On Sept 20, health authorities in Uganda declared an outbreak of Ebola virus after a 24-year-old was confirmed positive for the relatively rare Sudan strain of the virus, termed Sudan Ebola virus, for which there are currently no licenced vaccines or treatments available.According to the Ugandan Ministry of Health, a total of 60 confirmed cases, including 24 deaths and 24 recoveries, have been reported as of Oct 20. The current outbreak comes only 3 years since the last Ebola virus disease outbreak was announced in Uganda, back in 2019, and exactly a decade after the last outbreak owing to Sudan Ebola virus.Ebolavirus consists of six species, named Sudan, Zaire, Bundibugyo, Tai Forest, Bombali, and Reston. Zaire Ebolavirus (commonly known as the Ebola virus) and Sudan Ebolavirus are the dominant species that give rise to Ebola virus disease in humans.Ebola is a severe and often lethal haemorrhagic illness caused by the Ebola virus. Commonly believed to be of zoonotic origin, a typical Ebola virus disease outbreak starts with a single infected person, followed by transmission to other humans via direct contact or contact with infected bodily fluids or contaminated fomites. Ebolavirus belongs to the Filoviridae family of viruses, which also includes Cuevavirus and Marburgvirus. The genus The New England Journal of Medicine that was conducted during the same outbreak found that monoclonal antibody treatments mAb114  and REGN-EB3 (Inmazeb) were effective at reducing mortality from Ebola virus disease. Both are now included in the first guidelines recommended by WHO for Ebola virus disease therapeutics. However, similar therapies and vaccines to prevent the spread of the current Sudan Ebola virus outbreak are still in various stages of clinical development and no date has been issued for availability. This delay is in part due to the rarity of Sudan Ebola virus outbreak and researchers not being able to widely test some of these candidates.One of the largest epidemics ever reported took place in west Africa between 2013 and 2016 and was due to the Ebola virus. It resulted in over 28 600 suspected cases and more than 11 300 confirmed deaths. This epidemic was the first Ebola virus disease outbreak in which the ERVEBO vaccine, developed by Merck , was widely distributed among the affected communities, resulting in a prevention rate of over 80%. A clinical trial published in The New England Journal of Medicine assessed its safety and immunogenicity and found that the cAd3-EBO vaccine was well tolerated with no serious safety concerns, and vaccine-induced immune responses were similar to findings from previous challenge studies from non-human primates, and sustained responses were evident for up to 11 months. Several thousand doses of this vaccine, which is licensed to the Sabin Vaccine Institute  are anticipated for imminent potential deployment in Uganda, pending local regulatory approval for an efficacy trial during the current outbreak.Several vaccine candidates have been evaluated against Sudan Ebola virus in phase 1 human trials, including virus-like particles, recombinant modified vaccinia virus, DNA vaccines, human and chimpanzee adenoviruses, and viral vectors . As of October, three vaccine candidates have passed safety testing in humans, and six more are in the pipeline. But the efficacy of these candidates has yet to be tested in a large-scale study. The one furthest in development is a replication-defective recombinant chimpanzee adenovirus type 3-vectored ebolavirus vaccine (cAd3-EBO), encoding glycoprotein from the Ebola virus and Sudan Ebola virus. A phase 1 trial published on Nov 26, 2014, in The Lancet Infectious Diseases. The trial was conducted in Sierra Leone, a country previously affected by Ebola, extending their data collected from several other countries, including Uganda, Tanzania, Kenya, and the UK. This vaccine regimen received marketing authorisation for prophylactic use under exceptional circumstances in the EU as the trial results showed that Ebola virus glycoprotein-specific antibody binding at 21 days after the second dose were similar with levels observed after 21 days with rVSV-ZEBOV-GP vaccine. The latter is the only vaccine with demonstrable efficacy data in the world that has marketing authorisation in the EU, USA, and several African countries.Another strong contender that is being geared for efficacy testing during the current outbreak in Uganda is a two dose heterologous booster vaccine consisting of a prime dose of adenovirus type 26 vector encoding Ebola glycoprotein (Ad26.ZEBOV), followed by a secondary dose of modified vaccinia Ankara vector, encoding glycoproteins from the Ebola virus, Sudan Ebola virus, Marburg virus, and Tai Forest virus nucleoprotein (MVA-BN-Filo), with a 56-day gap. The vaccine was developed as part of the EBOVAC project, which is a research and pharmaceutical partnership aimed at forming a strategic alliance to accelerate the development of Ebola vaccines via clinical trials or following regulatory approval. Earlier phase 1 studies were conducted to assess the safety and tolerability of this vaccine regimen and potential long-term durability of the vaccine-induced immune responses. The latest results on the safety and long-term immunogenicity of this vaccine regimen were shown in a trial published in the January 2022 issue of While these vaccine trials are being set up, the Ugandan Government is mobilising several different surveillance and containment measures to curtail the virus from spreading to highly populated regions that are based on lessons learnt from previous Ebola virus disease outbreaks. On Oct 15, Uganda's President Yoweri Museveni introduced a 3 week lockdown in two affected districts of Uganda, along with a curfew to restrict movement of individuals, as a control measure to halt the transmission of Sudan Ebola virus to other regions and neighbouring countries. The country's Ministry of Health has ramped up training for health-care workers to handle Sudan Ebola virus case management, from contact tracing to safe burials of the dead and decontamination of affected dwellings, and training local volunteers to educate rural communities about the disease and its management. Contact tracing is being widely deployed to identify individuals who have been in close contact with affected individuals, who are then monitored for up to 21 days. Bordering countries, among others (including the USA), are on high alert and are implementing surveillance measures at border controls to curb international spread of the virus.The Lancet Infectious Diseases by Mukadi-Bamuleka and colleagues developed and evaluated the performance of three rapid diagnostic tests (RDT) for detecting viral antigens as a means to overcome some of these diagnostic challenges in a remote setting. Despite robust performance, all three RDTs, including the OraQuick, Coris EBOLA Ag K-SeT and QuickNavi-Ebola, failed to reach WHO's recommended threshold for sensitivity and specificity and did not show similar performance to the Cepheid GeneXpert Ebola assay reference test. These disappointing findings highlighted the need for the development of robust tests that are affordable, can provide a rapid diagnosis, and can be used with minimal training in a remote setting.A lesson learnt from the COVID-19 pandemic is also being put into practice, whereby a mobile laboratory has been set up in Mubende, where the outbreak first began, to enable on site, same day diagnosis. Several diagnostic methods, including RT-PCR, can be used to diagnose Sudan Ebola virus. However, the early signs of the disease mimic other infectious diseases, such as malaria, typhoid fever, and meningitis, which adds to the challenges of running diagnostics in remote low-resource setting. A study published in eBioMedicine, the authors developed an ELISA-based serodiagnostic assay, referred to as EBOV-Detect, that can differentiate Ebola virus disease-induced antibodies from those that have been raised in response several candidate vaccines (namely rVSV-ZEBOV and ChAd/MVA-EBOV) with 95% specificity and 96% sensitivity. The authors hope that this assay, and other similar diagnostic tools, will be implemented in later stages of the outbreak to facilitate epidemiological and surveillance efforts.In a study by Ravichandran and Khurana published in eBioMedicine, we encourage researchers working on all aspects of Ebola virus disease diagnostic, surveillance, and epidemiological and disease mechanisms to submit robust and early evidence to form studies to help fight the Ebola virus disease outbreak.In the absence of effective treatments and vaccines, the Ugandan Government, backed by international health authorities, can end the Sudan Ebola virus outbreak by applying all the lessons learnt through past experiences, such as surveillance strategies and timely response management tailored to the affected communities on the ground. At


A180V virus) showed poor cross-reactivity to immune sera from animals immunized with the SH/F/98 , SD/SS/94 (A180), JS/Y618/12 (T180), and rF/HAA180V (V180) viruses by microneutralization (MN) assay. The A180V substitution in the parent virus caused a significant decrease in cross-MN titres by enhancing the receptor binding activity, but it did not physically prevent antibody (Ab) binding. The strong receptor binding avidity prevented viral release from cells. Moreover, the A180V substitution promoted H9N2 virus escape from an in vitro pAb-neutralizing reaction, which also slightly affected the cross-protection in vivo. Our results suggest that the A180V mutation with a strong receptor binding avidity contributed to the low reactors in MN/HI assays and slightly affected vaccine efficacy but was not directly responsible for immune escape, which suggested that the A180V mutation might play a key role in the process of the adaptive evolution of H9N2 virus.Based on differences in the amino acid sequence of the protein haemagglutinin (HA), the H9N2 avian influenza virus (H9N2 virus) has been clustered into multiple lineages, and its rapidly ongoing evolution increases the difficulties faced by prevention and control programs. The HA protein, a major antigenic protein, and the amino acid mutations that alter viral antigenicity in particular have always been of interest. Likewise, it has been well documented that some amino acid mutations in HA alter viral antigenicity in the H9N2 virus, but little has been reported regarding how these antibody escape mutations affect antigenic variation. In this study, we were able to identify 15 HA mutations that were potentially relevant to viral antigenic drift, and we also found that a key amino acid mutation, A180V, at position 180 in HA (the numbering for mature H9 HA), the only site of the receptor binding sites that is not conserved, was directly responsible for viral antigenic variation. Moreover, the recombinant virus with alanine to valine substitution at position 180 in HA in the SH/F/98 backbone (rF/HA H9N2 avian influenza virus (AIV) has spread rapidly since its breakout in Hebei Province, China, in 1998, after which it rapidly became one of the most important epidemics in the poultry industry , which sOver 30 antigenic sites of the H9N2 virus have already been reported, most of which were precisely mapped by the use of monoclonal antibodies (mAbs) –14. ViraWe have previously reported that the H9N2 vaccine representative strain A/Chicken/Shanghai/F/1998 , which belonged to the BJ/94-like lineage, enabled antigenic variation continually when passaged in specific pathogen-free (SPF) chicken embryos or