Leishmania RNA Virus Research Articles

Detection of Leishmania RNA virus 2 in Leishmania species from Turkey.

Leishmania RNA virus (LRV) is a double-stranded RNA (dsRNA) virus infecting some Leishmania strains and triggering a destructive hyperinflammatory response in mammalian hosts in the New World. There is limited knowledge of the presence of this virus in Old World Leishmania species and its role in the outcome of the disease. We aimed to investigate the presence of LRV in Leishmania species/strains from Turkey. Twenty-nine previously identified Leishmania isolates (24 L. tropica, 2 L. infantum, 3 L. major) were examined for LRV positivity using dsRNA visualization in agarose gel after total nucleic acid extraction and RQ-deoxyribonuclease treatment and amplification of a 526 bp fragment of the LRV2-specific RNA-dependent RNA polymerase gene by reverse transcription polymerase chain reaction. Ten (7 L. tropica [24.13%], 3 L. major [10.34%]) of the 29 Leishmania strains gave positive results for LRV. Basic Local Alignment Search Tool analysis showed that all these viruses are LRV2-1. LRV2 was detected for the first time in L. tropica strains in the present study. The clinical manifestation and resistance status of the disease can be different depending on the host and parasite species/strains. The presence of LRV2 may be one of the factors contributing the course of disease. Further studies are needed to elucidate the specific role of LRV2, as it may be a potential target for effective treatment strategies.

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

Leishmania RNA virus (LRV) was first detected in members of the subgenus Leishmania (Viannia), and later, the virulence and metastasis of the New World species were attributed to this virus. The data on the presence of LRV in Old World species are confined to Leishmania major and a few Leishmania aethiopica isolates. The aim of this study was to survey the presence of LRV in various Iranian Leishmania species originating from patients and animal reservoir hosts. Genomic nucleic acids were extracted from 50 cultured isolates belonging to the species Leishmania major, Leishmania tropica, and Leishmania infantum. A partial sequence of the viral RNA-dependent RNA polymerase (RdRp) gene was amplified, sequenced and compared with appropriate sequences from the GenBank database. We detected the virus in two parasite specimens: an isolate of L. infantum derived from a visceral leishmaniasis (VL) patient who was unresponsive to meglumine antimoniate treatment, and an L. major isolate originating from a great gerbil, Rhombomys opimus. The Iranian LRV sequences showed the highest similarities to an Old World L. major LRV2 and were genetically distant from LRV1 isolates detected in New World Leishmania parasites. We could not attribute treatment failure in VL patient to the presence of LRV due to the limited number of specimens analyzed. Further studies with inclusion of more clinical samples are required to elucidate the potential role of LRVs in pathogenesis or treatment failure of Old World leishmaniasis.

An improved purification procedure for Leishmania RNA virus (LRV).

Leishmania RNA Virus (LRV, Totiviridae) infect Leishmania cells and subvert mice immune response, probably promoting parasite persistence, suggesting significant roles for LRV in host-parasite interaction. Here we describe a new LRV1-4 purification protocol, enabling capsid visualization by negatively stained electron microscopy representing a significant contribution to future LRV investigations.

MyD88 and TLR9 dependent immune responses mediate resistance to Leishmania guyanensis infections, irrespective of Leishmania RNA virus burden.

Infections with Leishmania parasites of the Leishmania Viannia subgenus give rise to both localized cutaneous (CL), and metastatic leishmaniasis. Metastasizing disease forms including disseminated (DCL) and mutocutaneous (MCL) leishmaniasis result from parasitic dissemination and lesion formation at sites distal to infection and have increased inflammatory responses. The presence of Leishmania RNA virus (LRV) in L. guyanensis parasites contributes to the exacerbation of disease and impacts inflammatory responses via activation of TLR3 by the viral dsRNA. In this study we investigated other innate immune response adaptor protein modulators and demonstrated that both MyD88 and TLR9 played a crucial role in the development of Th1-dependent healing responses against L. guyanensis parasites regardless of their LRV status. The absence of MyD88- or TLR9-dependent signaling pathways resulted in increased Th2 associated cytokines (IL-4 and IL-13), which was correlated with low transcript levels of IL-12p40. The reliance of IL-12 was further confirmed in IL12AB−/− mice, which were completely susceptible to infection. Protection to L. guyanensis infection driven by MyD88- and TLR9-dependent immune responses arises independently to those induced due to high LRV burden within the parasites.

