Epidemiology and Genetic Characterization of Leishmania RNA Virus in Leishmania (Viannia) spp. Isolates from Cutaneous Leishmaniasis Endemic Areas in Panama

Leishmania (Viannia) spp. can harbor a double-stranded RNA virus known as Leishmania RNA virus 1 (LRV-1), whose presence has been reported in nine countries across the Americas and seven Leishmania species. Here, we studied 100 Leishmania (Viannia) isolates from patients with cutaneous leishmaniasis collected from different endemic areas in Panama from 2016 to 2022. We identified L. (V.) panamensis, L. (V.) guyanensis, L. (V.) braziliensis/guyanensis hybrid, and L. (V.) panamensis sp.1. (genetic variant). LRV-1 was detected by RT-PCR in 9% of L. (Viannia) isolates (eight cases in L. (V.) panamensis, and one in L. (V.) guyanensis). Phylogenetic analysis based on sequencing data classified all LRV-1 isolates within genotype A, suggesting that LRV phylogenetic proximity is closely aligned with geographical distribution or to the phylogenetic proximity of the Leishmania host in the case of the L. (V.) panamensis and L. (V.) guyanensis in Panama.

The viral genome ranges in size from 4.9 to 5.3 kb and encodes the capsid protein (ORF2) and the RNA-dependent RNA polymerase (ORF3) [18].It also contains the so-called ORF1 located at the 5 ′ end that does not encode a protein but it presumably plays a role in RNA stability and translation [19].
Panama is considered an endemic country for cutaneous leishmaniasis (CL) with an average of 1000 new cases annually, predominantly caused by L. (V.) panamensis [32,33].According to the Pan American Health Organization (PAHO) report in 2023, there has been a notable increase in the risk of transmission in recent years, elevating the risk classification from moderate to high [33].Previously, we have confirmed the presence of LRV-1 in 11 L. (V.) panamensis isolates from CL endemic regions in Panama [34].However, a comprehensive analysis to characterize the virus at the genotype level remains pending.Furthermore, the evaluation of LRV presence in other Leishmania species circulating in Panama remains unexplored, even though the circulation of L. (V.) guyanensis, L. (V.) braziliensis, L. (V.) naiffi, L. (V.).braziliensis/guyanensis hybrids, and a genetic variant of L. (V.) panamensis named sp.1 have been recorded in the country [34][35][36][37].In this regard, LRV-1 has been already described in L. (V.) guyanensis, L. (V.) naiffi, and L. (V.) braziliensis in various South American countries [17,20,22,23].
In this work, we present a retrospective study of LRV-1 in clinical isolates of Leishmania (Viannia) collected in Panama.We demonstrate that Panamanian LRV-1 circulates in L. (V.) panamensis and L. (V.) guyanensis and belongs to the genotype A.

Study Design and Leishmania Isolates
This is a cross-sectional study with 100 isolates of Leishmania (Viannia) spp.sampled from skin lesions of patients who attended the Unidad de Diagnóstico, Investigación Clínica y Medicina Tropical of the Instituto Conmemorativo Gorgas de Estudios de la Salud (ICGES), Panama, between 2016 and 2022 (Table S1).These isolates were previously identified using the RFLP/PCR-Hsp70 method and were cryopreserved in the ICGES biobank [38].Additionally, the amplified products of the Hsp70 gene for the LRV-1-positive Leishmania isolates were sequenced to confirm the species by reconstructing a Neighbor-Joining phylogenetic tree in MEGA X software using the Kimura 2-parameter model and branch support assessed with 2000 bootstrap replicates [37,38].Nucleotide sequences of the LRV-1-positive Leishmania isolates are available in NCBI GenBank under the accession numbers PP796356 to PP796364.

Ethical Considerations
This study and the retrospective use of biological samples were carried out with the approval of the ICGES Institutional Bioethics Committee (code 267/CBI/ICGES/21).The study did not involve direct work with human subjects or animals.

Preparation of Isolates
Parasites from the biobank were thawed and grown in Schneider medium enriched with 25% fetal bovine serum (FBS).Once the stationary phase was reached, RNA was obtained from the parasite pellet using the QIAGEN RNeasy Mini kit (QIAGEN, Redwood City, CA, USA) according to the manufacturer's instructions.The extracted RNA was quantified using NanoDrop 2000 equipment (Thermo Fisher Scientific, Wilmington, DE, USA) and stored at −80 • C until use.

