Family Reoviridae Research Articles

The phosphoproteomic responses of duck (Cairna moschata) to classical/novel duck reovirus infections in the spleen tissue

Duck reovirus (DRV) is a fatal member of the genus Orthoreovirus in the family Reoviridae. The disease caused by DRV leads to huge economic losses to the duck industry. Post-translational modification is an efficient strategy to enhance the immune responses to virus infection. However, the roles of protein phosphorylation in the responses of ducklings to Classic/Novel DRV (C/NDRV) infections are largely unknown. Using a high-resolution LC–MS/MS integrated to highly sensitive immune-affinity antibody method, phosphoproteomes of Cairna moschata spleen tissues under the C/NDRV infections were analyzed, producing a total of 8,504 phosphorylation sites on 2,853 proteins. After normalization with proteomic data, 392 sites on 288 proteins and 484 sites on 342 proteins were significantly changed under the C/NDRV infections, respectively. To characterize the differentially phosphorylated proteins (DPPs), a systematic bioinformatics analyses including Gene Ontology annotation, domain annotation, subcellular localization, and Kyoto Encyclopedia of Genes and Genomes pathway annotation were performed. Two important serine protease system-related proteins, coagulation factor X and fibrinogen α-chain, were identified as phosphorylated proteins, suggesting an involvement of blood coagulation under the C/NDRV infections. Furthermore, 16 proteins involving the intracellular signaling pathways of pattern-recognition receptors were identified as phosphorylated proteins. Changes in the phosphorylation levels of MyD88, NF-κB, RIP1, MDA5 and IRF7 suggested a crucial role of protein phosphorylation in host immune responses of C. moschata. Our study provides new insights into the responses of ducklings to the C/NDRV infections at PTM level.

Safety and efficacy of inactivated African horse sickness (AHS) vaccine formulated with different adjuvants

African horse sickness virus (AHSV) is a virus species in the genus Orbivirus of the family Reoviridae causing African Horse Sickness (AHS) in equids with a mortality of about 95% in naïve horses. AHS causes serious losses in developing countries where horses play a central role in draft power and transportation. There are nine AHSV serotypes inducing no or low cross-neutralizing antibodies. AHSV is spread by biting Culicoides midges. AHS is endemic in sub-Saharan Africa, and a serious threat outside Africa, since Culicoides species in moderate climate conditions are spreading the closely related bluetongue virus. AHS outbreaks will be devastating for the equestrian industry in developed countries. Live-attenuated vaccines (LAVs) are licensed, marketed and in use in Africa. Their application is controversial with regard to safety issues. LAVs are not allowed in AHS-free countries. We here studied inactivated AHSV with different adjuvants in guinea pigs and horses. Subcutaneous and intramuscular vaccination were studied in horses. Local reactions were observed after prime and boost vaccination. In general, neutralizing antibodies (nAbs) titres were very low after prime vaccination, whereas boost vaccination resulted in high nAb titres for some adjuvants. Vaccinated horses were selected based on local reactions and nAb titres to study efficacy. Unfortunately, not all vaccinated horses survived virulent AHSV infection. Further, most survivors temporarily developed clinical signs and viremia. Further, the current prototype inactivated AHS vaccine is not suitable as emergency vaccine, because onset of protection is slow and requires boost vaccinations. On the other hand, inactivated AHS vaccine is completely safe with respect to virus spread, and incorporation of the DIVA principle based on NS3/NS3a serology and exploring a vaccine production platform for other serotypes is feasible. A superior adjuvant increasing the protective response without causing local reactions will be required to develop payable and acceptable inactivated AHS vaccines.

Identification and characterization of a novel orbivirus, Yonaguni orbivirus, isolated from cattle on the westernmost island of Japan.

A novel orbivirus (genus Orbivirus, family Reoviridae), designated Yonaguni orbivirus (YONOV), was isolated from bovine blood collected on a subtropical island of Japan in 2015. The YONOV genome (20,054 nucleotides in total) has a coding arrangement similar to those of mosquito-borne orbiviruses. YONOV has a close genetic relationship to mosquito-borne orbiviruses, especially to Mobuck virus (MBV), which was isolated in North America. However, YONOV and MBV share less than 74% nucleotide sequence identity in the major subcore protein (T2) coding sequence, which satisfies the criterion for species demarcation. It is still uncertain whether YONOV should be assigned to a novel species in the genus Orbivirus.

