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RESEARCH ARTICLE • Mem. Inst. Oswaldo Cruz 118 • 2023 • https://doi.org/10.1590/0074-02760230071   copy

First report of putative Leishmania RNA virus 2 (LRV2) in Leishmania infantum strains from canine and human visceral leishmaniasis cases in the southeast of Brazil

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

BACKGROUND

Leishmania RNA virus 1 (LRV1) is commonly found in South American Leishmania parasites belonging to the subgenus Viannia, whereas Leishmania RNA virus 2 (LRV2) was previously thought to be restricted to the Old-World pathogens of the subgenus Leishmania.

OBJECTIVES

In this study, we investigated the presence of LRV2 in strains of Leishmania (L.) infantum, the causative agent of visceral leishmaniasis (VL), originating from different hosts, clinical forms, and geographical regions.

METHODS

A total of seventy-one isolates were screened for LRV2 using semi-nested reverse transcription-polymerase chain reaction (RT-PCR) targeting the RNA-dependent RNA polymerase (RdRp) gene.

FINDINGS

We detected LRV2 in two L. infantum isolates (CUR268 and HP-EMO) from canine and human cases, respectively.

MAIN CONCLUSIONS

To the best of our knowledge, this is the first detection of LRV2 in the New World.

Key words:
Leishmania; Leishmania infantum; leishmaniasis; Leishmania RNA virus; Totiviridae

Leishmania (Kinetoplastea: Trypanosomatidae) are protistan parasites that cause leishmaniasis, a vector-borne disease prevalent in nearly 100 countries.11. PAHO - Pan American Health Organization. Leishmaniases. Epidemiological report of the Americas, December 2020 [Internet]. 2020. Available from: https://iris.paho.org/handle/10665.2/53090.
https://iris.paho.org/handle/10665.2/530... ,22. Bruschi F, Gradoni L. The leishmaniases: old neglected tropical diseases. 1st ed. Vol. 1. Berlim: Springer International Publishing; 2018. 245 pp. This disease exhibits a wide range of clinical manifestations resulting from complex interactions between the parasite and the host immune system.33. Desjeux P. The increase in risk factors for leishmaniasis worldwide. Trans R Soc Trop Med Hyg. 2001; 95(3): 239-43.,44. Mann S, Frasca K, Scherrer S, Henao-Martínez AF, Newman S, Ramanan P, et al. A review of leishmaniasis: current knowledge and future directions. Curr Trop Med Reports. 2021; 8(2): 121-32. However, despite the substantial efforts to elucidate the molecular mechanisms governing this intricate interface, they remain poorly understood. The presence of endosymbiotic Leishmania viruses has been suggested as an important component in this puzzle.55. Ives A, Ronet C, Prevel F, Ruzzante G, Fuertes-Marraco S, Schutz F, et al. Leishmania RNA virus controls the severity of mucocutaneous leishmaniasis. Science. 2011; 331(6018): 775-8.,66. Hartley MA, Ronet C, Zangger H, Beverley SM, Fasel N. Leishmania RNA virus: when the host pays the toll. Front Cell Infect Microbiol. 2012; 2: 99.,77. Kostygov AY, Karnkowska A, Votýpka J, Tashyreva D, Maciszewski K, Yurchenko V, et al. Euglenozoa: taxonomy, diversity and ecology, symbioses and viruses. Open Biol. 2021; 11(3): 200407.

Various representatives of the subgenera Leishmania, Mundinia, and Viannia have been screened for the presence of the virus, leading to the discovery of double-stranded Leishmania RNA viruses (LRVs, family Totiviridae) and negative-sense single-stranded RNA viruses, known as leishbuviruses (LBV, family Leishbuviridae, older name Leishbunyaviridae).88. Paranaiba LF, Pinheiro LJ, Macedo DH, Menezes-Neto A, Torrecilhas AC, Tafuri WL, et al. An overview on Leishmania (Mundinia) enriettii: biology, immunopathology, LRV and extracellular vesicles during the host-parasite interaction. Parasitology. 2018; 145(10): 1265-73.,99. Kostygov AY, Grybchuk D, Kleschenko Y, Chistyakov DS, Lukashev AN, Gerasimov ES, et al. Analyses of Leishmania-LRV co-phylogenetic patterns and evolutionary variability of viral proteins. Viruses. 2021; 13(11): 2305.,1010. Cantanhêde LM, Mata-Somarribas C, Chourabi K, da Silva GP, das Chagas BD, Pereira LOR, et al. The maze pathway of coevolution: a critical review over the Leishmania and its endosymbiotic history. Genes (Basel). 2021; 12(5): 657. LRVs encompass four species, namely LRV1 to LRV4. LRV1 and LRV2 have been found primarily in the subgenus Viannia (L. braziliensis, L. guyanensis, L. lainsoni, L. naiffi, L. panamensis, and L. shawi), and the subgenus Leishmania (L. aethiopica, L. infantum, L. major, and L. tropica), respectively.99. Kostygov AY, Grybchuk D, Kleschenko Y, Chistyakov DS, Lukashev AN, Gerasimov ES, et al. Analyses of Leishmania-LRV co-phylogenetic patterns and evolutionary variability of viral proteins. Viruses. 2021; 13(11): 2305.,1010. Cantanhêde LM, Mata-Somarribas C, Chourabi K, da Silva GP, das Chagas BD, Pereira LOR, et al. The maze pathway of coevolution: a critical review over the Leishmania and its endosymbiotic history. Genes (Basel). 2021; 12(5): 657.,1111. Zangger H, Hailu A, Desponds C, Lye LF, Akopyants NS, Dobson DE, et al. Leishmania aethiopica field isolates bearing an endosymbiontic dsRNA virus induce pro-inflammatory cytokine response. PLoS Negl Trop Dis. 2014; 8(4): e2836.,1212. Salinas G, Zamora M, Stuart K, Saravia N. Leishmania RNA viruses in Leishmania of the Viannia subgenus. Am J Trop Med Hyg. 1996; 54(4): 425-9. The other two species, LRV3 and LRV4, have been documented in a different trypanosomatid group known as the genus Blechomonas.1313. Grybchuk D, Kostygov AY, Macedo DH, Votýpka J, Lukes J, Yurchenko V. RNA viruses in Blechomonas (Trypanosomatidae) and evolution of Leishmaniavirus. mBio. 2018; 9(5): e01932-18. LBV representatives have been found to infect L. martiniquensis, of the subgenus Mundinia, in Martinique and Brazil.1414. Grybchuk D, Macedo DH, Kleschenko Y, Kraeva N, Lukashev AN, Bates PA, et al. The first non-LRV RNA virus in Leishmania. Viruses. 2020; 12(2): 168.,1515. Mendes Jr AAV, Filgueira CPB, Miranda LFC, de Almeida AB, Cantanhêde LM, Fagundes A, et al. First report of Leishmania (Mundinia) martiniquensis in South American territory and confirmation of Leishbunyavirus infecting this parasite in a mare. Mem Inst Oswaldo Cruz. 2023; 118: e220220.

