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Nature Communications Sep 2020Traditionally, molecular assembly pathways for viruses are inferred from high resolution structures of purified stable intermediates, low resolution images of cell...
Traditionally, molecular assembly pathways for viruses are inferred from high resolution structures of purified stable intermediates, low resolution images of cell sections and genetic approaches. Here, we directly visualise an unsuspected 'single shelled' intermediate for a mammalian orthoreovirus in cryo-preserved infected cells, by cryo-electron tomography of cellular lamellae. Particle classification and averaging yields structures to 5.6 Å resolution, sufficient to identify secondary structural elements and produce an atomic model of the intermediate, comprising 120 copies each of protein λ1 and σ2. This λ1 shell is 'collapsed' compared to the mature virions, with molecules pushed inwards at the icosahedral fivefolds by ~100 Å, reminiscent of the first assembly intermediate of certain prokaryotic dsRNA viruses. This supports the supposition that these viruses share a common ancestor, and suggests mechanisms for the assembly of viruses of the Reoviridae. Such methodology holds promise for dissecting the replication cycle of many viruses.
Topics: Animals; Capsid; Cell Line; Cryoelectron Microscopy; Electron Microscope Tomography; Orthoreovirus; Virion; Virus Assembly
PubMed: 32895380
DOI: 10.1038/s41467-020-18243-9 -
Science Advances May 2021Global expansion of aquaculture and agriculture facilitates disease emergence and catalyzes transmission to sympatric wildlife populations. The health of wild salmon...
Global expansion of aquaculture and agriculture facilitates disease emergence and catalyzes transmission to sympatric wildlife populations. The health of wild salmon stocks critically concerns Indigenous peoples, commercial and recreational fishers, and the general public. Despite potential impact of viral pathogens such as Piscine orthoreovirus-1 (PRV-1) on endangered wild salmon populations, their epidemiology in wild fish populations remains obscure, as does the role of aquaculture in global and local spread. Our phylogeographic analyses of PRV-1 suggest that development of Atlantic salmon aquaculture facilitated spread from Europe to the North and South East Pacific. Phylogenetic analysis and reverse transcription polymerase chain reaction surveillance further illuminate the circumstances of emergence of PRV-1 in the North East Pacific and provide strong evidence for Atlantic salmon aquaculture as a source of infection in wild Pacific salmon. PRV-1 is now an important infectious agent in critically endangered wild Pacific salmon populations, fueled by aquacultural transmission.
Topics: Animals; Aquaculture; Fish Diseases; Phylogeny; Reoviridae Infections; Salmo salar
PubMed: 34039598
DOI: 10.1126/sciadv.abe2592 -
Scientific Reports Jun 2021Mammalian orthoreovirus (MRV), a non-enveloped virus with a ten-segmented double-stranded RNA genome, infects virtually all mammals, including humans. Human infection...
Mammalian orthoreovirus (MRV), a non-enveloped virus with a ten-segmented double-stranded RNA genome, infects virtually all mammals, including humans. Human infection with MRV seems to be common in early childhood, but is rarely symptomatic. Despite the ubiquitous presence of MRV in mammals as well as in environmental waters, the molecular characterisation of the MRV genome remains to be fully elucidated. In this study, two novel strains, MRV-2 THK0325 and MRV-1 THK0617, were unintentionally isolated from wastewater in Japan via an environmental surveillance of enteric viruses. Homology and phylogenetic analysis demonstrated that all the segments of THK0325 were closely related to the MRV-2 Osaka strains, which were recently proposed to have existed for at least two decades in Japan. Most of the segments in THK0617 also showed a close relationship with the MRV-2 Osaka strains, but the M2, S1, and S3 segments belong to another MRV cluster. According to the S1 sequence, the determinant of serotype THK0617 was classified as MRV-1, and both the M2 and S3 segments were closely related to MRV-1 and -3 from the tree shrew in China. These results suggest that the MRV-2 Osaka-like strain spread widely throughout Japan, accompanied by intertypic reassortment occurring in East Asia.