Leishmania aethiopica field isolates bearing an endosymbiontic dsRNA virus induce pro-inflammatory cytokine response.

BackgroundInfection with Leishmania parasites causes mainly cutaneous lesions at the site of the sand fly bite. Inflammatory metastatic forms have been reported with Leishmania species such as L. braziliensis, guyanensis and aethiopica. Little is known about the factors underlying such exacerbated clinical presentations. Leishmania RNA virus (LRV) is mainly found within South American Leishmania braziliensis and guyanensis. In a mouse model of L. guyanensis infection, its presence is responsible for an hyper-inflammatory response driven by the recognition of the viral dsRNA genome by the host Toll-like Receptor 3 leading to an exacerbation of the disease. In one instance, LRV was reported outside of South America, namely in the L. major ASKH strain from Turkmenistan, suggesting that LRV appeared before the divergence of Leishmania subgenera. LRV presence inside Leishmania parasites could be one of the factors implicated in disease severity, providing rationale for LRV screening in L. aethiopica.Methodology/Principal FindingsA new LRV member was identified in four L. aethiopica strains (LRV-Lae). Three LRV-Lae genomes were sequenced and compared to L. guyanensis LRV1 and L. major LRV2. LRV-Lae more closely resembled LRV2. Despite their similar genomic organization, a notable difference was observed in the region where the capsid protein and viral polymerase open reading frames overlap, with a unique −1 situation in LRV-Lae. In vitro infection of murine macrophages showed that LRV-Lae induced a TLR3-dependent inflammatory response as previously observed for LRV1.Conclusions/SignificanceIn this study, we report the presence of an immunogenic dsRNA virus in L. aethiopica human isolates. This is the first observation of LRV in Africa, and together with the unique description of LRV2 in Turkmenistan, it confirmed that LRV was present before the divergence of the L. (Leishmania) and (Viannia) subgenera. The potential implication of LRV-Lae on disease severity due to L. aethiopica infections is discussed.

Controversial role of leishmania RNA virus as a determinant of pathogenicity in human leishmaniasis

Controversial role of leishmania RNA virus as a determinant of pathogenicity in human leishmaniasis

Severity of tegumentary leishmaniasis is not exclusively associated with Leishmania RNA virus 1 infection in Brazil

Leishmania RNA virus (LRV) has been shown to be a symbiotic component of Leishmania parasites in South America. Nested retro-transcription polymerase chain reaction was employed to investigate LRV1 presence in leishmaniasis lesions from Brazil. In endemic areas of Rio de Janeiro (RJ), no LRV1 infection was observed even with mucosal involvement. LRV1 was only detected in Leishmania (V.) guyanensis cutaneous lesions from the northern region, which were obtained from patients presenting with disease reactivation after clinical cure of their primary lesions. Our results indicated that the severity of leishmaniasis in some areas of RJ, where Leishmania (V.) brazi-liensis is the primary etiological agent, was not associated with Leishmania LRV1 infection.

