J Arthropod-Borne Dis, September 2016, 10(3): 383–393 M Mohammadian et al.: Molecular Assay on … 383 http://jad.tums.ac.ir Published Online: January 06, 2016 Original Article Molecular Assay on Crimean Congo Hemorrhagic Fever Virus in Ticks (Ixodidae) Collected from Kermanshah Province, Western Iran Maria Mohammadian 1, Sadegh Chinikar 2, *Zakkyeh Telmadarraiy 1, Hassan Vatandoost 1, Mohammad Ali Oshaghi 1, Ahmad Ali Hanafi-Bojd 1, Mohammad Mehdi Sedaghat 1, Mehdi Noroozi 3, Faezeh Faghihi 4, Tahmineh Jalali 2, Sahar Khakifirouz 2, Nariman Shahhosseini 2,5, Firoozeh Farhadpour 1 1Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Arboviruses and Viral Hemorrhagic Fevers Laboratory (National Reference Laboratory), Pasteur Institute of Iran, Tehran, Iran 3Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 4Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran 5WHO Collaborating Centre for Arbovirus and Haemorrhagic Fever Reference and Research, Department of Virology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany (Received 25 Dec 2013; accepted 10 Mar 2015) Abstract Background: Crimean-Congo Hemorrhagic Fever (CCHF) is a feverous and hemorrhagic disease endemic in some parts of Iran and caused by an arbovirus related to Bunyaviridae family and Nairovirusgenus. The main virus reser- voir in the nature is ticks, however small vertebrates and a wide range of domestic and wild animals are regarded as reservoir hosts. This study was conducted to determine the infection rate of CCHF virus in hard ticks of Sarpole- Zahab County, Kermanshah province, west of Iran. Methods: From total number of 851 collected ticks from 8 villages, 131 ticks were selected randomlyand investi- gated for detection of CCHF virus using RT-PCR. Results: The virus was found in 3.8% of the tested ticks. Hyalommaanatolicum, H.asiaticum and Rhipicephalus sanguineus species were found to have viral infection, with the highest infection rate (11.11%) in Rh. sanguineus. Conclusion: These findings provide epidemiological evidence for planning control strategies of the disease in the study area. Keywords: Ixodidae, CCHFV, Kermanshah, Iran Introduction Crimean Congo Hemorrhagic Fever (CCHF) is a viral disease with approximate mortality rate of 30% in humans (Ergonul 2006). The disease can be transmitted via contact with blood or secretions of infected animals and tick bite or manipulation and squishing of CCHF infected ticks. Human to human transmission i.e. nosocomial infec- tion is another main way for the disease transmission (Hoogstraal 1979, Charrel 2004, Appannanavarand Mishra 2011). CCHF dis- ease has a worldwide dissemination and is considered to be an endemic disease in many countries in Asia, Africa and Europe conti- nents (Charrel 2004, Appannanavar and Mish- ra 2011). Until now CCHF virus has been detected in 31 species of several species of hard and soft ticks (Hoogstraal 1979, Linthicum and Bailey 1994, Papa et al. 2002). The disease was first reported in Iran during 1970 (Chu- makov1972), and now is considered as an *Corresponding author: Dr Zakkyeh Telmadarraiy, Email: ztelma@yahoo.co.in J Arthropod-Borne Dis, September 2016, 10(3): 383–393 M Mohammadian et al.: Molecular Assay on … 384 http://jad.tums.ac.ir Published Online: January 06, 2016 endemic disease in many parts of the world. Previous studies confirmed CCHF cases. In Iran, many studies were conducted on disease carriers; however in 1978, the virus was separated from soft tick larvae, Ornithodoros lahorensis (Sureau 1980) for the first time. Since then, many studies were conducted in different