Topics: Animals; China; Chiroptera; Feces; Humans; Orthoreovirus, Mammalian; Phylogeny; Reassortant Viruses; Serogroup; Swine; Swine Diseases; Wastewater
PubMed: 34131201
DOI: 10.1038/s41598-021-92019-z -
The Journal of General Virology Oct 2020Mammalian orthoreovirus (MRV) has been identified in humans, livestock and wild animals; this wide host range allows individual MRV to transmit into multiple species....
Mammalian orthoreovirus (MRV) has been identified in humans, livestock and wild animals; this wide host range allows individual MRV to transmit into multiple species. Although several interspecies transmission and genetic reassortment events of MRVs among humans, livestock and wildlife have been reported, the genetic diversity and geographic distribution of MRVs in Africa are poorly understood. In this study, we report the first isolation and characterization of MRVs circulating in a pig population in Zambia. In our screening, MRV genomes were detected in 19.7 % (29/147) of faecal samples collected from pigs by reverse transcription PCR. Three infectious MRV strains (MRV-85, MRV-96 and MRV-117) were successfully isolated, and their complete genomes were sequenced. Recombination analyses based on the complete genome sequences of the isolated MRVs demonstrated that MRV-96 shared the S3 segment with a different MRV isolated from bats, and that the L1 and M3 segments of MRV-117 originated from bat and human MRVs, respectively. Our results suggest that the isolated MRVs emerged through genetic reassortment events with interspecies transmission. Given the lack of information regarding MRVs in Africa, further surveillance of MRVs circulating among humans, domestic animals and wildlife is required to assess potential risk for humans and animals.
Topics: Animals; Animals, Wild; Chiroptera; Feces; Genome, Viral; Host Specificity; Orthoreovirus, Mammalian; Phylogeny; Prevalence; Reassortant Viruses; Recombination, Genetic; Reoviridae Infections; Swine; Swine Diseases; Viral Proteins; Whole Genome Sequencing; Zambia
PubMed: 32706330
DOI: 10.1099/jgv.0.001476 -
Viruses Feb 2021Mammalian orthoreoviruses (MRVs) are emerging infectious agents that may affect wild animals. MRVs are usually associated with asymptomatic or mild respiratory and...
Mammalian orthoreoviruses (MRVs) are emerging infectious agents that may affect wild animals. MRVs are usually associated with asymptomatic or mild respiratory and enteric infections. However, severe clinical manifestations have been occasionally reported in human and animal hosts. An insight into their circulation is essential to minimize the risk of diffusion to farmed animals and possibly to humans. The aim of this study was to investigate the presence of likely zoonotic MRVs in wild ungulates. Liver samples were collected from wild boar, red deer, roe deer, and chamois. Samples originated from two areas (Sondrio and Parma provinces) in Northern Italy with different environmental characteristics. MRV detection was carried out by PCR; confirmation by sequencing and typing for MRV type 3, which has been frequently associated with disease in pigs, were carried out for positive samples. MRV prevalence was as high as 45.3% in wild boars and 40.6% in red deer in the Sondrio area, with lower prevalence in the Parma area (15.4% in wild boars). Our findings shed light on MRV occurrence and distribution in some wild species and posed the issue of their possible role as reservoir.
Topics: Animals; Animals, Wild; Artiodactyla; Disease Reservoirs; Italy; Liver; Orthoreovirus, Mammalian; Prevalence; RNA, Viral; Serogroup
PubMed: 33546342
DOI: 10.3390/v13020238 -
Experimental Biology and Medicine... Sep 2019Primary liver cancer is a major public health challenge that ranks as the third most common cause of cancer worldwide despite therapeutic improvement. Reovirus has been...
