Complete Genome Analyses of a Novel Flexivirus with Unique Genome Organization and Three Endornaviruses Hosted by the Mycorrhizal Fungus Terfezia claveryi.

The extensive use of high-throughput sequencing (HTS) has significantly advanced and transformed our comprehension of virus diversity, especially in intricate settings like soil and biological specimens. In this study, we delved into mycovirus sequence surveys within mycorrhizal fungus species Terfezia claveryi, through employing HTS with total double-stranded RNA (dsRNA) extracts. Our findings revealed the presence of four distinct members from the Alsuviricetes class, one flexivirus designated as Terfezia claveryi flexivirus 1 (TcFV1) and three endornaviruses (TcEV1, TcEV2, and TcEV3) in two different T. claveryi isolates. TcFV1, a member of the order Tymovirales, exhibits a unique genome structure and sequence features. Through in-depth analyses, we found that it shares sequence similarities with other deltaflexiviruses and challenges existing Deltaflexiviridae classification. The discovery of TcFV1 adds to the genomic plasticity of mycoviruses within the Tymovirales order, shedding light on their evolutionary adaptations. Additionally, the three newly discovered endornaviruses (TcEV1, TcEV2, and TcEV3) in T. claveryi exhibited limited sequence similarities with other endornaviruses and distinctive features, including conserved domains like DEAD-like helicase, ATPases Associated with Diverse Cellular Activities (AAA ATPase), and RNA dependent RNA polymerase (RdRp), indicating their classification as members of new species within the Alphaendornavirus genus. In conclusion, this research emphasizes the importance of exploring viral diversity in uncultivated fungi, bridging knowledge gaps in mycovirus ecology. The discoveries of a novel flexivirus with unique genome organization and endornaviruses in T. claveryi broaden our comprehension of mycovirus diversity and evolution, highlighting the need for continued investigations into viral populations in wild fungi.

A colorimetric reverse transcription-loop mediated isothermal amplification (RT-LAMP) method for the rapid detection of SARS-CoV-2 in Thailand.

COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global health threat. Timely identification of infected cases is important for appropriate patient management and the control of viral spread. Simple and cost-effective tests are required to increase access to testing and early case detection. Here, we describe a colorimetric reverse transcription-loop-mediated isothermal amplification (RT-LAMP) method to detect SARS-CoV-2. The RT-LAMP could amplify the orf1ab sequence detectable by visual color change within 45 min at 63 °C. The limit of detection (LoD) for SARS-CoV-2 RNA was less than 100 copies (13.36) per reaction with no cross-amplification with other related viruses. Clinical evaluation using leftover RNA samples extracted from 163 nasopharyngeal swab specimens showed perfect agreement in negative (n = 124) and positive samples with cycle thresholds (Ct) < 34 cycles (n = 33) detected by real-time reverse transcription-polymerase chain reaction (RT-PCR), targeting RdRp and N genes as a reference. Overall, the diagnostic accuracy, sensitivity, specificity, positive and negative predictive values of RT-LAMP in testing were 96.32% (95% CI: 92.16-98.64%), 84.62% (95% CI: 68.47-94.14%), 100% (95% CI: 97.07-100.0%), 100% (95% CI: 89.42-100.0%), and 95.38% (95% CI: 90.22-98.29), respectively. This RT-LAMP assay is simple and reliable, with the potential to be an alternative for the rapid detection of SAR-CoV-2 with minimal time and fewer resources compared to real-time RT-PCR.

Occurrence and characterization of viruses infecting Amorphophallus in Yunnan, China.

Viral diseases are becoming an important problem in Amorphophallus production due to the propagation of seed corms and their trade across regions. In this study, combined-High-Throughput Sequencing, RT-PCR, electron microscopy, and mechanical inoculation were used to analyze virus-like infected Amorphophallus samples in Yunnan province to investigate the distribution, molecular characterization, and diversity and evolution of Amorphophallus-infecting viruses including three isolates of dasheen mosaic virus and three orthotospoviruses: mulberry vein banding associated virus (MVBaV), tomato zonate spot virus (TZSV) and impatiens necrotic spot virus (INSV). The results showed that DsMV is the dominant virus infecting Amorphophallus, mixed infections with DsMV and MVBaV to Amorphophallus were quite common in Yunnan province, China. This is the first report on infection of Amorphophallus with MVBaV, TZSV, and impatiens necrotic spot virus (INSV) in China. This work will help to develop an effective integrated management strategy to control the spread of Amorphophallus viral diseases.

