Lloviu cuevavirus (LLOV) was the first identified member of Filoviridae family outside the Ebola and Marburgvirus genera. A massive die-off of Schreibers’s bats (Miniopterus schreibersii) in the Iberian Peninsula in 2002 led to its initial discovery. Recent studies with recombinant and wild-type LLOV isolates confirmed the zoonotic nature of the virus in vitro. We examined bat samples from Italy for the presence of LLOV in an area outside of the currently known distribution range of the virus. We detected one positive sample from 2020, sequenced the complete coding region of the viral genome and established an infectious isolate of the virus. In addition, we performed the first comprehensive evolutionary analysis of the virus, using the Spanish, Hungarian and the Italian sequences. The most important achievement of this study is the establishment of an additional infectious LLOV isolate from a bat sample using the SuBK12-08 cells, demonstrating that this cell line is highly susceptible to LLOV infection and confirming the previous observation that these bats are effective hosts of the virus in nature. This result further strengthens the role of bats as the natural hosts for zoonotic filoviruses. Filoviridae family can cause serious illness and mortality in humans, including Ebola and Marburg viruses2. However, some members, like the Reston virus, do not cause disease in humans4. While ebolaviruses have received significant attention from the public and health experts due to past outbreaks, including the West African Ebola outbreak in 2013–2016 and a recent outbreak in Uganda, other filoviruses also pose a potential threat7. Research has shown that certain bat species are suspected as natural reservoirs for the Ebola virus, while only the Marburg and Ravn viruses have been isolated directly from bats before the recent isolation of Lloviu virus11. The natural host for other filoviruses is still unknown, but bats are considered as the likely natural hosts for these viruses. In recent years, several new filoviruses have been described in bats from Asia13, Africa15, and Europe16.Some viruses in the Lloviu cuevavirus (LLOV) was the first identified member of Filoviridae family outside the Ebola and Marburgvirus genera. Widespread, consecutive die-off events of Schreibers’s bats (Miniopterus schreibersii) in the Iberian Peninsula in 2002 led to its discovery. Almost complete genome sequence of the virus was retrieved with various sequencing approaches, although the infectious virus was not isolated from these bats at that time17. It has been reported that Schreibers’s bats in Spain and Hungary are LLOV-seropositive, confirming that the virus circulates there19. Viral RNA was sequenced from Spanish and Hungarian samples, and recently, an infectious isolate was established from the blood sample of a naturally infected bat in Hungary20.21.Studies with recombinant and wild-type LLOV isolates confirmed the susceptibility of human-derived cell lines and primary human macrophages to LLOV infection in vitro. Based on these data, LLOV is now considered as a potential zoonotic virus with unknown pathogenicity to humans and bats22. The recent isolation of the virus from Schreibers’s bats in Hungary confirmed the role of these bats as hosts for LLOV and perhaps as a reservoir species. It is unclear if the first detection of the virus in Spain coincided with an initial introduction of LLOV to European M. schreibersii populations. Schreibers’s bats roost in large colonies and are migratory, making them ideal reservoir hosts for the rapid and effective spread of certain infectious agents within their geographic distribution. Thus, it is possible that LLOV was already broadly circulating in these bats across the continent prior to the first detection of the virus in Spain in 2002.The ability of LLOV infection to cause disease in bats is unclear. Although no causative relationship has been established between Schreibers’s bat die-off events and circulation of LLOV within bat populations to date, the correlation is noteworthyThe aim of this study is to gain a better understanding of the presence, evolution, and connection to Schreibers’s bats of LLOV in previously unexplored geographical regions by using retrospective samples.To further explore the occurrence of LLOV in the wider distribution range of these bats and better understand its ecology and evolution, we screened 276 samples from Schreibers’s bats collected from four different locations in northern and central Italy between 2018 and 2021  (GenBank: ON186772). Compared to the reference LLOV genome from Spain (GenBank: NC_016144.1) and the Hungarian LLOV sequence (GenBank: MW775011), the sequence from the Italian LLOV sample showed 99.13% and 99.86% identity at nucleotide level, respectively. The phylogenetic analysis showed that the Italian LLOV isolate is more closely related to the Hungarian isolate  had a ratio above 1, implying the presence of positive selection pressure. The highest rate of synonymous mutations was found in the VP24 gene. Using the Spanish LLOV sequence as a baseline, we observed a roughly linear mutational rate for the Hungarian and Italian LLOV sequences with respect to the time of sample collection . Non-synonymous mutations are summarized in Table 23. Another interesting coding change we identified was in amino acid position 16 (F16L) of VP24. A similar but inverted change was found in aa position 26 (L26F) for EBOV VP24. This mutation was described as a molecular determinant of virulence in guinea pig model, with this single amino acid change in EBOV proving to be sufficient to cause increased pathogenesis and death in guinea pigs24. This observation was further confirmed in another study where EBOV amino acid variations associated with increased pathogenesis were studied23. Considering the phylogenetic relatedness and functional similarities of LLOV to EBOV, these mutations are prominent targets of functional experiments in the future to better understand LLOV evolution and pathogenesis.Analysis of the mutational landscape gives the first insight into the evolution of n Figure B. Non-syn Figure B. Non-syMiniopterus-derived cell line SuBK12-0826. After the first passage of the supernatant, cytopathic effect (CPE) was observed in the cells, similar to previous LLOV isolation studies19. To verify the presence of replicative LLOV, we performed RNA fluorescent in situ hybridization (FISH) staining with LLOV RNA-specific probes. We observed accumulation of LLOV genomic RNA in virus-induced inclusion bodies and a diffuse distribution of VP35 mRNA throughout the cytoplasm of the infected bat cells which are hallmarks of active viral replication  or greater mouse-eared bats (M. myotis). These traits and habits make ideal circumstances for the spread of diseases between and within colonies, possibly causing serious conservational threats for the bats22.Lloviu virus has now been detected in three European countries, suggesting a more widespread occurrence of the virus than previously anticipated Fig. A17–20. SConsidering the zoonotic potential of LLOV, there is an urgent need to better understand the evolution, ecological background, and molecular features of LLOV. Our optimised qRT-PCR system combined with a novel primer set allows for the rapid and sensitive screening for LLOV viral RNA in various samples derived from bats. Large-scale screening of archived and newly collected samples would help to investigate our hypothesis about the wide geographic distribution of the virus among these bats. We verified our hypothesis about the broader geographic range of Schreibers’s bats infected with LLOV and widened the possible active transmission area of the virus to the geographic range of its currently known host.29. LLOV-positive bats were detected in September both in Hungary and in Italy, although it is not yet clear if this points to a potential seasonality of the virus or if this reflects a random occurrence pattern. In case of European Bat Lyssavirus type 2, the peak season of virus prevalence correlated with the autumn swarming season of the bats30.The high viral load in the blood sample of the single LLOV-positive bat is in accordance with recent reports on LLOV in Schreibers’s bats in Hungary and with previous observations for other filoviruses in bats19. This is consistent with a previous report which found these cells to be highly susceptible to Vesicular Stomatitis Virus pseudotyped with LLOV GP26.The most important achievement of this study is the isolation of an additional infectious LLOV isolate from a bat sample collected in Italy using the SuBK12-08 cells, demonstrating that this cell line is highly susceptible to LLOV infection19. Extensive surveillance and sequencing efforts are essential to understand the potential role of Schreibers’s bats in the natural circulation of this virus and its evolution. Notably, these bats are mainly considered to be cave-dwelling, although in rare cases they might respond and adapt to habitat disruption by moving closer to humans to artificial roost sites. Indeed, the recent discovery of roosts hosting hundreds of Schreibers’s bats within urban settings in Italy raises concerns about the increasing risk of both direct bat-human contact as well as indirect contact through the spillover to domestic animals31. This also emphasizes the importance of the conservation of bats and their habitats as a means for limiting potential zoonotic spillovers to humans32.Notably, there is still no evidence for actual spillover of LLOV from bats to humans, but the repeated isolation of infectious LLOV from Schreibers’s bats highlight the need for additional studies to better understand potential spillover mechanisms. Urine and faeces of LLOV-infected bats were found to be negative in a recent study22. This report describes the detection of LLOV from live-sampled animals that were apparently healthy, with no reported unusual mortality events among these bats in the area. Investigating the circulation of LLOV among populations of this bat species, including the estimation and evaluation of mortality events, is crucial not only for public health but also for the conservation of Schreibers’s bats that are classified as Vulnerable on the IUCN Red List32.Cases of mass die-offs of Schreibers’s bats have been reported across Europe, including areas where LLOV was detectedBats were captured during the day with hand-nets in full compliance with best practice and national and European regulations, in derogation to the Council Directive 92/43/EEC of 21 May 1992 on the conservation of natural habitats and of wild fauna and flora . Animal handling