Pseudogymnoascus destructans partitivirus-pa, had infected the North American isolates of Pseudogymnoascus destructans, the fungal pathogen that causes white-nose syndrome in bats. We showed that the diversity of the viral coat protein sequences is correlated to their geographical origin. Here we hypothesize that the geographical adaptation of the virus could be used as a proxy to characterize the spread of white-nose syndrome. We used over 100 virus isolates from diverse locations in North America and applied the phylogeographic analysis tool BEAST to characterize the spread of the disease. The strict clock phylogeographic analysis under the coalescent model in BEAST showed a patchy spread pattern of white-nose syndrome driven from a few source locations including Connecticut, New York, West Virginia, and Kentucky. The source states had significant support in the maximum clade credibility tree and Bayesian stochastic search variable selection analysis. Although the geographic origin of the virus is not definite, it is likely the virus infected the fungus prior to the spread of white-nose syndrome in North America. We also inferred from the BEAST analysis that the recent long-distance spread of the fungus to Washington had its root in Kentucky, likely from the Mammoth cave area and most probably mediated by a human. The time to the most recent common ancestor of the virus is estimated somewhere between the late 1990s to early 2000s. We found the mean substitution rate of 2 X 10−3 substitutions per site per year for the virus which is higher than expected given the persistent lifestyle of the virus, and the stamping-machine mode of replication. Our approach of using the virus as a proxy to understand the spread of white-nose syndrome could be an important tool for the study and management of other infectious diseases.Understanding the dynamics of white-nose syndrome spread in time and space is an important component for the disease epidemiology and control. We reported earlier that a novel partitivirus,  White-nose syndrome is a deadly fungal disease of North American bats, that has been spreading from the northeastern United States to Canada and to the south and west. Understanding the spread has been difficult because the fungus is nearly identical throughout North America, consistent with a recent introduction. The fungus is infected with an RNA virus that shows significant variation over time, especially in the gene for the coat protein. Here we used the variation in the virus to create a phylogenetic tree that could help us understand the spread of the fungal disease. We found that the fungus/virus combination was likely in North America since around 1999, earlier than previously thought. In addition, we found that the initial site of introduction was more likely Connecticut, rather than New York State, and that the recently identified fungal infections in bats in Washington state most likely came from Kentucky. This study also demonstrates that viruses of a pathogen can be used to understand the epidemiology of the pathogen. Psedogymnoascus destructans (Pd), is a highly pathogenic infection spreading among North American bats since around 2006 ) is within the order of magnitude for dsRNA viruses (~10−7 to 10−3) reported earlier .(PPTX)Click here for additional data file.S4 FigPseudogymnoascus destructans partitivirus-pa as 2X10-3 substitution per site per year with 95% HPD .The statistics from the analysis are summarized in an inset which showed the mean substitution rate for (PPTX)Click here for additional data file.S5 FigThe tree is annotated with colors coded for the locations that included 12 US states and New Brunswick, Canada. Standard abbreviations of US states are used in the figure legend except “Can” for New Brunswick province of Canada and “KYmc” for Mammoth cave locations in Kentucky. Nodes of the tree are denoted by solid black circles and the size of the circle corresponds to posterior supports shown in the legend. Branch lengths are in time, depicted by a horizontal time-scale. For detail description of the virus isolate ID, refer to (PPTX)Click here for additional data file.S6 FigThe lines connecting different locations represent branches in the maximum clade credibility tree. The bright to dark gradient of the line’s red color represent time from 1999 to 2018. Note higher number of connectivity in New York, West Virginia and Kentucky.(PPTX)Click here for additional data file.S7 FigThe line between the locations represent branches in the maximum clade credibility (MCC) tree. The dark to light color gradient of the circles indicate the relative age of the branch inferred (old to recent).(PPTX)Click here for additional data file.


Genetic sequencing of polioviruses detected through clinical and environmental surveillance is used to confirm detection, identify their likely origin, track geographic patterns of spread, and determine the appropriate vaccination response. The critical importance of genetic sequencing and analysis to the Global Polio Eradication Initiative has grown with the increasing incidence of vaccine-derived poliovirus (VDPV) infections in Africa specifically (470 reported cases in 2019), and globally, alongside persistent transmission of serotype 1 wild-type poliovirus in Pakistan and Afghanistan (197 reported cases in 2019). Adapting what has been learned about the virus genetics and evolution to address these threats has been a major focus of recent work. Here, we review how phylogenetic and phylogeographic methods have been used to trace the spread of wild-type polioviruses and identify the likely origins of VDPVs. We highlight the analysis methods and sequencing technology currently used and the potential for new technologies to speed up poliovirus detection and the interpretation of genetic data. At a pivotal point in the eradication campaign with the threat of anti-vaccine sentiment and donor and public fatigue, innovation is critical to maintain drive and overcome the last remaining circulating virus. The virus has only one natural host, humans, and is primarily spread between individuals by faecal-oral transmission. In rare cases poliovirus can enter the central nervous system, leading to paralytic poliomyelitis ). Although genomics was in its infancy when work on preventing poliomyelitis began, poliovirus was one of the earliest genomes to be sequenced in full, with two groups publishing serotype one sequences in 1981 . Followiin vitro poliovirus growth a possibility, and knowledge of the three serotypes, successful development of inactivated poliovirus vaccine (IPV) and live-attenuated oral poliovirus vaccine (OPV) followed in 1953 and 1956, respectively , which are OPV strains  that have regained transmissibility and pathogenicity and can cause outbreaks of paralysis similar to their wild counterparts . AlthougThis review describes how collection and analysis of increasing amounts of genetic data is transforming the long-standing poliovirus eradication programme, highlighting both current activities and future promise. It emphasises how our understanding of poliovirus genetics and evolution will be critical to success in tackling the major challenges facing the GPEI, allowing us to better describe transmission of virus and develop more efficient and stable vaccines.Identification of poliovirus in the laboratory has had a role in the eradication programme since its inception, with detection of viruses present in stool and environmental samples acting as the major surveillance tool.2.1Poliovirus surveillance has traditionally relied on detection and sequencing of poliovirus from stool samples taken from patients with AFP. Preceding the global eradication drive the Pan-American Health Organization recommended reporting all AFP cases in children under 15 years of age and in adults where poliovirus was the suspected cause of paralysis to ensure the detection of paralytic cases . This waAs only one in 200 − 1,000 individuals infected with poliovirus is paralysed, the majority of infections are asymptomatic or result in mild illness and are not detected by AFP surveillance. Analysis of the genetic relatedness of polioviruses isolated from AFP cases gives an indication of the sensitivity of AFP surveillance, with viruses found to be distantly genetically related to others referred to as ‘orphan’ viruses, which are indicative of virus circulation without detection. In Nigeria, a country removed from the endemic list in September 2015, cases of WPV were detected in 2016 that were directly linked to isolates from virus circulating in the same region in 2011 . This su2.2As the incidence of paralytic polio has fallen globally, sampling of sewage networks to look for the virus has expanded; especially in countries with continuing WPV1 transmission or long-term transmission of VDPVs. The majority (>99%) of poliovirus infections are asymptomatic and do not result in paralysis, and so are not detected by AFP surveillance, but poliovirus shed in stool by infected individuals can still be detected in environmental (sewage) samples. Environmental surveillance (ES) has been shown to be more sensitive than AFP surveillance in areas with convergent sewage networks . For exaSewage sampling is efficient as it allows fewer resources to be used to survey a larger population of possibly affected individuals than direct door to door or local stool surveys. A study in 1991 in Cartagena, Colombia was able to directly sample stool from 196 children in 3 days in a neighbourhood with approximately 5,300 children. Seventy per cent of households in this neighbourhood were served by the sewage network and in the same time period forty-two sewage samples were taken. These samples represent repeat samples of the same 70 per cent of the population but much more complete sampling than the 3.6 per cent coverage with stool surveys. When comparing rates of poliovirus detection, 21 per cent of sewage samples were positive compared with 8 per cent of stool samples .Despite the utility of poliovirus ES, it is not without challenges. Poliovirus survival in the environment is highly variable, with a 90 per cent infectivity loss observed in 26 days in sewage at 23°C . This af2.3The WHO recommended protocol for detection of poliovirus in stool or ES samples is to isolate the virus using two cell lines, one murine cell line (L20B) that is genetically modified to express the human poliovirus receptor (CD155) and selectively grows poliovirus, and one human rhabdomyosarcoma (RD) cell line which is susceptible to a broad range of enteroviruses . Viruses3.1−2 nucleotide changes per site per year in the VP1 region  , which can provide information on the geographic history of viral movement and associated rates. Starting from geolocated virus sequences, this approach allows inference of the location of internal nodes in phylogenetic trees. DTA has previously been applied to inform viral diffusion for several infectious diseases including rabies, influenza and Ebola . An illuC880521)  Fig. 2)KC880365–C880521) .Ongoing analyses using the DTA approach have been applied to