Development of an Indirect Fluorescent Antibody (IFA) Assay for the Detection of Leishmania RNA Virus 2 (LRV2) in Leishmania Parasites

Homa  Hajjaran; Maryam  Ebadizadeh; Angila  Ataei-Pirkooh; Mehdi  Mohebali; Katayoun  Samimi-Rad; Reza  Saberi; Saied Reza  Naddaf

doi:10.18502/ijpa.v17i3.10625

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 Scienc-es, Sari, Iran
Saied Reza Naddaf Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran

DOI: https://doi.org/10.18502/ijpa.v17i3.10625

Keywords: Leishmania RNA virus; Indirect fluorescence antibody; (RdRp) gene

Abstract

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.