was performed by experienced chiropterologists, no animals were harmed during field work. In vitro isolation procedures were performed under Biosafety Level 4 conditions within the laboratory of Szentágothai Research Centre, University of Pécs, Hungary.All methods were carried out in accordance with relevant guidelines and regulations, detailed in and approved by the previously mentioned permissions.33. During the regular colony checking occasions in 2018 and 2019, 54 Schreibers’s bat carcasses were collected from two sampling sites in North-eastern Italy. Also, in the same region but in a different cave a bat mortality among neonates were detected and 49 additional carcasses were collected in 2020. Altogether 103 lung samples were subjected to LLOV surveillance after the necropsy. Also, 173 samples from live animals involved in this study were collected from a single colony in Central Italy. Blood samples were obtained during four active surveillance campaigns between late summer 2020  and spring 2021 (April), to avoid disturbing the bat colonies during hibernation and the first month after birth. The detailed list of samples is available in the supplementary materials (S2).Bat species identification were performed according to morphological identification keysLung samples were homogenized in 300 µl Eagle’s Minimum Essential Medium  with the usage of a TissueLyser II . In the case of blood samples, the blood pellets were suspended in 300 µl EMEM. After a slight centrifugation (3 min – 8000 rpm), 200 µl supernatant was transferred for nucleic acid extraction.Lloviu cuevavirus isolate Hungary/2019/378 (GenBank: MZ541881.1). We used the following primer sequences: LLOV-Fw-scr1: 5′-AAGCATTTCCGAGTAATATGATGGTTG-3′, LLOV-Rev-scr1: 5′-TACATGGTCTCCTAGATTGCCCTG-3′, LLOV-Prob-scr1: 5′-FAM-CCTGATGAAGGAGAGTTTCTTTCTG-ZEN-3′ with the qRT-PCR Brilliant III Probe Master Mix  under these conditions: 50 °C for 10 min, 95 °C for 3 min and 50 cycles of 95 °C for 5 s, 60 °C for 30 s. All experiments were run on the MyGo Pro PCR system platform . Genomic copy number per millilitre was calculated from the Ct value and standard dilutions. Previously published LLOV specific targeted amplicon sequencing was used to sequence the native positive RNA sample. In brief, cDNA was generated using SuperScript IV  with random hexamers. Multiplex PCR reactions were conducted with Q5 HF Polymerase  using 3 different primer sets. Amplicon pools were end-repaired using NEBNext Ultra II End Repair/dA-Tailing Module . Barcodes from EXP-NBD196  were ligated to the amplicons with NEBNext Ultra II Ligation Module . For the sequencing, the LSK-110 kit  was used, and the AMX-F motor protein was ligated to the barcoding pool with NEBNext Quick Ligation Module . Cleaning steps between reactions were conducted with Ampure XP beads  using 75% ethanol except the last clean up where Small Fragment Buffer was applied. After the cleaning steps, the library was quantified using Qubit dsDNA HS Assay Kit  on a Qubit 4 fluorometer . The final library was sequenced on a R9.4.1 (FLO-MIN106D) flow cell.RNA was extracted from samples using Direct-Zol RNA Miniprep kit  according to the manufacturer’s recommendations. For LLOV RNA detection, a novel TaqMan PCR assay was developed and optimized using the 34. The processed reads were mapped to the reference genome (NC_016144.1) using Minimap2 (v. 2.23)35. The SAM to BAM conversion and consensus calling was conducted with samtools and bcftools (v. 1.17)36. The draft sequence was polished with Medaka (v. 1.72) Complete genomes from Filoviridae family were collected and aligned using Muscle algorithm37. Phylogenetic analyses were conducted in MEGA 1138.We ran the analysis under Ubuntu Linux 22.04 LTS operating system. The generated raw data was basecalled using Guppy basecaller (ONT guppy v6.3.2) with super-accurate model (dna_r9.4.1_450bps_sup config file). Demultiplexing was performed using Guppy barcoder with the necessity to find barcodes at both ends. For the quality check the NanoPlot (v1.33.0) software was used. We performed a quality (Q > 11) and length filtering with NanoFilt (v2.8.0) and 50 extra bases were trimmed2 in EMEM with 1% of L-Glutamine (200 mM) . The culture medium was supplemented with 2% heat inactivated Fetal Bovine Serum (FBS) , and 1% Penicillin/Streptomycin . The positive blood clot sample was used for inoculation, after adding 200 µl serum-free EMEM. After 1 h incubation at 37 °C and 5% CO2, the cells were washed once with serum-free cell culture media and the final supernatant, same as cell growth supernatant was added. The cells were monitored daily for cytopathic effects (CPE). After observing CPE, supernatant was subjected to nucleic acid extraction by Direct-Zol RNA MiniPrep Kit and analyzed for the presence of LLOV RNA by RT-PCR using LLOV-specific primers.SuBK12-08 cells were grown at 37 °C and 5% CO5 SubK12-08cells were seeded per well of 2-well chamber slides. One day later, the cells were mock-infected or infected with LLOV Italy. The cells were fixed 24 h post-infection in 10% formalin for at least 6 h. RNA FISH was performed using the RNAscope Multiplex Fluorescent V2 kit  with multiplexed, custom-designed probe sets in different imaging channels specific for LLOV VP35 mRNA and negative-sense genomic RNA in the nucleoprotein (NP) gene. Probes were designed based on the reference LLOV genome sequence from Spain (Genbank: NC_016144.1). LLOV mRNA was detected in channel 1 using probes targeting the VP35 transcripts and stained with Opal 520 fluorophore . LLOV genomic RNA was detected in channel 2 using probes targeting the negative-sense genomic sequences of the NP gene and stained with Opal 690 fluorophore . Staining was performed according to the manufacturer’s protocol for adherent cell samples, with the exception of an additional HRP blocking step following the signal development of the probes detecting viral mRNA as per the manufacturer’s recommendation. Nuclei were stained with kit supplied DAPI following the manufacturer's protocol. Coverslips were mounted onto the chamber slides using FluorSave mounting medium, and slides were subsequently stored at 4 °C prior to imaging. Images were acquired at 40× magnification using a Nikon Ti2 Eclipse microscope and Photometrics Prime BSI camera with NIS Elements AR software.6 × 10Supplementary Information.


Campylobacter enteritis (CE) remains on a high level in many parts of the world. The aim of this study was to analyze the health care utilizations and direct and indirect costs of CE and sequelae of patients insured by a large health insurance with 26 million members in Germany.The number of reported cases of Claims data of insurants with at least one CE diagnosis in 2017  were provided, of which 9,945 were included in the analysis of health care utilizations and costs. If medical services were not diagnosis-linked, CE-associated costs were estimated in comparison to up to three healthy controls per CE patient. Indirect costs were calculated by multiplying the work incapacities by the average labor costs. Total costs of CE in Germany were extrapolated by including all officially reported CE cases in 2017 using Monte Carlo simulations.Insurants showed a lower rate of 56 CE diagnoses per 100,000 than German surveillance data for 2017, but with a similar age, gender and regional distribution. Of those CE cases, 6.3% developed post-infectious reactive arthritis, Guillain-Barré syndrome (GBS), inflammatory bowel disease (IBD) and/or irritable bowel syndrome (IBS). Health care utilizations differed depending on CE severity, age and gender. Average CE-specific costs per patient receiving outpatient care were € 524 (95% CI 495–560) over a 12-month period, whereas costs per hospitalized CE case amounted to € 2,830 . The analyzed partial costs of sequelae ranged between € 221 (IBS) and € 22,721 (GBS) per patient per 12 months. Total costs of CE and sequelae extrapolated to Germany 2017 ranged between € 74.25 and € 95.19 million, of which 10–30% were due to sequelae.CE is associated with a substantial economic burden in Germany, also due to care-intensive long-lasting sequelae. However, uncertainties remain as to the causal relationship of IBD and IBS after CE. Campylobacter is a gram-negative bacterium that can cause acute gastrointestinal infections in humans. In the European Union, Campylobacter enteritis (CE) is the most reported gastrointestinal disease and the number of confirmed CE cases has stabilized on a high level in recent years [nt years . The cornt years –3.The symptoms associated with CE encompass diarrhea, abdominal pain, fever, nausea, vomiting and/or bloody stools, and are generally self-limiting within two weeks , 4. HoweCampylobacter contaminations in the broiler food chain [Risk factors for CE include the handling, preparation and consumption of chicken meat, drinking unpasteurized milk or untreated water, eating out, traveling, use of proton pump inhibitors (PPI) and animal contact, especially with poultry , 8. Variod chain . HoweverSeveral disease models were established to estimate the health and/or economic burden associated with CE and sequelae in different countries, and two of them were developed for Germany , 11. So In recent years, COI studies for CE were performed in Europe , New Zealand and North America, e.g. –17. TheyIn this study, we present an overview of the health care utilizations and associated costs of CE and sequelae in Germany over a 12-month period, which were analyzed based on the claims data of a large health insurance representing 26 million members. The study was designed and conducted by taking into consideration the recently published checklist for COI studies in the German context . In addiAnonymized claims data of a statutory health insurance (SHI) in Germany were used to assess the CE-specific health care utilizations and costs. The SHI in Germany is characterized by an universal health coverage with comprehensive benefits . The AOKth revision, German Modification [st of January 2017 and the 31st of December 2017 were provided by WIdO. However, the exact diagnosis date is usually not recorded within the claims data. Thus, insurants with CE were included either on the basis of the date of an outpatient CE-associated benefit, where available; otherwise, the first day of the regular quarterly diagnosis reports was used. In case of hospitalized insurants, the hospital discharge dates were taken . In this study, CE-associated claims were defined according to the positions of the doctor’s fee scale within the SHI scheme [Campylobacter, position 32723: stool test with at least five cultural media including