WPV1 sequence data from Pakistan and Afghanistan, where WPV1 persists with complex circulation dynamics and geographical spread due to mobile and hard to reach populations. These preliminary analyses have identified cycles of transmission between the south-west of each country (Kandahar/Quetta and Karachi). They also highlight repeated introductions of virus from the tribal regions (between Islamabad and Kabul) into the Punjab province of Pakistan and show uninterrupted virus circulation within these tribal regions. Analysis of the distribution of clusters of transmission without formal DTA analysis also highlights these three, key, high-burden regions but does not construct probable ancestral locations or virus movements .Phylogenetic visualisations that split trees into clusters of transmission based on those presented by Dudas et al. for the 2014 Ebola outbreak and interactive phylogenetic trees using the Nextstrain platform have also been developed, and are provided in internal WHO reports, but are not yet publicly available . RegularGiven the declaration of polio as a PHEIC and the value of poliovirus genetic sequence data to interpreting its epidemiology, there is an urgency to share poliovirus sequences and phylogenetic trees more rapidly and more widely to allow broad scrutiny by scientists and policy makers. This is being pursued with the planned creation of a global polio nucleotide sequence database (PONS) by the US CDC.Live-attenuated OPV strains replicate in the gut to induce both local mucosal and systemic immunity, the former being important to prevent transmission of wild viruses. This is a major benefit over injected IPV, which provides systemic immunity against paralysis but limited intestinal mucosal immunity. Replication of OPV strains is, however, associated with virus shedding and transmission to contacts. Selection for loss of the attenuating mutations which reduce OPV virulence occurs during virus replication through mutation and recombination. In rare cases, this can lead the vaccine virus to regain pathogenicity and transmissibility similar to that seen in wild-type polioviruses. Vaccine strains are classified according to their source and their relatedness to the Sabin strain and other polioviruses. Current reporting standards define serotype two VDPVs as those viruses with at least six nucleotides difference from the Sabin strain in the VP1 region (at least ten nucleotides for serotypes one and three). Vaccine-related viruses not reaching this threshold are classified as ‘Sabin-like’. Although it is possible for Sabin-like viruses to cause poliomyelitis, this occurs much less frequently than with viruses which have crossed the threshold to be classified as VDPVs .Replication and shedding of the vaccine virus in the intestine and throat can occur long term in individuals with a primary B cell immunodeficiency who have been vaccinated with OPV as they are not able to clear vaccine virus infection. These immunodeficient VDPVs (iVDPV) may pose a risk to the wider population and have required the introduction of a primary immunodeficiency surveillance programme by the GPEI . Long-teVDPVs are classed as ambiguous (aVDPVs) when they are not linked to ongoing chains of transmission or to any known immunodeficient individuals. When linked cases are subsequently identified, many aVDPVs are re-classed as circulating VDPVs (cVDPVs). cVDPVs may cause large outbreaks of poliomyelitis and are a major threat to the eradication of poliovirus. They are likely to emerge where population immunity is low, and sanitation is poor .A number of studies have highlighted key ‘gatekeeper’ mutations required for Sabin poliovirus to regain wild-type fitness in the human intestine. Recent studies of serotype two poliovirus agree on three specific codon positions for these reversions, two in the 5′ UTR and the third in the protein coding VP1 region  Famular, two of 4.1Since the 2016 switch from trivalent to bivalent OPV, the incidence of VDPV2 outbreaks has increased with 29 VDPV2 outbreaks in 15 countries reported between January 2018 and June 2019 compared with nine in six countries in the same period in 2017 − 8 . This incVDPV cases have been reported in fifteen countries between January 2017 and January 2020  World H. Using g4.2When the live-attenuated Sabin OPV was in development almost 70 years ago, potential vaccine strains were selected empirically based on their immunogenicity and neuropathogenicity in animals prior to testing in clinical trials. Current projects to update vaccines can now be informed by our knowledge of the structure and function of the genome. More genetically stable OPVs are in development with genomes edited to reduce the risk of reversion to neurovirulence . Human pWith an upsurge in the number of wild poliovirus cases in 2019, and nine countries affected by ongoing VDPV outbreaks at the beginning of 2020, the GPEI is at a critical stage. Recent improvements in genetic sequencing and analysis techniques are helping support efforts to achieve a polio-free world. The main current lines of research include the development of direct detection and sequencing protocols that allow rapid detection and typing of poliovirus from stool and environmental samples (thus reducing the time from sample collection to reporting); the implementation of intuitive pipelines for bioinformatics and genetic analysis that speed and standardise the production of genetic data; the implementation of phylogeographic analysis and development of visualisations tools that allow analysis and interpretation of the genetic data by planners and policymakers; and the production of new oral vaccines based on newly engineered strains that have a lower risk to revert and regain virulence.KC880365–KC880521).No new data is presented in this article. An illustrative example is presented using publicly accessible data from GenBank (


Citrus tristeza virus (CTV). A direct confirmation of such close protection came with the finding that “pre-immunization” of citrus plants with the variants of the T36 strain of CTV but not with variants of other virus strains was providing protection against a fluorescent protein-tagged T36-based recombinant virus variant. Under natural conditions close protection is functional and is closely associated both with the conservation of the CTV genome sequence and prevention of superinfection by closely similar isolates. It is suggested that the mechanism is primarily directed to prevent the danger of virus population collapse that could be expected to result through quasispecies divergence of large RNA genomes of the CTV variants continuously replicating within long-living and highly voluminous fruit trees. This review article provides an overview of the CTV cross-protection research, along with a discussion of the phenomenon in the context of the CTV biology and genetics.“Cross-protection”, a nearly 100 years-old virological term, is suggested to be changed to “close protection”. Evidence for the need of such change has accumulated over the past six decades from the laboratory experiments and field tests conducted by plant pathologists and plant virologists working with different plant viruses, and, in particular, from research on  Citrus tristeza virus (CTV) that provided satisfactory field protection against severe stem pitting isolates of the virus for “Marsh” seedless grapefruit (Citrus paradisi MacFadyen) failed to provide similar protection when applied under similar conditions to the “Star Ruby” variety of grapefruit plants [The variety and abundance of non-cultivated wild plants as well as the large numbers of cultivated crop plants continue to survive despite being continuously exposed to numerous plant pathogens, including viruses. Although plants lack systems which provide humoral immunity, they are able to successfully confront a wide range of disease agents, primarily, by using many different constitutive and acquired resistance strategies based on either single or multiple genetic components that interact and evade fatal interactions of pathogens and host plants. Additionally, the outcome of a pathogen infection often depends on pre-existing community of microorganisms in the same host, which may engage in the synergistic or antagonistic interactions and enhance or ameliorate the impact of the pathogen-induced disease. One of the outstanding examples of the latter scenario is viral cross-protection. The phenomenon essentially depicts a situation in which host plants previously infected with a symptomless or mild variant of a virus species are protected from disease imposed by a subsequent encounter with a closely related severe variant of the virus. Unlike the more commonly recognized plant protection systems based solely on host plant genetic resources, cross-protection is apparently the result of interactions between the genetic traits of the protecting and challenging viruses. This generalization may, however, be questioned. Past practical experience has suggested that certain mild isolates of t plants .Tobacco mosaic virus (TMV) prevented its subsequent infection by a TMV variant with a different phenotype. Salaman [Potato virus X were protected from the following infection by other more aggressive isolates of the same virus, although they continued to be susceptible to infection by two unrelated viruses, TMV and Potato virus Y. These results continued to encourage plant virologists in different geographical locations to explore the use of such a protection strategy for practical needs. Interest intensified particularly as a result of outbreaks of emerging viral diseases for which breeders were lacking genetic resources or the process of resistance breeding was difficult and/or lengthy. However, as indicated by Fulton [The phenomenon of cross-protection was first observed almost one hundred years ago by McKinney . He foun Salaman  extendedy Fulton , the majy Fulton ), were gC. aurantium L.) rootstock could be effectively controlled by changing to citrus rootstocks or citrus relatives conferring tolerance or resistance to CTV. However, the stem pitting syndrome of CTV affects the citrus scion segments, regardless of the rootstock reaction to the virus [C. sinensis (L.) Osbeck) variety–cv. Pera, white and red skin grapefruits, and Oroblanco, a hybrid of grapefruit and acid-less pummelo (C. grandis Osbeck x C. paradisi MacFadyen). Trees of sensitive citrus scion varieties, when infected with stem pitting CTV isolates, are severely stunted and perform poorly, producing fruits of small size and low quality. Indeed, the stem pitting disease of citrus trees, rather than the more commonly known tristeza decline, is considered to be the most significant and difficult to control manifestation of CTV.Among the outstanding examples of controlling a serious plant viral disease via cross-protection is the wide-scale use of mild CTV isolates to control the manifestation of devastating stem pitting disease, which could be induced by some aggressive virus isolates. The typical quick decline—“tristeza”-symptoms of citrus trees grafted on the sour orange  