173 SHORT COMMUNICATION Parole chiave Caratterizzazione molecolare, Culicoides imicola, Peste dei piccoli ruminanti, Trasmissione. Riassunto Ad ottobre 2015 un focolaio di Peste dei piccoli ruminanti (PPR) ha colpito Antalya, una provincia della Turchia caratterizzata da condizioni climatico ambientali ottimali per gli artropodi vettori. Dal momento che non ci sono dati su un possibile ruolo svolto dalle specie di Culicoides nel trasmettere il virus della PPR (PPRV), questo studio si è posto come obiettivo quello di valutare se Culicoides spp. possano effettivamente agire come vettori del virus. Numerosi Culicoides sono stati catturati da metà ottobre a metà dicembre 2015. Dodici pool di femmine non ingorgate sono stati predisposti ed esaminati per rilevare la presenza del virus attraverso una RT-PCR real time specifica per il gene nucleocapside del PPRV. Dei 12  testati, 7 pool sono risultati positivi. L'analisi della sequenza del gene della subunità  I della citocromo ossidasi mitocondriale, effettuata su Culicoides spp. per identificare la specie implicata, ha individuato C. imicola nei pool positivi. Il confronto tra le sequenze genomiche dei ceppi di PPRV responsabili dei focolai del 2015 e quelli rilevati in C. imicola suggerisce che C. imicola potrebbe aver avuto un ruolo nella trasmissione del PPRV nei focolai di Antalya. Virus della Peste dei piccoli ruminanti in Culicoides imicola (Diptera: Ceratopogonidae) in Turchia Keywords Peste des petits ruminants, Transmission, Culicoides imicola, Molecular characterization. Summary An outbreak of Peste des petits ruminants (PPR) occurred in the Antalya Province in Turkey during October 2015. The Antalya Province has suitable habitats for vectors. There is no information available on the role of Culicoides spp. in the transmission of Peste des petits ruminants virus (PPRV). In this study we investigated the potential role of the Culicoides spp. in the transmission of PPRV. Culicoides were trapped throughout middle of October and middle of December, 2015. A total of 12 pools of non-engorged females were analysed with real-time RT-PCR targeting the nucleocapsid (N) gene of the PPRV. PPRV RNA was detected in 7 of 12 Culicoides pools. These pools were negative for the bovine/ovine beta-actin mRNA. Culicoides spp. were identified to the species level by sequence analysis of the mitochondrial cytochrome oxidase subunit  I gene. The species of Culicoides found PPRV positive was Culicoides imicola. Molecular characterization of field isolates from recent outbreaks and pools of midges that tested positive for PPRV suggests that PPRV replication might occur in Culicoides imicola, and it may have played a role in transmitting PPRV. Veterinaria Italiana 2019, 55 (2), 173-177. doi: 10.12834/VetIt.1069.5784.2 Accepted: 08.08.2016 | Available on line: 30.06.2019 1Department of Virology, Veterinary Faculty, Hatay Mustafa Kemal University, Hatay, Turkey. 2Department of Molecular Microbiology, Konya Veterinary Control Institute, Turkey. *Corresponding author at: Department of Virology, Veterinary Faculty, Mustafa Kemal University, Hatay, Turkey. Tel.: +90 326 2455313 / 1561, Fax: +90 326 2455704, e‑mail: dr_muratank@hotmail.com. Murat Şevik1* and Mustafa Emin Oz2 Detection of Peste des petits ruminants virus RNA in Culicoides imicola (Diptera: Ceratopogonidae) in Turkey 174 Veterinaria Italiana 2019, 55 (2), 173-177. doi: 10.12834/VetIt.1069.5784.2 Peste des petits ruminants in C. imicola Şevik & Emin Oz Germany). RNA was extracted from the supernatant by using a QIAamp viral RNA Mini Kit (Qiagen) on a QIAcube (Qiagen). The real-time RT-PCR was performed to detect N gene of the PPRV (Batten et  al. 2011). Furthermore, Culicoides midges were tested for the recent intake of a blood meal by a quantitative real-time RT-PCR specific for beta-actin mRNA (Toussaint et  al. 2007). Nuclease free water (Qiagen, Hilden, Germany) was used as the negative control in all real-time RT-PCR assays. DNA was extracted from the supernatant of the Culicoides pools by using a QIAamp DNA Mini Kit (Qiagen). The species of Culicoides spp. of the PPRV positive pools was identified to the species level by sequence analysis of the mitochondrial cytochrome oxidase subunit I gene (COI) using the primers C1-J-1718 and C1-N-2191 (Dallas et al. 2003). PPRV RNA was detected in 7 of the 