SPF chickens with or without homologous vaccine antibodies , 25. In The SPF chickens and chicken embryos used in this study were purchased from Nanjing Biology Medical Factory, Qian Yuan-hao Biological Co., Ltd. The procedures that involved the care and use of animals were approved by the Jiangsu Administrative Committee for Laboratory Animals (permission number SYXK 2016-0020) and were performed by the Jiangsu Laboratory Animal Welfare and Ethics guidelines of the Jiangsu Administrative Committee of Laboratory Animals.2.The H9N2 virus F/98 was isolated in 1998 in Shanghai and stored at −70 °C at the Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou University. The GenBank accession numbers of the sequence of the F/98 strain are AY253750-AY253756 and AF461532 . Human eThe primers, which were synthesized by Tsingke Biological Technology , were used to amplify the DNA sequence used to add the single mutation in the F/98 virus HA protein and were designed using Primer 5.0 software  based on the HA gene sequence of the F/98 H9N2 avian influenza virus. The full-length HA genes containing the single mutation were amplified by PCR and inserted into the transcriptional/expression vector pHW2000  by using2 for 24 h, 2 µg/mL TPCK-trypsin  was added to the wells. Thirty hours after transfection, the supernatants were harvested and inoculated into 10-day-old SPF embryonated chicken eggs for virus propagation. The rescued virus was analysed with an HA assay, and the HA genes from the rescued virus were sequenced by Tsingke Biological Technology  to confirm the accuracy of the designed mutation.A total weight of 2.4 µg of the eight plasmid mixtures at a ratio of 1:1 was mixed with 100 µL of Opti-MEM medium . Next, 7 µL of PolyFect transfection reagent  was added, and the samples were incubated at room temperature for 10 min and then added to 70–80% confluent monolayers of 293 T cells in 24-well plates. After incubation at 37 °C and 5% CO50 assay was performed as previously described [–1 to 10–11 and then added to MDCK cells in 96-well plates. After incubation at 37 °C and 5% CO2 for 1 h, the supernatants were removed, the plates were washed twice with PBS, and 100 µL of DMEM was then added to each well. After incubation at 37 °C and 5% CO2 for 72 h, the HA titres of the cell supernatants were analysed, and they were calculated according to the Reed-Muench formula [The TCIDescribed , and the formula .A180V, as described previously [v/v) for 24 h at 37 °C. The booster vaccination was performed two weeks after the initial immunization, and the antisera were collected and pooled from the vaccinated SPF chickens three weeks after the booster vaccination.Six three-week-old SPF chickens were immunized twice by subcutaneous injection of 0.3 mL of an oil-emulsion containing inactivated whole virus vaccines of the viruses F/98 and rF/HAeviously , which wv/v) chicken erythrocytes [The antisera were treated with cholera filtrate  to remove nonspecific haemagglutination inhibitors before the HI assay, which was performed using 4 haemagglutination units (HAU) of H9N2 and 1%  chicken erythrocytes to confirm the existence of haemagglutination produced by the virus, with the MN titre being defined as the highest dilution of serum with an absence of haemagglutination.The MN assay was performed as previously described , and theS127N virus or the rF/HAA180V virus in chickens were then added in serial twofold dilutions with PBS containing 0.05% Tween 20 and incubated for 3 h at 37 °C.The ELISA was performed as previously described , and suc2SO4. The absorbance was recorded at 450 nm using an automated ELISA plate reader , and the area under the curve (AUC) of either virus was assessed for virus-Ab binding above that of the corresponding negative control with GraphPad Prism 8 software .After washing, goat anti-chicken horseradish peroxidase antibody  was added and incubated for 1.5 h at 37 °C, TMB   substrate was added, and the reaction was stopped by adding Hv/v) solutions) to 4 HAU of each virus (as determined by using nontreated chicken erythrocytes). The agglutination was measured after incubation for 1 h, and the virus with a higher binding avidity for the receptor was able to bind to chicken erythrocytes that were treated with high amounts of α2-3,6,8 neuraminidases.The receptor binding assay was performed as previously described , and theA180V viruses were serially diluted in PBS with 50 μL aliquots of these twofold dilutions incubated with 50 μL 1% (v/v) chicken erythrocytes in V‐bottom microtiter plates. The plates were placed at 4 °C for 30 min and then transferred to 37 °C. The decrease in HA titre was monitored for 6 h, which reflected elution from the chicken erythrocytes.The virus elution assay was performed as previously described , and the6 cells per group, and 1000 TCID50 H9N2 viruses were used as the control group. Then, each treatment group was treated with 1000 TCID50 H9N2 viruses, and the control group was incubated in an ice bath for 1 h. The first treatment group and the control group were incubated at 37 °C for 30 min, whereas the second treatment group was incubated on ice. After collecting the supernatant after centrifugation, the RNA was extracted from the supernatant using the TIANamp Virus RNA kit . Reverse transcription to generate cDNA was performed by using HiScript® II Reverse Transcriptase  . The number of copies of the M gene were determined by quantitative real-time reverse transcriptase PCR (qRT–PCR) following the instructions provided by ChamQ SYBR qPCR Master Mix  [MDCK cells were divided into 2 treatment groups, including approximately 10, China) . Each grA180V vaccine group, which included 12 chickens, and both groups were immunized with the F/98 virus emulsion vaccine and the rF/HAA180V virus. On Day 21 post-vaccination, chickens were bled from the wing vein for sera, and HI reactions against the F/98 virus or the rF/HAA180V virus were performed.A total of 24 three-week-old SPF chickens were divided into three groups: the F/98 vaccine group, which included 12 chickens, and the rF/HA3 TCID50 of the F/98 virus or the rF/HAA180V virus. Chickens were monitored daily for morbidity and mortality after the challenge, and tracheal and cloacal swabs from challenged chickens were collected in 1 mL of PBS that contained antibiotics on Days 3 and 5 after the challenge. After one freeze–thaw cycle, the swabs were centrifuged at 3000 rpm for 10 min, and 0.2 mL supernatant was taken to inoculate 10-day-old SPF chicken eggs. Viral shedding in the trachea and cloacal was evaluated via HA titres of the allantoic cavity of post-inoculated SPF chicken eggs at Day 5 according to the HA standard of ≥ 23.Six chickens from each group were then challenged intranasally and intratracheally with 10SD for all assays, and Student’s t test analysis was used to compare different groups and was executed with GraphPad Prism 8 software, from which differences were considered statistically significant when the P value was < 0.05.The data are shown as the mean ± K113R, rF/HAQ115H, rF/HAS127N, rF/HAQ146L, rF/HAA150T, rF/HAG163E, rF/HAA180V, rF/HAM206K, rF/HAQ216L, rF/HAY246H, rF/HAG252R, rF/HAG256R, rF/HAK260E, rF/HAI368V, and rF/HAK381N.We previously reported that 15 mutations  passaged in SPF embryonated chicken eggs [A180V , A/Chicken/Jiangsu/YZ618/2012  [A180V in chickens to confirm whether a single A180V mutation would cause low serological reactivity against antisera from other viruses possessing 180 A, T or V. The results showed that the rF/HAA180V virus displayed fourfold lower MN titres for anti-GD/SS/94 serum than those of the GD/SS/94 virus  , and rF/A180V bound to chicken erythrocytes treated with 32-fold higher α2-3,6,8 neuraminidase concentrations than those of the F/98 strain. The viruses rF/HA128, rF/HA168, rF/HA268, and rF/HA47 carrying HA V180 bound to chicken erythrocytes were treated with at least fourfold higher α2-3,6,8 neuraminidase concentrations than those of the viruses F/98, rF/HAK113R, rF/HAS127N, rF/HAG163E, and rF/HA126 possessing HA A180   to further evaluate the effect of the A180V HA mutation, which possessed a strong receptor binding avidity and caused the rF/HAA180V virus had any impact on viral infection and release from the cell surface. The results showed that the A180V mutation did not influence viral infection  -PCR assay in MDCK cells to investigate whether the rF/HAn Figure B, and th) Figure C. These A180V were collected for the HI assay 21 days after immunization. While the anti-F/98 serum against the F/98 virus was 15.3-fold higher than that against the rF/HAA180V virus (P < 0.001), the anti-rF/HAA180V serum against the F/98 virus was 14.4-fold higher than that against the rF/HAA180V virus (P < 0.001). The serum HI titre from the chickens vaccinated with the rF/HAA180V virus was lower than that of the chickens that were vaccinated with the F/98 virus , and the A180V mutation, which promotes strong receptor binding, seems to disrupt the functional balance between the affinity of HA receptor binding and NA activity. Naturally, compensatory mutations that increase avidity or modulate NA function are required to restore viral fitness [In our study, the A180V substitution promoted viral escape from a pAb-neutralizing reaction, which was related to different viruses possessing either A, T or V 180, by promoting significant receptor binding activity, while A or V at the HA 180 position did not prevent virus-antibody binding, which was consistent with the results reported by Hensley et al., who found a positive correlation between the avidity of receptor binding and escape from antiserum . In the  fitness , which i fitness , which mA180V virus, whereas the antibody induced by the rF/HAA180V vaccine could provide 83.3% protection against the challenge posed by either the F/98 virus or the rF/HAA180V virus.Sealy et al. found an association in Pakistan during 2014–2016 between the increased avidity of receptor binding and escape from antibody-based immunity by characterizing isolated H9N2 viruses, confirming that HA A180T/V substitutions with an enhanced avidity of receptor binding resulted in antigenic variation , which wA180V virus from the antibodies induced by the vaccine of the paternal virus F/98 or its own antibodies. The slight contribution of A180V to this viral immune escape further suggests that this mutation could not be directly responsible for viral immune escape, while it might also be related to the phenomenon that homologous vaccine antibodies could not provide acceptable protection for vaccinated chicken flocks in recent years [Thus, although the single A180V mutation slightly affected in vivo cross-protection, it broke down the full protective efficiency of the inactivated vaccine of the paternal F/98 virus, which suggests that the single A180V mutation, which possessed a strong avidity of receptor binding, facilitated the escape of the rF/HAnt years .In summary, our findings reveal that the mechanism of the A180V mutation promoted viral escape from in vitro pAbs, and they also showed that this mutation makes a slight contribution to the escape from antibody-based immunity. While the A180V substitution promoted H9N2 virus escape from a pAb-neutralizing reaction by enhancing the receptor binding activity, it also did not physically prevent Ab binding, which led to significantly decreased readouts of cross-MN titres, slightly affecting in vivo cross-protection. These data suggest that HA mutations with strong avidity of receptor binding should be given more attention when monitoring the emergence of new antigenic variations.


