After the nationwide, massive winter losses of honey bees in Korea during the winter of 2021, samplings were conducted from live honey bees in colonies and dead honey bees nearby colonies in the same bee-farms in six regions in Korea. Each sample was subjected to virome analysis using high-throughput sequencing technology. The number of viral reads was the lowest in the live honey bee group sample with 370,503 reads and the highest in the dead honey bee group sample with 42,659,622 reads. Viral contigs were matched with the viral genomes of the black queen cell virus, deformed wing virus, Israeli acute paralysis virus, and sacbrood virus, all of which have been previously reported in Korea. However, Apis rhabdovirus 5, bee macula-like virus, Varroa orthomyxovirus-1, Hubei partiti-like virus 34, Lake Sinai virus 2, 3, and 4, and the Ditton virus, were also discovered in this study, which are the first records in Korea. Plant viral sequences resembling those of Arabidopsis latent virus 1, and a novel viral sequence was also discovered. In the present study 55 complete viral genome sequences were identified. This study is the first virome analysis of domestic honey bees and provides the latest information on the diversity of honey bee viruses in Korea. Under current beekeeping conditions, tens of thousands of honey bees live together in a narrow cavity nest or hive; consequently, honey bees are highly susceptible to diseases . Of the Apis mellifera in 1913  technology have increased access to virome analysis and played a major role in the discovery of viruses. HTS technology has opened new horizons in genetic analysis by considerable reducing the extensive cost and time of the previously used Sanger sequencing technology . Even ifResearch on honey bee viruses using HTS has been actively conducted; as a result, many viral mutations and novel viruses have been discovered. Apis cerana from 2009 in Korea  proposed by ® NP80 .Honey bee samples were collected between March 31 and April 3, 2022, from Yeongwol in Gangwon-do, Yeongdong in Chungcheongbuk-do, Gunwi in Gyeongsangbuk-do, Geochang in Gyeongsangnam-do, and Hwasun and Gangjin in Jeollanam-do  was ensured using a 2100 Bioanalyzer , and libraries were constructed using TruSeq Stranded Total RNA with Ribo-Zero H/M/R Gold . After the libraries were created libraries, their quality was checked using the 2100 Bioanalyzer. Samples that passed QC were analyzed using an Illumina NovaSeq 6000 (Illumina).2.3Apis mellifera) was downloaded from the NCBI Genome database .Raw reads obtained through HTS technology were analyzed using the CLC Genomics workbench software . Unnecessary parts of raw reads of each sample were first trimmed through ‘Trim reads’. After that, the host genome of the honey bee  at 90 V under 100 VAC for 25 min, and a 100 bp DNA ladder  was used as the size marker. After electrophoresis, the gel was read using GelDoc Go .Based on the viral sequences found in the virome analysis, primer sets were designed using the NCBI Primer-BLAST tool  and the complete sequence information of each virus was registered in the NCBI GenBank. Phylogenetic trees were created using the maximum likelihood method , and 1002.6TCS analysis  was perf33.1From the virome analysis, the average number of reads in the sample after trimming was 61,428,161. In the case of viral reads, the average of group A was 1,402,119, and the largest read number among group A was identified in Gangjin . The average read number of group B was 11,478,776, and the sample from Hwasun had the highest number of reads  as a result of BLASTn. A tBLASTx of the RdRp region showed 71.99% identity with Vespa velutina associated partiti-like virus 2 ’.A contig found in Yeongdong of group B was 1,325 nt in length and showed 96% similarity to the uncultured virus (JF732915) belonging to the 3.3To confirm the analysis of the virome results from HTS, RT-PCR was performed using primers designed based on the viral genome sequences identified in this study. As a result of performing RT-PCR on viruses found in samples from each region, bands at the target size were confirmed in all samples, except for samples in which some reads were read low ’.From tBLASTx, four contigs showed the highest similarity with Arabidopsis latent virus 1 , belonging to the subfamily ectively Table S33.5Regarding the four viruses that have been previously reported in Korea , phylogenetic trees and pairwise comparison tables were constructed using CLC Genomics Workbench with each complete viral sequence reported to NCBI GenBank by October 2022 S13. The3.6Similarity comparisons were also conducted between the unreported seven virus species  and isolates reported abroad. ARV5 has been reported only in China thus far, and the sequences of Yeongdong and Yeongwol of group B (OP972869 and OP972870) confirmed in this study were 99.77% similar compared to each other and 97%-98% similarity with the isolate reported in Chinese  were also analyzed and compared to foreign isolates. For LSV2, Yeongdong of group A (OP972884) had the highest similarity with the China isolate  and Gangjin of group A (OP972922) had the highest similarity with the China Shanxi isolate  but both the Korean isolates showed 85.80% similarity when compared with each other S19. For3.7The TCS haplotype network and phylogenetic tree were verified by referring to the pairwise comparison table for the LSV group, which confirmed that LSV1, 2, 3, 4, and 8 were associated with each virus 4. The c4The health of honey bees is an important topic related to worldwide food production in a rapidly changing climate. Substantial threats from habitat losses, malnutrition, pesticides, pests and diseases and other environmental factors were reported associated with the colony collapse disorder . PesticiIn Korea, there has been little interest in novel viruses as only PCR-based investigations of seven major viruses have been conducted on honey bees. Therefore, starting with several honey bee viruses discovered in this study, it is necessary to investigate unrecorded viruses and novel viruses in Korea by including more regions in future studies.In the present study, 55 complete viral genome sequences were identified. Among them, there is a virus believed to be a plant virus. Four major viruses  have been detected for diagnosis in Korea. Among them, DWV and IAPV should be noted. Researchers have suggested that DWV would highly associated with the winter losses since this is mainly vectored by the parasitic varroa mites and indicating the varroa mite pressure . It has Drosophila suzukii. However, the previously reported DV did not have a complete ORF with a partial sequence, but the contig identified in this study could confirm the complete ORF.DV are first discovered in The LSV group  is the most frequently detected virus group in this study and have received considerable attention in recent studies . LSV3 waViruses identified by HTS based virome analysis were assayed using RT-PCR. However, no bands were identified using RT-PCR for DWV (Gangjin B) and BeeMLV (Hwasun B). It was impracticable to confirm the RT-PCR results because of the small number of reads corresponding to the target virus using virome analysis. In addition, all Gunwi A RNA samples were used for HTS analysis, and it was impossible to test for viruses identified in the analysis.The honey bees used in this study were collected from the live honey bee in colonies and dead honey bee nearby colonies from only six regions in spring. The sampling amount would not be sufficiently enough to visualize the distribution of honey bee viruses by region or period. In addition, since virus level varies according to the season , a studyThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/E-JK and CJ conceived and designed the study. MK performed the experiments. MK conducted the bioinformatics and data analysis. MK and E-JK wrote the manuscript. All authors contributed to manuscript revision and read and approved the submitted version.


