Segmented Double-stranded RNA Genome Research Articles

The recruitment of TRiC chaperonin in rotavirus viroplasms correlates with virus replication.

The replication of rotavirus takes place in cytosolic inclusions termed viroplasms. In these inclusions, the distinct 11 double-stranded RNA genome segments are co-packaged to complete a genome in newly generated virus particles. In this study, we show for the first time that the tailless complex polypeptide I ring complex (TRiC), a cellular chaperonin responsible for the folding of at least 10% of the cytosolic proteins, is a component of viroplasms and is required for the synthesis of the viral negative-sense single-stranded RNA. Specifically, TRiC associates with NSP5 and VP2, the cofactor involved in RNA replication. Our study adds a new component to the current model of rotavirus replication, where TRiC is recruited to viroplasms to assist replication.

Recombinant rotavirus expressing the glycosylated S1 protein of SARS-CoV-2.

Reverse genetic systems have been used to introduce heterologous sequences into the rotavirus segmented double-stranded (ds)RNA genome, enabling the generation of recombinant viruses that express foreign proteins and possibly serve as vaccine vectors. Notably, insertion of SARS-CoV-2 sequences into the segment seven (NSP3) RNA of simian SA11 rotavirus was previously shown to result in the production of recombinant viruses that efficiently expressed the N-terminal domain (NTD) and the receptor-binding domain (RBD) of the S1 region of the SARS-CoV-2 spike protein. However, efforts to generate a similar recombinant (r) SA11 virus that efficiently expressed full-length S1 were less successful. In this study, we describe modifications to the S1-coding cassette inserted in the segment seven RNA that allowed recovery of second-generation rSA11 viruses that efficiently expressed the ~120-kDa S1 protein. The ~120-kDa S1 products were shown to be glycosylated, based on treatment with endoglycosidase H, which reduced the protein to a size of ~80 kDa. Co-pulldown assays demonstrated that the ~120-kDa S1 proteins had affinity for the human ACE2 receptor. Although all the second-generation rSA11 viruses expressed glycosylated S1 with affinity for the ACE receptor, only the S1 product of one virus (rSA11/S1f) was appropriately recognized by anti-S1 antibodies, suggesting the rSA11/S1f virus expressed an authentic form of S1. Compared to the other second-generation rSA11 viruses, the design of the rSA11/S1f was unique, encoding an S1 product that did not include an N-terminal FLAG tag. Probably due to the impact of FLAG tags upstream of the S1 signal peptides, the S1 products of the other viruses (rSA11/3fS1 and rSA11/3fS1-His) may have undergone defective glycosylation, impeding antibody binding. In summary, these results indicate that recombinant rotaviruses can serve as expression vectors of foreign glycosylated proteins, raising the possibility of generating rotavirus-based vaccines that can induce protective immune responses against enteric and mucosal viruses with glycosylated capsid components, including SARS-CoV-2.

A rapid and affordable amplicon-based method for next-generation genome sequencing of the infectious bursal disease virus

The infectious bursal disease virus (IBDV) causes a severe immunosuppressive disorder in young chickens. IBDV evolution resulted in the emergence of strains with divergent genetic, antigenic, and pathogenic characteristics. Genetic classification is typically performed by sequencing the coding region of the most immunogenic region of the viral protein 2 (VP2). Sequencing both double-stranded RNA genome segments is essential to achieve a more comprehensive IBDV classification that can detect recombinants and reassortments. Here, we report the development and standardization of a tiled PCR amplicon protocol for the direct and cost-effective genome sequencing of global IBDV strains using next-generation technology. Primers for tiled PCR were designed with adapters to bypass expensive and time-consuming library preparation steps. Sequencing was performed on Illumina MiniSeq equipment, and fourteen complete genomes of field strains were assembled using reference sequences. The PCR-enrichment step was used to obtain genomes from low-titer biological samples that were difficult to amplify using traditional sequencing. Phylogenetic analyses of the obtained genomes confirmed previous strain classification. By combining the enrichment methodology with massive sequencing, it is possible to obtain IBDV genomic sequences in a fast and affordable manner. This procedure can be a valuable tool to better understand virus epidemiology.

Identification and molecular characterization of novel duck reoviruses in Henan Province, China.

