In addition, the adsorption capacity further increased to 0.192–0.320 mmol/g by treating the GOPTES-treated silica beads with triethylenetetramine, pentaethylenehexamine, or PEI. These empirical, equilibria, and kinetic aspects obtained in this study support that the porous silica-based adsorbents prepared in this study can be applied to the removal of Cr(VI) ions.The hexavalent chromium (Cr(VI)) ion adsorption properties were conferred to porous silica beads by introducing alkylamine chains through functionalization with an aminosilane coupling agent, [3-(2-aminoethylamino)propyl]triethoxysilane (AEAPTES), or with an epoxysilane coupling agent, (3-glycidyloxypropyl)triethoxysilane (GOPTES), and polyfunctional amine compounds or poly-ethylenimines (PEIs). The presence of amino groups on the silica beads was confirmed by XPS and the amount of amino groups increased to 0.270 mmol/g by increasing the AEAPTES concentration and/or reaction time. The adsorption capacity of the silica beads functionalized with AEAPTES was the maximum at the initial pH value of 3.0 and the initial adsorption rate increased with an increase in the temperature. The adsorption capacity increased with an increase in the amount of amino groups at pH 3.0 and 30 °C. The adsorption behavior obeyed the pseudo-second order kinetic model and was well expressed by the Langmuir isotherm. These results support that Cr(VI) ion adsorption is accomplished through the electrostatic interaction between protonated amino groups and HCrO Chromium (Cr) is the seventh most abundant element on earth and naturally occurs in the environment in the Cr(III) and Cr(VI) oxidation states. The main anthropogenic sources include metallurgical industries, refractory brick production, electroplating, combustion of fuels, waste incineration, and the production of Cr-containing chemicals, mainly chromates and dichromates, pigments, Cr trioxide, and Cr salts. Natural sources include volcanic eruptions and the erosion of soils and rocks . Cr(III)Conventional methods, such as chemical precipitation, ion exchange, reverse osmosis, coagulation, and adsorption, have been reported for the removal of Cr(VI) ions from aqueous media. Among these methods, adsorption is viewed as superior to other methods because of its simplicity, ability for regeneration, cost-effectiveness, and enabling large-scale applications. Moreover, the greatest advantage of the adsorption method is that by-products, such as sludge, are not generated .VI) ion removal trimethoxysilane (AEAPTES). The Cr(VI) ion adsorption behavior of AEAPTES-modified silica beads (AEAPTES-silica beads) was estimated as a function of the pH value, the temperature, and the amount of amino groups, and then kinetically and thermodynamically analyzed. In addition, a further increase in the adsorption capacity was attempted by the pretreatment of the silica beads with an epoxysilane coupling followed by functionalization with polyfunctional amine compounds or polyethylene-imines (PEIs).2/g) were purchased from Sigma Aldrich . As aminosilane coupling agents, AEAPTES , N-(6-aminohexyl)aminomethyl-triethoxysilane , and 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane  were used. An epoxysilane coupling agent, GOPTES, was purchased from Sigma Aldrich. Polyfunctional amine compounds, ethylenediamine (EDA), diethylenetriamine (DETA), triethylenetetramine (TETA), and pentaethylenehexamine (PEHA), were purchased from FUJIFILM Wako Pure Chemical . The chemical structure of the silane coupling agents used in this study was shown in The porous silica beads, Davisil Grade 636,  were mildly stirred in the polyfunctional amine compound solutions for 6 h or PEI solutions for 24 and 72 h at 70 °C to introduce amino groups on their surfaces ,18.NH, was calculated from the concentration difference in the HCl solution using Equation (1):NHSB is the weight of the NH-silica beads immersed.The silica beads functionalized with the aminosilane coupling agents (NH-silica beads) and GOPTES-silica beads functionalized with the polyfunctional amine compounds or PEIs were immersed in a 20 mM NaOH solution so as to neutralize the protonated amino groups on the alkylamine chains and then washed with water several times. Then, 0.2–1.0 g of the NH-silica beads were immersed in 20 mL of a 20.0 mM HCl solution and the solutions were mildly stirred for at least 24 h under a nitrogen atmosphere. The HCl solutions were titrated with a 20.0 mM NaOH solution with bromothymol blue as an indicator ,19. The NH value by the molar mass of an alkylamine chain .In addition, the amount of alkylamine chains introduced to the silica beads was determined by dividing the A1s, O1s, N1s, and Si2p were recorded under reduced pressure lower than 5 × 10−5 Pa on a Shimadzu ESCA 3400 X-ray photoelectron spectrometer  with the MgKα (1253.6 eV) source operating at 8 kV and 20 mA for the untreated and functionalized silica beads. The electron energy calibration was performed by taking the Au4f core level peak at 83.8 eV as the reference.The XPS core level spectra of C3/mol·cm) [2Cr2O7 solution at 0.20 mM was adjusted to 3.0 with HCl. First, 10 mg of NH-silica beads were immersed in a pH 3.0 HCl solution for at least 12 h. Then, the NH-silica beads were placed in a 0.20 mM K2Cr2O7 solution (50 cm3) at 30 °C to initiate the adsorption experiments. The absorbance at 338.2 nm was measured at predetermined intervals to determine the Cr(VI) ion concentration. The adsorbed amount was calculated from Equation (3):t and C0 are the Cr(VI) ion concentration at time t and the initial concentration, respectively, and WNHSB is the weight of the NH-silica beads.The calibration curve of Cr(VI) ions was prepared at the isosbestic point of 338.2 nm  ,21. The 4Cl at 0.10 M, and NaOH at 0.50 mM were prepared as an eluent for desorption of Cr(VI) ions [Aqueous solutions of NaCl at 0.50 M, NHVI) ions . The Cr untreated silica beads and (b) AEAPTES-silica beads. For the AEAPTES silica beads, in addition to a main peak, which was composed of a peak at 284.3 eV assigned to C-Si and a peak at 285.0 eV assigned to -CH2-CH2-, a small peak assigned to -C-NH- and -C-NH2 at 286.5 eV was observed in the C1s spectrum [1s peak assigned to -NHR- and -NH2 emerged and the O1s and Si2p peaks decreased. These results confirmed that amino groups were introduced on the surfaces of the silica beads according to the reaction scheme shown in The strong peaks at 1080 cmca beads ,21. Howespectrum ,27. A N11/2, which is defined as the time required for the adsorption to take up half as much Cr(VI) ions as its equilibrium values, was also calculated [The time course of the adsorption of Cr(VI) ions on the AEAPTES-silica beads with different amino group amounts at 30 °C and pH 3.0 is shown in lculated .1/2 value with the initial pH value. The adsorption capacity and initial adsorption rate had the maximum values at pH 3. The t1/2 value was at most 12.4 min at pH 3.0, indicating that the adsorption behavior is relatively fast.The effects of the pH value and temperature on the Cr(VI) ion adsorption were estimated for the AEAPTES-silica beads with the amount of amino groups of 0.220 mmol/g. 4−) ions. When the pH value moves away from 3, the fraction of HCrO4− ions decreases and the fraction of H2CrO4 increases. In addition, the concentration of Cl ions added to adjust the pH value increases. Meanwhile, the pH value at the adsorption equilibrium increased to 4.20 at pH 4.0 and to 5.28 at pH 5.0, respectively. In the pH range higher than 3, as the pH value increases, the fraction of CrO42− ions increases. In addition, an increase in the pH value results in an increase in the deprotonation of -NH2+- or NH3+ groups, because the dissociation constant (pKa) values of AEAPTES are 6.8 and 9.3 [Next, the adsorption behavior will be explained in terms of chromium species present in water ,29,30,31 and 9.3 . The abo1/2 value with the temperature. When the temperature increased, the initial adsorption rate increased and the t1/2 value decreased. On the other hand, the adsorption capacity remained unchanged against the temperature. These results suggest that the Cr(VI) ion adsorption more successfully proceeded at higher temperatures. There are some systems in which the adsorption capacity is dependent on the temperature [2 values from the pseudo-second order equation in the following section to discuss the temperature dependence of the Cr(VI) ion adsorption in more detail.Based on the above results, the temperature dependence was investigated at pH 3.0. perature  and otheperature . The eff2Cr2O7 solution and the adsorbed amount was measured against the immersion time. 1/2 value with the amount of amino groups. The adsorption capacity and initial adsorption rate increased with the amount of amino groups and then leveled off higher than 0.12 mmol/g. In addition, the adsorption ratio was calculated by dividing the adsorption capacity by the amount of amino groups introduced on the AEAPTES-silica beads [1/2 value remained almost unchanged at higher amounts of amino groups.At pH 3.0 and 30 °C, 10 mg of AEAPTES-silica beads with different amino groups were immersed in a 0.20 mM Kca beads . As showIn addition, to further increase the adsorption capacity, the functionalization of silica beads was also performed with other aminosilane coupling agents with two or three amino groups, AHAMTES and AEAEAPTMS. The adsorption capacity of AHAMTES-functionalized silica beads (AHAMTES-silica beads) and AEAEAPTMS-functionalized silica beads (AEAEAPTMS-silica beads) was summarized in t and Qeq denote the adsorbed amount at time t and at equilibrium, respectively, and k1 is the pseudo-first order rate constant.The adsorption data of the AEAPTES-silica beads with the amount of amino groups of 0.220 mmol/g were analyzed with the pseudo-first and pseudo-second order models. The pseudo-first order rate equation is generally expressed by:2 is the pseudo-second order rate constant [On the other hand, the pseudo-second order equation is expressed as:constant ,33,34.2 values were also calculated from the adsorption data at 20–50 °C discussed in the above section. Since the k2 values increased with an increase in temperature, the activation energy was calculated by plotting ln k2 against the temperature reciprocal.a is the activation energy, and R is the gas constant (8.314 J/K·mol). The activation energy was calculated to be 19.6 kJ/mol from the slope of the linear relationship of the ln k2 value with the reciprocal temperature  ion adsorption. The Weber and Morris intraparticle diffusion model ,40 is ex2Cr2O7 solution at pH 3.0 and 30 °C. The adsorption process was divided into three straight lines  with regression coefficients higher than 0.99. The kid values obtained