Japanese soil-borne wheat mosaic virus (JSBWMV) can lead to substantial losses in the grain yield of barley and wheat crops. While genetically based resistance to this virus has been documented, its mechanistic basis remains obscure. In this study, the deployment of a quantitative PCR assay showed that the resistance acts directly against the virus rather than by inhibiting the colonization of the roots by the virus’ fungal vector Polymyxa graminis. In the susceptible barley cultivar (cv.) Tochinoibuki, the JSBWMV titre was maintained at a high level in the roots during the period December–April, and the virus was translocated from the root to the leaf from January onwards. In contrast, in the roots of both cv. Sukai Golden and cv. Haruna Nijo, the titre was retained at a low level, and translocation of the virus to the shoot was strongly suppressed throughout the host’s entire life cycle. The roots of wild barley (Hordeum vulgare ssp. spontaneum) accession H602 responded in the early stages of infection similarly to those of the resistant cultivated forms, but the host was unable to suppress the translocation of the virus to the shoot from March onwards. The virus titre in the root was presumed to have been restricted by the action of the gene product of Jmv1 (on chromosome 2H), while the stochastic nature of the infection was suppressed by the action of that of Jmv2 (on chromosome 3H), a gene harbored by cv. Sukai Golden but not by either cv. Haruna Nijo or accession H602.Infection by the  Japanese soil-borne wheat mosaic virus (JSBWMV), which belongs to the genus Furovirus and family Virgaviridae, can infect barley (Hordeum vulgare), wheat (Triticum spp.), and certain other related species. The virus was first detected in 1973 in Japan  is a pathogen against which several genes for resistance in bread wheat (T. aestivum), durum wheat (T. durum), and barley are known. Sbm1, which confers resistance in bread wheat against both SBWMV and soil-borne cereal mosaic virus (SBCMV), has been mapped to a location on the long arm of chromosome 5D  and Jmv2 (chromosome 3H) present in cv. Sukai Golden act together to confer complete resistance  of four ways: firstly, by blocking the exit of the virus from the vector ; secondlH. vulgare ssp. spontaneum) accession H602 are susceptible  was sampled from each plant. The cotton blue staining procedure adopted for visualizing the propagules has been published elsewhere  step, which was followed by 50 cycles of 95°C/10 s and 55°C/30 s using a CFX96 real-time system device (Bio-Rad). The baseline threshold was set to 100 for both FAM and Texas Red. Two technical replicates were used for each plant, along with three biological replicates per treatment. The Tukey–Kramer method was applied to find multiple-sample means that are significantly different from each other.DNA was extracted from 100 mg of fresh roots using DNeasy Plant Mini Kit  using the suppliers’ protocols. The TaqMan assay used to quantify lsewhere  and listE. coli DH5α competent cells (Invitrogen) for sequencing using a Hitachi 3130X Genetic Analyzer.Total RNA was extracted from each sample using a Qiagen RNeasy Plant Mini Kit using the suppliers’ protocols then converted to cDNA using SuperScript III reverse transcriptase  and SBWMV-UNIR . TaqMan The RNAs required to conduct a TaqMan assay of JSBWMV were extracted from 100 mg fresh plant material using a Qiagen RNeasy Plant Mini Kit fitted with a column DNase Digestion Kit (Takara Bio inc.) using the suppliers’ protocols. The RNA concentrations were estimated spectrophotometrically and adjusted to 50 ng/µl. The primers and probe used in the TaqMan assay were named, respectively, SBWMV_CP_Fw/_Rv and SBWMV_CP_Prove, and their sequences are given in Each 6.5-µl reverse transcription reaction contained 150 ng total RNA, 20 nmol dNTP, 5 pmol SBWMV_CP_Rv, 125ng oligo (dT)12-18 primer , and 0.25 µl RNase free water. The reaction was held at 65°C for 5 min and then quenched on ice before adding 5× First-Strand buffer, 0.5 µl 0.1 M DTT (Invitrogen), 20 U RNaseOUT (Invitrogen), and 100 U SuperScript III reverse transcriptase (Invitrogen). The reactions were incubated for 5 min at 25°C, then for 1 h at 55°C, and then stopped by holding at 75°C for 15 min. The resulting cDNA preparations were diluted by the addition of 15 µl 0.1× Tris-EDTA buffer.t-test for unequal variances was applied to see if two sample means are significantly different.Each 10-µl TaqMan reaction contained 2× SsoFast Probe Supermix (Bio-Rad), 300 nM of the JSBWMV or barley actin primer pairs, 300 nM of either the JSBWMV or the barley actin TaqMan probe, and 6 ng cDNA. The reactions were subjected to an initial denaturing step of 95°C/30 s, followed by 50 cycles of 95°C/10 s, 55°C/30 s, using a Step One System Device . Ct values were automatically analyzed by v2.3 StepOne™ software. The baseline threshold was set to 0.1 and 0.15 for each of FAM and Cy5. Two technical replicates were included for each plant, along with five biological replicates for each treatment. Welch’s P. graminis was able to successfully colonize the roots in each case: Mature sporosori containing large numbers of resting spores were visible within the root cortex cells (P. graminis colonization. The quantitative PCR assay of infected roots suggested the absence of any significant (Tukey–Kramer method) variation in the number of vector spores present in the host roots, which further supported the notion that the hosts’ JSBWMV resistance, rather than inhibiting the entry of the vector, results from the suppression of the virus’ in planta multiplication  than that in the roots grown in sterile soil   than in the non-exposed plants’ leaf tissue but significantly lower  than in cv. Tochinoibuki leaf tissue all the way from January to April. The implication is that the resistance displayed by cv. Sukai Golden acted by suppressing the replication of the virus in the root. The behavior of cv. Haruna Nijo plants was largely similar to that of cv. Sukai Golden plants. During March and April, a lower proportion of cv. Haruna Nijo plants showed a titre above 3.0 in their root or leaf tissue than was the case for accession H602 plants.In the roots of cv. Sukai Golden plants, the viral titre was consistently lower  and a few Hordeum spp. can do so  in wheat, where two dominant WYMV resistance genes (Ym1 and Ym2) are known  are responsible for conferring resistance against barley yellow mosaic virus  must reflect the presence/absence of resistance mechanisms acting only in the roots.For each of the three non-immune hosts, the observable presence of virus in the root  was accompanied by a comparable titre in the leaf. Thus, the translocation of the virus from the root to the shoot was concluded to be largely dependent on a sufficient titre developing in the root. It is therefore unlikely that the gene product of The quantitative assay JSBWMV described here suggests that the translocation of virus from the root to the shoot appears to be largely dependent on a sufficient titre developing in the root. The assay proved to be both sensitive and stable. It was able to highlight genotypic differences in the accumulation of JSBWMV in various parts of the host plant, which allowed for direct comparisons to be drawn between the viral titre present across time and space. Its application was able to characterize the process of JSBWMV infection, multiplication, and translocation from the root to the shoot and to identify at what point in time and space in the host’s life cycle the two known major resistance genes are functional.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/KO, TKa, TKo, and KN conceived the research, KO and KM carried out the microscopic analysis, KO, KM, YO, and KN carried out the qRT-PCR experiments, KO, WX, KM, and TKo the analyzed data, and KO, WX, and TKo wrote the paper. All authors contributed to the article and approved the submitted version.


