Detection of SARS-CoV-2 using machine learning-enabled paper-assisted ratiometric fluorescent sensors based on target-induced magnetic DNAzyme.

The development of an advanced analytical platform with regard to SARS-CoV-2 is crucial for public health. Herein, we present a machine learning platform based on paper-assisted ratiometric fluorescent sensors for highly sensitive detection of the SARS-CoV-2 RdRp gene. The assay involves target-induced rolling circle amplification to generate magnetic DNAzyme, which is then detectable using the paper-assisted ratiometric fluorescent sensor. This sensor detects the SARS-CoV-2 RdRp gene with a visible-fluorescence color response. Moreover, leveraging different fluorescence responses, the ResNet algorithm of machine learning assists in accurately identifying fluorescence images and differentiating the concentration of the SARS-CoV-2 RdRp gene with over 99% recognition accuracy. The machine learning platform exhibits exceptional sensitivity and color responsiveness, achieving a limit of detection of 30 fM for the SARS-CoV-2 RdRp gene. The integration of intelligent artificial vision with the paper-assisted ratiometric fluorescent sensor presents a novel approach for the on-site detection of COVID-19 and holds potential for broader use in disease diagnostics in the future.

Characterization of a novel gammapartitivirus infecting the phytopathogenic fungus Pyricularia oryzae.

In this study, we identified a novel double-strand RNA (dsRNA) mycovirus in Pyricularia oryzae, designated "Magnaporthe oryzae partitivirus 4" (MoPV4). The genome of MoPV4 consists of a dsRNA-1 segment encoding an RNA-dependent RNA polymerase (RdRP) and a dsRNA-2 segment encoding a capsid protein (CP). Phylogenetic analysis indicated that MoPV4 belongs to the genus Gammapartitivirus within family Partitiviridae. The particles of MoPV4 are isometric with a diameter of about 32.4 nm. Three-dimensional structure predictions indicated that the RdRP of MoPV4 forms a classical right-handed conformation, while the CP has a reclining-V shape.

Optimized Recombinant Expression and Purification of the SARS-CoV-2 Polymerase Complex.

An optimized protocol has been developed to express and purify the core RNA-dependent RNA polymerase (RdRP) complex from the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The expression and purification of active core SARS-CoV-2 RdRp complex is challenging due to the complex multidomain fold of nsp12, and the assembly of the multimeric complex involving nsp7, nsp8, and nsp12. Our approach adapts a previously published method to express the core SARS-CoV-2 RdRP complex in Escherichia coli and combines it with a procedure to express the nsp12 fusion with maltose-binding protein in insect cells to promote the efficient assembly and purification of an enzymatically active core polymerase complex. The resulting method provides a reliable platform to produce milligram amounts of the polymerase complex with the expected 1:2:1 stoichiometry for nsp7, nsp8, and nsp12, respectively, following the removal of all affinity tags. This approach addresses some of the limitations of previously reported methods to provide a reliable source of the active polymerase complex for structure, function, and inhibition studies of the SARS-CoV-2 RdRP complex using recombinant plasmid constructs that have been deposited in the widely accessible Addgene repository. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression and production of SARS-CoV-2 nsp7, nsp8, and nsp12 in E. coli cells Support Protocol: Establishment and maintenance of insect cell cultures Basic Protocol 2: Generation of recombinant baculovirus in Sf9 cells and production of nsp12 fusion protein in T. ni cells Basic Protocol 3: Purification of the SARS-CoV-2 core polymerase complex.

Interleukin-2 enhancer binding factor 2 negatively regulates the replication of duck hepatitis A virus type 1 by disrupting the RNA-dependent RNA polymerase activity of 3D polymerase.

