Zoonotic viruses threaten the lives of millions of people annually, exacerbated by climate change, human encroachment into wildlife habitats, and habitat destruction. The Powassan virus (POWV) is a rare tick-borne virus that can cause severe neurological damage and death, and the incidence of the associated disease (Powassan virus disease) is increasing in the eastern United States. The mechanisms by which POWV is maintained in nature and transmitted to humans are complex and only partly understood. This review provides an overview of what is known about the vector species, vector-host transmission dynamics, and environmental and human-driven factors that may be aiding the spread of both the vector and virus. Borrelia burgdorferi, is the most prevalent vector-borne disease in the US, resulting in an estimated 476,000 cases a year , su, suIxode. cookei . Moreoved as DTV  19,34],,34,Ixodeountains . With thfor POWV , it is uA number of field-collected vertebrates were found with neutralizing antibodies for POWV, indicating prior infection, including striped skunks, short-tailed and long-tailed weasels, racoons, porcupines, red squirrels, gray squirrels, groundhogs, opossums, some birds, and chipmunks ,29,36,37I. cookei and groundhogs and mustelids, and, to a lesser extent, I. marxi and squirrels, conclusive evidence for a wildlife reservoir of DTV is lacking. It is known that larval and nymphal I. scapularis frequently parasitize white-footed mice (Peromyscus leucopus) in the US, and this vector-host relationship robustly sustains B. burgdorferi in nature. It is often assumed in the literature that P. leucopus is also the reservoir for DTV. One study found seropositive wild-caught P. leucopus and DTV-infected I. scapularis in the same site, but the seroreactivity could have been from either POWV-LB or DTV [Peromyscus spp. also had DTV antibodies in New Mexico, but this could not be confirmed in the absence of any vectors, isolates, or sequence data [I. scapularis-infested P. leucopus were found to frequently enter groundhog burrows at night, fostering the ability for vectors and potential reservoirs to converge, no testing was done on any vertebrates or ticks to determine infection status [I. scapularis collected from P. leucopus tested negative for DTV [Peromyscus spp. to effectively implicate this host as a reservoir. While the enzootic cycles sustaining POWV-LB are purported to be between B or DTV . Two Pernce data . Moreoven status . Further for DTV . To dateB. burgdorferi, DTV appears focally, even though it is transmitted by the same vector species. Because DTV is understudied, a lot of what is known is extrapolated from our knowledge about tick-borne encephalitis virus (TBEV), a flavivirus closely related to POWV that circulates in Europe. Specifically, POWV and DTV are members of the TBEV complex. In Europe, there is strong evidence that voles (Myodes spp.) are the natural reservoirs for TBEV. TBEV was isolated from the brain of voles in Slovakia [Myodes population displaying seropositivity for TBEV [It is possible that potential reservoirs for DTV are small burrowing rodents such as voles because, unlike Slovakia  and fromSlovakia . In FinlSlovakia . Additiofor TBEV ,44. Sincfor TBEV . There ifor TBEV . HoweverI. cookei and POWV-LB since humans have more exposure to I. scapularis and DTV. With the assumption that both lineages have similar transmission mechanisms, this section will discuss POWV-LB and DTV concurrently as POWV. Concerningly, a unique feature of POWV is that it can transmit to the mammalian host in as little as 15 min of tick attachment [B. burgdorferi which transmits between 24–48 h [B. burgdorferi spirochete which is housed in the midgut before migrating to the salivary glands [As with other tick-borne viruses, multiple modes of transmission exist for POWV . Since btachment , compare 24–48 h . While t 24–48 h , as oppoy glands . Furthery glands . Due to y glands ,51, and y glands , and thiy glands ,54. One hypothesis for the focality of and low prevalence of POWV is that the virus is sustained predominantly through cofeeding transmission where aggregating uninfected and infected ticks feed in close proximity and transmit the virus to one another. Cofeeding transmission can still occur if a host is non-viremic , and eviB. burgdorferi and Babesia microti will suppress the host’s immune response and can worsen clinical outcomes [B. burgdorferi may also increase the incidence of B. microti in ticks, aiding in its geographic expansion [B. burgdorferi is widespread [Ticks are capable of being infected with multiple pathogens simultaneously, and previous research has demonstrated that pathogens may behave synergistically, facilitating transmission. For example, the presence of both outcomes ,61, and xpansion . Mosquitxpansion ,64,65. Hdespread , and morI. scapularis is a generalist and will feed on a wide array of vertebrates. It is likely that humans will be incidental hosts, as seen with the growing number of Lyme disease cases [I. cookei was predominantly nidicolous, remaining in burrows and being highly host-specific [I. cookei have been collected through public passive surveillance in New York , Maine [I. cookei does have the potential to cause virus spillover to humans, I. scapularis is likely more of a threat due to its wider distribution range and more frequent human encounters [Transmission potential is influenced by many factors, but it largely begins with host preference. se cases . Previouspecific . However), Maine , and Pen), Maine . While, counters ,69. HoweI. cookei and I. scapularis, discussions of environmental associations will focus on these two species. However, compared to I. scapularis, research on I. cookei is sparse since it historically is encountered less by the public. I. scapularis and I. cookei are found throughout the eastern half of the US, with concentrations in the Northeast, Upper Midwest, and Great Lakes regions, and the distribution range is expanding [I. scapularis has a larger distribution range compared to I. cookei, which may assist its ability to vector POWV more effectively to a larger population.Since POWV is maintained in nature by Ixodid ticks, the presence of POWV depends on the geographic location where the vectors are found and suitable environmental factors that support the vector population. With transmission cycles primarily sustained by xpanding ,70,71. II. scapularis has a two-year life cycle where adult ticks quest in spring and late fall , nymphs in early spring (June–July), and larvae in early fall , retain humidity which is favorable for I. scapularis [Meteorological variables, like temperature, humidity, and rainfall, affect moisture content in microhabitats, influencing tick reproduction rate, desiccation status, and host food availability. as lower , and hots nymphs . Certainapularis . Furtherapularis . While m2 TBEV focus in Germany revealed two circulating clades of virus that occupied different habitats (forest-meadow ecotone or forest). Over a decade, one clade showed more annual stability while the other appeared more sporadically. Moreover, nymphal counts varied annually. However, the minimum infection rate remained stable [Multiple POWV strains exist in nature, but what causes certain strains to present in specific foci remains speculative. It has become evident that POWV is highly focal and stable . In Connd stable . Similard stable . It is pI. cookei northward into Canada and a decreasing presence southward in the US, while I. scapularis is predicted to expand northward and westward [I. cookei population density or POWV prevalence is unknown.The changing climate and landscape are impacting the presence of tick and host species, and, subsequently, are affecting the presence of tick-borne pathogens. Global climate change is leading towards rising annual temperatures, caused largely in part by the excess release of carbon dioxide and methane . Resultiwestward , exposinwestward , and thiwestward . It is pwestward . HoweverI. scapularis populations [I. scapularis distribution [I. scapularis, hosts, and virus should be examined.In the early 1800s, Japanese barberry was introduced from Japan to the US, and it became a popular ornamental plant that has since invaded much of the Northeast and Upper Midwest . Found iulations ,90, it cribution ,89, and I. scapularis [I. cookei is undetermined since the tick may effectively be sustained in burrows. Furthermore, without knowing the reservoir for DTV, we cannot be sure how habitat loss directly impacts DTV prevalence.With the encroachment of humans into wildlife habitats, large tracts of land are becoming fragmented for urbanization and agriculture. Habitat fragmentation inadvertently leads to the loss of biodiversity and the concentration of adaptable hosts into pockets of natural vegetation. Specifically, white-footed mice and voles  are successful at adapting to the fragmented landscape ,92, whicapularis , though While humans are contributing to global climate change and habitat reduction, increasing tick-borne disease prevalence, there are a number of ways that people can physically reduce tick densities and personal tick-borne pathogen acquisition, including POWV. Personal preventative measures for reducing pathogen attainment include frequently checking for ticks when outdoors, staying in the center of maintained trails, wearing EPA-approved repellents, wearing light-colored clothing, and tucking one’s pants into their socks . PreventI. scapularis and I. cookei. Both ticks will bite and transmit the virus to humans, and the distribution range of these ticks is growing. The relationship between pathogen, host, vector, and environment is complex and highly interdependent. Research is ongoing to elucidate what sustains POWV in localized areas as well as environmental variables that may facilitate the maintenance and spread of vector and pathogen. The best practices for reducing POWV disease include practicing personal tick prevention methods to decrease tick bites.Tick-borne pathogens are rising in incidence, and there are currently no vaccines in the US to curb the spread of these pathogens to humans. POWV is a rare and deadly virus causing a disease that is growing in incidence with two major tick species supporting the virus in nature:


