Respiratory viruses, such as SARS-CoV-2, preliminarily infect the nasopharyngeal mucosa. The mechanism of infection spread from the nasopharynx to the deep lung–which may cause a severe infection—is, however, still unclear. We propose a clinically plausible mechanism of infection spread to the deep lung through droplets, present in the nasopharynx, inhaled and transported into the lower respiratory tract. A coupled mathematical model of droplet, virus transport and virus infection kinetics is exercised to demonstrate clinically observed times to deep lung infection. The model predicts, in agreement with clinical observations, that severe infection can develop in the deep lung within 2.5–7 days of initial symptom onset. Results indicate that while fluid dynamics plays an important role in transporting the droplets, infection kinetics and immune responses determine infection growth and resolution. Immune responses, particularly antibodies and T-lymphocytes, are observed to be critically important for preventing infection severity. This reinforces the role of vaccination in preventing severe infection. Managing aerosolization of infected nasopharyngeal mucosa is additionally suggested as a strategy for minimizing infection spread and severity. Respiratory viruses, like the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which causes the disease COVID-19, are transmitted mainly through virus-laden droplets . The dro(∼65%) . The LRT(∼65%) . In addi(∼65%) . As suchOnce the infection is seeded in the nasopharynx, the virus releases its ribonucleic acid (RNA) and utilizes the host cell machinery to create and release hundreds of new virions resulting in rapid progression of infection. Severity of infection and associated health complications ), however, depend on whether the virus can gain access to the distal regions of the thorax and the lung, particularly the alveoli. Autopsy studies on patients with COVID-19 who had developed respiratory failure has confirmed the existence of SARS-CoV-2 in the alveolar epithelial cells as well as in alveolar macrophages . The mec2/diffusivity)! Hematogenous viremia is another possible route for the virus to reach the distal lungs. However, it only explains a small fraction of the fatalities associated with SARS-CoV-2  could be through virus diffusion in respiratory mucus if mucociliary clearance is impaired . However∼0.28 m , while dbe 60 nm ], resultRS-CoV-2 . SimilarRS-CoV-2 . AnotherRS-CoV-2 . HydrodyRS-CoV-2 . While mIn order to explore the plausibility of this mechanism, one needs a mathematical model which includes droplet (in airways) and virus (in mucus) transport within the LRT, along with virus infection kinetics. Extensive independent studies have been carried out over the years on all these aspects . A few sThe primary goal is to use this mathematical model to understand the onset of SARS-CoV-2 infection in different regions of the LRT, particularly the deep lung, through inhalation of carrier droplets present in the pharynx, and the subsequent infection progression in the presence of mucociliary clearance and virus diffusion. Different situations are analysed through pertinent dimensionless parameters with respect to their impacts on infection severity and infection resolution. The role of immune responses and vaccination is particularly highlighted. In addition, the time required post symptoms onset for development of pneumonia from a severe infection is also estimated from the results obtained.trumpet model, with appropriate modifications, to approximate the dichotomous structure of the LRT in a human being . These droplets may be a combination of the droplets inhaled during respiration (which have not deposited in the URT) or droplets that are formed through ANM. No additional aerosolization of the mucosa or droplet source are considered. The inhaled droplets are either deposited or washed out of the LRT. Major droplet deposition mechanisms viz. diffusion, sedimentation and impaction have been taken into account while calculating droplet deposition in the LRT  can remain small. Q represents the volume flow rate of air during breathing and is modelled such that Q = Q0q(τ), q(τ) being a sinusoidal function accounting for airflow variation during breathing. L0 and A0 are the airway length and cross-sectional area at N = 0, respectively.The following scaling parameters are used to obtain Eq. relation . kB, ϕv, Pev