dsRNA Genome Segments Research Articles

Detection and Characterization of Cucumis melo Cryptic Virus, Cucumis melo Amalgavirus 1, and Melon Necrotic Spot Virus in Cucumis melo.

Three RNA viruses—Cucumis melo cryptic virus (CmCV), Cucumis melo amalgavirus 1 (CmAV1), and melon necrotic spot virus (MNSV)—were identified from a melon (Cucumis melo) transcriptome dataset. CmCV has two dsRNA genome segments; dsRNA-1 is 1592 bp in size, containing a conserved RNA-dependent RNA polymerase (RdRp), and dsRNA-2 is 1715 bp in size, and encodes a coat protein (CP). The sequence alignment and phylogenetic analyses of the CmCV RdRp and CP indicated CmCV clusters with approved or putative deltapartitiviruses in well-supported monophyletic clade. The RdRp of CmCV shared an amino acid sequence identity of 60.7% with the closest RdRp of beet cryptic virus 3, and is <57% identical to other partitiviruses. CmAV1 is a nonsegmented dsRNA virus with a genome of 3424 bp, including two partially overlapping open reading frames (ORFs) encoding a putative CP and RdRp. The sequence alignment and phylogenetic analyses of CmAV1 RdRp revealed that it belongs to the genus Amalgavirus in the family Amalgaviridae. The RdRp of CmAV1 shares 57.7% of its amino acid sequence identity with the most closely related RdRp of Phalaenopsis equestris amalgavirus 1, and is <47% identical to the other reported amalgaviruses. These analyses suggest that CmCV and CmAV1 are novel species in the genera Amalgavirus and Deltapartitivirus, respectively. These findings enrich our understanding of new plant dsRNA virus species.

Quantitative RT-PCR assays for identification and typing of the Equine encephalosis virus.

Equine encephalosis (EE) is an acute, arthropod-borne, noncontagious, febrile disease of equids. The clinical signs of EE are similar to milder forms of African horse sickness (AHS) and the two diseases can be easily confused. The Equine encephalosis virus (EEV) is a distinct virus species within the genus Orbivirus, family Reoviridae, with ten linear segments of dsRNA genome. Seven distinct serotypes of EEV have been recognised on the basis of sequence analyses of Seg-2. The need for differential diagnosis of similar forms of EE and AHS warranted the development of molecular diagnostic methods for specific detection and identification of EEV. We report the development of quantitative real-time RT-PCR assay for detection of any member of the EEV species targeting the highly conserved EEV Seg-9. Similar serotype-specific qRT-PCR assays were designed for each of the seven EEV serotypes targeting genome Seg-2, encoding the serotype determining VP2 protein. These assays were evaluated using different EEV serotypes and other closely related orbiviruses. They were shown to be EEV virus species-specific, or EEV type-specific capable of detecting 1 to 13 copies of viral RNA in clinical samples. The assays failed to detect RNA from closely related orbiviruses, including AHSV and Peruvian horse sickness virus (PHSV) isolates.

In Situ Structures of the Polymerase Complex and RNA Genome Show How Aquareovirus Transcription Machineries Respond to Uncoating.

