One Open Reading Frame Research Articles

Characterization and phylogenetic analysis of the complete mitochondrial genome of the red alga, Gracilaria eucheumatoides Harvey 1860 (Gracilariales: Gracilariaceae) from Vietnam

The marine red alga, Gracilaria eucheumatoides, is economically significant for its agar production and pharmacologically active compounds. This study reveals its complete mitochondrial genome (mitogenome), sequenced using Illumina’s next-generation technology. The mitogenome is a 25,909 bp circular molecule with a G + C content of 27.21%, comprising 24 protein-coding genes, two ribosomal RNA genes, 24 transfer RNA genes, and one open reading frame (ORF) with an unidentified function. Both gene structure and composition are highly conserved within Gracilaria. The phylogenetic analyses fully support a close relationship of G. eucheumatoides with other Gracilaria species, as well as its sister relationship with G. urvillei. This mitogenome sequencing effort of G. eucheumatoides provides crucial data for future phylogenetic research on marine red algae.

ICTV Virus Taxonomy Profile: Qinviridae 2023.

Qinviridae is a family of negative-sense RNA viruses with genomes of 7.3-8.2 kb that have been associated with crustaceans, insects, gastropods, and nematodes. The qinvirid genome consists of two segments, each with at least one open reading frame (ORF). The large (L) segment ORF encodes a large protein containing an RNA-directed RNA polymerase (RdRP) domain. The small (S) segment ORF encodes a nucleocapsid protein. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Qinviridae, which is available at ictv.global/report/qinviridae.

Isolation and genomic characterization of a cypovirus from Clanis bilineata.

Clanis bilineata Walker, soybean hawkmoth, belongs to the subfamily Ambulicinae (Sphingidae, Lepidoptera) and is an edible insect that usually grows on soybean leaves. In this study, we isolated a new cypovirus from naturally diseased Clanis bilineata larvae (named CbCPV), scanned its structure, sequenced its genome, and studied its phylogenetic relationship to other cypoviruses. Microscopy showed that CbCPV polyhedral occlusion bodies were about 1.878μm on average and contained many virions in the ultrathin sections. The complete genome sequence of CbCPV is 22,812bp comprising 10 segmented double-stranded RNAs. Apart from segment 1 containing one open reading frame (ORF) and one sub-ORF, the other nine segments all contain one open reading frame and encoded one putative protein. The non-coding regions contained conserved sequences at 5' termini (AGUCAAA) and 3' termini (AGC), except segment 4 containing a different 5' termini (AUGUUUA). The whole sequence of the polyhedrin gene in CbCPV contained 892 nucleotides, encoding a protein of 246 amino acids. Based on amino acid sequences of polyhedrin or RNA dependent RNA polymerase (RdRp), the phylogenetic analysis indicated that CbCPV was closely related to DnCPV-23. The putative function of all segments differed from each other, but the most closely related species of segments were DnCPV-23 with 98.2-99.8% nucleotide identity. Overall, the evidence of morphology, protein analysis and nucleic acids (genomic pattern) showed that CbCPV is a new isolate in the cypovirus-23 type and can be termed Clanis bilineata cypovirus type 23 (CbCPV-23).

Four distinct isolates of a novel polymycovirus identified in Setosphaeria turcica.

Isolation and analysis of double-stranded RNA (dsRNA) from the phytopathogenic fungus Setosphaeria turcica f. sp. zeae revealed the presence of a new double-stranded RNA (dsRNA) virus, tentatively named "Setosphaeria turcica polymycovirus 2" (StPmV2). The genome of StPmV2 consists of five segments (dsRNA1-5), ranging in size from 965 bp to 2462 bp. Each dsRNA contains oneopen reading frame (ORF) flanked by 5' and 3' untranslated regions (UTRs) with conserved terminal sequences.The putative protein encoded by dsRNA1 shows 64.52% amino acid sequence identity to the RNA-dependent RNA polymerase (RdRp) of the most closely related virus, Cladosporium cladosporioides virus 1, which belongs to the family Polymycoviridae. dsRNAs 2-4 encode the putative coat protein, methyltransferase (MTR), and proline-alanine-serine-rich protein (PASrp), respectively, and dsRNA5 encodes a protein of unknown function. Phylogenetic analysis based on the RdRp protein indicated that StPmV2 clustered with members of the family Polymycoviridae and is therefore a new mycovirus belonging to the genus Polymycovirus in the family Polymycoviridae. In addition, three other distinct isolates of StPmV2 were identified: one isolated from S. turcica f. sp. zeae and two from S. turcica f. sp. sorghi. To our knowledge, this is the first report of a polymycovirus infecting both S. turcica f. sp. zeae and S. turcica f. sp. sorghi.