Betacoronavirus from bank voles (Myodes glareolus) in Grimsö, Sweden, and this virus is designated as Grimso virus. Repeated detection over three years and an overall prevalence of 3.4% suggest that the virus commonly occurs in bank voles. Furthermore, phylogenetic analyses indicate that the Grimso virus belongs to a highly divergent Embecovirus lineage predominantly associated with bank voles. Given that bank voles are one of the most common rodent species in Sweden and Europe, our findings indicate that Grimso virus might be circulating widely in bank voles and further point out the importance of sentinel surveillance of coronaviruses in wild small mammalian animals, especially in wild rodents.The unprecedented pandemic COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with bats as original reservoirs, has once again highlighted the importance of exploring the interface of wildlife diseases and human health. In this study, we identified a novel  Coronaviruses (CoVs) have a high plasticity in host infection which produces a great diversity of CoVs and a complexity evolution. Zoonotic resources of human pathogenic CoVs are mostly from bats or rodents. Coronavirus (CoV) belongs to the Coronaviridae family in the Nidovirales order. They are the largest known RNA viruses with a genome size of 26–32 kb. CoVs have the ability of zoonotic transmission to humans or livestock, and they can cause respiratory or enteric diseases. There are four genera within the coronavirus family, the alpha-, beta-, gamma- and delta-coronaviruses. The genera alpha- and beta-coronaviruses are usually associated with human health of importance .Before the emergence of SARS-CoV, MERS-CoV and SARS-CoV2, four human CoVs HCoV-OC43, HCoV-HKU1, HCoV-NL63, and HCoV-229E, have been adapted to the human population, which were responsible for approximately 30% of endemic common colds in human [M. glareolus) is one of the most common rodent species in Europe and a known reservoir for several zoonotic pathogens, such as Puumala orthohantavirus and Francisella tularensis. Previous studies have detected various Alphacoronavirus and Betacoronavirus in bank voles in the UK, Poland, Germany, and France [Bank voles  between 2015 and 2017. All trapping and sampling were approved by the Animal Experiment Ethical Committee, Umeå (Reference: A13–14), and followed the Swedish Board of Agriculture regulations. Animal species were identified in the laboratory, and lung tissues were harvested and subsequently stored at −80 °C until further investigation, including molecular species identification as described in [https://dna.macrogen-europe.com/eng/ (accessed on 1 June 2021)).Total RNA was extracted using Qiagen RNeasy mini kit . Specific primers targeting the spike protein gene  were designed based on a virome investigation of Swedish bank voles previously. This primer set was used for screening coronaviruses in 266 bank voles captured from Grimsö between 2015 and 2017. In parallel, we also screened the same samples using a published pan-coronavirus RT-PCR [https://en.novogene.com/ (accessed on 1 May 2021)). The number of raw reads reached around 50 million pair-end reads of 150 base-pairs (bp). A data analysis pipeline was used to trim and assemble the reads from the sequencing results as described in [Two positive RNA samples extracted from lung tissues (Grimso215 and Grimso2306) were sent for RNA sequencing, at the Illumina NovaSeq 6000 sequencing platform, from Novogene Hong Kong (ribed in . We obtaribed in . Using thttps://www.uppmax.uu.se/ (accessed on 20 April 2022) (Project ID: SNIC 2019/8-68)).For the recombination and phylogenetic analyses, the reference rodent CoVs were downloaded from the NCBI RefSeq database, and multiple sequence alignment was obtained using MAFFT v7.490, which was refined using trimAl v.1.4.1 . PairwisTo analyze possible receptor usages, the sequences of spike protein and hemagglutinin esterase genes were examined by searching in the blastx with default settings.Initially, we screened 266 bank voles collected during 2015–2017 at the same site in Grimsö, Sweden  for coronaviruses using an in-house PCR method based on custom primers targeting the spike gene . We deteThereafter, we selected two samples collected in 2015 (Grimso215) and 2017 (Grimso2306) for RNA-sequencing to characterize the complete genome. We obtained a full-length coronavirus sequence from the bank vole sample Grimso215  and a near-complete coronavirus sequence from the bank vole sample Grimso2306 . The in-depth phylogeny of Grimso virus is demonstrated in Pairwise sequence identity of Grimso virus (strain Grimso215) with other rodent CoVs ranged from 55 to 79% at the nucleotide level and 44 to 67% at the amino acid level, confirming that the Grimso virus is divergent and genetically distinct from earlier described rodent CoVs .Alphacoronavirus and Betacoronavirus genera (Alphacoronavirus) (Embecovirus) and (2) a cluster of CoVs isolated from bank voles in Germany and France, along with strains Grimso215 and Grimso2306 from this study, which formed separate divergent lineage within the subgenus Embecovirus  gene from 101 CoV genome sequences. The results indicated that bank voles harbor at least three virus species across the s genera A,B. Specnavirus) A. In addnavirus) B: (1) a ecovirus B.We examined the spike protein, hemagglutinin esterase and possible receptor usages by searching in the blastx. It had only specific hits in the S2 region but not in the S1 or receptor-binding regions .Luchacovirus and Embecovirus from genera Alphacoronavirus and Betacoronavirus, respectively [Rodents are the primordial hosts of CoVs, and rodent CoVs constitute at least two subgenera, ectively ,15. In tThe Grimso virus is highly divergent and genetically distinct from earlier described rodent CoVs based on the RNA-sequencing results, which also explains that we failed to detect any CoVs by using a published pan-coronavirus RT-PCR. By using the specific primers targeted to the spike gene, we have discovered nine samples which were positive in the PCR screening, with a continuous positivity for three years, which are 2/48 in 2015, 1/61 in 2016, and 6/157 in 2017.We recovered two genome sequences from two strains of Grismo virus. The genomes of strains Grimso215 and Grimso2306 shared 95.6% sequence identity at the nucleotide level, with 1338 site differences. This divergence is notably higher than the expected differences based on a typical substitution rate for coronaviruses of 0.001 substitutions per site per year ,17, whicThe wild animals provide pools of divergent virus species for interspecies transmission. The studies on the discovery of animal CoVs have been especially important since the emergence of human coronaviruses. The hidden diversity of CoVs has been explored in at least 37 rodent species distributed in Asia and Europe ,19,20,21To understand the zoonotic risks of Grismo virus, we analyzed the protein domain of the spike protein and hemagglutinin esterase. Due to the high divergence and the lack of isolated live virus, we could not yet identify the host receptor usage for this novel rodent CoV. However, we cannot neglect the zoonotic potential of Grimso virus to livestock or humans. Together with mapping and continuous monitoring of the Grimso virus, further studies will aim to isolate the virus and assess the pathogenic profile.Embecovirus, named the Grimso virus, in Swedish bank voles. Our analyses suggest that multiple distinct viral strains co-circulate in this population, although further investigation will be necessary to fully understand the transmission ecology. Furthermore, we found that closely related coronaviruses are broadly distributed across Europe and exclusively associated with bank voles and other vole species, indicating that bank voles are likely natural reservoirs of the Grimso virus. While the potential threat posed by the virus to human and animal health is unknown, our findings underscore the importance of longitudinal surveillance of CoVs in wild rodents in advancing current knowledge on the ecology of CoVs in reservoir populations.We identified a highly divergent


Cations influenced the gel strength of the wet gel beads in the following order (least strong) Ca2+ < Zn2+ < Fe3+~Al3+ (most strong). The swelling degree of the CaPG beads after 24 h of incubation in the RPMI-1640 medium was 104%, whereas the ZnPG, FePG, and AlPG beads swelled by 76, 108, and 134%, respectively. The strength of the pectin gel decreased significantly after incubation in the RPMI-1640 medium for 24 h, regardless of the cross-linking cation, although the FePG beads remained the strongest. All the pectin beads adsorbed serum proteins to a low degree, however the serum protein adsorption by the ZnPG and FePG beads (1.46 ± 0.87 and 1.35 ± 0.19 µg/mm2) was more than the CaPG and AlPG beads (0.31 ± 0.36 and 0.44 ± 0.25 µg/mm2). All the pectin beads reduced the production of TNF-α and IL-10 by hPBMCs in response to LPS stimulation. The IL-1β response of cells to LPS was significantly reduced by the CaPG, ZnPG, and FePG beads, whereas the AlPG beads enhanced it twofold. The CaPG, FePG, and AlPG beads had no cytotoxicity. The viability of hPBMCs and human fibroblasts incubated with ZnPG beads was 5.3 and 7.2%, respectively. Thus, the use of different cross-linking cations changed the properties of the pectin gel, which is important for biocompatibility.The study aimed to compare the in vitro biocompatibility of pectin gels formed by different cross-linking cations. Hydrogel beads named CaPG, ZnPG, FePG, and AlPG were prepared from 4% solutions of apple pectin using ionotropic gelling with CaCl In the branched areas of pectin, side chains composed of arabinose and galactose residues, predominantly, link to rhamnose residues inserted in the galacturonic backbone  when the pectin gels were immersed in protein solutions during incubation.The adsorption kinetics were estimated as the relative protein concentration [CE. coli 055:B5 . In all adsorption experiments, pectin gel beads were inserted into glass vials and incubated at 37 °C with 100 rpm shaking. The amount of free LPS in the sample aliquots was analyzed after pre-defined time intervals of 1, 6, and 24 h of incubation. Upon incubation, samples were centrifuged 1000× g for 20 min at 4 °C , and the supernatant containing free LPS was collected. The concentration of LPS was measured using the kinetic chromogenic Limulus amoebocyte lysate test .The adsorption of LPS was investigated by immersing pectin gel beads in RPMI-1640 medium supplemented with 10% FBS and 10 μg/mL LPS Venous blood was collected from healthy volunteers in vacuum tubes  after obtaining written informed consent. The protocol was approved by the Ethical Committee of the Komi Science Centre of the Russian Academy of Sciences. Complement activation evaluation was performed by measuring the C3a levels using an enzyme-linked immunosorbent assay (ELISA) kit  as previously described .v/v) with PBS at room temperature, and 30 mL of the mixture was layered onto 15 mL of Ficoll-1077 in 50 mL tubes. After centrifugation at 500× g for 30 min at 20 °C without a break, buffy coats were collected, pooled, resuspended