Despite recent advancements in the field, the extent to which the virus affects parasite virulence or host immune responses during Leishmania infection remains unclear. In the case of LRVs, it is known that the endosymbiotic virus confers an adaptive advantage to the parasite by suppressing anti-leishmanial immunity in the vertebrate host, thereby facilitating the parasite’s survival.66. Hartley MA, Ronet C, Zangger H, Beverley SM, Fasel N. Leishmania RNA virus: when the host pays the toll. Front Cell Infect Microbiol. 2012; 2: 99.,1616. Brettmann EA, Shaik JS, Zangger H, Lye LF, Kuhlmann FM, Akopyants NS, et al. Tilting the balance between RNA interference and replication eradicates Leishmania RNA virus 1 and mitigates the inflammatory response. Proc Natl Acad Sci USA. 2016; 113(43): 11998-2005. The exacerbation of the LRV1-mediated disease appears to be dependent on TLR3 limiting the activation of the NLRP3 inflammasome in macrophages, resulting in elevated parasitaemia and destructive mucosal inflammation.55. Ives A, Ronet C, Prevel F, Ruzzante G, Fuertes-Marraco S, Schutz F, et al. Leishmania RNA virus controls the severity of mucocutaneous leishmaniasis. Science. 2011; 331(6018): 775-8.,1717. de Carvalho RVH, Lima-Junior DS, da Silva MVG, Dilucca M, Rodrigues TS, Horta CV, et al. Leishmania RNA virus exacerbates leishmaniasis by subverting innate immunity via TLR3-mediated NLRP3 inflammasome inhibition. Nat Commun. 2019; 10(1): 5273.

18. Rossi M, Castiglioni P, Hartley MA, Eren RO, Prével F, Desponds C, et al. Type I interferons induced by endogenous or exogenous viral infections promote metastasis and relapse of leishmaniasis. Proc Natl Acad Sci USA. 2017; 114(19): 4987-92.

19. Hartley MA, Bourreau E, Rossi M, Castiglioni P, Eren RO, Prevel F, et al. Leishmaniavirus-dependent metastatic leishmaniasis is prevented by blocking IL-17A. PLoS Pathog. 2016; 12(9): e1005852.-2020. Eren RO, Reverte M, Rossi M, Hartley MA, Castiglioni P, Prevel F, et al. Mammalian innate immune response to a Leishmania-resident RNA virus increases macrophage survival to promote parasite persistence. Cell Host Microbe. 2016; 20(3): 318-28. LRV2 from L. aethiopica also modulates the host immune response, leading to a TLR3-dependent hyperinflammatory response.1111. Zangger H, Hailu A, Desponds C, Lye LF, Akopyants NS, Dobson DE, et al. Leishmania aethiopica field isolates bearing an endosymbiontic dsRNA virus induce pro-inflammatory cytokine response. PLoS Negl Trop Dis. 2014; 8(4): e2836. Additionally, LRV1 can be shed via exosomes and this process may also govern Leishmania virulence.2121. Atayde VD, Lira Filho AS, Chaparro V, Zimmermann A, Martel C, Jaramillo M, et al. Exploitation of the Leishmania exosomal pathway by Leishmania RNA virus 1. Nat Microbiol. 2019; 4(4): 714-23.,2222. Olivier M, Zamboni DS. Leishmania viannia guyanensis, LRV1 virus and extracellular vesicles: a dangerous trio influencing the faith of immune response during muco-cutaneous leishmaniasis. Curr Opin Immunol. 2020; 66: 108-13.,2323. Lafleur A, Olivier M. Viral endosymbiotic infection of protozoan parasites: how it influences the development of cutaneous leishmaniasis. PLOS Pathog. 2022; 18(11): e1010910. Interestingly, LRV1 and LRV2 may elicit different responses in various Leishmania species.2424. Saura A, Zakharova A, Klocek D, Gerasimov ES, Butenko A, Macedo DH, et al. Elimination of LRVs elicits different responses in Leishmania spp. mSphere. 2022; 7(4): e0033522.

