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Arboviruses such as chikungunya virus (CHIKV) and dengue virus (DENV) collectively afflict millions of individuals worldwide particularly in endemic countries like India, leading to substantial morbidity and mortality. With the lack of effective vaccines for both CHIKV and DENV in India, the search for antiviral compounds becomes paramount to control these viral infections. In line with this, our investigation was focused on screening natural compounds for their potential antiviral activity against CHIKV and DENV. Using different assays, including plaque assay, immunofluorescence, and reverse transcription-quantitative real-time PCR (qRT-PCR), out of 109 natural compounds tested, we confirmed lycorine's in vitro antiviral activity against CHIKV and DENV at low micromolar concentrations in different cell types. Time of addition assays indicated that lycorine does not impede viral entry. Additionally, qRT-PCR results along with time of addition assay suggested that lycorine interferes with the synthesis of negative strand viral RNA. Molecular docking analysis was done to understand the mode of inhibition of viral replication. The results revealed that the most likely binding site with the highest binding affinity of lycorine, was at the palm and finger domains, in the vicinity of the catalytic site of CHIKV and DENV RNA-dependent RNA polymerase (RdRp). Collectively, our data underscores the potential of lycorine to be developed as a direct acting inhibitor for DENV and CHIKV, addressing the critical need of requirement of an antiviral in regions where these viruses pose significant public health threats., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

An effective strategy for accurately detecting single nucleotide variants (SNVs) is of great significance for genetic research and diagnostics. However, strict amplification conditions, complex experimental instruments, and specialized personnel are required to obtain a satisfactory tradeoff between sensitivity and selectivity for SNV discrimination. In this study, we present a CRISPR-based transistor biosensor for the rapid and highly selective detection of SNVs in viral RNA. By introducing a synthetic mismatch in the crRNA, the CRISPR-Cas13a protein can be engineered to capture the target SNV RNA directly on the surface of the graphene channel. This process induces a fast electrical signal response in the transistor, obviating the need for amplification or reporter molecules. The biosensor exhibits a detection limit for target RNA as low as 5 copies in 100 μL, which is comparable to that of real-time quantitative polymerase chain reaction (PCR). Its operational range spans from 10 to 5 × 10 5 copy mL -1 in artificial saliva solution. This capability enables the biosensor to discriminate between wild-type and SNV RNA within 15 min. By introducing 10 μL of swab samples during clinical testing, the biosensor provides specific detection of respiratory viruses in 19 oropharyngeal specimens, including influenza A, influenza B, and variants of SARS-CoV-2. This study emphasizes the CRISPR-transistor technique as a highly accurate and sensitive approach for field-deployable nucleic acid screening or diagnostics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Hepatitis C virus (HCV) is a common blood-borne infection that can lead to long-term illnesses such as hepatocellular cancer and liver cirrhosis. Early diagnosis is crucial for effective management, as no vaccine is available for preventing HCV infection. However, the high cost and complexity of current molecular diagnostic tools hinder efforts to achieve early diagnosis and prevent transmission, particularly in resource-limited settings. We developed a novel electrochemical biosensor for point-of-care testing (POCT) of HCV RNA. The sensor utilizes a strand displacement method, where the target RNA displaces a gold nanoparticle-labeled reporter probe (AuRP) from a pre-hybridized duplex with a magnetic nanoparticle (MNP)-labeled capture probe. The amount of displaced AuRP, detected using differential pulse anodic stripping voltammetry (DPASV), is directly proportional to the target RNA concentration. The biosensor exhibited excellent analytical performance, with a detection limit of 4 fM for synthetic targets and 43 ng/µL for RT-PCR products. Importantly, it successfully detected HCV RNA directly in clinical plasma samples without the need for RNA extraction or amplification. The sensor was used to analyze 30 RNA samples from HCV-positive patients, 20 cDNA samples from viral RNA, 30 HCV-positive plasma samples, and 22 HCV-negative plasma samples. The sensor results show good concordance with the RT-PCR results, demonstrating the sensor's potential for detecting HCV in clinical samples., (© 2024. The Author(s).)

The replication of RNA viruses relies on the activity of RNA-dependent RNA polymerases (RdRps). Despite large variations in their genomic sequences, viral RdRps share a common architecture generally known as a closed right hand. The P2 polymerase of cystovirus φ6 is currently among the best characterized viral RdRps. This polymerase is responsible for carrying out both replication and transcription of the viral double-stranded RNA genome using de novo initiation. Despite the extensive biochemical and structural studies conducted on φ6 P2, further structural information on other cystoviral RdRps is crucial to elucidate the structural and functional diversity of viral RdRps. Here, we have determined the atomic X-ray structure of the RdRp P2 from the φ6-related cystovirus φ8 at 3Å resolution. This structure completes the existing set of structural information on the φ8 polymerase complex and sheds light on the difference and similarities with related cystoviral RdRps., (© 2024. The Author(s).)

In this study, a novel positive-sense single-stranded RNA (+ ssRNA) mycovirus, Alternaria tenuissima mitovirus 1 (AtMV1), was identified in Alternaria tenuissima strain YQ-2-1, a phytopathogenic fungus causing leaf blight on muskmelon. The genome of AtMV1 is a single RNA molecule that is 3013 nt in length with an A + U content of 66.58% and contains a single open reading frame (ORF) using the fungal mitochondrial genetic code. The ORF was predicted to encode a 313-amino-acid RNA-dependent RNA polymerase (RdRp) with a molecular mass of 35.48 kDa, which contains six conserved motifs with the highly conserved GDD tripeptide in motif IV. The 5' and 3' untranslated regions were predicted to fold into stem-loop and panhandle secondary structures. The results of a BLASTp search revealed that the amino acid (aa) sequence of RdRp of AtMV1 shared the highest sequence similarity (51.04% identity) with that of Sichuan mito-like virus 30, a member of the genus Duamitovirus within the family Mitoviridae. Phylogenetic analysis based on the aa sequence of the RdRp suggested that AtMV1 is a novel member of the genus Duamitovirus. To our knowledge, this is the first report of the complete genome sequence of a new mitovirus infecting A. tenuissima., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Isothermal nucleic acid amplification tests (NAATs) are a vital tool for point-of-care (POC) diagnostics. These assays are well-suited for rapid, low-cost POC diagnostics for infectious diseases compared to traditional PCR tests conducted in central laboratories. There has been significant development of POC NAATs using paper-based diagnostic devices because they provide an affordable, user-friendly, and easy to store format; however, the difficulties in integrating separate liquid components, resuspending dried reagents, and achieving a low limit of detection hinder their use in commercial applications. Several studies report low assay efficiencies, poor detection output, and poorer limits of detection in porous membranes compared to traditional tube-based protocols. Recombinase polymerase amplification is a rapid, isothermal NAAT that is highly suited for POC applications, but requires viscous reaction conditions that has poor performance when amplifying in a porous paper membrane. In this work, we show that we can dramatically improve the performance of membrane-based recombinase polymerase amplification (RPA) of HIV-1 DNA and viral RNA by employing a coin cell-based vibration mixing platform. We achieve a limit of detection of 12 copies of DNA per reaction, nearly 50% reduction in time to threshold (from ∼10 minutes to ∼5 minutes), and an overall fluorescence output increase up to 16-fold when compared to unmixed experiments. This active mixing strategy enables reactions where the target and reaction cofactors are isolated from each other prior to the reaction. We also demonstrate amplification using a low-cost vibration motor for both temperature control and mixing, without the requirement of any additional heating components.