The suspension for bead fabrication contained sodium alginate as gelling agent and was added dropwise into a calcium chloride solution to trigger the consolidation process. After debinding in air, sintering of the composite beads was performed under inert atmosphere. Samples in green and sintered state were analyzed by digital light microscopy and scanning electron microscopy equipped with energy dispersive X-ray spectroscopy. Investigations by mercury intrusion porosimetry revealed that pure alumina beads featured smaller pores compared to composite beads, although the open porosities were comparable. The fracture strength was evaluated on single beads. Contrary to the pure alumina, the composite beads showed a clear plastic deformation. Pure niobium beads showed a ductile behavior with very large deformations. XRD analyses revealed the presence of calcium hexaluminate and beta-alumina as minor phases in the alumina beads, while the composite ones contained about 25 wt% of impurities. The impurities comprised NbO arising from the oxidation, and β-Nb Ceramic-metal composites benefit from the combination of high melting point, hardness and chemical stability of ceramics and high toughness and ductility of metals. Usually, the application temperature of such composites is limited by the relatively low melting point of the used metals or by the reaction between the metal and ceramic phase and/or environment. Regarding the melting point, so-called refractory metals, such as Zr, Mo, Nb, W, and Ta (to name the most abundant), can help extending the application to higher temperatures. In particular, niobium and tantalum exhibit a similar thermal expansion coefficient as alumina in a wide temperature range, which allows to produce composites with a high thermal shock resistance ,2,3,4. HIn a recent work, Zienert et al. reported for the first time the production of coarse-grained refractory composites consisting of refractory metals and refractory ceramic materials . Refract 1500 °C . The shrEspecially in the case of ceramic materials, the ability to produce near-net shaped parts is very beneficial since it reduces the need for final machining, which is usually limited for sintered bodies (due to their brittleness) or often results in rejected green pieces (due to their weakness). Among these shaping methods, aqueous gel casting is one of the most common technique. In general, a ceramic suspension with high solid contents is mixed with an additive allowing for a direct sol-gel transition. Originally, toxic chemicals, such as acryl amide, were utilized as gelling agents ,8,9,10. 2+ or Ba2+, which results in the bonding between two G-blocks of different polysaccharide chain segments and leads to a 3D crosslinking and, thus, in the gelation. This phenomenon occurs stepwise and depends on the ratio between divalent ion (M2+) and G monomers [A particular class of natural compounds commonly used in gel casting is that of alginates, which are derived from brown seaweeds. They are widely applied as thickeners, stabilizers and gelling agents, especially in the food and pharmaceutical industry . Alginatmonomers . The addmonomers . The shamonomers ,18. Altemonomers .Recently, hollow and full beads based on zirconia and steel were fabricated using the alginate gelation technique . In part2 · 2H2O  was used as solidifying agent.The main raw material for the beads production was niobium powder  with measured The fabrication process was based on the gelation of alginate in contact with bivalent ions in an aqueous solution as solidifying agent. The raw powders  were dry mixed for 5 min using a homogenizer DIAX 600 . Similarly, the liquid additives were dissolved in deionized water. Next, the two mixtures were filled into a polypropylene barrel together with alumina milling balls (3 cm in diameter) and mixed for 4 h. The formulations of the different slurries are given in After mixing, the suspension was loaded into a 50 mL syringe and poured dropwise into a water solution (1 mass%) of calcium chloride from a height of approximately 3 cm. The bead formation occurred instantly. The process was automated by means of an infusion pump Perfusor Secura FT , consisting of a computer-controlled motor turning a screw that pushes the plunger on the syringe. Different nozzle gauges allowed to control the size of the produced beads, as shown in After gelation, the wet green beads were removed from the calcium chloride solution with the aid of a sieve, washed with deioinzed water to remove any solution residues, and, finally, dried at 50 ic peaks . However−1, and it was then kept constant for 90 s and, finally, decreased again.The rheological behavior of the slurries was evaluated by means of a rheometer HAAKE MARS 60 , using a coaxial cylinder measuring system with rotor CC38/Ti/SE and cup Z40. The measurements were performed under controlled-rate/steady-state conditions at a temperature of 20 Mercury intrusion porosimetry was used to evaluate the open porosity and pore size distribution (PSD) of the sintered beads according to the International standard ISO/DIS 15901-1. The morphology and fracture surfaces of the samples were analyzed with aid of a digital light microscope . The average diameter of each sample batch in dried, as well as in sintered, condition and the resulting shrinkage were estimated through image analysis on the micrographs. In addition, scanning electron microscopy coupled with EDS  was used.P is the load at fracture, and The fracture strength of the beads in sintered state was estimated with the aid of an universal testing machine TT2420  and a measuring test device for single beads equipped with a load cell of 1 kN. Thirty beads per batch were loaded to fracture in a diametral compression mode between two parallel steel plates at a displacement rate of 0.05 mm/s. The fracture stress was estimated from the force-displacement data according to the method reported by Kschinka et al., who investigated the mechanical behavior of glass spheres :(1)σf=2.pecimens . After cThe 3D-macrostructure of selected large (L) beads was analyzed before and after mechanical loading using microfocus X-ray computed tomography (µ-CT). The analyses were performed with a CT-ALPHA  equipped with a transmission X-ray tube FXE-160.20/25  and a flat panel X-ray detector Dexela 1512 . The µ-CT was operated with at 150 kV and 80 µA using a 0.3 mm copper filter in order to reduce beam hardening artifacts. The exposure time was set to 1 s for alumina samples and to 2 s for niobium containing samples. The volume data were reconstructed using the software Volex 6.0 . The resulting voxel size was 5.5 µm for all investigated samples. The reconstructed volume data were visualized using the software VG Studio MAX 2.2 .Beads in sintered state were manually ground into fine powder and analyzed by means of XRD. The XRD diffractometer Empyrean  was operated in Bragg-Brentano geometry, with standard Cu Kα radiation (−1, together with a slight thixotropy. The shear thinning behavior is a fundamental feature for slurries which need to be pumped or sprayed through a nozzle. Despite the lower solids content, the Al2O3_100 slurry showed an overall higher dynamic viscosity compared to the others, hence requiring a higher shear stress to be stirred. This behavior was likely due to the particle size and morphology of the raw materials used in the preparation of the slurries: the tabular alumina had a The results of the rheological investigation are plotted in t et al. .2O3_100-L batch. Larger beads have a smaller surface-to-volume ratio, which might result in less effective debinding and sintering steps. The apparent density obtained through mercury intrusion was similar for batches from the same slurry and with different sizes. As expected, the Nb_100-L batch showed a higher value of apparent density compared to the Nb_95-L one, due to the lack of alumina in the corresponding slurry. The mercury intrusion curves for all batches are presented in 2O3 addition entailed no remarkable effect in the pore size distribution of large composite beads.The main results from mercury intrusion porosimetry are reported in Some selected load-displacement curves for the different batches are presented in m was slightly higher for the alumina samples, as presented also in m were within the expected range of partially sintered alumina [m of the composite beads can be attributed to the interaction of two separate phases within the samples, which possess significantly different mechanical properties. However, the main factor was related to the average particle size and particle size distribution of the used raw materials. As mentioned in The Weibull diagrams for all batches are presented in  alumina . The lowm.The parameters of the fracture strengths obtained from the Weibull analysis are given in Reconstructed volume images from the CT analyses on the sintered beads are presented in SEM micrographs of the surface of pure alumina beads are presented in XRD analysis of the corundum powder revealed 98.9 mass% α-Al ICSD 737 with lata of CA6 remained almost the same as in the corundum raw material, whereas c increased to Besides niobium  (ICSD 76% β-Nb2C ,29 (adopIn this study, metal-ceramic composite beads containing niobium and alumina, particularly 100 vol% alumina, 100 vol% niobium, and 95/5 vol% niobium/alumina, were produced by means of gel casting with sodium alginate as binder. The slurries were poured dropwise through nozzles with different diameters into a calcium chloride solution in order to obtain beads with distinct sizes. The samples containing Nb were carefully debound in air and subsequently sintered at 1600 2C were found in the composite beads due to the reaction with the residual carbon and oxygen from the alginate binder.Full beads with good spherical shape and average final diameters in the range 1.75–2.9 mm were obtained. The linear shrinkage after debinding and sintering was quite high, reaching more than 10% for the AlThis study demonstrated that the gel casting process allows to reliably produce spherical grains with defined properties, which can be used as aggregate fraction in new coarse-grained refractory castables, among others. Such products are being currently investigated and will be the object of an upcoming study. In addition, the particle size distribution of the slurries will be optimized to obtain the best possible particle packing, in order to improve the material densification during thermal treatment.


