This study presents a user-friendly model for assessing the value of dairy bulls in artificial insemination (AI) companies. The model, a Markov Chain model, considers important factors for replacement decisions, such as a bull’s age, expected semen production, and predicted genetic merit. Data from a leading AI company were used to apply this model, which calculates a bull’s net present value based on various costs and revenues. The model also calculates the value of a bull compared to a potential young replacement. Findings showed that nearly half of the bulls were recommended for replacement due to a negative value comparison. The value of a bull was primarily influenced by market distribution and pricing, as well as the interaction of semen production with genetic merit.Deciding when to replace dairy bulls presents a complex challenge for artificial insemination (AI) companies. These decisions encompass multiple factors, including a bull’s age, predicted semen production, and estimated genetic merit. This study’s purpose was to provide a practical, objective tool to assist in these decisions. We utilized a Markov Chain model to calculate the economic valuation of dairy bulls, incorporating key factors such as housing costs, collection and marketing expenses, and the bull’s probable tenure in the herd. Data from a leading AI company were used to establish baseline values. The model further compared a bull’s net present value to that of a potential young replacement, establishing a relative valuation . The range of BullVal$ observed spanned from −USD 316,748 to USD 497,710. Interestingly, the model recommended culling for 49% of the bulls based on negative BullVal$. It was found that a bull’s net present value was primarily influenced by market allocation and pricing, coupled with the interaction of semen production and genetic merit. This study offers a robust, data-driven model to guide bull replacement decisions in AI companies. Key determinants of a bull’s valuation included market dynamics, semen production rates, and genetic merit. Dairy genetics companies seek to provide top genetics from elite bulls to accelerate genetic progress and enhance farm profitability of their customers. Most companies have well-established protocols for acquiring and raising young bulls and collecting, processing, and selling their semen. However, the decision of when to replace a bull with a new selection candidate can be subjective and may be influenced by many factors and their interactions.Currently, replacement decisions typically involve many individuals, with competing interests, within a given artificial insemination (AI) company, including sire analysts (who acquire the bulls), veterinarians, inventory managers, sales and marketing staff, and barn managers. These individuals must use data regarding genetic potential, semen production, health, temperament, and other factors to decide whether a given bull is likely to: (1) achieve “premium” status in the marketplace and generate millions of dollars in revenue and tens of thousands of offspring; (2) reach (or remain in) “cash cow” status and contribute a modest revenue stream for the foreseeable future, or (3) lag behind its herd mates in revenue-generating potential due to poor semen production and/or a genetic profile that is no longer competitive or marketable. Each bull must be evaluated relative to its existing herd mates at the AI company, as well as potential replacement bulls that may be younger and have higher genetic merit , while simultaneously considering the fixed and variable costs associated with keeping the bull or acquiring a replacement.While objective methods to combine semen production, genetic potential, age, and other factors for valuation of dairy bulls in an AI company context are lacking, such methodologies and decision-support tools are well-established for dairy cows ,2,3. TheThe most common index used for ranking dairy cattle today is lifetime net merit (NM$), which considers the predicted genetic merit of cows, bulls, heifers, and calves for production, type, health, longevity, fertility, and calving traits relative to an average animal of the same breed . HoweverAnother, possibly most important, defining factor of a bull’s profitability for an AI company is his market appeal. Globally, even within a country, farmers’ needs for genetics and types of bulls differ ,9,10.Therefore, the objectives of this study are as follows: (1) provide a user-friendly Markov Chain (MC) model of economic valuation for dairy bulls, focusing on the most important factors contributing to replacement decisions, and (2) describe the features and outcomes of this model when applied to data from a leading AI company.The replacement problem of a bull was solved by MC as the difference between net present value (NPV) of a bull (NPV bull) and its replacement (NPV replacement), hence:This simple algorithm is the aggregation of a MC model, in which a user can define parameters of a bull and compare with those of a potential replacement, with considerations of age, total sperm production (TSp), and genetic merit.A dairy bull herd was represented by a 4-month age bin MC model as a matrix. Four months was chosen to model three rounds of replacement decisions per year, based on the current frequency of Council on Dairy Cattle Breeding  genetic evaluations of US dairy cattle. One state defines potential bull ages: AGE .AGE) were simulated through MC following Cabrera (2012) . A A bull’s (or replacement’s) probabilistic life was represented from the time the bull entered the analysis (Age Start) until a point in the future when the bull and its potential replacements had reached the MC condition of steady state ,11,12. TThe model was solved through recursive iterations until the probability distribution of a bull across all states of model reached steady state. In each iteration, aggregated discounted net returns of all probabilities of the bull were estimated for the given 4-month time period. Steady state of the Markov chain was reached after 310 iterations.To create the MC, assumptions were made to establish a base herd of bulls. Bulls were assumed to enter production at 10 months of age. Once they entered collection status, bulls were collected eight times per month until culled. In practice, bulls removed from collection rarely return; exceptions include injury, illness, or bulls that have not yet reached puberty. In the model, any remaining bulls were culled at age 85 months (7 years 1 month). TSp for a given bull in a 4-month period was treated as a deviation from the mean sperm production for bulls of that age. In a production setting, many factors may impact predicted and actual TSp, including collection frequency, barn personnel, temperament, environmental conditions, semen quality, and processing regimen.The NPV of a bull or its replacement was the aggregated 4-month discounted (∂) net value over 330 iterations  that resulted in NPV bull  or NPV replacement . Economic factors used in this calculation were the incomes and revenues incurred in the maintenance, production, and culling of a bull: (1) income from straw units produced (Si) according to the bulls’ age, predicted TSp deviation, genetic merit, and market; (2) maintenance cost (Mc), including housing, veterinary care, labor, and feed; (3) costs associated with involuntary culling (Cc), including cost of replacement and depreciation, and (4) income generated from involuntary culling a bull (Ci) and salvage value. Therefore,AGE. Age bin defined age of bull at the starting point (iteration = 1) in the MC model. As explained previously, AGE contained 19 four-month bins, from 10 to 85 months of age.Age Class. To obtain price per unit in the income equation (explained below), age bins were grouped into 3 classes: young (1–4 AGE), in-waiting (5–12 AGE), and proven (12–19 AGE). In-waiting signified a bull that is older than genomic (young) bulls that entered the AI stud recently and younger than proven (old) bulls that already have offspring with performance data. This was done to reduce dimensionality, allow more samples within each grouping, and achieve a more stable price.Arrival Age. Age in months at which a bull arrived at the AI stud. Used to calculate depreciation value within culling cost variable of NPV . A bull could arrive at the AI stud at 1 to 15 months of age and might not enter the production herd immediately. Costs associated with rearing bulls prior to production were assumed to be constant across all bulls and are not considered within the NPV calculation.Expected TSp percent deviation from mean. The NPV bull could be calculated assuming a bull’s average TSp production. A bull’s expected production capability, Pdev, was the percent deviation from mean TSp, and was multiplied by the aggregated income generated (Inc) based on average TSp production.Net Merit decile bin. The genetic contribution of a bull was considered using the decile of NM$ for the bull, compared with the herd’s distribution of NM$ values. This value was used to find the price per unit, which was a function of age, market, and genetic merit, in the income calculation.Aggregated discount. The aggregated discount was defined as: int was the interest rate.Income from straws produced. Income generated by unit sales was defined as:Market pricing and distribution of semen units. Bulls (and units) were valued differently depending on where the semen was sold. The income equation had the capability to calculate Inc based on the amount of product distributed to each market group (market share).TSp per straw unit (packing rate). Average number of cells packaged into a unit. The value divided TSp expected per bull into units, which was then multiplied by price per unit to get income from straw units produced.Income generated from unit sales. Money generated from the sale of product destined to different markets, based on age, TSp production, and genetic merit was the product of price per unit (PU), market share percentage (MS), and number of units produced (U):Maintenance cost. The maintenance cost variable incorporated prices for housing, maintenance, and veterinary costs for a bull.Culling cost. Cost of culling a bull Depreciation. Value assigned to a bull for insurance purposes, based on age of bull, arrival age, and term length.Depreciation cost within the NPV was calculated as Depr = Depreciation Value— ∗ ), where Depreciation Value is the original value assessed for a bull, Depreciation term is the length of depreciation realization, and age is the age that is currently being evaluated within the aggregated NPV.Depreciation term. The length for depreciation realization, based on age (month). Depreciation was the remaining cost that must be paid if a bull was culled prior to the depreciation term.Culling income. Income generated from culling the bull, which was simply the product of salvage value  and proportion of culled animals: Culling percentage. Involuntary culling percentage (CULL) per age bin. It was assumed that all bulls were culled at the end of AGE 19.Model performance was demonstrated using production data, sales records, health events, and bull demographics of Holstein bulls at two collection facilities of a commercial AI company . Bulls in production from April through November 2020 were used in this illustration (reflective of two trimesters). The MC model for each bull was calculated using age of the bull in April 2020. Genetic predictions for NM$ from the December 2020 CDCB genetic evaluation were used to classify each bull into a decile, and its arrival age was used to calculate depreciation. Each