Bacteroides thetaiotaomicron and the butyrate producer Roseburia intestinalis. By combining machine learning and flow cytometry, we found that the number of viable B. thetaiotaomicron cells decreases with glucose consumption due to acid production, while R. intestinalis survives post-glucose depletion by entering a slow growth mode. Both species attach to mucin beads, but only viable cell counts of B. thetaiotaomicron increase significantly. The number of viable co-culture cells varies significantly over time compared to those of monocultures. A combination of targeted metabolomics and RNA-seq showed that the slow growth mode of R. intestinalis represents a diauxic shift towards acetate and lactate consumption, whereas B. thetaiotaomicron survives glucose depletion and low pH by foraging on mucin sugars. In addition, most of the mucin monosaccharides we tested inhibited the growth of R. intestinalis but not B. thetaiotaomicron. We encoded these causal relationships in a kinetic model, which reproduced the observed dynamics. In summary, we explored how R. intestinalis and B. thetaiotaomicron respond to nutrient scarcity and how this affects their dynamics. We highlight the importance of understanding bacterial metabolic strategies to effectively modulate microbial dynamics in changing conditions.Bacterial growth often alters the environment, which in turn can impact interspecies interactions among bacteria. Here, we used an in vitro batch system containing mucin beads to emulate the dynamic host environment and to study its impact on the interactions between two abundant and prevalent human gut bacteria, the primary fermenter  The human gut is populated by hundreds of microbial species interacting with each other and with their host. Gut bacterial species differ in their distribution along the human digestive tract and favor different microhabitats, such as the lumen of the large intestine, colonic crypts and mucus layers , 2. DiveBacteroides is one of the most abundant but also most variable genera across human fecal samples [Bacteroides thetaiotaomicron (B. thetaiotaomicron) holds a large repertoire of genes involved in sensing and hydrolyzing many different polysaccharides, and B. thetaiotaomicron was reported to strongly express genes involved in the degradation of dietary plant glycans [B. thetaiotaomicron can alter its metabolic response to mucin glycans in the absence of dietary nutrients, to colonize the mucus layer and maintain its persistence in the human gut [An efficient way to acquire nutrients is essential for microbial persistence in the human gut. In addition to dietary fiber in the lumen, host-derived mucin glycans provide an alternative source of nutrients for members of the gut microbiota . The col samples . Its mos glycans , 16. As uman gut .Faecalibacterium, Eubacterium and Roseburia, are able to convert acetate and/or lactate into butyrate using the prevalent butyrate-producing pathway via butyryl-CoA:acetate CoA-transferase [Roseburia intestinalis  during growth on glucose [Short-chain fatty acids (SCFAs) including acetate, propionate and butyrate, are the major end products of the anaerobic fermentation of polysaccharides by the human gut microbiota. All these SCFAs exert multiple health benefits, in particular butyrate, which is used preferentially as an energy source to fuel colonic enterocytes and may trigger signals regulating mucin synthesis and secretion, as well as to maintain the integrity of epithelial barriers , 19. Metnsferase , 22. Lacnsferase . In addinsferase , 25. Upo glucose –28.R. intestinalis colonized mucin-covered carriers [Roseburia but not Bacteroides decreased in relative abundance with mucin supplementation [B. thetaiotaomicron and R. intestinalis , DSM 24846, DSM 108160, DSM 108161 and R. intestinalis L1-82 (DSM 14610T) were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen . The strains were frozen in Wilkins–Chalgren Anaerobe Broth  plus 20% glycerol and maintained at −80 °C until use.Bacterial strains of 2 solution (adjusted to pH 5.2) for 20 min at room temperature. Beads with a diameter of 500 μm were selected by wet sieving and stored at 4 °C in aseptic conditions until use.Mucin beads were generated based on a protocol previously described . BrieflyB. thetaiotaomicron and R. intestinalis. Briefly, each bottle was first filled up with 50 ml WC medium, and 3 g wet mass of 500 μm mucin beads in 10 ml WC were pipetted into each bottle under sterile conditions. The bottles were transferred to the anaerobic workstation  to be deoxygenated for 120 h. Oxygen-free conditions in liquid and in the beads were confirmed through the color change of redox indicator resazurin. The bottles were closed with rubber stoppers and sealed with aluminum caps that were UV sterilized for 1 h in a laminar flow chamber before use. For bottle experiments without mucin beads, bottles were filled with 60 ml WC medium that was prepared in the same way as described above.Serum bottles (120 ml) containing 60 ml WC medium and around 8000 mucin beads were used as a spatially structured system to grow B. thetaiotaomicron or R. intestinalis from frozen stocks was first inoculated in a 10-ml serum vial (Wheaton) containing 5 ml WC medium as preculture. These serum vials were prepared by adding autoclaved medium and were deoxygenated in the anaerobic workstation for 24 h. The vials were closed with rubber stoppers and sealed with aluminum caps before use. After 16 h, each species’ preculture was diluted to an OD600 of 0.1 determined by a portable spectrometer  and used as the inoculum. The serum bottles were inoculated with 1 ml of the diluted preculture and were incubated at a constant stirring rate of 170 rpm at 37 °C in a shaker . Cultivations of the monoculture and co-culture were followed for 120 h after inoculation, with samples taken from both the liquid and beads every four hours for the first 48 h and every 12 h afterwards. For further investigation of the B. thetaiotaomicron and R. intestinalis growth and their interactions in the long term, an experiment was designed for the co-culture to follow for 288 h with samples taken every 24 h since 120 h after inoculation. These batch fermentation experiments were performed in closed bottles, i.e., metabolites and gas composition were not kept constant. All experiments were performed at least in three biological replicates and always had a negative control bottle without inoculation but with sampling for each time, to verify its sterility.To prepare the inoculum, 100 μl of 600, and to profile metabolites as well as quantify bacterial cell counts by live/dead staining followed by flow cytometry.For each time point, 2 ml of the fermentation broth containing the suspended mucin beads were sampled to 2-ml tubes (Eppendorf). The serum bottles were opened and closed carefully each time to avoid contamination in the anaerobic workstation. During sampling the beads settled downward to the bottom within seconds. The supernatant was carefully pipetted into new 2-ml tubes in the anaerobic workstation, and subsequently was used to measure pH  and ODB. thetaiotaomicron and R. intestinalis interact with each other, and how abiotic factors influenced their growth. First, we grew a culture of B. thetaiotaomicron in WC for 48 h without mucin beads and sterilized the spent medium through a filtration with 0.2-μm sterile syringe filters . In addition, the filtered medium was autoclaved and deoxygenated for 24 h in the anaerobic workstation. Four conditions were prepared to cultivate B. thetaiotaomicron in the spent medium: (i) the original spent medium without adjusting pH (pH 5) and without adding carbon source, (ii) spent medium with pH adjusted to 6.5, (iii) spent medium with glucose and pyruvate added in the same concentrations as in WC at pH 5 and (iv) spent medium with added glucose and pyruvate plus pH adjusted to 6.5. We grew B. thetaiotaomicron in three biological replicates in the serum bottles for a period of 48 h and sampled every 8 h for measuring pH, OD600, metabolites and live/dead cell counts. Second, we grew B. thetaiotaomicron in WC plus mucin beads in different periods of 24, 48 and 72 h, as well as in WC without mucin beads for 48 h. The cell- and oxygen-free spent media were prepared in the same way as described above, and the pH was adjusted to 6.5 before autoclaving.Several spent media assays were conducted in serum bottles to clarify how B. thetaiotaomicron was followed for 48 h, with samples taken at 0, 4, 8, 12, 24, 36 and 48 h and measured as described above.We further adjusted the pH of WC medium to the specified values of 4.5, 5.0, 5.5 and 6.0 by using 1 M HCl solution, and also included the original WC of pH 6.7 for testing in serum bottles. The growth of B. thetaiotaomicron cells were added. We first grew B. thetaiotaomicron culture in a serum bottle containing 60 ml WC plus mucin beads, with a same inoculum size as described: 1 ml of OD600 0.1. After 24 h cultivation, 2 ml of the fermentation broth containing the suspended mucin beads were sampled to each 2-ml tube. 0.5 ml of phosphate buffer  was added to each tube after washing with 1 ml PBS. The beads in PBS were transferred to a 12-well plate , with 1.5 ml sterile WC medium in each well. For comparison, we included wells of fresh WC medium and WC medium supplemented with sterile mucin beads, both of which were inoculated with B. thetaiotaomicron in a volume of 25 µl of OD600 0.1. The experiment was performed in four replicate wells for each group. The plates were incubated at a constant stirring rate of 170 rpm at 37 °C, with liquid samples taken at 0, 30, 60, 90, 150 and 240 min for live/dead cell staining followed by flow cytometry.To estimate the rate at which bacterial cells detach from particles, we followed the population change of planktonic cells in a fresh WC medium, into which mucin beads with attached N-acetylneuraminic acid (Neu5Ac), N-acetylgalactosamine, N-acetylglucosamine, fucose, mannose and galactose were tested for their effects on the growth of B. thetaiotaomicron and R. intestinalis. The tests for each species were performed by using 24-well plates  filled with two types of media: the standard WC and WC without carbon sources glucose and pyruvate. Each medium was split into eight aliquots, each of which was supplemented with either one of those mucin sugars in 1 mM or a mixture of all six sugars together in 1 mM or without any sugar added as the control. We first inoculated 0.5 ml preculture with