Emerging and re-emerging zoonotic viral diseases have historically posed a serious threat to animal, environmental, and public health and have impacted economies, trade, and tourism around the world. Highly impactful viral diseases such as COVID-19, SARS, MERS, Ebola, avian influenza and Mpox are associated with multifactorial determinants. Crucially important determinants include pathogen spillover from animals to humans, wide host range, multiple cellular tropisms, and changing host-agent-environment dynamics. These disease-causing factors are cumulatively described as “One Health,” which addresses the intricate interrelationships between human, animal, plant, and environmental health . The OneKhalifa et al.) provided evidence for receptor-mediated spillover of rabies virus in different host species and found the absence of the integrin plexin domain and signaling domain in the integrin β1 (ITGB1) receptor in black fruit bats. It was found that the glycoprotein (G) ectodomain of the rabies virus is indispensable for interaction with ITGB1 in humans, dogs, and bats and for virus entry into different cells/hosts. This molecular docking and structural biology study provided insight into rabies virus transmission dynamics, virulence, and vaccine development insights. Another study  demonstrated that rabies virus triggered activation and polarization of human macrophages with upregulation of macrophage-derived antiviral genes . Furthermore, in vitro rabies virus infection stimulated a unique virus-induced polarization of macrophages from M0 to M1, M2a, and M2c phenotypes and antiviral interferon pathways. This study suggests a potential antiviral role for macrophages in controlling the rabies virus at the cellular and molecular levels.One first study  elucidated the effect of dexamethasone (a steroid) in a combined stress and infectious bronchitis virus (IBV) infection model in chickens. The metabolomics-based study concluded that dexamethasone decreased immunity, caused weight loss, and increased viral load in chicken kidneys in association with increased serum cholesterol, fatty acids, amino acids, and sugar metabolites. This study demonstrated altered persistence and clearance of IBV in the chicken host under induced stress, which has clinical application for the use of dexamethasone during viral infections.One research  found that mucomycosis co-infection in COVID-19 patients in India was associated with an increased mortality rate and pre-existing co-morbidities such as diabetes, cancer, steroid therapy, and elevated iron levels. At the cellular level, mucosal co-infection in COVID-19 patients was mediated by overexpression of glucose-regulated protein 78 (GRP78), availability of hepcidin-dependent free iron, and compromised host immune responses. SARS-CoV-2 entry into host cells was mediated by the angiotensin-converting enzyme-II (ACE2) receptor, which is primarily expressed in lung epithelial cells, the gastrointestinal tract (GIT), hepatocytes, and the reproductive tract  disease mechanisms, as shown by Saeed et al.. This study suggested that the expression of the ACE2 receptor in adipose tissue may lead to the progression of liver injury to non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) in COVID-19-affected patients.The economic and health losses and mortality associated with COVID-19 are attributed to SARS-CoV-2 virulence, variant emergence, demographics, host characteristics, pre-existing infections, and herd immunity. The increased death rate in COVID-19 patients was significantly correlated with age and co-infections. One of the studies  identified the feline panleukopenia virus (FPV) in giant pandas (Ailuropoda melanoleuca) and described its pathogenicity and phylogenetic clusters. The study showed a unique point mutation substitution at G299E residue in the capsid/VP2 protein of FPV, which increased the antigenicity and host range of FPV and posed a threat to the life of the endangered giant panda species. Finally, a case study was reported about a Chinese patient who had co-infections of HEV, E. coli, and Clonorchis sinensis  in biliary drainage. The patient was found to have elevated bilirubin, aminotransferases , HEV-IgM, HEV-RNA, and a positive bacterial culture.Another study (in silico studies that provide evidence for immunopathogenesis, spillover, immune-prophylaxis and clinical insights for the prevention of viral diseases.In conclusion, this Research Topic summarizes findings on rabies virus, FPV, IBV, HEV, SARS-CoV-2, and co-infections in animals and humans, in addition to All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


Especially in many online markets, consumers can readily observe prices, but may need to inspect products further to assess their suitability. We study the effects of product differentiation and search costs on competition and market outcomes in a tractable model of price-directed consumer search. We find that: (i) firms’ equilibrium pricing always induces efficient search behavior; (ii) for relatively large product differentiation, welfare distortions still occur because some consumers (may) forgo consumption; and (iii) lower search costs lead to stochastically higher prices, which increases firms’ expected profits and decreases their frequency of sales. Consumer surplus often falls when search costs decrease. The advent of the Internet has drastically improved consumers’ ability to shop around and compare different offerings. Access to price comparison websites and product search engines enables consumers to obtain price quotes quickly from many different sellers. Yet, while the Internet has substantially reduced the search frictions that consumers face, in many cases it is still costly to gather product information in order to find a suitable product. This motivates research on price-directed consumer search when products are differentiated.However, solving such models can be intricate: If competing firms are ex-ante symmetric, it is optimal for consumers to sample cheaper products first. Firms thus have an incentive to reduce their prices to gain a favorable position in consumers’ search process, which eliminates a pure-strategy equilibrium. Unfortunately, the corresponding mixed-strategy equilibrium turns out to be quite intractable under standard assumptions,match values—are perfectly negatively correlated in the former and are all-or-nothing in the latter, which undermines an understanding of the role of product differentiation on market outcomes. Moreover, two important features are ruled out in these models: Consumers never return to previously sampled firms—so-called returning demand does not exist—and classic deadweight losses that stem from market power cannot arise.2Another route, taken by Armstrong and Zhou  and Dingpartial matches, our contribution is to set up a simple and tractable model of price-directed search through which the role of product differentiation and its interaction with search costs can be studied. We characterize three types of mixed-strategy pricing equilibria, two of which are novel to the literature and give rise to returning demand.By making consumers’ match values binary and allowing for We also analyze the effects of lower search costs—such as caused by the advancement of information technologies—on market outcomes. We show that lower search costs have a perverse effect on firms’ pricing: They lead to stochastically higher prices, which results in higher expected profits. In terms of welfare, there are two conflicting effects: Lower search costs directly reduce the search friction that is incurred by consumers; but lower search costs also lead to higher prices, which may increase the expected deadweight loss. Still, we establish that the former positive effect almost always dominates. This is quite different for consumers, however: Due to the induced higher prices, they are often harmed as search costs decrease.The remainder of this article is structured as follows: In Sect. 4Our paper joins an extensive literature on costly consumer search, which studies the effects of frictions and incomplete information about product characteristics and/or prices on market outcomes.In departures from models of random search, there have been efforts to describe environments in which consumers search firms according to some order. The first papers in this vein focused on predetermined orders, arising, e.g., due to spatial features  decrease in search costs is shared with most concurrent models of price-directed consumer search, including Armstrong and Zhou , Shen 2, Haan etWe study the following market: There are i perfectly suits her needs—the product is “a full match”—with probability i is only “a partial match”, for which a consumer’s willingness to pay is given by There is a unit mass of risk-neutral consumers with unit demand and an outside-option value normalized to zero. All consumers freely observe the prices of all products. However, there is horizontal differentiation in the sense that the consumers do not initially know how well each product fits their tastes: For each individual consumer, product mentclasspt{minimaImportantly, as is common in the literature on sequential consumer search with horizontally differentiated products, we assume that the match values i  after having searched In order to find out their match values, consumers have to incur a search cost The timing of the game is as follows: First, firms simultaneously set prices In order to make the problem interesting, we finally assume that the search cost is not too large: Optimal Search. Since, apart from their prices, firms’ products appear ex-ante identical, consumers clearly find it optimal to search firms in ascending order of prices.The following lemma fully characterizes consumers’ optimal search behavior:If If Optimal Search:i for which The first part follows trivially from the fact that partial matches are irrelevant in the considered case (since they provide a negative net utility); hence consumers who have not found a full match so far find it optimal to search, in increasing order of prices, exactly those firms The second part is true because for Preliminary Equilibrium Results. Having characterized consumers’ optimal search behavior, one may first note that for very high search costs—If See the argument above. Given We will subsequently refer to the parameter region where Proposition If N of the consumers. However, unlike the case where c). This is because, when all rival firms price at c, setting a price in the non-empty range A symmetric pure strategy-equilibrium at any price level above marginal cost cannot exist because firms would have an incentive to undercut marginally, so as to be searched first by all consumers, rather than just by 1/Hence, any symmetric equilibrium must be in mixed strategies. The respective equilibrium pricing CDF must be bounded away from marginal cost because firms can guarantee a positive profit. It must be atomless because otherwise, transferring probability mass from the atom(s) to prices marginally below would pay because this avoids ties. The argument for why no asymmetric pure-strategy equilibrium exists is relegated to the “Appendix”. Preview of Mixed-Strategy Equilibria. It turns out that the symmetric mixed-strategy equilibrium for A “high-price equilibrium”  pricing in a high range strictly above The gap above Proposition Suppose that See “Appendix”. Remarkably, the gap equilibrium price distribution has the property that the spread of its support—mentclass2pt{minimThe described property is in fact used to pin down the explicit functional form of the equilibrium price distribution: From An interesting feature of the characterized equilibrium is that profits are independent of the (common) marginal costs of production. The reason is that higher marginal costs have a similar effect on firms’ incentive to compete as a higher degree of product differentiation  welfare losses in the high-price and gap equilibrium, Corollary As a corollary and for future reference, we may also derive explicit expressions for the social welfare in the different equilibria. In the Bertrand equilibrium, this is trivially given by mentclass2pt{minimThe total social welfare in the market is given byThe surge of the Internet, the emergence of a wide array of price-comparison websites and product search engines, as well as the ongoing improvement of smartphones and mobile applications has arguably led to a steady decline in consumers’ costs of searching and comparing products. In this section, we therefore study the comparative effects of a reduction of search costs within our model framework.We will subsequently define “sales” as price draws that do not exceed Suppose that See “Appendix”. The intuition is that lower search costs make consumers more willing to continue to search after having obtained only partial matches so far, which allows firms to attract these consumers even when they charge higher prices and thereby competition is relaxed. As a direct consequence, firms’ expected prices and profits increase and they may reduce their propensity to engage in sales.As mentioned earlier, the finding that lower search costs unambiguously increase prices and profits is also featured in the models of price-directed search by Armstrong and Zhou , Shen 2, Choi etWe turn finally to the effect of lower search costs on total welfare and consumer surplus:A decrease in search costs: (i) strictly increases the expected total social welfare whenever See “Appendix”. s has two effects on welfare: On the one hand, it directly reduces the aggregate search friction. On the other hand, as was documented above, it makes pricing less competitive, which shifts the equilibrium price distribution to the right. However, since the prices paid are mere redistributions, we need to examine only the effect of lower search costs on the expected consumption surplus net of search costs in order to evaluate their impact on welfare.A decrease in search costs s; thus a decrease in search costs unambiguously improves welfare. In the gap equilibrium, a decrease in search costs actually decreases the expected consumption surplus, since the probability that at least one firm engages in a sale decreases (compare with Proposition In the Bertrand, high-price, and low-price equilibria, the consumption surplus is deterministic and independent of s is smaller when there is a larger number of firms, as it becomes more important to be searched early and hence price competition is generally more aggressive. Moreover, while a decrease in s increases the probability that a single firm prices above all firms price above N is larger. As it turns out, the detrimental welfare effect may dominate only when Still, also for the gap equilibrium, we can show that the reduced search friction outweighs the expected loss of consumption surplus for almost all parameter combinations; the only exception is when Interestingly, the expected consumer surplus instead often decreases after a reduction of search costs. The reason is that consumers have to pay higher prices on average due to the strategic effect on firms’ pricing, which may dominate their gains stemming from less costly search. In particular, we show in the proof of Proposition A partial intuition for this is that for small s for the cases of two, three, and four firms. It can clearly be seen that a reduction of s may indeed decrease the expected consumer surplus in the market over a wide range of search costs.Figure We have developed a tractable model of price-directed search in which consumers observe prices, but need to engage in costly sequential search to discover whether products fully or only partially match their needs. We have characterized consumers’ optimal search behavior and the set of symmetric pricing equilibria that are induced by different degrees of product differentiation.While it turns out that consumers’ equilibrium search behavior is always efficient from a social point of view, welfare losses still occur, as all firms may price above consumers’ valuation for partial matches. If this happens, a fraction of the consumers inefficiently drop out of the market. Investigating the impact of lower search costs on market outcomes, we establish that these lead to higher prices and profits, but typically also to higher total social welfare. In contrast, consumer surplus may well fall when search costs decrease.For future work, it might be interesting to generalize the model by allowing for more general match-value distributions—e.g., by also incorporating a zero match utility—or by including a share of informed consumers who know their match values in advance, as in Ding and Zhang . HoweverAnother promising route may be to introduce various forms of observable or unobservable firm heterogeneity into tractable models of price-directed search, and examine the effects on equilibrium pricing and market outcomes. In particular, the impact of unobservable quality differences on the interaction between firms’ pricing and consumers’ search behavior does not seem to be well understood.Ultimately, we hope that our model will serve both as a useful building block for applied researchers studying markets with price-directed search, and as a starting point for further modeling developments.