Modern taxonomy of viruses of vertebrates

The modern taxonomy of viruses of vertebrates is presented according to the information of ICTV issue 07.2019, ratification 03.2020. The leading criteria of taxonomy of viruses are named: type and structure of viral genome, mechanism of replication and morphology of virion. The periods of formation of taxonomic ranks of viruses are characterized: in 1966–1970 genera of viruses were formed, in 1971–1975 – families and subfamilies, since 1990 – orders, in 2018–2019 – realms, kingdoms, phylums, subphylums, classes, suborders, subgenеres. The nomenclature of viruses is described. Viruses belong to the Viruses domain. Viruses of vertebrates (1878 species) belong to 4 realms, 5 kingdoms, 10 phylums, 2 subphylums, 20 classes, 26 orders, 3 suborders, 45 families (of which 15 – DNA-genomic and 30 – RNA-genomic), 33 subfamilies, 345 genera and 49 subgenera. Taxonomic ranks of DNA- and RNA-genomic viruses of vertebrates are described. The DNA-genome family Anelloviridae and the unclassified RNA-genomic genus Deltavirus are not included in any realm. The family Birnaviridae is not classified within the kingdom Orthornavirae. The family of DNA-genomic Hepadnaviridae is included in the realm of RNA-containing viruses Riboviria on the grounds that the replication of hepadnaviruses occurs through the stage of RNA on the principle of reverse transcription, as in the family Retroviridae. The main taxonomic features of DNA- and RNA-genomic viruses of vertebrates are described: type and structure of viral genome (DNA or RNA, number of strands, conformation, fragmentation, polarity), shape and size of virions, presence of outer lipoprotein shell, type of capsid symmetry (spiral, iсosahedral). Some families, in addition to viruses of vertebrates, contain viruses of invertebrates and plants, in particular: families Poxviridae, Iridoviridae, Parvoviridae, Circoviridae, Smacoviridae, Genomoviridae, Rhabdoviridae, Nyamiviridae, Peribunyaviridae, Phenuiviridae, Nairoviviridae, Nodaviridae, Reoviridae and Birnaviridae – viruses of insects; families Genomoviridae, Rhabdoviridae, Phenuiviridae and Reoviridae – viruses of plants; family Nyamiviridae – viruses of nematodes, cestodes, sipunculidеs and echinoderms; family Rhabdoviridae – viruses of nematodes; family Reoviridae – Eriocheir sinensis reovirus; family Birnaviridae – viruses of tellines and rotifers.

Characterization of the lipidomic profile of BmN cells in response to Bombyx mori cytoplasmic polyhedrosis virus infection

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV)that belongs to the genus Cypovirus in the family of Reoviridae is one of the problematic pathogens in sericulture. In our previous study, we have found that lipid-related constituents in the host cellular membrane are associated with the BmCPV life cycle. It is important to note that the lipids not only affect the cellular biological processes, they also impact the virus life cycle. However, the intracellular lipid homeostasis in BmN cells after BmCPV infection remains unclear. Here, the lipid metabolism in BmCPV-infected BmN cells was studied by lipidomics analysis. Our results revealed that the intracellular lipid homeostasis was disturbed in BmN cells upon BmCPV infection. Major lipids constituents in cellular membrane were found to be significantly induced upon BmCPV infection, which included triglycerides, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, phospholipids, glucoside ceramide, monoetherphosphatidylcholin, ceramide, ceramide phosphoethanolamine and cardiolipin. Further analysis of the pathways related to these altered lipids (such as PE and PC) showed that glycerophospholipid metabolism was one of the most enriched pathways. These results suggested that BmCPV may manipulate the lipid metabolism of cells for their own interest. The findings may facilitate a better understanding of the roles of lipid metabolic changes during virus infection in future studies.