12 pools assayed. The Ct-values of the real-time RT-PCR ranged from 29.8 to 34.1. Reported Ct values generated by using the same assay from blood of experimental infected goats with a Moroccan strain of PPRV were 25.7-34.5 (Hammouchi et  al. 2012). Generally, 100-200  µl of sheep or goat blood are used for detection of PPRV, whereas less than 1  μl of blood remains in a midge after a blood meal. Therefore, Ct values should have been 6-7 units higher when the biting midge is tested by real-time RT-PCR (Hoffmann et al. 2009). Additionally, bovine/ovine beta-actin mRNA was not determined in positive pools. This result showed that the detection of PPRV RNA within the Culicoides midges didn't result from recent blood meals from infected animals. It has been reported that sub-transmissible infections are common in Culicoides and Ct values can be used to define transmissible and sub-transmissible infections (Mellor 2000, Veronesi et  al. 2013). Comparison of the Ct-values (29.8 to 34.1) obtained from the PPRV positive Culicoides pools in this study with predicted quantities (25.7-34.5) of PPRV in the experimental infection study (Hammouchi et  al. 2012) demonstrated that PPRV replication may occur in the biting midges. PPRV-positive Culicoides pools (n  =  7) and one positive sheep blood sample from each district (Aksu, Alanya, Döşemealtı, Korkuteli, Manavgat and Muratpaşa), where PPR outbreaks (n  =  6) have been recently reported, were selected for sequence analysis. The primers described by Forsyth and Barrett (Forsyth and Barrett 1995) were used to amplify the fusion (F) gene of the PPRV, whereas N gene was amplified with the primer pairs N1/N2 (Kerur et al. 2008). Nucleotide sequences of the F and N genes were obtained from 2 of the 7 PPRV positive Culicoides pools (pool-c1 and pool-c4). The two positive nucleotide sequences obtained from F and N genes Peste des petits ruminants (PPR) is a transboundary small ruminant disease characterised by high fever, nasal and ocular discharges, erosive stomatitis, conjunctivitis, pneumonia, and severe diarrhoea (Gibbs et al. 1979, Lefevre and Diallo 1990). Peste des petits ruminants virus (PPRV), classified within the genus Morbillivirus in the family Paramyxoviridae, is the causative agent of the disease (Gibbs et al. 1979). Although PPRV has also been reported in cattle and pigs, sheep and goats are considered the natural hosts of PPRV (Anderson and Mckay 1994). Vaccination of lambs and kids has been used to control PPR in Turkey, however, sporadic outbreaks have been observed. In October 2015, an outbreak of PPR occurred in the Antalya Province, which is located in the Mediterranean region of Turkey. It is known that PPRV needs close contact between infected and susceptible animals for transmission (Lefevre and Diallo 1990). The Antalya Province has suitable climatic circumstances for the growth and distribution of Culicoides. Several species of biting midges of the genus Culicoides play a role in the transmission of viral diseases of ruminant livestock, including Bluetongue (BT), Epizootic hemorrhagic disease (EHD) and Schmallenberg virus (SBV) (Paweska et  al. 2002, Ruder et  al. 2012, Balenghien et  al. 2014). However, there are no evidence of vectorial transmission of PPR in affected (endemic) regions of Africa, Middle East and India. The purpose of this study therefore was to investigate the potential role of the Culicoides spp. in the transmission of PPRV. This study was conducted in the Antalya Province (29°20’-32°35’ E, 36°07’-37°29’ N) in South-West Anatolia. The Antalya Province has Mediterranean climate characterised by mild and rainy winters and hot, dry summers. During 1981-2014, the annual mean temperature was 18.6  °C and annual precipitation was 783.8 mm (Turkish State Meteorological Service). Its elevation is 30 metres, and the average temperature during the study was 17.8  °C. Three sheep farms within a radius of 2  km from the PPRV-infected flocks were selected for the collection of Culicoides. Two of the three farms were located in Manavgat district and one in Döşemealti district where the highest number of PPR cases occurred. Culicoides were trapped at biweekly intervals between middle of October and middle of December 2015 by using Onderstepoort-type blacklight traps. Culicoides spp. were separated from other insects using