bull’s valuation was relative to a replacement animal of AGE = 1, Arrival age = 6, and NM = 9. It was assumed that a replacement bull would be a young bull at the beginning of its productive life, with the average arrival age, and NM just below that of the most elite bulls used to create the next generation .Involuntary Culling Percentage. Involuntary culling percentages, used as transition probabilities, were derived from health records of bull deaths, recommended culling decisions, or actual culls .Expected TSp percent deviation from mean . This was derived using company collection records from 2018 to 2020, aggregated to an average TSp per age bin, such that each month a bull was collected 8 times . For theMarket share and pricing. Twenty-nine countries that received more than 200,000 semen units according to 2018 to 2020 sales records were split into 6 market classes. To identify trends in types of products used in each country, PTAs of bulls sold in each country were averaged. The countries were then ranked for each PTA value, providing an estimate of importance that trait has on overall selection by country. Similar countries were grouped together manually. Market A contained 6 countries, in which fertility traits had high importance and milk composition traits had low importance. Market B contained 3 countries, in which milk production traits were of high importance and fertility and type traits had low importance. Market B also contained the domestic market. Market C contained 5 countries, in which milk yield and type composites were of moderately high importance. Market D contained 3 countries with high importance for type composites. Market E contained 6 countries, in which PTA Milk, Productive Life, SCS, and Net Merit were of high importance. Lastly, Market F contained 6 countries that did not fit into above groupings and lacked a discernable pattern in traits of high importance. Once grouped, average price per group was calculated per age bin. A smoothing function was applied to each market to limit the influence of outliers (see below). Market share was calculated as the percentage of total units per age group directed to each market. Unit price smoothing function. Price per unit of semen was estimated based on age class , market class, and NM$ decile . Age clai = interpolation factorβ2i/niµ = population meaniMaintenance cost. The AI company used in this case study assigns an estimate of USD 30/bull/day for covering all physical maintenance costs, such as feeding, housing, and veterinary costs. This value was adjusted to fit 4-month age bin and remains the same across all bulls in the herd.Depreciation value and term length. The company insured bulls for USD 54,000 for 36 months (depreciation term).AGE = 1. The SD between the 310th and 330th iterations for AGE = 1 was 0.137%, showing that there was minimal variability in the proportions between iterations. The replacement bull  had a discounted NPV of USD 250,951. This was broken down into maintenance cost of USD 63,600, culling cost of USD 7264, income from culling of USD 532, and income from semen sales of USD 321,283. Adjusting any input values of a replacement bull would change his NPV and the BullVal$ of the herd, but it would not change the overall ranking of bulls within the herd.The base scenario was derived from the case study, subsequently all results pertain to the case study. Across 330 iterations, steady state was reached around the 310th iteration, based on BULLMarket prices  were estA total of 396 Holstein bulls collected from April to November 2020 made up the herd. i = 1 . This model and case study had limitations and challenges. First, the sales data available for this study were average sales prices for orders, which could contain multiple bulls, all averaging to the same price. This does not accurately portray the actual sales price of a bull semen. Moreover, a company may sacrifice on sales price to foster a budding relationship with a new market, undervaluing bulls and losing present revenue for (hopeful) future gain. Additional business relationships, contracts, and government regulations, among other constraints are not considered in this study, but would play significant roles in pricing and market distribution. Lastly, the adoption of an objective tool can be a challenge when competing interests exist, and greater insight into the performance of this bull valuation tool may have been gained if the authors had access to data from several commercial AI companies. It would be beneficial for the tool to be modified or updated to reflect market changes and bull herd demographics of specific companies. Future studies should consider differences in reliability between young, genome-tested bulls and older bulls with milking daughters, because this uncertainty could cause some bulls to change NM bins.The authors suggest that this tool would be most beneficial in culling decisions when tied into the product allocation and collection scheduling process. Bulls with negative BullVal$ should be culled before high BullVal$ bulls , to make way for more profitable replacements. An example of how this may fit into a collection scheduling process is in a situation where collection spots are limited, we would prioritize higher BullVal$ bulls for those spots. A similar case can be made with product allocation: assigning higher BullVal$ bulls to markets would capitalize on the potential net revenue. Again, this model would need to be updated routinely (2–3 times/year) to reflect the current bull population and market characteristics.The present study demonstrated that a Markov chain model can be used to provide economic valuation of dairy bulls, while focusing on the most important factors contributing to replacement decisions, such as age, predicted semen production, and predicted genetic merit. The Markov chain model allows for user-defined input based on current replacement policy and bull demographics. This model provides a new metric of ranking and valuing bulls based on their actual contribution to revenue of the company. A negative bull value indicates that the chosen bull is less profitable than the predicted discounted lifetime profit of a new young sire of average production capabilities, suggesting that the bull should be culled. A case study demonstrated the tool’s feasibility of valuing and ranking a herd and highlighted pitfalls with data availability. The range of BullVal$ encountered was −USD 316,748 to USD 497,710, with 49% of bulls recommended for culling based on negative BullVal. A bull’s NPV was influenced primarily by market allocation and pricing, as well as the interaction of sperm production with genetic merit. This decision support tool is contained within an Excel workbook, allowing individual bull valuation and whole-herd assessment.


The aim of this study was to fabricate celecoxib-loaded chitosan/guar gum (CS/GG) single (SC) and dual (DC) crosslinked hydrogel beads using the ionotropic gelation approach. The prepared formulations were evaluated for entrapment efficiency (EE%), loading efficiency (LE%), particle size and swelling studies. The performance efficiency was assessed by in vitro drug release, ex-vivo mucoadhesion, permeability, ex-in vivo swelling and in vivo anti-inflammatory studies. The EE% was found to be ~55% and ~44% for SC5 and DC5 beads, respectively. The LE% was ~11% and ~7% for SC5 and DC5 beads, respectively. The beads showed a matrix-like network with thick fibers. The particle size of beads ranged from ~2.74 to 1.91 mm. About 74% and 24% celecoxib was released from SC and DC hydrogel beads, respectively, within 24 h. The SC formulation showed higher %swelling and permeability than the DC counterpart, while the %mucoadhesion was relatively higher for DC beads. During the in vivo study, a significant decrease in the inflammation of the rat paw and inflammatory markers including C-reactive proteins (CRP) and interleukin-6 (IL-6) was observed following treatment with the prepared hydrogel beads; however, the SC formulation showed better therapeutic efficiency. In conclusion, celecoxib-loaded crosslinked CS/GG hydrogel beads can provide sustained drug release and act as potential candidates for managing inflammatory conditions. Celecoxib (CX), a selective cyclooxygenase-2 inhibitor non-steroidal anti-inflammatory drug (NSAID), is the drug of choice for chronic inflammatory diseases. However, its erratic absorption, reduced aqueous solubility and bioavailability require the administration of high doses (200–400 mg twice a day) to achieve the desired outcomes . The lonHydrogel beads, multi-particulate (0.2–3.0 mm) polymeric systems, swell in response to aqueous environments and release drugs smartly. Hydrogel beads have been fabricated by using several approaches including ionotropic gelation, polyelectrolyte complexation, syringe dropping/extruding, air atomization, emulsion gelation, the incorporation of both oils and waxes and impinging technology. Among these, ionotropic gelation is the simplest and most convenient method. Several natural and synthetic polymers such as acrylic acid, guar gum, chitosan and sodium alginate can be used for preparing hydrogel beads. Among these materials, natural polymers, being biocompatible, non-toxic and renewable, may offer better drug delivery characteristics ,6. Such Guar gum (GG), a polysaccharide, is made up of sugars, galactose and mannose. Chitosan (CS), a cationic biodegradable and biocompatible polymer, comprises repeated units of (2-amino-2-deoxy-D-glucopyranose). GG and CS can serve as potential candidates for formulating hydrogel beads due to their release extending properties, pH responsiveness and susceptibility to degradation by the intestinal microbes ,8.Sodium tripolyphosphate (STPP or TPP) interacts with positively charged amino groups of CS via its negatively charged counter ions. Glutaraldehyde (GLA) forms imine bonds with amine groups of CS via its aldehyde groups .The present work aimed to develop celecoxib-loaded hydrogel beads comprising GG and CS  and TPP and GLA (as crosslinkers) by using the ionotropic gelation technique. The fabricated beads were evaluated for an in vitro drug release study, ex vivo swelling, mucoadhesion, a permeation study and an in vivo anti-inflammatory study in a rat paw oedema model.4−) groups of TPP interact with protonated amino (NH3+) groups of CS in the acetic acid solution. TPP interacts with hydroxyl (-OH) groups of GG via hydrogen bonding, hence offering a complex linked structure [A schematic diagram of interaction between polymers and crosslinkers is represented in tructure . Amine gtructure . GG alsoThe particle size of all  wet SC beads ranged from 1.65 ± 0.018 mm to 3.64 ± 0.025 mm. For wet DC formulations , the particle size of all  wet DC formulations ranged from 1.28 ± 0.035 mm to 2.37 ± 0.013 mm. The DC beads showed a lower particle size as compared to the SC counterparts, possibly due to the high crosslinking density  . Dry SC w/v CS) compared to that of L beads (composed of 1.5% w/v CS). As compared to M samples, the relatively more compact and tightly packed structure of H beads (containing 2.5% w/v CS) led to a relatively lower water uptake. AJ Sami et al. presented that the swelling ratio depends upon the amount of CS used [The SC and DC beads showed B,C an in CS used .All the formulations showed higher swelling ratios at pH 7.5 than at pH 1.2. The higher swelling ratio at a high pH was probably due to the degradation of galactan and mannan units by phosphate buffer ions. Moreover, the hydrophilicity of CS and electrostatic repulsion between functional