Origin and evolution of emerging Liao ning Virus (genus Seadornavirus, family Reoviridae)

BackgroundLiao ning virus (LNV) is a member of the genus Seadornavirus, family Reoviridae and has been isolated from kinds of vectors in Asia and Australia. However, there are no systematic studies describe the molecular genetic evolution and migration of LNVs. With the development of bioinformatics, viral genetic data combining the information of virus isolation time and locations could be integrated to infer the virus evolution and spread in nature.MethodsHere, a phylogenetic and phylogeographic analysis using Bayesian Markov chain Monte Carlo simulations was conducted on the LNVs isolated from a variety of vectors during 1990–2014 to identify the evolution and migration patterns of LNVs.ResultsThe results demonstrated that the LNV could be divided into 3 genotypes, of which genotype 1 mainly composed of LNVs isolated from Australia during 1990 to 2014 and the original LNV strain (LNV-NE97–31) isolated from Liaoning province in northern China in 1997, genotype 2 comprised of the isolates all from Xinjiang province in western China and genotype 3 consisted the isolates from Qinghai and Shanxi province of central China. LNVs emerged about 272 years ago and gradually evolved into three lineages in the order genotype 1, genotype 2 and genotype 3. Following phylogeographic analysis, it shows genotype 1 LNVs transmitted from Australia (113°E-153°E,10°S-42°S) to Liaoning province (118°E-125°E,38°N-43°N) in Northeast Asian continent then further spread across the central part of China to western China (75°E-95°E,35°N-50°N).ConclusionLNVs were initially isolated from Liaoning province of China in the Northeast Asia, however, the present study revealed that LNVs were first appeared in Australia in the South Pacific region and transmitted to mainland China then rapidly spread across China and evolved three different genotypes. The above results suggested that LNV had the characteristics of long-distance transmission and there were great genetic diversity existed in the LNV population. Notably, current information of 80 strains of LNVs are limited. It is of great importance to strengthen the surveillance of LNVs to explore its real origin in nature and monitoring of the LNVs’ population variation and maintain vigilance to avoid LNV breaking through the species barrier and further clarify its relationship to human and animal infection.

Bacillus pumilus ribonuclease rescues mice infected by double-stranded RNA-containing reovirus serotype 1

Reoviruses (respiratory enteric orphan viruses) are nonenveloped viruses with segmented dsRNA genome. Viruses in the family Reoviridae are quite stable in the environment. Recently, they have been identified with various pathologies and physiologic dysfunctions in a wide range of organs and tissues, including the hepatobiliary system, the myocardium, lungs, and endocrine tissues. Although most cases of reovirus infection are mild or subclinical diseases, the prevention measures are currently needed, especially for young children suffering from dehydrating gastroenteritis. To inhibit viral replication, different RNases targeting viral RNA are proposed. Here, we first have shown that RNase from Bacillus pumilus (binase) acts as an antiviral agent at the level of the whole animal organism infected by Mammalian orthoreovirus 1 strain Lang (TL1). The results obtained on the mice model infected with 10 LD50 and 20 LD50 doses of reovirus indicate the restoration of mice physiological parameters under binase treatment at the dose of 50 μg/mouse. Thus, our research supports the relevance of binase as a promising antiviral agent that affects viral RNA.

Polycistronic Genome Segment Evolution and Gain and Loss of FAST Protein Function during Fusogenic Orthoreovirus Speciation.

The Reoviridae family is the only non-enveloped virus family with members that use syncytium formation to promote cell–cell virus transmission. Syncytiogenesis is mediated by a fusion-associated small transmembrane (FAST) protein, a novel family of viral membrane fusion proteins. Previous evidence suggested the fusogenic reoviruses arose from an ancestral non-fusogenic virus, with the preponderance of fusogenic species suggesting positive evolutionary pressure to acquire and maintain the fusion phenotype. New phylogenetic analyses that included the atypical waterfowl subgroup of avian reoviruses and recently identified new orthoreovirus species indicate a more complex relationship between reovirus speciation and fusogenic capacity, with numerous predicted internal indels and 5’-terminal extensions driving the evolution of the orthoreovirus’ polycistronic genome segments and their encoded FAST and fiber proteins. These inferred recombination events generated bi- and tricistronic genome segments with diverse gene constellations, they occurred pre- and post-orthoreovirus speciation, and they directly contributed to the evolution of the four extant orthoreovirus FAST proteins by driving both the gain and loss of fusion capability. We further show that two distinct post-speciation genetic events led to the loss of fusion in the waterfowl isolates of avian reovirus, a recombination event that replaced the p10 FAST protein with a heterologous, non-fusogenic protein and point substitutions in a conserved motif that destroyed the p10 assembly into multimeric fusion platforms.

