Routine skin cancer screening (SCS) is covered by the German statutory health insurance (SHI) since 2008. The objective of this study was to compare direct healthcare costs between patients in whom skin cancer was detected by routine SCS and patients in whom skin cancer was not detected by routine SCS.A retrospective observational study of administrative claims data from a large German SHI was performed. Patients with a diagnosis of malignant melanoma (MM) or non-melanoma skin cancer (NMSC) diagnosed in 2014 or 2015 were included. Costs were obtained for one year before and one year after diagnosis and analyzed in a difference-in-differences approach using regression models. Frequency matching was applied and risk adjustment was performed. Additional analyses were conducted, separately for specific age groups, excluding persons who died during the observation period and without taking costs for screening into consideration.A total of 131,801 patients were included, of whom 13,633 (10.3%) had a diagnosis of MM and 118,168 (89.7%) had a diagnosis of NMSC. The description of total costs (without risk adjustment) shows lower mean total costs among patients whose skin cancer was detected via routine SCS compared to patients in whom skin cancer was not detected by routine SCS  €5,073; €5,579) vs. €9,038 ; NMSC: €4,660  vs. €5,890 ). Results of the regression analysis show cost savings of 18.8%  through routine SCS for patients with a diagnosis of MM. These cost savings in MM patients were more pronounced in patients younger than 65 years of age. For patients with a diagnosis of NMSC, the analysis yields a non-substantial increase in costs ).Cost savings were detected for persons with an MM diagnosed by routine SCS. However, the study could not detect lower costs due to routine SCS in the large fraction of persons with a diagnosis of NMSC. These results offer important insights into the cost structure of the routine SCS and provide opportunities for refinements.The online version contains supplementary material available at 10.1186/s12913-022-08141-9. Skin cancer includes malignant melanoma (MM) and non-melanoma skin cancer (NMSC). MM is the most aggressive form of skin cancer . It affeIn Germany, around 22,890 persons were diagnosed with MM in 2018, while 199,400 persons were diagnosed with NMSC . For MM,According to the principle of economic efficiency (Wirtschaftlichkeitsgebot) which is anchored in § 12 Social Code Book V , health insurance services in Germany have to be sufficient, expedient and cost-effective; they may not go beyond what is necessary. Accordingly, the corresponding guideline (Krebsfrüherkennungs-Richtlinie) of the Joint Federal Committee (Gemeinsamer Bundesausschuss) stipulates regular evaluations of the quality and goal attainment of the routine SCS [A number of studies have analyzed the effectiveness of routine SCS in Germany. While results concerning the reduction of MM mortality are heterogeneous, they indicate a presumable increase in the detection of skin cancer in earlier stages –15. In aIn this study, we want to contribute to the emerging knowledge by analyzing direct healthcare costs for the year after diagnosis of skin cancer between patients in whom skin cancer was detected by routine SCS and patients in whom skin cancer was not detected by routine SCS based on a large cohort of statutory insured persons. Due to differences in prevalence, prognosis and course of disease, MM and NMSC will be analyzed separately. By this means, this analysis aims to provide differentiated information on the financial consequences of routine SCS. The study is part of a larger research project funded by the innovation fund .A retrospective observational study was performed. Nationwide claims data was provided in a anonymized form by the AOK Research Institute . The AOK is a large SHI provider covering about 27 million people in Germany, which represents around one third of the German population.A sample of 450,000 AOK-insured persons diagnosed with skin cancer was provided by WIdO. Due to data restrictions, these insureds were randomly drawn from a total number of 586,475 AOK insured persons diagnosed with skin cancer in 2014/15. Of these, patients with an initial inpatient or outpatient diagnosis of skin cancer in the years 2014 or 2015 were included. Initial diagnosis defines patients who did not have a suspected or confirmed diagnosis of skin cancer in the 24 months before diagnosis. Individuals with an age between 35 (lower age limit for routine SCS) and 100 years were included if they were insured by the AOK for at least five years. Furthermore, a tumor-associated surgical treatment and/or a tumor-associated medical treatment had to be performed in the twelve months before or the six months after diagnosis of skin cancer. A tumor-associated surgical treatment was defined if the patient received at least one surgical treatment from a list of pre-defined treatments. A tumor-associated medical treatment was defined if the patient received one or more prescriptions according to the pharmaceutical registration number  or the Anatomical Therapeutic Chemical (ATC) code from a list of pre-defined prescriptions. A complete list of included codes will be published elsewhere. Persons with diagnosis of MM  code C43) and NMSC (ICD code C44) were assessed separately. Persons who have been identified with both MM and NMSC were assigned to the cohort of the skin cancer which was diagnosed first. The date of diagnosis was defined as the date of discharge  or the date of service provision (outpatient care). Persons were assigned to the screening group if a routine SCS according to fee schedule item (Gebührenordnungsziffer) 01745 or 01746 was performed in the three months prior to diagnosis of skin cancer. Thus, patients were assigned to four different groups, depending on tumor entity  and the pattern of tumor detection.A calculation of the total costs in the year before and after diagnosis of skin cancer was carried out. The charges considered in the analysis comprise data on direct costs contained in the claims data in six areas, namely (i) inpatient hospitalization costs, (ii) outpatient hospitalization costs, (iii) costs for pharmaceuticals, (iv) outpatient healthcare costs, (v) remedy costs and (vi) costs for rehabilitation paid by the health insurance. Analysis was performed from the SHI perspective and thus, net costs without copayments were considered. Total costs in these six areas were analyzed. For pharmaceutical costs, the date of submission by the pharmacy was considered and for remedy costs, the invoice date was considered instead of prescription date. Due to the structure of the claims data, inpatient hospitalization costs, outpatient healthcare costs and costs for rehabilitation were only available for time periods . Only costs that occurred in the relevant observation period were included (on a pro-rata basis). For example, in cases that fell in both the pre- and post-observation periods, the costs were allocated proportionally over the days in the respective period. Differences between groups were tested for significance according to Mann–Whitney-U-test.A regression analysis was undertaken to predict the cost differences of patients who underwent routine SCS compared to patients diagnosed outside of the routine SCS. Statistical analysis was performed with SAS V9.4 . A difference-in-differences (DiD) approach was applied to control for possible background changes in outcomes that occur with time. For the DiD estimation, a pre-observation period of one year was defined, which ended one day before the date of skin cancer diagnosis. A generalized linear model with log link and gamma distribution was fitted to the data by means of maximum likelihood estimation and by estimating the parameters of the model via an iterative fitting process. The distribution was determined based on a modification of the Park’s test for heteroscedasticity proposed by Manning & Mullahy . For theth Revision of ICD (ICD-10) by Quan et al. [Frequency matching was applied. Included persons were matched by diagnosis , five-year age group, sex, and federal state of residence. To control for possible differences between the two groups and limit the potential for bias, the regression analysis adjusted for the Elixhauser Comorbidity Index and Pharmacy-based Metric. The Elixhauser Comorbidity Index is a risk adjustment measure based on distinctive ICD codes , which wn et al. . The Phan et al. . Both ElScr = dummy-variable to distinguish between routine SCS that has taken place (1) and no routine SCS (0)Per = dummy-variable to distinguish between pre- (0) and post-observation (1) periodPBM = Pharmacy-based MetricEH = Elixhauser Comorbidity Indexwhere.and coefficient estimator stimator :\documenAdditional analyses were conducted in which (i) persons who died in the observation period were excluded, (ii) only persons with an age of 35 to 64 years were included, (iii) only persons from 65 years of age onwards were included, and (iv) where screening costs for the individual persons diagnosed via routine SCS were subtracted.n = 271,613). A total of 7,474 patients were excluded because they were younger than 35 or older than 100 years of age. Further, 6,466 patients were excluded because information on federal state was missing or because they have been AOK-insured for less than five years. Finally, 32,646 patients have been excluded because no tumor-associated treatment was performed.The selection of the included study population will be reported in detail elsewhere. Patients without a relevant diagnosis of skin cancer and patients with a diagnosis of skin cancer before 2014 were excluded  underwent routine SCS. In addition, a total of 118,168 persons with a diagnosis of NMSC have been included, of which 43,308 (36.6%) persons underwent routine SCS. Patient characteristics including scores of the risk adjustment measures are shown in Table Descriptive statistics of costs for inpatient hospitalization, outpatient hospitalizations, pharmaceuticals, outpatient healthcare, remedies and rehabilitation as well as total costs as a sum of these components for the post-observation period are shown in Table Results of the main regression analyses are shown in Table Detailed results of the additional analyses are reported in Supplementary Table 4, Additional File Of the total number of 131,801 insured persons with skin cancer, 49,788 persons had undergone routine SCS. Based on the fact that the sample of 450,000 was randomly drawn from a sample of 586,475 insured persons with diagnosis of skin cancer, a total number of 64,888 AOK insured persons with positive SCS is estimated . According to the SHI frequency statistic (GKV-Frequenzstatistik), which depicts the services that have been billed for SHI-accredited healthcare services, a total of 4,900,618 routine SCS were performed in AOK insured persons in 2014 and 2015 (of which 53% were billed with EBM 01746 and 47% with EBM 01745), leading to total screening costs of €96,162,994. Applying these costs to the estimated 64,888 individuals with routine SCS would result in screening costs of €1,482 per detected skin cancer.n= 118,168) were diagnosed with NMSC, while 13,633 persons were diagnosed with MM. Almost half of the persons with a diagnosis of MM  underwent routine SCS while 36.6%  of NMSC patients underwent routine SCS. These numbers are similar to the proportions seen in the precursory pilot project SCREEN, in which 50.0% (585/1169) of MM and 37.8% of NMSC (2353/6218) patients were detected via routine SCS [A total of 131,801 persons with skin cancer were included in this study. The majority of persons (tine SCS .The cost-analysis of this study regarding the healthcare costs of patients diagnosed with skin cancer in Germany shows for MM lower healthcare costs for patients with a diagnosis of MM detected via routine SCS compared to patients who were diagnosed outside of the routine SCS program. Analysis of specific age groups indicates that this difference in costs was highest in patients younger than 65 years of age. In contrast, for patients diagnosed with NMSC there seems to be a tendency towards slightly higher costs in the group of persons with NMSC detected via routine SCS compared to the control group. However, both MM and NMSC are detected by routine SCS, with the program aiming to detect early stages. Considering the high proportion of NMSC in the total number of skin cancers diagnosed, the benefit of the routine SCS remains unclear. Since only cost analyses have been carried out so far, a need for a well-conducted cost-effectiveness analysis can be derived. Up to now, only one other study has assessed economic aspects of the German routine SCS program. Krensel et al.  includedBased on the results of 108,000 total skin examinations, Guther et al.  developeThe present study has several limitations. First, per definition, screening involves testing asymptomatic persons. Thus, individuals with suspected skin cancer who have participated in the SCS program must not be included in the routine SCS group. However, based on the available data, it was not possible to identify symptomatic patients in the routine SCS group who attended the SCS program to undergo diagnostic testing of their suspected skin cancer. This is due to the fact that in Germany, the medical documentation of dermatologists is anonymous and not contained in the claims data. Up to now, no data linkage is possible. This likely results in an overestimation of the true number of persons with routine SCS-detected skin cancer in the routine SCS group. This limitation was also mentioned in context of the precursory pilot program . Within Concluding, the results of this study indicate that routine SCS leads to lower costs in the year after diagnosis for persons diagnosed with MM. Cost differences were more pronounced in younger persons. The results do not provide a confirmation that routine SCS is viable from an economic perspective for persons diagnosed with NMSC. Methodological limitations inherent to the evaluated data have to be considered, due to which the screening costs of all insured persons were not taken into account. Future studies should aim to investigate the cost-effectiveness of the German routine SCS. These studies should seek to compare the specific patient benefits, such as an increase in quality of life or a reduction in the recurrence rate, with the additional costs of the routine SCS. Advancements of the German routine SCS program should address the problem that the majority of detected skin cancers do not clearly benefit from early detection. In addition, the question of whether a screening offer for specific risk populations would be an advisable alternative to the routine SCS program remains.Additional file 1. Additional file 2. Additional file 3. Additional file 4.


