Various viral diseases can be widespread and cause severe disruption to global society. Highly sensitive virus detection methods are needed to take effective measures to prevent the spread of viral infection. This required the development of rapid virus detection technology to detect viruses at low concentrations, even in the biological fluid of patients in the early stages of the disease or environmental samples. This review describes an overview of various virus detection technologies and then refers to typical technologies such as beads-based assay, digital assay, and pore-based sensing, which are the three modern approaches to improve the performance of viral sensing in terms of speed and sensitivity. Globalization has facilitated long-distance travel of people and goods. On the one hand, this promoted progress in technologies and the global economy due to technology transfer; on the other hand, this resulted in the rapid worldwide spreading of viruses. Some of these viruses are pathogenic, and they often cause a global pandemic with serious social disruption. In recent years, we have often heard of the damages due to not only viral infectious diseases that infect humans, such as the recent novel coronavirus infection COVID-19) [9 [1], seRapid virus detection technology is of great importance in sustaining global economics while preventing the spread of pathological viruses. The detection of viral infection at an early stage helps to contain viruses and limits its spreading, e.g., the early detection of the Ebola virus has limited the spreading of the Ebola virus mainly within West Africa in 2014–2015 , while f50) method [While virus detection technology is important in the public health and medical sectors, it is a challenging task due to the small size and obligate intracellular functions of viruses . Biologi) method ,13. The ) method . DespiteA representative example of virus detection technology that targets nucleic acids is polymerase chain reaction (PCR), particularly quantitative PCR (qPCR) . This meOn the other hand, another molecular fingerprint of virus, i.e., proteins, were detected and quantified mainly with immunoassays. These assays are also widely known as antigen tests ,19. AntiIn the development of virus detection technologies, progress has aimed mainly at two directions: the reduction of limit of detection (LOD) and the improvement of speed of detection. However, these two directions are in a trade-off relationship, as shown in This review describes an overview of efforts to improve the LOD and speed of detection of virus detection technologies, with particular attention to three approaches: bioassays using micro- or nanoparticles (bead-based assays), digital bioassays, and pore-based sensing. These approaches have a common characteristic: the use of nm-size or μm-size components, such as magnetic nanoparticles, μm-scale well-arrays, and nm-scale pores.The characteristics of viruses, as described in the previous section, is a factor which has limited the sensitivity and speed of detection. The small size of virus particles has limited the use of conventional biological detection methods, e.g., microscopic examination to detect virus particles in biological sample; its property with obligate intracellular functions has increased the difficulty for in vitro detection of viruses a. To addBead-based assays are indirect virus detection methods in which virus particles in biological samples were captured and enriched with microbeads (0.5–500 μm). Following that, the signals generated from these microbeads were detected.While the concept to apply particles in the biological study was introduced by Wagner et al. in 1964 , it was For the optical detection of viruses captured by microbeads, captured antibodies will be coated on fluorescence microbeads, e.g., Luminex microbeads. The coating can be achieved either by passive adsorption of antibody on the surface of microbeads, binding through biotin-avidin binding, or by the covalent binding through amine groups. Virus particles are captured by the capture antibody on the surface of microbeads and detected by detection antibody and fluorochrome. The beads–virus complex will be analyzed with flow cytometer by detecting the fluorescence signal emitted from the fluorochrome on detection antibody b. This m−10–5 × 10−7 IU/mL range with a detection limit of 10−11 IU/mL [In addition to optical detection, bead-based detection based on the electrochemical properties of materials was also exploited and used in virus detection. Some enzymes catalyze the oxidation of substrate, in which electrons will be released into its environment after enzymatic reaction. The flow of electrons into a reaction buffer can be measured as a current. By correlating the magnitude of current with the concentration of antigen, we could thereby quantify the concentration of viruses with the magnitude of current. However, the measurement of current in nA–μA magnitude is difficult with distance between analyte and electrode. Although it is possible to adsorb antibodies on electrodes by coating a layer of polymer on an electrode to facilitate the passive adsorption of proteins, the polymer might interfere with the detection of weak currents generated by enzymes when the concentration of the target virus is low. To address this problem, Gehring et al. proposed capturing the target analyte with antibody-coated magnetic nanoparticles and localizing these magnetic nanoparticles on electrodes with a magnet to increase the sensitivity of current detection c. This m11 IU/mL . The resIn addition to ELIME, label-free electrochemical bead-based assay, e.g., immunomagnetic impedance metric sensor, was also developed. An antigen-antibody reaction will result in changes in electrochemical impedance, which allows for label-free detection of the target. In this method, first, streptavidin-coated magnetic microbeads are immobilized on the surface of gold electrode with a magnetic bar to ease the process of regeneration of sensing surface when necessary. Next, the biotinylated antibody was coated on magnetic microbeads followed by introduction of the sample. The concentration of target was correlated to changes in electron transfer resistance differences antibody .Other than proteins, nucleic acid can also be captured and analyzed with bead-based assay. Either peptide nucleic acid or a fragment of complementary DNA was immobilized on the surface of magnetic beads to trap target DNA from samples. Magnetic beads were used instead of direct immobilization of complementary DNA on probes or electrodes, as it is difficult for long fragments of DNA to hybridize directly short fragment of nucleic acid attached on the surface of electrodes. After incubation, magnetic beads were collected with magnets, and the non-complementary DNA was removed by washing. Then, intercalators were introduced, the beads were collected after reaction, and the electrochemical signal from the intercalator was measured. This method is known as genomagnetic electrochemical bioassay ,30. The To further reduce the limit of detection of bead-based method to amol/L order, the bio-barcode method has been proposed ,33. The In bio-barcode assay, first, the target will be captured with magnetic beads. Then, nanoparticles with detection probes and barcode are incubated with magnetic beads–target complexes. Magnetic beads–target–nanoparticles complexes are isolated with magnetic field, and the barcode will be dehybridized from nanoparticles. Either barcode DNA or nanoparticles will be detected d. The am3 virus particles/mL. To address this issue, our group has an External Force-Assisted Near-field Illumination (EFA-NI) biosensor [In addition to the detection of targets captured by particles, motion-based sensing has also attracted considerable attention due to its potential in real-time detection and high spatial resolution ,39. Howeiosensor ,41 that In and near the evanescent field from near-field illumination, the EFA-NI biosensor uses an external force (magnetic force) to move the target which has bound to antibody-modified magnetic particles. This movement of particles is used to distinguish signal from noise. While various studies have demonstrated the use of magnetic particles for target manipulation, EFA-NI is the first to combine the movement of magnetic particles and optical signal detection to reduce false positivity. Our approach has enhanced the contrast between signal and noise by detecting the “moving optical signal”, thus increasing the signal-to-noise ratio ,41. SincFor the electric field enhancement of near-field light, the surface plasmon resonance based on a thin metal film on the surface (a leaking electric field with a height of approximately 100–200 nm) is often used for immunosensors. However, in the detection area up to about 200 nm in height, the detection target easily leaves the detection area with the EFA-NI biosensor that does not capture the detection target on the surface. Therefore, to use a wider space as the detection area, we adopted a waveguide mode excitation mechanism using a multi-layered waveguide, which is capable of forming a highly enhanced electric field with a height of approximately micrometers.A mixture that included the sample, the antibody-modified magnetic particles, and the antibody-modified optical markers was introduced into the liquid cell formed on the sensor chip. The targeted virus was detected as a moving optical signal by applying a magnetic field from a sandwiched complex with the antibody-modified magnetic particles and the antibody-modified optical signal marker. Contaminants, which are noise sources, do not move in the same manner as magnetic particles when a magnetic field is applied, so optical signals originating from contaminants can be easily distinguished as noise.2–104 NoVLPs/mL  ,43. Norod method ) and cand method ,41. Due To this point, the discussions revolve around virus detection technologies based on bulk analysis in a single reactor with large volume (>10 μL). These methods are known as analogue assays, in which the concentration of target,  I (I∝C) a. When tTwo methods have been proposed to address the issue of limited sensitivity of detection in bulk system: (1) increase sensitivity of sensors. However, the sensitivity of sensors is constrained by a trade-off among the physical principles of sensors and its fabrication process which has come to its limitations. (2) The compartmentalization of the reaction system to enrich the target molecules in each compartment, thus increasing signal intensity and probability of detection. Theoretically, all compartments with target molecules can be detected