groups were possibly responsible for the relatively loose/less compact structure. The same phenomenon was reported by K.M. Manjanna et al. for hydrogel mirobeads . HoweverM and H samples of both SC and DC beads exhibited appropriate particle sizes compared to L samples. The M samples showed a better swelling profile. Among the M samples, SC6 and DC6 presented good results in terms of swelling. Other characteristics such as the lesser shape uniformity, non-flexibility during reproduction (viscous solutions) and handling (sticky beads) and unstable nature during swelling were excluded while optimizing the best formulation. The swelling ratio of SC5 and DC5 (M) was not significantly different from that of SC6 and DC6 (M). Moreover, SC5 and DC5 presented good results regarding particle size, strength, shape uniformity and stability during relevant studies A,B. In tThe entrapment efficiency (EE%) was found to be 55.00 ± 0.028% and 43.75 ± 0.016% for SC5 and 43.75 ± 0.016% for DC5 beads, respectively. The loading efficiency (LE%) was found to be 11.00 ± 0.030% and 7 ± 0.018% for SC5 and DC5 beads, respectively. As the results indicate, LE% and EE% were higher for SC beads as compared to DC beads. A possible reason includes the decreased distance between molecules due to the high crosslinking density, which permitted a low amount of the drug to be entrapped within the DC hydrogel beads. Natalia Sedyakina et al. also reported that a high crosslinking density reduced the entrapment efficiency of bovine serum albumin in the polymer matrix .Scanning electron microscopy (SEM) images  revealed−1 and 3400 cm−1 due to -OH stretching vibrations and the presence of the -NH2 group, respectively. The bands at 2875 cm−1, 1374.04 cm−1, 1647.50 cm−1 and 1023.42 cm−1 represented -C-H, -C-N and -C=O stretching vibration (amide group) and presence of -C–O, respectively, thus, representing the polysaccharide structure of chitosan [−1 and 3400 cm−1 due to -OH stretching vibrations. The peaks between 3000 cm−1 and 2800 cm−1 represented -CH stretching bonds [−1, 1348 cm−1 and 1165 cm−1, which were assigned to NH, asymmetric SO2 and symmetric SO2 stretching vibrations, respectively [−1 representing its free -OH groups. The optimized SC5 beads showed spectral peaks from 2800 cm−1 to 3400 cm−1, indicating the reaction between TPP and amine groups [−1, depicting the overlapping of the -C-H bond with -CH2- groups of GLA. The stretch band at 1646 cm−1 related to the imine bond represented the crosslinking reaction between GLA and CS amino groups. The CX-specific peaks at 3340 cm−1, 1348 cm−1 and 1165 cm−1 appeared in both SC5 and DC5 beads. Therefore, the observation confirmed the successful loading of CX in SC5 and DC5 beads (The Fourier transform infrared (FTIR) spectrum of CS displayed a characteristic broad band between 3200 cmchitosan ,15. GG dectively . TPP shoe groups . DC5 beaC5 beads .θ = 19.94°, representing its crystalline structure [θ = 11.0°, 14.84°, 16.08°, 21.52° and 29.6°, confirming its crystalline nature. The SC5 and DC5 beads showed less intense peaks, indicating a considerable decrease in the crystallinity of the drug following incorporation within the polymeric hydrogel beads (The X-ray diffraction (XRD) scan of CS showed a characteristic peak at 2el beads .The differential scanning calorimetry (DSC) thermogram of CS manifested a shallow, broad endotherm around 90 °C, relating to dehydration. A more prominent exothermic peak at 280 °C was attributed to the thermal decomposition of the polymeric material . GG showThe TGA  thermogram of CS showed approximately 10% weight loss until 100 °C due to the removal of adsorbed moisture. Another weight loss of 45% was observed at 270 °C because of the thermal decomposition of the polymer . The the2 for both SC5 (0.939 at pH 1.2 and 0.986 at pH 7.5) and DC5 (0.915 at pH 1.2 and 0.961 at pH 7.5) beads depicted that the drug release profile followed the Korsmeyer Peppas model, with SC5 values closer to one at both pH values. The values of ‘n’ for SC5 (0.502 at pH 1.2 and 0.525 at pH 7.5) and DC5 (0.621 at pH 1.2 and 0.520 at pH 7.5) revealed that the beads follow non-Fickian diffusion.The results indicated that there is a direct relation between the swelling pattern and release of a drug from the hydrogel beads. The percentage of beads that remained adhered to the goat intestinal mucosa was 0 ± 0% and 8 ± 0.82% for SC and DC beads, respectively, after 24 h . The num−3 cm/min) as compared to that observed for SC5 (0.733 × 10−3 cm/min) and DC5 beads (0.643 × 10−3 cm/min). The amount of CX that permeated through the intestinal mucosa was higher in the case of prepared hydrogel beads when compared to the pure drug . The Papp value for pure CX was lower , orally administered formulations, i.e., SC5 and DC5 beads, remained intact in the 1st hour. After 2 h, the SC5 beads displayed swelling to a certain extent, while the DC5 beads showed negligible swelling. After 4 h, the SC5 beads showed significant swelling, while the DC5 beads did not show further swelling. After 6 h, the SC5 beads started to erode due to excessive swelling, while the DC5 beads were still intact . The excThe induction of oedema was confirmed by measuring the increase in the paw volume using a pleythysmometer. The reduction in paw volume was considered as an improvement of the disease state, which was interpreted by calculating the percentage of oedema inhibition. The results indicated that the paw volume of all groups increased after carrageenan (CG) administration, except for the NR  group. In the case of the NC (negative control) group, the maximum paw volume of 0.92 mL was observed at the 4th hour after CG injection, which was subsequently reduced to 0.82 mL at the 7th hour. The RF (reference) group showed a maximum paw volume of 0.47 mL at the 4th hour, which was subsequently reduced to 0.35 mL at the 7th hour. The PC (positive control) group showed a maximum paw volume of 0.48 mL at the 4th hour, which was subsequently reduced to 0.37 mL at the 7th hour. The SC (single crosslinked) and DC (double crosslinked) beads-administered groups showed maximum paw volumes of 0.55 mL and 0.65 mL, respectively, at the 4th hour, which were subsequently reduced at the 7th hour, i.e., 0.37 mL and 0.44 mL, respectively A. The SCThe C-Reactive protein (CRP) levels of the NR, SC, PC, DC, RF and NC groups were found to be 2.9 mg/L, 3.0 mg/L, 3.5 mg/L, 4 mg/L, 4.4 mg/L and 10.5 mg/L, respectively C. In theHistological images of the NR group A depicteThe oral route is the most preferred option for drug delivery due to the ease of administration and the good patient compliance . HoweverIn the present study, single  and dual  crosslinked CS- and GG-based hydrogel beads incorporated with celecoxib were prepared by using the ionotropic gelation technique. CS is a physiologically compatible, naturally occurring polysaccharide with mucoadhesive properties . Moreover, CS is degraded/metabolized by the enzymes released by colonic microbes, particularly N-acetyl-β-glucosaminidase, leading to the release of encapsulated active ingredient in the colon . LikewisDry DC beads displayed a relatively small particle size  as compared to the SC formulation . Swelling studies were performed at pH 1.2 and 7.5, relating to the pH of the stomach and ileo-colonic region in a fasting state, respectively. The ileo-colonic region offers a high retention time for orally administered beads and thus improves the drug absorption. Moreover, higher pH value as well as abundant microbial flora in this region significantly contribute towards the degradation of polymers  and thus favor the release of the loaded drug . DC formCS (low molecular weight (MW), ≥75% deacetylated), GG , TPP , GLA , CG, dipotassium hydrogen phosphate and sodium chloride were purchased from Sigma Aldrich, Steinheim, Germany. CX was kindly donated by Getz Pharma Private Limited, Karachi, Pakistan. Potassium phosphate monobasic was purchased from Duksan, Ansan, South Korea. Distilled water of an in-house facility  was used throughout the study. All other materials used were of analytical grade.Two types of formulations were prepared by using the ionotropic gelation technique , includiw/v) was dissolved in 1.5% v/v acetic acid solution with stirring at 3000 rpm at room temperature, i.e., 25 ± 3 °C (labelled as solution A). The GG at the concentration ranges of 0.15–1.25% w/v was dissolved in distilled water under stirring at 80 ± 5 °C (labeled as solution B). Solutions A and B were mixed at 40 ± 3 °C with continuous stirring at 2000 rpm. The homogenous mixture was added drop-wise into 1% w/v TPP solution using a 21G needle connected to a 20 cc syringe under constant stirring. The formed beads were gently stirred for 6 h, which ensured complete crosslinking and hardening at 2000 rpm and 25 ± 3 °C temperature. The beads were filtered, washed with distilled water for the complete removal of the unreacted polymers and crosslinker, dried at room temperature and stored in a moisture-free environment for further use (part A).For the preparation of SC hydrogel beads, CS (1.5–2.5% w/w (with respect to the dry mass of the polymers) GLA for 1 h. The beads were allowed to cure overnight, filtered, washed with distilled water, dried at 25 ± 3 °C and stored in air-tight containers for further analysis (Part B).DC hydrogel beads were prepared by immersing the beads formed in Part A into 10 mL of 2% w/v) and gently stirred for 24 h. The hydrogel beads were filtered, washed with distilled water to remove any free drug from the surface and dried at room temperature.The drug was loaded in the prepared hydrogel beads by using the sequential method (active loading technique). Optimized wet SC and DC beads (70 mg each) were individually immersed into a hydroalcoholic solution (three parts water and three parts ethanol) of CX . The sizes of the wet and dry beads were analyzed by using a measuring scale graduated in centimeters. The sizes were also determined by using an optical microscope equipped with a pre-calibrated optical micrometer. After complete drying, the %size reduction of all formulations was calculated by using the following formula.Swelling studies were carried out at pH 1.2 and 7.5 buffer media that relate to the stomach and intestine, respectively. The dried hydrogel beads were weighed (70 mg) and immersed in 30 mL of buffer media at room temperature. The swollen hydrogel beads were removed at discrete time intervals, weighed and re-immersed in the same buffer solutions. The equilibrium swelling ratio, S(EQ), was determined with the help of the following equation.3 design was applied for the comparison of the swelling ratio and size reduction [General factorial 2eduction . Input vv/v acetic acid, followed by sonication. The solution was filtered, and the content of CX was determined by using ultraviolet-visible spectrophotometric analysis at λmax 252 nm. EE% and LE% were calculated by using the following equations [A known amount of CX containing SC and DC hydrogel beads was immersed individually in 200 mL of 0.75% quations .EE% = ASEM  was performed to study the surface morphologies of fabricated hydrogel beads (SC and DC). The samples were placed on carbon stubs, coated with graphene, and images were taken at various magnifications (500× to 1500×) and 20.00 kV.