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Insects and plantsLarvae of fall armyworm (FAW, Spodoptera frugiperda) were cultured at the Department of Entomology at the Max Planck Institute for Chemical Ecology, and reared on a semi-artificial diet based on pinto bean59, and maintained under controlled light and temperature conditions (12:12 h light/dark, 21 °C).Feeding experiments3rd–4th instar FAW larvae were utilized for all experiments. Insects were starved overnight prior to feeding experiments. The following day insects were fed with a semi-artificial, pinto bean-based diet or put on maize leaves in small plastic cups and allowed to feed on the respective diets for a day. Insects were dissected in cold phosphate buffered saline (PBS, pH = 7.4) to harvest larval tissues (guts, Malphigian tubules, fat bodies, cuticle), which were stored at − 80 °C until further use. For droplet feeding, 12.5 mM DIMBOA was prepared by dissolving the compound in DMSO. This DIMBOA solution was further diluted in 10% aqueous sucrose solution. The larvae were stimulated with forceps to encourage regurgitation, and 2 μL DIMBOA-sucrose solution was administered directly to the larval mouthparts. Insects were then fed on semi-artificial diet for up to 6 h; following which gut tissue was dissected using cold phosphate buffer and the tissue samples were stored at − 80 °C until further use.Insect cell culturesSpodoptera frugiperda Sf9 cells and Trichoplusia ni Hi5 cells were cultured in Sf-900 II serum-free medium (Gibco) and ExpressFive serum-free medium (Gibco), respectively. Adherent cultures were maintained at 27 °C, and sub-cultured every 3–4 days.Cell treatmentsInsect cells were seeded in 6 well culture plates (Corning) and left at 27 °C overnight. For transcript stability tests, a fresh cycloheximide (CHX) stock (50 mg/mL) was prepared in ethanol and added to the cultured cells at a concentration of 50 µg/mL. Incubations with CHX were performed up to 6 h. For testing substrate specificity, cells were then treated with the following compounds for 1 h—DIMBOA (25–100 μM), indole (50–100 μM), quercetin (50–100 μM), and esculetin (50–100 μM). All the stocks were prepared in DMSO and cells treated with the corresponding volume of pure DMSO served as a control. The range of concentrations used for the substrates was based on previous work38.RNA extraction, reverse transcription and real time-PCR analysisTissue samples from the larvae were homogenized and total RNA extracted using the innuPREP RNA Mini Kit (Analytik Jena). Cell cultures used for RNA extraction were obtained during sub-culturing at full confluency, and centrifuged at 500×g for 5 min. The culture medium was discarded, and the fresh pellets were directly used for RNA extraction. RNA concentrations were measured with the NanoDrop 2000 UV–Vis Spectrophotometer (Thermo Scientific). First strand cDNA was synthesized from 1 μg total RNA using SuperScript III Reverse Transcriptase and OligodT primers from Invitrogen. Sequences were successfully amplified using Phusion High Fidelity DNA Polymerase (New England Biolabs) (PCR protocol: 30 s at 98 °C; 35 cycles of 10 s at 98 °C, 20 s at 55 °C, 45 s at 72 °C; and 5 min at 72 °C). The PCR products were purified with a PCR cleanup kit (Qiagen) and cloned into pCR-Blunt II-TOPO vector (Life Technologies) and transformed into NEB cells (Life Technologies), which were plated on selective LB agar medium containing 100 μg/mL ampicillin and incubated overnight at 37 °C. Positive colonies were identified by PCR using vector-specific M13 primers. Positive clones were confirmed by sequencing. Real time PCR analyses were carried out using Brilliant III SYBR Master Mix, employing SYBR Green chemistry. Relative quantification of the transcript levels was done using the 2−∆∆Ct method60. SfRPL10 was used as reference gene for all analyses. The primer pairs used for distinguishing between the variants are listed in Supplementary Table 1. As the expression of full-length and variants of SfUGT33F28 differed according to the strains, tissues, and treatments being analyzed, variant expression is reported as ratios relative to the canonical transcript to facilitate comparisons.Preparation of minigenes for