J Arthropod-Borne Dis, March 2021, 15(1): 126–132 H Staji et al.: The First Study of … 126 http://jad.tums.ac.ir Published Online: March 31, 2021 Short Communication The First Study of West Nile Virus in Feral Pigeons (Columba livia domestica) Using Conventional Reverse Transcriptase PCR in Semnan and Khorasane- Razavi Provinces, Northeast of Iran *Hamid Staji 1 ; Morteza Keyvanlou 2 ; Zeinab Geraili 3 ; Hedyeh Shahsavari 3 ; Elnaz Jafari 3 1Department of Pathobiology, Faculty of Veterinary Medicine, Semnan University, Semnan, Iran 2Department of Clinical Sciences, Faculty of Veterinary Medicine, Semnan University, Semnan, Iran 3Faculty of Veterinary Medicine, Semnan University, Semnan, Iran *Corresponding author: Dr Hamid Staji, E-mail: hstaji@semnan.ac.ir (Received 16 Nov 2019; accepted 13 Mar 2021) Abstract Background: West Nile Virus (WNV) is an arboviral infection continuing to be as major threat to human health as well as the livestock industry all around the world. Birds including pigeons are one of the potential reservoirs for WNV. This study aimed to detect the presence of WNV genome in feral pigeons circulating in Semnan and Khorasane-Razavi Prov- inces (Iran) including 10 urban and 10 suburban areas. Methods: Totally, 150 samples (brain and kidney) were collected equally from feral pigeons and the presence of WNV genome was evaluated in these samples after RNA extraction. Results: All the samples were negative for the presence of WNV-RNA in this investigation. Conclusion: Although obtained result indicated no evidence of WNV genome in feral pigeons but complementary stud- ies regarding serologic detection of WNV in vertebrate hosts as well as pigeons and identification of arthropod vectors seems necessary for comprehensive determination about infection status in these areas. Keywords: Feral Pigeons; West Nile Virus; RT-PCR; Iran Introduction West Nile virus (WNV) is a mosquito-borne virus related to genus Flavivirus (Flaviviri- dae) with nearly worldwide distribution and spread by mosquitoes belonging to the genera Aedes, Anopheles, Culex and Ochlerotatus (1- 3). Historically, WNV infection was first iden- tified from human cases with encephalitis in Africa (1937) and its significance as a zoono- tic virus was highlighted (1). West Nile Virus infection shows wide distribution characteris- tics throughout southwestern Asia, Russia, the Middle East, Europe, Australia, and Africa. The worldwide distribution of WNV has changed during the last decades by the synergism of predisposing factors such as modern transpor- tation, global warming, and demographic chang- es (4). Previous studies have demonstrated that the WNV has been detected in at least 27 prov- inces of Iran in different hosts including hors- es, humans and birds (5). In nature, WNV circulates in birds and mos- quitoes feeding from birds as biologic vectors and these hosts are characterized as its natural hosts at which the virus amplifies in their body. For WNV, humans and horses are expressed as accidental dead-end hosts notifying its zo- onotic potential more (6). West Nile Virus in- fection has been reported from different conti- nents as well Asia. The Iranian Semnan and Khorasan-Razavi Provinces are situated in the central and northeastern geographic parts of Iran and these two provinces are well-known for their Copyright © 2020 The Authors. Published by Tehran University of Medical Sciences. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited. mailto:hstaji@semnan.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, March 2021, 15(1): 126–132 H Staji et al.: The First Study of … 127 http://jad.tums.ac.ir Published Online: March 31, 2021 suitable conditions for migratory birds from oth- er countries in cold seasons. Migratory birds are one of the major reservoirs of WNV for other avian (pigeons) species and for arthropod hosts and virus distribution occurs via different roots of transmission such as feeding conditions (7). Previous studies confirm the existance of WNV in pigeons and their possible role in spreading of WNV to mammalian hosts (8-10). Cities and livestock farms are considered as two suitable places for pigeons to make colo- nies because of the existence of food and for nesting purposes. So, these birds are now well adapted to farms and cities. Recently, there has been some concern about zoonotic pathogens that can be transmitted via pigeons to humans and domesticated animals as well horses. Spe- cially, when such bird species co-exist with humans and livestock, they are considered to be a high risk factor for transmitting avian- derived pathogens such as WNV because of the strong chance of contact with mammalian hosts (8). In Iran, some investigations have confirmed the seroprevalence of WNV infec- tion in humans, equines and wild water birds (birds circulating in water like ducks) and ge- nome (WNV RNA) detection in mosquitoes but WNV RNA-detection found in these stud- ies are very limited (11, 12). However, despite the presence of WNV genome