regions of Iran to find the CCHF infection in ticks. Based on previous studies, CCHF infection was detected in 5 genera of soft and hard ticks including Hyalomma, Rhipicephalus, Der- macentor, Haemaphysalis and Ornithodoros (Shirani et al. 2004, Telmadarraiy et al. 2007). A diverse range of infection rates has been reported in these ticks from 0.2 to 33.3% (Shirani et al. 2004, Moradi et al. 2008, Nasiri 2008, Tahmasebi et al. 2010, Telmadarraiy et al. 2007, 2010, 2014, Salim-Abadi et al. 2011, Chinikar et al. 2012, Faghihi et al. 2015, Sarifinia 2012, Karimi 2013, Mehrava- ran et al. 2013, Champour et al. 2014). Kermanshah Province contains a big pop- ulation of nomads in west of the country, so it is an important region for the legal and illegal import/export of domestic animals from Iraq, the neighbor country at the bor- derline of Sarpole-Zahab City. Crimean- Congo hemorrhagic fever is reported from some parts of Iraq and seems to be an en- demic disease there, where during 1979– 1981, 63 cases of the disease were reported within number of 48 mortalities. Studies over 50% of Iraqi goat, sheep, and horse sera were positive for the presence of antibodies, in another sero-survey 29% of all animal breeders tested in Iraq were also reported to be positive for those antibodies (Defens Pest Managment Information Analysis Center Armed Forces Glen section Walter Reed Army Medical Center Washington 1999). According to the national monitory system of Iraq, 0–6 of CCHF cases were annually reported during 1998–2009, and in 2010, 11 confirmed cases of the disease with 36% mortality and 28 suspected cases with 4% mortality were reported (Majeed et al. 2012). Presence of more than 140,000 livestock in the county makes it as a major area of animal husbandry in Kermanshah province. Adjacent plains and mountainous areas are the major locations for nomad migration, which have large herds of livestock. Sarpole- Zahab has a long borderline with Iraq; make it a suitable area for legal/illegal livestock export/import trades between two countries. So risk of the disease transmission exists in both sides of the borderlines of the two neighboring countries where it can be passed from one side to another side of the border, periodically. This study was aimed to inves- tigate CCHF virus infection rate in ticks of domestic animals, as the main vectors of the disease. Materials and Methods Study area Kermanshah province is located at the western region of Iran. Sarpole-Zahab Coun- ty located at the western margin of the prov- ince, in coordinates of 34o27’40” N and 45 o 51’46” E, with an area of about 1.271 square kilometers. Sarpole-Zahab has a relatively warm and semi-arid climate with the mild winters and hot summers. The county has mountainous, plain, and foothill topographic areas. In this study, eight villages of Sarzal, Ghalee Vari, Mela Kabob, Salman Tape, Berimov and, Anzal, Dare Balut and Sare Baghe Golin located in different geograph- ical locations of the county were selected randomly (Table 1). Tick specimen collec- tion from the livestock was conducted as de- scribed below during the years 2012–2013 (Fig. 1). Based on statistical analysis of available data, 131 tick specimens were se- lected to determine the CCHF infection rate in ticks of the area study. J Arthropod-Borne Dis, September 2016, 10(3): 383–393 M Mohammadian et al.: Molecular Assay on … 385 http://jad.tums.ac.ir Published Online: January 06, 2016 Sample collection and preparation Tick specimens were collected seasonally based on the species diversity, type of host animals and geographic location of the area study. Samples were maintained individually in labeled tubes and transferred to the labor- atory of School of Public Health, Tehran Uni- versity of Medical Sciences in cool boxes. The specimens were identified to species level using the known morphologically keys (Hoogstraal 1979, Walker et al. 2003). 