dsRNA Segments Research Articles

Characterization of a Novel Pathogenic Reovirus in Grasshoppers.

Grasshoppers can swarm in the millions and destroy crops over wide areas, posing a major economic threat to agriculture. A wide range of insect-related viruses has recently been reported in the metagenomics of grasshoppers. Here, we identified and isolated a novel reovirus from grasshoppers, named Acrididae reovirus (ARV). The complete genome of ARV was composed of nine dsRNA segments. Phylogenetic analysis revealed that ARV formed a monophyletic lineage with unclassified insect-associated reoviruses and was sufficiently distinct from known genera of Reoviridae. ARV could replicate in its host Locusta migratoria and result in host death. Lower-dose ARV infection affected ovary development and resulted in a significant reduction in fecundity. The identification and characterization of a novel pathogenic reovirus could potentially promote the development of new biological control agents.

ICTV Virus Taxonomy Profile: Spinareoviridae 2022

Spinareoviridae is a large family of icosahedral viruses that are usually regarded as non-enveloped with segmented (9-12 linear segments) dsRNA genomes of 23-29 kbp. Spinareovirids have a broad host range, infecting animals, fungi and plants. Some have important pathogenic potential for humans (e.g. Colorado tick fever virus), livestock (e.g. avian orthoreoviruses), fish (e.g. aquareoviruses) and plants (e.g. rice ragged stunt virus and rice black streaked dwarf virus). This is a summary of the ICTV Report on the family Spinareoviridae, which is available at ictv.global/report/spinareoviridae.

Isolation and characterization of a duck reovirus strain from mature ducks in China

In 2018, a disease characterized by splenic hemorrhage and necrosis killed ducks in a duck farm in Guangxi province, China. A duck reovirus strain was isolated from the tissues of the dead ducks by inoculating duck embryos and BHK-21 cells. Electron microscopy of the cultured the isolate showed that the viral particles were nearly round in shape and approximately 70 nm in diameter, and they were designated DRV-GL18. Sequence analysis showed that the GL18 strain viral genome was 23,419 nt in length and had 10 dsRNA segments. Phylogenetic analysis of cDNA amplicons of segments encoding the protein σC which are outer capsid proteins showed that the isolate belongs to the branch of the epidemic strains of duck reovirus. The Recombination Detection Program (RDP) and SimPlot program analyses suggested potential genetic recombination events in the M2 segments. Pathogenicity experiments revealed that GL18 produced severe hemorrhaging in livers and necrosis in the spleen of infected SPF ducklings. A death rate of 50% in the experimental ducklings was calculated during the first 7 d, and the rest of the ducklings were observed to undergo spleen necrosis. These data suggested that GL18 is a duck reovirus isolate with severer pathogenicity, and it could be a candidate for development of vaccine. This is the first reported isolation of duck reovirus from mature ducks.

Genetic characterization of a novel pheasant-origin orthoreovirus using Next-Generation Sequencing.

A field isolate (Reo/SDWF /Pheasant/17608/20) of avian orthoreovirus (ARV), isolated from a flock of game-pheasants in Weifang, Shandong Province, was genetically characterized being a field variant or novel strain in our recent research studies in conducting whole genome sequencing by using Next-Generation Sequencing (NGS) technique on Illumina MiSeq platform. Among a total of 870,197 35-151-mer sequencing reads, 297,711 reads (34.21%) were identified as ARV sequences. The de novo assembly of the ARV reads resulted in generation of 10 ARV-related contigs with the average sequencing coverage from 1390× to 1977× according to 10 ARV genome segments. The complete genomes of this pheasant-origin ARV (Reo/SDWF /Pheasant/17608/20) were 23,495 bp in length and consist of 10 dsRNA segments ranged from 1192 bp (S4) to 3958 bp (L1) encoding 12 viral proteins. Sequence comparison between the SDWF17608 and classic ARV reference strains revealed that 58.1-100% nucleotide (nt) identities and 51.4-100% amino acid (aa) identities were in genome segment coding genes. The 10 RNA segments had conversed termini at 5' (5'-GCUUUU) and 3' (UCAUC-3') side, which were identical to the most published ARV strains. Phylogenetic analysis revealed that this pheasant ARV field variant was closely related with chicken ARV strains in 7 genome segment genes, but it possessed significant sequence divergence in M1, M3 and S2 segments. These findings suggested that this pheasant-origin field variant was a divergent ARV strain and was likely originated from reassortments between different chicken ARV strains.