OD600 0.1 to 20 ml of each aliquot, and then transferred 1.2 ml to each well in three replicates. The plates of B. thetaiotaomicron or R. intestinalis were incubated at a constant stirring rate of 120 rpm at 37 °C for 24 or 48 h, respectively, with samples taken every 12 h for pH and live/dead staining followed by flow cytometry measurements.The degradation products of mucin including B. thetaiotaomicron containing the modified plasmid pWW3452 (CTAATG in the start codon of GFP was replaced with CATATG), which can stably fluoresce when grown under anoxic conditions with subsequent exposure to oxygen for 60 min as described [DNA-based staining using SYBR Green I  in combination with propidium iodide  was applied to quantify and differentiate bacterial cells with intact and damaged cytoplasmic membranes following the protocols described before . Brieflyescribed .B. thetaiotaomicron and R. intestinalis cells from different growth phases in serum bottles containing WC medium without and with mucin beads to exclude most of the background events and to obtain accurate cell counts. In addition to the instrumental calibration with CytoFLEX Daily QC Fluorospheres, 0.5 μm and 1 μm green fluorescent beads  were applied to each batch of experiments as internal standards, to monitor the instrument stability and to make cytometric data from different batches more comparable.Flow cytometric counts of the fresh cells were obtained by using a benchtop CytoFLEX S flow cytometer , equipped with the identical lasers and detectors as described previously . Cell evB. thetaiotaomicron and R. intestinalis cells by clustering populations of flow cytometric events in a UMAP space [K-nearest neighbors vote classifier  to identify the species labels of the co-culture events based on their proximity to the monoculture events in the supervised UMAP space. Before filtering out the blanks, each sample was overlayed with their corresponding blank runs and manually inspected using clickable three-dimensional scatter plots, where we could visually assure that the populations classified as blank in our samples overlap with the populations in the blank files and not with the cell populations. Examples of these scatter plots are available at: bit.ly/3WNrslL. Similarly, we manually verified that each classified species population overlapped with their monoculture runs for similar time points. The manual inspection also showed that our parameter choices for UMAP and the K-nearest vote classifier were robust.We used an in-house pipeline to quantify the absolute abundance of AP space . The detB. thetaiotaomicron and R. intestinalis in WC with and without mucin beads, to confirm the abundance profile of B. thetaiotaomicron and R. intestinalis in co-culture with 16S rRNA gene amplicon sequencing . The supernatants were used for chemical analyses by applying external standards for calibration and quality control. For the calibration curves, all compounds (Merck) were in high purity and acidic form (except sugars), and were dissolved or diluted in ultrapure water for calibration. The concentrations of trehalose, glucose, pyruvate, succinate, formate, acetate, lactate, n-butyrate and Neu5Ac were determined by high-performance liquid chromatography (HPLC). Propionate was also measured but not detected in our tests since B. thetaiotaomicron requires vitamin B12 (absent in our medium) to produce propionate via the succinate pathway [2SO4 as the mobile phase at a flow rate of 0.6 ml/min.Liquid samples in 1 ml were collected and centrifuged for 20 min at 21,130 ×  pathway , 41. HPLConcentrations of mucin monomeric sugars galactose, fucose and mannose were relatively quantified by using gas chromatography, carried out by the Metabolomics Core of KU Leuven. The supernatant in 100 μl of each sample was diluted to 1 ml in 80% methanol, and left overnight to allow the precipitation of lipids and proteins. After centrifugation, 100 μl of the supernatant was transferred and evaporated to dryness on a speedvac. A 2% methoxyamine solution was prepared by dissolving methoxyamine hydrochloride  in pyridine . 25 μl of the methoxyamine solution was added to each sample and left to react for 1.5 h at 37 °C for methoximation of the hexoses. Afterwards, 80 μl N,O-bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane  was added and left to react for 30 min at 60 °C for trimethylsilylation of the methoximated hexoses. One microliter of each methoximated and trimethylsilylated sample was injected by 2:1 split injection by an Agilent Technologies 7693 autosampler  into an Agilent 7890 A gas chromatograph equipped with a fused silica capillary column (0.25 mm × 30 m) with a chemically bonded 0.25 μm HP-5MS stationary phase . The injector temperature was 250 °C, and the septum purge flow rate was 3 ml/min. The helium flow rate through the column was 0.9 ml/min. The column temperature was held at 160 °C for 23.5 min and then increased by 100 °C/min to 300 °C, with 1 min holding there. The column effluent then entered an Agilent 7000 triple quadrupole mass spectrometer. Temperatures of the transfer line and electron impact ion source were 250 °C and 230 °C, respectively. Ions were generated by a 70 eV electron beam. Spectra were acquired in a selected ion monitoring mode, with fragment masses based on the analyses of standards.B. thetaiotaomicron and R. intestinalis in three biological replicates for RNA sequencing, which were selected from the following six independent experiments: monoculture B. thetaiotaomicron in WC , monoculture R. intestinalis in WC , co-culture B. thetaiotaomicron and R. intestinalis in WC , monoculture B. thetaiotaomicron in WC plus mucin beads , monoculture R. intestinalis in WC plus mucin beads , and co-culture B. thetaiotaomicron and R. intestinalis in WC plus mucin beads .We included a total of 69 samples representing the different growth phases of β-mercaptoethanol. The content of PowerBead tube  was further added to the lysate prior to using a bead beater  for homogenizing the mixture for 5 min at a speed of 30 beats/s. Other steps were performed by following the instructions. The eluted RNA samples in nuclease free water were stored at −80 °C until use. RNA concentrations were determined with a Nanodrop ND 1000 spectrophotometer  and a Qubit 2.0 fluorometer using the Qubit RNA High Sensitivity Assay Kit (Invitrogen). Samples with a 260/230 absorbance ratio below 0.5 were purified using the MinElute PCR Purification kit (Qiagen) and re-measured afterwards, with all samples above 0.5 after purification. RNA integrity and yield were evaluated using RNA Nano and Pico 6000 LapChips  run on an Agilent 2100 Bioanalyzer (Agilent Technologies).Total RNA was extracted and purified from RNAprotect-treated  frozen liquid samples using the phenol-free RNeasy Plus Mini Kit (Qiagen) according to the instructions of the manufacturer. The cell pellets from −80 °C were first lysed by adding the lysis buffer RLT from the kit with 10 μl/ml Library preparation and sequencing of RNA-seq libraries were carried out by the Genomics Core of KU Leuven. All samples were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina , with an initial amount of 100 ng total RNA in 12 μl. Bacterial rRNA depletion was proceeded using the rRNA Depletion Kit (Bacteria) , and the residual mRNA was quality controlled and converted to the libraries that were sequenced on a NovaSeq 6000  on a v1.5 S4_300 kit in XP mode. The resulting data of paired-end reads in fastq file format were obtained and demultiplexed before further analysis.B. thetaiotaomicron and R. intestinalis were downloaded from the ensemble bacteria [The analysis of the raw sequencing reads was performed as follows: low quality reads and adapters were trimmed with fastp . High qubacteria  and BV-Bbacteria , respectB. thetaiotaomicron VPI-5482), GCA_000011065.ASM1106v1.cds.all.fa (B. thetaiotaomicron VPI-5482), GCA_900537995.1_Roseburia_intestinalis_strain_L1-82_genomic.fna , GCA_900537995.1_Roseburia_intestinalis_strain_L1-82_cds_from_genomic.fna . Raw transcript counts were extracted from salmon’s output and used for differential gene expression analysis. COG categories, COG numbers and KO numbers were extracted from NCBI COG and KEGG database [B. thetaiotaomicron. To obtain annotations for R. intestinalis, we mapped the respective transcripts to the eggNOG database v5.0 with the eggNOG-mapper 2.1.9 [GCA_000011065.ASM1106v1.dna.toplevel.fa .To measure gene expression levels, we used methods of transcripts per million and DESeq2’s median of ratios to normalize RNA read counts . A stricDetails of model definition and parameterization can be found in the Supplementary file B. thetaiotaomicron produced succinate, lactate, formate, and acetate during fermentation, which decreased the pH from 6.7 to 5.0. At 20 h, B. thetaiotaomicron reached its maximal density of 1.2 × 106 viable cells/µl and hereafter its density decreased. After the depletion of glucose and pyruvate, the acidic conditions led to a loss of viable B. thetaiotaomicron cells but did not affect the total cell count. Loss of viability at low pH (5.0) was further confirmed by spent media and pH experiments. We re-grew B. thetaiotaomicron in its spent medium (collected at 48 h cultivation when glucose is depleted), and found that an initial pH of 6.5 is required for growth  increase of butyrate concentration concurrent with decreasing acetate and, in some cases, lactate , resulting in a pH increase from 5.7 to 6.0. In brief, viable B. thetaiotaomicron cells decline primarily due to the lowered pH, whereas R. intestinalis maintains higher cell densities by entering a slow growth mode.Glucose and pyruvate were provided as the main carbon sources in WC medium Fig. . When gr6.7 Fig. . R. inte/µl Fig. . After tR. intestinalis grown in monoculture for several time points and replicates to investigate its metabolic mechanism. During the exponential growth period, genes associated with glycolytic and pyruvate-utilizing pathways were more highly expressed at mid-log phase of 12 h compared to at 4 h  and butyryl-CoA:acetate CoA-transferase (RIL182_00413) corroborates the finding that R. intestinalis converts lactate/acetate to butyrate after glucose depletion. The energy yielded by butyrate production from lactate/acetate (ΔfG0 = −46 kJ/mol) is much lower than that from glucose (ΔfG0 = −250 kJ/mol) [R. intestinalis grows more slowly on lactate/acetate. In summary, R. intestinalis maintains its slow growth in the absence of glucose by