Complete genome sequence and genetic characterization of a novel segmented RNA virus infecting Nilaparvata lugens.

The brown planthopper (BPH), Nilaparvata lugens, is a significant agricultural pest capable of long-distance migration and transmission of viruses that cause severe disease in rice. In this study, we identified a novel segmented RNA virus in a BPH, and this virus exhibited a close relationship to members of a recently discovered virus lineage known as "quenyaviruses" within the viral kingdom Orthornavirae. This newly identified virus was named "Nilaparvata lugens quenyavirus 1" (NLQV1). NLQV1 consists of five positive-sense, single-stranded RNAs, with each segment containing a single open reading frame (ORF). The genomic characteristics and phylogenetic analysis support the classification of NLQV1 as a novel quenyavirus. Notably, all of the genome segments of NLRV contained the 5'-terminal sequence AUCUG. The characteristic virus-derived small interfering RNA (vsiRNA) profile of NLQV1 suggests that the antiviral RNAi pathway of the host BPH was activated in response to virus infection. These findings represent the first documented report of quenyaviruses in planthoppers, contributing to our understanding of quenyaviruses and expanding our knowledge of insect-specific viruses in planthoppers.

Molecular characterization of birch toti-like virus, a plant-associated member of the new family Orthototiviridae.

A novel totivirus, named "birch toti-like virus" (BTLV), was discovered in European white birch (Betula pendula) plants. The genome of BTLV is 4,967 nucleotides long and contains two overlapping open reading frames (ORFs) coding for the capsid protein (CP) and an RNA-dependent RNA-polymerase (RdRP). The encoded CP and RdRP proteins shared 46.9% and 60.2% amino acid sequence identity, respectively, with those of Panax notoginseng virus B. The presence of a putative slippery heptamer signal 82 nt upstream of the stop codon of ORF1 suggests that a -1 translational frameshifting strategy is involved in the expression of ORF2, like in other totiviruses. Phylogenetic analysis based on the CP and RdRP amino acid sequences placed this virus within a clade of plant-associated totiviruses, with taro-associated virus as its closest relative. Hence, based on its distinct host and the amino acid sequence similarity between BTLV and its relatives, we conclude that birch toti-like virus is a new member of the genus Totivirus.

Serotype and genomic diversity of dengue virus during the 2023 outbreak in Pakistan reveals the circulation of genotype III of DENV-1 and cosmopolitan genotype of DENV-2.

Dengue, a mosquito-borne viral disease, poses a significant public health challenge in Pakistan, with a significant outbreak in 2023, prompting our investigation into the serotype and genomic diversity of the dengue virus (DENV). NS-1 positive blood samples from 153 patients were referred to the National Institute of Health, Pakistan, between July and October 2023. Among these, 98 (64.1%) tested positive using multiplex real-time PCR, with higher prevalence among males (65.8%) and individuals aged 31-40. Serotyping revealed DENV-1 as the predominant serotype (84.7%), followed by DENV-2 (15.3%). Whole-genome sequencing of 18 samples (DENV-1 = 17, DENV-2 = 01) showed that DENV-1 (genotype III) samples were closely related (>99%) to Pakistan outbreak samples (2022), and approx. > 98% with USA (2022), Singapore and China (2016), Bangladesh (2017), and Pakistan (2019). The DENV-2 sequence (cosmopolitan genotype; clade IVA) shared genetic similarity with Pakistan outbreak sequences (2022), approx. > 99% with China and Singapore (2018-2019) and showed divergence from Pakistan sequences (2008-2013). No coinfection with dengue serotypes or other viruses were observed. Comparisons with previous DENV-1 sequences highlighted genetic variations affecting viral replication efficiency (NS2B:K55R) and infectivity (E:M272T). These findings contribute to dengue epidemiology understanding and underscore the importance of ongoing genomic surveillance for future outbreak responses in Pakistan.

Identification of a novel waikavirus infecting Pittosporum tobira in China.

