Flavivirus infections, such as those caused by dengue and West Nile viruses, emerge as new challenges for the global healthcare sector. It has been found that these two viruses encode ion channels collectively termed viroporins. Therefore, drug molecules that block such ion-channel activity can serve as potential antiviral agents and may play a primary role in therapeutic purposes. We screened 2839 FDA-approved drugs and compounds in advanced experimental phases using three bacteria-based channel assays to identify such ion channel blockers. We primarily followed a negative genetic screen in which the channel is harmful to the bacteria due to excessive membrane permeabilization that can be relieved by a blocker. Subsequently, we cross-checked the outcome with a positive genetic screen and a pH-dependent assay. The following drugs exhibited potential blocker activities: plerixafor, streptomycin, tranexamic acid, CI-1040, glecaprevir, kasugamycin, and mesna were effective against dengue virus DP1. In contrast, idasanutlin, benzbromarone, 5-azacytidine, and plerixafor were effective against West Nile Virus MgM. These drugs can serve as future antiviral therapeutic agents following subsequent in vitro and in vivo efficacy studies. Aedes mosquitos and exacts an appreciable toll with ca. 40,000 annual deaths stemming from 390 million infections and half a million hospitalizations [The genus Flaviviruses belongs to the Flaviviridae family of enveloped positive-strand RNA viruses. It includes several important arboviral pathogens that represent a specific health burden, such as the dengue virus, West Nile virus, Zika virus, yellow fever virus, and tick-borne encephalitis, to name a few. Dengue fever, as an example, is transmitted by izations . Despiteizations .West Nile fever, transmitted by Culex mosquitos, is another cause of concern. Identified in Uganda in 1937, it is now spread globally . ConsequPrevention options for flavivirus infection focus on transmission containment. At the same time, vaccines are available against some members of the genus, such as yellow fever, Japanese encephalitis, tick-borne encephalitis, and dengue fever. However, the last vaccine is only recommended for individuals who have previously contracted dengue fever since it may increase the probability of a severe outcome for those who have never contracted the disease . FinallyThe non-segmented flavivirus genome is 10–11 kbp in length and codes for a single large protein proteolytically cleaved to approximately a dozen structural and non-structural proteins ,7. AmongOne such example is the anti-flu aminoadamantane drugs  that taractivity . Widespractivity , emphasiactivity .Considering the paucity of antiviral agents against flaviviruses, we set forth to identify blockers of viroporins from two flaviviruses: dengue virus and West Nile virus. We employed three bacteria-based assays to characterize the protein’s channel activity and then screened a drug repurposing library against each target. Our results have yielded four blockers against each channel that may represent a starting point for an antiviral drug discovery program.Three bacteria-based channel assays were employed to investigate the viroporin channel activity and blockers thereof. In all assays, the MBP  fusion purification system  was used, where dengue virus DP1 and West Nile virus MgM were expressed as a chimera by fusion to the carboxy-terminal of the maltose-binding protein. Previous results have shown that utilizing this construct results in functional expression in bacteria ,18,19,20600 reached 0.2. From the culture, 50 μL were added to 96-well flat-bottomed plates, pre-treated with 50 μL of required staff. Induction was achieved by employing different concentrations of isopropyl-β-d-1-thiogalactopyranoside. A multi-plate incubator  was used to incubate the plates for 16 h at 37 °C at a constant shaking rate (700 rpm). Bacterial growth was monitored by measuring O.D.600 every 15 min. Duplicates or triplicates were conducted for every measurement.Bacteria cultures (DH10B) were grown overnight and finally diluted 500-fold and again set to grow until their O.D.The positive assay employed the same protocol as the negative assay, but a was used . Additio600 of 0.6–0.8. Protein expression was induced by isopropyl-β-d-1-thiogalactopyranoside at different concentrations, as noted. After one hour of induction, the cells were diluted to an O.D.600 of 0.2 and pelleted at 3500 g for 10 min. Subsequently, the cells were resuspended in McILvaine Buffer [2HPO4 and 0.9% NaCl adjusted to pH 7.6 with 0.1 M citric acid. Then, 200 μL of cell suspension were added to a 96-well plate , each well containing 30 μL of McILvaine Buffer. The plate included three wells with McILvaine buffer and three with culture without induction as control. The fluorescent measurements were carried out at an ambient temperature in a microplate reader  with two pairs of bandpass filters: 520 nm emission filter and 390 and 466 nm excitation filters. At the starting point, 70 μL of 300 mM citric acid was added to the bacterial culture, and a fluorescent read-out was taken for 30 s for each wavelength. Finally, proton concentration was calculated from the ratio of two wavelengths [The acidity assay is based on bacteria expressing a chromosomal copy of a pH-sensitive GFP . Overnige Buffer , which celengths .d-1-thiogalactopyranoside was used to induce DP1 and MgM expression. Bacteria without isopropyl-β-d-1-thiogalactopyranoside  were used as a positive control, while bacteria that received only DMSO served as the negative control. Drugs that increased bacterial growth to a certain threshold were selected for further duplicate analyses. Subsequently, drugs that passed duplicate analysis in the negative assays were examined by positive assay, where 5 μM and 10 μM isopropyl-β-d-1-thiogalactopyranoside were used to induce DP1 and MgM expression, respectively. Compounds that passed the negative and positive assays were subjected to dose-response analyses and fluorescence-based assays. Finally, the negative assay tested the additive effects of drugs by employing equal concentrations of all possible combinations.A chemical library of 2839 compounds was purchased from MedChem Express . All chemicals were screened at 100 μM using the negative screening at first, whereby 100 μM isopropyl-β-a is the normalized activity and K2) are listed in the figure.The inhibitory constant calculation was conducted for the negative assay results using Prism 9 . The data were normalized by setting uninduced and induced bacteria growth rates to 1 and 0, respectively. Subsequently, the data are non-linearly fit to the following equation: d-1-thiogalactopyranoside was purchased from Biochemika-Fluka . All other chemicals were purchased from Sigma-Aldrich laboratories .The isopropyl-β-Lysogeny broth (LB) was used for most cases; only LBK was used for the positive assay, where NaCl was replaced with KCL at 10 gm/lt. All media contained ampicillin at 100 μg/mL.To evaluate the activity of both viroporins, we used bacteria-based assays in which the channel’s functionality changes the bacteria’s phenotype. The advantages of the assays are that they are amenable to high-throughput screening, and the ease of genetic manipulations in bacteria enables a rapid transition from one sequence/variant to another. Finally, these assays have been tested on numerous viroporins from various viruses ,18,19,20The first assay used to characterize both viroporins is one in which the protein is expressed at increasing levels in bacteria. As seen in other channels ,18,19,20As seen in The second bacteria-based assay we employed was reciprocal in nature to the negative assay listed above. otassium . HoweverResults shown in bacteria , as seenThe final that we employed measured tive GFP . This asuctivity . The resFollowing the confirmation of channel activity of both viral proteins, we sought to identify drugs that could block their function. We employed the negative assay described above and screened a 2839 repurposed drug library. We subsequently followed by cross-checking every hit using the positive and pH-based assays. In the negative assay, a successful hit is expected to increase bacterial growth by abrogating the detrimental impact the viroporin has. In contrast, in the positive assay, successful blockers would impair bacterial growth since they would counteract the beneficial effect of the viral channel.The reciprocal nature of both assays decreases the possibility of erroneous results. Specifically, compounds were classified as hits only if they increased bacterial growth in the negative assay while concomitantly reducing growth in the positive assay.The negative assay screen results are shown in Finally, the hits identified in the negative assay were examined in the acidity assay: Out of the seven blockers active against dengue virus DP1, six were confirmed in the acidity assay e. Once mThe final step in the screening process involved dose–response analyses of the compounds that passed the negative and positive assays for each viroporin: plerixafor, streptomycin, tranexamic acid, and CI-1040 against dengue virus DP1, and idasanutlin, benzbro-marone, 5-azacytidine, and plerixafor against West Nile virus MgM. The results are depicted in We next sought to determine the potential additivity or synergism between the different hits. To that end, we examined all possible combinations of active drugs against dengue virus DP1 and West Nile virus MgM channels in equal concentration. This study aimed to identify inhibitors of proteins belonging to two flaviviruses as a means of curbing infectivity. We chose viroporins unique to each virus as drug targets since ion channels as a family are highly amenable to inhibition . SubsequWe started by establishing a baseline of protein activity in the three aforementioned assays. Both proteins were detrimental to bacterial growth when expressed at increasing levels , as expeK+ media , as experansport . Finallybacteria , which iIn order to identify blockers, we screened a library of 2839 repurposed compounds, which includes FDA-approved drugs and those in advanced investigational stages. Drug repurposing has been proven a good option for new drug development, including antiviral research . Many reOur strategy employed positive and negative assays since their reciprocal behavior minimizes false positives. Hence, only compounds that score positively in both assays are considered by us a viable hit. Lastly, the acidity assay was used as a further independent check.Starting with the negative assay, we identified seven blockers against dengue virus DP1 and four against West Nile virus MgM. Subsequently, we analyzed the hits from the negative assay in the positive and acidity-based assays: All compounds identified by the negative assay against West Nile virus MgM were confirmed by the other two assays. In contrast, out of the seven hits against dengue virus DP1 identified by the negative assay, only four were confirmed by the positive assay, and the acidity assay confirmed six.Interestingly, the most efficacious blocker, plerixafor, inhibited both channels , perhapsThe differences between the efficacy of the drugs in the different assays may result from several factors: (i) The assays monitor similar albeit non-identical characteristics: prevention of membrane permeabilization versus blockage of Upon testing the additive effects of drugs in both channels using the negative assay , we obseIn conclusion, our findings provide a list of blockers that inhibit dengue virus DP1 and West Nile virus MgM. Further studies employing appropriate bio-safety facilities are required to examine if the said compounds and their combinations exhibit antiviral activity in vitro and in vivo. Results of an identical approach used on SARS-CoV-2 have proven fruitful , indicat