converting acetate and lactate to butyrate.Given the observed diauxic shift from glucose/pyruvate to lactate/acetate, we performed RNA-seq of 4 h Fig. . Althougent Fig. . We furtent Fig. . When co kJ/mol) . TherefoB. thetaiotaomicron survives longer in the presence of mucin beads than without them. After depleting glucose and pyruvate at 24 h, planktonic B. thetaiotaomicron retained a viable cell count higher than 1.0 × 106 cells/µl  increase of 0.12 mM in B. thetaiotaomicron monoculture after 120 h and no significant change in R. intestinalis monoculture  compared to the WC control  and hybrid two-component system (HTCS) [σ showed significantly high expression in the first 4 h , and genes of ECF-σ-linked loci were also expressed after the glucose/pyruvate depletion (48 and 120 h), indicating BT utilization of mucin O-glycan components. Specifically, after glucose/pyruvate ran out at 24 h, we observed a significantly  increased expression of genes encoding the extracellular galactosidase  and fucosidase (BT_1842), as well as genes involved in the utilization of galactose  and fucose . These results are consistent with B. thetaiotaomicron metabolizing mucin O-glycans including galactose and fucose  across the outer membrane. At a low glucose/pyruvate level, genes of lactate dehydrogenase (RIL182_00335) and butyryl-CoA:acetate CoA-transferase (RIL182_00413) were significantly down-regulated  . The genose Fig.  and S9b.um Figs.  and S4b.um Figs. , the preted Fig. . Thus, m5 viable cells/µl for B. thetaiotaomicron and R. intestinalis, respectively  but lower than in R. intestinalis monoculture (5.7). After depletion of glucose and pyruvate, B. thetaiotaomicron’s viable cell numbers gradually declined, as in monoculture. R. intestinalis presented a second peak of 4.1 × 105 viable cells/µl at 38 h. In addition, the net concentration of lactate in co-culture was significantly lower than in each monoculture . For the co-culture grown in the presence of mucin beads, B. thetaiotaomicron reached a two-fold higher maximal density than without beads (1.5 × 106 viable cells/µl at 16 h). In contrast, R. intestinalis in the mucin co-culture exhibited significantly lower cell densities than in WC co-culture (p < 0.01 by Student’s t test), accompanied by a lower maximal density of 1.8 × 105 viable cells/µl. The pH of 5.1 was nearly as acidic as in the B. thetaiotaomicron monoculture with or without mucin. A second peak of R. intestinalis density in mucin co-culture was observed at 120 h, corresponding to a roughly ten-fold increase in density from 96 h to 120 h  increase of galactose in the co-culture compared to the B. thetaiotaomicron monoculture suggests that both species hydrolyzed mucin  changed over time for both organisms with and without mucin beads  changed from negative to positive, likely by reducing the concentration of mucin degradation products that affect R. intestinalis’ growth negatively in the presence of glucose. In summary, changing conditions alter interaction mechanisms between the two species, which lead to varying interaction signs and strengths.To investigate the interaction dynamics of ads Fig. . In the R. intestinalis, B. thetaiotaomicron genes associated with the transport of glucose  were significantly down-regulated in WC without mucin  belonging to the membrane-associated, proton-translocating ferredoxin:NAD+ oxidoreductase (Rnf complex) were also more expressed with mucin  and the ATP generation via proton pump (RIL182_00203-10) were down-regulated at high and low glucose/pyruvate levels, respectively , pyruvate (RIL182_01689), galactose (RIL182_01067-69) and N-acetylglucosamine (RIL182_02688-89), as well as seven out of ten glycolysis genes were systematically down-regulated at high glucose/pyruvate levels with mucin . We simplified the model by treating glucose and pyruvate, acetate and lactate as well as mucin sugars as single entities . Parameter values including metabolite production and consumption as well as state switching rates were determined manually. The equations, parameter values and initial conditions are given in the Supplementary file To reproduce physiological observables such as total and viable cell counts as well as concentrations of key nutrients and metabolites, we developed a model with ordinary differential equations that summarizes our understanding of the interaction mechanisms Fig. . Since w acidity ). In addose Fig. . R. intelow Fig. . FinallyB. thetaiotaomicron at 32 h in WC monoculture, the long-term survival of R. intestinalis in WC and of B. thetaiotaomicron in the presence of mucin beads , coupled with the more beneficial effect of mucin on the growth of B. thetaiotaomicron than R. intestinalis, agrees with their known niches in the human gut: B. thetaiotaomicron thrives as a mucus resident, while R. intestinalis tends to inhabit the lumen [Here, we investigated the molecular mechanisms underlying the ecological interactions between two representative gut bacteria in the presence of simple and complex carbon sources within a spatially structured environment Fig. . The grehe lumen , 32.Fig.B. thetaiotaomicron and R. intestinalis used alternative strategies to cope with and anticipate nutrient scarcity. Growth data combined with temporal gene expression patterns suggest that B. thetaiotaomicron acts as a “funnel” with its central carbon/energy metabolism capable of tapping several monosaccharides. Independent entry points are funneled into glycolysis until phosphoenolpyruvate is produced, which is converted into oxaloacetate by fixing a CO2 molecule with the generation of an extra ATP [2 could be supplied from either the external environment or the conversion of pyruvate to acetyl-CoA. Although this strategy results in accelerated growth, it comes at the cost of significantly inferior performance when actively consumed nutrients are depleted. In contrast, R. intestinalis uses carbon chain elongation to generate additional energy and maintain a redox balance  gene. B. thetaiotaomicron is unable to catabolize Neu5Ac and did not grow on it in a minimal medium [B. thetaiotaomicron’s growth during glucose fermentation  particularly for mucin utilization [B. thetaiotaomicron consumed glucose. As B. thetaiotaomicron was reported to easily switch its metabolism from dietary input to mucin glycans [R. intestinalis does not grow well when glucose and mucin are supplied together  alters the composition and structural properties compared to the colonic mucins, which could potentially alter the attachment and further utilization abilities of gut bacteria. In addition, nutrient availability and pH may vary less in the colon than in our experiments since sorption across the epithelium reduces the accumulation of fermentation products. Lastly, we studied interaction mechanisms in a specific context using specific strains. It remains uncertain how these findings would apply to different strains of these two species or in varied environmental settings (e.g. different medium). Although context and strain-specific dynamics are widely expected in microbial ecosystems—with strains differentiated by as few as 100 bp SNPs possibly having markedly different biotic interactions —we obserins Fig.  and someeviously .A deeper understanding of alternative metabolic strategies and changing species interactions in gut microbiota is necessary to rationally design next-generation probiotics and to predict the outcome of fecal microbiota transplantation. By deciphering the interaction mechanisms between two common gut bacteria across changing conditions, we have taken another step towards this goal.Supplementary file 1Supplementary file 2Supplementary file 3Supplementary file 4Supplementary file 5


We report a case of occupational monkeypox virus infection from a needlestick injury in a healthcare worker in South Korea and review similar reports in the literature during 2022. Postexposure prophylactic treatment with a third-generation smallpox vaccine and antiviral agent tecovirimat inhibited local virus spread and alleviated lesion pain. In July 2022, the World Health Organization (WHO) declared the international mpox outbreak a global public health emergency . However, most infections of HCWs occurred in the community, rather than from occupational exposure. Further investigations are needed to determine the main risk of occupational exposure to monkeypox virus in hospitals and the best responses for prevention  as postexposure prophylaxis. The HCW had no history of smallpox vaccination, exposure to monkeypox virus, or recent sexual contact and had not traveled abroad.On November 14, 2022, a 33-year-old healthy female HCW was exposed to monkeypox virus through a needlestick (26G needle) injury on the left index finger during aspiration of an infected patient’s vesicle. The HCW wore personal protective equipment consisting of a disposable gown, double gloves, and powered air-purifying respirator. Bleeding occurred after the injury, and povidone-iodine was applied to the puncture site ≈20 min after the incident. Within 20 hours after injury, the HCW received a third-generation smallpox vaccine , the HCW noticed a small papule at the needlestick site , panel AOn day 6, the lesion was slightly larger and had a central umbilication , panel BOn day 8, a new lesion appeared immediately above the initial lesion and began to progress , panel COn day 18, the lesions formed a crust , panel DWe conducted a literature review to evaluate the status of and response to monkeypox virus infections among HCWs during the 2022 outbreak . TransmiAs recommended by WHO (On the basis of our case report and literature review, we recommend the following procedures for HCWs who treat patients with mpox. First, the literature review revealed differences in clinical manifestations depending on the infection route and vaccination status, similar to findings from previous reports from the prairie dog–associated mpox outbreak in the United States (In summary, we report a case of monkeypox virus infection in a HCW after a needlestick injury and a literature review of similar cases during the 2022 mpox outbreak. Although larger studies are needed to determine efficacy of postexposure vaccination prophylaxis, this case series indicates postexposure vaccination might have prevented dissemination of virus lesions. Therefore, clinicians should consider postexposure vaccination and tecovirimat or other antiviral drugs to inhibit local monkeypox virus spread and alleviate lesion pain.


