Recognition of RNA virus by RIG-I results in activation of CARD9 and inflammasome signaling for interleukin 1 beta production.

Interleukin 1 beta (IL-1 beta) is a potent proinflammatory factor during viral infection. Its production is tightly controlled by transcription of Il1b dependent on the transcription factor NF-kappaB and subsequent processing of pro-IL-1 beta by an inflammasome. However, the sensors and mechanisms that facilitate RNA virus-induced production of IL-1 beta are not well defined. Here we report a dual role for the RNA helicase RIG-I in RNA virus-induced proinflammatory responses. Whereas RIG-I-mediated activation of NF-kappaB required the signaling adaptor MAVS and a complex of the adaptors CARD9 and Bcl-10, RIG-I also bound to the adaptor ASC to trigger caspase-1-dependent inflammasome activation by a mechanism independent of MAVS, CARD9 and the Nod-like receptor protein NLRP3. Our results identify the CARD9-Bcl-10 module as an essential component of the RIG-I-dependent proinflammatory response and establish RIG-I as a sensor able to activate the inflammasome in response to certain RNA viruses.

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.

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.

Leishmania RNA virus controls the severity of mucocutaneous leishmaniasis.

Mucocutaneous leishmaniasis is caused by infections with intracellular parasites of the Leishmania Viannia subgenus, including Leishmania guyanensis. The pathology develops after parasite dissemination to nasopharyngeal tissues, where destructive metastatic lesions form with chronic inflammation. Currently, the mechanisms involved in lesion development are poorly understood. Here we show that metastasizing parasites have a high Leishmania RNA virus-1 (LRV1) burden that is recognized by the host Toll-like receptor 3 (TLR3) to induce proinflammatory cytokines and chemokines. Paradoxically, these TLR3-mediated immune responses rendered mice more susceptible to infection, and the animals developed an increased footpad swelling and parasitemia. Thus, LRV1 in the metastasizing parasites subverted the host immune response to Leishmania and promoted parasite persistence.

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.

Detection of Leishmania RNA virus in Leishmania parasites.

BACKGROUND: Patients 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 FINDINGS: This 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/SIGNIFICANCE: We 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.

Prevalence and Distribution of Leishmania RNA Virus 1 in Leishmania Parasites from French Guiana.

In South America, the presence of the Leishmania RNA virus type 1 (LRV1) was described in Leishmania guyanensis and Leishmania braziliensis strains. The aim of this study was to determine the prevalence distribution of LRV1 in Leishmania isolates in French Guiana given that, in this French overseas department, most Leishmania infections are due to these parasite species. The presence of the virus was observed in 74% of Leishmania spp. isolates, with a highest presence in the internal areas of the country.

Presence of Leishmania RNA Virus 1 in Leishmania guyanensis Increases the Risk of First-Line Treatment Failure and Symptomatic Relapse.

Treatment failure and symptomatic relapse are major concerns in American tegumentary leishmaniasis (TL). Such complications are seen frequently in Leishmania guyanensis infections, in which patients respond variously to first-line antileishmanials and are more prone to develop chronic cutaneous leishmaniasis. The factors underlying this pathology, however, are unknown. Recently, we reported that a double-stranded RNA virus, Leishmania RNA virus 1 (LRV1), nested within L. guyanensis parasites is able to exacerbate experimental murine leishmaniasis by inducing a hyperinflammatory response. This report investigates the prevalence of LRV1 in human L. guyanensis infection and its effect on treatment efficacy, as well as its correlation to symptomatic relapses after the completion of first-line treatment. In our cohort of 75 patients with a diagnosis of primary localized American TL, the prevalence of LRV1-positive L. guyanensis infection was elevated to 58%. All patients infected with LRV1-negative L. guyanensis were cured after 1 dose (22 of 31 [71%]) or 2 doses (31 of 31 [100%]) of pentamidine. In contrast, 12 of 44 LRV1-positive patients (27%) presented with persistent infection and symptomatic relapse that required extended therapy and the use of second-line drugs. Finally, LRV1 presence was associated with a significant increase in levels of intra-lesional inflammatory markers. In conclusion, LRV1 status in L. guyanensis infection is significantly predictive (P = .0009) of first-line treatment failure and symptomatic relapse and has the potential to guide therapeutic choices in American TL.

How the double spherules of infectious bronchitis virus impact our understanding of RNA virus replicative organelles

Powered by advances in electron tomography, recent studies have extended our understanding of how viruses construct "replication factories" inside infected cells. Their function, however, remains an area of speculation with important implications for human health. It is clear from these studies that whatever their purpose, organelle structure is dynamic (M. Ulasli, M. H. Verheije, C. A. de Haan, and F. Reggiori, Cell. Microbiol. 12:844-861, 2010) and intricate (K. Knoops, M. Kikkert, S. H. Worm, J. C. Zevenhoven-Dobbe, Y. van der Meer, et al., PLOS Biol. 6:e226, 2008). But by concentrating on medically important viruses, these studies have failed to take advantage of the genetic variation inherent in a family of viruses that is as diverse as the archaea, bacteria, and eukaryotes combined (C. Lauber, J. J. Goeman, M. del Carmen Parquet, P. T. Nga, E. J. Snijder, et al., PLOS Pathog. 9:e1003500, 2013). In this climate, Maier et al. (H. J. Maier, P. C. Hawes, E. M. Cottam, J. Mantell, P. Verkade, et al., mBio 4:e00801-13, 2013) explored the replicative structures formed by an avian coronavirus that appears to have diverged at an early point in coronavirus evolution and shed light on controversial aspects of viral biology.