128 specimens of the identified ticks were selected in random and transferred in cold chain to the Arbovirus and Viral Hemorrhagic Fevers Laboratory (National Ref. Lab) at Pasture Institute of Iran for molecular detection of CCHF virus. RNA Extraction and RT-PCR For RNA extraction, each tick was washed twice with PBS 1X and then crushed by a mortar and pestle in 300 µ l of PBS buffer. RNA extraction was performed using RNA easy mini kit (QIAGEN, Germany) based on the protocol recommended by the manufac- turer. The extracted RNA was dissolved in RNase-free water and kept at -70 ˚C until use. RT-PCR reaction was performed using One-Step RT-PCR Kit (QIAGEN, Germany) based on the protocol. In each PCR reaction, 5µ l of the extracted RNA and 1µ l of each specific primer (Forward: 5’-TGGACACC TTCACAAACTC-3’and Reverse: 5’-GAC AATTCCCTACACC-3’) were added to am- plify the small segment (S-segment) of the virus (Chinikar et al. 2004). At next step, 5 µ l of RT-PCR products were mixed with 1µ of loading buffer and the mixture was loaded on 1.5% agarose gel for electrophoresis. DNA bands were stained with ethidiumbromide and were visualized under UV trans illumi- nator (Chinikar et al. 2008, 2010). Sequencing and sequence analysis RT-PCR products were sequenced by ABI Genetic Analyzer 3130 machine using Big Dye Terminator V3.1 Cycle sequencing Kit and specific primers (Chinikar, Shah- Hosseini et al. 2013).The partial sequences around 500 bp of the S-segment were used for phylogenetic analysis (Table 2). The mul- tiple alignments were performed using Clus- tal W, for seven sequences of current study and some sequences from Gen Bank. Phyloge- netic tree was drawn by maximum-likelihood method with Kimura 2-parameter model using Mega 5.2 software. Bootstrap method with replications of 1000 was used for assessing confidence in phylogenetic tree results. Results A total number of 851 ticks were col- lected and identified in this study. Tick in- festation rate was accounted as much as 84.2%, 10.53% and 5.27%, in sheep, cows and goats respectively. Three genera of Ix- odid ticks including 10 species Hyalom- maanatolicum, Hy. asiaticum,Hy. drome- darii, Hy. marginatum, Hy. detritum, Hy. sp, Rhipicephalus sanguineus, Rh. bursa, Rh. sp and Haemaphysalissulcata were identified. A subsetof 131 ticks (15.4%) out of 851 ticks was examined to detect CCHF virus transcripts (Fig. 2). RT-PCR amplification of S-segment of CCHF virus produced a PCR band of 536bp (Table 1). The results of RT- PCR showed an infection rate of 3.8% (n=5) among the tested specimens. The infected species were found to be Hy. anatolicum (4.1%), Hy. asiaticum (4.54%) and Rh .sanguineus (11.11%). The infected ticks were collected from cow and sheep. Molecu- lar results showed CCHF virus genome in 6.38% (3/47) and 2.85% (2/70) of ticks from cowand sheep, respectively, while all ticks collected from goat were negative (Fig .3). Phylogenetic analysis The Phylogenetic relationship of the iso- lated CCHF sequences from ticks of Sar- J Arthropod-Borne Dis, September 2016, 10(3): 383–393 M Mohammadian et al.: Molecular Assay on … 386 http://jad.tums.ac.ir Published Online: January 06, 2016 pole-Zahab with genbank available sequenc- es was drawn using Maximum Likelihood method and