According to previous research, borderline personality disorder (BPD) is associated with high cost-of-illness. However, there is still a shortage of cost-of-illness-studies assessing costs from a broad societal perspective, including direct and indirect costs. Further, there are considerable differences in the results among the existing studies. In the present study, 167 German men and women seeking specialized outpatient treatment for BPD were included. We assessed societal cost-of-illness bottom-up through structured face-to-face interviews and encompassed a wide range of cost components. All costs were calculated for the 2015 price level. Cost-of-illness amounted to € 31,130 per patient and year preceding disorder-specific outpatient treatment. € 17,044 (54.8%) were direct costs that were mostly related to hospital treatment. Indirect costs amounted to € 14,086 (45.2%). Within indirect costs, costs related to work disability were the most crucial cost driver. The present study underlines the tremendous economic burden of BPD. According to the present study, both the direct and indirect costs are of significant importance for the societal costs associated with BPD. Besides the need for more disorder-specific treatment facilities for men and women with BPD, we assume that education and employment are topics that should be specifically targeted and individually supported at an early stage of treatment.Trial Registration: German Clinical Trial Registration, DRKS00011534, Date of Registration: 11/01/2017, retrospectively registered. Borderline personality disorder (BPD) is a severe and prevalent mental disorder . AccordiHowever, BPD-related costs vary considerably among existing studies ranging from € 9174 per patient and year in a German study  and € 56First, cost-of-illness analysis can be conducted from several different perspectives , and the perspective determines which costs are included in the analysis. The most comprehensive perspective is the societal perspective taking into account all costs irrespectively of who bears them. At the same time, the cost analysis conducted from the same perspective can still considerably differ in the number of cost components included in the study.bottom-up studies, costs are assessed on the patient level based on self-reports or individualized claims data of payers such as health insurance providers. In top–down studies, total costs per health care sector are derived from databases such as national registers and health reports and are assigned to a specific diagnosis. While top–down studies can rely on large samples, costs per patient must be calculated based on prevalence data or several assumptions since little disease-specific information is available in public databases.Second, there are different methodological approaches to obtain cost data . In bottIn sum, BPD has substantial impacts on every area of life, and costs accrue far beyond the health care sector. However, there is still a shortage of cost-of-illness-studies assessing BPD-related costs from a societal perspective.In the present study, we aim to expand and further clarify the knowledge on the cost-of-illness of BPD. We assessed annual costs bottom-up from a societal perspective in a relatively large sample of 167 German men and women with BPD seeking specialized outpatient treatment. We included a wide range of direct costs  and indirect costs (costs due to loss of productivity).A clear picture of the economic burden of BPD is crucial to inform decisions on resource allocation in a budget-constrained healthcare system and to develop and implement specific strategies in BPD treatment leading to both a higher quality of life for patients but also a reduction of costs for society.The present cost-of-illness study was conducted as part of the PROgrams for Borderline Personality Disorder study (Pro*BPD), a randomized controlled trial comparing the clinical effectiveness and cost-effectiveness of outpatient Dialectical Behaviour Therapy (DBT) and Schema Therapy (ST)   they had a primary diagnosis of BPD according to DSM-IV—diagnosed with the Structured Clinical Interview for DSM-IV for Axis II (SCID-II)—, (ii) had a severity score higher than 20 points on the Borderline Personality Disorder Severity Index Version 4 (BPDSI) , (iii) iResource consumption and productivity loss were assessed from a societal perspective. We used patient-level data and assessed resource consumption and productivity loss through structured face-to-face interviews (“Cost Interview”) covering the previous 12 months.The Cost Interview used in the current study was validated afore and used in a previous study on costs of BPD . Before It is essential to mention that the assessment of resource consumption and productivity loss was exclusively based on patients’ self-reports. This also applied to direct non-medical resource consumption. For example, in the case of informal care, we asked patients to what extent relatives helped them with household chores. Similarly, in the case of traffic offenses, patients estimated the amount of the corresponding fines. Information on resource consumption and productivity loss was collected in a two-stage process: Patients received the Cost-Interview before the interview appointment for an initial self-evaluation. In the interview appointment, missing items were assessed by the interviewer. In the rare cases when information was missing, the patient was called again.During the interview appointment, it was also determined whether resource consumption and productivity loss were due to mental disorders or medical diseases. In the current study, we only distinguished between the costs due to psychological disorders and somatic diseases. Wagner et al.  found thTo calculate direct costs, we multiplied resources by their corresponding unit cost . Unfortunately, there is no obligatory unit cost list in Germany covering all medical and non-medical services used by men and women with BPD. At the same time, standardized unit costs for many areas have been calculated for Germany from the societal perspective and recently been published.For medical services such as hospital treatments and outpatient medical care, we referred to the unit costs calculated by Bock et al. . For comSince the above publications do not include unit costs for the areas of psychotherapy and medication, we calculated the corresponding costs from the perspective of the payers. Accordingly, unit costs for individual and group psychotherapy were calculated based on the current physician's fee schedule for psychotherapy .To calcuUnit costs related to delinquent behavior were dependent on the consequences of a specific behavior. In the case of property damage, thievery, and traffic offenses, unit costs were based on the patient's information about the value of the damaged item or the fine. Treatment costs related to bodily injury were calculated in the above-mentioned medical unit costs. If the police or an emergency ambulance was involved in an incident, we also added mean costs for a police operation or emergency ambulance operation. Unit costs for a police operation were calculated based on the assumption that two police officers are involved in an operation and that an operation takes one hour and based on the relevant mean labor costs for police officers . Unit coAll unit costs were adjusted for the 2015 price level with the consumer price index  since daThe calculation of indirect costs was initially carried out in the primary analysis with the Human Capital Approach and in a sensitivity analysis with the Friction Cost Approach .According to the Human Capital Approach, we counted productivity loss due to days of incapacity to work and productivity loss due to work disability. To calculate costs related to incapacity to work, we considered those employees engaged in an employment relationship that carries obligatory social insurance. We multiplied days of incapacity to work by the individual labor costs for each patient with a paid labor, including the individual gross wage plus non-wage labor costs . Work diAccording to the Friction Cost Approach, only the costs due to incapacity to work were considered calculating indirect costs. Here, 80% of individual labor costs were included in the calculation, and a friction period of 49 working days or 72 calendar days was assumed . The friData entry and calculation of costs were performed with IBM SPSS statistic version 26. We used a non-parametric bootstrap resampling method with 1000 replications as an alternative to the geometric means as the cost data were skewed and not normally distributed. All costs are expressed in Euros with the price level of the year 2015.The socio-demographic and clinical characteristics of participants are shown in Table Table Of the total mean annual cost-of-illness, 49.2% were direct medical costs, 5.5% were direct non-medical costs, and 45.3% were indirect costs.Table Patients used a wide range of direct medical and direct non-medical resources. By far, the most critical cost driver was costs associated with hospital treatment, which almost accounted for three-quarters of total direct costs. Ninety-four patients (56.3%) were at least once treated in hospital due to psychiatric reasons in the year before the Pro*BPD trial started. Among these patients, mean annual hospital days were 70.5 (SD = 46.1) days, and mean annual costs amounted to € 22,058 .In contrast, costs due to outpatient psychotherapy only made up 7.2% of total direct costs. The majority of patients (86.2%) used individual outpatient psychotherapy mainly in low frequency in the observation period. Within this subgroup, patients received an average of 13.0 (SD = 8.9) sessions per year.Psychotropic drugs were taken by 133 patients (79.6%), which, on average, took 2.6 (SD = 1.5) psychotropic drugs.Most patients (97.6%) visited a physician during the investigation period. When considering both visits due to psychological and somatic reasons, the total mean annual number of visits to physicians increased to 19.3 (SD = 19.8).In the area of direct non-medical costs, resources were used by a small proportion of patients. Regarding informal care, 34 patients (20.4%) received help from relatives and friends due to psychological problems. On average, they received 240.7 h (SD = 419.5) of informal care amounting to mean annual costs of € 3430 (€ 5978).In addition, 54 patients (32.3%) were involved in delinquent behavior in the investigation period, with, on average, 7.4 (SD = 12.8) incidents and related costs of € 381 (SD = € 851). Thereof, 54.5% of all incidents were attributable to property damage, 29.8% to traffic offenses, and 15.6% to police operations, thefts, and bodily injury.In Table Within indirect costs, costs related to work disability showed to be the most significant cost driver. Fifty-three patients (31.7%) were work-disabled throughout the year due to a combination of BPD symptoms and chronic somatic problems.Costs related to incapacity to work were related to 36 patients (21.6%) in employment at any time during the investigation. On average, those patients had a gross monthly earning of € 2094 (SD = € 977). Mean annual sick days were 75.3 days (SD = 119.5) in this patient subgroup. Of those, 68.5 days (SD = 121.9) were due to psychological problems, and the corresponding mean annual costs amounted to € 6882  within the subgroup of working patients.According to the Friction Cost Approach, costs due to incapacity to work, and thus total indirect costs, amounted to € 436 (SD = € 1508) per patient and year across the entire sample. The corresponding mean annual days of incapacity to work related to psychological problems were 5.5 (SD = 17.5). The significantly lower costs due to incapacity to work compared to the Human Capital Approach were related to the fact that the number of days of incapacity to work exceeded the friction period of 49 days for seven patients.In the present study, we assessed societal cost-of-illness in a sample of 167 German men and women with BPD seeking specialized outpatient treatment and included a wide range of cost components. Cost-of-illness amounted to € 31,130 per patient in the year preceding disorder-specific outpatient treatment. Thereof, € 17,054 (54.7%) were direct costs, while indirect costs amounted to € 14,085 (45.3%). Several significant findings emerge from our study.First, our study underlines the tremendous economic burden of BPD. Mean annual BPD-related costs calculated in the present study exceed those in patients with many other mental diseases by far. By comparison, societal cost-of-illness calculated bottom-up in a German sample of patients with depression recruited in a primary care setting was € 7139 per patient and year . ThereofSecond, the costs found in the present study exceed the costs calculated in the majority of previous studies in men and women with BPD. The two German cost studies by Wunsch et al.  and BodeFrom a methodological point of view, the approach chosen in our study is comparable to those in the cost studies by Wagner et al.  and van Wagner et al. found a total cost-of-illness of € 30,220 per patient and year in a sample of 47 German men and women with BPD. Thereof, 67.9%  were direct costs, and 32.1% (€ 9701) were indirect costs. In the study by van Asselt et al., the total cost-of-illness amounted to € 22,849 per patient and year in a sample of 88 Dutch patients. Here, only 21.7% (€ 4958) of total costs were direct healthcare costs, while 26.5% (€ 6055) were direct non-medical costs and more than 50%  of total costs were indirect costs.The third significant finding of our study is that hospital treatment costs were by far the most crucial cost driver, accounting for almost 75% of direct costs. Patients in our study were, on average, 39.7 days in the hospital due to BPD in the year preceding the assessment. This finding is in line with the other German cost-of-illness-study  and undeAt the same time, compared to the German study by Wagner et al., mean annual hospital days were lower by almost 12 days in the present study. This result initially is surprising since we included severely ill men and women with BPD and many comorbid problems. We assume that this finding is linked to specific characteristics of the treatment setting in Lübeck. The Department of Psychiatry and Psychotherapy has a long tradition and expertise in treating men and women with BPD. Treatment structures are tailored to the needs of patients with severe illness and aim at avoiding short-term inpatient admissions.Our study underscores the great importance of indirect costs within societal costs in men and women with BPD as a last major finding. Within indirect costs, costs related to work disability were the most significant cost driver. Almost one-third of the sample was disabled for work, and almost 30% of the sample was unemployed, pointing to deficient occupational functioning of the patients investigated.M = € 12,602) were higher compared to those calculated in the Dutch study by van Asselt et al. (M = € 9162) and the German study by Wagner et al. (M = € 7999). Similar to our study, 32% of the patients in the Dutch study were fully disabled, and further, 9% were partially disabled. Higher costs related to work disability calculated in the present study are primarily related to the fact that a higher proportion of our sample was male than in the Dutch study (21% vs. 8%). This is because the calculation of work disability-related costs is based on gender-adjusted average gross income, and men, on average, have a higher income. In contrast, in the study by Wagner et al., only 18% of the sample was work-disabled, resulting in lower work-disability-related costs than in the present study.Costs related to work disability calculated in our study  are both part of the diagnostic criteria of BPD as well as of the diagnostic criteria of an Axis-1-disorder. Notably, the distinction between costs due to mental disorders and medical diseases might also have led to underestimating BPD-related costs in the present study as BPD is associated with physical health problems , 8, 41.Future cost analyses should further examine the direct and indirect costs of men and women with BPD in different recruitment settings to gain an even more differentiated picture of the cost-of-illness of BPD. For example, it is desirable to examine the costs of patients seeking treatment in more rural areas and away from specialized treatment centers. Also, the costs of treatment-seeking patients should be compared to patients who do not seek treatment. In addition, there are several disorder-specific and clinically effective treatments for BPD  that areThe main objective of a cost-of-illness analysis is to determine the economic relevance of a disease and not primarily inform decision-making in health care. However, suggestions for better care of men and women with BPD can be derived from the results of this study. For example, the patients we studied used inpatient stays intensively, and most patients took at least one psychotropic drug, although drug treatment of BPD is not supported by the current evidence . In contFurthermore, while pointing to deficient occupational functioning of the majority of the BPD-patients investigated, our study is in line with previous publications showing that the low psychosocial functioning of men and women with BPD remained stable over extended periods of time . AccordiFinally, in the field of prevention, early detection and treatment of BPD in youth and early adulthood and early interventions in high-risk families are of pivotal importance .In the present study, societal cost-of-illness was assessed comprehensively in a sample of men and women with BPD seeking specialized outpatient treatment in the clinical setting of a University psychiatric hospital. In conclusion, our study underlines the high social and economic relevance of BPD. Costs related to hospital treatment are the most crucial cost driver within direct costs in German patients. Furthermore, indirect costs are of significant importance within societal costs in patients. By now, the finding of the high cost-of-illness in men and women with BPD that are both due to high direct and indirect costs can be viewed as a robust finding across different recruitment settings, national social welfare systems, and despite methodological differences.