The interaction between viral components and cellular proteins plays a crucial role in viral replication. In a previous study, we showed that the 3'-untranslated region (3'-UTR) is an essential element for the replication of duck hepatitis A virus type 1 (DHAV-1). However, the underlying mechanism is still unclear. To gain a deeper understanding of this mechanism, we used an RNA pull-down and a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay to identify new host factors that interact with the 3'-UTR. We selected interleukin-2 enhancer binding factor 2 (ILF2) for further analysis. We showed that ILF2 interacts specifically with both the 3'-UTR and the 3D polymerase (3Dpol) of DHAV-1 through in vitro RNA pull-down and co-immunoprecipitation assays, respectively. We showed that ILF2 negatively regulates viral replication in duck embryo fibroblasts (DEFs), and that its overexpression in DEFs markedly suppresses DHAV-1 replication. Conversely, ILF2 silencing resulted in a significant increase in viral replication. In addition, the RNA-dependent RNA polymerase (RdRP) activity of 3Dpol facilitated viral replication by enhancing viral RNA translation efficiency, whereas ILF2 disrupted the role of RdRP in viral RNA translation efficiency to suppress DHAV-1 replication. At last, DHAV-1 replication markedly suppressed the expression of ILF2 in DEFs, duck embryo hepatocytes, and different tissues of 1 day-old ducklings. A negative correlation was observed between ILF2 expression and the viral load in primary cells and different organs of young ducklings, suggesting that ILF2 may affect the viral load both in vitro and in vivo.

Bactericera cockerelli Picorna-like Virus and Three New Viruses Found Circulating in Populations of Potato/Tomato Psyllids (Bactericera cockerelli).

An investigation of viruses circulating in populations of field and laboratory potato/tomato psyllids (Bactericera cockerelli) was conducted using high-throughput sequencing (HTS) technology and conventional RT-PCR. Three new viruses were discovered: one from the family Tymoviridae and two from the family Solemoviridae. A tymo-like virus sequence represented a nearly complete 6843 nt genome of a virus named Bactericera cockerelli tymo-like virus (BcTLV) that spanned five open reading frames (ORFs) which encoded RNA-dependent RNA polymerase (RdRP), helicase, protease, methyltransferase, and a capsid protein. Phylogenetic analyses placed the RdRP of BcTLV inside a divergent lineage of the viruses from the family Tymoviridae found in insect and plant hosts in a sister clade to the genera Tymovirus, Marafivirus, and Maculavirus. Four solemo-like virus sequences were identified in the HTS outputs, representing two new viruses. One virus found only in field-collected psyllids and named Bactericera cockerelli solemo-like virus 1 (BcSLV-1) had a 5479 nt genome which spanned four ORFs encoding protease and RdRP. Three solemo-like sequences displayed 87.4-99.7% nucleotide sequence identity among themselves, representing variants or strains of the same virus named Bactericera cockerelli solemo-like virus 2 (BcSLV-2). The genome of BcSLV-2 spanned only two ORFs that encoded a protease and an RdRP. Phylogenetic analysis placed the RdRPs of BcSLV-1 and BcSLV-2 in two separate lineages as sister clades to viruses from the genus Sobemovirus found in plant hosts. All three new psyllid viruses were found circulating in psyllids collected from potato fields in southern Idaho along with a previously identified Bactericera cockerelli picorna-like virus. Any possible role of the three viruses in controlling populations of the field psyllids remains to be elucidated.

BAG6 inhibits influenza A virus replication by inducing viral polymerase subunit PB2 degradation and perturbing RdRp complex assembly.

The interaction between influenza A virus (IAV) and host proteins is an important process that greatly influences viral replication and pathogenicity. PB2 protein is a subunit of viral ribonucleoprotein (vRNP) complex playing distinct roles in viral transcription and replication. BAG6 (BCL2-associated athanogene 6) as a multifunctional host protein participates in physiological and pathological processes. Here, we identify BAG6 as a new restriction factor for IAV replication through targeting PB2. For both avian and human influenza viruses, overexpression of BAG6 reduced viral protein expression and virus titers, whereas deletion of BAG6 significantly enhanced virus replication. Moreover, BAG6-knockdown mice developed more severe clinical symptoms and higher viral loads upon IAV infection. Mechanistically, BAG6 restricted IAV transcription and replication by inhibiting the activity of viral RNA-dependent RNA polymerase (RdRp). The co-immunoprecipitation assays showed BAG6 specifically interacted with the N-terminus of PB2 and competed with PB1 for RdRp complex assembly. The ubiquitination assay indicated that BAG6 promoted PB2 ubiquitination at K189 residue and targeted PB2 for K48-linked ubiquitination degradation. The antiviral effect of BAG6 necessitated its N-terminal region containing a ubiquitin-like (UBL) domain (17-92aa) and a PB2-binding domain (124-186aa), which are synergistically responsible for viral polymerase subunit PB2 degradation and perturbing RdRp complex assembly. These findings unravel a novel antiviral mechanism via the interaction of viral PB2 and host protein BAG6 during avian or human influenza virus infection and highlight a potential application of BAG6 for antiviral drug development.