While dsRNA viruses have been detected in various Leishmania spp., their prevalence varies among isolates of a given species.2525. Saberi R, Fakhar M, Mohebali M, Anvari D, Gholami S. Global status of synchronizing Leishmania RNA virus in Leishmania parasites: a systematic review with meta-analysis. Transbound Emerg Dis. 2019; 66(6): 2244-51. In the New World, LRV1s are predominant in clinical isolates from Bolivia, Colombia, Ecuador, French Guiana, Peru, and Suriname,1212. Salinas G, Zamora M, Stuart K, Saravia N. Leishmania RNA viruses in Leishmania of the Viannia subgenus. Am J Trop Med Hyg. 1996; 54(4): 425-9.,2626. Adaui V, Lye LF, Akopyants NS, Zimic M, Llanos-Cuentas A, Garcia L, et al. Association of the endobiont double-stranded RNA virus LRV1 with treatment failure for human leishmaniasis caused by Leishmania braziliensis in Peru and Bolivia. J Infect Dis. 2016; 213(1): 112-21.,2727. Kariyawasam R, Grewal J, Lau R, Purssell A, Valencia BM, Llanos-Cuentas A, et al. Influence of Leishmania RNA Virus 1 on proinflammatory biomarker expression in a human macrophage model of American tegumentary leishmaniasis. J Infect Dis. 2017; 216(7): 877-86.,2828. Ginouvès M, Simon S, Bourreau E, Lacoste V, Ronet C, Couppié P, et al. Prevalence and distribution of Leishmania RNA virus 1 in Leishmania parasites from French Guiana. Am J Trop Med Hyg. 2016; 94(1): 102. while being rare in southeastern Brazil.2929. Macedo DH, Menezes-Neto A, Rugani JM, Rocha AC, Silva SO, Melo MN, et al. Low frequency of LRV1 in Leishmania braziliensis strains isolated from typical and atypical lesions in the State of Minas Gerais, Brazil. Mol Biochem Parasitol. 2016; 210(1-2): 50-4.,3030. Zabala-Peñafiel A, Fantinatti M, Dias-Lopes G, da Silva JL, Miranda LFC, Lyra MR, et al. First report of Leishmania RNA virus 1 in Leishmania (Viannia) braziliensis clinical isolates from Rio de Janeiro State - Brazil. Mem Inst Oswaldo Cruz. 2022; 117: e210107. LRV2 has been detected in Central Asian and Middle Eastern countries, with a higher prevalence in L. major compared to other susceptible species.1111. Zangger H, Hailu A, Desponds C, Lye LF, Akopyants NS, Dobson DE, et al. Leishmania aethiopica field isolates bearing an endosymbiontic dsRNA virus induce pro-inflammatory cytokine response. PLoS Negl Trop Dis. 2014; 8(4): e2836.,3131. Scheffter SM, Ro YT, Chung IK, Patterson JL. The complete sequence of Leishmania RNA Virus LRV2-1, a virus of an old world parasite strain. Virology. 1995; 212(1): 84-90.

32. Hajjaran H, Mahdi M, Mohebali M, Samimi-Rad K, Ataei-Pirkooh A, Kazemi-Rad E, et al. Detection and molecular identification of Leishmania RNA virus (LRV) in Iranian Leishmania species. Arch Virol. 2016; 161(12): 3385-90.

33. Nalçaci M, Karakus M, Yilmaz B, Demir S, Özbilgin A, Özbel Y, et al. Detection of Leishmania RNA virus 2 in Leishmania species from Turkey. Trans R Soc Trop Med Hyg. 2019; 113(7): 410-7.

34. Kleschenko Y, Grybchuk D, Matveeva NS, Macedo DH, Ponirovsky EN, Lukashev AN, et al. Molecular characterization of Leishmania RNA virus 2 in Leishmania major from Uzbekistan. Genes (Basel). 2019; 10(10): 830.-3535. Yurchenko V, Chistyakov DS, Akhmadishina LV., Lukashev AN, Sádlová J, Strelkova MV. Revisiting epidemiology of leishmaniasis in Central Asia: lessons learnt. Parasitology. 2022; 150(2): 129-36. Interestingly, LRV2-positive strains have not yet been detected in the New World, despite the presence of certain Old-World species of the subgenus Leishmania, known to be virus-positive in Asia. This suggests the independent co-evolution of LRV1 and LRV2 with their hosts in the New and Old Worlds, respectively.99. Kostygov AY, Grybchuk D, Kleschenko Y, Chistyakov DS, Lukashev AN, Gerasimov ES, et al. Analyses of Leishmania-LRV co-phylogenetic patterns and evolutionary variability of viral proteins. Viruses. 2021; 13(11): 2305.,1010. Cantanhêde LM, Mata-Somarribas C, Chourabi K, da Silva GP, das Chagas BD, Pereira LOR, et al. The maze pathway of coevolution: a critical review over the Leishmania and its endosymbiotic history. Genes (Basel). 2021; 12(5): 657.,3636. Widmer G, Dooley S. Phylogenetic analysis of Leishmania RNA virus and Leishmania suggests ancient virus-parasite association. Nucleic Acids Res. 1995; 23(12): 2300-4. In this work, we analysed L. (L.) infantum strains from Brazil and Honduras for the presence of LRV2.