Dermatobia hominis is the most common fly species responsible for this parasitic infection. Genital piercing is an ornamental practice used in certain social circles. At placement, it transverses the skin surface and, as such, may be related to complications.  Myiasis is caused by larval infestation that usually occurs in exposed wounds.  We report a case of a 31-year-old man with a history of wound infection secondary to genital piercing who was exposed to an environment with flies, leading to myiasis. Mechanical removal and systemic antiparasitic drugs are possible treatments for myiasis. However, prevention that includes wound cleaning and dressing is the best way to avoid this disease.  Genital piercing can lead to potential complications and myiasis may occur when skin lesions are not properly treated. Although adornment with genital piercings has been increasing over recent years, the clinical implication of this practice is still under debate.Dermatobia hominis is the most common species of fly that parasitizes humans. The larvae are more frequently found in infected areas that present necrotic tissue.Myiasis is a parasitic infestation in body tissues caused by flies’ larvae. It occurs in healthy tissues (primary myiasis) or wounded tissues (secondary myiasis). The treatment for myiasis may range from local extraction of larvae to systemic or topical use of antiparasitic drugs, depending on the severity of the infestation. We sought to report a case of secondary myiasis in a patient who developed wound infection after genital piercing. To the best of our knowledge, this is the first case reporting this medical condition .A 31-year-old man came to the emergency room (ER) complaining about genital ulcers and diffuse erythema and edema in the pubic and genital area. The patient had a history of piercing placement at the base of the penis one month previously. He reported that a wound infection at the piercing site developed seven days after the procedure. At that time, the patient extracted the piercing at home and did not take any medication to treat the infection. He started then to go to sleep without clothes because of pain at the site of infection. He also reported that he kept the windows of his bedroom opened to be able to tolerate the bad smell. Two weeks later, the patient noticed an erythematous area at the pubis and genitalia that itched. He also saw some black spots and small ulcers at the previous piercing site. He described episodes of acute stabbing pain, in these areas.During physical examination, his vital signs  were within normal limits. Examination of his genitals showed that there was a large area of erythematous plaque in the pubic region with desquamation and pustules. Four necrotic ulcers were found; the largest was at the base of the penis with a diameter of 4 cm, where the urogenital piercing was previously inserted . Inside The patient was taken to the operating room for surgical debridement and extraction of the myiasis under regional anesthesia. Given the large number of larvae, systemic ivermectin was prescribed after the procedure, along with antibiotics and local dressings. The patient was discharged on day seven with use of topical antibiotics and corticoids to treat and avoid new infection and decrease inflammatory reaction .Genital myiasis is a rare medical condition caused by infestation with fly larvae. It is related to poor hygiene conditions and is more prevalent in rural zones. Although uncommon, its association with genital wounds, such as those caused by genital piercing, needs to be taken into account nowadays.,,In addition to genital myiasis, genital piercing can also lead to other types of complications such as bleeding, skin irritation, urinary and sexual problems.,,Some studies have demonstrated that genital myiasis can mimic inflammatory or ulcerous lesions.Although cases of penile myiasis had previously been published, the correlation with genital skin lesions caused by piercing had never been reported before. Genital piercing is a form of cultural expression that can lead to several complications. Furthermore, some cases of sexually transmitted disease, like human immuno-deficiency virus infection and hepatitis, have been associated with this practice.There are several ways to treat myiasis. Regarding genital myiasis, mechanical removal of larvae is the most frequent method.The purpose of this case report was to draw attention to possible medical issues relating to genital piercing, which is a form of cultural expression. Firstly, genital piercing can lead to potential complications. A recent case series reported that almost 20% of subjects who underwent piercing in the genitalia experienced complications.In conclusion, among subjects who undergo genital piercing, myiasis infestation may potentially be associated with wound infection secondary to the procedure. Doctors need to be aware of this complication so as to be able to provide the best treatment for this condition and give medical counseling about the potential problems of genital piercing.


By virtue of their lack of motility, viruses rely entirely on their own temperature(Brownian motion) to position themselves properly for cell attachment. Spiked viruses useone or more spikes  to attach. The coronavirus uses adjacent peplomerpairs. These peplomers, identically charged, repel one another over the surface of theirconvex capsids to form beautiful polyhedra. We identify the edges of these polyhedra withthe most important peplomer hydrodynamic interactions. These convex capsids may or may notbe spherical, and their peplomer population declines with infection time. These peplomersare short, equidimensional, and bulbous with triangular bulbs. In this short paper, weexplore the interactions between nearby peplomer bulbs. By interactions, we mean thehydrodynamic interferences between the velocity profiles caused by the drag of thesuspending fluid when the virus rotates. We find that these peplomer hydrodynamicinteractions raise rotational diffusivity of the virus, and thus affect its ability toinfect. I.peplomers) to attach  to position themselves properly for cell attachment.nearby, we mean beads whose centers terminate a common edge of thepolyhedral solution to the Thomson problem  dimensionless andmassless rods represent the rigidly fixed distances between adjacent or nearby beads. Inthis concise paper, we consider only the hydrodynamic interferences between nearby beads. Byinteractions, we mean the hydrodynamic interferences between the velocityprofiles caused by the drag of the suspending fluid when the virus rotates. We focusspecifically on the average number of peplomers on a coronavirus particle immediately afterinfection,In this short paper, we explore the interactions between nearby peplomer bulbs. Byminus the imaginary parts of the coronavirus contribution to the complexviscosity, we get [Eqs. (23) and (24) of Ref. For general rigid bead-rod theory with hydrodynamic interaction, for the real andFrom the fitted values of We find that for General rigid bead-rod theory distinguishes itself from theories for a suspension ofspheres, about which general rigid bead-rod theory is silent. General rigid bead-rod theory,after all, arrives at the complex viscosity through the orientation distribution function insmall-amplitude oscillatory shear flow.II.other, we mean peplomer hydrodynamic interactions other than thosebetween ends of each polyhedral edge.In this paper, we employ the recent method of Secs. II of Refs.  blue in  with fiv blue in  with sixFrom The III.minus the imaginary part of the complex viscosity ofthe coronavirus suspension increases with hydrodynamic interaction. Otherwise put,hydrodynamic interaction makes the coronavirus suspension more non-Newtonian in this sensetoo. However, from minus the imaginary part of the complex viscosity increases only slightlywith frequency (witness the ordinate gradation magnitude). By non-Newtonian, we thus meaneither the rise of minus the imaginary part or the fall of the real part ofthe complex viscosity.From IV.minus the imaginary part ofthe complex viscosity of the coronavirus, suspension increases with hydrodynamic interaction(In this short paper, we explore the interactions between nearby peplomer bulbs Sec. . By inteeraction . Otherwieraction. OtherwiIn this work, we focus entirely on We have restricted this work to spherical capsids, but we know coronavirus to bepleomorphic,We have further restricted this work to the lowest energy peplomer arrangement,


Virus infestation can seriously harm the host plant’s growth and development. Turnip yellows virus (TuYV) infestation of host plants can cause symptoms, such as yellowing and curling of leaves and root chlorosis. However, the regulatory mechanisms by which TuYV affects host growth and development are unclear. Hence, it is essential to mine small RNA (sRNA) and explore the regulation of sRNAs on plant hosts for disease control. In this study, we analyzed high-throughput data before and after TuYV infestation in Arabidopsis using combined genetics, statistics, and machine learning to identify 108 specifically expressed and critical functional sRNAs after TuYV infection. First, comparing the expression levels of sRNAs before and after infestation, 508 specific sRNAs were significantly up-regulated in Arabidopsis after infestation. In addition, the results show that AI models, including SVM, RF, XGBoost, and CNN using two-dimensional convolution, have robust classification features at the sequence level, with a prediction accuracy of about 96.8%. A comparison of specific sRNAs with genome sequences revealed that 247 matched precisely with the TuYV genome sequence but not with the Arabidopsis genome, suggesting that TuYV viruses may be their source. The 247 sRNAs predicted target genes and enrichment analysis, which identified 206 Arabidopsis genes involved in nine biological processes and three KEGG pathways associated with plant growth and viral stress tolerance, corresponding to 108 sRNAs. These findings provide a reference for studying sRNA-mediated interactions in pathogen infection and are essential for establishing a vital resource of regulation network for the virus infecting plants and deepening the understanding of TuYV virus infection patterns. However, further validation of these sRNAs is needed to gain a new understanding. Arabidopsis thaliana serves as a key model organism in the plant kingdom. Turnip yellows virus, formerly known as Beet western yellows virus (BWYV) [s (BWYV) , affectss (BWYV) ,3. It iss (BWYV) ,5. Recens (BWYV) ,7.A sophisticated innate immune system, similar to the non-viral pathogenic system and broadly separated into the pathogen-associated molecular pattern (PAMP)-triggered immunity and effect-triggered immunity protects plants from viral infections . FurtherArabidopsis thaliana through machine learning