Reaching movements are often used to assess selective trunk control in people with neurological conditions. Also, it is known that reaching performance after stroke is increased through training on a mobile seat compared to conventional physical therapy. However, the effect of a mobile seat on joint kinematics has not yet been investigated. This study aimed to quantify differences in the range of motion of the hip and trunk during reaching exercises on a mobile and stable sitting surface. Fifteen healthy participants performed reaching beyond arm’s length on a mobile and a stable seat in four different directions: ipsilateral, anterior, contralateral, and contralateral diagonal. Biomechanical data were collected, including kinematics of the hip and trunk, and surface electromyography of the trunk muscles. The mobile sitting surface led to a higher range of motion in the trunk and the hip in the frontal and sagittal plane, but not in the rotational plane. Differences between reaching directions were found in all joint directions, except that of trunk flexion. Hence, movement patterns of the hip and trunk differ during reaching on different sitting surfaces and in different directions. A larger range of motion in the frontal or sagittal plane while training on the mobile seat provides added neuromuscular stimuli to the trunk muscles (= a higher demand on trunk muscles), which could result in more efficient training and therefore, increased trunk control after stroke. However, this has to be investigated in a future study with people after stroke. Many activities of daily living, such as putting on socks, tying a shoelace, or reaching for the telephone during office work, are performed in sitting and involve reaching movements , 2. To pDecreased trunk coordination and limited muscle strength are associated with impairments after stroke , 7. FollDiminished selective trunk control in people with neurological conditions could be trained using trunk exercises, such as reaching in sitting . SystemaThe aims of this study were twofold. Firstly, to quantify the differences in the range of motion (ROM) of the hip and trunk between the mobile and stable sitting surface. Secondly, to investigate whether the factors of muscle activity, maximal reaching distance, or reaching direction could explain possible differences in the ROM of the hip and trunk.-2, and being able to understand verbal and written instructions in German. The exclusion criteria were the presence of acute or chronic musculoskeletal, neurological, or cardiopulmonary diseases, scoliosis, and pregnancy. Participant characteristics are provided in Between September 2020 and March 2021, 15 healthy participants were recruited at the university campus  via mail. Prior to enrolment, screening was made by verbal interview using preselected criteria. The inclusion criteria were over 18 years of age, a body mass index of 18–28 kgmThe medical Ethics Committee of the Canton of Zurich juristically verified the study (Req-2020-00569), which complied with the tenets of the Declaration of Helsinki. Each participant provided written informed consent prior to the start of the study.The mobile seat used in this study was developed to support chronic stroke patients during their sitting balance rehabilitation. Through U-shaped rails, it has the ability to move in the sagittal and frontal plane, or a combination thereof, thus allowing a 3D range of movement . The seaThe starting position for each reaching task was sitting upright, with the hip and knees at a 90° angle, feet hip-width apart and placed on a footstool . For conTime standardization of the task was performed as following. Starting position was considered as 0% of task time, while returning to the starting position after the reach was defined as 100% of task time.Data collection took place in the movement laboratory of Zurich University of Applied Sciences. Arm length was measured from the acromion to the pointing finger with the arms hanging loosely next to the body. Reflective markers were placed on the dominant side of the participant’s upper arm and pointing finger, as well as on the thigh, pelvis, and trunk. The markers on the thigh, pelvis, and upper arm were placed according to the cluster marker model of List et al. 2013). The marker model of Rast et al. (2016)  was used13. The mth order Butterworth filter with a cut-off frequency of 7 Hz was applied to the marker data. Post-processing of sEMG-data was performed as in a previous study using the mean of the five repetitions as outcome [After shaving and cleaning of the skin, bipolar electrodes  were placed bilaterally at the M. Multifidi (MF), M. Erector Spinae (ES), and M. Obliquus Externus (OE). Electrodes were placed according to the recommendations of the surface electromyography (sEMG) of the non-invasive assessment of muscle project and Ng and colleagues , 22. The outcome , 24. MaxThe mean ROM of the five repetitions of each angle was used as the outcome variable for statistical analysis. In hip kinematics, the dominant side was used for statistical analysis since it can be assumed that the movement of one side of the hip depends on the other side’s movement. Included co-variates were maxreach as well as maximal RMS sEMG in percentage of the static sitting trial. Linear-mixed models were used for all outcome variables with a significance level of p < .05. Firstly, all covariates  were included in the model. Then, through backwards optimization, the model with the best fit  for all three directions of the joint angle was found and the same models were applied to all joint directions. An overview of the tested models can be found in the After application of the statistical model, marginal means were estimated  to predict the ROM of future data using the function emmeans. R Version 4.10 , includiDescriptive values as well as joint angle illustration plots for the ROM of the hip and trunk in all four reaching directions, can be found in the R2 = .82).Differences in the ROM of hip adduction between the mobile and the stable seat condition were small and not significant, although the ROM tended to be slightly higher on the mobile seat. While in the anterior and ipsilateral directions the predicted ROM was smaller than 6°, in the contralateral and the contralateral diagonal directions the predicted ROM was between 10° and 15° . The preR2 = .80).The predicted ROM for hip flexion was higher during mobile sitting in all reaching directions. Anterior reaching showed the most prominent differences between the mobile and stable seats. In addition, anterior reaching showed the highest predicted ROM . IpsilatR2 = .54). On the one hand, differences between the seat conditions in all reaching directions were small, but on the other hand, the predicted ROM was higher in the ipsilateral reaching direction compared to the other directions  = 9.57, p < .001 and maxreach F  = 15.13, p < 0.001 (rections .R2 = .85). The highest predictive ROM can be seen during contralateral reaching, followed closely by contralateral diagonal reaching. A few degrees less were predicted for ipsilateral and for anterior reaching  = 43.21, p < .001 and maxreach F  = 7.17, p = .009 (reaching .R2 = .77). Reaching direction showed no significant main effect. A similar ROM was predicted for contralateral, anterior, and ipsilateral directions. The diagonal contralateral direction showed a smaller predicted ROM (Table 3).Differences in trunk flexion between the seat conditions were around 3–5° for all reaching directions, with a greater predicted ROM during mobile sitting. The main effect of trunk flexion was significant for seat condition F  = 12.67, p = .002 and maxreach F  = 9.61, p = .003 (R2 = .94). Overall, the greatest ROM was predicted for contralateral reaching, followed by contralateral diagonal, ipsilateral and anterior reaching directions  than in anterior reaching . Hof et Combining the predicted ROM of the trunk and hip results in distinct movement patterns for each reaching direction. The ROM of the hip during ipsilateral reaching differs between the mobile and stable seat in the lateral and rotational components, in the anterior component it is the trunk. Therefore, the mobile seat is compensated by either the hip or the trunk. Rotation of the trunk and hip, as well as hip adduction and trunk lateral flexion, were probably smallest during anterior reaching because the movement is unidirectional. While flexion of the trunk during anterior reaching is similar to other directions, flexion of the hip is high. During anterior reaching the BoS through the feet is high, which leads to higher muscle activation of the legs, lower muscle activation of the trunk, but higher stability , 32, 36.Possible limitations of this study were the uncontrolled target height and speed of the reaching movement. Since, however, reaching was performed to a pylon with a standardized height and the task time was normalized, it can be assumed that this did not affect the ROM of the hip and trunk. Due to the Covid-19 pandemic only a small, young and physically fit sample size could be recruited. This does not represent the general world population. It is known from literature that the average distance reached, as well as the lateral pelvic tilt range, decreases with increasing age . Due to A mobile sitting surface leads to a higher ROM of the trunk and the hip in the frontal and sagittal planes, but not in the rotational plane. When a larger range of motion in the frontal or sagittal plane is desired, the mobile seat would be a good training tool for trunk rehabilitation since there would be additional training stimuli. However, this has to be investigated in a future study of kinematics on the mobile and stable seat in people after stroke.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.