kimura2 parameter (Fig. 4). All the isolates were clustered in Asia I clade, closely to Matin and SR3 strains of Pakistan and newly released strain from Afghanistan (Ölschläger et al. 2011). Overall mean dis- tance computation of this study isolates show just 0.3% divergence between them. Fig. 1. Map of the Study area in Kermanshah Province, Iran Fig. 2. RT-PCR products of CCHF S-segment (536 bp band) found in tick specimens collected in Sarpole-Zahab County, Kermanshah Province. Lad: 100 bp ladder, PC: positive control, NC: negative control, S1, S2, S3, S4, S5, S6, S7 and S9: negative samples; S8 and S10: positive samples. Sh ee p Co w Go at 0 20 40 60 80 Negative Positive Host N o of t ic ks t es te d Fig. 3. Details of CCHF infected ticks and their animal hosts in Sarpole-Zahab County, Kermanshah, Western Iran J Arthropod-Borne Dis, September 2016, 10(3): 383–393 M Mohammadian et al.: Molecular Assay on … 387 http://jad.tums.ac.ir Published Online: January 06, 2016 Fig. 4. Maximum Likelihood phylogenetic tree retrieved from 500bp of CCHFV partial S-segment sequences obtained in this study (CCHFM14, CCHFM16, CCHFM18, CCHFM22 and CCHFM24) and the available data from Genbank. Only boot strap values more than 70% are shown. J Arthropod-Borne Dis, September 2016, 10(3): 383–393 M Mohammadian et al.: Molecular Assay on … 388 http://jad.tums.ac.ir Published Online: January 06, 2016 Table 1. Details of the tick species were collected and examined for the presence of CCHF virus genome, Sarpole-Zahab County, Western Iran Animal Location Hy. anatolicum (tested /+) Hy. asiaticum (tested/+) Hy. dromedarii (tested/+) Hy. marginatum (tested/+) Hy. detritum (tested/+) Hy. sp (tested /+) Rh. sanguineus (tested /+) Rh. Bursa (tested/+) Rh. Sp (tested/ +) Ha. sulcata (tested/+) Total Sheep SZ 7/0 3/0 0/0 0/0 1/0 0/0 0/0 1/0 0/0 0/0 12/0 GV 6/0 0/0 0/0 0/0 0/0 1/0 8/0 0/0 1/0 1/0 17/0 MK 5/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 5/0 ST 7/1 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 7/1 BV 3/0 2/0 4/0 2/0 2/0 0/0 1/1 0/0 0/0 0/0 14/1 AN 1/0 2/0 1/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 4/0 DB 4/0 0/0 1/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 5/0 SBG 1/0 3/0 0/0 0/0 2/0 0/0 0/0 0/0 0/0 0/0 6/0 Cow SZ 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 GV 2/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 2/0 MK 7/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 7/0 ST 3/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 3/0 BV 2/0 0/0 2/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 4/0 AN 9/1 4/1 5/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 18/2 DB 6/0 4/0 2/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 12/0 SBG 1/1 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 1/1 Goat SZ 3/0 2/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 5/0 GV 0/0 3/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 3/0 MK 1/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 1/0 ST 1/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 1/0 BV 2/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 2/0 AN 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 DB 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 SBG 2/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 2/0 Total 73/3 23/1 15/0 2/0 5/0 1/0 9/1 1/0 1/0 1/0 131/5 SZ: Sarzal, GV: GhaleeVari, MK: Mela Kabob, ST: Salman Tape, BV: Berimovand, AN: Anzal, DB: DareBalut, SBG: SareBagheGolin. J Arthropod-Borne Dis, September 2016, 10(3): 383–393 M Mohammadian et al.: Molecular Assay on … 389 http://jad.tums.ac.ir