as endpoint entity and are thus categorized as positive and negative compartments. Then, the number of both positive and negative compartments will be collected and statistically analyzed. Absolute quantitation of target molecule is defined as the product of average number of target molecules per compartment,  sensors b.Bioassay through compartmentalization was first introduced by Lwoff and Gutmann in their study of lysogeny of Bacillus megatherium in 1950 . Lwoff a6 copies/mL), and its property as a tool for relative quantitation has resulted in large interlaboratory variation  . TherefoCombining the methodology of limiting dilution and compartmentalization, as proposed by Lwoff and Gutmann, Vogelstein and Kinzler developed a method which could detect the presence of specific DNA fragment as either negatives or positives in 1999, which they named the method “digital PCR (dPCR)” . As the to ≈0.1% . As we ito ≈0.1% ,58.Today, there are generally two approaches in which compartmentalization was conducted: (1) chip digital PCR (cdPCR) based on microwell array, and (2) droplet digital PCR (ddPCR) based on microdroplets suspended in oil generated by microfluidic chips or vortex mixing.Both approaches have been commercialized. The Fluidigm BioMark system and Thermo Fisher Quantstudio 3D are commonly used systems for cdPCR; Bio-Rad QX100/200, the RainDance RainDrop system, and the Stilla Naica system are commonly used ddPCR systems a.Technically, cdPCR has larger compartment volume compared to ddPCR as the cost of fabrication increases exponentially with the reduction in size of the microwell. ddPCR also offers higher dynamic ranges (5–6 log) compared to cdPCR (2–5 log), and sensitivity for rare mutation detection. RainDance RainDrop dPCR system could detect 0.071 copies/μL , while tAlthough the method demonstrated by Vogelstein and Kinzler relied on limiting dilution, it is unnecessary for digital assays. To ensure a sufficient number of empty compartments for quantification described in Equation (1), the range of 001 to 6 . In otheA concern that remained for ddPCR compared to cdPCR is the volume uncertainty of droplets. Unlike microwell array, the volume of droplets is not constrained by the volume of container, and therefore there is higher volume variation in droplets (2–8%) compared to the microwell array (<1.0%). To address this issue, Pinheiro et al. have revised an equation (Equation (3)) which could counteractively correct the result of measurement with volume uncertainty .(3)u=K√With the recent development of vortex ddPCR , the cos2–106) compartment, statistic collection becomes a cumbersome task. Therefore, it is important to combine high-throughput analysis technology with digital assays for the practical application of digital assays. Before the advent of microscale analytic systems, various methods were developed to quantify colloidal particles aligned as single particles in fluid flow, based on photoelectric effect [When a portion of suspension is compartmentalized into a vast amount  and reovirus (60–80 nm) could be distinguished from background noise . By chanThe progress of flow virometry has been accelerated by the development of stable nucleic acid stain, which allows for the labelling and identification of DNA viruses . The invFurthermore, the invention of flow virometry has also changed the paradigm of virus’s characterization. It has always troubled researchers and health workers that viral stocks generally contain large fractions of defective viruses compared to infectious viruses. Analogue assays usually measured the sample as bulk, which does not provide useful information on the composition of proteins in viruses. With flow virometry, El Bilali et al. demonstrated that it is possible to sort Herpes simplex virus 1 by infectivity based on tegument proteins VP16 and VP22 .On the other hand, the development of microfluidic devices since the beginning of the 21st century has enabled customizable fluid manipulation in microelectromechanical systems. The successful generation of droplets in microfluidic devices  has allorescence , fluoresrescence , absorbarescence ,82, scatrescence ,84, Ramarescence , X-ray [rescence , and UV-rescence , which aWith the help of enrichment technology, e.g., bead-based enrichment ,89, enriProtein analysis is another important approach for virus identification and quantitation. Among various methods for protein quantitation, ELISA has emerged as the golden standard for protein quantitation. As analogue assays, the LOD of analogue assays of ELISA is pmol/L. This LOD does not meet the sensitivity required for the early detection of a virus . Therefore, a single-molecule detection method with high sensitivity is required to detect proteins at amol/L. In 1961, Rotman reported a method to encapsulate single β-galactosidase molecule into emulsion and successfully measured the activity of individual molecules . By obse3–105 of wells with femtoliter volumes) with fluorogenic substrate  has developed and reported the first digital ELISA (dELISA) system that employed targeted molecules capturing by immunomagnetic beads and enzyme labeling in 2010 . The schubstrate d. When tubstrate e. By usidELISA technology flourish around the Simoa series, and various progress has been obtained, e.g., Kim et al. increased the number of microwell in dELISA by 1 order of magnitude, which lowered the LOD of dELISA to 2 amol/L ; Leirs e6 wells; reducing the number of magnetic beads could also improve the sensitivity of dELISA as it increased the fraction of immunocomplex analyzed, e.g., reducing the number of magnetic beads by 100-fold could improve sensitivity by 189-fold [Further studies have been conducted to improve the LOD of dELISA to zmol/L. While increasing the number of microwells is effective to improve LOD as a larger volume of suspension can be sampled , the fie189-fold . Neverth189-fold .Balancing the trade-off of sensitivity and speed of detection is a challenging task in dELISA with the current design, which allows for only single bead per well. A breakthrough is needed to achieve high sensitivity, such as rapid dELISA.As mentioned above, dELISA for virus detection ,104,105 8 particles/mL) antibody-modified magnetic particles can quickly capture viruses in the sample. Next, the enzyme-labeled antibodies are bound to the virus and unbound enzyme-labeled antibodies are removed. By rapidly guiding the complexes into the well array using a magnetic field, the viruses sandwiched with the magnetic particles and the labeled antibodies are concentrated in a short time. Almost all the particle–virus complexes and magnetic particles can be accommodated in the well array. Furthermore, they are enclosed in the well array together with fluorogenic substrates that react with the enzyme label. Fluorophore was released, and fluorescent signals are detected only in the wells, including the viruses. Viruses in the sample can be detected and quantified by counting the wells that emit fluorescent signals.To address this issue, our group has proposed a lysis-free method that could simplify the complex and cumbersome sample preparation process, while achieving similar sensitivity to the PCR method, which we call a multiparticle-concentrated digital immunoassay (MCDIA) ,117. Fig50/mL in a reaction time of 3 min, for which the concentration is approximately the lowest virus concentration (titer) that can be detected in the saliva of SARS-CoV-2 patients [Theoretically, the MCDIA can achieve detection within a few to a few tens of minutes with a sensitivity equal to or greater than that of the PCR method. Both rapid virus-capturing and high sensitivity can be realized by introducing a multi-particle concentration method into the digital detection technology. We have successfully developed an influenza A virus (IAV) detection system  and a SApatients ,120,121.In future works, while maintaining sensitivity, we aim to shorten the entire detection time by automating and improving the efficiency of the procedure part and will achieve both the highest-level sensitivity and fastest-level sensitivity required for screening tests for asymptomatic people and for evaluating the risk of infection in a certain space. If a highly sensitive and reliable rapid on-site test is put into practical use, it is possible to control the spreading of viruses before it evolves into a pandemic by screening asymptomatic patients and reducing the risk of aerosol transmission. In the event of an infectious disease pandemic, we can also expect to avoid situations that cause huge social and economic losses, such as declaring a state of emergency or pre-emergency measures in the global pandemic of viral infection.Pore-based sensing is a promising candidate for the early detection of extremely small particles such as molecules, proteins, and viruses (50–200 nm). This method is known as “Coulter principle” or “resistive pulse sensing”. Pores usually consist of a nano- to micrometer hole within a substrate which forms a barrier between two electrolyte-filled reservoirs. A pair of electrodes are placed on each side of substrate, and voltage is applied to the pore and measures the ion current flowing through it . ParticlNanopores are widely used in biochemical and single molecule detection, and could be made of biomacromolecule or synthetic materials. For example, molecular measurements using nanopores, in which α-hemolysin with cyclodextrins inside is embedded in lipid membranes, was reported ,124, andparamecium bursaria chlorella virus 1 with a diameter of 175–190 nm [An important aspect of nanopore measurement is techniques for making pores. There are many ways to penetrate thin membranes for the fabrication of pores such as ion beam drilling, electron beam lithography, electrochemical etching, etc. ,128,129.5–190 nm ,132. Oth5–190 nm ,133,134,5–190 nm . This meAn advantage to using pores for particle measurement is that not only the size of the particles can be estimated, but also the waveform of electric current varies with the shape of nanoparticles. Therefore, the preparation of pores with precisely controlled geometry is also of great importance to obtain highly reproducible measurement. For example, precisely uniformed low-aspect ratio pore prepared by electron beam lithography and reactive ion etching can obtain the specific current waveform derived from the shape of measured particles ,137,138.The conventional technique to measure viruses does not depend on the amount of virus particles but on infection titer such as hemagglutination inhibition assay and plaque assay, as viruses cannot be detected directly. PCR is also a conventional assay by measuring