alternative splicing studiesGenomic DNA was isolated from S. frugiperda larvae using the cetyl trimethyl ammonium bromide (CTAB) protocol61. DNA concentration was measured with the NanoDrop 2000 UV–Vis Spectrophotometer (Thermo Scientific). The minigene was amplified using Phusion High Fidelity DNA Polymerase (New England Biolabs) (PCR protocol: 30 s at 98 °C; 35 cycles of 10 s at 98 °C, 30 s at 55–60 °C, 1 min 30 s at 72 °C; and 10 min at 72 °C), cloned into a pCR-Blunt II-TOPO vector (Life Technologies) and sequenced using M13 primers. The confirmed sequence was eventually cloned into a pIB/V5-His-TOPOvector (Life Technologies) and transformed into NEB cells (Life Technologies). Positive colonies were identified by colony PCR using vector-specific OpIE2 primers, sub-cultured overnight at 37 °C in liquid LB medium containing 100 μg/mL ampicillin and used for plasmid DNA purification with the NucleoSpin Plasmid kit (Macherey-Nagel). Concentration and purity of the obtained construct was assessed by the NanoDrop 2000 UV–Vis Spectrophotometer (Thermo Scientific) and the correct orientation of the PCR products was confirmed by DNA sequencing.Nuclear protein isolationNuclear proteins were isolated from insect cells62 using the protocol originally described with few modifications. Cells grown to concentrations of up to 1 × 106 cells/well were harvested and washed with PBS (pH 7.4). The extracts were centrifuged at 12,000×g for 10 min and pellets were re-suspended in 400 μL cell lysis buffer (10 mM HEPES, pH 7.5, 10 mM KCl, 0.1 mM EDTA pH 8.0, 1 mM DTT, 0.5% Nonidet-40 and 10 μL protease inhibitor cocktail). Cells were allowed to swell on ice for 20 min with intermittent mixing. Suspensions were vortexed to disrupt the cell membranes and then centrifuged at 12,000×g for 10 min at 4 °C. Pelleted nuclei were washed thrice with cell lysis buffer, re-suspended in 50 μL nuclear extraction buffer (20 mM HEPES pH 7.5, 400 mM KCl, 1 mM EDTA pH 8.0, 1 mM DTT, 10% glycerol and protease inhibitor) and incubated on ice for 30 min. Nuclear fractions were collected by centrifugation at 12,000g for 15 min at 4 °C. Protein concentrations were measured by Bradford and extracts were stored at − 80 °C until further use.Electrophoretic mobility shift assay (EMSA)EMSA was performed using the LightShift Chemiluminescent EMSA kit (Thermo Scientific) following the manufacturer’s instructions. Genomic DNA fragments of 20–25 bp corresponding to the 5′ flanking region of UGT33F28 exon 1 (with and without AhR-ARNT motif deletion) were synthesized with covalently linked biotin (Sigma). The DNA probes used in the experiment are listed in Supplementary Table 6. EMSA was performed in 20 µL reactions containing 20 fmol biotinylated DNA probe with 3.5–4 µg nuclear protein extracted from insect cells, according to manufacturer’s instructions. A reaction comprising the above along with the excess of unlabeled canonical DNA probe (200 molar excess) was further employed as a control. The reaction was assembled at room temperature and incubated for 30 min. The reactions were separated on a 5% TBE gel in 0.5X TBE at 100 V for 60 min. The samples were then transferred to a positively charged nylon membrane (Hybond N+, Amersham Bioscience) using semi-dry transfer at 15 V for 30 min. The membrane was cross-linked for 1 min using the auto cross-link function on the UV cross-linker (Stratagene). The biotinylated DNA–protein complex was detected by the streptavidin–horseradish peroxidase conjugated antibody provided in the kit. The membrane was washed and incubated with the chemiluminescence substrate for 5 min and the signals were developed by exposing the membrane to an X-ray film for 1 min.Streptavidin affinity purificationStreptavidin agarose (Sigma-Aldrich) was employed for protein purification. Briefly, 50–100 μL of agarose was packed into a 1.5 mL Eppendorf tube for each sample. The agarose was allowed to settle with a short centrifugation (500×g, 5 min) and the supernatant was discarded. The agarose was washed 4–5 times with binding buffer (PBS containing 1 mM EDTA, 1 mM DTT, 4 µg poly dI. dC as non-specific competitor DNA and protease inhibitor). Simultaneously, the binding reaction with the nuclear protein fraction and the