A Novel Virus Alters Gene Expression and Vacuolar Morphology in Malassezia Cells and Induces a TLR3-Mediated Inflammatory Immune Response.

Most fungal viruses have been identified in plant pathogens, whereas the presence of viral particles in human-pathogenic fungi is less well studied. In the present study, we observed extrachromosomal double-stranded RNA (dsRNA) segments in various clinical isolates of Malassezia species. Malassezia is the most dominant fungal genus on the human skin surface, and species in this group are considered etiological factors of various skin diseases including dandruff, seborrheic dermatitis, and atopic dermatitis. We identified novel dsRNA segments, and our sequencing results revealed that the virus, named MrV40, belongs to the Totiviridae family and contains an additional satellite dsRNA segment encoding a novel protein. The transcriptome of virus-infected Malassezia restricta cells was compared to that of virus-cured cells, and the results showed that transcripts involved in ribosomal biosynthesis were downregulated and those involved in energy production and programmed cell death were upregulated. Moreover, transmission electron microscopy revealed significantly larger vacuoles in virus-infected M. restricta cells, indicating that MrV40 infection dramatically altered M. restricta physiology. Our analysis also revealed that viral nucleic acid from MrV40 induced a TLR3 (Toll-like receptor 3)-mediated inflammatory immune response in bone marrow-derived dendritic cells, suggesting that a viral element contributes to the pathogenicity of MalasseziaIMPORTANCEMalassezia is the most dominant fungal genus on the human skin surface and is associated with various skin diseases including dandruff and seborrheic dermatitis. Among Malassezia species, Malassezia restricta is the most widely observed species on the human skin. In the current study, we identified a novel dsRNA virus, named MrV40, in M. restricta and characterized the sequence and structure of the viral genome along with an independent satellite dsRNA viral segment. Moreover, expression of genes involved in ribosomal synthesis and programmed cell death was altered, indicating that virus infection affected the physiology of the fungal host cells. Our data also showed that the viral nucleic acid from MrV40 induces a TLR3-mediated inflammatory immune response in bone marrow-derived dendritic cells, indicating that a viral element likely contributes to the pathogenicity of Malassezia This is the first study to identify and characterize a novel mycovirus in Malassezia.

Individual monitoring of immune response in Atlantic salmon Salmo salar following experimental infection with piscine myocarditis virus (PMCV), agent of cardiomyopathy syndrome (CMS).

Piscine myocarditis virus (PCMV) is a double-stranded RNA virus structurally similar to the Totiviridae family. PCMV is the causative agent of cardiomyopathy syndrome (CMS), a severe cardiac disease that affects farmed Atlantic salmon (Salmo salar). A recent study characterized the host immune response in infected salmon through a transcriptome immune profiling, which confirmed a high regulation of immune and anti-viral genes throughout infection with PCMV. Previously we developed a novel model based on repeated non-lethal blood sampling, enabling the individual monitoring of salmonids during an infection. In the present work, we used this model to describe the host immune response in the blood cells of Atlantic salmon after intramuscular infection with PCMV-containing tissue homogenate over a 77-day period. At the final stage heart samples were also collected to verify the PCMV load, the pathological impact of infection and to compare the transcript profiles to blood. The expression level of a range of key immune genes was determined in the blood and heart samples by real-time PCR. Results indicated selected immune genes (mx, cd8α and γip) were up-regulated in the heart tissue of infected animals at the terminal time point, in comparison to the non-infected fish. When analyzing the blood samples over the course of infection, a significant n up-regulation of mx gene was also observed. The time and number of peaks in the kinetics of expression was different between individuals. The PCMV load and CMS pathology was verified by real-time PCR and histopathology, respectively. No pathogen and no pathology could be detected during the course of the experiment except at the terminal stage (viral load by qPCR and pathology by histology). This study emphasizes the value of non-lethal monitoring for evaluating the health status of fish at early stages of infection and in the absence of clinical signs.

Characterization of myocardial lesions associated with cardiomyopathy syndrome in Atlantic salmon, Salmo salar L., using laser capture microdissection.