testing for Yersinia (enterocolitica) and Campylobacter spp., position 32707: detection of bacterial antigens, and/or position 32001: provision or arrangement of laboratory services in general) or ii) mere medical suspicion of a reportable disease (position 32006). Subsequent CE diagnoses were considered as separate (new) CE diagnoses, if the timespan between the two diagnoses exceeded 90 days [Data sets of insurants with at least one diagnosis of CE , 10fication ) betweenI scheme  either a 90 days . In the st of December 2019. For a few hospitalized patients, the study period started with the quarter of their discharge from hospital in 2018. Insurants who were not continuously insured throughout the study period were excluded from the analysis. This also applies to insurants, which had passed away.The individual study period started with the quarter of the first CE diagnosis in 2017 and lasted until the 31The data sets comprised detailed information about health care utilizations and costs covered by the health insurance as well as co-payments made by insurants themselves referring to i) outpatient medical care, ii) inpatient medical care, iii) prescribed medication and iv) inpatient rehabilitation measures. For most medical services, details regarding the underlying diagnoses were available. Additionally, notifications and duration of incapacities for work as well as sociodemographic characteristics of insurants  could be obtained. Insurants were divided into six age groups: <5, 5 to 14, 15 to 29, 30 to 44, 45 to 64 and ≥65 years in consideration of i) the reported CE incidence rates, which are highest among small children and young adults .Disease severity was categorized based on the health care utilizations in the first year of the individual study period into:moderate: insurants seeking outpatient medical care and/or inpatient medical care with a secondary CE diagnosis, orsevere: insurants seeking inpatient medical care with a principal CE diagnosis.Insurants with asymptomatic CE or with mild CE symptoms, which had not made use of any (claimable) medical services, were not considered in the claims data analysis.In addition, probable sequelae of CE were also evaluated. Diseases as listed below were considered to be CE-associated in accordance with other established disease models of campylobacteriosis , 11:reactive arthritis (REA), ICD-10 code M02.1,Guillain-Barré syndrome (GBS), ICD-10 code G61.0,inflammatory bowel disease (IBD): Crohn’s disease (CD) and ulcerative colitis (UC), ICD-10 codes K50.0, K50.1, K50.9 and K51.-,irritable bowel syndrome (IBS), ICD-10 code K58.-,provided that these diagnoses were made concurrently with or subsequent to a CE diagnosis. Preexisting REA, GBS, IBD or IBS were not considered as sequelae, if they were already diagnosed within 12 months prior to the individual study period. The disease model is displayed in patient), three randomly selected insurants (control) matching in terms of age, gender and place of residence  were included. Controls were required to have no history of campylobacteriosis, i.e. no CE diagnoses within 12 months prior to and during the study period, and they had to be continuously insured. The beginning of the individual study period in controls was set to be identical to that of the matched patients.In order to compare insurants with and without CE regarding i) the occurrence of sequelae, ii) the presence of comorbidities and iii) the costs of medical services not diagnosis-linked, data were also provided for a control group: For each insurant with at least one CE diagnosis in 2017  and specialists associated with (confirmed) CE diagnoses or (confirmed or suspected) diagnoses of sequelae or consultations provided in hospitals,inpatient medical care: hospital stays associated with principal diagnoses of CE or sequelae,inpatient rehabilitations: rehabilitations of non-working patients associated with diagnoses of sequelae.Additionally, to determine productivity losses, the frequency and average duration of disease-related temporary work incapacities were assessed. The proportion of patients that had received prescriptions for medicines were also evaluated. As those prescriptions were not linked to diagnoses, the information given in national medical treatment guidelines was used instead. Medication for the treatment of CE recommended in the above-mentioned guidelines include antibiotics such as azithromycin or ciprofloxacin (but not erythromycin), analgesics and spasmolytics as paracetamol, opioids, metamizole and/or butylscopolamine, and oral rehydration solutions in special severe cases, when children are involved , 31. A pThe possibility that the use of PPI such as omeprazole and pantoprazole may increase the susceptibility to enteric diseases including CE is under discussion . TherefoCampylobacter enteritis and sequelae were calculated by analyzing direct and indirect costs [The costs of ct costs , 19. Thect costs . Therefoct costs , was disThe costs of medical care in hospitals, of rehabilitations and of work incapacities linked to diagnoses of CE or sequelae were assessed separately for each patient. The costs of outpatient medical care and of prescribed medication are usually not directly diagnosis-linked in the claims data. Therefore, CE-specific costs were estimated as additional costs in comparison to basic costs as incurred in the respective controls in the first year of the study period via two-part regression models: First, logistic regression was used to fit the probability of non-zero costs in moderate and severe CE. Then, the costs were estimated using an ordinary least squares regression of log-transformed non-zero costs. Age group and gender were included as explanatory variables. Regression analyses were performed in the subgroup of patients and controls matched by comorbidities. A similar estimation of sequelae-specific costs of outpatient medical care and of prescribed medication was not performed due to small case numbers.Total costs of CE cases in Germany in 2017 were extrapolated following an incidence approach: Based on the costs obtained over 12 months, total costs of all 2017 officially reported CE cases in Germany according to surveillance data [Average patient groups with increasing age range were formed and additionally separated according to gender (see above). However, in order to reflect differences in case numbers, mortality and the remaining life expectancy of the elderly, the age group ≥65 years was subdivided into two groups (65 to 74 and ≥75 years).For each patient group, age- and gender-specific numbers of reported CE cases, hospitalizations as well as deaths due to CE in Germany in 2017 were retrieved from the official statistics , 37. TheIt was assumed that the probability to develop sequelae derived from the AOK insurants would be equally applicable to all CE cases (base case model), while in a separate sensitivity analysis, probabilities from published meta-analyses , 39 wereFor GBS, age- and gender-specific parameters were incorporated into the model: The distribution of cases among the patient groups was based on published GBS incidence rates in Germany . Group-sIn order to extrapolate the total amount of expenses for CE in Germany, the number of reported moderate and severe CE cases were valued with the age- and gender-specific costs obtained from the claims data. An adjustment was included for multiple CE infections per patient over 12 months to approximate the costs per case.Similarly, each case of sequela was valued with the obtained partial costs over the assumed disease duration. As the 36-month observation period was too short to analyze the duration of long-lasting sequelae like GBS, IBD and IBS, published reports on the duration of sequelae were reviewed –50. For The CE-specific health care utilizations and costs were analyzed in the first year of the individual study period; for sequelae, the average utilizations and costs over a 12-month period were determined considering the individual time between the first and the last diagnosis of the sequelae within the study period.Health care cost data often show a right-skewed distribution due to a few outliers with extreme values, many zero costs and no costs less than zero. Therefore, non-parametric 95% confidence intervals of the mean costs were estimated –54 basedFor the extrapolation of total costs in Germany in 2017 a Monte Carlo simulation was performed, in which parameters varied simultaneously depending on a priori defined distribution functions  patients were available for the analysis of health care utilizations and costs.In 43.4% of the patients, the CE diagnosis was made in hospitals. In 8.7% of the patients, the (outpatient) diagnoses were recorded as About half of the study population was female; the median age was 47 years . The stuThe use of PPI was positively associated with a CE diagnosis: The adjusted odds of a pharmacy dispensing of prescribed PPI within the last four weeks was 3.2 times (95% CI 2.9–3.5) higher in patients than in controls  after the first diagnosis. In the first year of the study period, there was an average of 1.11 and 1.01 CE diagnoses per patient with moderate and severe CE, respectively.Previous CE diagnoses within 12 months prior to the study period were reported for 11.8 and 0.7% of the patients with moderate and severe CE, respectively. In general, the prevalence of previous or persistent REA, GBS, IBD and IBS was low . MoreoveHealth care utilizations associated with CE diagnoses differed significantly depending on disease severity . The perFor moderate CE patients aged 15 to 64 years, work incapacities lasted an average of eight days, which was longer than the three days for hospitalized patients. However, the proportion of patients with work incapacities was higher in the case of patients with severe CE than in the case of patients with moderate CE (57.5 vs. 18.9% of patients aged 15 to 64 years).CE-associated health care utilizations and incapacities for work differed significantly between age group and gender. A comprehensive listing of age- and gender-specific utilizations is provided in Table D in About half of the IBD and IBS patients had sought outpatient GP consultations; in the case of REA and GBS patients, the proportions were lower . InterniThe estimation of CE-specific costs of outpatient medical care and prescribed medication was based on 4,036 patients with at least one control matched by age, gender, state of residence and existing comorbidities . Insurants included in the regression analyses were younger and had fewer comorbidities and chronic conditions than the total study population . The regTotal costs of CE summed up to € 524 (95% CI € 495–560) per patient with moderate CE and € 2,830  per patient with severe CE over a 12 month-period. Total cost of illness was lowest for patients between five and 14 years of age and highest for the age group 30 to 64 for both moderate and severe CE . Direct The costs of hospital medical care and rehabilitations as well as the indirect costs associated with a diagnosis of GBS summed up to € 22,721  per patient over a 12-month period. Mean disease-related costs for REA, CD, UC and IBS were considerably lower  later on, even if positive laboratory results were made available. By taking only confirmed CE diagnoses into account, the rate of CE diagnoses was considerably lower . Moreover, in some cases, it seems possible that medical services were not included in the claims data, as they were not relevant for accounting or were provided outside the scope of the SHI-accredited care.Interestingly, AOK insurants appeared to be more frequently hospitalized: While 25.6% of the claimed CE diagnoses in 2017 were principal diagnoses made in hospitals, official statistics revealed 13,159 hospitalizations due to principal CE diagnoses in Germany corresponding to 18.9% of the officially reported 69,476 CE cases in 2017 . TherefoWe investigated whether the AOK insurants can be considered representative for Germany–both in general and in terms of CE cases: No major differences in the distribution of age groups and gender between the 26 million AOK insurants and the general population of 83 million were found, although the proportion of inhabitants insured by AOK varied across the Federal States  recommended in medical treatment guidelines and ii) occurred within the same annual quarter. However, this might not necessarily be the case. Moreover, there might be discrepancies between the recommended and the actual treatment of CE. For example, erythromycin was still a frequently taken antibiotic in a survey of CE cases in Germany between 2011 and 2014 [The analysis of and 2014 . A separThe analysis also showed that the median duration of work incapacities of severe CE patients was shorter than their hospital stays, which seems like a self-contradictory result. This underestimation can be explained by i) certificates of incapacity for work are usually issued to employees only , ii) it is possible that not all employees submitted a separate copy of the certificate to the health insurance as required, and iii) depending on the employer, no certificate is needed until the fourth day of sick leave. Short-term absences that were not recorded within the claims data are particularly likely in the case of not severely affected patients. Therefore, the duration of work incapacities in patients with moderate CE might be overestimated due to the missing short-term absences, which also explains their longer median duration compared to patients with severe CE. The true proportion–and in the case of severe CE also the duration–of sick leaves are most probably higher than the numbers extracted solely from the claims data. This assumption is supported by a recent survey of 1,800 CE cases in Germany, in which a significantly higher percentage of self-reported work absences of 79%, including those of working parents with sick children, was observed .Beside the absences from work, presenteeism–i.e. going to work despite reduced productivity for example due to a disease–is known to increase the indirect costs. Presenteeism is rarely included in cost of illness studies until now, also because there is no consensus on how it can be measured appropriately . The samCampylobacter enteritisdirect and indirect costs of  in other countries range from a total of € 350 per CE case (not differentiating between moderate and severe) in the Netherlands to € 540 in Sweden to $ 1,020 in the USA [Estimates of the  the USA , 13, 15. the USA , and oveSevere CE caused 4.4 times higher costs than moderate CE. In contrast, the modeling approach for the USA suggests 23 times higher costs in severe compared to moderate CE cases  with a similar definition of disease severity . These dHowever, some assumptions had to be made in order to evaluate disease-specific utilizations and costs, e.g. the CE-attribution of benefits that are not diagnosis-linked in the claims data. To prevent an overestimation of hospital stays due to other–probably more care- and cost-intensive–diseases, hospital stays associated with secondary CE diagnoses were not considered disease-related and consequently not analyzed.confirmed diagnoses, we could not identify outpatient consultations nor laboratory tests for some of the moderate CE patients. Possible explanations for this observation could be that i) not all outpatient medical services were claimed separately due to lump sum compensations, and ii) some codes related to single benefits were not identifiable, as different additional regional contracts exist between the association of SHI physicians and the health insurance, and/or due to potential entry errors.Although one would expect that detection of the pathogen or respective antibodies or antigens following a physician’s consultation is a prerequisite for making Campylobacter. Although the subgroup was younger than the total study population, the excess costs could be estimated for all six age groups. Unfortunately, a comparable approach for sequelae was not possible due to small case numbers. This is also the reason why there was no subgroup analysis by age for sequelae, although symptoms and treatment of GBS, IBD and IBS differ between children and adults [A major limitation of analyzing the COI according to German claims data is the missing link between the underlying diagnoses and the costs of outpatient medical care and of prescribed medication. The CE-specific costs were therefore estimated in a subgroup of patients and controls with matching comorbidities. The additional risk adjustment was necessary, as the randomly selected controls seemed to be healthier according to the Elixhauser comorbidity index and the pharmacy-based metric. The difference in comorbidities might also be induced by the health-seeking behavior of patients: Insurants with comorbidities are more likely to consult a physician due to other health problems, which also increases the probability of getting tested for d adults , 61, 62.societal perspective. However, most costs reflect the payer’s perspective, as claims data were used. By including patients’ co-payments where available and by monetizing the incapacities for work, an approximation of the societal costs could be achieved [Health care costs were analyzed from a achieved . Neverthachieved  are not achieved , 64 and achieved , 65, 66.Campylobacter-associated GBS seems to be more severe and of longer duration than other GBS [In a recent claims data analysis from Germany, mean total costs of CD and UC per year were € 10,100 and € 8,770, respectively, and medication costs were responsible for around 50% of total COI . Howeverther GBS , 70. Thecourse of CE and the development of sequelae. The proportion of patients who developed GBS and IBS are similar to previously published rates of 0.07 and 4.0%, respectively [The study period was limited to 36 months to generate current estimates of costs incurred in 2017/2018, but also to obtain some information about the ectively . In contectively , 39. Witectively . Diagnosectively .Campylobacter and an initial manifestation of IBD may be difficult [Campylobacter enteritis and a sequela supports a causal relationship, but this might not always be the case.The observed new IBD diagnoses could overestimate the actual number of CE-associated cases, as the distinction between a self-limiting gastrointestinal disease caused by ifficult , 46. Morifficult , 69, whiifficult , 11. In There was a high number of CE patients with preexisting IBD (n = 170) and IBS (n = 181), which were not considered to be CE-related in our analysis. This could explain the significantly higher proportion of patients taking medication for chronic IBD according to the pharmacy-based metric compared to controls.st of December 2019. Moreover, some providers might claim health services with a delay, so that sequelae-related services from 2019 may be missing in the dataset. Therefore, published results and assumptions on the duration of sequelae were used for the extrapolation of total COI in Germany. The total costs associated with CE cases in 2017 varied considerably, depending on the assumed probability to develop sequelae. The observed higher frequencies of CD and UC for AOK insurants compared to published figures [The duration of the respective sequelae was approximated by using the first and the last day of the disease-specific utilizations available from the claims data. However, this probably does not reflect the average disease duration of sequelae due to small case numbers, the limited study period of up to 36 months and the possibility of missing diagnosis dates in the claims data. While for REA the timespan between the first and the last diagnosis was found to be maximally 20 months, the treatment of other sequelae might have continued after the 31 figures  led to s figures , 13, 15.Campylobacter were not included in the extrapolation. In Germany, the estimated number of cases might be four to nine times as high as the officially reported cases [To estimate the total societal economic burden of campylobacteriosis in 2017, we assumed that all officially reported CE cases in Germany caused the same average amount of expenses according to their assignments into the respective patient groups . This might not completely reflect the situation, as 12.8% of the population in Germany is not statutorily insured  and medied cases , 74. Mosmedication with PPI, which explained 10% of CE cases in a German case-control study with an adjusted odds ratio of 1.9 [It is estimated that 20 to 30% of the human CE cases can be attributed to the preparation, handling and consumption of contaminated chicken . Anothero of 1.9 . The clao of 1.9 . In the o of 1.9 , 75.In this study, detailed information on the health care utilizations and associated costs of CE patients in Germany are presented for the first time. The results show that CE is associated with a substantial economic burden–even if IBD and/or IBS are not considered as sequelae of CE. As far as we know, this study is the largest claims data analysis regarding CE, with almost 10,000 cases included. It is even more noteworthy that PPI could be confirmed as a risk factor for CE on the basis of this large data set.S1 File(PDF)Click here for additional data file.