technology to verify its sequence specificity.Machine learning is vital for understanding plant virus pathogenesis and host-virus interactions. Machine learning allows accurate and rapid analysis of high-throughput data from virus-infected plants to identify gene regulatory networks (GRNs) and novel host factors involved in host-virus interactions . Few stuCurrent studies on the regulation of TuYV infestation have focused on the molecular level. The interactions with the host are still not fully understood, e.g., Marion Clavel et al. revealed the complexity of antiviral RNA silencing of phloem-restricted polerovirus TuYV and prompted a re-evaluation of the role of its inhibitors in silencing P0 during actual infection . FurtherThe results showed that SVM and CNN worked best and that sRNA expression levels before and after infection, if 10 times different, already had an apparent specificity. We selected several machine learning techniques for medium abundance testing.Processing features using the SVM is easier and quicker, and we were able to calculate the evaluation scores for the variance of the SVM at each fold using the medium abundance see A,C. Our Following target gene prediction, we show partial results in The functional enrichment results included 38 biological processes (GO), 18 molecular functions (GO), 22 cellular components (GO), and three KEGG pathways. According to GeneRatio sorting, we primarily mapped the top 10 biological processes (GO) and three KEGG pathways, as shown in Five biological processes, including protein phosphorylation, protein autophosphorylation, immune system process regulation, regulation of defensive responses, and regulation of defense responses to other organisms, are all related to defense-related regulation.The regulation of viral gene transcription, viral intracellular/intercellular motility, viral genome replication, viral infectivity of host plants, plant innate immunological response and counter-defense, and viral particle assembly, in particular, is greatly influenced by phosphorylation .Four biological processes regulate growth and development: the regulation of developmental processes, cellular elements in biogenesis, one-dimensional cell growth, and regulation of the root-shoot system’s development.Two functional protein kinase activities and calmodulin assemblies associated with viral action show enrichment in molecular function. During plant–virus interactions, protein phosphorylation is a frequent post-translational alteration that regularly takes place. Usually, host protein kinases control viral pathogenicity and infectivity by phosphorylating viral proteins . Some reAmong cellular components, vesicles are linked to elements involved in defense. Recent studies have demonstrated the relationship between vesicle acidification and plant immunity and the necessity of vesicle acidification for plant antiviral immunity. By interfering with vacuolar acidification, viral proteins prevent autophagic breakdown and advance viral infection .In KEGG analysis, pathways related to defense may involve plant hormone signal transduction. The primary hormones that control a plant’s ability to defend itself against infections are salicylic acid (SA), jasmonic acid (JA), ethylene (Et), and abscisic acid (ABA) .According to the study, pathogenic processes may have developed in Arabidopsis due to TuYV infection. In this study, we noticed that 247 sRNAs might be virus-derived and predicted these 247 sRNAs to target host Arabidopsis genes, of which 108 sRNAs affected Arabidopsis growth and defense. The lack of degradation data halted further study. In order to better understand the 108 sRNAs that may play a vital role in the regulation of Arabidopsis and determine whether they also exist in other plants infected with the TuYV virus, further research requires additional experimental data.In the realm of virus-infected plants, there are currently no unquestionable and consistent selection criteria for differential sRNAs. For TuYV-infected Arabidopsis differential sRNAs, we chose medium abundance RPM values to limit random drift and get more sRNAs. Discoveries may result from different screening techniques or differential sRNAs acquired at various RPM abundances.We trained several machine learning models to verify the sequence specificity of sRNAs. However, these models predicted infection only for sRNAs expressed at levels above medium abundance RPM values, even though sRNAs expressed at other abundance values may be critical to the infection process. The method screens out sRNAs based on medium abundance at different folds to generate positive and negative samples and can achieve a prediction accuracy of approximately 96.8%. Most of the machine learning models used in this study performed well on the medium abundance specific sRNA dataset, with the best SVM outcomes coming from samples with a 10-fold change in 30 RPM value and the best CNN outcomes coming from samples with a 10-fold change in 50 RPM value. Multiple models were used to confirm that sRNAs are sequence-specific and may even be functional. Functional sRNAs of TuYV viruses have received minimal study, and more research is required to comprehend this subject thoroughly.http://www.ncbi.nlm.nih.gov/geo/; accessed on 10 November 2021) [https://www.ncbi.nlm; accessed on 15 March 2018). The Arabidopsis Information Resource  [We retrieved TuYV-infested Arabidopsis sRNA HTS dataGSE176378) from GEO  . Samples6378 fromly 2019)  was used6 [The expression levels of each sRNA before and after infection, or the total number of sequences in the data file, are necessary to identify sRNAs with appreciable changes in expression. Researchers often use read count and read per million (RPM) values to assess sRNA expression. RPM values were employed in the statistics to account for the slight variance in sRNA length and to remove the sequencing depth bias. Normalized counting of an sRNA =  × 106 . We remo6 . After TIn the pre-processing stage, a script is used to control the length and count the RPM values to produce a file that only contains sequences and RPM values with a sequence length of 18–25 nt and only one occurrence.For constructing specific sRNA datasets before and after infection, random drift and identifying precise expression changes  are two At present, there is no unified standard for qualitative differential expression. Common methods include the log2 ratio, multiple comparisons ,35,36,37We conducted data pre-processing to get the sRNA files before and after infection, including sequences and RPM values. The total number of sRNAs present before and after infection was 1,279,983 after statistics.After script screening, we obtained sRNA samples at each fold change above medium abundance and used the samples above and below each time as positive and negative samples, respectively, with the positive and negative samples’ labels set to 1 and 0. The training and test sets were split up and trained using various machine learning models to ensure that the resulting sRNAs were sequence-specific.We used the pseudo-ribonucleic acid composition features of RNA sequences to generate 104-dimensional feature vectors using the SC-PseDNC-General method of Pse-in-one  for inpuThe feature vector is normalized to lie between  in each machine learning method using maximum-minimum normalization. The formula states the following:N, inC, H, W) and output  of the convolutional layer can be precisely described as follows:N is the batch size, C denotes the number of channels, H is the height of input planes in pixels, and W is the width in pixels.Machine learning is trained on the parameters using tenfold cross-validation and grid search and implemented in scikit-learn 0.24.2 , where tSupport vector machine : regularization parameter (2.91), kernel (RBF), kernel coefficient (0.91);RandomForest (RF): number of trees (100), the minimum number of samples for splitting a node (2), minimum number of samples at a leaf node (1);DecisionTree (DT): criterion (gini), minimum number of samples for splitting a node (2), minimum number of samples at a leaf node (1);K-Nearest Neighbor (KNN): number of nearest data points (5);9);GaussianNB (NB): portion of the largest variance of all features (1 × 10LogisticRegression (LR): penalty (L2), max iteration (3000);XGBoost: booster (gbtree), learning_rate (0.3);CNN: Parallel convolution filter widths , Convolution dimension , Pooling layer width (uniformly 2), Pooling layer calculation (uniformly choose the maximum pooling layer), Batch normalization between the convolution and pooling layers, use dropout2D after batch normalization (dropout rate 0.5), Activation function (chosen uniformly among ReLU and Sigmoid), use dropout1D in the last two fully connected layers (Dropout rate 0.5), Learning rate (0.001).We explored multiple classification models with the following parameters:After choosing sRNAs and confirming the sequence specificity of those sRNAs using machine learning, we aim to identify their sources and investigate their functions, revealing their regulatory roles through genome matching and predicting them against host target genes.We used Bowtie  for genoWe obtained GO and KEGG biological processes by additional enrichment analysis after using the DAVID database . We seleArabidopsis thaliana at the sRNA level. Specific sRNA datasets were constructed by reducing random drift and obtaining more sRNAs, with a recommended RPM value of medium abundance. Multiple machine learning models were combined to validate the sequence-level specificity of sRNAs and achieved approximately 96.8% accuracy, and we assessed SVM and 2D convolutional CNN as demonstrating optimal performance for this study. The 108 functional sRNAs, possibly from the TuYV virus, were targeted and predicted to affect four biological processes of growth and five biological processes of defense in Arabidopsis. Due to the lack of degradation group data, we cannot further verify these sRNAs, but the occurrence of these sRNAs in other plants infected by the TuYV virus is also worth exploring in more experiments. Our highlight is that from the perspective of machine learning, we have verified the sequence specificity of some sRNAs produced by TuYV infecting Arabidopsis, obtained 108 sRNAs, and analyzed the potential role of these sRNAs in Arabidopsis. These sRNAs may not have been taken seriously in previous studies.This study explored the regulation of TuYV infection in