Novel Duck reovirus (NDRV) is an ongoing non-enveloped virus with ten double-stranded RNA genome segments that belong to the genus Orthoreovirus, in the family Reoviridae. NDRV-associated spleen swelling, and necrosis disease have caused considerable economic losses to the waterfowl industry worldwide. Since 2017, a significant number of NDRV outbreaks have emerged in China. Herein, we described two cases of duck spleen necrosis disease among ducklings on duck farms in Henan province, central China. Other potential causative agent, including Muscovy duck reovirus (MDRV), Duck hepatitis A virus type 1 (DHAV-1), Duck hepatitis A virus type 3 (DHAV-3), Newcastle disease virus (NDV), and Duck tembusu virus (DTMUV), were excluded by reverse transcription-polymerase chain reaction (RT-PCR), and two NDRV strains, HeNXX-1/2021 and HNJZ-2/2021, were isolated. Sequencing and phylogenetic analysis of the σC genes revealed that both newly identified NDRV isolates were closely related to DRV/SDHZ17/Shandong/2017. The results further showed that Chinese NDRVs had formed two distinct clades, with late 2017 as the turning point, suggesting that Chinese NDRVs have been evolving in different directions. This study identified and genetic characteristics of two NDRV strains in Henan province, China, indicating NDRVs have evolved in different directions in China. This study provides an insight into the ongoing emerged duck spleen necrosis disease and enriches our understanding of the genetic diversity and evolution of NDRVs.

ICTV Virus Taxonomy Profile: Hadakaviridae 2023.

The family Hadakaviridae, including the genus Hadakavirus, accommodates capsidless viruses with a 10- or 11-segmented positive-sense (+) RNA genome. Currently known hosts are ascomycetous filamentous fungi. Although phylogenetically related to polymycovirids with a segmented double-stranded RNA genome and certain encapsidated picorna-like viruses, hadakavirids are distinct in their lack of a capsid ('hadaka' means naked in Japanese) and their consequent inability to be pelleted by conventional ultracentrifugation; they show ribonuclease susceptibility in host tissue homogenates. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Hadakaviridae, which is available at ictv.global/report/hadakaviridae.

Reovirus Activated Cell Death Pathways.

Mammalian orthoreoviruses (ReoV) are non-enveloped viruses with segmented double-stranded RNA genomes. In humans, ReoV are generally considered non-pathogenic, although members of this family have been proven to cause mild gastroenteritis in young children and may contribute to the development of inflammatory conditions, including Celiac disease. Because of its low pathogenic potential and its ability to efficiently infect and kill transformed cells, the ReoV strain Type 3 Dearing (T3D) is clinical trials as an oncolytic agent. ReoV manifests its oncolytic effects in large part by infecting tumor cells and activating programmed cell death pathways (PCDs). It was previously believed that apoptosis was the dominant PCD pathway triggered by ReoV infection. However, new studies suggest that ReoV also activates other PCD pathways, such as autophagy, pyroptosis, and necroptosis. Necroptosis is a caspase-independent form of PCD reliant on receptor-interacting serine/threonine-protein kinase 3 (RIPK3) and its substrate, the pseudokinase mixed-lineage kinase domain-like protein (MLKL). As necroptosis is highly inflammatory, ReoV-induced necroptosis may contribute to the oncolytic potential of this virus, not only by promoting necrotic lysis of the infected cell, but also by inflaming the surrounding tumor microenvironment and provoking beneficial anti-tumor immune responses. In this review, we summarize our current understanding of the ReoV replication cycle, the known and potential mechanisms by which ReoV induces PCD, and discuss the consequences of non-apoptotic cell death—particularly necroptosis—to ReoV pathogenesis and oncolysis.

RNA Packaging in the Cystovirus Bacteriophages: Dynamic Interactions during Capsid Maturation.

The bacteriophage family Cystoviridae consists of a single genus, Cystovirus, that is lipid-containing with three double-stranded RNA (ds-RNA) genome segments. With regard to the segmented dsRNA genome, they resemble the family Reoviridae. Therefore, the Cystoviruses have long served as a simple model for reovirus assembly. This review focuses on important developments in the study of the RNA packaging and replication mechanisms, emphasizing the structural conformations and dynamic changes during maturation of the five proteins required for viral RNA synthesis, P1, P2, P4, P7, and P8. Together these proteins constitute the procapsid/polymerase complex (PC) and nucleocapsid (NC) of the Cystoviruses. During viral assembly and RNA packaging, the five proteins must function in a coordinated fashion as the PC and NC undergo expansion with significant position translation. The review emphasizes this facet of the viral assembly process and speculates on areas suggestive of additional research efforts.

