J Arthropod-Borne Dis, December 2019, 13(4): 391–398 V Moin-Vaziri et al.: A Molecular Screening of … 391 http://jad.tums.ac.ir Published Online: December 31, 2019 Original Article A Molecular Screening of Mosquitoes (Diptera: Culicidae) for Flaviviruses in a Focus of West Nile Virus Infection in Northern Iran Vahideh Moin-Vaziri1; Remi N Charrel2; Mehdi Badakhshan3; Xavier de Lamballerie2; Nourina Rahbarian1; Mulood Mohammadi Bavani3; *Shahyad Azari-Hamidian4 1Department of Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran 2Unité des Virus Emergents (UVE: Aix Marseille Univ, IRD 190, INSERM 1207, IHU Méditerranée Infection), Marseille, France 3Department of Medical Entomology and Vector Control, School of Public Health, Urmia University of Medical Sciences, Urmia, Iran 4Department of Health Education, Research Center of Health and Environment, School of Health, Guilan University of Medical Sciences, Rasht, Iran (Received 05 Dec 2018; accepted 05 Dec 2019) Abstract Background: Mosquito-borne arboviruses such as West Nile (WN), dengue, Rift Valley fever, and Sindbis viruses are reported in Iran, but large-scale studies have not been performed on mosquitoes to find their vectors. A molecular study of the adult mosquitoes (Diptera: Culicidae) for flaviviruses was carried out in a focus of WN infection, Guilan Province, northern Iran. Methods: Mosquito collections were carried out in five stations of two counties (Anzali and Rasht) using light traps, hand catch by manual aspirators and night landing catch during August–September 2013 and 2014. Molecular screening of WN virus and more widely for Flavivirus RNA was carried out using a specific PCR technique. Results: In total, 1015 adult mosquitoes were collected including eight species representing four genera. The most preva- lent species were Aedes vexans (33.2%), Culex tritaeniorhynchus (22%), Cx. pipiens (20.7%), and Anopheles maculi- pennis s.l. (15.6%). Molecular screening was carried out on the 1015 mosquitoes after they were organized as 38 pools according to sex, species and trapping location. None of the pools were positive. Conclusion: Surveillance should be continued while increasing the sampling campaigns due to the presence of wetlands in the region and abundant species which are considered as vectors, feeding on both birds and humans. Keywords: Aedes; Anopheles; Coquilletidia; Culex; Flavivirus Introduction Based on the latest classification, mosquitoes (Diptera: Culicidae) include two subfamilies, 11 tribes, 41 or 113 genera (depending on the ge- neric classification of the tribe Aedini) and 3563 species (1). Mosquitoes are involved in the trans- mission of several arboviruses belonging to dif- ferent families such as Flaviviridae, Phenuiviri- dae and Togaviridae (2). The family Flaviviridae comprises 58 viruses that are of greatest con- cerns for human health. The genus Flavivirus includes 53 virus species, of which 39 are trans- mitted by mosquitoes or ticks (3). West Nile virus (WNV) (Flaviviridae: Flavivirus) is distrib- uted in Eurasia, Africa, North and Central Amer- ica and Australia. Mosquitoes are the principal vectors of the virus and some virus isolations have been reported from soft and hard ticks (Arachnida: Ixodida). Wild birds, especially wet- land species, are the principal vertebrate hosts and also the virus has been isolated from mam- mals and frogs (4). The most recent checklist of Iranian mos- quitoes includes seven genera and 64 species (5, 6). Subsequently, Anopheles superpictus was *Corresponding author: Dr Shahyad Azari- Hamidian, E-mail: azari@gums.ac.ir http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2019, 13(4): 391–398 V Moin-Vaziri et al.: A Molecular Screening of … 392 http://jad.tums.ac.ir Published Online: December 31, 2019 found to be a complex including two species in Iran based on the internal transcribed spacer 2 (ITS2) sequences of rDNA (7) which later were listed as species A and B (8). A new species of the An. hyrcanus group (An. hyrcanus spIR) was recognized from southwestern Iran, also based on ITS2 sequence data (9). More recent- ly, the occurrence of Aedes albopictus [Stego- myia albopicta] and Ae. unilineatus [Stegomyia