Penaeus chinensis. In order to enrich the epidemiological information of the World Organisation for Animal Health (WOAH)-listed and emerging diseases in wild P. chinensis, we collected a total of 37 wild P. chinensis from the Yellow Sea in the past three years and carried out molecular detection tests for eleven shrimp pathogens. The results showed that infectious hypodermal and hematopoietic necrosis virus (IHHNV), Decapod iridescent virus 1 (DIV1), yellow head virus genotype 8 (YHV-8), and oriental wenrivirus 1 (OWV1) could be detected in collected wild P. chinensis. Among them, the coexistence of IHHNV and DIV1 was confirmed using qPCR, PCR, and sequence analysis with pooled samples. The infection with YHV-8 and OWV1 in shrimp was studied using molecular diagnosis, phylogenetic analysis, and transmission electron microscopy. It is worth highlighting that this study revealed the high prevalence of coinfection with YHV-8 and OWV1 in wild P. chinensis populations and the transmission risk of these viruses between the wild and farmed P. chinensis populations. This study enriches the epidemiological information of WOAH-listed and emerging diseases in wild P. chinensis in the Yellow Sea and raises concerns about biosecurity issues related to wild shrimp resources.At present, there are few studies on the epidemiology of diseases in wild Chinese white shrimp  Penaeus monodon and Indian white shrimp P. indicus [P. chinensis.The seed industry is a fundamental core supporting modern aquaculture. Viral diseases have been considered a vast hazard to the shrimp farming industry worldwide . Wild sh indicus ,5,6,7. N indicus ,9,10. Va indicus . Shrimp  indicus ,13. HoweOkavirus of the family Roniviridae, of which three were accepted as virus species by the International Committee on Taxonomy of Viruses (ICTV). Yellow head virus  is assigned to genotype 1 (YHV-1) [Gill-associated virus) is assigned to genotype 2 [Okavirus 1) is assigned to genotype 8 [P. monodon in Asia [P. chinensis and P. vannamei in China and South Korea in recent years [P. chinensis, is a novel bunyavirus in the genus Wenrivirus of the family Phenuiviridae with a negative-stranded and 4-segmental RNA virus [P. chinensis, its pathogenicity is still unclear [Penstyldensovirus in the family Parvoviridae, was first identified from P. stylirostris in Hawaii, where mortality has been notifiable to be greater than 90% [Decapodiridovirus of the family Iridoviridae [There are eight genotypes in yellow head complex viruses (YHVs) in the genus  (YHV-1) . Gill-asnotype 2 . Yellow notype 8 . Other gnotype 8 . Infectinotype 8 , has cau in Asia ,17,18,19nt years ,21,22,23NA virus . Althoug unclear . IHHNV, than 90% . Infectithan 90% , can leathan 90% ,27,28. Mthan 90% ,29. Decaoviridae . DIV1 caoviridae ,32,33.P. chinensis populations, this study collected wild P. chinensis individuals captured from the Yellow Sea for three consecutive years. Using molecular diagnostic methods, we confirmed the existence of IHHNV and DIV1 and coinfection with YHV-8 and OWV1 in wild P. chinensis populations. Consequently, the study provides epidemiological information for consideration of the biosecurity strategy related to wild P. chinensis resources as broodstock sources in hatching for the farmed shrimp larvae and the stock enhancement of natural resources.In facing the limited epidemiological information for WOAH-listed and emerging diseases in wild P. chinensis individuals of about 18–21 cm were sampled in the Yellow Sea, nine shrimp in November 2020, eight in November 2021 and twenty shrimp in March 2022. We pretreated the selected shrimp individuals on ice in advance, and all efforts were made to minimize the suffering of animals accordingly.Chinese white shrimp P. chinensis by TIANamp Marine Animal DNA Kit and RNAprep pure Tissue Kit , respectively, according to the manufacturer’s instructions. The concentration and quality of DNA and RNA were measured by NanoDrop 2000 .The cephalothorax was snap-frozen in liquid nitrogen to extract DNA and RNA and then used for routine pathogen detection. Total DNA and RNA were extracted from 20–30 mg individual cephalothorax tissue of Enterocytozoon hepatopenaei (EHP), covert mortality nodavirus (CMNV), infectious myonecrosis virus (IMNV), and Taura syndrome virus (TSV). Both PCR and qPCR methods targeting different sequence locations of each pathogen published in papers or recommended by the WOAH Aquatic Manual were used for molecular detection of the above seven pathogens [We mixed DNA or RNA extracted from different batches of shrimp and detected seven pathogens, including WSSV, DIV1, IHHNV, athogens ,45,46,47P. chinensis, we detected GAV, YHV-1, YHV-8, and other YHVs by RT-PCR and OWV1 by RT-qPCR in every sampled P. chinensis [In order to explore whether there was coinfection with YHVs and OWV1 in wild hinensis ,48. WhenFor transmission electron microscopy (TEM), samples were prepared in two ways: purifying viruses from gills for negative staining and embedding gills and lymphoid organs for ultrathin sections.3) of the lymphoid organ and gills were sampled and fixed in the TEM fixative . Ultrathin sections were cut, mounted on collodion-coated grids, and stained with aqueous uranyl acetate/lead citrate using standard procedures as we previously published [Small pieces  with 0.5 mM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) . The tissue was homogenized by a homogenizer in an ice bath at a speed of 10,000 rpm for 5 s. These steps were repeated several times (> 3) until a uniform tissue homogenate was obtained. The homogenate was centrifuged at 1400 g for 15 min at 4 °C . A 20 mL of SM buffer was added to the pellet and homogenized in an ice bath at 10,000 rpm for 10 s, followed by centrifugation at 6000 g for 15 min at 4 °C. These two supernatants obtained in the above steps were combined, filtered through a 38 μm mesh, and the filtrate was centrifuged at 10,000 g for 25 min at 4 °C. The final supernatant was again filtered through a 38 μm mesh, mixed with an equal volume of SM buffer, and centrifuged at 40,000 g for 2 h at 4 °C  to pellet viral particles [Fifteen grams of articles . FinallyThe supernatant and the pellet suspension were dropped on grids, negatively stained with 2% phosphotungstic acid , and observed under a TEM  at 80 kV.Okavirus, the approach proposed by Mohr was employed using primers YH30 m/31 m to get a partial sequence of the open reading frame 1b (ORF1b) region to clarify the genotype [Wenrivirus, we constructed four pairs of primers  was determined as the best model for Okavirus. Then based on the lowest Bayesian information criterion score, the LG with frequencies model gamma distributed with invariant sites (LG+G+I+F) was determined as the best model for Wenrivirus. The maximum likelihood phylogenetic tree was constructed using the best model for Okavirus and Wenrivirus. Phylogenetic testing was performed using the bootstrap method with 1000 replicates. All relevant sequences have been submitted to NCBI.For the genus genotype . For the primers  with ovees, etc. ). Multipsoftware , and “FiP. chinensis populations, we detected the pooled samples for seven known pathogens, including DIV1, IHHNV, CMNV, EHP, WSSV, IMNV, and TSV. The PCR and qPCR results  of OWV1 in the cytoplasm of the gill tissue (TEM performed with ultrathin sections of lymphoid organs and gills of l tissue A,B. Howel tissue C,D.P. chinensis populations [P. chinensis collected in this study.To unambiguously identify the genotype of YHVs, we performed a phylogenetic analysis of the samples collected in this study. The products of the YHV ORF1b region obtained were sent for sequencing. After NCBI Nucleotide-BLAST , it showulations  and are P. chinensis cluster with the OWV1 reference sequence previously isolated from farmed P. chinensis [Wenrivirus (The obtained product of OWV1 RdRp region was sent for sequencing and translated into the amino acid sequence using Geneious version 2022.1.1. After NCBI Protein-BLAST , it can hinensis  and thennrivirus .P. chinensis populations in the Yellow Sea. Furthermore, we observed coinfection with YHV-8 and OWV1 in all P. chinensis individuals by molecular diagnosis, phylogenetic analysis, and TEM. As the P. chinensis farming industry still largely relays on the wild P. chinensis broodstock [P. chinensis reveal significant risks in the shrimp farming industry and the stock enhancement program for fishery resources of P. chinensis in East Asia.Shrimp diseases have become an important factor hindering the shrimp industry’s green development of the shrimp farming industry . This stoodstock , the conUnlike human health, surveillance for aquatic animal health usually emphasizes the population-based approach. Detection of a lower prevalence in a larger population requires a larger sample size , which rP. chinensis is much more difficult due to the declined fishery resources and uncertainty of seasonal migration caused by climate change [Compared with the farmed samples, the accessibility of captured wild e change ,56. MeanP. monodon. Notably, YHV-8 and GAV belong to Okavirus, and MoV and OWV1 belong to Wenrivirus. The similarity of the virus taxonomy and the coinfection in Spann et al.’s and our results raise the interesting issue of whether the Okavirus and Wenrivirus have a synergistic mechanism in the concomitance of coinfection. Teixeira-Lopes et al. [P. chinensis populations may help to discover the deep infection mechanism of shrimp viruses.Unlike the research on the viral diseases of farmed crustaceans, the reports on the spread of wild crustacean viruses and coinfection with multi-pathogen are still very limited . Spann es et al.  revealeds et al. ,59. FurtP. chinensis in the Yellow Sea has been infected with IHHNV and DIV1. IHHNV natively originated from the Western Hemisphere. It was first detected from farmed P. vannamei in 2001 in China [P. chinensis has not been listed as a susceptible species of IHHNV [P. chinensis in the National Target Surveillance Program of China in 2019 [P. chinensis implies the transmission risk of viruses between farmed and wild shrimp populations. DIV1 was detected in the wild P. monodon captured from the northeastern Indian Ocean [P. chinensis indicated a broader virus spread in the wild. We did not find viral particles like DIV1 or IHHNV with TEM, which may be related to the selected tissues, centrifugal force, and low viral load. After subsequent virus purification, a large number of suspected nucleocapsid complex structures, likely YHV-8 virions, were observed in the supernatant. However, no complete complex structure was found, which might be caused by a large centrifugal force [The confirmatory diagnosis of a suspected case of infection with a specific pathogen is generally based on meeting the criterium of two or more independent tests in the WOAH Manual . The diain China . P. chinof IHHNV . However in 2019 . IHHNV dan Ocean . DIV1 deal force .Okavirus isolates from captured wild P. chinensis cluster with YHV-8 previously identified from farmed P. chinensis [P. chinensis. Conclusively, the sequence similarity of the isolates from farmed and wild P. chinensis for both viruses implies the transmission risk of viruses between farmed and wild shrimp populations.hinensis  and sepahinensis  cluster P. chinensis, which reminds pathogenic risks in wild shrimp populations are nonnegligible. Wild shrimp used for broodstock must be strictly inspected and quarantined to select specific pathogen-free stocks [This study has detected multiple pathogens in wild e stocks ,29,63. De stocks . On the P. chinensis populations and the transmission risk of viruses between wild and farmed populations. The findings imply that we should pay attention to the epidemiology of diseases in wild P. chinensis, implement disease surveillance on wild shrimp, and introduce biosecurity policies and measures to prevent disease risks both in shrimp farms and hatcheries for stock enhancement.This study is the first to report the coexistence of multiple viruses, including DIV1, IHHNV, YHV-8, and OWV1. Notably, the results reveal a consistently high prevalence of coinfection with YHV-8 and OWV1 in wild