Chapter 2a: Virology

TBEV is the most medically important member of the tick-borne serocomplex group within the genus Flavivirus, family Flaviviridae. Three antigenic subtypes of TBEV correspond to the 3 recognized genotypes: European (TBEV-EU), also known as Western, Far Eastern (TBEV-FE), and Siberian (TBEV-SIB). An additional 2 genotypes have been identified in the Irkutsk region of Russia, currently named TBE virus Baikalian subtype (TBEV-BKL) and TBE virus Himalayan subtype (Himalayan and “178-79” group; TBEV-HIM). TBEV virions are small enveloped spherical particles about 50 nm in diameter. The TBEV genome consists of a single-stranded positive sense RNA molecule. The genome encodes one open reading frame (ORF), which is flanked by untranslated (non-coding) regions (UTRs). The 5′-UTR end has a methylated nucleotide cap for canonical cellular translation. The 3′-UTR is not polyadenylated and is characterized by extensive length and sequence heterogeneity. The ORF encodes one large polyprotein, which is co- and post-translationally cleaved into 3 structural proteins (C, prM, and E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). TBEV replicates in the cytoplasm of the host cell in close association with virus-induced intracellular membrane structures. Virus assembly occurs in the endoplasmic reticulum. The immature virions are transported to the Golgi complex, and mature virions pass through the host secretory pathway and are finally released from the host cell by fusion of the transport vesicle membrane with the plasma membrane.

Recombinant Pichinde viral vector expressing tuberculosis antigens elicits strong T cell responses and protection in mice.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) remains a major global health threat. The only available vaccine Bacille Calmette-Guérin (BCG) does not prevent adult pulmonary TB. New effective TB vaccines should aim to stimulate robust T cell responses in the lung mucosa to achieve high protective efficacy. We have previously developed a novel viral vaccine vector based on recombinant Pichinde virus (PICV), a non-pathogenic arenavirus with low seroprevalence in humans, and have demonstrated its efficacy to induce strong vaccine immunity with undetectable anti-vector neutralization activity. Using this tri-segmented PICV vector (rP18tri), we have generated viral vectored TB vaccines (TBvac-1, TBvac-2, and TBvac-10) encoding several known TB immunogens (Ag85B, EsxH, and ESAT-6/EsxA). A P2A linker sequence was used to allow for the expression of two proteins from one open-reading-frame (ORF) on the viral RNA segments. The immunogenicity of TBvac-2 and TBvac-10 and the protective efficacy of TBvac-1 and TBvac-2 were evaluated in mice. Both viral vectored vaccines elicited strong antigen-specific CD4 and CD8 T cells through intramuscular (IM) and intranasal (IN) routes as evaluated by MHC-I and MHC-II tetramer analyses, respectively. The IN inoculation route helped to elicit strong lung T cell responses. The vaccine-induced antigen-specific CD4 T cells are functional, expressing multiple cytokines as detected by intracellular cytokine staining. Finally, immunization with TBvac-1 or TBvac-2, both expressing the same trivalent antigens (Ag85B, EsxH, ESAT6/EsxA), reduced Mtb lung tissue burden and dissemination in an aerosol challenge mouse model. The novel PICV vector-based TB vaccine candidates can express more than two antigens via the use of P2A linker sequence and elicit strong systemic and lung T cell immunity with protective efficacy. Our study suggests the PICV vector as an attractive vaccine platform for the development of new and effective TB vaccine candidates.