Published Online: January 06, 2016 Table 2. Details of sequence data used in this study Isolate Name or Accession Number Country City Host isolated Reference CCHF14 Iran Sarpole-Zahab Sheep This study CCHF16 Iran Sarpole-Zahab Sheep This study CCHF18 Iran Sarpole-Zahab Cow This study CCHF22 Iran Sarpole-Zahab Cow This study CCHF24 Iran Sarpole-Zahab Cow This study HM452305 Afghanistan NS Human Ölschläger et al. 2011 AJ538198 Pakistan Karachi Human Hewson et al. 2004 AY366379 Iran Sistan-Baluchistan Human Chinikar et al. 2004 AY366378 Iran Sistan-Baluchistan Human Chinikar et al. 2004 DQ446213 Iran NS NS Direct submission DQ446212 Iran NS NS Direct submission AY366377 Iran Sistan-Baluchistan Human Chinikar et al. 2004 AY366374 Iran Sistan-Baluchistan Human Chinikar et al. 2004 AY366373 Iran Sistan-Baluchistan Human Chinikar et al. 2004 DQ446214 Iran NS NS Direct submission U88414 NS NS NS Direct submission AY366376 Iran Sistan-Baluchistan Human Chinikar et al. 2004 AY366375 Iran Qom Human Chinikar et al. 2004 AF527810 Pakistan NS NS Direct submission GU456725 Iran Hamedan Tick Chinikar et al. 2010 GU456728 Iran Hamedan Tick Chinikar et al. 2010 GU456724 Iran Hamedan Tick Chinikar et al. 2010 GU456727 Iran Hamedan Tick Chinikar et al. 2010 GU456726 Iran Hamedan Tick Chinikar et al. 2010 AY905662 Pakistan Quetta NS Burt and Swanepoel 2005 AY905663 Pakistan Quetta NS Burt and Swanepoel 2005 AY905661 Pakistan Quetta NS Burt and Swanepoel 2005 NS: Not Stated J Arthropod-Borne Dis, September 2016, 10(3): 383–393 M Mohammadian et al.: Molecular Assay on … 390 http://jad.tums.ac.ir Published Online: January 06, 2016 Discussion Our results in this study showed the pres- ence of CCHF virus in different regions of Sarpol-e-Zahab City, Kermanshah Province. This infection was confirmed in Hy. anatoli- cum, Hy. asiaticum, R. sanguineus. Findings of this study are consistent with the results obtained other groups in different regions of the country (Tahmasebi et al. 2010, Telma- darraiy et al. 2010). Although Hyalomma ticks are the main carriers of the virus in Africa, Asia, Europe and the Middle East (Swa- nepoel et al. 1987, Whitehouse 2004, Ergönül 2006) the virus was also detected in other genera of both soft and hard ticks (White- house 2004) as we found in R. sanguineus. In other studies conducted in western part of Iran, CCHF virus infection was re- ported in Hy. marginatum, Hy. anatolicum, Hy. detritum, Hy. dromedarii, Hy. asiaticum, Haemaphysalispunctata, R. sanguineus, and R. bursa, (Moradiet al. 2008, Tahmasebi et al. 2010, Sharifinia 2012, Fakoorziba et al. 2012, Nasiri Unpublished data). According to our previous study conducted in Ardabil Province, northwest of Iran, viral infection was detected in Hy. schulzei, Hy. margina- tum, Hy. aegyptium and R. bursa (Telmadar- raiy et al. 2010). Studies conducted in west part of Iran showed that the genome of CCHF virus exists in different species of three genera of hard ticks including Hy- alomma, Rhipicephalus and Haemaphysalis. The infection rate of ticks in this study was 3.8%. This rate has been reported from 0.2 to 33.3% in previous studies in Iran (Shi- rani et al. 2004, Telmadarraiy et al. 2006, 2007, 2010, 2014, Moradi et al. 2008, Nasiri 2008, Tahmasebi et al. 2010, Salim-Abadi et al. 2011, Chinikaret al. 2012, Fakoorziba et al. 2012, Sarifinia 2012, Karimi 2013, Mehrava- ran et al. 2013, Champour et al. 2014). Ticks of Hyalomma genus showed an infection rate of 3.36%, while the species of this genus were found to be infected 1.57% to 20% to CCHF virus in other studies (Shiraniet al. 2004, Telmadarraiy