Tehran University of Medical Sciences Iranian Journal of Parasitology 1735-7020 17 3 2022 09 05 349 357 Homa Hajjaran Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Maryam Ebadizadeh Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Angila Ataei-Pirkooh Department of Virology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran Mehdi Mohebali Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Katayoun Samimi-Rad Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Reza Saberi Toxoplasmosis Research Center, Department of Parasitology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran Saied Reza Naddaf Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran 2021 11 19 2022 09 05 Background: Detection of Leishmania RNA virus (LRV) in Old World Leishmania species and their possible role in the disease prognosis requires sensitive and specific methods, preferably independent of the viral genome. We aimed to develop an indirect immunofluorescence antibody (IFA) assay to detect LRV in the Old World Leishmania parasites. Methods: Clinical samples were collected from 86 cutaneous leishmaniasis (CL) patients in different endemic areas of CL in Iran, during 2017-2019. For antibody preparation, the viruses were obtained from sediment of an LRV-infected L. major culture-using freeze and thaw cycles followed by gradient cesium chloride centrifugation. The purified viruses were used to immunize a male 3-4 months rabbit. Various dilutions of the LRV-immunized rabbit's serum and a conjugated antibody were deployed to detect LRV in 48 isolates by IFA assay. Results: LRV virus was detected in four of the 48 CL cases using IFA method. Amplification of a partial fragment of RNA-dependent RNA polymerase (RdRp) gene from the isolates confirmed the IFA results. In phylogeny, the generated RdRp sequences from four isolates were grouped with the other Old World LRVs, but separate from L. aethiopica LRVs, which appeared as a highly supported distinct clade. Conclusion: Further optimization of this approach to detect the LRV directly in lesion scrapings can make it a more reliable tool for field studies and disclosing the virus's possible role in disseminating and unusual clinical features. https://ijpa.tums.ac.ir/index.php/ijpa/article/view/3507 https://ijpa.tums.ac.ir/index.php/ijpa/article/download/3507/1230