New insights into the genetic diversity of <i>Leishmania RNA Virus 1</i> and its species-specific relationship with <i>Leishmania</i> parasites

<div><p>Cutaneous leishmaniasis is a neglected parasitic disease that manifests in infected individuals under different phenotypes, with a range of factors contributing to its broad clinical spectrum. One factor, <i>Leishmania RNA Virus</i> 1 (LRV1), has been described as an endosymbiont present in different species of <i>Leishmania</i>. LRV1 significantly worsens the lesion, exacerbating the immune response in both experimentally infected animals and infected individuals. Little is known about the composition and genetic diversity of these viruses. Here, we investigated the relationship between the genetic composition of LRV1 detected in strains of <i>Leishmania</i> (<i>Viannia</i>) <i>braziliensis</i> and <i>L</i>. (<i>V</i>.) <i>guyanensis</i> and the interaction between the endosymbiont and the parasitic species, analyzing an approximately 850 base pair region of the viral genome. We also included one LRV1 sequence detected in <i>L</i>. (<i>V</i>.) <i>shawi</i>, representing the first report of LRV1 in a species other than <i>L</i>. <i>braziliensis</i> and <i>L</i>. <i>guyanensis</i>. The results illustrate the genetic diversity of the LRV1 strains analyzed here, with smaller divergences detected among viral sequences from the same parasite species. Phylogenetic analyses showed that the LRV1 sequences are grouped according to the parasite species and possibly according to the population of the parasite in which the virus was detected, corroborating the hypothesis of joint evolution of the viruses with the speciation of <i>Leishmania</i> parasites.</p></div

NeighborNet presenting the relationship among LRV1 sequences from different <i>Leishmania</i> (<i>Viannia</i>) species.

<p>The network was computed using SplitsTree software. EqualAngle was employed for splits transformation. Text colors refer to groups from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198727#pone.0198727.g004" target="_blank">Fig 4</a>: red = cluster I, blue = cluster II, and green = cluster III. For sample details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198727#pone.0198727.t001" target="_blank">Table 1</a>.</p

Minimum spanning network displaying the connections among LRV1 sequences from different <i>Leishmania</i> (<i>Viannia</i>) species.

<p>I = LRV1 sequences from <i>L</i>. <i>guyanensis</i> groups A, B, C and E, and <i>L</i>. <i>shawi</i>; II = LRV1 sequences from <i>L</i>. <i>braziliensis</i> group; III = LRV1 sequences from <i>L</i>. <i>guyanensis</i> group D. For sample details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198727#pone.0198727.g001" target="_blank">Fig 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198727#pone.0198727.t001" target="_blank">Table 1</a>.</p

Maximum likelihood tree of <i>Leishmania</i> RNA Virus 1 detected in <i>Leishmania</i> (<i>Viannia</i>) species from South American countries.

<p>The analysis involved 43 nucleotide sequences. There was a total of 687 positions in the final dataset (Dataset I). Boxes indicate the clustering of M5313, 1398, 3542, and 2169 after analysis involving 47 nucleotide sequences, with a total of 351 positions. Groups A-F were defined as previously reported [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198727#pone.0198727.ref023" target="_blank">23</a>]. The tree was inferred based on the Tamura 92 model with a Gamma distribution and invariable sites (I). Bootstrap values (after 10,000 replicates) above 70% are shown. Sites containing gaps and missing data were excluded from the analysis. L.b. = <i>L</i>. <i>braziliensis</i>; L.g. = <i>L</i>. <i>guyanensis</i>; L.s. = <i>L</i>. <i>shawi</i>. For details of samples see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198727#pone.0198727.t001" target="_blank">Table 1</a>.</p

Neighbor-net constructed on SplitsTree software employing the chord distance values among the <i>Leishmania infantum</i> genotypes.