Reoviruses carry out genomic RNA transcription within intact viruses to synthesize plus-sense RNA strands, which are capped prior to their release as mRNA. The in situ structures of the transcriptional enzyme complex (TEC) containing the RNA-dependent RNA polymerase (RdRp) and NTPase are known for the single-layered reovirus cytoplasmic polyhedrosis virus (CPV), but not for multilayered reoviruses, such as aquareoviruses (ARV), which possess a primed stage that CPV lacks. Consequently, how the RNA genome and TEC respond to priming in reoviruses is unknown. Here, we determined the near-atomic-resolution asymmetric structure of ARV in the primed state by cryo-electron microscopy (cryo-EM), revealing the in situ structures of 11 TECs inside each capsid and their interactions with the 11 surrounding double-stranded RNA (dsRNA) genome segments and with the 120 enclosing capsid shell protein (CSP) VP3 subunits. The RdRp VP2 and the NTPase VP4 associate with each other and with capsid vertices; both bind RNA in multiple locations, including a novel C-terminal domain of VP4. Structural comparison between the primed and quiescent states showed translocation of the dsRNA end from the NTPase to the RdRp during priming. The RNA template channel was open in both states, suggesting that channel blocking is not a regulating mechanism between these states in ARV. Instead, the NTPase C-terminal domain appears to regulate RNA translocation between the quiescent and primed states. Taking the data together, dsRNA viruses appear to have adapted divergent mechanisms to regulate genome transcription while retaining similar mechanisms to coassemble their genome segments, TEC, and capsid proteins into infectious virions.IMPORTANCE Viruses in the family Reoviridae are characterized by the ability to endogenously synthesize nascent RNA within the virus. However, the mechanisms for assembling their RNA genomes with transcriptional enzymes into a multilayered virion and for priming such a virion for transcription are poorly understood. By cryo-EM and novel asymmetric reconstruction, we determined the atomic structure of the transcription complex inside aquareoviruses (ARV) that are primed for infection. The transcription complex is anchored by the N-terminal segments of enclosing capsid proteins and contains an NTPase and a polymerase. The NTPase has a newly discovered domain that translocates the 5' end of plus-sense RNA in segmented dsRNA genomes from the NTPase to polymerase VP2 when the virus changes from the inactive (quiescent) to the primed state. Conformation changes in capsid proteins and transcriptional complexes suggest a mechanism for relaying information from the outside to the inside of the virus during priming.

Genome Sequence of Chiqui Virus, a Novel Reovirus Isolated from Mosquitoes Collected in Colombia.

We report here the complete genome sequence of a novel reovirus, designated Chiqui virus (CHQV) strain CoB38d, that was isolated from a pool of unidentified mosquitoes collected in northern Colombia in 2013. CHQV has nine double-stranded DNA (dsRNA) genome segments and has similarity to viruses belonging to the family Reoviridae, subfamily Spinareovirinae.

Oncolytic Reovirus Inhibits Immunosuppressive Activity of Myeloid-Derived Suppressor Cells in a TLR3-Dependent Manner.

Oncolytic reovirus, which possesses 10 segments of dsRNA genome, mediates antitumor effects via not only virus replication in a tumor cell-specific manner, but also activation of antitumor immunity; however, the mechanism(s) of reovirus-induced activation of antitumor immunity have not been fully elucidated. Recent studies have demonstrated that overcoming an immunosuppressive environment in tumor-bearing hosts is important to achieve efficient activation of antitumor immunity. Among the various types of cells involved in immunosuppression, it has been revealed that myeloid-derived suppressor cells (MDSCs) are significantly increased in tumor-bearing hosts and play crucial roles in the immunosuppression in tumor-bearing hosts. In this study, we examined whether reovirus inhibits the immunosuppressive activity of MDSCs, resulting in efficient activation of immune cells after in vivo administration. The results showed that splenic MDSCs recovered from PBS-treated tumor-bearing mice significantly suppressed the Ag-specific proliferation of CD8+ T cells. In contrast, the suppressive activity of MDSCs on T cell proliferation was significantly reduced after reovirus administration. Reovirus also inhibited the immunosuppressive activity of MDSCs in IFN-β promoter stimulator-1 knockout (KO) mice and in wild-type mice. In contrast, the immunosuppressive activity of MDSCs in TLR-3 KO mice was not significantly altered by reovirus treatment. The activation levels of CD4+ and CD8+ T cells were significantly lower in TLR3 KO mice than in wild-type mice after reovirus administration. These results indicate that reovirus inhibits the immunosuppressive activity of MDSCs in a TLR3, but not IFN-β promoter stimulator-1, signaling-dependent manner.

ICTV Virus Taxonomy Profile: Chrysoviridae.

The Chrysoviridae is a family of small, isometric, non-enveloped viruses (40 nm in diameter) with segmented dsRNA genomes (typically four segments). The genome segments are individually encapsidated and together comprise 11.5–12.8 kbp. The single genus Chrysovirus includes nine species. Chrysoviruses lack an extracellular phase to their life cycle; they are transmitted via intracellular routes within an individual during hyphal growth, in asexual or sexual spores, or between individuals via hyphal anastomosis. There are no known natural vectors for chrysoviruses. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Chrysoviridae, which is available at www.ictv.global/report/chrysoviridae.