Complete genome sequence of a novel dsRNA virus from the phytopathogenic fungus Fusarium oxysporum.

Fusarium oxysporum is a widespread plant pathogen that causes fusarium wilt and fusarium root rot in many economically significant crops. Here, a novel dsRNA virus tentatively named "Fusarium oxysporum virus 1" (FoV1) was identified in F. oxysporum strain 3S-18. The genome of FoV1 is 2,944 nucleotides (nt) in length and contains two non-overlapping open reading frames (ORF1 and 2). The larger of these, ORF2, encodes an RNA-dependent RNA polymerase (RdRp) of 590 amino acids with a molecular mass of 67.52 kDa. ORF1 encodes a putative nucleocapsid protein consisting of 134 amino acids with a molecular mass of 34.25 kDa. The RdRp domain of FoV1 shares 60.00% to 84.24% sequence identity with non-segmented dsRNA viruses. Phylogenetic analysis further suggested that FoV1 is a new member of the proposed genus "Unirnavirus" accommodating unclassified monopartite dsRNA viruses.

Genome characterization of a novel narnavirus infecting the plant-pathogenic fungus Ustilaginoidea virens.

Mycoviruses are viruses that infect fungi and oomycetes. They are widespread in all major groups of plant-pathogenic fungi and oomycetes. To date, only the full genome of dsRNA mycoviruses and the contigs of positive-sense single-stranded RNA (+ssRNA) mycoviruses have been reported in Ustilaginoidea virens, which is the notorious causal agent of rice false smut (RFS). Here, we report the molecular characterization of a novel +ssRNA mycovirus, Ustilaginoidea virens narnavirus 4 (UvNV4), isolated from U. virens strain Uv418. UvNV4 has a genome of 3,131 nucleotides (nt) and possesses an open reading frame (ORF) predicted to encode an RNA-dependent RNA polymerase (RdRp) of 1,017 amino acids (aa) sequence with a molecular mass of 116.6 kDa. BLASTp analysis revealed that the RdRp showed 50.34% aa sequence identity to that of the previously described Zhangzhou Narna tick virus 1. Phylogenetic analysis indicated that UvNV4 is closely related to members of the family Narnaviridae. Taken together, these results clearly demonstrate that UvNV4 is a novel +ssRNA virus infecting U. virens.

BPR3P0128, a non-nucleoside RNA-dependent RNA polymerase inhibitor, inhibits SARS-CoV-2 variants of concern and exerts synergistic antiviral activity in combination with remdesivir.

Viral RNA-dependent RNA polymerase (RdRp), a highly conserved molecule in RNA viruses, has recently emerged as a promising drug target for broad-acting inhibitors. Through a Vero E6-based anti-cytopathic effect assay, we found that BPR3P0128, which incorporates a quinoline core similar to hydroxychloroquine, outperformed the adenosine analog remdesivir in inhibiting RdRp activity (EC50 = 0.66 µM and 3 µM, respectively). BPR3P0128 demonstrated broad-spectrum activity against various severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern. When introduced after viral adsorption, BPR3P0128 significantly decreased SARS-CoV-2 replication; however, it did not affect the early entry stage, as evidenced by a time-of-drug-addition assay. This suggests that BPR3P0128's primary action takes place during viral replication. We also found that BPR3P0128 effectively reduced the expression of proinflammatory cytokines in human lung epithelial Calu-3 cells infected with SARS-CoV-2. Molecular docking analysis showed that BPR3P0128 targets the RdRp channel, inhibiting substrate entry, which implies it operates differently-but complementary-with remdesivir. Utilizing an optimized cell-based minigenome RdRp reporter assay, we confirmed that BPR3P0128 exhibited potent inhibitory activity. However, an enzyme-based RdRp assay employing purified recombinant nsp12/nsp7/nsp8 failed to corroborate this inhibitory activity. This suggests that BPR3P0128 may inhibit activity by targeting host-related RdRp-associated factors. Moreover, we discovered that a combination of BPR3P0128 and remdesivir had a synergistic effect-a result likely due to both drugs interacting with separate domains of the RdRp. This novel synergy between the two drugs reinforces the potential clinical value of the BPR3P0128-remdesivir combination in combating various SARS-CoV-2 variants of concern.

