ORIGINAL ARTICLE

Detection and molecular identification of leishmania RNA virus(LRV) in Iranian Leishmania species

Homa Hajjaran1• Maryam Mahdi1 • Mehdi Mohebali1 • Katayoun Samimi-Rad2

•

Angila Ataei-Pirkooh3• Elham Kazemi-Rad4

• Saied Reza Naddaf4•

Reza Raoofian5

Received: 15 February 2016 / Accepted: 31 August 2016Ó Springer-Verlag Wien 2016

Abstract Leishmania RNA virus (LRV) was first detectedin members of the subgenus Leishmania (Viannia), andlater, the virulence and metastasis of the New World spe-cies were attributed to this virus. The data on the presenceof LRV in Old World species are confined to Leishmaniamajor and a few Leishmania aethiopica isolates. The aimof this study was to survey the presence of LRV in variousIranian Leishmania species originating from patients andanimal reservoir hosts. Genomic nucleic acids wereextracted from 50 cultured isolates belonging to the speciesLeishmania major, Leishmania tropica, and Leishmaniainfantum. A partial sequence of the viral RNA-dependentRNA polymerase (RdRp) gene was amplified, sequencedand compared with appropriate sequences from the Gen-Bank database. We detected the virus in two parasitespecimens: an isolate of L. infantum derived from a vis-ceral leishmaniasis (VL) patient who was unresponsive tomeglumine antimoniate treatment, and an L. major isolate

originating from a great gerbil, Rhombomys opimus. TheIranian LRV sequences showed the highest similarities toan Old World L. major LRV2 and were genetically distantfrom LRV1 isolates detected in New World Leishmaniaparasites. We could not attribute treatment failure in VLpatient to the presence of LRV due to the limited numberof specimens analyzed. Further studies with inclusion ofmore clinical samples are required to elucidate the poten-tial role of LRVs in pathogenesis or treatment failure ofOld World leishmaniasis.

Introduction

Leishmaniases are a variety of diseases caused by proto-zoan parasites of the genus Leishmania and are naturallytransmitted to humans and among reservoirs via theinfective bites of sand flies [1, 2]. In Iran, leishmaniasis is amajor health problem represented by two clinical forms,visceral leishmaniasis (VL) and cutaneous leishmaniasis(CL), which may sometimes appear as atypical forms withthe involvement of mucosa or dissemination to other partsof the body [3, 4]. The etiological agents of cutaneousleishmaniasis in the country are Leishmania major andLeishmania tropica, while Leishmania infantum causesvisceral leishmaniasis, except a few cases in which L.tropica was identified as the causative agent [3, 5].

Leishmania parasites are classified into two subgenera,Leishmania (Leishmania) and L. (Viannia), with the latterresponsible for mucosal (ML) or disseminated cutaneousleishmaniasis (DCL) in the New World [6]. In SouthAmerica, up to 10 % of CL cases, including thosewith asymptomatic primary infections, progress to muco-cutaneous leishmaniasis (MCL) forming destructive sec-ondary lesions in nasopharyngeal mucosa [7]. The

& Homa Hajjaranhajaranh@tums.ac.ir

& Mehdi MohebaliMohebali@tums.ac.ir

1 Department of Medical Parasitology and Mycology, Schoolof Public Health, Tehran University of Medical Sciences,Tehran, Iran

2 Department of Virology, School of Public Health, TehranUniversity of Medical Sciences, Tehran, Iran

3 Department of Virology, Faculty of Medicine, IranUniversity of Medical Sciences, Tehran, Iran

4 Department of Parasitology, Pasteur Institute of Iran, Tehran,Iran

5 Legal Medicine Research Center, Legal MedicineOrganization, Tehran, Iran

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Arch VirolDOI 10.1007/s00705-016-3044-z

aggressive manifestation of the disease in the SouthAmerican CL patients was attributed to the presence ofdouble-stranded (ds) Leishmania RNA viruses (LRVs) inthe members of the subgenus L. (Viannia) [8–11]. Thepresence of LRV in some strains is associated with ele-vated parasitaemia, destructive inflammation, and overallexacerbation of the disease [12, 13]. In the New World,LRVs have frequently been identified in members of thespecies L. (Viannia) braziliensis and L. (V) guyanensis[12], whereas in the Old World, until recently, the data onthe presence of LRV were confined to an L. major isolatefrom Turkmenistan that was not linked with any particularmanifestation [14]. More recently, Zangger and colleaguesidentified a new LRV member in a few Leishmaniaaethiopica isolates [13].