Rice black-streaked dwarf virus: From multiparty interactions among plant-virus-vector to intermittent epidemics.

Rice black‐streaked dwarf virus (RBSDV) (species Rice black‐streaked dwarf virus, genus Fijivirus, family Reoviridae) is the causal agent of rice black‐streaked dwarf and maize rough dwarf diseases, which occur in intermittent epidemics in East Asian countries and are responsible for considerable yield losses. Intermittency of epidemics make accurate forecasting and designing of effective management strategies difficult. However, recent insights into host–virus–vector insect interactions are now informing forecasting and disease control measures. Resistance genes are also being identified and mapped.Symptomatology and host rangeRBSDV induces extreme stunting, darkened, and stiff leaves of crops and weeds only in the family Poaceae, including Oryza sativa, Zea mays, and Triticum aestivum. Infected plants produce totally or partially deformed panicles and remain alive through harvest.Genome and gene functionThe nonenveloped virus particles comprise a double‐layered capsid, 50‐nm core with genomic double‐stranded RNA (dsRNA), and six proteins. The genome of RBSDV contains 10 segments of dsRNA, named S1 to S10 in decreasing order of molecular weight. Segments 1, 2, 3, 4, 6, 8, and 10 encode the RNA‐dependent RNA polymerase (RdRp), the major core structural protein, a protein with guanylyltransferase activity, an outer‐shell B‐spike protein, viral RNA‐silencing suppressor, the major capsid protein, and the outer capsid protein, respectively. Each of the segments 5, 7, and 9 encodes two proteins: P5‐1, a component of viroplasms; P5‐2 of unknown function; nonstructural protein P7‐1, involved in forming the structural matrix of tubular structures in infected tissues; P7‐2 of unknown function; P9‐1, the main component of viroplasms in infected cells and involved in viral replication; and P9‐2 of unknown function.Transmission and epidemiologyRBSDV is transmitted by Laodelphax striatellus in a persistent propagative manner. The vector insect is the only means of virus spread in nature, so its migration and transmission efficiency are obligatory for disease epidemics to develop. Susceptible varieties are widely planted, but efficient transmission by vectors is the primary reason for the epidemics. Cultivation system, pesticide overuse, and climatic conditions also contribute to epidemics by affecting the development of the vector insects and their population dynamics.Disease managementIn the absence of resistant varieties, integrated disease management aims at disrupting the cycle of virus transmission by the insect vector. Inheritance studies have indicated that resistance is mostly governed by quantitative trait loci or multiple genes. Genetic engineering through RNA‐interference and gene‐editing strategies are potential approaches for disease control.

A Perfect Ten—Decoy Maps Uncover Polymerase Complexes within Reoviridae Virion

In this issue of Structure, Kaelber etal. (2020) use cryo-EM and synthetic decoy maps to reveal the patterning of 10 polymerase complexes within FAKV, a Reoviridae family member containing 9 genome segments. Their findings support a model for FAKV assembly that has implications for the entire Reoviridae family.

Discovery and characterization of a novel insect-specific reovirus isolated from Psammotettix alienus.

A novel double-stranded RNA (dsRNA) virus designated Psammotettix alienus reovirus (PARV) was found in the leafhopper Psammotettix alienus in China. Spherical particles approximately 70 nm in diameter arranged in a crystalline array were observed in the salivary gland tissues of infected leafhoppers by transmission electron microscopy. Some viral particles were also encased in tubules, similar to those of previously described reoviruses. Whole-genome sequencing revealed that the dsRNA genome of PARV consists of 29 569 nucleotides (nt) divided into 10 segments ranging from 4403 to 1476 nt, with low G+C content (29.5-36.5 %). All segments contained conserved terminal sequences (5'AAC…GUCA3') and specific panhandle structures formed by inverted terminal repeats in the noncoding regions. Phylogenetic analysis based on the deduced RNA-dependent RNA polymerase (RdRp) revealed that PARV was in the fijivirus clade, but in a monophyletic lineage with an unassigned insect reovirus (Hubei insect virus 2, HBIV-2), although PARV and HBIV-2 are distinct enough to represent a new group within the genus Fijivirus. Biological assays showed that PARV infects P. alienus but not wheat plants, implying that it is a new insect-specific reovirus in the leafhopper. Given these features, PARV should be considered as a new species in the genus Fijivirus, family Reoviridae.