The self-administered neck mobility assessment tool (S-ROM-Neck) is the subjective cervical region range of motion (S-ROM) assessment scale. The study aimed to demonstrate the reliability and validity of the Turkish version of the S-ROM-Neck in patients with chronic neck pain.A cross-sectional study was conducted with a total of 60 chronic neck pain patients in the Physical Therapy and Rehabilitation Clinic of Pamukkale University Hospital between January and August 2021. The mean age of the individuals was 34.1 ± 9.9 years. Patients were assessed with S-ROM-Neck twice to prove the test-retest reliability. In addition, Visual Analogue Scale (VAS), Neck Disability Index (NDI) and bubble inclinometer measurement were used to analyze the construct validity of S-ROM-Neck.The intraclass correlation coefficients of the S-ROM-Neck were higher than 0.80 . The internal consistency of the S-ROM-Neck total score was within the acceptable limits (α = 0.754). Construct validity was high regarding the correlation analysis .Turkish S-ROM-Neck is a valid and reliable tool to assess the S-ROM of individuals with chronic neck pain. Cervical region range of motion (S-ROM) is a frequent evaluation method in clinical practice . SeveralS-ROM is measured with various methods and tools in clinical practice 10, 11). Goniometers are sensitive to assess the S-ROM with various devices or additional tools. Wolan-Nieroda et al. demonstrated the reliability and validity of S-ROM evaluation with a head-fixed universal goniometer . The uni, 11. GonLangenfeld et al. developed the self-administered neck mobility assessment tool (S-ROM-Neck) due to the lack of a subjective assessment tool. S-ROM-Neck is a patientreported outcome measure (PROM) to evaluate S-ROM in painful conditions . To our ClinicalTrials.gov Identifier: NCT04575129).A cross-sectional study was conducted with a total of 60 chronic neck pain patients in the Physical Therapy and Rehabilitation Clinic of Pamukkale University Hospital between January and August 2021. Sixty chronic neck pain patients were included. Informed consent from the patients was obtained. The ethics committee of Pamukkale University approved the study protocol (No: 13.10.2020-19). The study protocol was also registered  native Turkish speakers, (2) individuals >18 years, and (3) patients with neck pain >3 months. The exclusion criteria of the study were; (1) history of neck surgery, (2) specific neck conditions or comorbidities: e.g., posttrauma, fracture, disc herniation, spasm, tumor, infection, (3) rheumatologic and neurological conditions, (4) pregnancy, and (5) cognitive problems.The permission to translate the S-ROM-Neck into Turkish was received from Dr Langenfeld. International translation and cross-cultural adaptation guidelines were used , 21. FirA single physiotherapist evaluated the patients in a face-to-face session. A detailed information was given to the individuals during the recruitment process.Patients were assessed with S-ROM-Neck twice to prove the test-retest reliability.Visual Analogue Scale (VAS), Neck Disability Index (NDI) and bubble inclinometer measurements were used to analyze the construct validity of S-ROM-Neck.S-ROM-Neck includes six questions targeting the primary movements of the neck joint motion . Patients were asked to report the restriction  during neck movements. S-ROM-Neck includes a visual analog scale. The left and right side of the analog scale indicates severe ROM restriction and maximum ROM, respectively. S-ROM also contains iconographic content to inform the patients about neck movements. The total score ranged from “0 to 600”. A lower score represents better ROM  Table 1Table 1.VAS is a frequently used pain assessment tool. A minimum [0] and maximum score [100] indicate no pain and most severe pain, respectively. The validity and reliability of this scale have been demonstrated .NDI contains ten sections: pain intensity, personal care, weight lifting, reading, headaches, concentration, work-life, driving, sleep, and leisure activities. Each section is scored from “0” (positive status) to “5” points (negative status). A high score indicates a high disability .Flexion, extension, lateral flexion and rotation of the cervical joint were evaluated with the Baseline Bubble Inclinometer . The inclinometer is valid and reliable for evaluating the S-ROM. The flexion, extension, and lateral flexion were assessed with the procedure (1). Rotation was evaluated with the procedure (2).An inclinometer was placed on the top of the patient’s head as the pivot point, and the participant was asked to complete the movement to the last point and return to the initial position .The inclinometer was placed on the forehead of the participant in the supine position, and the patient was asked to rotate. The measured value in the inclinometer was recorded. Each movement was evaluated three times separately, and the average score was noted. .95) and minimum detectable change-minimal detectable change (MDC95) were calculated. The relationship between S-ROM-Neck and other measures was analyzed with the Spearman correlation analysis since the data was not normally distributed. A correlation coefficient >0.5 indicates excellent validity [“IBM SPSS Statistics ” computer package program was used for all statistical analyses. Descriptive statistical information was given as “mean ± standard deviation (x ± SD)”, “median (IQR)” and “number, percent” . Kolmogorov–Smirnov/Shapiro–Wilk tests were used to determine the homogeneity of the data distribution. The skewness and kurtosis coefficients were considered to ensure the data normality. The intraclass correlation coefficient (ICC) was used to assess test-retest reliability. ICC > 0.80 indicates an excellent test-retest reliability . In addivalidity . The staThe mean age of the individuals was 34.1 ± 9.9 years, and 51.7% (31) of the participants were women. The median (IQR) of the Neck pain duration was 12 (19) months. Characteristics and clinical data of the participants are presented in During the cross-cultural adaptation process, no modifications were required. Besides, comprehensibility was excellent regarding the pilot study reports.95 and MDC95 values were 6.9 and 19.3, respectively . The test-retest reliability was excellent. The internal consistency of the S-ROM-Neck total score was within the acceptable limits (α = 0.754). Besides, S-ROM-Neck’s SEMectively .Construct validity was high regarding the correlation analysis . The correlation between S-ROM-Neck with VAS, NDI, and inclinometer-based total ROM was −0.563, −0.677, and 0.904, respectively. The correlation between the S-ROM-Neck total score with inclinometer-based measurements of flexion, extension, lateral flexion and rotation ranged from 0.579 to 0.807 . On the 95 and MDC95 values of S-ROM-Neck can provide essential reference data for patient monitoring in clinical practice. Several PROMs provide essential clinical data in patients with neck pain [The study results proved the reliability and validity of the Turkish S-ROM-Neck. SEMeck pain . S-ROM-Neck pain .The ICC results demonstrated an excellent test-retest reliability of S-ROM-Neck (ICC > 0.80). The development study (ICC = 0.718) also reported similar results in terms of test-retest reliability. Regarding these results, the Turkish S-ROM-Neck was considered a reliable tool in different measures. Contrary to the development study, we also calculated the ICC value of each item separately . The ICCCronbach’s alpha values were within the acceptable limits both in development study and Turkish S-ROM-Neck . Interna95 and MDC95 values as 3.1 and 8.7, respectively [95 and MDC95 values were 6.9 and 19.3, respectively. The difference in these values may have been caused by the difference in the clinical group and the severity of the symptoms. For instance, the VAS value was approximately 35 in the development study and around 56 in our study. Therefore, The MDC95 value of both studies is acceptable. Clinicians can use these values as a reference value to observe the lowest detectable change when using the S-ROM-Neck measurement in the treatment follow-up of their patients. Clinicians can decide based on symptom severity for MDC95 reference here [Langenfeld et al. calculated the SEMectively . The Turnce here .The higher correlation coefficient indicated the higher validity level of the S-ROM. The S-ROM-Neck was highly related to NDI, VAS, and inclinometer measurements. This result revealed a high validity of S-ROM-Neck. On the other hand, high correlations were also observed in comparing inclinometer and S-ROM-Neck item-based measurements. Considering that S-ROM measurement with bubble inclinometer, NDI and VAS are widely used, valid, and reliable gold standard neck assessment tools , 24, 29,First, responsiveness was not analyzed since this evaluation includes a follow-up process (30). Second, our patients did not include elderly individuals. Therefore, the validity and reliability of this iconographic tool in older adults is also a matter of interest. Moreover, further research would be more potent with data obtained from other neck pathologies and different age groups. Third, S-ROM-Neck could not assess combined/complex movements than six simple neck motions . Since combined movements are used in some tasks of the individual’s daily routine, more specific tools are required to holistically evaluate the S-ROM in terms of complex movements. Fourth, accurate measurement of the cervical range of motion is due to the complex and compensatory movements. Using a more reliable method, such as an S-ROM measurement device consisting of three separate inclinometers, an electronic sensor-based system, or radiography, may provide more reliable results in measuring the cervical range of motion.Turkish S-ROM-Neck is a valid and reliable tool to assess the S-ROM of individuals with chronic neck pain. S-ROM-Neck is a unique subjective tool with its iconographic content. Further studies should focus on responsiveness analysis of S-ROM-Neck.


Syndesmotic screw removal following acute syndesmotic injury is a commonly performed procedure. However, recent studies suggest that the removal does not result in improved patient reported outcome, while the procedure has proved not to be without complications. The aim of this study was to present a health-economic evaluation of on-demand removal (ODR) compared to routine removal (RR) of the syndesmotic screw.Data were collected from the RODEO trial, a randomized controlled non-inferiority trial comparing functional outcome of ODR with RR. Economic evaluation resulted in total costs, costs (in Euro) per quality adjusted life year (QALY) and costs per point improvement on the Olerud Molander Ankle Score (OMAS). This included both direct and indirect costs.p < 0.001). The difference in QALY was not significant. The difference in OMAS at 12 months was 1.79 with an incremental cost-effectiveness ratio (ICER) of €-1763 (p = 0.512). The ICER was well below the willingness to pay. Although unit costs might vary between hospitals and countries, these results provide relevant data of cost-effectiveness.Total costs for ODR were significantly lower with a mean difference of 3160 euro compared to RR (The clinical effectiveness of both ODR and RR can be considered equal. The costs are lower for patients treated with ODR, which leads to the conclusion that ODR is cost-effective.The online version contains supplementary material available at 10.1007/s00068-022-02158-9. Syndesmotic screw removal is a commonly performed procedure within the field of orthopaedic trauma surgery. Syndesmotic screws are placed in order to fixate syndesmotic injuries in unstable ankle injuries, either with or without a fracture –3. AlthoSurgical site infections (SSI) occur frequently following implant removal . SSIs arData were collected from the RODEO trial, a randomized controlled non-inferiority trial comparing on-demand removal to routine removal of the syndesmotic screw, of which the full study protocol has been previously reported . In summThe trial was registered in the Netherlands Trial Register (NTR5965) and Clinicaltrials.gov (NCT02896998) and sponsored by ZonMw, a Dutch governmental organization (grant number: 843002705).The economic evaluation consisted of a cost-utility analysis (CUA) and a cost-effectiveness analysis (CEA) from a societal perspective with the costs per quality adjusted life year (QALY) and the costs per point functional outcome improvement as the primary economic outcomes. The costs were also compared to the willingness to pay. Both direct and indirect costs were used, based on average unit costs of 2018. The analyses were based on the intention-to-treat groups, using a time horizon of 12 months, since relevant differences in outcome and health care costs were expected to occur within that period.Data on resource utilization were collected through questionnaires at 3, 6 and 12 months after syndesmotic fixation. The questionnaires used to evaluate in and out of hospital resource use were the iMTA Medical Consumption Questionnaire (iMCQ) and the iMTA Productivity Cost Questionnaire (iPCQ). In addition to this, patients’ medical records were screened after follow-up completion (12 months) to check for complications and revisions procedures. Resources were classified as either direct medical, direct non-medical or indirect non-medical costs. Direct medical costs were further subdivided into (1) regular in hospital costs, related to the index admission , (2) additional in hospital costs, related to complications or readmissions , (3) out of hospital (para-)medical costs . Indirect non-medical costs resulting from work absence or decreased productivity were determined using the iPCQ. To estimate loss of productivity, the friction costs method was used. Total costs per patient and per group were calculated by multiplying resources by the associated unit costs.Quality of life was measured by the single value calculated from the EQ-5D-5L questionnaire on each time point , using the standard Dutch tariff values. QualityCosts of the various resources were extracted from the Dutch guideline on unit costs for health care, which was based on average health care prices in 201418]. To . To 18].Results are reported as means and mean difference along with their 95% confidence intervals and tested using independent sample t test. Differences in costs and effectiveness between the two groups were drawn and displayed graphically with cost-effectiveness planes. Incremental cost-utility (ICUR) and cost-effectiveness (ICER) ratios were calculated by dividing the mean difference in total costs per patient between treatment groups by the mean difference in QALYs and in OMAS, respectively. Therefore, ICER is the price in euro’s per point difference on the OMAS.Missing data were handled according to the recent paper by Brand et al. . Costs, The confidence intervals of the ICER and the cost-effectiveness plane were based on bias-corrected and accelerated non-parametric bootstrapping of 1000 bootstrap samples after combining the multiple data sets into one mean single imputation .In addition to the primary CUA and CEA analyses, preplanned subgroup-analyses were performed for patients younger/older than 60 years old and for patients with/without a SSI. Sensitivity analyses were performed by varying medical costs of screw removal, outpatient clinic visits, emergency room visits and revalidation clinic admissions by 30%. Analyses were performed using R version 3.6.1 .Between January 2017 and April 2019, 197 patients were included in the RODEO trial, of which 93 randomized for RR and 104 for ODR of the syndesmotic screw. Baseline characteristics of all patients incorporated in the current economic evaluation are shown in Table p < 0.001), in favour of ODR. This difference was made up by the difference in direct costs; 1. Costs for syndesmotic screw removal, which were significantly lower in the ODR group, as expected , 2. More readmissions and surgical debridements in RR, leading to a mean difference per patient of -€1,274 , and 3. A shorter duration of revalidation and home care in the ODR group . Also included in the difference were the indirect non-medical costs, which were €503,985 for RR and €482,891 for ODR (Table Table p = 0.474). This difference was not statistically significant, nor clinically relevant. Figure The mean QALYs in 1 year was 0.795 (95% CI 0.727–0.862) in the group with on-demand removal and 0.782 (95% CI 0.753–0.811) in the group with routine removal, which lead to a difference in QALYs of 0.013 . This is shown in Fig. The mean difference in functional outcome, measured by the OMAS at 12 months, was 1.79 (95% CI − 4.92 to 8.50), with a mean OMAS of 78.62 (95% CI 66.90–90.34) in the ODR group and 76.83 (95% CI 71.82–81.83) in the RR group. The ICER was in favour of ODR: − €1.763  showed cost-effectiveness for ODR when it comes to functional outcome for patients under 60 y/o. For every point difference on the OMAS, there was a difference of − €3,333  between ODR and RR with less expenses in the ODR group , mean difference in functional recovery by OMAS at 12 months of 1.34 (− 5.35 to 8.04) and a mean difference in QALY over 12 months of 0.014 (− 0.026 to 0.054). Costs, functional recovery and QALY were more favourable in the ODR group. The ICUR was -€230,542 (€− 219.210.538 to €54.613).Following sensitivity analyses, medical costs all showed a difference in costs between RR and ODR, in favour of ODR Table .Table 3SThe mean difference in total costs was €3,160 per patient in favour of the ODR group. Even though the upper limit of the 95% confidence interval of the ICER was marginally higher than zero, it was well below the level of willingness to pay, which is 50.000 euro, which makes ODR a cost-effective intervention .This is the first cost analysis study to focus solely on screw fixation for syndesmotic injury, comparing cost-effectiveness of syndesmotic screws removed routinely or on demand. Previously performed cost analyses have focussed on the cost-effectiveness of screw versus dynamic implants, such as the suture button –25. NearIn this study, the direct costs were higher following RR, mainly because of the costs of higher frequency of surgery and more readmissions at the surgical ward. In addition, we expected the costs to be higher due to the higher rate of SSIs in the RR group, resulting in higher health care costs. Therefore, we also calculated the difference in costs excluding patients with SSI, which had no relevant effect on the results. Thus, the difference in costs cannot be explained solely by the amount of SSIs in the RR group.For indirect costs, the iPCQ and the friction costs method with age-adjusted mean daily wages were used to estimate loss of productivity. Indirect costs, defined as lost hours of productivity, were higher in the RR group. Because this cohort was part of a RCT, we can assume that the hourly wage and therefore lost productivity costs were equally divided between both groups and that this does not account for the difference.The functional outcome after 12 months, measured with OMAS, did show a difference of 1.79 with higher scores following ODR . This leIn the subgroup analysis comparing patients aged < 60 years and patients ≥ 60 years, we found that the costs gained when choosing for ODR are higher in the younger group, which can be attributed to the fact that the younger group has less indirect costs following ODR because these costs include costs made by work deprivation. The results of this subgroup analysis could be taken into account in daily practice.This study compared ODR and RR following syndesmotic injury, and the removal was performed at the operation room. Sugi et al. recently state that it is safe to remove the syndesmotic screw in a clinical setting out of the operating room based on a complication rate of 2 SSIs in 170 patients . This miA limitation of our study was that unit costs might vary between hospitals due to overhead costs, which was not accounted for in the current study, leading to an estimation of the costs per patient and in total. However, due to the randomized set-up of the study, uncertainties are expected to be equally divided. Additionally, in this study, only costs made within 12 months after syndesmotic fixation were incorporated. The results thereby do not include removal of screws after this period of time, or potential late complications of retaining the syndesmotic screw. We know that if the retained syndesmotic screw breaks, the location of breakage affects the chance of cortical erosion due to the broken syndesmotic screw . But to The clinical effectiveness of both ODR and RR can be considered equal. The costs are lower for patients treated with ODR, which leads to the conclusion that ODR is cost-effective.Supplementary file1 (DOCX 18 KB)Below is the link to the electronic supplementary material.


Vaccinia virus antibodies, and electron-microscopically. Notably, the early macular stage only showed subtle viropathic changes; it did not express of Orthopoxvirus proteins in immunohistochemistry and therefore can easily be missed histologically. In later stages, immunohistochemistry with anti-Vaccinia virus antibodies might be useful to distinguish mpox from differential diagnoses such as herpes virus infections. In the ulcerative stages, the identified occlusive vasculopathic changes could be an explanation for the severe pain of the lesions reported by some patients. Despite the small number of samples examined, our analysis suggests that the histological findings of mpox are highly dependent on the stage of the biopsied lesion. Therefore, knowledge of all different stages of histology is necessary to reliably diagnose mpox histologically, especially when molecular testing is not available.Monkeypox (mpox), a former rare viral zoonosis, has increasingly made it into the public eye since the major outbreak that started in May 2022. Mpox presents with skin lesions that change over time and go through different stages . In this study, we evaluated skin biopsies of all stages. Therefore, five biopsies from four patients were analyzed histologically, immunohistochemically with anti- Mpox (formerly named monkeypox), a rare viral disease caused by an orthopox virus, was first described in 1970 in the Democratic Republic of Congo ,3,4,5. DAccording to systematic investigations of the current outbreak in larger patient collectives, the infection shows a staged course: After a short prodromal stage with fever, fatigue, and lymphadenopathy, a few days later, the first skin lesions appear, progressing through various characteristic stages before healing within 14–21 days ,23,24. CBeyond the typical clinical presentation, the diagnosis of mpox needs to be confirmed by further molecular testing. Currently, the WHO recommends PCR testing as the only method to be used and thus the gold standard. Skin lesion material, including (i) swabs from the lesion surface and/or exudate, (ii) roofs from more than one lesion, or (iii) lesion crusts, is the recommended specimen material to perform appropriate testing. Confirmation of MPXV infection is then based on nucleic acid amplification testing (NAAT) using a real-time or conventional polymerase chain reaction (PCR) to detect unique sequences of viral DNA. Several protocols exist, including two-step protocols in which the first PCR reaction detects only orthopoxvirus (OPXV) but does not identify the exact species. MPXV can then be specifically detected in a subsequent step utilizing PCR or sequencing. Alternatively, generic monkeypox detection (to confirm etiology) can be performed directly, followed by additional PCR testing for specific clade differentiation ,28,29.Although not part of the routine WHO diagnostic algorithm, histopathologic and immunohistochemical examinations of skin lesions may also be useful, especially when molecular methods are not available or mpox has not been considered as a differential diagnosis ,30. HistIn July 2022, four patients presented to the Department of Dermatology, University Hospital Muenster, Germany, with skin lesions clinically suspicious for mpox. The diagnosis of mpox was confirmed by molecular detection of the virus (PCR) in lesional swabs according to WHO guidelines. As part of the routine diagnostic algorithm in our department, after informed consent, photographs and skin biopsies of representative lesions were taken. Each patient presented with multiple skin lesions in different anatomical regions at different stages of development. Therefore, lesions corresponding to the clinically described stages of mpox were specifically selected for biopsy. In three of the four patients, one biopsy was taken; and in one patient, two skin lesions were biopsied. Retrospectively, the biopsies were assigned to the different clinical stages of mpox. Patients’ characteristics and further clinical information such as time between sexual risk contact, appearance of first skin lesion, and medical consultation as well as symptoms during the course of infection among others were collected retrospectively using the medical records. Detailed information can be found in Vaccinia virus antibody: citrate buffer pH 6.1 ). The following antibodies were used in respective dilutions: CD4 ; CD8 ; CD68 ; MPO , and rabbit polyclonal anti-Vaccinia virus antibody . Counterstaining was performed with hematoxylin according to Mayer, and the slides were mounted with Aquatex . Histology was systematically evaluated using a list of criteria for epidermal and dermal inflammatory and vascular changes (Tissue sections (3 µm) were cut from formalin-fixed and paraffin-embedded tissue blocks and stained with hematoxylin and eosin (H&E). For immunohistochemistry, tissue was deparaffinized and rehydrated with distilled water. Subsequently, ‘heat-induced epitope retrieval’ was performed with the respective buffers : EDTA buffer pH 9.1 , CD68, and anti- changes . The invSpecimens were processed with Karnovsky’s fixative followed by 1% osmium tetroxide, dehydrated, and embedded in EPON resin mixture . Ultra-thin sections were cut with diamond knives, and after mounting on copper grids, the sections were stained with uranyl acetate and lead citrate.The QIAamp