Hybridization, defined as breeding between two distinct taxonomic units, can have an important effect on the evolutionary patterns in cross-breeding taxa. Although interspecific hybridization has frequently been considered as a maladaptive process, which threatens species genetic integrity and survival via genetic swamping and outbreeding depression, in some cases hybridization can introduce novel adaptive variation and increase fitness. Most studies to date focused on documenting hybridization events and analyzing their causes, while relatively little is known about the consequences of hybridization and its impact on the parental species. To address this knowledge gap, we conducted a systematic review of studies on hybridization in mammals published in 2010–2021, and identified 115 relevant studies. Of 13 categories of hybridization consequences described in these studies, the most common negative consequence (21% of studies) was genetic swamping and the most common positive consequence (8%) was the gain of novel adaptive variation. The total frequency of negative consequences (49%) was higher than positive (13%) and neutral (38%) consequences. These frequencies are biased by the detection possibilities of microsatellite loci, the most common genetic markers used in the papers assessed. As negative outcomes are typically easier to demonstrate than positive ones , they may be over-represented in publications. Transition towards genomic studies involving both neutral and adaptive variation will provide a better insight into the real impacts of hybridization. Until recently, hybridization was considered as a rare phenomenon in the animal kingdom and thus its role in animal evolution has been underappreciated . GrowingCross-breeding between species with limited postzygotic isolation can often lead to an intake of genetic variation typical of one species into another species’ gene pool—a process called introgression. Introgressive hybridization can affect creation, maintenance and loss of biodiversity . In someOn the other hand, hybridization can be also considered as a potential threat to species survival ,23,24. AHybridization may be particularly common in widespread, abundant species and in non-indigenous species that were intentionally or unintentionally introduced into a new habitat by humans ,36. HybrIn recent decades, several review papers on hybridization have been published. They were focused on specific aspects of hybridization and/or particular taxonomic groups, for example the evolutionary importance of natural hybridization , the rolWe focused on papers on hybridization in mammals published between 2010 and 2021. The database search for papers published in 2021 was completed on the 3rd of December of that year, so papers published after that date are excluded from the results. For finding relevant papers in the Web of Science, we employed the following string:  AND  NOT . With these keywords we found limited numbers of relevant papers (49 papers), we therefore applied different sets of keywords:  AND  NOT . We combined the search results based on these two sets of keywords.Felis silvestris silvestris and domestic cat Felis silvestris catus), as well as wild vs captive/farmed populations of the same species. Although we designed the search terms to be as comprehensive as possible in the detection of studies on mammalian hybridization, some relevant studies could have been missed. Nevertheless, the resulting set of papers is free of human bias (except for the choice of keywords) and therefore provides a reliable overview of the current knowledge on the consequences of hybridization.We excluded from the search books, review papers, theses, annuals or meeting reports. We also excluded papers published in journals that were outside of the following categories: Evolutionary Biology, Genetics and Heredity, Ecology, Biology, Zoology, Biodiversity Conservation, Multidisciplinary Science and Biochemistry and Molecular Biology. Abstracts of the papers that were identified after applying this automatic exclusion  were read and these papers that were not focused on the hybridization process in mammals were removed. We also removed one of the two copies of papers that overlapped between the paper sets resulting from the search with each set of keywords . We thenAccording to the inclusion criteria defined above, a total of 49 and 87 published papers were selected based on the first and second set of keywords. This was a small subset of papers initially identified with each set of keywords; we tried multiple versions of keywords and did not manage to improve the accuracy of results. After excluding overlapping papers between the two sets of keywords (15.4%), 115 papers were retained for the subsequent analysis . These pWe calculated the frequency of mammalian orders and families involved in hybridization process in the surveyed studies . 39.13% We compared the frequencies of species representing different mammalian orders in the surveyed studies on hybridization with the frequencies of all currently recognized contemporary mammalian species  represenWe classified the hybridization outcomes described in the 115 papers included in the systematic review into 13 categories . These cOf 115 studies considered, 21 (18.26%) identified hybrids using microsatellite loci as the only genetic markers, 18 studies (15.65%) used mtDNA fragments, 35 (30.43%) studies used both microsatellite loci and mtDNA, 12 studies (10.43%) used genome-wide single nucleotide polymorphisms (SNPs) or whole genome sequencing, two studies (1.73%) used all these three types of markers, and the remaining studies used another method of hybrid detection. Altogether, 50.42% of studies used microsatellite loci as either the only type of genetic markers or together with other types.To assess whether the analysis of genome-wide data may affect the type of hybridization outcomes observed, we calculated the frequencies of different outcomes based on 14 papers that used genome-wide SNPs or whole genome sequencing. As more than half of these papers (eight studies) focused on hybridization between domestic animals and their wild relatives, introgression from a domesticated lineage was the most common negative effect (36% of studies), followed by genetic swamping (18%). Novel adaptive variation was the only positive impact of hybridization and was reported in five studies (23%). In total, negative outcomes were reported more frequently than positive ones . The frequencies of both negative and positive outcomes were higher than in the entire dataset (49% and 13%), while the frequency of neutral outcomes was smaller. However, these frequencies should be treated with caution due to the small number of studies and overrepresentation of hybridization with domestic animals among the studies considered.Mus. Several studies published in the previous decade  detected signatures of hybridization in several bat species e.g., [The frequency of different mammalian orders in the studies included in this systematic review does not reflect the number of species within each order. Representatives of orders Carnivora and Cetartiodactyla prevail among the species studied, with the frequencies of 39% and 27%, respectively. The frequency of species from these orders among all mammalian species are 5% and 6%, respectively . In contes e.g., ,69,70,71es e.g.,  or a stres e.g., . As batses e.g.,  and duries e.g., , reproduAccordingly, overrepresentation of orders Carnivora and Cetartiodactyla in hybridization studies may result from more relaxed reproductive barriers between congeneric species from these orders compared with other mammals, overrepresentation of studies focused on these orders, or a combination of both. High interest in studying species from these orders may result from their important roles in ecosystems and in some cases their high commercial value. Large species from the order Carnivora are keystone species in their ecosystems and are frequently subject to active management and conservation strategies ,77,78,79In theory, the proportion of species within each mammalian order that are subject to introgressive hybridization could be used as a measure of the strength of reproductive barriers between species within each order. However, to achieve a reliable comparison between mammalian orders, several sources of bias would have to be accounted for, including the above-mentioned differences in intensity of research on different mammalian orders as well as differences in criteria used to define species. Therefore, in practice the frequency of detected hybridization cases is not a reliable measure of the strength of reproductive isolation.Canis lupus familiaris) and the domestic cat (F. s. catus), respectively. Among the studies included in this systematic review, most (68%) of studies on the Canidae family were focused on hybridization between gray wolves and domestic dogs [The frequency of families within each mammalian order that are subject to hybridization studies is biased as well. For example, the Canidae family is represented in 62% of studies on the order Carnivora and 24% of all 115 studies on mammalian hybridization assessed in this systematic review. The second most frequently represented family within Carnivora is the Felidae family, represented in 18% of studies on this order. Canidae and Felidae are the only families within the order Carnivora that include domesticated species, the domestic dog  and domestic pig (Sus scrofa domesticus). Although free-roaming domestic pigs are rare, hybridization with wild boars may occur in open domestic boar farms [Bos grunniens) and wild yaks (Bos mutus) [Within Artiodactyla, Cervidae, Bovidae and Suidae were the most studied families. All hybridization cases described in the family Suidae were between the wild boar   , was deliberately introduced to many European countries for hunting [Capreolus capreolus), is known to hybridize with Siberian roe deer (Capreolus pygargus) [Capreolus c. italicus) [Rupicapra rupicapra tatrica) and introduced Alpine chamois (Rupicapra rupicapra rupicapra) [In particular, the Cervidae family includes multiple valuable game species. One of them, the sika deer  ,113 and talicus) . Althougtalicus) . In Bovipicapra) . In thatBalaenoptera physalus) and blue whale  showed that the hybrid individuals can reproduce and survive to adulthood in specific circumstances [In cetacean species, hybridization has been documented both in captive breeding sites and in the wild ,116, witmstances .Depending on species and environmental conditions, hybridization may have either negative or positive impacts, and sometimes there may be very limited consequences. In some cases, hybridization can drive species toward extinction, while in others it provides an opportunity to create new species . GeneticOf 13 categories of hybridization outcomes identified in the studies considered in this review , geneticThe frequencies of different outcomes of hybridization may be affected by the specific sets of keywords that were employed in the literature search. We included keywords such as “Genetic swamping”, “Hybrid zone”, “Hybrid speciation”, “Extinction”, and “Outbreeding depression”, and therefore studies focusing on these topics may be overrepresented. Furthermore, information about the consequences of hybridization is missing from some studies, which could also affect the result. Nonetheless, we also identified outcomes that were not included in the keywords, and some which were included had very low frequencies among the selected papers .Genetic swamping refers to the process where genotypes of one or both parental species are partially replaced by hybrid genotypes . GeneticNearly half (48%) of studies included in this systematic review that reported genetic swamping were focused on hybridization between domesticated mammals and their wild relatives, including wolf and domestic dog ,81,135, Reproductive barriers between closely related species that evolved in geographic isolation may be weak, and therefore after the secondary contact due to translocation, cross-breeding and production of fertile offspring may be common. In such cases, continuous cross-breeding across generations may result in considerable genetic swamping . AccordiSus verrucosus), which is endangered with extinction via genetic swamping from the common Indonesian banded pig (Sus scrofa vitattus), because of high hybridization rates resulting from the breakdown of reproductive barriers and reduced fertility of hybrids [C. c. italicus) due to extreme genetic swamping from the introduced European roe deer (Capreolus c. capreolus) [Lepus timidus) and European hare (Lepus europaeus), showing