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

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

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

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

Pre-pandemic antibodies screening against SARS-CoV-2 and virus detection among children diagnosed with eruptive fevers.

OBJECTIVES: This study aims to assess the seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG antibodies against the spike (S) and nucleocapsid (NP) proteins, as well as neutralizing antibodies against the receptor-binding domain (RBD). Additionally, it aims to detect viral RNA of SARS-CoV-2 in pre-pandemic archival pediatric specimens collected before the announcement of the COVID-19 pandemic spread on March 20th, 2020, in Morocco. The objective is to investigate the existence of pre-pandemic immunity to SARS-CoV-2. METHODS: We conducted a cross-sectional study, to analyze IgG antibody levels in a cohort of 106 pre-pandemic pediatric participants. Using an indirect enzyme-linked immunosorbent assay (ELISA), we measured the IgG levels against the S and NP proteins of SARS-CoV-2. Additionally, we staged a competitive ELISA assay to evaluate the neutralizing capability of these antibodies. We used reverse transcription polymerase chain reaction (rRT-PCR) to detect viral NP and ORF1ab genes of SARS-CoV-2 in oropharyngeal swabs. Moreover, we conducted on the same specimens a multiplexed RT-PCR to detect RNA of the most common 27 pathogens involved in lower respiratory tract infections. RESULTS: Among the 106 serum samples, 13% (nn = =14) tested positive for SARS-CoV-2 IgG antibodies using ELISA. Temporal analysis indicated varying IgG positivity levels across 2019. Neutralizing antibodies were found in 21% of the 28 samples analyzed, including two with high inhibition rates (93%). The SARS-CoV-2 RNA was detected using rRT-PCR in 14 samples. None of the samples tested positive for the other 27 pathogens associated with lower respiratory tract infections, using multiplexed RT-PCR. CONCLUSION: Our study addresses the possibility, that COVID-19 infections occurred in Morocco before the recognized outbreak. On the other hand, some of the cases might reflect cross-reactivity with other coronaviruses or be influenced by previous viral exposures or vaccinations. Understanding these factors is crucial to comprehending pediatric immune responses to newly emerging infectious diseases.

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

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

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

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

[How do 2'-O-methylations within Human Immunodeficiency Virus type 1 (HIV-1) genome regulate its replication?] / Effets de la 2'-O-méthylation de l'ARN génomique du VIH-1 sur la réplication virale.

The genomic RNA of HIV-1 is modified by epitranscriptomic modifications, including 2'-O-methylations, which are found on 17 internal positions. These methylations are added by the cellular methyltransferase FTSJ3, and have pro-viral effects, since they shield the viral genome from the detection by the innate immune sensor MDA5. In turn, the production of interferons by infected cells is reduced, limiting the expression of interferon-stimulated genes (ISGs) with antiviral activities. Moreover, 2'-O-methylations protect the HIV-1 genome from its degradation by ISG20, an interferon-induced exonuclease. Conversely, these methylations also exhibit antiviral effects, as they impede reverse-transcription in vitro or in quiescent cells, which are known to contain low nucleotide concentrations. Altogether, these observations suggest a balance between the proviral effect of 2'-O-methylations, related to the protection of the viral genome from detection by MDA5 and degradation by ISG20, and the antiviral effect, associated with the negative impact of 2'-O-methylations on the viral replication. These findings pave the way for further optimization of therapeutic RNA, by selective methylation of specific nucleotides.