MATERIALS AND METHODS

Parasite culture, DNA and RNA isolation, and complementary DNA (cDNA) synthesis - A panel comprising 71 strains of L. infantum, including World Health Organization reference strains and field isolates (Fig. 1, Table), was initially cultured in M199 medium supplemented with 10% foetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, USA), penicillin (200 u/mL), and streptomycin (200 μg/mL) (all from Merck, Darmstadt, Germany) at 25ºC for one to three weeks. DNA was extracted from 5 × 107 cells using the PureGene Core Kit A (Qiagen, Hilden, Germany). The hsp70 gene was amplified and digested with HaeIII to confirm the parasite’s identity3737. Garcia L, Kindt A, Bermudez H, Llanos-Cuentas A, De Doncker S, Arevalo J, et al. Culture-independent species typing of neotropical Leishmania for clinical validation of a PCR-based assay targeting heat shock protein 70 genes. J Clin Microbiol. 2004; 42(5): 2294-7. [Supplementary data (Figure)].

Fig. 1:
origin (Honduras and Brazil) and number of New World strains of Leishmania infantum analysed in this study for the presence of Leishmania RNA virus 2 (LRV2). Brazilian states marked in orange indicate LRV2 positive strains.

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TABLE
New World Leishmania (Leishmania) infantum strains prospected in this study for the presence of Leishmania RNA virus 2 (LRV2)

RNA was isolated from 1 x 108 cells using the Trizol method according to the manufacturer’s protocol (Thermo Fisher Scientific). One hundred ng of each RNA sample was treated with DNAse I prior to complementary DNA (cDNA) synthesis. Reverse transcription was performed using the Super Script III- First Strand Synthesis Kit (Invitrogen), with random hexamer primers according to the manufacturer’s specifications.

Detection of Leishmania RNA virus 2 and sequencing - Semi-nested reverse transcription-polymerase chain reaction (RT-PCR) was performed to target the RNA-dependent RNA polymerase (RdRp) gene. In the first stage, a 526 bp fragment was amplified using the primers LRVF-HR (5′-TGTAACCCACATAAACAGTGTGC-3′) and LRVR-HR (5′-ATTTCATCCAGCTTGACTGGG -3′). In the second stage, a 315-bp internal fragment of RdRp was amplified using the primers LRVF2-HR (5′-AGGACAATCCAATAGGTCGTGT-3′) and LRVR-HR.3232. Hajjaran H, Mahdi M, Mohebali M, Samimi-Rad K, Ataei-Pirkooh A, Kazemi-Rad E, et al. Detection and molecular identification of Leishmania RNA virus (LRV) in Iranian Leishmania species. Arch Virol. 2016; 161(12): 3385-90. To ensure cDNA integrity, β-tubulin locus was used as a positive control. It was amplified using the primers bTUBf (5′-ACTGGATCCATGCGTGAGATCGTTTCCTGCC-3′) and BtubR (5′-GACAGATCTCATCAAGCACGGAGTCGATCAGC-3′).1111. Zangger H, Hailu A, Desponds C, Lye LF, Akopyants NS, Dobson DE, et al. Leishmania aethiopica field isolates bearing an endosymbiontic dsRNA virus induce pro-inflammatory cytokine response. PLoS Negl Trop Dis. 2014; 8(4): e2836. In all experiments, cDNA from the reference LRV2-positive strain of L. major (MHOM/SU/1973/5-ASKH) was used as a positive control. PCR products were purified and sequenced according to the manufacturers’ protocols.

Sequence analysis and phylogenetic inference - Partial RdRp sequences obtained from the positive isolates (HP-EMO, 305 nt and CUR268, 293 nt) were used as queries in BLASTN searches against the NCBI nt database, resulting in 52 LRV2 sequences. These were aligned iteratively using an MAFFT v. 7.511 program (g-INS-i algorithm).3838. Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013; 30(4): 772-80. The alignment was used directly (without trimming) for phylogenetic inference using an IQTree2 program with an automatically selected model (GTF + F + I + G4) and 1,000 thorough bootstrap replicas for statistical support.3939. Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, Von Haeseler A, et al. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Mol Biol Evol. 2020; 37(5): 1530-4. The LRV2 from Ethiopia were chosen as the closest outgroup based on previous studies.1111. Zangger H, Hailu A, Desponds C, Lye LF, Akopyants NS, Dobson DE, et al. Leishmania aethiopica field isolates bearing an endosymbiontic dsRNA virus induce pro-inflammatory cytokine response. PLoS Negl Trop Dis. 2014; 8(4): e2836.,3434. Kleschenko Y, Grybchuk D, Matveeva NS, Macedo DH, Ponirovsky EN, Lukashev AN, et al. Molecular characterization of Leishmania RNA virus 2 in Leishmania major from Uzbekistan. Genes (Basel). 2019; 10(10): 830. Tree rendering was done using MEGA11.4040. Tamura K, Stecher G, Kumar S. MEGA11: molecular evolutionary genetics analysis version 11. Mol Biol Evol. 2021; 38(7): 3022-7. The RdRp sequences obtained in this study were deposited in the GenBank database under accession numbers OR208212 and OR208213.