<p>Identical genotypes for the 14 microsatellite markers were grouped and are represented by “TYPEs” (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036242#pone.0036242.s003" target="_blank">Table S1</a>). The distribution of the splits shows the same populations that were determined by the STRUCTURE analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036242#pone-0036242-g002" target="_blank">Figure 2</a>); some genotypes from POP2 are closer to POP1, and others are closer to POP3.</p

<i>Leishmania infantum</i> populations inferred from STRUCTURE analysis based on profiles of 14 microsatellites.

<p>The barplot was generated in Excel using the results of the aligned distribution of Q values from 10 for <i>K</i> = 3, which was generated using CLUMPP software. Evanno's method predicted that three was the most likely number of populations (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036242#pone.0036242.s001" target="_blank">Figure S1</a>). The POP1 (n = 111) is composed of strains from all of the foci (with the exception of PA), and most of the strains contain traces from POP2. POP2 (n = 31) includes strains from 10 states, but it is predominantly observed in MT. POP3 (n = 31) is composed primarily of MS strains and of four strains from other states of Northeast Brazil. Abbreviations: AM, Amazonas; BA, Bahia; CE, Ceará; DF, Distrito Federal; ES, Espírito Santo; MA, Maranhão; MG, Minas Gerais; MT, Mato Grosso; MS, Mato Grosso do Sul; PA, Pará; PE, Pernambuco; PI, Piauí; RJ, Rio de Janeiro; RN, Rio Grande do Norte; RS, Rio Grande do Sul; SE, Sergipe; SP, São Paulo; PY, Paraguay.</p

The distribution of MLMT genotypes shared among <i>Leishmania infantum</i> strains by hosts and geographic origins.

<p>POP1, ‘TYPE1’ to ‘TYPE14’; POP2, ‘TYPE15’ to ‘TYPE19’; POP3, ‘TYPE20’ to ‘TYPE22’. Brazilian States: AM, Amazonas; BA, Bahia; CE, Ceará; DF, Distrito Federal; ES, Espírito Santo; MA, Maranhão; MG, Minas Gerais; MT, Mato Grosso; MS, Mato Grosso do Sul; PA, Pará; PE, Pernambuco; PI, Piauí; RJ, Rio de Janeiro; RN, Rio Grande do Norte; RS, Rio Grande do Sul; SE, Sergipe; SP, São Paulo. Paraguay: ASU, Asunción.</p><p>The number of strains per state for each genotype is shown in brackets.</p><p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036242#pone.0036242.s003" target="_blank">Table S1</a> for more details.</p

Factorial correspondence analysis (FCA) of the <i>Leishmania infantum</i> genotypes.

<p>Spacial distribution of strains after FCA of microsatellite genotypes. Blue dots, POP1; yellow dots, POP2; grey dots, POP3.</p

The geographic origin of <i>Leishmania infantum</i> strains and populations of STRUCTURE analysis.

<p>The yellow dots represent the locations of the collections of each analyzed strain. The graphics indicate the proportion numbers of the strains (Y axis) in each population (X axis). The assignment of the strains to a population was performed in the STRUCTURE analysis that was based on the profiles of 14 microsatellite markers. POP1 is a widespread population, and it is predominant in most of the foci. POP2 and POP3 are predominant in Central West Brazil where <i>Lutzomyia longipalpis</i> and <i>Lutzomyia cruzi</i> are involved in the transmission cycle of Visceral Leishmaniasis. The abbreviations for the Brazilian states are as follows (in bold): AM, Amazonas; BA, Bahia; CE, Ceará; DF, Distrito Federal; ES, Espírito Santo; MA, Maranhão; MG, Minas Gerais; MT, Mato Grosso; MS, Mato Grosso do Sul; PA, Pará; PE, Pernambuco; PI, Piauí; RJ, Rio de Janeiro; RN, Rio Grande do Norte; RS, Rio Grande do Sul; SE, Sergipe; SP, São Paulo. International country codes: AR, Argentina; BO, Bolivia; CL, Chile; CO, Colômbia; GF, French Guiana; GY, Guyana; PY, Paraguay; PE, Peru; SR, Suriname; UR, Uruguay; VE, Venezuela. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036242#pone.0036242.s003" target="_blank">Table S1</a> for more details.</p