Complete genome sequence of a novel partitivirus with a dsRNA3 segment, isolated from Fusarium commune strain CP-SX-3 causing strawberry root rot.

A novel partitivirus, Fusarium commune partitivirus 1 (FcoPV1), was identified in Fusarium commune strain CP-SX-3 isolated from diseased roots of strawberry with symptoms of root rot. The complete genome of FcoPV1 comprises three double-stranded RNAs (dsRNAs): dsRNA1 (1,825 nt), dsRNA2 (1,592 nt), and dsRNA3 (1,421 nt). dsRNA1 contains a single open reading frame (ORF1) encoding an RNA-dependent RNA polymerase (RdRp), and dsRNA2 contains a single ORF (ORF2) encoding a coat protein (CP). dsRNA3 is a possible satellite RNA that does not appear to encode a known protein. BLASTp analysis revealed that RdRp (86.59%) and CP (74.13%) encoded by the two ORFs (ORF1 and ORF2) had the highest sequence similarity to their counterparts in Fusarium equiseti partitivirus 1 (FePV1). Phylogenetic analysis based on the complete amino acid sequence of RdRp suggested that FcoPV1 should be considered a member of a new species in the proposed genus "Zetapartitivirus" within the family Partitiviridae. To the best of our knowledge, this is the first report of a zetapartitivirus infecting phytopathogenic F. commune.

Phenolic compounds of Theobroma cacao L. show potential against dengue RdRp protease enzyme inhibition by In-silico docking, DFT study, MD simulation and MMGBSA calculation.

BACKGROUND: Currently, there is no antiviral medication for dengue, a potentially fatal tropical infectious illness spread by two mosquito species, Aedes aegypti and Aedes albopictus. The RdRp protease of dengue virus is a potential therapeutic target. This study focused on the in silico drug discovery of RdRp protease inhibitors. METHODS: To assess the potential inhibitory activity of 29 phenolic acids from Theobroma cacao L. against DENV3-NS5 RdRp, a range of computational methods were employed. These included docking, drug-likeness analysis, ADMET prediction, density functional theory (DFT) calculations, and molecular dynamics (MD) simulations. The aim of these studies was to confirm the stability of the ligand-protein complex and the binding pose identified during the docking experiment. RESULTS: Twenty-one compounds were found to have possible inhibitory activities against DENV according to the docking data, and they had a binding affinity of ≥-37.417 kcal/mol for DENV3- enzyme as compared to the reference compound panduratin A. Additionally, the drug-likeness investigation produced four hit compounds that were subjected to ADMET screening to obtain the lead compound, catechin. Based on ELUMO, EHOMO, and band energy gap, the DFT calculations showed strong electronegetivity, favouravle global softness and chemical reactivity with considerable intra-molecular charge transfer between electron-donor to electron-acceptor groups for catechin. The MD simulation result also demonstrated favourable RMSD, RMSF, SASA and H-bonds in at the binding pocket of DENV3-NS5 RdRp for catechin as compared to panduratin A. CONCLUSION: According to the present findings, catechin showed high binding affinity and sufficient drug-like properties with the appropriate ADMET profiles. Moreover, DFT and MD studies further supported the drug-like action of catechin as a potential therapeutic candidate. Therefore, further in vitro and in vivo research on cocoa and its phytochemical catechin should be taken into consideration to develop as a potential DENV inhibitor.

The RdRp genotyping of SARS-CoV-2 isolated from patients with different clinical spectrum of COVID-19.

