Full-length Genome Characterization Research Articles

The Comparative Full-Length Genome Characterization of African Swine Fever Virus Detected in Thailand.

African swine fever virus (ASFV) has been responsible for the globally devastating epidemics in wild and domesticated pigs. Of the 24 identified ASFV genotypes, genotype II is the primary cause for the pandemic occurring in Europe and Asia since its emergence in Georgia in 2007. The current study aimed to characterize the full-length genomic pattern of the ASFV strain from Thailand, TH1_22/CR (Accession No. PP915735), which was then compared with genomic diversity across other Asian isolates using Georgia 2007/1 (Accession No. FR682468) as the reference. Viral DNA was isolated from the pig spleen sample following library preparation and paired-end sequencing using the MiSeq Illumina platform. The sequenced TH1_22/CR isolate spanned 189,395 nucleotides encoding 193 open reading frames (ORFs), exhibiting maximum nucleotide similarity (99.99%) with Georgian (Georgia 2007/1) and Chinese (Wuhan 2019-1 and China HLJ) isolates. Based on phylogenetic analysis, the TH1_22/CR isolate (Accession No. PP915735) was characterized as genotype II, serogroup 8, and IGR-II due to the presence of three tandem repeat sequences (TRSs). Genetic variations including SNPs and single and polynucleotide indels were identified in TH1_22/CR in agreement with other Asian isolates. For comprehensive analysis, the genome was divided into four regions (I-IV) based on gene location. Overall, the TH1_22/CR isolate demonstrated eight SNPs and indels in its genome. Two unique SNPs were reported in the coding regions of the TH1_22/CR isolate, out of which, a C-591-T substitution was seen in MGF 360-4L and a C-297-T was found in A238L, and four unique SNPs were reported in non-coding regions (NCRs). Furthermore, a 29 bp deletion was observed in the IGR between MGF 110-13La and MGF 110-13Lb, as well as 52 bp deletion in the ASFV G ACD 00350 gene. This comparative analysis establishes the foundational information for future studies on the diversity and phylogeography of this regionally significant genetic sub-group of ASFV.

Identification and full-length genome characterization of a novel mitovirus hosted by the truffle species Tuber rufum

Identification and full-length genome characterization of a novel mitovirus hosted by the truffle species Tuber rufum

Detection and genetic characterization of chicken astrovirus and avian nephritis virus from hatchery to farm

ABSTRACT Astroviruses are an emerging hazard in hatchery management, and they are responsible for great economic losses in poultry farms worldwide. Chicken astrovirus (CAstV) and avian nephritis virus (ANV) are involved in decreased hatching rate, mainly due to embryo deaths, and pale and runted chicks too weak to hatch. Hatchery diseases occur through direct egg infection or vertical transmission. However, the specific role of astroviruses in these diseases is still largely unknown. To elucidate this a high-quality Piedmont hatchery (Italy) was selected based on farm clinical signs and the astrovirus serological positivity in the breeder farm. The viral dynamics were followed from the hatchery to the farm. The presence of both CAstV and ANV was monitored by evaluating the viral load in environmental and bird samples. Interestingly, both viruses were detected in chick yolk sacs at 18 days of incubation, and in the gut contents of 1-day chicks, whereas in the hatchery environmental samples, the eggs' external surfaces and the embryos’ intestinal contents were negative. The CAstV copy number was higher in bird tissues from the hatchery, while a greater ANV load was detected in bird tissues from the farm. Moreover, our data suggest vertical transmission of both viruses. Frequent co-infection was also observed in samples from both the hatchery and the farm. Lastly, the first Italian full-length genome and molecular characterization of ANV was obtained. RESEARCH HIGHLIGHTS Identified the role of the hatchery in astrovirus transmission. Sequenced the avian nephritis virus complete genome. Investigated tissue distribution of astrovirus from egg to chicks. Demonstrated co-infection of ANV/CAstV.

Near Full-Length Genome Characterization of Two Novel Unique Recombinants (CRF01_AE/CRF07_BC) in Beijing, China.