and Stm denotes the dimensionless virus concentration in mucus, virus Peclet number and mucus Strouhal number, respectively. The quantity The only source of viruses in the LRT is the carrier droplets that deposit in the mucosa of the LRT. The deposited viruses diffuse in the mucosa and are also subjected to mucociliary advective transport. The corresponding transport equation of the deposited viruses in the mucosa is formulated considering these transport mechanisms in addition to kinetics of the virus infection  and it varies with time asλg,i and λd,i are the dimensionless growth rate and decay rate of interferons, respectively. τp,i is the dimensionless time at which amount of interferon reaches its maximum.Interferons are assumed to attenuate virus replication from the infectious cells. The virus replication rate  see Eq.  and f iscl in Eq. Ab is the fraction of antibodies which varies with time asλg,a is the dimensionless growth rate of antibodies and Ab0 is the initial fraction of antibodies present.The presence of antibodies increases the probability of neutralising the viruses present in the body. The clearance rate of viruses . The trachea (N = 0) is assumed to be exposed to virus-laden droplets, presumably a combination of inhaled droplets (which have not deposited in the URT) or those formed due to ANM, for a specific inhalation period (τinh). The droplets are transported deeper into the LRT, along with the airflow, during inhalation (Eq. N = 0) along with mucus, irrespective of inhalation/exhalation (Eq. N = 23), the total advection-diffusion flux of both droplets and viruses is assumed to be zero  or those formed due to ANM. In either case, these droplets are transported with airflow deeper into the LRT during which they may also get deposited in the respiratory mucosa. The viruses, thus deposited, diffuse in the mucosa and are also transported upstream by advective mucociliary clearance. At the same time, the deposited viruses undergo replication forming virus colonies and are also simultaneously removed by various mechanisms .τexp = 5). Extrapolation to longer exposure times and its impact on infection will be discussed separately. It is observed that the virus concentration in the mucus (ϕv), at the end of the inhalation, qualitatively follows droplet deposition characteristics  are transported upstream towards the trachea (N = 0) by mucociliary advection and are eventually washed out. This is evident from the spatial change in ϕv with time (see ϕv in the upper airways (lower N) is due to smaller mucus volume in those regions. The viruses continue to be washed out of the lungs as long as washout dominates over virus replication. After a certain time period , virus replication starts to dominate over washout causing ϕv to again increase with time, as can be observed in ϕv  are transported only through diffusion due to absence of mucociliary advection. This leads to longer persistence of viruses deposited in the deep lung. The dynamics of ϕv in the deep lung is, thus, determined by the weak diffusive transport and virus kinetics only. It can be observed that ϕv reduces considerably in the initial period post-deposition due to larger virus clearance as compared to virus replication. Once virus replication starts to dominate, ϕv starts to increase as time progresses , thus obtained, corresponds to the peak infectious state. The corresponding time is denoted as τpinf,.In contrast, viruses deposited in the deep lung . Ped is defined as the ratio of advective airflow in the lung to diffusive transport of the droplets in air  in the deep lung follows the droplet deposition characteristics  even if a small fraction of the inhaled droplets reach the deep lung. Transport of viruses to the deep lung, thus, needs to be minimised.The effects of inhaled droplet size and airflow rate are studied by varying the droplet Peclet number  and a substantially larger ϕv, max . Pev is defined as the ratio of advective mucociliary transport and diffusive transport of the deposited viruses in the mucus layer  and vice versa. Weaker diffusive transport can be the result of a larger virus or more viscous mucus.The impact of virus size, mucociliary advection and mucus viscosity is studied by varying the virus Peclet number ( see Eq. . A largePev (stronger mucociliary advection) leads to relatively faster washout of viruses from the upper airways of the lung where mucociliary