that under climate change scenarios, increased hybridization rate can lead to the mountain hare’s extinction via genetic swamping [In extreme cases, extensive genetic swamping may lead a population or an entire species towards extinction ,140. End hybrids . The secpreolus) . The thiswamping . These sNotably, identifying key factors involved in the extinction process is very challenging. In many cases, interaction of different forces such as environmental stress, low genetic diversity and small population size, may lead to the extinction vortex ,143. TheOutbreeding depression occurs when cross-breeding between two species or populations that are adapted to different environmental conditions results in a loss of local adaptations and reduction of fitness in hybrid individuals . In the On the other hand, captive breeding programs for conservation purpose have become a conservation tool to prevent extinctions and support reintroductions in cases when remaining wild populations are small and have low genetic diversity ,150. ForAmong the papers discussed in this systematic review, five studies were focused on admixture between wild and captive-bred populations of the same species. Two of these studies did not detect any signatures of hybridization, and the remaining three studies reported both outbreeding depression and genetic swamping. One of them reported genetic swamping for the native population and increase in genetic diversity for the captive-bred population studied.Capra ibex ibex) acquired one of its two MHC DRB alleles from domestic goats (Capra aegagrus hircus), which critically increased diversity of this genetically impoverished species at the key component of the immune system [Hybridization between wild species and their domesticated relatives frequently results in the introgression of gene variants typical of domesticated animals to wild populations. Although there is a considerable overlap between the studies included in this category with other categories, we consider it separately due to specific conservation problems resulting from this type of introgression . Domestie system .Among 25 studies from our systematic review that fit in this class, 15 studies focused on hybridization between the grey wolf or the dingo and the domestic dog, four studies focused on the wild boar and the domestic pig, four studies on the wild cat and the domestic cat, one study on the wild sheep and the domestic sheep, and one study on the wild yak and the domestic yak. These studies show that the introgression from domestic animals into their wild relatives is more frequent than in the opposite direction. Population sizes of domestic animals are dependent on the human population size, and therefore human population growth combined with the fragmentation of natural habitats increases both the numbers of domestic animals and the probability of encounters with their wild relatives. Given that wild populations are typically considerably smaller than populations of domestic animals, a single hybridization and back-crossing event will have a larger effect on gene pools of wild populations compared with domestic ones.Studies focused on hybridization between domestic animals and their wild relatives constituted 36% of studies considered in this systematic review, and thus they had significant impact on the overall proportions of different consequences of hybridization. By default, they were responsible for all the cases of introgression from domesticated lineages, which were reported in 44% of studies focused on hybridization with domestic animals. Further 24.5% of studies reported genetic swamping; in this case, the difference between these two consequences is only in wording, with the exception of introgression cases from wild to domestic populations. The most common positive consequence was gaining novel adaptive variation (7%). Overall, the frequency of hybridization outcomes considered as negative (70%) was considerably higher than the frequency of positive outcomes (9%). This suggests that studies on hybridization between domestic animals and their wild relatives have a disproportional contribution to the negative hybridization outcomes in the overall assessment. However, this is based on the assumption that introgression from domesticated lineages and genetic swamping are negative outcomes by default, which has rarely been tested. Given low divergence between domesticated animals and their wild relatives, it may be expected instead that introgression will rarely lead to increased mortality and infertility of admixed individuals, but the presence of atypical phenotypic traits may result in reduced fitness.Interspecific hybridization influences phenotypic traits and may create novel or unusual traits ,158. MorPhodopus campbelli and Phodopus sungorus) [Connochaetes taurinus and Connochaetes gnu) [The divergence of phenotypic traits of admixed individuals from average traits within each of the cross-breeding species increases with their divergence time ,161,162.ungorus)  and skultes gnu) .Microtus hartingi lydius and Microtus hartingi strandzensis produces viable and prolific F1 hybrids, while in the F2 generation, males are sterile and the mortality rate is high [Hybridization can change reproductive output by leading to changes in mating behavior  or by wa is high . FallingR. r. rupicapra) was shown to improve genetic diversity of Tatra chamois (R. r. tatrica), an endangered endemic population in the Tatra Mountains that suffered from a high level of inbreeding depression [In a specific case when genetic diversity of a population is very low and the rate of inbreeding is high, introgression from a non-native population or species can increase genetic diversity without any signs of outbreeding depression. This can be considered as a positive consequence of hybridization. Moreover, in small and fragmented populations that have low genetic diversity and experience inbreeding depression, hybridization can restore population viability ,166. Genpression .Neovison vison) increased genetic diversity of the invasive populations of these species in Europe, which could increase their adaptive potential and therefore compromise management efforts to control them [Although introgression from domesticated lineages can be considered as a threat for wild populations, in some circumstances it can increase genetic diversity and viability of wild populations. For example, increase in genetic diversity has been reported in European wild boars that cross-bred with domestic pigs . Accordirol them . AlthougC. pygargus) allowed the European roe deer (C. capreolus) to adapt better to severe winters, which are an important contributing factor of roe deer mortality [Canis latrans) and the grey wolf (Canis lupus) in Canada has resulted in the introduction of novel adaptive variation to the coyote populations, allowing them to increase in body size, which in turn improved their success in hunting deer [In some cases, creation of novel genetic diversity via hybridization can facilitate species adaptation to variable or novel environmental conditions, without a loss of its genetic integrity ,168. Admortality . Furthering deer .EPAS1 haplotype that confers an adaptive advantage in high altitude environments [In several mammalian species, including humans, the presence of adaptive variation from their extinct relatives has been detected ,174,175,ronments ,177. AltHybrid speciation refers to the process in which hybridization results in the creation of a new species, which is characterized by mixed ancestry and distinct genetic composition from its parental species . HybridiThree criteria should be met to demonstrate speciation via hybridization; first, confirmed evidence of a past hybridization event in the putative hybrid species, second, reproductive isolation between the hybrid species and its parental species, and finally the presence of isolating impacts of hybridization . Hybrid Cercopithecus is one of the key mechanisms driving hybrid speciation in this genus [P. campbelli and P. sungorus, were suggested to play an important role in speciation by contributing to reproductive isolation between these recently diverged species [Mus musculus), which is a model species in genetics, provided an insight into the molecular mechanisms underlying hybrid speciation. Dysfunction in the Mecp2 protein in the house mouse resulting from introgressive hybridization within the hybrid zone may induce changes in the expression of thousands of genes, which may initiate the speciation process [In this review we found four studies that reported hybrid speciation ,181,182.is genus . Abnorma species . Despite species . HoweverTamias spp.) in the Sierra Nevada, USA, was associated with 14% error rate, which was in part attributed to sporadic hybridization among these species [Although in some cases interspecific hybridization may create deleterious morphological anomalies or novel adaptive traits (see above), hybrid individuals frequently show intermediate phenotypic traits compared to their parents . The add species . The fitMartes martes and the sable Martes zibellina in Western Siberia, a vast hybrid zone has formed between these species after the Last Glacial Maximum [Neotoma bryanti and Neotoma lepida), a hybrid zone has been maintained as a result of sporadic cross-breeding between these species and hybrid fertility [Mus musculus musculus and Mus musculus domesticus) [Hybrid zones are areas where two genetically distinct linages meet, mate and create viable offspring . These g Maximum . Feature Maximum . In woodertility . Among sesticus) , differeesticus) ,199,200,esticus) , primateesticus) , artiodaesticus) ,202, Dipesticus) ,204 and esticus) ,205.Some studies included in this review showed that despite hybridization, populations maintained their genetic distinctiveness . For insThis category includes studies that have not found any signs of hybridization in the populations studied or found only very limited evidence e.g., ,214,215.Myotis myotis and Myotis blythii has been reported in Europe, but in Turkey no signs of hybridization between these species have been detected [In some cases, efficient conservation management may result in low rate of hybridization ,227. Fordetected .Altogether, 38% of the studies assessed in this systematic review reported neutral or unspecified outcomes of hybridization. Within this group, “no or rare evidence of hybridization” was the most common hybridization outcome, which occurred in 17% of all the studies on hybridization. This result suggests that one of the five categories of hybridization outcomes delineated for the purpose of species conservation , “negligOnly 18% of studies considered in this review were focused on threatened species (having the IUCN Red List categories of Near Threatened (NT), Vulnerable (VU), Endangered (EN), Critically Endangered (CR), Extinct in the Wild (EW)). Negative consequences  were reported in 38% of these studies and positive consequences  were reported in 14% of them. In the remaining studies, the authors did not mention any positive or negative consequences or did not find any evidence of hybridization. The frequency of the positive consequences reported for all the studies assessed in this systematic review was very similar (13%), while the frequency of the negative consequences was higher (49%) compared to those observed in the studies on threatened species. This suggests that the negative consequences of hybridization are not intensified in endangered species, at least the mammalian species considered in this review.Among the papers included in this systematic review, hybridization outcomes typically considered as negative had considerably higher frequency (49%) than those considered as positive 13%). However, these frequencies could have been biased by several factors and therefore should be treated with caution. For instance, two most frequent outcomes of hybridization, genetic swamping and introgression of variants from domestic animals are typically considered as negative, but this is not always the case. In some circumstances, moderate levels of genetic swamping or introgression of domestication-related variants may result in increased fitness and genetic rescue ,230. The%. HoweveIt is also important to stress that the detection of different outcomes of hybridization depends on the type of molecular markers applied. Microsatellite markers enable identification of first-generation hybrids and recent back-crosses, but cannot reliably detect more distant hybridization events . Since mA combination of neutral loci and those located within coding genes is better suited to provide an unbiased insight into the relative frequencies of positive and negative hybridization outcomes and identify factors that affect them. Single Nucleotide Polymorphisms (SNPs) can be genotyped in large numbers (hundreds of thousands to millions) using arrays or next generation sequencing, which makes them suitable for identification of adaptive loci ,232 as wIn some circumstances, hybridization can be used as a conservation tool to facilitate adaptation of populations to changing habitat conditions and to increase individual fitness in populations experiencing inbreeding depression ,13. To u