were able to protect sour orange seedlings against the severe seedling yellows symptoms induced by the CTV isolates VT and Mor-T . Initially, the Miqveh-T mild isolate also showed outstanding protection for sweet orange trees on the sour orange rootstock, which were planted in T strain  due to iT strain ,25 and OPhytopathology [A. gossypii was only noticed about three decades after the introduction (in the 1920′s) of citrus varieties infected with exotic CTV variants. The spread of the introduced variants was first observed in 1969, with circumstantial evidence suggesting that natural infection and tree decline began at least 2–3 years earlier. The major difference between the originally introduced isolates vs. the isolates recovered from the local spread of the virus was evident. The introduced isolates were non- or poorly transmitted by A. gossypii, while isolates collected from recent incidences of natural spread were highly transmissible by different A. gossypii colonies. This was an indication of the emergence of a mutant CTV variant(s) well adapted to the local aphid species. Given a large number of CTV virions being produced continuously in the field citrus trees and an abundance of aphids, the emergence of A. gossypii-transmissible CTV variants and the shift in the CTV populations would have been expected to occur at least two decades earlier. The long lag period pointed to a mechanism that prevented the timely emergence of CTV variants adapted to transmission by A. gossyppi. In order to test a possibility that conservation of the non-transmissible isolate resulted from cross-protection of the parental isolate against a transmissible one, we challenged plants carrying a poorly transmissible isolate ST using a highly transmissible isolate VT. The result was a marked reduction in A. gossypii transmission of the recovered virus isolates [Cross-protection was mostly tested between isolates showing phenotypic differences in symptomatology. In 1978, a Letter to the Editor published in athology  reportedisolates .It is interesting to note that effective cross-protection was obtained using ST rather than the other, much more poorly transmissible CTV isolates described by Bar-Joseph . Both CTC. aurantifolia) indicator plants were less aggressive, compared with those of VT. Unlike CTV VT, which showed a strong seedling yellows reaction, the CTV HT infections were apparently symptomless. Instead of the classical cross-protection testing based on prevention of differential symptoms, we used a new approach in which the RNA extracted from the infected plants was hybridized with an isolate-specific cDNA probe. While CTV VT gave a strong hybridization signal, CTV HT was not recognized by the VT D1 probe [C. macrophylla with the HT isolate, followed by topping plants after 2–3 weeks, and, upon symptom appearance, challenging those by inoculation with the CTV VT-carrying budwood, along with a similarly inoculated group of healthy control plants. The two groups underwent a second topping after about a month and were tested by hybridization, soon after showing virus symptoms. The hybridization results were similar: both groups showed clear and strong hybridization signals with the VT-specific D1 probe due to the presence of CTV VT, including all plants, which were pre-inoculated with CTV HT. We considered these results to be negative and not worth publishing, since it was already well known from the seminal work of Costa and Muller [Rosner et al.  describeD1 probe . The crod Muller  that crod Muller , and, bad Muller  analyzedC. meyeri) MT isolate, showed strikingly different genetic composition of their coat protein genes as compared with isolates of the VT strain [Interestingly, two of the sequenced CTV isolates, including CTV HT and the Meyer lemon  and the 19.3 kb single-stranded positive-sense non-segmented RNA genome are among the largest of the RNA viruses [CTV has several peculiarities, which set it apart not just among the elongated plant viruses, but even within the  viruses ,34,35,36 viruses  : T3, T30, T36, VT, T68, RB, and HA 16-5 ,49,50. S−4 to 10−5 nucleotides per site per year, thus, placing CTV in the 10th percentile of the most slowly evolving viruses [C. macrophylla tree under the greenhouse conditions for seven years revealed only nine nucleotide substitutions, which equals an evolutionary rate of 6.67 × 10−5 nucleotide alterations per site per year [A remarkable characteristic of CTV is the slow evolutionary rate, which ranges from 10 viruses . Strong  viruses ,58. Analper year . The sloper year ,60, whicper year , which pper year ,63.Another peculiarity of the CTV-plant host interactions is the continuous and repeated failure to obtain durable transgenic resistance of citrus plants using cDNA constructs corresponding to viral genes ,65 ) ,20 were Examination of the CTV SIE mechanism revealed that it is mediated by multiple components. Remarkably, sequences in the 3′-half of the CTV genome did not appear to have any effect on SIE . Hybrid −3 to 10−5 substitutions per nucleotide copied. It was suggested that, on average, replication of RNA viruses with the genome length between 3 kb and 32 kb results in 0.1–1 mutation introduced per RNA template copied. Some of those may be lethal and impede further replication of a mutant genome. Nevertheless, a continuous input of mutant virus variants would be expected [−4 and 10−5 nucleotides per site per year. Closteroviruses lack the proof-reading exonuclease reported in coronaviruses [It has been accepted that viral RNA polymerases possess low copying fidelity. Calculations of mutation frequencies for a number of RNA viruses supported the values ranging from 10expected . On the aviruses ,62. HencTurnip crinkle virus (TCV), a small (ca. 4 kb) positive-sense RNA virus in the family Tombusviridae, which infects cruciferous and some non-cruciferous plants. In this study, Zhang et al. [SIE, which operates between closely related virus variants, could be a two-function mechanism that (i) regulates genomic stability of CTV by preventing co-replication of newly produced mutated progeny arising from error-prone replication process and (ii) prevents implantation of new variants with closely similar 5′-terminal genomic regions. Indeed, experiments with animal alphaviruses have demonstrated that the phenomenon of SIE could be established within a fraction of an hour after the primary virus enters the cell ,79. Furtg et al.  showed tg et al. ,85.Beet yellows closterovirus (BYV) [Lettuce infectious yellows virus, another member of the family Closteroviridae, inoculation of plant protoplasts with a replicon of CTV carrying only replication-associated genes (ORFs 1a and 1b) led to active replication of the introduced virus construct and concominant formation of the characteristic double-membrane vesicles and multivesicular bodies   and thenus (BYV) ,34. The  bodies , CTV infects only a proportion (one-third or less) of susceptible cells, leaving the majority of the plant cells free of the virus [With CTV, the role of SIE in maintaining the integrity of the founder virus genome is especially important. CTV infects long-living citrus trees in which the virus can persist for the whole tree lifespan that could be more than 50 years. Because of so lengthy infections, the ‘resident’ virus also needs to protect itself from repeated introductions of highly similar variants mediated by vectoring insects. Importantly, even in the most susceptible citrus hosts  provided effective protection against severe PRSV isolates only in the geographical areas where the protective mild variant originated and failed in other locations harboring different virus strains [“Cross-protection”, a nearly 100 years-old virological term, could be now changed to “close protection”. Evidence for the need of such change accumulated before the genome sequencing era with finding that a mild nitrous acid-induced variant of  strains ,101. The


Whether hybridization generates or erodes species diversity has long been debated, but to date most studies have been conducted at small taxonomic scales. Salamanders (order Caudata) represent a taxonomic order in which hybridization plays a prevalent ecological and evolutionary role. We employed a recently developed model of trait-dependent diversification to test the hypothesis that hybridization impacts the diversification dynamics of species that are currently hybridizing. We find strong evidence supporting this hypothesis, showing that hybridizing salamander lineages have significantly greater net-diversification rates than non-hybridizing lineages. This pattern is driven by concurrently increased speciation rates and decreased extinction rates in hybridizing lineages. Our results support the hypothesis that hybridization can act as a generative force in macroevolutionary diversification. The prevailing view in the animal literature is that hybridization constrains lineage diversification because hybrid lineages are often documented to be less fit than parentals7. Under this scenario, hybridization is predicted to increase extinction rates. Further, introgressive hybridization has the potential to “wash away” accumulating divergence among incompletely isolated lineages10, leading to a prediction of decreased speciation rates. In contrast, the prevailing view in the plant literature is that hybridization enhances adaptive potential by introducing novel genetic and phenotypic variation13. Reinforcement, or the accumulation of post-zygotic reproductive isolation through selection against hybrids14, has long been considered to expedite the speciation of diverging lineages9. Reinforcement has the potential to act even when heterosis occurs, as hybrids may be largely sterile, such as in Triturus newts16. Additionally, hybridization-mediated shuffling of old genetic variants may fuel rapid diversification, as outlined by the combinatorial view of speciation17. Accordingly, hybridization is predicted to increase speciation rates and/or decrease extinction rates.A leading unresolved question in evolutionary biology is whether hybridization, defined as the interbreeding between two genetically distinct lineages21. In allopolypoid hybrid speciation, a mode of speciation common in plants but rare in animals, nearly complete reproductive isolation may evolve in a single generation due to a change in ploidy11. In contrast, homoploid hybrid speciation typically has to occur in the face of continued gene flow, which acts to homogenize the diverging hybrid lineages10.Whereas evidence of hybrid speciation in plants has long been abundant, evidence for widespread hybrid speciation in animals is relatively scarce28. Additionally, studies of hybridization often occur along different stages of the speciation continuum29, whereby hybridization can appear as a force that either facilitates or impedes speciation. Recent work highlights this uncertainty by demonstrating that the outcomes of hybridization depend on the underlying nature of selection pressures and demography30. We suggest that studies at broad phylogenetic and macroevolutionary