The LRVs from the New and Old World, due to morethan 60 % difference in their genome, have been arbitrarytermed as LRV1 and LRV2, respectively [12, 13, 15].LRVs are members of the family Totiviridae with smallvirions composed of a double-stranded RNA (dsRNA)genome (5.3 kb), a capsid protein (CP), and an RNA-de-pendent RNA polymerase (RdRp) [16, 17].

The aim of the present study was to detect potentialLRVs in Leishmania isolates belonging to prevalent spe-cies in Iran and to demonstrate their homology with otherLRVs from the Old and New World Leishmania species.

Mater ials and methods

Leishmania isolates were obtained from 43 cutaneous (L.tropica, n = 19; L. major, n = 24) and seven visceral (L.infantum n = 4; L. tropica n = 3) leishmaniasis patients orreservoir hosts. The cutaneous samples were obtained frompatients referred to the leishmaniasis laboratory at TehranUniversity of Medical Science, and the visceral sampleswere actively collected from patients and dogs during fieldtrips to VL endemic areas from 2007 to 2015. The isolateswere identified by analysis of nagt and ITS1 genes asdescribed by others [3, 5]. The cryopreserved parasiteswere cultured in RPMI1640 (Gibco, Invitrogen) supple-mented with 20 % fetal bovine serum.

Genomic nucleic acid extr action

Stationary-phase promastigotes (4 x 106 cells) were pel-leted by centrifugation at 990 g, 4 °C, for 10 min andresuspended in Tris/HCl buffer, pH 7.4. Nucleic acidscontaining parasitic genomic DNA and LRV dsRNA wereextracted with phenol-chloroform followed by precipitationwith 5 M NaCl in absolute ethanol and centrifugation at990 g, 4 °C, for 30 min. Nucleic acids were resuspended inTNE (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM

EDTA, pH 8) and visualized on 1.2 % agarose gels con-taining ethidium bromide (0.5 l g/ml) under UV light. One-microliter samples of extracted nucleic acids were treatedwith the DNase I enzyme (Fermentas, Life Sciences) asrecommended by the manufacturer.

Total RNA extr action and cDNA synthesis

Total RNA from 108 promastigotes in the stationary phasewas extracted using TriPure Isolation Reagent (Roche,Germany) according to the manufacturer’s instructions.The quantity of the extracted RNAs was analyzed using aNanoDrop spectrometer (Thermo Scientific Fisher, USA).Synthesis of cDNA was performed using a cDNA Syn-thesis Kit (Roche, Germany) with random hexamer primersaccording to the protocol provided by the manufacturer.

Semi-nested RT-PCR

Semi-nested RT-PCR was performed using primers thatwere designed using Primer3 software, version 0.4.0(http://frodo.wi.mit.edu/). The primers for the first stage(LRV F-HR, 50-4304tgt aac cca cat aaa cag tgt gc-30; LRVR-HR, 50-4809att tca tcc agc ttg act ggg-30) amplified a526-bp external partial sequence of the RdRp gene. Thesecond stage, performed with the primers LRV F2-HR(50-4515agg aca atc caa tag gtc gtg t-30) and LRV R-HR,amplified a 315-bp internal fragment of the RdRp gene.The 25-l l reactions included 10 l l of master mix (Am-pliqon, Denmark), 10 pmol each of forward and reverseprimers, and 1 l l of cDNA in the first stage, and the sameamount of the resulting amplicon in the second stage.Amplification in both stages contained an initial denaturingstep of 94 °C for 4 min, followed by 35 repetitions ofdenaturation at 94 °C for 35 s, annealing at 60 °C for 35 s,and extension at 72 °C for 45 s with a final extension at72 °C for 5 min. The PCR products were run along a100-bp DNA marker (Fermentas, Life Sciences) on 1.2 %agarose gels containing ethidium bromide (0.5 l g/ml) andphotographed under UV light.

Sequencing of the RdRp gene

The amplicons from the first stage were sequenced by theSanger method in both directions with the same primersused for amplification by Pouya Gostar Gene Company(pggene.com) in a Genetic Analyzer 3500 (AppliedBiosystems, Carlsbad, USA). The DNA sequences werechecked manually using BioEdit software (version 7.1) andcompared with similar sequences from the GenBankdatabase (http://www.ncbi.nlm.nih.gov). The two & 520 bpsequences were aligned with appropriate sequences fromthe GenBank database using ClustalW (http://www.ebi.ac.