Sogatella furcifera. Autophagy is usually initiated as an adaptive response for cell survival in unfavorable conditions, such as pathogen invasion. The invasion of the virus in the vector may elicit autophagy and may subsequently function to suppress or promote virus propagation and transmission in the insect vector. Here, we annotated two key autophagy-related genes, SfATG3 and SfATG9, in the planthopper vector and found that the genes are conserved among various insects. Interestingly, exposure of the vector to the virus activated the expression of SfATG3 and SfATG9, and silencing SfATG3 or SfATG9 promoted propagation and transmission rates of the virus. These results indicate that the expression of autophagy-related genes is initiated in the vector following exposure to the virus and that autophagy-related genes functions to suppress SRBSDV propagation and transmission.Plant viruses are mostly transmitted by insects and cause severe damage to crops. The southern rice black-streaked dwarf virus is a rice virus exclusively transmitted by a planthopper, ATG3 and ATG9 in the white-backed planthopper (Sogatella furcifera) during the process of transmitting the southern rice black-streaked dwarf virus (SRBSDV). In this study, we annotated two autophagy-related genes, SfATG3 and SfATG9, from the female S. furcifera transcriptome. The cDNA of SfATG3 and SfATG9 comprised an open reading frame (ORF) of 999 bp and 2295 bp that encodes a protein of 332 and 764 amino acid residues, respectively. SfATG3 has two conserved domains and SfATG9 has one conserved domain. In S. furcifera females exposed to SRBSDV, expression of autophagy-related genes was significantly activated and shared similar temporal patterns to those of SRBSDV S9-1 and S10, all peaking at 4 d post viral exposure. Silencing the expression of SfATG3 and SfATG9 promoted SRBSDV propagation and transmission. This study provides evidence for the first time that S. furcifera autophagy-related genes ATG3 and ATG9 play an antiviral role to suppress SRBSDV propagation and transmission.Autophagy plays diverse roles in the interaction among pathogen, vector, and host. In the plant virus and insect vector system, autophagy can be an antiviral/pro-viral factor to suppress/promote virus propagation and transmission. Here, we report the antiviral role of autophagy-related genes  The survival, transmission, and spread of vector-borne viruses depend on the interplay among insect vector, host, and virus ,2. InsecBemisia tabaci) vector system, where autophagy activated by TYLCV infection led to the degradation of viral particles and suppressed virus transmission [Laodelphax striatellus) system, autophagy induced by RBSDV infection in L.striatella resulted in suppression of RBSDV invasion and propagation [Recilia dorsalis) by infection of two plant viruses, rice gall dwarf virus (RGDV) and rice dwarf virus (RDV), facilitated viral propagation and transmission [Laodelphax striatellus did not directly affect the propagation or transmission of the rice stripe virus (RSV), but increased the phosphorylation of c-Jun N-terminal kinase [Autophagy is an important innate cellular immune response in eukaryotes. It is a highly conserved catabolic process that mediates the clearance of long-lived proteins and damaged organelles via a lysosomal degradative pathway ,13,14,15smission . In ricepagation . Autophasmission . Besidesl kinase , which fl kinase .Bactericera cockerelli, whose expression is activated by pathogenic infection [Many genes participate in autophagy for promoting or inhibiting pathogens, for example, autophagy-related genes (ATGs). Under normal circumstances, autophagy keeps at a low level, but it can be activated by stress signals such as pathogens, which are known to induce the expression of some ATGs . Numbersnfection . Thus, tThe southern rice black-streaked dwarf virus (SRBSDV) causes severe yield losses in Asia . The virATG3 and ATG9 from the WBPH transcriptome data and analyzed the sequences of SfATG3 and SfATG9. The expression patterns of autophagy-related genes and SRBSDV genomes were detected in WBPH females exposed to SRBSDV in comparison to those exposed to healthy rice plants by qPCR. Finally, the role of autophagy-related genes in SRBSDV propagation, acquisition, and inoculation was determined by silencing SfATG3 and SfATG9.Given the varying roles of vector autophagy in virus transmission, the present study was conducted to determine the role of ATGs in the transmission of SRBSDV.ATG3 catalyzes the conjugation of ATG8 and phosphatidylethanolamine, APG9  is the only integral membrane ATG protein that is essential for autophagosome formation. Therefore, as a start point, we annotated Potted seedlings of rice  were cultured in 80-mesh cages (50 by 50 by 50 cm) in a greenhouse . A WBPH population was maintained using caged rice seedlings (var. TN1). SRBSDV originated from rice seedlings collected from Xing’an , and a virus stock culture was maintained in caged rice plants in the greenhouse.To obtain SRBSDV-infected rice plants, SRBSDV-free 30 d old rice seedlings were caged with viruliferous WBPHs at a density of five or seven insects per rice for 5 d. Fifteen days thereafter, the SRBSDV infection status of these rice seedlings was detected individually by one-step RT-PCR based on SRBSDV S10 . SRBSDV-ATG3 and ATG9 coding genes in autophagy, we identified SfATG3 and SfATG9 coding genes from female WBPH transcriptome data (Accession number: PRJNA781429). The deduced amino acid sequences of SfATG3 and SfATG9 genes were obtained using the Translate tool provided by the Swiss Institute Bioinformatics . Domain analysis of the retrieved protein sequence was executed by NCBI Conserved-Domain Tool  and Prosite  to predict the conserved domains in the open reading frame (ORF) of SfATG3 and SfATG9. The phylogenetic relationship of ATG3 between WBPH and 14 other species and of ATG9 between WBPH and 9 other species were established by constructing a phylogenetic tree using MEGA7.0, where phylogenetic analysis was conducted by the neighbor-joining method with P-distance under the default parameters. Bootstrap values were obtained by the bootstrap method using 5000 repetitions. The species name, gene name, and GenBank accession involved in the phylogenetic analysis were listed in Due to their indispensable function of SfATG3 and SfATG9 and SRBSDV genomes, including the first part of the 9th RNA segment of the genome (52–1095 nt) that encodes a key viroplasm protein and the 10th RNA segment of genome that encodes the structural protein .Newly emerged 500 WBPH females were fed on SRBSDV-infected rice seedlings for 2 d for virus acquisition, which resulted in more than 80% of WBPH females being SRBSDV-positive, and then the females were transferred to healthy plants. At 0, 2, 4, 8, or 12 days post-exposure (dpe), 30 females were randomly sampled for 3 duplicates to determine the temporal patterns of viral infection influence on the expression of several key autophagy-related genes SfATG3 and SfATG9, total RNA was extracted from ten WBPH females using the Total RNA Extraction Kit  and reverse-transcribed into cDNA using PrimeScript RT Master Mix . Besides, the SRBSDV genomic RNA abundance was also measured with S9-1 and S10 to determine the propagation of the virus in WBPH. The housekeeping gene elongation factor 1-α (EF1α) of WBPH was measured in parallel as an internal control [To determine the expression of  control . Real-tiATG3, dsATG9, and dsGFP using the T7 transcription Kit  according to the manufacturer’s instructions. The size of dsRNA products was confirmed by electrophoresis in 1% (w/v) agarose gels  mixed with 15% sucrose (v/v = 50/50) in a 50-mm diameter cylindrical container for 24 h. WBPH females fed with dsGFP were used as the control for those insects fed with dsRNAs. Then the insects were confined with SRBSDV-infected rice seedlings for 2 d and thereafter transferred to healthy rice plants. At 5 dpe, the insects were individually detected for SRBSDV infection status. The test of SRBSDV acquisition was performed on a batch of more than 15 females and repeated in three batches for treatment or control. The SRBSDV acquisition rate was calculated as the number of viruliferous insects in a batch/number of tested insects in the batch.Newly emerged SRBSDV nonviruliferous WBPH females were fed with dsRNA , and normalized against S. furcifera EF1α. Analysis of variance (ANOVA) was used to detect the significance of treatment effects on gene expression level and SRBSDV transmission rate. Where there was a significant effect, the Tukey test was used to detect a difference when equal variance was assumed, or Games-Howell test was used when equal variance was not assumed. Virus acquisition and inoculation rates were transformed by the arcsine of square root before ANOVA. The means and standard error (S.E.) shown in the figure were based on untransformed data. All data were analyzed in GraphPad Prism 8.0.The data of qPCR was calculated using the comparative CT method (2SfATG3 (Genbank: OL539544) and SfATG9 (Genbank: OL539545) are complete-length genes encoding 332 and 764 amino acids, respectively. The identity values of ATG3 and ATG9 between S. furcifera and Nilaparvata lugens are 86.06% and 84.07%, respectively  and SfATG9 has one conserved domain (APG9)  . The phyectively A,B.SfATG3, SfATG9, and SRBSDV S9-1 and S10 was measured in WBPH females exposed to SRBSDV in comparison to those exposed to healthy rice plants. The expression of SRBSDV S9-1 (p < 0.001). The temporal expression of the ATG genes followed a similar pattern to that of the SRBSDV S9-1 and S10, also peaking at 4 dpe in SRBSDV-exposed WBPH females  than those to healthy plants (Control)  dsATG3 for 24 h, the expression of SfATG3 was significantly suppressed in comparison with the insects fed with dsGFP  and 22.10% , respectively; and increased SRBSDV acquisition rate by 31.43%  and 35.91% , respectively. The SRBSDV acquisition rate  and inoculation rate  showed no significant difference between dsATG3 and dsATG9 treatment.When the WBPH females were fed with 400 ng/μL  system, autophagy is activated by virus infection or virus protein and functions to suppress the propagation and transmission of the virus [L. striatellus system, virus infection also activates the expression of ATG genes in the vector, but virus infection is promoted instead of suppressed [R. dorsalis system, autophagy is also activated by virus infection whereas RGDV propagation in R. dorsalis is promoted [Activation of autophagy in vectors by virus infection has been reported in many vector-virus systems, but plays differential pro- or anti-viral roles. In he virus ,17. Howeppressed . In RGDVpromoted . From thpromoted ,43,44,45promoted ,46. FurtSfATG3 and SfATG9 are autophagy-related genes. And our results do show that silencing ATG3 and ATG9 promoted SRBSDV propagation and transmission in the planthopper. It is reasonable to argue that autophagy has occurred in S. furcifera. More studies are needed to provide direct evidence showing the role of autophagy in the propagation and transmission of SRBSDV.In this study, we did not measure autophagy signals and observe autophagic vesicles, as did in previous studies ,17,18,19SfATG3 and SfATG9 function to suppress SRBSDV propagation and transmission, which provides a new insight into the control of SRBSDV by using autophagy activator, although there is a long way to go before field application.Taken together, the current results indicate that autophagy-related genes ATG3 and ATG9 from S. furcifera. The temporal expression patterns of autophagy-related genes ATG3 and ATG9 in S. furcifera exposed to SRBSDV-infected rice plants follow that of SRBSDV S9-1 and S10, indicating the propagation of SRBSDV is regulated by autophagy-related genes. The results of ATG3 and ATG9 RNAi demonstrate for the first time that autophagy-related genes in WBPH females suppress SRBSDV transmission. Therefore, autophagy-related genes SfATG3 and SfATG9 in WBPH play an antiviral role against SRBSDV.In summary, we identified the autophagy-related genes