Since September 2020, Germany has experienced the first ever outbreak of African swine fever (ASF). The first known cases occurred exclusively in wild boar in forest areas in Brandenburg and Saxony; in July 2021, infected domestic pigs were also confirmed for the first time. As wild boar are considered the main reservoir for the virus in the European region, an effective interruption of this infection chain is essential. In particular, the removal and safe disposal of infected carcasses and the direct disinfection of contaminated, unpaved ground are priorities in this regard. For the disinfection, highly potent as well as environmentally compatible disinfectants must be used, which are neither influenced in their effectiveness by the soil condition nor by increased organic contamination. Thus, in this study, slaked lime, milk of lime and quicklime (1% to 10% solutions) were selected for efficacy testing against the test virus recommended by the German Veterinary Society (DVG), Modified Vaccinia Ankara virus (MVAV), and ASF virus (ASFV) in conjunction with six different forest soils from Saxony in two different soil layers  each. In summary, 10% of any tested lime type is able to inactivate both MVAV and ASFV under conditions of high organic load and independent of the water content of the soil. At least a 4 log reduction of the virus titer in all tested forest soil types and layers and by all applied lime types was observed. In conclusion, the high efficacy and suitability of all tested lime products against both viruses and in the presence of high organic load in forest soil can be confirmed and will help to control ASF spread. African swine fever virus (ASFV) is one of the most important emerging animal diseases, which spreads rapidly worldwide. From 2007, ASFV spread through Georgia and Russia to the EU in the Baltic states. From there, it continuously spread westwards and in September 2020 the first case in Germany was diagnosed in the state of Brandenburg . Within Asfarviridae, which is enveloped and can be easily inactivated by commonly used disinfectants like NaOH or formaldehyde [Orthopoxvirus Modified Vaccinia Ankara virus (MVAV) as a representative of enveloped viruses [The stability of ASFV in the environment is well documented ,8,9. ASFaldehyde , but mayaldehyde ,12. Nevealdehyde ,14,15,16 viruses . The aim2), quicklime (CaO) and lime milk (Ca(OH)2 in water) were tested. We have focused on six soil types in one of the most affected states in Germany, Saxony, and followed the guidelines of the DVG of using MVAV for screening the disinfectants. The most effective concentration was then tested on ASFV contaminated soil under appropriate high containment conditions. In addition, the best water/lime ratio was determined. The experimental layout is presented in In order to determine effective concentrations to inactivate enveloped viruses in contaminated soil, various concentrations of slaked lime (Ca(OH)As illustrated in 8 TCID50/mL for MVAV or 106.75 TCID50/mL for ASFV Armenia ∆258L GFP huCD4 [Three different lime products were examined in a suspension form: powdery slaked lime , powdery quicklime  and lime milk (made by mixing the slaked lime with water at various concentrations). The lime milk was freshly prepared to a stock solution before using in the experiment, representing 3.4-fold the desired final concentration of either 1%, 5% or 10%. A volume of 2.7 (diameter) + 1.5 (height) cm of TS and MS of each of the 6 soil types corresponding to about 3 mL soil were filled in 50-mL centrifuge tubes, and 3 mL of virus (10FP huCD4 ) and 4 m50) at a constant DNA genome copy number concentration.The experiment for each concentration of the lime products in each soil type and layer were done in duplicates. For each control, the experimental setup remained the same but without the disinfectant. The final virus dilution of the control suspensions at the end of the experiment corresponds to that of the suspensions with disinfectant. Quantitative real-time PCR to determine the total amount of viral DNA for both MVAV and ASFV were done in all disinfection experiments as described previously . This waTo determine the amount of water needed to activate the powdery lime products (quicklime and slaked lime), various water contents of soil were tested. The same experimental layout for MVAV was performed as above for one MS and one TS with little modifications . BrieflyThe experimental use of highly potent disinfectants in conjunction with cell cultures can lead to pronounced cell-toxic effects that can severely compromise the validity of results, especially for the exclusive evaluation via cytopathogenic effects (CPE) like in the case of MVA.While the CPE of MVA appears in the form of swollen, blistered and rounded cell bodies, toxic effects present themselves in the form of black staining (necrosis) and detachment of the cells. Only in case of high-grade cell toxicity, no more healthy cells remain to identify the CPE, which would make correct evaluation impossible.Extensive preliminary tests were carried out to investigate the toxicity of the different types and amounts of lime on the corresponding cell cultures.For this purpose, the respective lime concentrations  in the final dilutions used in the experiment were titrated without virus and added to the corresponding cell culture in 96-well plates. The cell culture was incubated and tested for toxicity over 2 h. If cell toxicity (necrosis/detachment of cells) was observed during this time in one or more dilution levels, the respective dilution level was added in addition to a 24-well plate containing cell culture. Again, incubation and observation of toxicity was performed for 2 h. If toxicity was no longer detectable here in the respective dilutions, inoculation of the critical dilution levels could be performed in larger cell culture plates in the main experiment (containing disinfectant and virus). Throughout the experiments, at least one control was titrated per experiment  to ensure a visual comparison between potential cell toxicity and pure CPE. This allowed optimal discrimination between toxic effects and CPE.Various types and concentrations of lime were used to deactivate MVAV in the presence of high organic contents (FCS). For lime milk, quicklime and slaked lime, 10% was sufficient to reduce the viral titer of MVAV by at least 4 logs . Same efThe influence of water contents on the disinfectant’s potency was tested. A complete inactivation was observed, when no extra water or up to 16 times the volume of the lime powder extra water was added. The amount of added water had no influence on the inactivation of MVAV in soil . The vir10 TCID50/mL inactivation was achieved with 10% for all three tested lime types at 10 °C and an exposure time of two hours. To simulate a field situation, the experiments were done with high organic soiling. In a previous study, 0.1% peracetic acid completely inactivated ASFV in various soil types, while citric acid had only limited efficacy [Decontamination of soil beneath and around carcasses can play an important role in preventing spread of ASFV. Many studies were conducted on the efficacy of the disinfectants on contaminated solid floor and walls of stables ,22,23, befficacy , lime waefficacy . Powderyefficacy ,29. Limeefficacy ,31 or poefficacy ,31.The powder form of lime has an advantage of ease of transportation to the affected area and to the target zone in forest, where carcasses are found. Ambient temperature has little to no effect on lime decontamination characteristics . Less orThe mechanism behind this class of disinfectants is the alkalization of the matrix (pH up to 12 or higher) ,33. ThisDVG in Germany has recommended the MVAV as a representative for enveloped viruses for testing the efficacy of disinfection solutions . A previIn conclusion, lime  are effective for the disinfection of forest soil contaminated with ASFV, especially in the presence of high organic soiling.