Evolution of Animal South American RVA Told by the NSP4 Gene E12 Genotype.

Rotavirus A (RVA) possesses a genome of 11 double-stranded (ds) RNA segments, and each segment encodes one protein, with the exception of segment 11. NSP4 is a non-structural multifunctional protein encoded by segment 10 that defines the E-genotype. From the 31 E-genotypes described, genotype E12 has been described in Argentina, Uruguay, Paraguay, and Brazil in RVA strains infecting different animal species and humans. In this work, we studied the evolutionary relationships of RVA strains carrying the E12 genotype in South America using phylogenetic and phylodynamic approaches. We found that the E12 genotype has a South American origin, with a guanaco (Lama guanicoe) strain as natural host. Interestingly, all the other reported RVA strains carrying the E12 genotype in equine, bovine, caprine, and human strains are related to RVA strains of camelid origin. The evolutionary path and genetic footprint of the E12 genotype were reconstructed starting with the introduction of non-native livestock species into the American continent with the Spanish conquest in the 16th century. The imported animal species were in close contact with South American camelids, and the offspring were exposed to the native RVA strains brought from Europe and the new RVA circulating in guanacos, resulting in the emergence of new RVA strains in the current lineages' strongly species-specific adaption. In conclusion, we proposed the NSP4 E12 genotype as a genetic geographic marker in the RVA strains circulating in different animal species in South America.

Molecular characterization of a novel polymycovirus identified in the phytopathogenic fungus Colletotrichum gloeosporioides.

A novel polymycovirus isolated from the plant-pathogenic fungus Colletotrichum gloeosporioides was identified. The viral genome is composed of nine double-stranded RNA segments, ranging in size from 699 bp to 2,444 bp. With the exception of dsRNA5, which contains two open reading frames (ORF5-1 and ORF5-2), the other dsRNA segments each contain one ORF. The proteins encoded by ORFs 1-8 are homologous to the proteins encoded by ORFs 1-8 of Colletotrichum camelliae filamentous virus 1 (CcFV-1). The amino acid sequences of the RNA-dependent RNA polymerase (RdRp) encoded by ORF1 and the viral methyltransferase encoded by ORF3 share 87.6% and 83.3% identity with CcFV-1. The proline-alanine-serine-rich protein (PASrp) encoded by ORF4 shares 86.6% sequence identity with that of CcFV-1. The proteins encoded by ORFs 2, 5 - 1, 6, 7, and 8 share 86.6%, 82.5%, 89.0%, 45.7%, and 95.5% sequence identity, respectively, with the corresponding proteins of CcFV-1. dsRNA9 is a defective copy of dsRNA2 that lacks a stretch of 1556 bp (nt 519 to nt 2074). Phylogenetic analysis based on the RdRp protein indicated that the novel virus clustered with members of the family Polymycoviridae, and based on the above results, we have tentatively named it "Colletotrichum gloeosporioides polymycovirus virus 1" (CgPmV1). To our knowledge, this is the first report of a polymycovirus with a defective dsRNA genome in C. gloeosporioides.

ICTV Virus Taxonomy Profile: Sedoreoviridae 2022.

Sedoreoviridae is a large family of icosahedral viruses that are usually regarded as non-enveloped with segmented (10-12 linear segments) dsRNA genomes of 18-26 kbp. Sedoreovirids have a broad host range, infecting mammals, birds, crustaceans, arthropods, algae and plants. Some of them have important pathogenic potential for humans (e.g. rotavirus A), livestock (e.g. bluetongue virus) and plants (e.g. rice dwarf virus). This is a summary of the ICTV Report on the family Sedoreoviridae, which is available at ictv.global/report/sedoreoviridae.

Molecular characterization and transcriptomic analysis of a novel polymycovirus in the fungus Talaromyces amestolkiae.