in PBS, and centrifuged at 500× g for 20 min at 20 °C without a break. The pellets were resuspended in 60 mL of PBS and again layered onto Ficoll-1077 as described previously. Buffy coats were aspirated, washed with ice-cold PBS, and centrifuged at 500× g for 8 min at 4 °C without a break. One half of the harvested PBMCs were cultured overnight in RPMI-1640 supplemented with 1% penicillin/streptomycin and 10 vol% heat-inactivated FBS in 12-well plates in a humidified atmosphere .Venous blood was collected from healthy volunteers in vacuum tubes  after obtaining written informed consent. Isolation of PBMCs from human blood was performed by density gradient centrifugation into Ficoll-1077. Briefly, 300 mL of freshly drawn blood was mixed at a ratio of 1:1 (6/mL) were added to the wells of a 12-well plate and 10 gel beads were added to the cell suspension. LPS was used for stimulation at a final concentration of 2 µg/mL. Cultivation was carried out in 1 mL of RPMI-1640 nutrient medium with 10% heat-inactivated FBS and 1% penicillin/streptomycin under standard conditions . Twenty-four hours later, the conditioned medium was removed, aliquoted, and stored at −70 °C. For the cytokine profile, IL-1β, IL-10, and TNF-α were determined using the BD Cytometric bead array (CBA) human soluble protein master Kit.Isolated hPBMCs (1 × 10The human cytokine kit BD Cytometric bead array (CBA) human soluble protein master Kit was used to measure the concentration of interleukin IL-1β, IL-10, and TNF-α in blood human plasma. This array kit provided a mixture of capture beads with distinct fluorescent intensities that were coated with capture antibodies specific for each cytokine. The tests were performed according to the manufacturer’s instructions. About 50 μL of assay beads, 50 μL of detection reagent, and 50 μL of the studied sample or standard were added consecutively to each sample tube and incubated for 3 h at room temperature in the dark. Next, the samples were washed with 1 mL of wash buffer and centrifuged. The supernatant was discarded. The pellet was resuspended in 300 μL of buffer and acquired in the flow cytometer. Samples were measured using a BD FACSCanto II flow cytometer and analyzed with the FCAP Array™ Software . Individual cytokine concentrations were indicated by their fluorescent intensities. Cytokine standards were diluted serially to construct calibration curves to determine the concentration of the analyte.5/mL, after which 10 gel beads were added to the cell suspension. Cultivation was carried out in 1 mL of Dulbecco’s Modified Eagle Medium (DMEM) with 10% FBS and 1% penicillin/streptomycin under standard conditions . A day later, the culture medium was removed, and adherent cells were separated using a tr-EDTA solution, after which the cells were washed three times in 10 mL of PBS and centrifuged for 5 min at 400× g.A cell culture of human fibroblasts  was introduced into the wells of a 12-well plate in the amount of 6–7 × 106 cells/mL. A total of 100 µL of the solution was transferred to a 5 mL culture tube and 5 µL of FITC Annexin V and 5 µL of propidium iodide were added. The cells were gently vortexed and incubated for 15 min at room temperature in the dark. A total of 400 µL of binding buffer was added to each tube and analyzed by flow cytometry within 1 h.An FITC Annexin V Apoptosis Detection Kit  was used to estimate the number of apoptotic cells. The procedure was carried out according to the manufacturer’s protocol. The cells washed in PBS were resuspended in binding buffer at a concentration of 1 × 10p ≤ 0.05 were considered statistically significant.The results were presented as the arithmetic mean ± standard deviation. One-way ANOVA with Tukey’s honest significance test was applied to determine statistically significant differences in gel characterization, swelling, protein adsorption, and complement activation. ANOVA for repeated measurements was used to determine statistically significant differences in experiments that involved human cell cultures. Values of 2+, Zn2+, Fe3+, and Al3+ ions. Trivalent cations (Fe3+ and Al3+) provided a stronger pectin gel formation than divalent cations (Ca2+ and Zn2+), probably because of their additional ionic bonding in the egg-box structure. The cations influenced the swelling degree of the dried gel beads in the RPMI-1640 medium in the following order: Zn2+ < Ca2+ = Fe3+ < Al3+. The small size and high density of the dry ZnPG beads may explain their lowest swelling, while the greatest swelling of the AlPG beads may be because of the smallest ionic radii of Al3+ ions. The type of cross-linking cation was found to influence the in vitro biocompatibility of the pectin gel beads. All the pectin beads adsorbed serum proteins to a low extent and reduced TNF-α and IL-10 production by hPBMCs in response to LPS stimulation. A feature of the biocompatibility of the ZnPG and FePG beads was their serum protein adsorption, which was four times higher than that of the CaPG and AlPG beads. The main finding regarding the ZnPG beads was their extremely high cytotoxicity towards both hPBMCs and fibroblasts, which may limit the use of Zn2+-cross-linked pectin gels for engineered cell delivery. The main finding regarding the AlPG beads was their stimulating effect on IL-1β production by hPBMCs, which was consistent with the known use of aluminum salts as immunoadjuvants. For the first time, ionotropic pectin gel was found to increase the number of apoptotic fibroblasts, where the effect of the Al3+-cross-linked gel was stronger than that of the Ca2+-, Zn2+-, and Fe3+-cross-linked gels.This study revealed the differences in the mechanical properties and swelling behaviors of pectin gel beads cross-linked with CaThus, the use of different cross-linking cations changed the properties of the pectin gels, which are important for biocompatibility. Variation in cross-linking cations, besides the variation in the polysaccharide’s structure, is proposed to modify the interaction of pectin-based biomaterials with biological tissues.


Peste des petits ruminants (PPR) is a highly contagious disease of small ruminants. The causal agent, PPR virus (PPRV), is classified into four genetically distinct lineages. Lineage IV, originally from Asia, has shown a unique capacity to spread across Asia, the Middle East and Africa. Recent studies have reported its presence in two West African countries: Nigeria and Niger. Animals are frequently exchanged between Mali and Niger, which could allow the virus to enter and progress in Mali and to other West African countries. Here, PPRV samples were collected from sick goats between 2014 and 2017 in both Mali and in Senegal, on the border with Mali. Partial PPRV nucleoprotein gene was sequenced to identify the genetic lineage of the strains. Our results showed that lineage IV was present in south‐eastern Mali in 2017. This is currently the furthest West the lineage has been detected in West Africa. Surprisingly, we identified the persistence at least until 2014 of the supposedly extinct lineage I in two regions of Mali, Segou and Sikasso. Most PPRV sequences obtained in this study belonged to lineage II, which is dominant in West Africa. Phylogenetic analyses showed a close relationship between sequences obtained at the border between Senegal and Mali, supporting the hypothesis of an important movement of the virus between the two countries. Understanding the movement of animals between these countries, where the livestock trade is not fully controlled, is very important in the design of efficient control strategies to combat this devastating disease. It is one of the most economically important animal diseases in areas that rely on small ruminants for their livelihoods. The main clinical signs of PPR are the rapid rise in body temperature, the appearance of nasal and ocular secretions which become mucopurulent, necrotizing stomatitis, profuse, haemorrhagic diarrhoea in cases of parasitism and death. Signs of pneumonia , abortions that can be observed among pregnant animals, are important clinical aspects of PPR; because of its high contagiousness, high morbidity and mortality, PPR is a World organization for Animal Health (OIE) notifiable disease and since 2015 targeted for global eradication by 2030  based on N and F genes partial sequences  of PPR virus strains identified in most of the administrative regions of Mali between 2014 and 2017. Mali is a crossroads for the passage of small ruminants from West African border countries. Recurrently, there are many exchanges of animals between Mali and Niger, which could allow the virus to enter and let lineage IV continue to progress across Mali. From Mali, it could spread to other West African countries. We explored this hypothesis by carrying out sampling at major crossing points at the border between Mali and Senegal, a major endpoint for livestock in West Africa.22.1Samples were collected between 2014 and 2017 from domestic small ruminants (goat) showing clinical signs suggesting PPR infection by Mali's Central Veterinary Laboratory (LCV). These were collected in different towns in the regions of Kayes, Segou, Sikasso and Mopti  (Invitrogen) and 2 mm glass beads. The swabs were processed in 1 ml of MEM and vortexed. All the sample suspensions were then centrifuged for 3 min at 1,000 g and the supernatant collected. RNA extraction was carried out using the NucleoSpin RNA virus extraction kit (Macherey‐Nagel) and according to the instructions of the manufacturer. A 351 base pair (bp) fragment of the PPRV N gene was amplified by RT‐PCR carried out using the qScript XLT One‐Step RT‐PCR kit  using the protocol described Tounkara et al., .2.2Positive PCR products were cleaned up and sequenced in both forward and reverse directions by the Genewiz sequencing platform (United Kingdom) and the sequences submitted to GenBank. Sequence assemblage and trimming were performed using Geneious v. 8.1.6 . Clean sequences were aligned using MEGA 7 with 53 other sequences available in GenBank, including representatives of the four genetic lineages identified in Africa   was placed within lineage IV Figure . The seqMost samples obtained belonged to lineage II, the most abundant lineage in the region (Souley et al., The great diversity of strains found in Mali provides further support for the extensive circulation of the virus in the region (Tounkara et al., In conclusion, our study suggests that three different PPRV lineages are currently co‐circulating in Mali. Despite the dominance of lineages II and IV, lineage I may continue to circulate in isolated regions. We detected the presence of PPRV lineage IV in Mali, and it is possible that this lineage is now spreading across the country and in other West African countries through animal trade and transhumance. Understanding the animal movements in the African States belonging to ECOWAS where free movements for the livestock trade are allowed through regional regulations is very important for the PPR eradication programme. This would improve our capacity to design efficient control strategies against this devastating disease.The authors declare that there is no conflict of interest.App S1Click here for additional data file.