LRR-RLP) disease resistance gene family in a wild banana. We identified 78 LRR-RLP genes in the banana genome. Remarkably, seven MaLRR-RLPs formed a gene cluster in the distal part of chromosome 10, where resistance to Fusarium wilt caused by Foc race 1 has been previously mapped. Hence, we proposed these seven MaLRR-RLPs as resistance gene candidates (RGCs) for Fusarium wilt. We also identified seven other banana RGCs based on their close phylogenetic relationships with known LRR-RLP proteins. Moreover, phylogenetic analysis of the banana, rice, and Arabidopsis LRR-RLP families revealed five major phylogenetic clades shared by these plant species. Finally, transcriptomic analysis of the MaLRR-RLP gene family in plants treated with Foc race 1 or Foc TR4 showed the expression of several members of this family, and some of them were upregulated in response to these Foc races. Our study provides novel insights into the structure, distribution, evolution, and expression of the LRR-RLP gene family in bananas as well as valuable RGCs that will facilitate the identification of disease resistance genes for the genetic improvement of this crop.Banana is the most popular fruit in the world, with a relevant role in food security for more than 400 million people. However, fungal diseases cause substantial losses every year. A better understanding of the banana immune system should facilitate the development of new disease-resistant cultivars. In this study, we performed a genome-wide analysis of the leucine-rich repeat receptor-like protein ( Fusarium oxysporum f. sp. cubense (Foc) and Pseudocercospora fijiensis, respectively [Fusarium causing Fusarium wilt on bananas have been described [P. fijiensis infects susceptible banana cultivars throughout the stomata, causing leaf necrosis and premature ripening of the fruits [P. fijiensis, up to 66 applications per year in some producing regions, which is expensive and environmentally unsound [Banana is a staple food or source of income for more than 400 million people in many developing countries in the tropics. It is the most exported fruit in the world, and more than 145 million tonnes are produced annually, with an estimated economic value of US$52 billion [ectively . Foc infescribed . Banana e fruits . An inte unsound . This siP. fijiensis exist in wild bananas, and resistance gene candidates of the nucleotide-binding site and leucine-rich repeat (NBS-LRR) class of resistance (R) genes have been isolated [RGA2 (a.k.a. RGC2) [NBS-LRR gene family has been recently reported [R genes in bananas should facilitate the identification of novel R genes for Foc or P. fijiensis, which could be used to genetically improve the current susceptible banana cultivars, including the forgotten Gros Michel cultivar.Sources of resistance for Foc and isolated ,9,10,11.a. RGC2) . Moreovea. RGC2) ,14, and reported . The genThe knowledge gained in the plant immunity field over the past three decades has shown that disease resistance proteins of the receptor-like protein (RLP) and receptor-like kinase (RLK) classes located at the plasma membrane are involved in the perception of pathogens that colonize the extracellular space and activation of defense responses to halt pathogen spread . This plP. fijiensis are hemibiotrophic pathogens that colonize the xylem vessels and apoplastic spaces of leaf tissue, respectively [R gene of the NBS-LRR class is involved in the immune response of banana [P. fijiensis. In this sense, the recent discoveries made in tomato and its fungal pathogens Fusarium oxysporum f.sp. licopersici (Fol) and Cladosporium fulvum, which are taxonomically closely related to Fol and P. fijiensis, respectively [P. fijiensis. The study of the pathosystem tomato–Fol has uncovered remarkable insights into the identity of R proteins that confer resistance to multiple Fol races. The first tomato R gene identified to confer resistance to Fol was I-2, which belongs to the NBS-LRR class and confers resistance to Fol race 2 [I-3 gene, which belongs to the SRLK class of R genes and confers resistance to Fol race 3, was discovered [I and I-7 genes belong to the LRR-RLP class of R genes; the I gene confers resistance to Fol race 1, while I-7 confers broad-spectrum resistance to different Fol races, including races 1, 2, and 3 [F. oxysporum. On the other hand, the pathosystem tomato–Cladosporium fulvum reveals a dominant role of the LRR-RLP class of R proteins in providing protection against different races of this fungal pathogen [LRR-RLP gene could be involved in resistance to P. fijiensis since the tomato Cf-4 protein that confers resistance to C. fulvum race 4 belongs to the LRR-RLP class and is capable of detecting the P. fijiensis effector protein MfAVR4, an ortholog of the C. fulvum CfAVR4 effector, and mounts a hypersensitive-like response in Nicotiana benthamiana and banana leaves [LRR-RLP gene involved in immunity to P. fijiensis, to date, this hypothesis has not been proven.Foc and ectively ,7. In thf banana ; howeverectively ,19, can l race 2 . Later, scovered . Subsequ2, and 3 ,23. Thespathogen . It has a leaves . AlthougLRR-RLP genes cloned thus far confer resistance to extracellular pathogens and encode proteins with an extracellular LRR domain and transmembrane spanning domain anchored at the cell membrane [LRR-RLP class of R genes to provide protection to extracellular fungal pathogens related to Foc or P. fijiensis, a genome-wide analysis of the LRR-RLP gene family in the wild banana Musa acuminata ssp. malaccensis [P. fijiensis, would be of great value to the search for potential R genes related to these pathogens. Therefore, in the present study, we carried out a genome-wide annotation of LRR-RLP family members in M. acuminata ssp. malaccensis and determined their structure and location on the chromosomes. Moreover, we inferred their phylogenetic relationships with known R proteins of the LRR-RLP class and with the LRR-RLP families of the model plants Arabidopsis and rice, and finally, we analyzed the expression of several members of this family. The insights gained in this study provide a valuable platform for the systematic functional analysis of the LRR-RLP gene family in bananas that will facilitate the cloning of resistance genes for the genetic improvement of this crop.Most membrane . Taking accensis , which iM. acuminata ssp. malaccensis [https://banana-genome-hub.southgreen.fr/, accessed on 28 May 2020). Sequences with or without a signal peptide at the N-terminus followed by an LRR domain fused to a transmembrane domain and predicted to be located at the cell membrane were considered LRR-RLPs. These criteria were chosen based on the structure and subcellular localization of known LRR-RLP proteins  with the default parameter settings. Then, the C3-D HMM profile was used to search the banana proteome using the hmmsearch program of the HMMER package v3.3 with the default parameter settings . A second HMM search was carried out using the C3-D region of banana LRR-RLPs following the previous procedure. The retrieved sequences were analyzed and annotated using a combination of programs. The SMART [http://smart.embl-heidelberg.de/, accessed on 21 July 2021) and InterProScan v5.54-87 [https://www.ebi.ac.uk/interpro/search/sequence/, accessed on 21 July 2021) programs were used to identify the banana LRR-RLPs using the default parameter settings. The isoelectric point and molecular weight were determined with the Compute pI/Mw program [https://web.expasy.org/compute_pi, accessed on 21 July 2021). Finally, the DeepLoc v1 program [https://services.healthtech.dtu.dk/service.php?DeepLoc-1.0, accessed on 21 July 2021) was used to predict the subcellular localization of banana LRR-RLPs. The banana LRR-RLP genes were named with the prefix ‘Ma’ indicating M. acuminata, followed by numbers designated based on their positions from top to bottom on the 11 chromosomes.The banana genome and proteome sequences v2 of accensis  were obtproteins . To retrproteins  using thhe SMART   was used to identify exons, introns, and untranslated regions (UTRs) in the LRR-RLP genes. Then, the MEME v5.4.1 program [https://meme-suite.org/meme/tools/meme, accessed on 23 August 2021) was employed to identify conserved motifs in the LRR-RLP protein sequences, which were displayed with the GSDS program. Finally, 3D protein modeling was carried out using the SWISSMODEL suite [https://swissmodel.expasy.org/, accessed on 19 January 2022), and 3D models were edited with the PyMol program v2 .The Gene Structure Display Server   v2 program [http://mg2c.iask.in/mg2c_v2.0/, accessed on 19 March 2022 ). Physical clustering among LRR-RLPs was defined following the criteria of Jupe et al. [LRR-RLP genes was required to be less than 200 kb.Banana  program   was used to align known LRR-RLP proteins and banana LRR-RLP RGCs with the default parameter settings. Identical and similar amino acids were indicated with black and gray shading, respectively, using the Color Align Conservation program [https://www.bioinformatics.org/sms2/color_align_cons.html, accessed on 2 February 2022). For phylogenetic tree construction, the C3-F region of LRR-RLP proteins was used as proposed by Fritz-Laylin et al. [https://www.megasoftware.net/, accessed on 20 March 2022). Then, the maximum likelihood method based on the Jones–Taylor–Thornton (JTT) model implemented in the MEGA package was used for the construction of phylogenetic trees. The nodes were tested by bootstrap analysis with 100 replicates, and trees were edited with the FigTree v1.4.4 program . The phylogenetic relationships of banana LRR-RLPs with other plant LRR-RLP families were inferred using 57 and 109 LRR-RLPs from Arabidopsis and rice, respectively [The Clustal Omega program  . To assess the expression of some banana LRR-RLP RGCs in different tissues, three in vitro plants of M. acuminata ssp. burmannica accession Calcutta 4 were used to extract total RNA from leaf and root tissues. The in vitro plants were grown on MS medium supplemented with 3% sucrose under a 16 h light/8 h dark regime at 25 °C. Total RNA was extracted using the Norgen Plant/Fungi Total RNA purification kit  according to the manufacturer's protocol. Total RNA (5 μg) was used for first-strand cDNA synthesis with SuperScript III reverse transcriptase following the manufacturer's protocol . For RT–qPCR, Applied Biosystems™ SYBR™ Green PCR Master Mix (Thermo Fisher Scientific) was used. Three technical replicates were performed for each independent biological replicate. The banana 25S gene, amplified with primers reported by van den Berg et al. [−ΔΔCt method [The expression data reported by Li et al.  in Tableg et al. , was selt method . RT–qPCRM. acuminata ssp. malaccensis proteome v2 revealed 78 MaLRR-RLPs (MaLRR-RLP genes were divided into two groups: the first group comprised 59 members (76%) with a signal peptide motif at the N-terminus, LRR domain, and transmembrane domain (SP-LRR-TM), while the second group comprised 19 members (24%) with the LRR and transmembrane domains (LRR-TM) (The C3-D HMM profile was built from 20 known LRR-RLPs  and usedLRR-RLPs . A secon(LRR-TM) . All 78 MaLRR-RLP gene family showed