RESULTS

Out of the 71 samples analysed, two L. infantum isolates (CUR-268 and HP-EMO) were found to be positive for the 315 bp fragment at the 3′-end of RdRp using semi-nested RT-PCR analysis (Fig. 2). The resulting sequences showed a high degree of similarity to previously described LRV2 isolates from various regions in Asia (Fig. 3). Specifically, the sequences of both isolates were identical to LRV2 isolated from L. tropica in Turkey (isolate LRV2/EP01/TR/Lt01), with only a single nucleotide substitution compared to LRV2 from L. infantum in Iran (isolate LRV2/IR/2014/HM-1). A slightly higher degree of divergence (2-7 substitutions) was observed when compared with LRV2 sequences from Uzbekistan.3434. Kleschenko Y, Grybchuk D, Matveeva NS, Macedo DH, Ponirovsky EN, Lukashev AN, et al. Molecular characterization of Leishmania RNA virus 2 in Leishmania major from Uzbekistan. Genes (Basel). 2019; 10(10): 830. Phylogenetic analysis placed both isolates within the Asian clade of LRV2. However, due to the lack of complete genomic sequences for most isolates, it is challenging to determine the precise relationships between the viruses within this clade. All strains tested positive for β-tubulin (Fig. 2).

Fig. 2:
detection of Leishmania RNA virus 2 (LRV2) in L. infantum isolates by targeting the RNA-dependent RNA polymerase (RdRp) encoding gene. (A) 526 bp and 315 bp amplicons from the first and second round, respectively, of semi-nested amplification. (B) 396 bp product of the β-tubulin encoding gene reverse transcription-polymerase chain reaction (RT-PCR) assessing the integrity of cDNA synthesis. Lanes: MM, molecular weight marker (100 bp); 1, LRV2+ strain of L. major (MHOM/SU/1973/5-ASKH); 2 and 3, L. infantum strains positive for LRV2 (MCAN/BR/2004/CUR268 and MHOM/BR/2001/HP-EMO, respectively); 4 and 5, L. infantum negative strains for LRV2 (MHOM/BR/1970/BH46 and MHOM/BR/1989/Ba262, respectively); NC, non-template control.

Fig. 3:
phylogenetic tree based on nucleotide sequences of Leishmania RNA virus 2 (LRV2) viruses isolated in different countries. Clades were collapsed for better visibility with the number of samples indicated in brackets. Numbers at branches are thorough bootstrap supports, values smaller than 75 were hidden, dots indicate absolute support (100). Sequences reported in this study are highlighted in black. LRV2 virus isolated from L. infantum in Iran is highlighted in grey. Branches with triple crossing were contracted three times of their actual length. Tree was rooted on mid-point.

DISCUSSION

Endosymbiotic double-stranded RNA viruses (dsRNA) have been identified in various protistan parasites and the presence of LRVs in Leishmania spp. is considered one of the major factors associated with the severity of leishmaniasis.55. Ives A, Ronet C, Prevel F, Ruzzante G, Fuertes-Marraco S, Schutz F, et al. Leishmania RNA virus controls the severity of mucocutaneous leishmaniasis. Science. 2011; 331(6018): 775-8.,66. Hartley MA, Ronet C, Zangger H, Beverley SM, Fasel N. Leishmania RNA virus: when the host pays the toll. Front Cell Infect Microbiol. 2012; 2: 99.,4141. Gupta V, Deep A. An insight into the Leishmania RNA virus. Indian J Med Microbiol. 2007; 25(1): 7-9. In this study, we report the first detection of two putative LRV2 strains of L. infantum in Brazil. These strains were isolated from cases of VL in dogs and humans in the states of Minas Gerais and Espírito Santo, respectively, in southeastern Brazil. Until now, LRV2 has been detected in L. aethiopica,1111. Zangger H, Hailu A, Desponds C, Lye LF, Akopyants NS, Dobson DE, et al. Leishmania aethiopica field isolates bearing an endosymbiontic dsRNA virus induce pro-inflammatory cytokine response. PLoS Negl Trop Dis. 2014; 8(4): e2836., L. major,3131. Scheffter SM, Ro YT, Chung IK, Patterson JL. The complete sequence of Leishmania RNA Virus LRV2-1, a virus of an old world parasite strain. Virology. 1995; 212(1): 84-90.,3333. Nalçaci M, Karakus M, Yilmaz B, Demir S, Özbilgin A, Özbel Y, et al. Detection of Leishmania RNA virus 2 in Leishmania species from Turkey. Trans R Soc Trop Med Hyg. 2019; 113(7): 410-7. Old World L. infantum3232. Hajjaran H, Mahdi M, Mohebali M, Samimi-Rad K, Ataei-Pirkooh A, Kazemi-Rad E, et al. Detection and molecular identification of Leishmania RNA virus (LRV) in Iranian Leishmania species. Arch Virol. 2016; 161(12): 3385-90. and L. tropica.4242. Saberi R, Fakhar M, Hajjaran H, Ataei-Pirkooh A, Mohebali M, Taghipour N, et al. Presence and diversity of Leishmania RNA virus in an old zoonotic cutaneous leishmaniasis focus, northeastern Iran: haplotype and phylogenetic based approach. Int J Infect Dis. 2020; 101: 6-13. This finding represents the first report of LRV2 in the New World.