Unexpected Genetic Diversity of Two Novel Swine MRVs in Italy.

Mammalian Orthoreoviruses (MRV) are segmented dsRNA viruses in the family Reoviridae. MRVs infect mammals and cause asymptomatic respiratory, gastro-enteric and, rarely, encephalic infections. MRVs are divided into at least three serotypes: MRV1, MRV2 and MRV3. In Europe, swine MRV (swMRV) was first isolated in Austria in 1998 and subsequently reported more than fifteen years later in Italy. In the present study, we characterized two novel reassortant swMRVs identified in one same Italian farm over two years. The two viruses shared the same genetic backbone but showed evidence of reassortment in the S1, S4, M2 segments and were therefore classified into two serotypes: MRV3 in 2016 and MRV2 in 2018. A genetic relation to pig, bat and human MRVs and other unknown sources was identified. A considerable genetic diversity was observed in the Italian MRV3 and MRV2 compared to other available swMRVs. The S1 protein presented unique amino acid signatures in both swMRVs, with unexpected frequencies for MRV2. The remaining genes formed distinct and novel genetic groups that revealed a geographically related evolution of swMRVs in Italy. This is the first report of the complete molecular characterization of novel reassortant swMRVs in Italy and Europe, which suggests a greater genetic diversity of swMRVs never identified before.

Codon Usage Bias Analysis of Bluetongue Virus Causing Livestock Infection

Bluetongue virus (BTV) is a double-stranded RNA virus with multiple segments and belongs to the genus Orbivirus within the family Reoviridae. BTV is spread to livestock through its dominant vector, biting midges of genus Culicoides. Although great progress has been made in genomic analyses, it is not fully understood how BTVs adapt to their hosts and evade the host’s immune systems. In this study, we retrieved BTV genome sequences from the National Center for Biotechnology Information (NCBI) database and performed a comprehensive research to explore the codon usage patterns in 50 BTV strains. We used bioinformatic approaches to calculate the relative synonymous codon usage (RSCU), codon adaptation index (CAI), effective number of codons (ENC), and other indices. The results indicated that most of the overpreferred codons had A-endings, which revealed that mutational pressure was the major force shaping codon usage patterns in BTV. However, the influence of natural selection and geographical factors cannot be ignored on viral codon usage bias. Based on the RSCU values, we performed a comparative analysis between BTVs and their hosts, suggesting that BTVs were inclined to evolve their codon usage patterns that were comparable to those of their hosts. Such findings will be conducive to understanding the elements that contribute to viral evolution and adaptation to hosts.

Detection of Piscine orthoreoviruses (PRV-1b AND PRV-3a) in farmed Coho salmon with jaundice syndrome from Chile

Piscine orthoreoviruses (PRVs) represent a group of marine viruses belonging to Reoviridae family, which are associated to emerging diseases in farmed Atlantic salmon, Rainbow trout and Coho salmon around the world. In Chile, the prevalence and deleterious effects of PRV infection in farmed salmonids had been studied recently, showing the occurrence of PRV-1 (formerly PRV-Ss) in Atlantic salmon with Heart and Skeletal Muscle Inflammation (HSMI), PRV-3 (formerly PRV-Om) in Rainbow trout with HSMI-like disease, and both viruses (PRV-1 and PRV-3) in Coho salmon with HSMI-like disease. In 1997, Jaundice Syndrome in Coho salmon was first described in Chile associated to outbreaks with high mortality, which occur during autumn season. Then, it was reported in 2006 that the jaundice condition in Coho salmon is consistent with an infectious form of hemolytic anemia and probably a virus was the causative agent, however the identification of the putative virus was not possible. Currently, the etiology of this syndrome is unknown, but several of the clinical signs exhibited by diseased Coho salmon are comparable to the signs showed by salmonids affected with some disease which are caused by PRVs. Therefore, it was of interest to determine if some PRVs could be related to Jaundice Syndrome in Coho salmon. In this manner, heart and head kidney tissue samples obtained from Coho salmon mortalities were analyzed in search of viral pathogens, where the presence of both PRV-1 and PRV-3 was confirmed by RT-qPCR and Sanger sequencing. Moreover, full-length genomic segment S1 sequences were obtained to determine the phylogenetic relationship with others PRV isolates previously described in Europe, America and Japan. One group of sequences found in Coho salmon with Jaundice Syndrome clustered into Sub-genotype Ib together with PRV-1 isolate described in Coho salmon with HSMI-like condition from Chile, and other group of sequences clustered into Genotype II with the first PRV-3 detected in Rainbow trout from Norway into previously described PRV-3a clade. Our results also suggest that PRVs has developed an infection cycle, which involves to Atlantic salmon, Rainbow trout and Coho salmon.