that the majority of its members, 42 genes out of 78 (54%), were predicted to be intronless . The role of RLKs in plant immunity makes them obvious resistance gene candidates (RGCs) to start the search for a Foc race 1 resistance gene within this group of 19 RLKs. Since our study found seven MaLRR-RLPs  in the distal part of chromosome 10 . The seven Foc race 1 RGCs formed a phylogenetically closely related group. Considering that one or more of these seven RGCs could be involved in Foc race 1 resistance, it was intriguing that they were positioned next to clade I, where there is a gene (I) involved in resistance to F. oxysporum. Apart from the Foc race 1 RGCs, we also considered as RGCs those MaLRR-RLPs that showed a close phylogenetic relationship with known disease resistance LRR-RLPs. Therefore, we found a total of 14 MaLRR-RLPs that we proposed to be RGCs , one from Brassica napus (RLM2), and three from Arabidopsis . Although clade I showed a major expansion of LRR-RLPs in Arabidopsis and rice, which was not the case for bananas, only one MaLRR-RLP clustered in this clade. In total, five clades  were shared by banana, rice, and Arabidopsis LRR-RLP proteins, suggesting an ancient origin for these phylogenetic clades. Clade VI comprised MaLRR-RLPs belonging to banana only, and this clade showed the largest expansion of MaLRR-RLPs, with 31 sequences.The ML phylogenetic analysis of 78 MaLRR-RLP members revealed nine major clades and two phylogenetic singletons . Clades  be RGCs . Only onelopment . Moreover clades . Clade IMaLRR-RLPs in response to biotic stress and particularly to Foc, we used expression data from a previous study by Li et al. [MaLRR-RLP genes present in the 11,412 gene transcripts identified in the Cavendish cultivar roots  was higher in Foc race 1-infected plants than in those challenged with Foc TR4. Conversely, the expression of three genes  was higher in Foc TR4-infected plants than in the Foc race 1-infected plants. Interestingly, two RGCs, MaLRR-RLP58 and MaLRR-RLP78, which clustered in clades I-7 and I (MaLRR-RLP74) of the seven Foc race 1 RGCs was assessed by RT–qPCR in leaf and root tissues of the wild banana M. acuminata ssp. burmannica accession Calcutta 4, which is resistant to Foc race 1 and Foc TR4. The expression levels of MaLRR-RLP58 and MaLRR-RLP78 were higher in root tissue than in leaf tissue; notably, MaLRR-RLP78 showed a 26-fold increase, whereas the expression of MaLRR-RLP74 was higher in leaf tissue than in root tissue  from Fol, which is phylogenetically closely related to Foc race 1 and Foc TR4 [MaLRR-RLP74 and MaLRR-RLP75 genes, as they showed a similar size and 3D structure to I and I-7 deduced proteins that confer resistance to F. oxysporum in tomato. In fact, the identification of the I and I-7 resistance genes followed a similar rational approach to reduce the complexity of the number of RGCs. The I gene was mapped in a region of 1 Mb with 98 genes, including three TIR-NBS-LRR and one LRR-RLP gene. Since the three TIR-NBS-LRR genes were associated with viral resistance, the LRR-RLP gene was chosen instead as the most likely Fol resistance gene for the functional assay, resulting in the identification of the I gene [I-7 gene was located in a region of 210 kb with 29 genes, of which 18 were root-expressed, and only 1 of them resembled a resistance gene with an LRR-RLP structure, resulting in the identification of the I-7 gene in the functional assay [MaLRR-RLP family should facilitate the cloning of a Foc race 1 resistance gene. A genetic complementation approach using a banana Foc race 1 susceptible genotype such as Lady Finger will be useful to conduct transformation experiments with the seven LRR-RLP RGCs to assess their role in Foc race 1 resistance. Lady Finger has been shown to be a tractable genotype to study the role of antiapoptotic transgenes in Foc race 1 resistance [NBS-LRR RGA2 [More than 60 years have passed since the introduction of the Foc race 1-resistant cultivar Cavendish as a replacement for the highly susceptible Gros Michel cultivar. The resistance shown by Cavendish against Foc race 1 has been quite robust and durable; however, the molecular identity of the resistance gene has remained unknown. The mapping by Ahmad et al.  of the Fd et al. . Accordid et al.  did not  Foc TR4 . This in Foc TR4 ,59. Homo Foc TR4 , which r Foc TR4  to sevene I gene . The I-7al assay . Interesal assay  and our LRR RGA2  will opeMaLRR-RLPs that we considered RGCs based on their close phylogenetic relationships with LRR-RLPs with known functions in plant immunity. Interestingly, these known LRR-RLP proteins were not scattered in several clades but formed a few clusters, each containing at least one MaLRR-RLP. The LRR-RLPs of these clades may have a common signal transduction pathway to trigger immunity, causing them to cluster together in the phylogenetic tree. In bananas, an RGC of the NBS-LRR class of disease resistance genes known as RGA2 (a.k.a. RGC2) clustered in a phylogenetic clade shared by the tomato I-2 and melon Fom-2 genes that confer resistance to F. oxysporum [RGA2 showed that this gene was capable of mounting an effective immune response against Foc TR4 in the field [MaLRR-RLP RGCs represent a valuable platform on which to perform functional assays that may lead to the identification of novel resistance genes to combat the most damaging diseases of bananas, such as Fusarium wilt and black Sigatoka diseases. New technologies based on cisgenesis or gene editing should facilitate the introduction of new disease resistance traits into banana cultivars using resources from the same banana germplasm.In addition to the Foc race 1 RGCs located on chromosome 10, we found seven other he field . This fiArabidopsis and rice. We found five clades that were shared by banana, rice, and Arabidopsis, suggesting an ancient origin for these phylogenetic clades that may predate the divergence of monocot and eudicot plants that occurred approximately 200 million years ago [Arabidopsis and rice, unlike banana, which showed a radical contraction of members in this clade with just one LRR-RLP protein. Conversely, clade VIII showed a major expansion and diversification of LRR-RLPs only from bananas. The contraction or expansion of LRR-RLPs in these phylogenetic clades may be the result of different selection pressures from microbes that shape the immune receptor diversity in these plant species after having separated from the common ancestor [To analyze the phylogenetic relationships of the banana LRR-RLP family members with the complete set of LRR-RLPs from other plant genomes, we chose the LRR-RLP families of the model plants ears ago . Moreoveancestor ,63.MaLRR-RLP family in response to biotic stress showed that the expression of 18 genes was present in banana root tissue, and a few genes were upregulated in response to Foc race 1 or Foc TR4, suggesting the presence of cis-acting regulatory elements in the promoters of these genes that respond to biotic stress signals. A recent transcriptional study of the LRR-RLP family in Arabidopsis showed that biotic stresses perturbed the expression of the largest proportion (87%) of 51 AtLRR-RLP genes [Erysiphe orontii caused an upregulation of 50% of the AtLRR-RLP family members, indicating that a significant number of AtLRR-RLPs can respond to a particular pathogen [MaLRR-RLPs in the transcriptomic data of Li et al. [MaLRR-RLP genes with the most abundant transcripts in root tissue, while the remaining genes were not detected due to their low expression levels that escaped detection at the sequencing coverage used. Our RT–qPCR results point in this direction, since the expression of the RGCs MaLRR-RLP58 and MaLRR-RLP78 in root tissue was shown to be higher than that of Foc race 1 RGC MaLRR-RLP74, whose expression was not present in the transcriptomic data of Li et al. [MaLRR-RLP58 and MaLRR-RLP78 in root tissue challenged with Foc TR4 was intriguing since these two genes grouped with the Fusarium wilt resistance genes I-7 and I genes, respectively. Further expression analysis of the MaLRR-RLP58 and MaLRR-RLP78 genes in Foc TR4-resistant and Foc TR4-susceptible banana genotypes will provide valuable insights into their role in Foc resistance. The increased expression of MaLRR-RLP78 in root tissue in comparison to leaf tissue indicates that it may function as a root-specific receptor. Overall, this transcriptomic analysis has provided a foundation for further expression and functional studies of the LRR-RLP family in bananas.Transcriptional analysis of the i et al. ; this pei et al. . The uprMaLRR-RLP genes that were resolved in nine phylogenetic clades, and 60% of them were organized into gene clusters on six chromosomes. Phylogenetic analysis of banana, rice, and Arabidopsis LRR-RLP families revealed five major phylogenetic clades shared by these plant species, suggesting an ancient origin that may predate the monocot-eudicot split. Furthermore, evidence of expansion and contraction of MaLRR-RLP gene numbers in particular phylogenetic clades was found. In addition, our data analysis showed that seven MaLRR-RLPs formed a gene cluster in the distal part of chromosome 10, where resistance to Foc race 1 has been previously mapped. Therefore, we considered these seven MaLRR-RLPs as RGCs for Foc race 1. We also identified seven other MaLRR-RLP RGCs based on their close phylogenetic relationships with known LRR-RLPs. Finally, we detected the expression of several members of this family in transcriptomic data of plants treated with Foc race 1 and Foc TR4, where some MaLRR-RLP genes were shown to be upregulated in response to these Fol races. Taken together, the comprehensive analysis of the MaLRR-RLP family in a wild banana provides valuable resources that will facilitate the cloning of a Foc race 1 resistance gene and other disease resistance genes for the genetic improvement of banana cultivars and perhaps other crops.In this study, we identified 78