A novel waikavirus, tentatively named "Pittosporum tobira waikavirus" (PtWV), was identified in Pittosporum tobira plants exhibiting mosaic and ringspot symptoms on foliage in Yunnan, China. The full-length genomic sequence was determined by high-throughput sequencing and rapid amplification of cDNA ends. The genome of PtWV is 12,709 nt in length and has a large open reading frame (ORF) of 11,010 nt, encoding a polyprotein, and a small ORF that encodes a 13.2-kDa bellflower vein chlorosis virus (BVCV)-like protein. Phylogenetic analysis and sequence alignment revealed that PtWV is closely related to actinidia yellowing virus 1 (AcYV1), which shares the highest amino acid (aa) sequence similarity (50.1% identity) in the Pro-RdRp region. To the best of our knowledge, this is the first report of a novel waikavirus in P. tobira.

Shedding of measles vaccine RNA in children after receiving measles, mumps and rubella vaccination.

BACKGROUND: Measles, mumps, and rubella(MMR) vaccination is critical to measles outbreak responses. However, vaccine reactions and detection of measles vaccine RNA in recently immunized persons may complicate case classification especially in those presenting with another respiratory viral illness. We aim to characterize cases of measles vaccine shedding in recently vaccinated children presenting with respiratory viral symptoms. METHODS: Children who were tested with a multiplex respiratory panel <30 days after receiving MMR were identified. Remnant nasopharyngeal(NP) samples were tested for measles vaccine by PCR. Medical records were reviewed for demographics, presenting symptoms, and test results. RESULTS: From January 2022 to March 2023, 127 NP from children who received MMR were tested. Ninety-six NP were collected after the first dose, of which 33(34.4 %) were positive for vaccine RNA. The median interval between MMR and detection was 11.0 days. Thirty-one NP were collected after the second MMR and 1(3.2 %) was positive; time between the vaccination and detection was 18.9 days. Median cycle threshold(Ct) value of the measles PCR for vaccine shedding was significantly higher than median Ct in children with wild-type infection. CONCLUSION: Shedding of measles vaccine RNA is not uncommon and vaccine RNA can be detected up to 29 days post MMR; the amount of vaccine RNA shedding is low indicated by high Ct values. Clinicians and public health officials should consider performing measles vaccine testing on those testing positive for measles within one month of MMR vaccination, especially if the Ct value is high and definitive epidemiological links are absent.

Genetic determinants of host- and virus-derived insertions for hepatitis E virus replication.

Hepatitis E virus (HEV) is a long-neglected RNA virus and the major causative agent of acute viral hepatitis in humans. Recent data suggest that HEV has a very heterogeneous hypervariable region (HVR), which can tolerate major genomic rearrangements. In this study, we identify insertions of previously undescribed sequence snippets in serum samples of a ribavirin treatment failure patient. These insertions increase viral replication while not affecting sensitivity towards ribavirin in a subgenomic replicon assay. All insertions contain a predicted nuclear localization sequence and alanine scanning mutagenesis of lysine residues in the HVR influences viral replication. Sequential replacement of lysine residues additionally alters intracellular localization in a fluorescence dye-coupled construct. Furthermore, distinct sequence patterns outside the HVR are identified as viral determinants that recapitulate the enhancing effect. In conclusion, patient-derived insertions can increase HEV replication and synergistically acting viral determinants in and outside the HVR are described. These results will help to understand the underlying principles of viral adaptation by viral- and host-sequence snatching during the clinical course of infection.

Epitranscriptomic cytidine methylation of the hepatitis B viral RNA is essential for viral reverse transcription and particle production.

Epitranscriptomic RNA modifications have emerged as important regulators of the fate and function of viral RNAs. One prominent modification, the cytidine methylation 5-methylcytidine (m5C), is found on the RNA of HIV-1, where m5C enhances the translation of HIV-1 RNA. However, whether m5C functionally enhances the RNA of other pathogenic viruses remains elusive. Here, we surveyed a panel of commonly found RNA modifications on the RNA of hepatitis B virus (HBV) and found that HBV RNA is enriched with m5C as well as ten other modifications, at stoichiometries much higher than host messenger RNA (mRNA). Intriguingly, m5C is mostly found on the epsilon hairpin, an RNA element required for viral RNA encapsidation and reverse transcription, with these m5C mainly deposited by the cellular methyltransferase NSUN2. Loss of m5C from HBV RNA due to NSUN2 depletion resulted in a partial decrease in viral core protein (HBc) production, accompanied by a near-complete loss of the reverse transcribed viral DNA. Similarly, mutations introduced to remove the methylated cytidines resulted in a loss of HBc production and reverse transcription. Furthermore, pharmacological disruption of m5C deposition led to a significant decrease in HBV replication. Thus, our data indicate m5C methylations as a critical mediator of the epsilon elements' function in HBV virion production and reverse transcription, suggesting the therapeutic potential of targeting the m5C methyltransfer process on HBV epsilon as an antiviral strategy.