DNA probe was assembled as described above. A 100 μg amount of total nuclear protein was incubated with 4 μg of biotinylated DNA probe at room temperature for 20 min. The reaction was loaded onto the streptavidin column equilibrated with the binding buffer and incubated for another 1 h at room temperature with gentle shaking. Subsequently, the agarose was washed 4–5 times with the binding buffer. After the final wash, the supernatant was aspirated and 10 μL was left above the beads. For protein separation, 20–30 μL pf the SDS loading buffer was added onto the agarose, boiled at 95 °C for 5 min and the sample thus obtained was utilized for electrophoresis.Deletion mutagenesisFor deletion mutagenesis, a pair of primers flanking the sequence to be deleted (non-overlapping) was designed. The pCR-Blunt II-TOPO vector (Life Technologies) clone for the SfUGT33F28 exon 1–2 minigene was utilized as a template. Sequence was successfully amplified using Phusion High Fidelity DNA Polymerase (New England Biolabs) (PCR protocol: 30 s at 98 °C; 20 cycles of 10 s at 98 °C, 30 s at 55–60 °C, 4 min at 72 °C; and 10 min at 72 °C). A DpnI digest was performed to remove the background DNA, followed by ligation and transformation into fresh cells. The sequence of the mutant TOPO clone was then confirmed and utilized as a template for cloning into pIB/V5-His-TOPO vector (Life Technologies) for transfection into insect cells.Cloning and heterologous expression of SfUGTsSequences were amplified from S. frugiperda gut cDNA samples using Phusion High Fidelity DNA Polymerase (New England Biolabs) (PCR protocol: 30 s at 98 °C; 35 cycles of 10 s at 98 °C, 20 s at 55–60 °C, 45 s at 72 °C; and 5 min at 72 °C). The resulting amplified products were purified with a PCR cleanup kit (Qiagen) and incubated with GoTaq DNA polymerase (Promega) for 15 min at 72 °C in order to add A overhangs. The products were cloned into the pIB/V5-His-TOPO vector (Life Technologies) and transformed into NEB cells (Life Technologies), which were plated on selective LB agar medium containing 100 μg/mL ampicillin and incubated overnight at 37 °C. Positive colonies were identified by PCR using vector-specific OpIE2 primers, sub-cultured overnight at 37 °C in liquid LB medium containing 100 μg/mL ampicillin and used for plasmid DNA purification with the NucleoSpin Plasmid kit (Macherey-Nagel). Concentration and purity of the obtained constructs were assessed by NanoDrop 2000 UV–Vis Spectrophotometer (Thermo Scientific) and the correct orientation of the PCR products was confirmed by DNA sequencing.Insect cell transfectionFor transfection, Sf9 cells and Hi5 cells were sub-cultured at full confluency in a 6-well plate in a 1:3 dilution and left overnight to adhere to the flask surface. The medium was replaced, and transfections were carried out using FuGENE HD Transfection Reagent (Promega) in a 1:3 plasmid/lipid ratio (1.7 μg plasmid and 5.0 μL lipid for 3 mL medium). Cells were incubated for 48–72 h at 27 °C and re-suspended in fresh medium containing 50 μg/mL blasticidin for 2 weeks. Stable cell cultures were subsequently maintained at 10 μg/mL blasticidin.Cell lysate preparationCells were obtained from cultures 2 weeks post transfection growing stably on 50 μg/mL blasticidin. A 1 mL quantity of cells was harvested for each construct and re-suspended into 100 µL buffer. Protein concentrations were measured using the Bradford reagent, and 1–2 μg of the cell lysate was used for enzyme assays.Microsome preparationFor microsome extraction, confluent, stably transfected cells from five T-75 flasks (10 mL culture) per recombinant plasmid were harvested by scraping the cells off the bottom using a sterile cell scraper (Sarstedt AG, Nuembrecht, Germany). The obtained cell suspensions were combined into a 50 mL falcon tube and centrifuged at 1000×g for 15 min at 4 °C (AvantiTM J-20 XP Centrifuge, Beckman Coulter, Krefeld, Germany). The supernatant was discarded, the cells were washed twice with ice-cold PBS buffer (pH 7.4) and centrifuged at 1000×g for 15 min. The resulting cell pellet was re-suspended in 10 mL hypotonic buffer (20 mM Tris, 5 mM EDTA, 1 mM DTT, 20% glycerol, pH 7.5), containing 0.1% BenzonaseR nuclease and 100 μL Protease