clearance is substantial (see N = 12). Faster washout reduces the residence time of the viruses in the upper airways. This inhibits virus replication and reduces ϕv, max in the upper airways. Similar effects are observed in the upper airways for the entire range of Ped considered in this analysis. In contrast, only a minor impact is observed in the deep lung (N = 23) for the range of Pev considered. This can be primarily attributed to the negligible mucociliary clearance from the deep lung. A change in Pev in the deep lung, thus, implies modification of virus diffusivity. However, the time-scale of virus diffusion is too long for it to have any substantial impact on the infection time-course in the deep lung.A larger tial see , N = 12.While the size of inhaled viruses cannot be controlled, the rate of mucociliary clearance and mucus viscosity can be therapeutically modified. This provides a viable approach for controlling infection progression in the upper airways of the lung. A similar viable approach for deep lung infection is, however, not feasible.Sta) and the mucus Strouhal number (Stm). Sta is defined as the ratio of time scale of advective airflow in the LRT to the breathing time period  leads to a larger volume of droplets inhaled into the lung, keeping all other parameters constant. This allows a greater portion of the inhaled droplets to reach the deep lung  and a corresponding higher ϕv, max  from the upper airways of the lung at lower Stm. This can be observed from Stm is inversely dependent on Tb. Thus, faster virus washout takes place from the upper airways at larger Stm and vice versa. This leads to a steeper concentration gradient between the deep lung and the upper airways resulting in faster diffusive transport in the deep lung. Thus, infections get resolved relatively faster in the deep lung at larger Stm. In addition, a larger Tb (for lower Stm) also allows the deposited viruses to replicate more leading to relatively higher ϕv, max at lower Stm  leads to larger ϕv in the deep lung throughout the time-course of infection  is observed to affect ϕv as well as τpinf,  and also delays attainment of the peak infectious state. The reverse happens when cl is reduced.In contrast, the virus clearance rate , innate response (interferons) and cellular response (cytotoxic T-lymphocytes). As such, it becomes necessary to distinguish between these responses in order to identify the key effects of the individual responses on virus concentration in the lung and infection time-course. This is achieved by varying the parameters of a specific response model, while keeping the other response models constant.Ab0 and the antibody growth rate λg,α). Results (see Ab0. The antibody concentration builds up over time and it is only after the antibody concentration becomes large enough that the impact on infection becomes apparent. A larger Ab0 (more antibody availability) allows initial targeting of greater number of viruses which, in turn, reduces the virus replication. This results in a lower ϕv, max and relatively shorter τpinf, . These antibodies bind with the pathogens (viruses) and neutralize them, thereby enhancing the virus clearance rate. The efficacy of this response is, thus, dependent on the rate at which the antibodies neutralize the pathogens ults see , 9C indilung see . Similarf,p see . The infp0). f) on the infection time-course in the deep lung. f is defined such that, for f = .5, the virus growth rate is halved when interferon concentration is at 50% of its peak concentration. A larger f, therefore, has a smaller impact on the virus concentration in the lung and vice versa. ϕv, max is also observed to reduce significantly and τpinf, becomes relatively longer with decreasing f.The innate immune response mechanism works by production of interferons which inhibits virus replication through various secondary mechanisms. The effects of such secondary mechanisms are not considered in detail in the present model and the sole effect of interferons is modeled to be reduction in virus growth rate  is also observed to influence the infection time-course  reduces ϕv during the initial stages of infection, but does not have any significant influence during the later stages. As τp,i becomes longer, the impact on virus production during the initial stages of infection becomes delayed resulting in relatively larger ϕv. Beyond a certain τp,i, however, no