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uk/clustalw2). The distances between sequences were cal-culated, and a phylogenetic tree was constructed using theTamura 3-parameter distance option of the neighbor-join-ing method in a complete deletion procedure usingMEGA5 software (http://www.megasoftware.net). Weused the Trichomonas vaginalis RNA virus (TVV1) RdRpsequence (accession no. U57898.1) as an outgroup.

Results

Out of 50 isolates subjected to nucleic acid extraction, onlytwo, an L. infantum isolate and an L. major isolate, showeda distinct band of [ 5 kbp indicative of viral dsRNA. Theband was not eliminated by treatment with DNase (Fer-mentas, GMBH, Germany). The two isolates yielded theexpected 526-bp and 315-bp products in the first and sec-ond rounds of nested RT-PCR amplification, respectively(Fig. 1). The L. infantum isolate was derived from a bonemarrow aspirate of a 13-year-old VL patient residing in thenorthwest of Iran [18]. The other isolate of L. major wasobtained from the ear lobe of a great gerbil (Rhombomysopimus) [19].

Sequence analysis and phylogeny

The similarities between seven LRVs, based on the 502-bpRdRp sequence, ranged from 16.10 % to 99.40 %, with thehighest similarity between the Iranian L. infantum LRV2isolate and the L. major LRV2 isolate from Turkmenistan(accession no. U32108) and the lowest similarity between L.braziliensis guyanensis isolate LRV1-4 (accession no. NC-003601) and L. aethiopica LRV2 (accession no. NC-024115). The LRVs of the two Iranian Leishmania isolatesshowed 96.80 % sequence identity, as shown in Table 1.

Phylogenetic analysis showed that the LRVs of the OldWorld isolates clustered into two clades, L. aethiopica andL. major/L. infantum, separate from the New World LRVs.The topologies of trees generated by the NJ method werealmost identical when using different options (pairwisedeletion, partial deletion, and complete deletion). The Ira-nian L. infantum LRV grouped with the L. major LRV fromTurkmenistan and was separate from the Iranian L. majorLRV (Fig. 2). The sequence data for the two LeishmaniaRNA viruses obtained in this study were deposited in theGenBank database under the accession numbers KP054244for L. infantum LRV (LRV2/IR/2014/HM-1) and KP054245for L. major LRV (LRV2/IR/2014/HM-2).

Discussion

Many protozoan parasites carry endosymbiotic dsRNAviruses. These viruses do not replicate in the vertebratehost, but their genomic dsRNA, gene products or wholevirions shed by the parasites can trigger the immune sys-tem, leading to inflammatory complications [20]. LRVswere first detected in promastigotes of L. guyanensis(1988) and L. braziliensis (1996), the causative agents ofNew World mucocutaneous leishmaniasis (MCL) [21]. TheMCL phenotypes in the New World are not only associatedwith the presence of LRV but are also correlated with thevirus burden in the parasites [12]. Similarly, in the OldWorld, a metastatic form was observed in golden hamstersharboring LRV-infected L. aethiopica, suggesting thatLRV might play a role in parasite pathogenesis [13]. Here,we report detection and characterization of LRVs in twodifferent Leishmania isolates belonging to the species L.infantum and L. major. The LRV-infected L. infantum wasderived from bone marrow of a VL patient residing in the

Fig. 1 Detection of LRV in Leishmania isolates by targeting theRNA-dependent RNA polymerase (RdRp) gene using a semi-nestedRT-PCR assay. (a) 526-bp amplicons from the first round ofamplification. Lanes 1 and 2, LRV-positive L. infantum; lanes 3and 4, LRV-positive L. major; lane 5, LRV-negative sample; M,

1-kbp DNA marker (Fermentas, Life Sciences); NTC, non-templatecontrol. (b) Internal 315-bp fragment. Lanes 1, 2 and 3, LRV-positiveL. infantum; lanes 5, 6 and 7, LRV-positive L. major; M, 1-kbp DNAmarker (Fermentas, Life Sciences)

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northwest of Iran. The patient was unresponsive to threecourses of systemic administration of Glucantime, and anin vitro assay confirmed that the parasite was resistant tothe compound [18]. In L. braziliensis-infected patientsfrom Peru and Bolivia, the presence of LRV1 was shown tobe significantly associated with an increased risk of treat-ment failure. However, there was no significant associationwith intrinsic SbV resistance among parasites, suggestingthat treatment failure arises from LRV1-mediated effectson host metabolism and/or parasite survival [22]. Also, theLRV1 status in L. guyanensis infection has been shown tobe significantly prognostic of first-line treatment failure[23]. In the present study, treatment failure of the VLpatient could not be attributed to the presence of LRV dueto the limited number of specimens analyzed. The other