in mosquitoes and human cases with encephalitis from Iran (11, 13), there is no information about the ex- istance of WNV in free-living pigeons in Iran. Therefore, a cross-sectional (5 years) study was carried out to investigate the molecular prevalence of WNV in feral pigeons from Sem- nan and Khorasane-Razavi Provinces (Iran) to identify the probable role of these birds as reservoirs of WNV in this two regions. Materials and Methods Sample collection In this cross-sectional study, the sample size was calculated using the following equation: n= 4 PQ/L2, where n represents the minimum sample size needed for the prevalence estima- tion, P: prevalence (assumed prevalence of WNV in pigeons of the screened areas was considered 50% because of unavailability of previous data regarding its prevalence), Q: 100P, and L: allowable error or precision (con- sidered 0.1 in the present study). Then, the min- imum required sample size became 100 sam- ples from birds. Brain and kidney samples were taken based on sterile procedure from deeply injured, eu- thanized with ketamine (30mg/kg) or freshly dead wild pigeons which had been admitted to different vet clinics located in Semnan and Khorasene-Razavi Provinces during a 5-year period. The study was performed on 150 pigeons from different regions of (including 70 sam- ples from 10 urban and 80 samples from 10 suburb areas) Semnan and Khorasane-Razavi provinces (Fig. 1) in north-eastern Iran during for autumn 2014 to winter 2019. In the present study, the 70 samples from urban regions were collected from six cities located in Semnan Province including: Semnan (35.5537810°N, 53.3791795°E), Damghan (36.1747867°N, 54. 3542161°E), Shahroud (36.3917606°N, 54. 9941698°E), Biyarjomand (36.0971499°N, 55. 8126512°E), Mayamey (36.4227077°N, 55. 6451097°E), Forumad (36.5198894°N, 56. 7492357°E) and four cities located in Khorasane- Razavi Province including: Sabzevar (36. 2301945°N, 57.6391283°E), Davarzan (36. 3431047°N, 56.8838182°E), Sheshtemad (35. 94831686°N, 57.7599779°E) and Rudab (36. 0283218°N, 57.3040453°E). Also, the 80 sam- ples from suburbs were collected from five re- gions located in Semnan Province including: Ebrahim Abad (36.4160772°N, 55.7220140°E), Abkhori (35.8064424°N, 53.8563982°E), Ala (35.5431662°N, 53.4924760°E), Delazian (35. 4978981°N, 53.4066454°E), Armian (36. 3630742°N, 55.4031629°E) and five regions located in Khorasane-Razavi Province includ- ing: Kahak (36.3584244°N, 56.7727503°E), Mazinan (36.3156334°N, 56.8212518°E), Sadkharve (36.3119113°N, 57.0695328°E), J Arthropod-Borne Dis, March 2021, 15(1): 126–132 H Staji et al.: The First Study of … 128 http://jad.tums.ac.ir Published Online: March 31, 2021 Chesham (36.1717463°N, 57.0416171°E) and Karrab (36.3553765°N, 57.5003219°E). These birds were not vaccinated against any viral agents, previously. Generally, sampling was accidental and kidney and brain samples were collected from mentioned birds and stored at −70 °C in sterile falcon tubes until performing RNA extraction. Viral RNA extraction and RT-PCR ampli- fication The experiment was carried out using con- ventional Reverse Transcriptase PCR (RT-PCR) technique. Total RNA was extracted from brain and kidney samples using RNX- Plus Solution (Sinaclon, Iran) as described by the manufac- turer’s instructions. Then, cDNA was synthe- sized from 500ng of total RNA using CycleScript Reverse Transcriptase kit (Bioneer, Korea). De- tection of WNV was carried out by amplifica- tion of the 3-untranslated region (3-UTR) us- ing previously described primer pair (14). For all RT-PCR reaction sets, a plasmid contain- ing the 3-UTR region of WNV was used as positive control and distilled H2O as negative control. Each 25μL PCR reaction consisted of 2× concentration mastermix containing poly- merase, dNTPs, MgCl2, reaction buffer, and stabilizers (Jena Bioscience, Germany) by add- ing 0.8μL of each primer (10μmol/L; forward primer, CAGACCACGCTA CGGCG; reverse primer, CTAGGGCCGCGT GGG) and 50ng of template cDNA quantified by Nanodrop® (Smart Nano, Canada). Reaction mixes were overlaid with mineral oil and subjected to one step of 94 °C for 3min and then 35 cycles of amplification in a thermocycler (BIOER XP Cycler, China). The cycling condition was as follows: denaturation at 94 °C for 3min, an- nealing at a temperature specific (achieved by gradient temperature conditions) to the primer pairs 55 °C for 40s, and extension at 72 °C for 60s. Then, a final extension step was followed at 72 °C for 5min for completion of the last PCR cycle. Polymerase chain reaction ampli- cons (8–10μL) were electrophoresed in 3% aga- rose (Sigma-Aldrich) gel, stained with ethidi- um bromide (Sigma-Aldrich), visualized and photographed under a UV illuminator (Nano- lytik™, England). Midrange DNA ladder (100 bp, Jena Bioscience) was used for fragment size determination. Results The molecular experiment amplified the expected fragment of DNA (536bp) in the positive control but all the 150 kidney and brain samples tested for