BACKGROUND: The evolution of SARS-CoV-2 has been observed from the very beginning of the fight against COVID-19, some mutations are indicators of potentially dangerous variants of the virus. However, there is no clear association between the genetic variants of SARS-CoV-2 and the severity of COVID-19. We aimed to analyze the genetic variability of RdRp in correlation with different courses of COVID-19. RESULTS: The prospective study included 77 samples of SARS-CoV-2 isolated from outpatients (1st degree of severity) and hospitalized patients (2nd, 3rd and 4th degree of severity). The retrospective analyses included 15,898,266 cases of SARS-CoV-2 genome sequences deposited in the GISAID repository. Single-nucleotide variants were identified based on the four sequenced amplified fragments of SARS-CoV-2. The analysis of the results was performed using appropriate statistical methods, with p < 0.05, considered statistically significant. Additionally, logistic regression analysis was performed to predict the strongest determinants of the observed relationships. The number of mutations was positively correlated with the severity of the COVID-19, and older male patients. We detected four mutations that significantly increased the risk of hospitalization of COVID-19 patients (14676C > T, 14697C > T, 15096 T > C, and 15279C > T), while the 15240C > T mutation was common among strains isolated from outpatients. The selected mutations were searched worldwide in the GISAID database, their presence was correlated with the severity of COVID-19. CONCLUSION: Identified mutations have the potential to be used to assess the increased risk of hospitalization in COVID-19 positive patients. Experimental studies and extensive epidemiological data are needed to investigate the association between individual mutations and the severity of COVID-19.

A Marine Natural Product, Harzianopyridone, as an Anti-ZIKV Agent by Targeting RNA-Dependent RNA Polymerase.

The Zika virus (ZIKV) is a mosquito-borne virus that already poses a danger to worldwide human health. Patients infected with ZIKV generally have mild symptoms like a low-grade fever and joint pain. However, severe symptoms can also occur, such as Guillain-Barré syndrome, neuropathy, and myelitis. Pregnant women infected with ZIKV may also cause microcephaly in newborns. To date, we still lack conventional antiviral drugs to treat ZIKV infections. Marine natural products have novel structures and diverse biological activities. They have been discovered to have antibacterial, antiviral, anticancer, and other therapeutic effects. Therefore, marine products are important resources for compounds for innovative medicines. In this study, we identified a marine natural product, harzianopyridone (HAR), that could inhibit ZIKV replication with EC50 values from 0.46 to 2.63 µM while not showing obvious cytotoxicity in multiple cellular models (CC50 > 45 µM). Further, it also reduced the expression of viral proteins and protected cells from viral infection. More importantly, we found that HAR directly bound to the ZIKV RNA-dependent RNA polymerase (RdRp) and suppressed its polymerase activity. Collectively, our findings provide HAR as an option for the development of anti-ZIKV drugs.

PB2 residue 473 contributes to the mammalian virulence of H7N9 avian influenza virus by modulating viral polymerase activity via ANP32A.

Since the first human infection reported in 2013, H7N9 avian influenza virus (AIV) has been regarded as a serious threat to human health. In this study, we sought to identify the virulence determinant of the H7N9 virus in mammalian hosts. By comparing the virulence of the SH/4664 H7N9 virus, a non-virulent H9N2 virus, and various H7N9-H9N2 hybrid viruses in infected mice, we first pinpointed PB2 as the primary viral factor accounting for the difference between H7N9 and H9N2 in mammalian virulence. We further analyzed the in vivo effects of individually mutating H7N9 PB2 residues different from the closely related H9N2 virus and consequently found residue 473, alongside the well-known residue 627, to be critical for the virulence of the H7N9 virus in mice and the activity of its reconstituted viral polymerase in mammalian cells. The importance of PB2-473 was further strengthened by studying reverse H7N9 substitutions in the H9N2 background. Finally, we surprisingly found that species-specific usage of ANP32A, a family member of host factors connecting with the PB2-627 polymorphism, mediates the contribution of PB2 473 residue to the mammalian adaption of AIV polymerase, as the attenuating effect of PB2 M473T on the viral polymerase activity and viral growth of the H7N9 virus could be efficiently complemented by co-expression of chicken ANP32A but not mouse ANP32A and ANP32B. Together, our studies uncovered the PB2 473 residue as a novel viral host range determinant of AIVs via species-specific co-opting of the ANP32 host factor to support viral polymerase activity.IMPORTANCEThe H7N9 avian influenza virus has been considered to have the potential to cause the next pandemic since the first case of human infection reported in 2013. In this study, we identified PB2 residue 473 as a new determinant of mouse virulence and mammalian adaptation of the viral polymerase of the H7N9 virus and its non-pathogenic H9N2 counterparts. We further demonstrated that the variation in PB2-473 is functionally linked to differential co-opting of the host ANP32A protein in supporting viral polymerase activity, which is analogous to the well-known PB2-627 polymorphism, albeit the two PB2 positions are spatially distant. By providing new mechanistic insight into the PB2-mediated host range determination of influenza A viruses, our study implicated the potential existence of multiple PB2-ANP32 interfaces that could be targets for developing new antivirals against the H7N9 virus as well as other mammalian-adapted influenza viruses.