a stereomicroscope, followed by species identification (Dyce 1969). Subsequently, they were grouped into males, non-engorged and blood-fed females (engorged). Non-engorged females were used for detection of PPRV RNA. Twelve pools of non-engorged females, 10 midges per pool, were homogenized in nuclease-free water (400  µl) by using a tissueruptor (Qiagen, Hilden, 175Veterinaria Italiana 2019, 55 (2), 173-177. doi: 10.12834/VetIt.1069.5784.2 Şevik & Emin Oz Peste des petits ruminants in C. imicola The network analysis based on F gene sequences showed that isolates from this study and previously characterised Turkish isolates clustered in the same taxon (Figure 1). The isolates Culicoides-PPRV  F  01 and Culicoides-PPRV  F  02 differentiated into two nodes with field isolates of the present study and previously characterised Turkish isolates. These two nodes were separated from each other with a single mutation. A previously characterized Turkish isolate (KF478918) from the Antalya Province in 2012 was placed on the same node with the isolate Culicoides-PPRV F 02. The network analysis based on N gene sequences showed that eight isolates of the present study clustered in two taxa (Figure 2). The isolates Culicoides-PPRV  N  01 and Culicoides-PPRV  N  02 formed a node with isolate of Alanya district (KU325487). The field isolates of Döşemealti district (KU325485 and KU325492) were the closest to this node with a single mutation. A Turkish isolate characterised in 2009 (KF478928) was the most distant previously characterised Turkish isolate from this node. A second node with six mutations was formed by three field isolates (KU325489, KU325491 and KU325493) of the present study. Network analyses showed that the number of mutations, among the isolates of the present study, was higher in N genes compared with F genes of corresponding isolates. This is expected because PPRV is more in Culicoides were from the same samples (pool-c1 and pool-c4). Furthermore, nucleotide sequences of the F and N genes were obtained from PPRV positive field samples from the Alanya, Döşemealtı, Manavgat and Muratpaşa districts. Nucleotide sequences of the F gene were not obtained from PPRV positive field samples from the Aksu and Korkuteli districts, only nucleotide sequences of the N gene were obtained from these two field samples. The analysis of the F gene sequences revealed that the homology between the four field isolates in the present study ranged between 99.3% and 100%, whereas the similarity between two isolates from Culicoides midges (KU175170 and KU1751702) and four field isolates ranged from 98.9-99.6%. The two sequences from Culicoides midges showed 98.9% nucleotide homology with each other. The deduced amino acid homology of the isolates of the present study was 100%. The analysis of the N gene sequences revealed that the homology between the six field isolates in the present study ranged between 97.2% and 100%, whereas the similarity between two isolates from Culicoides midges (KU175171 and KU325486) and six field isolates ranged from 97.6-99.4%. The two sequences from Culicoides midges showed 99.8% nucleotide homology with each other. The deduced amino acid homology of the isolates of the present study ranged from 96.4-100%. 216 279 231 16 48 78 572145120144 273 60 81192021237 42 24 297 242 265 237 219 174 156 160 287 282 267 263 243 226 207 204 195 165 150 132 96 93 75 57 27 18 287 270 243 222 166 132 96 90 75 63 57 33 30 19 93 271 288 288 42 123 102 186 270 180 84 126 189 9 160 255 264 93 157 159 168 183 8 213 249 156 120 111 87 54 39 36 252 53 102 123 129 141 234 276 Nigeria/75/1 (X74443) Oman/Ibri/83 (FR667553) Qatar/A37/10 (FN995206) Yemen/01b (FN995999) Ethiopia/96 (FN995997) Sudan/KSUD71 (HQ131956) Iran/R22/10 (FN995204) China/Tibet/07-1 (EU816772) Iraq/02 (FN995440) Pakistan/09 (FN996973) TR/KONYA/Cihanbeyli/2009 (KF478920) TR/Konya/Guneysinir/2011 (KF478921) TR/Konya/Kulu/2011 (KF478922) TR/Dosemealti/Culicoides-PPRV F 01 (KU175170) TR/Antalya/2015/PPRV F1464 (KU325484) TR/Antalya/2015/PPRV F1545 (KU325488) TR/Antalya/2015/PPRV F1578 (KU325490) TR/Isparta/Yalvac/2013 (KF478919) Nepal/09 (FN996974) Nepal/95 (FR667648) Bangladesh/Narayangonj/09 (JX094440) Saudi Arabia/94 (FR667645) India/Sungri/96 (GQ452015) Bangladesh/00 (FR667556) Pakistan/94 (FR667646) Kuwait/99 (FR667644) TR/Manavgat/Culicoides-PPRV-F 