Identification of Newcastle disease virus (NDV) genotype VIIi in wild birds in Turkey

Background: Newcastle disease viruses (NDVs) can spread across continents via migratory birds. Hence, we investigated the frequency of NDV in both nonmigratory and birds migrating on the South East European flyway, in Istanbul, Turkey. Birds were trapped using nets placed around the Kucukcekmece lake Avcilar, Istanbul, in spring seasons of 2016 and 2018. In total, 297 birds belonging to 42 different species were trapped, categorised according to species and sex, and oropharyngeal swabs were collected. Swabs from 115 mallards caught by hunters in Edirne and from 207 birds belonging to 31 species which had been treated in the Veterinary Faculty of Istanbul were also collected. Tissue samples were taken from dead wild birds brought by public to Veterinary Faculty of Istanbul. A total of 619 oropharyngeal swabs were pooled into 206 samples. RNA was extracted from the swabs and tissue samples. Real-time RT-PCR prob assay was used to detect NDV-RNA in samples. Results: There was no amplification in real time RT-PCR in samples taken from wild birds caught by traps. However, amplification of NDV-F gene was observed in oropharyngeal swabs taken from 2 waterfowls, and in tissue samples taken from 2 little owls and 1 common kestrel. Sequencing and phylogenetic analyses of these 5 samples for NDV-F gene showed great similarity with NDV lineage VIIi viruses. One (waterfowl) of the five NDV-F gene sequences obtained in this study was slightly different than previously isolated Turkish NDVs and NDVs from Bulgaria and Georgia. Remaining four NDV-F gene sequences were similar to NDVs that were previously isolated in Turkey, Bulgaria and Georgia. Although the strains found in this study are closely related, there is a relatively small degree of molecular divergence within F gene of the Turkish NDVs and strains from Iran Pakistan, Israel and Belgium. Conclusions: Our findings show that first time in Turkey, genotype VIIi of NDVs is predomiant in wild birds in the Eurasia region and some degree of molecular evolution when compared to the earlier NDV-VIIi isolates in Multiple alignments of the NDV F gene region sequences were made using the MEGA–7 software. Phylogenetic analyses were carried out using the criterion of neighbor-joining trees based on genetic distance model by Tamura and others [14]. The reference sequences from the GenBank for NDV classification were used to reconstruct the topology of the F gene sequences generated in this study. The partial NDV F gene sequences obtained in this study were submitted to GenBank (MK210596, MK210597, MK210598, MK210599, MK210600).

Turkey. Also, NDV viruses prevailing in the migratory birds pose a serious risk to poultry in Turkey and other countries.

Background
Newcastle disease virus (NDV) can potentially infect all species of birds and widely circulates in poultry and wild birds [1]. NDV exists in different pathotypes (lentogenic, mesogenic, and velogenic), and carries distinguishing genetic markers in the virus surface glycoproteins hemagglutinin-neuraminidase (HN) and fusion (F). The different pathotypes can cause a range of morbidities in avian hosts; infection in chickens can vary from inapparent subclinical to severe disease with 100% mortality and in some cases genetic phenotype of NDV does not translate into its pathotype potential [2]. Therefore, in some cases, determining virus virulence has been equally important together with identification of genotypic virulence markers (presence of multi-basic amino acids) at the cleavage site of the F) protein for implementation of appropriate disease control measure.
Turkey is located in the palearctic bio-geography on the flyways of the main migratory bird's route [3]. Istanbul and Bosphorus areas are seasonally populated by birds migrating from eastern Europe [4]. In a recent study, 352 bird species were identified in the Istanbul area [5].
A range of bird species are reservoirs for the NDV strains with enormous genetic diversity [2; 6; 7; 8]. There is likely a dynamic population for NDV which is carried along the transcontinental flyways for transmission to domestic poultry [7; 9]. However, depending on virus genotypes and pathotypes NDV rarely causes severe disease in wild birds [2]. Therefore, it is critical to monitor the virus population diversity in wild birds. Knowledge on the extent of viral burden and their genotypic and pathotypic characteristics can provide real time risk assessment about the emerging threats posed. This would allow development of appropriate disease control tools and implementation of informed disease control strategies. To achieve these goals, we analysed the presence, prevalence and molecular diversity of NDV within migratory and nonmigratory wild birds in Kucukcekmece lake as well as in ducks in Edirne which borders Greece and Bulgaria.

Results
Clinical findings in birds caught in the field and birds submitted to Wildlife Rehabilitation Clinic Neither respiratory signs nor evidence of diarrhoea were observed in the birds caught by traps. All trapped birds looked clinically healthy. In contrast, birds submitted to Veterinary Faculty of Istanbul exhibited a variety of clinical symptoms including exhaustion, diarrhea, emaciation and torticollis (Fig. 2). The common kestrel and 2 little owls submitted to the Wildlife Rehabilitation Clinics died after 2 days of clinical examination.

Necropsy
Tracheal hemorrhages which indicate tracheitis were observed in the dead common kestrel and little owls. Heamorhages and necrosis were seen in the proventriculus of these birds (Fig. 3). The walls of the intestines were thickened and there was green mucoid diarrhoea. Patchy heamorrhages were present in the small intestine. The liver and kidneys were slightly congestive. Pale areas in the liver were noticed. Blood vessels at the surface of the cerebrum and cerebellum were conspicious.