Establishment of dual reverse transcriptase-polymerase chain reaction for detection system for Areca palm velarivirus 1.

Areca palm velarivirus 1 (APV1) is one of the main pathogen causing yellow leaf disease, and leading to considerable losses in the Areca palm industry. The detection methods for APV1 are primarily based on phenotype determination and molecular techniques, such as polymerase chain reaction (PCR). However, a single PCR has limitations in accuracy and sensitivity. Therefore, in the present study, we established a dual RT-PCR APV1-detection system with enhanced accuracy and sensitivity using two pairs of specific primers, YLDV2-F/YLDV2-R and YLDV4-F/YLDV4-R. Moreover, two cDNA fragments covering different regions of the viral genome were simultaneously amplified, with PCR amplicon of 311 and 499 bp, respectively. The dual RT-PCR detection system successfully amplified the two target regions of the APV1, demonstrating high specificity and sensitivity and compensating for the limitations of single-primer detection methods. We tested 60 Areca palm samples from different geographical regions, highlighting its advantages in that the dual RT-PCR system efficiently and accurately detected APV1 in samples across diverse areas. The dual RT-PCR APV1 detection system provides a rapid, accurate, and sensitive method for detecting the virus and offers valuable technical support for research in preventing and managing yellow leaf diseases caused by APV1 in Areca palms. Moreover, the findings of this study can serve as a reference for establishing similar plants viral detection systems in the future.

A Statovirus-like virus from respiratory tracts of patients, China.

The emerging evidence of human infections with emerging viruses suggests their potential public health importance. A novel taxon of viruses named Statoviruses (for stool-associated Tombus-like viruses) was recently identified in the gastrointestinal tracts of multiple mammals. Here we report the discovery of respiratory Statovirus-like viruses (provisionally named Restviruses) from the respiratory tracts of five patients experiencing acute respiratory disease with Human coronavirus OC43 infection through the retrospective analysis of meta-transcriptomic data. Restviruses shared 53.1%-98.8% identities of genomic sequences with each other and 39.9%-44.3% identities with Statoviruses. The phylogenetic analysis revealed that Restviruses together with a Stato-like virus from nasal-throat swabs of Vietnamese patients with acute respiratory disease, formed a well-supported clade distinct from the taxon of Statoviruses. However, the consistent genome characteristics of Restviruses and Statoviruses suggested that they might share similar evolutionary trajectories. These findings warrant further studies to elucidate the etiological and epidemiological significance of the emerging Restviruses.

The question of screening organ donors for hepatitis e virus: a case report of transmission by kidney transplantation in France and a review of the literature.

BACKGROUND: Hepatitis E is a potentially serious infection in organ recipients, with an estimated two-thirds of cases becoming chronic, and with a subsequent risk of cirrhosis and death. In Europe, transmission occurs most often through the consumption of raw or undercooked pork, more rarely through blood transfusion, but also after solid organ transplantation. Here we describe a case of Hepatitis E virus (HEV) infection transmitted following kidney transplantation and review the literature describing cases of HEV infection transmitted by solid organ transplantation. CASE PRESENTATION: Three weeks after kidney transplantation, the patient presented with an isolated minimal increase in GGT and hepatic cytolysis 6 months later, leading to the diagnosis of genotype 3c hepatitis E, with a plasma viral load of 6.5 log10IU/mL. In retrospect, HEV RNA was detected in the patient's serum from the onset of hepatitis, and in the donor's serum on the day of donation, with 100% identity between the viral sequences, confirming donor-derived HEV infection. Hepatitis E had a chronic course, was treated by ribavirin, and relapsed 10 months after the end of treatment. DISCUSSION: Seven cases of transmission of HEV by solid organ transplantation have been described since 2012 without systematic screening for donors, all diagnosed at the chronic infection stage; two patients died. HEV organ donor transmission may be underestimated and there is insufficient focus on immunocompromised patients in whom mild liver function test impairment is potentially related to hepatitis E. However, since HEV infection is potentially severe in these patients, and as evidence accumulates, we believe that systematic screening of organ donors should be implemented for deceased and living donors regardless of liver function abnormalities, as is already the case in the UK and Spain. In January 2024, the French regulatory agency of transplantation has implemented mandatory screening of organ donors for HEV RNA.