Spodoptera litura (Fabr.). Zeolite nanoparticles were prepared using the beads-milling method. The statistical analysis was carried out by a description exploration method with six replications. The occlusion bodies’ concentration in the virus formulation was 4 × 107 OBs in 1 mL medium. Zeolite nanoparticles formulation sped up the lethal time significantly (7.67 days) compared to micro-size zeolite (12.70 days) and only nucleopolyhedrovirus (8.12 days) and received acceptable mortality (86.4%). The zeolite nanoparticles delivery system provides an alternative formulation for nucleopolyhedrovirus with a significantly improved speed of killing the virus while maintaining suitable efficacy of the virus preparation in terms of the prevalence of mortality.Synthetic insecticides frequently cause pest resistance and destroy non-target organisms. Thus, virus formulation is an issue that deserves considerable attention in developing virus-based insecticides. The hindrance of using nucleopolyhedrovirus alone as a virus-based insecticide is due to slow lethal time, though its mortality remains high (100%). This paper reports the formulation of zeolite nanoparticles as a delivery system to accelerate lethal time in controlling  Spodoptera litura (Fabr.) (Lepidoptera: Noctuidae), also known as armyworm, is a polyphagous and widely distributed pest of many vegetables, fields, and ornamental crops [S. litura may lead to the development of resistant pest populations and negatively affect non-target organisms [S. litura, to reduce the use of synthetic insecticides. [al crops ,2. The lrganisms . In the ticides. ,3.Microbial agents  or their bioactive agents can be used as active substances and therefore are referred to as microbial pest control agents (MPCA) . They arHelicoverpa armigera [S. litura larvae called Helicoverpa armigera nucleopolyhedrovirus subculture (HaNPV1).Viruses as biocontrol agents are an essential component of integrated pest management. This control method has been used for significant insect pests . For exaarmigera . This coarmigera . Howeverarmigera ,10 becauarmigera ,8 and faarmigera ,11. In aZeolites are microporous, chemically inactive, crystalline aluminosilicates with promise as crop protection agents ,13,14. ZSuaeda maritima-based herbal coils as potential biopesticides against the dengue vector Aedes aegypti (Diptera: Culicidae) and the tobacco cutworm Spodoptera litura (Lepidoptera: Noctuidae) [S. litura, by increasing the effective use of pesticides and decreasing pesticide residue [The use of nanotechnology to design and manufacture targeted pesticides with an environmentally benign, controlled release through chemical modifications and compounds has been suggested to offer great potential for creating new formulations (citation). Nanopesticides for plant protection is an emerging field of research, and nanopesticide-based formulations can help the development of more effective and safer pesticides and biopesticides  ,16. For ctuidae) . Thus, t residue .HaNPV1 (control) by assessing the mortality and lethal time of S. litura exposed to these products.Nano-sized zeolites have been found to be more reactive than conventional micro-sized zeolites . HoweverS. litura larvae have been taken from the Indonesian Vegetables Research Institute, in 517 Tangkuban Parahu, Lembang, West Java, Indonesia 40,391 . These insect larvae were kept in 1000 mL plastic containers covered with insect screen cloth and reared in a growth chamber at 28.1 °C and 12:12 h (light:dark) photoperiod [Fourth instars toperiod . Insectitoperiod .S. litura larvae called Helicoverpa armigera nucleopolyhedrovirus subculture (HaNPV1).The larval were reared until the pupal stage. The pupae were placed in a rearing container with soil as pupal substrate until they hatched into the imago. The imago cage is equipped with ovitrap, which is fed with 10% honey solution. The eggs are separated in a different container until they hatched into the first instar larvae . Larval Virus preparation materials using sodium dodecyl sulphate , Tris buffer , sodium chloride , Aquadest , and sodium azide . Natural zeolite is used as a delivered medium for virus formulation.HaNPV was isolated from H. armigera larvae as the main host with a production rate of 9.5 × 109 OBs mL−1. HaNPV1 is a subculture of HaNPV in S. litura as an alternate host with enhanced OBs production 1.55 × 1011 OBs mL−1 compared to HaNPV produced in H. armigera as the main host.Originally, HaNPV1 culture was obtained from the Laboratory of Applied Microbiology Department of Biology, Faculty of Mathematics and Natural Sciences Universitas Padjadjaran. The HaNPV1 was produced by propagating the HaNPV in S. litura as an alternate host. The virus was isolated after one passage subculture in S. litura larvae. Spodoptera litura third instar larval were infected by a virus suspension with a concentration of 4 × 105 occlusion bodies (OBs) mL−1. The infected larval was collected in a 100 mL glass chamber and stored at 4 °C. Then, the cadavers of 40 larvae in the same cohort were crushed by mortar and mixed with 20 mL Tris buffer solution  and 20 mL 0.1% sodium dodecyl sulfate (SDS) solution. This concentrate was stored at 4 °C for 24 h [The subculture of the for 24 h .After storage, the concentration of the virus was filtered with two layers of muslin cloth in a sterilized 100 mL glass chamber. The 8 mL suspension of the virus was transferred to a 10 mL centrifuge tube with a pipette. Tubes were placed in a centrifuge, and the virus suspension was processed at 3500 rpm for 15 min at 4 °C. After 15 min of the first centrifugation, the virus suspension was divided into a supernatant solution and a solid precipitated polyhedral. Then, the solid precipitated polyhedral was washed three times with the following procedure. The supernatant solution was discarded, and the solid precipitated polyhedral was removed with a pipette to the centrifuge tube. The solid precipitated polyhedral was resuspended in 5 mL Tris buffer solution  and mixed with 5 mL 0.1% SDS solution. The mixture was centrifuged at 3500 rpm for 15 min at 4 °C. After washing three times, the solid precipitated polyhedral was resuspended into a mixture of Tris buffer solution  and 0.1% SDS solution by adding 0.2% sodium azide.9 OBs/mL solution. The virus suspension with 4 × 107 OBs/mL concentration in the suspension medium was used for bioassay.To count the numbers of the virus OBs, 0.1 mL of resuspended virus pellet was mixed by adding 0.9 mL mixed solution of Tris buffer  and 0.1% SDS with a 1:1 ratio. Counts were done with a Neubauer hemocytometer by a light microscope with a magnification of 400. The presence of OBs in the suspension was the cuboidal shape and green color. The virus structure was observed by scanning electron microscope (SEM/JEOL-JSM 6360LA) magnifier 30,000×. The concentration of stock virus suspension is 2.6 × 10Zeolite nanoparticles were prepared by the beads-milling method. Bead milling is one of the equipment or top-down processes for the size reduction of bulk particles into fine particles ,23. CommAfter dispersion, the suspension was pumped from the dispersing section to the separation region where centrifugal force was used to separate the zirconia beads from the particle suspension. The zeolite suspension was then recycled back to the dispersing section. To keep the temperature of the system constant, the vessel was supported by a water jacket system and was completely sealed. The PEG 400 content in suspension was 150 weight fraction of zeolites. The water-suspended ZNPs with weight fractions of 0.1 wt%, milling time of 120 min, and addition of acetic acid solution (1 vol%) after milling to promote the positive surface change were used for optimal technical conditions for beads milling.The particle size distribution of ZNPs was measured using dynamic light-scattering (DLS) equipment with A DelsaTM nano series instrument . Micro-sized zeolite powder was mixed in water, and the concentration of ZNPs in the suspension was 1%.HaNPV1: ZNPs) as an adjuvant was made by mixing 15 mL stock virus solution and 1000 mL ZNPs suspension. Virus formulation in Z and ZNPs can be used for larval infection. The virus suspension concentration formulated in Z and ZNPs was 4 × 107 OBs gram powder or mL−1 solution. Both Z and ZNPs suspension as an adjuvant without virus were used for bioassay test larvae.Virus formulation with zeolite (Z) as an adjuvant was made by mixing 15 mL stock virus solution and 1000 g zeolite. The mixture was stirred evenly and dried at 22–28 °C for a week. Virus formulation in ZNPs  was defined as the number of infected larvae that died out of the total number of larvae tested during observation. The lethal time was defined as the time required for larvae to die after a virus infection.The third instar larvae of N is the number of polyhedral counted, V is the area counted (Neubauer counting chamber), and P is the dilution factor.The total number of polyhedral was calculated using the formula as follows:The average lethal time was calculated using the formula in Equation (1) for 14 days of observation, according to Prasetio et al.  as folloW is the average lethal time, iW is the lethal time on day i (and not day 1), iZ is the number of insects that died on day i (and not day 1), and Y is the total number of insects that died. The data were collected and organized using Microsoft Excel . Data distribution showed homogeneity. The obtained data were analyzed by ANOVA using IBM SPSS Statistics (version 22). Their significance was determined by Duncan’s multiple distance test (DMRT) with p-values < 0.05 considered significant.HaNPV and HaNPV1. The morphology of HaNPV OBs, as shown in HaNPV1 OBs in an alternate host showed a spherical shape and porous structure. SEM images observed the subculture polyhedral’s spherical and porous structure shape with magnification of 8500 , alumina (Al2O3), or aluminosilicate (AlSi). This Z also contained alkaline earth metals  and other elements, such as Mg, Na, and Fe.HaNPV1 suspension were used as the control and resulted in 100% mortality. The mortality of larvae exposed to Z was 37%, while ZNPs caused 49% mortality. Larval mortality of 78.16% was observed for larvae treated with HaNPV1 + Z and of 86.40% for HaNPV1 + ZNPs. In this condition, the existence of ZNPs as an adjuvant in the formulation causes a decrease in the viability of the virus. However, the obtained larval mortality still meets the minimum standard of efficacy of the national pesticides commission [In this study, larvae infected by mmission .Many researchers reported the use of ZNPs in virus formulation showed higher mortality compared to Z ,28,29. TS. litura larval cadaver non-infected and infected by HaNPV1 is shown in HaNPV1 + ZNPs showed liquified bodies, abnormal body size, and stinky differences with noninfected larvae  and ZNPs were applied, the synergetic mechanism occurred, as shown in HaNPV1 + ZNPs biocontrol agent provided faster lethal time and possessed high mortality compared to only HaNPV1 or ZNPs. The ZNPs have a strong affinity to target such proteins and initiate the abrading to membrane cells, causing the virus to easily enter the cell [HaNPV1 reduce the lethal time of larvae due to effectively damaging cell membranes and creating fast viral replication in midgut cells [In contrast, when both virus  with a lethal time of up to 8.12 days. The mortality of larva against virus and micro size zeolite (HaNPV1 + Z) was very low (80%) with a longer lethal time of up to 14.6 days. In contrast, the additional ZNPs included in the virus formulation (HaNPV1 + ZNPs) caused 86.40% mortality and reduced the lethal time to 6.2 days. We conclude that ZNPs acted as an adjuvant and decreased the lethal time of S. litura significantly when infected by HaNPV1 with acceptable mortality. Thus, based on the mortality and lethal time, the best formulation was HaNPV1 + ZNPS. Therefore, the proposed formulation has the potential to be used as an alternative to synthetic insecticide to address the critical issue of the slow lethal time of the application of HaNPV1.The larvae mortality was 100% when infected only by virus formulation (