Medicago sativa L.) is one of the most important quality forages worldwide and is cultivated throughout China. Alfalfa is susceptible to a variety of viral diseases during its growth, which has caused huge amounts of commercial losses. However, the profile of the alfalfa virus in China remains ambiguous and the viruses transmitted by Odontothrips loti , dominant insect pests in alfalfa, are also poorly understood. In the present study, virus diversity was investigated in the primary alfalfa-growing areas in China. A total of 18 alfalfa viruses were identified through RNA-sequencing (RNA-seq) and reverse transcription-polymerase chain reaction (RT-PCR). Two new plant viruses, Medicago sativa virus 1 (MsV1) and Medicago sativa luteovirus 1 (MsLV1), were detected for the first time. Another four viruses, including the Alfalfa ringspot-associated virus (ARaV), Alfalfa virus F (AVF), Alfalfa enamovirus 1 (AEV1), and Alfalfa deltaparitivirus (ADPV), were reported in China for the first time as well. Both Alfalfa mosaic virus (AMV) and Medicago sativa alphapartitivirus 2 (MsAPV2) are the dominant pathogens, with an infection incidence of 91.7–100%, and 74.4–97.2%, respectively. Additionally, O. loti with first- and second-instar nymphs were shown to acquire the AMV within 0.25 h of feeding on a virus-infected alfalfa. Transmission by thrips to healthy alfalfa plants was also demonstrated. Additionally, we clarified the dynamic changes in the AMV in pre-adult stages of O. loti, which indicated that the AMV is propagated in the nymph stage of O. loti. These findings provide valuable information for understanding the alfalfa virome, confirm the role thrips O. loti plays in alfalfa virus transmission, and improve our fundamental knowledge and management of diseases in China.Alfalfa ( Medicago sativa L.) is able to be grown under a range of conditions, has high protein value, and is important in the security of livestock and poultry production systems subject to increasing climate variability [2 of alfalfa was being grown, and increased 91.3% compared to the 2.3 million ha2 in 2016 [Alfalfa mosaic virus (AMV), followed with a 21.5% reduction in the pollen pollination rate and a 31–67% decrease in the number of nodules [Alfalfa  has become undoubtedly the best way to unravel the virome of plants and animals currently ,12,13,14Bean leafroll virus (BLRV), and Alfalfa leaf curl virus (ALCV) [Odontothrips loti  being the most significant, with infestations resulting in a more than 20% loss in alfalfa production, as well as a 36.9% and 37.9% decrease in plant height and leaf area, respectively [Tomato spotted wilt virus (TSWV) by thrips, including Frankliniella occidentalis (Pergande) and Thrips tabaci (Linderman), is the most well-known with its persistent manner by which the virus replicates and circulates throughout the various developmental stages of these thrip species [O. loti has been elucidated, the information involving its ability to transmit alfalfa viruses is poorly understood.As most plant viruses are transmitted by insect vectors, especially insects with piercing-sucking mouthparts which facilitate the efficient transmission of viruses ,19. Aphis (ALCV) ,21,22,23s (ALCV) ,25. Thris (ALCV) ,27. Moreectively ,29,30,31ectively . Transmi species . While tO. loti can successfully transmit the virus. The results obtained in this study provide valuable information for understanding the profile of the alfalfa virome and the main insects involved in virus transmission. These results contribute to the development of effective management strategies to manage viruses and their insect vectors.To investigate the occurrence and distribution of viral species present in alfalfa across China, alfalfa samples were collected from major alfalfa-growing regions, and viruses were identified by RNA-sequencing (RNA-seq), reverse transcription-polymerase chain reaction (RT-PCR), and quantitative real time PCR (qRT-PCR). The transmission efficiency of AMV by thrips was also explored to determine whether later@ Solution  at 4 °C overnight, and then stored at −20 °C until further used.A total of 569 leaf samples were collected from the eight main alfalfa-growing provinces  in China from 2018 to 2020. The details for sample collection are shown in cv. Zhongmu No. 1 used for the transmission test was cultivated in pots (15 cm high and 10 cm in diameter) containing vegetative soil and vermiculite. The pots were kept in an insect-proof environmental chamber at 26 ± 1 °C with a Light 16 h: Dark 8 h (L16:D8) photoperiod. Plants had developed through to the three true leaf stage before being used in the bioassay.Alfalfa O. loti colony was initially obtained from the alfalfa field at Shangzhuang Experimental Station at the China Agricultural University  and maintained on hyacinth bean (Lablab purpureus L.) pod in transparent plastic jars (10 cm high and 10 cm in diameter) with 200 mesh nylon mesh covering the jar lids in an environmental chamber at 25 °C, 70% relative humidity (RH), and a L16:D8 photoperiod. The hyacinth bean pods were sourced from a commercial supplier. Before being placed with the thrips, the pods were soaked in 0.1% NaOH solution for 1 h, followed by a triple rinse in distilled water, then air dried for 1 h.The Alfalfa samples (100 mg) were ground in liquid nitrogen, and total RNA was extracted by using RNAprep Pure Plant Kit (Polysaccharides & Polyphenolics-rich)  according to standard manufacturer’s instructions. Total RNA from thrips samples was extracted using TRIzol reagent  following Manson’s protocol . ExtractTo understand the alfalfa virome, fifteen alfalfa leaves from five different geographical regions  in autumn 2019 A, were p−5 to identify viruses. We blasted the assembled contigs against the viral database [The quality of reads generated was checked using FastQC (v 0.11.9). To obtain clean reads, adapters and low-quality reads were filtered out using the Sickle program (v 1.33) with the parameters “-q 30-l 50”. The clean reads were initially mapped to two reference genomes of alfalfa ,36 usingdatabase . The candatabase ). To undhttps://www.ncbi.nlm.nih.gov/orffinder/, accessed on 15 January 2022) for viruses were analyzed based on the reference viral genome. Nucleotide sequences were aligned using MAGE X [For the phylogenetic study, the open reading frames (ORFs) predicted by ORFfinder  were placed in plastic jars containing hundreds of mixed-sex adults. Previous methods  had shoO. loti to acquire AMV, first- and second-instar nymphs and adults were provided with an AMV-infected alfalfa leaf in a Petri dish (14 cm-diameter) containing a moist filter paper. The insects were confined for 11 different acquisition access period (AAP): 0.25, 0.5, 1, 2, 4, 8, 12, 16, 24, 36, and 48 h, respectively. Because sourcing pure AMV-free alfalfa seeds proved to be problematic, fresh hyacinth bean pods was used as the control treatment. The AMV titers in the thrips were determined using qRT-PCR, with the treatments represented by five replicates per time period, each with five thrips.To study the ability of To determine virus propagation dynamics, newly hatched nymphs, which had been provided with a 0.25 h AAP, were transferred to hyacinth beans for 0, 6, 24, 48, 96, 144, 288, 336 h, respectively. The nymphs that had not been exposed to AMV were maintained on hyacinth beans as controls. Nymphs were placed in an incubator under the condition of 26 ± 1 °C and a photoperiod of L16:D8. The AMV titers in the thrips were determined using qRT-PCR, with the treatments represented by five replicates per time period, each with five thrips.2), one of which was used as a control. The leaf was grouped together on a moist piece of filter paper and placed in a 5 mL centrifuge tube containing absorbent cotton moistened to minimize desiccation. The centrifuge tube was covered with 200 mesh nylon mesh to prevent the escape of thrips. After 24 h inoculation access period (IAP), thrips were removed and the leaves were incubated in the centrifuge tube for three days. qRT-PCR was used to evaluate if the leaf had been infected with AMV. There were 12 replicates per instar and five thrips per replicate.Thrips were transferred to healthy alfalfa leaves after feeding on AMV-infected alfalfa leaves for the best AAP to understand their ability to transmit AMV. A healthy alfalfa leaf was sliced into four identical square leaves  of forward and reverse primers,12.5 μL of GS AntiQ qRT-PCR SYBR Green Master Mix  and 1 μL cDNA, was used. The following PCR conditions were used: 5 min at 95 °C, followed by 40 cycles of 10 s at 95 °C, and 30 s at 60 °C. At the end of each qRT-PCR, the melting curve of each gene was recorded. The absolute gene expression was analyzed using the geometric mean of threshold cycle (Ct) values with the standard curve method. The protocol of construct the standard curve of each virus was showed in t method .To calculate the incidence, the samlpe’s Ct value with none was considered as the healthy sample based on the result of qRT-PCR. The incidence of virus (%) is the number of healthy samples / the sum of samples. For each of measurement dates, the difference in viral accumulation in the leaves was examined by the Kruskal-Wallis test using nonparametric analysis of variance of the SPSS Statistics v. 22.0 . The difference in the relative expression of AMV among the treatments was examined by one-way ANOVA tests using SPSS Statistics v. 22.0 .Medicago sativa alphapartitivirus 1 (MsAPV1), Medicago sativa alphapartitivirus 2 (MsAPV2), Medicago sativa deltapartitivirus 1 (MsDPV1), Medicago sativa amalgavirus 1 (MsAV1), Cnidium vein yellowing virus 1 (CnVYV1), Pea streak virus (PeSV), Alfalfa dwarf virus (ADV), Alfalfa deltaparitivirus (ADPV), Alfalfa leaf curl virus (ALCV), Bean leafroll virus (BLRV), Alfalfa enamovirus 1 (AEV1), Lucerne transient streak virus (LTSV), Alfalfa ringspot-associated virus (ARaV), Medicago sativa marafivirus 1 (MsMV1), Alfalfa virus F (AVF), Medicago sativa virus 1 (MsV1), Medicago sativa luteovirus 1 (MsLV1), and Lettuce mosaic virus (LeMV). Following clean data mapping of the reference viruses and confirmation by RT-PCR  and Strawberry virus 3  belonging to the Mononegavirales order. All 19 contigs were assembled and predicted six ORFs including nucleocapsid (N), phosphoprotein (P), hypothetical protein (P3), matrix protein (M), glycoprotein (G), and the large multi-functional RNA-dependent RNA polymerase (L). Contigs obtained by the RNA-seq covered were not adequate to assemble into the complete genome