−1.FTIR spectroscopy was performed to determine the drug–polymer interaction, polymer/crosslinker crosslinking and functional groups. Individual constituents and fabricated beads were placed on the stage of the spectrophotometer , and the spectra were obtained over a wave number range of 650–4000 cmθ range of 06–90° and a step size of 0.04°, with the help of a diffractometer .XRD analysis was performed to assess the phase of the drug, polymers and fabricated hydrogel beads. The samples were placed in the holder and scanned at a 2DSC and TGA  were used to determine the thermal stability of the pure ingredients and prepared beads. For DSC analysis, the samples  were added to alumina pans and heated from 50 to 400 °C at a rate of 10 °C/minute, and enthalpic changes were recorded. The calorimeter was calibrated for temperature and heat flow under nitrogen purging (50 mL/min) by using nickel as the standard. For TGA, the samples were heated from 30 to 500 °C, and the % weight/mass loss was determined.A known amount of optimized hydrogel beads was added into a dialysis bag and placed into a type 1 dissolution apparatus containing 900 mL of buffer media of two different pH values  at 37 °C and 100 rpm. After predetermined time intervals, a 5 mL sample was withdrawn, and the same volume of the buffer was added to maintain sink conditions. The concentration of the drug was measured by ultraviolet-visible spectrophotometric analysis at a λmax of 252 nm.The studies involving animals were approved by the ethical committee of Bahauddin Zakariya University, Multan, Pakistan (letter no. 213/PEC/2021).Mucoadhesion studies of optimized hydrogel bead formulations were performed by using the wash-off method, with a few modifications. The goat intestine  was cut into two pieces. Two sections of the mucosal membrane (dimensions 3 × 3 cm) were stored in normal saline. Mucosal membranes were individually placed in a petri dish, and a known mass of SC and DC beads (70 mg each) was added. An assembly was maintained in order to perform the experiment. The mounted tissues were suspended into a beaker containing 100 mL of phosphate buffered saline  maintained at 37 °C and agitated at 100 rpm. The beads that shed off at specified time intervals were collected and weighed. The beads that remained attached to the mucosal membrane indicated %mucoadhesion . The %mu2, rinsed with Krebs solution and tied into a sac [The goat intestinal mucosa was sliced longitudinally into three equal segments of 4 × 2 cmto a sac . The pouIn this study, eight healthy rabbits were used. These rabbits were divided into two groups, i.e., A and B, each comprising four rabbits. The rabbits were not given access to food for 12 h; however, they were allowed to have free access to water. Groups A and B were administered with optimized SC and DC beads, respectively. The rabbits in each group were sacrificed at 1, 2, 4 and 6 h. The stomach and the intestine were removed, and the ex-in vivo swelling behavior of the beads was observed.w/v CG solution (0.1 mL) into the plantar region of the right hind paw.Anti-inflammatory activity was performed by using a CG-induced rat paw oedema model , in whicThe animals were weighed (average weight 200 ± 6.3 g) and randomly divided into six groups labeled as NR, NC, RF, PC, SC and DC beads-treated . After 3For all groups, the in vivo anti-inflammatory activity was terminated after 7 h from the initial induction of the rat paw oedema using CG injection. At this time point, the blood sample was withdrawn by the retro-orbital technique from each rat and transferred to a sterile glass vacuum tube (BIO-VAC) containing the gel and clot activator. The samples were centrifuged at 1000 rpm for 10 min. The serum was isolated for the measurement of inflammatory markers including CRP and IL-6. These inflammatory markers were measured by an Invitrogen Rat ELISA Kit  with a pre-coated 96-well plate standard. The samples were placed into these wells. These samples were bound between an antibody pair (a specific antibody pre-coated and a second detector antibody), hence presenting a sandwich. The intensity of the signals produced by this complex was measured. The intensity represented the concentration of the CRP and IL-6 present in the sample .At the end of the in vivo anti-inflammatory study, the rat paw tissues of all groups were removed and stored in formalin solution. The tissue samples were cut into thin slices by using a microtome. The sliced tissues were fixed on glass slides and stained with haematoxylin and eosin, and histopathological examination was performed under an optical microscope  at 40× magnification .p ˂ 0.05.All the experiments were performed at least three times. The results were analyzed by using various statistical parameters. The results were expressed as the mean ± SD. The results were compared by using a t-test and two-way ANOVA, followed by Dunnett’s test. The software employed for this purpose included Microsoft Excel version 2016, SPSS  version 15.0 and GraphPAD Prism version 8.0. The results were considered significant at Celecoxib-loaded single and dual crosslinked chitosan/guar gum-based hydrogel beads with desired features were fabricated. The prepared formulations released the loaded drug in a sustained manner. Single crosslinked beads released ~74% of the drug within 24 h, while in the case of the dual crosslinked formulation, only ~24% drug release was observed. The dual crosslinked formulation showed better mucoadhesion; however, single crosslinked hydrogel beads showed a higher swelling% and permeability. Moreover, the SC formulation showed a relatively better therapeutic performance in the rat paw oedema model. It was concluded that celecoxib-loaded crosslinked chitosan/guar gum-based hydrogel beads are capable of providing a sustained release of the drug and treating inflammatory conditions.


Traditional Chinese medicine (TCM) is a widely used method for treating dengue fever in China. TCM improves the symptoms of patients with dengue, but there is no standard TCM prescription for dengue fever. This real-world study aimed to evaluate the effects of Chai-Shi-Jie-Du (CSJD) granules for the treatment of dengue fever and the underlying mechanisms. in vitro assay and network pharmacology analysis. Patients from 5 hospitals in mainland China who received supportive western treatment in the absence or presence of CSJD were assigned to the control and CSJD groups between 1 August and 31 December 2019. Propensity score matching (PSM) was performed to correct for biases between groups. The clinical data were compared and analyzed. The antidengue virus activity of CSJD was tested in Syrian baby hamster kidney (BHK) cells using the DENV2-NGC strain. Network pharmacological approaches along with active compound screening, target prediction, and GO and KEGG enrichment analyses were used to explore the underlying molecular mechanisms.  We implemented a multicenter real-world study, an P < 0.05) and the disease course  were significantly shorter in the CSJD group than those in the control group. CSJD showed no anti-DENV2-NGC virus activity in BHK cells. Network pharmacology analysis revealed 108 potential therapeutic targets, and the top GO and KEGG terms were related to immunity, oxidative stress response, and the response to lipopolysaccharide.  137 pairs of patients were successfully matched according to age, sex, and the time from onset to presentation. The time to defervescence (1.7 days vs. 2.5 days,  CSJD granules exhibit high potential for the treatment of dengue fever, and the therapeutic mechanisms involved could be related to regulating immunity, moderating the oxidative stress response, and the response to lipopolysaccharide. Dengue fever is one of the most prevalent mosquito-borne infectious diseases globally; it is endemic in more than 100 countries and has an incidence of 400 million infections per year . In 2019Traditional Chinese medicine (TCM) may provide an alternative treatment option for dengue. First, TCM has been used in China for thousands of years to treat warm diseases, which refer to acute infectious diseases. To date, TCM is still extensively used by Chinese people and encouraged by the Chinese government, especially after the outbreak of COVID-19. WHO has recognized and promoted TCM as safe and effective for use in existing healthcare systems . Second,Chai-Shi-Jie-Du (CSJD) granules were first used in the 2014 dengue outbreak in Guangzhou, and they have been modified and improved during subsequent years. They have been proven to be effective in relieving fever and other symptoms of dengue virus infection  and to hChiCTR2100049922). The recruited patients were diagnosed by using a DENV-specific reverse transcription-polymerase chain reaction (RT-PCR) assay or nonstructural protein 1- (NS1-) specific ELISA [This retrospective multicenter real-world study was carried out in 5 tertiary hospitals located in 3 provinces in Southern China from 1 August to 31 December 2019. The study was approved by the Institutional Research Ethics Committee of Guangzhou Eighth People's Hospital, Guangzhou Medical University (20170885), and registered in the Chinese Clinical Trial Registry  using SPSS 23.0. Clinical data were collected before treatment and on the last day of the treatment. The disease course was defined as the number of days between the first and the last days of the treatment. The primary outcomes were the time to defervescence (from presentation to defervescence and lasting for at least 24 h), the disease course, and the rate of progression to SD. The secondary outcomes were the laboratory examination results, symptoms, and signs. The safety survey was used to record adverse effects according to guidelines from the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. All of the data were retrospectively collected by the researchers at each site through the Cloud Platform for Traditional Chinese Medicine Fever Specialist Alliance (203.86.79.218:8188). The study flowchart is shown in Gypsum Fibrosum (Shi gao), Scutellariae Radix (Huang qin), Radix Bupleuri (Chai hu), Pogostemon cablin (Blanco) Benth. (Guang huo xiang), Radix Puerariae (Ge gen), Talc (Hua shi), Bubalus bubalis (Shui niu jiao), Codonopsis Radix (Dang shen), Coicis Semen (Yi yi ren), and licorice (Gan cao). It was manufactured by E-FONG Pharmaceutical, Foshan, Guangdong, China, strictly following the technical requirements for quality control and the standard formulation of Chinese medicine granules developed by the National Administration of Traditional Chinese Medicine (NATCM). CSJD is a brown powder (9.1 g per packet). Patients took one packet twice a day for five days.CSJD comprised 10 TCM drugs, including µL of CellTiter-Glo solution was directly mixed with the culture medium, followed by incubation at room temperature for 15 min. Then, the luminescence intensity was recorded, and the 50% cytotoxic concentration (CC50) was calculated by using GraphPad Prism 7.0 software.CSJD was dissolved in dimethyl sulfoxide (DMSO) to achieve a concentration of 500 mg/mL, then, the fully dissolved mixture was centrifuged, and the supernatant was stored at 4°C prior to use. Syrian baby hamster kidney (BHK) cells were adopted to evaluate the cytotoxic effects. The cytotoxic effects of CSJD were evaluated using luminescent CellTiter-Glo®  according to the manufacturer's instructions. Briefly, monolayers of BHK cells in 96-well plates were rinsed with PBS followed by incubation with the indicated concentrations of CSJD. After 96 h of incubation, 100 50) assay. BHK-cell monolayers were grown in 96-well plates and inoculated with 100 TCID50 of DENV-2 NGC at 37°C for 1 h. The inoculum was removed, and the cells were rinsed with PBS twice and subsequently incubated with the indicated concentrations of CSJD. Following 96 h of incubation, the cell viability was evaluated using luminescent CellTiter-Glo®, and IC50 was calculated as described in the