Correspondingly, the same hybridization inducessubstantial variations in the ability of the Co/C60 interfaceto support spin-polarized currents and can bring out a spin-filteringeffect. The knowledge of the effects at both sides allows for a betterand more complete understanding of interfacial physics. In this paperwe investigate the Co/C60 bilayer in the role of a spin-polarizedelectrode in the La0.7Sr0.3MnO3/SrTiO3/C60/Co configuration, thus substituting the bareCo electrode in the well-known La0.7Sr0.3MnO3/SrTiO3/Co magnetic tunnel junction. The studyrevealed that the spin polarization (SP) of the tunneling currentsescaping from the Co/C60 electrode is generally negative:i.e., inverted with respect to the expected SP of the Co electrode.The observed sign of the spin polarization was confirmed via DFT calculationsby considering the hybridization between cobalt and molecular orbitals.Orbital hybridization at the Co/C Orbital hybridizationat the interface between a C60 overlayer and an epitaxialultrathin film of Co(0001) leads to a considerable change in the magneticanisotropy of the Co,6 able to induce amagnetization reorientation transition from in-plane to out-of-planein the ferromagnetic layer and a magnetic hardening.7 Correspondingly, the nonmagnetic C60 moleculeis also modified,8 becoming spin activeby acquiring a net magnetic moment as a consequence of coupling ofthe carbon atoms closest to the underlying Co.The formation of a hybridized layer atthe interface between ferromagneticmetals and organic semiconductors has been proven to be efficientin the modulation of the magnetic and spin properties of both components.60 interfaceas a fascinating element for the modulation of the spin functionalityin solid-state devices: the magnetic hardening of a FM layer, obtainedby interfacing it with an organic molecule, is of technological interestin spintronic memories9 and also the inducedspin selectivity at the interface is fundamental for the spin injection.10 In this regards, spin transport has been provedto depend on the coupling of C60 molecules on the magneticsurface in case of fcc-Co(111)/C6011 and Cr(001)/C60.12These unusualfeatures liven up the Co/C60 interface can be integratedin the prototypical magnetic tunnel junction (MTJ) La0.7Sr0.3MnO3/SrTiO3/Co (LSMO/STO/Co),13 resulting in a change of the spin tunnelingcurrent of the device. We address this issue by inserting an ultrathinC60 molecular layer at the interface with the ferromagneticCo layer acting as a spin-polarized electrode and by comparing themagnetotransport properties of LSMO/STO/C60/Co MTJ withthose of the reference LSMO/STO/Co MTJ. Given that spin-polarizedtunneling in an MTJ depends on the band structure of the insulatingbarrier, on the properties of ferromagnetic layers, and even morecritically on those of their interfaces, an investigation of TMR valuesand sign together with a calculation of spin conductance in an MTJthus provides a direct method to test the spin polarization at theC60/Co interface, allowing unlocking of potential applicationsof the Co/C60 interface in solid-state spintronic devices.Herewe prove that Co/C60/Co were obtained by a shadow-masking technique on single-crystalNdGaO3 (NGO) (110) substrates. An LSMO layer that was 15nm thick was deposited by a channel spark ablation method followingthe procedure described by Graziosi et al.14 The STO tunneling barrier (5 nm) was grown with the same CSA techniqueunder an O2 atmosphere (10–2 mbar), keepingthe substrate at 700 °C. The Co top contact was obtained by e-gunevaporation under UHV (P < 10–9 mbar) at room temperature. The 2 nm thick C60 layer wasdeposited on STO by thermal evaporation with a ultralow flux MBKomponentecell with a growth rate of 0.15 Å/s. The Co top contact was obtainedby e-gun evaporation under UHV (P < 10–9 mbar) at room temperature. C60 was not damaged by thefabrication of the top Co electrode.15 Metalliccontacts were provided by gold pads evaporated on the LSMO and theCo electrodes. Junctions were 500 μm × 500 μm insize. The overall structures are depicted in Cross-bar LSMO/STO/Co and LSMO/STO/CTransmission electron microscopy (TEM) was employedfor structuralcharacterization, using a probe-corrected JEOL ARM200cF instrumentthat was operated at 200 kV and was equipped with a cold field emissionelectron gun and a Gatan Quantum electron energy loss spectrometer.Cross-sectional samples were prepared using standard “lift-out”procedures on an FEI Nova Nanolab Focused Ion Beam instrument.60 films was investigated by usingan AFM Smena microscope  in noncontact mode(NCM) under ambient conditions. Silicon cantilevers were employed.The topography of C0H = 0.9 T. The LSMO electrode was biased, while the Co was grounded.Spin transport measurements were performed using an exchange gascryostat, equipped with an electromagnet. A van der Pauw configurationwas adopted to minimize the contributions from the electrode resistances.The TMR was measured by sweeping the magnetic field in the plane ofthe device, while applying various constant biases through the junctionby using a Keithley 2400 SMU in the temperature range 100–300K, with a maximum applied field of μab initio calculations using the planewave electronic structure package Quantum ESPRESSO16 in the framework of density functional theory (DFT). Weused Perdew–Burke–Ernzerhof parametrization (PBE) forexchange-correlation functionals and the ultrasoft pseudopotentialformalism. Energy cutoffs of 30 and 300 Ry were employed for the wavefunctions and the charge density, respectively. The C60/ferromagnetic interface was simulated using a seven-layer slab ofhcp-Co (0001) and a 4 × 4 in-plane periodicity. The full systemwas first relaxed, fixing four Co bottom layers at their bulk positionsand using a 2 × 2 k-point mesh; then the electronicproperties of the relaxed structure were studied using a finer 6 ×6 mesh of k-points. The same computational parametersas in ref (We performedspin-polarized s in ref  wereuseA high-resolution transmissionelectron microscopy (HR-TEM) cross-sectionimage of LSMO (15 nm)/STO (5 nm)/Co (50 nm) confirms the excellentmorphology of the MTJ. 60 layer is obtainedby depositingthe organic layer onto the LSMO/STO. The STO tunnel barrier exhibitsa smooth surface . Afterthe growth of 2 nm C60, the molecules form clusters distributedon the STO surface with an overall roughness of about 1 molecularlayer (RMS = 0.7 ± 0.1 nm), as shown in section SI1) indicate a quite uniformcoverage of the surface with an estimated coverage of 98%.The insertion of a C60 is unavoidable, given the RMSof the C60 layer, but several works pointed out that thequite compact nature of the C60 molecule prevents the diffusionof the Co ion into the molecule.18 Moreover,during the Co growth, the C60 molecule maintains its integrity19 and molecular clusters tend to be incapsulatedbeneath the Co film. It is worth noting that, even in case of submonolayerdeposition,20 a C60 layer hasbeen used as buffer layer in organic light-emitting diodes to efficientlyimprove the qualities of the interface with metals by changing theinterfacial work functions and the energy level alignment.21Subsequently, a Co deposition was performed. Thepartial intermixedlayer between Co and C60 as decouplinglayer between Coand STO was addressed, we addressed transport data measured in thecurrent-perpendicular-to-plane (CPP) geometry. Conduction across theLSMO/STO/Co heterostructure exhibits the typical features expectedfor a tunneling conduction process (see section SI2), ruling out any possible Ohmic path. The magnetoresistanceresponse at 100 K measured under a bias of V = −0.1V corresponds to a typical MTJ butterfly curve  so that TMR = 25 that turn out to beof fundamental importance in the description of tunneling phenomenaincluded in the LSMO/STO/Co MTJ.13 Consideringthat the densities of states for Co and LSMO are both positive,22 the inverse TMR at negative bias has been interpretedin terms of interfacial hybridization between Co atoms and the STObarrier, producing a change in the Co tunneling spin polarization.The hybridization at the Co/STO interface due to the formation ofcovalent bonding of Co and O atoms induces a magnetic moment on theinterfacial Ti atoms which are aligned antiparallel to the magneticmoment of the Co layer.26 This leads toa negative spin polarization of tunneling across the STO barrier fromthe Co electrode, inverting the spin polarization at the LSMO/STOinterface and hence the TMR signal.Once the role of Cly curve 4a. The m60 layeracting as adecoupling layer between Co and STO does not change the MR sign, asthe hybrid LSMO/STO/C60/Co MTJ still has a negative TMR, in agreement with most studies performed on other MTJswith LSMO and Co electrodes,28 which couldbe a result of either a decrease in the spin polarization of LSMOat the interface with STO29 and/or spin-independenttunneling through impurity levels in the barrier activated upon increasingthe temperature.30The TMR effect in both cases decreases with increasing temperatureand disappears at nearly room temperature (Vb = −0.1 V) and the TMR featuresa crossover from negative to positive magnetoresistance above the Vb = +0.2 V threshold value, as we observed in 31 This peculiar bias dependence of the referenceMTJ has been ascribed to the structure of the DOS of the d band ofCo as described by De Teresa et al.22 andto the contribution of nonresonant tunneling events trough specificdefect states induced by the O vacancies in the barrier.24In addition tothe sign, interfaces were found to be critical forthe definition of the bias dependence of TMR. In the reference LSMO/STO/CoMTJ, the maximum magnetoresistance value is at a negative bias voltage at the metal–ferromagnetic interface.This finding is in agreement with the experimental observation ofa higher TMR signal for a C60-based MTJ. In order to betterunderstand the transport properties of the C60/Co interfaceand to make a better connection to the experiment, we compute thespin-resolved conductance  dE/U. This simplified approachallowed is to make tractable our complex problem of spin-polarizedtransport across the full LSMO/STO/C60/Co junction, assumingthat all spin dependence comes from the Co/C60 interface.Two main assumptions were therefore made: (i) the DOS of LSMO wassupposed to be constant in energy ; (ii) similarly, the tunneling rate of all electronicstates across the STO barrier is assumed to be the same and energy-independent.These assumptions, expected to work well at a small bias, can be lessjustified farther from the Fermi energy , whichmay explain a worse agreement between experimental and theoreticalresults for increasing bias. Considering the LSMO/STO as a perfectspin-up injector,22 the TMR of the full junctionshould depend on the ratio of spin up/down conductances, as representedin U >4V). Nevertheless, this simulation does not reproduce the positionof the TMR maximum, which has been experimentally found at V = −0.2 V while calculations place it at positivebias. These discrepancies can be ascribed to the approximations madein our calculations. Indeed, the employed LSMO/STO and Co/C60 band structures were based on ideal interfaces without defects anddisorder, preventing a precise quantitative comparison with the experimentaldata collected in polycrystalline samples. Also, considering the absenceof TMR inversion and the TMR intensity, the decoupling of STO andCo by the insertion of C60 should limit the role of O vacanciesin STO in the tunneling process. This could prevent the scatteringand the loss of parallel angular momentum conservation23 also observed in a defective amorphous STO barrier33 and be plausibly responsible for the increaseof TMR signal in our devices.To better clarifythe role of Co/Cdin ref . Figure .Notably, the differences between the shapes of calculated and measuredvoltage dependences of SP are expected to be at least partially causedby the polycrystalline and defective nature of the investigated interfaces,but the presence of more complex and still unknown interfacial effectscannot be ruled out.In this work, we have shown that the insertionof an ultrathinlayer of COur results demonstrate that hybrid ferromagnetic/molecularinterfacesoffer versatile routes for tuning of the TMR strength and sign inMTJs, enhancing the choice of spintronic device solutions for logicand memory applications.