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.

The complete genome sequence of a double-stranded RNA mycovirus from Fusarium graminearum strain HN1.

The complete nucleotide sequence of a double-stranded RNA (dsRNA) mycovirus, Fusarium graminearum dsRNA virus 5 (FgV5), was identified and characterized. The FgV5 genome comprises two dsRNA genome segments of 2030bp and 1740bp. FgV5 dsRNA1 contains a single open reading frame (ORF1), which is predicted to encode a protein of 613 amino acids (aa) with a molecular mass of 70.4kDa and has a conserved RNA-dependent RNA polymerase (RdRp) motif. FgV5 dsRNA2 is predicted to contain two discontinuous ORFs (ORF2 and ORF3) that code for products of unknown function. Sequence comparisons showed that FgV5 has the highest aa sequence identities to Fusarium graminearum virus 4 (FgV4) (83.01% for ORF1, 78.70% for ORF2, and 76.27% for ORF3), suggesting that FgV5 and FgV4 should be regarded as members of different species. Phylogenetic analysis indicated that FgV5 belongs to a taxonomically unassigned dsRNA mycovirus group that is related to the families Amalgaviridae and Partitiviridae. Here, we propose that FgV5 and related viruses are members of a yet to be named and formally recognized new family.

Characteristics of siRNAs derived from Southern rice black-streaked dwarf virus in infected rice and their potential role in host gene regulation

BackgroundVirus-derived siRNAs (vsiRNAs)-mediated RNA silencing plays important roles in interaction between plant viruses and their hosts. Southern rice black-streaked dwarf virus (SRBSDV) is a newly emerged devastating rice reovirus with ten dsRNA genomic segments. The characteristics of SRBSDV-derived siRNAs and their biological implications in SRBSDV-rice interaction remain unexplored.MethodsVsiRNAs profiling from SRBSDV-infected rice samples was done via small RNA deep sequencing. The putative rice targets of abundantly expressed vsiRNAs were bioinformatically predicted and subjected to functional annotation. Differential expression analysis of rice targets and RNA silencing components between infected and healthy samples was done using RT-qPCR.ResultsThe vsiRNA was barely detectable at 14 days post infection (dpi) but abundantly present along with elevated expression level of the viral genome at 28 dpi. From the 28-dpi sample, 70,878 reads of 18 ~ 30-nt vsiRNAs were recognized (which mostly were 21-nt and 22-nt), covering 75 ~ 91% of the length of the ten genomic segments respectively. 86% of the vsiRNAs had a <50% GC content and 79% of them were 5’-uridylated or adenylated. The production of vsiRNAs had no strand polarity but varied among segment origins. Each segment had a few hotspot regions where vsiRNAs of high abundance were produced. 151 most abundant vsiRNAs were predicted to target 844 rice genes, including several types of host resistance or pathogenesis related genes encoding F-box/LRR proteins, receptor-like protein kinases, universal stress proteins, tobamovirus multiplication proteins, and RNA silencing components OsDCL2a and OsAGO17 respectively, some of which showed down regulation in infected plants in RT-qPCR. GO and KEGG classification showed that a majority of the predicted targets were related to cell parts and cellular processes and involved in carbohydrate metabolism, translation, and signal transduction. The silencing component genes OsDCL2a, OsDCL2b, OsDCL4, and OsAGO18 were down regulated, while OsAGO1d, OsAGO2, OsRDR1 and OsRDR6 were up regulated, significantly, upon SRBSDV infection.ConclusionsSRBSDV can regulate the expression of rice RNA silencing pathway components and the virus might compromise host defense and influence host pathogenesis via siRNA pathways.

Genome Sequence of Spinach Cryptic Virus 1, a New Member of the Genus Alphapartitivirus (Family Partitiviridae), Identified in Spinach.

A distinct double-stranded RNA (dsRNA) cryptic virus, named spinach cryptic virus 1 (SpCV1), was identified from spinach transcriptome datasets. The SpCV1 genome has two dsRNA genome segments. The larger dsRNA1 has an open reading frame for a conserved RNA-dependent RNA polymerase (RdRp). The smaller dsRNA2 encodes a putative coat protein (CP). The sequence identity of SpCV1 RdRp and CP to the closest cryptic virus is 81% and 60%, respectively. Phylogenetic analysis indicates that SpCV1 is a novel member of the genus Alphapartitivirus (family Partitiviridae).