Our analysis revealed only two LRV2-positive strains (2.8%) out of 71 samples analysed. The low frequency of LRV2 detection could be influenced by a variety of factors, including the potential impact of successive in vitro passages on the presence and stability of the endosymbiotic virus.2424. Saura A, Zakharova A, Klocek D, Gerasimov ES, Butenko A, Macedo DH, et al. Elimination of LRVs elicits different responses in Leishmania spp. mSphere. 2022; 7(4): e0033522.,4343. Kuhlmann FM, Robinson JI, Bluemling GR, Ronet C, Fasel N, Beverley SM. Antiviral screening identifies adenosine analogs targeting the endogenous dsRNA Leishmania RNA virus 1 (LRV1) pathogenicity factor. Proc Natl Acad Sci USA. 2017; 114(5): E811-9.,4444. Robinson JI, Beverley SM. Concentration of 2'C-methyladenosine triphosphate by Leishmania guyanensis enables specific inhibition of Leishmania RNA virus 1 via its RNA polymerase. J Biol Chem. 2018; 293(17): 6460-9. In this study, a panel of L. infantum strains was used to assess the presence of LRV2, and this may have contributed to the limited detection of the endosymbiotic virus. However, the low frequency observed in our study is in line with the overall prevalence of LRV in Brazil, suggesting that LRV2 is indeed less common in this region. LRV1 is more frequently found in the Amazon Basin (Northern region) and less common in the other regions.2929. Macedo DH, Menezes-Neto A, Rugani JM, Rocha AC, Silva SO, Melo MN, et al. Low frequency of LRV1 in Leishmania braziliensis strains isolated from typical and atypical lesions in the State of Minas Gerais, Brazil. Mol Biochem Parasitol. 2016; 210(1-2): 50-4.,4545. Cantanhêde LM, da Silva Jr CF, Ito MM, Felipin KP, Nicolete R, Salcedo JMV, et al. Further evidence of an association between the presence of Leishmania RNA virus 1 and the mucosal manifestations in tegumentary leishmaniasis patients. PLoS Negl Trop Dis. 2015; 9(9): e0004079. In southeastern Brazil, only a few cases of LRV1 have been documented, including biopsies of patients with cutaneous leishmaniasis in the municipality of Caratinga, Minas Gerais4646. Ogg MM, Carrion R, Botelho ACC, Mayrink W, Correa-Oliveira R, Patterson JL. Short report: quantification of Leishmaniavirus RNA in clinical samples and it possible role in pathogenesis. Am J Trop Med Hyg. 2003; 69(3): 309-13. and in Rio de Janeiro.3030. Zabala-Peñafiel A, Fantinatti M, Dias-Lopes G, da Silva JL, Miranda LFC, Lyra MR, et al. First report of Leishmania RNA virus 1 in Leishmania (Viannia) braziliensis clinical isolates from Rio de Janeiro State - Brazil. Mem Inst Oswaldo Cruz. 2022; 117: e210107.,4747. Pereira LOR, Maretti-Mira AC, Rodrigues KM, Lima RB, de Oliveira-Neto MP, Cupolillo E, et al. Severity of tegumentary leishmaniasis is not exclusively associated with Leishmania RNA virus 1 infection in Brazil. Mem Inst Oswaldo Cruz. 2013; 108(5): 665-7.

In our study, the Brazilian LRV2 isolates showed a high degree of similarity with LRV2 isolates from various parts of Asia. However, it is important to interpret this result with caution due to the relatively small size of the amplified fragment (~300 bp). Therefore, a comprehensive analysis of the whole genome would be desirable, to determine the phylogenetic relationships accurately. We believe that our study contributes to the understanding of the geographical distribution of LRV2s in Leishmania (Leishmania) and provides new avenues for investigating the evolutionary relationships between LRV-infected Leishmania parasites and their vertebrate hosts.

ACKNOWLEDGEMENTS

To the Fiocruz Network of Technological Platforms at Instituto René Rachou - Fiocruz Minas for the DNA sequencing facility, and their assistance and DNA sequencing services. We are grateful to Cristiane P Gomes and Patrícia PN Miranda, from project support services at Fiocruz Minas (SAPRO), for project and resource management. We thank Dr Frédéric Frézard and Cristiano CP dos Santos, Universidade Federal de Minas Gerais, for their collaboration on providing clinical samples of the canine CVLs that were analysed in this work.