The pilot catalytic systems we useare perovskites having the general structure ABO3 withlanthanides or group II elements at the A sites and transition metalsat the B sites. Depending on the chemical substitutions of ABO3, their catalytic activity for OER can be tuned by varyingthe composition.Water splitting is intensively studied for sustainableand effectiveenergy storage in green/alternative energy harvesting–storage–releasecycles. In this work, we present our recent developments for combiningliquid jet microtechnology with different types of soft X-ray spectroscopyat high-flux X-ray sources, in particular developed for studying theoxygen evolution reaction (OER). We are particularly interested inthe development of in situ RIXS studiesof the manganese L-edge of perovskites during OER. We have developedvarious X-ray spectroscopy approaches like transmission zone plate-,reflection zone plate-, and grating-based emission spectroscopy techniques.Combined with tunable incident X-ray energies, we yield complementaryinformation about changing (inverse) X-ray absorption features ofthe perovskites, allowing us to deduce element- and oxidation-state-specificchemical monitoring of the catalyst. Adding liquid jet technology,we monitor element- and oxidation-state-specific interactions of thecatalyst with water adsorbate during OER. By comparing the differenttechnical spectroscopy approaches combined with high-repetition-frequencyexperiments at synchrotrons and free-electron lasers, we concludethat the combination of liquid jet with low-resolution zone-plate-basedX-ray spectroscopy is sufficient for element- and oxidation-state-specificchemical monitoring during OER and easy to handle.In this work, we present our For an in-depthstudy of OER mechanisms, however, including thecharacterization of catalyst–water adsorbate in terms of theircharge transfer properties and especially valence intermediates formedduring OER, high-resolution spectroscopy tools based on a combinationof liquid jets with gratings bear bigger potential since they allowresolution of otherwise-overlapping X-ray spectroscopy transitions.Common for all of these experimental approaches is the conclusionthat without the versatile developments of liquid jets and liquidbeam technologies, elaborate experiments such as high-repetition experimentsat high-flux X-ray sources (like synchrotrons or free-electron lasers)would hardly be possible. Such experiments allow sample refreshmentfor every single X-ray shot for repetition frequencies of up to 5MHz, so that it is possible (a) to study X-ray-radiation-sensitivesamples and also (b) to utilize novel types of flux-hungry X-ray spectroscopytools like photon-in/photon-out X-ray spectroscopy to study the OER. HallmannJ.; GrübelS.; RajkovicI.; QuevedoW.; BusseG.; ScholzM.; MoreR.; PetriM.; TechertS.First steps toward probing chemical reaction dynamicswith free-electron laser radiationradiation—case studies atthe FLASH facility. J. Phys. B: At., Mol.Opt. Phys.2010, 43, 194009.1Thisexperimental research paper invents the liquid jet technology forfree-electron lasers and demonstrates that the liquid jet can be usedin X-ray scattering but also in X-ray spectroscopy setups .BusseP.; YinZ.; MierwaldtD.; ScholzJ.; KressdorfB.; GlaserL.; MiedemaP.S.; RothkirchA.; ViefhausJ.; JoossC.; TechertS.; RischM.0.6Sr0.4MnO3 in water vapor by in situ photon-in/photon-outspectroscopyProbing the surface of La. J. Phys. Chem. C2020, 124, 7893–7902.2Thisexperimental research paper introduces resonant inelastic X-ray scatteringat synchrotron sources for surface studies of the electronic propertiesof complex catalysts. The studies concentrate on perovkites without/withwater vapor and confirm former X-ray absorption studies.RaabeS.; MierwaldtD.; CistonJ.; UijttewaalM.; SteinH.; HoffmanJ.; ZhuY.; BlöchlP.; JoossC.In situ electrochemical electron microscopystudy of oxygen evolution activity of doped manganite perovskites. Adv. Funct. Mater.2012, 22, 3378–3388.3In this work, in situ electronmicroscopy is introduced for studying perovskite catalysts for theoxygen evolution reaction. Complementary to in situ X-ray spectroscopy,local information about the catalytic activities of the perovskitesurface under in situ conditions can be derived.13 In the field of high-flux X-ray sources, for both synchrotrons andfree-electron lasers,14 this means developingnew measurement techniques and methods that provide an alternate andadditional view on HER or OER alongside existing measurement techniques,leading to improvements in electrochemically and photoelectrochemicallyactive catalysts and their mode of action.One of the greatestchallenges of our current time lies in thesystematic research and characterization of chemical reactions thatallow the storage and output of energy in a highly efficient mannerwhile conserving natural resources. In the field of the productionof so-called “green hydrogen”, the hydrogen evolutionreaction (HER) as well as the oxygen evolution reaction (OER), i.e.,the production of hydrogen and oxygen by water splitting and its backreaction, belong to the most important and currently most studiedchemical reactions of water utilization for energy storage and production.in situ(operando.19 The studies concentrate on detecting the interacting X-rays in absorptionmode either by direct transmission absorption experiments or by inverseabsorption techniques derived from emission spectra (as explainedin the following paragraph). All in all, the X-ray spectroscopiesallow monitoring of element-specific oxidation state changes duringOER. Insight into hole and electron generation in relation to theband gap properties of the catalytic materials can also be obtained.In soft X-ray spectroscopy,various groundbreaking techniques havebeen developed for studying OER, focusing on X-ray absorption techniques in situ,16 and o1 The development of flux-hungry X-ray spectroscopytechniques to high resolution (12 is a photon-in/photon-out spectroscopy techniquewhere the outgoing energy of photons scattered by the sample is detectedas a function of the incident X-ray energy. 0.6Sr0.4MnO3 (LSMO), i.e., the manganese L-edge transition features, sincewe expect their oxidation state changes during the OER. The manganeseatoms are highlighted as violet points in the LSMO crystal structureshown in in situ (or even operando) X-ray methodsallows changes of the population of the electronic states to be deducedin a dynamic way. Taking into account that RIXS belongs to the corehole clock spectroscopies (With the presented resonant inelastic X-ray scattering (RIXS) experimentswe want to add another precision step by determining intermediatespecies between the catalyst and the water on the surface of the catalyst.To follow the line of already developed techniques, over the lastyears we have paid particular attention to the development of time-dependentX-ray methods such as time-resolved and ultrafast X-ray diffractionor time-resolved multidimensional soft X-ray spectroscopy for studyingchemical reactions beyond X-ray absorption spectroscopy.solution 1A has beoscopies 1B, it isBy tuning the incident X-rayenergy and collecting the X-ray emissionintegrally, or—in other words—inverting XES by monitoringparticular X-ray emission spectroscopy (XES) transitions and tuningthe incident X-ray energy, it is possible to collect the partial  fluorescence yield (PFY) and total fluorescenceyield (TFY) as functions of the incident energy. The spectra collectedare comparable to the ones obtained from X-ray absorption spectroscopyin transmission mode.1 e.g.,when the PFY of the 3s to 3d transition is monitored.2 It is then possible to study specific selected sheets oflayered compositions. An example would be layers on target materials,where the spectroscopic properties of the target materials are outof the monitoring window and thus do not contribute to the backgroundof the collected signals. Furthermore, liquid jets can be added (asanother layer), and complex reactions like the OER can be investigated.This finally leads to the possibility of utilizing the full advantagesof high-flux X-ray sources , allowing for the development ofhigh-resolution and multidimensional X-ray spectroscopy tools likeXES or RIXS.27 This can enable more detailed investigations of the mechanisms drivingthe OER. Its technical realization is schematically drawn in 23 In order to reduce background noise in the X-ray spectra, the wholeexperimental chamber is set to 10–3 mbar vacuum,which is a challenge for running the liquid jet smoothly. Radiationdamage when monitoring chemical reactions with soft X-ray photonsis an additional challenge. To avoid or minimize it, we have developeda vacuum-compatible liquid jet technology.However, since the X-ray photons are collectedin fluorescenceand scattering mode and the X-ray penetration depth sets the monitoredsample thickness 1A, the s23 ChemRIXS has been operated atPETRA-III, FLASH-I, and FLASH-II. The liquid jet is used as a megahertzsample exchange unit in the high-resolution Heisenberg RIXS apparatusat the SCS beamline of the European XFEL.24 The liquid jet technology can avoid typical soft X-ray radiationdamage processes that allow otherwise atypical reaction pathways tooccur in electrochemistry, which is especially important when studyingthe reactions of water splitting, namely, HER and OER.Since the liquidjet technology 1D runs uin situ and operando studies  in materials, thus contributing to the “film of chemicalreactions” from an electron energy, electron orbital, and electrondistribution point of view for molecular and homogeneous catalyticsystems.Due tothe high-repetition-frequency performance of the liquidmicrojet and its compatibility with high-flux X-ray sources, we developedthe ChemRIXS chamber for time-resolved  studies 2.2,20−23quid jet 2A combinquid jet 2B. For pntiostat 2C,D.2,2029 Our time-resolved