Identification of a bisegmented double-stranded RNA virus (picobirnavirus) in calf faeces

To determine the incidence of rotavirus infection among dairy herds in the State of Sdo Paulo, Brazil, 576 faecal samples obtained from calves aged 1-45 days with and without diarrhoea, reared on 63 dairy cattle farms, were analyzed. Polyacrylamide gel electrophoresis (PAGE) identified 28 samples positive for group A rotavirus, while four samples, two diarrhoeic and two non-diarrhoeic, showed a bisegmented genome with a typical picobirnavirus pattern. Electron microscopy revealed spherical virus particles with a diameter of 37 nm and without a defined surface structure. The present study is the first report of a bisegmented virus identified in cattle in Brazil. (C) 2003 Elsevier B.V. Ltd. All rights reserved.

Detection of Leishmania RNA virus in Leishmania parasites.

BACKGROUND Patients 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 FINDINGS This 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/SIGNIFICANCE We 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.

The Host Nonsense-Mediated mRNA Decay Pathway Restricts Mammalian RNA Virus Replication

In addition to classically defined immune mechanisms, cell-intrinsic processes can restrict virus infection and have shaped virus evolution. The details of this virus-host interaction are still emerging. Following a genome-wide siRNA screen for host factors affecting replication of Semliki Forest virus (SFV), a positive-strand RNA (+RNA) virus, we found that depletion of nonsense-mediated mRNA decay (NMD) pathway components Upf1, Smg5, and Smg7 led to increased levels of viral proteins and RNA and higher titers of released virus. The inhibitory effect of NMD was stronger when virus replication efficiency was impaired by mutations or deletions in the replicase proteins. Consequently, depletion of NMD components resulted in a more than 20-fold increase in production of these attenuated viruses. These findings indicate that a cellular mRNA quality control mechanism serves as an intrinsic barrier to the translation of early viral proteins and the amplification of +RNA viruses in animal cells.

Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome

The construction of cDNA clones encoding large-size RNA molecules of biological interest, like coronavirus genomes, which are among the largest mature RNA molecules known to biology, has been hampered by the instability of those cDNAs in bacteria. Herein, we show that the application of two strategies, cloning of the cDNAs into a bacterial artificial chromosome and nuclear expression of RNAs that are typically produced within the cytoplasm, is useful for the engineering of large RNA molecules. A cDNA encoding an infectious coronavirus RNA genome has been cloned as a bacterial artificial chromosome. The rescued coronavirus conserved all of the genetic markers introduced throughout the sequence and showed a standard mRNA pattern and the antigenic characteristics expected for the synthetic virus. The cDNA was transcribed within the nucleus, and the RNA translocated to the cytoplasm. Interestingly, the recovered virus had essentially the same sequence as the original one, and no splicing was observed. The cDNA was derived from an attenuated isolate that replicates exclusively in the respiratory tract of swine. During the engineering of the infectious cDNA, the spike gene of the virus was replaced by the spike gene of an enteric isolate. The synthetic virus replicated abundantly in the enteric tract and was fully virulent, demonstrating that the tropism and virulence of the recovered coronavirus can be modified. This demonstration opens up the possibility of employing this infectious cDNA as a vector for vaccine development in human, porcine, canine, and feline species susceptible to group 1 coronaviruses.

Rescue of a segmented negative-strand RNA virus entirely from cloned complementary DNAs

We provide the first report, to our knowledge, of a helper-independent system for rescuing a segmented, negative-strand RNA genome virus entirely from cloned cDNAs. Plasmids were constructed containing full-length cDNA copies of the three Bunyamwera bunyavirus RNA genome segments flanked by bacteriophage T7 promoter and hepatitis delta virus ribozyme sequences. When cells expressing both bacteriophage T7 RNA polymerase and recombinant Bunyamwera bunyavirus proteins were transfected with these plasmids, full-length antigenome RNAs were transcribed intracellularly, and these in turn were replicated and packaged into infectious bunyavirus particles. The resulting progeny virus contained specific genetic tags characteristic of the parental cDNA clones. Reassortant viruses containing two genome segments of Bunyamwera bunyavirus and one segment of Maguari bunyavirus were also produced following transfection of appropriate plasmids. This accomplishment will allow the full application of recombinant DNA technology to manipulate the bunyavirus genome.

Methionine-independent initiation of translation in the capsid protein of an insect RNA virus

Protein synthesis is believed to be initiated with the amino acid methionine because the AUG translation initiation codon of mRNAs is recognized by the anticodon of initiator methionine transfer RNA. A group of positive-stranded RNA viruses of insects, however, lacks an AUG translation initiation codon for their capsid protein gene, which is located at the downstream part of the genome. The capsid protein of one of these viruses, Plautia stali intestine virus, is synthesized by internal ribosome entry site-mediated translation. Here we report that methionine is not the initiating amino acid in the translation of the capsid protein in this virus. Its translation is initiated with glutamine encoded by a CAA codon that is the first codon of the capsid-coding region. The nucleotide sequence immediately upstream of the capsid-coding region interacts with a loop segment in the stem–loop structure located 15–43 nt upstream of the 5′ end of the capsid-coding region. The pseudoknot structure formed by this base pair interaction is essential for translation of the capsid protein. This mechanism for translation initiation differs from the conventional one in that the initiation step controlled by the initiator methionine transfer RNA is not necessary.