Mycobacterium tuberculosis (MtCM) has only low activityon its own. Remarkably, its catalytic efficiency kcat/Km can be boosted morethan 100-fold by complex formation with a partner enzyme. Recently,an autonomously fully active MtCM variant was generated using directedevolution, and its structure was solved by X-ray crystallography.However, key residues were involved in crystal contacts, challengingthe functional interpretation of the structural changes. Here, weaddress these challenges by microsecond molecular dynamics simulations,followed up by additional kinetic and structural analyses of selectedsets of specifically engineered enzyme variants. A comparison of wild-typeMtCM with naturally and artificially activated MtCMs revealed theoverall dynamic profiles of these enzymes as well as key interactionsbetween the C-terminus and the active site loop. In the artificiallyevolved variant of this model enzyme, this loop is preorganized andstabilized by Pro52 and Asp55, two highly conserved residues in typical,highly active chorismate mutases. Asp55 stretches across the activesite and helps to appropriately position active site residues Arg18and Arg46 for catalysis. The role of Asp55 can be taken over by anotheracidic residue, if introduced at position 88 close to the C-terminusof MtCM, as suggested by molecular dynamics simulations and confirmedby kinetic investigations of engineered variants.Unlike typical chorismatemutases, the enzyme from  In fact, the first discovered AroQδ enzyme, the intracellular CM from Mycobacteriumtuberculosis (MtCM),21 is on itsown only a poor catalyst (kcat/Km = 1.8 × 103 M–1 s–1),21 despite itscrucial role for producing the aromatic amino acids Tyr and Phe. However,this low activity can be boosted more than 100-fold to a kcat/Km of 2.4 × 105 M–1 s–1 through formationof a noncovalent complex with the first enzyme of the shikimate pathway,3-deoxy-d-arabino-heptulosonate 7-phosphate(DAHP) synthase (MtDS)  1A.2121 or its corresponding cationic residues in other CMs (of both AroHand AroQ families).22 However, high catalyticprowess is only achieved when this cationic residue is optimally positionedsuch that it can stabilize the developing negative charge at the etheroxygen in the transition state  twiceexceeding that of wild-type MtCM in the MtCM–MtDS complex,and can no longer be activated further through the addition of MtDS.12 The biggest gains in catalytic activity weredue to replacements T52P and V55D in the H1–H2 loop and R87P,L88D, G89A, and H90M at the C-terminus , followed by T52P (6-foldgain).12 Combined, these two changes, discussedin detail in a previous publication, gave a kcat/Km that was 22 times highercompared to wild-type MtCM.12 The fourC-terminal amino acid replacements together increased the activitymore modestly (by a factor of 4), and the five exchanges introducedin the two final evolutionary rounds yielded an additional factorof 5. The resulting combination of large-impact and more subtle residuesubstitutions in MtCMV  and differs considerably from that observed in free MtCM simulations to investigatethe behavior of MtCM in the absence or presence of ligands and toanalyze whether the protein is able to interconvert between activatedand nonactivated conformations in the absence of the MtDS partnerenzyme. We also compared the wild-type MtCM with the evolved MtCME. coli DH5αor XL1-Blue . All cloning techniquesand bacterial culturing were performed according to standard procedures.29 Oligonucleotide synthesis and DNA sequencingwere performed by Microsynth AG .General cloningwas carried out in 6 tag, first 5 residues missing). Partsof the MtCM gene (Gene Rv0948c) were amplified using oligonucleotides412-MtCM-N-V55D (5′-GTTCGCTAGCGGAGGTACACGTTTGGATCATAGTCGGGAGATGAAGGTCATCGAAC)or 413-MtCM-N-T52P (5′-GTTCGCTAGCGGAGGTCCGCGTTTGGTCCATAGTCGGGAGATGAAGGTCATCGAAC)together with oligonucleotides 386-LpLib-N2 (5′-GGTTAAAGCTTCCGCAGCCACTAGTTATTAGTGACCGAGGCGGCCACGGCCCAAT)on template pMG24812 to create a 163 bpPCR product. The PCR products were restriction digested with NheI and HindIII and the resulting 148bp fragments were individually ligated to the accordingly cut 2873bp fragment from acceptor vector pKTNTET-0.12 The ligation was performed with T4 DNA ligase  overnight at 16 °C. The ligation productswere transformed into chemically competent E. coli XL1-Blue cells. The cloned PCR’ed DNA fragments were confirmedby Sanger sequencing. Subsequently, the genes for MtCM-T52Pand MtCM-V55D were isolated by restriction digestion using enzymes XhoI and SpeI followed by a preparativeagarose gel, yielding corresponding 260 bp fragments. pKTCMM-H21 was used as acceptor vector and was accordinglyrestriction digested with XhoI and SpeI, yielding a 4547 bp acceptor fragment. The fragments were ligatedovernight at 16 °C, using T4 DNA ligase. The ligation productswere transformed into chemically competent E. coli KA12 cells23 and the inserts were analyzedby Sanger sequencing. The gene for variant PHS10-3p3,12 carrying an N-terminal His6-tag and missingthe first five residues, was recloned into the native format providedby plasmid pKTCMM-H. Acceptor vector pKTCMM-Hand pKTNTET-PHS10-3p3 were restriction digested with XhoI and SpeI, and the fragments were isolated frompreparative agarose gels. The 4547 bp and 260 bp fragments were ligatedovernight at 16 °C with T4 DNA ligase and transformed into chemicallycompetent XL1-Blue cells. The relevant gene sequence was confirmedby Sanger sequencing.Forthe construction of expression plasmids pKTCMM-H-V55D and pKTCMM-H-T52Pfor the native MtCM single variants, the individual site-directedmutants were first constructed in the pKTNTET background  anddifferent reverse primers  on different DNA templates. The gene encoding MtCM L88Dwas produced by PCR with primers LB5 (5′-TCCGCACATATGAACCTGGAAATG) and LB4 (5′-TAAGCAACTAGTTATTAGTGACCGTCGCG) on the template plasmid pKTCMM-H carrying thewild-type gene.21 The gene for the triplevariant MtCM (T52P V55D L88D) was assembled with primers LB5 and LB4on a pKTCMM-H derivative containing MtCM variant 3p3 (T52P V55D).12 The gene for MtCM variant PNAM (D88N) was generatedwith primers LB5 and LB6 (5′-TAAGCAACTAGTTATTACATAGCATTCGGA), and for the MtCM variant PLAM (D88L) with primersLB5 and LB7 (5′-TAAGCAACTAGTTATTAGTGACCAAGCGGA),in both cases using a version of the template plasmid pKTCMM-H, intowhich the gene for the top-evolved s4.15 variant had been inserted.12 The resulting 296 bp PCR fragments containing NdeI and SpeI restriction sites at the5′ and 3′ ends of the MtCM gene, respectively, weredigested with the corresponding enzymes to yield 278 bp fragments.These fragments were ligated to the 4529 bp NdeI–SpeI fragment of pKTCMM-H yielding the final 4807 bp plasmids.Different C-terminal variants of the MtCMgene were generated byPCR mutagenesis. DNA fragments were amplified with the same forwardprimer (containing an E. coli strain KA1330 carrying anendogenous UV5 Plac-expressed T7 RNA polymerase gene wasused to overproduce the (untagged) MtCM variants. KA13 cells weretransformed by electroporation with the appropriate pKTCMM-H plasmidderivative that carries the desired MtCM gene variant.T52P) and V55D(MtCMT52P), the transformed cells were grown in baffledflasks at 30 °C in LB medium containing 100 μg/mL sodiumampicillin until the OD600 reached 0.5. Gene expressionwas induced through the addition of isopropyl-β-d-thiogalactopyranoside(IPTG) to a final concentration of 0.5 mM, and incubation was continuedovernight. The cells were harvested by centrifugation (6500g for 20 min at 4 °C) and frozen at −80 °Cbefore being resuspended in a buffer suitable for ion exchange chromatography,supplemented with DNase I (Sigma), 150 μM phenylmethanesulfonylfluoride (PMSF) and cOmplete protease inhibitor cocktail(Roche). The cells were lysed using BeadBeater , with four times 30 s pulses with a 60 s wait betweeneach pulse. Insoluble debris was removed by centrifugation .For thetwo crystallized MtCM variants T52P (MtCMN-morpholino)ethanesulfonicacid (MES), pH 6.5. MtCM V55D has a pI of 6.74; therefore, the pelletwas resuspended in 50 mM acetic acid, pH 5.25. After lysis and centrifugation,the soluble lysate was loaded onto a HiTrap XL SP column for cation exchange chromatography and eluted with a 0–0.5M NaCl gradient. The purity of the eluted fractions was gauged bysodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)analysis and sufficiently pure fractions were pooled and concentratedusing concentrator tubes with a 5 kDa molecular mass cutoff (VivaspinMWCO 5K). The proteins were then further purified by size-exclusionchromatography using a Superdex 75 300/10 column  withrunning buffer 20 mM 1,3-bis[tris(hydroxymethyl)methylamino]propane(BTP), pH 7.5, 150 mM NaCl. Finally, the proteins were concentrated(Vivaspin MWCO 5K), frozen, and stored at −80 °C.The resuspension bufferwas selected based on the theoretical isoelectricpoint (pI) of the protein. MtCM T52P has a pI of 8.14, so the pelletwas resuspended in 50 mM 2- and washed once with 100mM tris(hydroxymethyl)aminomethane(Tris)–HCl, pH 7.5. The cells were pelleted again, and thecell pellet was either frozen for storage at −20 °C ordirectly resuspended in 80 mL of sonication buffer . The cells were disrupted by sonication on ice. The crude lysate was clearedby centrifugation .The supernatant was supplemented with sonication buffer to 100 mL,42 g of ammonium sulfate was added, and the solution was stirred at4 °C for 1.5 h. The precipitate was pelleted by centrifugation, dissolved in 8mL of low-salt buffer , and dialyzed against1 L of low-salt buffer overnight. Dialysis was repeated against another1 L of low-salt buffer for 3 h before application to a MonoQ  FPLC column .The sample was eluted over 80 mL in 20 mM piperazine by applying agradient from 0 to 30% of a high-salt buffer .The cells were harvested by centrifugation was determined using the Bradford assay.31The MonoQ fractions containing the proteinof interest were pooledand concentrated to less than 1 mL. The concentrated sample was directlyapplied to a gel-filtration column  and eluted in 20 mM BTP, 150 mM NaCl, pH 7.5. Proteinidentity was confirmed by liquid chromatography–mass spectrometry(LC-MS) ,with the observed mass being within 1 Da of the calculated mass. Proteinpurity was assessed by SDS-PAGE  and the enzyme concentration () ranging from 10 to 1500 μM were usedat 274 nm (ε274 = 2630 M–1 cm–1) or 310 nm (ε310 = 370 M–1 cm–1). Chorismate disappearance upon enzyme additionwas monitored to determine the initial reaction velocity (v0). The obtained data were fitted to the Michaelis–Mentenequation with the program KaleidaGraph  to obtain the catalytic parameters kcat and Km.Michaelis–Menten kinetics of the untaggedpurified MtCM variants were determined by a continuous spectroscopicchorismate depletion assay .The purified enzymes were diluted into 20 mM potassium phosphate,pH 7.5, containing 0.01 mg mL2VKL)21 or from the structure of the CM polypeptide in the apoMtCM–MtDS complex .The malate complex was chosen over ligand-free MtCM (PDB ID: 2QBV)41 due to its higher resolution and better refinement statistics.Both simulations gave essentially the same result; therefore, we willnot refer to the second data set any further. For the highly activeevolved MtCM variant (MtCMV), we used the recent crystalstructure (PDB ID: 5MPV).12 The MtCM–ligand complex (MtCMLC) was taken from PDB ID: 2W1A,21 excludingthe MtDS partner protein, where MtCM was co-crystallized with a transitionstate analog (TSA) in its active site (Table S1). Residues that were not fully defined were addedto the models using (often weak) electron density maps as referencein Coot.37 When no interpretabledensity was visible, geometric restraints  were applied during model building, to ensurestable starting geometries. The N-termini of all of the models wereset at Glu13, corresponding to the first defined residue in almostall of the resolved structures available. Glu13 was capped with anacetyl group to imply the continuation of the H1 helix. CM dimerswere generated by 2-fold crystallographic symmetry.Molecular dynamics (MD)simulations were carried out on a number of representative structuresfor CM. They included two independent sets of simulations for apoMtCM, starting either from the X-ray crystal structure of MtCM incomplex with malate   and 55 (V55D), we set outto determine the