in situ and operando XES/RIXS studies can therefore beeither bulk- or surface-sensitive, as shown in Due to the flexible geometricdesign of the various components,it is possible to run the RIXS spectrometers of ChemRIXS in transmissionor reflection geometry or in the grazing incidence (GI) geometry asGIRIXS.34 The exchangeable soft X-ray spectrometer units are equipped witha transmission zone plate (TZP), a reflection zone plate (RZP), anda classical grating spectrometer (The X-ray beamhits the sample (solid/liquid beam).The interaction with the X-rays increases the electronenergy in the material/sample under investigation in an element-specificmanner.The sample emits photons of specificenergy.The emitted photons hit an opticalgrating and are separatedaccording to their energies.Photonsseparated according to energy hit the detector.A spectrum of energies can be measured via the distributionof photons on the detector.Depending on the information aboutthe OER which we want to gather,different X-ray spectrometer types can be combined with liquid microjetsin the ChemRIXS chamber.trometer 3. CommonIn ChemRIXS the liquid jet can be combined with a conventionalX-ray grating spectrometer, where the incident X-ray energy is wavelength-selectedby a monochromator of the beamline and the outgoing X-ray photonsare recorded as XES spectra as a function of incident X-ray energy3A. In thin situ studies of LSMO in water vapor (LSMO*H2O) and with alkalinesolution (LSMO*H2O/KOH). LSMO perovskite can be used asa catalyst for either the oxygen reduction reaction (ORR) or the OER.Its perovskite crystal structure is shown in Depending on the scientific question, with thesethree differenttypes of spectrometers coupled to liquid microjets, different questionsof the OER activity and the OER reaction mechanism are addressed.In the following we will present our comparative 3, wherecommonly lanthanides or group II elements are found at the A sitesand transition metals at the B sites.35 Complete or partial substitution of the A sites and/or B sites changesthe physicochemical properties of the perovskite oxide, often withoutaffecting the perovskite structure significantly. Chemical substitutionsin perovskite oxides have been shown to impact their catalytic activityfor the OER, making them a good choice for systematic studies.39 Highly crystalline thin films with defined orientation have beeninsightful model systems studied in water vapor, e.g., by ambientpressure in situ soft XAS,15in situ XPS/XANES,40 environmental TEM,43 or ultrafasttime-resolved optical studies.44 Here wefocus on photon-in/photon-out synchrotron spectroscopy using a liquidjet setup.Perovskite oxides have the generalstructure ABOin situ and operando studies:(i)A study of the OER under insitu conditions with low-resolution X-ray emission spectroscopy ,we name the experiments in situ rather than operando according to the common definitions.troscopy 4 and 5. (ii)A study of the electronicinfluence of the water adsorbate on the LSMO surface with high-resolutionX-ray emission spectroscopy coupled to a liquid jet  resembles a technical preparation study confirmingthat LSMO can be studied under in situ (or even operando) conditions with high-flux X-ray sources. It confirmsthat spectroscopic analysis deduces trends in manganese oxidationstates under in situ conditions. The more sophisticatedhigh-resolution in situ X-ray emission studies (ii),which are prepared to reduce radiation damage from the high-flux X-raysources, can be built on the results obtained in (i) and add informationabout the electronic properties of LSMO when water layers are addedfor the in situ experiments.In this work we will present the scientific resultsof two typesof proof-of-concept OER studies on LSMO which resemble two complementaryX-ray emission spectroscopy capabilities for Both types ofsoft X-ray spectrometry allow recording of the changesin the oxidation state or the element-specific chemical compositionof the bulk electrocatalyst or water, the surface of the electrocatalyst,or the water adsorbate. In general, the highest surface sensitivityis reached during recording with the grazing incidence geometry.iin situ studies, we combined TZP X-ray spectroscopywith a potentiostat and monitored the Mn valence and oxidation statesof LSMO with water upon voltage stepping. With the TZP-based X-rayspectroscopy, the changes in the oxidation states of various elementsin LSMO can then be quickly recorded. Integrating in particular themanganese L-edge emission features (3d → 2p1/2 RIXS)for different applied voltages yields the manganese L-edge partialfluorescence yields as 3d-PFY. For the iPFY signals we applied thesame data procedure as been described in the general procedure ofintegration of the RIXS features of LSMO:2 we obtained the iPFY of the Mn L-edge by integrating the regionof interest of the O 2p–1s emission feature and inverting thesignal intensity. In the following we will concentrate on the iPFYsignals. Changes in (and confirmation of) catalytic activity are additionallymonitored by coupling a mass spectrometer to the setup (which is notconsidered in this work).For our 2 by the release of electrons, whichare given to the LSMO anode surface. At this particular moment ofelectron admission, higher-valence intermediate Mn states (Mn3+/Mn4+) are formally reduced toward lower-valencestates by the admission of electrons, leading to a lower mixed orintermediate valence state (Mn2+/3+/4+). Note that theoccurrence of mixed or intermediate valence states of Mn can be distinguished.Intermediate valence states are present in the metallic phases ofLSMO, where 3d electrons in the conduction band are delocalized. Thisis typically the case for valence states in between Mn3.2+ and Mn3.5+ in highly crystalline LSMO with low defectdensity. Mixed valence states are present in semiconducting or insulatingphases of LSMO, where electric charge is rather localized and mixedvalence states of Mn2+, Mn3+, and Mn4+ can evolve. This can be triggered by, e.g., point defects, changeof doping, strain, and surface reconstruction. In 48 ranging over the manganese valence states of Mn2+, Mn3+, Mn2+/Mn3+, and Mn4+. Inthe current work, the beamline and part of the spectrometers havebeen calibrated on well-defined argon and xenon lines of soft X-raygas detectors.20 This sets the energy of the spectrometersto 0.1–0.5 eV energy precision. The best energy resolutionof the TZP, however, is about 0.8–1 eV. Independent of theinitial oxidation state, applying Mosley’s law yields a +1.6eV relative shift/oxidation state in our PFY spectra in 2+, 641.4 eV for Mn3+ and 643.2 eVfor Mn4+. Additional mixed-state transitions are assignedto the transition shoulders at 639.6 eV (Mn2+/Mn3+), 640.5 eV (Mn2+), 642 eV (Mn2+/Mn3+), and 643.8 eV (Mn4+), also for mixed valences like inMn3O4.46 The assignmentsof the respective calibration points are marked as vertical linesin the PFY spectra in On the chemical side, during the OER,water is oxidized to O2+ to Mn4+ in LSMO before the OER and alsoon the surface. Since also Mn2+/Mn3+ mixed valencetransitions are found at around 639.4 eV and the reference is thezero voltage (vs ground) LSMO form, we interpret the existence ofthe shoulder and its variation during voltage sweep or surface coverageas a change in the manganese valence state toward smaller values,i.e., from Mn3+/4+ to Mn3+ or even mixed Mn2+/3+ states, etc. It is safe to state that activating LSMOby increasing the applied voltage leads also to a slight reductionof the surface Mn valence states in LSMO, which are not fully recoveredby the charge flow back from the bulk and which we will use for thefollowing interpretations as a type of intrinsic LSMO anode featurewithout water adsorbate and electrolyte around.The present OER studies of LSMO (as the anode)under more realisticoxidation conditions reveal also a more precise monitoring of thedynamic LSMO oxidation state changes 4 and sugin situ inverted partial fluorescence yield (iPFY)of LSMO*H2O, and in situ studies ofthe iPFY of LSMO*H2O/KOH when the water vapor in the chamberforms a KOH/water mixture with a KOH-covered LSMO film and under voltagesweep. In 2O decreases the first639 to 643 eV iPFY transition band when the voltage is increased from0 to 3.9 V, and the second iPFY transition band with its maximum at652.5 eV increases.51 and shown in in situ PFY when the voltage is ramped up.Within the resolution of the recorded data, the found behavior isquite similar to that of LSMO covered with water  degrees offreedom. Therefore, the studies have been extended from water to alkalinesolution covering LSMO. Alkaline solution can be created by eitheradding water to the salt covering the LSMO surface or using mixingunits precoupled to the nozzle and transfer tube for water in a microjetmixing unit that can mix different solution channels, as describedpreviouslyth water 4A, sugge52The current spectrometers are not sensitive to eventually fullydissolved Mn ions in water or in alkaline solution (with KOH). Thephase evolution at different pH and during oxidation has been previouslyreported for the Mn-based OER catalysts, and they are found to exhibithigh structure flexibility that relates to their OER behavior.3 to Mn-L2 integral intensities assuming that the Mn-L3 edge heightis reduced and that other ratios are thus expected for undistorteddata. This method has successfully been developed for in situ electron energy loss spectroscopy of LSMO for getting a deeper insightinto in situ studies.41 Since ourPFY spectra are quite distorted (the 3d-PFY even more than the iPFYpresented in this work), this analysis can only be seen as a firstzeroth-order approach emphasizing the proof of concept of the presentstudy. 