were summarized in id values in this region were much lower than those in the first region (kid1 > kid2). The final third region is the equilibrium stage (kid3 = 0) [id3 = 0) .eq is the Cr(VI) ion concentration at equilibrium, Qmax is the maximum adsorption capacity, and KL is the Langmuir adsorption constant related to the affinity of binding sites.F is the Freundlich constant related to adsorption capacity and 1/n is the empirical parameter corresponding to adsorption intensity, depending on the heterogeneity of the adsorbents.The adsorption capacity was determined in the range of the initial Cr(VI) ion concentrations of 0.05–0.30 mM at pH 3.0 and 30 °C for the AEAPTES-silica beads with the amount of amino groups of 0.220 mmol/g and applied to the Langmuir and Freundlich isotherm models, represented by Equations (8) and (9), respectively ,42,43,444− ion and a protonated amino group as shown in Figure 13 in ref [The obtained parameters by both isotherms were summarized in 4Cl, and 0.50 mM NaOH. These eluents were used for the desorption of Cr(VI) ions in our previous papers [4− ions with Cl− ions on protonated dimethylamino groups, -NH+(CH3)2, in NaCl and NH4Cl and through the deprotonation from -NH+(CH3)2 to -N(CH3)2 in NaOH as shown in ref. [4Cl and 0.50 mM NaOH was effective for the desorption of Cr(VI) ions because high desorption % values were obtained, and the desorption was faster.The desorption of Cr(VI) ions was of great importance for the regeneration or reuse of adsorbents. Here, the desorption of Cr(VI) ions from AEAPTES-silica beads with the amount of amino groups of 0.220 mmol/g was measured in 0.50 M NaCl, 0.10 M NHs papers ,45. The  in ref. ,45. Of tC-O- bond in an epoxy ring was observed at 287 eV as shown in 1s peak appeared at 399 eV, indicating the introduction of alkylamino chains or PEI chains on the silica surfaces.To further increase the Cr(VI) ion adsorption capacity, amino groups were introduced to the silica beads through the pretreatment with GOPTES and the subsequent functionalization of epoxy groups with polyfunctional amine compounds or PEIs. When the silica beads were treated with GOPTES, an overlapped small peak assigned to the -1s peak at 287 eV and a N1s peak was also observed for the functionalization with the other polyfunctional amine compounds and PEIs. Therefore, the amount of amino groups for these NH-silica beads was determined by the back titration, and then their adsorption capacity was measured at 30 °C and pH 3.0. As shown in The C2Cr2O7 solution, vary from article to article. However, it can be safely said that the adsorption capacity obtained in this study was comparable to, or higher than some of the other adsorbents, despite the Cr(VI) ion concentration being considerably lower than in the other articles cited. Our next goal is to prepare the adsorbent for Cr(VI) ions with better adsorption behavior through the functionalization with an epoxysilane coupling agent and polyfunctional amine compounds or PEIs.In addition, the Cr(VI) ion adsorption capacity of the NH-silica beads obtained in this study was compared with those of various particles functionalized by silane coupling reactions in In this study, the NH-silica beads were prepared through functionalization with different aminosilane coupling agents and through the pretreatment with an epoxysilane coupling agent; followed by the functionalization with polyfunctional amine compounds or PEIs. The kinetic, isotherm, and equilibrium characteristics of the Cr(VI) ion adsorption were investigated as a function of the experimental parameters, such as the pH value, the temperature, and the amount of amino groups in detail; desorption behavior was also discussed.4− ion and a protonated amino group. Cr(VI) ions were successfully released in 0.50 M NaCl, 0.10 M NH4Cl, and 0.50 mM NaOH after a relatively short immersion time. In addition, the adsorption capacity was further increased by the pretreatment with GOPTES and the subsequent functionalization with PEHA to 0.201 mmol/g and with 0.6KPEI to 0.320 mmol/g, supporting that the introduction of PEI chains or longer alkylamine chains to the silica beads is effective in producing a high-capacity adsorbent for Cr(VI) ions.For the AEAPTES-silica beads, the adsorption capacity and initial adsorption rate had the maximum values at pH 3.0. On the other hand, the initial adsorption rate increased with an increase in the temperature, although the adsorption rate remained almost constant against the temperature. At 30 °C and pH 3.0, the adsorption capacity increased and the adsorption ratio decreased with an increase in the amount of amino groups. The kinetics of Cr(VI) ion adsorption obeyed the pseudo-second order model and the equilibrium data were well described by the Langmuir isotherm model. These results support that the Cr(VI) ion adsorption will occur through the electrostatic interaction between a HCrOThe results obtained emphasize that the introduction of amino groups to the silica beads above-mentioned is an effective procedure to confer the Cr(VI) ion adsorption properties on the silica beads. The NH-silica beads exhibit significant potential as an adsorbent in the removal of Cr(VI) ions from aqueous media and wastewater. Therefore, we will aim for better adsorption behavior and higher adsorption capacity of Cr(VI) ions in the porous silica beads.


What a mess! Clusters of COVID-19 cases here, outbreaks there, catastrophic epidemics somewhere else, and all the while asymptomatic individuals  allowing the virus (SARS-CoV-2) to spread silently between unsuspecting individuals. COVID-19  is racing around the world and then racing around the world again. Vaccines have been produced and most are effective in most people. The number of reported cases and deaths in Africa are very low but we do not know why. We have evidence that anthroponotic transmission (human to non-human vertebrate) is known to occur in chimpanzees, gorillas, giant anteaters, bottlenose dolphins, horses, alligators, bats (not likely to be transmitted to bats from humans), sheep, pangolins, house cats, tigers, lions, minks, deer, and other forms of wildlife and domestic vertebrates. Experimental infections conducted by several research groups have shown that monkeys, hamsters, ferrets, cats, dogs, pigs, tree shrews, various rodents, and fruit bats are permissive, some of them only experimentally, while poultry seem to be resistant (Neovison vison) and white-tailed deer (Odocoileus virginianus) may also serve as reservoirs of SARS-CoV-2 compounds the devastating problems caused by asymptomatic infections of some humans. If these animals infected with a strain, or strains, of SARS-CoV-2 that infects humans, can produce sufficient loads of virus to infect humans, and now may serve as reservoirs of the virus, we will not rid ourselves of it unless and until they are vaccinated or otherwise immunized against it, an unlikely scenario. Other wild vertebrates may also cause the same problems.SARS-CoV- to spreaAs long as there is a single person who is not immune to SARS-CoV-2, which causes COVID, there is a possibility for human infection by the virus and then mutation of that virus, and then of course there are all the susceptible newborns to follow. People who are not immune to variants of the virus may serve as its reservoirs but, worse, replication of the virus sooner or later results in production of mutants that are somewhere between slightly different to astonishingly different from the parental  virus, whatever that was. If all humans and other vertebrates were infected and immune to this virus the process would end. That is unlikely to occur, so we now race to prevent random, unpredicted mutations. But how can we predict the unpredictable?The bad news is that we await the time when SARS-CoV-2 becomes less pathogenic and more endemic. One piece of good news for some, particularly students, is that all this unknowing provides job opportunities for young people interested in virology, in vaccine manufacturing, in viral ecology, and in much more. Henry David Thoreau said, “People talk about Bible miracles because there is no miracle in their lives. Cease to gnaw the crust. There is ripe fruit over your head.” This epidemic is an example of such “ripe fruit;” the prevention of or response to the occurrence of random mutations. Thus far, there is no evidence that we can prevent anything that is random, so how might we begin suppressing random mutations? Burke  has specWhat is the origin of SARS-CoV-2? This is the focus of political and virological efforts and arguments. The answer may matter politically but not virologically. We have survived nicely without knowing the definitive origin of yellow fever virus, of tick-borne encephalitis virus(es), of hepatitis A virus, of smallpox virus. These are questions the answers to which would build very nice careers for the person or people who find them but, at this point, few care and, really, at this late date, it does not matter epidemiologically.Students, or others who want to change careers, might find that the current agonies offer a rare opportunity. Jobs now in existence but declining as opportunity: coal miner, taxi driver, surveyor or mapping technicians, print media, herder, travel agent, even diagnosticians; these occupations are or will soon be replaced by automated machinery  and the like.Surely many governmental and non-governmental organizations soon will awaken and be pouring funds into even more research regarding virus evolution and ecology. There being no real rush, it might even be a good idea if students would take an extra year of pre-graduation studies to become more informed about the humanities, geology, psychology, writing, Great Books readings, logic, etymology, cooking, accounting and personal finance, world history, physical education, political science, statistics, gardening, or even taxonomy. These would provide opportunities for the student to become a well-rounded person, one with an education, rather than one who is trained. As importantly, graduate work in virology, evolution, ecology, and other specialties would prepare one for a well-paying job and opportunities to travel to places you have never wanted to visit and to eat food you never wanted to eat.https://www.youtube.com/watch?v=FVsbvFkhzY4.It will cost a large fortune to rebuild Ukraine and part of the funds surely will go to rebuilding schools, laboratories, places for administrators to sit, and grocery stores, as well as libraries, restaurants, and petrol stations. Of course, if all else fails, everyone is welcome to come to the USA, arguably the largest lunatic asylum in the world. Start preparing by listening to the Eagles:


Development of efficient virus aerosol monitoring and removal devices requires aerosolization of the test virus using atomizers. The number concentration and size measurements of aerosolized virus particles are required to evaluate the performance of the devices. Although diffusion dryers can remove water droplets generated using atomizers, they often fail to remove them entirely from the air stream. Consequently, particle measurement devices, such as scanning mobility particle sizer (SMPS), can falsely identify the remaining nanosized water droplets as virus aerosol particles. This in turn affects the accuracy of the evaluation of devices for sampling or removing virus aerosol particles. In this study, a plaque-forming assay combined with SMPS measurement was used to evaluate sufficient drying conditions. We proposed an empirical equation to determine the total number concentration of aerosolized particles measured using the SMPS as a function of the carrier air flow rate and residence time of the particles in the diffusion dryers. The difference in the total number concentration of particles under sufficient and insufficient diffusion drying conditions was presented as a percentage of error. Developing new devices and techniques for applications in an ambient environment requires numerous tests and accurate device performance evaluations in controlled environments.Virus aerosol exposure is associated with adverse health effects. Characterizing and quantifying airborne pathogens is crucial to exposure and risk assessments. Various aerosol instrument systems, such as air samplers and antimicrobial filtering devices, have been developed3. The air flow rate (AFR), particle concentration in the liquid solution of the atomizer, and geometry of the atomizer affect aerosolized particle generation rate4. Since most aerosol instruments are sensitive to moisture, a series of diffusion dryers consisting of a circular tube with absorbing walls surrounded by silica-gel desiccant beads are used to remove moisture. Excess moisture within an aerosol moves into the dry environment due to diffusion. This leads to dehumidification, which is an adsorption process that results from the pressure difference of water vapor between the desiccant surface and surrounding air6. Hence, the residence time, dimensions of the dryer, and AFR, which is utilized in optimizing aerosol drying8 govern the design of a diffusion dryer.An airblast atomizer aerosolizes virus particles suspended in a liquid solution. When compressed air enters the atomizer, the pressure from the air jet promotes aerosolization of the water droplets containing virus particles10, then SMPS data can be incorrectly interpreted and used in calculations. Inadequate moisture removal interferes with the measurement of virus aerosol particles if the moisture particles are within the same size range as the pure water droplets generated.During aerosolization experiments, the aerosol number concentration is measured using a particle measurement device, such as a scanning mobility particle sizer (SMPS). If the diffusion dryers do not entirely remove moisture from the airstreamThe size distribution and total number concentration of aerosol particles generated from solutions with and without virus particles were measured for different AFRs and residence times (τ) in the diffusion dryers. Plaque-forming assay data and SMPS data were used to determine sufficient drying conditions. The difference in the total number concentrations of aerosolized particles at sufficient and insufficient drying conditions was presented as a percentage of the error. Further, we proposed an empirical equation to determine total number concentration of aerosolized particles.Experiments were performed using an atomizer solution without virus particles. Path A of Fig. –5 (min m3/L s).The residence time, τ, of the air flow in the diffusion dryers calculated using the following equation:Figure 4 cm–3 (τ = 2.64 s), 3 × 104 cm–3 (τ = 4.90 s), and 1.6 × 104 cm–3 (τ = 7.16 s), which correspond to 60%, 80%, and 89% decrease in total number concentration, respectively. Table The details of the results in Fig. –3) and decay constant (1/s), respectively; both are linearly proportional to the flow rate:5 min/L cm3 and 0.251 min/L s, respectively. The results are listed in Table Conversely, for each case Table , the totτ = 0 cm– and decaThe test results for the MS2 bacteriophage aerosols are shown in Fig. 6 plaque forming units per milliliter (PFU/mL) for each case , the error in the total number concentration measurement is approximately 151% when considering 1.8 × 105 cm−3  is evaluated via their collection efficiencies, which are calculated using the aerosolized virus number concentration. This can be measured upstream and downstream of a device by using particle counters such as SMPS16. Given that many of the routine laboratory procedures used to study and process virus samples  exhibit high potential for producing aerosols18, it is important to adopt strict safety precautions when artificially aerosolizing virus particles using atomizers. Hence, it is preferable to generate aerosolized viruses at low concentrations to reduce health risks19 and mimic the actual environment in which airborne virus particles are present as low concentrations21. Several studies indicate that SMPS results are affected by particle shape and morphology and by the upper and lower size limits set by the instrument22. It is well known that the performance of SMPS may vary with respect to each particle size23. Most atomization processes generate virus particles of interest along with nanosized water droplets that can be mistakenly detected by an SMPS as virus particles. Along with Fig. In recent years, there has been an increasing interest in air monitoring and aerosol measurements. Specifically, numerous studies have examined airborne virus transmission routes during the current COVID-19 pandemic as well as continuously emerging virus mutations, which facilitate virus infection even at low concentrations3 to 1.2 × 106 cm−3 when using different AFRs and residence times , whereas the total number concentration measured by the SMPS decreased with increasing τ and was maintained constant (1.8 × 105 virus particles per 1 cm3 of air volume) for τ > 7.16 s  of each diffusion dryer for 2 L/min AFR was calculated with the following equation. The results are presented in Fig. Whether diffusion loss could affect the decrease in nanoparticle concentration, the following Gormley and Kennedy equation (Eq. 6)mentclass2pt{minimThe diffusional particle loss of Case 4 was less than 10% for 20 nm particle size and decreased to almost zero with the increase of diameter see Fig. , it is aThe percentage of error calculated using Eq.  indicateTherefore, to increase the data accuracy from number concentration measurements and reduce uncertainties, we suggest using sufficient diffusion residence times, thus realizing maximum droplet removal when using the atomization method. Furthermore, another approach can be utilized, which involves diluting the air stream exiting from the atomizer with a clean air stream, thus realizing a lower air RH. In this study, the effect of nano droplet generation and diffusion drying was investigated for pressure type atomization. For other methods such as electrostatic and ultrasonic/sonic (whistle)-based atomization, further study is needed to explore the droplet generation characteristics and appropriate diffusion drying conditions.Sterile ultrapure DNase/RNase-Free, Protease-Free deionized (DI) water  (50 mL) was poured into a conventional atomizer  and aerosolized according to the manufacturer’s protocols. Clean air with an AFR of 2–5 L/min was directed into the atomizer. The AFR was controlled using a mass flow meter . Subsequently, the moisture and charge of the aerosol particles were removed via a series of diffusion dryers and an aerosol charge neutralizer . A freshly replaced adsorbent was used in each experiment .The size distribution of the aerosolized water droplets was measured using an SMPS . The SMPS consists of a classifier controller , condensation particle counter , differential mobility analyzer , and an aerosol charge neutralizer . The RH for each experiment was measured using a capacitive humidity sensor . A schematic of the experimental design is shown in Fig. 26. The MS2 bacteriophage uses Escherichia coli as a host for reproduction. An aliquot of the MS2 bacteriophage virus  solution (10–100 μL) was added to 50 mL of sterilized DI water. Different concentrations of MS2 bacteriophage (5.14 × 103–4.11 × 104 PFU/mL) were tested. Each virus solution was transferred to a TSI atomizer. The air was then directed into the atomizer at a flow rate of 2 L/min, thereby creating an aerosolized mixture of water and viruses. Subsequently, the air flow passed through a series of diffusion dryers for pure water droplet removal.The experiments described above were repeated using an atomizer containing a virus solution. The MS2 bacteriophage was selected because it is not pathogenic to humans and is similar in structure and function to many human enterovirusesThe size distribution and total number concentration of the aerosolized particles were measured via SMPS. The aerosolized particles were sampled on a Sartorius gelatin filter  with a diameter of 25 mm using an SKC Button aerosol sampler . The sampling time was set to 5 min, and the experiments were conducted in triplicate. Immediately after sampling, the gelatin filter was removed from the SKC Button sampler and immersed in 10 mL of sterile DI water for virus recovery, and the virus sample solution was used for plaque assay analysis. A schematic of the experimental design is shown in Fig. E. coli C-3000 (ATCC 15597) hosted bacteria and 29 mL of tryptic soy agar . The mixture was poured into a Petri dish and incubated at 37 °C under aerobic conditions in an atmosphere of 95% RH for 24 h. The viral PFU number was then counted. Finally, the following equation was used to calculate the concentration of infectious virus particles (PFU/mL):p denotes the average number of plaques counted, D denotes the number of dilutions, and V denotes the volume of the virus solution. Further details are provided in Fig. In the study, PFU/mL concentrations were obtained after serial dilution of the sampled virus particles. Briefly, each gelatin filter containing the sampled virus was dissolved in 10 mL of DI water. Next, 0.1 mL of the serially diluted virus solutions were mixed with 0.3 mL of the All experiments were performed in triplicate, as indicated in the figure legends. Data are expressed as the mean ± standard error of the mean. Statistical analyses were performed using GraphPad Prism version 8.Supplementary Information.