With the prevalence of human immunodeficiency virus type 1 (HIV-1) CRF01_AE and CRF07_BC subtypes in China, the co-circulation of multiple subtypes in the HIV-1-positive population may result in dual infection or superinfection in the population, leading to the emergence of unique recombinant forms (URFs) of the HIV-1 virus. In this study, two second-generation unique recombinant strains, BI0114 and BI0116, were identified, and their near full-length genome sequences were obtained. Recombination analysis showed that both sequences were isoforms of URF_0107, and they were second-generation unique recombinant strains formed by the recombination of CRF01_AE and CRF07_BC, with the isoforms being CRF01_AE and CRF0107_BC, respectively. The continued emergence of novel CRF01_AE/CRF07_BC recombinant strains suggests that the epidemiological, preventive, and control situation of HIV-1 is complex and that the relevant health authorities urgently need to establish responses to the challenges posed by changes in the pattern of strain recombination.

Full-length genome characterization of a novel mitovirus isolated from the root rot fungus Armillaria mellea.

Members of the genus Armillaria belong to the group of pathogenic and facultative saprotrophic fungi that are generally known as one of the causative agents of white root rot in infected plants including deciduous and evergreen trees and shrubs. Although several single-stranded RNA mycoviruses were previously described in different Armillaria species, there is no report on mitoviruses (one of the simplest RNA viruses of fungal hosts) known to infect Armillaria taxa. In this study, a new mitovirus denominated "Armillaria mellea mitovirus 1" (AmMV1) was identified in the sporophore samples of Armillaria mellea, commonly known as honey mushroom. AmMV1 has a genome length of 4440 nucleotides and a G + C content of 48%. It encompasses a single open reading frame (ORF) that encodes an RNA-dependent RNA polymerase (RdRp). Comparison through BLASTp analysis revealed that the RdRp domain of AmMV1 shares a sequence identity ranging from 33.43% to 43.27% with RdRp domains of Duamitovirus genus members, having the highest similarity (43.27%) to Rhizoctonia solani mitovirus 94. According to phylogenetic analysis, AmMV1 is classified as a member of the genus Duamitovirus belonging to the Mitoviridae family. This marks the initial instance of a mitovirus identified in Armillaria spp..

HIV-PULSE: a long-read sequencing assay for high-throughput near full-length HIV-1 proviral genome characterization.

A deep understanding of the composition of the HIV-1 reservoir is necessary for the development of targeted therapies and the evaluation of curative efforts. However, current near full-length (NFL) HIV-1 proviral genome sequencing assays are based on labor-intensive and costly principles of repeated PCRs at limiting dilution, restricting their scalability. To address this, we developed a high-throughput, long-read sequencing assay called HIV-PULSE (HIV Proviral UMI-mediatedLong-readSequencing). This assay uses unique molecular identifiers (UMIs) to tag individual HIV-1 genomes, allowing for the omission of the limiting dilution step and enabling long-range PCR amplification of many NFL genomes in a single PCR reaction, while simultaneously overcoming poor single-read accuracy. We optimized the assay using HIV-infected cell lines and then applied it to blood samples from 18 individuals living with HIV on antiretroviral therapy, yielding a total of 1308 distinct HIV-1 genomes. Benchmarking against the widely applied Full-Length Individual Proviral Sequencing assay revealed similar sensitivity (11vs 18%) and overall good concordance, although at a significantly higher throughput. In conclusion, HIV-PULSE is a cost-efficient and scalable assay that allows for the characterization of the HIV-1 proviral landscape, making it an attractive method to study the HIV-1 reservoir composition and dynamics.

Canine respiratory coronavirus in Thailand undergoes mutation and evidences a potential putative parent for genetic recombination.

Canine respiratory coronavirus (CRCoV) is associated with canine infectious respiratory disease complex. Although its detection has been reported worldwide, the genomic characteristics and evolutionary patterns of this virus remain poorly defined. In this study, 21 CRCoV sequences obtained from dogs in Thailand during two episodes (2013-2015, group A; 2021-2022, group B) were characterized and analyzed. The genomic characteristics of Thai CRCoVs changed from 2013 to 2022 and showed a distinct phylogenetic cluster. Phylogenetic analysis of the spike (S) genes divided the analyzed CRCoV strains into five clades. The full-length genome characterization revealed that all Thai CRCoVs possessed a nonsense mutation within the nonstructural gene located between the S and envelope genes, leading to a truncated putative nonstructural protein. Group B Thai CRCoV strains represented the signature nonsynonymous mutations in the S gene that was not identified in group A Thai CRCoVs, suggesting the ongoing evolutionary process of Thai CRCoVs. Although no evidence of recombination of Thai CRCoV strains was found, our analysis identified one Thai CRCoV strain as a potential parent virus for a CRCoV strain found in the United States. Selective pressure analysis of the hypervariable S region indicated that the CRCoV had undergone purifying selection during evolution. Evolutionary analysis suggested that the CRCoV was emerged in 1992 and was first introduced in Thailand in 2004, sharing a common ancestor with Korean CRCoV strains. These findings regarding the genetic characterization and evolutionary analysis of CRCoVs add to the understanding of CRCoVs. IMPORTANCE Knowledge of genomic characterization of the CRCoV is still limited and its evolution remains poorly investigated. We, therefore, investigated the full-length genome of CRCoV in Thailand for the first time and analyzed the evolutionary dynamic of CRCoV. Genomic characterization of Thai CRCoV strains revealed that they possess unique genome structures and have undergone nonsynonymous mutations, which have not been reported in previously described CRCoV strains. Our work suggests that the Thai CRCoVs were not undergone mutation through genetic recombination for their evolution. However, one Thai CRCoV strain PP158_THA_2015 was found to be a potential parent virus for the CRCoV strains found in the United States. This study provides an understanding of the genomic characterization and highlights the signature mutations and ongoing evolutionary process of CRCoV that could be crucial for monitoring in the future.