Talaromyces amestolkiae is an important fungal species owing to its ubiquity in soils, plants, air, and food. In this study, we identified a novel six-segmented polymycovirus, Talaromyces amestolkiae polymycovirus 1 (TaPmV-1). Each of the double-stranded (ds) RNA segments of TaPmV-1 contained a single open reading frame, and the proteins encoded by dsRNA1, dsRNA2, dsRNA3, and dsRNA 5 shared significant amino acid identities of 56, 40, 47, and 43%, respectively, with the corresponding proteins of Aspergillus fumigatus polymycovirus-1(AfuPmV-1). DsRNA1, dsRNA3, and dsRNA5 of TaPmV-1 encoded an RNA-dependent RNA polymerase (RdRp), a viral methyltransferase, and a PAS-rich protein, respectively. The functions of the proteins encoded by dsRNA2, dsRNA4, and dsRNA6 have not been elucidated. Comparison of the virus-infected strain LSH3 with virus-cured strain LSHVF revealed that infection with TaPmV-l may reduce the production of red pigments and induce the clustering of fungal sclerotia. Furthermore, transcriptomic analyses demonstrated that infection with TaPmV-l downregulated the expression of transcripts related to metabolism, and may correlate with the reduced production of red pigments and clustering of sclerotia in T. amestolkiae. These results of this study provide novel insights into the mechanism of fungal gene regulation by polymycovirus infections at the transcriptome level, and this study is the first to report a novel polymycovirus of T. amestolkiae.

Extending the Host Range of Fusarium Poae Virus 1 from Fusarium poae to other Fusarium Species in the Field.

Fusarium poae virus 1 (FpV1, a betapartitivirus) is one of the mycoviruses which is discovered earlier. Due to the vegetative incompatibility barrier that often exists between different species or strains of filamentous fungi, FpV1 has been thought to be limited to its host, F. poae, as a non-hypovirulence mycovirus in the past 20 years in the field. Here, a novel strain of FpV1 (FpV1-Fa) with two dsRNA segments (2157-and 2080-nt) was consistently identified in F. asiaticum isolates in the field. FpV1-Fa induced abnormal morphology and hypovirulence of F. asiaticum, along with a high viral load. FpV1-Fa was detected only from the F. asiaticum and F. tricinctum strains at a FpV1-Fa sampling site (119.014289, 33.8261), while the other strains from other sites were not identified FpV1-Fa. A horizontal transmission experiment showed that FpV1-Fa can transfer from F. asiaticum to F. poae and F. tricinctum, but not to F. graminearum. The selection analysis of FpV1-Fa revealed RdRP and CP were under strong purifying selection, and the C-terminal side of RdRP was under positive selection. In these regions, 9 amino acid mutations in RdRP and 21 mutations in CP appeared to cause the variation of host range and virulence in FpV1-Fa.

In vitro particle-associated uridyltransferase activity of the rotavirus VP1 polymerase

Rotaviruses are 11-segmented, double-stranded RNA (dsRNA) viruses with a unique intra-particle RNA synthesis mechanism. During genome replication, the RNA-dependent RNA polymerase (VP1) performs minus-strand RNA (-ssRNA) synthesis on positive-strand RNA (+ssRNA) templates to create dsRNA segments. Recombinant VP1 catalyzes -ssRNA synthesis using substrate NTPs in vitro, but only when the VP2 core shell protein or virus-like particles made of VP2 and VP6 (2/6-VLPs) are included in the reaction. The dsRNA product can be labeled using [α32P]-UTP and separated from the input +ssRNA template by polyacrylamide gel electrophoresis. Here, we report the generation of [α32P]-labeled rotavirus +ssRNA templates in reactions that lacked non-radiolabeled NTPs but contained catalytically-active VP1, 2/6-VLPs, and [α32P]-UTP. Non-radiolabeled UTP competed with [α32P]-UTP to decrease product levels, whereas CTP and GTP had little effect. Interesting, ATP stimulated [α32P]-labeled product production. These results suggest that rotavirus VP1 transferred [α32P]-UMP onto viral + ssRNA in vitro via a particle-associated uridyltransferase activity.