The avian leukemia virus causes avian leukemia (AL), a severe immunosuppressive disease in chickens (ALV). Since the 1990s, the diversity of ALV subpopulations caused by ALV genome variation and recombination, and the complexity of the infection and transmission, with currently no effective commercial vaccine and therapeutic for ALV, has resulted in severe economic losses to the chicken business in various parts of the world. Therefore, as a key means of prevention and control, an effective, rapid, and accurate detection method is imperative. A new real-time reverse transcription recombinase-aided amplification (RT-RAA) assay for ALV with rapid, highly specific, low-cost, and simple operational characteristics have been developed in this study. Based on the amplification of 114 base pairs from the ALV P12 gene, real-time RT-RAA primers and a probe were designed for this study. The lowest detection line was 10 copies of ALV RNA molecules per response, which could be carried out at 39°C in as fastest as 5 min and completed in 30 min, with no cross-reactivity with Marek's disease virus, avian reticuloendothelial virus, Newcastle disease virus, infectious bronchitis virus, infectious bursal disease virus, infectious laryngotracheitis virus, and avian influenza virus. Furthermore, the kappa value of 0.91 (>0.81) was compared with reverse transcription–polymerase chain reaction (RT-PCR) for 44 clinical samples, and the coefficients of variation were within 5.18% of the repeated assays with three low-level concentration gradients. These results indicate that using a real-time RT-RAA assay to detect ALV could be a valuable method. Retroviridae  belong to the family type C Most infected chickens show subclinical symptoms on farms, such as decreased egg production, immunosuppression, or growth retardation Witter, . The ALVIn China, exogenous ALV infection was dramatically reduced at major representative breeding farms when the Nationwide Eradication Program (NEP) was launched in 2008 , disease pathogens, and other fields. For instance, researchers had applied the RAA detection in zoonosis of L37202) vaccine . Wens Foodstuff Group Co., Ltd supplied the avian reticuloendotheliosis virus  . Newcastle disease virus , infectious bronchitis virus , infectious bursal disease virus , infectious laryngotracheitis virus , subgroup A avian leukemia virus , subgroup J avian leukemia virus , subgroup K avian leukemia virus , subgroup H9N2 avian influenza virus , and chicken infectious anemia virus  were stored in our laboratory. The P12 gene of subgroup B avian leukemia virus , subgroup C avian leukemia virus , subgroup D avian leukemia virus , and subgroup E avian leukemia virus  were synthesized by Sangon Biotech (Shanghai) Co., Ltd. .Harbin Pharmaceutical Group Bio-Vaccine Co., Ltd provided the Marek's disease virus . The cells were inoculated with 44 clinical plasma samples of suspected ALV infection provided by Guangdong Ihealth Biotechnology Co., Ltd  after they grew over 80%. After 7 days, we collected culture supernatant fluid.Cell culture DF-1 cells were purchased from the American Type Culture Collection. The cells were cultured in Dulbecco's modified Eagle medium  containing 10% fetal bovine serum  and 1% penicillin/streptomycin  at 37°C with 5% COTM19-T Vector Cloning Kit . In this investigation, a real-time fluorescence quantitative PCR apparatus  was employed.Guangzhou Suyuan Biotechnology Co., Ltd.  provided the AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit. The RT-fluorescence nucleic acid amplification reagent (RAA method) was purchased from Nanning Zhuangbo Bio & Tech Co., Ltd. . Guangzhou Qiyun Bio & Tech Co., Ltd. provided the pMDRecombinase was bound to primers to form complexes, and the complexes searched for a specific binding site on the target DNA at a constant temperature. By utilizing single-stranded binding proteins, the double strand of template DNA is opened, and a single-stranded molecule is formed. In the next step, DNA polymerase was initiated at the 3′ end of the primer and synthesized toward the 5′ end of the target DNA. Hence, two new double-stranded DNA molecules were formed, and the cycle was repeated to achieve amplification  and used DNAMAN  and DNASTAR  to analyze and align multiple sequences. Based on the sequence alignment results, we designed primers and probes (Z46390) of the P12 gene. PCR primers . After extraction, the concentration of this plasmid was evaluated, and the copy number of the ALV P12 gene was calculated to be around 2.41 × 1010 copies/μl.Targeted P12  gene fragments of ALV were amplified by the PCR primers and are shown in We extracted MDV, CAV, IBV, NDV, IBDV, H9N2, ILTV, REV, and the 44 culture supernatant fluid samples using the AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit . The nucleic acids were then stored in the ultralow-temperature freezer at −80°C in our laboratory.2O. The RT-PCR system required RAA-F (10 μM) 1.0 μl, RAA-R (10 μM) 1.0 μl, RNase-free ddH2O 5.0 μl, One Step Enzyme Mix 1 μl, 2 One Step Mix 10.0 μl, and RNA 2 μl. The RT-PCR was performed at 50°C for 30 min, 94°C for 3 min, 94°C for 10 s, 58°C for 30 s, 35 cycles, and 72°C for 5 min. On a 1.5 percent agarose gel, the RT-PCR products were analyzed.In testing the size of the real-time RAA primers, the positive control was ALV nucleic acid, and the negative control was ddH2O and 2–5 μl of templates. The fluorescence signals were acquired after one cycle of 39°C for 60 s and 40 cycles of 39°C for 30 s of real-time RT-RAA. After the reaction, the positive control had a smooth amplification curve, and the Ct value was ≤39; the negative control had no amplification curve, or the Ct value was >39, showing the assay had valid results.The real-time RT-RAA reaction system is made up of the following components: RAA-F (10 μM) 2.0 μl, RAA-R (10 μM) 2.0 μl, RAA-P (10 μM), 2.0 μl, RT-RAA reaction unit 1 tube, A buffer 25 μl, and B buffer 2.5 μl. The total volume was 50 μl, with 12.9–15.9 μl of ddH2O. We employed blood nucleic acid of SPF chickens as the dilution solution of the standard plasmid (complex plasmid solutions) of ALV P12 to more effectively apply to clinical sample detection. A total of six concentration gradients served as templates, which were 105 copies/μl, 104 copies/μl, 103 copies/μl, 102 copies/μl, 101 copies/μl, and 100 copies/μl. The sensitivity assays of two different dilution methods were then repeated three times.Serial dilutions of the standard plasmid of ALV P12 were carried out 10 times by ddHThe nucleic acid of ALV-A, ALV-B, ALV-C, ALV-D, ALV-E, ALV-J, and ALV-K was a positive control and ddH2O was a negative control. For specificity analysis of MDV, CAV, IBV, NDV, IBDV, H9N2, ILTV, and REV nucleic acids, 2 μl of templates were obtained and repeated three times.3 copies/μl, 102 copies/μl, and 101 copies/μl were taken at 5 μl, and repeating assays were three times respectively.The complex solution plasmids and standard plasmids dilution gradients were 10Nucleic acids from the 44 culture supernatant fluid samples suspected of being infected with ALV were used to detect by RT-PCR and real-time RT-RAA. We compared and analyzed the results of RT-PCR and real-time RT-RAA to evaluate the method we established in this study.A PCR experiment was conducted to verify the size of the real-time RAA primers. The results are shown in 1 copies/μl .The detection time of real-time RT-RAA was as fast as 5 min, and within 30 min the assay was completed, which could specifically detect ALV-A, B, C, D, E, J, and K, and without cross-reaction with MDV, CAV, IBV, NDV, IBDV, H9N2, ILTV, and REV. Because this method can be combined with a portable GENCHEK fluorescence detector, it improves analysis reliability in complex and poor environments. The reported assay reliably has a limit of detection of approximately 10 copies of the virus per reaction both in pure standard plasmids and complex plasmid solutions, with dilutions ranging from approximately 10In reality, most breeding industries choose ELISA when performing NEP because it does not need a specific environment and complex operation. However, the accuracy of the result needs to be improved .XW conducted the experiments, analyzed the data, and wrote the manuscript. FChu, SC, HZ, and HH conducted the experiments. LZ, LG, JW, and MC analyzed part of the data. FChe and ZX checked and finalized the manuscript. QX and XW designed the experiments, modified the manuscript, and supervised the whole work. All authors read and approved the final manuscript.This work was funded by the Key Research and Development Program of Guangdong Province (grant number 2020B020222001), the National Natural Science Foundation of China (grant numbers 31902252 and 31972659), Guangdong Basic Research Foundation , the Chief Expert Project of Agricultural Industry Technology System in Guangdong Province (grant numbers 2019KJ128 and 2020LJ128), and the Special Project of National Modern Agricultural Industrial Technology System (grant number CARS-41).