BK virus‐associated hemorrhagic cystitis is a significant complication of hematopoietic stem cell transplantation. Although severe BK virus‐associated hemorrhagic cystitis is associated with treatment‐related mortality, sufficient evidence regarding its management is lacking.A 14‐year‐old boy presented with BK virus‐associated hemorrhagic cystitis and bladder clot retention after hematopoietic stem cell transplantation. Various urological interventions failed to improve cystitis. While bladder clot retention frequently recurred, surgical intervention was difficult because of the underlying hematological disorder. Hence, bilateral single‐J ureteral stenting followed by Foley catheter placement was performed as a urinary diversion. The bladder clot completely disappeared 27 days after stenting. No additional procedure was required. BK virus‐associated hemorrhagic cystitis did not recur after the blood clot disappeared.Bilateral single‐J ureteral stenting followed by Foley catheter placement is a simple and effective treatment method and should be considered before surgical intervention for severe BK virus‐associated hemorrhagic cystitis. BKV‐HCBK virus‐associated hemorrhagic cystitisCBIcontinuous bladder irrigationCRPC‐reactive proteinHbhemoglobinHChemorrhagic cystitisHSCThematopoietic stem cell transplantationPCypercutaneous cystostomyPltplateletPNpercutaneous nephrostomyRCCred cell concentrateTUEtransurethral electrocoagulationThe authors report the first case of BK virus‐associated severe hemorrhagic cystitis successfully treated with bilateral single‐J ureteral stenting, followed by Foley catheter placement. As the proposed method is simple and effective, it should be considered before surgical intervention for severe BKV‐HC.BK viruria can lead to BKV‐HC, a well‐recognized and significant complication in up to 40% of recipients of HSCT.9 copies/mL, while adeno and JC virus levels were normal. No bacteriuria was observed. A Foley catheter was placed for the diagnosis of BKV‐HC, and urological intervention was needed as bladder retention occurred on day 12 due to a blood clot. The purchase of Cidofovir (not approved in Japan), which was reported to be effective in several reports,A 14‐year‐old boy with acute lymphocytic leukemia developed slight hematuria 4 days after HSCT at our hospital. Urine tests revealed significantly increased BK virus levels of 5.0 × 108 copies/mL), at 8 months post‐HSCT, BKV‐HC has not recurred.The BKV‐HC with bladder clot retention persisted for 4 months with temporary improvement and recurrence; hence, frequent manual bladder washout and CBI were performed each time. TUE performed under general anesthesia on days 84 and 117 also failed to improve BKV‐HC. The bladder wall was diffusely edematous and hemorrhagic Fig. . A bilatTherapy for severe HC is stressful for patients because of recurrent clot retention and the necessity for bladder irrigation.Our decision to place bilateral single‐J ureteral stents was based on the following possible mechanisms: (i) decreased bladder distension by urine, thereby reducing microtrauma; (ii) protection from urinary urokinase, which prevents clot formation; (iii) bladder packing by the clot and subsequent bladder mucosal healing and bleeding cessation.Urologists do not have sufficient evidence to support the management of severe HC after HSCT. We have summarized retrospective studies and a non‐randomized control study on urologic intervention for severe HC after HSCT Table . CBI aftTo our knowledge, this is the first report of successful treatment of BKV‐HC after HSCT using bilateral single‐J ureteral stenting. The mechanism of hemostasis is similar to that of PN; therefore, similar effects to PN can be expected without the risk of kidney bleeding. Our method is simple and less invasive; in addition, it does not impair the patients' activities of daily living compared to the methods in previous reports. This procedure should be attempted before surgical intervention for severe BKV‐HC after HSCT in combination with appropriate pain control. The age of the patient is an important consideration for our procedure; at the very least, patients should be old enough to undergo cystoscopy. The risk of developing BK virus‐related nephropathy due to the placement of ureteral stents should be consideredBilateral single‐J ureteral stenting followed by Foley catheter placement is a simple and effective method for the treatment of BKV‐HC. Hence, it should be considered before surgical interventions.Akira Fujita: Conceptualization; data curation; visualization; writing – original draft. Kohei Kobatake: Conceptualization; writing – review and editing. Takafumi Fukushima: Investigation. Kenshiro Takemoto: Investigation. Syunsuke Miyamoto: Investigation. Hiroyuki Kitano: Validation. Kenichiro Ikeda: Methodology. Keisuke Goto: Methodology. Keisuke Hieda: Methodology. Shuhei Karakawa: Resources. Tetsutaro Hayashi: Methodology. Jun Teishima: Supervision. Nobuyuki Hinata: Supervision.The authors declare no conflict of interest.Not applicable.The patient involved provided informed consent for the publication of this study.Not applicable.


Pseudorabies virus (PRV) is the pathogen of pseudorabies (PR), which belongs to the alpha herpesvirus subfamily with a double stranded DNA genome encoding approximately 70 proteins. PRV has many non-essential regions for replication, has a strong capacity to accommodate foreign genes, and more areas for genetic modification. PRV is an ideal vaccine vector, and multivalent live virus-vectored vaccines can be developed using the gene-deleted PRV. The immune system continues to be stimulated by the gene-deleted PRVs and maintain a long immunity lasting more than 4 months. Here, we provide a brief overview of the biology of PRV, recombinant PRV construction methodology, the technology platform for efficiently constructing recombinant PRV, and the applications of recombinant PRV in vaccine development. This review summarizes the latest information on PRV usage in vaccine development against swine infectious diseases, and it offers novel perspectives for advancing preventive medicine through vaccinology. Herpesviridae family, primarily affects pigs and can occasionally be transmitted to cattle, goats, sheep, cats, and dogs [Pseudorabies virus (PRV) is the etiological agent of pseudorabies (PR), belonging to the and dogs ,2,3,4,5.and dogs ,7,8. Theand dogs ,10. The and dogs . Therefoand dogs . Live vector vaccine can stimulate humoral immunity and solid cellular immunity, which is the main goal for developing a swine infectious disease vaccine. Scientists have generated PRV vaccine strains with few side effects and good immunity by inserting foreign genes or knocking out self-virulence genes. This review summarizes the biological characteristics of PRV, the methodological principle and technology platform for constructing characteristics of PRV, the methodological principle and technology platform for constructing PRV recombinants efficiently, and the applications of PRV recombinants in vaccine development. This review is intended to serve as a reference for PRV live vector vaccine research and development. There are 10 glycoproteins encoded by PRV, which are divided into two groups  according to the degree to which viral replication depends on them ,14. The Bartha-K61, the original PRV vaccine strain, contains the deletion of US2, gE, gI, and US9 genes. This vaccine strain has been widely used to control PR worldwide. Since the 1990s, few swine PR cases have been reported due to PRV Bartha-K61 ,29,30,31There are some non-essential regions in the large genome of PRV. As a result of this character, several kb of foreign DNA can be accommodated by PRV without compromising its stability. Reverse genetics plays a crucial role in PRV gene modification, and with the development of molecular biology techniques, many reverse genetic techniques have been used to construct the recombinant PRV.In the past, PRV recombinants were generated by homologous recombination (HR) in permissive cells. The construction of recombinant viruses based on the principle of HR can be divided into two steps: construction of a transfer plasmid and screening of the recombinant virus. Usually, the virus genome is used as a template to amplify the two gene fragments used as recombinant homologous arms by PCR technology, and then the foreign gene expression cassette is inserted between the homologous arms to construct the transfer vector containing the foreign gene expression cassette and the left and right homologous arms. Gene deletion or replacement is achieved by homologous recombination of the transfer vector and parent strain in eukaryotic susceptible cells. At present, there are two methods used for transfection. One method is co-transfection of the transfer vector and the parent virus genome, and another method is transfection of the transfer vector first and then inoculation of the virus. The reporter gene carried on the transfer vector is used to screen the plaque of the recombinant virus. Generally, the recombinant virus with infective activity can be obtained after 5–10 generations of screening. By using this method, Tong et al. and Wang et al., constructed a recombinant PRV strain expressing the E2 protein of CSFV in a gE-deleted PRV variant strain ,50; Yan Escherichia coli, which can be divided into two steps [In comparison with homologous recombination, bacterial artificial chromosomes (BAC) are more efficient. Red/ET recombination technology can be used to modify BAC in wo steps . The firwo steps . Furtherwo steps .E coli. Since the fosmid only contains part of the PRV genome, the efficiency of obtaining virus mutants is greatly improved. Modification of fosmid in E. coli can also utilize Red/ET recombination technology. A fosmid library based on the PRV-TJ strain was constructed by Zhou et al., and recombinant PRV was rescued by transfecting a group of fosmid directly into Vero cells. Furthermore, the green fluorescent protein-expressing reporter virus was rescued successfully by the Red/ET system [The traditional HR method is not efficient in obtaining recombinant viruses. Creating recombinant BAC constructs is a labor-intensive and time-consuming process. When the whole genome PRV is cloned to the BAC plasmid, fragment deletion often occurs during the electroporation process, affecting recombination efficiency. In contrast, fosmid library generation is more efficient. The huge genome of PRV is randomly sheared into various fragments, then each segment is ligated with a fosmid vector to screen out the various combinations covering the whole genome of PRV, and the fosmid combination was co-transfected into virus-susceptible eukaryotic cells to rescue the virus ,56. ConsT system . Abid etT system . In addiMolecular biotechnology has dramatically contributed to studying viruses’ replication and vaccines. The CRISPR /Cas9-mediated genome editing system has been widely applied as a powerful tool to construct PRV mutants. The researchers used CRISPR/Cas9 technology to construct PRV gE, gI, or TK gene-deleted recombinants ,43,46. FEscherichia coli. The homologous recombination method is labor-intensive due to several rounds of plaque purification. The BAC system was more efficient than homologous recombination, but the generation of recombinant BAC construct is time-consuming, and the huge PRV genome is easily broken. The fosmid library and CRISPR/Cas9-based genetic manipulation platform for PRV offer several advantages over the conventional technology, but some disadvantages still appeared during the