Rice Black-streaked Dwarf Virus Preparation and Infection on Rice.

Rice black-streaked dwarf virus (RBSDV), a member of genus Fijivirus in the family Reoviridae, infects rice, maize, barley and wheat, and can seriously affect crop yields. RBSDV is transmitted by the small brown planthopper (Laodelphax striatellus, SBPH) in a persistent manner. RBSDV has 10 linear dsRNA genomic segments, making it difficult to construct infectious clones for functional studies in plants. Here we describe a method for inoculating and maintaining RBSDV on rice in a greenhouse for use in laboratory research. The protocol uses SBPHs mass reared in the laboratory. We also describe in detail the propagation of a healthy planthopper population, the preparation of plant material, RBSDV inoculation and the evaluation of the rice after inoculation.

Full-Genome Sequencing and Confirmation of the Causative Agent of Erythrocytic Inclusion Body Syndrome in Coho Salmon Identifies a New Type of Piscine Orthoreovirus.

Erythrocytic inclusion body syndrome (EIBS) causes mass mortality in farmed salmonid fish, including the coho salmon, Onchorhynchus kisutchi, and chinook salmon, O. tshawytscha. The causative agent of the disease is a virus with an icosahedral virion structure, but this virus has not been characterized at the molecular level. In this study, we sequenced the genome of a virus purified from EIBS-affected coho salmon. The virus has 10 dsRNA genomic segments (L1, L2, L3, M1, M2, M3, S1, S2, S3, and S4), which closely resembles the genomic organization of piscine orthoreovirus (PRV), the causative agent of heart and skeletal inflammation (HSMI) in Atlantic salmon and HSMI-like disease in coho salmon. The genomic segments of the novel virus contain at least 10 open reading frames (ORFs): lambda 1 (λ1), λ2, λ3, mu 1 (μ1), μ2, μNS, sigma 1 (σ1), σ2, σ3, and σNS. An additional ORF encoding a 12.6-kDa protein (homologue of PRV p13) occurs in the same genomic segment as σ3. Phylogenetic analyses based on S1 and λ3 suggest that this novel virus is closely related to PRV, but distinctly different. Therefore, we designated the new virus ‘piscine orthoreovirus 2’ (PRV-2). Reverse transcription–quantitative real-time PCR revealed a significant increase in PRV-2 RNA in fish blood after the artificial infection of EIBS-naïve fish but not in that of fish that had recovered from EIBS. The degree of anemia in each fish increased as the PRV-2 RNA increased during an epizootic season of EIBS on an inland coho salmon farm. These results indicate that PRV-2 is the probable causative agent of EIBS in coho salmon, and that the host acquires immunity to reinfection with this virus. Further research is required to determine the host range of PRV species and the relationship between EIBS and HSMI in salmonid fish.

Molecular Characterization of a Trisegmented Mycovirus from the Plant Pathogenic Fungus Colletotrichum gloeosporioides.

A novel double-stranded RNA (dsRNA) mycovirus, consisting of three dsRNA genome segments and possibly belonging to the family Chrysoviridae, was isolated from the filamentous phytopathogenic fungus Colletotrichum gloeosporioides and designated as Colletotrichum gloeosprioides chrysovirus 1 (CgCV1). The three dsRNAs of the CgCV1 genome with lengths of 3397, 2869, and 2630 bp (dsRNAs1–3) were found to contain a single open reading frame (ORF) putatively encoding the RNA-dependent RNA polymerase (RdRp), a capsid protein, and a protease, respectively, all of which exhibited some degree of sequence similarity to the comparable putative proteins encoded by the genus Chrysovirus. The 5′- and 3′-untranslated regions in each dsRNA segment contained similar sequences that were strictly conserved at the termini. Moreover, isometric virus-like particles (VLPs) with a diameter of approximately 40 nm were extracted from fungal mycelia. Phylogenetic analysis based on the conserved dsRNA1-encoded RdRp showed that CgCV1 is a new virus belonging to the Chrysoviridae family. BLAST analysis revealed the presence of CgCV1-like sequences in the chromosomes of Medicago truncatula and Solanum tuberosum. Moreover, some sequences in the transcriptome shotgun assembly (TSA) library and expressed sequence tag database (ESTdb) of other eudicot and monocot plants were also found to be related to CgCV1.