Inhibitor Cocktail (Serva) followed by incubation on ice for 30 min. After cell lysis, the cells were homogenized by 20–30 strokes in a Potter–Elvehjem tissue grinder (Kontes Glass Co., Vineland, USA) and were subsequently mixed with an equal volume of sucrose buffer (20 mM Tris, 5 mM EDTA, 1 mM DTT, 500 mM sucrose, 20% glycerol, pH 7.5). The homogenate was centrifuged at 1200×g and 4 °C for 10 min (AvantiTM J-20 XP Centrifuge, Beckman Coulter), and the supernatant was transferred into Beckman polycarbonate ultracentrifugation bottles (25 × 89 mm) (Beckman Coulter) and centrifuged at 100,000×g and 4 °C for 1.5 h in a fixed angle Type 70 Ti rotor (OptimaTM L-90K Ultracentrifuge, Beckman Coulter). After ultracentrifugation, the clear supernatant, containing the cytosolic fraction, was aliquoted into 1.5 mL Eppendorf tubes. The pellet, containing the microsomal fractions, was re-suspended in 1 mL of phosphate buffer (100 mM K2HPO4, pH 7.0), containing 10 μL Protease Inhibitor Cocktail (Serva) and stored at − 80 °C until further use. Typically, 5–10 μg of the microsome fraction so obtained was utilized for the enzyme assays.Cross-linking assaysCross-linking assays were performed using dimethyl suberimidate (DMS) as the cross-linking agent. A fresh stock of DMS (5 mg/mL) was prepared in 0.2 M triethanolamine (pH 8.0) at the start of each assay. DMS was added to a final concentration of 2.5 mg/mL to insect cell microsomes with gentle shaking up to 3 h, and samples were subsequently stored at − 20 °C until further use. All protein samples were electrophoresed using a 12% Mini-PROTEAN tris glycine gel, blotted onto PVDF membrane using wet transfer at 70 V for 30–45 min, followed by detection using the V5-HRP conjugate.V5-based affinity purificationAnti-V5 agarose affinity gel (Sigma-Aldrich) was employed for protein purification. Briefly, 50–75 μL of the agarose was packed into a 1.5 mL Eppendorf tube for each sample. The agarose was allowed to settle with a short centrifugation and the supernatant was discarded. The agarose was washed 4–5 times with PBS (pH 7.4). Samples to be purified were incubated with 5% digitonin on ice for 20 min and subject to centrifugation at 16,000×g for 30 min. Clarified cell lysate or microsomal extract was added onto the resin (up to 200 μL, volume adjusted by addition of PBS) and incubated for 1.5 h on a shaker. Subsequently, the agarose was washed 4–5 times with PBS. After the final wash, the supernatant was aspirated and 10 μL was left above the beads. This fraction was used for both protein electrophoresis and enzyme assays (separate purifications). For SDS-PAGE, 20–30 μL pf the SDS loading buffer was added onto the agarose, boiled at 95 °C for 5 min and sample thus obtained was utilized for electrophoresis.LC–MS/MS peptide analysisProtein bands of Coomassie Brilliant blue R250 stained gels were cut from the gel matrix and tryptic digestion was carried out63. For LC–MS/MS analysis of the resulting peptides, samples were reconstituted in 20 μL aqueous 1% formic acid, and 1 μL was injected onto an UPLC M-class system (Waters, Manchester, UK) coupled to a Synapt G2-si mass spectrometer (Waters, Manchester, UK). Samples were first pre-concentrated and desalted using a Symmetry C18 trap column (100 Å, 180 µm × 20 mm, 5 µm particle size) at a flow rate of 15 µL/min (0.1% aqueous formic acid). Peptides were eluted onto a ACQUITY UPLC HSS T3 analytical column (100 Å, 75 µm × 200 mm, 1.8 µm particle size) at a flow rate of 350 nL/min with the following gradient: 3–15% over 3 min, 15–20% B over 7 min, 20–40% B over 30 min, 40–50% B over 5 min, 50–70% B over 5 min, 70–95% B over 3 min, isocratic at 95% B for 1 min, and a return to 1% B over 1 min. Phases A and B were composed of 0.1% formic acid and 100% acetonitrile in 0.1% formic acid, respectively). The analytical column was re-equilibrated for 10 min prior to the next injection. The eluted peptides were transferred into the mass spectrometer operated in V-mode with a resolving power of at least 20,000 full width at half height FWHM. All analyses were performed in a positive ESI mode. A 100 fmol/μL sample of human Glu-Fibrinopeptide B in 0.1% formic acid/acetonitrile (1:1 v/v) was infused at a flow rate of 1 