timescales can help overcome these limitations by providing a phylogenetic context in which to view repeated hybridization events over evolutionary timescales and the consequent impact on lineage diversification rates.Interest in hybrid-mediated speciation has recently burgeoned, but studies have typically been limited in taxonomic scope. Studies of the effect of hybridization on diversification have most commonly been conducted among closely related pairs or small clades of taxa, and results have been equivocal31), which are particularly suitable for this study because hybridization is pervasive and has been studied extensively . Many salamander groups have diversified through primarily non-ecological means of speciation (i.e.48). Salamander species have often diverged in allopatry/parapatry following restriction to refugial, isolated populations during periods of climatic fluctuations or via orogeny of mountain ranges (e.g.51). Following the evolution of incomplete reproductive isolation, young, diverging lineages may then come into secondary contact and hybridize (e.g.23). If salamanders are indeed predisposed to the evolution of incomplete reproductive isolation, then hybridization may play an important role in their diversification.Salamanders present a valuable case study of hybridization, as numerous groups are comprised of speciose, yet morphologically and ecologically conservative species52, because our experimental design cannot address this (See supplement). We replicate this test across four datasets to investigate the robustness of our results: (1) including all available data; (2) excluding species that do not exhibit sympatry (defined as <10% geographic range overlap) thereby lacking the opportunity to hybridize; (3) only the family Plethodontidae, which are the most widely hybridizing and diverse of the 10 salamander families; (4) all (nine) salamander families except the Plethodontidae.If hybridization plays a meaningful role in the diversification process, differences in diversification rates among hybridizing and non-hybridizing taxa are expected. Thus, we test the hypothesis that there is a difference in diversification rates (speciation and extinction rates) between contemporaneously hybridizing and non-hybridizing salamander lineages. Note that we are simply testing whether contemporary hybridization influences diversification rates, not whether ancient hybridization facilitated the present radiation as postulated by the hybrid swarm hypothesis32 as the source for downstream analyses. This tree32 was constructed using nine nuclear genes and three mitochondrial genes as data using RAxMLv7.2.853, and time-calibrated using treePL54. Using the APE package (v.3.455) in R, we extracted the subtree containing salamanders for subsequent analyses. This approach yielded a tree containing 469 of 716 extant species of salamanders , as we did not perceive these cases to meet the criteria of substantial and replicated genealogical discordance. Although inference of hybridization via the detection of genealogical discordance warrants caution, our narrow definition of hybridization does not recognize these species as hybridizable. In total, we retrieved 56 papers  in the narrow dataset and two families  in the broad dataset.Each species was scored as ‘non-hybridizable’ (NH) or ‘hybridizable’ (H), based on an extensive literature review of hybridization using the search engines Google Scholar and ISI Web of Knowledge. To do so we paired each species with the terms ‘hybrid’ and ‘introgress’ as well as dialectical and structural variants thereof  to search for cases of hybridization. Criteria for hybridizability under the first, “narrow” definition were the documentation of hybridization among natively distributed species, specifically the observation of heterospecific mating or hybrid offspring in the wild as detected by substantial and repeated morphological and molecular intermediacy. Criteria for hybridizability under the second, “broad” definition included those criteria described previously, as well as: 1) the observation of hybridization occurring in laboratory settings or among introduced and native species, and/or; 2) inference of historical introgression as determined using molecular lines of evidence . This latter definition is less conservative than the former. We excluded cases where one or two individuals exhibited cyto-nuclear discordance between markers such as allozymes and mtDNA , we compared diversification rates using three additional datasets, produced using both the narrow and broad datasets . In the first dataset, we required that “non-hybridizable” taxa were sympatric (>10% range overlap) with another species, thus possessing sufficient opportunity to hybridize. Consequently, species that were not sympatric with any other salamander taxa were excluded from the dataset. Therefore, species classified as non-hybridizing in this analysis may have limited opportunity to hybridize but have not been observed to do so. Although we cannot account for historical species distributions (e.g. species’ ranges may have previously overlapped), our primary (narrow) definition of hybridizability necessitates contemporary hybridization. Thus, our designation of sympatry occurs at the same time scale as our designation of hybridizability.In addition to analyzing the entire salamander subclade from the Pyron37, the greatest number of instances of hybridization have been documented in this group (Table Plethodon in the Eastern United States have been described as species on the basis of a threshold genetic distance (e.g.42). Coincidentally, nearly half of these species have been observed to hybridize in nature. To test for an effect of this relative taxonomic over splitting, we excluded the Plethodon glutinosus group  may be found in Supplementary Table In the second and third datasets, we tested for a family-specific effect of hybridization on diversification rates. As plethodontids are the most diverse family of salamanders Fig.  and haveup Table . Additiooup Fig.  and repe61) trait-dependent diversification model to test for differences in speciation and extinction rates between hybridizing and non-hybridizing lineages. HiSSE infers speciation and extinction rates for a binary character while allowing for heterogeneity in diversification rate to exist within each character state. In the HiSSE model, hidden states are co-distributed with the trait of interest and account for unsampled traits that may simultaneously contribute to the diversification process. The hidden state need not be associated with any single trait but may instead be associated with a set of traits or suite of traits. Inclusion of the hidden state thus ameliorates the confounding effects of unsampled traits on diversification rate estimations by allowing for greater rate heterogeneity in the tree than in previous SSE models61. Thus, we are in essence measuring the impact of contemporary hybridization while controlling for other, correlated traits on diversification rate. Finally, by accounting for increased rate heterogeneity in character-dependent and character-independent (null) models through the inclusion of hidden states, model rejection properties are greatly improved61 relative to previous SSE models.We applied the HiSSE .We evaluated a total of 14 competing models using HiSSE, half of which represent a model of character-dependent diversification and the remaining half represented models of character independence . To explore the effect this assumption had on our results, we repeated these analyses assuming (1) we have sampled all extant hybridizing species . That is, parameter estimates obtained from each fitted model are averaged together such that their contribution to the average is proportional to their relative support (Akaike weights) among the set of candidate models  were analyzed using two datasets (narrow & broad definitions of hybridization). Each data/tree-set combination was tested assuming the three aforementioned differences in prevalence of hybridization in nature. In all, a total of 32 rounds of model testing were performed, comparing seven models of trait-independent diversification and seven models of trait-dependent diversification for each sampling fraction. Further information on how these data/tree-sets were produced and analyzed may be found in the Supplementary Materials. Lastly, a description of our test of sensitivity to phylogenetic uncertainty may also be found in the Supplement , we conducted sister clade comparisons. Specifically, using all comparisons of three or more taxa , we used the method of Barraclough, Harvey and Nee67.To test for possible circularity of causality between diversification rates, species richness and opportunity to hybridize, we quantified the relationships between the three. Specifically, we tested for a relationship between 1) mean diversification rates and the proportion family hybridizing, 2) mean diversification rates and species richness, and 3) proportion family hybridizing and species richness  assuming 20% of species hybridize, three of four analyzed phylogenies consistently found that hybridizing species experience increased speciation rates, decreased extinction rates, and therefore increased net-diversification rates  is significantly greater than the average decrease in extinction rate (95% CI: 0.0145 ± 0.0037 species/MY), we conclude it is primarily differential speciation that is driving the increase in net-diversification in hybridizing lineages. Interpreted as a waiting time, this means that hybridizing species, on average, speciate every 9.2 (95% CI: 8.84–9.62) million years and go extinct every 160.7 (118.97–247.96) million years.r = λ − μ), and turnover rate (τ = λ + μ) were greater in hybridizing than non-hybridizing lineages, whereas extinction rate (μ) and extinction fraction (Ɛ = μ ÷ λ) were lower. Increases were on average 125% (95% CI: 117–133%) for speciation, 576% (486–699%) for net diversification and 43% (41–45%) for turnover. In contrast, decreases were on average 305% (292–317%) for extinction and 945% (893–997%) for extinction fraction  found between states for all five parameter estimates Figs.  – S7, rer), and 120%  respectively in hybridizing versus non-hybridizing lineages. In contrast, extinction rate and the extinction fraction decreased in hybridizing lineages by 1003%  and 3653%  respectively.Analysis of the tree including only sympatric taxa regardless of the hybridization criterion (narrow versus broad) datasets recovered patterns identical to those obtained using the complete phylogeny Figs. -S7. Specr), and 78%  greater than those experienced by non-hybridizing lineages. Extinction rate and extinction fraction were reduced by 3186%  and 9000%  in hybridizing lineages relative to non-hybridizing lineages.Similarly, plethodontids exhibited the same patterns described above Figs. -S7. Hybrr) greater in hybridizing lineages, whereas extinction rate and extinction fraction were reduced by 1964%  and 4060%  in hybridizing lineages relative to non-hybridizing lineages. Analysis of the broad dataset revealed no significant differences and parameter estimates between hybridizing and non-hybridizing species.In contrast, analysis of non-plethodontids revealed greater ambiguity as to the impact of hybridization on diversification rates. Whereas the narrow dataset inferred trait-dependent diversification in which hybridization drove increased diversification, the broad dataset did not Figs.  and S7. Sister clade contrasts broadly supported results of the HiSSE analysis. That is, hybridizing clades were found to have significantly greater species richness than non-hybridizing clades for all datasets except for plethodontid salamanders. Details on significance of these tests may be found in Supplemental Table R2: λ = −0.095; μ = −0.084; r = −0.092) and proportion of family hybridizing (Adjusted R2 = −0.112) are decoupled from family species richness, despite a significant correlation  between mean diversification rate and proportion family hybridizing  that have been documented in salamanders (e.g.71) and the process of reinforcement which has long been recognized to contribute to the diversification process74. We outline the potential contribution of each below.Here, we show strong support that contemporary hybridization is correlated with elevated diversification rates in the order Caudata. Net-diversification of hybridizing species tends to be significantly greater than that of non-hybridizing species, driven primarily by a coincident increase in speciation and decrease in extinction rates Figs. , 2. The 75) owing to limited dispersal abilities and low rates of gene flow76, thus leading to an abundance of opportunities to evolve in allopatry. Additionally, terrestrial species such as the Plethodontid salamanders of the southeastern United States experience elevational range expansions and contractions associated with climatic change71. Perhaps this combination of the primarily sessile nature of many salamander species and frequent repeated secondary contact leads to hybridization occurring regularly across evolutionary timescales. Under these scenarios, hybridization may then play a creative evolutionary role in the diversification process similar to that observed in haplochromine cichlids77. Allopatric speciation of haplochromine cichlids has occurred in lakes that frequently have dried, split, and reformed, whereas sympatric speciation has occurred within lakes in which lineages exhibited extreme habitat specificity and have been reproductive isolated at fine spatial scales78. Under these circumstances, hybridization may have afforded genetic rescue from the consequences of small population size by providing increased standing genetic variation and thus expedited adaptation to novel stressors, as in Lake Victorian cichlids post-colonization25.Salamanders often exhibit substantial genetic differentiation at small geographic distances . However, while periodic geographic range expansion and contraction may initiate speciation, the reproductive isolation that evolves may be incomplete, predisposing the young species/diverging lineages to hybridization (i.e.23). As a result, hybridization may commonly occur in salamanders during periods of climatic fluctuations.Such hypotheses of repeated contractions, expansions, and secondary contact of salamander populations have been well supported, across both North91 and a consequent increase in frequency of hybridization among previously isolated taxa95. An informed understanding of the influence of hybridization on macroevolutionary diversification may thus provide invaluable context for contemporary processes. This possibility of climate-change mediated hybridization has already been demonstrated in plethodontid salamanders , as well as in ecologically divergent subspecies of salamandrid salamanders . Thus, it seems likely that salamander species worldwide, particularly those found at high elevations due to their more limited potential geographic distributions, may experience a heightened frequency of hybridization as climate change advances. While generalizations regarding the outcome of hybridization should be made with caution30, our study indicates that perhaps speciation reversal99 need not be the expectation. Rather, our study implies that hybridization may facilitate adaptation to novel conditions under climate change, leading to diversification of new salamander lineages.There is now clear evidence for latitudinal and elevational range shifts mediated by climate change14, is intrinsically intertwined with hybridization. Reinforcement has been documented both experimentally72 and observationally74 to accelerate the initiation and/or completion of the speciation process101. For instance, reinforcement is likely to play an important role in the speciation process due to strong interspecific sexual selection and mate choice in plethodontids102. Indeed, patterns of sexual isolation among populations of Plethodon jordani and P. teyahalee match expectations of reinforcement14, with sexual selection being stronger in sympatry than in allopatry103. Although we cannot currently quantify the contribution of reinforcement to diversification rate differences using our data, we urge further research measuring the degree of association between contemporaneous hybridization and reinforcement among taxa. Nonetheless, were reinforcement to play a role in the production of the patterns observed in this study, the very occurrence/process of hybridization would be the ultimate driver . Under such a scenario, our study design is well-suited to identify such a signal.Here, we show the novel result of a strong correlation of contemporary hybridization with elevated speciation and net diversification at a large taxonomic scale. However, reinforcement, defined as the strengthening of prezygotic reproductive isolation in sympatryPlethodontidae is more limited . Thus, our observation of hybridization facilitating the diversification process applies to the majority of salamanders and implies that, at a minimum, contemporaneous hybridization does not impede the diversification process of extant salamanders.Although a generative role of hybridization is robustly supported across three of our four datasets, evidence for such a role outside of the ed Table -S14. We 104). For example, for trees of 300 species, BiSSE attains a power of at most 50%, with power dropping below 15% of trees of 100 taxa104. Our phylogeny of non-plethodontids includes only 167 species; that we detected a positive relationship between hybridization and speciation rates using our narrow (most conservative) dataset despite such reduced power is a testament to the strength of the signal in our data. Whereas our larger datasets  have greater power, our lack of detection of a relationship between hybridization and diversification rates in non-plethodontids using our broad definition of hybridization is perhaps unsurprising, given the low power of the analysis . Although the power of HiSSE under such scenarios has not been specifically established, accuracy of parameter estimation does decay with decreasing tree size61. Consequently, we cautiously interpret the results of the analysis of non-plethodontids (163 spp). Interestingly, sister clade comparisons consistently supported a positive relationship between hybridization and species richness in non-plethodontids, despite not supporting such a relationship in plethodontids . Whereas much of the subsequent studies focused on ancient hybridization, our study instead focuses on the effects of contemporary hybridization.An important distinction between our study and most previous studies investigating the influence that hybridization exerts on the diversification process is that of the time-scale at which hybridization is being assessed. Following Seehausen’set al.23, tested the hybrid swarm hypothesis in the Plethodon glutinosus group  hybridizing polyploid taxa (none of which are plethodontids). Our study adds to the growing evidence that hybridization may fuel rapid diversification (e.g.52) and is a compliment to speciation genomics studies characterizing the genomic basis of this process (e.g.111). Herein we have shown that hybridization may act as a generative force across a phylogenetic order, and additional studies at such macroevolutionary scales are needed to determine if this pattern holds more generally across the tree of life.We explicitly tested the hypothesis that contemporary hybridization plays a creative role in the diversification process in the broadest taxonomic and temporal scale study to date, and our observations strongly supported the predictions of this hypothesis. Specifically, hybridization was found to be correlated with both increased speciation rates and decreased extinction rates, resulting in increased net diversification rates relative to non-hybridizing lineages. Although other factors certainly contribute to the observed diversification dynamics, we have shown that hybridization plays a significant role, while accounting for hidden, correlated states in our analysis. Nearly all studies of hybridization have focused on individual case studies in which hybridization results in species collapseSupplementary Information.


African swine fever (ASF) causes huge economic losses and is one of most dangerous diseases of pigs. The disease is known for almost 100 years, an effective vaccine or treatment is still unavailable, only proper biosecurity measures, including disinfection, are being applied, in order to prevent disease outbreaks. Eight active substances, i.e., formaldehyde, sodium hypochlorite, caustic soda, glutaraldehyde, phenol, benzalkonium chloride, potassium peroxymonosulfate and acetic acid, were tested, in order to confirm their effectiveness against African swine fever virus (ASFV). This specific selection was done based on the World Organisation for Animal Health (OIE)’s recommendation and previous disinfectant studies on surfaces. The result of our study shows that most of them inactivate the virus, in recommended concentrations. In order to reduce the cytotoxicity of the four substances, Microspin S-400 HR columns were applied, therefore making it possible to demonstrate four logarithms virus titer reduction. Sodium hypochlorite, glutaraldehyde, caustic soda and potassium peroxymonosulfate showed the best ASFV inactivation rates, achieving titer reductions over 5 logs. Despite microfiltration, the virucidal activity of formaldehyde was not assessable, due to its high cytotoxicity. Our results showed that cleaning is particularly important, because removal of the soiling provides improved effectiveness of the tested chemical compounds. Today, in the face of the Covid-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), nearly everyone knows what biosecurity and disinfection rely on. Moreover, despite intensive research efforts, a commercially available vaccine has not been developed against Covid-19 or African swine fever (ASF) [Suidae family, mainly domestic pigs, wild boars and warthogs [ASF, caused by the African swine fever virus (ASFV), is one of the most dangerous  