Argonaute-independent, Dicer-dependent antiviral defense against RNA viruses.

Antiviral RNA interference (RNAi) is conserved from yeasts to mammals. Dicer recognizes and cleaves virus-derived double-stranded RNA (dsRNA) and/or structured single-stranded RNA (ssRNA) into small-interfering RNAs, which guide effector Argonaute to homologous viral RNAs for digestion and inhibit virus replication. Thus, Argonaute is believed to be essential for antiviral RNAi. Here, we show Argonaute-independent, Dicer-dependent antiviral defense against dsRNA viruses using Cryphonectria parasitica (chestnut blight fungus), which is a model filamentous ascomycetous fungus and hosts a variety of viruses. The fungus has two dicer-like genes (dcl1 and dcl2) and four argonaute-like genes (agl1 to agl4). We prepared a suite of single to quadruple agl knockout mutants with or without dcl disruption. We tested these mutants for antiviral activities against diverse dsRNA viruses and ssRNA viruses. Although both DCL2 and AGL2 worked as antiviral players against some RNA viruses, DCL2 without argonaute was sufficient to block the replication of other RNA viruses. Overall, these results indicate the existence of a Dicer-alone defense and different degrees of susceptibility to it among RNA viruses. We discuss what determines the great difference in susceptibility to the Dicer-only defense.

Identification and characterization of Varicella Zoster Virus circular RNA in lytic infection.

This study investigates the role of circular RNAs (circRNAs) in the context of Varicella-Zoster Virus (VZV) lytic infection. We employ two sequencing technologies, short-read sequencing and long-read sequencing, following RNase R treatment on VZV-infected neuroblastoma cells to identify and characterize both cellular and viral circRNAs. Our large scanning analysis identifies and subsequent experiments confirm 200 VZV circRNAs. Moreover, we discover numerous VZV latency-associated transcripts (VLTs)-like circRNAs (circVLTslytic), which contain multiple exons and different isoforms within the same back-splicing breakpoint. To understand the functional significance of these circVLTslytic, we utilize the Bacteria Artificial Chromosome system to disrupt the expression of viral circRNAs in genomic DNA location. We reveal that the sequence flanking circVLTs' 5' splice donor plays a pivotal role as a cis-acting element in the formation of circVLTslytic. The circVLTslytic is dispensable for VZV replication, but the mutation downstream of circVLTslytic exon 5 leads to increased acyclovir sensitivity in VZV infection models. This suggests that circVLTslytic may have a role in modulating the sensitivity to antiviral treatment. The findings shed new insight into the regulation of cellular and viral transcription during VZV lytic infection, emphasizing the intricate interplay between circRNAs and viral processes.

Wide Real-Life Data Support Reduced Sensitivity of Antigen Tests for Omicron SARS-CoV-2 Infections.

With the continuous spread of new SARS-CoV-2 variants of concern (VOCs), the monitoring of diagnostic test performances is mandatory. We evaluated the changes in antigen diagnostic tests' (ADTs) accuracy along the Delta to Omicron VOCs transition, exploring the N protein mutations possibly affecting ADT sensitivity and assessing the best sampling site for the diagnosis of Omicron infections. In total, 5175 subjects were enrolled from 1 October 2021 to 15 July 2022. The inclusion criteria were SARS-CoV-2 ADT combined with a same-day RT-PCR swab test. For the sampling site analysis, 61 patients were prospectively recruited during the Omicron period for nasal and oral swab analyses by RT-PCR. Next-Generation Sequencing data were obtained to evaluate the different sublineages. Using RT-PCR as a reference, 387 subjects resulted in becoming infected and the overall sensitivity of the ADT decreased from 63% in the Delta period to 33% in the Omicron period. This decrease was highly statistically significant (p < 0.001), and no decrease in viral load was detected at the RNA level. The nasal site presented a significantly higher viral load than the oral site during the Omicron wave. The reduced detection rate of Omicron infections by ADT should be considered in the global testing strategy to preserve accurate diagnoses across the changing SARS-CoV-2 variants.