μL/min through the reference sprayer every 45 s to compensate for mass shifts in MS and MS/MS fragmentation mode. Data were acquired using data-dependent acquisition (DDA). The acquisition cycle for DDA analysis consisted of a survey scan covering the range of m/z 400–1800 Da followed by MS/MS fragmentation of the ten most intense precursor ions collected at 0.5 s intervals in the range of 50–2000 m/z. Dynamic exclusion was applied to minimize multiple fragmentations for the same precursor ions. MS data were collected using MassLynx v4.1 software (Waters, Manchester, UK).Data processing and protein identificationDDA raw data were processed and searched against a sub-database containing common contaminants (human keratins and trypsin) using ProteinLynx Global Server (PLGS) version 2.5.2 (Waters, Manchester, UK). Spectra remaining unmatched by database searching were interpreted de novo to yield peptide sequences and subjected to homology-based searching using the MS BLAST program64 installed on a local server. MS BLAST searches were performed against a Spodoptera frugiperda database obtained by in silico translation of the S. frugiperda transcriptome37 and against arthropoda database (NCBI). PKL-files of MS/MS spectra were generated and searched against Spodoptera frugiperda database combined with NCBI nr (downloaded on May 24, 2020) using MASCOT software version 2.6.2. The following searching parameters were applied: fixed precursor ion mass tolerance of 15 ppm for the survey peptide, fragment ion mass tolerance of 0.1 Da, 1 missed cleavage, fixed carbamidomethylation of cysteines and possible oxidation of methionine.Enzymatic assaysFor UGT assays, samples from insect cell cultures (transient or stable) were prepared in phosphate buffer (pH 7.0, 100 mM). Typical enzyme reactions included 5–10 µg cell microsomal extracts, 2 μL of 12.5 mM DIMBOA in DMSO (25 nmol), 4 μL of 12.5 mM UDP-glucose in water (50 nmol), and phosphate buffer (pH 7.0, 100 mM) to give an assay volume of 50 μL. Controls containing either boiled enzymatic preparation, or only the protein suspension and buffer were included. After incubation at 30 °C for 60 min, the enzyme reactions were interrupted by adding 50 μL of 1:1 (v:v) methanol/formic acid solution. For enzyme assays involving resin purified microsomal extracts, equal amounts of extracts were employed for resin purification and the enzyme assay (buffer + substrate) was pipetted directly onto the resin. Post incubation, samples were centrifuged, supernatant was collected, and reaction was stopped by addition of methanol/formic acid solution. Assays were centrifuged at 5000g for 5 min and the obtained supernatant was collected and analyzed by LC–MS/MS.Chromatographic methodsFor all analytical chromatography procedures, formic acid (0.05%) in water and acetonitrile were used as mobile phases A and B, respectively, and the column temperature was maintained at 25 °C. Analyses of enzymatic assays and plant samples used a Zorbax Eclipse XDB-C18 column (50 × 4.6 mm, 1.8 μm, Agilent Technologies) with a flow rate of 1.1 mL/min and with the following elution profile: 0–0.5 min, 95% A; 0.5–6 min, 95–67.5% A; 6.02–7 min, 100% B; 7.1–9.5 min, 95% A. LC–MS/MS analyses were performed on an Agilent 1200 HPLC system (Agilent Technologies) coupled to an API 6500 tandem spectrometer (AB Sciex) equipped with a turbospray ion source operating in negative ionization mode. Multiple reaction monitoring (MRM) was used to monitor analyte parent ion to product ion conversion with parameters from the literature for DIMBOA65 and DIMBOA-Glc16. Analyst (version 1.6.3, Applied Biosystems) software was used for data acquisition and processing.Statistical analysisAll statistical analyses were carried out using SigmaPlot 12.0 and R studio (version 3.6.3). Data were tested for homogeneity of variance and normality and were appropriately transformed to meet these criteria where required. The specific statistical method used for each data set is described in the figure legends. More