Sofosbuvir Suppresses the Genome Replication of DENV1 in Human Hepatic Huh7 Cells.

Dengue virus (DENV) causes dengue fever and dengue hemorrhagic fever, and DENV infection kills 20,000 people annually worldwide. Therefore, the development of anti-DENV drugs is urgently needed. Sofosbuvir (SOF) is an effective drug for HCV-related diseases, and its triphosphorylated metabolite inhibits viral RNA synthesis by the RNA-dependent RNA polymerase (RdRp) of HCV. (2'R)-2'-Deoxy-2'-fluoro-2'-methyluridine (FMeU) is the dephosphorylated metabolite produced from SOF. The effects of SOF and FMeU on DENV1 replication were analyzed using two DENV1 replicon-based methods that we previously established. First, a replicon-harboring cell assay showed that DENV1 replicon replication in human hepatic Huh7 cells was decreased by SOF but not by FMeU. Second, a transient replicon assay showed that DENV1 replicon replication in Huh7 cells was decreased by SOF; however, in hamster kidney BHK-21 cells, it was not suppressed by SOF. Additionally, the replicon replication in Huh7 and BHK-21 cells was not affected by FMeU. Moreover, we assessed the effects of SOF on infectious DENV1 production. SOF suppressed infectious DENV1 production in Huh7 cells but not in monkey kidney Vero cells. To examine the substrate recognition of the HCV and DENV1 RdRps, the complex conformation of SOF-containing DENV1 RdRp or HCV RdRp was predicted using AlphaFold 2. These results indicate that SOF may be used as a treatment for DENV1 infection.

Marine Brown Algae-Derived Compounds as Potential Inhibitors of Japanese Encephalitis Virus RNA-Dependent RNA Polymerase.

The Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that primarily affects people in Asia and seriously threatens public health. Considering the rising occurrence rates and lack of targeted antiviral treatments, it is essential to comprehend and tackle obstacles related to JEV in order to lessen its influence on world health. This investigation explores compounds derived from marine brown algae (Phaeophyceae) as potential inhibitors of JEV RNA-dependent RNA polymerase (RdRp), a critical enzyme in the virus's replication cycle. Employing the computational virtual screen approach, four compounds, i.e., CMNPD16749, CMNPD2606, CMNPD27817, and CMNPD23662, with favorable binding energies ranging from -15.7 Kcal/mol to -13.9 kcal/mol were identified. Subsequently, through molecular docking analysis, the interactions responsible for the binding stability between the target protein and hit molecules compared to the reference molecule Galidesvir were studied. Further, through extensive molecular dynamic (MD) simulation studies at 200 ns, it was confirmed that each docked complex showed acceptable dynamic stability compared to the reference molecule. These findings were further validated using MM/PBSA free binding energy calculations, PCA analysis and free energy landscape construction. These computational findings suggested that the brown algae-derived compounds may act as an antiviral drug against JEV infection and lay a crucial foundation for future experimental studies against JEV.

Complete genome sequence of a novel gammapartitivirus from Penicillium oxalicum RCEF7482.

Penicillium oxalicum, an important biocontrol fungus in China, has been a subject of extensive study due to its role in combating various pathogenic fungi. Despite the prevalence of mycoviruses with double-stranded (ds) RNA genomes in filamentous fungi, there has been no screening of mycoviruses in P. oxalicum. In this report, we describe the identification and characterization of a novel dsRNA virus isolated from P. oxalicum, designated as "Penicillium oxalicum partitivirus 1" (PoPV1). The genome of PoPV1 consists of two dsRNA segments, dsRNA1 (1,770 bp) and dsRNA2 (1,584 bp), each containing a single open reading frame (ORF): ORF1 and ORF2. Comparative analysis revealed that the RdRp and CP amino acid sequences of PoPV1 share the highest identity (89.18% and 73.97%, respectively) with those of Penicillium aurantiogriseum partitivirus 1 (PaPV1). Motif analysis based on RdRp amino acid sequences places PoPV1 in the genus Gammapartitivirus within the family Partitiviridae, with a distinctive motif VI (R/KV/ILGDD). Phylogenetic analysis further established a close relationship of PoPV1 to PaPV1, forming a unique clade among the gammapartitiviruses. Consequently, we propose that Penicillium oxalicum partitivirus 1 represents a new species in the genus Gammapartitivirus. This is the first report of a dsRNA virus in P. oxalicum.