Droplet digital polymerase chain reaction (ddPCR) stands out as a highly sensitive diagnostic technique that is gaining traction in infectious disease diagnostics due to its ability to quantitate very low numbers of viral gene copies. By partitioning the sample into thousands of droplets, ddPCR enables precise and absolute quantification without relying on a standard curve. However, current ddPCR systems often exhibit relatively low levels of integration, and the analytical process remains dependent on elaborate workflows for up-front sample preparation. Here, we introduce a fully-integrated system seamlessly combining viral lysis, RNA extraction, emulsification, reverse transcription (RT) ddPCR, and fluorescence readout in a sample-to-answer format. The system comprises a disposable microfluidic cartridge housing buffers and reagents required for the assay, and a centrifugal platform that allows for pneumatic actuation of liquids during rotation, enabling automation of the workflow. Highly monodisperse droplets (∼50 μm in diameter) are produced using centrifugal step emulsification and automatically transferred to an integrated heating module for target amplification. The platform is equipped with a miniature fluorescence imaging system enabling on-chip read-out of droplets after RT-ddPCR. We demonstrate sample-to-answer detection of SARS-CoV-2 N and E genes, along with RNase P endogenous reference, using hydrolysis probes and multiplexed amplification within single droplets for concentrations as low as 0.1 copy per μL. We also tested 14 nasopharyngeal swab specimens from patients and were able to distinguish positive and negative SARS-CoV-2 samples with 100% accuracy, surpassing results obtained by conventional real-time amplification.

Soybean dwarf virus (SbDV; family Tombusviridae, genus Luteovirus, species Luteovirus glycinis) is an RNA plant virus that is transmitted solely by aphids in a persistent, circulative and non-propagative manner. SbDV causes significant losses in cultivated Fabaceae, especially in subterranean clover (Trifolium subterraneum) pastures of mainland Australia. SbDV isolates are classified into four phenotypically distinguishable strains: YP, YS, DP, and DS. Y and D strains differ primarily in their host range, and P and S strains in their primary vector species. Genetically, Y and D strains separate into two clades in every genomic region except for the N-terminal region of the readthrough domain (N-RTD), in which P and S strains separate. SbDV diversity in Australia has yet to be investigated, so in this study, 41 isolates were collected from six different host species across two production regions of Australia: the south coast of Western Australia ('south-west') and northern New South Wales/southern Queensland ('north-east'). A near-complete genome sequence of each isolate was obtained, and together with all 50 whole-genome sequences available in the GenBank database, underwent phylogenetic analysis of the whole genome nt and the N-RTD aa sequences. At the whole-genome level, the isolates separated into D and Y clades. At the N-RTD level, most of the isolates separated into P and S clades. All south-west isolates and 11 of the 31 north-east isolates were in the Y clade, and the remaining 20 north-east isolates were in the D clade. Except for one isolate that fell outside the P and S clades, all south-west and north-east isolates were in the P clade, suggesting that they are transmitted by Acyrthosiphon pisum and Myzus persicae. Available biological data largely supported the phenotypic inferences made from the phylogenetic analysis, suggesting that genetic data can provide critical epidemiological insights, provided that sufficient biological data have been collected., (© 2024. Crown.)

Diagnostic methods based on CRISPR technology have shown great potential due to their highly specific, efficient, and sensitive detection capabilities. Although the majority of the current studies rely on fluorescent dye-quencher reporters, the limitations of fluorescent dyes, such as poor photostability and small Stokes shifts, urgently necessitate the optimization of reporters. In this study, we developed innovative quantum dot (QD) reporters for the CRISPR/Cas systems, which not only leveraged the advantages of high photoluminescence quantum yield and large Stokes shifts of QDs but were also easily synthesized through a simple one-step hydrothermal method. Based on the trans-cleavage characteristics of Cas12a and Cas13a, two types of QD reporters were designed, the short DNA strand and the hybridization-based QD reporters, achieving the detection of DNA and RNA at the pM level, respectively, and validating the performance in the analysis of clinical samples. Furthermore, based on the unique property of QDs that allowed multicolor emission under one excitation, the application potential for simultaneous detection of diseases was further investigated. Taken together, this work proposed novel QD reporters that could be applied to the various CRISPR/Cas systems, providing a new toolbox to expand the diagnosis of bioanalytical and biomedical fields.

The Nipah virus (NiV), a member of the Paramyxoviridae family, is notorious for its high fatality rate in humans. The RNA polymerase machinery of NiV, comprising the large protein L and the phosphoprotein P, is essential for viral replication. This study presents the 2.9-Å cryo-electron microscopy structure of the NiV L-P complex, shedding light on its assembly and functionality. The structure not only demonstrates the molecular details of the conserved N-terminal domain, RNA-dependent RNA polymerase (RdRp), and GDP polyribonucleotidyltransferase of the L protein, but also the intact central oligomerization domain and the C-terminal X domain of the P protein. The P protein interacts extensively with the L protein, forming an antiparallel β-sheet among the P protomers and with the fingers subdomain of RdRp. The flexible linker domain of one P promoter extends its contact with the fingers subdomain to reach near the nascent RNA exit, highlighting the distinct characteristic of the NiV L-P interface. This distinctive tetrameric organization of the P protein and its interaction with the L protein provide crucial molecular insights into the replication and transcription mechanisms of NiV polymerase, ultimately contributing to the development of effective treatments and preventive measures against this Paramyxoviridae family deadly pathogen., (© 2024. The Author(s).)

Respiratory pathogen infections are seasonally prevalent and are likely to cause co-infections or serial infections during peak periods of infection. Since they often cause similar symptoms, simultaneous and on-site detection of respiratory pathogens is essential for accurate diagnosis and efficient treatment of these infectious diseases. However, molecular diagnostic techniques for multiple pathogens in this field are lacking. Herein, we developed a microfluidic LAMP and real-time fluorescence assay for rapid detection of multiple respiratory pathogens using a ten-channel microfluidic chip with pathogen primers pre-embedded in the chip reaction well. The microfluidic chip provided a closed reaction environment, effectively preventing aerosol contamination and improving the accuracy of the detection results. Its corresponding detection instrument could automatically collect and display the fluorescence curve in real time, which was more conducive to the interpretation of results. The results showed that the developed method could specifically recognize the nucleic acid of influenza A(H1N1), Mycoplasma pneumoniae , respiratory syncytial virus type A, and SARS-CoV-2 with low detection limits of 10 4 copies per mL or 10 3 copies per mL. The test results on clinical samples demonstrated that the developed method has high sensitivity (92.00%) and high specificity (100.00%) and even has the capability to differentiate mixed-infection samples. With simple operation and high detection efficiency, the present portable and simultaneous detection assay could significantly improve the efficiency of on-site detection of respiratory infectious diseases and promote the accurate treatment, efficient prevention and control of the diseases.

We report a virus infecting Entomophthora muscae, a behavior-manipulating fungal pathogen of dipterans. The virus, which we name Berkeley Entomophthovirus, is a positive-strand RNA virus in the iflaviridae family of capsid-forming viruses, which are mostly known to infect insects. The viral RNA is expressed at high levels in fungal cells in vitro and during in vivo infections of Drosophila melanogaster, and virus particles can be seen intracellularly in E. muscae. This virus, of which we find two closely related variants in our culture of E. muscae, is also closely related to three different viruses reported from metagenomic surveys, two of which were isolated from wild dipterans, and a third isolated from wild ticks. By analyzing sequencing data from these earlier reports, we find abundant reads aligning to E. muscae specifically in the samples from which viral reads were sequenced. These data establish a wide and perhaps obligate association with E. muscae in the wild, consistent with our laboratory data that E. muscae is the host for these closely related viruses. Because of this, we propose the name Entomophthovirus (EV) for this group of highly related virus variants. As other members of the iflaviridae have been reported to cause behavioral changes in insects, we speculate on the possibility that EV plays a role in the behavioral manipulation of flies infected with E. muscae., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Immune evasion represents a crucial milestone in the progression of cancer and serves as the theoretical foundation for tumor immunotherapy. In this study, we reveal a negative association between Human Papillomavirus (HPV)-encoded circular RNA, circE7, and the infiltration of CD8 + T cells in head and neck squamous cell carcinoma (HNSCC). Both in vitro and in vivo experiments demonstrate that circE7 suppresses the function and activity of T cells by downregulating the transcription of LGALS9, which encodes the galectin-9 protein. The molecular mechanism involves circE7 binding to acetyl-CoA carboxylase 1 (ACC1), promoting its dephosphorylation and thereby activating ACC1. Activated ACC1 reduces H3K27 acetylation at the LGALS9 gene promoter, leading to decreased galectin-9 expression. Notably, galectin-9 interacts with immune checkpoint molecules TIM-3 and PD-1, inhibiting the secretion of cytotoxic cytokines by T cells and promoting T cell apoptosis. Here, we demonstrate a mechanism by which HPV promotes immune evasion in HNSCC through a circE7-driven epigenetic modification and propose a potential immunotherapy strategy for HNSCC that involves the combined use of anti-PD-1 and anti-TIM-3 inhibitors., (© 2024. The Author(s).)

