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Unlabelled: Early biochemical studies of viral replication suggested that most viruses produce double-stranded RNA (dsRNA), which is essential for the induction of the host immune response. However, it was reported in 2006 that dsRNA could be detected by immunofluorescence antibody staining in double-stranded DNA and positive-strand RNA virus infections but not in negative-strand RNA virus infections. Other reports in the literature seemed to support these observations. This suggested that negative-strand RNA viruses produce little, if any, dsRNA or that more efficient viral countermeasures to mask dsRNA are mounted. Because of our interest in the use of dsRNA antibodies for virus discovery, particularly in pathological specimens, we wanted to determine how universal immunostaining for dsRNA might be in animal virus infections. We have detected the in situ formation of dsRNA in cells infected with vesicular stomatitis virus, measles virus, influenza A virus, and Nyamanini virus, which represent viruses from different negative-strand RNA virus families. dsRNA was also detected in cells infected with lymphocytic choriomeningitis virus, an ambisense RNA virus, and minute virus of mice (MVM), a single-stranded DNA (ssDNA) parvovirus, but not hepatitis B virus. Although dsRNA staining was primarily observed in the cytoplasm, it was also seen in the nucleus of cells infected with influenza A virus, Nyamanini virus, and MVM. Thus, it is likely that most animal virus infections produce dsRNA species that can be detected by immunofluorescence staining. The apoptosis induced in several uninfected cell lines failed to upregulate dsRNA formation., Importance: An effective antiviral host immune response depends on recognition of viral invasion and an intact innate immune system as a first line of defense. Double-stranded RNA (dsRNA) is a viral product essential for the induction of innate immunity, leading to the production of type I interferons (IFNs) and the activation of hundreds of IFN-stimulated genes. The present study demonstrates that infections, including those by ssDNA viruses and positive- and negative-strand RNA viruses, produce dsRNAs detectable by standard immunofluorescence staining. While dsRNA staining was primarily observed in the cytoplasm, nuclear staining was also present in some RNA and DNA virus infections. The nucleus is unlikely to have pathogen-associated molecular pattern (PAMP) receptors for dsRNA because of the presence of host dsRNA molecules. Thus, it is likely that most animal virus infections produce dsRNA species detectable by immunofluorescence staining, which may prove useful in viral discovery as well., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Tilapines are important for the sustainability of ecological systems and serve as the second most important group of farmed fish worldwide. Significant mortality of wild and cultured tilapia has been observed recently in Israel. The etiological agent of this disease, a novel RNA virus, is described here, and procedures allowing its isolation and detection are revealed. The virus, denominated tilapia lake virus (TiLV), was propagated in primary tilapia brain cells or in an E-11 cell line, and it induced a cytopathic effect at 5 to 10 days postinfection. Electron microscopy revealed enveloped icosahedral particles of 55 to 75 nm. Low-passage TiLV, injected intraperitoneally in tilapia, induced a disease resembling the natural disease, which typically presents with lethargy, ocular alterations, and skin erosions, with >80% mortality. Histological changes included congestion of the internal organs (kidneys and brain) with foci of gliosis and perivascular cuffing of lymphocytes in the brain cortex; ocular inflammation included endophthalmitis and cataractous changes of the lens. The cohabitation of healthy and diseased fish demonstrated that the disease is contagious and that mortalities (80 to 100%) occur within a few days. Fish surviving the initial mortality were immune to further TiLV infections, suggesting the mounting of a protective immune response. Screening cDNA libraries identified a TiLV-specific sequence, allowing the design of a PCR-based diagnostic test. This test enables the specific identification of TiLV in tilapines and should help control the spread of this virus worldwide., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Viruses with positive-strand RNA genomes amplify their genomes in replication complexes associated with cellular membranes. Little is known about the mechanism of replication complex formation in