crystal structures of these two enzyme variants.Together, these two substitutions led to an increase in kcat/Km by 22-fold comparedto the parent enzyme.12The fact that MtCM exhibitsonly low natural catalytic activityprovided us with a perfect opportunity to probe features that optimizeCM catalysis by directed evolution.T52P crystals had the same spacegroup (P43212) and similarcell parameters as the wild-type enzyme , where the asymmetric unit contained the biologicaldimer. The MtCMT52P structure was refined to 1.6 Åand Rwork/Rfree values of 24.0/26.5% , whereas MtCMV55D diffraction data yielded lower-qualityelectron density, particularly for the H1–H2 loop . Consistent with this, the Wilson B-factor of MtCMV55D is high (57.8 Å2), indicating structural disorder. Refinement of the 2.1 ÅMtCMV55D model stalled at Rwork/Rfree values of 27.6/34.9%, with veryhigh B-factors for H1–H2 loop residues, especiallyfor protomer B. For both structures, residues preceding residue Glu13and C-terminal to Leu88 showed poorly defined electron density. Therefore,the terminal residues were not included in the final model.Whereas MtCMgroup P432 and sim2VKL(2QBV), with oT52P (PDBID: 6YGT) andMtCMV55D are very similar to the structure of substrate-freewild-type MtCM (PDB ID: 2QBV),41 with RMSD = 0.3 and0.4 Å, respectively. However, the H1–H2 loops (47MASGGPRLDHS57) of both protomers of MtCMV55D adopt a different conformation (RMSD = 2.3 Å compared to PDBID: 2QBV), whichmost closely resembles the kinked conformation in the MtCM–MtDScomplex  . This interaction preorganizesthe active sites of both MtCM variants for catalytic activity, mimickingMtCM in the complex with MtDS , differs significantly between the structures  2A. In th compare S1E,G,H. LC ). In particular, helix H2 showed a tendencyto unravel  from the original crystal structure geometry forthe CM core regions (RMSD = 2.8 ± 1.2 Å (MtCM) or 3.4 ±1.5 Å . The fact that the fluctuationsof the MtCMV H1–H2 loop are contained within theconformational basin of the catalytically competent geometry . Interestingly,ligand presence in one of the two binding pockets was sufficient tostabilize the structure of the whole dimer. Nonetheless, the H1–H2active site loop of the apo protomer retained its intrinsic flexibility. This result reflects the expected rigidification occurring uponsubstrate binding due to additional protein–ligand interactionsin MtCMLC. MtCMV accomplishes rigidificationas a direct consequence of its evolved sequence. Interestingly, alsoMtCMV55D shows generally dampened fluctuations, possiblydue to the extended helical motif observed in that structure.Overall, MtCMThe MtCM activesite contains four arginine residues 1F, amongWT, the two variants MtCMV and MtCMV55D exhibited lower RMSF values for all active site Arg residues.A crucial factor for the enhanced activityof MtCM in the MtCM–MtDScomplex is an MtDS-induced interaction between MtCM’s H1–H2loop and its C-terminus.Figure S4B,C).Our 1 μs-long simulations detected persistent, multiple interactionsinvolving the C-terminal carboxylate. In contrast, the hydrophobiccontacts between Leu54 and Leu88 were disrupted in the first nanoseconds,and almost never observed again during the rest of the simulationtime are substituted with Pro–Asp–Ala–Met (PDAM) atpositions 87–90, which include another carboxylate, introducedthrough Asp88. Our MD simulations show that the Asp88 carboxylatein the evolved variant MtCMV offers an alternative modeof interaction with Arg53 of the H1–H2 loop (S5), which is not possible for wild-type MtCM. This allowsfor a persistent interaction of C-terminal residues with the H1–H2loop throughout the simulation, while maintaining a highly flexibleC-terminus. Moreover, in MtCMV, Arg46 is topologicallydisplaced from its original position with respect to the loop andno longer able to engage in a catalytically unproductive salt bridgewith the C-terminus.In MtCM–H2 loop 6B and S5V, is a proline residue (RLGHto PDAM).12 However, in contrast to Pro52,Pro87 did not appear to have a major influence on the simulations.While Pro52 is likely contributing to H1–H2 loop rigidity,with an average RMSF of 1.6 Å in MtCMV compared to2.5 Å (MtCM) for this region, the C-termini showed similarlyhigh RMSF values in the two models (>3 Å). Although Pro87induceda kink at the C-terminus, this did not appear to affect the flexibilityof the three terminal residues Asp88–Ala89–Met90.Another interesting substitution, whichemerged within the four C-terminal residues during the laboratoryevolution toward variant MtCMV, the L88D replacement was only acquired after theH1–H2 loop-stabilizing substitutions T52P and V55D were alreadyintroduced. Guided by the outcome of the MD simulations, we thereforeprobed the kinetic impact of the innocuous single L88D exchange inthe context of three different sets of MtCM variants to experimentallyassess the benefit of the introduced negative charge for fine-tuningand optimizing catalytic efficiency. We looked at (i) changing Asp88in the MtCMV sequence 87PDAM90 intoAsn88 or Leu88, (ii) directly introducing Asp88 into the MtCM wild-typesequence, and (iii) the triple variant T52P V55D L88D (MtCM Triple).All variants were obtained in their native format, i.e., with their native N-terminus and without a His-tag, to allow foroptimal comparison with the structural and computational results.The variants were purified by ion-exchange and size-exclusion chromatographyfrom the E. coli host strain KA13,which is devoid of CM genes to rule out contamination by endogenousCMs.30 Subsequently, the enzymes’ kineticparameters were characterized by a spectrophotometric chorismate depletionassay.In the course of the directed evolution ofMtCMV leads toa 2.5-fold drop in the catalytic efficiency kcat/Km to 1.7 × 105 M–1 s–1. This decrease is dueboth to a slightly lower catalytic rate constant (kcat) as well as a reduced substrate affinity (doubled Km). When residue 88 is further changed to thesimilarly sized but nonpolar wild-type residue Leu88 in variant MtCMPLAM, the catalytic parameters essentially remain the same as forthe Asn88 variant , a trend for anincrease in catalytic activity upon replacing Leu88 by Asp88 was observed51 in thecomplex with MtDS (PDB ID: 2W1A)21 . This kink is much less prominent in wild-typeMtCM, or even MtCMT52P (S1B), andcompletely lost during the simulations of MtCMWT (we didnot carry out simulations on the single variant MtCMT52P). Thus, this conformation may well be a crystallization artifactrather than a prerequisite for an active MtCM.Both MtCMV.12 This residue is located onthe C-terminal side of the H1–H2 loop . During the MD simulations of MtCMV and the single variant MtCMV55D, the presence of Asp55reduced the mobility of active site residues. By interacting withArg18′ and Arg46, this residue helps to preorganize the activesite for catalysis and reduce unfavorable conformational fluctuationscaused by electrostatic repulsion in the absence of a substrate. Thisis supported by the lower RMSF values of MtCMV comparedto uncomplexed wild-type MtCM  measurements; preliminary data). By decreasingthermal fluctuations in the active site, Asp55 likely also reducesthe entropic penalty associated with substrate binding. Pro52 appearsto exert a similar stabilizing effect on the protein, despite therather small structural changes, as suggested by a 2 °C increasein melting temperature of MtCMT52P in DSF experiments comparedto MtCM (preliminary data). This single substitution alone raisesthe kcat/Km value of the enzyme by a factor of six.12 It is worth noting that the simultaneous substitution of T52P andV55D increased the melting temperature by 6 °C (monitored bycircular dichroism spectroscopy) and boosted kcat/Km by 22-fold.12 The top-evolved MtCMV even showed a meltingtemperature of 83 °C compared to 74 °C for the parent MtCM.12Nevertheless,preorganization and prestabilization appear to beof crucial importance for the catalytic prowess of MtCM. The largestboost in catalytic efficiency (12-fold enhancement) by a single substitutionwas observed for the V55D replacement found in the evolved MtCM–H2 loop 1D,E and –H2 loop 5A, an inype MtCM 1 and by 21 Specifically,a salt bridge is formed between the C-terminal carboxylate of MtCM(which is repositioned upon MtDS binding) and loop residue Arg53,possibly bolstered by a newly formed hydrophobic interaction betweenLeu88 and Leu54 . The 1 μssimulations suggest that salt bridge formation with Arg53 occurs insolution in all tested cases, whereas the hydrophobic contact is lessimportant.MtCMactivation by MtDSinvolves a change in conformation of the C-terminus of MtCM and itsactive site H1–H2 loop.26 Conserved positions emerged only when probingfor an intact activation mechanism by MtDS.26 Still, when residues 87–90 of MtCMV were evolvedfrom Arg–Leu–Gly–His to Pro–Asp–Ala–Met.Our simulations indicate that in free wild-typeMtCM, an interactionof the C-terminal carboxylate with the key active site residue Arg46is possible but infrequent due to fluctuations 1. In conc pocket 5 and 2B,ket V in 7. In theδ subclass CM of M. tuberculosis to the evolutionary distinct AroHclass CMs, or even to the structurally and functionally divergentAroQα, AroQβ, and AroQγ subclasses.53 Neither of those groupsof CMs have evolved to be deliberately poor catalysts that becomeproficient upon regulatory interaction with a partner protein suchas MtDS.21 To be amenable to ‘inter-enzymeallosteric’ regulation,28 the H1–H2loop in MtCM must be malleable and allow for conformational switchingbetween a poorly and a highly active form. In contrast, this regionis rigidified in a catalytically competent conformation in the overwhelmingmajority of CMs from other subclasses. This is exemplified by theprototypic EcCM (AroQα subclass) and the secreted*MtCM (AroQγ), which possess the sequence 45PVRD48 and 66PIED69, respectively, at the positioncorresponding to the malleable H1–H2 loop sequence 52TRLV55 of wild-type MtCM.12 Remarkably, the two most impactful substitutionsT52P and V55D occurring during the evolution of MtCMV haveled to the tetrapeptide sequence 52PRLD55, with both Pro and Asp being conserved in naturallyhighly active CMs.12It is obviouslyimpossible to directly transfer our findings of critical detailedmolecular contacts from the AroQδ subclass CM from Corynebacterium glutamicum is another structurally well-characterized poorly active CM (kcat/Km = 110 M–1 s–1) that requires complex formationwith its cognate DAHP synthase for an impressive 180-fold boost incatalytic efficiency.54 In that case, inter-enzymeallosteric regulation involves a conformational change of a differentmalleable segment between helices H1 and H2. Thus, while the moleculardetails important for the activation of a particular AroQδ CM cannot be transferred directly from one system to another, ourfindings suggest as a general regulatory principle the deliberateand reversible destabilization of a catalytically critical loop conformation.The AroQM. tuberculosis with the top-performing MtCM variant MtCMV (kcat/Km = 4.3 × 105 M–1 s–1), which emerged from directed evolution experiments. Both in MtDS-activatedwild-type MtCM and in MtCMV, high activity