A putative novel virus was identified in Agastache rugosa in China and tentatively named "Agastache rugosa-associated varicosavirus" (ARaVV). The nearly complete genome sequence of ARaVV was obtained through RNA sequencing (RNA-seq) and subsequent RT-PCR. The ARaVV genome consists of two negative-sense single-stranded RNA segments that are 6428 and 3862 nucleotides (nt) in size, respectively. RNA1 encodes a large polymerase (L), and RNA2 encodes a putative nucleocapsid protein (N), protein 2 (P2), and protein 3 (P3). The L protein shared the highest amino acid (aa) sequence identity (51.3%) with Erysimum virus 1 (EryV1, BK061766.1). The N, P2, and P3 shared the highest aa sequence identity (33.1%, 14.0%, and 24.2%) with Leucanthemum virus 1, Raphanus virus 1, and Spinach virus 1, respectively. Phylogenetic analysis based on amino acid sequences of the L protein showed that ARaVV clustered in a clade with the varicosaviruses, indicating that ARaVV is a putative new member of the genus Varicosavirus., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

RNA is a central molecule in RNA virus biology; however, the interactions that it establishes with the host cell are only starting to be elucidated. In recent years, a methodology revolution has dramatically expanded the scope of host-virus interactions involving the viral RNA (vRNA). A second wave of method development has enabled the precise study of these protein-vRNA interactions in a life cycle stage-dependent manner, as well as providing insights into the interactome of specific vRNA species. This review discusses these technical advances and describes the new regulatory mechanisms that have been identified through their use. Among these, we discuss the importance of vRNA in regulating protein function through a process known as riboregulation. We envision that the elucidation of vRNA interactomes will open new avenues of research, including pathways to the discovery of host factors with therapeutic potential against viruses., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The packaging of genomic RNA (gRNA) into retroviral particles relies on the specific recognition by the Gag precursor of packaging signals (Psi), which maintain a complex secondary structure through long-range interactions (LRIs). However, it remains unclear whether the binding of Gag to Psi alone is enough to promote RNA packaging and what role LRIs play in this process. Using mouse mammary tumor virus (MMTV), we investigated the effects of mutations in 4 proposed LRIs on gRNA structure and function. Our findings revealed the presence of an unsuspected extended LRI, and hSHAPE revealed that maintaining a wild-type-like Psi structure is crucial for efficient packaging. Surprisingly, filter-binding assays demonstrated that most mutants, regardless of their packaging capability, exhibited significant binding to Pr77Gag, suggesting that Gag binding to Psi is insufficient for efficient packaging. Footprinting experiments indicated that efficient RNA packaging is promoted when Pr77Gag binds to 2 specific sites within Psi, whereas binding elsewhere in Psi does not lead to efficient packaging. Taken together, our results suggest that the 3D structure of the Psi/Pr77Gag complex regulates the assembly of viral particles around gRNA, enabling effective discrimination against other viral and cellular RNAs that may also bind Gag efficiently., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Prabhu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The co-circulation of mosquito-borne Japanese encephalitis virus (JEV), Murray Valley encephalitis virus (MVEV), and West Nile virus (WNV) has impacted human and animal health in multiple countries worldwide. To facilitate early warnings and surveillance of the presence of these viral infectious agents in the environment, a triplex reverse transcription-quantitative PCR (RT-qPCR) was developed for simultaneous quantification of JEV, MVEV, and WNV in potential hotspots such as piggery and urban wastewater and environmental water samples. The performance of the developed triplex RT-qPCR assay was compared with that of simplex counterparts, all using the same primer and probe sequences. The quantifiable results showed a concordance rate of 93.9%-100% (Cohen's kappa) between the triplex and simplex assays. The mean concentrations of exogenous JEV, MVEV, and WNV using the triplex and simplex RT-qPCR assays were remarkably similar in piggery/urban wastewater and environmental water samples. However, the impacts of the matrix effects (i.e., sample composition and PCR inhibition) of environmental water samples on the accurate quantification of these viruses need to be considered. Taken together, this newly developed triplex RT-qPCR assay of JEV, MVEV, and WNV will allow for a more rapid and cost-efficient sample analysis and data interpretation. The application of the triplex assay for environmental surveillance may be a valuable tool to complement the existing disease and mosquito surveillance approaches used to safeguard the health of both humans and animals.IMPORTANCEThe co-circulation of mosquito-borne Japanese encephalitis virus (JEV), Murray Valley encephalitis virus (MVEV), and West Nile virus (WNV) poses significant threats to human and animal health globally. In this study, a triplex RT-qPCR assay was developed for simultaneous quantification of these viruses in wastewater and environmental water samples. Results demonstrated high concordance and sensitivity of the newly developed triplex RT-qPCR assay compared to simplex assays, indicating its efficacy for environmental surveillance. This cost-effective and rapid assay offers a vital tool for timely monitoring of mosquito-borne viruses in environmental samples, enhancing our ability to mitigate potential outbreaks and safeguard public health., Competing Interests: The authors declare no conflict of interest.

Metagenomic shotgun sequencing (mNGS) can serve as a generic molecular diagnostic tool. An mNGS proficiency test (PT) was performed in six European veterinary and public health laboratories to detect porcine astroviruses in fecal material and the extracted RNA. While different mNGS workflows for the generation of mNGS data were used in the different laboratories, the bioinformatic analysis was standardized using a metagenomic read classifier as well as read mapping to selected astroviral reference genomes to assess the semiquantitative representation of astrovirus species mixtures. All participants successfully identified and classified most of the viral reads to the two dominant species. The normalized read counts obtained by aligning reads to astrovirus reference genomes by Bowtie2 were in line with Kraken read classification counts. Moreover, participants performed well in terms of repeatability when the fecal sample was tested in duplicate. However, the normalized read counts per detected astrovirus species differed substantially between participants, which was related to the different laboratory methods used for data generation. Further modeling of the mNGS data indicated the importance of selecting appropriate reference data for mNGS read classification. As virus- or sample-specific biases may apply, caution is needed when extrapolating this swine feces-based PT for the detection of other RNA viruses or using different sample types. The suitability of experimental design to a given pathogen/sample matrix combination, quality assurance, interpretation, and follow-up investigation remain critical factors for the diagnostic interpretation of mNGS results., Importance: Metagenomic shotgun sequencing (mNGS) is a generic molecular diagnostic method, involving laboratory preparation of samples, sequencing, bioinformatic analysis of millions of short sequences, and interpretation of the results. In this paper, we investigated the performance of mNGS on the detection of porcine astroviruses, a model for RNA viruses in a pig fecal material, among six European veterinary and public health laboratories. We showed that different methods for data generation affect mNGS performance among participants and that the selection of reference genomes is crucial for read classification. Follow-up investigation remains a critical factor for the diagnostic interpretation of mNGS results. The paper contributes to potential improvements of mNGS as a diagnostic tool in clinical settings., Competing Interests: The authors declare no conflict of interest.