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Autophagy, a conserved lysosomal degradation pathway, can degrade intracellular waste, such as damaged organelles or misfolded proteins, to meet the metabolic needs of cells and maintain the homeostasis of the intracellular environment . In addivia endocytosis and thus remains enclosed in the endosomes during the early stages of infection, making it difficult to identify by the host cellular autophagy. There is unknown how the virus can be specifically identified as selective autophagy cargo so as to be eliminated by host cellular autophagy and how does virus-induced autophagy occur in mammalian cells. Recently, Dong et al. found that the endosomal protein sorting nexin 5 (SNX5) is critically required for virus-induced, but not for other factor induced autophagy  domain and carboxy-terminal Bin, Amphiphysin, Rvs (BAR) domain, both of which are evolutionarily conserved domains. PX domain mainly recognizes the sorting motif of cargo (proteins to be sorted), and can help SNX5 to localize to the endosomal membrane of endocytosis pathway , 7. SNX via an endocytic vesicle. Only a small number of viruses infect host cells by means of genetic material injection, such as phages, and the targeted cells are generally cells with the cell wall and outer membrane  mice were more susceptible to lethal infection with SIN, Chikungunya virus (CHIKV), or West Nile virus (WNV) virus than their wild-type littermates (Snx5+/+) (in vivo (Snx5+/+) . Whereas, they will get some cues, such as exposure to low pH or proteolytic cleavage, which will trigger changes in the virus particle, and the activated viruses can penetrate the vacuolar membrane, enter the cytoplasm or nucleus to replicate their genetic material, synthesize virus protein, and so on . The cytAlthough, SNX5 is essential for virus-induced autophagy and can inhibit the transmission of the virus and reduce the harm of the virus to the host. It may be a double-edged sword for the hosts, considering that some viruses may kidnap the host’s autophagy process to serve its replication and transmission. Therefore, we should take this into account when we want to target SNX5 to against the virus. Our understanding of SNX5 in virus-induced autophagy is far from enough. For example, how does SNX5 only recognize endosomes that contain virus particles rather than other substances? In the future, it will be interesting to investigate how SNX5 can specifically “recognizes” virus-infected endosomes and promote the activity of autophagy. This will be an important step towards understanding how specificity is achieved in selective autophagy and how can we use this to against and to combat the spread of the virus, thereby reducing its damage to the host.D-YL, J-HW, SL, and J-XT wrote the first draft of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by Natural Science Foundation of Guangdong Province (2019A1515110152) and Discipline construction project of Guangdong Medical University (4SG21229G).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Eyelash trichomegaly is a rare disorder in which normal features of eyelashes such as length, color, thickness, or curling changes. It may occur due to many causes, such as the presence of other disorders such as HIV infection, congenital anomalies like oculocutaneous albinism, or Oliver–McFarlane syndrome. It may be linked to the use of certain drugs and can also be present as an isolated trait by birth. Here, we report a rare case of eyelash trichomegaly in a 19-year-old female having diffused alopecia, diagnosed with systemic lupus erythematosus. Eyelash trichomegaly is defined as a condition in which eyelashes exceed their normal length (12 mm or more), curling, pigmentation, or thickness [Gray, in 1944, for the first time, used the term “trichomegaly” while observing a lymphoma patient with abnormally long eyelashes . This diHere, we present a case of eyelash trichomegaly associated with systemic lupus erythematosus. Hair loss in a diffused pattern is also present in the patient. All information presented here were obtained with the permission of the patient's parents.For nine months, a Jordanian girl, 19 years old , had typHer medical record for the last six months showed that she had taken no medicine except prednisolone orally (20 mg/day). Her family history shows no cases of hypertrichosis. Mild signs and symptoms such as erythema in the paranasal region and diffused hair loss in the scalp were discovered during the examination . Trichomegaly was observed as her eyelashes measured 17 mm long .3, ESR 72 mm/h, and the number of platelets present were 105,000/mm3 indicative of thrombocytopenia, which were all found on her hematological reports. No proteins or casts were seen in the urine examination. Antinuclear antibodies  and anti-dsDNA antibodies were detected. The patient was diagnosed with SLE and was directed to consult a rheumatologist for treatment.Hb 9.8 g/dL, TLC 2800/mmth week of gestation. Their growth completes in a cycle of three stages in six months: anagen phase (30 days), catagen phase (15 days), and telogen phase (over 100 days).During development at embryonic stages, eyelashes are the first hair to grow. They appear roughly around the 12The normal length of eyelashes of the eyelid is 8–12 mm. An increase of this length or thickening, curling, and pigmentation is known to be eyelash trichomegaly [It may also show up as an isolated benign family feature in rare cases. These changes also develop as a characteristic feature of other syndromes developed later in life, such as atopic dermatitis, uveitis, vernal keratoconjunctivitis, and HIV infection. In rare cases, it may also be associated with other acquired syndromes such as alopecia areata, metastatic renal adenocarcinoma, dermatomyositis, and systemic lupus erythematosus.Certain drugs are also key players in the development of these changes. The drugs to which this condition is linked most commonly include cyclosporine, inhibitors of epidermal growth factor receptor (EGFR), cetuximab, and frequent use of prostaglandin analogs such as latanoprost and travoprost , 4 Tabl.SLE is a chronic autoimmune disease involving multiple systems. The autoantibodies which are produced cause a wide spectrum of medical complications. Hair growth patterns change as a result of SLE. In the classification criteria, nonscarring alopecia has also been included . DiffuseThe pathophysiology of eyelash trichomegaly has been inadequately explored because of a lack of histology and organ culture data for eyelash hair. A molecular mechanism for trichomegaly development has been proposed. According to this theory, stem cell inactivation in different regions of the hair follicle by the nuclear factor of activated T cells (NFAT) may lead to eyelash trichomegaly. EGFR regulates the growth cycle of eyelash hairs, and its inhibition may lead to trichomegaly .In this report, we document a case of trichomegaly associated with SLE along with diffused hair loss. The development of eyelash trichomegaly in our patient may have been caused by EGFR inhibition.


Astroviridae, Circoviridae, Coronaviridae, Dicistroviridae, Herpesviridae and Retroviridae were detected, with unclassified viruses. Retroviral sequences were prevalent; 74.1% of all samples were positive, with distinct correlations between virus, site and host bat species. Detected retroviruses comprised Myotis myotis, Myotis ricketti, Myotis daubentonii and Galidia endogenous retroviruses, murine leukemia virus-related virus and Rhinolophus ferrumequinum retrovirus (RFRV). A near-complete genome of a local RFRV strain with identical genome organization and 2.8% nucleotide divergence from the prototype isolate was characterized. Bat coronavirus sequences were detected with a prevalence of 24.1%, where analyses on the ORF1ab region revealed a novel alphacoronavirus lineage. Astrovirus sequences were detected in 25.9%of all samples, with considerable diversity. In 9.2% of the samples, other viruses including Actinidia yellowing virus 2, bat betaherpesvirus, Bole tick virus 4, Cyclovirus and Rhopalosiphum padi virus were identified. Conclusions: Further monitoring of bats across Kenya is essential to facilitate early recognition of possibly emergent zoonotic viruses.Background and Methods: To investigate virus diversity in hot zones of probable pathogen spillover, 54 oral-fecal swabs were processed from five bat species collected from three cave systems in Kenya, using metagenome sequencing. Results: Viruses belonging to the  Spillover of a pathogen from the original source to intermediate or definitive recipient hosts often precede the onset of emerging infections . For virBats are globally distributed mammals with high diversification ,4. They Metagenome sequencing allows for an unbiased characterization of target nucleic acids in the sample and has become a powerful tool for identifying known and novel pathogens, especially viruses, in a wide range of samples ,14. It aWith highly diverse geography and climate, Kenya harbors a large diversity of wildlife species with potential as virus reservoirs, including several bat species ,16. PrevAs part of a collaboration between Maasai Mara University. Smithsonian Institution and the Institute of Primate Research of the National Museums of Kenya, bat samples were obtained from species captured in three cave systems in Kenya’s Rift Valley in 2020. The collection sites comprised the religious cave at Menengai Crater, an abandoned diatomite mine at Soysambu Conservancy and the volcanic tunnels and caves at Mount Suswa . The bats were captured using mist and hand nets. The 6- and 12-m nylon mist nets were deployed in probable flyways and monitored continuously, typically from dusk to about midnight. Mist nets are useful in identifying areas used by bats in foraging or commuting while hand nets are best employed to capture bats that roosts in caves, mines, and tunnels. The latter method is particularly informative as it sheds light on the social groupings as well as roosting preferences of bats. Captured bats were placed in individual cloth bags (to prevent cross-contamination) and transported to preparation areas, where they were examined, measured and identified to species by a trained field biologist. Subsequently, oral and rectal swabbing were performed in compliance with field protocol and the samples were transported in Trizol  on dry ice from the field to −80 °C storage before further processing. Oral and rectal swabs from individual bats were pooled prior to further processing. Sampled bats were released following sample collection. No bats were excluded from the study. The study protocol was approved by the National Zoological Park Institutional Animal Care and Use Committee (NZP-IACUC #20-02) and by Kenya Wildlife Service. Sample collection, experiments and reporting of the results follow the recommendations described in the ARRIVE guidelines. All collection, sampling and subsequent experiments were performed in accordance with these guidelines and approved protocols.Nucleic acid purification was conducted using the Direct-zol RNA MiniPrep Kits (Zymo Research USA) at IPR and purified nucleic acids were converted to complementary DNA (cDNA) using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific), according to the manufacturers’ recommendations. The cDNA was quantified using a NanoDropTM 2000/2000c Spectrophotometer (Thermofisher Scientific) and samples were shipped to the Walter Reed Biosystematics Unit  for second strand cDNA synthesis, library preparation and further downstream processing. Double-stranded cDNA was prepared using NEBNext Non-Directional RNA Second Strand Synthesis