Hypovirulence caused by mycovirus in Colletotrichum fructicola

Colletotrichum fructicola is a pathogenic fungus causing leaf black spot and fruit rot disease in a wide variety of crops. Some mycoviruses that cause detrimental effects on fungal hosts could be useful in studying the pathogenesis of fungal hosts. In this study, we reported two mycoviruses, Colletotrichum fructicola ourmia-like virus 1- Colletotrichum gloeosporioides ourmia-like virus 1 (CfOLV1-CgOLV1) and Colletotrichum fructicola ourmia-like virus 2 (CfOLV2), from a C. fructicola fungus. The complete genome sequences of CfOLV1-CgOLV1 and CfOLV2 contain 2,516 bp and 2,048 bp, respectively. Both of these viruses contain only one open reading frame (ORF), which encodes an RNA-dependent RNA polymerase (RdRp). CfOLV1-CgOLV1 was identical as the previously reported virus CgOLV1. Phylogenetic analysis showed that CfOLV2 is closely related to Scleroulivirus and Magoulivirus in the family Botourmiaviridae. Virus elimination and horizontal transmission experiments proved that the associated mycoviruses could reduce the pathogenicity of the host C. fructicola. In addition, we found that the virus-containing strains showed a much higher percentage of appressorium formation and more melanin production compared to isogenic virus-free strain, and the presence of the virus is detrimental to the growth of host fungi and regulates the integrity of the cell wall. Transcriptomic analysis showed that mycovirus infection caused various abnormal genes expression in C. fructicola. To the best of our knowledge, this is the first report of a hypovirulence-associated ourmia-like mycovirus in C. fructicola.

Molecular characterization of a novel virga-like virus associated with wheat.

In this work, we report the detection of a novel single-strand RNA virus from wheat, tentatively named "Triticum aestivum-associated virga-like virus 1" (TaAVLV1). Further characterization revealed that the complete genome of TaAVLV1 is divided into two segments, RNA1 and RNA2, which are 3530 and 3466 nt in length, excluding their respective polyA tails, and each contains only one open reading frame (ORF). The ORF of RNA1 encodes an RNA-dependent RNA polymerase (RdRp), while the ORF of RNA2 encodes a putative protein with methyltransferase and helicase domains. Phylogenetic analysis showed that the RdRp of TaAVLV1 is closely related to those of members of the unclassified virga-like virus group in the family Virgaviridae. Thus, we have identified TaAVLV1 as a putative novel virga-like virus belonging to the family Virgaviridae.

HPV16 and HPV18 Genome Structure, Expression, and Post-Transcriptional Regulation.

Human papillomaviruses (HPV) are a group of small non-enveloped DNA viruses whose infection causes benign tumors or cancers. HPV16 and HPV18, the two most common high-risk HPVs, are responsible for ~70% of all HPV-related cervical cancers and head and neck cancers. The expression of the HPV genome is highly dependent on cell differentiation and is strictly regulated at the transcriptional and post-transcriptional levels. Both HPV early and late transcripts differentially expressed in the infected cells are intron-containing bicistronic or polycistronic RNAs bearing more than one open reading frame (ORF), because of usage of alternative viral promoters and two alternative viral RNA polyadenylation signals. Papillomaviruses proficiently engage alternative RNA splicing to express individual ORFs from the bicistronic or polycistronic RNA transcripts. In this review, we discuss the genome structures and the updated transcription maps of HPV16 and HPV18, and the latest research advances in understanding RNA cis-elements, intron branch point sequences, and RNA-binding proteins in the regulation of viral RNA processing. Moreover, we briefly discuss the epigenetic modifications, including DNA methylation and possible APOBEC-mediated genome editing in HPV infections and carcinogenesis.

Complete nucleotide sequence of a novel mycovirus infecting Rhizoctonia fumigata AG-Ba isolate C-314 Baishi.

The complete nucleotide sequence of a novel mycovirus, designated as "Rhizoctonia fumigata bipartite virus 1" (RfBV1), from Rhizoctonia fumigata AG-Ba isolate C-314 Baishi was determined. The genome of RfBV1 is composed of two double-stranded RNAs (dsRNA). dsRNA-1 (2311 bp) contains one open reading frame (ORF), which codes for the putative RNA-dependent RNA polymerase (RdRp) of the virus. dsRNA-2 (1690 bp) contains one ORF, which encodes a putative protein whose function is unknown. Phylogenetic analysis indicated that the RdRp of RfBV1 clustered with several unassigned bipartite viruses belonging to the CThTV-like viruses group, but not the family Amalgaviridae or Partitiviridae. Our study suggests that RfBV1 is a novel mycovirus related to the CThTV-like viruses.

Identification of Novel Micropeptides Derived from Hepatocellular Carcinoma-Specific Long Noncoding RNA.