substantial change is observed on the virus concentration.The time taken for interferon concentration to reach its peak  in the body.The cellular response mechanism is dependent on the action of cytotoxic T − lymphocytes on ϕv remains negligible during the initial period of infection mainly due to low Tl in the body  on ϕv in the deep lung. τp,t is defined as the time taken by Tl to reach its peak. A shorter τp,t results in faster buildup of Tl and vice versa  caused faster infection spread with milder symptoms in the infected individuals. Other variants  often caused more severe health effects . The tim variant .Our results indicate that the reason for such difference in symptom severity and infection time-course can be correlated to the relative replication and neutralization of the virus in the host cells see . For a gB − lymphocytes and T − lymphocytes (or memory cells) within the body  suppress virus replication from the onset of infection itself leading to lower ϕv, max and hence, reduces the probability of disease severity. Similarly, a larger initial antibody presence in the body allows targeting  of a larger number of viruses. This slows down virus replication and enables other immune effects to act effectively, thereby decreasing disease severity  and those formed due to aerosolisation of the mucus layer during respiratory motions (coughs etc.). More the availability of droplets, longer is the duration for which these may be inhaled into the LRT and greater is the probability of these droplets reaching the deep lung causing infection see . ClinicaThe severity of a deep lung infection is determined by the binding affinity of the virus with cells in the deep lung as well as the replication/neutralization characteristics of the virus. Binding affinity is lower and replication becomes attenuated for the Omicron variant of SARS-CoV-2 in the deep lung cells  resultinOnset of pneumonia is a common yardstick used to gauge the severity of any infection since it can progress rapidly to respiratory failure. Clinical studies have found that the time taken for the development of respiratory failure from pneumonia in a human body from a SARS-CoV-2 infection is typically 6–14 days after the initial onset of symptoms , primaricv,cr) required for onset of pneumonia. Virus concentration in the pharyngeal region (N = 0) is assumed to be the clinically observed viral load in mild infections in pharyngeal samples, where SARS-CoV-2 is initially registered   is assumed to be the clinically observed median viral load in sputum samples (7 × 106 copies/mL) of symptomatic individuals exhibiting pneumonia . It is further assumed that the pharyngeal viral load remains constant throughout the inhalation duration. It is calculated that the time required for onset of pneumonia in a SARS-CoV-2 infection can vary from cv,cr and associated parameters. The results presented above are used to estimate the time taken for the SARS-CoV-2 concentration in the deep lung to reach the critical magnitude (pies/mL) . The crineumonia  (see Supdv), volume flow rate of air (Q0) and length geometry (L0). A relatively larger change dd), while a substantial change is observed when the breathing period vice versa, with T − lymphocytes. However, a fast enough production of T − lymphocytes resisted pneumonia onset.These estimates suggest no significant change rate see . PneumonRespiratory viruses, such as SARS-CoV-2, are primarily transmitted through the airborne route through respiratory droplets. When a subject inhales these droplets, they are deposited in respiratory mucus of the upper respiratory tract, where the virus infects and replicates in the nasopharyngeal epithelial cells. Some patients may develop severe pneumonia and acute respiratory distress syndrome if the infection spreads to the deep lung . This work discusses whether a nasopharyngeal infection can spread to the deep lung through inhalation of droplets, present in the nasopharynx, into the lower respiratory tract and the degree of severity of such an infection. A coupled mathematical model of droplet and virus transport is solved computationally considering virus infection kinetics and the effect of key dimensionless parameters is investigated. Different conditions are analysed through these dimensionless parameters based on which necessary remedies and public health recommendations can be suggested.vice versa. Thus, adequate measures