Given the recent outbreaks of the Marburg (MARV) virus within the first quarter of the year 2023, interest in the MARV virus has been re-ignited given its shared phylogeny with the dreadful Ebola virus. This relation gives some insight into its virulence, associated morbidities, and mortality rates. The first outbreak of MARV recorded was in Germany, in 1967, of which seven died out of 31 reported cases. Ever since, subsequent cases have been reported all over Africa, a continent replete with failing healthcare systems. This reality impresses a need for a contemporary and concise revision of the MARV virus existing publications especially in the areas of vaccine research. A functional MARV vaccine will serve as a panacea to ailing communities within the African healthcare landscape. The objective of this scoping review is to provide pertinent information relating to MARV vaccine research beginning with an outline of MARV’s pathology and pathogenesis in addition to the related morbidities, existing therapies, established outbreak protocols as well as areas of opportunities. IntroductionMarburg (MARV) virus belongs to the Filoviridae family which is known to cause hemorrhagic fever . Due to MARV has been listed on the Priority Disease List in 2018  given itEfforts to continue ongoing vaccine research have been reignited since the recent outbreak in 2023 concerning which the Center for Disease Control (CDC) issued a travel alert against traveling to Equatorial Guinea and Tanzania. The outbreak was declared on February 13, 2023, in Equatorial Guinea with at least nine confirmed cases and seven deaths as well as 20 suspected deaths caused by the virus . AnotherMaterials and methodsSearch StrategyPubmed and Google Scholar were the search engines used for this scoping review from the start of the study till date of publication. MeSH terms relating to MARV were used and include MARV vaccine, MARV therapies, MARV treatments, MARV, MARV pathology, and pathogenesis to name a few. The websites of the WHO and CDC were consulted for established outbreak protocol using such MeSH terms as virus outbreak and MARV precautions. Sections of the paper were selected by each reviewer and the said reviewer was tasked with conducting independent search and selection of the relevant studies related to the chosen section. The separate body of work was joined together and critiqued by all reviewers for relevance before a final draft was established. All duplicate studies were removed and the lead reviewer made the final edits. Study Design and Inclusion CriteriaThis scoping review included only MARV vaccine research of which Phase 1 clinical trials had commenced. The selected studies administered a vaccine regimen to healthy participants and measured the strength of protection conferred over weeks to months. Given the limitations in available literature surrounding MARV vaccines, the chosen studies were not assessed for any risk of bias. A comprehensive scoping review was conducted using sources that allowed access to peer-reviewed literature as well as publicly available health guidelines. HistoryMARV was first discovered during a series of outbreaks of hemorrhagic fever in the German cities of Marburg and Frankfurt in 1967; the name of the virus came from the city that had the highest number of infections . A totalMARV reintroduced itself with two substantial outbreaks in the Democratic Republic of the Congo (DRC) in 1998-2000 and in Angola (Western Africa) in 2004-2005 -13. The By 2008, there had been 452 total cases reported and 368 confirmed deaths, but experts believe that these numbers are likely higher in reality . AccordiRisk factorsRousettus aegyptiacus) or their secretions particularly when visiting the caves or mines in the Sub-Saharan region of Africa , headache, sore throat, muscle aches, joint pain, and malaise 2-21 days after the initial infection , 28-29. Phase 2Also known as the early organ phase which lasts from days 5 to 13 after the initial onset of symptoms. Infected individuals may develop conjunctival injection, shortness of breath, viral exanthem, and edema . PatientPhase 3Also called the late organ phase or convalescent phase. This phase usually has one of two clear outcomes; patients either recover from their illness completely or die due to internal bleeding and multiple organ failure , 38. Pat MARV pathophysiology in humansThere are limited clinical studies available for MARV due to the rural conditions as well as the critical phenomenon of most of the MARV outbreaks in Africa, and the collection of laboratory and pathological evidence from individuals infected with the virus has been traditionally insufficient. Our knowledge of clinical symptoms and physical exam findings is derived from the information linked to the first epidemic in Germany, with the South African outbreak and smaller outbreaks in other regions of Africa .MARV primarily affects the phagocytic system which was found during the immunochemical and electron microscopic investigations of a case in Kenya in 1987, which detected MARV antigen and virions in both circulating and associated macrophages . Flow cyIn MARV infection, the liver has the most distinct histopathological characteristics with hepatocytes demonstrating intracellular eosinophilic and filamentous or ovoid inclusions of the virus . It can Patients usually have disseminated intravascular coagulation (DIC), along with the overall systemic spread of the virus that leads to the multiorgan failures seen in severe and lethal MHF -57.MARV hemorrhagic feverMARV primarily targets macrophages and dendritic cells -59. It lMARV impairs the adrenocortical cells in the adrenal gland, thereby resulting in the impaired release of steroid-synthesizing enzyme production and release which causes hypovolemia and hypotension, leading to shock .Clinically significant hemorrhage has been observed among MARV-infected individuals at both the macroscopic  and microscopic  level . During Clinical presentationMARV disease (MVD) cannot be clinically diagnosed with high certainty and this creates a unique difficulty when it comes to early detection . The sigManagement and treatmentAt this time, no treatment currently exists for MVD. Management primarily involves addressing the symptoms and providing supportive care in response to metabolic derangements, to prevent further complications or death. After exposure to the virus, it is crucial to manage pain if reported, replenish fluids and electrolytes to prevent dehydration, stabilize oxygen levels and blood pressure, replace blood or clotting factors in the case of hemorrhaging, and treat any secondary infections or complications .Preventative measures and recommendationsMARV can be transmitted from its natural host, the Egyptian fruit bat (Rousettus aegyptiacus), to both human and non-human primates . InfectePrimary transmission of the virus from fruit bats and non-human primates can be greatly limited or eliminated by reducing direct contact with these animals . SecondaIn addition to the aforementioned measures, proper education of communities of both healthcare and non-healthcare personnel will improve awareness and stop the spread of the virus -71. SuchVaccines and researchAs of this writing, no vaccine or specific therapy (aside from supportive care) has been approved worldwide or specific therapy for MARV disease. Many MARV candidate platforms for non-human primates (NHPs) have been demonstrated to provide protection against viruses such as recombinant vesicular stomatitis virus (rVSV), virus-like particles (VLPs), and adenovirus (AdVs) . The thrVaccines using DNA vectors are promising, but their comparative efficacy as a stand-alone platform has been limited in primates due to suboptimal gene expression or cell targeting -76. A MASince 1967, there have been only 16 outbreaks of MVD, including that involving over 100 cases. Most outbreaks are relatively small and need to be controlled quickly using existing infection control and public health measures. Therefore, it is suggested that multiple outbreaks could be used to conduct Phase 3 trials for MVD vaccines until sufficient endpoints have been accumulated to assess vaccine efficacy (VE) and can be estimated by estimating the number of outbreaks required . Several adenovirus-based vaccine studies are limited to MARV. Phase 1 clinical trials have demonstrated that an experimental MARV vaccine called the cAd3 Marburg vaccine displays a GP found on the surface of MARV to induce immune responses . ClinicaIn 2016 a Phase 1 clinical trial was published where 87 participants were randomized to receive either Zabdeno  or the MVA-BN-Filo vaccine, encoding GPs common among viruses found within the same phylogeny as MARV , 81. FolThe WHO states that the odds are against a successful trial because the necessary measures to combat an outbreak (e.g. quarantine) could potentially end the outbreak before a single vaccine can be administered. Even if a trial has commenced, there may be an inadequate number of cases to accurately judge the vaccine’s effectiveness. Evidence points to creating an effective vaccine by gathering information from multiple outbreaks . DiscussionThe recent COVID-19 pandemic has brought all the potential pandemic-causing pathogens to the limelight. Even though the epidemiological spread of respiratory viruses is much easier than contact-spread viruses like the viruses of the Filoviridae family, the high mortality rate of these viruses always draws focus and cannot be ignored. The average mortality percentage of the MARV infection is estimated to be about 50%, compared to the estimated mortality rate of the SARS-CoV-2 (Covid-19) virus at only 0.5%-2% , 84. SoaOnly a handful of MARV outbreaks have been reported but the devastation it has caused has been enormous. Although infected fruit bats act as the primary host, the virus has been reportedly found in other animals like pigs and nonhuman primates furthering the transmission chain . Hence, Foremost importance has to be given to improving awareness of MVD among the general population of the endemic areas as well as healthcare personnel. It is crucial that they are educated not only about the modes of viral transmission but also on how to identify the early symptoms of MVD. This ensures early identification and isolation of the infected individuals and may help contain the outbreak. Moreover, definitive government policies and protocols need to be forged to minimize or prevent human-to-human transmission, with particular attention on limiting exposure to bodily fluids. This can include hygienic and dignified burial practices, safer sexual practices, and environmental hygiene. Nosocomial spread of the virus must be taken extremely seriously and is necessary to limit the interaction of healthcare personnel with the infected individual. Furthermore, all healthcare personnel have to be educated about self-protective practices, respiratory hygiene, safe injection practices, proper hand washing techniques as well as proper contaminated waste disposal techniques in order to protect themselves and other patients in the hospital . Deeply rooted cultural or traditional practices and lack of healthcare education amongst the masses can sometimes lead to people acquiring the virus. An example of this is unsafe cultural burying practices in the endemic regions of Africa which may cause significant exposure to the dead bodies who fell victim to the MARV . The govThe MARV disease has no known cure or approved vaccine; hence, all the research is limited to only a handful of highly fortified laboratories around the world. Various types of vaccines are in different stages of development but novel vaccine platforms like the viral vector vaccine and DNA vaccine have shown protective efficacy in rodents and nonhuman primates . The mosEffortless exchange of information about the virus between governments and multinational organizations like WHO is crucial. This will not only help other countries screen their own people but also ensure the victim countries receive international help as soon as possible. International protocols and regulations must be set up in order to be prepared for an anticipated outbreak. Governments around the world along with the UN and WHO must bring about policies for pandemic education, containment measures, and of healthcare personnel training. Anticipating an outbreak and having the infrastructure and resources ready, and trained manpower to overcome is the best strategy/blueprint for the future.Recurrent outbreaks and the high mortality risk of the Filoviridae family of viruses make it very essential to continue MARV vaccine research. Concrete governmental policies are needed to limit the exposure of people to zoonotic hosts and minimize human-to-human transmission if an outbreak is confirmed. It is crucial to increase awareness and education in the general population about the animal hosts of the virus, viral transmission, and precautionary measures that need to be taken to prevent the transmission of the virus. Healthcare personnel must strictly adhere to the regulations and protocols set up by governmental and international agencies like WHO to curb any possible nosocomial spread. It is the need of the hour for extensive research into developing a cure or a vaccine that can potentially save hundreds if not thousands of lives in the future.