Successful full-length genomic cloning and characterization of site-specific nick structures of Phytophthora endornaviruses 2 and 3 in yeast, Saccharomyces cerevisiae.

Two endornaviruses, Phytophthora endornavirus 2 (PEV2) and Phytophthora endornavirus 3 (PEV3), have been discovered in pathogens targeting asparagus. In this study, we analyzed the nick structure in the RNA genomes of PEV2 and PEV3 in the host oomycetes. Northern blot hybridization using positive and negative strand-specific RNA probes targeting the 5' and 3' regions of PEV2 and PEV3 RNA genomes revealed approximately 1.0 kilobase (kb) RNA fragments located in the 5' regions of the two genomes. 3' RACE analysis determined that the size of the RNA fragments were 958 nucleotides (nt) for PEV2 and 968 nt for PEV3. We have successfully constructed full-length cDNA clones of the entire RNA genomes of PEV2 and PEV3 using a homologous recombination system in the yeast, Saccharomyces cerevisiae. These full-length cDNA sequences were ligated downstream of a constitutive expression promoter (TDH3) or a galactose-inducing promoter (GAL1) in the shuttle vector to enable the production of the full-length RNA transcripts of PEV2 and PEV3 in yeast cells. Interestingly, a 1.0 kb RNA fragment from the PEV3 positive-strand transcript was also detected with a 5'-region RNA probe, indicating that site-specific cleavage also occurred in yeast cells. Further, when PEV2 or PEV3 mRNA was overexpressed under the GAL1 promoter, yeast cell growth was suppressed. A fusion protein combining EGFP to the N-terminus of the full-length PEV2 ORF or C-terminus of the full-length PEV3 ORF was expressed, and allowed PEV2 and PEV3 ORFs to be successfully visualized in yeast cells. Expression of the fusion protein also revealed presence of heterogeneous bodies in the cells.

Isolation and pathogenicity of porcine circovirus type 2 in mice from Guangxi province, China

BackgroundPorcine circovirus type 2 (PCV2), a member of the genus Circovirus and family Circoviridae, is a closed, small, circular, and single-stranded DNA virus, and it is a crucial swine pathogen of porcine circovirus-associated diseases (PCVADs). PCV2 was first detected in PK-15(ATCC-CCL) cells in 1974, which has caused significant economic loss to the swine industry throughout the world. And the first case of PCV2 was reported in China in 2000. At present, PCV2d is the main genotype circulating widely in China.MethodsLymph samples were obtained from piglets with emaciation and respiratory disease in Guangxi province, China. The main pathogens were detected via PCR from lymph samples, and then PCV2-single positive samples were used to inoculate with PK-15 cells. After successive generations, the isolate was subsequently identified by polymerase chain reaction (PCR), immunofluorescence assay (IFA), Western blot (WB), and transmission electron microscopic (TEM). The full-length genome and genetic characterization of isolates were analyzed by Sanger sequencing. The TCID50 of the PCV2-GX-6 was determined by IFA, and the pathogenicity of PCV2 in BALB/c mice was analyzed via the mouse model.ResultsThe isolates were successfully isolated from clinical samples. The complete genome of PCV2-GX-4, PCV2-GX-6, PCV2-GX-7, PCV2-GX-11 and PCV2-GX-16 have been amplified, sequenced, and deposited in GenBank (accession no.: OR133747, OQ803314, OR133748, OR133749, OR133750). Homology and phylogenetic analysis with reference strains showed that the isolates belonged to the PCV2d genotype. The PCV2-GX-6 could be stably passaged more than 30 times in PK-15 cells. PCV2-GX-6 was identified by PCR, IFA, WB and TEM. The results of homology showed that PCV2-GX-6 was closely related to the reference strains PCV2-JS17-8 (GenBank accession no.: MH211363). Pathogenicity studies in mice have shown that PCV2-GX-6 can lead to growth inhibition of mice. Meanwhile PCV2-GX-6 caused the typical lesions of spleen, lung and kidney. The results of qPCR showed that PCV2 can effectively proliferate in the liver, spleen, lung, and kidney.ConclusionPCV2-GX-6 can successfully infect BLAB/c mice, effectively proliferate in major organs, and possessed high pathogenicity. In conclusion, combined with the genotype and pathogenicity of PCV2d currently prevalent, PCV2-GX-6 can be used as a candidate vaccine strain.