REFERENCES

  • 1
    PAHO - Pan American Health Organization. Leishmaniases. Epidemiological report of the Americas, December 2020 [Internet]. 2020. Available from: https://iris.paho.org/handle/10665.2/53090
    » https://iris.paho.org/handle/10665.2/53090
  • 2
    Bruschi F, Gradoni L. The leishmaniases: old neglected tropical diseases. 1st ed. Vol. 1. Berlim: Springer International Publishing; 2018. 245 pp.
  • 3
    Desjeux P. The increase in risk factors for leishmaniasis worldwide. Trans R Soc Trop Med Hyg. 2001; 95(3): 239-43.
  • 4
    Mann S, Frasca K, Scherrer S, Henao-Martínez AF, Newman S, Ramanan P, et al. A review of leishmaniasis: current knowledge and future directions. Curr Trop Med Reports. 2021; 8(2): 121-32.
  • 5
    Ives A, Ronet C, Prevel F, Ruzzante G, Fuertes-Marraco S, Schutz F, et al. Leishmania RNA virus controls the severity of mucocutaneous leishmaniasis. Science. 2011; 331(6018): 775-8.
  • 6
    Hartley MA, Ronet C, Zangger H, Beverley SM, Fasel N. Leishmania RNA virus: when the host pays the toll. Front Cell Infect Microbiol. 2012; 2: 99.
  • 7
    Kostygov AY, Karnkowska A, Votýpka J, Tashyreva D, Maciszewski K, Yurchenko V, et al. Euglenozoa: taxonomy, diversity and ecology, symbioses and viruses. Open Biol. 2021; 11(3): 200407.
  • 8
    Paranaiba LF, Pinheiro LJ, Macedo DH, Menezes-Neto A, Torrecilhas AC, Tafuri WL, et al. An overview on Leishmania (Mundinia) enriettii: biology, immunopathology, LRV and extracellular vesicles during the host-parasite interaction. Parasitology. 2018; 145(10): 1265-73.
  • 9
    Kostygov AY, Grybchuk D, Kleschenko Y, Chistyakov DS, Lukashev AN, Gerasimov ES, et al. Analyses of Leishmania-LRV co-phylogenetic patterns and evolutionary variability of viral proteins. Viruses. 2021; 13(11): 2305.
  • 10
    Cantanhêde LM, Mata-Somarribas C, Chourabi K, da Silva GP, das Chagas BD, Pereira LOR, et al. The maze pathway of coevolution: a critical review over the Leishmania and its endosymbiotic history. Genes (Basel). 2021; 12(5): 657.
  • 11
    Zangger H, Hailu A, Desponds C, Lye LF, Akopyants NS, Dobson DE, et al. Leishmania aethiopica field isolates bearing an endosymbiontic dsRNA virus induce pro-inflammatory cytokine response. PLoS Negl Trop Dis. 2014; 8(4): e2836.
  • 12
    Salinas G, Zamora M, Stuart K, Saravia N. Leishmania RNA viruses in Leishmania of the Viannia subgenus. Am J Trop Med Hyg. 1996; 54(4): 425-9.
  • 13
    Grybchuk D, Kostygov AY, Macedo DH, Votýpka J, Lukes J, Yurchenko V. RNA viruses in Blechomonas (Trypanosomatidae) and evolution of Leishmaniavirus. mBio. 2018; 9(5): e01932-18.
  • 14
    Grybchuk D, Macedo DH, Kleschenko Y, Kraeva N, Lukashev AN, Bates PA, et al. The first non-LRV RNA virus in Leishmania. Viruses. 2020; 12(2): 168.
  • 15
    Mendes Jr AAV, Filgueira CPB, Miranda LFC, de Almeida AB, Cantanhêde LM, Fagundes A, et al. First report of Leishmania (Mundinia) martiniquensis in South American territory and confirmation of Leishbunyavirus infecting this parasite in a mare. Mem Inst Oswaldo Cruz. 2023; 118: e220220.
  • 16
    Brettmann EA, Shaik JS, Zangger H, Lye LF, Kuhlmann FM, Akopyants NS, et al. Tilting the balance between RNA interference and replication eradicates Leishmania RNA virus 1 and mitigates the inflammatory response. Proc Natl Acad Sci USA. 2016; 113(43): 11998-2005.
  • 17
    de Carvalho RVH, Lima-Junior DS, da Silva MVG, Dilucca M, Rodrigues TS, Horta CV, et al. Leishmania RNA virus exacerbates leishmaniasis by subverting innate immunity via TLR3-mediated NLRP3 inflammasome inhibition. Nat Commun. 2019; 10(1): 5273.
  • 18
    Rossi M, Castiglioni P, Hartley MA, Eren RO, Prével F, Desponds C, et al. Type I interferons induced by endogenous or exogenous viral infections promote metastasis and relapse of leishmaniasis. Proc Natl Acad Sci USA. 2017; 114(19): 4987-92.
  • 19
    Hartley MA, Bourreau E, Rossi M, Castiglioni P, Eren RO, Prevel F, et al. Leishmaniavirus-dependent metastatic leishmaniasis is prevented by blocking IL-17A. PLoS Pathog. 2016; 12(9): e1005852.
  • 20
    Eren RO, Reverte M, Rossi M, Hartley MA, Castiglioni P, Prevel F, et al. Mammalian innate immune response to a Leishmania-resident RNA virus increases macrophage survival to promote parasite persistence. Cell Host Microbe. 2016; 20(3): 318-28.
  • 21
    Atayde VD, Lira Filho AS, Chaparro V, Zimmermann A, Martel C, Jaramillo M, et al. Exploitation of the Leishmania exosomal pathway by Leishmania RNA virus 1. Nat Microbiol. 2019; 4(4): 714-23.
  • 22
    Olivier M, Zamboni DS. Leishmania viannia guyanensis, LRV1 virus and extracellular vesicles: a dangerous trio influencing the faith of immune response during muco-cutaneous leishmaniasis. Curr Opin Immunol. 2020; 66: 108-13.
  • 23
    Lafleur A, Olivier M. Viral endosymbiotic infection of protozoan parasites: how it influences the development of cutaneous leishmaniasis. PLOS Pathog. 2022; 18(11): e1010910.
  • 24
    Saura A, Zakharova A, Klocek D, Gerasimov ES, Butenko A, Macedo DH, et al. Elimination of LRVs elicits different responses in Leishmania spp. mSphere. 2022; 7(4): e0033522.
  • 25