cytotoxic assay.The DENV2-NGC virus was propagated in C6/36 cells (<4 generations), and the virus titers were determined based on a 50% tissue culture infectious dose (TCIDhttps://www.tcmsp-e.com) and BATMAN-TCM [https://bionet.ncpsb.org.cn/batman-tcm/). Gypsum Fibrosum was not included in any of the databases. The ingredients of Gypsum Fibrosum were obtained from articles [https://stitch.embl.de/) with “Homo sapiens” as the species. The molecules with oral bioavailability (OB ≥ 30%) and druglikeness (DL ≥ 0.18) were identified as candidate compounds for analysis except for molecules of Bubalus bubalis, Talc, and Gypsum Fibrosum, because their OB and DL data were not available in the database. All the targets obtained above were standardized according to their gene names by searching the UniProtKB (https://www.UniProt.org/) database with “Homo sapiens” as the species.The compounds and targets of the constituents in CSJD were retrieved from the online public databases TCMSP  , and the intersection of the drug targets and disease targets were regarded as the potential therapeutic target proteins.The CSJD-related proteins and the disease genes were mapped employing the Venny2.1 online tool  was used to generate a protein-protein interaction (PPI) network of the potential target proteins. The species was set to “Homo sapiens”; the lowest mutual action threshold was set to “highest confidence” (>0.9), and other parameters were set to their default settings. Next, the network topology parameters were analyzed by Cytoscape 3.9.0, and the hub target proteins were screened depending on the value of degree.The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING)   or Fisher's tests. Continuous data are presented as the means ± standard deviations and were analyzed with independent t-tests or Mann–Whitney U tests. When the p value was less than 0.05, the result was considered to be statistically significant.Data management and statistical analysis were performed by using SPSS 23.0. Categorical data were expressed as the frequency and percentage and analyzed using the chi-squared (P < 0.05)  .P < 0.05). The disease course in the CSJD group was significantly shorter than that in the control group . Each group had a patient who progressed to SD due to acute kidney injury, and the rates of progression to SD were the same in the two groups  (P=0.077)  . The mosP=0.077) .μg/mL, and TC50 was 3545 μg/mL . This finding could be related to the shorter disease course of the CSJD group. The present study showed that CSJD could help relieve fever and assist in disease recovery. However, the in vitro experiment showed that CSJD could not inhibit dengue virus replication in its safe dose, with an IC50 of 5954 μg/mL and a therapeutic index of 0.6. Thus, we explored the underlying mechanisms of CSJD in treating dengue fever through network pharmacology.The disease course in the CSJD group was 2 days shorter than that in the control group, which was of great significance for patients, not only by saving time but also reducing healthcare costs. Part of the reason for the shortened disease course in the CSJD group was that patients usually felt better after defervescence and were discharged from the hospital. The most frequent adverse events were level 2 digestive system discomfort, which was higher in the CSJD group than that in the control group with no significant difference, which could be related to the taste of CSJD. Usually, TCM decoctions are very bitter and smelly, which may cause nausea and vomiting. Chinese patent medicines that already on the market are usually made to taste sweet, but our preparation has not yet improved the taste. The AST level after treatment in the CSJD group was significantly higher than that in the control group , MMP, and IFN-γ, is considered a key factor contributing to DHF pathogenesis [CSJD had a strong influence on the immune response according to network pharmacological analysis. Many cytokines and immune regulation-related proteins, such as IL-1, IL-2, IL-4, IL-6, CXCL-8, CXCXL-11, MMP-9, INF-analysis . KEGG enanalysis . Monocytogenesis . GeneralGypsum, Scutellariae Radix, Radix Bupleuri, Radix Puerariae, and Bubalus bubalis might exert antipyretic effects together, among which Gypsum might be the most important one. One of the main indications for Gypsum is in treating febrile diseases caused by exogenous pathogens [Gypsum was reported to reduce the elevation of body temperature in rats and cats, and this effect was mediated by its influence on thermosensitive neurons in the PO/AH region [κB signaling pathway; and reduction in the levels of TNF-α, interleukin-1 (IL-1), and interleukin-6 (IL-6) [Scutellariae Radix, Radix Bupleuri, Radix Puerariae, and Bubalus bubalis are also antipyretic herbal medicines used in TCM, and their antipyretic effects have been confirmed in animal experiments [Scutellariae Radix were found to be associated with reducing the levels of TNF-α, IL-1β, IL-6, and other pyrogenic cytokines in the serum, hypothalamus, and cerebrospinal fluid [κB activation [Radix Bupleuri antipyretic mechanism is related to the regulation of the synthesis and secretion of cyclic adenosine monophosphate (cAMP) and arginine vasopressin (AVP) [Radix Puerariae is due to the regulation of the synthesis and release of endogenous pyrogen via the inhibition of NF-κB activation and the MAPK pathway [Radix Bupleuri and Radix Puerariae could play an immunomodulatory role and reduce the levels of cytokines, such as IL-1, IL-6, IL-17, TNF-α, and IFN-γ [Bubalus bubalis has been shown to exert antipyretic activity by enhancing antioxidation enzyme activities and decreasing prostaglandin E2 (PGE2) production [Pogostemon cablin (Blanco) Benth., can suppress the T-cell response and attenuate TNF-α-induced injury in A549 cells by inhibiting NF-kappa B and activating Nrf2 pathways [Codonopsis Radix [licorice [Changes in the immune response, cytokines, and chemokines influence body temperature. athogens . It is aathogens , 35. GypH region , 37; sup6 (IL-6) . Scutelleriments –44. The al fluid , which mtivation . The Radin (AVP) , 44. The pathway . Radix Bnd IFN-γ , 44. Buboduction . COX2, opathways , 48. Codlicorice , 51 alsoSeveral oxidative stress-related pathways were identified in the GO and KEGG enrichment analyses. An imbalance of reactive oxygen species (ROS) triggers the sudden release of cytokines, which can aggravate existing cases of disease and lead to the apoptosis of microvascular endothelial cells , 53. As The top 2 terms in GO enrichment were related to lipopolysaccharide (LPS). It is a potent stimulus for the production of platelet-activating factors (PAF) and other inflammatory cytokines from monocytes . Serum Lin vitro analyses. Nevertheless, we demonstrated the true effectiveness of CSJD in treating dengue fever and the potential underlying mechanisms, providing a basis for future research.There were some limitations in this study. First, the study was a retrospective study rather than a prospective and randomized blind trial. Second, the conventional details of western treatment were not recorded or analyzed. Third, cytokines were not detected in the clinical or in vitro and network pharmacological analyses, the potential mechanisms were likely related to regulating immunity, moderating the oxidative stress response, and the response to lipopolysaccharide.This multicenter real-world study showed that CSJD granules might shorten both the time to defervescence and the disease course of dengue fever. Based on the results of


Emerging and re-emerging swine coronaviruses (CoVs), including porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome-CoV (SADS-CoV), cause severe diarrhea in neonatal piglets, and CoV infection is associated with significant economic losses for the swine industry worldwide. Reverse genetics systems realize the manipulation of RNA virus genome and facilitate the development of new vaccines. Thus far, five reverse genetics approaches have been successfully applied to engineer the swine CoV genome: targeted RNA recombination, in vitro ligation, bacterial artificial chromosome-based ligation, vaccinia virus -based recombination, and yeast-based method. This review summarizes the advantages and limitations of these approaches; it also discusses the latest research progress in terms of their use for virus-related pathogenesis elucidation, vaccine candidate development, antiviral drug screening, and virus replication mechanism determination. Coronaviridae family, and they are represented by four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. All CoVs have similar genomic organization; they have a genome that is approximately 26–32 kilobases (kb) in length and contains at least six open reading frames (ORFs): ORF1a, ORF1b, spike (S), envelope (E), membrane (M), and nucleocapsid (N) , a poly(A) tail, hepatitis delta virus ribozyme (HDVr), and a bovine growth hormone (BGH) polyadenylation sequence into the pBeloBAC11 vector. The remaining AJ1102 genome was divided into five fragments and amplified through a reverse transcription (RT) polymerase chain reaction (PCR). The five DNA fragments were assembled into a pBAC-M-PEDV vector through homologous recombinations to obtain an infectious clone of AJ1102. The recombinant virus could be recovered through transfection of 6 μg of the recombinant BAC plasmid into Vero cells and by replacing the medium with DMEM containing 5 μg/mL trypsin 6 h after transfection (The CoV genome is large (26–32 kb) and has a replicase gene with cDNA sequences, which are unstable in bacterial cloning systems. The BAC vector is based on the bacterial F’ factor, which regulates the DNA synthesis so that its copy number is kept at a low level; this ensures the stability of its cloned genes and increases its tolerance for >300-kb genes . As suchsfection .BstBI restriction site, a partial C-terminal region of PDCoV genome , a 25 nt poly(A) tail, HDVr, and a BGH sequence. Then, five DNA fragments encompassing the genome of PDCoV CHN-HN-1601 were amplified through RT-PCR, and the first two DNA fragments were ligated into pBeloBAC11-M by seamless cloning using an NEBuilder HiFi DNA Assembly Cloning kit . Similarly, the other three DNA fragments were also ligated into pBeloBAC11-M through seamless cloning. Finally, the infectious clone plasmid can be obtained through restriction digestion and ligation with BstBI and MluI. Virus recovery can be performed through transfection of recombinant BAC plasmid into LLC-PK1 cells, followed by replacement of the medium with 10 μg/mL trypsin and 37.5 μg/mL pancreatin. The limitation of this approach is that tedious cloning procedures are needed to obtain a recombinant BAC plasmid encoding the full-length CoV genome. Due to the low copy number of BAC in a bacterium, extracting a certain amount of BAC plasmid from bacteria is time-consuming. However, several advantages, including high stability of exogenous genes, high efficacy of cDNA transfection into susceptible cells, and easy manipulation for genes, make it is an attractive method to establish CoV reverse genetics system [The BAC system has also been applied to other swine CoVs. For example, Zhou et al. constructed an infectious clone for PDCoV CHN-HN-1601 using a BAC plasmid as the cloning vector . The autAn advantage of using the yeast system for constructing a CoV reverse genetics system is that >10 cDNA fragments can be ligated simultaneously such that the total fragment length exceeds 110 kb. In 2022, Zhou et al. reported the construction of a PEDV reverse genetics system using the yeast system . The seq5) were infected with the