The heterogeneous nuclear ribonucleoprotein (hnRNP A2B1) is a key component of the hnRNP complex involving RNA modulation in eukaryotic cells and it has also been reported to be involved in the replication of the hepatitis E virus, influenza A virus, and hepatitis B virus. However, it is not clear whether the role of the hnRNP A2B1 in viral replication is conserved among RNA viruses and what is the mechanism of hnRNP A2B1 in RNA virus replication. In this study, we first used severe fever with thrombocytopenia syndrome virus (SFTSV), a tick-borne RNA virus that causes a severe viral hemorrhagic fever as well as other RNA viruses including VSV-GFP, SeV, EV71, and ZIKV to demonstrate that knockout hnRNPA2B1 gene inhibited viral RNA replication and overexpression of hnRNP A2B1 could restore the RNA levels of all tested RNA viruses. These results suggest that hnRNPA2B1 upregulation of viral replication is conserved among RNA viruses. Next, we demonstrated that hnRNP A2B1 was translocated from the nucleus to the cytoplasm under RNA virus infection including SFTSV, VSV-GFP, SeV, EV71, and ZIKV, suggesting translocation of hnRNP A2B1 from the nucleus to the cytoplasm is crucial for RNA virus replication. We then used SFTSV as a model to demonstrate the mechanism of hnRNP A2B1 in the promotion of RNA virus replication. We found that overexpression of SFTSV nucleoprotein can also cause hnRNP A2B1 translocation from the nucleus to the cytoplasm and that the SFTSV NP interacted with the RNA recognition motif 1 domain of hnRNP A2B1. We further demonstrated that the hnRNP A2B1 interacted with the 5' UTR of SFTSV RNA. In conclusion, we revealed that the hnRNP A2B1 upregulation of viral RNA replication is conserved among RNA viruses; the mechanism of hnRNP A2B1 in promotion of SFTSV viral RNA replication is that SFTSV NP interacted with the hnRNPA2B1 to retain it in the cytoplasm where the hnRNP A2B1 interacted with the 5' UTR of SFTSV RNA to promote the viral RNA replication.IMPORTANCESevere fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne RNA virus with a high mortality rate of up to 30%. In this study, we first used SFTSV as a model to demonstrate that the role of hnRNPA2B1 in viral replication is conserved in SFTSV. Then we used other RNA viruses, including VSV-GFP, SeV, EV71, and ZIKV, to repeat the experiment and demonstrated the same results as SFTSV in all tested RNA viruses. By knocking out the hnRNPA2B1 gene, SFTSV RNA replication was inhibited, and overexpression of hnRNPA2B1 restored RNA levels of SFTSV and other tested RNA viruses. We revealed a novel mechanism where the SFTSV nucleoprotein interacts with hnRNPA2B1, retaining it in the cytoplasm. This interaction promotes viral RNA replication by binding to the 5' UTR of SFTSV RNA. The findings suggest that targeting hnRNPA2B1 could be a potential strategy for developing broad-spectrum antiviral therapies, given its conserved role across different RNA viruses. This research provides significant insights into the replication mechanisms of RNA viruses and highlights potential targets for antiviral interventions., Competing Interests: The authors declare no conflict of interest.

Rotavirus group C is an important cause of sporadic cases and outbreaks of gastroenteritis worldwide. Whole-Genome sequences of human rotavirus C (RVC) in public databases are limited. We performed genome sequencing to analyze a RVC outbreak of acute gastroenteritis in China. Samples from 22 patients were screened for pathogens using RT-PCR, and six samples were positive for rotavirus. Whole-Genome sequencing analysis showed that the outbreak strain SJZ217 belongs to the G4-P[2]-I2-R2-C2-M3-A2-N2-T2-E2-H2 genotype and shares almost identical genomic sequences with Chungnam isolated in Korea. Phylogenetic analysis revealed strain SJZ217 also fell into a cluster with rotavirus C strains from Japan and Europe. Reassortment in the VP4 fragment was observed. These results helped to understand the genetic diversity and possible spread of RVC strains., (© 2024. The Author(s).)

Globally, porcine rotavirus is a leading cause of gastroenteritis in nursing and post-weaning piglets, as well as adult pigs. Between February 2015 and June 2016, 156 fecal samples were collected from pigs in the Northeastern part of Accra, Ghana, and screened for Group A rotavirus using the Proflow TM Kit. Here, we describe different extraction methods that were employed to recover high-quality RNA for downstream analysis, with emphasis on a novel hybrid extraction method. The hybrid approach with a kit and manual extraction method led to a 10-fold greater RNA yield versus the kit-based method alone. The new extraction method gave an average purity ratio (A 260 /A 280 ) of 1.8, which was also significantly higher than that obtained solely from the manual or kit-based extraction methods. Our novel hybrid approach will be useful in the extraction of rotavirus from animal fecal samples, thus improving the yield of RNA for downstream analysis. © 2024 Wiley Periodicals LLC. Basic Protocol: Hybrid 2: A double lysis method for RNA extraction from animal stool samples Support Protocol 1: The GenElute extraction method Support Protocol 2: Hybrid 1 extraction method., (© 2024 Wiley Periodicals LLC.)

TRIM25 is an RNA-binding ubiquitin E3 ligase with central but poorly understood roles in the innate immune response to RNA viruses. The link between TRIM25's RNA binding and its role in innate immunity has not been established. Thus, we utilized a multitude of biophysical techniques to identify key RNA-binding residues of TRIM25 and developed an RNA-binding deficient mutant (TRIM25-m9). Using iCLIP2 in virus-infected and uninfected cells, we identified TRIM25's RNA sequence and structure specificity, that it binds specifically to viral RNA, and that the interaction with RNA is critical for its antiviral activity., (© 2024. The Author(s).)

Summary: RNA viruses are ubiquitous across a broad spectrum of ecosystems. Therefore, beyond their significant implications for public health, RNA viruses are also key players in ecological processes. High-through sequencing has accelerated the discovery of RNA viruses. Nevertheless, many of these viruses lack taxonomic annotation, posing a challenge to functional inference and evolutionary study. In particular, virus classification at the genus level remains difficult due to the limited reference data and ambiguous boundaries between some closely related genera. We introduce VirTAXA, a robust classification tool that combines remote homology search and tree-based validation to enhance the genus-level taxonomic classification of RNA viruses. VirTAXA is able to predict the genus label of an assembled viral contig and provide evidence type for each prediction. It achieves comparable accuracy to state-of-the-art methods while assigning genus labels to a greater number of sequences. Specifically, on the Global Ocean RNA metatranscriptomic data, VirTAXA can assign genus labels for 18% more contigs than the second-best classification tool. Furthermore, we demonstrated that VirTAXA can be conveniently extended to other types of viruses., Availability and Implementation: The source code and data of VirTAXA are available via https://github.com/JudithEllyn/VirTAXA., (© The Author(s) 2024. Published by Oxford University Press.)

We explored how a simple retrovirus, Mason-Pfizer monkey virus (M-PMV) to facilitate its replication process, utilizes DHX15, a cellular RNA helicase, typically engaged in RNA processing. Through advanced genetic engineering techniques, we showed that M-PMV recruits DHX15 by mimicking cellular mechanisms, relocating it from the nucleus to the cytoplasm to aid in viral assembly. This interaction is essential for the correct packaging of the viral genome and critical for its infectivity. Our findings offer unique insights into the mechanisms of viral manipulation of host cellular processes, highlighting a sophisticated strategy that viruses employ to leverage cellular machinery for their replication. This study adds valuable knowledge to the understanding of viral-host interactions but also suggests a common evolutionary history between cellular processes and viral mechanisms. This finding opens a unique perspective on the export mechanism of intron-retaining mRNAs in the packaging of viral genetic information and potentially develop ways to stop it., Competing Interests: Competing interests statement:The authors declare no competing interest.

The individual detection of human immunodeficiency virus (HIV) virions and resolution from extracellular vesicles (EVs) during analysis is a difficult challenge. Infectious enveloped virions and nonviral EVs are released simultaneously by HIV-infected host cells, in addition to hybrid viral EVs containing combinations of HIV and host components but lacking replicative ability. Complicating the issue, EVs and enveloped virions are both delimited by a lipid bilayer and share similar size and density. The feature that distinguishes infectious virions from host and hybrid EVs is the HIV genomic RNA (gRNA), which allows the virus to replicate. Single-particle analysis techniques, which provide snapshots of single biological nanoparticles, could resolve infectious virions from EVs. However, current single-particle analysis techniques focus mainly on protein detection, which fail to resolve hybrid EVs from infectious virions. A method to simultaneously detect viral protein and internal gRNA in the same particle would allow resolution of infectious HIV from EVs and noninfectious virions. Here, we introduce SPIRFISH, a high-throughput method for single-particle protein and RNA analysis, combining single particle interferometric reflectance imaging sensor with single-molecule fluorescence in situ hybridization. Using SPIRFISH, we detect HIV-1 envelope protein gp120 and genomic RNA within single infectious virions, allowing resolution against EV background and noninfectious virions. We further show that SPIRFISH can be used to detect specific RNAs within EVs. This may have major utility for EV therapeutics, which are increasingly focused on EV-mediated RNA delivery. SPIRFISH should enable single particle analysis of a broad class of RNA-containing nanoparticles.