Brassica rapa, Arabidopsis thaliana, and Oryza sativa were compared. Moreover, the roles of BrCC–NBS–LRR genes involved in pathogenesis-related defense were studied and analyzed. First, the expression profiles of BrCC–NBS–LRR genes were detected by inoculating with downy mildew and black rot pathogens. Second, sensitive and resistant Chinese cabbage inbred lines were screened by downy mildew and black rot. Finally, the differential expression levels of BrCC–NBS–LRR genes were monitored at 0, 1, 3, 6, 12 and 24 h for short and 0, 3, 5, 7, 10 and 14 days for long inoculation time. Our study provides information on BrCC–NBS–LRR genes for the investigation of the functions and mechanisms of CC-NBS-LRR genes in Chinese cabbage.The nucleotide-binding site–leucine-rich repeat (NBS–LRR) gene family is the largest group of plant disease resistance (R) genes widespread in response to viruses, bacteria, and fungi usually involved in effector triggered immunity (ETI). Forty members of the Chinese cabbage CC type NBS–LRR family were investigated in this study. Gene and protein characteristics, such as distributed locations on chromosomes and gene structures, were explored through comprehensive analysis. CC–NBS–LRR proteins were classified according to their conserved domains, and the phylogenetic relationships of CC–NBS–LRR proteins in  Plants have developed specific mechanisms to protect themselves from abiotic or biotic stress ,2. Planttoll/interleukin-1 receptor-NBS-LRR (TNL) genes are rare in monocotyledons [Arabidopsis thaliana interacted directly with PopP2, which was the pathogen protein of bacterial wilt [Arabidopsis were in response to Pseudomonas syringae by indirect interaction with AvrRpm1 and AvrB [Arabidopsis RPW8 enhanced the resistant ability to powdery mildew in grapevine [NBS–LRR proteins are divided into two types according to their conservative functional domains. One contains a toll/interleukin 1 receptor (TIR) domain at the N terminal and the other is the non-TIR(CC–NBS–LRR type), which assembles a coiled-coil (CC) domain at the N terminal instead of a TIR domain. Other domains in the N-terminal of CC–NBS–LRR replace or coexist with CC domains, particularly zinc fingers or RPW8 domains ,10. CC–Ntyledons , revealityledons . The R gtyledons . Additioial wilt . RPS2 anand AvrB ,15. The rapevine .Hyaloperonospora parasitica, DM is one of the three major diseases of Chinese cabbage and even the entire cruciferous family. BR is a bacterial disease caused by Xanthomonas campestris pv. campestris dowson, which infects many Brassica species, such as cabbage (Brassica oleracea var), Chinese cabbage (Brassica pekinensis), and oil seed rape (Brassica campestris) [Chinese cabbage is a popular vegetable worldwide, especially in Asia. The leaf heading, which forms at the late heading stage, is the main edible part and commodious organ and is directly associated with the yield and quality of Chinese cabbage . Downy mpestris) ,20. ThusBrassica rapa were identified without a reference genome [Brassica rapa ssp. Pekinensis [CC–NBS–LRR genes of Chinese cabbage were identified and analyzed in a genome-wide range. The basic information of the BrCC–NBS–LRR family was listed, including Genome position, exon No., the length of CDS and Protein, etc. A phylogenetic tree of CC–NBS–LRR genes in Brassica rapa, Arabidopsis thaliana, and Oryza sativa was built to elucidate the evolution. The expression profiles of BrCC–NBS–LRR genes in different tissues and in response to diseases were detected and analyzed, showing that BrCC–NBS–LRR genes were enriched in leaf, and their responses were quite different due to the different types of pathogen and time. Furthermore, the sensitive and insensitive inbred lines were obtained and inoculated with Hyaloperonospora parasitica and Xanthomonas campestris to explore the predicting functions of BrCC–NBS–LRR genes. These results suggest that they play an opposite or consistent function to diseases. Our study provides information on BrCC–NBS–LRR genes. This information is useful to further investigate the gene functions and mechanisms of CC–NBS–LRR genes in Chinese cabbage.A total of 92 NBS-encoding resistance genes of e genome , and thekinensis . CC–NBS–CC–NBS–LRR family genes were searched using the Brassica rapa genome on Brassica Database (BRAD). A total of 40 CC–NBS–LRR family candidates were found, and their protein sequences were verified using online tools for protein functional domains. The tools were PfamScan , InterPro , and ScanProsite . Finally, 40 genes were screened, all of which have intact CC, NBS, and LRR domains. The CC–NBS–LRR family genes of Chinese cabbage were named according to their genes that were ID-searched on BRAD. Blast searches for the homologous genes of BrCC–NBS–LRR in Arabidopsis thaliana were conducted using TAIR , and 20 genes were homologous to CC–NBS–LRR members shown in CC–NBS–LRR genes in Brassica rapa are summarized and shown in The CC–NBS–LRR family usually encodes two typical domains, including the CC domain at the C terminal and the NBS–LRR domain at the N terminal. All BrCC–NBS–LRR family genes can be found on BRAD , and are drawn and shown in Brassica rapa from A1 to A10. The majority of BrCC–NBS–LRR genes were located on A09 with 12 members (30%), followed by A06 with 8 (15%) BrCC–NBS–LRR members. However, only one member was located on A02. Two members were located on A05 and A10, and four, five, and six members were located on A3, A01, and A08, respectively. A total of 24 CC–NBS–LRR family genes were found in Arabidopsis thaliana, 16 (67%) of which were located on Chr.1 and eight were located on Chr.5 . Then, the structures were drawn with Gene Structure Display Server, as shown in BrCC–NBS–LRR genes had only one exon (17 of 39), and nine genes had two exons and one intron. However, Bra026094 had most exons up to 12. The rest of the CC–NBS–LRR genes contained more than three exons. Specifically, six members had three exons, two had four, and four had five  genes [CC–NBS–LRR members contained WRKY71OS elements, except Bra019754. A total of 38 BrCC–NBS–LRR members contained GT1GMSCAM4 in their promoters, the expression of which is induced by pathogens and mediated in part by a GT-1 box that interacts with a GT-1-like transcription factor. WBOXATNPR1 and WBBOXPCWRKY1, a salicylic acid (SA)-induced WRKY DNA binding protein and a “W box” WRKY protein that binds specifically to DNA sequence motifs [BrCC–NBS–LRR gene promoters with a total of 96 and 20 items , which regulates defense-related gene expression assisted by GCC box and non-GCC box cis elements [Bra026682 promoter, and only one TL1ATSAR was detected in the promoter regions of 13 NPR1-responsive ER-resident genes on the Bra019754 promoter. Only one AGCBOXNPGLB was conserved in most PR-protein genes on the Bra037139 promoter. Furthermore, 40 cis-elements were present on Bra011432 and Bra034631, and only five cis-elements were present on Bra026924 gene promoters listed in BrCC-NBS-LRR genes might be regulated by different mechanisms, and these genes play different roles in disease resistance in Chinese cabbage.The promoter sequences of rt sites . They wele genes . GT1CONSe motifs , appeare20 items B. A totaelements . Two GCCBrCC–NBS–LRR genes in the roots, stems, leaves, flowers, and siliques, the total mRNA of the different tissues of the Chinese cabbage cultivar A160 were isolated and reversed-transcribed into cDNAs, which were amplified with specific primers  or distilled water as a control. They were harvested after 1, 3, 6, 12, and 24 h for short inoculations and 3, 5, 7, 10, and 14 days for long inoculations for the detection of the expression levels of BrCC–NBS–LRR genes through real-time PCR. The expression characteristics were analyzed, as shown in CC–NBS–LRR family genes were up-regulated after 1 h of inoculation. This result indicated that they quickly respond to DM. For example, Bra034631 expression was induced and increased 11-fold within 1 h. As for Bra031482, the expression was up-regulated 10-fold. The expression level of Bra026978 increased with inoculation time, showing that Bra026978 was continuously induced by DM. The expression levels of numerous BrCC–NBS–LRRs dramatically changed after 12 h of inoculation. However, the expression levels of BrCC–NBS–LRRs were relatively evenly up-regulated 3, 5, 7, and 10 days after inoculation  showed decreased expression 14 days after the inoculation of DM relative to the expression levels detected at day 0 of inoculation  and 3, 5, 7, 10, and 14 days (long inoculations)). Seven of the 40 BrCC–NBS–LRR genes (18%) were up-regulated more than twofold after 1 h of inoculation relative to the expression at 0 h, and the expression levels of Br018245, Bra030778, and Bra017572 increased 3.5-, 7.3-, and 2.2-fold after 3 h of BR inoculations. Six hours after inoculation, only Bra016785 showed an increase in expression level. The expression of the others showed no change or was down-regulated. However, more BrCC–NBS–LRRs (15 of 40) were up-regulated after 12 h of inoculation. The expression levels of CC–NBS–LRRs were smooth and steady compared with those after the short inoculations. Bra030778 was up-regulated in the long inoculations , and the expression levels of Bra029405, Bra019063, Bra017572, and Bra015597 increased remarkably 3 days after inoculation. The expression levels of Bra030779 and Bra015597 remarkably increased 5 days after inoculation. After 7 days of BR inoculation, 17 CC–NBS–LRR genes showed up-regulated expression, but 93% of the BrCC–NBS–LRR genes showed down-regulated expression 14 days after inoculation. Most of the Chinese cabbages showed numerous disease spots. Compared to long time inoculation, these dramatically differential expressions of BrCC-NBS-LRR genes in the 24 h short inoculation suggested that they respond quickly to BR.Expression levels after exposure to BrCC-NBS-LRR in response to fungal and bacterial disease, 24 inbred lines of Chinese cabbage were applied to screen the sensitive and resistant lines to DM and BR. On the basis of statistics , particularly Bra013947, Bra026368, Bra029405, Bra030778, Bra030779, Bra036995, Bra027097, Bra018863, Bra019752, Bra019755, Bra036845, Bra037139, and Bra015597. The trends of Bra026368, Bra030778, Bra018863, Bra019755, and Bra015597 in the 1 day short inoculation were consistent with those in the 14 day long inoculation. By contrast, Bra009882 and Bra026978 expression in A24 and A95 decreased obviously after 1 day of DM inoculation.Then, the expression levels of inbred lines A95 and A24 were detected when they were exposed to DM for 1 day and 14 days, as shown in Xanthomonas campestris, and the expression levels of the BrCC–NBS–LRR family were detected through qPCR 1 and 14 days after inoculation, as shown in BrCC–NBS–LRR genes were induced by BR after 1 day of inoculation in A167 and A96, and six BrCC–NBS–LRR genes were induced after 14 days of inoculation. In addition, the expression levels in A96 were even higher than those in A167 after inoculation. Especially, the expression levels of Bra035424 and Bra030778 in A167 were up-regulated more than 