infectious viral diseases of pigs, mainly due to enormous socioeconomic consequences. The disease affects animals of the warthogs ,3. The cwarthogs . Poland,warthogs . Up to twarthogs . With thwarthogs . Due to Asfarviridae and the genus Asfivirus. It is a large, enveloped DNA-virus. Its virions have a specific icosahedral structure of a protein capsid, which surrounds an internal membrane and a nucleoprotein core [Ornithodoros genus and indirect contact through contaminated feed, pork meat, people, vehicles or fomites [ASFV is a member of the family ein core ,8. The c fomites ,10. In t fomites . Irrespo fomites ,13,14. A fomites . Biosecurity is defined as the implementation of a set of measures that reduces the risk of infection, amongst other, through cleaning and disinfection . The fulFormaldehyde;Sodium hypochlorite;Caustic soda solution;Glutaraldehyde;Phenol;Chemical compounds based on lipid solvents, e.g., benzalkonium chloride;Multi-constituent compounds, e.g., potassium peroxymonosulfate;Organic acids, e.g., acetic acid, etc.The aim of the study was to prove the effectiveness of the following representative chemical compounds:10 reduction in the initial viral titer, which is equivalent to a loss infectivity level of 99.99% [The above-listed compounds are widely recommended as being effective against ASFV and commonly used in the production of commercial disinfectants. Despite the extreme importance, little information is available regarding the in vitro suspension testing of disinfectants against ASFV . Virusesf 99.99%   and was subcultured in a Minimum Essential Medium , a 10% Fetal Bovine Serum (FBS)  and a 1% Antibiotic Antimycotic Solution (100×) . The cultures were grown at 37 °C, in a humidified atmosphere of air containing 5% CO2. The Vero-adapted BA71V strain was obtained from the European Union Reference Laboratory, in Spain. A Vero cell line was obtained from ATCC (ATCC6 tissue culture infectious dose (TCID50/mL) of the virus and were incubated at 37 °C, until a 100% cytopathic effect was observed, usually after 4–5 days. In order to provide a sufficient virus titer (at least 106.5 TCID50/mL), allowing for demonstration of a 4 log reduction of titer, after disinfectant treatment, the obtained viruses were subjected to 3 freeze/thaw cycles and precipitated, using the following buffer: 20% Polyethylene glycol (PEG) and 2.5 M sodium chloride in a 2:3 buffer:virus ratio. The virus–buffer solution was agitated overnight at 4 °C, subsequently ASFV was pelleted by centrifugation at 13,000 g for 90 min, at 4 °C, and resuspended in 1/10 volume of the initial medium. The obtained virus stocks were titrated and aliquoted, and stored at −80 °C. Virus titers were determined by a TCID50/mL titration, using the Spearman–Kärber method [Subconfluent monolayers of Vero cells were infected with 10r method .Three selected concentrations of each active substance were prepared immediately before use by dilution in hard water. The basic concentrations were selected, based on OIE recommendation as being effective against ASFV . MoreoveWe used the following concentrations: formaldehyde : 0.4%, 0.8% and 1.6%; sodium hypochlorite : 0.3%, 1% and 1.5%; caustic soda : 1%, 2% and 3%; glutaraldehyde : 0.1%, 0.5% and 1%; phenol  in 0.5%, 1% and 2%; benzalkonium chloride : 0.5%, 1% and 2%; potassium peroxymonosulfate : 0.5%, 1% and 2%; acetic acid : 1%, 2% and 3% concentrations.+, Ca2+, Cl−, HCO3− anions (pH 7). The hard water was prepared according to the PN-EN 14675:2015 European Standard. The suspension test was prepared with interfering substances: BSA—bovine albumin 3.0 g/L (low-level soiling) and BSA + YE—bovine albumin 10 g/L, plus yeast extract 10 g/L (high-level soiling) were prepared, according to the PN-EN 14675: 2015 European Standard.All tested chemical compounds were diluted with water, of standardized hardness, containing defined concentration of Mg2 and examined daily for the appearance of cytopathic effect (CPE). All plates were ultimately scored for cytopathic effect upon microscopic examination, for 7 days. A minimum 6.5 log10 (TCID50/mL) of virus titer in the control sample was required to demonstrate a ≥4 log reduction.Each concentration was tested in triplicate. One part of the virus suspension was mixed with one part of the interfering substances, respectively, with low level soiling or high level soiling and incubated at 10 ± 1 °C for 2 min ± 10 s. Subsequently, eight parts of the disinfectant diluted to 1.25-fold of each tested concentration was added. The obtained mixture of virus, disinfectant and interfering substances was incubated at 10 ± 1 °C for 30 min ± 10 s; afterwards, the test tubes were placed on crushed ice (4 °C). The samples were immediately, serially diluted (in quadruplicates) 10-fold  in a Vero cell culture, in 96-well plates. The plates were incubated for 7 days, at 37 ± 2 °C, in air containing 5% CO10 titer reduction. Microspin S-400 HR columns  were used, in order to remove the cytotoxic agent from the samples, right after 30 min incubation of the tested and control samples. Virus controls with and without micro-filtration were compared, to detect losses in the virus titer. Several chemical agents turned out to be cytotoxic to the Vero cells, therefore precluding in proper test performance and demonstration of a 4 logBoth standard and cytotoxicity controls were processed in the same manner as the chemical compounds, but the chemical agent was replaced with hard water. Statistical analyses were performed, using the GraphPad Prism . Analyses of the mean differences between each disinfectant were shown with a standard deviation.10 titer reduction, in comparison to the control, was demonstrated. A summary of the results of the first suspensions in vitro tests of chemical compounds are presented in The efficacy of chemical compounds was determined by comparing mean log reduction in the mixture of virus and tested substances, with the log titer of virus control. Disinfectant was considered virucidal, when a ≥4 log10 loss of the initial virus titer (data not shown); however, cytotoxicity was reduced from 1 log10 to ≥3 log10, therefore allowing for the assessment of the antiseptic effectiveness of the three compounds. Microfiltration was insufficient to reduce the cytotoxicity of formaldehyde, which showed the highest cytotoxicity in the initial testing.Initial testing showed the high cytotoxicity of formaldehyde, glutaraldehyde, benzalkonium chloride and acetic acid, therefore the test was repeated, using Microspin S-400 HR filtration, and these results are included in 10 reduction  was performed at concentrations from 0.03%, 0.01% to 0.0075%, which correspond to 0.0045%, 0.0015% and 0.001125% of active chlorine, respectively, but none of these concentrations were effective (data not shown). On this account, the test of the sodium hypochlorite was repeated, using higher concentrations, e.g., 0.3%, 1% and 1.5% . This time, all concentrations, except 0.3% (at the high soiling condition) resulted in the virucidal effect, even reaching 5.58 (±0.47) mean logeduction . Sodium eduction . The greDuring our tests, it was observed that the control virus titer, under high-level soiling conditions, was significantly higher than that under low level soiling . It was,ASF is one of the most dangerous animal diseases, decimating pig populations almost worldwide; therefore, disinfection plays a key role in the prevention of the disease. Correct selection of a disinfectant and a properly applied disinfection process are crucial, to provide the highest effectiveness of the chemical substance, which depends also on the proper use, mechanism of action and application conditions, such as concentration, contact time, pH, temperature and the presence of organic matter.To our knowledge, this is the first in vitro suspension test reporting virucidal activity of chemicals compounds against ASFV, based on a modified PN-EN 14675:2015 European Standard. Seven out of the eight tested chemicals  caused effective ASFV inactivation. Each of them was effective at specific concentrations, usually recommended by the OIE, and at different soiling conditions. Our studies showed that the control virus titer, in high soiling conditions, was higher in all tested compounds . It prov10 titer reduction, i.e., by 5.58 (±0.47), thus being effective at almost every tested and presented concentration. The effectiveness of sodium hypochlorite has already been confirmed by Krug et al., during tests on surfaces, where, despite drying, 0.05% hypochlorite effectively inactivates ASFV [10. Potassium peroxymonosulfate is the main active substance in commonly used disinfectants, approved by the OIE and the United States Department of Agriculture (USDA) [The virucidal efficacy of formaldehyde could not be determined, due to high cytotoxicity, which was not reduced by microfiltration. Nevertheless, ASFV titer reduction by 2.83 * ±0.31) at 0.4% concentration of formaldehyde was still observable, suggesting its moderate effectiveness. Nevertheless, using formaldehyde as a disinfectant has been limited, due to it is carcinogenicity, based on the International Agency for Research on Cancer (IARC) concluding that using it heightens the risks of nasopharyngeal cancer and leukemia. Glutaraldehyde showed very high virucidal efficiency, even at high soiling conditions, despite the cytotoxicity neutralization by microfiltration being required. Similar to previous reports, our study confirmed that glutaraldehyde, caustic soda and potassium peroxymonosulfate showed a wider ASFV inactivation spectrum ,24,31. S at 0.4% tes ASFV . Its efftes ASFV . Caustice (USDA) ,36. Cause (USDA) . With reOur previous studies of commercial disinfectants showed high cytotoxity in disinfectants based on QUATs and glutaraldehyde, which did not allow for proper result assessment . TherefoIn summary, the highest virucidal activity against ASFV was shown in sodium hypochlorite, glutaraldehyde, caustic soda and potassium peroxymonosulfate. The wide range of effective disinfectants against ASFV is not surprising, since the virus belongs to the enveloped variant vulnerable to disinfectants interrupting its lipid-envelope structure. Moreover, the provided studies showed that ASFV may be also inactivated by pH manipulation, in the presence of a strong alkaline (caustic soda) or acid (acetic acid). Our research proved that the presence of organic matter is a crucial challenge, which may support the virus-replication process or lead to a reduction of the disinfection effectiveness. Therefore, cleaning with soap and water seems to be crucial, in order to reduce soiling and, therefore, improve disinfection efficacy cannot be omitted. In the presence of organic matter, ASFV multiplies more efficiently and is more resistant to inactivation. Despite using the proper disinfectants and appropriate contact time, the chemical compound may turn out to be ineffective.In the future, it is worth getting involved in searching for eco-friendly disinfectants, showing reduced cellular cytotoxicity and a less harmful impact on humans and the environment.