Genome constellations of rotavirus a isolated from avian species in Brazil, 2008-2015.

Rotaviruses are members of the family Reoviridae and are a common cause of acute diarrhea in many mammalian and avian species. They are non-enveloped icosahedral particles and their genome comprises 11 segments of double-stranded RNA, which encodes six structural proteins (VP1-4, VP6-7) and six nonstructural proteins (NSP1-6). Genotypes are defined based upon the diversity found in these genes and viral characterization plays a central role on epidemiological studies and prevention. Here we investigate the distribution of Brazilian RVAs genotypes in 8 chicken samples collected between 2008 and 2015 from different regions by RT-PCR, partial (Sanger) nucleotide sequencing and phylogenetic analysis from all rotavirus genes. Although the identified genotypes were typical from avian host species, when analyzed together, they form novel genetic constellations: G19-P[31]-I11-R6-C6-M7-A16-N6-T8-E10-H8 and G19-P[31]-I4-R4-C4-M4-A16-N4-T4-E4-H4. This study highlights that avian rotaviruses are widespread among commercial farms in Brazil, and the co-circulation of at least two different genomic constellations indicates that may present a way bigger genetic variability, that can be increased by the possible transmission events from other birds, lack of specific preventive measures, as well as the different viral evolution mechanisms.

Development of a Digital RT-PCR Method for Absolute Quantification of Bluetongue Virus in Field Samples.

Bluetongue (BT) is a major Office International des Epizooties (OIE)-listed disease of wild and domestic ruminants caused by several serotypes of Bluetongue virus (BTV), a virus with a segmented dsRNA genome belonging to the family Reoviridae, genus Orbivirus. BTV is transmitted through the bites of Culicoides midges. The aim of this study was to develop a new method for quantification of BTV Seg-10 by droplet digital RT-PCR (RTdd-PCR), using nucleic acids purified from complex matrices such as blood, tissues, and midges, that notoriously contain strong PCR inhibitors. First, RTdd-PCR was optimized by using RNAs purified from serially 10-fold dilutions of a BTV-1 isolate (105.43TCID50/ml up to 10−0.57 TCID50/ml) and from the same dilutions spiked into fresh ovine EDTA-blood and spleen homogenate. The method showed a good degree of linearity (R2 ≥ 0.995). The limit of detection (LoD) and the limit of quantification (LoQ) established were 10−0.67TCID50/ml (0.72 copies/μl) and 100.03TCID50/ml (3.05 copies/μl) of BTV-1, respectively. Second, the newly developed test was compared, using the same set of biological samples, to the quantitative RT-PCR (RT-qPCR) detecting Seg-10 assay widely used for the molecular diagnosis of BTV from field samples. Results showed a difference mean of 0.30 log between the two assays with these samples (p < 0.05). Anyway, the analysis of correlation demonstrated that both assays provided similar measurements with a very close agreement between the systems.

In Vitro Anti-cancer Activity of Adipose-Derived Mesenchymal Stem Cells Increased after Infection with Oncolytic Reovirus.