cells infected with Nodamura virus. This virus is unique in its ability to lethally infect both mammals and insects. In mice and in larvae of the greater wax moth (Galleria mellonella), Nodamura virus-infected muscle cells exhibit mitochondrial aggregation and membrane rearrangement, leading to disorganization of the muscle fibrils on the tissue level and ultimately in hind limb/segment paralysis. However, the molecular basis for this pathogenesis and the role of mitochondria in Nodamura virus infection remains unclear. Here, we tested the hypothesis that Nodamura virus establishes RNA replication complexes that associate with mitochondria in mammalian cells. Our results showed that Nodamura virus replication complexes are targeted to mitochondria, as evidenced in biochemical, molecular, and confocal microscopy studies. More specifically, we show that the Nodamura virus RNA-dependent RNA polymerase interacts with the outer mitochondrial membranes as an integral membrane protein and ultimately becomes associated with functional replication complexes. These studies will help us to understand the mechanism of replication complex formation and the pathogenesis of Nodamura virus for mammals., Importance: This study will further our understanding of Nodamura virus (NoV) genome replication and its pathogenesis for mice. NoV is unique among the Nodaviridae in its ability to infect mammals. Here we show that NoV establishes RNA replication complexes (RCs) in association with mitochondria in mammalian cells. These RCs contain newly synthesized viral RNA and feature a physical interaction between mitochondrial membranes and the viral RNA-dependent RNA polymerase (RdRp), which is mediated by two membrane-associated regions. While the nature of the interaction needs to be explored further, it appears to occur by a mode distinct from that described for the insect nodavirus Flock House virus (FHV). The interaction of the NoV RdRp with mitochondrial membranes is essential for clustering of mitochondria into networks that resemble those described for infected mouse muscle and that are associated with fatal hind limb paralysis. This work therefore provides the first link between NoV RNA replication complex formation and the pathogenesis of this virus for mice.

Cardiomyopathy syndrome (CMS) of farmed and wild Atlantic salmon (Salmo salar L.) is a disease of yet unknown etiology characterized by a necrotizing myocarditis involving the atrium and the spongious part of the heart ventricle. Here, we report the identification of a double-stranded RNA virus likely belonging to the family Totiviridae as the causative agent of the disease. The proposed name of the virus is piscine myocarditis virus (PMCV). On the basis of the RNA-dependent RNA polymerase (RdRp) sequence, PMCV grouped with Giardia lamblia virus and infectious myonecrosis virus of penaeid shrimp. The genome size of PMCV is 6,688 bp, with three open reading frames (ORFs). ORF1 likely encodes the major capsid protein, while ORF2 encodes the RdRp, possibly expressed as a fusion protein with the ORF1 product. ORF3 seems to be translated as a separate protein not described for any previous members of the family Totiviridae. Following experimental challenge with cell culture-grown virus, histopathological changes are observed in heart tissue by 6 weeks postchallenge (p.c.), with peak severity by 9 weeks p.c. Viral genome levels detected by real-time reverse transcription (RT)-PCR peak earlier at 6 to 7 weeks p.c. The virus genome is detected by in situ hybridization in degenerate cardiomyocytes from clinical cases of CMS. Virus genome levels in the hearts from clinical field cases correlate well with the severity of histopathological changes in heart tissue. The identification of the causative agent for CMS is important for improved disease surveillance and disease control and will serve as a basis for vaccine development against the disease.

Cucumber mosaic virus (CMV) systemically infects both tobacco and zucchini squash. CMV capsid protein loop mutants with single-amino-acid substitutions are unable to systemically infect squash, but they revert to a wild-type phenotype in the presence of an additional, specific single-site substitution. The D118A, T120A, D192A, and D197A loop mutants reverted to a wild-type phenotype but did so in combination with P56S, P77L, A162V, and I53F or T124I mutations, respectively. The possible effect of these compensatory mutations on other, nonsystemically infecting loop mutants was tested with the F117A mutant and found to be neutral, thus indicating a specificity to the observed changes.