3 to Mn-L2 integral intensities as a function ofapplied potential. The iPFY ratio (492 and L3 edges for LSMO covered with water(LSMO*H2O) and LSMO covered with alkaline solution (LSMO*H2O/KOH). For LSMO*H2O (red curve) and LSMO*H2O/KOH (blue curve), a clear tendency to a thresholdlike behaviorhas been found.Formally, the valence state change ofMn can be monitored by calculatingthe iPFY intensity ratio of the Mn-LFY ratio 5B is comFY ratio 5A.49Figu2O is set to 1 for no voltage applied,and that of LSMO*H2O/KOH is slightly smaller (0.8). Thefound voltage-independent abscissa offset decreases in the integralintensity ratio of LSMO*H2O and LSMO*H2O/KOHcan be explained by the decrease of the emitted photon signal dueto the added water/alkaline solution layer and the energy-dependentabsorption of the added water/alkaline water layer to LSMO.In 2O and LSMO*H2O/KOH, a voltagethreshold of >2.2 V is found, from where the ratio then monotonicallydecreases as the voltage increases from 2.2 to 4 V vs ground. We hypothesizethat the reduction of Mn is due to water/hydroxide oxidation and thuscorrelate the onset of this reaction with the onset of the OER onLSMO in rotating ring–disk electrode (RRDE) laboratory experimentsat 1.65–1.7 V vs RHE, shown in 49 The RRDEexperiments were performed on the same LSMO system, synthesized inthe same way and with the same doping level. The deviations betweenour found in situ studies and the ones from the laboratoriescan be explained in the following way: first, our proof-of-conceptstudies presented here are technically not as much optimized as thering electrode experiments, as already the statistics of both experimentssuggest. Second, our applied voltages are not electronically experimentallyreferenced in the same way as the ring electrode experiments. Third,the ratio method utilizing TZP spectroscopy misses some sensitivity which adds an insensitivity on the ratioscaling . Fourth, mechanistic differences may also influencethe voltage threshold value since the LSMO sample environment slightlydiffers in comparison with a ring electrode experiment and the LSMOwater layers produced in this experiment. Overall, in situ LSMOactivity. However, it also emphasizes that more detailed electronicand electronic–structural studies are needed to understandcompletely the LSMO water coverage.For both, LSMO*Hii2+ or Mn3+ or intermediate Mn2+/Mn3+ valence states. The discussed inconsistencies and normalizationfurthermore suggest that there may be more modifications in particularon the LSMO surface that we have not taken into account yet.When the liquid jet/LSMO is coupled to our high-resolutionX-ray emission spectrometer, we can monitor in detail with greatersensitivity the formation of water layers adsorbed on LSMO. From theTZP-based PFY studies explained in the last paragraph, we know thatwhen water is added to the surface but no voltage potential is applied,the valence states of surface Mn are not reduced to pure MnWith grating-based X-ray spectroscopy and utilizing the megahertzrepetition frequency of the synchrotron, RIXS maps of the system canbe recorded in a reasonable time. Utilizing the liquid jet reducesthe probability of radiation damage. Consequently one RIXS map withthe X-ray emission features versus the incident X-ray energy 6 can be 2O recorded with a grating spectrometer.The XES spectrum can directly be energy-calibrated to the elasticpeak (yellow line). Additional different mixed/intermediate Mn3+/Mn4+ valence states can be assigned: the resolvedtransition bands belong to the local d–d transition (orangetransition bands), the Mn 3d → 2p1/2 fluorescence(gray transition bands), and some Mn3+/Mn4+ emissionbands in the strong crystal field (cyan transition bands). These transitionshave also been found in other LSMO XES studies.48 Upon addition of water to LSMO, in the XES spectrum of 2O water adsorbate additional transition bandshave been observed that were not recorded in our earlier studies ofdry LSMO in vacuo.2 Weassign these additional O 2p→ Mn 2p emission transition bandsto charge transfer from the oxygen atom of the adsorbed water to themanganese atoms of the LSMO surface, abbreviated as MLCTaq and highlighted with orange and purple transition bands in aq states alter the activation properties of the surface Mnsites in LSMO even before application of a potential.in situ (and operando) studies since it reduces the probability of accumulating radiationdamage. Depending on the X-ray spectrometer added, qualitative orquantitative chemical analysis, like oxidation state changes or element-specificcomposition changes, can be monitored in situ. Dueto additional mixing capabilities, liquid jet technologies add furtherdegrees of freedom for the chemical composition. For an in-depth mechanisticstudy, however, high-resolution X-ray spectroscopy is of advantage.For studying the water-splitting OER electrocatalytic reaction ofLSMO perovskite, high-resolution grating studies allow monitoringof transient charge transfer states, the filling of empty LUMOs ofLSMO through the adsorption of water, etc. However, all of these novelexperimental approaches are only possible through the versatile developmentsof liquid jets and liquid beam technologies.We can conclude that the current advances ofliquid jet technologycombined with state-of-the-art spectroscopy at high-flux X-ray sourcesallow the study of the electronic, structural/electronic, and mechanisticproperties of metastable states as well as very short-lived intermediates.Liquid jet technology not only contributes to time-resolved studiesbut can also enhance


The chicken astrovirus (CAstV) is a ubiquitous enteric RNA virus that has been associated mainly with conditions, such as the runting-stunting syndrome, severe kidney disease, visceral gout, and white chick syndrome, in broiler-type chickens worldwide. Sequence analysis of the capsid genes’ amino acids of the strains involved in these conditions reveals a genetic relationship and diversity between and within the CAstV genogroups and subgroups based on phylogenetic analysis, genetic distance (p-dist), and pathogenicity. While the two genogroups (A and B) are demarcated phylogenetically, their pairwise amino acid sequence identity is 39% to 42% at a p-dist of 0.59 to 0.62. Group-A consists of three subgroups  with an inter- and intra-subgroup amino acid identity of 78% to 82% and 92% to 100%, respectively, and a p-dist of 0.18 to 0.22. On the other hand, the six subgroups  in Group-B, with a p-dist of 0.07 to 0.18, have an inter- and intra-subgroup amino acid identity of 82% to 93% and 93% to 100%, respectively. However, these groupings have little to no effect on determining the type of CAstV-associated pathology in chickens. Undoubtedly, for over five decades, the poultry industry has witnessed rapid growth, contributing significantly to the provision of animal protein and creating a global niche in food economics. Driven by the growing demand for poultry meat and eggs worldwide, it is projected that the industry will continue to witness a modest pace of growth for the next 10 years . Among tKnown to be among the leading causative pathogens of diarrhea in birds and mammals, and ubiquitous in both sick and healthy humans, animals, and birds, astroviruses were first discovered in 1975 during an electron microscopic (EM) examination of diarrheic stool samples from young children of 2 years old and below that were suffering from gastroenteritis . AstroviPresently, the two recognized and characterized astroviruses of economic importance in the poultry industry are the avian nephritis virus (ANV) and chicken astrovirus (CAstV) ,13. ThesThe name astrovirus originates from the ultrastructural morphological presentation of the virus under the electron microscope with five spikes of a star (astron in Greek) . It is wGenerally, astrovirus virion sizes under the electron microscope (EM) are within the range of 28 to 35 nm in diameter ,25. The ORF-1a, ORF-1b, and ORF-2, which are bound by untranslated regions (UTRs) on both the 5′- and 3′-ends [ORF-1a and ORF-1b), upstream of the genome, are composed of non-structural proteins (NSPs): trypsin-like serine protease, a viral protein genome-linked (VPg) at the end of the 5′-end possessing a TEEEY-like residue (SEEEY), and RNA-dependent RNA polymerase (RdRp) genes, which are all responsible for replication and virion generation [The genome of chicken astrovirus is similar to that of other astroviruses and consists of a linear, positive-sense single-stranded (ss) RNA (+ssRNA) with a length that varies from 7 to 7.6 kb without the polyadenylated (poly-A) tail at the 3′-end. The genome is composed of three serially arranged genes in the form of open reading frames (ORFs):  3′-ends . The 5′- 3′-ends . The firneration  , six possible transmembrane (TM) domains, a coiled-coil (CC) domain, and a helicase (HEL) domain [ORF-1b has a start codon (ATG), which makes it a true ORF by definition; it is highly conserved and has a consistent length in CAstVs [ORF-1b in CAstV starts through a start codon near the 5′-end of the heptanucleotide region [ORF-2) encodes a structural protein and a viral capsid protein precursor that is expressed from a sub-genomic (sg) RNA, and it is situated at the 3′-end of the astrovirus genome. The region is documented to be within a range of 670 to 744 amino acids (aa) in length across various avian astroviruses [ORF-2 of all astroviruses encodes a sizeable polyprotein that generates capsid protein when cleaved by cellular protease. The capsid protein gene has been studied extensively; much of what is currently known about the astroviruses, and their relatedness, comes from its analyses. The capsid protein has three significant domains: the RNA-binding, N-terminal, and C-terminal domains. The N-terminal domain is more conserved than the RNA-binding domain and the C-terminal domain. The N-terminal domain contains a preserved motif of repeats, SRSRSRSRSRSR, and highly basic residues similar to those of DAstV and TAstv-2 [These two