the number of viral nucleic acid, which can measure viruses quantitatively at first glance. However, viral integrity must be maintained for infectivity, and the detection of viral nucleic acid does not necessarily indicate risk of infection. Although the infectivity of the counted virus particles cannot be measured, the virus can be quantified from a different perspective than PCR because particles that have the shape of a virus can be counted.A main challenge of pore-based sensing is that the detection opportunity depends on a stochastic factor, that is, it relies on the probability that a viral particle in the vicinity of a pore will pass through the pore. The frequency of passing particles increases with the density of particles. Therefore, this concentration range is the dynamic range of the pore sensor. Note that a passing efficacy is not only depends on a particle concentration but characteristics of particles. The main driving force for particles to pass through the pores is electrophoresis caused by the application of voltage. Accordingly, particles with higher or lower surface potential relative to their environment are more likely to pass through the pores, while particles with no charge rarely pass through the pores by electrophoresis. Electroosmotic flow also allows particles to pass through the pore , but the7 particles/mL viral suspension was applied, which is roughly viral concentration at the limit of detection by single pore-based sensing. Although this value requires more virus than the PCR detection limit of 100 copies/mL, it should be considered that the viral particles are measured as they are without any manipulation, such as gene extraction or gene amplification. The fact that PCR involves dozens of amplification operations also indicates that pore-based sensing is inherently capable of detecting very small amounts of virus. Apart from this, the upper limit should also be considered. According to this report, the limit of linearity between counting rate and concentration of virus is approximately 1.0 × 1010 particles/mL viruses [The problem of pore-based sensing can be improved by external forces. Pore sensors, in which a pore is provided in a stretchable membrane and the size of the pore is changed by stretching, are called tunable resistive pulse sensing and are commercially available under the product names qNano and Exoid ,141,142. viruses . This isPore-based sensing is a useful technique to measure the virus in the suspension quantitatively. Since the parameter of physical quantity of virus is essentially different from virus titer and number of nucleic acids, these different parameters should be matched at a viewpoint of infection risk. It is a challenging problem to quantify the risk of infection, and virus evaluation by pore-based sensing may be one tool to solve this.In the previous subsection, we discussed the property of virus counting quantitatively. However, an actual sample, including viruses, certainly has other impurities, which sometimes prevents a viral particle measurement. While pore-based sensing is an attractive method for evaluating each measured particle individually, the properties of the particles obtained are limited to physical parameters such as size and charge density. Therefore, pore-based sensing performance must be advanced to selectively measure biological particles such as viruses. Clogging of the pore, which is one of the most troubling problems for pore-based sensing, should also be solved, because the impurities contained in virus suspension could prevent the viruses from passing through the pore.Hydrophilic plasma treatment is commonly used to prevent the clogging of pores. However, facilities for plasma irradiation are required, and the effect is lost over time. Surface functionalization of a pore is one effective technique to improve the performance of pore-based sensing. For example, preventing unwanted adhesion is a required technique in the field of biomaterials and biosensing, which is mostly solved by surface functionalization. It is known that polyethylene glycol can give the non-fouling character on a surface by preventing impurity approaches because of excluded volume effects derived from a hydrophilic chain . ZwitterThere are some methods for the specific detection of target particles. One technique is to use a specially shaped pore for sensing. As mentioned previously, a low aspect ratio pore can obtain a detail of particle shape based on current waveform. For example, several kinds of viruses, such as vesicular stomatitis virus, tobacco mosaic virus , bafinivAnother approach is surface functionalization to capture the target. Conventional biosensors such as surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) sensors use the ligands on the surface of the sensor for specific detection. Since ligands are provided on the surface of the pore or near pore and measuring the passing time of particles significantly reveals how the target is captured through molecular recognition , pore-baApart from this, the conjugation approach has also been suggested to identify the virus. For example, when the virus and antibody interact specifically, the volume of the virus increases because the virus is covered by the antibody due to complex formation. Therefore, the presence of the virus can be determined by changing the size of the viral particle . It has As a recent trend, artificial intelligence (AI) technologies for pore-based sensing have been investigated ,160,161.In this chapter, pore-based sensing technologies for virus detection have been discussed. This method is still an emerging technology and needs further development to become a general sensing technology. However, the method of detecting and analyzing viruses in their particle form is unprecedented and is expected to lead to rapid diagnosis. Since pore-based sensing only measures current values, it does not require optical devices, etc., and can be miniaturized. It is anticipated that pore-based sensing will be widely used as a particulate measurement method in the future.The COVID-19 pandemic has caused tremendous damage to society and the economy around the world. While we hope that the tragedy can be prevented, unfortunately, the development of human society with dense traffic and the nature of cross-species virus transmission through random mutation  has dictIn this article, we provided an overview of various virus detection technologies . As showOn the other hand, physical approaches focus on the measurement of native signals generated from viruses, e.g., light scattering and electrical impedance. The label-free nature of physical approaches has made them an attractive approach in virus detection, as reagents to label viruses, e.g., antibodies are not basically necessary. However, it should be noted that viruses are not completely isomorphic and have a wide range of physical properties. Therefore, physical approaches exhibit less specificity compared to chemical approaches. For the reason above, the advanced techniques such as molecular recognitions and machine learning technology, mentioned in the relevant section, might help to improve the specificity of virus detection based on physical approaches by extracting and clustering the signal pattern specific to certain types of viruses.In recent years, the introduction of digital measurement technology for virus detection has contributed to increasing the effective SNR of signals and reducing the LODs. In addition, the practical application of an extreme imaging device that realizes single photon counting has been announced ,164. TheIn this way, various technological innovations have been boosted in recent years. By combining the innovations of these technologies, there is no doubt that the ideal virus testing method, which has both high enough detection sensitivity equivalent to that of PCR and quick testing time such as within 1 min, would be established in the near future.


Plants are vulnerable to the challenges of unstable environments and pathogen infections due to their immobility. Among various stress conditions, viral infection is a major threat that causes significant crop loss. In response to viral infection, plants undergo complex molecular and physiological changes, which trigger defense and morphogenic pathways. Transcription factors (TFs), and their interactions with cofactors and cis-regulatory genomic elements, are essential for plant defense mechanisms. The transcriptional regulation by TFs is crucial in establishing plant defense and associated activities during viral infections. Therefore, identifying and characterizing the critical genes involved in the responses of plants against virus stress is essential for the development of transgenic plants that exhibit enhanced tolerance or resistance. This article reviews the current understanding of the transcriptional control of plant defenses, with a special focus on NAC, MYB, WRKY, bZIP, and AP2/ERF TFs. The review provides an update on the latest advances in understanding how plant TFs regulate defense genes expression during viral infection. Crop growth and productivity are negatively impacted by a variety of abiotic and biotic stresses in the environment, including high temperatures, low-temperature, salinity, drought, pathogen infection, insect attack, and weeds ,2. TheseBiotic stress, caused by living organisms such as viruses, bacteria, fungi, arachnids, and weeds, directly deprives plants of nutrients, resulting in reduced plant vigor and, in severe cases, plant death . HoweverViruses, which are submicroscopic infectious agents, can infect all living organisms, including plants. They use a variety of strategies to replicate and spread within the plant’s cellular environment ,8. ViralPlants have evolved defense mechanisms that involve the recognition of pathogens and the production of a specific response in the plant’s cells. Cell wall receptors are responsible for this recognition, activating internal signaling components that initiate transcriptional and physiological changes within host cells . Plants According to the Baltimore virus classification system , virusesArabidopsis thaliana activating factor 1/2), and CUC2 (cup-shaped cotyledon 2), which share the same DNA-binding domain [http://planttfdb.gao-lab.org, accessed on 31 January 2022) [The NAC gene family is named after three TFs: NAM , ATAF1-2  ,43. Arabry 2022) . Many NAry 2022) . Recent ry 2022) ,46,47. Hry 2022) .rice stripe virus (RSV), rice tungro spherical virus (RTSV), rice black-streaked dwarf fijivirus (RBSDV), rice grassy stunt tenuivirus (RGSV), rice ragged stunt oryzavirus (RRSV), and rice transitory yellowing virus (RTYV). Microarray analysis revealed that nine SNAC genes were upregulated in response to RBSDV, RGSV, and RRSV, while the most upregulated SNAC genes were observed in response to RGSV, and the least expressed ones were observed in response to RTYV [In a genome-wide analysis of rice NAC TFs, researchers identified a total of 151 non-redundant NAC genes . Within  to RTYV . turnip crinkle virus (TCV) capsid protein (CP) has been found to interact with the TCV-interacting protein (TIP), an Arabidopsis NAC transcriptional activator [wheat dwarf virus (WDV) are necessary for interaction with the N-terminal domain of GRAB proteins. These findings suggest that GRAB proteins may play a role in inhibiting the viral replication cycle. However, the underlying mechanism(s) of the inhibition remains to be investigated. It is possible that the interaction between host GRABs and viral RepA may trigger the transcriptional activation of other defense-related genes, or that the viral RepA protein might be sequestered by the host GRABs and viral replication might thus be interfered.Although interactions between viral proteins and NAC TFs are rare, there have been some notable cases reported. For example, the ctivator . Loss ofctivator . In wheactivator . These pmungbean yellow mosaic India virus (MYMIV) [tomato yellow leaf curl virus (TYLCV) infection [NAC083 is an essential component of geminivirus rolling circle replication in Arabidopsis, as it interacts with the replication initiator protein (Rep) of  (MYMIV) . Howevernfection , with ditobacco mosaic virus (TMV) 126/183 kDa replicase protein [The Arabidopsis NAC TF, ATAF2, interacts with the helicase domain of the  protein . ATAF2 irice dwarf virus Multiplication 1 (RIM1) gene in rice encodes a novel NAC-domain protein that belongs to the NTL subfamily and plays a role in the multiplication of rice dwarf virus (RDV) [rim1-1 mutant, with an inserted retrotransposon Tos17 in the RIM1 gene intron, did not exhibit disease symptoms when infected with RDV and had a reduced accumulation of RDV CP. No similar effects were observed for RTYV and RSV infections. Therefore, it was suggested that RIM1 negatively regulates rice resistance to RDV by acting as a host factor that is necessary for the multiplication of the virus [tomato leaf curl virus (TLCV) infection upregulates the expression of SlNAC1, a member of the ATAF subfamily of NAC genes in tomato [SlNAC1 induction is mediated by geminivirus replication enhancer (REn) proteins, indicating its potential involvement in stress responses. Overexpression of SlNAC1 resulted in a significant increase in viral DNA accumulation, suggesting its crucial role in the process of REn-mediated enhancement of TLCV replication. In regal lily , LrNAC35, a member of the ONAC022 subgroup in NAC family, was identified during cucumber mosaic virus (CMV) and TMV infection [lily mottle virus (LMoV) and lily symptomless virus (LSV) infections also triggered a significant increase in LrNAC35 transcripts in resistant and susceptible Lilium species. Overexpression of LrNAC35 in petunia enhanced resistance to CMV and TMV infections and promoted the accumulation of lignin in cell walls by regulating the Ph4CL lignin biosynthetic gene. NbNAC089, an endoplasmic reticulum (ER) membrane localized protein, with a transmembrane domain at the C-terminus, was discovered in Nicotiana benthamiana [N. benthamiana. NbNAC089 is suggested to be a negative regulator of unfolded protein response (UPR) and a positive regulator of programmed cell death (PCD), playing a crucial role in the defense of N. benthamiana to viral infection.The us (RDV) . The rimhe virus . The tomn tomato . SlNAC1 nfection . The lilthamiana . Upon TMCapsicum annuum NAC1 (CaNAC1) is a nuclear protein that contains the plant-specific NAC domain motif and has been identified as a TF involved in plant defense responses resulting in HR-cell death under pepper mild mottle virus (PMMV) invasion [cucumber green mottle mosaic virus (CGMMV), 15 differentially expressed NAC TFs were observed, with 13 upregulated and 2 downregulated, possibly playing a role in the adaptation to CGMMV-induced stress in leaf tissues [maize Iranian mosaic virus (MIMV) and suspected to modulate maize defense responses to MIMV infection [sonchus yellow net virus (SYNV) in N. benthamiana [Passiflora edulis) during the infection of CMV [groundnut bud necrosis virus (GBNV) and tomato leaf curl New Delhi virus (ToLCNDV) infection [sugarcane mosaic virus (SCMV)-resistant (B-48) genotype of sugarcane compared to those of the susceptible (Badila) genotype [N. benthamiana [bamboo mosaic virus (BaMV) infection, inducing the activity level of NbAGO5, and leading to antiviral defense  [COLORED1 (C1), was found two decades ago, and its expression of MYB domain protein is necessary for anthocyanin synthesis in the maize aleurone [MYB TFs represent one of the largest gene families in plants and play a critical role in various biological processes, including plant growth and development, cell morphology, primary and secondary metabolic reactions, physiological activity metabolism, and responses to abiotic and biotic stresses ,69,70. Tus (AMV) , and subaleurone . The MYBaleurone .The DBD of MYB consists of about 52 amino acid residues that fold into three α-helices, which are named R1, R2, and R3 due to their resemblance to the c-Myb protein. Based on their DBD structure, MYB TFs in plants are divided into four subfamilies: 1R-MYB, R2R3-MYB (2R-MYB), R1R2R3-MYB (3R-MYB), and 4R-MYB  Figure A. The C-N. tabacum), the induction of NtMYB1 has been observed, leading to HR and development of SAR in the resistant tobacco cultivar due to the rise of endogenous SA [tomato leaf curl virus (ToLCV), lower levels of MYB33 TF have been observed, which is due to the two-fold higher accumulation of miR159a levels [cauliflower mosaic virus (CaMV) gene VI (P6) expression in Arabidopsis, upregulation of AtMYB96 was reported, and a subgroup of PR genes was also upregulated due to AtMYB96 overexpression [Citrullus lanatus), 18 differentially expressed MYB TFs have been discovered during CGMMV invasion [During TMV infection in tobacco  and potato virus X (PVX) in Arabidopsis and N. benthamiana, respectively. TEV is a popular model system for studying positive-sense RNA viruses in plants, and changes in gene expression profiles have been observed upon TEV infection [The pression . The infnfection . tobacco necrosis virus [OsMYB4 plays a critical role in the interplay of stress signaling pathways, as it activates many components, but its activity depends on the host’s genetic makeup [Saccharum officinarum) revealed the identification of a total of 202 R2R3-MYB genes  [Overexpression of OsMYB4 in rice induced the expression of multiple genes in Arabidopsis and tomato involved in resistance to is virus . Functioc makeup . In cucuc makeup . The expalleles) . After Salleles) . Thinopyrum intermedium MYB TF (TiMYB2R1) in wheat demonstrated its involvement in disease resistance by gradually increasing defense-correlated genes within the transgenic plant [N. benthamiana, the MYB4-like TF, coupled with the ethylene pathway, contributes to viral resistance. During TMV infection, NbMYB4L transcription is upregulated, and silencing the gene increases susceptibility to TMV replication [grapevine leaf-roll-associated virus-3 (GLRaV-3) infection in Vitis vinifera [The elevated expression of ic plant . In Arabic plant . In N. blication . The keyvinifera .Phalaenopsis aphrodite subsp. formosana was studied [N. benthamiana and P. aphrodite subsp. formosana. The upregulation of hormonally related TFs, such as NbMYB30, was suggested to control CymMV infection by increasing levels of JA and its marker genes, LOX2 and AOS2  is a highly conserved DNA binding domain found in WRKY protein families. It consists of a WRKY-like motif, WRKYGQK, at the N-terminus, followed by diverse downstream zinc finger motifs . WRKY TFWRKY TFs have been shown to play an important role in responding to biotic stress and influencing plant defense mechanisms, as shown in isochorismate synthase (ICS), which is required for the synthesis of SA, through the interaction with W-box in the ICS promoter. WRKY80 interacted with MAPK5, which can activate several TFs involved in resistance against pathogens. These findings suggest that these WRKY TFs are involved in regulating defense mechanisms against viral pathogens. During South African cassava mosaic virus (SACMV) infection, several WRKY TFs expressed differentially in cassava (Manihot esculenta Crantz) [Mulberry mosaic dwarf-associated virus (MMDaV)-encoded RepA was found to trigger HR through the upregulation of NbWRKY1 transcript [N. benthamiana, suggesting that responses of plant to RepA might be used to increase plant immunity to geminivirus attacks in crops [While most studies have concentrated on the role of WRKY in response to bacterial or fungal attack, an increasing amount of research indicates that WRKY can also react to viral infection via a conventional signaling pathway, making it another crucial determinant in the accumulation of viruses. A comparative transcriptome analysis of TYLCV-resistant and -susceptible tomato cultivars, Zheza-301 and Jinpeng-1, respectively, revealed that six group III WRKY TFs responded to TYLCV infection: WRKY41, WRKY42, WRKY53, WRKY54, WRKY80, and WRKY81 . In infe Crantz) . WRKY11 anscript . Althougin crops .Soybean mosaic virus (SMV) [N. tabacum, the WRKY TF TIZZ was identified as a TMV-induced, HR-responsive gene that contains a C2H2 ZF motif and a single WD. TIZZ could regulate downstream defense genes in an SA-independent signaling pathway, as exogenous applications of SA, MeJA, or ET did not activate its expression [The significance of WRKY TFs in the regulation of plant defense is well established. WRKYs are involved in both TIR (Toll-interleukin-like receptor) and non-TIR resistance genes, which encode proteins containing NBS-LRR domains . In tobaus (SMV) . The silpression .N. benthamiana, the overexpression of NbWRKY40 resulted in a decrease in tomato mosaic virus (ToMV) accumulation, activation of the W-box promoter of ICS1 that facilitates SA synthesis, and upregulation of SA marker genes, PR1 and PR2 [Research has shown that WRKY TFs can have a negative effect on viral infections. In  and PR2 . This is and PR2 ,118. Sim and PR2 .Poncirus trifoliate) and pummelo (Citrus grandis) plants infected with Citrus tristeza virus (CTV), the expression of different WRKYs was altered [C. annuum, the expression CaWRKY-α was activated by several signal molecules, including SA, MeJA, and ethylene, and was hypothesized to regulate pathogen-induced defense