Purpose: Reovirus type 3 Dearing (ReoT3D), a wild type oncolytic virus (OV) from the Reoviridae family, kills KRAS mutant cancer cells. However, the use of OVs has faced with some limitations such as immune responses, and delivery of OVs to the tumor sites in systemic therapy. To solve this, and also to increase the anti-cancer effects of these OVs, mesenchymal stem cells (MSCs) might be used as an effective vehicle for OVs delivery. In this study, we examined the anti-cancer effects of human adipose derived-MSCs (AD-MSCs) as a vehicle of ReoT3D against human glioblastoma cells. Methods: Here, AD-MSCs were characterized and toxicity of ReoT3D on them was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Then, capability of AD-MSCs for virus production was assessed by real-time polymerase chain reaction (PCR), and different in vitro anti-cancer experiments were applied for our anti-cancer purposes.Results: Our results from toxicity assay revealed that the isolated and provoked AD-MSCs were resistant to nontoxic concentration multiplicity of infection (MOI) >1 pfu/cells of ReoT3D. In addition, the results indicated that AD-MSCs were susceptible for virus life cycle complementation and were capable for production of virus progenies. Furthermore, our results showed that AD-MSCs had oncolysis effects and increased the anti-cancer effects of ReoT3D. Conclusion: AD-MSCs as a susceptible host for oncolytic reovirus could increase the anti-cancer activity of this OV against glioblastoma multiforme (GBM) cell line.

Serological survey of bluetongue virus in sheep from Minas Gerais

ABSTRACT: Bluetongue is an infectious, non-contagious disease that affects domestic and wild ruminants, caused by a virus from the Orbivirus genus, Reoviridae family, transmitted by arthropod vectors of the Culicoides genus. This paper aims to be the first serological survey of bluetongue in sheep from the Meso-regions of Campo das Vertentes and South and Southeast of Minas Gerais. Samples were collected from sheep from different properties. The serum samples were submitted to Agar Gel Immunodiffusion (AGID) and competitive Enzyme-Linked Immunosorbent Assay (cELISA). 303 serum samples were submitted to AGID and cELISA. In these samples, 164 (54.13%) were positive in the AGID technique, and 171 (56.44%) positive in the cELISA technique, with an almost perfect agreement between the techniques (kappa index = 0.887). In all visited properties, positive animals have been found in the herd. Animals acquired from properties of the studied mesoregions were more likely to be positive in IDGA and cELISA tests than animals acquired from properties in other regions of Brazil (p&lt;0.001). These results suggest that bluetongue virus (BTV) is widespread in the mesoregions of Campo das Vertentes and South and Southeast of Minas Gerais.

Differential Localization of Structural and Non-Structural Proteins during the Bluetongue Virus Replication Cycle.

Members of the Reoviridae family assemble virus factories within the cytoplasm of infected cells to replicate and assemble virus particles. Bluetongue virus (BTV) forms virus inclusion bodies (VIBs) that are aggregates of viral RNA, certain viral proteins, and host factors, and have been shown to be sites of the initial assembly of transcriptionally active virus-like particles. This study sought to characterize the formation, composition, and ultrastructure of VIBs, particularly in relation to virus replication. In this study we have utilized various microscopic techniques, including structured illumination microscopy, and virological assays to show for the first time that the outer capsid protein VP5, which is essential for virus maturation, is also associated with VIBs. The addition of VP5 to assembled virus cores exiting VIBs is required to arrest transcriptionally active core particles, facilitating virus maturation. Furthermore, we observed a time-dependent association of the glycosylated non-structural protein 3 (NS3) with VIBs, and report on the importance of the two polybasic motifs within NS3 that facilitate virus trafficking and egress from infected cells at the plasma membrane. Thus, the presence of VP5 and the dynamic nature of NS3 association with VIBs that we report here provide novel insight into these previously less well-characterized processes.

Development and optimization of a DNA-based reverse genetics systems for epizootic hemorrhagic disease virus.

Epizootic hemorrhagic disease virus (EHDV) is a member of the genus Orbivirus, family Reoviridae, and has a genome consisting of 10 linear double-stranded (ds) RNA segments. The current reverse genetics system (RGS) for engineering the EHDV genome relies on the use of in vitro-synthesized capped viral RNA transcripts. To obtain more-efficient and simpler RGSs for EHDV, we developed an entirely DNA (plasmid or PCR amplicon)-based RGS for viral rescue. This RGS enabled the rescue of infectious EHDV from BSR-T7 cells following co-transfection with seven helper viral protein expression plasmids and 10 cDNA rescue plasmids or PCR amplicons representing the EHDV genome. Furthermore, we optimized the DNA-based systems and confirmed that some of the helper expression plasmids were not essential for the recovery of infectious EHDV. Thus, DNA-based RGSs may offer a more efficient method of recombinant virus recovery and accelerate the study of the biological characteristics of EHDV and the development of novel vaccines.