Viruses and for expert researchers in the related scientific areas. The sustainable methodologies tackled by basic researchers could hopefully pave the way for highly valid preventions and/or treatments for infectious diseases caused by various human RNA viruses. Numerous species of RNA viruses pathogenic for humans are present worldwide. They are highly divergent in biological and molecular properties, and are therefore classified into distinct viral families. Of particular note, these parasitic entities can diversify significantly in their own groups to resist natural and artificial environmental changes that happen to be hostile to their existence. These adaptation events in pathogenic RNA viruses that currently represent major medical issues, sometimes deadly and highly transmittable infectious diseases, should be settled as promptly as possible. Through extensive experimental studies, we virologists have found and are presently learning how ingeniously RNA viruses of various species generate their genetic variant progenies for survival. In order to fully understand their pathogenic potentials and to effectively fight against them, some kinds of demonstrative model studies are prerequisite for successful establishment of anti-RNA virus strategies, such as vaccines and antiviral drug development. In this Special Issue named “Cell, Organoid and Animal Models to Study Pathogenic Human RNA Viruses”, we focus on these model studies to provide useful relevant information on the topic for devoted readers of Retroviridae), measles virus (Paramyxoviridae), and Zika virus (Flaviviridae). Review articles: Rotavirus (Reoviridae), influenza virus (Orthomyxoviridae), norovirus , astrovirus (Astroviridae), coronavirus SARS-CoV-2 (Coronaviridae), and human immunodeficiency virus (HIV) (Retroviridae). The authors have clearly described or summarized the details of the experimental systems used for the studies and the results obtained by the studies.Original research and review articles are included in this Special Issue. Target viruses for studies are as follows. Original research articles: Simian immunodeficiency virus (SIV) (Family Finally, we editors greatly appreciate all contributors to this Special Issue for their excellent works. We are happy with the final version of the issue and are very proud of the contributed articles.


A small-scale study with Mosi-guard Natural spray, an insect repellent containing Citriodiol, was performed to determine if it has virucidal activity against SARS-CoV-2. A liquid test examined the activity of the insect repellent and the individual components for virucidal activity. A surface contact test looked at the activity of the insect repellent when impregnated on a latex surface as a synthetic skin for potential topical prophylactic application. Both Mosi-guard Natural spray and Citriodiol, as well as other components of the repellent, had virucidal activity in the liquid contact test. On a latex surface used to simulate treated skin, the titre of SARS-CoV-2 was less over time on the Mosi-guard Natural-treated surface but virus was still recovered. Citriodiol is a commercial distillation of Eucalyptus citriodora oil, whose major component is reported to be p-menthane-3,8-diol (PMD), alongside other constituents . SeveralSARS-CoV . We dete50 assay as previously described [SARS-CoV-2 England-2 isolate was provided by Public Health England. Passage 3 material was grown in Vero C1008 cells and enumerated by TCIDMosi-guard Natural pump spray and Citriodiol  were used as provided and were tested alongside aqueous solutions of ethanol and isopropanol .6 TCID50 ml−1) in a polystyrene 24-well culture plate . For positive controls and to determine the maximum virus recovery, 25 µl tissue culture medium (TCM) was added to 25 µl SARS-CoV-2 stock. As negative controls, 25 µl of the test components was added to 25 µl TCM (no virus). All samples were tested in triplicate. After 1 min, 1 ml=TCM was added, samples were vortexed twice for 10 s and then centrifuged at 14 000 r.p.m. for 10 mins. After centrifugation the liquid was discarded and 1 ml fresh TCM was added to the tube. This was repeated twice. After the final centrifugation, 2 ml TCM was added to the tube and a TCID50 assay performed on each sample in duplicate. The centrifugation steps were required to dilute the chemical components that would otherwise cause toxicity in the cell-culture-based enumeration assay, and is similar to methods described previously [To test virucidal activity of Mosi-guard Natural and its main components (representative of the upper range indicated in the spray ), 25 µl 50 assay was performed the remainder of each replicate sample was pooled and inoculated onto confluent T12.5 flasks, which were passaged twice as described previously [8 TCID50 ml−1) for 1 min and processed as above to test Mosi-guard Natural spray at a final concentration of 90 %.After the TCIDTo test the efficacy of Mosi-guard Natural spray on a surface, a latex surface  of medical grade latex, Hytone HPN-09814 variant, 42’ wide, 0.020’ thick to simulate skin was treated with Mosi-guard Natural using a SprayCraft SP50K air brush and cut into discs, which were placed in a 24-well cell-culture plate. A second 24-well cell-culture plate was air brush-sprayed with Mosi-guard Natural. Untreated discs of latex and untreated 24-well plates were used as surface control samples to determine the natural decay over time of SARS-CoV-2. Approximately 1 h after the Mosi-guard Natural-treatment of surfaces, a single 10 µl droplet of concentrated SARS-CoV-2 was applied to each of the four surfaces with no further spreading. TCM only was also applied as a no virus control. Immediately after virus addition and at 30, 60, 120, 180 and 240 mins after virus addition, triplicate virus samples and one no-virus control were recovered and processed for enumeration and cell-culture passage as described above.T test with a Welch correction. For the time-course study, linear regression analysis on the logarithm-transformed data was used to model the data. Analysis of covariance was used to compare data-sets. Further details on the methods used can be found online [Statistical analysis was performed in Graphpad PRISM v8. For initial tests looking at individual components, statistical significance was determined by one-way ANOVA and Dunnett’s tests or a d online .4 TCID50 ml−1 whilst SARS-CoV-2 mixed with other components resulted in reduced titres  had the least activity against the virus with only a tenfold reduction in viable virus recovered. Ethanol, tested at 40 % (v/v) final concentration resulted in no viable virus being recovered by TCID50 assay  but infection did occur in the second cell passage indicating low levels of virus remained. The combination of ethanol and isopropanol  resulted in low levels of virus being recovered and flasks were also positive for infection.In the basic liquid test, the mean titre of recovered SARS-CoV-2 and TCM controls was 1.88×10d titres . Mosi-gu50 assay or in flask passage when concentrated virus in TCM  was mixed with 90 % Mosi-guard Natural (v/v)  , resultiP<0.001).Statistical analysis using one-way ANOVA and Dunnett’s tests showed that all treatments tested gave a significant decrease in viral titre compared to the SARS-CoV-2 and TCM controls  were treated with Mosi-guard Natural spray approximately 1 h before virus was applied. There was a notable difference on the wetting characteristics of insect repellent sprayed on the different surface types followed by addition of virus, which may have affected efficacy. The Mosi-guard Natural spray gave a relatively uniform coverage of liquid on the latex surface, and when virus was added it remained as discreet droplets. However, when applied to the polystyrene surface, an uneven distribution of Mosi-guard Natural spray was observed and the virus dispersed and wetted the whole surface of the well.On both latex and plastic surfaces, there was minimal change in titre over 4 h on the untreated surfaces, but a decrease in titre, with immediate effect, on the Mosi-guard Natural-treated surfaces was observed .10 in viral titre of SARS-CoV-2 England-2 isolate (P<0.001) at all time-points and an approximate 1.5 log10 reduction after 4 h on the surface, however, virus was recoverable at all time points (P=0.547 for latex synthetic skin and P=0.113 for 24-well plate) whilst on both Mosi-guard Natural-treated surfaces decay was observed (P<0.001 for both).Recovery efficacy from the latex synthetic skin was lower than from the 24-well plate, but pre-application of Mosi-guard Natural spray to a latex synthetic skin resulted in a reduction of at least 1 loge points . On the e points . Linear A parallel experiment was carried out to estimate evaporation by weighing Mosi-guard Natural-treated latex sections over time. Up to 44 % total weight loss was measured but the mean weight loss was 10 % and the median weight loss was 12 % (data not shown). Most weight loss occurred within an hour of treatment ; likely due to the more volatile compounds  evaporating. There was no significant weight loss on the Mosi-guard Natural-treated surface after 1 h (Data S1). This suggests that there should be no solvent present that could affect the virology results and the non-volatile components of Mosi-guard Natural spray left on the latex surface would have remained constant throughout the duration of the parallel 4 h experiment with SARS-CoV-2 .10 respectively, but not below the limit of quantification. At a higher concentration (90 % v/v), Mosi-guard Natural spray gave a significant decrease of over 4 log10 resulting in no recoverable virus. Unsurprisingly, ethanol alone (used at 40 %) had virucidal activity in our assay, and when used at 20 % in combination with isopropanol. Efficacy of ethanol against SARS-CoV-2 has already been reported [Two studies were performed to assess the virucidal potential of an insect repellent against SARS-CoV-2. Firstly the insect repellent was tested at two different concentrations alongside its main constituents  in a standard test. Mosi-guard Natural spray (50 % v/v) and Citriodiol (50 % v/v) decreased viral titre by approximately 2 and 3 logreported . In our reported . To datereported  and the reported .We have shown that Mosi-guard Natural spray, which is intended to be sprayed onto skin to repel insects, also has potential to reduce the level of SARS-CoV-2 on exposed skin. This was done to test the potential for topical application of insect repellent as a potential prophylactic barrier against contact transmission or exposure to droplets. This was the first time we have performed such an experiment looking at the survival of virus on an impregnated surface. Significantly less SARS-CoV-2 virus was recovered from Mosi-guard Natural-treated surfaces compared to untreated surfaces, but viable virus was still recovered. The methodology developed during this study could be extended to other types of surfaces and/or other compounds with virucidal activity.There were limitations to our study, particularly in the treated surface experiments where we tried to replicate how the insect repellent might have virucidal activity when on human skin. There are a number of further experiments that could be performed. We used latex as a synthetic skin at room temperature (19–22 °C), which is lower than the temperature at the surface of human skin. A study using pig skin determined a half-life of SARS-CoV-2 of 3.5 h at 22 °C and recovered virus at 96 h, but at 37 °C viable virus was detected at 8 h but not 24 h . On autoIn conclusion we have shown that in a 1 min liquid suspension test Mosi-guard Natural spray has virucidal activity against SARS-CoV-2 England-2 isolate. Additionally, viral studies on latex indicated that the insect repellent also has virucidal activity against SARS-CoV-2 on a Mosi-guard Natural-treated surface used to simulate skin. This offers the potential that topical application of the insect repellent could have some prophylactic potential. These small-scale studies can inform the basis of further research in this area.Click here for additional data file.