et al. 2006, 2007, 2010, 2014, Moradi et al. 2008, Nasiri 2008, Tah- masebi et al. 2010, Salim-Abadi et al. 2011, Chinikar et al. 2012, Fakoorziba et al. 2012, Sarifinia 2012, Karimi 2013, Mehravaran et al. 2013, Champour et al. 2014). Although 9.09% of Rhipicephalus ticks were infected in this study, other groups reported the infection rate between 1.8% to 55% (Tel- madarraiy et al. 2006, 2010, Moradiet al. 2008, Tahmasebi et al. 2010, Karimi 2013), therefore it can be concluded that although the Hyalomma ticks are usually introduced as the vectors of CCHF, this potential exists in other genera as well. Comparison of results obtained from dif- ferent regions of the county showed that ticks collected from central regions were more infected than southern and northern regions. The reason of such infection may be due to the condensation of livestock and the quality of breeding management including poor hy- gienic conditions of livestock breeding sites. People of Sarpol-e-Zahab area work in high risk professions in close contact with ticks and animals’ tissue and blood. There- fore, it is instructed to prevent these people from being subjected to different ticks in- fected with the virus and or infected ani- mals’ blood or tissue. This study indicates that CCHF must be considered as a critical health problem in health centers of Sarpole- Zahab as well as Kermanshah and other neigh- boring provinces, and appropriate strategies must be used for controlling carrier ticks. Future research should be focused on the population of carriers and their infection rate, presence of the virus in domestic ani- mal populations and also humans in other regions of the province in order to present a better picture of the dissemination and epi- demiology of the virus in the province. J Arthropod-Borne Dis, September 2016, 10(3): 383–393 M Mohammadian et al.: Molecular Assay on … 391 http://jad.tums.ac.ir Published Online: January 06, 2016 Conclusion The prevalence of infected to CCHF was higher in plain region of the county rather than mountain region. Teaching and prevention programs are recommended to people who are in close contact with ticks and animals’ tissue and blood to prevent from infection. This study indicates that CCHF must be considered as a critical health problem in health centers of Sarpole-Zahab and other neighbouring county, and appropriate strate- gies must be used for controlling carrier ticks. Acknowledgement The authors appreciate very much for kind collaboration of all the staff of Veteri- nary Office of Sarpole-Zahab County. This study has been done by financial supports of Tehran University offered by Medical Sci- ences and this work is part of Project No. 23859. References Appannaanvar SB, Mishra B (2011) An update on Crimean Congo hemor- rhagic Fever. J Global Infect Dis. 3 (3): 285. Champour M, Chinikar S, Mohammadi G, Razmi G, Shah-Hosseini N, Kha- kifirouz S, Mostafavi E, Jalali T (2014) Molecular epidemiology of Crimean-Congo hemorrhagic fever vi- rus detected from ticks of one humped camels (Camelus dromedarius) pop- ulation in northeastern Iran. J Parasit Dis. pp. 1–6. Charrel R, Attoui H, Butenko A, Clegg J, Deubel V, Frolova T, Gould E, Grit- sun T, Heinz F, Labuda M (2004) Tick‐borne virus diseases of human interest in Europe. Clin Microbiol Infect. 10(12): 1040–1055. Chinikar S, Persson SM, Johansson M, Bladh L Goya M, Houshmand B, Mirazimi A, Plyusnin A, Lundkvist A Nilsson M (2004) Genetic analysis of Crimean- Congo Hemorrhagic Fever virus in Iran. J Med Virol. 