Histopathological findings
Nonsuppurative meningioencephalitis was observed in all birds. The histologic changes included mononuclear perivascular cuffing, edema, congestion, necrosis of purkinje cells and meningitis. Mononuclear perivascular cuffing was the most severe lesion both in the cerebrum and cerebellum (Fig. 4-A). Necrotic hepatitis and diffuse paranchyme degeneration was prominent in the liver (Fig. 4-B). The sinusoids were dilated due to congestion. There was mild infiltration of mononuclear cells in portal regions. Hyperplasia in the bile ducts was observed. The main findings in the kidneys were congestion, mild interstitial nephritis and tubular degeneration. There was necrosis, mononuclear cell infiltration and foci of vacuolation in the glandular acinar tissue of pancreas ( Fig. 4-C).Alveolar vessels were congestive in the lungs. Interstitial pneumonia (Fig. 4-D), chronic myocarditis and chronic catarrhal enteritis were observed. Hemorhages in the small intestine were prominent.

Real time RT-PCR probe assay
During optimization of the asssay, optimal amplification signals were obtained when F and R primers were used in a concentration of 10 pmol/µl with 4 µl cDNA when using the positive controls. There was no positive amplification signal in samples taken from birds caught by traps as well as in the negative controls. However, amplification of NDV F gene was obsered in oropharyngeal swab samples taken from 2 waterfowl, and in tissue samples taken from 2 owls (Athene noctua) and 1 common kestrel (Falco tinnunculus)..

Sequencing and phylogenetic analysis
All 5 samples that were found positive for NDV by real time RT-PCR were subjected to NDV showed some degree of molecular evolution when compared to the earlier isolates (15).
Although strains detected in this study were very close to previously isolated Turkish strains, NDVs detected in a waterfowl, common kestrel and 2 little owls formed a different cluster from those NDVs isolated previously in Turkey, Georgia and Bulgaria (Fig. 6).
Importantly, all recent Turkish NDV isolates carry an F gene that is closely realated to genotype VIIi of NDV class II viruses. Poultry outbreaks with this genotype VIIi have been reported in Iran, Pakistan, Israel and Belgium. Although the strains found in this study are closely related, there is a relatively small degree of molecular divergence within 543 bp of Results of virus isolation has shown that AMPV-1 was prevalent between 0.5 and 2.5% in waterfowl including ducks [17; 18]. However, serological prevalence was reported up to 60% [18; 19]. At present, there is no report on monitoring wild birds for NDV in Turkey.
However, in Turkey, 4 and 81 domestic avian cases were reported to OIE in 2016 and 2017, respectively. There has been no report in 2018.
In a similiar study in Sanjiang natural reserve of Heilongjiang Province of China, migratory waterfowls were monitored for NDV. NDVs were isolated from waterfowls (mallard, goose, common teal and mandarin duck) [20]. In the North Sea, 543 passerine birds were investgated and the lentogenic strain of AMPV-1 was detected in 1.1% of birds [21]. In the USA, virulent strains of NDV have been found in wild birds but more frequently in pigeons, doves and doublecrested Cormorants. Research on NDV in wild ducks, gulls, and shorebirds found novel viral diversity, but no fusion gene sequences associated with high pathogenicity in poultry [7; 8]. However, it has been reported that most prevalent virulent genotype VII causing the endemics in Asia are co circulating into the ducks and chicken [22; 23]. Different genotypes of NDV viurses are prevalent in both poultry and wild birds.
For example, F gene of 47 NDV isolates analysed from poultry outbreaks in Bulgaria were belong to genotypes II, IV, V and VIIb [24]. The subgenotype VIIb was also found in the Middle East [25]. Later sstudy revealed that genotype VIId is circulating in Bulgaria and Ukraine [16]. This subgenotype from Bulgaria and Ukraine may have been part of a broader epizootic process in Eastern Europe rather than separate introductions from Asia or Africa. Similarly, analysis of 2 velogenic strains of NDV from ducks in China showed closer identity with genotype VII [22]. In the last few decades, genotype VI and genotype VII of NDV have been causing sporadic disease outbreaks in many countries in Asia and Europe including Denmark, Sweden, Switzerland, Austria, Hungary, Greece Germany, Belgium, Netherlands, Spain, Italy, Middle East, the Indian subcontinent and Indonesia [26]. Isolates of velogenic NDVs from domestic and synanthropic birds (pigeons, crows, and jackdaws) in Kazakhstan, Kirghizia, Ukraine, and Russia in 1993 to 2007 was sequenced and they were clustered in genotype VII comprising VIIa, VIIb, VIId [27].
In the past, NDV-II, VI and VII lineageas were found in domestic poultry. This is the first study indicating the NDV lineage VIIi is circulating amongst wild birds and can spread virus in and amongst countries. Lately virulent strains of NDV belonging to genotype VII have been causing severe diseases outbreaks in poultry in many neigbouring countries of Turkey. Genotype VIId has been isolated from Bulgaria and Ukraine between 2002 and 2013. Iran has reported poultry outbreaks with genotype VIIb [25] and VIId [27] and VIIi [28; 29]. These studies conclude that genotype VII is a domiant strain in poultry and wild migratory birds and gradually undergoing adaptive changes, retaining fitness to suvive in both immune and naturally exposed birds and having the ability to spread via migatory birds.
Our study validates these findings, 4 out of the 5 F gene sequences (MK210596.1, (KT585631.1, KP271974.1, KP271975.1, KP271976.1, KP271979.1) that were previously isolated in Turkey, Bulgaria (KP271973) and Georgia (KP271972.1) suggesting that this genotype remains endemic. However, they were slightly different than previously isolated Turkish strains (KT585617.1 and KT585629.1). Our data indicate that recent Turkish isolates of this study showed some degree of molecular evolution when compared to the earlier isolates [15]. Interestingly, one (waterfowl) of the 5 NDV-F gene sequences (MK210599.1) obtained in this study was slightly different than previously isolated Turkish