mechanisms and participate in phytohormone crosstalk [C. annuum, has been found to be stimulated by TMV and other phytohormones involved in plant defense. The suppression of CaWRKYd resulted in a decrease in HR cell death induced by TMV-P0 and an increase in virion accumulation. Additionally, CaWRKYd silencing led to a notable reduction in the expression of several genes related to HR and PR, such as CaBPR1, CaDEF1, CaAlaAT1, CaHIN1, and CaHIN2. The results indicate that CaWRKYd plays a critical role in resistance against TMV-P0 infection [Allium cepa) infected with onion yellow dwarf virus (OYDV), AcWRKY32 was found to participate in transcriptomic reprogramming during dormant release and serve as a host factor adopted by OYDV for symptom development [WRKY TFs have also been reported to exhibit antiviral activity in economically important crops. In citrus , and PVX [WRKY TFs from different plant species, including MtWRKY from  tabacum ,124. Ove tabacum . In toba tabacum , which i tabacum . The sil tabacum . Similar tabacum , while A tabacum . Further and PVX . The funLike other plant TFs, the basic leucine zipper (bZIP) belongs to a large family that modulates numerous physiological activities to help plants overcome biotic or abiotic stresses. The family was named based on the evolutionarily conserved bZIP domain, which is characterized by an N-terminal nuclear localization signal, an N-X7-R/K motif, and heptad repeats of leucines and other hydrophobic amino acids that form an amphipathic helix A. The coThe role of bZIP in pathogen defense mechanisms has been increasingly studied. Octopine synthase (OCS)-like elements can be recognized by the ocs-element-binding factor (OBF) group of bZIP TF, and their association with EREBPs suggests their involvement in regulating ethylene-induced PR genes ,140,141.Previous research revealed that AtNPR1 could interact with TGA2, TGA3, TGA5, and TGA6. Specifically, TGA2 and TGA3 were shown to recognize the SA-responsive element of the PR1 promoter, which includes the TGACG core sequence, a TGA-binding site. Mutations in npr1 negatively impact SA signaling as well as the interaction between NPR1 and TGAs, as reported in ,154. By Capsicum chinense. PPI1 transcripts were found to be upregulated during an incompatible interaction between pepper and pathogens, but not in response to abiotic compounds. The induction of PPI1 significantly inhibited viral or bacterial pathogen infection in capsicum leaves. The findings suggest that PPI1 responds specifically to biotic stress and participates in the plant defense-signaling pathway [rice tungro bacilliform virus (RTBV), a persistent pathogen that mainly infects rice fields in South Asia, has been identified. RF2a and RF2b from rice, which also share high sequence identity with the bZIP family, were shown to act as homo- and hetero-dimers in a distinct interaction with Box II. Their predominant localization in vascular tissue may be involved in the regulation of phloem-specific expression of the RTBV promoter. Further, silencing of RF2 in transgenic rice produces similar symptoms to those caused by RTBV. The results suggest that RTBV inhibits RF2a and RF2b expression in the phloem, while the presence of RF2b may disturb the normal physiological state and affect the symptom manifestation of rice tungro disease [Despite the limited number of investigations, some bZIPs have been identified as playing a role in virus resistance. For example, pepper-PMMV interaction 1 (PPI1), which encodes a protein with a DNA-binding domain that shares a high amino acid sequence similarity with the bZIP family, was differentially expressed in PMMV-infected  pathway . Additio disease ,159,160.plantago asiatica mosaic virus (PlAMV) accumulation in Arabidopsis, whereas both IRE1a/IRE1b and bZIP60 play critical roles in resistance against TuMV accumulation. Moreover, the triple gene block 3 (TGB3) from PlAMV, and 6K2 protein from TuMV and Potato virus Y (PVY), were recently reported to induce the expression of bZIP17, bZIP28, and bZIP60 [Positive-stranded RNA viruses establish a viral replication complex (VRC) from a membranous organelle, such as the endoplasmic reticulum (ER), to facilitate their replication and cell-to-cell movement. The sensors on the ER membrane can detect the increasing need for viral protein synthesis, triggering the unfolded protein response (UPR) to manage cytosol perturbations and activate cellular defenses ,162. Sevd bZIP60 ,163,164,Homeodomain-leucine zipper protein 1 (HAT1) of Arabidopsis, belonging to the hdZIP family of TFs, has been shown to be critical in plant defense against viruses. Overexpression and knockout analyses of HAT1, during infection with CMV, demonstrated that the knockout mutants exhibited greater tolerance to infection than did the overexpressing lines. Additionally, antioxidant systems and gene transcription involved in defense were downregulated in HAT1 overexpression lines but upregulated in mutant lines. Therefore, these findings suggest that HAT1 serves as a negative regulator of plant defenses against CMV . A recenApetala2/ethylene-responsive factor (AP2/ERF) is a critical TF in plants that plays a versatile role in helping plants survive harsh environmental conditions such as heat, drought, and salinity ,168. BasN. tabacum, ERF5 was found to be induced by wounds and infection from several pathogens, including TMV. The overexpression of ERF5 was reported to provide specific resistance against the replication and movement of TMV, through an N gene-independent mechanism [N. benthamiana, ERF5 (NbERF5) was characterized as a factor whose overexpression reduced viral accumulation. Protein–DNA interaction analysis revealed that NtERF5 interacted with the GCC box cis-elements, suggesting potential regulation of PR genes. Plants overexpressing ERF5 under the constitutive promoter, CaMV 35S, exhibited a higher level of resistance, a reduced HR response, and impaired systemic virus spread [Previous studies have demonstrated that AP2/ERF TFs play a role in pathogen defense, in response to biotic stress. In echanism . A comprechanism  revealedechanism . In N. bs spread .GLRaV-2 is a common virus associated with grapevine leafroll disease. The protein p24, which is an RNA-silencing suppressor (RSS) encoded by GLRaV-2, promotes the accumulation of GLRaV-2 by interacting with the B3 DNA-binding domain of grapevine related to abscisic acid insensitive3/viviparous1 (VvRAV1) transcription factor. Salicylic acid inducible VvRAV1 positively regulates the grapevine PR1 (VvPR1) gene by directly binding its promoter, suggesting that VvRAV1 may be involved in the regulation of host basal defense responses . HoweverThe important roles of AP2/ERF TFs in plants are widely recognized. With an increasing number of plant genomes being accessible in public databases, genome-based studies on AP2/ERF are becoming more prevalent. However, recent studies on AP2/ERF mainly focus on verifying gene function. The continued development of molecular biology techniques, including genome editing, will facilitate more convenient and in-depth research on AP2/ERF family factors.This review summarizes the current advances in the understanding of the roles of major plant TFs in the defense against biotic, particularly viral, stresses, and highlights the potential applications of these resistance/tolerance mechanisms. One of the most significant challenges facing humanity is guaranteeing food security. To meet the growing demand for food and sustain the expanding population, stress-tolerant crop varieties are critical. Plant biotic stress resistance not only enhances food security but also reduces the use of pesticides during crop production, leading to reduced production costs. TFs play a critical role at the transcriptional level, activating or inhibiting genes in response to various stresses. Among thousands of TFs identified in plants, the most important ones are distinct signal transduction pathways mediated by different TF families, such as NAC, WRKY, MYB, bZIP, AP2/ERF, etc. Crops have been exposed to various biotic and abiotic stress conditions over their evolutionary history, and they have developed physiological and molecular mechanisms, especially TFs, to cope with these challenges. Functional analysis of existing TFs has revealed that they can serve as targets for gene expression to control phytopathogenic viruses through using advanced disease management approaches. TFs can play a role in regulating various genes associated with defensive mechanisms, which have been studied in several model plants. Genetic and molecular techniques have greatly aided in the functional characterization of TFs. While detailed characterization tests have been carried out on Arabidopsis, tobacco, or rice, only a few members of the TFs have been functionally characterized in other species. In addition, there are still significant knowledge gaps between our understanding of these TFs and the actual applications. For example, the mechanisms for the crosstalks among the different pathways, regulated by various TFs under stress conditions, or the first elements of the surveillance system that sense the specific patterns or elicitors of the invading viruses in plants. As specific viruses seemed to only activate/repress the expression of specific TFs, there appear to be certain recognition systems in plants, like those in the immune systems of animals. These knowledge gaps require further studies to lead the way to practical applications. Nonetheless, the current investigations have provided excellent insights into viral stress signaling and response networks, including TFs.Various TF-regulated signal transduction pathways have been examined with the goal of alleviating viral stresses through control and modification. The use of TFs enables the plant to modify its surroundings, protecting it from a range of insects and pathogens. Although viral infections are challenging, TF regulation can be an important tool in protecting plants from viral stress. Tight control and fine-tuning of TFs during plant stress responses, involving interactions between TF proteins, downstream targets, and upstream regulators, contribute to the formation of complex signaling webs that play a role in stress modulation. For decades, breeders have attempted to improve the quality of crops through different approaches. The current development in the modulation of TFs may present novel tools and strategies for dealing with viral stresses and biotic stresses in general. Over time, as more and more elements are being discovered, the applications of TF expression manipulation in crop improvement may finally become a reality.