Here we have used existing surveillance to detect an emerging wildlife disease and appraise its impact by combining traditional host and vector screening with utilisation of national datasets generated by citizen scientists. Following the detection of USUV in the UK in 20207, whilst national surveillance identified no further cases of USUV infection in wild birds that year, we discovered a significant cluster of blackbird DIRs and an overlapping regional reduction in reported blackbird observations, possibly indicating disease-mediated population decline. Our investigation also identified mosquito vectors at the index site that were positive for USUV RNA, suggesting that ongoing virus transmission was likely.The most prevalent and notable histological changes in the blackbirds and house sparrow with confirmed USUV infection were those in the liver and spleen, consisting of necrosis and lymphohistiocytic inflammation along with moderate to abundant virus antigen labelling. Whilst neurotropism resulting in brain necrosis and lymphohistiocytic inflammation has been reported in studies which examined large numbers of wild blackbirds with USUV infection in continental Europe4,12, we found minimal evidence of neural lesions in the five wild birds examined in this study. Although, histopathological changes in other tissues were generally non-specific, immunolabelling demonstrated widespread virus antigen distribution in both bird species, which is similar to reports of USUV infection elsewhere4,13,14. Immunolabelling was disproportionately greater in the brain and heart in contrast to the minimal or absent histological changes observed in these organs: similar contrasting results of histological and immunohistochemical examinations of USUV-infected wild birds have previously been reported12. Although only brain and kidney samples were examined using USUV RT-PCR, our findings, together with earlier reports4,14, demonstrate that viral antigen can be detected in abundance in the heart and liver, suggesting that these organs could be useful for molecular diagnostic sampling.A differential for necrotising lesions in European passerines, and a comorbidity detected in blackbirds with USUV infection, is Plasmodium spp. infection4,8,15. DNA of the same Plasmodium spp. as detected in the tissues of USUV-positive blackbirds from the ZSL London Zoo site in 2020 was identified in Cx. pipiens s.l. that fed on blackbird hosts at this site previously in 2015, supporting endemic avian haemoparasite infection of this wild bird species at this location. In contrast to the results reported from USUV-positive blackbirds in the Netherlands4, no exo-erythrocytic stages of haemoprotozoa indicative of avian malaria were observed histologically in the two UK blackbirds positive for Plasmodium DNA. Since histological examination has limited sensitivity, in situ hybridisation could be used to further appraise the clinical significance of this co-infection in the future16.Zoological collections are ideally placed to form part of wildlife disease surveillance networks and have already contributed to flavivirus detection in mainland Europe10,13,17,18. The collection grounds at ZSL London Zoo are well monitored for evidence of morbidity or mortality in synanthropic wildlife; this unusually high level of vigilance is considered the likely explanation for detection of USUV at such a location. Recent import of infected captive birds can be excluded as a potential route of USUV introduction as the COVID-19 pandemic had led to suspension of animal movements into the zoological collection. Following USUV detection in synanthropic wildlife, preemptive management practices were employed to safeguard the health of captive animals (Supplementary Materials 1); there was no evidence of USUV-associated disease in the collection animals.The majority of mosquitoes trapped in 2020 were primarily ornithophagic Cx. pipiens s.l., a known vector for USUV1 and a common species in temperate urban habitats. This mosquito species was also the most frequently detected at the ZSL London Zoo site in 2015, during historical trapping sessions19 and at two zoological collections in northern England20. Bloodmeal analyses from mosquitoes at ZSL London Zoo in 2015 and 2020 demonstrate that this species feeds on both wild and collection birds, as would be expected for a generalist ornithophagic mosquito21. In addition, targeted mosquito surveillance