PMC9870268_01
lesion appearance and parasitic classification of the isolated and cultivated Leishmania strain. A) Image of cutaneous lesion and nearby area, noticing the lymph node involvement. B) Neighbour-joining tree based on genetic distances (p-distance) between each pair of sequence for the leishmanial hsp70 region. Reference strains were obtained from publicly available leishmanial sequences and compared to the positive sample, marked with a black arrow (GenBank  accession ON075820).
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On the right side of the neck the patient had a 5 cm (Fig. 1A), 3 months old, deep ulcer, with pruritic and sanguine-purulent tissue. The obtained sequence (GenBank  accession ON075820) was aligned with 32 other Leishmania hsp70 sequences retrieved from GenBank and clearly clustered with L. (V.) guyanensis sequences (Fig. 1B).
RT-PCR products, genetic distance and colour-coded geographic grouping between Leishmania RNA virus (LRV) sequences. A) RT-PCR fragment of the orf 1 and 2 regions (850 bp) lane 1: size marker, 100-1,000 base pairs (bp); 2: Leishmania (Viannia) guyanensis M4147; 3: negative control; 4: sample #108-19. In the size ladder, the more intense bands correspond to 1,000 bp and 500 bp, from top to bottom. B) Neighbour-joining tree based on genetic distances (p-distance) between each pair of sequences, for the orf 1 and 2 fragments of LRV. Corresponding Leishmania species can be seen to the right of the tree. Reference strains were obtained from publicly available LRV1 and two sequences and compared to the positive sample (GenBank  accession OM140825). Colour-coded dots correspond to Fig. 1C colour code. C) Grouping of samples by geographic location, either country of origin or state of origin in the case of Brazilian samples. The colour code can be seen in the bottom right corner.
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One typical RT-PCR product gel is shown in Fig. 2, illustrating the confirmation of one, out of the 24 strains, as positive for the endosymbiont. The amplified product chosen for sequencing was the longest one (corresponding to Fig. 2A), since this fragment displays approximately 850 base pairs (bp) corresponding to part of the orf1 region and the beginning of the orf2 region, including the portion responsible for encoding the viral capsid protein.  Assessing the relationship between sample and reference strains we produced a tree (Fig. 2B) that shows two distinct groups separating the main Leishmania (Viannia) species harbouring virus: L. (V.) guyanensis and L. (V.) braziliensis. The #108-19 LRV1 sequence grouped with other LRV1 sequences from the Northeast of the Brazilian Amazon region and from the French Guiana (Fig. 2C).