Dengue virus (DENV) is one of the most significant mosquito-borne diseases in Nepal. In 2023, DENV outbreaks began in Eastern Nepal, near the border with India, and rapidly spread nationwide. The study aims to describe the outbreak's epidemiological pattern, laboratory characteristics, DENV serotypes, and genotypes. A hospital-based cross-sectional study was conducted in four hospitals in Jhapa, Eastern Nepal, in 2023. Acute serum samples were obtained from dengue suspected patients within 7 days of illness and subjected to virus isolation, conventional and real-time polymerase chain reaction (RT-PCR), and phylogenetic analysis. Out of 60 samples, 42 (70 %), 11 (18.3 %) and 7 (11.7 %) were primary, secondary and non-dengue infection, respectively. Among 53 dengue confirmed patients, 46 (86.7 %) were positive for NS1 and 12 (22.6 %) were positive for both NS1 and IgM. Out of 42 dengue isolates, a new clade of the cosmopolitan genotype of DENV-2 was the most prevalent (28, 66.7 %), followed by genotype III of DENV-3 (11, 26.2 %) and genotype V of DENV-1 (3, 7.1 %). Genotype III of DENV-3 was first introduced in 2022-2023 in Nepal. Phylogenetic analysis of the E gene revealed the DENV-2 isolates from Nepal had 98 % homologous nucleotide similarity with the strains from India and Bangladesh. To our knowledge, this is the first report of circulating serotypes and genotypes of DENV in Jhapa. Integrating molecular findings into the dengue control plan can enhance surveillance efforts, monitor disease trends, and implement proactive measures to reduce the burden of dengue and prevent fatalities in future outbreaks., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Protein-RNA interactions are crucially important for numerous cellular processes and often involve coupled folding and binding of peptide segments upon association. The Nut-utilization site (N)-protein of bacteriophages contains an N-terminal arginine-rich motif that undergoes such a folding transition upon binding to the boxB RNA hairpin loop target structure. Molecular dynamics free energy simulations were used to calculate the absolute binding free energy of the N-peptide of bacteriophage P22 in complex with the boxB RNA hairpin motif at different salt concentrations and using two different water force field models. We obtained good agreement with experiment also at different salt concentrations for the TIP4P-D water model that has a stabilizing effect on unfolded protein structures. It allowed us to estimate the free energy contribution resulting from restricting the molecules' spatial and conformational freedom upon binding, which makes a large opposing contribution to binding. In a second set of umbrella sampling simulations to dissociate/associate the complex along a separation coordinate, we analyzed the onset of preorientation of the N-peptide and onset of structure formation relative to the RNA and its dependence on the salt concentration. Peptide orientation and conformational transitions are significantly coupled to the first contact formation between peptide and RNA. The initial contacts are mostly formed between peptide residues and the boxB hairpin loop nucleotides. A complete transition to an α-helical bound peptide conformation occurs only at a late stage of the binding process a few angstroms before the complexed state has been reached. However, the N-peptide orients also at distances beyond the contact distance such that the sizable positive charge points toward the RNA's center-of-mass. Our result may have important implications for understanding protein- and peptide-RNA complex formation frequently involving coupled folding and association processes., Competing Interests: Declaration of interests The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Given the worldwide risk for the outbreak of emerging/re-emerging respiratory viruses, establishment of new antiviral strategies is greatly demanded. In this study, we present a scheme to identify gapmer antisense oligonucleotides (ASOs) targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA that efficiently inhibit viral replication. We synthesized approximately 300 gapmer ASOs designed to target various SARS-CoV-2 RNA regions and evaluated their activity in cell-based assays. Through a multistep screening in cell culture systems, we identified that ASO#41, targeting the coding region for viral main protease, reduced SARS-CoV-2 RNA levels in infected cells and inhibited virus-induced cytopathic effects. Antiviral effect of ASO#41 was also observed in iPS cell-derived human lung organoids. ASO#41 depleted intracellular viral RNAs during genome replication in an endogenous RNaseH-dependent manner. ASO#41 showed a wide range of antiviral activity against SARS-CoV-2 variants of concern including Alpha, Delta, and Omicron. Intranasal administration to mice exhibited intracellular accumulation of ASO#41 in the lung and significantly reduced the viral infectious titer, with milder body weight loss due to SARS-CoV-2 infection. Further chemical modification with phosphoryl guanidine-containing backbone linkages provided an elevation of anti-SARS-CoV-2 activity, with 23.4 nM of 50% antiviral inhibitory concentration, one of the strongest anti-SARS-CoV-2 ASOs reported so far. Our study presents an approach to identify active ASOs against SARS-CoV-2, which is potentially useful for establishing an antiviral strategy by targeting genome RNA of respiratory viruses., Competing Interests: Declaration of competing interest TY., M.S., H.T., T.H., Y.K., H.Y., and T.N. are employees of Otsuka Pharmaceutical Co., Ltd., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Echinacea is an herbaceous plant originating from North America that is cultivated for gardening and landscaping because of its showy flowers. Using high-throughput sequencing, we identified two viral contigs from echinacea seeds that were related to the family Tombusviridae. These two viruses were similar to oat chlorotic stunt virus (OCSV) and other unassigned tombusviruses; therefore, we tentatively named them Echinacea-associated tombusviruses 1 and 2 (EaTV1 and EaTV2, respectively). The EaTVs represent putative readthrough sites and have no poly(A) tails, aligning with the common features of family Tombusviridae. The EaTVs are included in a monophyletic group of OCSV and several unassigned tombusviruses. Because OCSV is the only member of Avenavirus to date, EaTVs are tentative members of Avenavirus, or they are close sister species to OCSV with several unassigned tombusviruses. RNA-dependent RNA polymerases and coat proteins were well conserved among EaTVs and unassigned tombusviruses; however, their similarities were not correlated, implying divergent and complex evolution., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Epstein-Barr virus (EBV) is the first human oncogenic virus known to express microRNAs (miRNAs), which are closely associated with the development of various tumors, including nasopharyngeal and gastric cancers. Stearoyl-CoA Desaturase 1 (SCD1) is a key enzyme in fatty acid synthesis, highly expressed in numerous tumors, promoting tumor growth and metastasis, making it a potential therapeutic target. In this study, we found that SCD1 expression in EBV-associated gastric cancer (EBVaGC) was significantly lower than in EBV-negative gastric cancer (EBVnGC) at both cellular and tissue levels. In addition, EBV-miR-BART20-5p targets the 3'-UTR of SCD1, downregulating its expression. Moreover, overexpression of SCD1 in EBVaGC cells promoted cell migration and proliferation while inhibiting autophagy. These results suggest that EBV-encoded miRNA-BART20-5p may contribute to EBVaGC progression by targeting SCD1., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

HIV-1, the causative agent of AIDS, is a retrovirus that packages two copies of unspliced viral RNA as a dimer into newly budding virions. The unspliced viral RNA also serves as an mRNA template for translation of two polyproteins. Recent studies suggest that the fate of the viral RNA (genome or mRNA) is determined at the level of transcription. RNA polymerase II uses heterogeneous transcription start sites to generate major transcripts that differ in only two guanosines at the 5' end. Remarkably, this two-nucleotide difference is sufficient to alter the structure of the 5'-untranslated region and generate two pools of RNA with distinct functions. The presence of both RNA species is needed for optimal viral replication and fitness., Competing Interests: Declaration of competing interest None., (Published by Elsevier Ltd.)