Purification and Sequencing
The amplified products were purified using ExoSAP-IT Express PCR Product Cleanup, and the Sanger sequencing reaction was performed using the BigDye Terminator v3.1 cycling kit using the primers previously described [13,39].Sanger reaction products were purified with the BigDye XTerminator kit (Applied Biosystems, Waltham, MA, USA) and analyzed using the 3130xl Genetic Analyzer sequencer (Applied Biosystems, Foster, CA, USA).Nucleotide sequences were confirmed using Sequencher version 5.4.6DNA sequence analysis software and aligned using the BioEdit v7.0.9.0 program.

Construction of Phylogenetic Tree
Nucleotide sequences were aligned using MAFFT v7.490 [40] and visualized with Aliview v1.28 [41].The nucleotide substitution model for each dataset was selected using ModelFinder implemented within IQ-TREE v2.2.0 [42].Bayesian phylogenetic trees were constructed using MrBayes v3.2.7 with two parallel runs of four Monte Carlo Markov Chains (MCMC) with 10 million generations sampled every 10,000 generations, resulting in a total of 1000 trees [43].The MCMC convergence was evaluated in TRACER v.1.5with an effective sample size (ESS) > 200; the initial 10% of the run length was discarded as burn-in.The consensus tree was summarized from sampled trees and visualized with FigTree v.1.4.4.Clades with posterior probability <0.6 were collapsed.Nucleotide sequences are available in NCBI GenBank under the accession numbers listed in Table 1.
Assessment of disease severity concerning LRV-1 presence demonstrated that tw Nine isolates were positive for LRV-1 (9/100: 9%), of which eight were found in L. (V.) panamensis and one in L. (V.) guyanensis (Figure S1).All positive isolates corresponded to male patients.The age of the patients ranged between 27 and 62 years, with a mean of 42 years.Most of the patients had single lesions (7/9: 77.78%), with a range of 1 to 11 lesions per patient.The mean of the development time of the lesion was 42 days, with a range of 30-60 days.The lesions were mainly distributed on the arms (4/9, 44.44%) and legs (4/9, 44.44%).Only one patient presented with 11 ulcerated lesions: 7 on the face, 1 on the leg, 1 on the arm, and 2 on the back.Of the 91 LRV-1-negative isolates, the majority were men (65%).The age of the patients ranged between 1 and 75 years, with a mean of 31 years.Most of the patients had single lesions (81/91: 89.01%), with a range of 1 to 3 lesions per patient.The mean of the development time of the lesion was 35 days, with a range of 15-120 days.The lesions were mainly distributed on the arms (72/91, 79.12%), legs (17/91, 18.68%), back (1/91, 1.1%), and face (1/91, 1.1%).
Three of the LRV-1-positive L. (V.) panamensis isolates originated from Colon, two from Bocas del Toro, and three from Panama Oeste, Panama, and Darien.Additionally, one isolate of L. (V.) guyanensis was obtained from Cocle (Figure 1B).
Assessment of disease severity concerning LRV-1 presence demonstrated that two L. (V.) panamensis isolates were associated with complicated CL cases: one with 11 lesions, and one with treatment failure despite undergoing three complete rounds of pentavalent antimonial treatment (Table 1).In contrast, no complicated CL cases were observed in LRV-1-negative isolates.The association of complicated CL cases with LRV presence (2/9: 22.22% vs. 0/91: 0%) was statistically significant according to the N-1 chi-square test for (p < 0.0001) [44].

Phylogenetic Analysis of LRV-1 Detected in Panama
Our sequencing efforts allowed us to characterize a 240nt fragment of the ORF1 of all LRV-1 from the L. (V.) panamensis isolates.For the L. (V.) guyanensis isolate, we were able to reconstruct a partial genome including 790nt of ORF1 and the beginning of ORF2, 678nt within ORF2, and 942nt of ORF3 [13,29,39].
The Bayesian phylogenetic tree for the 240nt ORF1 fragment, including the same region from all LRV-1 sequences available in the NCBI GenBank database (up to March 2024), revealed a well-defined clustering pattern associated with the Leishmania host.Notably, all LRV-1 identified in Panama consistently clustered within genotype A, regardless of whether they infected L. (V.) panamensis or L. (V.) guyanensis (Figure 2).It is noteworthy that all LRV-1 sequences detected in Panama between 2016 and 2022 (from this study and the previous one) exhibited close evolutionary relationships with the sequence of L. (V.) guyanensis collected in Costa Rica in 2019 (OM140825) and two sequences from the Northeast of Brazil (KX808487 and U01899).Additional analysis of the L. (V.) guyanensis partial genome confirmed its association with the sequences of genotype A (Figure S2).
of whether they infected L. (V.) panamensis or L. (V.) guyanensis (Figure 2).It is noteworthy that all LRV-1 sequences detected in Panama between 2016 and 2022 (from this study and the previous one) exhibited close evolutionary relationships with the sequence of L. (V.) guyanensis collected in Costa Rica in 2019 (OM140825) and two sequences from the Northeast of Brazil (KX808487 and U01899).Additional analysis of the L. (V.) guyanensis partial genome confirmed its association with the sequences of genotype A (Figure S2).