need to be adopted to prevent conditions which promote larger viral load deposition in the lower respiratory tract, particularly the deep lung. One such measure is prevention of self-aerosolization of nasopharyngeal mucus layer which provides an additional source of virus-laden droplets in the nasopharynx for further inhalation into the lower respiratory tract. Another measure is avoiding longer breaths which reduces the volume of droplets inhaled.Results indicate that fluid dynamics play an important role only in transporting the droplets from the nasopharynx to various regions of the lower respiratory tract (including the deep lung), where the droplets deposit and release the viruses causing further infection. Progress of this infection is independent of the viral load deposited. However, infection severity depends on the deposited viral load - a smaller viral load causing a milder infection and vice versa. Specifically, the model predicts, in agreement with clinical observations, that a severe infection (pneumonia) can develop in the deep lung within 2.5–7 days of initial symptom onset, when nasopharyngeal droplets are inhaled into the lower respiratory tract. The immune responses, particularly antibodies and T − lymphocytes, are observed to be critically important for preventing infection severity and achieving quicker infection resolution. Stronger immune responses—which may be due to a prior infection or induced by vaccination—significantly lowers the chance of a severe infection. This reinforces the need of vaccination in preventing severe infections from SARS-CoV-2.Once an infection initiates, growth and resolution of the infection is determined by the infection kinetics and immune responses. A larger virus replication rate  increases the chance of a severe infection leading to pneumonia onset, and


Yadokariviridae) and an unrelated double-stranded (ds) RNA virus YnV1 (proposed family “Yadonushiviridae”) in a phytopathogenic ascomycete, Rosellinia necatrix. YkV1 has been proposed to replicate in the capsid provided by YnV1 as if it were a dsRNA virus and enhance YnV1 replication in return. Recently, viruses related to YkV1 (yadokariviruses) have been isolated from diverse ascomycetous fungi. However, it remains obscure whether such viruses generally show the YkV1-like lifestyle. Here, we identified partner viruses for three distinct yadokariviruses, YkV3, YkV4a, and YkV4b, isolated from R. necatrix that were coinfected with multiple dsRNA viruses phylogenetically distantly related to YnV1. We first established transformants of R. necatrix carrying single yadokarivirus cDNAs and fused them with infectants by single partner candidate dsRNA viruses. Consequently, YkV3 and YkV4s replicated only in the presence of RnMBV3 (family Megabirnaviridae) and RnMTV1 (proposed family “Megatotiviridae”), respectively. The partners were mutually interchangeable between the two YkV4 strains and three RnMTV1 strains but not between other combinations involving YkV1 or YkV3. In contrast to YkV1 enhancing YnV1 accumulation, YkV4s reduced RnMTV1 accumulation to different degrees according to strains. Interestingly, YkV4 rescued the host R. necatrix from impaired growth induced by RnMTV1. YkV3 exerted no apparent effect on its partner (RnMBV3) or host fungus. Overall, we revealed that while yadokariviruses generally require partner dsRNA viruses for replication, each yadokarivirus partners with a different dsRNA virus species in the three diverse families and shows a distinct symbiotic relation in a fungus.We have previously discovered a virus neo-lifestyle exhibited by a capsidless positive-sense (+), single-stranded (ss) RNA virus YkV1 (family  Rosellinia necatrix. Therein, a capsidless, (+)ssRNA virus termed yadokari virus 1 (YkV1) borrows the capsid of a dsRNA virus termed yadonushi virus (YnV1) and enhances YnV1 replication in return (trans-encapsidating yadokariviruses (Pisviricota)  . YnV1 isshifting , 13. YkVlication . Based olication , 10, 14.Yadokarivirales and a family Yadokariviridae in the phylum Pisuviricota (picornavirus supergroup) to the International Committee on Taxonomy of Viruses ICTV; https://ictv.global/taxonomy/taxondetails?taxnode_id=202112467, ICTV approved. These members can further be divided into two genera Alphayadokarivirus and Betayadokarivirus, based on the distinct phylogenetic clades. Examples of alphayadokariviruses include YkV1 and