respective recombinant vaccinia virus at 1 multiplicity of infection; at 1 h after infection, 5 μg of plasmid DNA was transfected using Lipofectamine 2000 (Invitrogen). After 2 days, plaque purification was performed to isolate the recombinant vaccinia virus after three rounds of plaque purification with GPT-positive or -negative selection as a selection marker.As a cloning vector, the vaccinia virus enables stable propagation of full-length CoV genomic cDNA in cell culture, as well as genetic manipulation through vaccinia virus-mediated homologous recombination . KristenHowever, even after the full-length cDNA of PEDV-MN was cloned into vNotI/tk vaccinia virus genome, an attempt to recover the virus failed. The recombinant PEDV was successfully recovered only after the authors modified the genome by substituting two nucleotides within the 5′UTR and two additional nucleotides within the S gene based on the sequence of cell-adapted PEDV CV777 . The vacSaccharomyces cerevisiae VL6-48N. This was followed by the extraction of yeast artificial chromosome (YAC) from the yeast culture medium and cleavage of YAC-containing viral cDNA at the unique restriction site downstream of the 3′-end poly(A) tail. After transcription in vitro, the infectious mRNA was electroporated along with the N gene transcript into susceptible cells to recover the recombinant virus (A 2020 study from Switzerland and Germany used a transformation-associated recombination (TAR) cloning platform to construct a CoV reverse genetics system. The authors constructed a recombinant novel CoV SARS-CoV-2 virus using the GFP protein to replace ORF7a, an accessory protein . First, nt virus . AlthougThe recently reported circular polymerase extension reaction (CPER)-based method may be an attractive method for recombinant-positive RNA virus recovery . CPER isIdentification of the virulence factors in CoVs is essential for the rational design of live-attenuated vaccine candidates. To date, an extensive effort has been made toward understanding the virulence factors of the emerging and re-emerging swine CoVs ,53,54,55In addition to structure proteins, nonstructural proteins, including nsp1, nsp15, and nsp16, are also linked to the virulence of PEDV. Niu et al. mutated two amino acids (N93A and N95A) in PEDV nsp1 and noted that the recombinant virus increased viral sensitivity to the host immune response and demonstrated an attenuation phenotype in vivo . We alsoThe aforementioned virulence factors for swine CoVs can be the target for manipulation by reverse genetics systems for rapid virus attenuation. The attenuated virus particles can then serve as attenuated vaccine candidates. By using the reverse genetics system of PEDV AH2012/12, we recently proved that the C-terminus of the S2 subunit contributes to PEDV virulence. We further confirmed that compared with the killed vaccine, the recombinant PEDV with seven-amino-acid deletion at the C-terminus of S protein elicits increased immunoglobulin (Ig) G and IgA, neutralization antibody production, and better protection effects against virulent virus challenge.720–892 aa with a trypsin-independent strain or the furin cleavage site in S protein induces trypsin independence during PEDV replication. Notably, replacement of S2894–1386 aa in G2 PEDV with the trypsin-independent G1 strain can confer protective effects against both G1 and G2 PEDV challenges and enable propagation to high viral titers in vitro. Changing the biological characteristics of the swine CoVs, such as adding trypsin independence and increasing viral titer in vitro by using reverse genetics systems, can improve the swine CoV vaccine manufacturing processes.The efficient replication of many swine CoVs, such as PEDV and PDCoV, requires the presence of trypsin. By using the reverse genetics system, some studies have identified the S protein of PEDV as the determinant of this trypsin dependency ,69,70. SMoreover, the reverse genetics system has the potential to be an expression vector that delivers antigens of interest. For example, Pascual-Iglesias et al. generated a recombinant chimeric TGEV that expresses PEDV S protein, which could protect against challenges with a virulent PEDV strain . BecauseBinding to cognate receptors is a prerequisite for the initiation of virus infection. For instance, the binding of S protein from SARS-CoV-2 to different species’ angiotensin-converting enzyme 2 (ACE2) is the molecular basis of the broad host range for SARS-CoV-2 ,74. We pCoV infection is initiated by the interaction between S protein and specific cellular receptors; this biological process largely determines a CoV′s host spectrum and tissue tropism. Phylogenetic analysis reveals that PDCoV is closely related to sparrow CoV HKU17, which supports the hypothesis that PDCoV evolved from avian deltacoronavirus ,78. Niu Considering the high pathogenicity of most emerging and reemerging swine CoVs, many scientists screened antiviral drugs that can inhibit pathogenic swine CoV replication. Edwards et al. synthetically constructed a recombinant SADS-CoV with a gene encoding tomato red fluorescent protein rather than ORF3 and applied it to understand whether remdesivir can efficiently inhibit SADS-CoV in vitro by calculating the florescence area . In 2021Accessory and structural proteins are expressed by a series of nested subgenomic RNAs, which are regulated by transcription regulatory sequences (TRSs) . The assDespite the challenges associated with establishing the reverse genetics systems for swine CoVs, several research teams have generated the infectious cDNA clones of swine CoVs. The availability of reverse genetics systems has enabled precise genetic manipulation of the viral genome. The reverse genetics systems of swine CoVs have been used extensively; for instance, they have been used to investigate the biological function of CoV proteins, elucidate CoV transcriptional regulatory mechanism, screen antiviral drugs for CoVs, and identify virulence factors and cell tropism related to CoVs. The studies reported thus far deepen our understanding of the biology of swine CoVs and assist in the rational design of a new generation of vaccines with improved safety and efficacy for the emerging and re-emerging swine CoVs.


This research project aimed to generate basic data forspecifying the trace mineral requirements of Fleckvieh bulls. Hence, the concentrations of the trace minerals iron (Fe), zinc (Zn), copper (Cu),and manganese (Mn) in the empty-body and body tissue fractions of growingFleckvieh bulls slaughtered at 120–780 kg live weight were determined.Results were used to calculate trace mineral accretion rates. Fe and Znrepresented the largest shares in the animals' bodies. The Zn accretionincreased, while Mn accretion steadily declined during cattle growth. Feaccretion attained a maximum at 400 kg live weight. Cu accretion declineduntil 600 kg live weight and then increased slightly afterwards. The provideddata may be used to adjust the recommendations with respect to the trace mineralrequirements of growing Fleckvieh bulls. Despite their low content, adequate tracemineral nutrition is essential to maintain physiological body functionsand ensure animal growth. A deficient trace mineral supply results invarious symptoms. Iron (Fe) deficiency entails anemia; this commonly occurs in calves, whichhave high iron requirements for physiological growth but must cope with the lowiron concentration in milk . Mohri et al. (2010)demonstrated that an additional administration of iron to newborn calvespromoted their growth. Impaired animal growth, skin lesions, delayed woundhealing, hair and coat impairment, diarrhea, and increased susceptibility toinfections are indicators of zinc (Zn) deficiency .Regarding copper (Cu) supply, Abramowicz et al. (2021) reported Cu deficiencyto result in anemia due to reduced red blood cell count and hemoglobinconcentration in dairy cattle. Furthermore, the authors found a reduction inlymphocytes and neutrophils, which was also associated with Cudeficiency and an impaired immune function by Cerone et al. (1998). Amanganese (Mn) shortage in cattle manifests itself in skeletal malformation andreduced growth . Trace mineral malnutrition andassociated deficiency symptoms can occur when animals are not fed accordingto their specific requirements, and this should be avoided to ensure goodlivestock performance and welfare.Has tracemineral accretion also changed to an equivalent degree during the past decades?Should feedingrecommendations be adjusted to the requirements of today's growingbulls?An important step in calculating the trace mineral requirements for growingcattle within the factorial requirement calculation method is to determinethe animals' trace mineral accretion at various stages of growth. Hence,this research project examines trace mineral concentrations and accretionrates in the empty body  and individual body tissues ofgrowing Fleckvieh  bulls. The Fleckvieh breed is a commondual-purpose cattle breed in southern Germany and Austria and simultaneously provides highmilk , according to the European guidelines for animal experiments, and approved by the LfL Committee for Ethics ofAnimal Experiments. Material and methods employed during calf rearing andbull fattening have been previously described by Honig et al. (2020).The trial included 72 male Fleckvieh calves  No. 1099/2009 (2009). Slaughter andtissue-sampling methods have been previously described by Honig et al. . In short, bulls from both feeding groups were slaughtered atfinal live weights of 120, 200, 400, 600, and 780 kg. During slaughter, thebulls' empty-body weights (EBWs) were determined as the final live weight minusthe contents of the urinary bladder and gastrointestinal tract (GIT).Subsequently, the entire empty body was dissected to individual body tissuefractions , which were then homogenized, sampled, and analyzed for their tracemineral concentration. The experimental design allowed for the analysis of the empty-body and individual-body-tissue trace mineral compositions at differentstages of the animals' maturity. Feeding varying energy concentrationsreflected the range of different growth intensities under practicalconditions.2.3The trace mineral analyses were conducted at the LfL Department ofLaboratory Analytics according to the methods of the Association of GermanAgricultural Analytic and Research Institutes . Threedifferent samples of individual feedstuffs and body tissues were analyzedfor their Fe, Zn, Cu, and Mn contents.For this, feedstuff and body tissue samples were homogenized and dissolvedin nitric acid and hydrogen peroxide. Trace minerals were subsequentlyextracted by pressure digestion, using a microwave pressure digestion system andanalyzed using inductively coupled plasma optical emission spectrometry. Applying a daily five-point calibration method based on certifiedstandard solutions  ensured good analysisquality. Analytical results were verified through reference materials andcontrol samples.The trace mineral concentrations of feedstuffs and TMRs given during calfrearing and the fattening period are presented in Table 1 and werecalculated based on the TMRs' compositions and the mineral content in theindividual feed components. The bulls' empty-body mineral contents werecalculated based on their body tissue composition  and themineral contents in the individual body tissues.2.4Statistical analysis of feed and body mineral contents was performed usingthe PROC MIXED procedure of SAS and the Kenward–Roger method to provide corrected degrees of freedom. Theanalysis included a two-way analysis of variance (ANOVA) with