73(3): 404–411. Chinikar S, Shayesteh M, Khakifirouz S, Jalali T, Rasi-Varaie FS, Rafigh M, Mostafavi E, Shah-Hosseini N (2013) Nosocomial infection of Crimean– Congo Hemorrhagic fever in eastern Iran: case report. TravelMedInfect Dis. 11(4): 252–255. Chinikar S, Shah-Hosseini N, Bouzari S, Jalali T, Shokrgozar AM, Mostafavi E (2013) New circulating genomic variant of Crimean-Congo Hemor- rhagic fever virus in Iran. Arc Virol. 158(5): 1085–1088. Chinikar S, Ghiasi S, Hewson R, Moradi M, Haeri A (2010) Crimean-Congo hem- orrhagic fever in Iran and neighbor- ing countries. J Clin Virol. 47(2): 110–114. Chinikar S, Ghiasi SM, Naddaf S, Piazak N, Moradi M, Razavi MR, Afzali N, Haeri A, Mostafavizadeh K, Ataei B (2012) Serological evaluation of Cri- mean-Congo hemorrhagic fever in humans with high-risk professions living in enzootic regions of Isfahan Province of Iran and genetic analysis of circulating strains. Vector Borne Zoonotic Dis. 12(9): 733–738. Chinikar S, Goya M, Shirzadi M, Ghiasi S, Mirahmadi R, Haeri A, Moradi M, Afzali N, Rahpeyma M, Zeinali M (2008) Surveillance and Laboratory Detection System of Crimean‐Congo Haemorrhagic Fever in Iran. Trans boundary and Emerging Dis. 55(5– 6): 200–204. Chumakov M (1972) Detection of antibod- ies to CHF virus in wild and domes- tic animal blood sera from Iran and Africa. pp. 367–368. J Arthropod-Borne Dis, September 2016, 10(3): 383–393 M Mohammadian et al.: Molecular Assay on … 392 http://jad.tums.ac.ir Published Online: January 06, 2016 Defens Pest Managment Information Anaiysis Center. Armed Forces Glen section Walter Reed Army Medical Center Washington (1999) Regional Disease Vector Ecology Profile the Middel East. Ergonul O (2006) Crimean-Congo haemor- rhagic fever. The Lancet Infect Dis. 6(4): 203–214. Faghihi F, Sadegh Chinikar, Zakkyeh Tel- madarraiy, Hasan Bakhshi, Sahar Kha- kifirooz, Tahmineh Jalali, Gidiglo God- win Nutifafa (2015) Crimean-congo hemorrhagic fever: A seroepidemio- logical and molecular survey in north of Iran. J Entomol Zool Stud. 3(1): 1–4. Hoogstraal H (1979) The epidemiology of tick-borne Crimean-Congo hemorrhag- ic fever in Asia, Europe, and Africa. J Med Entomol. 15(4): 307–417. Karimi F (2013) Determination of viral in- fection rate of hard ticks to Crimean- Congo hemorrhagic fever (CCHF) and their distribution pattern using Geographic Information System (GIS) in the Kashan District, Isfahan Prov- ince. Master of Science Medical En- tomology. Teheran University of Med- ical Sciences, School of public Health and Institute of Public Health Re- search Department of Medical Ento- mology and Vector Control. Linthicum KJ, Bailey CL (1994) Ecology of Crimean-Congo hemorrhagic fever. Ecological dynamic of tick-borne zoonoses (Sonenshine and Mather, eds). pp. 392–437. Majeed B, Dicker R, Nawar A, Badri S, Noah A, Muslem H (2012) Morbidity and mortality of Crimean-Congo hem- orrhagic fever in Iraq: cases reported to the National Surveillance System, 1990–2010. Trans Royal Society Tropical Med Hyg. 106(8): 480–483. Mehravaran A, Moradi M, Telmadarraiy Z, Mostafavi E, Moradi AR, Khakifirouz S, Shah-Hosseini N, Varaie FSR, Jalali T, Hekmat S (2013) Molecular de- tection of Crimean-Congo haemor- rhagic fever (CCHF) virus in ticks from southeastern Iran. Ticks and Tick-borne Dis. pp. 35–38. Moradi A, Chinikar S, Oshaghi M, Vatan- doost H, Holakoui-Naini K, Zahirnia A (2008) Molecular detection of Crimean-Congo Hemorrhagic Fever (CCHF) virus in Ticks (Ixodidae, Argasidae) of Hamedan Province, Iran. Biochem Cell Arch. 8(1): 119–123. Nasiri A (2009) Molecular study on tick in- fectivity to CCHF virus and sero- logical investigation on sheep and human in Abdanan region, Ilam Prov- ince, Iran. Master of Science Medical Entomology. Teheran University of Medical Sciences, School of Public Health and Institute of Public Health Research, Department of Medical Entomology and Vector Control. Ölschläger S, Gabriel M, Schmidt-Chanasit J, Meyer M, Osborn E, Conger NG, Allan PF, Günther S (2011) Com- plete sequence and phylogenetic char- acterisation of Crimean-Congo hem- orrhagic fever virus from Afghani- stan. J ClinVirol. 