Identification of cancer-specific target molecules and biomarkers may be useful in the development of novel treatment and immunotherapeutic strategies. We have recently demonstrated that the expression of long noncoding (lnc) RNAs can be cancer-type specific due to abnormal chromatin remodeling and alternative splicing. Furthermore, we identified and determined that the functional small protein C20orf204-189AA encoded by long intergenic noncoding RNA Linc00176 that is expressed predominantly in hepatocellular carcinoma (HCC), enhances transcription of ribosomal RNAs and supports growth of HCC. In this study we combined RNA-sequencing and polysome profiling to identify novel micropeptides that originate from HCC-specific lncRNAs. We identified nine lncRNAs that are expressed exclusively in HCC cells but not in the liver or other normal tissues. Here, DNase-sequencing data revealed that the altered chromatin structure plays a key role in the HCC-specific expression of lncRNAs. Three out of nine HCC-specific lncRNAs contain at least one open reading frame (ORF) longer than 50 amino acid (aa) and enriched in the polysome fraction, suggesting that they are translated. We generated a peptide specific antibody to characterize one candidate, NONHSAT013026.2/Linc013026. We show that Linc013026 encodes a 68 amino acid micropeptide that is mainly localized at the perinuclear region. Linc013026-68AA is expressed in a subset of HCC cells and plays a role in cell proliferation, suggesting that Linc013026-68AA may be used as a HCC-specific target molecule. Our finding also sheds light on the role of the previously ignored ’dark proteome’, that originates from noncoding regions in the maintenance of cancer.

Immunity Evaluation of an Experimental Designed Nanoliposomal Vaccine Containing FMDV Immunodominant Peptides.

Foot-and-mouth disease (FMD) is a highly contagious viral disease affecting cloven-hoofed animals. The particular virus causing FMD disease is called FMD virus and is a member of the Aphthovirus genus in the Picornaviridae family. The FMD virus has an 8500 nt long single strain positive RNAgenome with one open reading frame (ORF) trapped in an icosahedral capsid protein. This virus genome doesn't have proofreading property which leads to high mutagenesis. It has seven serotypes, including O, A, ASIA, SAT1, SAT2, and C serotypes, as well as many subtypes. Iran is an endemic region for foot-and-mouth disease. Vaccination of susceptible animals with an inactivated whole-virus vaccine is the only way to control the epidemic in many developing countries. Today, conventionally attenuated and killed virus vaccines are being used worldwide. In Iran, animals have been vaccinated every 105 days with an inactivated FMD vaccine. Although commercially available FMD vaccines are effective, they provide short-term immunity requiring regular boosters. A new FMD vaccine is needed to improve immunization, safety, and long-term immune responses. A synthetic peptide vaccine is one of the safe and important vaccines. Peptide vaccine has low immunogenicity, requiring strong adjuvants. Nanoliposomes can be used as new adjuvants to improve immune response. In the current study, nanoliposomal carriers were selected using Dimyristoylphosphatidylcholine (DMPC), dimyristoyl phosphoglycerol (DMPG), and Cholesterol (Chol) as an adjuvant containing two immunodominant synthetic FMDV peptides. The liposomal formulations were characterized by various physicochemical properties. The size, zeta potential, and encapsulation efficiency were optimized, and the obtained nanoliposome was suitable as a vaccine. The efficacy of vaccines has been evaluated in guinea pigs as animal models. Indirect ELISA was used to detect FMDV-specific IgG. The obtained results indicated that although antibody titer was observed, the amount was lower compared to the groups that received inactivated virus-containing liposomes. In addition, the results showed that liposome was an appropriate adjuvant, compared to other adjuvants, such as Alum and Freund, and can act as a depot and induce an immune response.

A novel polymycovirus with defective RNA isolated from the entomopathogenic fungus Beauveria bassiana Vuillemin.

A novel double-stranded RNA virus was isolated and identified from Beauveria bassiana Vuillemin, derived from the muscardine cadaver of an Ostrinia furnacalis larva in China. The virus contains six dsRNAs, and each viral dsRNA contains only one open reading frame (ORF). As in other polymycoviruses, dsRNA1 encodes an RNA-dependent RNA polymerase (RdRp), dsRNA3 encodes a methyltransferase (MTR), and dsRNA4 encodes a proline-alanine-serine-rich protein. A BLASTp search revealed that the viral RdRp domain showed 79.43%, 79.04%, and 59.05% sequence identity to Beauveria bassiana polymycovirus 2 and 3 (BbPmV-2, BbPmV-3) and Magnaporthe oryzae polymycovirus 1 (MoPmV-1), respectively. Phylogenetic analysis based on RdRp sequences showed that the phylogenetically closest relatives of this virus are BbPmV-2, BbPmV-3, and MoPmV-1. This virus, along with previously ill-defined polymycoviruses (BbPmV-2 and BbPmV-3), appears to belong to an as-yet-unestablished species. The findings further suggest that the virus is a new member of the genus Polymycovirus within the family Polymycoviridae, and we have named it "Beauveria bassiana polymycovirus 4" (BbPmV-4). However, the sixth dsRNA is a defective RNA with the same sequence as that of dsRNA4 except for a deletion of 312 bp from nt 185 to nt 496, but it still contains a complete ORF. To our knowledge, this is the first report of the existence of a defective RNA in a polymycovirus.