Bemisia tabaci (Hemiptera: Aleyrodidae) is one of the most important virus vectors, transmitting more than four hundred virus species, the majority belonging to begomoviruses (Geminiviridae), with their ssDNA genomes. Begomoviruses are transmitted by B. tabaci in a persistent, circulative manner, during which the virus breaches barriers in the digestive, hemolymph, and salivary systems, and interacts with insect proteins along the transmission pathway. These interactions and the tissue tropism in the vector body determine the efficiency and specificity of the transmission. This review describes the mechanisms involved in circulative begomovirus transmission by B. tabaci, focusing on the most studied virus in this regard, namely the tomato yellow leaf curl virus (TYLCV) and its closely related isolates. Additionally, the review aims at drawing attention to the recent knowhow of unorthodox virus—B. tabaci interactions. The recent knowledge of whitefly-mediated transmission of two recombinant poleroviruses (Luteoviridae), a virus group with an ssRNA genome and known to be strictly transmitted with aphids, is discussed with its broader context in the emergence of new whitefly-driven virus diseases.Many plant viruses depend on insect vectors for their transmission and dissemination. The whitefly  Transmission by insects accounts for the spread of over 75% of the known plant-infecting viruses . The mecBemisia Quaintance and Baker, Trialeurodes Cockerell, and Aleurodicus Russell are known transmitters of over four hundred diverse viruses (Bemisia tabaci Gennadius (B. tabaci), a species complex with morphologically indistinguishable species, stands out among them as the most versatile and important insect vector of plant viruses [B. tabaci transmits diverse plant pathogenic viruses with either DNA or RNA as the genetic material in a non-persistent, semi-persistent, or persistent mode  fauna comprises over 160 different genera , but onl viruses . Bemisia viruses ,11. B. tent mode . Howevere manner ,13. ReceThe transmission process of begomoviruses has been studied in detail and remains the main stay of our understanding of whitefly-mediated, persistent transmission of viruses. Although many other virus genera are transmitted by whiteflies in different transmission modes, little attention has been paid to the transmission mechanisms of other viruses. In this review, we thus use the known circulation process of the tomato yellow leaf curl virus (TYLCV) to describe the route of persistent, circulative viruses inside the whitefly. The transmission process of persistent viruses by whiteflies involves the following stages .Virus Ingestion: Virus particles mixed with the phloem sap from infected plants are ingested by the piercing-sucking mouthparts to reach the cibarium. The cibarium of the whitefly attaches and accumulates the non-persistently and semi-persistently transmitted viruses, and sorts them from the circulative viruses that continue through the esophagus to reach the midgut lumen. Circulative viruses can be PCR-detected in the cibarium and midgut of the whitefly after a minimum of 5 and 40 min of feeding, respectively [ectively .Virus attachment in the midgut: Recognition of the capsid protein [ protein  of only  protein ,28,29. T protein . Specifi protein , whereas protein ,32,33,34 protein . Ingeste protein , especia protein ,37.Virus entry and translocation across the basal membrane: Successful recognition of specific begomoviruses triggers reorganization of the midgut actin filaments [ilaments , resultiilaments ,39 into ilaments . Severalilaments . The minThe infection of midguts with TYLCV alters the expression of genes that regulate cargo-receptor activity and ATP-binding cassette transporters, indicating their role in the translocation of the virus across the gut epithelial cells . A recenB. tabaci [B. tabaci midguts [B. tabaci were found to induce upregulation of apoptosis marker genes such as caspase-1/3 and downregulation of inhibitors of apoptosis such as bcl-2 [B. tabaci [Virus entry inside the midgut, in addition to altering whitefly gene expression for facilitating its spread, triggers an immune response. Genes encoding antimicrobial peptides such as knottins and defensins, humoral and cell surface receptors regulating cell defense, and intracellular signaling molecules involved in defense response are upregulated in the midguts following virus acquisition . Activat. tabaci . Interes midguts ,59,62,63. tabaci . The aut. tabaci ,62. Acti. tabaci ,57,62. HRetention and circulation in the hemocoel: The hemolymph of the whitefly remains the most understudied barrier, although it is thought to be a hostile environment that challenges the stability of virions. No interactions of the virus with any hemolymph proteins, hemocytes other than with the whitefly vitellogenin protein [ protein , or prot protein ,53,54,56 protein . Direct  protein ,65,66. T protein ,53,54 un protein ,68 and a protein  circulatTranslocation across the membranes of the salivary gland: After circulating in the hemocoel, the virions cross the basal lamina of the primary salivary glands (PSG) of the whitefly [whitefly ,67,70. Twhitefly . The PSGwhitefly . Howeverwhitefly ,72. The whitefly .Until recently, begomoviruses were not known to replicate within their insect vectors, and no virus-encoded helper protein is known to be essential for transmission. The role of the capsid protein (CP) of begomoviruses has thus been the most studied in relation to whitefly transmission, and evidence of its criticality in persistent whitefly transmission is provided in multiple studies involving diverse virus–whitefly combinations ,74,75,76Plasmodium, the malarial parasite, is regulated by histone modifications in the chromatin of the mosquito [Plasmodium infection induces histone modifications in genes regulating vector competence [B. tabaci and are implied to regulate temperature tolerance [Multiple pieces of evidence for replication of TYLCV inside whiteflies ,62 implymosquito , and Plampetence . Enzymesolerance ,98. FutuB. tabaci [B. tabaci, and translocates across the midgut onto the hemolymph; however, it circulates inside the whitefly for a longer duration (≥120 h) before transmission [B. tabaci are highly identical to those of other poleroviruses transmitted by aphids, but the factors that determine the specific transmission of poleroviruses by aphids or whiteflies remain unknown. Moreover, no common pattern linked to the whitefly transmission can be identified by comparison of the virus-encoded proteins of PeWBVYV and CWBYV [Poleroviruses (Luteoviridae) comprising at least 31 species were erstwhile known to be strictly transmitted by aphids in a persistent non-propagative manner ,23,99. H. tabaci . The trasmission . Whethernd CWBYV . The twond CWBYV  and trannd CWBYV , respectnd CWBYV . Complemnd CWBYV . Moreovend CWBYV ,103. AltB. tabaci can have severe ramifications on global crop production. For example, the spread of PeWBVYV by whiteflies in Israel has forced the growers to completely abandon open-air cultivation and re-equip net-houses with whitefly-proof mesh. The lack of resistant cultivars against PeWBVYV has led to the heavy usage of insecticides from the beginning of the season to control whitefly, resulting in diminished economic returns. PeWBVYV was absent in the Arava valley region, the main pepper-growing area of Israel until the 2019 season, but has been recently detected in sporadic places (unpublished) and continues to threaten pepper cultivation in Israel. However, the evolutionary pressure necessitating the development of this new B. tabaci–polerovirus relationship is yet to be understood. A recent study suggests that the transmission of highly similar poleroviruses by different insect vectors is a strategy of competing viruses to escape competitive exclusion [B. tabaci transmitted poleroviruses is independent of the crop and geographical location. Crops belonging to diverse families such as cucurbits, beet, solanaceous crops , sweet potato, and cotton support high whitefly populations and are known hosts for multiple poleroviruses, putting them at high risk for the potential emergence of B. tabaci transmitted poleroviruses. Moreover, the worldwide spread of the polyphagous Middle-East Asia Minor 1 (MEAM1) species of B. tabaci intensifies the threat, and warrants monitoring for the association of whitefly incidence and viral disease symptoms. Lack of awareness of the whitefly as a possible vector can lead to rapid spread of the virus disease, leading to the introduction of the virus into new areas.The emergence of poleroviruses transmitted by xclusion . The bacB. tabaci [B. tabaci in India, bean crops in Tanzania, and multiple crops in China, suggesting widespread existence of B. tabaci transmitted rhabdoviruses [The recent report of the transmission of the legume-infecting bean-associated cytorhabdovirus (BaCV) belonging to Rhabdoviridae in Brazil by . tabaci  is anoth. tabaci . Furtheroviruses . Future B. tabaci is highly polyphagous, prevalent in all the continents (except Antarctica), has moderate to high levels of resistance to insecticides [B. tabaci. Interestingly, the Mediterranean (MED) species of B. tabaci, which is prevalent in Israel in addition to MEAM1, was reported as a non-transmitter of PeWBVYV [B. tabaci would shed more light on whether MEAM1 has a special contribution toward the emergence of B. tabaci as a vector of new viruses.The MEAM1 species of cticides , and is  PeWBVYV . PeWBVYVB. tabaci cell-line has been a major stumbling block for the conclusive identification of receptors or mechanisms of intracellular trafficking of the virus inside midgut or salivary glands. Understanding the regulation of gene expression of B. tabaci by acquired viruses can be key to deciphering the genes governing antiviral immunity. New relationships have evolved between whiteflies and viruses belonging to families conventionally transmitted by other insects, resulting in the increased threat of whiteflies as a quarantine pest. Prompt risk-assessment of whiteflies as non-conventional transmitters of viruses is required to mitigate the spread and evolution of emerging whitefly-transmitted virus diseases.The specific transmission of circulative viruses by specific whitefly species involves intricate interactions between the virus and the whitefly. The minimal requirement of interactions between the virus and the whitefly receptors to ensure whitefly transmissibility is yet to be deciphered. Several whitefly proteins identified to interact with the virus are implied to serve a role in transmission; yet, comparative studies with the identified protein–protein interactions in different whitefly–virus systems are required to understand the minimal requisite interactions to effectuate virus transmission. Moreover, the absence of a