50(1): 90–92. Papa A, Bozovi B, Pavlidou V, Papadi- mitriou E, Pelemis M, Antoniadis A (2002) Genetic detection and isola- tion of Crimean-Congo hemorrhagic fever virus, Kosovo. Emerging Infect Dis. 8(8): 853. Salim-Abadi Y, Chinikar S, Telmadarraiy Z, Vatandoost H, Moradi M, Oshaghi MA, Ghiasi-Seyed M (2011) Cri- mean-Congo hemorrhagic fever: a molecular survey on hard ticks (Ix- odidae) in Yazd province, Iran. Asian Pacific J Trop Med. 4(1): 61–63. Sharifinia N, Rafinejad J, Hanafi-Bojd AA, Biglarian A, Chinikar S, Baniardalani J Arthropod-Borne Dis, September 2016, 10(3): 383–393 M Mohammadian et al.: Molecular Assay on … 393 http://jad.tums.ac.ir Published Online: January 06, 2016 M, SharifiniaF, Karimi F (2013) Knowledge and Attitudes of the Ru- ral Population and Veterinary and Health Personnel Concerning Cri- mean-Congo Hemorrhagic Fever in Western Iran in 2012. Florida Ento- mologist. 96(3): 922–928. Shirani M, Asmar M, Chinikar S, Mirah- madi R, Piazak N, Mazaheri V (2004) Detection of CCHF virus in soft ticks (Argasidae) by RT-PCR method. J Infec Dis Trop Med. 9 (24): 11–15. Sureau P, Klein J, Casals J, Digoutte J, Salaun J, Piazak N, Calvo M (1980) Isolation of Thogoto, Wad Medani, Wanowrie and Crimean-Congo haem- orrhagic fever viruses from ticks of domestic animals in Iran. Annales de Virolo. 131(2): 185–200. Swanepoel R, Shepherd A, Leman P, Shep- herd S, McGillivray G, Erasmus M, Searle L, Gill D (1987) Epidemio- logic and clinical features of Cri- mean-Congo hemorrhagic fever in southern Africa. The American J Ttrop Med Hyg. 36(1): 120–132. Tahmasebi F, Ghiasi S, Mostafavi E, Moradi M, Piazak N, Mozafari A, Haeri A, Fooks A, Chinikar S (2010) Molecu- lar epidemiology of crimean-congo hemorrhagic fever virus genome iso- lated of ticks from Hamadan Prov- ince of Iran. J Vector Borne Dis. 47: 211–216. Telmadarraiy Z, Davari A, Vatandoost H (2006) Ruminant animal ticks and their role in CCHF transmission in Ghaen, south Khorasan Province, Iran during 2005. In ICOPA XI11th Inter Con Parasito. pp. 6–1. Telmadarraiy Z, Chinikar S, Vatandoost H, Holakoui K, Faghihi F, Zarei Z, Oshaghi M (2007) P1044 Serology and immunological study on the in- fectivity of host animals and ticks (Ixodidae, Argasidae) to CCHF virus in Ardabil Province, Iran. International JAntimicrobial Agents. 29: S280. Telmadarraiy Z, Ghiasi SM, Moradi M, Vatandoost H, Eshraghian MR, Faghihi F, Zarei Z, Haeri A, Chini- kar S (2010)A survey of Crimean- Congo haemorrhagic fever in live- stock and ticks in Ardabil Province, Iran during 2004–2005. Scandina- vian J Infect Dis. 42(2): 137–141. Telmadarraiy Z, Saghafipour A, Farzinnia B, Chinikar S (2014) Molecular De- tection of Crimean-Congo Hemor- rhagic Fever Virus in Ticks in Qom Province, Iran, 2011–2012. Iranian J Virolo. 6(3): 13–18. Walker AR, Bouattur A, Camicas J, Estrada- Pena A, Horak I, Latif A, Pegram R, Preston P (2003) Ticks of domestic animals in Africa: A guide to iden- tification of species. Bioscience re- ports Edinburgh. pp. 572–577 Whitehouse CA (2004) Crimean-Congo Hem- orrhagic fever. Antiviral research. 64: 145–60.