Aspergillus foetidus slow virus 2 (AfSV2)  . Exampleivirales . The othivirales . However10.1128/mbio.01685-22.1FIG S1Ghabrivirales. The phylogenetic relationships were analyzed based on the amino acid sequences of RdRPs listed in FIG S1, PDF file, 0.04 MB.Phylogenetic relationships of selected viruses in the order Copyright © 2022 Sato et al.2022Sato et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.01685-22.6TABLE S1Yadokariviridae. Download Table S1, DOCX file, 0.04 MB.Viruses in the family Copyright © 2022 Sato et al.2022Sato et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.01685-22.8TABLE S3Table S3, DOCX file, 0.03 MB.Accession numbers for viral sequences used in this study. Download Copyright © 2022 Sato et al.2022Sato et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the R. necatrix were diverse, from mutualistic, commensal, to parasitic. We will provide the catalog of three-layered symbiotic relationships among yadokariviruses, diverse dsRNA viruses, and a host fungus.In this study, we identified partner dsRNA viruses for the betayadokariviruses YkV3, YkV4a, and YkV4b and performed partner swapping assays among them and YkV1. Our results suggest that these yadokariviruses commonly have the yadokari nature but each partners with different dsRNA viruses in diverse families. Furthermore, the effects of these yadokariviruses on their partner viruses and host R. necatrix isolates, namely, Japanese W1032 and Spanish Rn95-16 and Rn454, that were coinfected with diverse RNA viruses (Rosellinia necatrix fusagravirus 3 (RnFGV3), in addition to another YkV4 isolate (YkV4b). YkV4a and YkV4b represent two different strains of YkV4, which share 96.0% global amino acid sequence identity in RdRP ssRNA hypovirus  were reported from three  viruses   Detection of viral RNA by RT-PCR. Host endogenous gene expression (Rntub2 mRNA) was detected as an internal control. The R. necatrix strains Rn480 and Rn30 were included as positive controls for viral detection. (B) Detection of viral RNA by Northern hybridization of ssRNA-enriched total RNA. Host ribosomal RNA (rRNA) was detected as an internal control. (C) Comparative data showing the presence or absence of viruses in fungal strains Rn454, Rn95-16, and Rn430. The detection patterns for 2018 were reported by Arjona-Lopez JM, Telengech P, Jamal A, Hisano S, et al. , while those in the current study are based on panels A and B and Rosellinia necatrix fusagravirus 2, RnFGV2) and capsidless (+)RNA fusagriviruses in Rn95-16 were undetectable in this study. Although Rn430 was supposed to be additionally infected by a fusagravirus strain (RnFGV3), this virus was also not detected in this study. These viruses possibly disappeared during subculture as reported elsewhere . (D) Electrophoretic profiles of total dsRNA purified from R. necatrix W97 singly transfected with RnMBV3, RnMTV1s, or RnFGV3. The virus source fungal strains  were analyzed in parallel. The bracket on the right side indicates the migration positions of the genomic dsRNA of RnMBV3, RnMTV1, and RnFGV3. The open red triangle indicates the migration positions of yadokarivirus dsRNA. The asterisk denotes the migration positions of defective dsRNA of RnMTV1. (E) Confirmation of single RnMTV1c infection of the R. necatrix W97 transfectant by RT-PCR. Strain W97 is a virus-free control. The field-collected fungal strain Rn430 is the RnMTV1c source in transfection. Rntub2 (R. necatrix β-tubulin gene) mRNA served as an internal control. Download FIG S2, PDF file, 2.0 MB.Recharacterization of viruses harbored in Copyright © 2022 Sato et al.2022Sato et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the n  in the figures, were cocultured with each of the transfectants singly infected with a partner candidate dsRNA virus  received a partner candidate and the transfectant sides (antibiotic susceptible) were each subcultured with or without antibiotics. Under the conditions where yadokarivirus can replicate, its replicative form dsRNA should be detected in both of the subcolonies from transformant and transfectant sides.To examine the autonomous-replication ability or determine partner viruses of the yadokariviruses, full-length infectious cDNA clones to betayadokariviruses, YkV2, YkV3, YkV4a, and YkV4b were constructed, as in the case for YkV1 . These bNA virus . After tAmong combinations of viruses from the fungal strain Rn454, only the coculture of transformants