Title: Effets de la 2'-O-méthylation de l'ARN génomique du VIH-1 sur la réplication virale. Abstract: Les ARN du virus de l'immunodéficience humaine sont décorés par des marques épitranscriptomiques, dont des 2'-O-méthylations internes. Ces marques ajoutées par une enzyme cellulaire, FTSJ3, sont des marqueurs du « soi ¼. Elles ont des effets proviraux en protégeant l'ARN viral de la détection par le senseur de l'immunité innée MDA5, et en limitant sa dégradation par l'exonucléase cellulaire ISG20, induite par l'interféron. Ces méthylations ont également un effet antiviral, dans la mesure où elles perturbent la rétrotranscription du génome ARN du virus, in vitro et dans des cellules quiescentes. Un équilibre subtil existe donc entre les effets proviraux et antiviraux des 2'-O-méthylations, assurant ainsi une réplication optimale du virus. Ces découvertes ouvrent des perspectives d'optimisation des ARN thérapeutiques à effet antiviral, par la méthylation sélective de certains nucléotides.

Nanoscale cellular organization of viral RNA and proteins in SARS-CoV-2 replication organelles.

The SARS-CoV-2 viral infection transforms host cells and produces special organelles in many ways, and we focus on the replication organelles, the sites of replication of viral genomic RNA (vgRNA). To date, the precise cellular localization of key RNA molecules and replication intermediates has been elusive in electron microscopy studies. We use super-resolution fluorescence microscopy and specific labeling to reveal the nanoscopic organization of replication organelles that contain numerous vgRNA molecules along with the replication enzymes and clusters of viral double-stranded RNA (dsRNA). We show that the replication organelles are organized differently at early and late stages of infection. Surprisingly, vgRNA accumulates into distinct globular clusters in the cytoplasmic perinuclear region, which grow and accommodate more vgRNA molecules as infection time increases. The localization of endoplasmic reticulum (ER) markers and nsp3 (a component of the double-membrane vesicle, DMV) at the periphery of the vgRNA clusters suggests that replication organelles are encapsulated into DMVs, which have membranes derived from the host ER. These organelles merge into larger vesicle packets as infection advances. Precise co-imaging of the nanoscale cellular organization of vgRNA, dsRNA, and viral proteins in replication organelles of SARS-CoV-2 may inform therapeutic approaches that target viral replication and associated processes.

Molecular insights into the Ebola virus life cycle.

Ebola virus and other orthoebolaviruses cause severe haemorrhagic fevers in humans, with very high case fatality rates. Their non-segmented single-stranded RNA genome encodes only seven structural proteins and a small number of non-structural proteins to facilitate the virus life cycle. The basics of this life cycle are well established, but recent advances have substantially increased our understanding of its molecular details, including the viral and host factors involved. Here we provide a comprehensive overview of our current knowledge of the molecular details of the orthoebolavirus life cycle, with a special focus on proviral host factors. We discuss the multistep entry process, viral RNA synthesis in specialized phase-separated intracellular compartments called inclusion bodies, the expression of viral proteins and ultimately the assembly of new virus particles and their release at the cell surface. In doing so, we integrate recent studies into the increasingly detailed model that has developed for these fundamental aspects of orthoebolavirus biology.

Natural evidence of coronaviral 2'-O-methyltransferase activity affecting viral pathogenesis via improved substrate RNA binding.

Previous studies through targeted mutagenesis of K-D-K-E motif have demonstrated that 2'-O-MTase activity is essential for efficient viral replication and immune evasion. However, the K-D-K-E catalytic motif of 2'-O-MTase is highly conserved across numerous viruses, including flaviviruses, vaccinia viruses, coronaviruses, and extends even to mammals. Here, we observed a stronger 2'-O-MTase activity in SARS-CoV-2 compared to SARS-CoV, despite the presence of a consistently active catalytic center. We further identified critical residues (Leu-36, Asn-138 and Ile-153) which served as determinants of discrepancy in 2'-O-MTase activity between SARS-CoV-2 and SARS-CoV. These residues significantly enhanced the RNA binding affinity of 2'-O-MTase and boosted its versatility toward RNA substrates. Of interest, a triple substitution (Leu36 → Ile36, Asn138 → His138, Ile153 → Leu153, from SARS-CoV-2 to SARS-CoV) within nsp16 resulted in a proportional reduction in viral 2'-O-methylation and impaired viral replication. Furthermore, it led to a significant upregulation of type I interferon (IFN-I) and proinflammatory cytokines both in vitro and vivo, relying on the cooperative sensing of melanoma differentiation-associated protein 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2). In conclusion, our findings demonstrated that alterations in residues other than K-D-K-E of 2'-O-MTase may affect viral replication and subsequently influence pathogenesis. Monitoring changes in nsp16 residues is crucial as it may aid in identifying and assessing future alteration in viral pathogenicity resulting from natural mutations occurring in nsp16.

