Molecular characterization of Peste des petits ruminants viruses in the Marmara Region of Turkey

Abstract Recent outbreaks of Peste des petits ruminants (PPR) in the Marmara region of Turkey including the European part of Thrace is important due to its proximity to Europe (Greece and Bulgaria) and the potential threat of spread of PPR into mainland Europe. In order to investigate the circulation of PPRV in the region suspect clinical and necropsy samples were collected from domestic sheep (n = 211) in the Marmara region of Turkey between 2011 and 2012. PPR virus (PPRV) genome was detected in 10.4% (22 out of 211) of sheep samples by real‐time RT‐PCR, and PPR virus was isolated from lungs of two sheep that died from infection. Of the 22 positive samples nine were used for partial N‐gene amplification and sequencing. The phylogenetic analyses indicated that the virus belongs to lineage IV, the same lineage that is circulating in eastern and central part of Turkey since its first official report in 1999. In addition, samples from 100 cattle were collected to investigate potential subclinical circulation of PPRV. However all were found to be negative by real‐time RT‐PCR, and also in serological tests indicating the large ruminants were likely not exposed or infected with the virus. The impact of these findings on the potential threat of spread of PPR to Europe including the first PPR outbreak in Europe in Bulgaria on 23rd June 2018 is discussed.

Turkey is located in the eastern Mediterranean and is a bridge between the continents of Europe and Asia. Turkey is among the highest small ruminant farming countries in the world, with an estimated sheep population of 31.5 million heads and a goat population of 10.4 million heads in 2016 (http://www.fao.org/faostat/ en/?#data/QA retrieved on 24th of October, 2018). Europe is free of PPR. While this paper was at revision stage the first PPR outbreak was reported in Bulgaria on 23rd June 2018. In Turkey PPRV infection was first officially reported in southern and eastern Anatolia in 1999 (OIE, 1999). OIE has reported approximately 1,000 (997)  Africa, outbreaks of PPR in Turkey pose a significant threat to Europe for the incursion of the disease (Parida et al., 2016).
Therefore this study was designed to investigate the circulation of PPRV in domestic small ruminants (sheep) in the Marmara region of Turkey. In addition attempts were also made to look into the large ruminant population in the same region which may provide a snapshot of virus infection within populations where mild disease is present or where small ruminants are regularly vaccinated (Abubakar et al., 2017).

| Ethics statement
This study was approved by the Animal Experiments Ethics Committee of Istanbul University, Istanbul, Turkey and performed in strict accordance with the recommendations of the Animal Experiments Ethics Committee.

| Study area and sample collection
The Marmara region of Turkey bordering Europe was selected for this study. Samples were collected from sheep from 10 administrative districts (out of 11, except Bilecik province) in the Marmara region of Turkey, between June 2011 and March 2012 ( Figure 2). A two stage sampling design was followed in which the first stage was taken forward through questionnaires to select the farms to sample, and the second stage was to select the animals to sample within each farm.
The farms mainly contained sheep with a flock size of 50-300 animals; no goats were encountered in these farms during the course of the study. The animals were over 6 months of age and reported to be unvaccinated by the farmer. Within the districts, some farms practiced mixed farming involving the housing and maintenance of large and small ruminants in close contact. Following this design, a total of 19 farms from seven administrative districts, namely Canakkale (n = 2), Edirne (n = 1), Istanbul (n = 6), Kirklareli (n = 1), Kocaeli (n = 2), Sakarya (n = 1) and Tekirdag (n = 6) were selected and biological samples were collected from a total of 111 animals (both male and female) selected randomly from each farm taking blood and nasal swabs from each animal. From each farm, a maximum number of 15 animals were sampled depending on the flock size. Within the district where PPR like symptoms (fever, respiratory distress, ocular and nasal discharge, weakness, diarrhoea etc.) were observed in some farms, attempts were made to collect samples from other farms where animals were not F I G U R E 1 (a) PPR outbreaks reported in different regions of Turkey from 1999 to 2018 (2018: January to June). All data were taken from OIE official web site (http://www.oie.int/wahis_2/public/wahid.php/Diseaseinformation/statusdetail). showing any PPR like symptoms with the exception of Edirne and Kirklareli. In addition, lung samples were collected from 100 sheep (about 2-12 months old) from 10 administrative districts at post-mortem that died with respiratory distress symptoms or slaughtered for meat purpose (Table 1) making it a total of 211 samples.
Cattle and buffalo can also be infected with PPRV without showing any clinical symptoms and sero-convert (Ozkul et al., 2002;Abraham et al., 2005;Abubakar et al., 2017). Following the same sampling strategy blood and nasal swabs were collected from 50 cattle from 13 farms from seven administrative districts, namely Canakkale (n = 2), Edirne (n = 1), Istanbul (n = 4), Kirklareli (n = 1), Kocaeli (n = 2), Sakarya (n = 1) and Tekirdag (n = 2) to account for subclinical infection. The farms had a flock size of 30-200 animals and were different to the farms where samples from sheep were collected. In each farm, a maximum number of 6 animals were sampled depending on the flock size. In addition, lung samples from 50 slaughtered cattle originating from the same region were also collected making it a total of 100 samples (Table 1).
Samples were transported to the laboratory on ice (4-8°C). The swabs and tissues were stored at −70°C until used while blood samples were processed immediately. The buffy coats from the blood samples with EDTA were collected and stored at −70°C for RNA extraction. Serum was separated from clotted blood and stored at −20°C until tested.