Near Full-Length Genomic Characterization of Two Novel HIV Type 1 Second-Generation Recombinant Forms (CRF01_AE/CRF07_BC) from Guangxi, China.

CRF01_AE and CRF07_BC are the two predominant HIV-1 subtypes currently circulating in China. We identified here a novel CCR5-tropic HIV-1 second-generation recombinant form virus found in two individuals, (GX19017 and GX19032), which were isolated from two HIV-1-positive people in Guangxi, southwest China. Phylogenic analyses indicated that these two sequences were all composed of two well-established circulating recombinant forms (CRFs) CRF07_BC and CRF01_AE, with four recombinant breakpoints observed in the pol, vpu/env, and env gene regions, respectively. The recombinant CRF01_AE region was clustered with the previously described CRF01_AE subcluster 2 lineage, which was characterized by the susceptibility to phenotypic transfer. The genome structure is significantly different from other previously reported CRFs and unique recombination forms. The emergence of a series of novel recombinant strains is indicative of the burgeoning complexity of the HIV-1 epidemic among the sexually transmitted population. Meanwhile, it may furnish significant insights into the dynamics and intricacy of the HIV-1 epidemic in China.

FRI386 - Full-length genome characterization of inherently resistant african HCV genotype 1, subtype 1l in patients failing DAA-based therapy

FRI386 - Full-length genome characterization of inherently resistant african HCV genotype 1, subtype 1l in patients failing DAA-based therapy

Discovery of novel DNA viruses in small mammals from Kenya.

Emergence and re-emergence of infectious diseases of wildlife origin have led pre-emptive pathogen surveillances in animals to be a public health priority. Rodents and shrews are among the most numerically abundant vertebrate taxa and are known as natural hosts of important zoonotic viruses. Many surveillance programs focused more on RNA viruses. In comparison, much less is known about DNA viruses harbored by these small mammals. To fill this knowledge gap, tissue specimens of 232 animals including 226 rodents, five shrews and one hedgehog were collected from 5 counties in Kenya and tested for the presence of DNA viruses belonging to 7 viral families by PCR. Diverse DNA sequences of adenoviruses, adeno-associated viruses, herpesviruses and polyomaviruses were detected. Phylogenetic analyses revealed that most of these viruses showed distinction from previously described viruses and formed new clusters. Furthermore, this is the first report of the discovery and full-length genome characterization of a polyomavirus in Lemniscomys species. This novel polyomavirus, named LsPyV KY187, has less than 60% amino acid sequence identity to the most related Glis glis polyomavirus 1 and Sciurus carolinensis polyomavirus 1 in both large and small T-antigen proteins and thus can be putatively allocated to a novel species within Betapolyomavirus. Our findings help us better understand the genetic diversity of DNA viruses in rodent and shrew populations in Kenya and provide new insights into the evolution of those DNA viruses in their small mammal reservoirs. It demonstrates the necessity of ongoing pathogen discovery studies targeting rodent-borne viruses in East Africa.

Near Full-Length Genomic Characterization of Two Novel HIV-1 Unique Recombinant Forms (CRF01_AE/CRF07_BC) Among Men Who Have Sex with Men in Shijiazhuang City, Hebei Province, China.