Ixodes ricinus, which is known to be a vector for many viral, bacterial, and protozoan pathogens, is the most prevalent tick species in Europe. For this study, we decided to investigate the virosphere of Belgian I. ricinus ticks. High-throughput sequencing of tick pools collected from six different sampling sites revealed the presence of viruses belonging to many different viral orders and families, including Mononegavirales, Bunyavirales, Partitiviridae, and Reoviridae. Of particular interest was the detection of several new reoviruses, two of which cluster together with members of the genus Coltivirus. This includes a new strain of Eyach virus, a known causative agent of tick-borne encephalitis. All genome segments of this new strain are highly similar to those of previously published Eyach virus genomes, except for the fourth segment, encoding VP4, which is markedly more dissimilar, potentially indicating the occurrence of a genetic reassortment. Further polymerase chain reaction–based screening of over 230 tick pools for 14 selected viruses showed that most viruses could be found in all six sampling sites, indicating the wide spread of these viruses throughout the Belgian tick population. Taken together, these results illustrate the role of ticks as important virus reservoirs, highlighting the need for adequate tick control measures.Recent metagenomics studies have revealed several tick species to host a variety of previously undiscovered RNA viruses.  Ticks from Gierle, Chimay, and Heverlee were kept at −20°C for long-term storage, while ticks from Torhout, Moerbeke, and Zoersel were stored at room temperature in 100 per cent ethanol. A total of 767 ticks were collected, including 33 adults, 513 nymphs, and 221 larvae. All ticks were sorted based on their location of origin and their developmental stage, and all were identified as 2. 2Each adult tick or pool (1–10) of nymphs/larvae (see Supplementary Table S1) was added to a tube containing 100 µl phosphate buffered saline and zirconium oxide beads and subsequently homogenized for 150 s at 4,000 g in a Bertin Minilys homogenizer. Total RNA was extracted from the homogenate using the RNeasy Mini Kit  according to the manufacturer’s instructions. DNA carry-over was further depleted by DNase treatment using 2U TurboDNase , and the remaining RNA was re-purified using the RNeasy MinElute Cleanup Kit .2. 3l-glutamine, 1 per cent gentamicin, and 0.2 per cent fungizone . BHK21s were kept in Minimal Essential Medium Rega-3 (Thermo Fisher Scientific), supplemented with +10 per cent FBS and 200 mM l-glutamine (Thermo Fisher Scientific). To attempt virus rescue, cells were seeded in 6-well plates and allowed to grow overnight. The medium was removed from the resulting monolayers, which were subsequently inoculated with 200 µl fresh tick homogenate for 1 h, after which 2 ml 2 per cent FBS-supplemented medium was added. Cells were observed daily for cytopathogenic effect for 2 weeks, and the cell medium was refreshed every 3–4 days. Collected supernatant was subjected to RNA extraction using the RNeasy Mini Kit (QIAGEN), followed by reverse transcription PCR (RT-PCR) screening as described below.Virus rescue was attempted using African green monkey kidney cells  and hamster kidney fibroblasts (BHK21). Vero E6 cells were maintained in Dulbecco’s Modified Eagle Medium  supplemented with 10 per cent fetal bovine serum , 1 per cent sodium bicarbonate, 1 per cent penicillin-streptomycin-2. 4https://blast.ncbi.nlm.nih.gov/Blast.cgi).RNA extracted from a single female adult tick was converted to complementary DNA (cDNA) using the Complete Whole Transcriptome Amplification Kit . Purification of cDNA was done using the MSB Spin PCRapace kit . The resulting cDNA was prepared for nanopore sequencing using the SQK-LSK108 kit  and loaded on an R9.4.1 flow cell. Reads corresponding to tick ribosomal RNA (rRNA) were removed, and following size selection, reads between 200 and 2,000 nucleotides were assembled using Canu v1.5 . Identif2. 5www.hmmer.org) was used to identify divergent virus segments that could not be classified by Diamond.Ten pools were made for Illumina sequencing, containing three to five RNA extracts each. An overview of the ten pools can be found in 2. 6The Qiagen OneStep RT-PCR kit was used to screen for the different viruses and to complete gaps in the obtained genome sequences. The following cycling conditions were used: 50°C for 30 min, 95°C for 15 min, 40 cycles of 94°C for 30 s, a primer-specific annealing temperature for 30 s and 72°C for 60 s, and a final extension step at 72°C for 10 min. Completion of genome ends was done using the Roche 5ʹ/3ʹ RACE kit, 2nd generation. An overview of all used screening primer sets and annealing temperatures can be found in Supplementary Table S2. PCR products were visualized by 2 per cent agarose gel electrophoresis and Sanger sequenced to confirm their identity. Sequencing was done by Macrogen Europe  after the products were purified in-house with PureIT ExoZAP .2. 7Rhabdoviridae and Reoviridae. For the rhabdovirus phylogenetic tree, amino acid sequences for all currently recognized International Committee on the Taxonomy of Viruses species, supplemented with all unclassified rhabdoviruses >5,500 bp (NCBI: txid35303), were downloaded from NCBI-Nucleotide (https://www.ncbi.nlm.nih.gov/nuccore). Seqkit was used to keep only protein sequences >1,800 amino acids, which were aligned using MAFFT   sequence of members of the families gorithm) . Based ogorithm) . The res3.3. 1MG407659-MG407660). Genome sequences similar to this virus (98.5–99.1 per cent nucleotide identity) have recently also been discovered in northern European ticks, under the name Norway phlebovirus 1 . The genome has the typical rhabdovirus genome architecture, encoding five open reading frames (ORFs): 3ʹ-N-P-M-G-L-5ʹ and clusters alongside Blacklegged tick rhabdovirus 1 and IRE CTVM19–associated rhabdovirus, both tick-derived viruses (Chimay betaricinrhavirus) in a novel genus (Betaricinrhavirus) in the family Rhabdoviridae.We reported previously the discovery of a novel nairovirus, Grotenhout virus, in a pooled library of ten adult tick RNA extracts . Even thovirus 1 . All fouovirus 1 , but for viruses . Chimay 3. 2Botourmiaviridae, Chrysoviridae, Endornaviridae, Hypoviridae, Polymycoviridae, and Quadriviridae) especially in Pool 6. This pool also displayed the largest diversity, but this is most likely attributable to the presence of fungi in the sample used for RNA extraction. Whether these fungi were infecting the ticks in this sample or should be considered environmental contaminants remains to be determined.The finding of three separate viruses in the RNA isolated from a single tick prompted us to further investigate the viral diversity in Belgian ticks. Therefore, we decided to collect additional ticks of different developmental stages and from different locations in Belgium and used next-generation sequencing for the detection of known and novel viruses in these ticks. Ten tick pools, made from pooling three to five RNA extracts, were sequenced on an Illumina 4000 HiSeq, yielding 48,976,538–83,201,118 reads per pool. Variable numbers of ticks from mixed locations of origin were used in the different pools to maximize the overall observed diversity. However, this approach also means that the pools should not be compared quantitatively with each other, as such comparisons would be biased due to the differences in sample handling and pooling. Following adapter trimming, between 4,874 and 74,219 contigs could be assembled for each pool . Althoug3. 3I. ricinus in Norway (MW874081-MW874092), as well as two previously undescribed viruses, named Gierle tick virus (GenBank ID: MW874093-MW874104) and Zoersel tick virus (GenBank ID: MW874105-MW874114), respectively. For Eyach virus strain Heverlee, a combination of Illumina and Sanger sequencing was used to obtain the complete sequence of all 12 genome segments. While almost all segments are highly similar (>94 per cent nucleotide identity) to previously identified strains of Eyach virus , a colti-like virus distantly related to the recently discovered Fennes virus, but only three partial segments could be reconstructed for this virus.While for most detected viruses only fragments of the genome were retrieved, some were present more abundantly, allowing the reconstruction of near-complete genome sequences. These include Grotenhout virus and the aforementioned Leuven phlebovirus, which have also recently been observed in n Norway . With thch virus , the foual virus . Lastly, viruses . By look3. 4I. ricinus from Norway , a common hard-bodied tick species in the east of North America, has been reported to carry nairo-, phlebo-, and rhabdoviruses that are closely related to Grotenhout virus, Leuven phlebovirus, and Chimay rhabdovirus, respectively  do seem to support this association. For many other viruses of which we find traces, which only share similarities with unclassified viruses or viruses belonging to virus lineages with mixed hosts, the origin remains largely unknown. Nonetheless, they do provide further evidence that virus diversity in ticks (and other arthropods) is much higher than until recently known. Additional studies are needed to further map this diversity, allowing a better understanding of tick-associated virus evolution. A better characterization of the virus diversity in tick populations can also facilitate studying the spread of known and novel zoonotic viruses, allowing more targeted pre- and post-exposure countermeasures for tick-borne diseases to be taken where needed.For several of the viruses we detected in our sequencing data, highly similar or related viruses have previously been reported in different tick species from various locations, strengthening the hypothesis that these viruses are truly tick-associated. As noted by Pettersson and colleagues, this association can sometimes be difficult to claim based only on sequencing data, especially if the viruses in question are only found infrequently or in low abundance . IntriguI. ricinus from Belgium. We show that Belgian ticks carry a wide variety of viruses and that several of these viruses are found in different locations and different tick developmental stages. We also managed to reconstruct (near-)complete genome sequences for several novel viruses, including a new strain of the pathogenic Eyach virus, a new coltivirus species, and a highly divergent reovirus that remains to be classified. This research further illustrates that many tick-associated viruses remain to be discovered and that additional studies are needed to expand our knowledge of virus diversity in ticks and to further characterize these novel viruses.In conclusion, we present the first study looking at virus diversity in veab066_SuppClick here for additional data file.