A novel alternavirus with three dsRNA segments from Fusarium pseudograminearum, the pathogen of Fusarium crown rot in wheat.

Three dsRNA segments were detected in Fusarium pseudograminearum strain CF14029, a pathogen causing Fusarium crown rot in China. Characterization and sequence analysis confirmed that these dsRNA sequences originated from the same virus. The viral genome consists of three dsRNA segments: dsRNA1 (3,560 nt in length), encoding an RNA-dependent RNA polymerase (RdRp), dsRNA2 (2,544 nt in length), encoding a hypothetical protein, and dsRNA3 (2,478 nt in length), encoding a putative coat protein (CP). Phylogenetic analysis based on the RdRp and CP amino acid sequences revealed a high degree of similarity of this virus to members of the genus Alternavirus, family Alternaviridae, isolated from other Fusarium fungi. As a novel member of the genus Alternavirus, this virus was provisionally named "Fusarium pseudograminearum alternavirus 1" (FpgAV1). Like other alternaviruses found in Fusarium species, the positive-sense strand of each genomic dsRNA of FpgAV1 possesses a poly(A) tail and a distinctive 5'-terminal octamer sequence (5'-GCT GTG TG-3'). This is the first report of the genomic sequence of an alternavirus identified in F. pseudograminearum.

Identification and molecular characterization of a novel totivirus from Mangifera indica.

In this study, we determined the complete genome sequence of a novel totivirus, tentatively named "Mangifera indica totivirus 1" (MiTV1), identified in 'Apple' mango in China. The double-stranded RNA genome of MiTV1 is 4800 base pairs (bp) in length and contains two open reading frames (ORFs) encoding a putative coat protein (CP) and an RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis based on RdRp and CP amino acid sequences showed that MiTV1 is closely related to members of the genus Totivirus in the family Totiviridae. To our knowledge, this is the first report of a totivirus found in Mangifera indica.

The complete genome sequences of a negative single-stranded RNA virus and a double-stranded RNA virus coinfecting the entomopathogenic fungus Beauveria bassiana Vuillemin.

Beauveria bassiana Vuillemin is an entomopathogenic fungus that has been developed as a biological insecticide. B. bassiana can be infected by single or multiple mycoviruses, most of which are double-stranded RNA (dsRNA) viruses, while infections with single-stranded RNA (ssRNA) viruses, especially negative single-stranded RNA (-ssRNA) viruses, have been observed less frequently. In the present study, we sequenced and analyzed the complete genomes of two new different mycoviruses coinfecting a single B. bassiana strain: a -ssRNA virus which we have named "Beauveria bassiana negative-strand RNA virus 1" (BbNSRV1), and a dsRNA virus, which we have named "Beauveria bassiana orthocurvulavirus 1" (BbOCuV1). The genome of BbNSRV1 consists of a single segment of negative-sense, single-stranded RNA with a length of 6169 nt, containing a single open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) with 1949 aa (220.1 kDa). BLASTx analysis showed that the RdRp had the highest sequence similarity (59.79%) to that of Plasmopara viticola lesion associated mononegaambi virus 2, a member of the family Mymonaviridae. This is the first report of a -ssRNA mycovirus infecting B. bassiana. The genome of BbOCuV1 consists of two dsRNA segments, 2164 bp and 1765 bp in length, respectively, with dsRNA1 encoding a protein with conserved RdRp motifs and 70.75% sequence identity to the putative RdRp of the taxonomically unassigned mycovirus Fusarium graminearum virus 5 (FgV5), and the dsRNA2 encoding a putative coat protein with sequence identity 64.26% to the corresponding protein of the FgV5. Phylogenetic analysis indicated that BbOCuV1 belongs to a taxonomically unassigned group of dsRNA mycoviruses related to members of the families Curvulaviridae and Partitiviridae. Hence, it might be the member of a new family that remains to be named and formally recognized.