Objectives: We evaluated the extent of virus heterogeneity in PeV infected infants in the UK, Canada and Australia., Methods: Samples were collected from PeV infected infants during 2013-16. Next generation sequencing was used to obtain sequencing data and construct phylogenetic trees based on analysis of the VP1 region. Comparison was made with sequencing data available from an outbreak in Australia., Results: We amplified and sequenced 58 samples. All obtained PeV sequences were genotype 3 apart from one UK sample which was PeV-A5. Phylogenetic analysis revealed that all strains clustered together on the same clade and showed no significant genetic variation. We saw no significant evidence of association between sequence and either clinical severity (defined by admission to paediatric intensive care), geographical origin (compared between Canada and U.K) or year of sample collection (samples sequenced during 2013 - 2018)., Conclusions: In this small cohort, sequencing data indicate that PeV circulating in the UK and Canada from 2013 to 18 are derived from a common ancestor. No association between disease severity and genetic sequence was seen in the UK or Canadian cohorts. Larger studies are required to support these findings., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: HIV-1 resistance testing is recommended in clinical management and next-generation sequencing (NGS) methods are now available in many virology laboratories., Objectives: To evaluate the diagnostic performance of Long-Read Single Molecule Real-time (SMRT) sequencing (Sequel, PacBio) for HIV-1 polymerase genotyping., Study Design: 111 prospective clinical samples (83 plasma and 28 leukocyte-enriched blood fraction) were analyzed for routine HIV-1 resistance genotyping using Sanger sequencing, Vela NGS, and SMRT sequencing. We developed a SMRT sequencing protocol and a bio-informatics pipeline to infer antiretroviral resistance on both haplotype and variant calling approaches., Results: The polymerase was successfully sequenced by the three platforms in 98 % of plasma RNA samples for viral loads above 4 log copies/mL. The success rate decreased to 83 % using Sanger or Vela sequencing and to 67 % using SMRT sequencing for viral loads of 3 to 4 log copies/mL. Sensitivities of 50 %, 54 % and 61 % were obtained using SMRT, Vela, and Sanger sequencing, respectively, in cellular DNA from patients with prolonged undetectable plasma HIV-1 RNA. Ninety-eight percent of resistance-associated mutations (RAMs) identified with Sanger sequencing were detected using SMRT sequencing. Furthermore, 91 % of RAMs (> 5 % threshold) identified with Vela NGS were detected using SMRT sequencing. RAM quantification using Vela and SMRT sequencing was well correlated (Spearman correlation ρ = 0.82; P < 0.0001)., Conclusions: SMRT sequencing of the full-length HIV-1 polymerase appeared performant for characterizing HIV-1 genotypic resistance on both RNA and DNA clinical samples. Long-read sequencing is a new tool for mutation haplotyping and resistance analysis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Early screening for pathogens is crucial during pandemic outbreaks. Nucleic acid testing (NAT) is a valuable method for keeping pathogens from spreading. However, the long detection time and large size of the instruments involved significantly limited the efficiency of detection. This work described an integrated NAT microsensor that facilitated rapid and extremely sensitive detection based on nucleic acid amplification (NAA) on a chip. The biochip consisted of two layers incorporating a heater, a thermometer, an interdigital electrode (IDE) and a reaction chamber. The Pt electrode based heater and thermometer were utilized to maintain a specific temperature for the sample in the chamber. The thermometer exhibited a good linear correlation with a sensitivity of 9.36 Ω/°C and the heater achieved a heating efficiency of approximately 6.5 °C/s. Multiple ions were released during NAA, resulting in a decrease in the impedance of the amplification system solution. A large signal of impedance was generated by the released ions due to its linear correlation with the logarithm of the ion concentration. With this detection principle, IDE was employed for real-time monitoring of the in-chip reaction system impedance and NAA process. Specific nucleic acids from two pathogens (SARS-CoV-2, Vibrio vulnificus) were detected with this microsensor. The samples were qualitatively analyzed on microchip within 3 min, with a limit of detection (LOD) of 10 3 copies/μL. The proposed sensor presented several advantages, including reduced NAT time and increased sensitivity. Consequently, it has shown significant potential in rapid and high-quality nucleic acid testing for the field of epidemic prevention., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and transmissible gastroenteritis virus (TGEV) are three clinically common coronaviruses causing diarrhea in pigs, with indistinguishable clinical signs and pathological changes. Rapid, portable and reliable differential diagnosis of these three pathogens is crucial for the prompt implementation of appropriate control measures. In this study, we developed a triplex nucleic acid assay that combines reverse transcription recombinase-aided amplification (RT-RAA) with lateral flow assay (LFA) by targeting the most conserved genomic region in the ORF1b genes of PEDV, PDCoV and TGEV. The entire detection process of the triplex RT-RAA-LFA assay included 10-min nucleic acid amplification at 42 °C and 5-min visual LFA readout at room temperature. The assay could specifically differentiate PEDV, PDCoV and TGEV without cross-reaction with any other major swine pathogens. Sensitivity analysis showed that the triplex RT-RAA-LFA assay was able to detect the viral RNA extracted from the spiked fecal samples with the minimum of 1 × 10 0 TCID 50 PEDV, 1 × 10 4 TCID 50 PDCoV, and 1 × 10 2 TCID 50 TGEV per reaction, respectively. Further analysis showed that the 95 % detection limit (LOD) of triplex RT-RAA-LFA for PEDV, PDCoV, and TGEV were 22, 478, and 205 copies of recombinant plasmids per reaction, respectively. The diagnostic performance of triplex RT-RAA-LFA was compared with that of PEDV, PDCoV and TGEV respective commercial real-time RT-PCR kits by testing 114 clinical rectal swab samples in parallel. The total diagnostic coincidence rates of triplex RT-RAA-LFA with real-time RT-PCR kits of PEDV, PDCoV and TGEV were 100 %, 99.1 % and 99.1 %, respectively, and their Kappa values were 1.00, 0.958 and 0.936, respectively. Collectively, the RT-RAA-LFA assay is a powerful tool for the rapid, portable, visual, and synchronous differential diagnosis of PEDV, PDCoV, and TGEV., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The California sea hare (Aplysia californica) is a model for age associated cognitive decline. Recent researched identified a novel nidovirus, Aplysia Abyssovirus 1, with broad tropism enriched in the Aplysia nervous system. This virus is ubiquitous in wild and maricultured, young and old animals without obvious pathology. Here we re-evaluated gene expression data from several previous studies to investigate differential expression in the nervous system and gill in response to virus and aging as well as the mutational spectrum observed in the viral sequences obtained from these datasets. Viral load and age were highly correlated, indicating persistent infection. Upregulated genes in response to virus were enriched for immune genes and signatures of ER and proteostatic stress, while downregulated genes were enriched for mitochondrial metabolism. Differential expression with respect to age suggested increased iron accumulation and decreased glycolysis, fatty acid metabolism, and proteasome function. Interaction of gene expression trends associated with viral infection and aging suggest that viral infection likely plays a role in aging in the Aplysia nervous system. Mutation analysis of viral RNA identified signatures suggesting ADAR and AID/APOBEC like deaminase act as part of Aplysia anti-viral defense., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Many functions of ribonucleic acid (RNA) rely on its ability to assume specific sequence-structure motifs. Packaging signals found in certain RNA viruses are one such prominent example of functional RNA motifs. These signals are short hairpin loops that interact with coat proteins and drive viral self-assembly. As they are found in different positions along the much longer genomic RNA, the formation of their correct structure occurs as a part of a larger context. Any changes to this context can consequently lead to changes in the structure of the motifs themselves. In fact, previous studies have shown that structure and function of RNA motifs can be highly context sensitive to the flanking sequence surrounding them. However, in what ways different flanking sequences influence the structure of an RNA motif they surround has yet to be studied in detail. We focus on a hairpin-rich region of the RNA genome of bacteriophage MS2-a well-studied RNA virus with a wide potential for use in biotechnology-and systematically examine context-dependent structural stability of 14 previously identified hairpin motifs, which include putative and confirmed packaging signals. Combining secondary and tertiary RNA structure prediction of the hairpin motifs placed in different contexts, ranging from the native genomic sequence to random RNA sequences and unstructured poly-U sequences, we determine different measures of motif structural stability. In this way, we show that while some motif structures can be stable in any context, others require specific context provided by the genome. Our results demonstrate the importance of context in RNA structure formation and how changes in the flanking sequence of an RNA motif sometimes lead to drastic changes in its structure. Structural stability of a motif in different contexts could provide additional insights into its functionality as well as assist in determining whether it remains functional when intentionally placed in other contexts., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Background: Whole genome sequencing (WGS) of respiratory viruses from rapid antigen tests (RAT-WGS) is a novel approach to expanding genomic surveillance of respiratory infections. To date however, there are limited data on the genomic stability of these viruses on RATs. In this study, we investigated the effect of storage conditions and nucleic acid preservatives on the ability to enhance stability and improve recovery of respiratory virus genomes from RATs., Methods: A mixture of common respiratory viruses was used to inoculate RATs at different environmental temperatures (4°C, 20°C and 36°C), with two preservative reagents (RNALater and DNA/RNA shield) Nucleic acid was extracted from RATs at two different timepoints (72 h and seven days) and subject to real-time multiplex respiratory PCR to detect a range of respiratory viruses. WGS was performed using target-enrichment with the TWIST Comprehensive Viral Research Panel. Defined metrics from an automated in-house bioinformatic pipeline were used to assess and compare viral genome recovery under different conditions., Results: Nucleic acid degradation (indicated by relative change in PCR cycle threshold and WGS-based metrics) was most notable at 20 °C and 36 °C. Storage in either RNALater or DNA / RNA shield improved genome recovery for respiratory viruses across all temperature conditions, although this was most pronounced for RNALater. Subtyping of Influenza viruses demonstrated the applicability of RAT-WGS in downstream genomic epidemiological surveillance., Conclusions: Under simulated conditions, RAT-WGS demonstrated that (i) viral genomes were generally stable at 4°C at 72 h and 1 week, (ii) RNALater has a more significant preservation of nucleic acids compared to DNA/RNA Shield and (iii) genome recovery can be achieved using a sequencing depth of 500,000 reads per sample in RNALater, across all respiratory viruses and conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier B.V. All rights reserved.)