Rhabdoviridae comprises over 140 different viral species, the most well-known being the Rabies lyssavirus, the principle cause of rabies. There are, however, a large number of other viruses within the Rhabdoviridae family that are less well studied but may also represent human pathogens. Modern techniques such as metagenomic next-generation sequencing have facilitated the discovery of new and previously neglected viruses. This review describes the evidence for human infection by rhabdoviruses and highlights the gaps in our knowledge of the contribution of this large viral family to human disease.The viral family Rhabdoviridae is a large viral family, with members infecting a diverse range of hosts including, vertebrate species, arthropods, and plants. The predominant human pathogen within the family is Rabies lyssavirus, the main cause of human rabies. While rabies is itself a neglected disease, there are other, less well studied, rhabdoviruses known to cause human infection. The increasing application of next-generation sequencing technology to clinical samples has led to the detection of several novel or rarely detected rhabdoviruses associated with febrile illness. Many of these viruses have been detected in low- and middle-income countries where the extent of human infection and the burden of disease remain largely unquantified. This review describes the rhabdoviruses other than Rabies lyssavirus that have been associated with human infection. The discovery of the Bas Congo virus and Ekpoma virus is discussed, as is the re-emergence of species such as Le Dantec virus, which has recently been detected in Africa 40 years after its initial isolation. Chandipura virus and the lyssaviruses that are known to cause human rabies are also described. Given their association with human disease, the viruses described in this review should be prioritised for further study. Rhabdoviridae constitutes a group of viruses infecting a diverse range of hosts, including vertebrates , invertebrates, plants, fungi, and protozoans. Rhabdoviridae sits within the order Mononegavirales, with over 230 viruses currently assigned to the family [Alpharhabdovirinae, Betarhabdovirinae, and Gammarhabdovirinae. In addition, there are several more divergent viral genera currently unassigned to a subfamily. Viruses in Alpharhabdovirinae infect both vertebrates and invertebrates; those in Betarhabdovirinae infect plants and are transmitted by arthropod vectors. Viruses within Gammarhabdovirinae infect fish. Several pathogens of medical and veterinary importance exist within Alpharhabdovirinae, the foremost being Rabies lyssavirus (RABV) [The family e family . The famRhabdoviruses display a characteristic bullet-shaped morphology when visualised by electron microscopy (EM). Virions range between 100 and 430 nm in length and 45 and 100 nm in diameter [The prototypic rhabdovirus vesicular stomatitis virus (VSV) and the predominant pathogen RABV have been the focus of studies on rhabdovirus structural biology ,5. ThereThe basic genomic organisation is conserved across the family and consists of a single strand of negative sense RNA coding for the five structural proteins: from the 3′ to 5′ direction nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and the large protein (L) . The genThe replication cycle is largely consigned to the cytoplasm. The viral polymerase binds to the N-RNA complex at its 3′ end and transcribes the viral genes in decreasing abundance from the 3′ to the 5′ end of the genome by variable dissociation of the polymerase at each intergenic transcription stop signal ,21. The Rhabdoviridae has undergone significant expansion in the last decade. Viral taxonomy is increasingly determined by sequence information alone, with biological characteristics not required for phylogenetic assignment [Alpharhabdovirinae from the genera Lyssavirus, Tibrovirus, Vesiculovirus, and Ledantevirus are notable for their association with human disease  and European bat 2 lyssavirus (EBLV-2). EBLV-1 is hosted by the European serotine bat Eptesicus serotinus, whilst EBLV-2 has been detected in two bat species: n tissue . The pren tissue . The twon tissue ,32.Crocidura species and concurrently linked to two fatal human cases [Lophuromys [In Africa, Duvenhage virus (DUVV) and Mokola virus (MOKV) have been implicated in human rabies. MOKV is particularly noteworthy, as vaccines against RABV do not confer protection against MOKV . MOKV waan cases ,35. Subsphuromys . Owing tphuromys . DUVV waphuromys . The virphuromys . He did phuromys .Pteropus and the insectivorous Saccolaimus flaviventris [bat, Saccolaimus flaviventris [Pteropus species) in Queensland in 1998 [The Australian bat lyssavirus (ABLV) is the only endemic lyssavirus in Australia. There have been three fatal human cases since its discovery in 1996 as part of a wildlife surveillance programme for the detection of the Hendra virus. Further surveillance of Australian wildlife suggests it predominantly circulates in fruit bats of the genus iventris . There iiventris . The firiventris . She hadiventris . She sub in 1998 . The lat in 1998 . There i in 1998 .Murina leucogaster bats. One human case has been recorded of a 20-year-old female who was bitten on the lip by a bat in the Russian Far East [Irkut virus (IRKV) is a bat-associated lyssavirus that has been isolated in China and Russia from Far East . IRKV maFar East ,49. It hLedantevirus [Le Dantec virus (LEDV) is the type species of the genus ntevirus ,50. It wntevirus .Sequence analysis shows LEDV to contain the five canonical rhabdovirus proteins as well as an additional open reading frame coding for an accessory gene, designated U1, between the G and L proteins, a property shared by the closely related Kern canyon virus (KCV), Vaprio virus (VAPV), and KEUV ,52. A fuEptesicus isabellinus, the meridional serotine bat in Spain [E. isabellinus has a range extending from southern Spain into North Africa. It is a largely sedentary species, suggesting LEDV may be established in southern Europe. KEUR, the closest relative of LEDV, was originally isolated from several rodent species in Senegal, including gerbils and Mastomys natalensis.To date, the reservoir and vector hosts of LEDV remain unknown. A viral RNA sequence similar to LEDV was isolated from an oropharyngeal swab from in Spain . These bLedantevirus. KCV was isolated from Californian bats in the 1950s. Several more distantly related viruses within the genus were also found in bats, including Mount Elgon bat virus and Fikirini virus, both isolated in East Africa [Further evidence of a link between LEDV and bats is suggested by the host associations of other viruses within t Africa ,57. Somet Africa . The Kant Africa . The autLedantevirus. Kumasi virus (KURV) was isolated from the spleen of a fruit bat, Eidolon helvum, captured in an urban area of Ghana [Eretmapodites leucopous mosquitoes and from a human case in Cameroon in 1965 [There is evidence of human infection by two other viruses within of Ghana . PCR scrof Ghana . The ant in 1965 . UnfortuLyssavirus was related to a cluster of three cases of hemorrhagic fever in a forest region of the Democratic Republic of the Congo (DRC) in 2009 [Tibrovirus. The virus was named Bas-Congo virus (BASV) after the province where it was isolated. Neutralising antibodies to the BASV glycoprotein were demonstrated in both the surviving patient and a close contact who had delivered nursing care during his acute illness. Further serological testing of the local population and PCR screening of stored sera from other outbreaks of undiagnosed hemorrhagic-fever illness in the DRC were negative. The virus failed to grow when acute serum was inoculated into various cell lines, an issue the authors attributed to inadequate cold chain infrastructure in the DRC [The most concerning episode of human disease associated with a rhabdovirus outside the genus  in 2009 . The ini the DRC .BASV is most closely related to the Tibrogargan virus (TIBV), isolated from cattle and mosquitoes in Australia ,64, and Vesiculovirus alongside the well-characterised VSV and demonstrates the canonical genome arrangement conserved across the Rhabdoviridae [Phlebotomus papatasi sand flies and Aedes aegypti [Chandipura virus (CHPV) was first isolated in 1965 from two patients with a mild illness characterised by fever and myalgia in the Indian city of Nagpur . It sitsoviridae . Transov aegypti ,69,70,71 aegypti ,73.CHPV received little attention until 2003, when it was associated with several outbreaks of childhood encephalitis in India ,75. In tLyssavirus, are capable of infecting humans (There is growing evidence that rhabdoviruses, in addition to those from the genus g humans . While tWhilst some rhabdoviruses appear to commonly cause human infection, others, such as LEDV, are considered to only emerge in rare spillover events from a presumed natural reservoir. Whilst there is strong evidence that vesiculoviruses such as CHPV are arthropod-vectored, the ecology of the ledanteviruses is more obscure. The detection of various ledanteviruses in humans, bats, rodents, and arthropods may indicate a broad host range within the genus. It is also possible that bat species may be the natural reservoir, with humans and other animals experiencing infection in spillover events vectored by arthropods such as bat flies or ticks. Transmission may also occur via human exposure to bat excreta, especially where bats reside in highly urbanised areas, as in the case of KURV. If this is the case, more human cases may be predicted as the population expands into new ecosystems. Further studies are required to fully understand the ecological relationships among this diverse group of viruses.