Cryptococcus neoformans and C. gattii species complexes. The C. neoformans species complex classically comprises four major molecular types , and the C. gattii species complex comprises another four  and the newly identified molecular type VGV. These major molecular types differ in their epidemiological and ecological features, clinical presentations, and therapeutic outcomes. Generally, the most common isolated types are VNI, VGI, and VGII. The epidemiological profile of cryptococcosis in domestic cats is poorly studied and cats can be the sentinels for human infections. Therefore, the present study aimed to determine the molecular characterization of Cryptococcus spp. isolated from domestic cats and their dwellings in the metropolitan area of Rio de Janeiro, Brazil. A total of 36 Cryptococcus spp. strains, both clinical and environmental, from 19 cats were subtyped using multilocus sequence typing (MLST). The ploidy was identified using flow cytometry and the mating type was determined through amplification with specific pheromone primers. All strains were mating type alpha and 6/36 were diploid . Most isolates (63.88%) were identified as VNII, a rare molecular type, leading to the consideration that this genotype is more likely related to skin lesions, since there was a high percentage (68.75%) of cats with skin lesions, which is also considered rare. Further studies regarding the molecular epidemiology of cryptococcosis in felines are still needed to clarify the reason for the large proportion of the rare molecular type VNII causing infections in cats.Cryptococcosis is a systemic fungal disease acquired from contaminated environments with propagules of the basidiomycetous yeasts of the  Cryptococcus neoformans species complex, which is a cosmopolitan agent. Species of the C. gattii species complex mainly affect immunocompetent patients and are mostly restricted to tropical and subtropical climates, although in the late 1990s, they expanded to areas with a temperate climate [C. neoformans/C. gattii complexes classically comprise eight major molecular types: C. neoformans var. grubii—VNI and VNII; C. neoformans var. neoformans—VNIV and the hybrid AD–VNIII; C. gattii—VGI, VGII, VGIII, and VGIV [C. neoformans and VGV of C. gattii [Cryptococcosis is a systemic mycosis that affects a wide variety of mammalian hosts . Most hu climate ,6. The Cand VGIV . A recenand VGIV ,9. Althoand VGIV  of C. ne. gattii . For thiC. neoformans/C. gattii complexes shows a predominance of VNI in North America (41%), Central America (78%), South America (64%), Africa (72%), Europe (45%), and Asia (77%) [C. gattii. Among all genetic lineages of the cryptococcosis agents, VGI is the most common in Oceania (43%) and the second most isolated in Asia (15%). The molecular type VGII is the second most common in the Americas, with 26% in North America, 21% in South America, and 11% in Central America [These major molecular types differ in their epidemiological and ecological features, clinical presentations, and therapeutic outcomes . The gloia (77%) . The molia (77%) . The mol America .C. neoformans species complex is widely dispersed in the environment, especially in soil enriched with the excretion of pigeons or gregarious birds, while the environmental isolation of the members of the C. gattii complex has been related to several species of trees. Nevertheless, the isolation of these species from the dwellings of humans has been reported [The reported ,16, emphThere is no opportunistic characteristic reported in animal cryptococcosis. In domestic cats, infections with the feline immunodeficiency virus (FIV) and the feline leukemia virus (FELV) are not predisposing factors for the occurrence of cryptococcosis . The infCryptococcus spp. from domestic cats in the metropolitan area of Rio de Janeiro, Brazil.The epidemiological profile of cryptococcosis in domestic cats is poorly studied. Epidemiological studies of cryptococcosis often include isolates from human hosts and the environment, but rarely isolates from animals. In general, animal cryptococcosis is under-reported. Most of the available literature consists of case reports with some atypical clinical presentations describing the clinical signs, diagnosis, and treatment, but rarely identifying the molecular type, or even the species complex. Additionally, very few animal cases of cryptococcosis have been documented in Latin America ,22,23. TC. neoformans and C. gattii strains recovered from cats assisted at the Laboratory of Clinical Research on Dermatozoonoses in Domestic Animals (Lapclin-Dermzoo), Fiocruz, Rio de Janeiro, Brazil, from 2000 to 2019, were included in this study. The owners were asked to investigate the possible environmental sources of infection at their residence and surroundings for the cases that occurred between 2016 and 2019. Clinical and epidemiological data from cats were obtained from the medical records at Lapclin-Dermzoo.All viable C. neoformans and C. gattii were differentiated on a canavanine-glycine-bromothymol blue medium (CGB).The environmental samples were processed according to the protocol described by Passoni et al. . BrieflyC. neoformans and C. gattii was applied [CAP59, GPD1, LAC1, PLB1, SOD1, and URA5) and the IGS1 region were amplified  using the PCR conditions previously described [http://www.megasoftware.net (accessed on 1 January 2021)). The allele types (ATs) and the sequence types (STs) were identified via sequence comparisons with the C. neoformans/C. gattii MLST database at http://mlst.mycologylab.org/ (accessed on 1 January 2021). The sequences of all the newly identified allele types were submitted to the C. neoformans MLST database. The haplotype and nucleotide diversity were calculated using DnaSP v5.10 (http://www.ub.edu/dnasp/ (accessed on 1 January 2021)).The ISHAM consensus multilocus sequence typing (MLST) scheme for  applied  to identescribed . SequencThe mating type was determined by PCR using a couple of mating type-specific (Mat) oligonucleotide primers: Mat-α  and Mat-a , which generated 101 bp and 117 bp amplification products, respectively. Amplification was performed in a final volume of 25 µL according to Chaturvedi et al. [URA5-RFLP analysis using the enzymes HhaI and Sau96I to verify the molecular type of the isolate with dubious results on the MLST [URA5-RFLP patterns were assigned visually by comparison with the banding patterns obtained from the standard strains WM 148 (VNI), WM 626 (VNII), WM 628 (VNIII), WM 629 (VNIV), WM 179 (VGI), WM 178 (VGII), WM 175 (VGIII), and WM 779 (VGIV).Identification of the major molecular types was performed by the MLST . URA5-RFFACSCalibur™, and CFRVS 70297 (WM 628) and CFRVS 70302 (WM 178) were used as diploid and haploid standard strains, respectively. The wavelength of the laser beam was 488 nm and 10,000 cells were counted and their fluorescence intensities were measured. The data are shown as histograms, where the abscissa represents the channel numbers in the proportion of their fluorescence intensities and the ordinate shows the number of cells.Flow cytometry analysis was performed to identify the ploidy of isolates according to Sia et al. . DNA ploDuring the study period (2000–2019), 26 cases of feline cryptococcosis and 4 cases of colonization were diagnosed at Lapclin-Dermzoo/INI/Fiocruz. Of these, 16 cases of cryptococcosis and 3 cases of colonization in the oral cavity had the isolates preserved and were included in the study. All 19 studied cases were from the metropolitan area of Rio de Janeiro, Brazil, and most of the cats were mixed breeds (n = 17). More than one strain was isolated from six cats, from different anatomical sites and/or from serial samples. For the other 10 cases, one isolate per cat was selected. The clinical samples included were fragments of skin lesions (3), oral secretions (3), nasal secretions (4), exudates of skin lesions (10), fragments of lymph nodes (2), an exudate from a conjunctival lesion (1), exudates of nasal lesions (3), a fragment of the lung (1), and fragments of the liver (1) .Of the 16 symptomatic cases, 11 had skin lesions (68.75%), i.e., with the lesions being located not near to any mucosa, and 8 had mucosal lesions. The most affected anatomical site was the nasal region (n = 9), followed by the limbs (n = 8). Lesions in other locations were observed to a lesser extent in the face, ear, neck, chest, lips, back, head, abdomen, tail, conjunctiva, and periocular region.Cryptococcus spp. were isolated from two residences , wood (n = 7), and cage dirt (n = 1). C. neoformans and 7 C. gattii strains. After identification, all isolates were deposited in the Collection of Pathogenic Fungi (CFP)/INI/Fiocruz.Thirty-six isolates, including twenty-eight clinical and eight environmental isolates, were analyzed. The CGB medium discriminated 29 C. neoformans VNII, 6 VNI, and 4 C. gattii VGII. From environmental origin, five isolates were VNII and three VGII. The MLST analysis identified 17 sequence types (STs) amongst the 35 strains studied and 6 of these had not yet been described: ST616, ST618, ST619, ST620, ST621, and ST639 , which led to the fact that no ST could be assigned. Flow cytometry analysis confirmed the diploidy status of the strain  corresponding to the molecular type VNII  and decomposing wood. Colonization of the nasal cavity by C. neoformans in cats has been previously reported [Cryptococcus spp. in a serology test (latex particle agglutination) with a negative nasal swab, suggesting that the colonization of the oral cavity may be another gateway to the disease in animals, in addition to the nasal cavity. Furthermore, there are reports of unique gastrointestinal lesions by Cryptococcus spp. in dogs and cats [Species of the genus  of dogs ,28. In treported ,29, but reported ,31,32. Dand cats ,34,35, aThe most frequent infection site in cats is