Chapter 2a: Virology

TBEV is the most medically important member of the tick-borne serocomplex group within the genus Flavivirus, family Flaviviridae. Three antigenic subtypes of TBEV correspond to the 3 recognized genotypes: European (TBEV-EU), also known as Western, Far Eastern (TBEV-FE), and Siberian (TBEV-SIB). An additional 2 genotypes have been identified in the Irkutsk region of Russia, currently named TBE virus Baikalian subtype (TBEV-BKL) and TBE virus Himalayan subtype (Himalayan and “178-79” group; TBEV-HIM). TBEV virions are small enveloped spherical particles about 50 nm in diameter. The TBEV genome consists of a single-stranded positive sense RNA molecule. The genome encodes one open reading frame (ORF), which is flanked by untranslated (non-coding) regions (UTRs). The 5′-UTR end has a methylated nucleotide cap for canonical cellular translation. The 3′-UTR is not polyadenylated and is characterized by extensive length and sequence heterogeneity. The ORF encodes one large polyprotein, which is co- and post-translationally cleaved into 3 structural proteins (C, prM, and E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). TBEV replicates in the cytoplasm of the host cell in close association with virus-induced intracellular membrane structures. Virus assembly occurs in the endoplasmic reticulum. The immature virions are transported to the Golgi complex, and mature virions pass through the host secretory pathway and are finally released from the host cell by fusion of the transport vesicle membrane with the plasma membrane.

Limitation of nutrients stimulates musty odor production by Streptomyces sp. isolated from a tropical environment.

Musty odor production by actinomycetes is usually related to the presence of geosmin and 2-methylisoborneol (2-MIB), which are synthesized by enzymes encoded by the geoA and tpc genes, respectively. Streptomyces spp. strain S10, which was isolated from a water reservoir in Malaysia, has the ability to produce geosmin when cultivated in a basal salt (BS) solid medium, but no 2-MIB production occurred during growth in BS medium. Strain S10 could produce higher levels of geosmin when the phosphate concentration was limited to 0.05 mg/L, with a yield of 17.53 ± 3.12 ✕ 105 ng/L, compared with growth in BS medium. Interestingly, 2-MIB production was suddenly detected when the nitrate concentration was limited to 1.0 mg/L, with a yield of 1.4 ± 0.11 ✕ 105 ng/L. Therefore, it was concluded that phosphate- and nitrate-limiting conditions could induce the initial production of geosmin and 2-MIB by strain S10. Furthermore, a positive amplicon of geoA was detected in strain S10, but no tpc amplicon was detected by PCR analysis. Draft genome sequence analysis showed that one open reading frame (ORF) contained a conserved motif of geosmin synthase with 95% identity with geoA in Streptomyces coelicolor A3 (2). In the case of the tpc genes, it was found that one ORF showed 23% identity to the known tpc gene in S. coelicolor A3(2), but strain S10 lacked one motif in the N-terminus.

Genome sequence of a novel member of the order Picornavirales from the endoparasitoid wasp Diversinervus elegans.

Here, we report a novel RNA virus from an encyrtid endoparasitoid wasp (Diversinervus elegans). This virus has a genome of 8845 nucleotides in length with a poly(A) tail. It contains one open reading frame (ORF) encoding a single polyprotein that shares the most significant similarity to the polyproteins of dicistroviruses. Phylogenetic analysis suggested that this virus belongs to the family Dicistroviridae from the order Picornavirales, but its genomic organization is distinct from that of the other known dicistroviruses, which have two ORFs. Consequently, we propose that this virus is a member of a new species in the order Picornavirales, and have named it "Diversinervus elegans virus" (DEV).