in 2020 confirmed circulating USUV in multiple Cx. pipiens s.l. pools at the index site over a three-week period subsequent to the detection of USUV-associated wild bird mortality. This further demonstrates that local mosquito trapping combined with PCR screening is useful as part of an integrated surveillance programme22 and provides evidence that native vectors in the UK may facilitate the onward transmission of USUV to susceptible hosts following an emergence event.Wild bird flavivirus surveillance in Great Britain integrates submissions from three schemes, each with a different taxonomic focus. These convenience samples inevitably lead to skews in species coverage. Although a common garden bird, the number of blackbirds tested for USUV was modest at 2–8 per annum over the period 2012–2019. A communication programme to raise awareness of blackbirds as a sentinel species for USUV, involving a range of stakeholder communities (e.g. non-governmental organisations, wildlife rehabilitators and veterinary surgeons) could help to increase the volume of submissions and, by extension, the ability to rapidly identify the occurrence of USUV in this species. The potential value of target species as sentinels within wild bird surveillance networks has been highlighted for other pathogens, e.g. highly pathogenic H5N1 avian influenza and West Nile virus23,24. In addition to this passive surveillance focused on disease detection in avian hosts, active targeted serosurveys could be conducted to identify cryptic exposure of subclinically affected birds in the future. Given the logistical challenges around active serosurveys in wild birds, screening of archived samples from captive birds in the zoological collection may provide a means to further appraise the extent of USUV circulation, as has previously been undertaken at other collections in mainland Europe25,26.Local reductions of blackbird populations have been reported following USUV outbreaks in mainland Europe27,28,29, but numbers recorded by the BBS have been stable in the UK and Greater London since 2011 when USUV incursion would be predicted most likely to have occurred on the basis of spatio-temporal patterns of spread in mainland Europe3 until the latest data are available from 2019 (Supplementary Figure 5). Whilst our index site detection of USUV is unlikely to represent the incursion event, and earlier sporadic or localised USUV incidents prior to 2020 may have occurred7, based on historical blackbird population trends it seems plausible that the existing surveillance system enabled rapid detection of this emerging infectious disease.Significant clustering of blackbird DIRs was observed in the Greater London, South East and East of England regions in 2020. These results should be interpreted with care given the potential for biases with these opportunistic data and the absence of confirmed aetiology for the DIRs, however, these findings are consistent with a regional increase in blackbird morbidity and mortality in summer 2020 around the USUV index site. Consequently, it is likely that further blackbirds, in addition to those recovered for PME, were infected and died with USUV. Whilst no evidence of an increase in generalised ill health or neurological disease category blackbird DIRs was found in 2020, particular attention should be paid to early detection of clusters of DIRs of these categories as a potential signal of USUV occurrence in the future.One indicator, the dead bird ringed recovery dataset, did not support increased scale of blackbird mortality in Greater London; however, the dataset is small and vulnerable to variation in observer bias (e.g. related to COVID-19 induced lockdown and travel restrictions). In contrast, using the GBW dataset, we identified a substantial seasonal decline in the blackbird weekly reporting rate which was associated with a concomitant reduction in weekly count in gardens, but not in ecologically similar control species, which was contemporaneous with the period of detected USUV activity in Greater London. These population trends are consistent with a hypothesis of disease-mediated decline. Alternative explanations, such as variation in climate, food availability or bird movement need