AGL gene, and may develop various types of pulmonary hypertension (PH). Here, we report a case of 24-year-old man with GSD-IIIb with two novel null variants in AGL (c.2308 + 2T>C and c.3045_3048dupTACC). He developed multi-drug-resistant pulmonary veno-occlusive disease (PVOD) and was registered as a candidate for lung transplantation. No pathogenic variants were detected in previously known causative genes for pulmonary hypertension and the underlying mechanism of coincidence of two rare diseases was unknown. We discuss the association of the loss of glycogen-debranching enzyme with incident PVOD.Glycogen storage disease type III (GSD-III) is an autosomal recessive metabolic disorder caused by mutations in the  AGL gene, which encodes a glycogen debranching enzyme, is known to cause GSD-III. Glycogen debranching enzymes degrade phosphorylase-limited dextrin, a form of glycogen broken down by phosphorylase α, to glucose 1-phosphate  is a rare autosomal recessive hepatic disease in which glycogen abnormally accumulates in the liver. This inherited disorder is rare, with an estimated prevalence of 1:100,000 . The muthosphate . Enzymathosphate . To datehosphate , while ohosphate  (Table 1Pulmonary veno-occlusive disease (PVOD) is commonly one of the causes of group 1 PH and accounts for 5%–25% of all PH cases, with a prevalence of 1-2 per million people . The proAGL, the causative gene for GSD-III, presumably present in the compound heterozygous state. We present the clinical course of this case and discuss the possibility that the newly discovered AGL mutations provoke PVOD. GSD-IIIb is considered to have good prognosis but can be complicated by severe PH requiring careful follow-up.There are very few cases of PVOD associated with GSD, with only one report of PVOD in GSD type I and no report of PVOD in GSD-III . In this2 86% and pO2 58.8 mmHg on the arterial blood gas test, and the intensity of the pulmonic component of the second heart sound was enhanced. Blood tests revealed mildly elevated levels of transaminases (aspartate aminotransferase (AST), 82 IU/L; alanine aminotransferase (ALT), 96 IU/L) and brain natriuretic peptide . Although mild elevations in antinuclear and anti–Sjögren syndrome-related antigen A (anti-SSA) antibody levels were observed, autoimmune diseases were carefully excluded through physical examination by rheumatologists. Chest radiography revealed dilatation of both pulmonary arteries (1) of 3.66 L, diffusing capacity for carbon monoxide (DLCO) of 8.62 mL/min/mmHg, and %DLCO of 32.5%. Based on these results, the patient was clinically diagnosed with PVOD and treated with riociguat (7.5 mg), macitentan (10 mg), and home-based oxygen therapy.A 24-year-old male was admitted to our hospital for close examination of shortness of breath on exertion. He was clinically diagnosed with GSD-III due to hepatomegaly and elevated liver enzyme levels at 6 months old; however, his subsequent growth and development were asymptomatic. Since he has no muscular symptoms nor cardiac symptoms at that time, his GSD was considered to be type Ⅲb. The patient had no family history of patients with GSD or PH. At 22 years of age, he became aware of the shortness of breath on exertion, which gradually worsened. Hypoxemia was observed at rest with SpOarteries , and elearteries . Echocararteries . Right harteries . Pulmonaarteries . RespiraAGL, a known causative gene for GSD-III , at the locus of for PVOD , or in aTo the best of our knowledge, this is the first case report of GSD-IIIb complicated by PH due to PVOD. There have been several reports describing cases of GSD complicated by PH, but most of them were reports of GSD type I or II, and only two cases of GSD-III accompanied by PH have been reported  Pizzo, . In the AGL gene, c.2308 + 2T>C and c.3045_3048dupTACC. No pathological variants were found in the known genes associated with the development of PH. This result gives rise to the possibility that these AGL gene mutations are common cause of the pathogenesis of GSD-IIIb and PVOD. c.2308 + 2T>C variant disrupts the conserved GT donor splice site of intron 17 with a change of Shapiro–Senapathy (S&S) splice scores from 74.25 to 57.09 and is predicted to cause exon 17 skipping (151 bp) and c.3045_3048dupTACC is an out-of-frame variant in exon 23 (p.THr1017TyrfsX54), and thus these two mRNAs are presumably degradated by non-sense-mediated mRNA decay, leading to loss of all enzymatic activity of AGL and contributing to the onset and progression of GSD- IIIb and PVOD.In this case, whole-exome sequencing identified two previously unreported null variants in the AGL gene is expressed in these cell types  were not previously reported in literature or databases we used in the analysis.Genomic DNA was isolated from whole blood using the Qiagen QiaAmp Mini Prep Kit (Qiagen United States) according to manufacturer’s standard protocol. The whole-exome library was prepared using Sure Select All Exon V6 kit  according to manufacturer’s standard protocol. Sequencing was performed on Illumina Novaseq6000 platform  with 150 bp paired-end reads. Reads were trimmed using Trimmomatic v0.33 and aligned to reference genome hg38 by UCSC  along with BWA v0.7.17. PCR duplicates were marked using Picard tools v2.18.2-SNAPSHOT. Variations were called using GATK tool (v4.0.5.1) and filtered variants were annotated using SnpEff (vSnpEff 4.3t 2017-11-24) and filtered with dbSNP(v151) and SNPs from the 1,000 genome project. In-house program and SnpEff are used to annotate with additional databases, including Exome Sequencing Progject (ESP6500SI_V2), ClinVar, dbNSFP (v3.5c), Exome Aggregation Consortium (ExAC), Genome Aggregation Database (gnomAD), 1,000 Genomes, and ACMG information. Mean depth of target regions was 122.4%x and 95.8% of the target regions had over 20x read depth. We filtered variants which are rare (MAF< 0.01 in the databases or not reported) and protein-altering. Two


A 10-μL volume of virus in tripartite soil  was combined with 50 μL of disinfectant in suspension for 0.5, 1, 5, or 10 min at 20–25 °C. Neutralized test mixtures were quantified by GFP expression to determine log10 reduction. Remaining material was passaged on Vero cells to confirm absence of residual infectious virus. Input virus titers of 6.6–8.0 log10 per assay were completely inactivated by each disinfectant within 1–5 min contact time. The rapid and substantial inactivation of LASV suggests the utility of these microbicides for mitigating spread of infectious virus during Lassa fever outbreaks.The World Health Organization’s R&D Blueprint list of priority diseases for 2022 includes Lassa fever, signifying the need for research and development in emergency contexts. This disease is caused by the arenavirus Lassa virus (LASV). Being an enveloped virus, LASV should be susceptible to a variety of microbicidal actives, although empirical data to support this expectation are needed. We evaluated the virucidal efficacy of sodium hypochlorite, ethanol, a formulated dual quaternary ammonium compound, an accelerated hydrogen peroxide formulation, and a  One of the listed diseases is Lassa fever, which is caused by Lassa virus (LASV), an enveloped virus from the Arenaviridae family (genus Mammarenavirus). This rapidly expanding RNA virus family has been established for a number of genetically related viruses, including several that, like LASV, frequently cause lethal zoonoses. These include Chapare virus, Lujo virus, Machupo virus, Junín virus, Guanarito virus, and Sabiá virus2.The World Health Organization (WHO)’s R&D Blueprint list of priority diseases3. The few manuscripts that have been published on the inactivation of LASV by microbicides  addressed only the efficacy in rendering laboratory specimens safe for handling within diagnostic and histology laboratories. In addition, review of this literature3 has indicated that the secondary literature sources for efficacy of more commonly used surface-hygiene microbicides for inactivating arenaviruses, including LASV, have not supported efficacy claims with primary literature citations.One of the concerning knowledge gaps for the diseases highlighted in the WHO R&D Blueprint list of priority diseases, including Lassa fever, is the lack of empirical data for the effectiveness of virucides against the infectious agents that might be used for applications, including air sanitization, skin sanitization, liquid inactivation, and inanimate surface hygiene4 to express green fluorescent protein (GFP). Using standardized methodologies described in ASTM E-1052-205, we have evaluated the virucidal efficacy of commonly used microbicidal actives (sodium hypochlorite and ethanol), and formulations containing a dual quaternary ammonium compound (QAC), accelerated hydrogen peroxide (AHP), and p-chloro-m-xylenol (PCMX) against LASV in suspension in the presence of a tripartite soil load6. Organic soil loads are used as the challenge matrix to model virus inactivation by microbicides in relevant matrices, such as human sputum or blood. Use of hard water as diluent for specific actives was included in the study design, because it is a known antagonist to microbicidal activity and is commonly available in the field7.Resolving this particular knowledge gap for LASV and many of the other viruses causing priority list diseases has been limited, in part, due to the need for manipulating such viruses within maximum containment (termed in Northern America biosafety level 4 [BSL-4]) laboratories. In the present study, we were able to leverage the BSL-4 laboratory at Public Health Agency of Canada, Winnipeg, MB, Canada, and a LASV engineeredChlorocebus aethiops kidney epithelial (Vero) cells and passaging any cultures found to be negative for GFP at least twice. This was done to evaluate the virucidal efficacy test for the possibility of any residual virus being present at levels lower than the limit of detection of the 50% tissue culture infectious dose (TCID50) titration assay performed in Vero cells per the ASTM standard.In addition to the methodologies in ASTM E-1052-20, our study was made more stringent by decreasing the volume of the microbicides used during testing and by ruling out the presence of residual infectious LASV in the post-disinfection/post-neutralization samples through use of a safety test (performed in addition to the standard quantification of titer reduction). This safety test involved inoculation of 650 µL of undiluted neutralized test sample onto six-well plates containing Chlorocebus aethiops) kidney epithelial (Vero) cells  were maintained at 37 °C with 5% carbon dioxide (CO2) in minimum essential medium  supplemented with 5% heat-inactivated fetal calf serum  and 10 units per mL penicillin/streptomycin . Lassa virus (Josiah strain) expressing green fluorescent protein (LASV-GFP) was amplified and used, as previously described4, for all assays. All culture manipulations involving LASV-GFP were performed in a BSL-4 laboratory at the Canadian Science Centre for Human and Animal Health, Winnipeg, MB, Canada.Grivet (g) for 10 min. The resulting supernatants were