UNLABELLED
Primary liver cancer is a major public health challenge that ranks as the third most common cause of cancer worldwide despite therapeutic improvement. Reovirus has been emerging as a potential anti-cancer agent and is undergoing multiple clinical trials, and it is reported that reovirus can preferentially cause the cell death of a variety of cancers in a manner of apoptosis. As few studies have reported the efficacy of oncolytic activity and safety profile of avian reovirus, in our study, LDH assay, MTT assay, DAPI staining, and flow cytometry assay were performed to demonstrate the oncolytic effects of avian reovirus against the HepG2 cells, and quantitative real-time PCR (qRT-PCR) and animal experiments were conducted to investigate the dynamic distribution of avian reovirus in infected mice and then illustrate the safety and tissue tropism of avian reovirus. LDH assay, DAPI staining, and flow cytometry assay confirmed the efficacy of the oncotherapeutic effects of avian reovirus, and MTT assay has indicated that avian reovirus suppressed the proliferation of HepG2 cells and decreased their viability significantly. qRT-PCR revealed the dynamic distribution of avian reovirus in infected mice that avian reovirus might replicate better and have more powerful oncolytic activity in liver, kidney, and spleen tissues. Furthermore, histopathological examination clearly supported that avian reovirus appeared non-pathogenic to the normal host, so our study may provide the new insights and rationale for the new strategy of removing liver cancer.
IMPACT STATEMENT
We demonstrated the efficacy of oncolytic activity of avian reovirus (ARV) by LDH assay, MTT assay, DAPI staining, and flow cytometry assay, and also investigated the dynamic distribution of ARV in infected mice and then illustrated the safety and tissue tropism of ARV by quantitative real-time PCR (qRT-PCR) and animal experiments. Collectively, our study may provide the new insights and rationale for the new strategy of removing liver cancer.
Topics: Animals; Hep G2 Cells; Humans; Mice; Oncolytic Virotherapy; Oncolytic Viruses; Orthoreovirus, Avian; Reoviridae Infections
PubMed: 31299861
DOI: 10.1177/1535370219861928 -
Journal of Virology Jan 2022Although a broad range of viruses cause myocarditis, the mechanisms that underlie viral myocarditis are poorly understood. Here, we report that the M2 gene is a...
Although a broad range of viruses cause myocarditis, the mechanisms that underlie viral myocarditis are poorly understood. Here, we report that the M2 gene is a determinant of reovirus myocarditis. The M2 gene encodes outer capsid protein μ1, which mediates host membrane penetration during reovirus entry. We infected newborn C57BL/6 mice with reovirus strain type 1 Lang (T1L) or a reassortant reovirus in which the M2 gene from strain type 3 Dearing (T3D) was substituted into the T1L genetic background (T1L/T3DM2). T1L was nonlethal in wild-type mice, whereas more than 90% of mice succumbed to T1L/T3DM2 infection. T1L/T3DM2 produced higher viral loads than T1L at the site of inoculation. In secondary organs, T1L/T3DM2 was detected with more rapid kinetics and reached higher peak titers than T1L. We found that hearts from T1L/T3DM2-infected mice were grossly abnormal, with large lesions indicative of substantial inflammatory infiltrate. Lesions in T1L/T3DM2-infected mice contained necrotic cardiomyocytes with pyknotic debris, as well as extensive lymphocyte and histiocyte infiltration. In contrast, T1L induced the formation of small purulent lesions in a small subset of animals, consistent with T1L being mildly myocarditic. Finally, more activated caspase-3-positive cells were observed in hearts from animals infected with T1L/T3DM2 than T1L. Together, our findings indicate that substitution of the T3D M2 allele into an otherwise T1L genetic background is sufficient to change a nonlethal infection into a lethal infection. Our results further indicate that T3D M2 enhances T1L replication and dissemination , which potentiates the capacity of reovirus to cause myocarditis. Reovirus is a nonenveloped virus with a segmented double-stranded RNA genome that serves as a model for studying viral myocarditis. The mechanisms by which reovirus drives myocarditis development are not fully elucidated. We found that substituting the M2 gene from strain type 3 Dearing (T3D) into an otherwise type 1 Lang (T1L) genetic background (T1L/T3DM2) was sufficient to convert the nonlethal T1L strain into a lethal infection in neonatal C57BL/6 mice. T1L/T3DM2 disseminated more efficiently and reached higher maximum titers than T1L in all organs tested, including the heart. T1L is mildly myocarditic and induced small areas of cardiac inflammation in a subset of mice. In contrast, hearts from mice infected with T1L/T3DM2 contained extensive cardiac inflammatory infiltration and more activated caspase-3-positive cells, which is indicative of apoptosis. Together, our findings identify the reovirus M2 gene as a new determinant of reovirus-induced myocarditis.