Comparative Genome Analysis of All Nine African Horse Sickness Serotypes Isolated From Equine Fatalities in Kenya and South Africa

African horse sickness (AHS) is a viral disease of equids, caused by a virus of the genus Orbivirus, family Reoviridae. The African horse sickness virus (AHSV) genome is made up of ten double-stranded RNA (dsRNA) segments that together code for seven structural and four nonstructural proteins. AHS is endemic in sub-Saharan countries. The efficacy and safety of inactivated AHS vaccines containing all nine serotypes, produced at the Central Veterinary Research Laboratory (CVRL) in Dubai, United Arab Emirates have been proven in the past. All nine AHSV serotypes were isolated from 102 samples collected in the last 20 years from horse fatalities in seven different area of Kenya, Africa. CVRL inactivated AHS vaccines are used in a few African countries defining the importance of this present study to compare the genome sequences of the nine AHSV serotypes isolated from horse fatalities in Kenya and nine AHSV serotypes isolated in South Africa. The hypothesized serotypes of the newly sequenced AHSV field strains from Kenya were likewise confirmed in this investigation, and they show substantial sequence homologies with recently isolated AHSV field strains.

Complete genome sequence of a novel chrysovirus infecting Talaromyces neofusisporus.

A double-stranded RNA (dsRNA) mycovirus was isolated from Talaromyces neofusisporus isolate HJ1-6 and named "Talaromyces neofusisporus chrysovirus 1" (TnCV1). It was found to consist of four dsRNA segments (TnCV1-1, TnCV1-2, TnCV1-3, and TnCV1-4) with lengths of 3595 bp, 3063 bp, 3054 bp, and 2876 bp, respectively. Sequence analysis showed that TnCV1-1 contains an open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) of 1136 amino acids (aa), TnCV1-2 contains an ORF encoding a hypothetical protein of 906 aa, TnCV1-3 contains an ORF encoding a putative capsid protein (CP) of 938 aa, and TnCV1-4 contains an ORF encoding a hypothetical protein of 849 aa. The 5' and 3' untranslated regions (UTRs) of TnCV1-1, TnCV1-2, TnCV1-3, and TnCV1-4 showed a high degree of sequence similarity to each other. Phylogenetic analysis based on RdRp sequences suggested that TnCV1 is a new member of the genus Alphachrysovirus in the family Chrysoviridae. This is the first chrysovirus isolated from T. neofusisporus.

Molecular characterization of a novel partitivirus isolated from Rhizoctonia solani.

Rhizoctonia solani is a widely distributed plant pathogen that can damage many crops. Here, we identified a novel mycovirus tentatively named Rhizoctonia solani partitivirus 433 (RsPV433) from an R. solani (AG-3) strain which caused tobacco target spot disease on flue-cured tobacco. RsPV433 was consisted of two dsRNA segments with lengths of 2450 and 2273 bp, which encoded an RNA-dependent RNA polymerase and a coat protein, respectively. BLASTP results of RsPV433 showed that the closest relative of RsPV433 was Sarcosphaera coronaria partitivirus (QLC36830.1), with an identity of 60.85% on the RdRp amino sequence. Phylogenetic analysis indicated that RsPV433 belonged to the Betapartitivirus genus in the Partitiviridae family. The virus transmission experiment revealed that RsPV433 can be transmitted horizontally. We further tested the biological effect of RsPV433 on R. solani strains and found that the RsPV433-infected R. solani strain grew slower than the RsPV433-free strain on the PDA medium and RsPV433 seemed to have no obvious impact on the lesion inducing ability of R. solani.

A Satellite dsRNA Attenuates the Induction of Helper Virus-Mediated Symptoms in Aspergillus flavus.