NSPs overlap with a nucleotide sequence with a length of between 12 and 45 nucleotides (nt) in avian astroviruses and contain a heptanucleotide “slippery sequence” (5′-AAAAAAC-3′) region, which is part of a ribosomal frameshift structure similar to the frameshift structures found in retroviruses and coronaviruses ,32. Puta) domain ,30  exhibit lengths similar to those of other sequenced bird astroviruses. In comparison, the 5′-end UTR of CAstV is between 7 and 21 nt, while the 5′-end UTRs of TAstV and DAstV are 10 and 23 nt, respectively. The 3′-end UTR is between 200 and 282 nt, which is within the range of ANV with 305 nt and TAstV with 192 nt . A coronGenerally, mutations within the s2ms restore base pairing via several compensatory mutations. The extensive conservation of this motif across many viruses indicates that the region plays a vital role in the replication of positive-sense RNA viruses . AlthougORF-1b and ORF-2 are in the +1-reading frame of ORF-1a, thus resembling the genome arrangement of TAstV-1  recognized the Astroviridae family based on a consistent phylogenetic reconstruction that divided it into two genera: the genus Mamastrovirus and the genus Avastrovirus, infecting mammalian and avian species, respectively ,27. Howehe virus ,26. HowePresently, the ICTV recognizes 19 species of mamastrovirus (MAstV-1 to MAstV-19) belonging to two major groups: genogroup I and genogroup II, with roughly 14 new strains awaiting grouping, of which some are tentatively identified as species ,40. On tThus, as usual for most avian diseases, records on the classification or variety of viruses most often emanate from poultry studies due to an understandable lack of evidence from wild species ,43. RemaORF-2 (capsid) gene amino acid sequences [Currently, in line with the above, the diversity and classification of CAstVs is based on phylogenetic analysis and the genetic distance (p-dist) between equences  , which shared a 100% amino acid capsid identity. The three isolates clustered and formed a new subgroup (Bv) in Group-B CAstV based on the earlier described share of inter- and intra-identity . The isoTherefore, conveniently, except for the members of subgroups Bii and Ai, a capsid gene amino acid sequence identity percentage of ≥94% and a p-dist of 0.00 to 0.04 to a reference or prototype strain indicate the emergence of a distinct variant or subgroup within the CAstV genogroups.ORF-1b sequences , and approximately 34% (117) are for the partial or complete capsid gene. This limited number (117) of capsid gene sequences poses some difficulties for classification and the detection of recombination events, especially with the emergence of new incomplete capsid sequences. Similarly, events between different types of CAstVs may lead to variations in host or tissue tropism and are more difficult to investigate when only partial genes or genomes are used, as most recombination processes do not occur exclusively in antigenic protein-encoding regions of astrovirus genes but rather in the complete genome [Currently, there are 347 sequences of CAstV deposited in GenBank, of which approximately 9% (30) are for near-complete or complete genomes, approximately 58% (200) are for partial e genome .Chicken astrovirus (CAstV) is a relatively recently emerged virus that is originally associated with malabsorption syndrome, which includes RSS in broiler chickens, uneven performance in growth, and enteritis in flocks. It is also a ubiquitous virus that is commonly isolated from the gut of clinically diseased and healthy chickens ,25,49. SAs a member of the RNA enteric viruses , CAstV uLike other known astroviruses, CAstV infects mostly young animals of commercially reared chickens and turkeys ,54,55. TPermissive and susceptible young chickens within the first 10 days of age exhibit varying illnesses because of different degrees of infection. The observable clinical signs consist of diarrhea, maldigestion, growth retardation, or mild runting, while severe clinical signs can lead to death after infection ,15,55. TPresently, three to four types of CAstV-related illnesses or conditions exist based on infectious virus strains : RSS or Identified in the broiler industry in the 1970s as a transmissible disease of broiler chickens of doubtful etiology ,69,70,71Compared with their cage mates, clinically affected birds exhibit ruffled feathering and growth retardation ,29. AddiGenerally, intestinal lesions are strain dependent based on experimental infection. Isolate 612 (Group-A CAstV (Ai)) of South Africa  and UPM1Recently, metagenomic findings have demonstrated that aside from being ubiquitous in healthy broiler chickens, RNA viruses in birds have a community composition that continues to diversify as the birds age . NeverthWith a diverse origin that includes infectious bronchitis virus (IBV) and management and nutritional factors , severe As with most other CAstV infections, clinical signs are overall dullness, drowsiness, and diarrhea within 3 to 4 dpi in very young broilers and in experimentally infected 1-day-old SPF chickens ,30,79. EORF-2 amino acid similarity, especially between the Indian and Middle East strains, strongly support the involvement of CAstV. In line with this premise, CAstV should equally be considered in the list of differentials in cases of kidney disease and gout in chickens. Similarly, based on the lesions observed in broiler flocks across Malaysia between 2017 and 2018, and with the subsequent chicken trial with CAstV isolate UPM1019/2018, there exists palpable evidence for the nephropathogenic potential of the circulating Malaysian CAstV strains [Consequently, the extensive variety of circulating CAstV strains in the UK, India, the Middle East, and Europe, causing severe renal pathology leading to gouty lesions, and the level of  strains . For over 40 years, CAstV has been associated with a hatchability condition referred to as “white chick” ,68. The A temporary but significant decrease of 4% to 68% in hatchability, and mid to late deaths of in-shell embryos, have been reported in broiler breeder flocks in Finland and Poland ,53,67,68Histopathologic lesions may include bile duct proliferation, cholangiofibrosis, cholestasis, hepatic necrosis with glycogen accumulation, a rise in hepatic extramedullary granulopoiesis, and heterophilic and lymphocytic interstitial nephritis ,20,67.In line with Koch’s postulate, the condition has been reproduced by inoculating embryonated chicken eggs, leading to associated hatching problems and a high mortality rate, with deaths recorded a few hours after hatching at a 100% mortality rate ,68,80. CPresently, strains belonging to the Aiii of Polish origin and members of the Biv subgroup that includes strains from Brazil and Canada ,18,53 haBased on serological profiling, it is possible to produce and keep birds free of astrovirus diseases. However, the ubiquity of avian astroviruses in domestic birds suggests that eradicating the virus from birds, particularly commercial poultry, is challenging given the virus’s ability to be transmitted vertically . In addiPoultry wastes, including poultry droppings, must be collected and removed on a regular basis in such a way that passages within the poultry facilities are free of contamination. The virus is efficiently and effectively removed by peroxymonosulfate (MPS) product, Virkon-S 1.5%, 0.3% formaldehyde, 0.1% propriolactone, and 90% methanol .Multiple age farming should be avoided, as it prolongs low performance by allowing infected hens to recover without showing clinical manifestations but yet shedding the virus .There are currently no prophylactic drugs or vaccines available to treat and prevent CAstV in poultry. The transfer of adequate maternal antibodies from breeder hens to chicken embryos is critical for limiting vertical virus transmission and providing timely defense against horizontal virus transmission. Although the exact age of the initial infection is unknown, the severity of the sickness is determined by the age of the first exposure; therefore, timely immunity is recommended . It has Compared with other infectious avian viruses, namely the avian influenza virus, avian coronavirus, fowl adenovirus, and avian paramyxovirus, there exists a dearth of information on the impact of CAstV on the poultry industry. A key factor for this has largely been due to the lack of standardized propagation systems of either cell culture or animal model that will allow biological characterization. Furthermore, genetic diversity, type of chicken affected, and the variations in clinical disease caused by the different strains of the CAstV complicate the capacity to develop a solid story similar to that of other members of Astroviridae. However, in the face of these challenges, over the past 15 years, significant progress has been made. Of all the avian astroviruses, CAstV is second only to human astroviruses in terms of characterization, thus allowing the understanding of the virus’s pathogenic mechanism and immunity characteristics. Yet, more research is needed.Importantly, there is an urgent need to develop a standard protocol for naming and grouping existing and future genus members. A standard method for comparing molecular characteristics would improve the understanding of the virus relationships within genera and identify potential virulence determinants. In trying to better understand CAstV, the avian virology community should try to characterize representative members of each genogroup, and subgroup fully. This would entail the development of a sequencing procedure, as well as the development of antibodies and other reagents. All these studies would contribute to a better understanding of the cellular, physiological, and immunological alterations CAstV causes in their host species so that we can develop more effective prevention, control, and therapeutic strategies.