Complete nucleotide sequence of Magnaporthe oryzae partitivirus 1.

A novel double-stranded RNA (dsRNA) mycovirus, designated Magnaporthe oryzae partitivirus 1 (MoPV1), was isolated from a strain of the plant pathogenic fungus Magnaporthe oryzae. The MoPV1 genome has two dsRNA genome segments. The larger segment (1763bp) has a single open reading frame (ORF) with a conserved RNA-dependent RNA polymerase (RdRp) domain. The smaller segment (1491bp) contains a single ORF encoding a putative coat protein (CP). Homology searches and phylogenetic analysis indicated that MoPV1 is a new member of the genus Gammapartitivirus. This is the first report of a mycovirus of the family Partitiviridae identified in Magnaporthe oryzae.

Genetic linkage of capsid protein-encoding RNA segments in group A equine rotaviruses.

Rotavirus virions are formed by three concentric protein layers that enclose the 11 dsRNA genome segments and the viral proteins VP1 and VP3. Interactions amongst the capsid proteins (VP2, VP6, VP7 and VP4) have been described to play a major role in viral fitness, whilst restricting the reassortment of the genomic segments during co-infection with different rotavirus strains. In this work we describe and characterize the linkage between VP6 and VP7 proteins based on structural and genomic analyses of group A rotavirus strains circulating in Argentinean horses. Strains with the VP7 genotype G3 showed a strong association with the VP6 genotype I6, whilst strains with G14 were associated with the I2 genotype. Most of the differences on the VP6 and VP7 proteins were observed in interactive regions between the two proteins, suggesting that VP6 : VP7 interactions may drive the co-evolution and co-segregation of their respective gene segments.

Isolation of a Novel Fusogenic Orthoreovirus from Eucampsipoda africana Bat Flies in South Africa.

We report on the isolation of a novel fusogenic orthoreovirus from bat flies (Eucampsipoda africana) associated with Egyptian fruit bats (Rousettus aegyptiacus) collected in South Africa. Complete sequences of the ten dsRNA genome segments of the virus, tentatively named Mahlapitsi virus (MAHLV), were determined. Phylogenetic analysis places this virus into a distinct clade with Baboon orthoreovirus, Bush viper reovirus and the bat-associated Broome virus. All genome segments of MAHLV contain a 5' terminal sequence (5'-GGUCA) that is unique to all currently described viruses of the genus. The smallest genome segment is bicistronic encoding for a 14 kDa protein similar to p14 membrane fusion protein of Bush viper reovirus and an 18 kDa protein similar to p16 non-structural protein of Baboon orthoreovirus. This is the first report on isolation of an orthoreovirus from an arthropod host associated with bats, and phylogenetic and sequence data suggests that MAHLV constitutes a new species within the Orthoreovirus genus.

A duck reovirus variant with a unique deletion in the sigma C gene exhibiting high pathogenicity in Pekin ducklings

A novel strain of duck reovirus (DRV) associated with a high mortality in Pekin ducklings in China, 2013, was isolated and characterized. This strain (designated as HN5d) grew well in Vero cells and produced marked cytopathic effects. HN5d contains 10 dsRNA genome segments, a typical feature of avian orthoreovirus. Following cloning, sequencing, and sequence analysis of the genome segments, a unique deletion of 18 amino acids was found in the sigma C protein of HN5d when compared with that of the recent Chinese waterfowl reoviruses (e.g., DRV 091). Phylogenetic analysis of cDNA amplicons of segments encoding for the outer capsid proteins revealed that HN5d is a novel genotype 2 waterfowl reovirus isolate. Inoculation of Pekin ducklings with HN5d resulted in splenic necrosis, a typical feature of “Duck spleen necrosis disease” (DSND) discovered in China in 2006. Unlike the typical DSND, HN5d produced severer hemorrhagic and/or necrotic lesions in livers of experimentally infected ducklings. 20–30% of death was observed during the first 7 day in the experimentally exposed birds. These findings suggest that HN5d is a novel duck reovirus isolate with severer pathogenicity in Pekin ducklings.