Condensation of RNase L promotes its rapid activation in response to viral infection in mammalian cells.

Oligoadenylate synthetase 3 (OAS3) and ribonuclease L (RNase L) are components of a pathway that combats viral infection in mammals. Upon detection of viral double-stranded RNA (dsRNA), OAS3 synthesizes 2'-5'-oligo(A), which activates the RNase domain of RNase L by promoting the homodimerization and oligomerization of RNase L monomers. Activated RNase L rapidly degrades all cellular mRNAs, shutting off several cellular processes. We sought to understand the molecular mechanisms underlying the rapid activation of RNase L in response to viral infection. Through superresolution microscopy and live-cell imaging, we showed that OAS3 and RNase L concentrated into higher-order cytoplasmic complexes known as dsRNA-induced foci (dRIF) in response to dsRNA or infection with dengue virus, Zika virus, or West Nile virus. The concentration of OAS3 and RNase L at dRIF corresponded with the activation of RNase L-mediated RNA decay. We showed that dimerized/oligomerized RNase L concentrated in a liquid-like shell surrounding a core OAS3-dRIF structure and dynamically exchanged with the cytosol. These data establish that the condensation of dsRNA, OAS3, and RNase L into dRIF is a molecular switch that promotes the rapid activation of RNase L upon detection of dsRNA in mammalian cells.

Epstein-Barr virus-encoded BART9 and BART15 miRNAs are elevated in exosomes of cerebrospinal fluid from relapsing-remitting multiple sclerosis patients.

Epstein-Barr virus (EBV) infection is approved as the main environmental trigger of multiple sclerosis (MS). In this path, we quantified ebv-miR-BART9-3p and ebv-miR-BART15 in exosomes of cerebrospinal fluid (CSF) of untreated relapsing-remitting MS (RRMS) patients in comparison with the control group. Interestingly, patients displayed significant upregulation of ebv-miR-BART9-3p (18.4-fold) and ebv-miR-BART15 (3.1-fold) expression in CSF exosomes. Moreover, the expression levels of hsa-miR-21-5p and hsa-miR-146a-5p were found to be significantly elevated in the CSF samples obtained from the patient group compared to those obtained from the HC group. The levels of Interferon-gamma (IFN-γ), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-17 (IL-17), interleukin-23 (IL-23), transforming growth factor beta (TGF-ß), and tumor necrosis factor-alpha (TNF-α) were observed to be significantly elevated in the serum and CSF exosomes of the patients. The highest increase was observed in TGF-ß (8.5-fold), followed by IL-23 (3.9-fold) in CSF exosomes. These findings are in agreement with the association between EBV infection and inflammatory cytokines induction. Furthermore, the ratios of TGF-ß: TNF-α and TGF-ß: IFN-γ attained values of 4 to 16.4 and 1.3 to 3.6, respectively, in the CSF exosomes of the patients, in comparison to those of the control group. These findings show EBV activity in RRMS patients is different from that of healthy ones. Elevation of ebv-miR-BART9-3p, ebv-miR-BART15, and inflammatory cytokines expression in CSF exosomes in RRMS patients provides a substantial link between EBV activity and the onset of the disease, as well as the transition from EBV infection to MS.

Analytical validation of a semi-automated methodology for quantitative measurement of SARS-CoV-2 RNA in wastewater collected in northern New England.