correlated witha kinked H1–H2 loop conformation and an interaction of thisregion with the C-terminus of MtCM. The autonomously fully activevariant MtCMV had amino acid changes in both of these regionsthat augment these structural features. In this report, we focussedon substitutions T52P, V55D, and L88D.MDgreatly aided the analysis of crystal structures that were compromisedor biased by extensive crystal contacts at the most interesting structuralsites. Our aim was to obtain insight into the crucial factors underlyingCM activity by comparing the structure and dynamics of the poorlyactive wild-type MtCM  are responsible for binding and rearranging the doubly negativelycharged substrate chorismate. Only one of these residues (Arg35) isfirmly in position before the substrate enters the active site. Ofcritical importance for catalysis is Arg46. During the MD simulations,Arg46 competes with another arginine residue (Arg53) for binding tothe C-terminal carboxylate 6A and adof Arg46 7,explaiV, namely, T52P, V55D, and L88D by investigating singlevariants as well as combinations with other residue replacements thatwere found to augment catalysis. The variants were characterized bycrystallography, MD simulations, and enzyme kinetics. The two residuesPro52 and Asp55 exert a major impact by prestabilization and preorganizationof catalytically competent conformations of active site residues,while Asp88 contributes to fine-tuning and optimizing the catalyticprocess. By expanding on the previous directed evolution studies,we have shown here how the accumulated set of amino acid substitutionsfound in MtCMV has resulted in an activity level matchingthat of the most active CMs known to date.12In summary, we tested our hypotheses on the specificimportanceof critical substitutions acquired during the directed evolution ofMtCM


Caliciviridae is a family of viral pathogens that naturally infects vertebrates, including humans, and causes a range of highly contagious infectious diseases. Caliciviruses are not well studied because of the lack of a universal approach to their cultivation; however, the development of molecular genetics and bioinformatics methods can shed light on their genetic architecture and evolutionary relationships. Here, we present and characterize the complete genome sequence of calicivirus isolated from a sandpiper—Temminck’s stint , preliminarily named Temminck’s stint calicivirus (TsCV). Its genome is a linear, non-segmented, single-stranded (+sense) RNA with genome organization typical of avian caliciviruses. Comparative studies have shown significant divergence of the nucleotide sequence of the TsCV genome, as well as the amino acid sequence of the major capsid protein from all publicly available genomic and protein sequences, with the highest genome sequence similarity to unclassified Ruddy turnstone calicivirus A (43.68%) and the lowest pairwise divergence of the major capsid protein with unclassified goose calicivirus (57.44%). Phylogenetic analysis, as well as a comparative analysis of the homologous proteins, showed evidence of another separate genus within the Caliciviridae family—previously proposed, but not yet accepted by International Committee on Taxonomy of Viruses (ICTV)—the Sanovirus genus, which combines seven previously unclassified genomic sequences of avian caliciviruses, including the newly discovered TsCV, which we propose to consider as a separate species. Caliciviridae family [Caliciviridae family—Lagovirus, Norovirus, Nebovirus, Recovirus, Sapovirus, Valovirus, Vesivirus, Bavovirus, Nacovirus, Minovirus and Salovirus [Caliciviridae is based on the protein sequence of the major capsid protein (VP1) with isolates with less than 60% sequence identity being assigned to different genera [Sanovirus [Secalivirus [Caliciviruses are small non-enveloped pathogens with a single-stranded RNA genome varying from 6.4 to 8.5 kb in length belonging to the e family . These ve family ,3. Accoralivirus .Norovirus, Recovirus and Vesivirus genera, three open reading frames (ORFs) are present. ORF1 of murine norovirus encodes a polyprotein, which is cleaved into six to seven nonstructural proteins (NS): N-term (NS1/2), NTPase , 3A-like (NS4), VPg , viral protease (NS6) and RNA-dependent RNA polymerase  [Lagovirus, Nacovirus, Nebovirus, Sapovirus and Valovirus, on the other hand, only two ORFs are present, with VP1 and the nonstructural polyprotein being encoded together by ORF1 and VP2 being encoded by ORF2. The 3′ end of the RNA genome is polyadenylated and the 5′ end is linked to VPg [The genomic RNA of caliciviruses is organized in one of two ways. In the genomes of representatives of Rp, NS7) . SimilarRp, NS7) . The calRp, NS7) ,11; the Rp, NS7) ; proteolRp, NS7) ; and theRp, NS7) . A majorRp, NS7) . In gened to VPg . Cup-shaped depressions located on the capsid surface of caliciviruses are considered to be a unique morphological feature of the group . The capBavovirus, Nacovirus and Norovirus. Bird-infecting caliciviruses are pathogens of known importance, since they are able to infect poultry, including chicken [Caliciviridae family, is more than 60% diverged from any other classified caliciviruses, suggesting that the newly identified virus does not belong to any of the ICTV-accepted genera, and shows the highest similarity (57.4%) to currently unclassified goose calicivirus (NCBI accession number KY399947.1), which was previously described as a founding member of the proposed Sanovirus genus [Birds are known hosts for caliciviruses from genera  chicken , turkey  chicken  and gees chicken . Additio chicken ,22,23, J chicken  and seve chicken ,26,27. A chicken ,29, whicCalidris temminckii), a small-sized shorebird of the Sandpipers family (Scolopacidae). The biological sample was collected without direct contact with animals; no invasive interventions on animals were carried out. The birds have been observed and identified by qualified zoologists at close distance from a camouflaged hideout. Bird droppings were collected immediately after discharge, taking only the surface part of the fecal pellet to avoid contamination. The sample under the study was collected in 2017 on the banks of the Yenisei River in Krasnoyarsk Region   and beloTo ensure the preservation of the nucleic acids of viral pathogens, fecal samples were placed in sterile tubes containing the transport medium . After transportation to the laboratory, the sample was stored in a low-temperature refrigerator.For the extraction of nucleic acids, an Allprep DNA/RNA mini kit  was used. All manipulations were carried out in accordance with the manufacturer’s instructions. Preliminary screening for avian viral pathogens was carried out as described earlier .Library preparation for high-throughput sequencing was performed with a NEBNext Ultra II RNA Library Prep Kit  following the manufacturer’s recommendations for partially degraded samples. No additional steps involving depletion and any kind of enrichment were used. Sequencing with MiSeq Reagent Kit v2 (500-cycles)  on the Illumina MiSeq platform  resulted in 1.276 M paired-end reads per the sample under the study.Caliciviridae family were used to obtain draft whole-genome assembly using the SeqMan NGen program .SPAdes software v.3.15.3  CAB SPb. ContigsAdditionally, the original raw reads were mapped to the draft whole-genome assembly to perform errors correction. BWA v.0.7.17  was usedAn NCBI open reading frame finder  was usedThe annotated genome assemblies used in the comparative analyses were retrieved from the GenBank database  [date ofCaliciviridae genus  is a linear, non-segmented, single-stranded positive-sense RNA, comprised of 8575 bases with an average G + C content of 51.73% . Ruddy turnstone calicivirus A  [Ruddy turnstone calicivirus A genome. Open reading frames (ORFs) prediction showed genome structure typical of avian caliciviruses—TsCV coding regions are organized into two ORFs: ORF1 of 6849 bases and short ORF2 of 711 bases separated by 1 nucleotide frameshift   . The ca526GPPGIGKT533 and 603KRKLFTSKLILATTN617; VPg motif 992DEYDTW997; protease motif 1169GDCGLP1174; RdRp motifs 1379KDELL1383, 1453DYSKWDST1460, 1556YGDD1559 and 1603FLKR1606; and VP1 (major capsid protein) motifs 1859PPG1861 and 1944FCLLKEP1950 , 48.5 kDa for NTPase (441 aa), 18 kDa for NS3 protein (160 aa), 8.3 kDa for VPg protein (76 aa), 75.8 kDa for Pro-Pol (689 aa) and 58.2 kDa for major capsid protein (546 aa) —the one(546 aa) .Caliciviridae family, the amino acid sequence of the major capsid protein (VP1) is used for taxonomic classification wherein the criterion for species demarcation is the divergence of the VP1 amino acid sequence of more than 60% [According to the International Committee on Taxonomy of Viruses (ICTV), in the than 60% . Caliciviridae family, since all the given divergence values, with their standard deviations considered, are more than 60% . Both of these genera include avian caliciviruses.We calculated the divergence of the VP1 amino acid sequence of TsCV and the VP1 sequences of representatives of each of the accepted genera. A set of representative sequences provided by ICTV was used for calculations . Thus, athan 60% . The VP1Nacovirus and Bavovirus according to the specified taxonomy. From each polyprotein sequence, a VP1 protein region was isolated based on sequence annotation and/or alignment with annotated members of the Nacovirus and Bavovirus genera .Goose calicivirus, (ARM65436.1 and QHW05885.1), duck calicivirus (AXF38657.1) and 2.Nacovirus (60.3 ± 0.8%) but is much closer to the proposed genus Sanovirus .Ruddy turnstone calicivirus A (AXF38726.1) has borderline divergence values from representatives of the genus 3.Sanovirus does not violate the demarcation criterion .The inclusion of ruddy turnstone calicivirus A (AXF38726.1) virus in the proposed genus 4.Nacovirus and with putative members of the proposed Sanovirus genus.Grey teal calicivirus (QDY92332.1) cannot be classified according to the accepted criterion, since the divergence values of its VP1 amino acid sequence are less than 60%, both in comparison with representative sequences of 5.Caliciviridae sp. QKN88796 appear to be misclassified to the genus Bavovirus and should be moved to the genus Nacovirus.Chicken caliciviruses accession numbers QXO14947, QXO14949, QXO14954, QXO14958, QXO14962, QXO14966, QXO14967, QXO14970, AFH89835.1 and Sequences belonging to unclassified caliciviruses are divided into two main clusters: a major one, which also includes representative sequences of 99947.1, ). With tCaliciviridae members. The tree was built on the basis of the amino acid sequences of major capsid proteins in accordance with the ICTV recommendations [Caliciviridae family.A phylogenetic analysis was performed to determine the evolutionary relationship between TsCV and other ndations , since tCaliciviridae family [Bavovirus and Nacovirus. Since all of the characterized members of the clade have been isolated from birds, the clade appears to represent a group of related bird-infecting caliciviruses.The topology of external nodes of the obtained phylogenetic tree  was stroe family . Genome Sanovirus sequences supports