construction of the recombinants  into Bartha K61, and subsequently vaccinating, significant clinical protection was achieved, and pathological lesions were reduced after the PRRSV challenge in piglets . FurtherSince 2011, farms immunized with Bartha-K61 have experienced PR outbreaks caused by PRV variants. Therefore, the researchers try to generate several gene mutant vaccines involving gE/TK, gI/gE, and gI/gE/TK deletion based on different PRV variants. These vaccines provided adequate protection against the PRV variant challenge. These gene mutant vaccines also are used as a vector system for foreign proteins to develop multivalent live virus-vectored vaccines against several swine infectious diseases. Tong et al. and Wang et al. constructed a recombinant expressing the CSFV E2 protein and evaluated its efficacy against both CSFV and PRV variant strains ,50. The Lymphokine-activated killer cells recognize infected cells associated with PRV gC during cell-mediated immunity, and PRV gC plays a role in cytotoxic T-lymphocytes recognition of infected cells ,66,67. TUp to now, the mechanism of PRV variant virulence enhancement is unknown. A reporter virus is a valuable tool for basic virology studies. Therefore, Fu et al. constructed a double reporter virus with stable expression of EGFP and firefly luciferase based on the PRV variant . The recThe genetically engineered live vector vaccine can effectively avoid the traditional vaccines’ defects, simplify the breeding industry’s immunization procedures, and improve the efficiency of epidemic prevention and control. Attenuated PRV-based vector vaccines do not induce any clinical signs of PR, but provide complete protection against PRV and other swine pathogens, and it has been widely used in the study of a recombinant multivalent vaccine against swine infectious disease. The BAC technology, CRISPR/Cas9 technology, and fosmid library were used to construct PRV recombinant, significantly improving the efficiency of exogenous gene expression . But we


Human monkeypox is an emerging zoonotic disease caused by a monkeypox virus. The monkeypox virus history originated in 1958 after the occurrence of a pox-like illness in monkeys. In September 1970, the first case of human monkeypox was identified in the Democratic Republic of Congo, Africa. This year, from January 01, to September 30, 2022, the virus swiftly spread from endemic to non-endemic counties, involving 106 states, infecting 68,017 people; 689 cases from 07 endemic African countries and 67,328 cases in 99 non-endemic countries in Europe, America, Asia and Oceania continents. The disease caused 27 deaths in 13 countries worldwide. The human monkeypox disease significantly affects the population in South, North and Central America 34767 (51.11%), Europe 32047 (47.11%), Africa 707 (1.03%), Asia 351 (0.51%), and Australia and Oceania continent 145 (0.21%). The occurrence of the disease is high in males with age ranges of 21-55 years. The common clinical features in monkeypox patients are skin rashes (95%), fever (72%), malaise (69%), chills (67%), pruritis (64%), headache (64%), enlarged lymph nodes (63%) myalgia (60%) and nausea and vomiting (20%). The transmission trends of the disease are rapidly changing; the virus is not limited to close contact with humans. It can spread through body fluids, respiratory droplets, and sexual contact. The disease can transmit during travelling, contact with soiled materials, infected cloths, bed linen, objects, air pollutants, and in various workplace environments. The monkeypox virus has adopted multiple transmission routes, and swiftly spreading and developing challenging and threatening situations worldwide. Orthopoxvirus, subfamily Chordopoxvirinae and family poxviridae”. The genomes of these family viruses are ≈200 kb long, with a dominant replication part and genetic material. The monkeypox family viruses are consisting of numerous pathogens and infect humans.Human monkeypox disease is caused by a monkeypox virus (MPXV), which is a zoonotic infection commonly found in African countries.The monkeypox virus was first found in 1958 after the occurrence of a pox-like ailment in monkeys, which were housed in a research institute in Denmark, hence the disease became popular with the monkeypox name.8In the same year, in October 1970 and May 1971, six cases of human MPXV were identified in “Liberia, Nigeria, and Sierra Leone”.3In the new millennium, the year 2003, the human monkeypox disease was first time spread from endemic African regions to non-endemic nations, including the United States of America (USA).12The possible route of transmission of MPXV is “animal-human and human-human”. The respiratory droplets, direct or indirect contact with body fluids, pieces of stuff, skin lesion of an infected person4The monkeypox virus DNA lies on the surfaces in hospitals and households, and is found on bed linen, air and dust samples in rooms used by monkeypox patients.This year, from May 2022, monkeypox diseases globally involved 106 countries.14In Nigeria, during 2017-2018, the outbreak involved 118 cases, and from September 2018 -May 2021, six people with a travelling history from Nigeria were diagnosed with monkeypox. The four people travelled to the “United Kingdom, one to Singapore and one to Israel. In July 2021, a man travelled from Nigeria to Dallas, Texas was diagnosed with monkeypox. Among 194 monitored contacts, 144 had a history of flight contacts.16In the Republic of Korea, in June 2022, a monkeypox case has been reported in a person with a travel history to Europe, day after his return to the Republic of Korea, the patient was diagnosed with monkeypox.This year, from January 2022, to September 30, 2022, the virus has adopted multiple transmission paths and swiftly spread from endemic to non-endemic counties, involving 106 states, infecting 68,017 people; 689 cases from 07 endemic African countries and 67,328 cases in 99 non-endemic countries in Europe, Central, South and North America, Australia, and Asia19The human monkeypox disease significantly affected the population in European countries and the United States of America , Fig.1. In Europe, the maximum number of cases reported from Spain is 7149, Germany 3621, United Kingdom 3635, France 3999, Netherlands 1223, Portugal 917, Italy 846, Belgium 770, Switzerland 513, and Austria 310.15The monkeypox virus history originated in 1958, and the first case of human monkeypox was identified in the Democratic Republic of Congo, Africa in September 1970. The virus has adopted multiple transmission paths and swiftly spreading from endemic to non-endemic countries. This year, from January 01, to September 30, 2022, the virus involved 106 countries, infecting 68,017 people; 689 cases from 07 endemic African countries, and 67,328 cases in 99 non-endemic countries worldwide. The human monkeypox disease sternly involved the entire European countries and the United States of America. The transmission trends of human monkeypox disease have rapidly changed, and the virus is not limited to close contact with humans. It can spread through body fluids, respiratory droplets, and sexual contact. The virus can spread through travel, contact with soiled materials, infected cloths, bed linen, objects, and through air pollutants and exposure to various workplace environments. The global health authorities must implement strict policies to control the burden of the disease both at regional and international levels. As the world has still not completely recovered from the major shake-up of COVID-19, society cannot afford another pandemic of human monkeypox.


Bats perform important ecological roles in our ecosystem. However, recent studies have demonstrated that bats are reservoirs of emerging viruses that have spilled over into humans and agricultural animals to cause severe diseases. These viruses include Hendra and Nipah paramyxoviruses, Ebola and Marburg filoviruses, and coronaviruses that are closely related to severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and the recently emerged SARS-CoV-2. Intriguingly, bats that are naturally or experimentally infected with these viruses do not show clinical signs of disease. Here we have reviewed ecological, behavioral, and molecular factors that may influence the ability of bats to harbor viruses. We have summarized known zoonotic potential of bat-borne viruses and stress on the need for further studies to better understand the evolutionary relationship between bats and their viruses, along with discovering the intrinsic and external factors that facilitate the successful spillover of viruses from bats.  Virology; Evolutionary biology Bats belong to the order Chiroptera and are one of the most abundant and geographically diverse vertebrates in the world, representing over 20% of all mammals . The ordDespite performing critical ecological roles, bats are an emerging reservoir host for important viruses that cause significant pathology in humans and animals. Seventy percent of emerging and re-emerging infectious diseases are from an animal origin or zoonotic , and batWhile recent reviews have summarized the diversity of viruses detected in bats and underlying mechanisms that may contribute to viral tolerance in these flying mammals , an updaMyotis lucifugus) rises to over 40°C, a temperature that is hypothesized to be less optimal for pathogen survival and is representative of a fever response in humans (Hypsignathus monstrosus), Buettikofer’s epauletted fruit bat (Epomops buettikoferi), the Egyptian fruit bat (Rousettus aegyptiacus), the Jamaican fruit bat (Artibeus jamaicensis), the Mexican free-tailed bat (Tadarida brasiliensis), and the big brown bat (Eptesicus fuscus), with virus propagation occurring when incubated at temperatures ranging from 37°C to 41°C (Epomorphorus wahlbergi) and flying foxes (Pteropus spp.) (Oryx leucoryx), southern flying squirrel (Glaucomys volans), and large treeshrews (Tupaia tana) that display an elevated body temperature of approximately 40°C  , suggestus spp.) A. Despitus spp.) . Moreoveus spp.) . Even soely 40°C .Figure 1T. brasiliensis are known to travel over 800 miles between their summer and overwintering sites  has re-evolved to utilize tongue clicking to navigate , have been identified to utilize a novel, cyclic, bradycardic state to reduce their daily expenditure of energy instead of torpor as the ambient temperature is too high for bats to sufficiently lower their body temperature   . Furtherrandtii) .Rhinolophus ferrumequinum) and the common bent-wing bat (Miniopterus schreibersii), but not in the free-living greater mouse-eared bats (Myotis myotis) (Rhinolophus spp.), the common vampire bat (Desmodus rotundus), long-eared bats (Plecotus spp.), and at least one Myotis lineage  . Wilkins lineage .Myotis myotis  and David’s myotis (Myotis davidii), suggesting the ability of these bats to resist DNA damage (Myotis brandtii), Desmodus rotundus, and Myotis myotis  that sense pathogen-associated molecular patterns (PAMPs) derived from various pathogens, such as viruses . FollowiPteropus alecto relies primarily on the innate antiviral response , a key antiviral transcription factor, is observed to have a wider cell-type and tissue-level distribution and expression pattern in P. alecto compared to humans   . Thus, datus L.) . Additioat cells . Some baat cells , which mat cells .P. alecto