Rab1b-GBF1-ARF1 Secretory Pathway Axis Is Required for Birnavirus Replication.

Birnaviruses are members of the Birnaviridae family, responsible for major economic losses to poultry and aquaculture. The family is composed of nonenveloped viruses with a segmented double-stranded RNA (dsRNA) genome. Infectious bursal disease virus (IBDV), the prototypic family member, is the etiological agent of Gumboro disease, a highly contagious immunosuppressive disease in the poultry industry worldwide. We previously demonstrated that IBDV hijacks the endocytic pathway for establishing the viral replication complexes on endosomes associated with the Golgi complex (GC). Here, we report that IBDV reorganizes the GC to localize the endosome-associated replication complexes without affecting its secretory functionality. By analyzing crucial proteins involved in the secretory pathway, we showed the essential requirement of Rab1b for viral replication. Rab1b comprises a key regulator of GC transport and we demonstrate that transfecting the negative mutant Rab1b N121I or knocking down Rab1b expression by RNA interference significantly reduces the yield of infectious viral progeny. Furthermore, we showed that the Rab1b downstream effector Golgi-specific BFA resistance factor 1 (GBF1), which activates the small GTPase ADP ribosylation factor 1 (ARF1), is required for IBDV replication, since inhibiting its activity by treatment with brefeldin A (BFA) or golgicide A (GCA) significantly reduces the yield of infectious viral progeny. Finally, we show that ARF1 dominant negative mutant T31N overexpression hampered IBDV infection. Taken together, these results demonstrate that IBDV requires the function of the Rab1b-GBF1-ARF1 axis to promote its replication, making a substantial contribution to the field of birnavirus-host cell interactions. IMPORTANCE Birnaviruses are unconventional members of the dsRNA viruses, with the lack of a transcriptionally active core being the main differential feature. This structural trait, among others that resemble those of the plus single-stranded (+ssRNA) viruses features, suggests that birnaviruses might follow a different replication program from that conducted by prototypical dsRNA members and the hypothesis that birnaviruses could be evolutionary links between +ssRNA and dsRNA viruses has been argued. Here, we present original data showing that IBDV-induced GC reorganization and the cross talk between IBDV and the Rab1b-GBF1-ARF1 mediate the intracellular trafficking pathway. The replication of several +ssRNA viruses depends on the cellular protein GBF1, but its role in the replication process is not clear. Thus, our findings make a substantial contribution to the field of birnavirus-host cell interactions and provide further evidence supporting the proposed evolutionary connection role of birnaviruses, an aspect which we consider especially relevant for researchers working in the virology field.

Bombyx mori Akirin hijacks a viral peptide vSP27 encoded by BmCPV circRNA and activates the ROS-NF-κB pathway against viral infection

Bombyx mori cypovirus (BmCPV), a member of the family Reoviridae, is a model of Cypovirus, has a 10 segmented double-stranded RNA genome. However, so far, only one viral small peptide vSP27 with negative regulation on viral infection was identified; the mechanisms underlying host-BmCPV interaction are still unknown. Here, we identified that vSP27 was translated from a BmCPV derived circular RNA (circRNA-vSP27). Subsequently, results showed that vSP27 induced generation of ROS activated the NF-κB signaling pathway, induced the expression of antimicrobial peptides, and suppressed BmCPV infection. On the other hand, we identified a nuclear protein Akirin that could hijack vSP27, positively regulate the NF-κB pathway, and lead to inhibiting the viral infection. Altogether, our data suggested that BmCPV derived circRNA-vSP27 with small peptide translation activity may be employed by the host immunity in defense against the BmCPV infection.

The M2 Gene Is a Determinant of Reovirus-Induced Myocarditis.

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 in vivo, which potentiates the capacity of reovirus to cause myocarditis. IMPORTANCE 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.

Genomic characterization of silvergrass cryptic virus 1, a novel partitivirus infecting Miscanthus sinensis.