Varroa destructor is an ectoparasitic mite of honeybees which vectors a range of pathogenic viruses, the most notable being Deformed wing virus (DWV). Mites parasitise bees during pupal development and male honeybees, drones, have a longer development cycle than female workers (24 versus 21 days), allow for more progeny mites to develop per foundress (1.6–2.5 compared to 0.7–1.45). How this longer exposure time influences evolution of the transmitted virus population is unknown. Using uniquely tagged viruses recovered from cDNA we investigated the replication, competition and morbidity of DWV genotypes in drones. Assays examining virus replication and morbidity revealed drones are highly susceptible to both predominant genotypes of DWV. In virus passage studies using an equimolar inocula of major DNA genotypes and their recombinants, the recombinant form dominated but did not reach 100% of the virus population within 10 passages. Using an in-silico model of the virus–mite–bee system we examined bottlenecks during virus acquisition by the mite and subsequent injection of viruses into the host, which may play a significant role in shaping virus diversity. This study furthers our understanding of the variables influencing DWV diversity changes and provides insight into areas of future research in the mite–virus–bee system. Together, Varroa and DWV are the most prevalent honeybee pathogens and are highly associated with global colony losses [Deformed wing virus (DWV) is an endemic single stranded positive sense RNA virus of honeybees which is transmitted by the ectoparasitic mite y losses , in party losses , and very losses ,4. Varroy losses –8. At thy losses .Varroa have evolved to preferentially enter drone cells—up to 11.6 times more frequently—when they invade colonies [colonies . The incet al. [in vitro passages [in vitro studies , may be a significant influence on the resulting virus population.There is a correlation between the level of mite infestation and an increase in the amount of DWV, but a decrease in the diversity of DWV at the colony level . This haet al.  demonstrpassages . This maIf certain DWV genotypes replicated in the mite vector they may gain a selection advantage in the virus population. Contrasting reports have shown evidence of DWV replication in mites, depending on the variants investigated, with evidence of Type B and recombinants  replicating in mites ,22, wherIn this study, we investigate the replication kinetics of clonal DWV populations, and demonstrate that all major DWV genotypes and recombinants achieve similar high viral loads in drones. In addition, the variants exhibit broadly similar morbidity rates and replicate to similar levels in coinfection experiments, as previously shown for worker pupae ,27. In s. 2 (a)Apis mellifera) used in this study were obtained from the University of St Andrews research apiary. Colonies were regularly and appropriately treated with miticides to manage Varroa levels and endogenous DWV levels were tested throughout the season. All pupae were removed from the comb and placed in an incubator 24 h prior to any injections.All honeybees ( (b)in vitro using a T7 Ribomax Express system (Promega). DWV was recovered following direct injection of honeybee pupae as described previously [The infectious cDNA copies of DWV, designated DDD, VVV and VVD, have been described previously ,28,29 an (c)3/106 GE for infectivity and 102 GE for morbidity, and incubated  for 48 h for the infectivity experiment, or until eclosion for the morbidity experiment. All bees were analysed in the morbidity experiment, including those that died prior to eclosion.For infectivity and morbidity studies pink eyed pupae were injected using an insulin syringe  with crude viral stocks, diluted in PBS to the concentrations of 103–1010 genomic equivalents (GE) per μg. The VVV and VVD constructs could not be distinguished by these primer sets, so are not co-infected in the infectivity or morbidity studiesTotal RNA was subsequently extracted following tissue homogenization  with a Genejet RNA extraction kit (Thermo Fisher) according to manufacturer's instructions. Approximately 1 µg of RNA per sample was used to generate approximately 1 µg cDNA following the manufacturer's protocol (Quanta scientific) with both oligo dT and random hexamer primers included in the reaction mixture. qPCR reactions were performed for DWV in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Reactions consisting of 1× Luna Universal qRT-PCR master mix (New England Biolabs), 0.25 µM forward and reverse primers and approximately 100 ng of total cDNA in a final volume of 20 µl. Type A and B variants were quantified using primer pairs unique to ‘Type A’ variants (DDD qPCR FP– 5′ GTCAAGAAGCAGGCGAATGTA 3′/RP – 5′ GCATAGGGGATCTAGAACACATAG 3′) and ‘Type B’ variants (VVV qPCR FP—5′ GAAAAGACGCGGGTGAGTTCG 3'/RP—5′ AACATTGGCGATCGATTACAAACG 3′) as per . Negativ (d)4 GE/μg RNA per 5 µl. For passage experiments pink eyed drone pupae were injected with 5 µl of the virus mix and incubated for 48 h. In each case the virus was injected into the first pupa and left for 16 h to create a ‘stock’ population of viruses . VirTaq polymerase under the following conditions: an initial denaturation step of 94°C for 20 s, 30 cycles of 94°C for 15 s, 57°C for 20 s and 68°C for 2 min, and a final extension at 68°C for 10 min. Amplicons were purified and each sample was barcoded for Illumina Miseq sequencing (data available at NCBI BioProject: PRJNA888084). Illumina reads were converted to fasta format, extracted and trimmed using Geneious Prime (v. 2019.1.3). To determine the proportions of each of the three variants present in any generation the haplotype diversity of the sequenced samples was determined using ShoRAH (short read assembly into haplotypes) [a).To analyse the virus population in each of the ten pupal generations, samples were prepared for next generation sequencing. One bee per generation in each of three of the four biological replicates was selected for sequencing, giving a total of 30 samples across the ten passaged generations . The three infectious clones could be distinguished by their sequence from one another and from any pre-existing infecting virus by the inclusion of translationally silent restriction sites. A region of approximately 1500 bp encompassing a known hotspot of recombination  and the introduced silent restriction site tags (AvrII and HpaI as per ) was amplotypes) . All set (e)The model to measure potential bottleneck events in the virus population was generated in R studio (V3) . Variablper os. This process repeats for 10 cycles of pupae before stopping. Each virus is calculated as a percentage of the total virus population and determined at the point where the amplified virus is mixed with the PupaVirusLoad in the new pupa. The full 10 cycle loop is repeated 10 times to give 10 populations, which are then used to create an average for each altered variable in Briefly, the for-loop takes the 1000 viruses generated from Variants and PupaVirusLoad  and rand. 3 (a)3 and 106 GE, selected as the lower and higher levels of mixed population infection levels [9 GE/μg RNA) except for one of the DDD samples (a). Equimolar co-infections of DDD/VVV and DDD/VVD, which could be distinguished using type-specific qPCR primers, revealed that both viruses reached similarly high levels. However, two low-level DDD/VVD samples had considerably lower DDD levels (DDD approx. 105 GE/μg RNA and VVD approx. 1010 GE/μg RNA).The three major variants of DWV  have all been shown to replicate similarly in  worker bee pupae and cause symptoms typically associated with DWV disease ,27. Howen levels ,21) of t samples a. Equimoa), but only approximately 80% have overt deformed wings upon eclosion. To examine whether morbidity differs in drones, white-eyed pupae were injected with 102 GE/μg RNA of the three variants individually or with two co-infections  and left until eclosion (8–10 days). Figure 3b shows the percentage morbidity of each group, with emerging drones classified as dead, deformed or apparently normal with figure 3c showing the final viral level after eclosion. Despite all drones injected with a single variant having similarly high genome equivalents of DWV, 5–20% of the bees had normal wings, displaying no obvious symptoms of DWV infection. Interestingly, 100% of co-infected pupae had deformed wings upon eclosion (figure 3b/c).Injected worker pupae all develop broadly equivalent viral loads, comparable to those recorded in drones in this study a, but on (b)As all variants produced similarly high genome equivalents by RT-qPCR as well as morbidity in drones, an experiment was designed to test the variants in competition during mixed infections across multiple generations of drone pupae by serial passage, broadly recapitulating mite-mediated virus transmission in successive generations of developing brood. Similar to observed bottlenecks in other vector-borne diseases like Dengue , we hypode novo generated recombinants, replaced all other variants in any of the three biological replicates.Population changes upon passage of the mixed inocula were determined using ShoRAH to analyse a region of the viral genome which contained two introduced unique translationally silent restriction sites (SRS) for VVV/DDD and VVD . The resulting haplotypes produced were compiled and the percentage change in each variant over time was calculated . In each (c)During serial passage no clonality within the 1500 bp window of the genome studied was observed despite the high virus loads transferred between bees. However, clonality has been reported in studies of natural virus transmission situations . Similarin silico model to investigate potential bottlenecks in the bee/mite/virus system at which the viral population could be significantly reshaped.Varroa feed on multiple pupae, moving from one cell to another for several cycles. This facilitates the spread of DWV and potentially acts as a bottleneck for virus replication, either through limited virus acquisition, or by being a site of selective replication of certain variants . Using RVarroa feeding on a pupa, (2) the amount of virus replication in the mite, (3) the number of infectious viruses introduced to the next parasitised pupa and (4) the resulting amplification of the virus during replication in the pupa. The role of each was modelled using field-realistic values derived from published literature (We reasoned that four key variables were (1) the number of infectious viruses ingested by terature , typicalterature . Followiterature .Figure in silico passage ten times. With only 5 viruses ingested by the mite, clonality was reached in 80% of cases (a).Logically, the fewer virus variants ingested by the mite, the more likely the recipient pupa would amplify a clonal virus population. If only one virus was ingested or transmitted by the mite, or the donor pupa already carried a clonal virus population, only one virus would become amplified in the recipient pupa. We modelled the influence of the number of virions ingested over the range 5–50 and observed that ingestion of greater than 5 viruses resulted in clonality in ≤50% of the final pupae after