11-fold. Only one member showed the same expression trend as above (Bra027866). However, the expression of Bra030779 was down-regulated after 1 day of BR inoculation. These results revealed that the CC–NBS–LRR gene family of Chinese cabbage may play different roles in response to a disease and the type of role depends on inoculation time and disease types.As for disease stress due to bacterial BR, the sensitive line A167 and resistant line A96 were also inoculated with TNL genes and non-toll/interleukin-1 receptor-NBS-LRR (nTNL) genes, which is composed CC–NBS–LRR [CC–NBS–LRR members of Chinese cabbage were searched and identified on the basis of the entire genome of Chinese cabbage. Although 47.1% NBS encodings of Brassica rapa appeared to have undergone tandem duplication and to be distributed in tandem arrays [BrCC–NBS–LRRs were unevenly distributed on A01–10, with five pairs of two copies  have mechanisms for recognizing and providing direct or indirect protection against pathogens in plants and play a critical role in effector triggered immunity (ETI) . Of all –NBS–LRR . In our m arrays , all BrCo copies  and two d on A05 , which sBrassica rapa shared high homology with model monocotyledonous plant Arabidopsis thaliana, based on the protein sequence the phylogenetic tree of the CC–NBS–LRR family was built to explore the homology among Arabidopsis thaliana, Brassica rapa and monocotyledon rice. To our surprise, not all these BrCC–NBS–LRRs were highly clustered in the same subgroup with their At orthologs; moreover, CC–NBS–LRRs of the same species were clustering together. Even two BrCC–NBS–LRRs shared high homology with rice instead of Arabidopsis, such as Bra013213 and Bra017572, as shown in Although Brassica rapa on IntrePro. The CC motif was analogous to the LRR motif in the critical core of the three-dimensional structure composed of hydrophobic chains and free solvent-exposed residues. The CC domain of a CC–NBS–LRR gene of LR10 in wheat directly recognizes pathogens; this result indicated that the CC domain function of CC–NBS–LRRs needs to be further explored and excavated [The protein structures of BrCC–NBS–LRRs were usually composed of three parts: one or more NBSs, an LRR domain at the C terminal, and a CC domain at the N terminal. NBS-encoding domains are necessary for the recognition of diverse pathogens in plants . Five kixcavated .BrCC–NBS–LRR promoters were downloaded from BRAD and analyzed with an online tool New PLACE for the searching of cis-acting regulatory elements. A total of 12 disease-resistance-related cis-acting elements were found in the promoters of BrCC–NBS–LRR. The GT1CONSENSU (consensus GT-1) elements dominated as investors and appeared on the promoters in tandem. It was first reported as the binding sites of many light-regulated genes in many species, for example, ribulose bisphosphate carboxylase small chain, phytase gene [PR-1a promoter can reduce TMV infection and SA treatment and influence the expression level of SA-inducible genes [BrCC–NBS–LRR genes, that is, the binding site of the BELL-type transcription factor of OsBIHD1 associated with resistance and response in rice [The cis-acting elements of gene promoters were essential for transcriptional expression, and the types of cis-acting elements indicated the potential functions of genes, particularly in response to pathogens . All thease gene ,39. The le genes . The funle genes ,40. “W-ble genes ,42. A to in rice . Some miBrassicaceae NBS–LRR genes were comprehensively analyzed and compared on the basis of established cross-species phylogenetic and syntenic relationships of NBS genes for the study of the R gene in Brassicaceae, particularly in Arabidopsis lyrata, Arabidopsis thaliana, Brassica rapa, Capsella rubella (127), and Thellungiella salsuginea [NBS-LRR genes exert critical effects in response to multiple pathogens, including viruses, bacteria, and fungi [Arabidopsis thaliana, rice, and wheat. However, they are rarely reported in Chinese cabbage in terms of response to DM and BR. In our study, the A160 inbred lines of Chinese cabbage were inoculated with Hyaloperonospora parasitica and Xanthomonas campestris, and short and long inoculations were performed for exploring the expression profile. In the short inoculations, heatmaps were drawn after 1, 3, 6, 12, and 24 h of inoculation , which enhances resistance during adult stages; TaRPM1, which is involved in resistance to Pst [BrCC-NBS-LRRs, the expression characteristics were carried out on the sensitive inbred lines A95 and A167 and resistant lines A24 and A96 to DM and BR for 1 day and 14 day inoculation, as shown in BrCC-NBS-LRR genes showing similar expression profiles induced by DM rather than the control; moreover, A24 was even higher than A95 DM for 1 day inoculation and for 13 for the long inoculation (14 days). The expression levels of BrCC–NBS–LRR genes after 1 day of inoculation with DM and BR were compared. Bra030778 and Bra034631 were induced and had the same expression level. In the long inoculation of DM and BR, three BrCC–NBS–LRRs  showed higher expression levels. These results revealed that BrCC–NBS–LRR family genes play various roles in response to phytopathogens, and the roles depend on disease type and inoculation time.An increasing number of CC–NBS–LRRs have been reported, such as Lr10, which is involved in (CC–NBS–LRR)-mediated resistance ; Yr10 /night (18 °C) and 16 h light/8 h dark cycles. For the expression profiles of DM and BR, the The inbred line A160 of Chinese cabbage was grown in a greenhouse until four leaves were moved to light incubator with 6°C for 25 days of vernalization, and then they were grown under conventional environment until flowering. In total, the tissues of root, stem, leaf, flower and silique were harvested, respectively, for RNA isolation.http://brassicadb.org/brad/, accessed on 27 February 2021), with the B. Rapa genome (version 1.5). They were identified with the NB–ARC domain according to the Hidden Markov Model of pfam00931 and the LRR domain of PLN03210 at the C terminal with the BLAST program of the NCBI database . The CC domain of BrCC–NBS–LRRs was confirmed using MPI Bioinformatics Toolkit  [Arabidopsis thaliana, with A thaliana genome: Araport11 and Oryza sativa were downloaded from TAIR  and Rice Genome Annotation Project , which were confirmed using the Basic Local Alignment Search Tool of NCBI. InterPro  and Pfam  were used for further confirmation [All the CC–NBS–LRR members of Chinese cabbage were obtained from BRAD (08–2021) . The othirmation .BrCC–NBS–LRR genes were searched on BRAD , and all of them were analyzed using New PLACE , which is a database containing plants used in the analysis of cis-acting regulatory elements [http://gsds.cbi.pku.edu.cn/, accessed on 27 February 2021. 2021) for the reconstruction of the gene structure [The promoter sequences of elements . Informatructure .Arabidopsis thaliana and Oryza sativa were downloaded from BRAD, TAIR, and Rice Genome Annotation Project. First, multiple sequences were aligned with MEGA6, then the phylogenetic trees were calculated and constructed using the neighbor-joining method within 1000 replicates [The CC–NBS–LRR protein sequences of Brassica rapa and plicates .The protein sequences were analyzed using the online tool MEME Suite with the following settings: site distribution, any number of repetitions; number of motifs, 10.The plants described in the Plant Materials section were harvested for RNA extraction with TRIzol™ reagent , and 2 mg of RNA was reversed-transcribed to cDNA with TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kits . Approximately 15 L of total volume was used for real-time PCR, containing 1 L of specific primers, 3 L of cDNA samples diluted 30–50-fold and 7.5 L of SYBR Green real-time PCR master mix . The reactions were amplified using a qTower3G  real-time PCR detection system for 40 cycles with unimodal dissolution curve and tubulin as an internal reference . The pri


How will the novel coronavirus evolve? I study a simple epidemiological model, in which mutations may change the properties of the virus and its associated disease stochastically and antigenic drifts allow new variants to partially evade immunity. I show analytically that variants with higher infectiousness, longer disease duration, and shorter latent period prove to be fitter. “Smart” containment policies targeting symptomatic individuals may redirect the evolution of the virus, as they give an edge to variants with a longer incubation period and a higher share of asymptomatic infections. Reduced mortality, on the other hand, does not per se prove to be an evolutionary advantage. I then implement this model as an agent-based simulation model in order to explore its aggregate dynamics. Monte Carlo simulations show that a) containment policy design has an impact on both speed and direction of viral evolution, b) the virus may circulate in the population indefinitely, provided that containment efforts are too relaxed and the propensity of the virus to escape immunity is high enough, and crucially c) that it may not be possible to distinguish between a slowly and a rapidly evolving virus by looking only at short-term epidemiological outcomes. Thus, what looks like a successful mitigation strategy in the short run, may prove to have devastating long-run effects. These results suggest that optimal containment policy must take the propensity of the virus to mutate and escape immunity into account, strengthening the case for genetic and antigenic surveillance even in the early stages of an epidemic. In the autumn of 2021 it has become clear, that even wide-spread access to vaccines has not (yet) eliminated the threat of COVID-19, as new variants prove to be fitter and better able to escape immunity  in which direction will the SARS-CoV2 virus causing COVID-19 evolve?, and b) which aggregate dynamics can we expect from an evolving virus in terms of infections and fatalities? In order to study these topics, i develop a parsimonious epidemiological model that simultaneously captures two types of viral evolution:a) Genetic variation: Mutations can change the characteristics of a virus in a way that alter its evolutionary fitness, e.g., by increasing its transmissibility  Antigenic variation . In this model, mutations cause a species  to move along an n-dimensional landscape. Each spot in this landscape represents a phenotype associated with a given fitness value, which may be static or change over time due to, for instance, environmental effects  of viral mutations and—more specifically—COVID-19 variants . The fourth section shows the results of a quantitative analysis of the ABM using Monte Carlo simulations. Finally, section five concludes.This section describes the baseline model using differential equations in order to investigate its basic properties analytically. In order to capture pre-symptomatic and asymptomatic infections, I extend the classical SIR model to incorporate the following compartments : Susceptible (S), exposed (E), pre-symptomatic (P), (permanently) asymptomatic (A), (symptomatic) infected (I), recovered (R) or dead (D). Susceptibles may become infected with the virus, if they meet a person who belongs to the compartments P, A or I. Once infected, a susceptible becomes exposed. This is known as a latent period, in which the person neither displays any symptoms, nor is infectious. After the latent period, a person becomes pre-symptomatic. In this case, agents are able to transmit the disease, but do not display any symptoms (yet). Pre-symptomatic agents may later become either symptomatic infected or stay permanently asymptomatic. Finally, permanently asymptomatic will recover, whereas symptomatic infected may either recover or die. A similar approach is used by Lee et al. . This raLike the standard SIR model, this model operates on the simplifying homogenous mixing assumption, i.e., every member of compartment i has an equal probability to meet a member of compartment j.Figure The laws of motion (omitting mutations and variants) of this model are given as follows, where The basic reproduction number is defined as the number of people infected by one infected person in an otherwise susceptible population. Since an infected person may belong to the compartments E (where they are not infectious), P, A or I (where they are infectious), it can be calculated by multiplying the number social contacts per time period with the time spent, i.e.,which collapses to:not only consist of susceptibles. Assuming that infectious contacts are not directed toward any group , the share of these contacts with susceptibles is given by the number of susceptibles in the population, i.e., The effective reproduction number If Differentiating From this analysis follows easily that those mutations are more successful, which increase the transmissibility and the infectious time. From the relationship with Uniform social distancing  reduces the number of social contacts (or their infectiousness) for all individuals in the same way.The basic reproduction number is now:With the new effective reproduction number given by:This does not change any of the considerations above. If, however, Another widespread measure to slow the spread of COVID-19 is to isolate symptomatic cases (or to encourage self-isolation upon developing symptoms). If we assume that all symptomatic cases are isolated, this changes the laws of motion regarding S and E accordingly , if we assume that this measure is not combined with uniform social distancing):Adapting Eq. Accordingly, the effective reproduction number is:In this case, the results regarding the evolutionary advantage of an increase in entclass1pt{minimaside effect of favoring asymptomatic infections. If, however, the survival chance is independent from the share of symptomatic infections, lethality is unaffected by evolutionary selection.If the lethality depends mechanically on the share of symptomatic infections, as suggested by above formulation  cEach agent may only be infected by one virus variant simultaneously.Whenever an individual is infected, the virus may mutate, creating a new variant which is an offspring of the infecting (“parent”) variant.A mutation randomly changes the properties of the virus  with the means given by the actual values of the parent variant see Eq. .Each virus variant belongs has an antigenic cluster that may change during a mutation, i.e., a mutation may be coupled with an antigenic drift.4A previous infection within the same antigenic cluster provides perfect cross-immunity between variants. It may provide cross-immunity between antigenic clusters depending on the antigenic (evolutionary) distance between two antigenic clusters.Each agent has an equal probability to meet another agent .In order to capture mutations, the interplay between the variants, while at the same time keeping the analysis as simple as possible, I make the following assumptions:Infected agents meet other agents and may infect them, a process in which mutations and antigenic drifts may occur , recover or die  records the time passed since becoming infected. Once it reaches Using a recursive function, the agent may also acquire immunity against the ancestor and/or descendants of this antigenic cluster with probability The simulation is initialized with the parameters described in Table This calibration allows for a basic reproduction number of the SARS-CoV2 wild type of 2.5 and a share of pre-symptomatic infections of 50% as estimated by the CDC . The incIn order to analyze the properties of the model, I rely on Monte Carlo simulations. Specifically, I run each scenario 100 times with fixed random seeds for 500 periods. I then analyze the results using the programming language R , levels of social distancing that bring the effective reproduction number of the wild type in a population inhabited by a very large share of susceptibles close to 1 can cause variants to evolve to higher fitness than lower levels of social distancing, if there is high cross-immunity between the antigenic clusters, as can be seen in the bottom right part of the figures.Figures m at time step 500,In order to present these results concisely, I compute mean Figures relative adapted relative adapted Figures see Fig. , adaptivThis subsection discusses the public health outcomes of an evolving virus and to which extent they can be used to identify the propensity of the virus to mutate. Figures There is no visible difference in mortality between the scenarios  for a given level of social distancing. The number of infected agents is more sensitive to changes, as the second wave of infection is visible for scenarios without any cross-immunity between antigenic clusters and very low levels of social distancing. In more realistic scenarios, however, we also cannot distinguish between the scenarios with regard to the mutation chance and the cross immunity between antigenic clusters by looking only at public health outcomes of the first 100 days.Things are different, however, if we look at a longer time horizon. Figure Moreover, the marginal burden associated with not preventing a full-scale outbreak  may drastically increase even in a scenario with high cross-protection and moderate cross-immunity, as mutations and antigenic drifts increase the chances of each individual to become infected at least once in their lifetime.Figures I developed a simple theoretical model to study the genetic and antigenic evolution of a virus under varying containment scenarios in a stylized way. The properties of the wild-type  are calibrated to resemble (infections with) the SARS-CoV2 virus causing COVID-19. Despite its limitations, some of which I outline below, I derived several crucial insights from my model that are empirically testable:First, containment policies have an impact on the speed of the evolution of the virus. All containment policies that successfully curb the number of infections reduce the number of mutations and thus also the rate at which the virus increases its fitness. If cross-immunity is high, however, the ultimate fitness of a virus may be higher if it circulates in a population engaging in medium-level social distancing than in a population not engaging in social distancing at all due to a slower, but longer, evolution of the virus.Second, containment policies may also affect the direction of viral evolution. Namely, if symptomatic individuals are isolated, viral fitness increases with the incubation time and the share of asymptomatic infections. Those traits then assert themselves via the endogenous process of evolutionary selection, thus making “smart” isolation policies less effective over time.Third, it is often not possible to distinguish between an “endemic” scenario, in which variations of the virus persist by continuously evolving and escaping immunity and a non-endemic scenario by looking only at public health outcomes during the first wave of infections. What seems to be a successful “herd immunity” strategy may plant the seeds for a long-term presence of a potentially lethal virus. Thus, it is crucial to monitor the number and severity of reinfections already in the early phases of an epidemic in order to assess a virus’ potential to become endemic and optimally design containment policies accordingly.My model is limited in various ways: First, it assumes that each property of the virus and associated disease may be subject to the same process of stochastic change, which is an obvious stylization. Second, it assumes a lethality rate, and, more generally, all properties of the disease which are uniform across the population. Relaxing this assumption in order to account for e.g., an age-dependent severity of the disease, may influence the results by reducing mortality even in case of lower levels of cross-protection gained from a past infection (or vaccination). Third, my model does not consider behavioral heterogeneity within the population (Mellacher Further research on this topic could go in several directions: First, it could address the limitations of this model by including endogenous viral evolution into a larger economic-epidemiological agent-based model (e.g., Basurto et al. 2020; Delli Gatti and Reissl


Biological indexing is the method generally recognized for the certification of propagative grapevines in many countries, and it is mandatory in the European Union. It consists of the evaluation of the plant material after grafting on indicators that are inspected for symptom development. This is a lengthy process that requires well-trained workers, testing field, etc. Alternative diagnostic methods such as serology and RT-qPCR have been discarded for certification because of their intrinsic drawbacks. In turn, high-throughput sequencing (HTS) of plant RNA has been proposed as a plausible alternative to bioassay, but before it is accepted, different aspects of this process must be evaluated. We have compared the HTS of small RNAs with bioassays and other diagnostic methods from a set of 40 grapevine plants submitted for certification. The results allowed the authors the identification of numerous grapevine viruses in the samples, as well as different variants. Besides, relationships between symptom expression and viromes were investigated, in particular leafroll-associated viruses. We compared HTS results using analytical and bioinformatics approaches in order to define minimum acceptable quality standards for certification schemes, resulting in a pipeline proposal. Finally, the comparison between HTS and bioassay resulted favorable for the former in terms of reliability, cost, and timing. Closteroviridae such as the closterovirus grapevine leafroll-associated virus 2 (GLRaV-2) and by a complex of different species of the genus Ampelovirus  for plant transcriptomics, the detection and identification of plant viruses by this technology has become widely used : ArMV, GFLV, GFkV, GLRaV-1, GLRaV-2, GLRaV-3, GLRAV-4 strains 4–9, and GLRaV-4 strain 6, following the indications of the manufacturer.RNAs were extracted from approximately 100 mg of phloem scrapings of the canes at the stage of vine ripening with the Spectrum Plant RNA kit . The RNAs were quantified using NanoDrop ND-1000 . For the