In the present study we report the identification of a novel partitivirus recovered from Miscanthus sinensis, for which the provisional name "silvergrass cryptic virus 1" (SgCV-1) is proposed. High-throughput sequencing (HTS) and rapid amplification of cDNA ends (RACE) allowed the assembly of the complete sequence of each double-stranded RNA genome segment of this novel virus. The largest dsRNA segment, dsRNA1 (1699 bp), was predicted to encode a viral RNA-dependent RNA polymerase protein (RdRp) with 478 aa, and dsRNA2 (1490 bp) and dsRNA3 (1508 bp) were predicted to encode putative capsid proteins (CPs) with 347 and 348 aa, respectively. SgCV-1 has the highest amino acid sequence identity (≤70.80% in RdPp and ≤34.5% in CPs) to members of the genus Deltapartitivirus, family Partitiviridae, especially to unclassified viruses related to members of this genus. Its genome segment and protein lengths are also within the range of those of deltapartitiviruses. Moreover, phylogenetic analysis based on RdRp amino acid sequences also showed clustering of this novel virus with the related unclassified deltapartitiviruses. An RT-PCR survey of 94 imported M. sinensis samples held in quarantine identified seven additional samples carrying SgCV-1. This new virus fulfils all ICTV criteria to be considered a new member of the genus Deltapartitivirus.

Unique Terminal Regions and Specific Deletions of the Segmented Double-Stranded RNA Genome of Alternaria Alternata Virus 1, in the Proposed Family Alternaviridae.

Alternaria alternata virus 1 (AaV1) has been identified in the saprophytic fungus Alternaria alternata strain EGS 35–193. AaV1 has four genomic double-stranded (ds)RNA segments (dsRNA1–4) packaged in isometric particles. The 3' end of each coding strand is polyadenylated (36–50nt), but the presence of a cap structure at each 5' end has not previously been investigated. Here, we have characterized the AaV1 genome and found that it has unique features among the mycoviruses. We confirmed the existence of cap structures on the 5' ends of the AaV1 genomic dsRNAs using RNA dot blots with anti-cap antibodies and the oligo-capping method. Polyclonal antibodies against purified AaV1 particles specifically bound to an 82kDa protein, suggesting that this protein is the major capsid component. Subsequent Edman degradation indicated that the AaV1 dsRNA3 segment encodes the major coat protein. Two kinds of defective AaV1 dsRNA2, which is 2,794bp (844 aa) in length when intact, appeared in EGS 35–193 during subculturing, as confirmed by RT-PCR and northern hybridization. Sequence analysis revealed that one of the two defective dsRNA2s contained a 231bp deletion, while the other carried both the 231bp deletion and an additional 465bp deletion in the open reading frame. Both deletions occurred in-frame, resulting in predicted proteins of 767 aa and 612 aa. The fungal isolates carrying virions with the defective dsRNA2s showed impaired growth and abnormal pigmentation. To our best knowledge, AaV1 is the first dsRNA virus to be identified with both 5' cap and 3'poly(A) structures on its genomic segments, as well as the specific deletions of dsRNA2.

Picobirnaviruses: prevalence, genetic diversity, detection methods.

This article presents a general overview of the prevalence, genetic diversity and detection methods of picobirnaviruses (PBVs), which are small, non-enveloped icosahedral viruses with a segmented double-stranded RNA genome consisting of two segments taxonomically related to the genus Picobirnavirus of the family Picobirnaviridae. This review of scientific papers published in 1988–2019 provides data on the PBV distribution in the nature and a broad host range. PBV infection is characterized as opportunistic, the lack of understanding of the etiological role of PBVs in diarrhea is emphasized, since these viruses are detected both in symptomatic and asymptomatic cases. The concept of PBV infection as a chronic disease caused by a long-lasting persistence of the virus in the host is considered. Such factors as stress syndrome, physiological conditions, immune status and host age at the time of primary PBV infection influence the virus detection rate in humans and animals. The possible zoonotic nature of human PBV infection is noted due to the capacity for interspecies PBV transmission acquired during evolution as a result of the reassortment of the genome segments of different viruses infecting the same host. Data providing evidence that PBVs belong to eukaryotes and a challenging hypothesis stating that PBVs are bacterial viruses are presented. The need to intensify work on PBV detection because of their wide distribution, despite the complexity due to the lack of the cultivation system, is emphasized. Two strategies of RT-PCR as main PBV detection methods are considered. The genomes of individual representatives of the genus isolated from different hosts are characterized. Emphasis is placed on the feasibility of developing primers with broader specificity for expanding the range of identifiable representatives of the genus PBV due to a huge variety of their genotypes. The importance of effective monitoring of PBV prevalence for studying the zoonotic and anthroponotic potential using metagenomic analysis is highlighted, and so is the possibility of using PBV as a marker for environmental monitoring.