interaction of Fleckvieh bulls with 200–650 kg liveweight fed ad libitum. For this purpose, a relation of the empty-body weight to live weight of 0.88 for 200 kg bulls and of 0.93 for 400–780 kgbulls was assumed as inferred from the present study.To calculate the bulls' mineral accretion, third-order polynomial regressionequations and their derivatives were used according to methods described byHonig et al. (2022b). The regression analyses were calculated using the PROCNLIN procedure in SAS and based on Eq. (1):The residuals of the fitted models for the NE and HE bulls were calculatedto estimate significant differences between the feed intake groups and toevaluate the goodness of fit of the regression equations. A two-way ANOVA. In brief,bulls in the NE and HE treatment groups exhibited respective daily weight gains of 1699and 1792 g d3.2Empty-body weights (EBWs) of the 120, 200, 400, 600, and 780 kg weight groups were 104, 176, 370, 553, and 734 kg, respectively .The average empty-body and body tissue trace mineral compositions in bulls indifferent weight groups are presented in Table 3. The trace mineralconcentrations in Table 3 refer to natural body tissue including moisture.As the dietary energy concentration had no significant effect on the bodytrace element concentration of bulls in the NE and HE treatment groups, themean values for both the NE and HE groups are shown. Results were comparedto recalculated empty-body trace mineral concentrations inFleckvieh bulls used in prior work by Kirchgessner et al. (1994b) that were fed ad libitum at defined live weights. The authors described the average empty-body and carcass tracemineral concentration in 54 Fleckvieh bulls with a 200–650 kg live weight.Therefore, the body tissues and parts  were ashed  and afterwards analyzed using a spectralphotometer. Hence, limitations in comparability are caused bydissimilarities in tissue collection during slaughter and carcass processingand different sample preparation and analysis methods.The bulls' empty-body trace mineral concentration changed during animalgrowth. Growing bulls showed increasing Zn but decreasing Cu and Mnconcentrations. The Fe concentration in the empty body was comparable in the lowestand highest weight groups. Comparing the results to recalculated empty-bodytrace mineral compositions showed lower Fe, Cu, and Mn concentrations but higher Znconcentrations in Fleckvieh bulls used in previous work . The mostnotable difference occurred in bulls with a 200 kg live weight used in this work, in whichCu and Mn concentrations were more than twice as high as in bulls used in former studies.Increasing amounts of Zn in the empty body resulted from the high Zn concentration inthe animals' muscle tissue. Fleckvieh bulls feature high proportions ofmuscle tissue, even at high live weights . Muscle Zn andFe concentrations increased by 75 % and 91 % from the lowest to thehighest weight group, respectively, while Cu and Mn concentrations did notchange during growth. This observation confirms research by Kirchgessner etal. (1994b), who reported increasing Zn and Fe concentrations but constantCu and Mn concentrations in muscle tissue of growing bulls fed ad libitum. However,the muscle Cu concentration in Fleckvieh bulls used in this work was twice as high as inbulls used in prior studies, which featured an average muscle Cu concentration of 0.6 mg per kilogram oftissue .Growth-associated changes in muscle mineral concentration were also observedby Giuffrida-Mendoza et al. (2007). Furthermore, the authors reporteddifferences in muscle P and Fe concentrations in buffalo and cattle species. Breed and muscle type can also affect themuscle trace mineral concentration . Moreover, musclemineral concentrations differ in different sexes within the same breed. Species-, breed-, sex-, and muscle-type-dependentdifferences in muscle mineral concentrations may be a result of differingintramuscular fat contents.The highest tissue Fe concentration was observed in the blood and organsfraction, where the Fe concentration increased by 69 % during growth.Furthermore, blood and organ tissue showed the highest tissue Cu concentration,whereas the lowest Cu concentration was observed in bone tissue of 780 kg bulls.The Fe, Cu, and Mn concentrations in bone tissue decreased from the lowest to thehighest weight group, which can be attributed to increasing bonemineralization. Former research has indicated decreasing Fe but constant Zn,Cu, and Mn concentrations in the bone tissue of growing bulls .Concentrations of Fe, Zn, and Mn in fat tissue decreased during animalgrowth as a result of increasing crude fat proportions. This observationpartly confirms research by Kirchgessner et al. (1994b), who reported that Feand Zn concentrations decreased from 12.8 to 4.7 and from 7.1 to 5.3 mg per kilogram of fattissue, respectively, while Cu and Mn concentrations remained constant at an average of0.4 and 0.09 mg per kilogram of fat tissue, respectively, in 200–650 kg bulls.It can be concluded that the empty-body trace mineral concentration in growingcattle is influenced by allometric growth and the respective stage of theanimals' maturity. Muscle tissue comprises the largest share of the animals'bodies  and shows increasing Fe and Zn concentrations,which are components of metabolically active proteins. Conversely,increasing bone mineralization and increasing crude fatproportions in fat tissue cause a reduction in the tissues' trace mineralconcentrations.3.3Third-order polynomial regressions were calculated to determine the empty-body trace mineral content in growing bulls, as illustrated in Fig. 1. Atwo-way ANOVA showed no significant differences in the residuals of bothfeed intake groups. Hence, the combined regression equations were used forboth groups and are displayed in Table 4.Trace mineral accretion rates, in milligrams per kilogram empty-body weight gain (EBWG), werecalculated using the first derivative of each regression equation, a methodpreviously applied by Honig et al. . Using regressionequations and their derivatives to calculate trace mineral accretionprovides the opportunity to calculate typical accretion rates within growinganimals. The results can be used to adjust feeding recommendations to fitthe growing Fleckvieh bulls' trace mineral requirements.The accretion rates of the trace minerals Fe, Zn, Cu, and Mn in growingbulls are presented in Table 5. Zn accretion per kilogram EBWG increases ingrowing bulls, which can be attributed to muscle growth and the increasing Znconcentration in muscle tissue (Table 3). Accretion rates of Mn decreaseduring growth, which is an effect of decreasing Mn concentrations in hide,fat, tendon, and bone tissues of growing bulls. Fe accretion attained amaximum at 400 kg live weight and declined afterwards. Cu accretion showed adecline until 600 kg live weight and increased slightly afterwards. Anincrease in Cu accretion in animals with a high live weight may be associatedwith increasing Cu accumulation in the liver. The liver serves as copperstorage in the animal's body and maintains Cu homeostasis until surplus Cucan be excreted via the bile. Peak Fe accretion in 400 kg bulls isattributable to a high Fe concentration and accretion in the blood and organsfraction.Previous research into trace mineral accretion determined the accretionrates of Fe, Zn, Cu, and Mn to range between 41.4 and 51.5, between 26.4 and 51.4,between 2.0 and 3.3, and between 1.5 and 2.7 mg per kilogram EBWG, respectively, in Fleckvieh bulls with a 200–650 kglive weight fed ad libitum . Hence, accretionrates were reported to increase. However, our study shows more variableaccretion patterns for the individual trace minerals, especially for Fe. Thetotal amount of trace mineral accretion depends on the animals' daily weightgain, which was not considered in Table 5.Increasing trace mineral accretion rates in growing Fleckvieh bulls areassociated with increasing mineral requirements, as specified by GfE (1995).In Germany, mean recommendations for dietary Fe, Cu, Zn, and Mnconcentrations are 50, 9, 40, and 40 mg per kilogram DM intake, respectively, for fattening cattlefrom a 175 kg live weight . The requirements reported by NRC (2016) for Fe, Cu, Zn, and Mn in beef cattle diets are 50, 10, 30,and 20 mg per kilogram DM intake, respectively. The dietary Cu supply recommended byGfE (1995) and NRC (2016) is in line with observations by Costa e Silva etal. (2015), who reported dietary Cu requirements to be 9.5 mg per kilogram DM intake.Furthermore, Costa e Silva et al. (2015) stated that dietary Fe, Zn, and Mnrequirements are 218, 61, and 9.6 mg per kilogram DM intake, respectively, whichwidely differs from the GfE and NRC recommendations.Differences in the dietary trace mineral supply recommendations can beattributed to differing trace mineral assessment and calculation methods. Inour study, the trace mineral supply per kilogram DM exceeded the requirementsrecommended by GfE (1995). Furthermore, HE-fed bulls showed significantlyhigher trace mineral intake than NE-fed bulls, as illustrated in Table 2.The trace mineral supply could have covered higher trace mineral accretion,but physiological homeostasis regulates the amounts of trace minerals in theanimals' body. Excessive trace mineral intake results in excretion, notaccretion, in body tissues. Therefore, an impairment of the animals' bodyfunction is not expected, but higher excretion rates may be related toincreased soil pollution. Hence, data covering trace mineral concentrationand accretion in bulls' body tissues and empty bodies correspond to theanimals' physiological growth process and can be used to derive the nettrace mineral requirements of growing bulls in their respective stages ofmaturity.Different trace mineral concentrations and accretion rates are expected inother cattle breeds if their body composition deviates from the presentanimals. Von Soosten et al. (2023) indicated lower protein but higher energyand phosphorus accretion in growing Holstein bulls, which showed higher bodyfat and ash concentrations in high final weights.4The empty-body trace mineral concentration is influenced by the allometric growth ofindividual body tissue fractions and changes related to the animals'respective stage of maturity. The trace minerals Fe and Zn show the highest empty-bodytrace mineral concentrations in cattle. Growing Fleckvieh bulls' bodiesexhibited increasing Zn but decreasing Cu and Mn proportions. The empty-bodyFe concentration was comparable in bulls with 120 and 780 kg live weights.Zn accretion increased while Mn accretion declined steadily duringcattle growth. Peak Fe accretion was observed in bulls with a 400 kg liveweight, whereas animals with a 600 kg live weight exhibited the lowest Cu accretionrates. Feeding high-concentrate rations did not alter the body trace mineralconcentration nor mineral accretion per kilogram EBWG.A comparison of trace mineral accretion in previous and present Fleckviehbulls shows more variable accretion patterns for the individual traceminerals in present bulls. Furthermore, the total trace mineral accretiondepends on the animals' daily weight gain, which was increased by HE feeding during certain stages of the fattening period. Hence, theprovided data on trace mineral accretion can be used to reassess and, ifnecessary, adjust the feeding recommendations with respect to the trace mineralrequirements of growing Fleckvieh bulls. The mean trace mineral accretionrates in growing bulls can also be applied to adjust the trace mineralbalance and excretion calculations with respect to the current practical conditions inGerman fattening bull farms.