02 (KU175172) TR/Antalya/2015/PPRV F1531 (KU325486) TR/Antalya/Ibradi/2012 (KF478918) TR/Konya/Kulu/2013 (KF478923) TR/Nigde/Ulukisla/2011 (KF478924) TR/Aksaray/2011 (KF478917) Guinea/91 (FR667554) Ivory Coast/89 (FR667555) Cote dIvoire/ICV89 (EU267273) 179 66 Lineage I Lineage II Lineage III Lineage IV Figure 1. Phylogenetic network analysis based on the F gene of the field isolates with corresponding sequences published in the GenBank. Numbers along the branches represent the nucleotide changes separating the nodes. Field isolates and isolates from Culicoides midges in this study are marked with round black spot () and black star (), respectively. 176 Veterinaria Italiana 2019, 55 (2), 173-177. doi: 10.12834/VetIt.1069.5784.2 Peste des petits ruminants in C. imicola Şevik & Emin Oz that C.  imicola plays a role in transmitting the Schmallenberg, Bluetongue and Epizootic hemorrhagic disease viruses (Paweska et  al. 2002, Ruder et  al. 2012, Balenghien et  al. 2014). However, to our knowledge, its role in the transmission of PPRV has not been previously reported. In conclusion, the absence of bovine/ovine beta-actin mRNA in PPRV positive pools and the results of sequence and phylogenetic network analyses suggest that PPRV might replicate in Culicoides midges and C.  imicola may have played a role in the transmission of PPRV. However, further experimental studies are needed to confirm PPRV field isolates within C. imicola. prone to mutations on the N gene compared to the F gene (Munir et al. 2015). Sequence comparisons and network analyses (based on F and N genes) showed that circulating PPRV isolates and isolates from Culicoides midges are very closely related and PPRV isolates from Culicoides midges seem to have diverged from previously characterised Turkish isolates (Figure  1 and Figure  2). These findings suggest that virus replication occurred in the biting midges. The sequence analysis of COI gene showed that the species of Culicoides in PPRV positive pools was C.  imicola. Previous studies have reported 243 178 223 195 157 78 45 187 85 24 14 217 220 157 7 13 137 223 247 115 109 68 58 48 175 44 82 122 92 17 142 220 169 223 140 48 97 113 253 31 35 74 25 135 85 46 19 43 63 64 91 126 153 172 179 193 211 214 133 105 79 57 46 37 1456473102109139147177187190198214217244217 223 223 182 182 148 193 184 113 107 65 19 13 135 122 76 99 99 13 21 22 35 38 90 113 120 136 140 151 157 175 191 223 153 18 59 68 85 121 165 176 34 18 35 36 41 43 52 72 83 88 93 107 113 124 132 140 141 166 169 172 179 186 193 235 238 103 77 96 102 106 152 13 215 241 198 188 157 113 100 91 69 63 45 18 103 223 223 57 Nigeria/76/1 (DQ840164) Nigeria/75/1 (DQ840160) Oman/Ibri/83 (DQ840168) United Arab Emirates/86 (DQ840169) Ethiopia/96 (DQ840183) Sudan/Gedarif-KSUD71 (HQ131918) Iran/98 (DQ840185) China/Tibet/0701 (EU360596) Pakistan/Faisalabad/2010 (JN009673) TR/Konya/Cihanbeyli/2009 (KF478928) TR/Konya/Guneysinir/2011 (KF478929) TR/Konya/Kulu/2011 (KF478930) Guinea/88 (DQ840170) Cote dIvoire/89 (DQ840199) Cote dIvoire/ICV89 (EU267273) Lineage I Lineage II Lineage III Lineage IV TR/Antalya/2015 PPRV N1464 (KU325485) TR/Antalya/2015 PPRV N1506 (KU325492) TR/Antalya/2015/PPRV N1545 (KU325489) TR/Antalya/2015/PPRV N1556 (KU325493) TR/Antalya/2015/PPRV N1578 (KU325491) TR/Dosemealti/Culicoides-PPRV N 01 (KU175171) TR/Manavgat/Culicoides-PPRV N 02 (KU325483) TR/Antalya/2015/PPRV N1531 (KU325487) India/Sungri/96 (AY560591) Tajikistan/04 (DQ840198) Saudi Arabia/99/7 (DQ840195) Bangladesh/09 (HQ131961) TR/Isparta/Yalvac/2013 (KF478932) TR/Aksaray/2011 (KF478925) Figure 2. Phylogenetic network analysis based on the N gene of the field isolates with corresponding sequences published in the GenBank. Numbers along the branches represent the nucleotide changes separating the nodes. Field isolates and isolates from Culicoides midges in this study are marked with round black spot () and black star (), respectively. 177Veterinaria Italiana 2019, 55 (2), 173-177. doi: 10.12834/VetIt.1069.5784.2 Şevik & Emin Oz Peste des petits ruminants in C. imicola Anderson J. & Mckay J.A. 1994. The detection of antibodies against peste des petits ruminants virus in cattle, sheep and goats and the possible implication to rinderpest control programme. Epidemiol Infect, 112, 225-231. 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