Expression of Separate Heterologous Proteins from the Rotavirus NSP3 Genome Segment Using a Translational 2A Stop-Restart Element.

The segmented 18.5-kbp dsRNA genome of rotavirus expresses 6 structural and 6 nonstructural proteins. We investigated the possibility of using the recently developed plasmid-based rotavirus reverse genetics (RG) system to generate recombinant viruses that express a separate heterologous protein in addition to the 12 viral proteins. To address this, we replaced the NSP3 open reading frame (ORF) of the segment 7 (pT7/NSP3) transcription vector used in the RG system with an ORF encoding NSP3 fused to a fluorescent reporter protein (i.e., UnaG, mRuby, mKate, or TagBFP). Inserted at the fusion junction was a teschovirus translational 2A stop-restart element designed to direct the separate expression of NSP3 and the fluorescent protein. Recombinant rotaviruses made with the modified pT7/NSP3 vectors were well growing and generally genetically stable, and they expressed NSP3 and a separate fluorescent protein detectable by live cell imaging. NSP3 made by the recombinant viruses was functional, inducing nuclear accumulation of cellular poly(A)-binding protein. Further modification of the NSP3 ORF showed that it was possible to generate recombinant viruses encoding 2 heterologous proteins (mRuby and UnaG) in addition to NSP3. Our results demonstrate that, through modification of segment 7, the rotavirus genome can be increased in size to at least 19.8 kbp and can be used to produce recombinant rotaviruses expressing a full complement of viral proteins and multiple heterologous proteins. The generation of recombinant rotaviruses expressing fluorescent proteins will be valuable for the study of rotavirus replication and pathogenesis by live cell imagining and suggest that rotaviruses will prove useful as expression vectors.IMPORTANCE Rotaviruses are a major cause of severe gastroenteritis in infants and young children. Recently, a highly efficient reverse genetics system was developed that allows genetic manipulation of the rotavirus segmented double-stranded RNA genome. Using the reverse genetics system, we show that it is possible to modify one of the rotavirus genome segments (segment 7) such that virus gains the capacity to express a separate heterologous protein in addition to the full complement of viral proteins. Through this approach, we have generated wild-type-like rotaviruses that express various fluorescent reporter proteins, including UnaG (green), mRuby (far red), mKate (red), and TagBFP (blue). Such strains will be of value in probing rotavirus biology and pathogenesis by live cell imagining techniques. Notably, our work indicates that the rotavirus genome is remarkably flexible and able to accommodate significant amounts of heterologous RNA sequence, raising the possibility of using the virus as a vaccine expression vector.

Discrete Virus Factories Form in the Cytoplasm of Cells Coinfected with Two Replication-Competent Tagged Reporter Birnaviruses That Subsequently Coalesce over Time.

The Birnaviridae family, responsible for major economic losses to poultry and aquaculture, is composed of nonenveloped viruses with a segmented double-stranded RNA (dsRNA) genome that replicate in discrete cytoplasmic virus factories (VFs). Reassortment is common; however, the underlying mechanism remains unknown given that VFs may act as a barrier to genome mixing. In order to provide new information on VF trafficking during dsRNA virus coinfection, we rescued two recombinant infectious bursal disease viruses (IBDVs) of strain PBG98 containing either a split GFP11 or a tetracysteine (TC) tag fused to the VP1 polymerase (PBG98-VP1-GFP11 and PBG98-VP1-TC). DF-1 cells transfected with GFP1-10 prior to PBG98-VP1-GFP11 infection or stained with a biarsenical derivative of the red fluorophore resorufin (ReAsH) following PBG98-VP1-TC infection, had green or red foci in the cytoplasm, respectively, that colocalized with VP3 and dsRNA, consistent with VFs. The average number of VFs decreased from a mean of 60 to 5 per cell between 10 and 24 h postinfection (hpi) (P < 0.0001), while the average area increased from 1.24 to 45.01 μm2 (P < 0.0001), and live cell imaging revealed that the VFs were highly dynamic structures that coalesced in the cytoplasm. Small VFs moved faster than large (average 0.57 μm/s at 16 hpi compared to 0.22 μm/s at 22 hpi), and VF coalescence was dependent on an intact microtubule network and actin cytoskeleton. During coinfection with PBG98-VP1-GFP11 and PBG98-VP1-TC viruses, discrete VFs initially formed from each input virus that subsequently coalesced 10 to 16 hpi, and we speculate that Birnaviridae reassortment requires VF coalescence.IMPORTANCE Reassortment is common in viruses with segmented double-stranded RNA (dsRNA) genomes. However, these viruses typically replicate within discrete cytoplasmic virus factories (VFs) that may represent a barrier to genome mixing. We generated the first replication competent tagged reporter birnaviruses, infectious bursal disease viruses (IBDVs) containing a split GFP11 or tetracysteine (TC) tag and used the viruses to track the location and movement of IBDV VFs, in order to better understand the intracellular dynamics of VFs during a coinfection. Discrete VFs initially formed from each virus that subsequently coalesced from 10 h postinfection. We hypothesize that VF coalescence is required for the reassortment of the Birnaviridae This study provides new information that adds to our understanding of dsRNA virus VF trafficking.