unilineata] in southeastern Iran was reported (10, 11). Finally, Orthopodomyia pulcripalpis was reported in northern Iran (12). Several pathogens, which are known to be transmitted by mosquitoes, are reported in Iran such as Dirofilaria immitis and D. repens (13), different Plasmodium spp. (9, 14), West Nile, dengue viruses (DENV) (Flaviviridae: Fla- vivirus), Sindbis virus (SINV) (Togaviridae: Al- phavirus) and Rift Valley Fever virus (RVFV) (Phenuiviridae: Phlebovirus) (15–20). It is note- worthy that there is the possibility of some other mosquito-borne arboviral outbreaks including Japanese encephalitis virus (JEV) (Flaviviri- dae: Flavivirus) in the World Health Organi- zation Eastern Mediterranean Region (21). Re- cently, infection to WNV has been confirmed by the polymerase chain reaction (PCR) tech- nique in Ae. caspius [Ochlerotatus caspius] and Culex pipiens in northwestern and northern Iran, respectively (22, 23). West Nile virus is reported in 26 provinces (Out of 31) of Iran in horses (17, 24, 25), hu- mans (15–17, 26–30) and birds (31). Guilan Province in Caspian Sea littoral, northern Iran, with vast wetlands is probably one of the foci of WNV where the infection is found in hu- mans (1.4–10%) (16, 17, 26), horses (2.2–25%) (17, 24) and birds (especially common coot as a main reservoir) (62.7%) (31). Surprisingly, despite the obvious importance in the emergence of viral diseases caused by fla- viviruses such as WNV and DENV, little data is published about their mosquito vectors in Iran and large-scale studies have not been performed. Also, there is no official vector surveillance for WNV. The aim of the present investigation was to inventory mosquitoes in Guilan Prov- ince and to screen these mosquitoes for WNV and more widely for flaviviruses using both virus-specific real-time RT-PCR and a real- time pan-flavivirus RT-PCR, respectively. Materials and Methods Study areas The study took place in Guilan Province (36o33'–38o27' N and 48o32'–50o36' E) during August–September 2013 and 2014. The prov- ince is located along the Caspian Sea and sur- rounded by Mazandaran Province in the east, Ardebil in the west and Zanjan and Qazvin in the south. It also borders the Republic of Azer- baijan in the north, as well as Russia across the Caspian Sea. The province is humid with mean annual rainfall ranging 1000–2000mm. Collec- tions were carried out in five sites including Saghalaksar of Rasht, Dehboneh of Sangar, Sar- avan Park and Chonchenan of Zibakenar (all in Rasht County) and Ghazian alongside Anzali Wetland, which is one of the few international Iranian wetlands (Anzali County) (Fig. 1). The collection sites are shown over the layers of minimum and maximum degree of temperature and annual rainfall of Guilan Province in Fig. 2. Mosquito sampling and processing Adult mosquitoes were collected by using CDC miniature light traps, operating overnight from sunset to sunrise, i.e. from 18:00PM to 6:00AM, hand catch by manual aspirators from hen shelters, barns and bathrooms, and night landing catch from human bate. Specimens were transferred alive to the laboratory where iden- tified using morphological-based keys (6) after anaesthetizing using an ice bag then trans- ferred to the portable nitrogen tank. Pooling of mosquitoes for viral RNA testing The mosquitoes were grouped into the pools according to sex, species and trapping location. Pools were homogenized in a final volume of 600μL as previously described (32). A 200μL http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2019, 13(4): 391–398 V Moin-Vaziri et al.: A Molecular Screening of … 393 http://jad.tums.ac.ir Published Online: December 31, 2019 volume was used for viral nucleic acid (NA) extraction using the BioRobot EZ1-XL Ad- vanced system (Virus Extraction Mini Kit, Qi- agen). Real-time RT-PCR for specific detection of West Nile virus RNA FiveμL of NA was used for RT-PCR. Sense (ProC-F1: CCTGTGTGAGCTGACAAACTT AGT) and reverse (ProC-R: GCGTTTTAGC ATATTGACAGCC) primers were combined with the fluorogenic TaqMan probe (ProC-TM: 6FAM-CCTGGTTTCTTAGACATCGAGAT CTTCGTGC TAMRA), and used with the Go Taq Probe 1-Step RT-qPCR (Promega) as pre- viously reported (33). This assay