In this study, we have designed an electrochemical biosensor based on topological material Bi 2 Se 3 for the sensitive detection of SARS-CoV-2 in the COVID-19 pandemic. Flake-shaped Bi 2 Se 3 was obtained directly from high-quality single crystals using mechanical exfoliation, and the single-stranded DNA was immobilized onto it. Under optimal conditions, the peak current of the differential pulse voltammetry method exhibited a linear relationship with the logarithm of the concentration of target-complementary-stranded DNA, ranging from 1.0 × 10 -15 to 1.0 × 10 -11 M, with a detection limit of 3.46 × 10 -16 M. The topological material Bi 2 Se 3 , with Dirac surface states, enhanced the signal-to-interference plus noise ratio of the electrochemical measurements, thereby improving the sensitivity of the sensor. Furthermore, the electrochemical sensor demonstrated excellent specificity in recognizing RNA. It can detect complementary RNA by amplifying and transcribing the initial DNA template, with an initial DNA template concentration ranging from 1.0 × 10 -18 to 1.0 × 10 -15 M. Furthermore, the sensor also effectively distinguished negative and positive results by detecting splitting-synthetic SARS-CoV-2 pseudovirus with a concentration of 1 copy/μL input. Our work underscores the immense potential of the electrochemical sensing platform based on the topological material Bi 2 Se 3 in the detection of pathogens during the rapid spread of acute infectious diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

With the prevalence of human immunodeficiency virus type 1 (HIV-1) CRF01&#95;AE and CRF07&#95;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&#95;0107, and they were second-generation unique recombinant strains formed by the recombination of CRF01&#95;AE and CRF07&#95;BC, with the isoforms being CRF01&#95;AE and CRF0107&#95;BC, respectively. The continued emergence of novel CRF01&#95;AE/CRF07&#95;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.

Since the onset of the COVID-19 pandemic, one productive area of research has focused on the intricate two- and three-dimensional structures taken on by SARS-CoV-2's RNA genome. These structures control essential viral processes, making them tempting targets for therapeutic intervention. This review focuses on two such structured regions, the frameshift stimulation element (FSE), which controls the translation of viral protein, and the 3' untranslated region (3' UTR), which is thought to regulate genome replication. For the FSE, we discuss its canonical pseudoknot's threaded and unthreaded topologies, as well as the diversity of competing two-dimensional structures formed by local and long-distance base pairing. For the 3' UTR, we review the evidence both for and against the formation of its replication-enabling pseudoknot., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2024. Published by Elsevier Ltd.)

Previous studies have emphasized the necessity of surveillance and control measures for hepatitis E virus (HEV) infection in wild boars, an important reservoir of HEV. To assess the current situation of HEV infection in wild boars in Japan, this study investigated the prevalence and genetic diversity of HEV among wild boars captured in 16 prefectures of Japan during 2018-2023. Serum samples from 968 wild boars were examined for anti-HEV IgG antibodies and HEV RNA. The prevalence of anti-HEV IgG varied geographically from 0 % to 35.0 %. HEV RNA was detected in 3.6 % of boars, with prevalence varying by prefecture from 0 % to 22.2 %. Genotype 3 was the most prevalent genotype (91.9 %), followed by genotype 4 (5.4 %), with one strain closely related to genotype 6. The prevalence of HEV infection among wild boars decreased from 2018/2019 to 2022/2023 with significant declines in levels of anti-HEV IgG antibodies (14.5 % vs. 6.2 %, P < 0.0001) and HEV RNA (7.6 % vs. 1.5 %, P < 0.0001). Regional analysis showed varying trends, with no HEV RNA-positive boars found in several regions in recent years. A plausible factor contributing to the decline in HEV infection is the application of countermeasures, including installing fences to prevent intrusion into pig farms, implemented in response to the emergence of classical swine fever virus (CSFV) infection in wild boars and domestic pigs, with incidents reported annually since 2018. Further investigation is warranted to explore the association between countermeasures to CSFV infection and the decrease in HEV infection among wild boars., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Purpose: To study the positivity rate of conjunctival realtime polymerase chain reaction (RT-PCR) testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)., Design: Systematic review and diagnostic accuracy meta-analysis., Methods: MEDLINE and EMBASE were queried using medical subject headings terms. Diagnostic accuracy meta-analyses and forest plots were obtained using the RevMan software., Results: After deduplication, appraisal of abstract titles and full-text analysis of 1441 articles, 42 articles with 3351 COVID-19 patients were included in this review. Of these, 412 conjunctival swabs/Schirmer paper strips tested positive for SARS-CoV-2 by RT-PCR. The pooled sensitivity of the RT-PCR tests across the 24 studies with laboratory-confirmed COVID-19 patients was 10.3%., Conclusions: Only 1 in 10 RT-PCR tests performed on conjunctival swabs were positive for SARS-CoV-2. Although this suggests that SARS-CoV-2 is likely present and detectable in the conjunctiva, this detection method has low diagnostic potential.