sequence, although they contained all six ORFs matching the Mononegavirales order. To fill these sequence gaps, bridging primers were designed for RT-PCR based on known contigs from RNA-seq data, and the amplicons were cloned and sequenced (Medicago sativa virus 1 (MsV1) was assembled, which comprised 14,041 nt (ON246246)  A. These N246246) . Meanwhithan 40% . The comtionship . The phyequences . Based oTombusviridae family. These contigs ranged in size between 408 nt and 812 nt and shared 59.3–66.7% aa sequence identity with the RNA-dependent RNA polymerase (RdRp) of several viruses. These belonged to the luteovirus including the Peach-associated luteovirus , Cherry-associated luteovirus , Soybean dwarf virus , Bean leafroll virus , Rose spring dwarf-associated virus , and Barley yellow dwarf virus  (Medicago sativa luteovirus 1 (MsLV1) as a new species.Three assembled contigs in Ningxia samples ZM1-NX1, 2, and 3 libraries were most similar to the members of luteovirus in the BY73679) . The aa BY73679) C. MultipBY73679) . SpeciesFimoviridae family. Of these, 18, 10, 3, and 3 contigs were blasted from RNA1 to RNA4, respectively. The three RNA3 contigs (ON409656~ON409658) identified ARaV in our study were 1079 nt–1245 nt long sequence encoding N of 299 aa and shared the 87.3% identity of the aa sequence with the ARaV-AU-VIC329 (QGX86471). Our isolates were identified as the isolates of ARaV by the phylogenetic analysis of the nucleocapsid (N) aa sequences  with a length of 5659 nt encoding five ORFs shared the highest genomic identity with AEV1 . ORF1 was located at 155 nt–1066 nt, encoding the putative RNA silencing suppressor (303 aa)—an ORF2 + 3 encoded fusion protein that was translated consequent to −1 programmed ribosomal frameshifting starting at 237 nt and ending at 3794 nt. This fusion protein was indicated by conserved sequence motifs encoding RdRp. ORF4 and ORF5 encoded CP  and the aphid transmission protein , respectively. The phylogenetic analysis of the deduced aa sequences of the CP genes showed that AEV1-XN2-XJ3 was an isolate of AEV1 .Two AVF isolates from Ningxia, namely AVF-ZM1-NX1 and 2, had the length of 6719 nt and 6084 nt encoding 2106 aa and 1957 aa protein sequences, respectively. Two isolates shared 78.6–78.9% genome identity and 93.4–100.0% CP identity with AVF (MG676465). According to the species demarcation in the marafivirus genus, which is based on genomic sequence differences of more than 20% and the aa of CP gene sequence difference of 10%, AVF-ZM1-NX1 and 2 were the isolates of AVF. The phylogenetic analysis of the deduced aa sequences of AVF, and the 13 other viruses belonging to the marafivirus revealed that AVF and MsMV1 were clustered together . Three MRed clover cryptic virus 3 (RcCV3) and ADPV was clustered with Dactylorhiza hatagirea deltapartitivirus (DhDPV1) . In addition, MsDPV1, also belonging to the deltapartitivirus genus, were identified from our samples and, therefore, we performed the phylogenetic analysis alongside with ADPV. There were ten RNA1 sequences of MsDPV1 in seven libraries from Xinjiang, Gansu and Jilin . The seq(DhDPV1) .cv. Gongnong No. 1 and 2 with higher copy number of CnVYV1 compared to others 27 alfalfa cultivars.AMV, MsAPV1, MsAPV2, MsDPV1, MsAV1, and CnVYV1 were widely distributed in all eight provinces with a high infection rate among the 18 viruses identified by RNA-seq and RT-PCR. Among the six viruses detected by qRT-PCR in the 569 samples, AMV was pervasive and severe in all alfalfa-growing regions, with virus infection incidences between 91.7–100.0% A,B. MeanO. loti to acquire and transmit AMV was investigated in the first- and second-instar nymphs and in adults. It was found that feeding on AMV-infected leaves for 0.25 h of AAP was enough for both the first- and second -instar nymphs of O. loti to acquire the AMV, with the highest titers in first-instar nymphs , which we report as the first record of their presence in China, and another twelve viruses previously reported in China. This study provides a more comprehensive understanding of alfalfa virus diversity in China and updates virus species in different regions.Rhabdoviridae family, MsV1, enveloped plant and animal viruses with segmented, linear, negative-sense RNA genomes [Rhabdoviridae family, based on the genus demarcation criteria for Rhabdoviridae [Tombusviridae family (Bean leafroll virus (BLRV), belonging to luteovirus, could also infect alfalfa, and be transmitted by aphids in a persistent manner [Meanwhile, the discovery of two new alfalfa viruses has the abundant alfalfa-virus database. One of the new viruses belonging to the  genomes , with a  genomes ,49. ADV- genomes . So far, genomes ,50,51. Aoviridae . The othe family C. The vit manner ,53. BLRVt manner ,53. BaseAphis craccivora (Koch) [Four viruses, AVF, AEV1, ADPV, and ARaV, were reported in China for the first time. The first virus was ARaV showing ringspot symptoms on the leaves, which was also newly identified in Australia with partial RNA1 and RNA3 . We detea (Koch) . In addia (Koch) . The fouPartitiviridae family [Partitiviridae), was transmitted vertically by the seed of Trifolium repens, which showed a 3–48% transmission rate in the variety cultivars and a 24% transmission rate after being stored for 50 years. Therefore, more surveillance for ADPV and MsDPV1 in alfalfa seeds is required. In addition, the ADPV sequence is incomplete, and future research may need to focus more on obtaining the complete genome sequence. For all these viruses, further studies are needed to clarify the effects on alfalfa and understand the prevalence in China.Except for ADPV, MsDPV1 also belonged to the deltaparitivirus, which has been so far only identified in China . Our rese family . White cTherioaphis trifolii (Monell) and Acyrthosiphon pisum (Harris) in Australia [Aphis glycines (Matsumura) in America [Picornavirales order which contains two segments and is linear with positive sense ssRNA, was firstly isolated from Cnidium officinale (Makino) in Korea in 2015 [cv. Gongnong No. 1 and 2, which were generally planted in the northeast of China and are cold-resistant. Therefore, the CnVYV1 may be an important virus in alfalfa crops growing in regions with low average annual temperature and is one to be monitored to reduce the virus becoming widespread.Based on the results of RNA-seq, the top six prevalent pathogens in those alfalfa planting regions were AMV, MsAPV1, MsAPV2, MsDPV1, MsAV1, and CnVYV1, with AMV and MsAPV2 being the most widespread and serious virus-infected alfalfa in China A,B. In tustralia , and Aph America . However America ,27. In t America , has rea America . This fi America . This is in 2015 . CnVYV1  in 2015 . It tendO. loti was shown to transmit the AMV, with first- and second-instar nymphs being effective vectors of the virus. First- and second-instar nymphs had a strong ability to pick up the AMV, with only 0.25 h feeding on infected leaves. At the same time, the AMV could be successfully transmitted to alfalfa after 24 IAP. Especially in the second-instar nymphs, AMV titers in leaves increased by 1.71 × 108 times compared to the healthy alfalfa. As the same with previous thrips-virus-transmission study, TSWV, after ingestion by first-instar nymphs, is efficiently transmitted by second-instar nymphs and adults of the thrips F. occidentalis [T. tabaci and F. occidentalis could also acquire TSWV in the adult stage but could not transmit TSWV [F. occidentalis infected by TSWV and Impatiens Necrotic spot virus (INSV), due to the virus titer decrease during the ecdysis stage of instar transition [O. loti. Furthermore, the male adult carried much more AMV than the females, while the opposite pattern was TWSV transmission in F. occidentalis, in which females adults were 2–3 times more virulent than males [F. occidentalis, which harbors a high titer of virus, is more likely to transmit TWSV multiple times [O. loti males will have a higher transmission rate than females. These results would indicate that O. loti is an important vector for horizontal transmission of AMV in the field.In our study, dentalis ,65. Howedentalis ,67. In tmit TSWV ,66. Remaansition ,69. Thisan males . Comparean males ,71,72. Ile times . TherefoO. loti was proved to transmit AMV. First- and second-instar nymphs could acquire AMV within 0.25 h, and the virus can be effectively transmitted after 24 h of feeding on healthy alfalfa. We clarified the dynamic changes of AMV in pre-adult stages of O. loti, and suggested AMV can be propagated in the nymph stage. With respect to the field scale impacts of these viruses, these and transmission pathways need to be confirmed and quantified, and effective mitigation options developed.In conclusion, a total of 18 alfalfa viruses were verified through RNA-seq and RT-PCR. Two new plant viruses, MsV1 and MsLV1, were detected. In addition, ARaV, AVF, AEV1, and ADPV were identified and reported in China for the first time. Six main alfalfa viruses were detected in eight alfalfa planting areas by qRT-PCR. AMV and MsAPV2 are the dominant prevalent and severe pathogens, with infection incidences 91.7–100.0%, and 74.4–97.2% respectively, in the alfalfa field in China. Additionally, it is the first time that


Discerning whether range of motion (ROM) is restricted by morphology or other pain sources is challenging in patients with femoroacetabular impingement syndrome (FAIS). Computed tomography (CT) motion simulation provides a hypothetical ROM based on morphology. This study aimed to explore associations between ROM measured using CT motion simulation and maximum passive ROM measured clinically using three dimensional (3D) motion analysis in patients with FAIS, prior to and post arthroscopic hip surgery.Eight males with FAIS  were included in this explorative feasibility study. Participants were examined using CT according to a low-dose protocol prior to and 7-months post arthroscopic surgery. Software was used to simulate at which ROM the impingement would occur. With the hip in 90 degrees’ flexion, maximum passive range of internal hip rotation, and maximum passive internal hip rotation coupled with adduction was examined clinically using 3D motion analysis pre- and postoperatively. Spearman rank correlation coefficients and linear regressions examined associations between methods.r = 0.71, p = 0.009). Linear regressions demonstrated that maximal internal rotation measured using CT motion simulation was predominantly larger than when measured using 3D motion analysis. Postoperatively, and when maximum internal rotation was coupled with adduction, no correlations were found between the two methods.Preoperatively, the correlation between maximum internal hip rotation measured using CT motion simulation and 3D motion analysis was strong (The hypothetical morphology restricted ROM is larger than clinically assessed pain restricted ROM, both prior to and post hip arthroscopy. These findings suggest that ROM is restricted by pain rather than mechanical, morphology-based impingement in individuals with FAIS. Femoroacetabular impingement (FAI), is a condition where the bones of the hip joint do not have a matching shape. Cam impingement or deformity, refers to a cam effect initiated by a non-spherical femoral head rotating inside the acetabulum , while pThe main symptoms of FAIS is motion-related or position-related