was developed and is routinely used by the French Reference Centre for Arboviruses. Molecular detection of Flavivirus RNA Total nucleic acid extraction was conduct- ed by using a Biorobot EZ1, with virus Mini Kit v2.O (Qiagen). Another 5µl aliquot of NA was used in a one-step Real-Time Quanti-Tec SYBER-GREEN RT-PCR assay (Qiagen) as previously described (34). This assay located in a highly conserved region of the polymerase gene allows detection of all recognized fla- viviruses, species identification is then achieved by sequencing the PCR product and comparing the sequence using BLAST NCBI program soft- ware and database. Results A total of 1015 adult mosquitoes were col- lected from Guilan Province including eight species representing four genera as follow: three species within subfamily Anophelinae (An. maculipennis s.l., An. pseudopictus, An. sacharovi) and five species within subfamily Culicinae (Ae. vexans [Aedimorphus vexans], Coquillettidia richiardii, Cx. pipiens, Cx. theileri, Cx. tritaeniorhyncus) (Table 1). The largest species diversity was observed in Ghazian, alongside Anzali Wetland, where all eight spe- cies were recorded. The most prevalent species were Ae. vexans (33.2%), Cx. tritaeniorhyn- chus (22 %), Cx. pipiens (20.7%), and An. maculipennis s.l. (15.6%), respectively. Culex pipiens was collected from all five sites fol- lowed by Ae. vexans and Cx. tritaeniorhynchus (four sites) and An. maculipennis s.l. (three sites) (Table 1). The composition percentages of species based on the collection methods were as follow: An. maculipennis s.l. [91.8% by night landing catch (NLC), 8.2% by aspira- tor (AS), An. pseudopictus (90.9% by NLC, 9.1% by AS), An. sacharovi (100% by AS), Ae. vexans (96.4% by NLC, 3.6% by AS), Cq. richiardii (100% by NLC), Cx. pipiens [55.7% by AS, 37.6% by NLC, 6.7% by light trap (LT)], Cx. theileri (100% by AS), Cx. tritaeniorhyn- chus (78.1% by AS, 21.9% by NLC). The 1015 mosquitoes were grouped into 38 pools according to sex, species and trapping loca- tion. None of the 38 pools tested by real-time RT-PCR either for flaviviruses or more spe- cifically for West Nile virus was positive. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2019, 13(4): 391–398 V Moin-Vaziri et al.: A Molecular Screening of … 394 http://jad.tums.ac.ir Published Online: December 31, 2019 Table 1. Details of collected specimens based on study areas and collection methods Species n Collected sites Collection methods Year total An. maculipennis s.l. 11 Rasht-Saghalaksar ASa 2013 158 (15.6%) 1 Anzali Wetland NLCb 2013 2 Sangar-Dehboneh AS 2013 144 Anzali Wetland NLC 2014 An. pseudopictus 4 Anzali Wetland AS and NLC 2013 22 (2.2%) 4 Anzali Wetland NLC 2014 14 Saravan Park NLC 2014 An. sacharovi 18 Anzali Wetland AS 2014 18 (1.8%) Ae. vexans 1 Rasht-Saghalaksar AS 2013 330 (32.5%) 8 Zibakenar-Chonchenan NLC 2013 57 Anzali Wetland AS and NLC 2013 255 Anzali Wetland NLC 2014 9 Saravan Park NLC 2014 Cq. richiardii 11 Anzali Wetland NLC 2013 35 (3.4%) 24 Anzali Wetland NLC 2014 Cx. pipiens 84 Rasht-Saghalaksar AS 2013 210 (20.7%) 30 Zibakenar-Chonchenan LTc and AS 2013 9 Anzali Wetland AS 2013 8 Sangar-Dehboneh AS 2013 51 Saravan Park NLC 2013 26 Anzali Wetland NLC 2014 2 Saravan Park NLC 2014 Cx. theileri 18 Anzali Wetland AS 2014 18 (1.8%) Cx. tritaeniorhyncus 30 Rasht-Saghalaksar AS 2013 224 (22.0%) 143 Anzali Wetland AS 2013 2 Sangar-Dehboneh AS 2013 29 Anzali Wetland NLC 2014 20 Saravan Park NLC 2014 Total 1015 (100%) aAS = Aspirator, bNLC = Night Landing Catch, cLT = Light Trap) Fig. 1. Map showing the sampling sites (Marked by signs) and the geographical location of Guilan Province in Iran http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2019, 13(4): 391–398 V Moin-Vaziri et al.: A Molecular Screening of … 395 http://jad.tums.ac.ir Published Online: December 31, 2019 Fig. 2. Sampling sites over the layers of different climatic condition of Guilan Province (A: Average maximum degree, B: Average minimum degree C: Annual rainfall) Discussion In total, eight different species of mosqui- toes were collected during this study, all found in Guilan Province before (9, 35). The most prevalent and widespread species of the