Hollow and floating gel-beads were designed using prilling/ionotropic gelation as the microencapsulation technique, zinc acetate in the gelling solution as the alginate external crosslinker, and calcium carbonate in the feed as the internal crosslinking agent able to generate gas when in contact with the acidic zinc acetate solution. To achieve this goal, drug/alginate solutions were opportunely combined with different amounts of calcium carbonate. The effect of the addition of calcium carbonate into the feed solution on buoyancy, encapsulation efficiency, morphology, size distribution, as well as in vitro drug release profile of the alginate particles was studied. Moreover, the ability of the floating beads to modulate in vivo the anti-inflammatory response was assayed using the carrageenan-induced acute oedema in rat paw. The proposed strategy allowed obtaining alginate beads with extremely high encapsulation efficiency values (up to 94%) and a very porous inner matrix conferring buoyancy in vitro in simulated gastric fluid up to 5 h. Moreover, in vivo, the best formulation, F4, resulted in the ability to prolong the anti-inflammatory effect up to 15 h compared with raw prednisolone.The aim of this research was to verify the application of alginate in combination with Ca In the field of oral controlled drug delivery, alginate is certainly one of the most explored natural polysaccharides owing to its cytocompatibility, biocompatibility, biodegradability, sol–gel transition properties, and chemical versatility that allows further changes to customize its drug release properties ,2,3,4,5.Alginate is a linear polysaccharide copolymer consisting of guluronic (G) and mannuronic acid (M) repeating units forming regions of M- and G-blocks and alternating structure (MG-blocks) . Some diThe prilling process (laminar jet break-up) has been widely used to produce crosslinked alginate micro-particles (gel-beads) acting as controlled drug delivery systems ,9. PrillMoreover, calcium alginate has often been used in combination with different gas-generating agents such as sodium bicarbonate, calcium carbonate, and citric acid or tartaric acid to obtain low-density floating formulations ,16,17. SAlthough some studies have been focused on the production of floating alginate beads with calcium in the gelling solution, few data are available on the simultaneous use of two different cations as cross-linking agents ,23. More2+) and zinc (Zn2+) could have a synergistic effect during the external ionotropic gelation, significantly improving the technological properties  of the resulting cross-linked alginate beads. This effect is because of both the ability of Ca2+ to establish quicker electrostatic interactions with guluronic groups and the ability of Zn2+ to bond in covalent-like manner carboxylate groups of guluronic and mannuronic moieties of alginate.As previously reported in our study , in fact2+ in the feed and Zn2+ in the gelling medium on the final beads’ performance. With this in mind, floating Ca-Zn alginate beads were designed as potential oral prolonged delivery systems of prednisolone as a model SAID, taking advantage of the efficient prilling/ionotropic gelation in the presence of zinc ions [In this paper, a double internal–external alginate gelation approach was investigated, verifying the combined effect of Cainc ions , widely inc ions ,25, calcw/v and drug/polymer ratio 1:5) [2 exploited during the particle manufacturing gave a porous alginate matrix with good results in terms of the encapsulation efficiency and morphology of the beads. The beads were shown to float in simulated gastric medium and to modulate in vivo the anti-inflammatory response of prednisolone, tested in a rat paw oedema model.Drug/alginate solutions   were opp3 in the polymer/drug feed solution as the gas-generating agent. When feed dripped in zinc acetate medium (pH 1.5), carbonate produced CO2 that was entrapped in the forming hydrogel matrix, reducing the system density [Six floating Ca-Zn alginate formulations loaded with prednisolone were produced, adding CaCO density ,26,27. T3/drug droplets produced by prilling apparatus fall down into the zinc acetate solution. Here, the released Ca2+ ions can promote an internal gelation, by cross-linking the alginate carboxyl group [2+ ions, prevented the leakage of the drug during the gel particle formation, with an increase of encapsulation efficiency (EE).The results reported in yl group ,27, whicw/w ratio).The presence of calcium in the matrix of F4 beads was confirmed by IR analyses  on calci−1) and symmetric (1412 and 1403 cm−1) stretching vibrations of carboxylate ion were detected [−1, νas(C-O)) cm−1) and symmetric methyl bending vibration ).In #F1_b and #F4_b spectra, asymmetric .3 displayed the formation of a macro-porous structure  was performed.3 from 1.25% to 2.5% w/v  resulted the inability to float in SGF, owing to a low calcium carbonate concentration giving a low porosity of the matrix.As a confirmation, #F2 and #F3 , a diffusional mechanism of Pred controlled by hydration, swelling, and then erosion phenomena, typically occurring in a cross-linked alginate matrix, has been previously demonstrated . The druThe formulation F4 was selected for the in vivo experiments, to verify whether its in vitro properties can induce a prolonged anti-inflammatory effect in rats.The in vivo anti-inflammatory effect of #F4, in comparison with raw prednisolone, was evaluated using a modified protocol of carrageenan-induced oedema in rat paw . Pred was administered at 0.5, 5, or 15 h before oedema induction and at the dose of 3.0 mg/kg. The dose of 3.0 mg/kg of Pred was selected based on dose-response experiments conducted in our previous research .The inflammation induced by carrageenan injection caused an increase in the rat paw volume (oedema) compared with the baseline one. According to literature data , the maxAs shown in Indeed, F4 beads with a very tough Ca-Zn-alginate matrix are an interesting delivery system, showing both the right porosity, required for buoyancy, and the ability to control the drug release in SGF, with an impressive extension of the in vivo anti-inflammatory activity.2O at 20 °C, MW ~240 KDa) was supplied from Carlo Erba .Sodium alginate  were purchased from Sigma-Aldrich . All other chemicals and reagents were obtained from Sigma Aldrich and used as supplied.w/v) and drug/polymer ratio  in the feed solution were fixed on the basis of our previous research, as well as zinc acetate dihydrate content (0.5 M) in the gelling solution [For the production of F1–F6 beads , the algsolution . In brie+2 aqueous solutions (pH adjusted to 1.5 adding an adequate amount of a 1 N solution of HCl), where they immediately gelified under gentle stirring.Beads were manufactured by a vibrating nozzle device , equipped with a syringe pump , pumping the drug/polymer/carbonate liquid feed through a 600 µm nozzle. The experiments were performed at a flow rate of 6 mL/min. The vibration frequency used to break up the laminar liquid jet was set at 350 Hz, at an amplitude of vibration of 100%. The distance between the vibrating nozzle and the gelling bath was fixed at 25 cm. The generated drops were observed as a stationary chain of drops by means of an LED-stroboscopic light, automatically synchronized from the control cabinet with the adjusted vibration frequency. Drug/polymer/carbonate droplets fell into the ZnIn detail, the syringe pump was stopped after processing 10 mL aliquots of the feed solution and the formed beads were kept in the gelling solution for a medium time of 2 min. Then, the crosslinked particles were recovered and thoroughly rinsed with distilled water. Thereafter, the syringe pump was re-started and the generated droplets were collected in a new gelling bath. Experiments were performed, processing few mL of feed solution to guarantee a gelling time as similar as possible for all particles. This procedure was repeated several times in order to obtain the desired amount of particles.After production, the gel beads were left to air dry at room conditions, until a constant weight was reached (about 12 h). As a comparison, unloaded beads (blank) were produced following the same protocol.Accurately weighed amounts of beads from each manufactured batch (about 10 mg each) were dissolved under vigorous stirring in PBS buffer  in order to disintegrate the polymer matrix and release the encapsulated drug. Afterwards, 23 mL of ethanol was added and the suspension was centrifuged at 6000 rpm for 15 min.The actual drug content (ADC) was determined by UV spectroscopy  at λ of 244 for prednisolone quantification using the following equation:Encapsulation efficiency (EE) was calculated as the ratio actual to theoretical drug content (TDC), that is, the weight of drug added (g)/weight of polymers/excipients and drug added (g) × 100. Each analysis was performed in triplicate; the results were expressed in terms of mean ± standard deviation.The dimensional distribution and morphology of dried beads were studied by scanning electron microscopy (SEM) using a Carl Zeiss EVO MA 10 microscope with a secondary electron detector  equipped with a LEICA EMSCD005 metallizator, producing a deposition of a 200–400 Å thick gold layer. The analysis was conducted at 20 KeV. The projection diameter of dried beads was obtained by image analysis . About one-hundred-bead micrographs were analysed for each preparation and for at least three different prilling processes. Inner structure images were obtained by cryofracture of dried beads and further analysis by SEM, as previously reported . In brie−1 were recorded using an FTIR spectrophotometer  equipped with a MIRacle ATR accessory with a ZnSe crystal plate. The samples were directly analyzed using 256 scans with a 1 cm−1 resolution step.FTIR spectra between 2000 and 600 cmFloating properties of prednisolone hollow beads were evaluated through the USP dissolution Apparatus II . About 100 mg (≈ 30 beads per formulation) were placed into 1 L glass vessels filled with 750 mL of simulated gastric fluid  and the time the beads took to surface and float on the SGF medium was considered as the floating lag time (FLT). Moreover, throughout the buoyancy test, the floating percentage (FP) was calculated as the ratio between the number of floating beads and the total number of beads inserted in each vessel .In vitro drug release tests were conducted in sink conditions on given amounts of bead formulations using a USP 27 dissolution apparatus II: paddle, 100 rpm, 37 °C  on line with a UV spectrophotometer  with an automated cuvette charger.3PO4 was added into the acidic medium and the pH was adjusted to 6.8, continuously monitoring Pred release.Briefly, floating beads were added to the simulated gastric fluid  for 5 h, the floating time evidenced for #F4–#F6 formulations. The release kinetic of the formulation F4, selected for the in vivo study, was further investigated via a pH-change assay: particles were initially plunged into 750 mL of a 0.1 M solution of HCl; after 5 h, 250 mL of 0.2 M NaData were analyzed spectrophotometrically at a λ of 244 nm for Pred quantification. Dissolution tests were conducted on six different batches of particles; mean values and standard deviation were reported.w/v) in the right hind paw, as previously reported [p-value < 0.05 was considered statistically significant. All the experiments were approved by Italian Health Ministry (authorization n° 805/2015-PR) and conducted according to institutional animal care guidelines, Italian Law 26/2014 based on the European Community Law for Animal Care 2010/63/UE.Male Wistar rats (180–220 g) were purchased from Charles River . To carry out the carrageenan oedema induction, rats were anaesthetized with isoflurane and oedema was induced by injecting 100 µL of carrageenan 1% (reported . The int−1) or with raw prednisolone [w/v at the time 0.5, 5, or 15 h before the injection of the phlogistic agent. Control groups received 1 mL of MC or blank beads in 1 mL of MC by oral gavage. In detail, rats were randomly divided into seven groups: (1) Control group (Ctr), receiving 1 mL of MC 0.5% w/v; (2) Group P (−0.5 h), receiving Pred half an hour before carrageenan injection; (3) Group P (−5 h), receiving Pred 5 h before carrageenan injection; (4) Group P (−15 h), receiving Pred 15 h before carrageenan injection; (5) Group (Ctr F1_b), receiving blank beads; (6) Group F4 (−5 h), receiving #F4 5 h before carrageenan injection; (7) Group F4 (−15 h), receiving #F4 15 h before carrageenan injection.To assess the prolonged in vivo anti-inflammatory efficacy of #F4 in comparison with raw prednisolone, rats were treated by oral gavage with #F4  with the addition of calcium carbonate, as the gas-generating agent and internal gelation promoter during the beads’ production. The opportune amount of the gas-generating and cross-linking agents allowed obtaining porous polysaccharide-based beads able to encapsulate a high amount of SAID (EE up to 94%) and to control prednisolone delivery efficiently. The prolonged anti-inflammatory effect observed up to 15 h in rats, compared with prednisolone raw, is a proof of the in vivo efficiency of the system.Hollow alginate particles able to float and at the same time to prolong prednisolone release in simulated gastrointestinal fluids were obtained by prilling, starting from an optimized Zn-alginate formulation  for many hours and successfully treat patient suffering from chronic inflammatory diseases, reducing the frequency of the oral administration.