Wastewater-based epidemiology (WBE) can be used to monitor the community presence of infectious disease pathogens of public health concern such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Viral nucleic acid has been detected in the stool of SARS-CoV-2-infected individuals. Asymptomatic SARS-CoV-2 infections make community monitoring difficult without extensive and continuous population screening. In this study, we validated a procedure that includes manual pre-processing, automated SARS-CoV-2 RNA extraction and detection workflows using both reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) and reverse transcriptase droplet digital PCR (RT-ddPCR). Genomic RNA and calibration materials were used to create known concentrations of viral material to determine the linearity, accuracy, and precision of the wastewater extraction and SARS-CoV-2 RNA detection. Both RT-qPCR and RT-ddPCR perform similarly in all the validation experiments, with a limit of detection of 50 copies/mL. A wastewater sample from a care facility with a known outbreak was assessed for viral content in replicate, and we showed consistent results across both assays. Finally, in a 2-week survey of two New Hampshire cities, we assessed the suitability of our methods for daily surveillance. This paper describes the technical validation of a molecular assay that can be used for long-term monitoring of SARS-CoV-2 in wastewater as a potential tool for community surveillance to assist with public health efforts.IMPORTANCEThis paper describes the technical validation of a molecular assay that can be used for the long-term monitoring of SARS-CoV-2 in wastewater as a potential tool for community surveillance to assist with public health efforts.

VLP-based model for the study of airborne viral pathogens.

The recent COVID-19 pandemic has underscored the danger of airborne viral pathogens. The lack of model systems to study airborne pathogens limits the understanding of airborne pathogen distribution as well as potential surveillance and mitigation strategies. In this work, we develop a novel model system to study airborne pathogens using virus-like particles (VLPs). Specifically, we demonstrate the ability to aerosolize VLP and detect and quantify aerosolized VLP RNA by reverse transcription-loop-mediated isothermal amplification in real-time fluorescent and colorimetric assays. Importantly, the VLP model presents many advantages for the study of airborne viral pathogens: (i) similarity in size and surface components; (ii) ease of generation and noninfectious nature enabling the study of biosafety level 3 and biosafety level 4 viruses; (iii) facile characterization of aerosolization parameters; (iv) ability to adapt the system to other viral envelope proteins, including those of newly discovered pathogens and mutant variants; and (v) the ability to introduce viral sequences to develop nucleic acid amplification assays. IMPORTANCE: The study and detection of airborne pathogens are hampered by the lack of appropriate model systems. In this work, we demonstrate that noninfectious virus-like particles (VLPs) represent attractive models to study airborne viral pathogens. Specifically, VLPs are readily prepared, are similar in size and composition to infectious viruses, and are amenable to highly sensitive nucleic acid amplification techniques.

Identifying cellular RNA-binding proteins during infection uncovers a role for MKRN2 in influenza mRNA trafficking.

Utilisation of RNA-binding proteins (RBPs) is an important aspect of post-transcriptional regulation of viral RNA. Viruses such as influenza A viruses (IAV) interact with RBPs to regulate processes including splicing, nuclear export and trafficking, while also encoding RBPs within their genomes, such as NP and NS1. But with almost 1000 RBPs encoded within the human genome it is still unclear what role, if any, many of these proteins play during viral replication. Using the RNA interactome capture (RIC) technique, we isolated RBPs from IAV infected cells to unravel the RBPome of mRNAs from IAV infected human cells. This led to the identification of one particular RBP, MKRN2, that associates with and positively regulates IAV mRNA. Through further validation, we determined that MKRN2 is involved in the nuclear-cytoplasmic trafficking of IAV mRNA potentially through an association with the RNA export mediator GLE1. In the absence of MKRN2, IAV mRNAs accumulate in the nucleus of infected cells, which may lead to their degradation by the nuclear RNA exosome complex. MKRN2, therefore, appears to be required for the efficient nuclear export of IAV mRNAs in human cells.

Major Group-B Enterovirus populations deleted in the noncoding 5' region of genomic RNA modulate activation of the type I interferon pathway in cardiomyocytes and induce myocarditis.