module, utilizing random primer mix  according to manufacturer recommendations. Library preparations were carried out using KAPA HyperPlus Kits , with a fragmentation step of 20 mins at 35 °C, ligation step of 90 mins and 20 cycles of amplification, as recommended by the manufacturer. Library quantification and quality control were performed using the TapeStation 4200 Automated Electrophoresis instrument . Excess adapters and fragments were removed using KAPA pure beads . Unbiased metagenomic sequencing was performed on the NovaSeq platform  (PE 2 × 150) at the Walter Reed Army Institute of Research (WRAIR), Silver Spring, MD, USA. Samples were run on one lane of a S4 flow cell with XP workflow, designed to maximize the overall raw reads output. Initial read quality assessment was carried out using fastqc .https://www.czid.org, accessed on 17 November 2022). Initial data analysis was performed using the built-in opensource pipeline (version 6.8), incorporating steps for validation, host and adaptor removal, quality assessment, alignment, assembly and taxonomic identification using National Center for Biotechnology Information (NCBI) nucleotide and protein databases. Briefly, adapter trimming and host filtration in different steps were carried out using Trimmomatic, STAR, Bowtie2 and GSNAP . Da. DaTadarWe detected astrovirus sequences in 14 bats (25.9%) collected at Menengai Crater and Soysambu Conservancy, but not at Mount Suswa . AstroviRhopalosiphum padi virus, bat betaherpesvirus B7D8, Bole tick virus 4 and Actinidia yellowing virus 2 . A partial sequence of the helicase/primase gene from bat betaherpesvirus was detected at Soysambu Conservancy, with 81.6% identity to B7D8 that was previously reported in Miniopterus schreibersii from Australia [We further detected virus sequences belonging to different families or unclassified viruses in five bat samples (9.2%) collected from the Menengai Crater and Soysambu Conservancy . These s virus 2 . A partiustralia . Virus sWe screened oral-rectal swabs obtained from individual bats from locations in Kenya’s Rift Valley, using metagenome sequencing. We targeted three bat dwelling sites with human and non-human primate hosts activity, illustrating a potential interface for pathogen spillover. The bat species documented in the study are widespread in Kenya and occur elsewhere outside the Rift Valley, in other roosting structures such as natural and coral caves, and in volcanic tubes and tunnels ,16. In tO. harrisoni bats at Mount Suswa. The sequences corresponded to the ORF1ab region of the coronavirus genome, encoding for the enzymes and accessory proteins required for virus genome replication. Phylogenetic analyses identified the sequences as a distinct alphacoronavirus lineage. High amino acid identities (>97%) further suggest these viruses to comprise a single alphacoronavirus species according to the current International Committee on Taxonomy of Viruses (ICTV) criteria, that set a demarcation threshold of 90% on conserved ORF1ab domains [Alphacoronavirus and Betacoronavirus genera [Chaerephon, Miniopterus and Rousettus species [O. harrisoni samples, which indicated structural regions of the novel bat alphacoronavirus to be significantly conserved among viruses from different individuals. The presence of the ORFx further suggested tropism for bats. Our findings contribute and expand on the observations that the African continent harbors an extensive bat coronavirus diversity. Hence, potential spillover of this coronavirus from bats to other susceptible vertebrate species must be closely monitored. We identified a variety of RNA and DNA viruses in the samples. Bat coronavirus sequences were detected with an overall prevalence of 24.1%, mainly in  domains . Previous genera ,21. Inte species ,21. We fMyotis spp. [M. tricolor bats examined. At Soysambu Conservancy, sequences related to RFRV were recovered in all R. landeri specimens as well as in M. natalensis and M. africanus samples, albeit with a lower prevalence. RFRV was initially identified by transcriptome sequencing of the brain tissues from a greater horseshoe bat (Rhinolophus ferrumequinum) [R. ferrumequinum samples as well as other bat species led to the assumption that RFRV may be present in a subset of the host gene pool and not yet fixed in the bat population [R. landeri samples clearly suggest a vertical route in this study, the lower prevalence observed in non-Rhinolophus bat species may be due to ongoing or recent horizontal infections. It remains to be determined whether any other vertebrates in the region carry RFRV-Kenya. We observed retroviruses as the most frequently detected viruses at all sites. At Menengai Crater, sequences closely related to those previously reported from different tis spp. ,42, wereequinum) . The neapulation . Therefopulation . Genome-pulation . While dGalidia elegans)—native to Madagascar—with bats implicated in spread [O. harrisoni samples herein is in line with pan-phylogenomic analyses indicating bats and rodents as major sources of origin and transmission of retroviruses to other mammals, significantly contributing to their spread and evolution [At Mount Suswa, we further recovered two retrovirus sequences (GERV and MLRV-like) in high prevalence and with frequent co-detection. Related to reticuloendotheliosis viruses, a group of amphotrophic bird retroviruses, GERV was described in tissues of ring-tailed mongooses  [Rhopalosiphum padi virus has previously been detected in bat guano from Hungary along with bee viral pathogens [Flaviviridae family [Finally, we detected various DNA and RNA viruses in samples from Menengai Crater and Soysambu Conservancy, including a bat betaherpesvirus. Bats distributed on every continent host a variety of herpesvirus sequences , presumai virus) , ticks (i virus)  and plani virus) , suggestathogens . Originae family . Recent e family . The same family . Similare family . TherefoO. harrisoni bats. Astroviruses were initially documented in bats from Kenya. Each sampling site was characterized by a distinct retrovirus genome signature, associated with different bat species. Further monitoring of similar sites is recommended to facilitate early recognition of emerging or re-emerging viruses. We detected a broad spectrum of viruses and described a novel coronavirus lineage in bat samples collected from locations with spillover potential. A novel alphacoronavirus is reported in


Transmissible gastroenteritis virus (TGEV) is a member of the alphacoronavirus genus, which has caused huge threats and losses to pig husbandry with a 100% mortality in infected piglets. TGEV is observed to be recombining and evolving unstoppably in recent years, with some of these recombinant strains spreading across species, which makes the detection and prevention of TGEV more complex. This paper reviews and discusses the basic biological properties of TGEV, factors affecting virulence, viral receptors, and the latest research advances in TGEV infection-induced apoptosis and autophagy to improve understanding of the current status of TGEV and related research processes. We also highlight a possible risk of TGEV being zoonotic, which could be evidenced by the detection of CCoV-HuPn-2018 in humans. In the past two decades, the continuous appearance of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2, swine acute diarrhea syndrome coronavirus (SADS-CoV), and porcine deltacoronavirus (PDCoV) warns us that the coronavirus is still one of the biggest threats to human and livestock health ,2,3,4,5.TGEV has made entirely new advances in the wave of research on various coronaviruses ,17,18,19TGEV is an alphacoronavirus with a positive-strand RNA genome of ~28.5 kb ,22. The TGEV was first reported in the United States in 1946 , and sinIn addition to the two classical TGEV subtypes, there is another related non-enteric pathogenic TGEV recombinant virus, porcine respiratory coronavirus (PRCV) ,69. PRCVThe lethality rate of TGEV in piglets is almost as high as 100%, and it has caused huge losses to the breeding industry in Europe, Asia, America, and other places ,62,63,64The transmission route of TGEV recombinant virus PRCV is similar to TGEV, and the main infection routes are the fecal–oral route and respiratory route through aerosol ,84,85. PThe detection rate of TGEV has declined in the decades following the outbreak due to the continuous development of means against TGEV, such as vaccines ,90, and The S protein is involved in receptor recognition and membrane fusion, delivering the nucleocapsid into host cells, which plays an important role in the virulence of the virus itself, tissue tropism in the host or cell tropism in vitro, and the pathogenesis of coronaviruses ,95,96,97We could observe a classical and attenuating mutation that is considered to be typical of the Purdue strain by summarizing some of the classic virulent and attenuated strains  66,67].,67.66,67Strains carrying ORF3 −75–−60nt and 195–223nt deletion are classic Miller strains, but the deletion of these sites has not been reported to be associated with virulence. There is an attenuated strain, Miller M60, that is missing 531nt in ORF3b in the Miller strain , and itsThe factors that affect the virulence of TGEV are far more than only caused by deletion. We also describe the effect of point mutations on the S gene on virus virulence . PRCV haThe nt1753 base of the S protein of the firstly isolated Purdue strain is thymine, and the strains carrying T at this site, such as Purdue, TS, Miller M6, and JS2012, are all virulent strains . HoweverNot limited to point mutations in the S protein, Carmen et al. showed that G637A (Gly108Asp) in ORF1 of TGEV affects papain-like protease-mediated cleavage in vitro, resulting in virus attenuation .The TGEV receptor is porcine APN (pAPN) . APN is These alphacoronaviruses mentioned above specifically recognize the APNs of their respective host cells through triglycan proteins on the virus surface . AfterwaInterestingly, the relationship between these alphacoronaviruses and APNs is not a one-to-one correspondence between viruses and animals. This means these viruses can bind to APNs from other species. FAPN is capable of being infected by a virus from different terminal hosts: FCoV from cats, TGEV from pigs, HCoV-229E from humans, and CCoV from dogs ,113. ThiAPN is also not