Hydrogels are essential in many fields ranging from tissue engineering and drug delivery to food sciences or cosmetics. Hydrogels that respond to specific biomolecular stimuli such as DNA, mRNA, miRNA and small molecules are highly desirable from the perspective of medical applications, however interfacing classical hydrogels with nucleic acids is still challenging. Here were demonstrate the generation of microbeads of DNA hydrogels with droplet microfluidic, and their morphological actuation with DNA strands. Using strand displacement and the specificity of DNA base pairing, we selectively dissolved gel beads, and reversibly changed their size on-the-fly with controlled swelling and shrinking. Lastly, we performed a complex computing primitive—A Winner-Takes-All competition between two populations of gel beads. Overall, these results show that strand responsive DNA gels have tantalizing potentials to enhance and expand traditional hydrogels, in particular for applications in sequencing and drug delivery. Hydrogels are fundamental in bioengineering, where they serve as scaffolds to support and steer the growth of tissues in-vitro, or in drug delivery where they serve as an excipient to carry drugs to target locations ,3,4,5,6.In that context, so-called stimuli-responsive gels have become an intense focus of research, as engineers seek to gain control over when, where and how gels can form ,16,17,18DNA gels—which gelify by the mutual hybridization of DNA nanostructures into an extended 3D network—have emerged as a new class of hydrogels with ideal properties for stimuli-responsiveness ,33,34,35Fabrication of microgels with microfluidics is now well established , enablinDNA gels that change their morphology in response to specific DNA stimuli have been reported . They opDNA strands  were purThe DNA hydrogel forms through the hybridization of 4 single stranded DNA  into a X-shaped Holliday junction (named X-motif) . The X-mWe also prepared two population of gel beads. One population of gel beads is made of (+) strands st1, st2, st3 and st4 (later referred to as the “(+) gels”). The other population is made of the exact complementary (-) strands st1*, st2*, st3* and st4* (referred to as the “(-) gels”). Since the complementary (-) strands have the same mutual connectivity as the native (+) strands, they also form Holliday junctions that gelify together B.We dynamically reconfigured the gels with DNA strands that triggered reversible swelling and shrinking C. Such mWe mixed the reagents as follows: 1× Tris-EDTA buffer pH 8.0, 150 mM NaCl, 1× Evagreen, and 100 μM of each strand. The solutions were put into a 96-well plate and fluorescently monitored with a CFX96 thermocycler . Starting from a short (3 min) incubation at 95 °C, the sample was cooled from 95 °C to 20 °C with a cooling rate of 0.1 °C/min.We produced microbeads of DNA gels with a previously reported droplet microfluidic device , we separated strands into distinct aqueous channels that mix just before meeting the junction. w/w of surfactant Span 80  for stabilizing the droplets . Tw. Tw75]. junction ,76. The We then experimentally demonstrated the generation of gel beads with droplets microfluidics, and their controlled dissolution with specific ssDNA . We prepWe then confirmed the selective dissolution of one population of gel beads while keeping the other population unaffected. To do that, we added a large excess of strands st1 and st3 to the solution. Those strands invade the (-) gel beads, binding to st1* and st3* by their toehold domain to initiate strand displacement that disrupts the X-motif and causes the dissolution of the (-) gel beads B. On theWe then exploited the complementary of (+) and (-) gel beads to “compare” their concentrations with a mechanism of mutual dissolution, implementing a Winner-Take-All computation. We start with two populations of (+) and (-) gel beads mixed in an unequal ratio, and we get them to mutually dissolve each other, until only one population of beads is left. This kind of Winner-Takes-All mechanism is highly nonlinear, and has been used to devise DNA-based classifiers . Since . HoweverWe reasoned that heating would “loosen up” the gel structures, partially breaking some sticky ends, unwinding some arms of the X-motif. If this happens when two (+) and (-) gel beads are close to each other, then complementary strands belonging to separate beads  could start to hybridize and drive the resolution of the Holliday junction into fully paired duplexes. This resolution is heavily favored thermodynamically for two reasons: the final duplexes have more base-pairing than the X-motif, and they lack the flexible linkers of the X-motif, which have an entropic cost. Since this process is thermodynamically downhill, it only needs to overcome small kinetics barriers. We chose to heat up the solution of 40 °C, which is halfway between room temperature and the gelation temperature (~50 °C).We experimentally validated those assumptions with 5 ratios of (+) and (-) gel beads . For a 3We experimentally tested the swelling and shirking of gel beads . StartinWe demonstrated the generation of DNA gel beads, as well as their controlled swelling, shrinking and dissolution. This degree of control over the gel—afforded by the programmability of DNA base pairing—could find direct applications in drug delivery. A drug payload could be entrapped in the mesh of a gel bead, and selectively released in presence of RNA biomarkers at specific sites in the human body ,80,81. AProgrammable DNA gels could also find applications in single-cell sequencing  for instance to extract from a cellular the transcripts belonging to a specific cellular type . In tiss


Polerovirus, family Luteoviridae. Typical viral symptoms of PeVYV including interveinal yellowing of leaves and upward leaf curling were observed in pod pepper plants (Capsicum frutescens) growing in Wenshan city, Yunnan province, China. The complete genome sequence of a virus from a sample of these plants was determined by next-generation sequencing and RT-PCR. Pod pepper vein yellows virus (PoPeVYV) (MT188667) has a genome of 6015 nucleotides, and the characteristic genome organization of a member of the genus Polerovirus. In the 5′ half of its genome (encoding P0 to P4), PoPeVYV is most similar (93.1% nt identity) to PeVYV-3 (Pepper vein yellows virus 3) (KP326573) but diverges greatly in the 3′-part encoding P5, where it is most similar (91.7% nt identity) to tobacco vein distorting virus  suggesting a recombinant origin. Recombination analysis predicted a single recombination event affecting nucleotide positions 4126 to 5192 nt, with PeVYV-3 as the major parent but with the region 4126–5192 nt derived from TVDV as the minor parent. A full-length clone of PoPeVYV was constructed and shown to be infectious in C. frutescens by RT-PCR and the presence of icosahedral viral particles.Pepper vein yellows viruses (PeVYV) are phloem-restricted viruses in the genus  Polerovirus, family Luteoviridae and are currently classified into six species , named Pepper vein yellows virus 1 to 6 , MCS- HDVRZ-NOS cassette was originally from the vector pHST40 [16]. Transcription Start site and Ribozyme cleavage site were based on vectors of pCass2 and pHST40 in references.Additional file 6. The strategy for constructing the full-length infectious clone by homologous recombination. Two overlapped PCR products of PoPeVYV were amplified with primers  and recombined with the linearized binary vector pCB301-MD which was amplified with primers pair Vec-pCB301 (Vec-pCB301 f/r).Additional file 7. The dataset used for the recombinant analysis.Additional file 8. NCBI-generated GenBank file showing the PoPeVYV sequence.