The virus formerly known as monkeypox virus, now called mpoxv, belongs to the Orthopoxvirus genus and can cause mpox disease through both animal-to-human and human-to-human transmission. The unexpected spread of mpoxv among humans has prompted the World Health Organization (WHO) to declare a Public Health Emergency of International Concern (PHEIC).We conducted a literature search to identify the gaps in biosafety, focusing on five main areas: how the infection enters the body and spreads, how much of the virus is needed to cause infection, infections acquired in the lab, accidental release of the virus, and strategies for disinfecting and decontaminating the area.The recent PHEIC has shown that there are gaps in our knowledge of biosafety when it comes to mpoxv. We need to better understand where this virus might be found, how much of it can spread from person-to-person, what are the effective control measures, and how to safely clean up contaminated areas. By gathering more biosafety evidence, we can make better decisions to protect people from this zoonotic agent, which has recently become more common in the human population. The World Organisation for Animal Health, The World Health Organization (WHO), and Chatham House are collaborating to improve the sustainable implementation of laboratory biological risk management, particularly in low-resource settings. The Biosafety Research Roadmap project aims at supporting the application of laboratory biological risk management and improving laboratory sustainability by providing an evidence base for biosafety measures (including engineering controls) and evidence-based biosafety options for low-resource settings.This will inform strategic decisions on global health security and investments in laboratory systems. This work involves assessing the current evidence base required for implementing laboratory biological risk management, aiming at providing better access to evidence, identifying research and capability gaps that need to be addressed, and providing recommendations on how an evidence-based approach can support biosafety.1 the current biosafety evidence, and available information regarding laboratory-acquired infections (LAIs) and laboratory releases.This manuscript presents the general characteristics of mpoxv, previously known as Monkeypox virus,A 15-member technical working group (TWG) was formed to develop a Biosafety Research Roadmap to support the application of laboratory biological risk management and improve laboratory sustainability by providing an evidence base for biosafety measures. The TWG conducted a gap analysis for a selected list of priority pathogens on the procedures related to diagnostic testing and associated research for those pathogens, including but not limited to sample processing, testing, animal models, tissue processing, necropsy, culture, storage, waste disposal, and decontamination.2 provided a detailed description of the materials and methods and an introduction to why the gap analysis was performed.To achieve this, databases, websites, publications, reviews, articles, and reference libraries were screened for relevant data. The main research domains used to perform the literature searches were the ABSA database, Belgian Biosafety Server, CDC reports, WHO reports, PubMed, and internet searches for terms related to biosafety matters, including, for example, inactivation, decontamination, LAIs, laboratory releases, and modes of transmission. Blacksell et al.Orthopoxvirus and family Poxviridae in risk group 3.3 Mpoxv is a 200 to 250 nm brick-shaped, enveloped virus with characteristic surface tubules and a dumbbell-shaped core component (adapted from reference 1). Mpoxv is divided into three clades: Clade I , Clade IIa, and Clade IIb (both formerly West African Clade).4Mpoxv is a viral zoonosis belonging to the genus 5–7 Signs and symptoms of mpox infection may include flu-like symptoms , swollen lymph nodes, and a rash. Symptomology and the severity of symptoms vary among infected individuals.Studies have shown that the Clade I mpoxv strain is genetically distinct from and more virulent than Clades IIa and IIb as distinguished by genetic sequencing and clinical signs and symptoms.8 Mpoxv usually circulates in Central and Western Africa;9 however, outbreaks were reported in 2003 in the United States and numerous non-mpox endemic countries in 202211 across five WHO regions.12 While a telltale symptom of infection by most viruses in the Orthopox genus is the formation of vesicular-pustular lesions, a clear distinction can be made between variola virus, vaccinia virus, cowpox virus, and mpoxv by genetic analysis.13–15The rash typically starts as flat red spots and progresses for 2–4 weeks through stages of forming hard red bumps, fluid-filled blisters, and blisters filled with pus, terminating with scabs that fall off.17 Tecovirimat is FDA-approved for treating smallpox but not other poxvirus diseases. It is available for clinical use under an expanded access Investigational New Drug (EA-IND) protocol and has demonstrated efficacy in treating the illness and improving disease outcomes.19At the time of this writing, no approved treatment is specific for mpoxv infections; treatment is supportive and based on alleviating symptoms.17 A Modified Vaccinia Ankara vaccine, JYNNEOS, is available under emergency authorization. It is a two-dose  used to protect against mpox and smallpox infection.21 While the ACAM2000 vaccine is approved for immunization against smallpox and was made available for use against mpox in 2022 in the United States under an EA-IND protocol,22 it is not as widely used as there are more known side effects and contraindications than the JYNNEOS vaccine.23The European Medicines Agency approved it for mpox treatment in 2022, but it has yet to be widely available.24–26 RT-qPCR and sequencing may not be feasible in resource-constrained settings due to equipment, reagent costs, and infrastructure capability .Tests that provide definitive and rapid diagnosis of mpox infection include nucleic acid amplification tests  and real-time quantitative PCR (RT-qPCR) alone or in conjunction with viral genome sequencing.Orthopoxvirus family.28 Mpox virus can also be detected in clinical samples by electron microscopy and procedures involving viral isolation.28Serological tests, including virus neutralization tests, hemagglutination-inhibition, immunofluorescence, enzyme-linked immunosorbent assays, and Western blot, may not provide a definitive diagnosis of mpox infection if there is cross-reactivity between reagents used to identify mpox viral antigens and viral antigens from other viruses in the 16 Historically, mpoxv infections in humans have been attributed to close contact with virus-carrying animals, and human-to-human transmission was considered rare.29Mpox is a zoonotic disease maintained in small animals. Reservoir species in central and west Africa include sun squirrels, giant pouched rats, African dormice, and other rodents.Cynomys spp.) that were housed with various exotic African rodents  shipped from Ghana to the United States as part of the pet trade.31 In this instance, another mode of transmission other than “close contact” occurred as the animals were in the same room but separately housed.In 2003, mpox infections were reported in the United States when patients were exposed to domesticated prairie dogs .34Research has indicated the presence of mpoxv on all surfaces touched by infected patients with high viral loads. Surface isolates tested demonstrated at least 1035–37There are no definitive data on the minimum infectious dose required to cause mpox infections in humans. Several studies in animal models have demonstrated infectious doses experimentally for reservoirs of rabbits, rats, pigs, non-human primates, and squirrels50 of mpoxv in Prairie dogs to be 5.9 × 103 pfu when administered via the intranasal route.38 The aerosol LD50 for non-human primates with Clade 1 has been reported as 7.8 × 104 pfu.39However, many studies related to vaccine challenge studies rather than determining the minimum infectious dose. A review of animal models cites the LD40–42 There have been multiple instances of laboratory-acquired exposures to other orthopoxviruses and subsequent infections, predominantly with the vaccinia virus.43There have not been any documented cases of mpoxv laboratory infections reported in the scientific literature. However, mpoxv infections involving sharps injuries have been reported in health care settings.44 In suspension tests, hydrogen peroxide (14.4%) and iodine (0.04–1%) were effective, whereas sodium hypochlorite , 2% glutaraldehyde (10 min), and 0.55% orthophthalaldehyde (5 min) were effective on artificially contaminated surfaces.44Vaccinia viruses could be inactivated by at least 4 log10 in suspension tests and on artificially contaminated surfaces by 70% ethanol (1 min) or 0.2% peracetic acid (10 min), as demonstrated primarily with various organic loads.®. Virkon used in the study contained 50% w/w potassium peroxomonosulfate, 5% sulfamic acid, and 15% sodium alkylbenzene sulfonate.45 After 3 min of exposure, copper surfaces (99.9%), such as those utilized in laboratories, lowered vaccinia virus and mpoxv titers by 4 log10, whereas stainless steel was substantially less efficient.46 Inactivation of vaccinia virus occurs in 2–3 h at 60°C or within minutes following exposure to 20 nM caprylate at 22°C; however, MPXV is more resistant than vaccinia to solvent-detergent treatment.47Vaccinia virus was demonstrated to be inactivated by Virkon48 , Zymo Research DNA/RNA Shield Buffer49 (SafeCollect Swab Tube), Qiagen Buffer ATL50 [Sodium dodecyl sulfate (≥1 < 10% w/w)], Roche Cobas PCR Media51 [≤40% (w/w) guanidine hydrochloride], Longhorn Vaccines & Diagnostics PrimeStore Molecular Transport Medium52 , Qiagen Buffer AVL53 (50–70% w/w guanidinium thiocyanate), 70% Ethanol,54 Thermo Scientific InhibiSURE Viral Inactivation Medium,55 Severn Biotech Ltd L6 Buffer,56 BioServ MagBead Viral RNA Lysis Buffer57 , Hologic Panther Fusion Specimen Lysis Tubes,58 and E&O Laboratories Ltd Molecular Sample Solution59 .The United Kingdom Health Security Agency has published evidence of the effectiveness of various commercial extraction buffers and transport mediums and the efficacy of inactivating mpoxv in clinical specimens . The fol60 , and the NeuMoDx Viral Lysis Buffer61  were not effective at complete mpoxv inactivation. A list of the evidence is provided in It was noted that the Cepheid Xpert CT/NG Swab Transport ReagentOrthopoxvirus are inactivated by heat (autoclaving and incineration).3 Autoclave decontamination procedures in most medical facilities inactivate the mpox virus in patient specimens.62 Complete inactivation of the closely related vaccinia virus occurs in 2–3 h at 60°C47  should be performed in an airborne infection isolation room. The PPE for health care professionals includes gloves, gowns, goggles or full-length face shields, and respiratory protection .64Work involving diagnostic samples should be conducted in a Biosafety Level 2 (BSL-2) laboratory , and using Class II Biosafety Cabinet (BSC) or other containment devices, especially if there is a potential to generate aerosols. The PPE may include solid-front gowns with cuffed sleeves, double gloves, eye protection  or face protection (face shield), and a particulate respirator .64A site-specific risk assessment that informs PPE use, primary containment, and practices should, at a minimum, consider the exposure risk posed by the procedure, proficiency of the individual conducting the work, availability of containment equipment, and whether staff have been vaccinated for smallpox or mpox.64Work on the open bench if a Class II BSC is not available is per a site-specific risk assessment and should include provisions for limiting the number of people in the laboratory and a combination of PPE and other containment devices  to create a barrier between the specimen and laboratory personnel.64 Staff working with mpoxv should be offered the vaccine as part of the occupational health program.Work involving in vitro mpoxv culture should be performed at BSL-3.It is still unclear where the mpoxv virus originates in the animal-to-human and human-to-human transmission routes. However, research has shown that the virus has been mainly found in a variety of hosts, including monkeys, rodents, squirrels, and prairie dogs, based on documented infection cases.33The infectious dose is not clearly defined, especially the minimum dose required to cause mpox infection via animal-to-human and human-to-human transmission.66According to the United States Environmental Protection Agency (EPA), there are no registered disinfectants for the mpox virus. However, the mpox virus is a group of viruses that are more sensitive to disinfectants than other viruses because the disinfectants can easily break the lipid envelope surrounding the virus, so the list of disinfectants published by the EPA can be used for mpox viruses based on their respective classifications .67 However, neither the CDC nor the EPA List Q for registered disinfectants for Emerging Viral Pathogens specifies contact time for use with mpoxv. Before disposal, the laboratory must confirm that the decontamination agent and process used have been validated.In addition, the Centers for Disease Control and Prevention (CDC) also recommends using disinfectants for mpoxv from the EPA.There were no reports that describe the effectiveness of gaseous chemical fumigants commonly used  in decontaminating spaces where work is being carried out involving pox viruses.As mpox has circulated throughout Central and Western Africa for decades, with patients hospitalized in settings ranging from poorly to well-equipped regarding patient isolation and barrier practices, more information should be needed regarding LAIs. Recent mpox infections in Europe and the United States, where the disease is neither endemic nor typically expected on initial patient presentation, have yet to result in the issuance of LAI reports.68 with only three sharps-related occupational infection reports by health care workers,40–42 but no reports of occupational-acquired infection of laboratory staff.There were 87,377 confirmed cases in over 111 countries between May 2022 and May 2023,Mpox is a highly contagious disease that can spread between animals and humans. Although it was previously confined to Africa and transmitted through person-to-person contact, it has recently spread to other regions, prompting the WHO to declare it a Public Health Emergency of International Concern in 2022.The primary mode of transmission is via close contact with infected humans or animals. To combat the disease, it is imperative that we conduct further research to understand its spread, develop effective treatments and vaccines, and provide clear guidance on diagnosis and patient care, especially in areas with limited resources.