Conclusions
Results of this study indicate that for the first time, NDV-VIIi is circulating amongst wild birds and risk of spreading to different countries and continents. This threatens commercial and backyard poultry thorughout the world, including Turkey, and as a result poultry production in many countries are experiencing economic loss. Therefore, continued surveillance of NDV in both migratory birds and poultry is critical for assessment of genetic traits of these viruses and their potential threats to poultry and wild birds. This can only be achieved through establishment of stronger national and international collaborations performing regional surveillance and improving disease control strategies.

Description of the wild bird trapping area
In the present study, field work was performed on the South East European migratory route of wild birds in the Marmara region of Turkey. Traps were placed around the Kucukcekmece Lake in Avcilar, Istanbul, in order to catch migratory and nonmigratory wild birds (Fig. 1). The lake contains brakish water and is about 16 km 2 in size, surrounded by villages, agricultural areas and forests with oaks, ash trees, shrubbery, and turpentine trees [5].

Bird traps, wild bird population and collection of samples
Mist nets were placed around Kucukcekmece lake (Fig. 1)  East European Network (SEEN) for researchers [10]. For sample collection, approval and permission were taken from the Ethics Committee of the Istanbul University-Cerrahpasa (Ethics No: 2016/39). Moreover, a legal permission to do field studies was taken from the Ministry of Forestry of Turkey and local legislation rules were strictly followed as well as international guidelines. The birds caught were categorised according to species and sex (in species having sexual dimorphism) as described previously [11]. A total of 297 birds belonging to 42 species were trapped (Supplementary document Table 1 In addition, tissues (brain, tarchea, lung, liver, pancreas and intestine) at necropsies were taken from dead birds including falcons, common kestrels submitted to Istanbul Veterinary Faculty of Istanbul (originally examined in the Wildlife Rehabilitation Clinic) and wild birds submitted to Ceyhan Veterinary Faculty.

Histopathology
Tissue samples (brain, lung, liver, pancreas and intestine) from the dead birds were analysed histopathologically. For this, samples were fixed in 10% neutral buffered formalin, embedded in paraffin blocks, cut into 4-5 µm sections, stained with hematoxylin and eosin (HE), and blindly examined in the Department of Pathology.

RNA extraction and reverse transcription
Tubes containing oropharyngeal swabs were vortexed individually after adding 500µl of nuclease free water. A total of 619 oropharyngeal swabs were pooled into 206 samples Real time RT-PCR probe assay for NDV All samples were analysed by real time RT-PCR assays for the matrix gene of NDV. Primers and probes used to detect NDV-RNA were described previously [12]. An optimized real- RT-PCR for sequencing partial NDV-F gene Primers used for sequencing parts of the NDV-F gene were designed based on a previosu study [13]. Samples found to be positive for NDV by real time RT-PCR were subjected to RT-PCR as described previously [13]. An optimisedRT-PCR reaction consisted of a 25 μl reactions, nuclease-free water was used as negative control in place of template as well as NDV specific RNA as positive control. After the PCR, the presence of the 534 bp product for NDV-F gene was confirmed by agarose gel (1.5%) electrophoresis. Products obtained by RT-PCR using the primers specific for the partial NDV-F gene were sequenced by a commercial company (MedSanTek, Istanbul, Turkey).

Phylogenetic analysis
Multiple alignments of the NDV F gene region sequences were made using the MEGA-7 software. Phylogenetic analyses were carried out using the criterion of neighbor-joining trees based on genetic distance model by Tamura and others [14]. The reference sequences from the GenBank for NDV classification were used to reconstruct the topology of the F gene sequences generated in this study. The partial NDV F gene sequences Consent was taken from all authors to participate in the study.

Consent for publication
Consent was taken from all authors for publication.

Availability of data and material
The data generated and/or analyzed during this study are available from the corresponding author upon request.     Neighbour-joining phylogentic tree based on partial NDV-F gene (534bp) sequences. Strain classification has been performed using the reference sequences submitted to GenBank. Red marks indicate strains detected in this study. Bootstrap supports reported near to the corresponding tree node.

Supplementary Files
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