DNA mini kit  was used for DNA extraction from 50 µm sections (5 × 10 µm per biopsy) of formalin-fixed paraffin-embedded tissue. Monkeypox virus (MPXV)-DNA detection was performed with MPXV-specific real-time PCR  on a LightCycler 480 II  according to the manufacturer’s instructions.All four patients were male and men who have sex with men (MSM). Their mean age was 41 years, ranging from 34 to 47 years. Three of the four patients were HIV-positive, and two had contracted syphilis in the past. The first skin lesions appeared between 12 and 17 days  after sexual risk contact. The number of lesions varied between a few (three lesions) to multiple (>twenty lesions). Clinically, all patients presented different stages of cutaneous manifestations of mpox: a macular stage a, a papuA total of five biopsies were taken from four different patients representing the different stages of the disease . The iniVaccinia virus antibodies marked the cytoplasm of keratinocytes in all samples except the early macular stage  were analyzed with electron microscopy, allowing for visualization of typical ovoid-shaped viral particles with a biconcave outline of the core within the cytoplasm of epidermal keratinocytes and hair follicle epithelia . In all five skin biopsies, MPXV-DNA was detectable by PCR using the above-mentioned protocol. The respective Ct values can be found in The current outbreak of mpox beginning in May 2022 has increasingly drawn the attention of medical professionals worldwide to this previously rare viral zoonosis . According to the current studies, MPXV seems to infect the host first via the mucous membranes and is then transported to the regional lymph nodes by Langerhans cells . After aThe histology of mpox lesions has been described by different groups before, all of them observing similar findings ,14,25,31Vaccinia virus antibodies can be useful. Our immunohistochemical analysis could confirm the results of previous studies showing that this antibody is suitable to detect mpox on histological material with strong reactivity in affected keratinocytes [Vaccina virus antibodies remained negative. In this stage, virus production is probably still below the detection limit of the antibody, as MPXV-DNA was already found by real-time PCR in the macular lesion. Hence, the early macular stage is associated with most differential diagnostic difficulties. Showing superficial and deep perivascular lymphocytic infiltrates, vacuolar alterations along the junctional zone, and only subtle keratinopathic changes, the histology of mpox at this early stage can easily be mistaken for drug reactions, cutaneous lupus erythematosus, pityriasis lichenoides, or cutaneous T-cell lymphoma. An accurate diagnosis of mpox at this very early stage therefore only seems to be possible with additional methods such as PCR.In the literature, it is frequently mentioned that the histology of mpox can be confused with herpes virus infections due to similar pallor of the epidermis with the ballooning and formation of multinucleated keratinocytes ,31,37. Hinocytes ,31. ThisVaccinia virus antibodies detected viral proteins not only in the keratinocytes of the interfollicular epidermis but also within hair follicle epithelia. This was also confirmed by electron microscopy and is in line with previous studies [Varicella zoster virus (VZV) [Notably, immunohistochemical staining with the anti- studies ,31. Of p studies ,31 but a studies . Folliclus (VZV) ,39. In tus (VZV) ,41. On tus (VZV) . This coWe are well aware that due to the small number of specimens that we have examined, only limited conclusions can be drawn. Nevertheless, our analysis of the inflammatory patterns, the composition of the infiltrate, and the subsequent vascular changes suggest the following immunological response: (i) The lesions are primarily triggered by a T-cell response dominated by CD4+ T-helper cells, probably directed against the infected keratinocytes. The skin lesions develop with a time delay because T-cell priming in the lymph node is probably required first. (ii) The subsequent cytotoxic CD8+ T-cell response results in the ballooning degeneration and necrosis of the infected keratinocytes, a known trigger for the recruitment of neutrophil granulocytes, which dominate in the lesions as they progress. (iii) The massive diapedesis of neutrophilic granulocytes from the postcapillary venules then probably leads to the vascular closures of the superficial vessels, which exacerbate to ulcerations that we observed in the late stages. This phenomenon is called immunothrombosis and has been described in other viral diseases, for example, in COVID-19 ,44,45. WDue to the retrospective nature and the small number of cases, our statements and conclusions are not generalizable. The largest study population examined histologically was described in Spain and included 20 patients . HoweverTo summarize, the histological changes of mpox are highly dependent on the stage of the clinical lesions. Especially, the early macular stage can be easily missed histologically. Therefore, clinicopathologic correlations and additional virologic testing with PCR as the diagnostic gold standard are required to establish the diagnosis of mpox with certainty. Since the further spread of mpox cannot be predicted and is still ongoing at present, mpox has to be included in the differential diagnostic repertoire. Knowledge of the histological changes is helpful in diagnostics, especially when molecular methods are not available.


The ICTV has approximately 180 members who vote by simple majority. Taxon-specific Study Groups established by the ICTV have a combined membership of over 600 scientists from the wider virology community; they provide comprehensive expertise across the range of known viruses and are major contributors to the creation and evaluation of taxonomic proposals. Proposals can be submitted by anyone and will be considered by the ICTV irrespective of Study Group support. Thus, virus taxonomy is developed from within the virology community and realized by a democratic decision-making process. The ICTV upholds the distinction between a virus or replicating genetic element as a physical entity and the taxon category to which it is assigned. This is reflected by the nomenclature of the virus species taxon, which is now mandated by the ICTV to be in a binomial format (genus + species epithet) and is typographically distinct from the names of viruses. Classification of viruses below the rank of species  is not within the remit of the ICTV. This article, authored by the ICTV Executive Committee, explains the principles of virus taxonomy and the organization, function, processes and resources of the ICTV, with the aim of encouraging greater understanding and interaction among the wider virology community.The taxonomy of viruses is developed and overseen by the International Committee on Taxonomy of Viruses (ICTV), which scrutinizes, approves and ratifies taxonomic proposals, and maintains a list of virus taxa with approved names ( Study GThe nature and origins of viruses have engaged virologists for decades . VirusesViruses are classified into taxa that are placed into hierarchical ranks; the primary ranks are named realm, kingdom, phylum, class, order, family, genus and species . AdditioRubivirus was included in the family Togaviridae along with members of the genus Alphavirus. However, rubiviruses (including rubella virus) differ considerably from alphaviruses such as Semliki Forest virus and chikungunya virus in their mode of transmission, virion structure and sequence relationships. Thus, in 2019, the genus was moved to a newly created family, Matonaviridae, which now includes both human and animal viruses [Taxonomy is a dynamic science that develops as analytical methods improve and new data become available. For example, there are six realms, each of which is inferred to represent a unique monophyletic evolutionary origin of its constituent viruses. However, recent evidence suggests that the number of independent and ancient evolutionary origins of viruses may be larger , implyinThe ICTV is charged with the development of a universal taxonomy that can be applied to all viruses irrespective of their hosts, whether they have been isolated and characterized phenotypically or if they have been identified only via sequences of nucleic acids in the metagenomic (or metatranscriptomic) analysis of viromes or environmental samples .Adnaviria, sub-ranks are not used . TaxoProps are submitted to the Subcommittee chair for the relevant group of viruses ; i.e. animal DNA viruses and retroviruses, animal dsRNA and (-)RNA viruses, animal (+)RNA viruses, archaeal viruses, bacterial viruses, fungal and protist viruses, and plant viruses ; ratified TaxoProps are archived at https://ictv.global/files/proposals/approved. TaxoProp templates and instructions can be downloaded from https://ictv.global/taxonomy/templates.When a TaxoProp is approved by the EC, it will enter a ratification vote by the full ICTV membership ; this voTaxoProps take the form of Microsoft Word documents summarizing the evidence for the proposed taxonomic changes or additions, and Excel spreadsheets itemizing any new taxa, as well as changes to existing taxonomic names or their place in the taxonomic hierarchy. TaxoProps must propose taxon names, provide supporting evidence for proposed taxonomic placements, and in the case of new species taxa, must provide nucleotide sequence information via GenBank accession numbers of an exemplar virus isolate. All TaxoProps must be based on the availability of at least one near-complete genome sequence of an exemplar virus. The ICTV does not classify viruses characterized only by partial genome sequences. Particular care must be taken with the accuracy of the Excel spreadsheets, because the process of correcting even an incorrectly spelled taxon name can be lengthy and may require submission of a corrected TaxoProp.One of the most important elements of a TaxoProp is the evidence that supports the assignment of a group of viruses into a taxon and the placement of that taxon in a ranking hierarchy that is built upon the inclusion principle (see Glossary). Thus, a proposal to classify a group of viruses for the first time must be clear about the criteria used to assign viruses to a taxon at the species rank and assign species to higher-rank taxa. At a minimum, species taxa must be assigned to a genus and, optionally, other higher-rank taxa. TaxoProps normally include a phylogenetic analysis of relevant nucleotide or amino-acid sequences, or present alternative evidence justifying monophyly. If the viruses are to be classified in an existing taxonomy, the established assignment criteria must be used or a convincing scientific argument to change these criteria must be presented.https://ictv.global/taxonomy/templates. The binomial format requirement is mandated for all new species taxon names and is being introduced retrospectively for existing names, with a deadline of the EC meeting of 2023.In virus taxonomy, the names of all taxa are written in italics. Names of taxa placed in ranks above species must end with a mandated suffix, as outlined in https://ictv.global/msl). A complementary list, called the Virus Metadata Resource (VMR), provides information about at least one virus assigned to each species taxon, including the virus name, an accession number for all or part of the virus genome, the nature of the virus genome , and the natural host , if known. This means that, by searching the VMR, it is possible to link virus names to the corresponding species and higher taxa and to link virus species taxa with the genome sequence of an exemplar virus . The VMR is updated several times a year and is available as a downloadable spreadsheet .The ICTV maintains a list of all virus taxa and their history, called the Master Species List (MSL). The MSL is updated each year following the ratification vote and is available from the ICTV website  Taxonomy Database . Howeverhttps://ictv.global/taxonomy/history). The site also provides information about the ICTV, recent news on virus taxonomy and forums for general and taxonomic discussions.The ICTV website provides access to the current taxonomy database in online and downloadable formats and maintains a complete history of virus taxa dating back to the first release in 1971 . It strives to be a comprehensive description of all virus taxa and includes extensive information on virion structure, genome structure and replication, biology and phylogeny. Chapters typically refer to a particular virus family and are written by ICTV Study Groups and edited by members of the EC. In addition, summaries of the individual ICTV Report chapters are published in a special section of the Journal of General Virology, called “ICTV Virus Taxonomy Profiles” (http://www.microbiologyresearch.org/content/ictv-virus-taxonomy-profiles). These Profiles provide concise summaries of the classification, structure and properties of members of the virus taxa included in the Report chapter. The Profiles are indexed in PubMed  and canArchives of Virology is