our hypothesis.Since the topology in the clade of interest was not supported by high bootstrap values , which can often be expected when constructing a phylogenetic tree of highly divergent sequences, we constructed a more accurate phylogenetic tree with a higher resolution on the basis of the whole genome nucleotide sequences of the TsCV and its closest relatives . The treCaliciviridae representatives was partitioned into 69 groups corresponding to different species. In addition, we applied the bPTP web interface, which uses the phylogenetic species concept to delimit species. Using both maximum likelihood and Bayesian approaches, 73 and 68 species partitions were identified, respectively. In both cases, the TsCV formed an independent operational taxonomic unit. Finally, using the GMYC method, 73 species groups with a single-threshold approach were identified . Various single-locus approaches based on the amino-acid sequence of the major capsid protein have been used to distinguish species. To create species partitions using paired patristic genetic distances, we employed the assemble species by automatic partitioning (ASAP) approach. The partition with the best ASAP score was selected. As a result of applying this method, the studied set of 96 Caliciviridae family. We propose assigning the TsCV virus to a new, previously undescribed species, preliminarily named Temminck’s stint calicivirus.The result shows that the TsCV genome is not partitioned with other genomes when using any of the listed models. Thus, we have shown the TsCV does not belong to a previously sequenced species of the 1.Bavovirus;Chicken calicivirus Q45/2013 (KM254171) and chicken calicivirus D62/2013 (KM254170) belonging the genus 2.Nebovirus;Bovine enteric calicivirus NB (AY082891) and Newbury-1 virus (DQ013304) belonging the genus 3.Norovirus;Chiba virus/GVIII (AJ844470) and Yuzawa virus GVIII (KJ196291) belonging the genus 4.Valovirus;Calicivirus pig/AB104/CAN (FJ355930), calicivirus_pig/AB90/CAN (FJ355928) and calicivirus_pig/F15-10/CAN (FJ355929) belonging the genus 5.Sapovirus.Sapovirus Angelholm virus SW278 (DQ125333), Ehime_virus (DQ058829) and Houston virus 7-1181 (AF435814) belonging the genus 6.Sapovirus;NongKhai-24 virus (AY646856) and Arg39 virus (AY289803) belonging the genus 7.Sapovirus;Sapovirus MT-2010/1982 (HM002617), sapovirus U65427 and Manchester virus (X86560) belonging the genus 8.Sapovirus;London_virus/29845 (U95645) and Bristol_virus (AJ249939) belonging the genus 9.Lagovirus;Rabbit_calicivirus-1 (X96868) and rabbit hemorrhagic disease virus-FRG (M67473) belonging the genus 10.Unclassified duck calicivirus 2 MN175552 and MN175556;11.Unclassified goose calicivirus KY399947 and MN068022;12.Nacovirus according to the ICTV Caliciviridae report [Bavovirus.Chicken calicivirus F10026n  belonging the genus 73715 bel2.Caliciviridae sp. OM469263.1 and OM469260.In addition, according to the species delimitation criteria used in this study, the following pairs of viruses should also be considered as the one species, since their major capsid protein sequences were exactly identical: We predicted the 3D structure of the following TsCV proteins: the VP1 protein, the proteinase–polymerase precursor protein (Pro-Pol) and the core domain of the VPg protein. All predictions were made using the machine learning approach AlphaFold 2  based onNorwalk virus [The predicted VP1 protein has a structure typical of the major capsid protein of caliciviruses and consists of short N-terminal arm and two main domains, shell (S) and protruding (P), linked by a flexible hinge A. P domalk virus ). The P1lk virus , folds iNorwalk virus [Norwalk virus, but misses a β-strand and a short α-helix in the proximal part of the domain. The polymerase pro-domain shows high structural similarity to that of both rabbit hemorrhagic disease virus (RHDV) [Norwalk virus [Norwalk virus  and misses a short α-helix present in RHDV. Several additional short β-strands compared to both RHDV and Norwalk virus are also found in the fingers domain. An additional α-helix in the C-terminal part of the thumb domain was predicted in the TsCV polymerase pro-domain.The predicted structure of the Pro-Pol complex contains proteinase and polymerase pro-domains A. Domainlk virus , but miss (RHDV)  and Norwlk virus , typicalThe core domain of the predicted 3D structure of VPg protein adopts a helical structure with N-terminus and C-terminus regions at two separate ends of the domain. The structure of the TsCV VPg core domain is highly similar to feline calicivirus  and porcFor the VP2 protein of TsCV, we were unable to obtain a 3D structure with acceptable pLDDT scores (the highest pLDDT score was 43.09); however, according to all the models obtained, the core part of the VP2 protein of TsCV consists of three consecutive alpha-helices—one long and two short. The predicted order of the secondary structures was in complete agreement with the structure of the VP2 protein of the feline calicivirus obtained with cryo-electron microscopy .Calidris temminckii captured in Russia, Siberia. Using metagenomic data, we were able to obtain the complete genome sequence of TsCV, annotate its CDS, describe its proteins and model their 3D structure, and carry out taxonomic classification and phylogenetic study. With the development of metagenomics, the inability to obtain a culture of microorganisms has ceased to be a problem that limits the ability to characterize the species diversity of an ecological niche and study the genomic features of its representatives. Here, we identified and characterized the complete genome of a novel Temminck’s stint calicivirus (TsCV), isolated from the wild bird Lagovirus, Nacovirus, Nebovirus, Sapovirus and Valovirus, for which the coding part is organized into two open reading frames encoding the polyprotein and VP1. The TsCV polyprotein has all the expected proteins, arranged in an order conservative among all caliciviruses [We showed that the structure of the TsCV genome corresponds to that of the accepted genera iviruses . Howeveriviruses ,78, as siviruses .The spatial similarity of the predicted 3D structures of TsCV proteins and the crystal structures of their homologues shows that machine-learning approaches can be successfully applied to model the caliciviral proteins and additionally confirms the CDS annotation. The ability to obtain the 3D structure of the caliciviral VP1 protein is a task of particular importance, since in caliciviruses, capsid-related functions, such as antigenicity and host specificity, are predominantly determined by its primary sequence and spatial configuration .Caliciviridae family. Then we applied the ICTV genus delimitation criterion to the unclassified viruses belonging to the Caliciviridae family and showed that TsCV can be assigned to the proposed Sanovirus genus [Caliciviridae sp. with accession numbers MT138017, MT138020 and MT138018.Metagenomic sequencing has provided researchers with a multitude of genomes, many of which require de novo classification. Development and improvement of bioinformatics methods and robust classification of metagenomic sequences allows a significant expansion of the formal taxonomy of viruses in the way of future studies of virus diversity ,81. Howeus genus  togetherus genus  and MN06us genus ) and CalCaliciviridae Study Group (CSG) for a caliciviral species was as follows: “A calicivirus species will be defined as a cluster of viruses that constitutes a major phylogenetic branch within a genus and is also distinguishable from other branch(es) by one or more of the following biologic properties: natural host range, natural cell and tissue tropism, and antigenicity” [The general definition developed by the enicity” . As can enicity”  and MN06We tested this assumption using several other single-locus methods for species delimitation. In this study, to classify species, we used GMYC and bPTP coalescent-based methods that combine population genetic and phylogenetic theory to provide an objective means for the delimitation of evolutionarily significant units of diversity ,54. In aBavovirus and Nacovirus genera, as well as unclassified caliciviruses, among which we localized the new TsCV virus. The tree demonstrates some minor differences in topology, e.g., the localization of branches MK204392.1 and MT138020.1. It should be noted that the nucleotide-based topologies are based on more phylogenetic information than amino acid-based topologies. In addition, it was shown that the use of alignment-editing methods allows the obtaining of a more correct topology, although sometimes with less robust supports [Despite the ICTV approach being traditional and widely used, we applied an additional method to obtain a more accurate phylogenetic tree with a higher resolution. For this, we applied the approach described in  with thesupports .Arenaria interpres) [Several viral species known to generate major disease burdens in people and animals, such as influenza viruses, West Nile virus, and Newcastle disease virus, have natural reservoirs in birds. Migratory birds, as a result, play a crucial role in the development and spread of dangerous viruses . Extensiterpres) . To our Temminck’s stint breeds in the north of Eurasia, mainly from Scandinavia to the east to Chukotka, Anadyr and Kamchatka, with more than 93% of the population occurring in Russia ,86. A ty


Orthopoxvirus, family Poxviridae. Many cases of MPVX infection are currently seen worldwide. Although the majority of cases reported are not severe, basic precautionary measures are key to preventing this infection from turning into a catastrophic pandemic. We report a case of a 54-year-old male with no high-risk behavior who developed a sudden uncharacteristic monkeypox virus rash while on a flight from Nigeria.Monkeypox virus (MPVX) is a double-stranded deoxyribonucleic acid (DNA) virus from the genus  Monkeypox virus (MPVX), a rare zoonotic infection, was first reported in 1958 as an outbreak of a pox-like disease in monkeys in Copenhagen, Denmark ,3. MPXV A 51-year-old African male presented with a rash Figures . The patAt the two-week follow-up, the patient was feeling better, and all his skin lesions have completely healed. There were no open lesions or scabs. He was asked to follow up if he noticed any similar lesions or a general worsening of his overall condition, which never happened.As our patient recently traveled from Nigeria, it is to be noted that MPXV originated from different parts of Central and West Africa . It is pBasgoz et al. described some of the common skin conditions that can have a variable dermatological clinical presentation like MPXV . It inclPolymerase chain reaction (PCR) is the definitive diagnostic method for MPXV , and itsRecently, a steep rise in MPXV individuals globally has been reported by the WHO. Varied clinical presentation and its prevalence in the uncontrolled HIV population warrant further research. Basic precautionary measures such as avoiding skin-to-skin contact with persons having a rash similar to monkeypox and maintaining good hand hygiene can have a huge impact in preventing this rare disease from turning into a pandemic.