| Antibody detection
Sera collected from sheep (n = 111) and cattle (n = 50) were tested for the presence of PPRV-specific antibodies using the anti-nucleoprotein (N) PPRV competitive ELISA (cN-ELISA, ID Screen ® PPR Competition ELISA, ID.Vet, France). The assays were performed and analysed following the manufacturer's instructions. Samples with percentage inhibition (PI) value <50% were considered positive. All the tests were carried out in duplicate, and the borderline positive samples were repeat tested to confirm results. The average of two results was used in subsequent analysis.

| Virus isolation
For isolation of PPR virus from lung tissues the tissues were processed following the method as previously described (Clarke, Mahapatra, Friedgut, Bumbarov, & Parida, 2017). The filtered lung homogenate was used for RNA extraction and also for virus isolation on Vero.DogSLAM (VDS) cells. The cells were checked every day for the appearance of PPRV-specific cytopathic effect (CPE) for 7 days, and at least 4 blind passages were carried out before the samples were declared negative.

Reaction (PCR) and nucleotide sequencing
Samples found positive by qRT-PCR were selected for PCR and sequencing. The viral RNA was reverse transcribed and the C-terminus of the N-gene was amplified as previously described (Baazizi et al., 2017). The PCR amplicons were purified using the QIAEXII PCR purification kit (Qiagen) according to the manufacturer's instructions and sent to a commercial company (REFGEN, Ankara, Turkey) for sequencing. Sequences were assembled and analysed using SeqMan II (DNAStar Lasergene 8.0). Alignments of the N-gene sequences were made using the Clustal W program and used for construction of distance matrices using the Kimura 2-parameter nucleotide substitution model (Kimura, 1980) as implemented in the programme MEGA 6.0 (Tamura, Stecher, Peterson, Filipski, & Kumar, 2013). A maximum-likelihood phylogenetic tree was then generated using MEGA 6.0, and the robustness of tree topology was assessed using 1,000 bootstrap replicates.

| Data analysis
The data analysis was carried out using Minitab 7.0 software.

| RESULTS AND DISCUSSION
In Turkey PPRV infection was first officially reported from Southeastern Anatolia, Eastern Anatolia and Mediterranean region in October 1999, and from the Aegean region in December of the same year (OIE, 1999) though there had been some previous reports of the presence of the virus in the country (Alcigir, Atalay Vural, & Toplu, 1996;Tatar, 1998;Ozkul et al., 2002). The following year   Out of 111 sheep sera analysed in this study, antibodies to PPRV were detected in 11.7% sheep. These sera originated from 4 different farms in the Marmara region, one from Kocaeli, two from Istanbul and one from Canakkale (Table 1) Of these nine (9) samples (blood: 2, nasal swabs: 4 and both blood and nasal swabs: 3) were biological samples collected from live animals whereas the remainder 13 were from the lung tissue samples (out of 100 samples) collected during necropsy (Table 1) Live infectious virus was isolated from two lung samples on 3rd day of inoculation using VDS cells and on 5th day using Vero cells (data not shown). Culture supernatants from those cells were tested in qRT-PCR and were found to be strongly positive.
No clinical symptoms were observed in cattle sampled in this study. During necropsy at the slaughter house no PPRV-specific pathological findings were observed. In serological tests no antibodies to PPRV were detected in cattle sera. All the samples were also negative in qRT-PCR assays. This indicates that the large ruminants  Figure S1). Stamping out policy included culling of all small ruminants reared in the village.  (Parida et al., 2016). There is also evidence of wild goats and white-tailed deer being either clinically or experimentally infected by PPRV (Hoffmann et al., 2012;Hamdy & Dardiri, 1976). Further from our unpublished data we have recorded wild boars and warthogs in Africa are positive for PPR antibodies. Recently, suids including wild boar have been reported to be experimentally clinically infected with PPRV (Schulz, Fast, Schlottau, Hoffmann, & Beer, 2018). However, at the moment the role of wildlife in the spread of the disease is unknown, and may warrant a survey in future. Therefore continuous monitoring of the disease, tracing of movement of animals in the Marmara region and maintaining a buffer zone in Thrace by regular vaccination is essential to prevent spread of the disease to Europe.

ACKNOWLEDGEMENTS
We would like to thank Professor Yusuke Yanagi and Professor Tuba Cig-

CONF LICT OF I NTEREST
Authors declare that they have no conflict of interest.

STATEMENT OF ANIMAL RIGHTS
There was no ethical issue concerning this study. Animals were humanly treated during sampling.

INFORMED CONSENT
Authors were informed and consent was approved.