repeating the of cases a.in silico passages that resulted in clonality, all of which were in the range 30–50% .We next modelled viral replication in the mite and virus injection into recipient pupae. Here, over the ranges tested, we found that neither variable dramatically altered the proportion of e 30–50% a,b.5 and 109) in the recipient pupa on virus diversity. Studies have shown very marked levels of virus replication within 24–48 of inoculation in workers [9 clonality was reached in 90% of cases (d). Given clonality was reached in some iterations of the model, but not all, the model recapitulates the stochastic processes that are observed in studies of virus diversity in the field and in laboratory-controlled experiments.The final variable tested was the influence of virus amplification . The extent and proportion of emerging drones that exhibited overt developmental deformities was similar to that seen previously in worker bees, with greater than 80% of drones presenting with deformed wings when injected as white eyed pupae (b). As with earlier studies with worker brood [c). This indicates that it is not virus level alone which necessarily causes deformities. Workers with normal wings, but similarly high virus levels, are also observed in naturally mite infested colonies [It is known that Varroa preferentially parasitise drone cells in the colony , where tt of DWV a. The exed pupae b. As witer brood ,27, a prcolonies . It is ncolonies . If highcolonies , which cReports of DWV transmitted by Varroa have indicated that over time, as Varroa levels increase in the colony, the diversity of the DWV population reduces at the colony level . HoweverWhen generating the model to examine changes in virus diversity over time, one bottleneck likely to shape the virus population was the number of viruses taken up by the mite during feeding . StudiesDifferent variants of DWV appear to have selective advantages in the mite, with evidence of Type B and recombinants thereof replicating in mites ,22, withDespite the unknown variables in the model system, we believe it provides a valuable tool for determining virus changes in the mite/bee/virus system, and has indicated target areas for future research. These include quantifying virus levels, diversity and replication in mite salivary glands and accurately measuring the amount of DWV transmitted by the mite through salivation experiments using feed packet systems to further our understanding of how much virus is passed from mite to pupa. A fluorescent-tagged DWV stock ,53 with


Nonvalvular atrial fibrillation (AF) is a major public health issue. The major complication of AF is an increased risk of stroke. Warfarin, long used for stroke prophylaxis in AF patients, has a narrow therapeutic window and numerous food and drug interactions necessitating regular laboratory monitoring. New oral anticoagulants  may meet the need for predictable anticoagulation with fixed, unmonitored dosing.To review costs of monitoring, bleeding, and stroke in AF patients to analyze costs of anticoagulants for stroke prophylaxis in AF patients.A literature search on the costs of treating AF used PubMed/MEDLINE databases (to April 2012) focusing on studies in the United States. Key words or MeSH terms were used, such as “observational studies,” “oral anticoagulants,” “warfarin,” “cost of bleeding,” “cost of stroke,” and “cost of INR monitoring.”The literature focused mainly on short-term, in-hospital expenditures and less on long-term care costs. Annual overall costs per patient for treating AF in the United States ranged from $18,454 to $38,270. Annual incremental costs of treating AF ranged from $8,705 to $16,311. Annual inpatient costs ranged from $7,841 to $22,582 per patient. Annual costs of anticoagulation monitoring ranged from $291 to $943 per patient. Intracranial hemorrhage and major gastrointestinal bleeding with oral anticoagulants were uncommon but expensive: 1-year costs ranged from $7,584 to $193,804. Annual direct costs of stroke in AF patients ranged from $23,143 to $37,620 .AF-associated direct costs are high and can be broken into costs of warfarin monitoring and direct costs of managing consequences of anticoagulant therapy—stroke and bleeding.


Aspergillus flavus strains. The comparative study showed that mycoviruses affect several cellular activities at the molecular level in a virus- and host strain-dependent manner. The virus-swapping experiment revealed that difference with only three bases in the virus genome led to different host fungal response at the transcriptional level. Our results highlighted highly specific relationship between viruses and their host fungi.Mycoviruses are viruses that infect fungi. Unlike mammalian infectious viruses, their life cycle does not generally have an extracellular stage, and a symbiosis-like relationship is maintained between virus and host fungi. Recently, mycoviruses have been reported to show effects on host fungi, altering biological properties such as growth rate, virulence, drug resistance, and metabolite production. In this study, we systematically elucidated the effects of viruses on host cells by comparing host phenotypes and transcriptomic responses in multiple sets of virus-infected and -eliminated   •Aspergillus flavusMycovirus was explored from clinical and environmental isolates of •Mycoviruses affected fungal cellular properties at the molecular level•The effects of mycovirus occurred in a virus- and host strain-dependent manner•Highly specific relationship was suggested between viruses and their host fungi Virology; Mycology; Most mycoviruses are RNA viruses, and they are vertically transmitted to newly generated cell spaces as the cells grow or during cell division. Mycoviruses do not destroy the host cells, and it is thought that they have no extracellular phase in their life cycle and no infection machinery.Cryphonectria parasitica, Alternaria alternata, Aspergillus fumigatus, and Pestalotiopsis theae,,,Fusarium graminearumFusarium pseudograminearum,Magnaporthe oryzae.Stemphylium lycopersici, viral infection reduced growth and altersolanol A production. Furthermore, genetic experiments revealed that viral open reading frame 3 (ORF3) suppressed host virulence.,,,,,,Functional analyses of mycoviruses can involve phenotypic comparison between mycovirus-infected strains and isolates in which the virus has been eliminated. Several viruses are known to affect host biology such as vegetative growth, pathogenicity, metabolite production, and stress tolerance. For example, virus-infected strains showed decreased pathogenicity in In elucidating the role of viruses in fungal ecology, an important challenge has been the elimination of the virus from the host fungus. Some virus-elimination methods have been developed using application of cycloheximide or the antiviral compound ribavirin. In general, however, the frequency of successful virus elimination is dependent on the virus species, and sometimes the removal of certain virus species has been unsuccessful.M. oryzae, have a high degree of sequence similarity; however, MoCV1-B shows greater effects on melanin biosynthesis, growth rate, and conidiation than MoCV1-A.Another challenge is the difficulty in extracting general insights from the reported virus effects across fungal or viral species since different strains of host and virus were used in these studies. For example, MoCV1-A and MoCV1-B, which infect different strains of ,,,Aspergillus flavus, a mycotoxin-producing fungus, and conducted comparative phenotypic and transcriptomic analyses across fungal strains and virus species. Multiple virus-infected strains were found, and isogenic virus-free isolates were generated using antiviral compounds. The fungal phenotypes were comprehensively compared, which revealed that the virus effects differed independently of viral and fungal strains. This finding was also supported by comparative transcriptomic analysis. Unexpectedly, the phenotypic and transcriptomic responses were generally inconsistent even between the virus-swapped isolates, which indicated that the effect of mycoviruses is highly unique to the combination of virus and host genotypes.Besides phenotypic evaluation, comparative transcriptome analyses have been conducted between virus-infected and -free isolates in several studies.A. flavus strains that were isolated from environmental and clinical samples  to RdRp of Sisal-associated virgavirus A . The seqtic tree . TherefoRegarding the fungal hosts, the genomes of virus-infected strains were sequenced. A phylogenetic tree was constructed, which revealed that there was a close relationship between IFM 65242 and IFM 65241, and IFM 63449g and IFM 63449w See . The strTo gain insights into viral effects on the host, virus-free isolates were established using nucleoside analogs  and confirmed by RT-PCR See . RegardiA. flavus, aflatoxin (AF) and cyclopiazonic acid (CPA), was investigated , IFM 63847 (partitivirus and polymycovirus), and IFM 61879 (vivivirus 1), SM profiles were significantly altered by virus infection , whereasated See . Accordiains See . In factains See .Figure 2To obtain deeper insights into virus effects on host biology, we conducted comparative transcriptome analysis between virus-infected and -free isolates for all sets. First, a heatmap was generated with the transcriptome data from all sets of virus-infected and -free isolates, and a phylogenetic tree was constructed based on the similarity of transcriptional pattern A. TranscA. flavus,,,Next, differentially expressed genes (DEGs) were extracted from the virus-infected and -free isolates in each set. The numbers of DEGs varied from 134 to 1507, in which IFM 65241 infected with the partitivirus showed the least and IFM 63847 co-infected with the partitivirus and polymycovirus showed the most DEGs C. From t,,,,A. flavus, genes involved in the RNA silencing system were not defined; thus, we searched for candidate genes by referring to the genes of A. nidulans and A. fumigatus.,Virus infection is known to trigger RNA silencing in plants and fungi.To gain a more comparative view of viral effects on host responses, DEGs were compared across the eight pairs. Although no DEGs were commonly included in all strains, several DEGs were shared in multiple strains. When compared among the 3 strains with partitivirus infection, only small proportions of the DEGs were common, indicating that a common transcriptional response to partitivirus infection was very limited A. In theFor a wider view of transcriptional responses to viral infection, correlation coefficients were calculated across the pairs based on the expression ratio of each gene C. Among To further analyze virus-host specificity, we focused on the IFM 65242 and IFM 65241 strains that are infected with partitiviruses. There are three single nucleotide variations (SNVs) between genomes of the two partitiviruses See A, which For a deeper understanding, transcriptomes were compared among the recipient isolates (Ha and Hb), homo-transmitted isolates (HaVa and HbVb), and hetero-transmitted isolates (HaVb and HbVa). PCA analysis revealed that the hetero-transmitted isolates showed different gene expression pattern