limited to alphacoronavirus-related receptor binding. A recent study on deltacoronaviruses showed that PDCoV can bind to pAPN through the C-terminal domain (CTD) of the S protein and use pAPN for cell entry . InteresBesides the classical TGEV receptor pAPN, the non-protein host factor sialic acid also acts as a receptor for binding to TGEV. Sialic acid is an acidic compound with a nine-carbon skeleton that can be used by the four coronaviruses of the alpha-, beta-, gamma-, and deltacoronavirus to bind and enter host cells ,133,134.Unlike TGEVs, which bind to extracellular receptors using S1-CTD, TGEVs bind to sialic acid through their S1-N terminal domain (NTD) . This abTGEV invades the small intestinal epithelium after infection, causing small intestinal villous atrophy and severe diarrhea, which are central events of piglet death caused by infection. This process is closely related to apoptosis. Apoptosis is a process of programmed cell death regulated by genes, which removes damaged or virus-infected cells and plays an important role in viral infectious diseases. Apoptosis pathways are mainly divided into the mitochondrial pathway (intrinsic pathway) and the death receptor pathway (extrinsic pathway) ,140. We The intrinsic apoptotic pathway is started by changes in mitochondrial membrane permeability, releasing cytochrome c (Cyt c) to activate caspase-9, and then caspase-9 activates caspase-3 to initiate the apoptotic program ,142,143.TGEV infection can activate not only intrinsic apoptotic pathways, but also extrinsic apoptotic pathways. After TGEV infection, caspase-8 indirectly activates caspase-9 by cleaving Bid, resulting in the release of Cyt c, or caspase-8 directly activating caspase- 3 to induce apoptosis . In addiMacroautophagy, also known as autophagy, is a conserved degradation process in eukaryotes. Under stress conditions (such as organelle damage or pathogen invasion), autophagy-related genes (ATGs) mobilize the immune system to capture and eliminate pathogens ,151,152.Several studies in recent years have demonstrated that the relationship between autophagy and viruses is not simply a positive or negative effect. With the technique called the genome-wide cellular knockout (GeCKO) library, researchers screened host factors for SARS-CoV-2 and other coronaviruses, and a brand new and poorly understood host factor in virus research, TMEM41B, came into view ,17,18,19TMEM41B is critical in multiple viral infections including TGEV in multiple independent studies on multiple viruses and is an essential gene for DMV formation during coronavirus replication ,18,167. In the past 20 years, coronaviruses have emerged with alarming speed and high virulence strains, and human diseases such as SARS-CoV, MERS-CoV, and SARS-CoV2 have caused serious public health problems. TGEV is also back in our sights in the pig breeding industry and study of zoonotic disease along with the coronavirus pandemic and outbreak in recent years.Although the detection rate of TGEV is now very low, it has been continuously detected in China ,7,8,9, STGEV is a cross-species infectious disease; we need to increase our focus on TGEV in this time of accelerated occurrence of zoonotic diseases. The canine–feline-like recombinant alphacoronavirus was recently reported for the first time in a case of pneumonia in Malaysia and was named CCoV-HuPn-2018 . This viIn the recent future, exploring the host factors of TGEV that have not been covered based on GeCKO library technology provides insight into the infection mechanism of this classic alphacoronavirus. It will be important to conduct deeper information mining based on the results and discover and identify these gene functions, which have hardly been studied in the virus field before. Meanwhile, the research results that have been reported may continue to provide new clues for the biology of coronaviruses or other naturally recombined novel coronaviruses in the future. Moreover, these clues allow us to revisit and gain a deeper understanding of this virus that has been quietly lurking in nature for some time, which has implications for the possible future (and possibly contemporary) emergence of a zoonotic recombinant alphacoronavirus and its impact on human public health.


N. benthamiana plants expressing multiple gRNAs simultaneously showed enhanced tolerance against CLCuV infection when compared to wild-type plants. T7 Endonuclease-I (T7EI) assay, showing indels in the CLCuV genome, confirmed the occurrence of double strand breaks (DSBs) in DNA at target sequence induced by Cas9 endonuclease. We observed that targeting CLCuV genome at multiple sites simultaneously resulted in better interference, also with inefficient recovery of altered virus molecules. Next, we tested multiplex construct in cotton to interfere CLCuV infection. We found significant decrease in virus accumulation in cotton leaves co-infiltrated with multiplex cassette and virus compared to cotton leaves infiltrated with virus only. The results demonstrate future use of CRISPR-Cas9 system for engineering virus resistance in crops. Moreover, our results also advocate that resistance to mixed virus infections can be engineered using multiplex genome editing.Advances in genome editing technologies have tremendous potential to address the limitations of classical resistance breeding. CRISPR-Cas9 based gene editing has been applied successfully in plants to tolerate virus infections. In this study, we successfully tested CRISPR-Cas9 system to counteract cotton leaf curl disease (CLCuD) caused by whitefly transmitted cotton leaf curl viruses (CLCuVs). We also analyzed the ability of CLCuV to escape the Cas9 endonuclease activity. Targeting overlapping genes of most prevalent CLCuVs with three gRNAs resulted in virus interference, as validated by low virus titer. Furthermore, multiplex CRISPR-Cas9 construct simultaneously targeting six genes of CLCuV, was found more effective to interfere with virus proliferation compared to targeting single region individually. Additionally, transgenic  Based on genome arrangement and phylogenetic studies, begomoviruses considered as highly dynamic group of viruses. Such viruses are classified into old world and the new world viruses.5 The new world begomoviruses are bipartite while the old world includes both bipartite and monopartite. The bipartite genome has two main components known as DNA-A and DNA-B, and an extra bipartite-like monopartite genome identical to DNA-A. Usually, DNA-A possesses six open reading frames (ORFs), while DNA-B includes two ORFs encoding movement protein and nuclear shuttle protein.Begomoviruses transmitted viral diseases threaten crop production worldwide. The situation has deteriorated in underdeveloped countries, especially in areas where crop-free seasons are unusual and plants are constantly exposed to vector-borne viral diseases.6 However, still there is yawning gap to combat challenges. Biologists are now more equipped to manipulate genes to enhance yield and resistance of crop varieties under different biotic and abiotic stresses.7 Advance genome engineering techniques offer maneuvering of the genome in precise manner i.e. editing, deleting or addition of genes site specifically.8 These genome modifications are more feasible alternative as compared to conventional methods of transgenic development.9 The ability to induce site-specific DSBs at the position of interest should be taken as rear-guard action before utilizing it at full scale. Recently, precise genome engineering approaches such as Zinc Finger Nucleases (ZFNs), Transcription Activators Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated-9 (CRISPR-Cas9) have been shown to induce favorable genetic alterations.10 The CRISPR-Cas9 system is a natural immune system in bacteria and archaea against invasion of viral or plasmid DNAs.11 It has been considered as a promising and versatile tool to reshape the genome engineering dimensions in agriculture.12 This approach is anticipated to result a broad range of applications in genome engineering experiments involving crop plants and devising multigenic disease resistance models. The programmable CRISPR-Cas9 system is a robust and inexpensive approach with great potential to bring a next-generation of gene editing experiments.Genome engineering has been on the forefronts to produce high yielding climate resilient crops.13 Initial reports of engineering plants with RNA-guided Cas9 targeting the highly destructive Geminiviruses have been shown successful virus genome interference.14–18 Other studies have recently demonstrated the deployment of the CRISPR-Cas9 system to target coding or non-coding viral DNA regions in plants with varying gRNA efficiencies.19–21 These studies showed delayed symptoms of the disease and reduced the virus accumulation in plants treated with CRISPR-Cas9 reagents. However, some reports described the emergence of virus variants capable of replicating and with different level of disease infections.22Since conventional breeding approaches have not been much successful against begomoviruses infection, due to multiple virus strains and complex interaction of host plant, whitefly vector and virus particles. However, potential use of CRISPR-Cas9 based virus genome editing can be effective to improve agricultural productivity.23 Multiple gRNAs should be required to target key DNA sequences at same time. Multiplexing potential gRNAs should be used to test the additive effects of multiple gRNAs on reduced virus accumulation and severity of the disease symptoms.23 In the present research, we initially analyzed the ability of the CRSIPR-Cas9 system to interfere individually with overlapping genes of three most prevalent CLCuVs in Pakistan; cotton leaf curl curl Multan virus (CLCuMuV), cotton leaf curl Burewala virus (CLCuBuV), and cotton leaf curl Kokhran virus (CLCuKoV). We found that three individual gRNAs designed to target AV2/AV1, AC2/AC3 and AC1/AC4 overlapping gene sequences targeted the respective sites in three CLCuV strains. Furthermore, we predicted that simultaneous targeting of six CLCuV genes would be a better choice to tackle virus proliferation inside the plants. We examined the ability of three gRNAs in a single construct to inhibit CLCuV replication in plants. Our results indicated that multiplexed CRISPR-Cas9 approach provided