splenocytes with Tioman virus preferentially induced IFNλ2, a type III IFN , have been detected in both unstimulated and stimulated P. alecto kidney, brain, and lung cells , a PRR that activates inflammatory mediators during virus infection  and pigs (for PEDV). Some bats have evolved multiple strategies to counteract an overt activation of the inflammatory response . A recennfection  and it infection . This danfection . In addinfection . Absent eukin-1β . Thus, tRhinolophus sinicus), Myotis davidii, and P. alecto, and has been linked to the loss of a serine residue at amino acid position 358 in STING  in response to surrogate virus infection [poly(I:C) stimulation] because of the activity of a TNFα promoter repressor protein, c-Rel  , a PRR tin STING . In addin, c-Rel . Overalln, c-Rel . It remaP. alecto and the large flying fox (Pteropus vampyrus) , IgG, IgA, and IgE have been detected in ampyrus) . Studiesralizing . Similar induced . Data frPteropus medius, previously Pteropus giganteus) have similar lymphoid development as other mammals  differ from other mammals as they are predicted to accommodate larger peptides and have consensus-binding motifs that have a C-terminal proline bias   , enhanciin vivo studies have been performed, and these studies demonstrate that bats do not exhibit signs of clinical disease when infected with Ebola and Marburg filoviruses, Nipah and Hendra paramyxoviruses, Dengue and West Nile flaviviruses, and coronaviruses such as MERS-CoV and SARS-CoV-2  excreted low levels of virus in urine, which may sustain transmission amongst bat species where there is regular urine contamination of the fur, mutual grooming, and where urine droplets are present within the environment   . The dif ferrets . The incresponse , as signobserved . MoreoveIII IFNs . IntriguIII IFNs . More woR. aegyptiacus with Sosuga virus did not lead to significant clinical illness; however, virus was detected in numerous tissues from two to thirteen days following inoculation   . Unlike perature . Transmiwas poor . Therefowas poor . AlthougA series of factors are required for bat-borne viruses to successfully spillover to humans, including ecological opportunity for contact, virus-host molecular and cellular compatibility, and a permissive or circumvented immune response . AlthougFactors that are directly related to the availability of a pathogen to humans, such as pathogen dynamics, survival, and dispersal outside of the reservoir comprise pathogen pressure. Pathogen dynamics is directly impacted by the distribution, density, and the prevalence of infection among the reservoir host(s). Due to increasing human population and urbanization, animals and humans are living in closer proximity than ever before. The effects of this have already been documented for rodents, where studies in Africa have demonstrated the increased risk of rodent-borne diseases as the loss of larger wildlife has released control on rodent density .Eidolon helvum) which are reservoirs of Lagos bat virus, henipaviruses, and Achimota virus , to prevent the next zoonotic outbreak.Cellular entry is the first step for a virus to be able to infect the host cell. Successful zoonotic viruses that have transmitted from bats use host molecules that are highly conserved amongst different species as receptors. For instance, bat-borne henipaviruses utilize ephrin-B2 and-B3 , filovirin vitro . Similarin vitro . These ePteropus scapulatus)  replication in co-infected M.lucifugus bats  . Co-infegus bats . Indeed,MERS-CoV . Additioin vitro and ex vivo models, such as primary cells and immunocompetent organoids, along with parallel studies in whole animal models will enable a better and wholesome understanding of the bat-virus relationship. There is also a lack of collaborative studies among bat wildlife ecologists and laboratory scientists. Inter- and multidisciplinary studies will help us better understand the implications of ecological impacts on bat biology and virus persistence using data from the field, along with complementary placebo-controlled laboratory studies. One way to facilitate this would be to mimic ecological stressors at the molecular level using synthetic chemical surrogates on bat cells or organoids, followed by assessment of the impact of these stress molecules on cellular immunity and virus replication. Multicellular models, such as organoid cultures will also allow researchers to perform longitudinal studies to identify cell-to-cell spread of virus infection and cell-type specificity of cytokine production and virus replication. Currently, very few bat organoid cultures exist. Commercially available species-specific cytokines also remain limited.Developing additional P.alecto, R.aegyptiacus, Rhinolophus spp., M.lucifugus and E.fuscus, with researchers having access to few high-quality annotated bat genomes. By increasing the availability of annotated bat genomes, such as through efforts by the Bat1K Consortium, we can achieve a better understanding of the mechanisms that facilitate a well-tolerated antiviral immune response in bats (Research on bats has been hampered by the lack of cross-reactive reagents . There a in bats . These sThere is a treasure trove of biological discoveries that are waiting to be uncovered once we develop the tools to mechanistically study bats and their viruses. For example, it remains unknown why bats only occasionally shed viruses. Understanding factors that lead to virus spillover from bats will enable the development of policies to prevent future zoonotic outbreaks and epidemics. In humans, aging is related to a deteriorating immune response . Bats haIt is also important to understand ecological and behavioral drivers of bat-virus interactions to understand and mitigate the spread of emerging and re-emerging bat-borne pathogens. Viruses that are found in bats, such as Nipah virus, continue to cause outbreaks in Bangladesh and Eastern India. However, in 2020, Nipah virus outbreaks were reported in Southern India for the first time , a regioIt is apparent that the ecological, behavioral, and molecular traits possessed by bats have influenced their ability to tolerate viral infection, making them unique reservoir hosts. Through the adaption of flight, bats may have acquired responses which allow for accelerated immune processes and therefore infection control which is more rapid than other mammals . MoreoveHibernating bats are also known to have an extended lifespan , where lThe continuous expansion of the human population is causing animals and humans to live in closer proximity and at higher density, thereby increasing the threat of zoonoses. Though certain countries may be at higher risk for the emergence and spread of zoonoses, the emergence of SARS-CoV-2 has made it clear that an outbreak that starts in one part of the world can quickly become global, affecting the health of both humans and animals. Overall, the adoption of a One Health approach across government organizations, in addition to a global surveillance system to monitor emerging infectious diseases will be an indispensable tool in our fight against zoonotic pathogens.


Emiliania huxleyi virus (EhV) is another member of the NCLDVs, which has a restricted host range limited to a species of marine algae called Emiliania huxleyi. This algal NCLDV has many similar morphological and physical characteristics to ASFV thereby making it a safe surrogate, with results that are applicable to ASFV and suitable for use in real-world experiments. Here we inoculated conventional soybean meal (SBMC), organic soybean meal (SBMO), and swine complete feed (CF) matrices with EhV strain 86 (EhV-86) at a concentration of 6.6 × 107 virus g−1, and then transported these samples in the trailer of a commercial transport vehicle for 23 days across 10,183 km covering 29 states in various regions of the United States. Upon return, samples were evaluated for virus presence and viability using a previously validated viability qPCR (V-qPCR) method. Results showed that EhV-86 was detected in all matrices and no degradation in EhV-86 viability was observed after the 23-day transportation event. Additionally, sampling sensitivity  rather than virus degradation best explains the variation of virus quantity observed after the 23-day transport simulation. These results demonstrate for the first time that ASFV-like NCLDVs can retain viability in swine feed matrices during long-term transport across the continental United States.African swine fever virus (ASFV) is a member of the nucleocytoplasmic large DNA viruses (NCLDVs) and is stable in a variety of environments, including animal feed ingredients as shown in previous laboratory experiments and simulations.  Foreign animal diseases, such as African swine fever virus (ASFV), pose a significant threat to the United States pork industry because contaminated feed ingredients, pork products, and humans can all be potential sources of disease introduction . Some RNAsfarviridae family which is part of a larger group of virus families that are classified as nucleocytoplasmic large DNA viruses (NCLDVs) and evolved from a common ancestor. These NCLDVs are found in a variety of environments, and can infect humans (Poxviridae), fish (Iridoviridae), insects (Ascoviridae), swine (Asfarviridae), amoeba (Marseilleviridae and Mimiviridae) and algae (Phycodnaviridae) (Emiliania huxleyi virus strain 86 (EhV-86) is an ecologically important NCLDV which controls blooms of the marine unicellular phytoplankton Emiliania huxleyi (African swine fever virus is a member of the viridae) . Until n huxleyi  and shar huxleyi . Both AS huxleyi . In factThere are many challenges involving various analytical methods and data interpretation when determining virus inactivation kinetics or survival in various types of feed matrices . A commoConsidering the challenges of determining ASFV survival in feed ingredients under commercial conditions, we chose to use EhV-86 as a suitable and safe surrogate for ASFV and employ the use of a new V-qPCR assay to quantify EhV-86 infectivity when recovered from selected feeds after an extended transport time period. This data can then be compared with the lab-based quantitative data on the half-life of ASFV Georgia 2007 in animal feed ingredients exposed to moderate temperature and humidity conditions simulating transoceanic shipment . The maiEmiliania huxleyi CCMP374  was grown in Alga-Gro® Seawater Medium  at 15°C with 18 h/6 h light/dark cycle (ca. 2400 lux) until the concentration of 2 × 105 cells mL−1 was reached. Isolate EhV-86  was added to E. huxleyi at a multiplicity of infection (MOI) of 1 and grown in a 15°C incubator until lysis was observed, which was usually after 4 d . For each feed matrix, four subsamples (30 g each) were weighed and placed into individual 50 ml mini-bioreactor tubes with vented caps. A total of three allotments per feed matrix were spiked with 2 ml EhV-86 . The truck carrying the inoculated feed samples departed from Minneapolis, Minnesota on November 30, 2020 and returned on December 22, 2020 (23 days). The route covered various regions of the United States including 29 states in the Midwest, Rocky Mountains, Southwest, Gulf Coast, Eastern Seaboard, New England region, and the Great Lakes region . The tem9 viruses mL−1) was added to 1 g of SBMC in a 50 ml Falcon tube , and held at room temperature for 5 min before