the nasal cavity, which can occur in 40% of the cases . The occC. neoformans isolated from cats were VNII [Cryptococcus spp. was observed in a study where cats were more affected by the molecular type VGIII and dogs by VNI [The most common molecular type globally is VNI, considering both clinical and environmental isolates . The VNIere VNII . The difs by VNI . Anothers by VNI . Thus, wC. neoformans/C. gattii species complexes. Animal exposure may be related to internal or external events at the residence [Restricting access to the street did not exempt cats from exposure to the species of the esidence . In the C. neoformans/C. gattii species complexes can share the same ecological niche [Cryptococcus spp. have already been reported in human patients [The species of the al niche . In viewpatients ,43. Our A single nucleotide polymorphism (SNP) was detected among the isolates from one of the cats (cat 6). The occurrence of microevolution in vitro was reported as a result of successive subcultures for long periods  or in viC. neoformans VNI and VNII, and the absence of the mating type a serves as additional evidence of the lack of recombination among the isolates. However, in the present study, six diploid strains, molecular type VNII MATα, were identified. Diploidy is usually transient and precedes several types of reproduction, such as sexual reproduction and same-sex mating type (endoreplication and hybridization) [The low molecular diversity supports the clonal population of ization) . NonetheC. neoformans VNI seem to be cosmopolitan and have also been identified in several countries. The ST5 is the dominant genotype in several populations in China [Despite the population clonality, the isolates demonstrated a high haplotypic diversity, with 17 sequence types being identified among the 35 strains studied. The sequence types ST5, ST23, and ST77 of in China  and is sin China ,47. ST23in China ,49,50. Tin China ,51,52,53ST20 (VGIIa) is considered a highly virulent subtype and it was the main sequence type of the Vancouver Island epidemic in 1999, which later expanded to the northwestern U.S. states, Washington and Oregon ,55. ST20Further studies regarding the molecular epidemiology of cryptococcosis in felines are still needed to clarify the reason for the large proportion of the rare molecular type VNII identified in this study; whether it is due to the preference of VNII isolates to cause skin lesions in cats or a greater susceptibility of cats to be infected by VNII isolates remains unknown.


Because of the catastrophic outbreak of global coronavirus disease 2019 (COVID-19) and its strong infectivity and possible persistence, computational repurposing of existing approved drugs will be a promising strategy that facilitates rapid clinical treatment decisions and provides reasonable justification for subsequent clinical trials and regulatory reviews. Since the effects of a small number of conditionally marketed vaccines need further clinical observation, there is still an urgent need to quickly and effectively repurpose potentially available drugs before the next disease peak. In this work, we have manually collected a set of experimentally confirmed virus-drug associations through the publicly published database and literature, consisting of 175 drugs and 95 viruses, as well as 933 virus-drug associations. Then, because the samples are extremely sparse and unbalanced, negative samples cannot be easily obtained. We have developed an ensemble model, EMC-Voting, based on matrix completion and weighted soft voting, a semi-supervised machine learning model for computational drug repurposing. Finally, we have evaluated the prediction performance of EMC-Voting by fivefold crossing-validation and compared it with other baseline classifiers and prediction models. The case study for the virus SARS-COV-2 included in the dataset demonstrates that our model achieves the outperforming AUPR value of 0.934 in virus-drug association’s prediction. In 2019, a new type of respiratory disease caused by the virus SARS-CoV-2 had been officially named the coronavirus disease 2019 (COVID-19), whose outbreak caused a disaster for people around the world, affecting economics, politics, and life in every way. It can cause fevers, fatigue, cough, muscle soreness, and progressive breathing difficulties . Also, iThe first category is the network-based model , which uSome hybrid models incorporate more than one approach . For exaThe workflow of this study mainly involves the following: (i) Collect and summarize the human virus-drug associations from public databases and calculate the integrated similarity for viruses and drugs, respectively. (ii) Test multiple prediction models, including machine learning–based models (supervised), matrix completion models (semi-supervised), and deep learning models, and select the ones with the best performance. (iii) Ensemble the selected models by the weighted soft voting strategy and predict a probability score list for all the virus-drug pairs. The flowchart of our model can be seen in There are numerous studies and experiments to confirm the associations between viruses and broad-spectrum antiviral drugs. According to the hypothesis, similar viruses tend to interact with similar drugs. This study attempts to predict potential virus-drug associations, especially those most related to the virus SARS-CoV-2, by combining known virus-drug association, virus-virus similarity, and drug-drug similarity. The existing virus-drug associations are extracted from public published databases. We construct a virus-drug adjacency matrix to represent binary interaction information. Virus similarity is integrated by virus function similarity and Gaussian similarity, and drug similarity is integrated by chemical structure similarity and Gaussian similarity.A ∈ ℜm×n to represent the known virus-drug associations, with rows representing viruses and columns representing drugs. If there is an association between virus vi (1≤i≤m) and drug dj (1≤j≤n), the element A in the adjacency matrix will be assigned 1, or else 0. We denoted the symbol Ω as a set of indices of partially known entries in the matrixA. The symbol PΩ(A) is defined as a linear projection operator subject to the Equation 1:The virus-drug association prediction problem can be modeled as a classification problem. By predicting, a list of scores corresponding to all the virus-drug pairs is obtained. Then, the virus-drug pairs are prioritized according to the predicted scores. Moreover, they are ranked to infer the potential associations between viruses and drugs. We use an adjacency matrix 1  and Hydroxychloroquine are recorded in the clinical phase III  represents the similarity score between virus vi and virus vj. Since not every pair of proteins has functional interaction, many pairs of viruses have no functional similarity. Thus, the matrix FS is extremely sparse, with many elements having a value of 0. In order to measure the virus-virus similarity more comprehensively, we calculated the Gaussian interaction profile (GIP) kernel similarity GIPv ∈ ℝm×m for all the virus-virus pairs by leveraging the known virus-drug association. The GIP kernel similarity GIPv between vi and vj can be calculated as the Equation 2.We calculate the virus functional similarity as that in reference . First, v is the normalized kernel bandwidth: IP(vi) is the i-th row of the association matrix A, denoting whether there is an association between vi and all the drugs. As a result, based on the above functional similarity FS and Gaussian kernel similarity GIPv, the integrated virus similarity Sv between virus vi and virus vj is calculated as Equation 3:where ||⋅|| denotes the norm of a vector. The symbol γ3 by the names of the drugs. Then, we input the IDs of all drugs into SIMCOMP2, which can calculate the structural similarity score between all drug-drug pairs in batches based on their structural information obtained by IDs. Finally, we denoted the structural similarity of all the drug-drug pairs as used as matrix DS ∈ ℝn×n. The value of the element DS represents the similarity score between drug di and drug dj.We calculated the drug chemical structure similarity by the method of SIMCOM2 . First, IP(vi), the drug interaction vector IP(dj) is the column vector of the association matrix A. The GIP similarity between di and dj, denoted as GIPd, also can be calculated by substituting the IP(di), IP(dj), and γd into Equation 2. As a result, the integrated drug similarity Sd between drug di and drug dj is calculated as Equation 4:Like the virus interaction vector Sd].According to the hypothesis, similar viruses will tend to be associated with similar drugs. Therefore, appropriate similarity measures will help predict potential virus-drug associations. In this study, we used the final integrated virus (drug) similarity [de novo virus (one without any known association) and vice versa.Since the number of known drug-virus associations is far less than the total number of virus-drug pairs, the virus-drug association adjacency matrix is highly sparse. Moreover, the virus-drug association matrix is highly unbalanced and biased due to the lack of negative samples (negative sample cannot be easily determined), just as with positive samples and unlabeled samples. For all these reasons, traditional supervised machine learning classifiers that both need positive labels and negative labels are not suitable for predicting potential virus-drug associations. Similarly, the deep learning model, which requires a large number of samples for training, is not suitable for this prediction scenario. Thus, this study takes advantage of the matrix completion method for prediction, a semi-supervised machine learning model that does not require negative samples and has made remarkable achievements in the recommender system. Most