Daucus carota ssp. sativus) and wild carrot (Daucus carota ssp. carota) populations, is particularly interesting with respect to comparisons of virus communities due to the low genetic barrier to virus flow since both population types belong to the same plant species. Using a highly purified double-stranded RNA-based HTS approach, we analyzed on a large scale the virome of 45 carrot populations including cultivated, wild and off-type carrots (carrots growing within the field and likely representing hybrids between cultivated and wild carrots) in France and six additional carrot populations from central Spain. Globally, we identified a very rich virome comprising 45 viruses of which 25 are novel or tentatively novel. Most of the identified novel viruses showed preferential associations with wild carrots, either occurring exclusively in wild populations or infecting only a small proportion of cultivated populations, indicating the role of wild carrots as reservoir of viral diversity. The carrot virome proved particularly rich in viruses involved in complex mutual interdependencies for aphid transmission such as poleroviruses, umbraviruses and associated satellites, which can be the basis for further investigations of synergistic or antagonistic virus-vector-host relationships.High-throughput sequencing (HTS) has proven a powerful tool to uncover the virome of cultivated and wild plants and offers the opportunity to study virus movements across the agroecological interface. The carrot model consisting of cultivated ( Agriculture has drastically changed biodiversity at the landscape level by converting natural habitats into intensively managed environments. As a result, an increasing proportion of natural habitats lie adjacent to agricultural lands, creating what is referred to as the agroecological interface, which integrates wild lands as highly fragmented patches within a matrix of crop-, pasture- and grasslands . Such inApiaceae comprises about 3,700 species consisting of wild plants and important crops [Daucus carota L) is the most widely grown crop within the Apiaceae family. In total, more than 30 viruses from 12 different families are known to infect carrots [Sequivirus) and the carrot motley dwarf (CMD) complex. CNDBV was originally described as the Anthriscus strain of parsnip yellow fleck virus  [Anthriscus sylvestris L.). In carrots, it causes leaf necrosis, severe stunting and dieback [Cavariella aegopodii. The CMD complex causes leaf reddening and yellowing, stunting and reduces sugar content of taproots [Chordovirus and Closterovirus genera have also been identified [The family nt crops . Carrot  carrots –11 with  dieback . The CMD dieback  and is c dieback . The umbtaproots , 15. Anotaproots . Additioentified . HoweverDaucus carota ssp. sativus) and its weedy relative, wild carrot (Daucus carota ssp. carota), belong to the same species and therefore represent a host pair with the lowest expected genetic barrier to virus flow. The aim of the present study was to characterize virus communities in wild and cultivated carrots using a double-stranded RNA (dsRNA)-based metagenomic approach. We have identified a rich and diverse carrot virome in different regions of France and central Spain, providing the basis for further studies on the role of wild plants as virus reservoirs and on the complex interplay and interdependencies of viruses in mixed infections as well as the underlying evolutionary processes in relation to their vectors and hosts.Recent advances in sequencing technologies such as high-throughput sequencing (HTS) have revolutionized the field of viral ecology over the past decade by moving from a "one host-one pathogen" perspective to analyzing the entire phytovirome at ecosystem level. HTS-based studies have highlighted the importance of wild plants as a source of virus diversity –21. MoreIn July-August 2019, 16 different carrot populations were sampled in south-west France (Gironde and Landes Départements of the Nouvelle Aquitaine region)  until use. For each population, a pool corresponding to the 50 sampled plants was assembled .In each sampled population, leaves from 50 individual plants were collected. Carrot plants were sampled regardless of the presence of viral symptoms, allowing to take into account asymptomatic viral infections, but plants with evident fungal infection or necrosis were excluded. Leaf samples were stored desiccated over anhydrous CaClDouble-stranded (ds) RNAs were purified from each pool consisting of 50 carrot plants. Samples were ground to a fine powder using liquid nitrogen and a mortar and pestle and dsRNAs purified by two rounds of CC41 cellulose chromatography as described . A negatde novo using a minimal contig length of 250 nt. Viral contigs were annotated by a comparison against the non-redundant sequence (nr) database in GenBank using BlastX [Sequencing reads were demultiplexed and trimmed on quality using CLC Genomic Workbench, version 21.0.3  using default settings and a minimum read length of 60 nucleotides (nt). Contigs were assembled g BlastX . Identifg BlastX  and multIn order to determine the geographic distribution of the identified viruses, reads from datasets, normalized to a common depth per sample using the read sampling tool in CLC-GW, were then mapped back to reference contigs (for novel viruses) and reference genomic sequences from GenBank (for known viruses) using CLC-GW and nt identity and length fraction set at a minimum of 90%. The mapped reads were then manually checked to ensure the identity of the detected virus. Two approaches were used to address the well-known HTS inter-sample cross talk issue. The first one considered the viral reads identified for each virus in the blank control for each run. The second one used data from individually analyzed plants for which we also had both virus-specific RT-PCR testing results, so that plants that tested negative in RT-PCR but showing a few reads for a given virus in the corresponding dataset could be used calculate an average number of background reads per million sequencing reads. With this strategy, a separate detection threshold could be calculated for those viruses that were screened by RT-PCR. For viruses not tested by RT-PCR, we used as a threshold the average of the threshold values determined for the RT-PCR tested viruses. Finally, the thresholds obtained for each virus using either the blank controls or the RT-PCR testing were compared and the highest value retained to establish the final HTS positive detection threshold for each virus and clean the reads mapping table for each sample/virus combination. Ultimately, identification of reads for a particular virus above the threshold translates in presence of this virus in the tested carrot population and at the site of that population.In some cases, genomes were completed by determining 5’ and 3’ genome ends by Rapid Amplification cDNA Ends (RACE) experiments using specific primers (S2 Table in Specific two-step RT-PCR assays  were devOpen reading frames (ORFs) were predicted using CLC-GW and confirmed using the ExPASy Translate Tool . MultiplRiboviria whose precise higher order taxonomic status remains to be established  in 2021. Details on the sampled carrot populations are shown in S1 Table in ablished . DetailsUmbravirus. All three umbraviruses known to infect carrots, including carrot mottle virus (CMoV), carrot mottle mimic virus (CMoMV) and the more recently reported wild carrot mottle virus (WCMoV) have been identified in the three different carrot population types . They were detected in more than 90% of all sampled populations in France and Spain. In addition, we identified in different French cultivated and wild populations a virus of uncertain taxonomic status related to CMoV and referred to here as carrot umbravirus 1 (CaUV1), and two novel umbraviruses, carrot umbravirus 2 (CaUV2) and carrot umbravirus 3 (CaUV3). In all cases, the genome organization was typical for the genus with four ORFs coding respectively from 5’ to 3’ for a replicase-associated protein, a RNA-dependent RNA polymerase  and long distance movement and cell to cell movement proteins encoded by overlapping 3’ ORFs. A phylogenetic tree based on a multiple nt alignment of the RdRp genes for known umbraviruses and for those novel ones reported here is shown in The French and Spanish carrot viromes were found to be rich in viruses of the genus Umbravirus genus. However, the available CaUV1 sequence covers only 67% of the full RdRp gene and it is therefore not possible to estimate the identity percentage that would be reached if full genomes were compared and, therefore, to decide whether CaUV1 should be regarded as a variant of PasUV1 or CMoV or as belonging to a new umbravirus species. A recombination analysis performed using the multiple nt sequence alignment used for phylogenetic analysis suggested the existence of a recombination event linking PasUV1, CMoV and CaUV1. Details of this recombination event, detected by all algorithms and with a combined Bonferoni corrected probability of 1.7xE-35 are shown in A 3.3kb scaffold with >3,000-fold coverage could be obtained for CaUV1, covering an estimated 79% of the full genome. This sequence shows a pairwise nt identity of 78% and 76% to pastinaca umbravirus 1  and CMoV (GenBank KF533714), respectively, in its partial RdRp gene  and in a smaller proportion of field carrot populations. CaUV2 was detected in 33.3% (5/15) of cultivated and 42.8% (3/7) of off-type ones, while the corresponding values for CaUV3 were respectively 13.3% (2/15) and 57.1% (4/7). These two novel umbraviruses were not detected in any of the six Spanish carrot populations.Nearly complete genomes of the other two novel umbraviruses, CaUV2 and CaUV3 could be assembled of respectively 4.25 and 4.06 kb and 605x and 1421x average coverage. These assembled genomes show highest nt pairwise identities of respectively 64% and 61% with carrot mottle mimic virus  and red clover umbravirus , based on full genome alignments  is a well-known carrot-infecting polerovirus that is part of the complex responsible for the CMD disease affecting carrot crops. CtRLV was detected in most of the sampled French populations  and in all Spanish cultivated populations , but not the wild one. The sequence of a distinct polerovirus, tentatively named wild carrot red leaf virus was deposited in 2020 in GenBank (LT615231). This virus was found exclusively in wild carrot populations, infecting 17/23 of wild populations sampled over two years in France. It was not detected in any of the sampled Spanish populations. A novel enamovirus, tentatively named carrot enamovirus 1 (CaEV1) and two novel poleroviruses tentatively named carrot polerovirus 1 (CaPV1) and carrot polerovirus 2 (CaPV2) were additionally discovered in various French carrot populations .Polerovirus with seven predicted ORFs encoding proteins P0 to P5 as well as a P3a  with CtRLV as major parent and CaPV1 as minor parent, the recombined region concerning part of the CP readthrough (CP-RT) domain (-9. A summary of both recombination events is given in Recombination events have been identified between members of the ) domain . AnotherEnamovirus genus. It was found more prevalent in wild carrot populations, being detected in 11/23 wild populations but in only 2/15 cultivated populations and one off-type population from France. It was not