LRV-infected isolate, L. major, originated from the ear of agreat gerbil (R. opimus) the main reservoir for zoonoticcutaneous leishmaniasis in the northeast and center of Iran[19]. In this study, LRVs were not detected in L. tropicisolates from canine visceral leishmaniasis (CVL) or in CLisolates penetrated into deeper layers of skin. The absenceof LRVs in laboratory strains should be viewed with cau-tion, as these viruses can be lost in laboratory culture [24].The homology between the LRV sequences generated inthis study and the others was independent of the Leish-mania species, as the similarity between the Iranian L.infantum LRV and the L. major LRV from Turkmenistanwas higher than between the two L. major LRVs (Table 1).Further studies with more clinical isolates from largepanels of clinical manifestations with the deployment of

Table 1 Similarities between the New and Old World LRVs based on the RdRp gene

KF256264(LRV2-L.aethiopica)

KF256265(LRV2-L.aethiopica)

KP054244(LRV2-L.infantum,IR)

KP054245(LRV2-L.major, IR)

NC-002063(LRV1-1, L.guyanensis.)

NC-003601(LRV1-4, L.braziliensisguyanensis)

NC-024115(LRV2-L.aethiopica)

U32108(LRV2-L. major)

KF256264(LRV2-L.aethiopica)

KF256265(LRV2-L.aethiopica)

76.50

KP054244(LRV2-L.infantum, IR)

64.40 60.20

KP054245(LRV2-L.major, IR)

65.40 60.50 96.80

NC-002063(LRV1-1, L.guyanensis)

32.40 24.10 28.90 28.90

NC-003601(LRV1-4, L.braziliensisguyanensis)

26.80 25.20 30.90 30.40 73.70

NC-024115(LRV2-L.aethiopica)

75.70 81.70 57.40 59.20 23.00 16.10

U32108 (LRV2-L.major)

64.10 59.90 99.40 96.60 29.40 31.40 57.10

LRV1, New World leishmania RNA virus

LRV2, Old World leishmania RNA virus KF256264 (LRV2-L. aethiopica); leishmania RNA virus strain MHOM/ET/2011/LDS303

KF256265 (LRV2-L. aethiopica); leishmania RNA virus strain MHOM/ET/2011/LDS327

KP054244 (LRV2-L. infantum, IR); L. infantum, LRV (LRV2/IR/2014/HM-1)

KP054245 (LRV2-L. major, IR); L. major, LRV (LRV2/IR/2014/HM-2)

NC_002063 (LRV1-1, L. guyanensis); leishmania RNA virus 1-1, complete genome

NC_003601 (LRV1-4, L. braziliensis guyanensis); leishmania RNA virus 1-4, complete genome

NC_024115 (LRV2-L. aethiopica); Leishmania aethiopica RNA virus isolate LRV2-Lae-L494, complete genome

U32108 (LRV2-L. major); leishmania RNA virus 2-1, complete genome

NC_027701 (TVV1-Trichomonas vaginalis); Trichomonas vaginalis virus 1 strain TVV1-UR1-1, complete genome

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improved methods for viral detection and quantitation andthe evaluation of the vertebrate host response to LRVs arerequired to decipher the potential role of these viruses inclinical features of Old World leishmaniasis.

Acknowledgments The authors would like to thank Dr. B. Akhoundiand Mrs. S. Charedar, for their kind cooperation. This work wasfinancially supported by the grant no. 25027-2014, Vice Chancellorfor Research, School of Public Health, Tehran University of MedicalSciences, Iran. The authors declare that there is no conflict of interest.

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Fig. 2 Phylogenetic analysis of the LRVs from the New and OldWorld Leishmania species based on the RdRp gene. The scale at thebottom represents the genetic distance in nucleotide substitutions persite. The tree was constructed by using the Tamura 3-parametermodel in MEGA software version V. The numbers above the

branches indicate the percentage of bootstrap replicates supportingthe branch. *, LRVs from the Iranian Leishmania isolates; LRV1,New World leishmania RNA virus; LRV2, Old World leishmaniaRNA virus; TVV1, Trichomonas vaginalis virus

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