currently an important freely accessible vehicle for communicating ICTV news to virologists and others . For example, previous VDN reviews and articles have encompassed the development of virus taxonomy, including official taxonomic changes [The Virology Division News (VDN) section of  changes , the sub changes  and info changes .Enterovirus A (family Picornaviridae) or (geno)types of avian hepatitis E viruses . Other viruses belonging to a single species taxon have been grouped using other biological characteristics, such as disease associations, host range or virion structure. In some cases, there may be a need to group viruses that are not congruent with evolutionary relationships, and these classifications may include viruses from several species taxa. This is often the case when the groupings have relevance for disease, bioregulation or biology. However, these alternative classifications cannot be regarded as virus taxonomies.The remit of the ICTV only extends to the assignment, placement and naming of taxa at and above the species rank. No general guidance can be given about classification below the species rank because criteria differ across groups of viruses. For example, viruses assigned to the same species taxon may be grouped into different serotypes or genotypes based upon cross-neutralisation or genetic divergence of their nucleotide or encoded amino-acid sequences. Examples include the numerous (sero)types of enteroviruses within the species Rubivirus rubellae (the species taxon name) in the family Matonaviridae (the family taxon name). Virus names are not regulated by the ICTV [An issue separate from classification below the level of the species taxon is the names given to viruses as physical entities or the diseases they produce. As an example of the use of these different kinds of names, rubella virus (the virus name), the etiological agent of rubella or German measles (the disease names), is a member of the species the ICTV  and are the ICTV .Alphainfluenzavirus influenzae, family Orthomyxoviridae), the isolate designation form is type/host (if not human)/country/strain number/year (haemagglutinin/neuraminidase type)—giving “influenza A virus, isolate A/swine/Italy/81220/2017(H1N1)”. Such names may simplify the computational analysis of virus sequences, but they can also become cumbersome. For bacterial viruses, a common naming pattern takes the form “Acinetobacter phage Apostate” or the formulaic “Acinetobacter phage vB_AbaM_Apostate”, consisting of the virus host genus followed by “phage” and a unique phage name, which can take the form “vB” (virus of bacteria), “Aba” (abbreviated host genus and species name), “M” , followed by the phage common name or identifier which should be unique [Another subsidiary issue concerns the naming of variants or strains of the same virus. There is no established convention for this within the field of virology and the ICTV has no remit in this matter. For some viruses, isolates are named according to a defined pattern. For example, for influenza A viruses . The inference of evolutionary history and relationships among or within groups of viruses.Polyphyletic. A polyphyletic taxon is defined as one that groups together the descendants of more than one most recent common ancestor.Ranking. In biological classification, the relative level of a group of organisms (a taxon) in an ancestral or hereditary hierarchy.Reassortment (or pseudorecombination). The exchange of one or more genetic segments that occurs in viruses with segmented genomes.Recombination. The combining of genetic information between nucleic acid molecules from divergent genomes. Recombination  occurs within a virus genome, between divergent co-infecting viruses or between virus and host (leading to gene acquisition).Taxon. A monophyletic category that includes viruses sharing certain characteristics related by descent from a common ancestor. The commonly accepted correct plural of “taxon” is the fake Greek “taxa”.Virome. The assemblage of viruses found associated with a particular ecosystem, organism or community of organisms.Virosphere. The pool of viruses in all hosts and all environments on planet Earth.Virion. Extracellular infectious virus particles that are capable of initiating a virus replication cycle.Virus. A replicative non-cellular entity that is obliged to depend on a host cell for replication.Virus taxonomy. The classification of viruses into taxa and taxon nomenclature.


Background: The years 2022–2023 witnessed a monkeypox virus (mpox) outbreak in some countries worldwide, where it exists in an endemic form. However, the number of infectious cases is continuously on the rise, and there has been an unexpected, drastic increase in cases that result from sustained transmission in non-endemic regions of the world. Under this scenario, it is pertinent for the world to be aware of healthcare threats to mpox infection. This review aimed to compile advanced data regarding the different aspects of mpox disease. Methods: A comprehensive strategy for the compilation of recent data was adopted to add data regarding mpox, biology, viral pathology, immune response, and brief details on the antiviral strategies under trial; the search was limited to 2016–2023. The aim is to make the scientific community aware of diverse aspects of mpox. Results: Consequently, detailed insights have been drawn with regard to the nature, epidemiology, etiology, and biological nature of mpox. Additionally, its host interaction and viral infectious cycle and immune interventions have been briefly elaborated. This comprehensively drawn literature review delivers brief insights into the biological nature, immune responses, and clinical developments in the form of therapeutics against mpox. This study will help scientists understand the biological nature and responses in hosts, which will further help clinicians with therapeutic handling, diagnosis, and treatment options. Conclusions: This study will provide updated information on mpox’s pathology, immune responses, and antiviral strategies. Moreover, it will also help the public to become educated on the healthcare-associated threat and take timely mitigation measures against expected mpox outbreaks in the future. Countries around the world have been undergoing an immense struggle against the healthcare burden put forth by the COVID-19 pandemic . During Mpox was discovered and identified in 1958 in the form of an outbreak within an animal facility in Denmark . The recAlthough much has been uncovered with regard to the aspects of mpox biology, structure, host–immune interaction, evolutionary characteristics, epidemiology, transmission characteristics, and ecological dimensions, a deep sense of understanding remains to be developed for this class of viruses . It is iThe data for this comprehensive review have been gathered from the most diverse research journals, recent publications, and most cited clinical studies related to monkeypox virus. The search strategy acquired for this purpose has been a systematic one to gather data from electronic sources, including mainly Google Scholar, while some other platforms like PubMed, NIH , and Web of Science, European database, Springer, and Embase databases have also been given consideration. Moreover, the official websites of WHO and UNAIDS have also been used to obtain the statistical results and latest updates regarding the MPXV outbreak and epidemiological episodes in recent years. As this study mainly covers mpox-related viral pathology, immunogenicity, and antiviral strategies, the major research items were viral pathology, immune response, and antiviral strategies; outbreak; poxviruses; vaccines; therapeutics; immune responses, and some other linked search terms.The inclusion and exclusion criteria simply revolved around gathering data from original research articles, sections from books, letters to the editors, short and lengthy reviews, and some case studies published recently. The available data were immense, but we tried limiting the data to around 50 studies, from which the interlinked data have been gathered. Thus, after a thorough analysis of the dates, abstracts, titles, and journals of research publications, they were included in this review. Moreover, this review has mostly reviewed studies conducted in last five years (2018–2023) to add only the most recent advances related to MPXV updates, especially during the latest endemic episodes of 2022 and 2023. It should be noted that studies of English origin have been made part of the review.The review was structured to initially incorporate the biology and natural history associated with mpox and related viruses, along with a brief discussion about the etiology, structure, replication, and epidemiological records of mpox. In the later sections, the clinical manifestations, pathogenicity, latest clinical case reports, and human studies are elaborated to present how the virus presents danger to human hosts. We also highlight the mpox host interactions, viral transmission, and life cycle, along with predictions of mpox host immune system interaction with monkeypox. In the last portion, we briefly discuss some potential vaccines, therapeutics, and antiviral strategies being devised against mpox, along with some related zoonotic poxviruses that affect humans. In the discussion portion, we focus more on the current mpox outbreak (2022) and future recommendations to deal with the expected outbreak of mpox in both non-endemic and endemic regions.Orthopoxvirus  and Vaccinia viruses), all of which belong to the family of Poxviridae. It is a diverse and widely distributed family of viruses. The separate types of mpox virus are referred to as clades or subclades. The recent 2022 outbreak has been accounted for by clade II of mpox, which is historically named the West African clade, or more specifically, the subclade IIb. Mpox multiply within the cytoplasm of their host cells with the help of their double-stranded (ds)DNA. The role of the host cell nucleus, however, remains elusive within the replication process [Entomopoxvirinae and Chordopoxvirinae. The latter subgroup Chordopoxvirinae mostly infects vertebrates and is further subdivided into 18 genera, whereas the genus Entomopoxviriane infects invertebrates and has a further 4 genera.Mpox is a zoonotic virus that is transmitted between humans and animals. This virus belongs to the genus  process . Two subPoxviridae, they exhibit the same biological characteristics as other Orthopoxviruses. They are ovoid-shaped particles ranging in size from 200 nm to 250 nm. The outer envelope is composed of a lipoprotein membrane encompassed by a capsid. Some distinct surface tubules with a well-established dumbbell-shaped core are part of its structural composition [Because mpox belongs to the family of position . Just liposition .Unlike the name, monkeys are not the natural hosts of mpox; instead, rodents, rats, and squirrels, among other related animals, are considered their principal reservoirs. That said, infections have also been reported in non-human primates such as monkeys  . WorldwiStatistical and epidemiological data from different studies help trace records of mpox outbreaks and transmission in past history, such as clade I emergence in DRC, and mpox clade IIa emergence in the US back in 2003, which might have been transmitted from Ghana, as indicated by epidemiological investigations . The EurAs of the start of 2022, WHO reported mpox cases from 112 member countries. The last update provided by WHO, cited on 10 July 2023 reported a total of 88,288 confirmed cases of mpox infection and 1084 suspected cases, as well as a death toll around 150 . These cThe clinical features of mpox infection appear mainly in the form of skin rashes and fever. The fever remains asymptomatic for an incubation period of 10–21 days, in which it increases up to 38–40 °C. During this period, cutaneous dermal petechia occasionally develops. Some other clinical manifestations may involve headaches, body aches, nausea, vomiting, prostration, fatigue, and weariness that develop after at least 1–3 weeks . MoreoveHigher lesion counts lead to severe complications of respiratory and gastrointestinal tracts, such as encephalitis, septicemia, and ocular infections, which may even lead to permanent vision loss. The virus infection is more severally manifested in the upper middle and lower air tracts due to them being the prime routes of infection . Skin raThe process by which infection from tissue-resident immune cells  progresses in the body remains a matter of in-depth debate for future vaccine implications. However, it is a matter of common observation in mpox infection that the lymphatic system acts as the primary source of viral dissemination, as it replicates extensively in the lymphoid tissues of the head and neck regions . Mouse mAlthough several studies have been conducted on animal models, there is a need to gather data from cases of real patients for studies of mpox-related implications. Apart from studying the clinical manifestations, scientists have gathered data regarding the implacability of the smallpox