consideration and are discussed next.Exploration of climate data indicates that, whilst the spring and early summer of 2020 was noteworthy with a high daily temperature average and low rainfall, at the time of USUV detection and the decline in the blackbird reporting rate, these parameters were within historical ranges (Supplementary Table 7). Consequently, while the climate may have been permissive for USUV transmission, there is no evidence to support variation in the weather alone as an explanation for the seasonal pattern of blackbird reporting rate decline; nor were declines observed in the robin or starling data, the control species with similar soil invertebrate diet and therefore similar vulnerability to summer drought. Blackbird, robin and starling populations in the UK are partially migratory; however, birds from mainland Europe do not migrate to overwinter in England until mid-October (i.e. after the decline in blackbird reporting rate occurred): consequently international bird movement does not offer an explanation for the observed regional blackbird decline. During the late summer season, short-distance movement from garden to non-garden habitats typically occurs, during the period of moult; however, the extent of the decline in blackbird reporting rate in gardens that occurred in Greater London in 2020 markedly exceeds that of the historical trend (2011–2019 inclusive; BTO unpubl. data). In summary, despite the fact that surveillance did not confirm further cases of wild bird USUV infection in 2020, and whilst it is not possible to ascribe causality, or exclude the chance that other factors may have contributed to the observed population trend, it remains possible that large-scale blackbird mortality due to USUV occurred in Greater London in summer 2020.Our study and others30 illustrate the need to integrate disease surveillance and long-term population monitoring schemes to evaluate disease impact, and to use control species to explore potential confounding drivers of population change (e.g. climate, food availability). Since GBW reporting rates are generated online in real-time, and nationwide, they offer a tool to rapidly detect changes in species presence (i.e. reporting rate) or flock size in gardens (i.e. weekly maximum count) that can be used to strategically enhance surveillance effort for disease detection. As wild bird ring recovery reports are also submitted online, there is also the potential to develop a complementary system that monitors for trends in occurrence of dead birds that might signal a disease outbreak. The BBS survey provides the most robust available data on population trends to appraise disease impact, however there is a delay of some months until data from this scheme become available. Since repeated incursions have occurred in mainland Europe following first detection1,17,31, it is likely that USUV will emerge in the UK again, either through overwintering or repeat incursion(s). Integrated disease surveillance in combination with bird population monitoring using the various available datasets, as we have capitalised on here, is required to assess whether USUV re-occurs, or becomes endemic, in UK wild birds and to identify any associated population impacts.By combining a range of professional and citizen science datasets our study approach facilitates the rapid detection of an emerging disease in free-living wildlife and enables insights into its incipient impact. We believe this multidisciplinary approach presents a framework for the early detection of disease outbreaks and incursion, thus helping to safeguard animal and public health. Such early warning systems could facilitate prompt mitigation action, for example targeted biosecurity measures and enhanced vigilance by medical and veterinary authorities. In addition, there is opportunity to further develop collaboration with ornithologists through active surveillance of wild birds, as was recently employed to detect West Nile virus in a migratory bird in the Netherlands32. Whilst population monitoring schemes are most developed for wild birds, lessons learned may be applied for the surveillance of diseases affecting other taxa. More

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