    Saberi R, Fakhar M, Mohebali M, Anvari D, Gholami S. Global status of synchronizing Leishmania RNA virus in Leishmania parasites: a systematic review with meta-analysis. Transbound Emerg Dis. 2019; 66(6): 2244-51.
  • 26
    Adaui V, Lye LF, Akopyants NS, Zimic M, Llanos-Cuentas A, Garcia L, et al. Association of the endobiont double-stranded RNA virus LRV1 with treatment failure for human leishmaniasis caused by Leishmania braziliensis in Peru and Bolivia. J Infect Dis. 2016; 213(1): 112-21.
  • 27
    Kariyawasam R, Grewal J, Lau R, Purssell A, Valencia BM, Llanos-Cuentas A, et al. Influence of Leishmania RNA Virus 1 on proinflammatory biomarker expression in a human macrophage model of American tegumentary leishmaniasis. J Infect Dis. 2017; 216(7): 877-86.
  • 28
    Ginouvès M, Simon S, Bourreau E, Lacoste V, Ronet C, Couppié P, et al. Prevalence and distribution of Leishmania RNA virus 1 in Leishmania parasites from French Guiana. Am J Trop Med Hyg. 2016; 94(1): 102.
  • 29
    Macedo DH, Menezes-Neto A, Rugani JM, Rocha AC, Silva SO, Melo MN, et al. Low frequency of LRV1 in Leishmania braziliensis strains isolated from typical and atypical lesions in the State of Minas Gerais, Brazil. Mol Biochem Parasitol. 2016; 210(1-2): 50-4.
  • 30
    Zabala-Peñafiel A, Fantinatti M, Dias-Lopes G, da Silva JL, Miranda LFC, Lyra MR, et al. First report of Leishmania RNA virus 1 in Leishmania (Viannia) braziliensis clinical isolates from Rio de Janeiro State - Brazil. Mem Inst Oswaldo Cruz. 2022; 117: e210107.
  • 31
    Scheffter SM, Ro YT, Chung IK, Patterson JL. The complete sequence of Leishmania RNA Virus LRV2-1, a virus of an old world parasite strain. Virology. 1995; 212(1): 84-90.
  • 32
    Hajjaran H, Mahdi M, Mohebali M, Samimi-Rad K, Ataei-Pirkooh A, Kazemi-Rad E, et al. Detection and molecular identification of Leishmania RNA virus (LRV) in Iranian Leishmania species. Arch Virol. 2016; 161(12): 3385-90.
  • 33
    Nalçaci M, Karakus M, Yilmaz B, Demir S, Özbilgin A, Özbel Y, et al. Detection of Leishmania RNA virus 2 in Leishmania species from Turkey. Trans R Soc Trop Med Hyg. 2019; 113(7): 410-7.
  • 34
    Kleschenko Y, Grybchuk D, Matveeva NS, Macedo DH, Ponirovsky EN, Lukashev AN, et al. Molecular characterization of Leishmania RNA virus 2 in Leishmania major from Uzbekistan. Genes (Basel). 2019; 10(10): 830.
  • 35
    Yurchenko V, Chistyakov DS, Akhmadishina LV., Lukashev AN, Sádlová J, Strelkova MV. Revisiting epidemiology of leishmaniasis in Central Asia: lessons learnt. Parasitology. 2022; 150(2): 129-36.
  • 36
    Widmer G, Dooley S. Phylogenetic analysis of Leishmania RNA virus and Leishmania suggests ancient virus-parasite association. Nucleic Acids Res. 1995; 23(12): 2300-4.
  • 37
    Garcia L, Kindt A, Bermudez H, Llanos-Cuentas A, De Doncker S, Arevalo J, et al. Culture-independent species typing of neotropical Leishmania for clinical validation of a PCR-based assay targeting heat shock protein 70 genes. J Clin Microbiol. 2004; 42(5): 2294-7.
  • 38
    Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013; 30(4): 772-80.
  • 39
    Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, Von Haeseler A, et al. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Mol Biol Evol. 2020; 37(5): 1530-4.
  • 40
    Tamura K, Stecher G, Kumar S. MEGA11: molecular evolutionary genetics analysis version 11. Mol Biol Evol. 2021; 38(7): 3022-7.
  • 41
    Gupta V, Deep A. An insight into the Leishmania RNA virus. Indian J Med Microbiol. 2007; 25(1): 7-9.
  • 42
    Saberi R, Fakhar M, Hajjaran H, Ataei-Pirkooh A, Mohebali M, Taghipour N, et al. Presence and diversity of Leishmania RNA virus in an old zoonotic cutaneous leishmaniasis focus, northeastern Iran: haplotype and phylogenetic based approach. Int J Infect Dis. 2020; 101: 6-13.
  • 43
    Kuhlmann FM, Robinson JI, Bluemling GR, Ronet C, Fasel N, Beverley SM. Antiviral screening identifies adenosine analogs targeting the endogenous dsRNA Leishmania RNA virus 1 (LRV1) pathogenicity factor. Proc Natl Acad Sci USA. 2017; 114(5): E811-9.
  • 44
    Robinson JI, Beverley SM. Concentration of 2'C-methyladenosine triphosphate by Leishmania guyanensis enables specific inhibition of Leishmania RNA virus 1 via its RNA polymerase. J Biol Chem. 2018; 293(17): 6460-9.
  • 45
    Cantanhêde LM, da Silva Jr CF, Ito MM, Felipin KP, Nicolete R, Salcedo JMV, et al. Further evidence of an association between the presence of Leishmania RNA virus 1 and the mucosal manifestations in tegumentary leishmaniasis patients. PLoS Negl Trop Dis. 2015; 9(9): e0004079.
  • 46
    Ogg MM, Carrion R, Botelho ACC, Mayrink W, Correa-Oliveira R, Patterson JL. Short report: quantification of Leishmaniavirus RNA in clinical samples and it possible role in pathogenesis. Am J Trop Med Hyg. 2003; 69(3): 309-13.
  • 47
    Pereira LOR, Maretti-Mira AC, Rodrigues KM, Lima RB, de Oliveira-Neto MP, Cupolillo E, et al. Severity of tegumentary leishmaniasis is not exclusively associated with Leishmania RNA virus 1 infection in Brazil. Mem Inst Oswaldo Cruz. 2013; 108(5): 665-7.

Publication Dates

  • Publication in this collection
    18 Sept 2023
  • Date of issue
    2023

History

  • Received
    17 Apr 2023
  • Accepted
    31 July 2023
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