The Role of the Host Cytoskeleton in the Formation and Dynamics of Rotavirus Viroplasms.

Rotavirus (RV) replicates within viroplasms, membraneless electron-dense globular cytosolic inclusions with liquid-liquid phase properties. In these structures occur the virus transcription, replication, and packaging of the virus genome in newly assembled double-layered particles. The viroplasms are composed of virus proteins (NSP2, NSP5, NSP4, VP1, VP2, VP3, and VP6), single- and double-stranded virus RNAs, and host components such as microtubules, perilipin-1, and chaperonins. The formation, coalescence, maintenance, and perinuclear localization of viroplasms rely on their association with the cytoskeleton. A stabilized microtubule network involving microtubules and kinesin Eg5 and dynein molecular motors is associated with NSP5, NSP2, and VP2, facilitating dynamic processes such as viroplasm coalescence and perinuclear localization. Key post-translation modifications, particularly phosphorylation events of RV proteins NSP5 and NSP2, play pivotal roles in orchestrating these interactions. Actin filaments also contribute, triggering the formation of the viroplasms through the association of soluble cytosolic VP4 with actin and the molecular motor myosin. This review explores the evolving understanding of RV replication, emphasizing the host requirements essential for viroplasm formation and highlighting their dynamic interplay within the host cell.

Deletion of 82-85 N-Terminal Residues in SARS-CoV-2 Nsp1 Restricts Virus Replication.

Non-structural protein 1 (Nsp1) represents one of the most crucial SARS-CoV-2 virulence factors by inhibiting the translation of host mRNAs and promoting their degradation. We selected naturally occurring virus lineages with specific Nsp1 deletions located at both the N- and C-terminus of the protein. Our data provide new insights into how Nsp1 coordinates these functions on host and viral mRNA recognition. Residues 82-85 in the N-terminal part of Nsp1 likely play a role in docking the 40S mRNA entry channel, preserving the inhibition of host gene expression without affecting cellular mRNA decay. Furthermore, this domain prevents viral mRNAs containing the 5'-leader sequence to escape translational repression. These findings support the presence of distinct domains within the Nsp1 protein that differentially modulate mRNA recognition, translation and turnover. These insights have implications for the development of drugs targeting viral proteins and provides new evidences of how specific mutations in SARS-CoV-2 Nsp1 could attenuate the virus.

Factor-Dependent Internal Ribosome Entry Site and -1 Programmed Frameshifting Signal in the Bemisia-Associated Dicistrovirus 2.

The dicistrovirus intergenic (IGR) IRES uses the most streamlined translation initiation mechanism: the IRES recruits ribosomes directly without using protein factors and initiates translation from a non-AUG codon. Several subtypes of dicistroviruses IRES have been identified; typically, the IRESs adopt two -to three overlapping pseudoknots with key stem-loop and unpaired regions that interact with specific domains of the ribosomal 40S and 60S subunits to direct translation. We previously predicted an atypical IGR IRES structure and a potential -1 programmed frameshift (-1 FS) signal within the genome of the whitefly Bemisia-associated dicistrovirus 2 (BaDV-2). Here, using bicistronic reporters, we demonstrate that the predicted BaDV-2 -1 FS signal can drive -1 frameshifting in vitro via a slippery sequence and a downstream stem-loop structure that would direct the translation of the viral RNA-dependent RNA polymerase. Moreover, the predicted BaDV-2 IGR can support IRES translation in vitro but does so through a mechanism that is not typical of known factorless dicistrovirus IGR IRES mechanisms. Using deletion and mutational analyses, the BaDV-2 IGR IRES is mapped within a 140-nucleotide element and initiates translation from an AUG codon. Moreover, the IRES does not bind directly to purified ribosomes and is sensitive to eIF2 and eIF4A inhibitors NSC1198983 and hippuristanol, respectively, indicating an IRES-mediated factor-dependent mechanism. Biophysical characterization suggests the BaDV-2 IGR IRES contains several stem-loops; however, mutational analysis suggests a model whereby the IRES is unstructured or adopts distinct conformations for translation initiation. In summary, we have provided evidence of the first -1 FS frameshifting signal and a novel factor-dependent IRES mechanism in this dicistrovirus family, thus highlighting the diversity of viral RNA-structure strategies to direct viral protein synthesis.