detected in Spanish carrot populations. In France, it was geographically limited to southwest of France including Nouvelle Aquitaine and one population sampled in the region Occitanie  due to their phylogenetic affinities with members of the Tombusviridae family [Several polerovirus-associated RNAs have been identified in the French and Spanish carrots virome including carrot red leaf virus-associated RNA (CtRLVaRNA), beet western yellows virus-associated RNA (BWYVaRNA), and two other such molecules detected for the first time in carrots, arracacha latent virus E-associated RNA (ALVEaRNA) and a new RNA, carrot red leaf virus-associated RNA 2 (CtRLVaRNA-2). These self-replicating subviral agents depend on helper viruses of the family e family . A detaie family . Moreovee family .Two linear single-stranded satellite RNAs of respectively 745 and 748 nt and named carrot mottle virus satellite RNA (CMoVsatRNA) and carrot mottle mimic virus satellite RNA (CMoMVsatRNA) are known . These tTwo chordoviruses, carrot chordovirus 1 (CChV1) and carrot chordovirus 2 (CChV2) have recently been described from carrot samples in the UK . Both viOK032418). Pairwise comparisons of CChV3 with the closest related sequences from GenBank show highest pairwise aa identities of 67% and 69% to hogweed virus 4 in the RdRp and CP gene, respectively , a capsid protein (Pfam PF05892) and a fourth protein (12.3 kDa) of unknown function  and carrot chordovirus 4 (CChV4) are proposed, have been identified in one off-type population , was identified in a single population of wild carrots sampled in 2020 in Gironde, France  or beet yellow stunt virus  (Carrot yellow leaf virus (CYLV), a known member of the  members  includin U51931) . This ge U51931) . In addi U51931) . Lastly,The two novel closteroviruses share respectively 83% and 61% aa identity in their RdRp and in the full ORF1a-ORF1b fusion protein, respectively. Pairwise comparisons in the HSP70h and the CP show respectively 80% and 70% aa identity between CtCV2 and CtCV3  and group in separate subclusters of a clade composed of rice ragged stunt virus  and unclassified plant-associated viruses, including grapevine Cabernet Sauvignon reovirus (GCSV) and raspberry latent virus (RpLV)  and carrot reovirus 2 (CaRV2), were identified in French wild carrot populations. Contigs for ten RNA genomic segments could be obtained for CaRV1, which was identified in three wild populations  . The late family . Eight oe family , with thPartitiviridae have been reported to infect carrots. A member of the genus Alphapartitivirus, carrot cryptic virus (CaCV), has been reported in Germany [Partitiviridae members have been reported in Japan [th Report of Virus Taxonomy [Alphapartitivirus genus and CteV3 in the genus Betapartitivirus. CaCV has been detected in a high proportion of cultivated  and off-type populations  but was only detected in one wild population sampled near Bordeaux  was detected in all three population types, infecting a higher proportion of wild carrot populations  than cultivated  or off type ones . It was geographically limited to the region Nouvelle-Aquitaine  and carrot cryptic virus 3 (CaCV3) and typically possess two RNA segments of about 1.5 kb each. RNA1 codes for the viral RdRp and RNA2 for the CP. CaCV2 was detected in 8/23 (34.8%) cultivated and 4/15 wild populations (26.7%), both in the north and southwest of France  were detected in several French wild and off-type populations in both sampling years. This virus was not identified from French cultivated populations or from Spanish carrots. A nearly complete genome of this new amalgavirus, referred to as carrot amalgavirus 1 (CaAV1), was obtained from a wild carrot population sampled in 2020 in France . Even though the sequence of CaAV2 is only partial, raising uncertainties about the divergence level when analyzing complete proteins, pairwise identity levels between recognized amalgavirus species in the region encompassed by the CaAV2 contig are similar to the CaAV1-CaAV2 divergence level, providing evidence that the two viruses likely correspond to different species. Both viruses cluster unambiguously within the Amalgavirus genus and away from Zygosaccharomyces bailii virus Z (GenBank NP_624325), the only member of the second genus recognized in the Amalgaviridae family (genus Zybavirus)  .Totiviridae family, and the encoded proteins show affinities to those of Toviviridae members, so that it is proposed to refer to the identified virus as carrot-associated toti-like virus (CaaTLV). In a BLASTN search, CaaTLV shares highest identity with black raspberry F virus . Pairwise comparisons of its CP and RdRp confirm BRVF as the most closely related virus with 65% and 73% aa identity in the CP and RdRp, respectively  and Dactylorhiza hatagirea beny-like virus (BK013327), which show pairwise RdRP aa identity levels of 43% and 39%, respectively  contigs were obtained from different French cultivated and off-type carrot populations in France. These contigs span the nearly complete genome of a novel virus showing homologies to members of the  members . In a BLe family . The virFurther identified putative novel Riboviria comprise carrot flavi-like virus 1 (CtFLV1) and carrot Ker-like virus (CaKLV). The molecular characterization of CtFLV1 and its geographical distribution has been recently described  while CaIn the past decade, several studies have begun to explore the largely underestimated viral diversity in unmanaged ecosystems and the role of wild plants as reservoirs of virus diversity –25. The Partitiviridae member distinct from those reported here. The range of carrot-infecting viruses with dsRNA genomes was extended through the identification of novel members of the families Amalgaviridae, Totiviridae, Spinareoviridae as well as two novel viruses of the Deltaparitivirus genus and one new host record for a divergent isolate of the betapartitivirus DiCV2. Most of the identified novel viruses are +ssRNA viruses that are aphid transmitted , that lack a CP gene, do not form virus particles and therefore rely on helper viruses, characteristically from the family Solemoviridae (formerly Luteoviridae) for encapsidation and aphid transmission [Umbravirus), tobacco distorting vein virus (genus Polerovirus) and their associated satellites in China’s Yunnan province [Enamovirus) was not observed in French or Spanish carrot viromes, arracacha latent virus E-associated RNA (ALVEaRNA) was frequently identified, often in coinfection with CtRLV, which likely fulfils a helper virus role, thus indicating some degree of plasticity in the association between different helper and dependent viruses [Polerovirus genus, including a statistically highly significant recombination in the CP-readthrough (CP-RT) protein involving WCtRLV, CtRLV and the novel CaPV1. The CP-RT domain of the polerovirus genome has been shown to be the most variable genome part, with a high frequency of sites subject to positive selection, and has been suggested to be a major player in mediating adaptation to host plants and to aphid vectors [In a limited screening of public carrot RNASeq transcriptomic data in GenBank sequence reads archives (SRAs), a total of seven viruses were identified, five of which are in common with the present study, with the other two being the potyvirus carrot thin leaf virus and a novel nsmitted , includismission . The carsmission . In addismission  were detsmission , 51 but smission . Moveoveprovince . To gain viruses . Co-infe vectors . Indeed,Cavariella spp. [Closteroviridae family but their order is not. A similar gene reshuffling is seen for example in the genera Ampelovirus and Crinivirus with a reversed order of the CPd and CP ORFs [Two closteroviruses were previously known from carrot, CYLV and CtCV1. CYLV was first isolated from carrots showing leaf yellowing in Japan  and is slla spp. . similarlla spp. . In complla spp. , five ge CP ORFs .Betaflexiviridae. The genus Chordovirus was recently accepted within the family Betaflexiviridae  and comprises carrot chordovirus 1 and 2 (CChV1 and CChV2), identified in the UK [Betaflexiviridae family have no known insect or fungal vector. However, carlaviruses are generally transmitted by aphid vectors, while some vitiviruses have been reported to be transmitted by mealybugs [Ovatus crataegarius and Cavariella spp. [Betaflexiviridae family members identified in carrot populations and their potential dependence on other viruses for transmission.Another group of +ssRNA viruses identified is this study involves the family n the UK , lettucen the UK  and hogwn the UK . We idenealybugs  and by tlla spp. . Multipllla spp. . No infePreviously unknown putative novel Riboviria have been found in different carrot populations including the recently described CtFLV1 , carrot-et al., submited). The failure to detect such negative-stranded RNA viruses might therefore mark either their absence/low prevalence in the studied carrot viromes or, possibly, a methodological bias.We did not identify any negative-stranded RNA viruses in the studied carrot viromes. While such viruses have been observed in several large virome studies –21, 25 tMost of the identified novel viruses showed preferential associations with wild carrots, either being exclusively identified from wild populations or infecting only a small proportion of cultivated populations . This suS1 FileS2 Table. RT-PCR and RACE primer pairs targeting known and novel viruses in the carrot virome. S3 Table. RNA-dependent RNA polymerase (RdRp) pairwise amino acid identities for carrot umbraviruses 1, 2 and 3 and selected genus Umbravirus members. S4 Table. RdRp pairwise amino acid identities for carrot poleroviruses 1 and 2, carrot enamovirus 1 and selected family Solemoviridae members. S5 Table. Geographical distribution of the various viruses identified in the carrot virome based on merged mapping of HTS data obtained from carrot populations from different regions of France and in Spain. Green squares indicate presence, red squares indicate absence of the virus in the respective locations. Novel viruses are indicated in red. S6 Table. RdRp and capsid protein (CP) pairwise amino acid identities for carrot chordoviruses 3 and 4, carrot vitivirus 1 and selected family Betaflexiviridae members. S7 Table. RdRp, heat-shock protein 70 (hsp70) and CP pairwise amino acid identities for carrot closteroviruses 2 and 3 and selected family Closteroviridae members. S8 Table. Full genome pairwise nucleotide identities and RdRp pairwise amino acid identities for carrot endornavirus 1 and selected family Endornaviridae members. S9 Table. RdRp pairwise amino acid identities for carrot reoviruses 1 and 2 and selected family Spinareoviridae members. S10 Table. RdRp pairwise amino acid identities for carrot cryptic viruses 2 and 3, dill cryptic virus 2 carrot isolate and selected family Partitiviridae members. S11 Table. RdRp fusion protein pairwise amino acid identities for Carrot_amalgaviruses 1 and 2 and selected family Amalgaviridae members. S12 Table. RdRp and CP pairwise amino acid identities for Carrot_associated_toti-like_virus and selected family Totiviridae members. S13 Table. RdRp pairwise amino acid identities for Carrot_associated_RNA_virus_1 and selected family Benyviridae members.Information on sampled carrot populations in France and Spain. (XLSX)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.