PCR-based reverse genetics strategy for bluetongue virus recovery

BackgroundBluetongue virus (BTV), an emerging insect vector mediated pathogen affecting both wild ruminants and livestock, has a genome consisting of 10 linear double-stranded RNA genome segments. BTV has a severe economic impact on agriculture in many parts of the world. Current reverse genetics (RG) strategy to rescue BTV mainly rely on in vitro synthesis of RNA transcripts from cloned complimentary DNA (cDNA) corresponding to viral genome segments with the aid of helper plasmids. RNA synthesis is a laborious job which is further complicated with a need for expensive reagents and a meticulous operational procedure. Additionally, the target genes must be cloned into a specific vector to prepare templates for RNA transcription.ResultIn this study, we have developed a PCR based BTV RG system with easy two-step transfection. Viable viruses were recovered following a first transfection with the seven helper plasmids and a second transfection with the 10 PCR products on the BSR cells. Further, recovered viruses were characterized with indirect immunofluorescence assays (IFA) and gene sequencing. And the proliferation properties of these viruses were also compared with wild type BTV. Interestingly, we have identified that viruses containing the segment 2 of the genome from reassortant BTV, grew slightly slower than the others.ConclusionIn this study, a convenient PCR based RG platform for BTV is established, and this strategy could be an effective alternative to the original available BTV rescue methods. Furthermore, this RG strategy is likely applicable for other Orbiviruses.

Bluetongue Virus Nonstructural Protein 3 Orchestrates Virus Maturation and Drives Non-Lytic Egress via Two Polybasic Motifs.

Bluetongue virus (BTV) is an arthropod-borne virus that infects domestic and wild ruminants. The virion is a non-enveloped double-layered particle with an outer capsid that encloses a core containing the segmented double-stranded RNA genome. Although BTV is canonically released by cell lysis, it also exits non-lytically. In infected cells, the BTV nonstructural glycoprotein 3 (NS3) is found to be associated with host membranes and traffics from the endoplasmic reticulum through the Golgi apparatus to the plasma membrane. This suggests a role for NS3 in BTV particle maturation and non-lytic egress. However, the mechanism by which NS3 coordinates these events has not yet been elucidated. Here, we identified two polybasic motifs (PMB1/PMB2), consistent with the membrane binding. Using site-directed mutagenesis, confocal and electron microscopy, and flow cytometry, we demonstrated that PBM1 and PBM2 mutant viruses retained NS3 either in the Golgi apparatus or in the endoplasmic reticulum, suggesting a distinct role for each motif. Mutation of PBM2 motif decreased NS3 export to the cell surface and virus production. However, both mutant viruses produced predominantly inner core particles that remained close to their site of assembly. Together, our data demonstrates that correct trafficking of the NS3 protein is required for virus maturation and release.

Identification of a Small Molecule That Compromises the Structural Integrity of Viroplasms and Rotavirus Double-Layered Particles.