RT-qPCR, we used 10 ng of total RNA extract from each plant that was added to the AgPath-ID One-step RT-PCR master mix  supplemented with 200 nM of the forward and reverse primers and 200 nM of the TaqMan probe to a final volume of 25 ml. For the qPCR, we used specific primers for GFLV, GFkV, GLRaV-1, GLRaV-2, GLRaV-3, GLRaV-3, GLRaV-4, GLRaV-4 strains 5, and GLRaV-4 strain 9 . ReversesRNA-enriched samples were also obtained from 100 mg of phloem tissue using the miRCURY RNA isolation kit  following the recommendations of the manufacturer. The samples were quantified using NanoDrop, and the quality was assessed with Bioanalyzer 2100 using RNA 6000 Nano Kit . The sRNA fraction  from the RNA extractions was eluted from polyacrylamide gels, and the corresponding cDNA libraries were prepared. The libraries were constructed for each extract using the TruSeq Small RNA kit , and paired-end 2 × 50 pb RNAseq was performed on the Hiseq 2500 Illumina deep sequencing platform of CRG . The 40 samples were processed in three batches, one consisting of six samples separating them using sequence barcodes (Batch #1: 29085-30123), for which the sequencing depth in a single run was >200 M reads, another consisting of 10 samples (Batch #2: 31004-31099) for which a sequence depth in a run was >250 M reads, and a third batch of 24 samples (Batch #3: 3200-33121) that were processed in a run of >450 M reads.de novo assembly function of Velvet v. 1.2.08 ; and for BLASTX, the parameter was adjusted for optimization. In addition to the BLASTX and BLASTN analysis, both the set of contigs and the 18–24 nt reads from each sample were aligned to sequences of reference virus genomes using the Map to Reference tool with default parameters as available in Geneious to search for matches indicative of specific virus sequences.The sequencing Illumina RNA adapter was removed from the HTS raw sequences, and the low-quality reads were discarded using Geneious prime v. 2019.2.3 . For the subsequent analysis, reads ranging from 18 to 24 nt were selected. These reads were assembled into contigs by the . 1.2.08  implemenRNA extractions performed with the Exiqon kit from grapevine phloem scrapings rendered specifically low-weight RNAs and sRNAs . Most ofE-value of BLASTX was 1e-3 , GFLV, GFkV, grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine rupestris vein feathering virus (GRVFV), grapevine Red Globe virus (GRGV), grapevine Pinot Gris virus (GPGV), grapevine virus A (GVA), grapevine virus B (GVB), and grapevine virus L (GVL) . Among tSix groups of GLRaV-2 variants have been identified by the workers so far, arranged in six distinct lineages represented by the type isolates 93/955, PN, PV20, RG, H4, and BD . These vAvailability of sRNA reads and contigs allowed the quantitation of alignments to reference sequences for the viruses found in the samples , 2. CompBLAST analysis of the contigs allowed the identification of viruses not previously reported in Spain, besides the different variants of GLRaV-2 described here. Sample 33109  showed contigs matching to vitiviruses . When thTo investigate the quality of the HTS process and its reliability for virus detection in grapevine, we explored some other parameters in addition to quality of the sRNA extraction and the total number of HTS reads obtained. The consistent presence of two viroids and GRSPaV in the samples provided internal references. Hence, we investigated the number of vsiRNAs matching to their genomes. Statistical analysis showed significant correlation between the specific vsiRNA amounts of the three species in the samples . There aV. rupestris du Lot . Besides, a minimum number of 18–24 nt reads will be necessary (>5 M reads). In addition, we recommend that the ratio of 21 nt vs. 18–24 nt siRNAs in the sample should be >30%. If there are viroids or GRSPaV in the sample, as frequently happens in grapevine, an additional quality parameter could be the titers and profile of vsiRNAs aligning to the viroid/GRSPaV genomes . In the pipeline, contigs will be generated from the 18–24 nt reads using Velvet  and submitted to BLASTN  and BLASTX  against the virus databases of NCBI as available in Viruseq. If there are hits with some of the viruses included in the certification standards, the material may be reported as non-compliant. If not, the 18–24 nt reads are aligned with reference genomic sequences. Next, if significant reads appear , then the presence of regulated virus species will be reported, and the material will be classified as unsuitable for certification. Negative results from the alignments would mean healthy plant material. Each diagnostic procedure could be validated by including in the analysis a control plant that has been previously analyzed. Finally, this certification scheme, provided all the steps are carried out properly, would take about 6 months after receipt of the plant material.We devised a flux diagram for virus certification using HTS of sRNAs in grapevine . In thisIn the last years, performing RNA-sequencing analysis for determination and discovery of plant viruses in crop plants has become widespread . More spde novo assembly to generate contigs using SPAdes, Trinity, Velvet, or some other tool appropriate for sRNAs. The algorithm of choice should be carefully evaluated for each particular situation to select the best option. Multiple k-mer mode tools such as the one used in Oases, a derivative of Velvet, and SPAdes are considered good for contig assembly of sRNAs , in the case of HTS it will probably decrease in the coming years, while bioassay costs are likely to remain the same, or may increase considering inflation. Another advantage of HTS is the ability to detect additional viruses and viroids in samples, such as those that are not included in the regulations but that may have effects on yield and quality. Finally, HTS offers the possibility of achieving a widely accepted standard and replacing bioassays.We have analyzed 40 grapevine plants in three sets at different HTS depths and quality of RNA extractions for virus detection. Numerous grapevine viruses could be identified in the samples, such as non-regulated viruses. Leafroll viruses, even non-regulated ones, resulted capable of inducing symptoms in indicator hosts in most of the cases. The results have shown the suitability of HTS in comparison with bioassays and other diagnostic tools. As a result, we propose a pipeline using HTS for virus certification in grapevine with the aim of addressing several problems identified by workers and breeders: timely results, identification of key parameters in sRNA extractions and bioinformatics processing, a user-friendly platform for BLAST analysis, and, finally, the procedure is susceptible of validation using different controls. The HTS pipeline is intended to be operated by non-scientific experts, so that interpretation of results can be performed by a technician with minimal training.https://www.ncbi.nlm.nih.gov/genbank/, PRJNA715068; MW715828; MW715829; MW715830; MW715831; MW715832; MW715833; MW715834; MW715835; MW715836.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: LV and CP: conceptualization, methodology, investigation, writing—review and editing, and funding acquisition. LV: bioinformatic analysis and writing—original draft preparation. Both authors read and agreed to the published version of the manuscript.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.


Psorophora ferox mosquitoes. While ROCV has remained mostly undetected since 1976, in 2011 it re-emerged in Goiás State causing a limited outbreak. Control of ROCV outbreaks depends on sustainable vector control measures and public education. To date there is no specific treatment or licensed vaccine available. Here we provide an overview of the ecology, transmission cycles, epidemiology, pathogenesis, and treatment options, aiming to improve our ability to understand, predict, and ideally avert further ROCV emergence.Rocio virus (ROCV) is a mosquito-borne flavivirus and human pathogen. The virus is indigenous to Brazil and was first detected in 1975 in the Sao Paulo State, and over a period of two years was responsible for several epidemics of meningoencephalitis in coastal communities leading to over 100 deaths. The vast majority of ROCV infections are believed to be subclinical and clinical manifestations can range from uncomplicated fever to fatal meningoencephalitis. Birds are the natural reservoir and amplification hosts and ROCV is maintained in nature in a mosquito-bird-mosquito transmission cycle, primarily involving  Flavivirus (family Flaviviridae). As with other members of the genus Flavivirus, ROCV has a linear, positive-sense single-stranded RNA genome, approximately 11 kilobases in length encapsulated in virions of 43 nm in diameter [Zonotrichia capensis) [Psorofora ferox [Sporophila caerulescens) and creamy-bellied thrush ) [Bubalus bubalis) [Rocio virus (ROCV) is an arthropod-borne virus (arbovirus) in the genus diameter , during diameter ,3. Untildiameter . The firdiameter . Howeverdiameter . The firdiameter . Nine ROdiameter , sentinediameter , an Andeapensis)  a pool ora ferox , and parra ferox . Subsequalinus)) ,11, horsalinus)) , water bbubalis)  and humabubalis) ,15, suggbubalis) . Interesbubalis)  were ovebubalis) ). These Flavivirus; spherical particles of 43 nm in diameter having an electron dense core surrounded by a host-derived lipid bilayer membrane [To date Tanaka et al.  remains membrane . The posmembrane ,18. The Flavivirus begins with the binding to host cell receptors and entering the cell through clathrin-mediated endocytosis. Following trafficking through endosomal compartments, envelope protein-mediated fusion of viral and cellular membranes, and dis-assembly of the virus particles, single-stranded viral RNA is released into the cytoplasm where translation occurs. Translation of the viral RNA into a polyprotein occurs at the ribosomes, which is further processed by host and viral proteases to non-structural (NS) and structural proteins, forming the replication complex within the endoplasmic reticulum (ER) to replicate the viral RNA. Viral particle assembly occurs on the membrane of the ER and particles bud into the ER as immature virus particles [Details of the ROCV replication cycle are limited , but thearticles . During articles  and matuarticles )  species at an ecologic gradient, spanning sylvatic, transition and peri-domestic ecotypes  [Sporophila caerulescens and Turdus amaurochalinus) [Passer domesticus), a common bird throughout the Americas, demonstrated their susceptibility to ROCV infection, with viremia peaking at 4.3 log10/mL and duration of 2–3 days, suggesting its low potential for ROCV transmission [The virus was isolated from rufous-collared sparrows (apensis)  in Sete apensis) ,48 and tapensis) . An addiapensis)    . Collectively, the transmission cycles of ROCV are not well understood and humans, horses and other vertebrates may serve as incidental dead-end hosts.More recent serological studies have also demonstrated evidence of ROCV infection in equines  and