the presence of West Nile Virus RNA by using conventional RT- PCR were negative as shown in Fig. 2. Fig. 1. Location of Semnan and Khorasane-Razavi Provinces in Iran (a). The magnified area of both provinces at which the sampling regions are distributed within the area (b) J Arthropod-Borne Dis, March 2021, 15(1): 126–132 H Staji et al.: The First Study of … 129 http://jad.tums.ac.ir Published Online: March 31, 2021 Fig. 2. Gel electrophoresis results of detected gene fragment regarding the West Nile Virus in the brain and kidney samples of screened pigeons. M: Marker (100bp), C+: Positive Control (3-UTR region of WNV weighting 536bp), B: Blank (Negative Control), 1-2: Samples showing no amplified fragments (negative samples) Discussion WNV genome was detected in none of the 150 screened pigeons in the present study. Previous publications have documented the role of migratory birds in the epidemiology of WNV. Also, field and experimental studies have shown that the pigeons have high WNV sero- prevalence rates in endemic areas and can re- produce significant levels of viraemia (15-17). Some investigations have confirmed the pres- ence of WNV genome (RNA) in pigeons in Asian countries such as Korea (8). However, to the best of our knowledge, no detailed study aimed at evaluation of the role of pigeons as reservoir for currently circulating Iranian WNV strains has been performed. According to the results regarding the absence of WNV genome in the pigeons of monitored areas in the pre- sent study, it is highly recommended to per- form a serologic survey on these bird species to find out their probable previous exposure to WNV and their seropositivity. The absence of WNV in these areas and in vertebrate hosts as well pigeons, the very low virus loads in the samples and region’s weather conditions in sampling seasons may provide unsuitable cir- cumstances for the presence of WNV in screened areas. West Nile virus is an emerging infectious pathogen for a variety of hosts including mam- mals, birds and even reptiles with a worldwide geographical distribution as well as Asian countries (18, 19). Migratory birds species play an important role in spreading WNV and specific species of birds are considered as crit- ical reservoirs for this viral agent because the ornithophilic arthropods especially Culex spp. feeding on birds play important role in the natural cycle of WNV infections (23, 25-26). Then, birds circulating around humans and farm animals can introduce WNV to these verte- brate hosts via infecting the Culex mosquitoes and their bites, subsequently. Previous investigations have confirmed the presence of WNV infection in Iran and neigh- J Arthropod-Borne Dis, March 2021, 15(1): 126–132 H Staji et al.: The First Study of … 130 http://jad.tums.ac.ir Published Online: March 31, 2021 boring countries via serologic and virological assays (11). The first report regarding the pres- ence of WNV in Iran is documented by Naficy and saidi (22) by serological assays in human cases and higher rates of seroprevalence have been observed in Central and Southwestern Iran. Because of the presence of different climatic conditions and theoretically suitable environ- ments for the establishment of WNV foci across Iran, different investigations have focused on the existence of WNV in arthropods species (11), humans and equine hosts (22-26), and migra- tory and water birds (19) representing a heter- ogeneous geographic distribution of WNV in different regions. However, despite the exten- sive distribution of feral pigeons as potential vector species, there is no information about the existence of WNV in this bird populations and monitoring of feral pigeons in Iran seems to be necessary as a factor involved in the cy- cle of this disease because previous studies have introduced pigeons as reservoirs of WNV (8). In the present study, WNV genome was not detected in screened birds in Semnan and Khorasane-Razavi regions of Iran. However, the reasons of these negative results can de- pend on various impacting factors. Conclusion In conclusion, although our result indicated no evidence of WNV infection in feral pigeons but serological surveillance of these birds as potential vectors for WNV is highly recom- mended to fully understand the actual statues of this infection. For this reason, our inves- tigations will continue for the detection of an- tibodies against WNV in vertebrate reservoirs including pigeons and determination of mos- quitos’ fauna regarding to this viral pathogen existing in these regions. Acknowledgements Authors are grateful to Dr Alireza Chavshin (Department of Medical Entomology and Vec- tor Control, School of Public Health, Urmia University of Medical Sciences) for providing the plasmid as positive control for RT-PCR protocol carried out in this study. No compet- ing financial interests exist. References 1. Trevejo RT, Eidson M (2008) West Nile virus. J Am Vet Med Assoc. 232(9): 1302–1309. 2. 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