eluting the virus from the soybean meal by adding 10 ml of Alga-Gro® Seawater Medium . The tube was vortexed repeatedly for 1 min before incubating in a water bath  set at 40°C for a total time of 30 min. The tube was removed and vortexed every 5 min for 30 s. At the end of the temperature exposure, the tube was centrifuged at 4700 rpm for 5 min to collect the soybean meal at the bottom of the tube. The supernatant (virus eluant) was removed and filtered through 0.22 μm syringe filter  into an Amicon centrifugal tube . The virus eluant was washed 3 times with 1X phosphate buffered saline  via centrifugation as per manufacturer’s instructions, and the final 200 μl volume obtained was split into two aliquots of 100 μl each. This process was repeated to create biologically independent replicate samples.Two hundred μL of EhV-86 filtrate  was added according to methods optimized by One μL from all the samples (virus eluants and DNA extractions) served as the DNA template in the subsequent 20 μl qPCR mix : 10 μl Master Mix, 0.1 μl QN ROX Reference Dye, 1.4 μl reverse primer , 1.4 μl forward primer , and 6.1 μl molecular grade water. The primers amplify part of the single copy major capsid protein (MCP) gene of EhV as described by Standards for the qPCR assays were created using EhV-86 as the template, with the MCP amplificon purity confirmed using E-Gel electrophoresis system  and extracted using Zymoclean™ Gel DNA Recovery Kit . The number of EhV-86 genomic copies that equate to MCP copies in our extracted MCP amplicon product was calculated using the following formula:23 is Avogadro’s number, 83454.93 Da is the molecular weight of our MCP amplicon as calculated using the Sequence Manipulation Suite , 6.022 × 10on Suite , and 1 ×One gram of each 30 g of feed matrix was sampled on d 23 and was used in the virus elution protocol as previously described. The qPCRs were carried out on DNA extracted from the eluant. The percentage of virus recovered was calculated as follows:−1) were normally distributed and compared using two-sample t-test assuming unequal variances in Excel.Visualization of data was performed using the ggplot2 package of RStudio environment  using R programming language . Viral quantity averages (virus μLy = 3.5926x + 7.0205) to convert the Ct values from the qPCR assay to EhV-86 genomic equivalents  and 1.26 × 101  EhV-86 μl−1 from the eluant in both standard (S) and viability (V) qPCRs, respectively, were obtained  the amount of virus that was detected via standard  and V-qPCR  (p = 0.81), suggesting that EhV-86 is stable in SBMC over the 5 min assay incubation period. Moreover, the elution efficiency of EhV-86 from SBMC was about 20% indicating that about 80% of the virus remained attached or associated to the feed matrix in some way.Before performing multiple extractions from feed samples, we first evaluated the EhV-86 elution protocol using a SBMC sample. We also determined if the eluants could be used directly as templates in the qPCR or if EhV-86 DNA needed to be extracted from the final eluant. Concentrations of 8.53 × 10obtained , which wCt 19.3) . Compari9 viruses mL−1, which resulted in an initial virus load of 6.6 × 107 viruses g−1 of feed matrix. For the inoculated CF samples, an average of 2.36 × 103 EhV-86 μl−1 of eluant (Ct 24.9) or 1.18 × 105 EhV-86 g−1 of CF was recovered on d 0, which represented only a 0.2% average recovery rate. In inoculated SBMC and SBMO samples, 6.83 × 103 EhV-86 μl−1 (Ct 23.3) or 3.41 × 105 EhV-86 g−1, and 8.61 × 103 EhV-86 μl−1 (Ct 23.2) or 4.31 × 105 EhV-86 g−1, respectively, were recovered. The average recovery rates for SBMC and SBMO were 0.52 and 0.65%, respectively . There was also greater EhV-86 deviation from the mean in SBMC  than in SBMO  or 3.41 × 105 EhV-86 g−1 was recovered from the CF matrix. This represents an average recovery rate of 0.52%, with a 289% increase in virus concentration compared to d 0 (3 EhV-86 μl−1 (Ct 25.3) or 9.37 × 104 EhV-86 g−1 was detected in SBMC (0.14% recovery or 28% more viruses) on d 23. For SMBO, an average of 6.25 × 103 EhV-86 μl−1 (Ct 24.0) or 3.13 × 105 EhV-86 g−1 was recovered, which equates to 0.47% recovery and a 73% increase in virus concentration compared to EhV-86 concentrations on d 0  compared to the CF  or 1.10 × 104 EhV-86 g−1 viable virus was detected with the V-qPCR assay from the CF matrix. These concentrations represent an average of 0.02% recovery rate and 9% viability at the start of the study when compared with the eluted virus counts on d 0. On d 23, an average of 7.46 × 102 EhV-86 μl−1 (Ct 25.5) or 3.73 × 104 EhV-86 g−1 was recovered representing an average recovery rate of 0.06%. This resulted in a range from 11 to 32% in virus viability depending on whether the d 23 or d 0 standard qPCR results were used as baselines, respectively. These results indicate that EhV-86 viability increased by 2 to 22%, and no loss in viability due to the 23-d transport event.On d 0, an average of 2.19 × 103 EhV-86 μl−1 (Ct 25.1) or 1.04 × 105 EhV-86 g−1 viable virus was detected on d 0 from SBMC. This represents an average 0.16% recovery rate and 31% viability at the beginning of the study. On d 23, 9.10 × 102 EhV-86 μl−1 (Ct 26.2) or 4.55 × 104 EhV-86 g−1 was measured, which equates to 0.07% recovery rate and a range in viability from 13 to 49%, depending on whether the d 0 or d 23 standard qPCR results were used as baselines, respectively  or 2.26 × 102 EhV-86 g−1 viable virus was detected on d 0 from SBMO, which represents an average 0.003% recovery rate and 0.05% viability at the start of the study. On d 23, 8.31 × 102 EhV-86 μl−1 (Ct 27.1) or 4.15 × 104 EhV-86 g−1 concentrations were obtained, which equate to a 0.06% recovery rate and a 10 to 13% range in viability depending on whether the d 0 or d 23 standard qPCR results were used as baselines, respectively. These results indicate that there was a gain of 10 to 13% in virus viability compared to standard qPCR results. As for the SBMC matrix, the large deviation from the mean for 1st and 3rd quartiles observed for both the standard and viability qPCR data  was significantly greater than in SBMC based on using both standard and viable qPCR methods, SBMO based on viable qPCR, and CF based on viable qPCR, with p = 0.006, 0.001, 0.002, and 0.001, respectively). The uninoculated negative control matrices had negative PCR results  as expected.There was a large range in EhV-86 concentrations in the SBM matrices compared with the F matrix . The avey = −1.528ln(x) + 42.55) was created by plotting virus quantity (virus g−1) against Ct values in the various feed matrices using both viable or standard qPCR  in samples was successfully quantified using the novel technology of the V-qPCR assay.In this study, the algal NCLDV EhV-86 was used as a surrogate for ASFV to study virus survival in selected feed matrices subjected to environmental conditions during a 23-days commercial trucking journey across the United States. Overall, viral load (virus μL50) tests, plaque assays, an EGFP-fluorescent technique, swine bioassays, median tissue culture infectious dose (TCID50) or cytopathic effect (CPE) assays, real-time RT-PCR and quantitative PCR (qPCR) (Current methods used to measure ASFV inactivation include hemadsorption (HADR (qPCR) . AlthougR (qPCR) . PropidiR (qPCR) . These dR (qPCR) . RecentlR (qPCR) .5 virus g−1), conventional soybean meal , and organic soybean meal  after 23-days transport, which implies that NCLDVs like ASFV, are relatively stable in certain feed matrices (5 EhV-86 g−1), conventional soybean meal , and organic soybean meal  after the 23-days transport period via viability qPCR. These results provide empirical evidence that the NCLDV ASFV-like E. huxleyi virus can remain viable in these three types of swine feed matrices for more than 3-wks. Among the three different feed matrices, the variation in the quantity of standard and viable DNA was greatest in the organic soybean meal and least in the complete feed. We also observed an increase in amount of virus detected in complete feed after the 23-days of transport time period compared with the amount on d 0 using the S-qPCR analysis, and an increase in the amount of viable virus detected in complete feed and organic soybean meal using the V-qPCR analysis. The factors contributing to these differences between feed matrices is unclear, but the likely reasons involve differences in their complexity and physiochemical properties . Limited evidence suggests that moisture content and water activity of feed matrices may play a role in survival of some viruses in some feed matrices (4 TCID50 that has been determined for ASFV demonstrates that ASFV can be transmitted orally through contaminated feed, and that repeated exposures exponentially increase the likelihood of infection (In this transcontinental United States transport scenario, EhV-86 viral DNA was present in the complete feed (average Ct value of 22.2 or 3.4 × 10matrices , 2020b. matrices  but no smatrices . Understmatrices . The minnfection , which eIn their recent 23-days bulk transport study, 5 EhV-86 daily. If these values are representative for ASFV, then an ASFV infection would most likely be observed based on current estimates of the minimum infective dose (in situ tests can be run in all real-world scenarios. Such experiments will provide invaluable information on the risk ASFV poses in current practices used in feed mills, refineries, processing plants and farm settings.In our study, a minimum of 1,500 viable EhV-86 per gram of organic soybean meal was detected at the end of a 23-days transport period, which indicates that a pig consuming 1.4 kg of feed a day would be exposed to 9.26 × 10ive dose . HoweverCurrently, chemical mitigation strategies involving formaldehyde, medium chain fatty acids, and glycerol monolaurate have been shown to reduce ASFV infectivity in feed matrices in laboratory settings . These mUse of the NCLDV EhV-86 as a surrogate for ASFV in experimentally inoculated conventional and organic soybean meal and complete feed based on corn and soybean meal was present in a viable form after a 23-days transcontinental truck transport journey. However, sampling sensitivity rather than virus inactivation best explains the variation of in EhV-86 quantity detected in feed matrices after the 23-days transport period. These results demonstrate for the first time that ASFV-like NCLDVs can retain viability in swine feed matrices during long-term transport across the continental United States, thereby providing evidence for the use of EhV as a surrogate for ASFV for evaluating virus survival and inactivation under real-world demonstrations.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.Emiliania huxleyi and EhV-86 stocks, performed the bioassays, ran the virus lysates on the flow cytometer, performed the viability qPCRs and wrote the manuscript. DS and HY designed the viability qPCR experiments. CB created the PCR EhV standards, optimized the viability qPCR, and prepared EhV-86 stock for the 23-day transport study. SD and AS designed and ran the 23-day transport study. DS supervised writing of the manuscript. All authors contributed to the article and approved the submitted version.DS, Jv, PU, and GS designed the use of EhV-86 as a surrogate for ASFV. AP maintained the This project was funded by SAM Nutrition.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.