Discussion
Cutaneous leishmaniasis is a public health problem of major concern in Panama, predominantly caused by L. (V.) panamensis [32,33].Since the discovery of the LRV-1 infecting different Leishmania species, several studies have tried to unravel the epidemiological importance of this virus and its relationship with Leishmania spp.[11,17,21,22,24,34,45,46].
Here, we present a surveillance study of Leishmania and LRV-1 in Panama from 2016 to 2022 to ascertain the LRV-1 positivity rate, genotypic classification, and host range.We studied 100 Leishmania (Viannia) isolates and found a 9% LRV-1 positivity rate, which is lower than the previous report in Panama (20%) [35].However, the N-1 chi-square test does not confirm significance of this difference (p = 0.0582).Apparently, the viruses have mosaic distribution in the populations of leishmaniae, and therefore, different studies have reported a wide range of virus detection rates-from 2% to 70% [17,21,24,47,48].Another reason may be the loss of the virus upon cultivation, leading to the underestimation of the virus infection rate.Analysis of parasites directly from the lesion is devoid of such a potential drawback [45].
Our analysis revealed the predominance of L. (V.) panamensis among leishmaniae in Panama but also demonstrated the presence of other species and genetic variants in provinces adjacent to the Panama Canal.However, LRV-1 was equally distributed throughout the country and was documented in Bocas del Toro alongside the previously documented Panama Oeste, Panama, Cocle, Colon, and Darien [34].It was not possible to detect the virus in the few cases of L. (V.) guyanensis/braziliensis hybrids and L. (V.) panamensis genetic variant sp.1.Notably, we documented for the first time LRV-1 infecting L. (V.) guyanensis isolates in the Cocle region.
The Panama isthmus is the geographical link between Central and South America, providing a biological corridor between Colombia and Costa Rica, allowing the entry to Panama of the LRV-infected Leishmania spp., in vectors and reservoirs [38].Another point to consider is that Panama currently serves as a significant transit point for migrants traveling through South America on their northward journey toward the USA or Canada.This transit poses a risk for the introduction of new Leishmania spp.harboring the LRV-1, potentially facilitating the establishment of these parasites and the virus they carry within Panama's natural transmission cycle [37,49].
Our analysis further revealed that LRV-1 sequences from Panama were evolutionarily close to those from Costa Rica and Northeast of Brazil, supporting previous hypotheses that the phylogenetic proximity of viral isolates aligns more closely with the geographical distribution of the Leishmania host [46].Additionally, there is a possibility that LRV-1 can infect L. (V.) guyanensis or L. (V.) panamensis indiscriminately, unlike what has been observed in other species such as L. (V.) braziliensis, L. (V.) shawi, or L. (V.) naiffi [18].In other words, the phylogenetic analysis of the LRV-1 partial genome from L. (V.) guyanensis in Panama reaffirmed its association with genotype A (Figure S2) [13,18].
LRV-1 is an important component in the dynamics of infection by parasites of the genus Leishmania, influencing the virulence of the parasite and the immune response of the vertebrate host [7,14,17,26,[57][58][59].In this context, the analysis of clinical characteristics among the LRV-1-positive cases showed that most of our evaluated patients with CL were classified as non-serious according to criteria established by the Infectious Diseases Society of America (IDSA).However, in the panel of evaluated parasites, we encountered two iso-lates of L. (V.) panamensis LRV+ associated with patients experiencing clinical complications: one with 11 lesions, while another exhibited treatment failure despite undergoing three cycles of pentavalent antimonial therapy for a single lesion.These findings are consistent with previous reports suggesting that, unlike in other species, the presence of LRV-1 in L. (V.) panamensis does not necessarily exacerbate clinical manifestations as observed in L. (V.) guyanensis and L. (V.) braziliensis [26,34,60].Likewise, our study did not observe an association between the presence of LRV-1 and complicated forms of CL in the single L. (V.) guyanensis LRV-1+ isolate analyzed.Although some authors have reported that LRV presence does not invariably lead to treatment failures or clinical complications, it is important to emphasize that the incidence of CL caused by L. (V.) guyanensis is markedly low in Panama compared to other endemic areas of South America [15,17,21].The study has limitations, including the small number of LRV-1-positive cases (although we analyzed a total of 100 L. (Viannia) isolates) and the inability to obtain viral complete genomes for all the isolates.Nevertheless, our study provides insights into the evolution and geographical distribution of LRV-1 and the role of coevolution with the Leishmania (Viannia) species.