Cryo-EM shows the polymerase structures and a nonspooled genome within a dsRNA virus.

Double-stranded RNA (dsRNA) viruses possess a segmented dsRNA genome and a number of RNA-dependent RNA polymerases (RdRps) enclosed in a capsid. Until now, the precise structures of genomes and RdRps within the capsids have been unknown. Here we report the structures of RdRps and associated RNAs within nontranscribing and transcribing cypoviruses (NCPV and TCPV, respectively), using a combination of cryo-electron microscopy (cryo-EM) and a symmetry-mismatch reconstruction method. The RdRps and associated RNAs appear to exhibit a pseudo-D3 symmetric organization in both NCPV and TCPV. However, the molecular interactions between RdRps and the genomic RNA were found to differ in these states. Our work provides insight into the mechanisms of the replication and transcription in dsRNA viruses and paves a way for structural determination of lower-symmetry complexes enclosed in higher-symmetry structures.

Characterization of a second open reading frame in genome segment 10 of bluetongue virus.

Viruses have often evolved overlapping reading frames in order to maximize their coding capacity. Until recently, the segmented dsRNA genome of viruses of the Orbivirus genus was thought to be monocistronic, but the identification of the bluetongue virus (BTV) NS4 protein changed this assumption. A small ORF in segment 10, overlapping the NS3 ORF in the +1 position, is maintained in more than 300 strains of the 27 different BTV serotypes and in more than 200 strains of the phylogenetically related African horse sickness virus (AHSV). In BTV, this ORF (named S10-ORF2 in this study) encodes a putative protein 50–59 residues in length and appears to be under strong positive selection. HA- or GFP-tagged versions of S10-ORF2 expressed from transfected plasmids localized within the nucleoli of transfected cells, unless a putative nucleolar localization signal was mutated. S10-ORF2 inhibited gene expression, but not RNA translation, in transient transfection reporter assays. In both mammalian and insect cells, BTV S10-ORF2 deletion mutants (BTV8ΔS10-ORF2) displayed similar replication kinetics to wt virus. In vivo, S10-ORF2 deletion mutants were pathogenic in mouse models of disease. Although further evidence is required for S10-ORF2 expression during infection, the data presented provide an initial characterization of this ORF.

Genomic and biological characterization of a new cypovirus isolated from Dendrolimus punctatus.

A novel cypovirus (designated DpCPV-MC) was isolated from the pine moth Dendrolimus punctatus using serial in vivo cloning procedures. DpCPV-MC occurs in typical polyhedral occlusion bodies, containing a number of spherical virions. Laboratory bioassays indicated that the infectivity of DpCPV-MC against second-instar Spodoptera exigua larvae does not differ significantly from that of Dendrolimus punctatus cypovirus 1. Full-length amplification of the DpCPV-MC cDNAs identified 16 dsRNA genome segments. Each segment encodes one open reading frame with unique conserved terminal sequences at the 5′ and 3′ ends, which differ from those of all previously reported cypoviruses. On a phylogenetic tree based on the amino acid sequences of the polyhedrin of 19 cypovirus species, DpCPV-MC was closest to the type-4 cypoviruses. Homology searches showed that ten segments of DpCPV-MC (S1, S2, S3, S4, S5, S7, S8, S9, S12, and S13) encode putative CPV structural and nonstructural proteins, three segments (S6, S10 and S14) encode putative insect proteins or other viral proteins, and the other three segments (S11, S15, and S16) encode proteins that have no obvious sequence similarity to any known protein. Based on RNA secondary structures analysis, two segments of them (S11 and S16) were predicted to possibly transcript less efficiently than the other segments. We speculate that DpCPV-MC is composed of several genotypes. The ten CPV-related segments constantly exist in all genotypes, and one or two of the six CPV-unrelated segments co-exist with the ten CPV-related segments in one DpCPV-MC genotype, thus each virion contains no more than 12 segments. Based on our results and the literature, DpCPV-MC is a new cypovirus (Cypovirus 22, strain DpCPV-22).