Detection of Leishmania RNA Virus in Leishmania Parasites

BackgroundPatients suffering from cutaneous leishmaniasis (CL) caused by New World Leishmania (Viannia) species are at high risk of developing mucosal (ML) or disseminated cutaneous leishmaniasis (DCL). After the formation of a primary skin lesion at the site of the bite by a Leishmania-infected sand fly, the infection can disseminate to form secondary lesions. This metastatic phenotype causes significant morbidity and is often associated with a hyper-inflammatory immune response leading to the destruction of nasopharyngeal tissues in ML, and appearance of nodules or numerous ulcerated skin lesions in DCL. Recently, we connected this aggressive phenotype to the presence of Leishmania RNA virus (LRV) in strains of L. guyanensis, showing that LRV is responsible for elevated parasitaemia, destructive hyper-inflammation and an overall exacerbation of the disease. Further studies of this relationship and the distribution of LRVs in other Leishmania strains and species would benefit from improved methods of viral detection and quantitation, especially ones not dependent on prior knowledge of the viral sequence as LRVs show significant evolutionary divergence.Methodology/Principal FindingsThis study reports various techniques, among which, the use of an anti-dsRNA monoclonal antibody (J2) stands out for its specific and quantitative recognition of dsRNA in a sequence-independent fashion. Applications of J2 include immunofluorescence, ELISA and dot blot: techniques complementing an arsenal of other detection tools, such as nucleic acid purification and quantitative real-time-PCR. We evaluate each method as well as demonstrate a successful LRV detection by the J2 antibody in several parasite strains, a freshly isolated patient sample and lesion biopsies of infected mice.Conclusions/SignificanceWe propose that refinements of these methods could be transferred to the field for use as a diagnostic tool in detecting the presence of LRV, and potentially assessing the LRV-related risk of complications in cutaneous leishmaniasis.

O013 Metastatic leishmaniasis—An IL-17 mediated response to Leishmania-virus

Introduction As veterans of infection, Leishmania guyanensis parasites have been plaguing humankind for centuries, provoking a deleterious hyper-inflammatory response, destroying host tissue and forming the ulcerating lesions, which typify most forms of the disease. About 15% of patients develop secondary lesions in the mouth and nose, where parasites metastasise to mucocutaneous tissues creating corrosive and exceptionally disfiguring inflammation. Our lab has recently linked disease severity in these infections to a virus naturally residing within the cytoplasm of some leishmania parasites. Here, Leishmania RNA virus (LRV) can act as an independently immunogenic entity, where its RNA-based nucleic acid acts as a potent innate immunogen, triggering a destructive hyper-inflammatory cascade through Toll-Like-Receptor 3 recognition [1] . The immune effectors underlying the devolution of stubborn and metastatic leishmaniasis of have largely focused on the Th1/Th2 (healing/non-healing) dogma: a paradigm, which neglects the role and interactions of other T-cell subsets, such as Th17. Methods Using Leishmania guyanensis clones, which are either naturally infected by LRV (V + ) or depleted in it (V − ), we set out to characterise the perpetrators of this chronic hyper-inflammation in a murine model of infection. These candidates were then used as targets for immunomodulatory intervention, with the aim of reaching a therapeutic end. Results Here, we found that V + parasites potently induced the production of IL-17A as compared to their V − equivalents, thus insinuating that this pro-inflammatory cytokine plays a destructive role in the devolution and severity of metastatic leishmaniasis. Indeed, IL-17A −/− mice infected with V + parasites have a significantly reduced disease severity and parasite burden compared to wild type controls. Further, in vitro studies revealed that IL-17A directly increases the parasitism of macrophages while simultaneously decreasing their ability to clear the infection. SR-1001: a recently described drug inhibiting the differentiation of Th17 cells, has been used in a pioneering study that inhibited the development of destructive inflammation in multiple sclerosis, we propose this immunomodulator (and its cheaper equivalent Digoxin), for trials in the treatment of complicated metastatic leishmaniasis. Conclusion Although IL-17 has a promiscuous role in immunity, stimulating both the innate and adaptive immune systems (as well as a plethora of non-immune cells), a major shared goal of these pathways is their self-propagation, where down-stream effects converge to create a hyper-inflammatory feedback loop. Indeed, the Th17 T-cell population has been thoroughly vilified as the architect of many chronic and destructive inflammatory processes. Determining the role of IL-17 in LRV-based virulence is essential to our understanding of its pathogenesis and would stand to guide and justify a much-needed immunotherapeutic revolution in the treatment of complicated leishmaniases.