This study was based on similar physicochemical characteristics of pseudorabies virus (PRV) and African swine fever virus (ASFV). A cellular model for evaluation of disinfectants was established with PRV as an alternative marker strain. In the present study, we evaluated the disinfection performance of commonly used commercialized disinfectants on PRV to provide a reference for the selection of good ASFV disinfectants. In addition, the disinfection (anti-virus) performances for four disinfectants were investigated based on the minimum effective concentration, onset time, action time, and operating temperature. Our results demonstrated that glutaraldehyde decamethylammonium bromide solution, peracetic acid solution, sodium dichloroisocyanurate, and povidone-iodine solution effectively inactivated PRV at concentrations 0.1, 0.5, 0.5, and 2.5 g/L on different time points 30, 5, 10, and 10 min, respectively. Specifically, peracetic acid exhibits optimized overall performance. Glutaraldehyde decamethylammonium bromide is cost effective but requires a long action time and the disinfectant activity is severely affected by low temperatures. Furthermore, povidone-iodine rapidly inactivates the virus and is not affected by environmental temperature, but its application is limited by a poor dilution ratio such as for local disinfection of the skin. This study provides a reference for the selection of disinfectants for ASFV. African swine fever (ASF) is an acute, severe, and highly contagious hemorrhagic viral disease with high morbidity and mortality caused by the African swine fever virus (ASFV) . It has ASFV is a member of the family Asfarviridae and the genus Asfivirus. It is a large, enveloped DNA virus. Its virions have a specific icosahedral structure of a protein capsid, which surrounds an internal membrane and a nucleoprotein core. It shows high similarity to other enveloped viruses in terms of particle size, genome size, and physicochemical properties ,13,14. TIn this context, the availability of data regarding the virucidal activity of chemical compounds against ASFV provides useful information to properly plan decontamination procedures aimed at preventing the introduction of ASFV and limiting its secondary spread . The ide2.The fluorescent-tag virus rPRV-ZJ was constructed and stored by our laboratory at the Zhejiang Academy of Agricultural Sciences (ZAAS) . BHK-21 Four disinfectants, including commercially available quaternary ammonium salts, iodine preparations, chloride preparations, and peroxides, were selected in this study. All products were approved by the authority and did not expire during the study.Glutaraldehyde decamethylammonium bromide solution : the recommended dilutions were 1:2000–1:4000, 1:500–1:1000, and 1:1500–1:3000 for conventional environmental spraying disinfection, environmental spraying disinfection during the epidemic, and immersion disinfection of instruments and equipment, respectively.Povidone-iodine solution  240041575): the recommended concentrations were 5, 0.5–1.0, and 0.1% for skin disinfection and treatment of skin diseases, immersion disinfection of cow teats, and flushing of mucosa and wound, respectively.2.Peracetic acid solution : the recommended concentrations were 2.0, 0.5, and 1.0 g/L for spray disinfection of animal houses and instrument, immersion disinfection of food utensils of livestock and staff clothes, and disinfection of epidemic focus, respectively.The neutralizers corresponding to glutaraldehyde decamethylammonium bromide solution, povidone-iodine solution, peracetic acid solution and sodium dichloroisocyanurate were 0.1% Tween 80 + 0.3% glycine (in PBS), 0.1 mol/L sodium thiosulfate (in PBS), 0.1 mol/L sodium thiosulfate (in PBS) and 0.2 mol/L sodium thiosulfate (in PBS), respectively ,24,25.2•6H2O into 1000 mL of distilled water.All disinfectants and neutralizers used in this study were prepared using standard hard water following the Technical Specifications for Disinfection. Specifically, the standard hard water was prepared by adding 0.034 g of CaCl2 and 0.139 g of MgClg for 10 min at 4 °C and the virus supernatant was collected. The virus suspension was aliquoted and stored at −80 °C. The virus suspension samples were diluted 10 times (gradient dilution) using serum-free DMEM cell culture solution and inoculated on a 96-well culture plate covered by a single layer of BHK-21 cells. Each gradient had eight replicates containing 100 µL sample suspension. For negative control, samples were diluted with DMEM cell culture solution containing 2% FBS and cultured at 37 °C and 5% CO2 for 96 h consecutively; cytopathic effect (CPE) changes (if any) of cells were recorded. Additionally, TCID50/100 μL titration of the virus was calculated using the Reed–Muench method.The rPRV-ZJ strain was cultured on BHK-21 cells for proliferation. After three freeze-thaw cycles, sample was centrifuged at 8000× According to the Technical Specifications for Disinfection by the Ministry of Health, the People’s Republic of China, the effects of neutralizer, disinfectant, and neutralized products on the growth of BHK-21 cells were evaluated. Herein, four groups were established, including the disinfectant group (Group A), the neutralizer group (Group B), the neutralized products group (Group C), and the negative control group (Group D). All reagents were inoculated on BHK-21 cells, mixed, and then cultured for 3–4 h. Cell growth was observed under a microscope .According to Technical Specifications for Disinfection 2002, a disinfectant solution was prepared of doubled concentration (2X), and then virus suspension of equal volume was added with mixing. Next, after the designated period of virus inactivation, a neutralizer solution of equal volume was added. After 10 min, the virus titer was measured to determine TCID50/100 μL. Herein, TCID50/100 μL of the original virus suspension and the sample solution is denoted by N0 and Nx, respectively.The disinfectant solution was mixed with the original virus suspension in equal volumes and kept at room temperature for 30 min. Then, a neutralizer solution of equal volume was added with mixing. After 10 min, TCID50 of the sample solution was measured and compared with that of the original virus suspension. The virus inactivation rate was calculated. Additionally, the disinfectant solution was diluted into the solutions of gradient concentrations and subjected to measurements as described above. The minimum effective concentration of the disinfectant is defined as the minimum disinfectant concentration corresponding to a 100% virus inactivation rate.We used the minimum effective concentration as the working concentration. Diluted disinfectant solutions were mixed with the original virus suspension in equal volumes and the mixtures were allowed to stand for 1, 5, 10, and 30 min, respectively. Next, an equal volume of neutralizer solution was added. After 10 min, the virus inactivation rates were measured.The measured minimum effective concentrations were used as working concentrations. The sample solutions were kept untouched at 4 and 37 °C for 30, 60, and 180 min, respectively. Next, an equal volume of neutralizer solution was added and the virus inactivation rates in BHK-21 cells were measured after 10 min.7.25/100 μL.The frozen rPRV-ZJ strain was sub-cultured in BHK-21 cells resulting in severe cytopathy  after 24 h A,B. The BHK-21 cells inoculated with different disinfectants, neutralizers, and neutralized products were characterized by an inverted microscope. Compared with the negative control group, inoculated BHK-21 cells exhibited regular morphology and good growth. This demonstrated that disinfectants, neutralizers, and neutralized products have negligible effects on cell growth suggesting no cellular toxicity.Following 24 h after virus inoculations, over 90% of the BHK-21 cells exhibited green fluorescence. However, the BHK-21 cells inoculated with both PRV and disinfectants showed significantly lower or negligible green fluorescence, indicating complete inactivation of the virus . The resNext, virus inactivation rates for the four disinfectants at different dilutions and their respective active components were investigated for 100% virus inactivation rate at room temperature for 30 min. The best dilution and effective content of glutaraldehyde decamethylammonium bromide solution were 1:1000 dilution and 0.1 g/L; the best dilution and effective contents of peracetic acid solution and sodium dichloroisocyanurate were 1:400 dilutions and 0.5 g/L, and the best dilution and effective content of the povidone-iodine solution was 1:20 dilution and 2.5 g/L, respectively. The peracetic acid solution with an effective content of 0.5 g/L had a virus inactivation rate of 66 and 100% at room temperature treatment for 1 and 5 min, respectively. Likewise, sodium dichloroisocyanurate (0.5 g/L) and povidone-iodine solution (2.5 g/L) had a virus inactivation rate of 100% at room temperature treatment for 10 min. The glutaraldehyde decamethylammonium bromide solution (0.1 g/L) with 1000 times dilution had PRV inactivation rates of 34, 77, and 100% after 5, 10, and 30 min treatments at room temperature, respectively .The results indicated that the disinfection performance of glutaraldehyde decamethylammonium bromide solution degraded drastically at low temperatures . After 3ASFV has possibly become the most relevant epizootic disease from an animal health perspective. Swine viral infections are acute and spread rapidly and severely, affecting the breeding industry and agricultural economy. Meanwhile, these diseases are also severe hazards to public health safety. Besides conventional vaccine immunization, the core prevention strategy against these viral infections remains the construction of an efficient biosafety system, such as the development of rational disinfection technology . The higEfficient, toxic-free, reliable, and cost-effective disinfectants may play a key role in blocking the spread of pathogenic microorganisms reducing the probability of animal infection . EspeciaAs a Cl-containing disinfectant, sodium dichloroisocyanurate was decomposed into hypochlorous acid and cyanuric acid in water. The structures of virus particles were destroyed by oxidation causing virus inactivation. The results indicated that sodium dichloroisocyanurate powder (1:400) exhibited good disinfection performance showing inactivation rates of 65% and 100% after 5 and 10 min treatments at room temperature, respectively. However, the disinfectant dilution was lower than that recommended in the instructions. This may be attributed to decomposition during storage suggesting that sodium dichloroisocyanurate powder performance decreases after long storage. Glutaraldehyde decamethylammonium bromide, a quaternary ammonium salt disinfectant, is a cationic surfactant that does not irritate animal skin and tissues. The study revealed that glutaraldehyde decamethylammonium bromide required low dosages. Specifically, the virus inactivation rate of glutaraldehyde decamethylammonium bromide (1: 1000) was 100% at room temperature treatment for 30 min. Hence, it is suitable for the disinfection of large sites. However, glutaraldehyde decamethylammonium bromide is highly sensitive to low temperatures. The virus inactivation rates decreased to 27 and 70% at 4 °C for 30 min and 3 h treatments, respectively. A 100% virus inactivation rate could be achieved only at 1:500 dilutions. Povidone-iodine, a loose complex of iodine and polyvinylpyrrolidone, has low toxicity which depolymerizes in the solvent to release iodine. This slow-release effect elicits a long-term disinfection effect ,34. PoviCompared with non-enveloped viruses, ASFV and PRV, which are both enveloped viruses, can be readily inactivated by chemical disinfectants ,37. AddiDue to the similar physiochemical characteristics of PRV with ASFV, the performances of four commonly used disinfectants without organic substances were evaluated using cell culture-based techniques, and PRV as an alternative marker strain. The results demonstrated that all disinfectants used in this study, under the conditions stated, inactivated PRV; the recommended dosages (optimized effective concentration and action time) of glutaraldehyde decamethylammonium bromide solution , peracetic acid solution , sodium dichloroisocyanurate , and povidone-iodine solution  were estimated. Among them, peracetic acid showed better action time, onset time, and reliability at both high and low temperatures. In practical applications, the use of disinfectant mixtures that are both effective and safe can further boost the overall efficacy of the disinfection process. This study provides a practical and theoretical reference for the establishment of ASFV disinfection standards.