Conclusions
Our study confirms the presence of LRV-1 in both L. (V.) panamensis and L. (V.) guyanensis isolates from CL endemic areas in Panama, with phylogenetic analysis classifying them within the genotype A. Sharing of the same viral lineage suggests that these two Leishmania spp.can exchange their viruses, which is apparently facilitated by the close relationship of both parasites.While most LRV-1+ isolates were associated with uncomplicated CL cases, we identified two cases of L. (V.) panamensis/LRV+ isolates linked to clinical complications and treatment failure.However, the association between LRV-1 presence and disease severity or treatment outcomes in Panama remains inconclusive.Further investigation is needed to clarify the impact of LRV-1 on the pathogenesis of Leishmania (Viannia) spp.infections.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/microorganisms12071317/s1,Table S1: General description of the Leishmania (Viannia) spp.isolates analyzed for the detection of LRV-1. Figure S1.Neighbor-Joining phylogenetic tree of the hsp70 sequences from Leishmania (Viannia) constructed with MEGAX software.Sequences from this study are highlighted in yellow and named, including the sample ID, the GenBank accession number, the Leishmania species, and the sequence ID.Reference sequences' names include the GenBank accession number, the Leishmania species, and the sequence ID.Bootstrap values above 50% (2000 replicates) are shown above for the node branches.All gapped positions were removed for each sequence pair (pairwise deletion option).Figure S2.Bayesian phylogenetic tree of LRV-1 genomes.It was inferred with the concatenated sequences of four genomic fragments representing all three ORFs of the LRV-1 isolate from L. (V.) guyanensis in Panama (highlighted in blue) and reference genomes of all genotypes of LRV-1 in L. (V.) guyanensis (LVRg).Genotype F (LRV-1 in L. (V.) braziliensis, LRVb) is selected as an outgroup to root the tree.Posterior probability >0.6 is reported at clade nodes.

Data Availability Statement:
The sequences used to support the findings of this study are available from GenBank with the accession numbers: PP504593-PP504601 and PP796356-PP796364.

Figure 1 .
Figure 1.Distribution of Leishmania (Viannia) isolates analyzed in this study.(A) All isolates 100).(B) Virus-positive isolates (n = 9).The colors show the Leishmania species, and the num inside the circle indicates the number of isolates analyzed.

Figure 1 .
Figure 1.Distribution of Leishmania (Viannia) isolates analyzed in this study.(A) All isolates (n = 100).(B) Virus-positive isolates (n = 9).The colors show the Leishmania species, and the number inside the circle indicates the number of isolates analyzed.

Figure 2 .
Figure 2. Bayesian phylogenetic tree partial ORF1 of the LRV-1.The phylogenetic tree was constructed from an alignment spanning approximately 240nt of the ORF1.Colored dots next to the sequence name indicate the Leishmania (Viannia) species.Sequences collected in Panama are

Figure 2 .
Figure 2. Bayesian phylogenetic tree partial ORF1 of the LRV-1.The phylogenetic tree was constructed from an alignment spanning approximately 240nt of the ORF1.Colored dots next to the sequence name indicate the Leishmania (Viannia) species.Sequences collected in Panama are highlighted in red for LRV-1 isolated from L. (V.) panamensis and in blue for LRV-1 isolated from L. (V.) guyanensis.Sequences obtained in this study are marked with green arrows and named by strain ID, accession number, host species, geographic region, and year of collection.Sequences from NCBI GenBank are identified by accession number, host species, country, and year of collection.Bars at the right of the phylogenetic clades denote the genotypes.Posterior probability >0.6 is shown at clade nodes.
* Leishmania LRV-positive isolated from a patient with three cycles of pentavalent antimonial.