watehttp://arquivo.ambiente.sp.gov.br/cpla/2018/05/proposta_zee_-valedoribeira_2014.pdf, accessed on 5 March 2021) and the rest is designated as a protected ecologic area (Atlantic rainforest). Most of the population (65.8%) lived in rural areas and 65.2% of the economically active population worked in rural activities . The study showed an increased prevalence to flavivirus-specific antibodies [The human epidemiology remains largely unknown, and the reasons of the virus periodic emergence remain a mystery. To date all the detected cases of ROCV in humans have been reported in three states , althougtibodies ,54 from tibodies . Criticatibodies ,56,57.Subsequent to the 1975–1977 outbreak, there was a precipitous decrease in the number of cases , with twCollectively, the data suggest an extended range of ROCV transmission in Brazil and reinforce the urgent need for improved diagnostic capabilities as well as sustainable arbovirus surveillance networks for the early and accurate detection of arboviral outbreaks.Clinical manifestations of infection include fever, malaise, severe headache, lower-extremity weakness, and severe neurologic disorders, with permanent sequelae observed in 20% of surviving patients ,9,15,61.Flavivirus, ROCV has been classified within the Japanese encephalitis virus (JEV) serogroup, whose members include St. Louis encephalitis virus (SLEV) and Ilhéus virus (ILHV). To date several rodent ) studies have been performed, seeking to elucidate ROCV pathogenesis [The pathogenesis of ROCV infection is not fully understood. Within the genus ogenesis ,64,65,66ogenesis , observaogenesis ,68,69. Vogenesis .wt) ROCV-infected animals, as well as significantly reduced inflammation and viral loads in the brains of the knockout mice, suggesting CCR5/MIP-1a mediates lymphocyte recruitment in the brain leading to disease severity [wt C57BL/6 mice they showed that ROCV infection induces the production of CCL2 in the blood and brain, resulting in the increased infiltration of macrophages and CD8+ T lymphocytes into the brain. Similarly, to the observations of de Barros et al [−/−Ccr2) C57BL/6 mice, demonstrated that CCR2 is required for efficient infiltration of macrophages into the brain, which is associated with a reduction in disease severity and mortality [ROCV-infected young adult BALB/c mice developed meningo-encephalomyelitis presenting with hind limb paralysis, muscle weakness, tremors, loss of balance and death 9 days post infection (d.p.i.) . Signifiseverity . Recentlseverity  investigos et al , virus ios et al . Conversortality .Collectively, the rodent models described above demonstrate the neurotropic  nature of ROCV infection, but they have not shed any light in the mechanism of neuro-invasion, reiterating the notion that ROCV pathogenesis remains poorly understood, and begging the urgent need of a suitable animal model mimicking human disease.The clinical presentation of ROCV infection is indistinguishable from the symptoms of other mosquito-borne flaviviruses, including neurotropic flaviviruses such as Japanese encephalitis, West Nile, and St Louis encephalitis viruses. As discussed earlier, ROCV infections have been often misdiagnosed with other arboviral infections endemic to Brazil, often as dengue or St. Louis encephalitis, therefore differential diagnosis of ROCV infection must be established in the laboratory. Diagnosis of infection with ROCV is possible with a variety of methods, including genetic  ,71 and sThere is no licensed vaccine or antiviral therapy available for ROCV infections, thus patient care protocols include stabilization and admission to the intensive care unit (ICU) for severe cases. Long term supportive care is recommended for survivors with long term sequalae. A three dose formalin inactivated vaccine developed in 1977 by Butantan elicited low rates of seroconversion in early phases of human trials and further development was discontinued . TherefoPsorophora. and Aedes species) and vertebrate and amplification hosts (Zonotrichia capensis) provide the required conditions for ROCV’s potential to emerge and become an urgent public health issue. Furthermore, this review reiterates the urgent need for the establishment of comprehensive surveillance networks that are geographically broad, encompass hotspots of biodiversity as well as human cohorts, and are well integrated with appropriate modeling techniques that can mitigate the threat posed by emerging zoonotic and resurging arboviruses. It is essential that sound public health policy to contain and control zoonotic pathogens must also invest in the development of more accurate diagnostic assays using new and affordable technologies and effective vaccine or antiviral countermeasures.ROCV is an emerging flavivirus responsible for the largest outbreak of arboviral encephalitis in Brazil. Despite several pioneering studies in the mid-1970s to late 1980s that provided some clarity on the epidemiology and clinical presentation of ROCV infections, its transmission cycles are not fully understood. Extensive serologic and ecologic surveillance studies on ROCV have not been carried out since then and the shortage and paucity of data has hindered our appreciation of the burden that ROCV infections may have on public health systems across Brazil and beyond. ROCV circulates in several regions of the country, infecting wildlife as well as animals important to agricultural and peri-domestic activities. As shown in the sections above, it is likely that the virus circulates undetected and most infections with mild symptoms are either underreported or attributed to other common arboviruses, such as dengue. The abundance of its mosquito vectors (


Plant viruses encounter a range of host defenses including non-host resistance (NHR), leading to the arrest of virus replication and movement in plants. Viruses have limited host ranges, and adaptation to a new host is an atypical phenomenon. The entire genotypes of plant species which are imperceptive to every single isolate of a genetically variable virus species are described as non-hosts. NHR is the non-specific resistance manifested by an innately immune non-host due to pre-existing and inducible defense responses, which cannot be evaded by yet-to-be adapted plant viruses. NHR-to-plant viruses are widespread, but the phenotypic variation is often not detectable within plant species. Therefore, molecular and genetic mechanisms of NHR need to be systematically studied to enable exploitation in crop protection. This article comprehensively describes the possible mechanisms of NHR against plant viruses. Also, the previous definition of NHR to plant viruses is insufficient, and the main aim of this article is to sensitize plant pathologists to the existence of NHR to plant viruses and to highlight the need for immediate and elaborate research in this area. The International Committee on Taxonomy of Viruses (ICTV) lists 1,000 different species of plant viruses, and among them, approximately 450 species are categorized as plant pathogenic viruses . Viral dViruses encode movement protein (MP), which aids in viral particle movement from cell to cell through the increased plasmodesmatal size exclusion limit . Host trGenerally, non-host plants do not allow the virus to replicate, which phenotypically fails to show any symptoms, systemic or local. In asymptomatic plant species inoculated with certain viruses, the progeny virus are not detectable, and such plants can be considered as non-hosts. The resistance owing to the early sensing of plant viruses, which completely restricts viral multiplication in the non-host plant, can be referred to as “true NHR”. However, recent evidence shows that some non-host plant species allow the multiplication of the virus till a minimal level in single or a few inoculated cells but further arrest the multiplication and cell-to-cell movement ; this caNon-host species mount efficient defense responses and possess diversified antiviral mechanisms that confer NHR to plant viruses. Through this brief review, we attempt to apprise plant scientists of the existence of NHR of plants to viruses and draw attention to the need to re-examine NHR to viruses from various angles. We also provide insights into NHR and “apparent NHR”, which can be exploited for providing broad-spectrum resistance. A clearer understanding of NHR will help in developing resistant crops and refining strategies and resources to achieve efficient control of plant viral diseases. Interestingly, this article delves into some critical and fundamental aspects that are often questioned by plant virologists. Except for R-gene resistance, virus transmission, insect vectors and others. Literature was retrieved and sorted based on the relevance of the topic. The literature was mined to extract data and information from relevant articles and was used to support the hypothesis behind the review article. Various citations from these articles were referenced to obtain elaborate information. Together, the compiled information was processed by the authors to write the manuscript. Relevant concepts were incorporated based on the author’s expertise in this field of research. The self and non-self recognition and topics under above key words were included. However, the literature related to animals in this regard were excluded.Several search engines including PubMed, Web of Science advanced search, Google scholar and specific journal web sites were used and search was performed based on key words specifically designed for the topic. The search keys included virus names, select plant species, non-host, virus resistance, virus co-evolution, RNA interference, silencing suppressors, effector and PAMP triggered immunity, host range, The presence of nucleic acid in a cellular compartment triggers immunity in mammals and plants. Therefore, the accumulation of non-self DNA and RNA cytosolic fragments indicates infection . In mammThe virus elicitors or PAMPs were recognized in a BRI1-associated kinase 1 (BAK1) manner, which believed to be the central regulator of pattern triggered immunity (PTI) . NHR agaRNA viruses interact strongly with the components of host translational machinery, such as host eIF-encoded genes . In bothViruses have limited host ranges, and adaptation to a new host is atypical phenomenon. Overcoming species barriers requires mutations in several viral-encoded proteins involved in cellular movement and replication. The adaptation of the virus to relatively new species is highly complex; therefore, a host jump by the virus is a very rare occurrence. Host jumps occur when pathogens encounter new hosts, followed by infection and successful multiplication in that host plant.Host jumps occur when pathogens encounter new hosts, followed by infection and successful multiplication in that host plant. Recently, a novel coronavirus (SARS-CoV-2) crossed species barriers  to infect humans and was efficiently transmitted among humans, leading to a global pandemic . TherefoMitovirus, a genus within the Narnaviridae.Virus transmitting insect vectors and the three-way interaction among virus-vector-plant also plays a role in virus host