Karabulut Cankut, Aksoy Tülay, Yıldırım Ahmet, Balcıoğlu I Cüneyt
Manisa Celal Bayar University Faculty of Medicine, Department of Medical Parasitology, Manisa, Türkiye.
Mikrobiyol Bul. 2023 Jul;57(3):463-472. doi: 10.5578/mb.20239938.
Leishmania RNA virus (LRV) is a double-stranded RNA (dsRNA) virus that is thought to contribute to the severe inflammatory response of the causative Leishmania parasite in the mammalian host by being present in many isolates of Leishmania spp. In our study, it was aimed to obtain data on the presence of Leishmania RNA Virus 2 (LRV2), which is thought to cause a change in the clinical course of leishmaniasis, in Leishmania major and Leishmania tropica isolates isolated from cutaneous leishmaniasis (CL) patients in Türkiye. Leishmania strains stored in liquid nitrogen tank by cryopreservation in Manisa Celal Bayar University Faculty of Medicine Parasite Bank were resuscitated under suitable conditions and cultivated in NNN and RPMI-1640 media. Then, the isolates were allowed to enter the logarithmic phase in a 26ºC incubator and DNA isolations were made using the "High Pure PCR Template Preparation Kit". Real-time polymerase chain reaction (Rt-PCR) melting analyzes were applied to the DNAs obtained by using primers and probes specific to the internal transcribed spacer-1 (ITS-1) gene region of Leishmania. After RNA isolation from promastigote suspension, cDNA synthesis was performed by reverse transcription. After gel electrophoresis with PCR amplification products, dsRNA band formation was evaluated in terms of LRV2 positivity under ultraviolet light. Among the 20 examined Leishmania spp. isolates (10 L.tropica and 10 L.major), four (three L.tropica, one L.major) were found to be positive for the presence of LRV2. Although the mechanism of LRV in recent studies has not been fully understood, it is known that it exacerbates the clinic of the disease and even has an effect on the formation of drug resistance by the parasite. It is important to obtain data on the presence of LRV in our country and to contribute to various clinical, drug development, prevalence studies, diagnosis and treatment of the disease in the future.
利什曼原虫RNA病毒(LRV)是一种双链RNA(dsRNA)病毒,由于存在于许多利什曼原虫属的分离株中,被认为会导致致病性利什曼原虫寄生虫在哺乳动物宿主中引发严重的炎症反应。在我们的研究中,旨在获取有关利什曼原虫RNA病毒2(LRV2)存在情况的数据,LRV2被认为会改变利什曼病的临床病程,该研究针对从土耳其皮肤利什曼病(CL)患者中分离出的硕大利什曼原虫和热带利什曼原虫分离株展开。保存在马尼萨切拉尔·贝亚尔大学医学院寄生虫库液氮罐中的利什曼原虫菌株在适宜条件下复苏,并在NNN和RPMI - 1640培养基中培养。然后,将分离株置于26℃培养箱中进入对数期,使用“高纯PCR模板制备试剂盒”进行DNA提取。对使用针对利什曼原虫内部转录间隔区1(ITS - 1)基因区域的引物和探针获得的DNA进行实时聚合酶链反应(Rt - PCR)熔解分析。从前鞭毛体悬液中分离RNA后,通过逆转录进行cDNA合成。用PCR扩增产物进行凝胶电泳后,在紫外线下根据LRV2阳性情况评估dsRNA条带的形成。在所检测的20个利什曼原虫属分离株(10个热带利什曼原虫和10个硕大利什曼原虫)中,发现有4个(3个热带利什曼原虫,1个硕大利什曼原虫)LRV2呈阳性。尽管近期研究中LRV的机制尚未完全明确,但已知它会加重疾病的临床症状,甚至对寄生虫产生耐药性的形成有影响。获取我国LRV存在情况的数据,并为该疾病未来的各种临床、药物研发、患病率研究、诊断和治疗做出贡献非常重要。
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