Topics: Animals; Animals, Newborn; Capsid Proteins; Inflammation; Mammalian orthoreovirus 3; Mice; Mice, Inbred C57BL; Myocarditis; Orthoreovirus, Mammalian; Reoviridae Infections; Viral Load; Virulence; Virus Replication
PubMed: 34757847
DOI: 10.1128/JVI.01879-21 -
Journal of Virology Oct 2019Avian reovirus (ARV) p17 protein continuously shuttles between the nucleus and the cytoplasm via transcription-dependent and chromosome region maintenance 1...
Avian reovirus (ARV) p17 protein continuously shuttles between the nucleus and the cytoplasm via transcription-dependent and chromosome region maintenance 1 (CRM1)-independent mechanisms. Nevertheless, whether cellular proteins modulate nucleocytoplasmic shuttling of p17 remains unknown. This is the first report that heterogeneous nuclear ribonucleoprotein (hnRNP) A1 serves as a carrier protein to modulate nucleocytoplasmic shuttling of p17. Both and studies indicated that direct interaction of p17 with hnRNP A1 maps within the amino terminus (amino acids [aa] 19 to 40) of p17 and the Gly-rich region of the C terminus of hnRNP A1. Furthermore, our results reveal that the formation of p17-hnRNP A1-transportin 1 carrier-cargo complex is required to modulate p17 nuclear import. Utilizing sequence and mutagenesis analyses, we have identified nuclear export signal (NES) LSLRELAI of p17. Mutations of these residues causes a nuclear retention of p17. In this work, we uncovered that the N-terminal 21 amino acids (aa 19 to 40) of p17 that comprise the NES can modulate both p17 and hnRNP A1 interaction and nucleocytoplasmic shuttling of p17. In this work, the interaction site of p17 with lamin A/C was mapped within the amino terminus (aa 41 to 60) of p17 and p17 colocalized with lamin A/C at the nuclear envelope. Knockdown of hnRNP A1 or lamin A/C led to inhibition of nucleocytoplasmic shuttling of p17 and reduced virus yield. Collectively, the results of this study provide mechanistic insights into hnRNP A1 and lamin A/C-modulated nucleocytoplasmic shuttling of the ARV p17 protein. Avian reoviruses (ARVs) cause considerable economic losses in the poultry industry. The ARV p17 protein continuously shuttles between the nucleus and the cytoplasm to regulate several cellular signaling pathways and interacts with several cellular proteins to cause translation shutoff, cell cycle arrest, and autophagosome formation, all of which enhance virus replication. To date the mechanisms underlying nucleocytoplasmic shuttling of p17 remain largely unknown. Here we report that hnRNP A1 and lamin A/C serve as carrier and mediator proteins to modulate nucleocytoplasmic shuttling of p17. The formation of p17-hnRNP A1-transportin 1 carrier-cargo complex is required to modulate p17 nuclear import. Furthermore, we have identified an NES-containing nucleocytoplasmic shuttling domain (aa 19 to 40) of p17 that is critical for binding to hnRNP A1 and for nucleocytoplasmic shuttling of p17. This study provides novel insights into how hnRNP A1 and lamin A/C modulate nucleocytoplasmic shuttling of the ARV p17 protein.