pain in the hip or groin . The onsComputed tomography (CT) provides additional information, compared to radiography, including 3D information of the joint. In 2007, a clinical pilot study examined a CT based method (‘HipMotion’) for noninvasive 3D assessment of FAI . HipMotiThe study was reported following the “Strengthening the Reporting of Observational Studies in Epidemiology” (STROBE) statement as a guideline . The stuHip joints of study participants were examined prior to and at median 7 months range 4–11) following arthroscopic hip surgery using a Toshiba Dual-Energy CT at Karolinska University Hospital, Solna, Sweden, according to a low-dose protocol. Motion simulation was conducted using the simulation software, Articulis software . The software automatically converts the CT scans to 3D models of the femur and pelvis. The software identifies the impinging area by 0.1 mm and calculates the amount of bone necessary to resect to create a hip configuration without impingement . A conventional biomechanical model, the Plug-in-Gait model, including 18 reflective markers was used. Good intra-sessional repeatability has been reported using the Plug-in-Gait model , and forDue to the nature of this explorative feasibility study, no sample size calculation was performed prior to the study. Statistical analyses were performed using IBM SPSS Statistics version 26 . A significance level was set at ɑ = 0.05. Mean, standard deviation, median, range, and percentage described variables. Spearman rank correlation coefficients examined the strength of the relationship between ROM measured using CT motion simulation and 3D motion analysis . CorrelaEight males with a mean age of 27.2 years (SD 7.3), mean height 176 cm (SD 7.7), and mean BMI 21.1 (SD 2.1), were included in the study. Four of the included participants had bilateral FAIS and thus, in total, measurements from 12 hip joints were examined and included in the analysis .At the preoperative test session of clinically assessed ROM, study participants rated their perceived pain on the VAS to median 65 mm (range 20–93). After surgery, pain was significantly reduced to median VAS 28 mm (range 0–65) . Evaluation of hip joint range of motion in all three planes simultaneously is complex and difficult to measure clinically. In combination with additional stress on soft tissues, this could be a contributing factor to the statistically non-significant correlations found when adduction was added as compared to just flexion and internal rotation. This may further be reflected in the larger discrepancy seen postoperatively between the simulated and clinically assessed ROM  full range of internal rotation (i.e. 30 degrees) was greater when measured with hip flexion only, and not coupled with adduction. This result may reflect that it is not always technically possible to resect the entire bony prominence of the femoral head circumference to create a hip configuration without impingement using arthroscopy.In the present study, ROM was re-evaluated at median 7 months after surgery. The timing of the postoperative evaluation may have impacted the results as pain was reduced, yet still present during test of passive maximal ROM. Preoperatively, study participants rated their perceived pain to be median VAS 65 (range 20–93). After surgery, pain was significantly reduced, although still present . An alternative approach, which possibly could have rendered a larger ROM during the clinical assessment, and perhaps a stronger correlation between methods, would have been to inject the hip joint with local anesthesia. In line with our results, a recent cross sectional study evaluating objective hip-related function in patients following hip arthroscopy reported less hip mobility 6 to 10 months after surgery as compared to controls . PreviouThis study holds several limitations. The study is based on a small number of observations, consequently, the risk for type two errors is evident, and the external validity as well as generalizability of the results are limited. The time period to follow-up may be considered too short as previously discussed, as further improvements during the first year postoperatively may be expected , 31. TheFindings of this study suggest that ROM is restricted by pain to a greater extent than mechanical, bone-on-bone, morphology-based impingement in individuals with FAIS both pre- and postoperatively. Prior to arthroscopic hip surgery, the correlation between clinically assessed maximum internal hip rotation and ROM measured using CT motion simulation is strong. CT motion simulation, exclusive labrum, cartilage and soft tissues, indicates a greater theoretically possible ROM, however, postoperatively no statistically significant correlation to clinically assessed ROM was found, indicating less predictable postoperative results on pain and function.


Eight olivine beads found at the Middle Chalcolithic site of Tel Tsaf , Jordan Valley, Israel, underscore a new facet of interregional exchange for this period. The current paper presents the olivine beads assemblage, its morphometric and technological characteristics, and chemical composition. The results of the chemical analysis suggest that all eight beads derive from the same source. By means of comparison with the chemical characteristics of known olivine sources, we argue for a northeastern African–western Arabian provenience and cautiously suggest Ethiopia as a probable origin. Finally, we discuss the significance of the assemblage, its possible origin, and the mechanisms that may have brought the beads to the site. In antiquity, beads and other items of personal adornment appreciated by the community were among the principal devices for transmitting social and economic information over large distances. By token of their small size, they could change hands easily, while their aesthetic and symbolic value rendered them economically effective . NotablyIn the prehistoric southern Levant, exchange and trade in beads was common practice for more than a hundred thousand years ,8, and wperidot or chrysolite , ±158 ppm Ni, ±41 ppm Ca, ±127 ppm Mn, ±18 ppm Al and ±24 ppm Cr.The beads were also subjected to elemental analysis at the Institute of Earth Sciences at the Hebrew University of Jerusalem. We employed a JEOL JXA-8230 electron probe microanalyzer (EPMA) equipped with four wavelength dispersive spectrometers. Across all analyses, the electron beam used was 1 μm in diameter with an acceleration voltage of 15 kV and a current of 120 nA. Peak counting rates were 30 s for the major elements  and 180 s for minor elements , while background counting rates were half the time for each background position . A set of reference materials were used to calibrate the EPMA for specifiable elements: San-Carlos olivine (NMNH# 111312–44) for Si, Mg, and Fe, SPI standard diopside . Furthermore, Mg/Fe ratio suggests a composition of 89.5%–91.5% forsterite (Fo#). Minor elements in the olivine beads have limited composition as well, varying between Ni = 2870±120 ppm, Mn = 880±100, Ca = 450±90, Cr = 100±30, and Al = 70±20 ppm.The Raman spectra collected from all beads are almost identical , consist90%–100% . This coWe hypothesized above, on the grounds of crystal size, that the olivine beads of Tel Tsaf derive from mantle rocks. Now, our chemical analyses provide further support: the beads’ Fo#, Ni, and Mn contents are well within the ranges of mantle olivine, which are 89%–94%, 2200–3400 ppm, and 450–1050 ppm, respectively –75. MoreThe eight olivine beads from Middle Chalcolithic Tel Tsaf are the earliest specimens of their kind in the Near East and a rare incidence in the southern Levant. As noted above, six potential olivine sources have been identified: Turkey, Pakistan, Egypt’s Eastern Desert and Zabargad, Ethiopia, Tanzania, and Saudi Arabia. Of these, the Fo# of Turkey’s and most of Tanzania’s olivines are higher . The beads had to travel via a long chain of hubs across far-flung and diffused systems of trade and exchange.Raw materials and artifacts of long-distance trade are a familiar feature of the south Levantine Chalcolithic period –82. The In all likelihood, the olivine beads constituted personal objects and were entwined with themes of value, desire, and social status. As a product, they boast high production value in terms of technology and distribution . FurtherThe abundance of long-distance traded artifacts and substances at Tel Tsaf indicates that it accommodated a community capable of obtaining and managing surplus to foster economic and social connections. Moreover, along with the circulation of tangible goods, exchange networks also provide an infrastructure for the flow of intangible cultural assets communicated by artifacts that convey coded information, interpreted or read as ‘value’ or social/economic significance. In the case of the olivine beads, the transmitted coded information pertains to their value that derives from acknowledging their distant origin and the difficulty obtaining them.However, modeling this multi-layered interaction system is nearly impossible due to numerous indeterminate agents and ambiguous factors. Nevertheless, we have good reason to presume that the value bestowed on exotic materials  is likel


The term virus ‘spillover’ embodies a highly complex phenomenon and is often used to refer to viral transmission from a primary reservoir host to a new, naïve yet susceptible and permissive host species. Spillover transmission can result in a virus becoming pathogenic, causing disease and death to the new host if successful infection and transmission takes place.The scientific literature across diverse disciplines has used the terms virus spillover, spillover transmission, cross-species transmission, and host shift almost indistinctly to imply the complex process of establishment of a virus from an original host (source/donor) to a naïve host (recipient), which have close or distant taxonomic or evolutionary ties. Spillover transmission may result in unsuccessful onward transmission, if the virus dies off before propagation. Alternatively, successful viral establishment in the new host can occur if subsequent secondary transmission among individuals of the same novel species and among other sympatric susceptible species occurred. As such, virus spillover transmission is a common yet highly complex phenomenon that encompasses multiple subtle stages that can be deconstructed to be studied separately to better understand the drivers of disease emergence. Rabies virus (RABV) is a well-documented viral pathogen which still inflicts heavy impact on humans, companion animals, wildlife, and livestock throughout Latin America due substantial spatial temporal and ecological—natural and expansional—overlap with several virus reservoir hosts. Thereby, the rabies disease system represents a robust avenue through which the drivers and uncertainties surrounding spillover transmission can be unravel at its different subtle stages to better understand how they may be affected by coarse, medium, and fine scale variables.The continued study of viral spillover transmission necessitates the elucidation of its complexities to better assess the cross-scale impacts of ecological forces linked to the propensity of spillover success. Improving capacities to reconstruct and predict spillover transmission would prevent