Chapter 2a: Virology

TBEV is the most medically important member of the tick-borne serocomplex group within the genus Flavivirus, family Flaviviridae. Three antigenic subtypes of TBEV correspond to the 3 recognized genotypes: European (TBEV-EU), also known as Western, Far Eastern (TBEV-FE), and Siberian (TBEV-SIB). An additional 2 genotypes have been identified in the Irkutsk region of Russia, currently named TBE virus Baikalian subtype (TBEV-BKL) and TBE virus Himalayan subtype (Himalayan and “178-79” group; TBEV-HIM). TBEV virions are small enveloped spherical particles about 50 nm in diameter. The TBEV genome consists of a single-stranded positive sense RNA molecule. The genome encodes one open reading frame (ORF), which is flanked by untranslated (non-coding) regions (UTRs). The 5′-UTR end has a methylated nucleotide cap for canonical cellular translation. The 3′-UTR is not polyadenylated and is characterized by extensive length and sequence heterogeneity. The ORF encodes one large polyprotein, which is co- and post-translationally cleaved into 3 structural proteins (C, prM, and E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). TBEV replicates in the cytoplasm of the host cell in close association with virus-induced intracellular membrane structures. Virus assembly occurs in the endoplasmic reticulum. The immature virions are transported to the Golgi complex, and mature virions pass through the host secretory pathway and are finally released from the host cell by fusion of the transport vesicle membrane with the plasma membrane.

Molecular characterization of a novel mycovirus infecting the phytopathogenic fungus Botryosphaeria dothidea.

In this study, we report the molecular characterization of a novel positive-sense single-stranded RNA (+ssRNA) mycovirus from Botryosphaeria dothidea strain G91, which we name "Botryosphaeria dothidea botourmiavirus 1" (BdBOV-1). The complete genome of BdBOV-1 is 2547 nucleotides (nt) long, contains one open reading frame (ORF) potentially encoding an RNA-dependent RNA polymerase (RdRp), and shows a close relationship to mycoviruses of the family Botourmiaviridae. Phylogenetic analysis based on the RdRp sequences confirmed that BdBOV-1 clustered together with the members of the family Botourmiaviridae. To the best of our knowledge, this is the first report of a +ssRNA mycovirus infecting B. dothidea.

Alternative ORFs and small ORFs: shedding light on the dark proteome.

Traditional annotation of protein-encoding genes relied on assumptions, such as one open reading frame (ORF) encodes one protein and minimal lengths for translated proteins. With the serendipitous discoveries of translated ORFs encoded upstream and downstream of annotated ORFs, from alternative start sites nested within annotated ORFs and from RNAs previously considered noncoding, it is becoming clear that these initial assumptions are incorrect. The findings have led to the realization that genetic information is more densely coded and that the proteome is more complex than previously anticipated. As such, interest in the identification and characterization of the previously ignored 'dark proteome' is increasing, though we note that research in eukaryotes and bacteria has largely progressed in isolation. To bridge this gap and illustrate exciting findings emerging from studies of the dark proteome, we highlight recent advances in both eukaryotic and bacterial cells. We discuss progress in the detection of alternative ORFs as well as in the understanding of functions and the regulation of their expression and posit questions for future work.

Isolation and genomic characterization of a cypovirus from the oleander hawk moth, Daphnis nerii.

The oleander hawk moth, Daphnis nerii, is a serious pest of plants belonging to the family Apocynaceae. Thus far, pathogen infection has not been reported in D. nerii. In this study, a new cytoplasmic polyhedrosis virus (cypovirus; CPV) was isolated from naturally diseased D. nerii larvae and named DnCPV-23. Virions were observed in ultrathin sections of DnCPV polyhedral bodies. Electrophoretic analysis revealed that the DnCPV genome consisted of 10 segments of double-stranded RNA (dsRNA). cDNA copies of these dsRNA segments were amplified using the method of full-length amplification of cDNAs (FLAC), cloned, and sequenced. Sequencing results showed that all segments contained one open reading frame (ORF); They shared the conserved terminal sequences AGUCAAA and AGC at 5' and 3' ends respectively, except segment 4, which is different from previously reported 22 cypoviruses. Phylogenetic analysis based on amino acid sequences of polyhedrin (encoded by segment 10) indicated that this CPV was closely related to CPV type 19. Altogether, DnCPV-23 is a new type of cypovirus.