Major 5'-terminally deleted (5'TD) RNA forms of group-B coxsackievirus (CVB-5'TD) has been associated with myocarditis in both mice and humans. Although it is known that interferon-ß (IFN-ß) signaling is critical for an efficient innate immune response against CVB-induced myocarditis, the link between CVB-5'TD RNA forms and type I IFN signaling in cardiomyocytes remains to be explored. In a mouse model of CVB3/28-induced myocarditis, major early-emerging forms of CVB-5'TD RNA have been characterized as replicative viral populations that impair IFN-ß production in the heart. Synthetic CVB3/28 RNA forms mimicking each of these major 5'TD virus populations were transfected in mice and have been shown to modulate innate immune responses in the heart and to induce myocarditis in mice. Remarkably, transfection of synthetic viral RNA with deletions in the secondary structures of the 5'-terminal CVB3 RNA domain I, modifying stem-loops "b", "c" or "d", were found to impair IFN-ß production in human cardiomyocytes. In addition, the activation of innate immune response by Poly(I:C), was found to restore IFN-ß production and to reduce the burden of CVB-5'TD RNA-forms in cardiac tissues, thereby reducing the mortality rate of infected mice. Overall, our results indicate that major early-emerging CVB3 populations deleted in the domain I of genomic RNA, in the 5' noncoding region, modulate the activation of the type I IFN pathway in cardiomyocytes and induce myocarditis in mice. These findings shed new light on the role of replicative CVB-5'TD RNA forms as key pathophysiological factors in CVB-induced human myocarditis.

Label-free Colorimetric Detection of Viral RNA Based on Clustered Regularly Interspaced Short Palindromic Repeats and Gold Nanoparticles with a Portable Device.

Viral infections, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are some of the most dangerous threats to humans. SARS-CoV-2 has caused a global pandemic, highlighting the unprecedented demand for rapid and portable diagnostic methods. To meet these requirements, we designed a label-free colorimetric platform that combines the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated proteins (Cas) 12a system for naked-eye detection (named LFP). This method utilizes reverse transcription loop-mediated isothermal amplification (RT-LAMP) and the trans-cleavage activity of the CRISPR/Cas12a system to increase the sensitivity and specificity of the reaction. This platform can detect as few as 4 copies/µL of RNA and produces no false positive results when tested against the influenza virus. To better meet the requirements of point-of-care (POC) detection, we developed a portable device that can be applied in resource-poor and densely populated regions. The LFP assay holds great potential for application in resource-limited settings, and the label-free gold nanoparticle (AuNPs) probe can reduce costs, making it suitable for large-scale screening. We expect that the LFP assay will be promising for the POC screening of COVID-19.

Porous Agarose Layered Magnetic Graphene Oxide Nanocomposite for Virus RNA Monitoring in Wastewater.

The detection of virus RNA in wastewater has been established as a valuable method for monitoring Coronavirus disease 2019. Carbon nanomaterials hold potential application in separating virus RNA owing to their effective adsorption and extraction capabilities. However, carbon nanomaterials have limited separability under homogeneous aqueous conditions. Due to the stabilities in their nanostructure, it is a challenge to efficiently immobilize them onto magnetic beads for separation. Here, we develop a porous agarose layered magnetic graphene oxide (GO) nanocomposite that is prepared by agglutinating ferroferric oxide (Fe3O4) beads and GO with agarose into a cohesive whole. With an average porous size of approximately 500 nm, the porous structure enables the unhindered entry of virus RNA, facilitating its interaction with the surface of GO. Upon the application of a magnetic field, the nucleic acid can be separated from the solution within a few minutes, achieving adsorption efficiency and recovery rate exceeding 90% under optimized conditions. The adsorbed nucleic acid can then be preserved against complex sample matrix for 3 days, and quantitatively released for subsequent quantitative reverse transcription polymerase chain reaction (RT-qPCR) detection. The developed method was successfully utilized to analyze wastewater samples obtained from a wastewater treatment plant, detecting as few as 10 copies of RNA molecules per sample. The developed aMGO-RT-qPCR provides an efficient approach for monitoring viruses and will contribute to wastewater-based surveillance of community infections.