a highly efficient virus genome interference and reliable approach for developing broad spectrum and durable plant tolerance against begomoviruses.Previously, several gRNAs were individually screened to thwart virus disease symptoms, limited or no efforts are reported of targeting plant virus genome at multiple sites with more than one gRNAs simultaneously. Developing durable and broad-spectrum tolerance under open-field conditions is a serious challenge because of mixed viral infections.http://www.multicrispr.net/multalign_input.html. Three potential and unique gRNA targets were found, present in consensus sequences of six overlapping genes of CLCuV DNA-A. These targets were also compared with CRISPR-P 2.0 recommended gRNA targets to validate the accuracy of CRISPR-Multitargeter software.We utilized the three CLCuV strains for DNA sequences analysis using CLUSTAL-W and T-Coffee online web tools. Consensus sequences in overlapping genes were selected to design potential gRNA targets using CRISPR-Multitargeter software, BsaI restriction sites in the pHSE401 vector were used for individual gDNA cloning. All gDNA oligos containing BsaI adapter sequences at the 5´ends were commercially synthesized for cloning. The pHSE401 was digested with BsaI, treated with alkaline phosphatase, and purified with a GeneJET gel extraction kit . Individual oligos . Positive clones were confirmed by sequencing using M13-F primers (https://en.vectorbuilder.com/.The pHSE401 plant expression vector (Addgene plasmid # 622010) was used for cloning and expression of both gRNA and Cas9 in plants. Two l oligos  were fir primers . The mulN. benthamiana and cotton were grown in small pots containing peat moss and soil at ambient temperature with 8 h dark and 16 h light photoperiod  and CRISPR-Cas9 constructs were infiltrated in the Agrobacterium tumefaciens (EHA105 strain) cells were transformed with the multiplex CRISPR-Cas9 (pHSE401-MV) plasmids by electroporation, and transformed single bacterial colony was used to transform In vitro grown N. benthamiana leaf discs, as described by UC Davis Plant Transformation Facility Center (Unpublished method). Cas9 gene specific primers were used to check the integration of multiplex cassette. Expression of gRNAs and Cas9 was measured by qPCR, actin gene of N. benthamiana was used as an internal control and reaction normalizer.To validate the activity of the CRISPR-Cas9 constructs on the CLCuV genome, mutation resulting from DNA DSB were verified by the non-homologous end joining [NHEJ) repair pathway, as described by.N. benthamiana plants. Next, Cas9-gRNAs infiltrated N. benthamiana plants were challenged with the infectious clones of CLCuKoV and helper betasatellite, CLCuMuB for symptoms development  as the selection marker and transferred to soil for acclimatization and seed harvesting. Five individual lines were selected for transgene screening. DNA was isolated from 5 well-rooted transgenic plants and Cas9 specific primers were used to check the integration of multiplex cassette. The qPCR of selected plants confirmed the expression of both Cas9 and gRNAs in transgenic N. benthamiana plants. A slight variation in the expression of Cas9 and gRNAs were observed among selected transgenic lines. Transgenic N. benthamiana line expressing highest level of Cas9 and all three gRNAs was selected for virus infectivity assays.We used Agrobacterium mediated leaf disc transformation method to express multiplex CRISPR-Cas9 construct (three distinct gRNAs and Cas9) . in the N. benthamiana line expressing multiplex CRISPR-Cas9 cassette was capable of interfering CLCuV genome, the virus infectious clones were infiltrated into the transgenic and wild-type N. benthamiana plants. Virus infiltrated transgenic N. benthamiana plants displayed less or no disease symptoms compared to non-transgenic plants. A virus titer analysis by qPCR clearly demonstrated very low virus accumulation (up to 95%) in systemic leaves  pathway for virus repair. Previously, it is reported that virus DNA molecules targeted by CRISPR-Cas9 machinery, capable of replicating and produced disease symptoms.ld type. . These fN. benthamiana) and screened highly efficient gRNAs targeting multiple CLCuV strains. Cotton plants were chosen to test the efficacy of multiplex construct against CLCuV infection. Multiplex CRISPR-Cas9 cassette was transformed into cotton plants by agroinfiltration and subsequently challenged with virus infectious clones. We observed virus symptoms on plants infiltrated with virus only and mild symptoms on plants infiltrated with CRISPR-Cas9/virus 10 dai. Virus titer analysis by qPCR showed reduced virus accumulation in multiplex CRISPR-Cas9 infiltrated plants when compared to control plants infiltrated with virus only. Next, we isolated the plant leaf DNA infiltrated with CRISPR reagents and virus infectious clones to check the Cas9 endonuclease activity to target virus DNA. Sequencing of PCR amplicons encompassing gRNA targets showed the indels induced by Cas9 endonuclease (Cotton leaf curl disease (CLCuD), caused by CLCuV complex, is a major factor affecting cotton production worldwide. Developing resistance cotton cultivars against CLCuD under natural field conditions is a major challenge due to occurrence of multiple CLCuV strains compared to controlled conditions. We first optimized the CLCuV interference in model plant . All infiltrated plants showed mild and delayed virus infection compared to control plants infiltrated with virus only. Virus accumulation in infiltrated leaves was quantified to further validate the results. The qPCR results demonstrated less virus accumulation in systemic leaves when compared with control plants as described in the results. Moreover, expression analysis of all three gRNAs and Cas9 in agroinfiltrated plants also confirmed the activity of the CRISPR-Cas9 constructs.We tested efficiencies of three gRNAs targeting six overlapping genes of three most prevalent strains of CLCuV to interfere with virus genome. 16 It was suggested that multiplexing several gRNAs in a single vector could potentially deliver its additive effect.29 Accordingly,30reported that, transgenic N. benthamiana plants expressing Cas9 and dual gRNAs that target different regions of the CLCuMuV genome confer complete resistance to virus infection, thereby demonstrating a novel approach for engineering resistance to geminiviruses. Therefore, to test inhibition virus proliferation after simultaneously targeting of all genes of CLCuV DNA-A, three efficient gRNAs causing low virus accumulation by qPCR were multiplexed in a single vector.Viral DNA accumulation reduced in all co-infiltrated plants when compared to control plants. Up to 70% reduction in virus accumulation was recorded when overlapping sequences of CLCuVs genome were targeted. As,N. benthamiana plants via agroinfiltration. Virus titer analysis revealed that virus accumulation was reduced significantly when we targeted virus DNA at multiple sites as compared to single gRNA target. Next, we tested the ability of stable multiplex cassette expressing N. benthamiana line to interfere with CLCuVs. A multiplex cassette was introduced into N. benthamiana by using Agrobacterium mediated leaf disc transformation method. Transgenic N. benthamiana line, expressing high level of Cas9 and gRNAs along with control (non-transgenic plants) was selected for infiltration with the virus infectious clones. Severe virus symptoms were recorded on the control non-transgenic plants, whereas very few virus symptoms were appeared on transgenic plants as compared to individual gRNAs, indicating higher efficacy of CRISPR-Cas9 multiplex vector to thwart virus infection. The qPCR with DNA extracted from both transgenic and non-transgenic plants further confirmed the efficiency of multiplex vector to inhibit CLCuVs proliferation in these plants. Moreover, DSB detection by T7EI assay also clearly demonstrated the ability of the CRISPR-Cas9 system to target CLCuVs genome. Silencing of the integrated gene cassette is common in crop plants, particularly when plants are engineered for virus resistance.3132 Therefore, we also tested CRISPR-Cas9 multiplex cassette to thwart virus infection in T1 and T2 generations. Results indicated the consistency and inheritance of multiplex cassette against virus interference. Finally, we also tested multiplex CRISPR-Cas9 cassette in cotton plants to interfere CLCuV infection. Virus titer determined by qPCR was found less in multiplex infiltrated cotton plants as compared to plants infiltrated with virus only. Moreover, sequencing results also confirmed the activity of Cas9 endonuclease in cotton plants.The ability of multiplex CRISPR-Cas9 cassette to inhibit CLCuV genome was tested in 17 targeting single region may lead to the emergence of mutated virus molecules capable of replication, proliferation, and systemic infection. Notably, ability of virus genome to replicate and escape Cas9 interference was earlier observed in ACMV, TYLCV, and HIV-1 viruses. Moreover, it is likely that targeting of viral DNA with multiple gRNAs would result long deletions rendering virus incapable of replication and systemic infection.17 Therefore, it would be a plausible strategy to achieve effective interference in which CRISPR-Cas9 system concurrently mutates several essential parts of virus.30 However, the role of targeting specific viral DNA sequences to promote recombination of virus itself under field conditions, is still to be determined.This study provides insight into the potential use of CRISPR-Cas9 system for establishing broad-spectrum and durable virus resistance in plants. Since, error prone NHEJ repair of the targeted regions of different Geminiviruses is very fast,The short genome of begomoviruses contains specific DNA sequences necessary for viral replication, host infection and interacting with host defense systems. For targeted viral DNA interference, focus should be on the sequences that can provide efficient and better interference. At the same time, targeting multiple regions of the virus genome would greatly facilitate to overcome virus ability to recover from DNA repair. These chopped viral DNA fragments would ultimately be degraded. In conclusion, multiplex gRNA approach would be considered for developing durable and broad-spectrum resistance against plant viral diseases.