Unique recombinant forms (URFs) are more likely developed among HIV-1 infections through men who have sex with men (MSM) because of cocirculation of multiple subtypes. In this study, two novel URFs deriving from two HIV-positive subjects (HB010014, HB010063) were identified in Shijiazhuang, Hebei province, China, and two sequences formed a distinct monophyletic cluster. Further recombination analysis showed that of two new URFs were consisted of circulating recombinant form (CRF)01_AE and CRF07_BC. The subregion phylogenetic analysis indicated that CRF01_AE segments were traced back to cluster 4 of CRF01_AE strains, which were prevalent among HIV-1 infections through MSM in China. New URFs being developing gradually and spreading released that more and more novel recombinant strains of HIV-1 could be developed, which means that the past prevention strategies need to be adjusted.

Molecular Characterization and Phylogenetic Analyses of full-length Viral Genomes From Iranian Patients With Chronic Hepatitis B Virus

Aim: Chronic hepatitis B infection is the main cause of liver complications such as hepatic failure, liver cirrhosis and hepatocellular carcinoma (HCC). In this study, we attempted to evaluate molecular characterization and phylogenetic analyses of full-length viral genomes from chronic hepatitis B virus (HBV)-infected patients. Methods: The full-length genomic sequence of the five HBV isolates from Ahvaz (city of Iran) patients was amplified, cloned in pTZ57R/T vector, sequenced and examined. Results: Phylogenetic analyses showed that all isolates belonged to genotype D (D1/D3). Serotyper tool identified ayw2 serotype in all HBV isolates. YMDE mutation was detected in an HBV isolate in the reverse transcriptase domain. Conclusion: In the present study, the analyses of full-length sequence of genome revealed that the HBV genotype D, sub-genotype D1/D3, and subtype ayw2 were predominant among Ahvaz HBV strains. As HBV genome replicates and is mediated via reverse transcription process, periodic investigations of full HBV genome are needed.

First complete genome sequence and molecular characterization of Canine morbillivirus isolated in Central Brazil

The Brazilian regions are still highly endemic areas for Canine morbillivirus [canine distemper virus (CDV)]. However, little is known regarding the genetic variability of the strain circulating in several Brazilian regions. Here, we report the first full-length genome and molecular characterization of CDV isolated from domestic dogs in the Brazilian Center-West region. Sequence alignment and phylogenetic analyses based on deduced amino acid and nucleotide sequences showed that the isolated strain is characterized as the South America-I/Europe genotype. However, it segregates into a CDV subgenotype branch. Interestingly, both H and F proteins have a gain of a potential N-glycosylation sites compared to the Onderstepoort vaccine strain. Therefore, this study provides a reference to further understand the epidemic and molecular characteristics of the CDV in Brazil.

Molecular characterization of an avian GA13-like infectious bronchitis virus full-length genome from Costa Rica.

We describe the first whole-genome sequence of a GA13-like isolate of avian infectious bronchitis virus CK/CR/1160/16 (MN757859), obtained in 2016 in the province of Alajuela, Costa Rica. This virus caused an outbreak with great economic impact to the local poultry industry. The genome sequence is 27 696 bp in length, with the following genome organization 5′-UTR-Pol-S-3a-3b-E-4b-4c-M-5a-5b-N-6b-3′-UTR. The complete genome sequence has the highest sequence identity (94.03%) with DMV/1639/GA9977/2019 (MK878536) from Georgia, USA, and the lowest identity (86.03%) with ck/CH/LHLJ/08-6 (KX252788), from China. Analysis of the S1 subunit indicates that the Costa Rican isolate belongs to genotype I, lineage 17 (GI-17) and displays 96.89% identity with the S1 subunit of Ga-13/14255/14 (KM087780) (USA). Possible recombination events in genes S, E, M, 4b y 4c were detected, with Massachusetts, Connecticut, Arkansas and MA5 as potential parental types. This study highlights the importance of the epidemiological and molecular surveillance of avian infectious bronchitis.

Near Full-Length Genomic Characterization of 16 HIV-1 CRF01_AE Primary Isolates from Guangxi, China.

Isolation and culture of human immunodeficiency virus (HIV) are an important basis for acquired immune deficiency syndrome (AIDS) etiology, immunology, drug screening, clinical treatment, and vaccine research. CRF01_AE is one of the predominant strains of HIV-1 in China. However, there are few HIV-1 CRF01_AE isolates that have been reported. In this study, 16 HIV-1 CRF01_AE strains from Guangxi, China, were isolated, and the near full-length genomes were reverse transcribed and amplified in two halves with the 1 kb overlapping region. The polymerase chain reaction products were sequenced directly. The phylogenetic analysis results showed that all of the 16 isolated strains were CRF01_AE recombinant form, and two clusters were set up in the phylogenetic tree. The tropic prediction of 16 strains showed that 2 isolates were CCR5 tropic, and the others are CXCR4 tropic. Eight of the isolated strains are drug resistant according to the genetic prediction. These 16 near full-length characterized CRF01_AE isolates obtained in this study will provide valuable genomic and phenotypic information on HIV-1 strains circulating in China for related researches.

