC staining of brain tissue from corvids is not the most reliable method for diagnosing WNV infection (Steele et al. 2000, Weingartl et al. 2004, Wünschmann et al. 2004).

WNV RNA was detected in human and equine cases caused by lineage 1 strains in Central Anatolia of Turkey (Ozkul et al. 2013). Ergunay et al. (2014) indicated a widespread WNV activity in provinces of Eastern, Mediterranean-Aegean, Southeastern, and Northeastern Anatolia of Turkey by demonstrating WNV partial sequences, characterized as lineage 1, obtained from various species of mosquito and also antibodies to WNV from infected hosts, such as horses, sheep, and ducks. To the authors' knowledge, WNV lineage 2 strain was only detected in the brain tissues of a mare in the Bursa region of Turkey (Monaco et al. 2016) and a mosquito pool in Thrace district of Turkey (Bilgin et al. 2020) but not in birds at present. The above findings indicate that WNV infections in humans and a variety of animal species pose a risk to public health.

EU member states and EU neighboring countries reported 463 human WNV infections in 2019. Fifty-three of these cases were from EU neighboring countries, and 10 of them were reported from Istanbul, Turkey (European Center for Disease Prevention and Control 2019b). In the present study, all the eight WNV-positive corvids were submitted between the beginning of July and mid-September 2019 indicating a possible temporal correlation in time between the human and avian cases.

Transmission season of the WNV in Turkey is estimated to be from May to November (European Center for Disease Prevention and Control 2020). The temperatures in Istanbul were higher than long-time averages in the winter 2019 (Bianet, 2020). To increase the sample size and assess if transmission period is prolonged with warmer temperatures, we extended the sampling period to April 2020. Eight of the 34 birds were sampled in this period, between November and April, and all were negative for WNV by PCR.

Incidence of disease after WNV infection depends on the virulence of the virus strain, environmental factors, and host genetics/immunity. Virulence of different isolates belonging to various lineages is highly variable and is associated with a specific virus genotype, not with a specific lineage (Sule et al. 2018, Zannoli and Sambri 2019). Isolates belonging to WNV lineages-1 and -2 have been predominantly responsible for outbreaks in people and animals (Aliota et al. 2012, David and Abraham 2016). Most of human and/or equine WNV infections were caused by lineage-1a viruses, which have a low virulence for birds (Calistri et al. 2010). However, since 2004, the WNV epidemiological pattern has changed with an increased mortality caused by lineage-2 viruses in certain avian species and a higher incidence of neurological signs in animals and humans (Bakonyi et al. 2005, Chaintoutis et al. 2013, Durand et al. 2017).

In the present study, phylogenetic analyses based on partial WNV genomic sequences revealed that all eight sequences clustered in WNV lineage-2 were 97% homologous to WNV lineage-2 sequences from Slovakia, Italy, Czechia, Hungary, Senegal, and 98.6% homologous to viruses from Austria, Serbia, Greece, Bulgaria, and Germany. This high homology of viruses circulating in Turkey with WNV isolates from the Balkan and central Europe indicates a spillover event from these regions to Turkey. In contrast, the Turkish WNV sequences showed some degree of divergence (87.94–94.46%) from WNV sequences reported from Romania, Central African Republic, South Africa, Madagascar, Israel, and Cyprus, which clustered into a different clade (Fig. 1).

Most WNV outbreaks and associated deaths in humans, horses, and birds have occurred due to infections with WNV lineage-1 viruses, which were responsible for outbreaks in Israel and North America (Lanciotti et al. 1999). The WNV lineage-2 was introduced into Europe in 2004 and later spread to other European countries. Lineage-2 was first detected in a goshawk in 2004 in Hungary and later in birds of prey (Bakonyi et al. 2006, 2013) and in raptors (Wodak et al. 2011) in Austria. WNV lineage-2 has been reported in humans and birds in Greece since 2010 (Chaintoutis et al. 2013) and it emerged in Italy in 2011 (Bagnarelli et al. 2011). The first introduction of WNV lineage-2 in Germany was around 2016, where it was detected in resident wild and aviary birds, including Eurasian blackbirds, northern goshawks and in 2018 in great gray owls (Ziegler et al. 2019). Outbreaks due to lineage 2 infections in birds and humans have been also reported in other European countries, such as Austria, Romania, and Serbia (Hernandez-Triana et al. 2014).

Conclusions

This study demonstrates that WNV lineage-2 viruses infect corvids in Istanbul, Turkey. Outbreaks with WNV lineage-2 viruses are also reported in Europe in various bird species and humans, indicating a possible spillover event from Europe to Turkey. The present study demonstrates that WNV lineage-2 is circulating in Istanbul and poses a risk to people living in this densely populated major city. The emergence of zoonotic WNV in Turkey needs to be addressed by the respective animal and public health authorities.

Ethical Approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Footnotes

Author Disclosure Statement

No conflicting financial interests exist.

Funding Information

This study was supported by the University of Istanbul-Cerrahpasa (BAP Project No.: 27352). This study was partially supported by the Department of Homeland Security Center of Excellence for Emerging and Zoonotic Animal Diseases (Grant No. HSHQDC-16-A-B0006) to J.A.R.

Supplementary Material

Supplementary Figure S1

Supplementary Figure S2

Supplementary Figure S3

Supplementary Figure S4

Supplementary Table S1

Supplementary Table S2

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