present study i.e. Ae. vexans, Cx. tritaeniorhynchus, Cx. pipiens and An. maculipennis s.l. were also found frequently as larvae in the previous in- vestigations in the province (35–37). Aedes vexans was the most abundant species (Table 1) mostly captured in the second year of study (2014) adjoining Anzali Wetland. This species is important as a vector of WNV, Snowshoe Hare virus (SSHV) and Tahyna virus (TAHV) (Bunyaviridae: Orthobunyavirus) (2, 4). The sec- ond prevalent species which collected in the current study was Cx. tritaniorhynchus (Table 1) is an important vector of WNV and JEV (4, 21). Culex pipiens was found in all collected sites (Table 1). This species is a domestic mos- quito which has a role in the transmission of some arboviruses including WNV, TAHV and SINV (2, 4). Culex theileri was collected only in 2014 from Anzali Wetland (Table 1). The species is known the vector of WNV (4). Some insect-specific flaviviruses (ISFs) were isolat- ed from this species in Portugal (38) and Tur- key (39) and designated Culex theileri flavivirus (CTFV). Coquilletidia richiardii was also found (Table 1), which is considered among the vec- tors of WNV, SINV and Batai Virus (BATV) (Bunyaviridae: Orthobunyavirus) (2, 4). Among the aforementioned species, Cq. richiardii and Cx. Pipiens in Europe and Cx. tritaeniorhyn- chus in Asia are the main vectors of WNV (4). Among anopheline mosquitoes there are some reports of isolation of WNV and BATV from An. maculipennis s.l. (2, 4), which also was col- lected during this study. Favorable climate of the region, including high precipitations, provides conditions that can lead to the emergence or re-emergence of mosquito-borne diseases (Fig. 2). The presence of rice fields, wetlands and lagoons used by migratory birds may cause new viral outbreaks (31). The area also provides suitable larval hab- itats for many mosquito species. Guilan Prov- ince is a well-known touristic place with thou- sands of visitors annually from different parts of the country. That can increase probable im- ported cases of mosquito-borne infections. Al- so the important vectors of WNV, Cx. pipiens and Cx. tritaeniorhynchus, are among the most abundant and widespread species in the prov- ince (37). As far as the authors know this study is the first one with the aim of Flavivirus screening in mosquitoes in Guilan Province. Most of the available data about the mosquitoes of the prov- ince are based on larval collections (35–37). Sampling adult mosquitoes in Guilan Province, using light traps and hand catch, can be an- other advantage of the present investigation. The http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2019, 13(4): 391–398 V Moin-Vaziri et al.: A Molecular Screening of … 396 http://jad.tums.ac.ir Published Online: December 31, 2019 collection, anaesthetizing, identification, and preparing of collected specimens based on mo- lecular protocols are time-consuming. That ef- fected sample size which could be one of the limitations of the present study to detect the virus. Using sentinel birds as bait may increase the possibility of sampling ornithophilic spe- cies which are WNV vectors and detect the vi- rus. That was not among the goals of this study, however that may be used in forthcoming sur- veys. In view of the absence of official vector surveillance for WNV and many other mos- quito-borne viruses in Iran, such investigations should be continued. Conclusion Although, the mosquito species found in this survey are among proven or potential Fla- vivirus vectors worldwide, our screening by RT-PCR did not reveal any Flavivirus prod- ucts. The total sample size could effect the out- comes. Despite this fact surveillance should be continued due to the presence of WNV infec- tions in humans, horses, birds and known vec- tors in the region. The probable forthcoming finding of WNV in mosquitoes will make a chance to compare the relativeness of detected virus strains in vectors and vertebrate hosts in the region. Also screening for other arbovirus- es other than Flaviviridae, which were not in- vestigated in the present study, should be con- sidered for future studies. 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