from the homo-transmitted isolates A and 5B.O-methyltransferase (AFLA_119000). These results suggested that the same species but different strains of the mycovirus (different at three nucleotides) are able to affect the host transcriptome response in a similar manner.When DEGs were compared between homo-transmitted and hetero-transmitted isolates, 67% (30/45 genes) of downregulated genes in HaVa were shared with HaVb, whereas 74% (73/98 genes) of upregulated genes were also shared D. In theRegarding the response of different fungal hosts to the identical virus, only 9% (4/45 genes) of downregulated genes in HaVa were shared with HbVa, whereas 4% (4/98 genes) of upregulated genes were also shared D. In theA. flavus, including a newly identified virus previously unassigned to a known virus species. Using these virus-infected strains, then we conducted a phenotypic study comparing virus-infected and -free isolates, and comparative transcriptome analysis to reveal virus effects. The results were then compared across the multiple virus-fungus combinations. Collectively, the different levels of comprehensive analyses highlighted a uniqueness of the viral effect that is highly dependent on both the viral and fungal strains. This is the first report to perform both phenotypic and transcriptomic analyses using multiple virus-fungus combinations, which helped to elucidate the range or magnitude of mycovirus effects on host fungi.In the present study, we identified multiple mycoviruses from environmental and clinical isolates of A. flavus isolates provided an interesting snapshot of the diversity of mycoviruses, and the FLDS method allowed us to identify several novel viral sequences from known mycovirus species. To determine if these novel sequences had been detected in the previous report, we retrieved the deposited metatranscriptome data, and the short reads were mapped to the novel sequences. As a result, these novel sequences were also present in the metatranscriptome data of the previous study.A survey of the virome of A. flavus. On the other hand, partitiviruses and narnaviruses have been widely isolated from fungi such as Aspergillus, Colletotrichum, Fusarium, Penicillium, and Saccharomyces. Nerva et al.Aspergillus ochraceus virus, a partitivirus, into the virus-free A. ochraceus isolate resulted in overproduction of ochratoxin A. Colletotrichum liriopes partitivirus reduced the pathogenicity and conidia production of C. liriopes, which was confirmed through virus elimination and transfection experiments.Fusarium equiseti partitivirus 1 was also reported to confer hypovirulence and reduce the growth of the host fungus.A. flavus isolate and was shown to influence colony morphology and conidia production of the host.Comparisons of phenotypes between virus-infected and -free isolates have shown that mycoviruses are able to affect host physiology. While visible phenotypic alterations were limited, any variation was highly unique to the combination of virus and host strain in our set of strains. Indeed, different SM compounds were produced in a virus-dependent manner . Interes,,,,Several reports so far have shown viral effects on the host transcriptome.A. flavus strains infected with viruses. This might be partly explained by the reports in which several mycoviruses act as RNA silencing suppressors.,A. flavus.Another contribution of this work is that a comparative view of fungal responses or adaptation to viral infection was provided using multiple strains. In particular, our data clearly indicated that RNA silencing was not activated in the The determinants of viral compatibility to its host and the ability to maintain infection are poorly understood. The frequency of virus elimination could be a clue to understanding how these relationships are established. Through a virus elimination experiment, we found that the partitivirus was more frequently removed by RBV treatment in IFM 63847 co-infected with partitivirus and polymycovirus compared with other partitivirus-infected strains. We also found that IFM 63847 with partitivirus infection showed greater effects on conidiation, aerial mycelia, conidial UV resistance, and SM production. From these results, we could conclude that there might be an association between virus effects on host physiology and virus compatibility with the host. This is a challenge for future work.To evaluate the uniqueness of virus isolates in their roles, partitiviruses were swapped in identical natural host isolates. Our results indicated that there were no differences in phenotypic effects between the viral isolates; however, they showed differential effects in the transcriptomic response. Therefore, a change of only three nucleotides led to transcriptomic differences in the host. Our finding suggested that if mutations occur in the viral genome during infection, the host response to the virus may be accordingly changed at the transcriptional level. In this study, the virus transmission experiment using an identical fungal strain clearly showed the uniqueness of the viral isolate. Virus transmission is a powerful and promising tool to assess the virus-host relationship and to identify novel ways in which mycoviruses affect host cells.We have sequenced RNA viruses by FLDS method in this study. FLDS method is unable to capture DNA viruses and detect “extremely” low titer RNA viruses. Therefore, the possibility that DNA viruses and extremely low-titer RNA viruses remain infected in the “virus-free” isolates could not be ruled out.Our results indicated that three SNVs in partitivirus genome differently affected host gene expression. However, the mechanism was remained to be resolved. This must be an open question for the next study.RNA used for RNA sequencing was extracted from fungus cultured under the same condition as that used for secondary metabolite extraction (conidia were spread on YESA medium and cultured at 25°C for 7 days). Therefore, the conditions are different from that for morphological observation (spreading the conidia on PDA medium and incubation at 25°C for less than 5 days). Therefore, the expression data may not match the results of the morphological observation.hagiwara.daisuke.gb@u.tsukuba.ac.jp).Further information should be directed to the lead contact, Daisuke Hagiwara , Japan, and can be distributed. Accession numbers and source of isolates are listed in The Nacalai Tesque, Japan), 1.5% agar ] for colony growth without conidiation and establishing isolates derived from single colonies, or YESA medium  for extraction of chemical compounds and transcriptome analysis. IFM 63449g and IFM 63449w were isolated from the same sample and stocked in a single tube; however, the strains were distinguishable by phenotype and genotype.Fungi were cultivated on PDA medium  for growth and conidiation, YPDA medium [yeast extract , peptone , glucose (We extracted dsRNA from mycelia cultured in liquid media and detected mycovirus-derived dsRNA by electrophoresis as previously reported.2 conidia were inoculated on the center of a PDA plate and incubated at 25°C. Colony diameter was measured every other day and growth rate (cm) per day was calculated. At 4 days after inoculation, the mycelia were cut with the agar medium attached and gross morphology was observed using a stereomicroscope. To compare conidia production, 103 conidia were first spread on a PDA plate and incubated at 25°C for 4 days. The produced conidia were harvested from the colony surface using 3 mL of 0.05% Tween 20  and the number of conidia were counted using a hemocytometer. For mycelial morphology, 102 conidia were inoculated onto the center of PDA slide culture medium and incubated at 25°C for 3 days. After incubation, morphology was observed using an optical microscope. Minimum inhibitory concentrations (MICs) were determined according to the Clinical & Laboratory Standards Institute (CLSI) standard. For UV resistance, 102 conidia were spread onto YPDA plates. The plates were UV irradiated  in a UV-box . After treatment, the plates were immediately moved to an incubator and incubated at 30°C. After 1 day, the number of colonies appearing on the plate was counted using 3 replicates. The survival rate was calculated by comparing the colony number on the UV treated plates with that on the control plates without UV treatment. In the phenotypic assay, we used three biological replicates with three technical replicates for each. The data for representative biological replicate was presented.For comparison of growth rate and observation of gross morphology, 10in vacuo. After dissolving in methanol , the samples were passed through a C18 column  and dried in vacuo again. The samples were dissolved in DMSO and analyzed using a 1260 Infinity LC system  with a Poroshell 120 EC-C18 column . The high-performance liquid chromatography (HPLC) analytical condition was a gradient elution of 5–100% acetonitrile containing 0.5% acetic acid for 18 min at a flow rate of 0.8 mL/min. For detection of ustiloxin, 105 conidia were incubated at 30°C in a mixture of 2.5 g cracked maize and 2.5 mL sterile water in a 50-mL tube for 7 days. A 10-mL aliquot of 70% (v/v) acetone was added to the tube, then the cracked maize and mycelia were removed. After vaporizing the acetone, 300 μL of the remaining water layer was mixed with an equal amount of ethyl acetate  for 1 h at room temperature. The water layer was analyzed using a 1260 Infinity LC system with a Poroshell 120 EC-C18 column. The HPLC analytical condition consisted of gradient elution of 2–30% acetonitrile containing 0.5% acetic acid for 20 min at a flow rate of 0.2 mL/min.Conidia (103) were inoculated in PDB liquid medium and cultured at 25°C for 4 to 7 days. Genomic DNA was extracted from the mycelia as previously reported.Conidia . Paired-end sequencing was performed by Novogene Co. (China). The data quality of genome sequencing is shown in Conidia . The data were retrieved from two technical replicates for the representative isolate.The quality and concentration of libraries were confirmed using an Agilent 2100 bioanalyzer. Paired-end sequencing was performed by Novogene Co. (China). The data quality of RNA-sequencing is shown in To generate phylogenetic trees, MEGA11,Differentially expressed genes (DEGs) between virus-infected and -free isolates were determined according to the following criteria: the genes were up- or down-regulated >2.5 times with a p value <0.05.Functional enrichment analysis was performed using FungiFun,ptrA, a selectable marker for pyrithiamine . Transformation of A. flavus was performed using the protoplast-PEG method, as previously reported . In brief, a plasmid carrying a pyrithiamine selectable marker, modified pPTRI , was introduced into protoplasts of A. flavus using PEG6000 . The pyrithiamine-resistant transformants were obtained after single spore isolation twice.For selection of recipient isolates with the transmitted virus, virus-free isolates were generated by transformation with An outline of virus transmission is shown in t test. p values less than 0.05 were considered to indicate a significant difference. ∗ means p value < 0.05, and ∗∗ means p value < 0.01.p values were calculated using Student’s