Aspergillus flavus is an important fungal pathogen of animals and plants. Previously, we reported a novel partitivirus, Aspergillus flavus partitivirus 1 (AfPV1), infecting A. flavus. In this study, we obtained a small double-stranded (ds) RNA segment (734 bp), which is a satellite RNA of the helper virus, AfPV1. The presence of AfPV1 altered the colony morphology, decreased the number of conidiophores, created significantly larger vacuoles, and caused more sensitivity to osmotic, oxidative, and UV stresses in A. flavus, but the small RNA segment could attenuate the above symptoms caused by the helper virus AfPV1 in A. flavus. Moreover, AfPV1 infection reduced the pathogenicity of A. flavus in corn (Zea mays), honeycomb moth (Galleria mellonella), mice (Mus musculus), and the adhesion of conidia to host epithelial cells, and increased conidial death by macrophages. However, the small RNA segment could also attenuate the above symptoms caused by the helper virus AfPV1 in A. flavus, perhaps by reducing the genomic accumulation of the helper virus AfPV1 in A. flavus. We used this model to investigate transcriptional genes regulated by AfPV1 and the small RNA segment in A. flavus, and their role in generating different phenotypes. We found that the pathways of the genes regulated by AfPV1 in its host were similar to those of retroviral viruses. Therefore, some pathways may be of benefit to non-retroviral viral integration or endogenization into the genomes of its host. Moreover, some potential antiviral substances were also found in A. flavus using this system.

Molecular characterization of a novel alternavirus infecting the entomopathogenic fungus Cordyceps chanhua.

In this study, a novel double-stranded (ds) RNA mycovirus, named Cordyceps chanhua alternavirus 1 (CcAV1), was detected in the entomogenous fungus Cordyceps chanhua in China and characterized. The complete genome of CcAV1 is composed of three dsRNA segments: dsRNA 1 (3,512 bp), dsRNA 2 (2,655 bp), and dsRNA 3 (2,415 bp). Each of the three dsRNAs possesses a single open reading frame (ORF). dsRNA 1 encodes a putative RNA-dependent RNA polymerase (RdRp), and dsRNA 2 and dsRNA 3 encode hypothetical protein 1 (HP 1) and hypothetical protein 2 (HP 2), respectively. The predicted amino acid sequences of the putative RdRp, HP 1, and HP 2 had the highest identity of 66.99%, 49.30%, and 56.91%, respectively, to those of Aspergillus foetidus dsRNA mycovirus. A maximum-likelihood phylogenetic tree based on RdRp amino acid sequences showed that CcAV1 clustered with members of the proposed family "Alternaviridae". Hence, we propose that Cordyceps chanhua alternavirus 1 is a novel member of the proposed family "Alternaviridae".

Characterization of a novel mitovirus isolated from the phytopathogenic fungus Fusarium pseudograminearum.

Mycoviruses are widespread in all major groups of plant-pathogenic fungi. So far, only one mycovirus has been reported to be associated with Fusarium pseudograminearum, the causal agent of Fusarium crown rot of wheat. In this study, a double-stranded RNA (dsRNA) segment was isolated from F. pseudograminearum strain JW2-1, and the sequence of its full-length cDNA (3077 nucleotides) was determined. Sequence analysis using the fungal mitochondrial genetic code (UGA coding for tryptophan) indicated that a single large open reading frame (ORF) is present on the positive strand of this dsRNA segment. The ORF encodes a putative RNA-dependent RNA polymerase (RdRp) of 748 amino acids (aa) with a molecular mass of 83.46 kDa. BLASTp analysis revealed that its aa sequence was 28.49-44.03% identical to those of viruses of the family Mitoviridae, with the most similarity to the corresponding RdRp sequences of Ophiostoma mitovirus 1c (44.03% identity) and Ophiostoma mitovirus 1b (40.33% identity). Phylogenetic analysis showed that this mycovirus, designated as "Fusarium pseudograminearummitovirus 1" (FpgMV1), should be classified as a member of a new species in the earlier proposed genus "Duamitovirus" within the family Mitoviridae. To our best of our knowledge, this is the first report of a mitovirus infecting F. pseudograminearum.

The RNA-dependent RNA polymerase of the infectious pancreatic necrosis virus is linked to viral mRNA acting as a cap substitute.