Chikungunya is a mosquito-borne viral disease caused by the chikungunya virus (CHIKV). CHIKV is expanding at an alarming rate, potentially spreading and establishing endemicity in new areas where competent vectors are present. The dramatic spread of CHIKV in recent years highlights the urgent need to take precautionary measures and investigate options for control. It is crucial in developing nations where diagnostic tools are limited, and symptoms are similar to other prevalent diseases such as malaria and dengue. The most reliable method for diagnosing chikungunya virus is viral gene detection by RT-PCR. Alternative methods like detecting human antibody and viral antigen can also be used, especially in areas where resources are limited. In this review, we summarize the limited data on antigen detection immunoassays. We further explain the essential structural elements of the virus to help comprehend the scientific concepts underlying the testing methods, as well as future methods and diagnostic approaches under investigation. Aedes [Chikungunya fever is a reemerging debilitating mosquito-borne disease characterized by severe arthritis . ClinicaAedes . This viAedes ,5. ConseAedes Togaviridae, genus Alphavirus. The Togaviridae family of viruses belong to group IV of the RNA viruses. The genus Alphavirus has a wide range of viruses and the medically relevant are: Sindbis (SINDV), Eastern Equine Encephalitis (EEEV), Semliki Forest virus (SFV), Western Equine Encephalitis (WEEV), Ross River (RRV) and o’nyong-nyong (ONNV) [CHIKV is an approximately 12-kb positive-sense RNA (+ssRNA) genome containing a 5′-methylguanylate cap, a 3′-polyadenylated tail, and two ORFs. It is a member of the family g (ONNV) . CHIKV ig (ONNV) . Figure g (ONNV) ,8,9. The structural proteins of CHIKV are synthesized by translation of subgenomic RNA located on the second 3′UTR-ORF. This region is translated to one polyprotein which is post-translationally cleaved to form five proteins. Glycoproteins E1 and E2 play an important role in the pathogenicity of the virus as they are involved during the fusion of the virus to the host cell (E1) and interaction with host receptors (E2) before fusion . The cryAedes aegypti. Although Aedes aegypti is the classical vector for CHIKV, the 2005 outbreak in La Réunion was associated with an atypical vector, Aedes albopictus. The geographic and temporal dispersion of the insect vectors, their growth rate, and the length of the viral incubation period inside them are only a few of the variables that affect how successfully arboviral illnesses are transmitted. Aedes albopictus is currently found in sub-Saharan Africa, several European nations, and around 25% of the United States [Aedes albopictus can successfully infect both humans and animals. It is thought to have started off as a zoophile before evolving into an anthropophile. The E1 Ala226Val mutation and other mutations that have recently been found in E2 also govern CHIKV adaptation to its mosquito hosts. These changes were seen in E1, a class II viral fusion protein that mediates viral entry at low pH [The main vectors of CHIKV are d States . Becauset low pH . The virt low pH . The virt low pH . Therefot low pH . CHIKV at low pH .Additionally, CHIKV seems to activate the interferon promoter stimulator 1 (IPS-1), which results in the accumulation of IRF3-dependent mRNAs and prevents these mRNAs from encoding proteins. Acute patients’ cutaneous rashes contain CD8+ lymphocytes, but chronic patients’ synovial effusions mostly contain CD4+ T cells. Bone marrow stromal antigen 2 (BST-2) may guard lymphatic tissues and control inflammatory reactions brought on by CHIKV in the host. A sudden onset of fever, typically accompanied by joint pain, is a defining feature of chikungunya. The joint discomfort, which often lasts for a few days but can last for weeks, months, or even years, is frequently excruciating. The virus can therefore lead to acute, subacute, or chronic illness. Muscle soreness, joint swelling, headaches, nausea, exhaustion, and rashes are other typical symptoms . Unlike Laboratory diagnosis is key to reduce the morbidity caused by CHIKV infection. Currently, several methods have been developed commercially or in-house for the detection of CHIKV . The labMolecular techniques such as RT-PCR and isothermal amplification are used for the detection of the CHIKV genome. The viral antigen detection is achieved by using an enzyme linked immunosorbent assay (ELISA), an immunofluorescence assay (IFA), a rapid diagnostic test (RDT), and immunoblotting methods. Plate reduction neutralization tests (PRNT) remain the gold standard for serology and confirm the presence of neutralizing antibodies but are seldom used routinely for diagnosis. This technique requires trained personnel working under specified experimental and biological safety level (BSL) conditions ,22,23.The diagnosis of CHIKV remains a challenge because its clinical manifestations are not specific and difficult to differentiate from other infections occurring in similar geographic regions, including malaria, typhoid, and dengue; which share common symptoms with CHIKV infection . The curThree types of tests are generally used for CHIKV antigen detection, ELISA, IFA, and RDT. ELISA is among the most common tests used in developing as well as developed countries. The first ELISA were developed to reduce the use of radioactive elements for revelation and create a fast, simple, and safe alternative . The anaWe review a limited number of articles describing the development and validation of CHIKV assays for the detection of antigens .p < 0.001) compared with IgM and IgG ELISAs. Antigen detection ELISA gave a positive confirmatory result in the early phase of the disease, and was also valuable in the subclinical stage and may be useful for field applications for the rapid detection of CHIKV infection [Various approaches to the development of CHIKV antigen detection have previously been described. A study by Kashyap et al. (2010) described a CHIKV diagnosis in an Indian population using an indirect ELISA for antigen detection. Antigens were prepared using an Indian strain of CHIKV, ISW HYD06 (GenBank accession number 876190), which belongs to the ECSA genotype and was previously isolated from a patient from Hyderabad, India, during the 2006 epidemic . Chikungnfection . This abRef.  describeRef.  evaluateRef.  used mouIn a study aiming at overcoming the limitations of molecular diagnostics in asymptomatic individuals, ref.  developeRef.  generateCross-reactions concern viruses of neighboring species and belonging to the same species; they are easily explained, because a virus is not a single antigen, but a real “mosaic of antigens”. When an animal is immunized by injecting whole particles, it reacts by producing multiple antibodies directed against all the injected antigens. If two viruses have an identical antigen in their mosaic, the immune sera obtained in the animal will give a cross-reaction between these two viruses . This phWhen developing these assays, researchers frequently focus on virus strains that are prevalent in their geographic area and ignore strains from other regions or continents. Future research should include additional related alphaviruses like the Ross River virus (RRV), which is prevalent in Australia and the Pacific Island regions, and the ONNV, which is found in Sub-Saharan Africa. Future research should examine how well assays perform with inactivated serum samples so that non-infectious material can be used for point-of-care testing, especially in regions with low resources.CHIKV is expanding at an alarming rate with the potential to spread and establish endemicity in new areas where competent vectors are present. The dramatic spread of CHIKV in recent years highlights the urgent need to take precautionary measures and investigate options for control. This is particularly important in developing nations where diagnostic tools are limited, and symptoms are like those of other diseases such as malaria and other medically relevant alphaviruses. The E1 and E2 glycoproteins are mainly responsible for membrane fusion and virus entry into host cells, where E2 interacts with the cellular receptor and functions in attachment to cells, and E1 participates in virus fusion to the cell membrane . Hence, The similarities between CHIKV and ONNV are higher than 90% for E1 and E2, and between 30–60% for other medically relevant alphaviruses (S1 and S2). From these observations, it is understandable that differentiating CHIKV and ONNV using antigen detection techniques will be challenging; however, a careful analysis of a non-conserved region could help differentiate CHIKV/ONNV from other viruses. This can be achieved by conducting structural studies of the target antigens and epitope mapping using X-ray co-crystallography and cryogenic microscopy (Cryo-EM), array-based oligo-peptide scanning, or site-directed mutagenesis. E1 and E2 are the main targets in the development of diagnosis. However, recent studies have been focusing on E2 most probably because of its lower similarities compared with E1 or simply because it is a better immunogen. The suitability of E2 as the preferred antigen is further highlighted in the study of Suzuki et al. (2020) where an increase of sensitivity and specificity was obtained after E1 was combined with E2 . The recFew studies have reported the development of monoclonal antibodies. Increasing the number of monoclonal antibodies isolated from humans and other species could help develop a more sensitive and specific serological assay. Three distinct domains have been identified in the E2 protein: A (16–134 aa), B 173–231 aa), and C (269–341 aa), which are involved in receptor binding and considered immunogenic ,23,47. A1 aa, andThe COVID-19 pandemic has proven the worth of rapid diagnostics in detecting emerging infectious diseases. The need for accurate RDT for infectious diseases has been highlighted by many and they are currently under development. The lateral flow assay developed by  presente