Background & Aims: Transcription termination fine-tunes gene expression and contributes to the specification of RNA function in eukaryotic cells. Transcription termination of HBV is subject to the recognition of the canonical polyadenylation signal (cPAS) common to all viral transcripts. However, the regulation of this cPAS and its impact on viral gene expression and replication is currently unknown., Methods: To unravel the regulation of HBV transcript termination, we implemented a 3' RACE (rapid amplification of cDNA ends)-PCR assay coupled to single molecule sequencing both in in vitro-infected hepatocytes and in chronically infected patients., Results: The detection of a previously unidentified transcriptional readthrough indicated that the cPAS was not systematically recognized during HBV replication in vitro and in vivo. Gene expression downregulation experiments demonstrated a role for the RNA helicases DDX5 and DDX17 in promoting viral transcriptional readthrough, which was, in turn, associated with HBV RNA destabilization and decreased HBx protein expression. RNA and chromatin immunoprecipitation, together with mutation of the cPAS sequence, suggested a direct role of DDX5 and DDX17 in functionally linking cPAS recognition to transcriptional readthrough, HBV RNA stability and replication., Conclusions: Our findings identify DDX5 and DDX17 as crucial determinants of HBV transcriptional fidelity and as host restriction factors for HBV replication., Impact and Implications: HBV covalently closed circular (ccc)DNA degradation or functional inactivation remains the holy grail for the achievement of HBV cure. Transcriptional fidelity is a cornerstone in the regulation of gene expression. Here, we demonstrate that two helicases, DDX5 and DDX17, inhibit recognition of the HBV polyadenylation signal and thereby transcriptional termination, thus decreasing HBV RNA stability and acting as restriction factors for efficient cccDNA transcription and viral replication. The observation that DDX5 and DDX17 are downregulated in patients chronically infected with HBV suggests a role for these helicases in HBV persistence in vivo. These results open new perspectives for researchers aiming at identifying new targets to neutralise cccDNA transcription., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Bovine viral diarrhea virus (BVDV) is a widespread pathogen of cattle and other mammals that causes major economic losses in the livestock industry. N4-TSC and 6NO 2 -TSC are two thiosemicarbazones derived from 1-indanone that exhibit anti-BVDV activity in vitro. These compounds selectively inhibit BVDV and are effective against both cytopathic and non-cytopathic BVDV-1 and BVDV-2 strains. We confirmed that N4-TSC acts at the onset of viral RNA synthesis, as previously reported for 6NO 2 -TSC. Moreover, resistance selection and characterization showed that N4-TSC R mutants were highly resistant to N4-TSC but remained susceptible to 6NO 2 -TSC. In contrast, 6NO 2 -TSC R mutants were resistant to both compounds. Additionally, mutations N264D and A392E were found in the viral RNA-dependent RNA polymerase (RdRp) of N4-TSC R mutants, whereas I261 M was found in 6NO 2 -TSC R mutants. These mutations lay in a hydrophobic pocket within the fingertips region of BVDV RdRp that has been described as a "hot spot" for BVDV non-nucleoside inhibitors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Usutu virus (USUV) is a mosquito-borne flavivirus originating from Africa, that belongs to the Japanese encephalitis virus (JEV) complex. In nature, USUV involves Culex spp. mosquitoes acting as vectors and birds as amplifying hosts. The virus has recently spread in Europe and is considered an emerging human pathogen. This is the first research study performed in Greece revealing the presence and circulation of USUV in Culex spp. mosquito populations. Out of the 1,500 mosquito pools tested with real-time RT-PCR, four (Roesch et al., 2019) were positive for USUV. All four pools were collected from the region of Central Macedonia, Northern Greece., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Japanese encephalitis (JE), a mosquito-borne zoonotic disease caused by the Japanese encephalitis virus (JEV), poses a serious threat to global public health. The low viremia levels typical in JEV infections make RNA detection challenging, necessitating early and rapid diagnostic methods for effective control and prevention. This study introduces a novel one-pot detection method that combines recombinant enzyme polymerase isothermal amplification (RPA) with CRISPR/EsCas13d targeting, providing visual fluorescence and lateral flow assay (LFA) results. Our portable one-pot RPA-EsCas13d platform can detect as few as two copies of JEV nucleic acid within 1 h, without cross-reactivity with other pathogens. Validation against clinical samples showed 100 % concordance with real-time PCR results, underscoring the method's simplicity, sensitivity, and specificity. This efficacy confirms the platform's suitability as a novel point-of-care testing (POCT) solution for detecting and monitoring the JE virus in clinical and vector samples, especially valuable in remote and resource-limited settings., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Single stranded RNA viruses are primary causative agents for pandemics, causing extensive morbidity and mortality worldwide. A pivotal question in pandemic preparedness and therapeutic intervention is what are the specific mutations which are more likely to emerge during such global health crises? This study aims to identify markers for mutations with the highest probability of emergence in these pandemics, focusing on the SARS-CoV-2 spike protein, an essential and therapeutically significant viral protein, starting from sequence information from the onset of the pandemic until July 2022. Quite consistently, we observed that emerged mutations tended to demonstrate a high genetic score, which reflects high similarity of the type of codon required for translation between an amino acid and to the mutated one. Further, this pattern is also observed in therapeutically significant proteins of other ssRNA pandemic viruses, including influenza (HA, NA), spike proteins of Ebola, envelope of Dengue and Chikungunya. We propose that the genetic score serves as an initial indicator, preceding the actual impact of the mutation on viral fitness. Finally, we developed a comprehensive computational pipeline to further explore and predict the subsequent effects of mutations on viral fitness. We believe that our pipeline can narrow down and predict future mutations in therapeutically important viral proteins during a pandemic., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Herpesviruses-encoded microRNAs (miRNAs) have been discovered to be essential regulators in viral life cycle, participating in viral replication, latent or lytic infection, and immunological escape. However, the roles of miRNAs encoded by duck plague virus (DPV) are still unknown. Dev-miR-D28-3p is a miRNA uniquely encoded by DPV CHv strain. The aim of this study was to explore the effect of dev-miR-D28-3p on DPV replication and explore the potential mechanisms involved. Our findings demonstrated that transfection of dev-miR-D28-3p mimic into duck embryo fibroblasts (DEFs) effectively suppressed viral copies, viral titers and viral protein expressions during DPV infection, while the results above were reversed after transfection with dev-miR-D28-3p inhibitor. Subsequently, we further discovered that dev-miR-D28-3p specifically bound to DPV-encoded UL27 and inhibited its expression, suggesting that UL27 was the target gene of dev-miR-D28-3p. Finally, we investigated the role of UL27 in DPV replication and found the overexpression of UL27 increased viral copies, viral titers, and viral protein expressions; whereas the opposite results appear when knockdown of UL27. Our findings illustrated a novel mechanism that DPV regulated itself replication via dev-miR-D28-3p, paving the way for exploring the role of DPV-encoded miRNAs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Competing interests The authors declare that they have no competing interests, (Copyright © 2024 Elsevier B.V. All rights reserved.)

Crimean-Congo Hemorrhagic Fever virus (CCHFV) is a tick-borne pathogen that causes severe acute fever disease in humans and requires a biosafety level 4 laboratory for handling. Hazara virus (HAZV), belonging to the same virus genus as CCHFV, does not exhibit pathogenesis in humans. To investigate host RNA-binding proteins (RBPs) that regulate CCHFV replication, we generated a series of mutant RAW264.7 cells by CRISPR/Cas9 system and these cells were infected with HAZV. The viral titers in the supernatant of these cells was investigated, and HuR (ELAVL1) was identified. HuR KO RAW264.7 cells reduced HAZV replication. HuR is an RBP that enhances mRNA stability by binding to adenyl-uridine (AU)-rich regions in their 3' non-coding region (NCR). HuR regulates innate immune response by binding to host mRNAs of signaling molecules. The expression of cytokine genes such as Ifnb, Il6, and Tnf was reduced in HuR KO cells after HAZV infection. Although HuR supports the innate immune response during HAZV infection, we found that innate immune activation by HAZV infection did not affect its replication. We then investigated whether HuR regulates HAZV genome RNA stability. HAZV RNA genome was precipitated with an anti-HuR antibody, and HAZV genome RNA stability was lowered in HuR KO cells. We found that HuR associated with HAZV RNA and stabilized it to enhance HAZV replication. Furthermore, HuR-deficiency reduced CCHFV minigenome replication. CCHFV is a negative-strand RNA virus and positive-strand RNA is produced during replication. HuR was associated with positive-strand RNA rather than negative-strand RNA, and AU-rich region in 3'-NCR of S segment was responsible for immunoprecipitation with anti-HuR antibody and minigenome replication. Additionally, HuR inhibitor treatment reduced CCHFV minigenome replication. Our results indicate that HuR aids replication of the CCHFV minigenome by associating with the AU-rich region in the 3'-NCR., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ikegawa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)