public health impacts on those most at risk populations across the globe. As  America , 13, swiin China , Nipah vngladesh , 16, Marm civets , Middle-dle East –20, Hendustralia , and sevast Asia –25 , sex, and age of the host reservoir could be linked to higher levels of viral shedding or higher contact rates to increase spillover transmission and successful virus establishment in a new species Fig. . At an iFurthermore, factors facilitating spillover transmission may act across scales and be interconnected. For example, paleontological evidence suggests that extinctions of large mammals due to climate change increased the endemicity of pathogens in the remaining wildlife community, which may conduce to increased spillover transmissions from wildlife to humans . FurtherPathogen virulence and pathogen-induced extinction risks are generally considered to be low in reservoir hosts . NeverthBox 1 Defining spilloverIn this opinion article, spillover transmission does not necessarily implies onward transmission after the new host is infected. Instead, spillover transmission refers to an specific moment in space and time when a given viral agent established in its primary reservoir host, circumstantially meets a new host. Thus, spillover transmission can be strictly referred as the moment of encounter, which implies a virus, somehow, enters a new host. Nevertheless, entering a new host does not necessary implies a virus will be successful to establish an infection and therefore be able to get transmitted intra-specifically  or inter-specifically . Both scenarios denote onward transmission, though with some subtle differences. Once spillover transmission occurs (the encounter), a onward transmission will depend on a successful infection . A successful infection will only happen if the virus finds an adequate permissive target cell and the virus possesses effective tools or mechanisms to evade the innate and adaptative host’s immune response of the host. Spillover could be considered as an stochastic event that may be governed by the probability a new host could get in touch with a virus from another species, regardless the virus’ ability to cause a successful infection with a consequent onward transmissionDifferent biological process act at different spatial scales . For exaBats are known to be major reservoirs hosts for many viruses, and consequently the primary source (donor or source host) of microorganisms that can successfully establish in naïve species (recipient host) , 53. TheViral establishment in the new host is dependent upon the frequency of spillover transmission events and demography of the new host . IntermeLyssavirus genus, and ranks among the best-understood and best-surveyed disease systems [Rabies is caused by all members of the  systems , 57. RAB systems . RABV is systems , 60. Rab systems , and dis systems . Further systems . Rabies  systems . In summDesmodus rotundus)  and birds . During feeding, D. rotundus can transmit rabies virus to their prey . Vampire bat-borne rabies virus (VB-RABV) outbreaks in humans suggest that mortality mainly occurs in tropical and subtropical regions. For example, Brazil reports about two cases of human rabies annually caused by bat-borne rabies viruses that have been laboratory-confirmed as antigenic variant 3, a RABV variant associated with D. rodundus. However, underreported cases that are not laboratory confirmed are probably much more common. Documenting how VB-RABV can spill from D. rotundus over to other species can inform and guide strategies to prevent VB-RABV spillover transmission to humans and domestic animals, and help prevent the potential spread of D. rotundus rabies from Latin America into the United States.While canine-rabies has been almost eradicated in the Americas, bat-borne rabies is now an emerging public health and agricultural problem , 63. In us) Fig. , one of rey Fig. , which mD. rotundus is one of the most well studied mammals in the Americas [D. rotundus, shows failure to establish onward transmission after spillover transmission to livestock. These elements make vampire bat borne rabies an extraordinarily unique wildlife-disease system for advancing the understanding of spillover transmission across large study areas.In summary, VB-RABV has been documented for over a century , and as Americas , 64, 67 Americas . For exabefore virus spillover transmission ends in disease emergence. The status quo in spillover transmission research reveals predictive limitations that fail to determine when a spillover transmission event could result in outbreaks, epidemics, and pandemics such as the COVID-19 pandemic caused by SARS-CoV-2.Virus spillover transmission, as the prelude for disease emergence, is a poorly understood highly complex phenomenon . AlthougD. rotundus to livestock is frequent and widespread in Latin America. Finally, VB-RABV primary spillover transmission to cattle results in end hosts, so that there are immense opportunities to better understand actual spillover transmission events that do not necessarily result in the establishment of new transmission cycles that may end up in long-lasting epidemics.Vampire-bat-borne rabies (VB-RABV) is an ideal biological system to study spillover transmission. For instance, RABV is well understood and there is an effective vaccine to reduce biosecurity risks for researchers. Additionally, VB-RABV spillover from e.g., lithium) or massive destruction after armed conflicts impact spillover transmission of wildlife viruses? These research lines are of critical public interest considering that the circulation of zoonotic viruses in wildlife is, in general, a threat to human health and social/economic development [Future research in this field should focus on five key questions. (1) What landscape conditions are needed for a spillover transmission event to occur in the first place? (2) How can biodiversity composition modulate the likelihood of spillover transmission? and (3) What type of mutations in viral genomes facilitate spillover transmission and subsequent disease spread across different species? (4) Are there any climatic drivers favoring or limiting spillover transmission? (5) How do landscape changes, such as changes due to emergent commodity or natural resources exploitation (elopment . By unde


Infectious diseases or communicable diseases are spread from person to person by various routs. So we have airborne diseases, bloodborne diseases, and contact diseases. In the work environment, pathogens can spread from employee to employee, often leading to the development of an occupational disease .According to the definition, an occupational disease is both acute and chronic disease, resulting from the performance of an occupation, resulting from the nature of the work or the conditions in which it takes place .-The possibility of causing disease in humans;-The degree of risk to workers;-Probability of spreading in the population;-Possibility of prophylaxis and/or effective treatment .Biological agents in the workplace are classified according to their infectious effect. The criteria for this breakdown include:Directive 2000/54/EC describes the principles of risk assessment, prevention, and control of biological agents at work. Biological agents are classified into four risk groups according to their level of risk of infection:Group 1—Biological agents for which there is a low probability of causing disease in humans, and, therefore, they practically do not pose a threat to workers ;Staphylococcus aureus causing skin infections, Borrelia burgdorferii causing Lyme Disease and Hepatitis A virus (HAV);Group 2—Biological agents with documented harmful effects on the human body. They can pose a risk to workers but are unlikely to spread to the human population. In addition, there are methods of effective prevention or treatment. Examples include Mycobacterium tuberculosis, Hepatitis B virus (HBV), Hepatitis C virus (HCV), and Human immunodeficiency virus (HIV). SARS-CoV-2 was also included in group 3;Group 3—Biological agents that are dangerous to humans and can cause serious diseases. They pose a serious threat to workers and are very likely to spread through the population. The current state of knowledge enables the implementation of effective prevention and/or treatment of these factors. This group includes: Ebola hemorrhagic fever virus, Lassa hemorrhagic fever virus and Variola virus [Group 4—Biological factors that cause severe disease in humans and most often lead to death. They pose a serious threat and their spread in the population of workers is very likely. At the same time, it is impossible to apply effective prophylaxis and treatment. This group includes only viruses: la virus .Infectious diseases are an important part of the diseases to which workers are exposed.In Poland, infectious diseases dominate among occupational diseases in recent years. Tuberculosis, HCV, and HBV are the most frequently diagnosed among healthcare workers. After all, not only medical personnel get sick. There are also teachers, officials, drivers, and all other employees who have contact with another person at the workplace  .The number of workers who become ill because they have had contact with the pathogen in the workplace is much greater. It is difficult to estimate how many because most of these events are unregistered. The ongoing COVID-19 pandemic has made this problem even worse.n = 577) outbreak related to staff reporting of infections. The number of healthcare workers infected with SARS-CoV-2 in connection with work in 2020 amounted to 20,697 people [In Poland, 2800 outbreaks of nosocomial infections were reported in 2020 (including 2265 outbreaks caused by SARS-CoV-2). Of the SARS-CoV-2 outbreaks, every fourth  is recommended for workers who have an occupational exposure to blood, tissue, or other body fluids that may contain HCV, HBV, HIV, but also for workers exposed to tetanus or rabies ,17,18.Unfortunately, the knowledge of employees about the risk of infection, as well as methods and possibilities of infection prevention, is insufficient, which is emphasized by the authors in their publications ,20.Prophylaxis in the workplace may include non-specific methods, such as the use of personal protective equipment , or washing and disinfecting hands, which is often emphasized as the most important method of interrupting the transmission of infectious agents . There aThe new epidemic challenges are related to the psychological burden on employees. It is true that the stress of the global epidemic threat affects society as a whole. However, medical staff experienced higher levels of anxiety, depression, and insomnia . These hFighting infections in the workplace requires legal bases that control behavior, describe the obligations of employers, regulate the rules of prevention, and, finally, regulate conflicts between the employee and the employer  Occupational infections in the workplace ; (2) Prevention of infections in the workplace; (3) Psychological problems of employees related to the risk of infection in the workplace; (4) Legal aspects of infections in the workplace.I have great pleasure to invite all authors to submit manuscripts for a Special Issue ”Infectious Diseases in the Workplace”. It will be a great compendium of the latest knowledge and developments in the field of workplace infection.