The infectious pancreatic necrosis virus (IPNV) is responsible for significant economic losses in the aquaculture industry. It is an unenveloped virus with an icosahedral capsid. Its viral genome comprises two dsRNA segments, A and B. Segment A contains a small ORF, which encodes VP5, and a large ORF, which encodes a polyprotein that generates the structural proteins and the viral protease. Segment B encodes the RNA-dependent RNA polymerase (RdRp), called VP1 in this free form, or Vpg when it covalently attaches to the viral RNA. The viral genome does not have cap or poly(A). Instead, each 5' end is linked to the Vpg. Recently, we demonstrated that mRNA-A contains an internal ribosome entry site (IRES) to command polyprotein synthesis. However, the presence of Vpg on IPNV mRNAs and its impact on cellular translation has not been investigated. This research demonstrates that IPNV mRNAs are linked to Vpg and that this protein inhibits cap-dependent translation on infected cells. Also, it is demonstrated that Vpg interacts with eIF4E and that rapamycin treatment partially diminishes the viral protein synthesis. In addition, we determined that an IRES does not command translation of IPNV mRNA-B. We show that VPg serves as a cap substitute during the initiation of IPNV translation, contributing to understanding the replicative cycle of Birnaviruses. Our results indicate that the viral protein VP1/Vpg is multifunctional, having a significant role during IPNV RNA synthesis as the RdRp and the primer for IPNV RNA synthesis and translation as the viral protein genome, acting as a cap substitute.

Investigation of avian rotavirus infections in broiler chicks from commercial flocks with different performance efficiency indexes.

The aim of this study was to investigate and compare the frequency of occurrence of avian rotavirus (AvRV) in poultry flocks according to its Performance Efficiency Index (PEI) scores. A total of 256 individual intestinal content samples of small sized-chicks (runts) with clinical signs of Runting Stunting Syndrome (RSS) and 24 clinically healthy chicks (control) were collected from twelve flocks in southern Brazil with different PEI scores: good (n = 4, PEI mean = 365); moderate (n = 4, PEI mean = 342) or poor (n = 4, PEI mean = 319). Silver-stained polyacrylamide gel electrophoresis (ss-PAGE) was used to detect and identify the AvRV species followed by RT-PCR and sequencing of the partial VP6 gene for species confirmation. AvRV was detected in 83% (10/12) of the flocks and 23.4% (60/256) of the chicks. The electrophoretic migration patterns of viral dsRNA segments were compatible with AvRV species A (AvRV- A), D (AvRV-D) and F (AvRV-F) in 9 (15%), 18 (30%), and 33 (55%) of the positive chicks fecal samples, respectively. The AvRV species identified by ss-PAGE were confirmed by RT-PCR and partial sequence analysis of the VP6 gene. The AvRV detection rate was statistically higher (p = 0.007) in chicks from flocks with poor PEI when compared to those with good PEI. The occurrence of AvRV-D and AvRV-F was statistically higher in 7 to 9 days old chicks, while AvRV-A was detected only in 13 to 14 days old animals.

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.

Characterization of a Novel Mycovirus from the Phytopathogenic Fungus Botryosphaeria dothidea.

Botryosphaeria dothidea is, globally, one of the most economically important phytopathogenic fungi worldwide, causing the canker and dieback of fruit trees. An increasing number of viruses infecting B. dothidea have lately been reported, several of which could confer hypovirulence. In this study, isolated from strain ZM170285-1 of B. dothidea, a novel double-stranded RNA (dsRNA) mycovirus, tentatively named Botryosphaeria dothidea partitivirus 2 (BdPV2), was identified well. The BdPV2 harbored three dsRNA segments (1–3) with lengths of 1751, 1568, and 1198 bp, which encoded an RNA-dependent RNA polymerase (RdRp), a capsid protein (CP), and a hypothetical protein of unknown function, respectively. BLASTp searches revealed that the predicted protein sequences of dsRNA1 and dsRNA2 had the highest identities (74.95% and 61.01%) with the corresponding dsRNAs of Penicillium stoloniferum virus S (PsV-S), whereas dsRNA3 shared the highest identity (32.95%) with the dsRNA3 of Aspergillus ochraceous virus 1 (AoV1). Phylogenetic analysis indicated that BdPV2 belonged to the Gammapartitivirus genus and Partitiviridae family. To our knowledge, this is the first report of a Gammapartitivirus in B. dothidea.