range. Determinants of host range have been identified in genomes of plant viruses in various studies . In spiti.e., virus replication and movement, is genetically controlled by host cellular factors (Plant viruses move long distances through vascular systems to infect tissues (roots and young leaves), and the entire infection cycle,  factors . Incompa factors . The ant factors    . In tobalication . These rSoybean mosaic virus-N (SMV-N) is capable of infecting cultivated soybean and wild soybean, whereas it seems to be undetectable in broad bean. In contrast, Clover yellow vein virus shows compatibility towards broad bean for systemic infection; whereas it is capable of infecting soybean locally  overcomes two layers of defense to move systemically in N. benthamiana non-hosts plants. Hence, the complete anti-BPMV defense in the non-host N. benthamiana consists of two layers of active defense: interference with RNA2 replication and RNA silencing  to infect N. tabacum, and of Potato virus Y to infect pea, could be due to inhibition of replication of viruses in mechanically inoculated cells (Cacao swollen shoot virus (CSSV) is able to complete its life cycle in the non-host plants belonging to Brassicaceae and Solanaceae families when CSSV viral genome is stably transformed into plant genome  and passive (lack of susceptibility factors) in nature . AntivirPotato virus X, which cannot infect Arabidopsis thaliana is capable of infecting Dicer-like mutants in Arabidopsis when co-infected with a Pepper ringspot virus. Moreover, Pepper ringspot virus has virus suppressor of RNA silencing (VSR), which contributes to the gained infectivity of PVX. This implies that RNA-mediated antiviral silencing is responsible for NHR against PVX (Tomato bushy stunt virus (TBSV) p19, has a crucial role in systemic invasion in the host. Moreover, Tobacco etch virus (TEV) helper component protease (HC-Pro) represses PTGS in tobacco but not in Arabidopsis, a non-host to TEV  proteins . For insinst PVX . A post-t to TEV .R genes grouped into two different classes of genes: The first class belong to the genes that encode proteins which are involved in regulation of different stages of virus life cycle. The other class belong to the genes encoding NLR (Nucleotide-Binding and Leucine-Rich Repeat domain) proteins, whichplays significant role in plant-viral defense  (Brome mosaic virus is due to Constitutive PR expression 5 (CPR5), a protein that represses R gene-mediated resistance   or non-a (ToLCV) . Also, Nsistance . The genethylase .Tomato chlorosis virus (ToCV)–an emerging virus that cause economic loss of tomato (Pea enation mosaic virus (PEMV), pea aphids (Acyrthosiphon pisum), and pea (Pisum sativum), wherein oxylipin signaling pathway was identified to be necessary for aphid attraction. Moreover, the CMV 2a RNA-directed RNA polymerase of Cucumber mosaic virus (CMV) seems to contain PAMP activity. The activated PTI may induce production of phytoalexins that negatively affect the host and insect vector interaction, which in turn affect CMV transmission (Tomato spotted wilt virus (TSWV) belonging to Bunyaviridae family changes the feeding behavior of thrips vector, Frankliniella occidentalis upon infection  and viral proteins . Non-hosSolanum habrochaites, a wild relative of tomato, and it cannot bind to TMV replication proteins or inhibit its multiplication (Cucumber mosaic virus 2b, and TuMV HC Pro proteins (e.g., SA-inducible RdRP from Nicotiana tabacum is required for defense against members of the Tobamovirus genus but not CMV or PVX (N. benthamiana provided protection against extreme weather conditions (The host range of viruses is a significant determinant of disease emergence and can also be used in designing strategies for disease control. A recent study unraveled the diversity and frequency of changes in non-host/host ranges over the evolutionary time scales of potyviruses and further determined the distribution of these changes among potyvirus and plant diversity . The stulication , 2009. Tlication . Severalproteins . MoreoveV or PVX . In anotnditions . Moreovenditions . The hosnditions . AdditioR gene mediated, and other mechanisms are part of NHR for plant viruses. The evolutionary interaction between virus, vector and plants prepares a plant to NHR or host jump. Understanding NHR against plant viruses and classifications are at the infancy stage and need much more systematic study. The apparent NHR looks promising for use in a resistance enhancement program, which can utilize recent genome editing tools like CRISPR on recessive host factors. We further need to understand the mechanistic diversity of antiviral defense in non-host plants and this article will help in building novel research strategies and resources for the utilization of NHR to achieve adequate control of viral infections.NHR against plant viruses differ from resistance to other plant pathogens. Lack of host factors, incompatible interaction of host and viral factors, restriction in movement and spread, RNAi silencing, to some extent


Poxviruses have evolved a variety of innate immunity evasion mechanisms, some of which involve poxvirus-encoded E3 ubiquitin ligases and adaptor proteins. Based on their functional domains and ubiquitin transfer mechanisms, these poxvirus-encoded E3 ubiquitin ligases and adaptor proteins can be divided into five categories: PRANC, ANK/BC, BBK, P28/RING, and MARCH proteins. Although the substrates of many poxvirus E3 ubiquitin ligases remain to be discovered, most of the identified substrates are components of the innate immune system. In this review, we discuss the current research progress on poxvirus-encoded E3 ubiquitin ligases and adaptor proteins to provide mechanistic insights into the interplay between these viruses and their hosts. Poxviridae are double-stranded DNA viruses that replicate in the cytoplasm of the host cell. After infecting the host, poxviruses usually cause local or systemic purulent skin damage. Poxviridae is divided into two subfamilies: Chordopoxvirinae and Entomopoxvirinae. The typical members of the Chordopoxvirinae subfamily are variola virus (VARV), cowpox virus (CPXV), monkeypox virus (MPXV), vaccinia virus (VACV), orf virus (ORFV), myxoma virus (MYXV), and ectromelia virus (ECTV) (Members of the family s (ECTV) .The host inhibits the replication and spread of the virus through both the innate and adaptive immune systems. Therefore, immune escape mechanisms are particularly important for the survival of the virus . Poxviruvia their Lys48 residues form the ubiquitin chain that triggers degradation by the 26S proteasome , ankyrin repeat (ANK)/Elongin B/Elongin C (BC), BTB/Kelch (BBK), P28/RING, and MARCH. Diagrams of the E3 ubiquitin ligases and adaptor proteins encoded by poxvirus are illustrated in Chordopoxvirinae, often functioning as a protein-protein interaction motif. Approximately 80% of ANK proteins have an F-box domain at the C-terminus, and this F-box domain is shorter than the typical F-box domain in the host cells (via the 26S proteasome (via the adaptor protein S-Phase Kinase Associated Protein 1 (SKP1)  was identified to trigger the ubiquitination and proteasome-mediated degradation of receptor interacting protein kinase 3 (RIPK3) and inhibit necroptosis .ORFV belongs to the genus Parapoxvirus and causes local skin infections in goats, sheep and humans . The fivECTV causes lethal mousepox in infected mice. The four proteins EVM002, EVM005, EVM154 and EVM165 encoded by ECTV all contain an F-box domain in the C-terminus that interacts with the host SCF complex . EVM005 VACV B18R is highly conserved in orthopoxviruses, containing ANK repeats and an F-box domain . B18R, tVACV C9 contains 6 ANK repeats and an F-box domain near the C-terminus, which antagonizes the antiviral state induced by type I interferons (IFNs) in the early stage of VACV replication . When VAvia leaky scanning translation from methionine 20 and is unable to block NF-κB activation  l BC box . Bioinfol BC box . Thus, iMCTV MC132 was identified to associate with NF-κB subunit p65 by unbiased affinity purification. MC132 targeted p65 for ubiquitin-dependent proteasomal degradation by recruiting p65 to a host Cullin-5 (Cul-5)/BC complex, leading to effective suppression of NF-κB activity and thus benefiting virus replication . PoxviruThe molecular composition and function of the Cul-3-dependent E3 ligase complex has been revealed. In this complex, the binding of BTB domain-containing proteins to their substrates is mediated by Cul-3. Poxvirus is the only virus family known to encode BBK proteins, indicating that poxvirus may interact with Cul-3 to regulate the host ubiquitination pathway.The BTB domains of the ECTV virus BBK proteins EVM150 and EVM167 bind to the N-terminal region of Cul-3 . In addiin vitro. The sheeppox virus BBK protein (SPPV)-019 is an important virulence factor. Calcium-independent cell adhesion is reduced in sheep infected with SPPV-019 knockout virus, indicating that SPPV-019 may regulate cell adhesion  and MYXV. P28 in ECTV, M143R in MYXV , P28 in During coevolution with their hosts, poxviruses have incorporated host cell genes into their genomes and adapted them to promote the viral life cycle. In recent years, with in-depth research on the mechanisms of host–virus interactions, research on the function of the E3 ubiquitin ligases encoded by poxviruses has made significant progress. Recently, many new proteins and protein substrates have been discovered using different approaches. For example, bioinformatics analysis and quantitative proteomics were applied to discover that ECTV protein 010 is a noncanonical ANK protein that binds to Cul-2 . In contHowever, the current understanding of poxvirus E3 ubiquitin ligases is limited. More E3 ligases encoded by poxviruses will be discovered, and many poxvirus E3 ubiquitin ligase substrates remain to be explored. The homologies between genes from different poxviruses and between poxvirus genes and human genes have aided the rational discovery of new poxvirus-encoded E3 ligases, which will facilitate future studies in this field.The known poxvirus E3 ubiquitin ligases involved in host immune invasion could be developed as potential inhibitors of the host immune system, providing new antiviral strategies. In view of the complex interactions between poxviruses and their hosts, the interplay between poxviruses and the ubiquitin system needs further exploration in the future. The identification of natural substrates of poxvirus E3 ubiquitin ligases will facilitate our understanding of host–virus interactions, and particularly the role of poxvirus E3 ubiquitin ligases in virus infection.LZ conceived the work. HC and YZ draft the manuscript. LZ modified the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by grants from National Natural Science Foundation of China [81871663 and 82072270], Academic promotion programme of Shandong First Medical University [2019LJ001], and Natural Science Foundation of Shandong Province [ZR2021QC095].The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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