Cardiomyopathy syndrome (CMS) in Atlantic salmon, Salmo salar L., is characterized by focal infiltration in the spongy myocardium and endocardium of the heart. The origin of the mononuclear infiltrate is unknown. Using experimentally infected fish, we investigated localization of the causative agent, piscine myocarditis virus (PMCV), within the heart and characterized the cell population associated with myocardial lesions. Cellular and transcriptional characteristics in the lesions were compared with adjacent non-infiltrated tissues using laser capture microdissection, RT-qPCR and immunohistochemistry. Our results reveal that PMCV is almost exclusively present in myocardial lesions. The inflammatory infiltrate comprises a variety of leucocyte populations, including T cells, B cells, MHC class II(+) and CD83(+) cells, most likely of the macrophage line. Correlation analyses demonstrated co-ordinated leucocyte activity at the site of the virus infection. Cellular proliferation and/or DNA repair was demonstrated within the myocardial lesions. Different cell populations, mainly myocytes, stained positive for proliferating cell nuclear antigen (PCNA). Densities of endothelial cells and fibroblasts were not significantly increased. The simultaneous presence of PMCV and various inflammatory cells in all myocardial lesions analysed may indicate that both viral lytic and immunopathological effects may contribute to the pathogenesis of CMS.

Leishmania RNA virus: when the host pays the toll.

The presence of an RNA virus in a South American subgenus of the Leishmania parasite, L. (Viannia), was detected several decades ago but its role in leishmanial virulence and metastasis was only recently described. In Leishmania guyanensis, the nucleic acid of Leishmania RNA virus (LRV1) acts as a potent innate immunogen, eliciting a hyper-inflammatory immune response through toll-like receptor 3 (TLR3). The resultant inflammatory cascade has been shown to increase disease severity, parasite persistence, and perhaps even resistance to anti-leishmanial drugs. Curiously, LRVs were found mostly in clinical isolates prone to infectious metastasis in both their human source and experimental animal model, suggesting an association between the viral hyperpathogen and metastatic complications such as mucocutaneous leishmaniasis (MCL). MCL presents as chronic secondary lesions in the mucosa of the mouth and nose, debilitatingly inflamed and notoriously refractory to treatment. Immunologically, this outcome has many of the same hallmarks associated with the reaction to LRV: production of type 1 interferons, bias toward a chronic Th1 inflammatory state and an impaired ability of host cells to eliminate parasites through oxidative stress. More intriguing, is that the risk of developing MCL is found almost exclusively in infections of the L. (Viannia) subtype, further indication that leishmanial metastasis is caused, at least in part, by a parasitic component. LRV present in this subgenus may contribute to the destructive inflammation of metastatic disease either by acting in concert with other intrinsic "metastatic factors" or by independently preying on host TLR3 hypersensitivity. Because LRV amplifies parasite virulence, its presence may provide a unique target for diagnostic and clinical intervention of metastatic leishmaniasis. Taking examples from other members of the Totiviridae virus family, this paper reviews the benefits and costs of endosymbiosis, specifically for the maintenance of LRV infection in Leishmania parasites, which is often at the expense of its human host.

Muco-cutaneous leishmaniasis in the New World: the ultimate subversion.

Infection by the human protozoan parasite Leishmania can lead, depending primarily on the parasite species, to either cutaneous or mucocutaneous lesions, or fatal generalized visceral infection. In the New World, Leishmania (Viannia) species can cause mucocutaneous leishmaniasis (MCL). Clinical MCL involves a strong hyper-inflammatory response and parasitic dissemination (metastasis) from a primary lesion to distant sites, leading to destructive metastatic secondary lesions especially in the nasopharyngal areas. Recently, we reported that metastasizing, but not non-metastatic strains of Leishmania (Viannia) guyanensis, have high burden of a non-segmented dsRNA virus, Leishmania RNA Virus (LRV). Viral dsRNA is sensed by the host Toll-like Receptor 3 (TLR3) thereby inducing a pro-inflammatory response and exacerbating the disease. The presence of LRV in Leishmania opens new perspectives not only in basic understanding of the intimate relation between the parasite and LRV, but also in understanding the importance of the inflammatory response in MCL patients.