pooled and layered onto 20% w/v sucrose cushions prepared in Tris-NaCl-EDTA buffer prepared in house. The pooled supernatants were subjected to centrifugation , and the viral pellets obtained were resuspended in MEM containing 2% FCS and 10 units per mL pen/strep to create virus culture medium (VCM). The virus pool obtained was aliquoted into small portions that were frozen at − 70 °C. The stock virus titer was found to be > 9.2 log10 per mL, as determined by TCID50 assay, using the method described by Reed and Muench8.A characterized stock of LASV-GFP was prepared by exposing ten T75 flasks of Vero cells at ≈80% confluency at a multiplicity of infection of 0.01 viral particles per cell. At 4 d post-infection, essentially all cells within the confluent cell monolayers were observed to express GFP. The flasks were then placed in a freezer at − 70 °C. The frozen flasks were thawed on the following day and the conditioned media was removed and clarified by low-speed centrifugation . The virus/tripartite soil load mixture (10 µL) was added to 50 µL of prepared microbicide or to 50 µL of VCM to create the positive virus control. A similar preparation was used to challenge the efficacy of the test microbicides. The virus/tripartite soil mixture was incubated with the test microbicide at room temperature for contact times of 30 s, 1 min, 5 min, and 10 min. At the end of each time point, the microbicides were neutralized, either by adding 940 µL of VCM , a mechanical filtration procedure via Amicon YM100 columns  was used during virucidal efficacy testing Fig. . After t50) were calculated according to the Reed-Muench method8.In either case , a 350-µL portion of neutralized test solution was assayed for residual infectious virus titer using a ten-fold dilution scheme in VCM, with 50 µL of each resulting dilution being added to 96-well plates of Vero cells (n = 5 replicate wells per dilution). The inoculated cell monolayers were scored 5 d post-infection for GFP, and virus titers  were added to Vero cells in wells of a six-well plate containing 4 mL of VCM. In addition, an inoculum containing < 5 TCID50 units of LASV-GFP was used in each six-well plate to serve as a positive control. All wells were scored 5 d post-infection for presence or absence of the fluorescence associated with GFP (indicative of LASV-GFP-infected cells). The confirmed ability of this assay to detect very low concentrations (< 5 TCID50 units) of the positive control virus indicates its sensitivity and provides confidence that a negative result in the separate TCID50 assay truly reflects the absence of residual infectious LASV-GFP in the neutralized test samples.In addition to the 96-well plate TCID50 titers for positive virus controls and neutralized microbicide test conditions were determined using the method of Reed and Muench8. The log10 reduction values achieved by the microbicides at given contact times were calculated by subtracting the post-disinfection log10 TCID50 values (titers) from the log10 TCID50 values obtained for the corresponding positive virus control. Statistical comparison of the mean (n = 5 replicates) viral titers obtained in the neutralization effectiveness studies  are provided in the Supplemental Materials. It was found that 0.5% sodium hypochlorite and 67% ethanol could be adequately neutralized using VCM alone. VCM plus mechanical neutralization using Amicon columns was required for dual QAC, AHP, and PCMX.10 TCID50 per mL (4.6 × 106 TCID50 per mL) was recovered for the positive control (no disinfectant)  contact time, representing a 6.2 log10 reduction. After 1, 5, and 10 min of contact with 0.5% sodium hypochlorite, complete inactivation (≥ 6.7 log10) of LASV-GFP was observed  sodium hypochlorite (NaOCl) Table  for inacnt) Fig. . The pos 0.5-min 0-s contaFurther evidence of the complete inactivation of LASV-GFP following the 1-, 5-, and 10-min contact times was obtained in the plate safety test Table . In this10 TCID50 per mL (4.4 × 106 TCID50 per mL) was recovered for the positive control (no disinfectant)  contact time and 0.42 log10 TCID50 per mL for the 1-min contact time. These correspond to reductions of 6.2 log10. After 5 and 10 min of contact with 67% ethanol, complete inactivation (≥ 6.6 log10) of LASV-GFP was observed  Table  for inacFurther evidence of the complete inactivation of LASV-GFP following the 5- and 10-min contact times was obtained in the plate safety test. One technical replicate from one assay of the 0.5 min contact time and three technical replicates from two assays of the 1-min contact time displayed GFP in this assay. No evidence of residual infectious virus was obtained from the technical replicates for the 5- and 10-min contact times Table .Table 3P10 TCID50 per mL was recovered for the positive control (no disinfectant)  for all contact times   contact time, representing a 7.1 log10 reduction. After 1, 5, and 10 min of contact with AHP, complete inactivation (≥ 7.3 log10) of LASV-GFP was observed  Fig. . The posved Fig. .Figure 6Further evidence of the complete inactivation of LASV-GFP following the 1-, 5-, and 10-min contact times for AHP was obtained in the plate safety test. Two technical replicates from one assay of the 0.5-min (30-s) contact time displayed GFP in this assay. No evidence of residual infectious virus was obtained from the technical replicates for the 1-, 5-, 10-min contact times Table .Table 4P10 TCID50 per mL, 7.3 log10 TCID50 per mL, and 7.3 log10 TCID50 per mL were recovered for the positive control (no disinfectant) conditions for the assay of the 0.12, 0.06, and 0.04% in-test concentrations of PCMX, respectively  of PCMX in a commercial formulation  contact time, representing log10 reductions of 6.9, 5.0, and 4.1 log10, respectively. Following a 1-min contact time, the 0.12% PCMX condition had no viable virus remaining post-exposure/neutralization, whereas for the 0.06 and 0.04% PCMX conditions 1.0 and 2.4 log10 TCID50 per mL, respectively, were recovered representing log10 reductions of 6.3 and 4.9 log10, respectively. Following a 5-min contact time, the post-exposure/neutralization titers for the 0.06 and 0.04% PCMX conditions were 0.97 and 0.94 log10 TCID50 per mL, respectively, representing log10 reductions of 6.3 and 6.4 log10, respectively. Complete inactivation (≥ 7.8 log10) of LASV-GFP was afforded by 0.12% PCMX at contact times of 1, 5, and 10 min. For the 0.06 and 0.04% PCMX concentrations, complete inactivation (≥ 7.3 log10) of LASV-GFP was observed at the 10-min contact time  are the primary reservoirs of the causal virus, LASV. Transmission to humans occurs primarily through contact with urine or feces of infected mastomys, but human-to-human transmission via direct contact with blood or bodily fluids may also occur, especially in hospital settings9. These considerations suggest that inanimate surface hygiene and liquid inactivation methods for LASV might limit dissemination of the virus to humans.Per WHO3 revealed that the limited published data pertained to the inactivation of laboratory specimens intended for diagnostic or histology applications14. The paucity of virucidal data of microbicides against LASV is also emphasized in a recent review of inactivation of emerging viruses in aqueous phase15. Our study was intended to resolve this knowledge gap, supplying efficacy information for commonly used microbicidal actives and formulations applicable to inactivation of LASV in liquid suspension.Partly because maximum containment is needed for conduct of inactivation studies on LASV, there is little published information on the efficacy of microbicides for inactivation of this arenavirus. A recent review of the available primary data on efficacy of microbicides against LASV16, the EPA has made note of the hierarchy of pathogen susceptibility to microbicides21 in recognizing that efficacy against one enveloped virus implies efficacy against other enveloped viruses. The EPA policy provides a “process that can be used to identify effective disinfectant products for use against emerging viral pathogens and to permit registrants to make limited claims of their product’s efficacy against such pathogens.”16. The guidance outlines “a voluntary two stage process, involving product label amendments and modified terms of registration and applies only to emerging viruses”16.The United States Environmental Protection Agency (US EPA) recognizes that microbicidal efficacy data may not be available for newly emerging viruses, especially those requiring BSL-4 laboratories for handling the viruses safely. The EPA, therefore, enacted a policy in 2016 enabling efficacy claims against emerging viruses to be made without having provided registration data specifically for those viruses. In its Guidance to Registrants3 derived from testing enveloped viruses , lipid-disrupting agents—including ethanol, quaternary ammonium compounds , and phenolics (such as PCMX)—were expected to be effective against other enveloped viruses, such as LASV. Certain microbicidal actives and formulations were considered mechanistically to be protein-denaturing agents  or genome-degrading agents . In fact, our study found that these agents caused rapid  and highly effective (≥ 6 log10) inactivation of LASV-GFP when tested in suspension in a tripartite soil load with hard water as diluent to simulate field use of dilutable products, including PCMX and sodium hypochlorite  and stainless steel surfaces for the Josiah isolate, and approximately 1.2 log10 per day on these surfaces for the Sauerwald isolate. These data indicate that surface contamination with infectious LASV could persist for days, depending on the initial titer of the deposited virus. Decay rates for the two isolates in deionized water (0.1 to 0.15 log10 per day) and wastewater (0.6 to 0.8 log10 per day) were observed25. Inactivation of the two LASV isolates by sodium hypochlorite  was concentration dependent, with the Sauerwald isolate displaying greater susceptibility to inactivation, with no reasons for this difference being offered in the paper25. Greater than 4 log10 inactivation of LASV occurred within 5 min contact time with 1 to 10 mg/L [ppm] sodium hypochlorite for each isolate25. These sodium hypochlorite concentrations are quite low, compared to the concentration used in our study (0.5% [5000 ppm]), and to concentrations proposed previously for use against LASV (0.5–1%)26.In a recently published preprint10, even in the presence of a tripartite organic load27. In future studies, we plan to explore the more stringent virucidal efficacy of a similar set of microbicidal actives and formulations against LASV in carrier-inactivation studies of the virus dried on a hard surface and performed in accordance with ASTM-E2197-117.In conclusion, we have provided empirical evidence of the virucidal efficacy of commonly employed microbicidal actives (ethanol and sodium hypochlorite) and formulations of microbicidal actives  for LASV. Each of these, at the appropriate concentration and contact time, was capable of reducing the titer of infectious virus by > 6 logSupplementary Information.