Near Full-Length Genomic Characterization of a Novel HIV-1 B/C Recombinant Form Identified in Guangdong Province, China.

The wide variety of new HIV-1 recombinant variants are a predominant challenge for understanding the molecular epidemiology and preventing the spread of the HIV-1 epidemic. In this study, we confirmed a novel HIV-1 unique B/C recombinant (ZLQ01186) isolated from a male patient infected with HIV-1 through injection drug use in Foshan city, Guangdong Province. The near full-length genome was amplified, and then the polymerase chain reaction products were sequenced by Sanger sequencing. The genomic sequence of the strain, with two subtype B segments inserted into the subtype C backbone, was 8,953 bp in length, extending from 647 to 9,599 bp according to the HXB2 genome. In addition, this B/C recombinant strain contained the non-nucleoside reverse transcriptase inhibitor resistance mutation K103N and the integrase strand transfer inhibitor other resistance mutation L74I according to the Stanford University HIV Drug Resistance Database program. The drug resistance profile indicates high-level resistance against efavirenz and rilpivirine. This study identified a recombinant between the main circulating strains, indicating a more complicated trend of the HIV-1 epidemic in Guangdong, China.

Full-length genome characterization of a novel alphapartitivirus detected in the ectomycorrhizal fungus Hygrophorus penarioides

Virus populations of ectomycorrhizal fungi remain poorly studied. In the present study, we characterized a new partitivirus isolated from the basidiomycetous, ectomycorrhizal fungus Hygrophorus penarioides, named "Hygrophorus penarioides partitivirus 1" (HpPV1). The whole genome of HpPV1, determined by merging deep sequencing and RLM-RACE approaches, comprised two dsRNA segments of 2053bp and 2072bp, respectively. Both dsRNA genome segments included a single open reading frame (ORF), encoding a putative RNA-dependent RNA polymerase (RdRp), and a capsid protein (CP), respectively. Based on BLASTp search, the sequences of the RdRp and CP exhibits the highest similarity (67.49% and 75.61% identity, respectively) to those of partitiviruses identified from an ascomycetous ectomycorrhizal fungus Sarcosphaera coronaria. Phylogenetic analyses performed based on the CP and RdRp sequences demonstrated that HpPV1 clusters within a clade that includes members of the genus Alphapartitivirus, belonging to the family Partitiviridae. Here, we propose that HpPV1 is a new member of the genus Alphapartitivirus. This is the first study reporting on a new partitivirus identified from the basidiomycetous, ectomycorrhizal fungus Hygrophorus penarioides.

Full-length genome characterization of a new fusagravirus hosted by the spring orange peel fungus Caloscypha fulgens.

Mycoviruses widely exist in diverse lineages of fungi, yet there are only a few studies on mycovirus infection in uncultivated fungi. We here report the presence of a dsRNA mycovirus in saprotrophic spring orange peel fungus Caloscypha fulgens. A novel dsRNA virus, named "Caloscypha fulgens fusagravirus 1" (CfFV1), was isolated from a single ascocarp of C. fulgens, and its molecular features were revealed. The full-length cDNA of CfFV1 comprises 9,548 nucleotides with a calculated GC content of 47.9% and two discontinuous open reading frames (ORF 1 and 2). A-1 ribosomal frameshift region with two distinctive elements, including a canonical slippery heptanucleotide (AAAAAAC) and a pseudoknot structure, predicted as a Recoding Stimulatory Element, was detected in the junction region of ORF1 and ORF2. The deduced amino acid sequence of ORF1 and ORF2 showed the highest similarity to the putative structural protein and RNA-dependent RNA polymerase (RdRp) of Rosellinia necatrix fusagravirus 4 (RnFGV4). Genome organization, sequence similarity, and phylogenetic analysis indicate that this virus belongs to a new member of the proposed family Fusagraviridae. This is the first report of the presence of a mycovirus in the spring orange peel fungus C. fulgens. Keywords: mycovirus; dsRNA; proposed Fusagraviridae; uncultivated fungi; Caloscypha fulgens.