The impact of single-stranded RNAs on the dimerization of double-stranded RNA-dependent protein kinase PKR.

The RNA-binding protein PKR serves as a crucial antiviral innate immune factor that globally suppresses translation by sensing viral double-stranded RNA (dsRNA) and by phosphorylating the translation initiation factor eIF2α. Recent findings have unveiled that single-stranded RNAs (ssRNAs), including in vitro transcribed (IVT) mRNA, can also bind to and activate PKR. However, the precise mechanism underlying PKR activation by ssRNAs, remains incompletely understood. Here, we developed a NanoLuc Binary Technology (NanoBiT)-based in vitro PKR dimerization assay to assess the impact of ssRNAs on PKR dimerization. Our findings demonstrate that, akin to double-stranded polyinosinic:polycytidylic acid (polyIC), an encephalomyocarditis virus (EMCV) RNA, as well as NanoLuc luciferase (Nluc) mRNA, can induce PKR dimerization. Conversely, homopolymeric RNA lacking secondary structure fails to promote PKR dimerization, underscoring the significance of secondary structure in this process. Furthermore, adenovirus VA RNA 1, another ssRNA, impedes PKR dimerization by competing with Nluc mRNA. Additionally, we observed structured ssRNAs capable of forming G-quadruplexes induce PKR dimerization. Collectively, our results indicate that ssRNAs have the ability to either induce or inhibit PKR dimerization, thus representing potential targets for the development of antiviral and anti-inflammatory agents.

Cryo-EM structures of Thogoto virus polymerase reveal unique RNA transcription and replication mechanisms among orthomyxoviruses.

Influenza viruses and thogotoviruses account for most recognized orthomyxoviruses. Thogotoviruses, exemplified by Thogoto virus (THOV), are capable of infecting humans using ticks as vectors. THOV transcribes mRNA without the extraneous 5' end sequences derived from cap-snatching in influenza virus mRNA. Here, we report cryo-EM structures to characterize THOV polymerase RNA synthesis initiation and elongation. The structures demonstrate that THOV RNA transcription and replication are able to start with short dinucleotide primers and that the polymerase cap-snatching machinery is likely non-functional. Triggered by RNA synthesis, asymmetric THOV polymerase dimers can form without the involvement of host factors. We confirm that, distinctive from influenza viruses, THOV-polymerase RNA synthesis is weakly dependent of the host factors ANP32A/B/E in human cells. This study demonstrates varied mechanisms in RNA synthesis and host factor utilization among orthomyxoviruses, providing insights into the mechanisms behind thogotoviruses' broad-infectivity range.

Real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay for detection of cassava brown streak viruses.

Cassava brown streak disease (CBSD) caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) is the most economically important viral disease of cassava. As cassava is a vegetatively propagated crop, the development of rapid and sensitive diagnostics would aid in the identification of virus-free planting material and development of effective management strategies. In this study, a rapid, specific and sensitive real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed for real-time detection of CBSV and UCBSV. The RT-RPA was able to detect as little as 2 pg/µl of purified RNA obtained from infected cassava leaves, a sensitivity equivalent to that obtained by quantitative real-time reverse transcription PCR (qRT-PCR), within 20 min at 37 °C. Further, the RT-RPA detected each target virus directly from crude leaf and stem extracts, avoiding the tedious and costly isolation of high-quality RNA. The developed RT-RPA assay provides a valuable diagnostic tool that can be adopted by cassava seed certification and virus resistance breeding programs to ensure distribution of virus-free cassava planting materials to farmers. This is the first report on the development and validation of crude sap-based RT-RPA assay for the detection of cassava brown streak viruses (UCBSV and CBSV) infection in cassava plants.