Topics: Active Transport, Cell Nucleus; Animals; Cell Nucleus; Cytoplasm; Heterogeneous Nuclear Ribonucleoprotein A1; Host-Pathogen Interactions; Humans; Lamin Type A; Models, Biological; Orthoreovirus, Avian; Protein Binding; Reoviridae Infections; Viral Matrix Proteins
PubMed: 31375578
DOI: 10.1128/JVI.00851-19 -
Virology Oct 2020Filamentous mammalian orthoreovirus (MRV) viral factories (VFs) are membrane-less cytosolic inclusions in which virus transcription, replication of dsRNA genome...
Filamentous mammalian orthoreovirus (MRV) viral factories (VFs) are membrane-less cytosolic inclusions in which virus transcription, replication of dsRNA genome segments, and packaging of virus progeny into newly synthesized virus cores take place. In infected cells, the MRV μ2 protein forms punctae in the enlarged region of the filamentous VFs that are co-localized with γ-tubulin and resistant to nocodazole treatment, and permitted microtubule (MT)-extension, features common to MT-organizing centers (MTOCs). Using a previously established reconstituted VF model, we addressed the functions of MT-components and MTOCs concerning their roles in the formation of filamentous VFs. Indeed, the MTOC markers γ-tubulin and centrin were redistributed within the VF-like structures (VFLS) in a μ2-dependent manner. Moreover, the MT-nucleation centers significantly increased in numbers, and γ-tubulin was pulled-down in a binding assay when co-expressed with histidine-tagged-μ2 and μNS. Thus, μ2, by interaction with γ-tubulin, can modulate MTOCs localization and function according to viral needs.
Topics: Animals; Cell Line; Chlorocebus aethiops; Fibroblasts; Gene Expression Regulation; Host-Pathogen Interactions; Microtubule-Organizing Center; Microtubules; Nocodazole; Orthoreovirus, Mammalian; Signal Transduction; Tubulin; Tubulin Modulators; Viral Proteins; Virus Replication
PubMed: 32805585
DOI: 10.1016/j.virol.2020.07.008 -
Cells Jan 2021Avian reovirus p17 (ARV p17) is a non-structural protein known to activate autophagy, interfere with gene transcription and induce a significant tumor cell growth...
Avian reovirus p17 (ARV p17) is a non-structural protein known to activate autophagy, interfere with gene transcription and induce a significant tumor cell growth inhibition in vitro and in vivo. In this study, we show that ARV p17 is capable of exerting potent antiangiogenic properties. The viral protein significantly inhibited the physiological angiogenesis of human endothelial cells (ECs) by affecting migration, capillary-like structure and new vessel formation. ARV p17 was not only able to suppress the EC physiological angiogenesis but also rendered ECs insensitive to two different potent proangiogenic inducers, such as VEGF-A and FGF-2 in the three-dimensional (3D) Matrigel and spheroid assay. ARV p17 was found to exert its antiangiogenic activity by upregulating transcription and release of the well-known tumor suppressor molecule dipeptidyl peptidase 4 (DPP4). The ability of ARV p17 to impact on angiogenesis is completely new and highlights the "two compartments" activity of the viral protein that is expected to hamper the tumor parenchymal/stromal crosstalk. The complex antitumor activities of ARV p17 open the way to a new promising field of research aimed to develop new therapeutic approaches for treating tumor and cancer metastasis.
Topics: Angiogenesis Inhibitors; Animals; Cell Movement; Chickens; Dipeptidyl Peptidase 4; Fibroblast Growth Factor 2; Human Umbilical Vein Endothelial Cells; Humans; Microvessels; Morphogenesis; Neovascularization, Pathologic; Orthoreovirus, Avian; Recombinant Proteins; Transduction, Genetic; Up-Regulation; Viral Nonstructural Proteins
PubMed: 33525607
DOI: 10.3390/cells10020259