importantly, the method of matrix completion can predict the drugs associated with a Various matrix completion (MC) models have been developed for association prediction and work well. However, each of them has its limitations and shortcomings. In order to overcome the shortcoming of one single model, ensemble learning is considered an effective method. Ensemble learning is a kind of hybrid model whose prediction is accurate and robust. There are also many ensemble strategies such as voting, RF, boosting, and stacking. In our study, weighted soft voting is utilized to ensemble multiple matrix completion models to predict potential associated drugs against the virus SARS-CoV-2. We fitted the known virus-drug associations, virus integrated similarities, and drug integrated similarities into seven matrix completion models, including the model of Graph Regularized Matrix Factorization (GRMF) , InductiA, assuming that the entries in the recovered matrix should be as close as possible to entries in the original adjacency matrix  ∈ Ω . The four basic matrix completion models selected and ensembled in our EMC-Voting model are described below.As discussed above, the association adjacency matrix is an incomplete binary matrix, so the purpose of matrix completion is to retrieve the missing items in the adjacency matrix y matrix . Combiney matrix . FurtherA ∈ ℝm×n into two low-rank feature matrices Lf ∈ ℝm×k and Rf ∈ ℝk×n for viruses and drugs, respectively, based on the principle of low-rank approximation (LRA). k is the number of the latent independent features in the feature matrix Lf and Rf. The objective function of GRMF is Graph Regularized Matrix Factorization (GRMF), one of the mathematical models of matrix completion, is proposed by l, λv, λd are the balance parameters, ai, bj are the i-th and the j-th row of the matrix Lf and Rf, respectively. Lf and Rf are minimized by Tikhonov regularization ×(m + n) is a computational model that has been used to predict the latent associations between lncRNAs and diseases . It is aR is the approximate matrix (the predicted/recovered matrix), rank(R) is the rank of R. Unfortunately, rank minimization is an NP-hard problem . PΩ(⋅) is a linear projection operator, the parameter α is used to balance the nuclear norm and the error. The model of BNNR can be solved by the Alternating Direction Method of Multipliers (ADMM) (where the matrix s (ADMM) . There aThe model of heterogeneous graph inference with matrix completion (HGIMC) achieves matrix completion at three steps : FirstlyThe parameters in the model of EMC-Voting were tuned on the training set by fivefold CVs using grid search. As shown in de novo virus, we deleted all known associations of the virus SARS-CoV-2 and used our model to predict associated drugs to see if the known associations were correctly predicted. At last, we conducted case studies on the top 40 predictive drugs against the virus SARS-COV-2. In our experiments, all the matrix completion models, including our EMC-Voting model, are conducted in the MATLAB 2017b, the operating system of windows10, with AMD Ryzen 5 3600 6-core, 3.59 GHz CPU, 32G memory. Furthermore, all the machine learning and deep learning comparison models are run on the system of python 3.7 with TensorFlow 2.2.0.In this section, we first introduced the performance evaluation metrics adopted in our model. Then, we compared our EMC-Voting model with several machine learning-based models and single MC-based models by fivefold CV on the same dataset to evaluate the predictive performance of our model. Furthermore, to test the prediction ability of EMC-Voting for the First, in order to make a fair comparison with machine learning classifiers (which require negative samples) and uniformly evaluate their prediction performance, we constructed negative samples with the same size as positive samples by the method proposed by F1 score can be interpreted as a weighted average of accuracy and recall rates:Sensitivity = TP/(TP + FN) = Recall. Specificity is the proportion of true negative: Specificity = TN/(TN + FP). PPV is the positive predictive value, which is equal to Precision: PPV = TP/(TP + FP) = Precision. NPV is the negative predictive value: NPV = TN/(TN + FN). AUC is the area under the ROC curve. Importing metrics from the sklearn library, we called the function roc_curve to draw the ROC curve, and the function auc is called to calculate the AUC value. AUPR is the area under the precision-recall (PR) curve. The function precision_recall_curve is called to calculate the Precision value and the recall value, and the function auc also can be called to calculate the AUPR value. The greater the metrics values are, the better the predictive performance is. So, the value of AUPR is employed as the main metric for performance evaluation. After one round fivefold CV, our EMC-Voting model achieves AUC values of 0.9280, 0.9135, 0.9415, 0.9502, 0.9303, and 0.9327 at each fold, respectively. The mean value of AUC of one round fivefold cross-validation is 0.9327. As shown in Sensitivity is the proportion of true positive: First, we compared our model with SVM, GBDT, NN, ERLX, and SPMCIIP models. The classifiers of SVM, GBDT, and NN were called with the method of “SVC,” “lightgbm,” and “mlp,” respectively. The model SPMCIIP ensembled SVM, GBDT, and NN classifiers with weights “0.3, 0.5, 0.2” to predict critical COVID-19 . The modTo further test the prediction performance for the virus-drug associations, we compare our EMC-Voting model with the state-of-the-art matrix completion-based models by fivefold CV on the virus-drug dataset. IMCMDA was first proposed by The results of our model and all the comparison models are shown in Although DMF and DLMC are deep learning–based methods that perform well in text recognition, they do not perform excellently on this DrugVirus dataset. Probably because the size of our dataset is small and the number of samples is not enough for adequate training. de novo virus, we deleted all associations between the virus SARS-CoV-2 and its associated drugs. After predicting through our EMC-voting model, the top 30 predicted drugs were compared with the list of drugs published in the database DrugVirus. Results are demonstrated in de novo virus, consistent with the advantages of the matrix completion algorithm.COVID-19 is a new disease, and there is currently no experimentally confirmed treatment for it. On the public database DrugVirus, 10 drugs under clinical studies have been considered to have great potential for subsequent clinical trials, such as Remdesivir, Ribavirin, Arbidol\Umifenovir, Cepharanthine, Chloroquine, Homoharringtonine, Hydroxychloroquine, Ivermectin, Lopinavir, and Mefloquine. To simulate a Finally, we leveraged our EMC-Voting model to predict potentially therapeutic drugs against the virus SARS-COV-2 and implemented a case study on the TOP 15 predicted drugs against SARS-COV-2. Although there are no experimentally confirmed drugs against SARS-COV-2, a few drugs such as Arbidol\Umifenovir, Cepharanthine, Chloroquine, Homoharringtonine, Hydroxychloroquine, Ivermectin, Lopinavir, Mefloquine, Remdesivir, and Ribavirin are already in clinical trials and are recorded as associated with the SARS-COV-2. All these associations published by the database DrugVirus were considered known and input into our ensemble model for prediction. After predicting, we obtained a predictive score matrix for all virus-drug pairs. Then, we deleted all known associated drugs for each virus (row in the score matrix) and sorted the predicted drugs in descending order. As a result, the TOP 15 predicted drugs for SARS-COV-2 are demonstrated in It can be seen that most of the predicted drugs by the EMC-Voting model have been successfully verified in current clinical treatments. For example, the antiviral efficacy of Favipiravir was evaluated using a Syrian hamster model. Favipiravir was found to have a strong dose effect when treatment was initiated before or at the same time as infection, leading to decreased titers of pulmonary infection and clinical relief of COVID-19 . Weiss eThe main contributions of this study are the following: (1) We have proposed a weighted soft voting ensemble model perceiving the accuracy of different matrix completion models. (2) Based on the DrugVirus dataset, we have screened several machine learning–based models belonging to different types and multiple deep learning models. Then we ensembled four matrix completion models as the optimal combination according to their performance indicators. (3) We have demonstrated reliable results for the EMC-Voting model through multiple cross-validation experiments and case studies. It can be found that our model has good predictive performance and can effectively assist clinicians in selecting possible therapeutic drugs.Based on the characteristics of the virus-drug database, with sparse, no negative samples and extremely unbalanced data, we screened several matrix completion models and ensembled four to predict drugs that may have therapeutic effects on SARS-COV-2, so it can assist clinical doctors to identify effective drugs at an urgent time. Furthermore, based on preclinical and clinical studies in which the database is being updated, the scope of BSAAs and their indications will be expanded in the future, and drug combinations for the treatment of emerging and re-emerging viral infections and co-infections will be identified .https://github.com/vivian457/COVID-19_drug_virus_association.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 below: WL, SW, and JX conceptualized the work and planned the procedure for experiments. JX collected the data. WL implemented all the experiments, analyzed the results, and drafted the manuscript. All authors have read and supported the final edition.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.