vaccine on mpox patients, especially during the recent outbreak . The conIn one study conducted in Peru, the highest number of mpox cases per million was reported in Latin Americans, with concentrations found in men having sex with men. Moreover, a viral co-infection has been reported in these patients, with approximately two-thirds of Peruvian patients suffering from mpox and HIV. In these patients, clinical symptoms of skin lesions in the anogenital area without preceding systemic symptoms were found to be common. Moreover, the majority of hospitalized mpox cases had HIV and were on antiretroviral treatment . In mostIn some recent studies conducted by scientists, the clinical data from cases of monkeypox infection were derived from a single center in Italy ,35. The These studies suggest that initially, the treatment is primarily supportive, although antiviral drugs may be beneficial in severe cases. Some studies also predicted the incidence of mpox in patients with previous vaccination with smallpox. These, along with some other studies, helped scientists infer that a history of smallpox vaccination may provide protection against mpox infection but not necessarily protect against symptomatic disease ,37,38. PStudies on the biology and host interaction of mpox are helpful for molecular virologists to find other linked disease-causing viral species and discover new approaches for therapeutics and drug targets for mpox and the related family of viruses. Conformational studies are still ongoing to pinpoint the exact routes of viral transmission in humans. A brief insight into the viral infectious cycle elaborates that close physical proximity with infected people may play a vital role in transmission . Mpox maAdditionally, studies have revealed that infections of mpox are differentiated into two versions due to the cause of two distinct forms of the virus: mature virion (MV) and enveloped virion (EV), which differ depending upon membrane layers and surface glycoproteins . MVs entThe replication, transcription, and translation mechanisms help assemble intracellular mature virion (IMV) after passing through the stages of immature viral DNA formation. IMV then acquires outer wrapping from the Golgi apparatus to adapt into intercellular enveloped virions (IEVs) . IEVs maImmune responses from host cells in animal and human models have not yet been established, even after decades of viral exposure to humans. Perhaps the virus remained less contagious and had limited infection rates up to this outbreak. After the declaration of the global health emergency (2022) of mpox, the scientific community largely became involved in figuring out the routes to deal with this healthcare emergency. Therefore, some insights are being drawn into the immune–virus responses against Vaccinia virus (VACV) and related viruses of the family Orthopoxviruses to improve our understanding of mpox. These viruses share the same process of infection, which is confirmed by similar genes and encoded proteins that interfere with host cell signaling responses, which ultimately reflect disrupted viral recognition, apoptosis, and immune responses. Mammalian cells can easily detect microbial interventions through pattern recognition receptors (PRRs) that undergo cellular signaling to activate transcription factors such as NF-κB and interferon regulatory factors (IRFs) and cellular and humoral responses from innate and adaptive immunity, which play a vital role in fighting foreign entities.The first line of defense in the form of innate immunity follows an active viral infection in the body, although there are instances where the same route provides a viral entry route. Monocytes have been identified as the major route of poxvirus entry as antigen representation by monocytes, and neutrophils predict the lethality generated by mpox . StudiesThe importance of B-cell-mediated immune responses in the form of the production of specific antibodies is of prime importance due to the successful vaccination of smallpox in history based upon B-cell-mediated immunity . Some stOrthopox viral infection are significant and could be considered for vaccine design. However, comprehensive and confirmatory studies are necessary to correlate the relationship between CD4+ and CD8+ T -cell responses with the severity of mpox infection in humans.As elaborated earlier, CD4+ T cells play a vital role in immunity in terms of mediating functional memory B-cell responses. These cells are related to the production of IFN-γ and TNF following vaccination with VACV. A study of patients with HIV has shown that a compromised CD4+ cell count leads to higher risks of developing a severe mpox infection. The results were more explanatory when studies on patients with HIV with higher CD4+ cell counts were observed to lack severe mpox infection development . NeverthThere is a certain level of similarity between viral biology; infection cycle; transmission mode; and clinical manifestations of VACV, mpox, and other members of the family Orthopoxviridae ,29. SpecSome clinical studies show that vaccines for the smallpox virus family also have functionality against mpox . Some vaAnother therapeutic intervention has come forward in the form of MVA, a Vaccinia virus strain Ankara-based vaccine also named JYNNEOS, which is also composed of live attenuated virus particles and belongs to a third-generation vaccine and shown elevated CD8+ T-cell responses compared to first-generation Drax immunization . Both ACAlthough these vaccines are advantageous in providing a certain grade of immunity, most of these vaccines are associated with some side effects, such as myocarditis and pericarditis, especially in vulnerable groups such as those with eczema and pregnant ladies . FactorsSimilar to the matching vaccination strategy between smallpox and mpox, some therapeutics developed for smallpox are also used against mpox infections. One such antiviral drug is tecovirimat, which does not affect the mature virus, but affects its lifecycle by targeting the VP37 membrane protein of mpox, inhibiting the maturation of the enveloped virus . Their eOrthopox virus DNA synthesis to prevent viral replication. It is approved for smallpox vaccination but is efficient against a large number of dsDNA viruses due to a similar notion of replication inhibition. Its efficacy has been determined in dog and mouse models, but its cytotoxicity and human experimentation remain to be determined [Another important antiviral drug is brincidofovir and its active ingredient, cidofovir, approved by the FDA, which works by inhibiting termined . Similartermined . These vtermined .CPXV), Variola , Vaccinia virus (VACV), Molluscum contagiosum virus, and Orf virus [The sudden outbreak of mpox highlights the need to look for other orthopox viruses that could cause severe human infection and could be a threat to the healthcare system. It is also necessary to be proactive and design drugs and vaccines before these diseases infect humans at a mass scale. Moreover, the understanding of viral, biology, ecology, epidemiology, etiology, and immune and host reaction will be helpful in the design of further antiviral drugs and to determine their co-efficacious potential . Virusesrf virus .Vaccinia virus (VACV) is the most prominent in this regard and causes severe infection in humans if the person is unvaccinated. Mpox, as explained in detail, is quite similar to variola in terms of structure, functional characteristics, and disease manifestation . SimilarThe outbreak of mpox (2022) is here, and the strategies for dealing with the viral spread should be well known in the scientific community and the public. This may include clinical trials and laboratory assessments, contact tracing, and effective social control measures. Healthcare authorities such as the WHO and CDC have presented some recommendations for dealing with mpox disease containment, which may include precautions regarding diagnosis, treatment, and patient isolation . MoreoveMoreover, there is a continuous need for researchers, public health workers, government authorities, and the public to coordinate in the form of a participatory approach to handle the further spread and work for mitigation and adaptation to the mpox epidemic. Affected nations must establish strategies for endemic handling to ensure global safety. For this reason, there is a need for international collaboration for training, prevention diagnostics, surveillance, and treatment strategies to avoid endemic conversion to a pandemic like that of COVID-19, which has been quite recently experienced.Recent outbreaks of infectious diseases such as mpox, dengue, ebola, and others have forced scientists to work on therapeutic strategies in more profound ways than ever before. For the scientific community, the past three years have been particularly crucial in terms of treatment strategies and vaccination protocols against such viruses, especially during the pandemic era. This is the very reason that so much research is being conducted on vaccination and antiviral drug protocols against such viral diseases. This literature contains several vaccination protocols and therapeutic strategies for mpox, which indicate that such trials can lead to improved diagnosis of mpox disease, improved treatment options, and an overall reduction in healthcare costs and associated economic burdens in mpox-affected regions. Mpox may present some hurdles for clinical trials due to its infectious nature, lack of funding, and poor coordination among clinicians and scientists in endemic countries.Under the described scenario, it is pertinent to encourage clinical experiments up to levels III and IV to obtain licensure vaccination in the market. Limited lab resources and technical glitches caused by a lack of clinical data are causing experiments to be delayed. Developed nations are well acquainted with the importance and maintenance of healthcare measures. There is a need for these advanced nations to keep on working with vaccine development in their special advanced labs and conduct studies on human subjects to establish statistical data in support of vaccine candidates. Furthermore, they should consider modern therapeutic approaches, such as those based on nanotechnology, to develop new vaccines and drugs. In developing nations, special focus should be kept on adaptive strategies to cope with mpox. Further confirmation of existing vaccination protocols should be sought, as well as incorporating some of the latest technologies into existing vaccination drives to create improved vaccination protocols, such as plant-driven organic compounds and nanomedical applications.In the future, standard methods of vaccination such as vector-based vaccination, live attenuated protocols, subunit vaccines, and others are likely to be advanced in terms of manufacturing techniques such as a combined application of gene-splicing, genetic engineering protocols, and nanobiotechnology, which can be helpful to enhance therapeutic manufacturing, dosages, outcomes, and clinical experimentation compared to previous drug protocols against mpox. In the next five to ten years, there is a need to provide more research funding to implement advanced research protocols and wide-scale clinical experiments against mpox. Moreover, there is a need to indulge the general population, healthcare workers, and educated populace in participatory approaches to health management and social awareness. This will allow endemic countries to adopt better mitigation and adaptive practices against mpox and related healthcare practices. Nonetheless, more scientific experimentation should continue to tackle the surge of mpox and similar infectious diseases to lessen the burden of the global healthcare system.The current outbreak of mpox is a reflection of the longstanding pathogenicity of poxviruses against humans. Rapid spread, alongside cross-genomic mutations, is a major threat to this endemic that may increase its intensity. It is critical to develop insights into mpox biology, pathological dimensions, epidemiology, rates of infection, and therapeutic hurdles to develop a robust strategy for handling the outbreak. Moreover, as the disease is still in its endemic state, it is better to devise and practice a broad-scale immunization drive in endemic regions to tackle the outbreak in its current form and prevent its further spread. Further, there is a threat of increased transmissibility, genomic changes, and augmented virulence exits. To deal with these risks, it is pertinent to ensure the availability of currently approved vaccines exclusively in endemic regions. In addition, there is a dire need to strengthen the healthcare management capabilities of affected countries to deal with this emerging epidemic. Moreover, the scientific rigor to discover preventive medicines and vaccines should be continued to discover the most effective therapeutic interventions to deal with an mpox outbreak. It is important to couple healthcare practices with public cooperation to create awareness, mitigation, and adaptation patterns against present and future outbreaks of a similar nature.