Despite the availability of two attenuated vaccines, rotavirus (RV) gastroenteritis remains an important cause of mortality among children in developing countries, causing about 215,000 infant deaths annually. Currently, there are no specific antiviral therapies available. RV is a nonenveloped virus with a segmented double-stranded RNA genome. Viral genome replication and assembly of transcriptionally active double-layered particles (DLPs) take place in cytoplasmic viral structures called viroplasms. In this study, we describe strong impairment of the early stages of RV replication induced by a small molecule known as an RNA polymerase III inhibitor, ML-60218 (ML). This compound was found to disrupt already assembled viroplasms and to hamper the formation of new ones without the need for de novo transcription of cellular RNAs. This phenotype was correlated with a reduction in accumulated viral proteins and newly made viral genome segments, disappearance of the hyperphosphorylated isoforms of the viroplasm-resident protein NSP5, and inhibition of infectious progeny virus production. In in vitro transcription assays with purified DLPs, ML showed dose-dependent inhibitory activity, indicating the viral nature of its target. ML was found to interfere with the formation of higher-order structures of VP6, the protein forming the DLP outer layer, without compromising its ability to trimerize. Electron microscopy of ML-treated DLPs showed dose-dependent structural damage. Our data suggest that interactions between VP6 trimers are essential, not only for DLP stability, but also for the structural integrity of viroplasms in infected cells.IMPORTANCE Rotavirus gastroenteritis is responsible for a large number of infant deaths in developing countries. Unfortunately, in the countries where effective vaccines are urgently needed, the efficacy of the available vaccines is particularly low. Therefore, the development of antivirals is an important goal, as they might complement the available vaccines or represent an alternative option. Moreover, they may be decisive in fighting the acute phase of infection. This work describes the inhibitory effect on rotavirus replication of a small molecule initially reported as an RNA polymerase III inhibitor. The molecule is the first chemical compound identified that is able to disrupt viroplasms, the viral replication machinery, and to compromise the stability of DLPs by targeting the viral protein VP6. This molecule thus represents a starting point in the development of more potent and less cytotoxic compounds against rotavirus infection.

Genetic determinants restricting the reassortment of heterologous NSP2 genes into the simian rotavirus SA11 genome

Rotaviruses (RVs) can evolve through the process of reassortment, whereby the 11 double-stranded RNA genome segments are exchanged among strains during co-infection. However, reassortment is limited in cases where the genes or encoded proteins of co-infecting strains are functionally incompatible. In this study, we employed a helper virus-based reverse genetics system to identify NSP2 gene regions that correlate with restricted reassortment into simian RV strain SA11. We show that SA11 reassortants with NSP2 genes from human RV strains Wa or DS-1 were efficiently rescued and exhibit no detectable replication defects. However, we could not rescue an SA11 reassortant with a human RV strain AU-1 NSP2 gene, which differs from that of SA11 by 186 nucleotides (36 amino acids). To map restriction determinants, we engineered viruses to contain chimeric NSP2 genes in which specific regions of AU-1 sequence were substituted with SA11 sequence. We show that a region spanning AU-1 NSP2 gene nucleotides 784–820 is critical for the observed restriction; yet additional determinants reside in other gene regions. In silico and in vitro analyses were used to predict how the 784–820 region may impact NSP2 gene/protein function, thereby informing an understanding of the reassortment restriction mechanism.

Molecular insights into RNA-binding properties of Escherichia coli–expressed RNA-dependent RNA polymerase of Antheraea mylitta cytoplasmic polyhedrosis virus

Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) is responsible for morbidity of the Indian non-mulberry silkworm, A. mylitta. AmCPV belongs to the family Reoviridae and has 11 double-stranded (ds) RNA genome segments (S1-S11). Segment 2 (S2) encodes a 123-kDa polypeptide with RNA-dependent RNA polymerase (RdRp) activity. To examine the RNA-binding properties of the viral polymerase, the full-length RdRp and its three domains (N-terminal, polymerase and C-terminal domains) were expressed in Escherichia coli BL21 (DE3) cells with hexahistidine and trigger factor tag fused consecutively at its amino terminus, and the soluble fusion proteins were purified. The purified full-length polymerase specifically bound to the 3' untranslated region (3'-UTR) of a viral plus-sense (+) strand RNA with strong affinity regardless of the salt concentrations, but the isolated polymerase domain of the enzyme exhibited poor RNA-binding ability. Further, the RdRp recognition signals were found to be different from the cis-acting signals that promote minus-sense (-) strand RNA synthesis, because different internal regions of the 3'-UTR of the (+) strand RNA did not effectively compete out the binding of RdRp to the intact 3'-UTR of the (+) strand RNA, but all of these RNA molecules could serve as templates for (-) strand RNA synthesis by the polymerase.