with YkV3 cDNA and transfectants with RnMBV3 resulted in YkV3 replication, showing mutual lateral transfer between the paired transformant and transfectant . SimilarR. necatrix W97 background (The band intensity ratio of YkV1 replicative form dsRNA to YnV1 dsRNA has been observed to be approximately 1:1 or higher in the ckground . In contckground . That ofckground  and E. Tn) side, which received the dsRNA virus after the coculture. As a result, YkV1 replication was shown to be supported only by coinfecting YnV1 but not by the other dsRNA viruses tested , three additional partnerships  were established in this study. To determine the partnership specificity, a swapping assay was carried out using fungal strains carrying single yadokarivirus infectious cDNA transgenes or dsRNA viruses as described above . The fous tested . Similars tested . YkV4a as tested  and D. TR. necatrix  or mutants that lacked the second segment  strain (#B1) and three mutant transfectants  were selected and analyzed, respectively. The original strains W779 and W97 were used as positive or negative controls for RnMBV1 detection, respectively. The top panel shows agarose gel electrophoresis patterns of total dsRNA isolated from the transfectants. The bottom two Northern panels show specific dsRNA bands hybridizing either of two genomic segments (L1 and L2) of RnMBV1. LS1 indicates rearranged L1 segment. Frequent rearrangement events of RnMBV1 dsRNA2 after transfection were reported earlier . The transfectant strain #B1 infected by wild-type RnMBV1 was used for the experiment in FIG S3, PDF file, 0.7 MB.Characterization of RnMBV1 transfectants and assay for their capacity to support YkV3 replication. (A) Electrophoretic profile of dsRNA from Copyright © 2022 Sato et al.2022Sato et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Taken together, each of the tested yadokariviruses showed relatively narrow, within-species partnership specificity .Yadonushiviridae,” Megabirnaviridae, and “Megatotiviridae”) in the order Ghabrivirales . In contrast, the other betayadokarivirus YkV3 made a partnership with a megabirnavirus RnMBV3 that belongs to the distinct suborder “Alphatotivirineae.” Thus, the partnership between yadokariviruses and partner dsRNA viruses is incongruent with their phylogenetic relationship.The partner dsRNA viruses determined in this study belong to the three phylogenetically distant families  or the ivirales . Phylogeivirales  and thosivirales  did not ivirales . An alphT = 1 capsid  fractions were obtained by cesium chloride or sucrose gradient ultracentrifugation and their dsRNA and protein components were analyzed . Both Yk1 capsid . Similar1 capsid . Sodium 1 capsid . The maj1 capsid . The N-t to 1652 , which a to 1652 . The thr to 1652 . This stR. necatrix strain W97 was coinfected with the yadokariviruses and their partner dsRNA viruses via hyphal fusion as described in The previous study reported that YkV1 makes a mutualistic relationship with YnV1 by enhancing YnV1 accumulation . To inve10.1128/mbio.01685-22.4FIG S4R. necatrix W97. See the legend for R. necatrix W97 on PDA media. The white bar represents 3 cm. For panels A and B, either 5- or 6-day-old cultures were photographed. (C) Spearman’s correlation coefficient (R) between the relative accumulation of RnMBV3 and the relative fungal colony area. The results of three independent experiments were analyzed. Relative RnMBV3 accumulation was estimated in FIG S4, PDF file, 0.2 MB.Repeated analyses to examine the effects of YkV3 on RnMBV3 and host fungus. (A) Viral RNA accumulation in the mycelia of Copyright © 2022 Sato et al.2022Sato et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.01685-22.5FIG S5R. necatrix W97. See the legend for R. necatrix W97 on PDA media. The white bar represents 3 cm. For each panel, either 5- or 6-day-old cultures were photographed. Download FIG S5, PDF file, 0.2 MB.Repeated analyses to examine the effects of YkV4s on RnMTV1s and host fungus. (A) Viral RNA accumulation in the mycelia of Copyright © 2022 Sato et al.2022Sato et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vitro growth of the host fungus (R. necatrix strain W97) (P < 0.05)  B and S5Bain W97) . In contain W97) . Notably < 0.05)  to F, wh < 0.05) . YkV4 st < 0.05) , almost Orthornavirae, the realm Riboviria. The five phyla include Duplornaviricota (dsRNA viruses), Kitrinoviricota [(+)ssRNA viruses], Lenarviricota [(+)ssRNA viruses], Negarnaviricota [(–)ssRNA viruses], and Pisuviricota   and Duplornaviricota . The dsRNA viruses identified as hosting yadokariviruses in this and the previous study are YnV1 for YkV1  in the hallmark of RNA viruses, RdRP , a biparticulate virus in the genus Botybirnavirus trans-enctnership , there mivirales , which sivirales , 36. Theivirales . This paivirales . Yadokar signals . A similA notable difference between newly and previously discovered partnerships is the ratio of contents of yadokarivirus dsRNA (replicative form) to that of its partner virus genomic dsRNA detected in purified particle preparations or mycelia  and C. TR. necatrix strain W97 and that coinfection of YkV1 and YnV1 in R. necatrix strain W1032 is associated with a fungal growth defect  or the three-layered virus (capsid borrower)-virus (capsid donor)-host interaction.R. necatrix strains Rn480 and Rn30. The fungal strains Rn480 and Rn30  and ExTaqHS (TaKaRa Bio Inc.) were used. The amplified fragments cloned into the pGEM-T Easy Vector (Promega Corp.) were sequenced after amplification by PCR with Quick Taq HS DyeMix . Sanger sequencing was performed by a BigDye Terminator v3.1 Cycle Sequencing kit (Thermo Fisher Scientific Inc.) with 3130xl DNA Analyzers . The sequences of virus-specific primers used for the PCRs are available upon request.The full-length genome sequence of RnMBV3 and RnMTV1b, previously only partially determined by next-generation sequencing (NGS) , was comescribed , 40. Theescribed  with thehttps://doi.org/10.1093/molbev/mst010) with the method L-INS-i  and the ultrafast bootstrap method in 1,000 iterations .The global identity of the amino acid sequence of viral proteins was estimated by Clustal Omega version 1.2.4 with default settings . The fulR. necatrix by ultracentrifugation as previously described  in the crude extracts and each of the fractions was extracted by a cellulose affinity column chromatography method as described below. The fractions containing viral dsRNA were diluted with 50 mM sodium phosphate buffer (pH 7.0) and further centrifuged to obtain concentrated pure virus particles. The purified virus particles were stained with EM stainer (Nissin EM Co.) and observed by a Hitachi H-7650 electron microscope. The purified virus fractions were subjected to SDS-PAGE followed by silver staining as previously described  of pCPXHY3 (Not I site of pCPXHY3 by the In-Fusion HD Cloning kit (TaKaRa Bio Inc.). The sequences of primers used for the PCRs are listed in Full-length cDNAs of YkV3 and YkV4a were synthesized by outsourcing  see . These c pCPXHY3 . The inf10.1128/mbio.01685-22.9TABLE S4Table S4, DOCX file, 0.03 MB.Primers used for cloning of the infectious cDNA clone of YkV4b. Download Copyright © 2022 Sato et al.2022Sato et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the R. necatrix virus-free strain W97 were prepared as previously described , followed by the clarification as described above. To obtain crude dsRNA, a constant amount  of the total nucleic acids was subjected to cellulose  column chromatography as previously described . The extFor genomic DNA extraction, the total nucleic acids suspension was treated with 10 μg/ml RNase A  at 37°C for 30 min. The enzyme was removed by PCIA and CIA extractions. Genomic DNA in the supernatant was precipitated with 2-propanol and sodium acetate and then rinsed with 70% ethanol. Dried pellets were dissolved into TE buffer and used for genotyping (see below).6] (TaKaRa Bio Inc.) according to the manufacturer’s instruction in a half scale. The cDNA was used as a template for PCR by Quick Taq HS DyeMix  on a 10-μl scale. PCR-based genotyping of R. necatrix was also performed by Quick Taq HS DyeMix  on a 10-μl scale with the host genomic DNA template. Primers are listed in Toward viral RNA detection by RT-PCR, the ssRNA-enriched fractions described above were used for cDNA synthesis by M-MLV Reverse Transcriptase  with the random primer [hexadeoxyribonucleotide mixture; pd (N)10.1128/mbio.01685-22.10TABLE S5Table S5, DOCX file, 0.04 MB.Primers used for RT-PCR, DIG-labeling PCR, or genotyping. Download Copyright © 2022 Sato et al.2022Sato et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The RT-PCR and genomic PCR products were electrophoresed in 1% (wt/vol) agarose in 0.5× TAE. The nucleic acids were visualized with ethidium bromide (EtBr) by postgel staining. GeneRuler 1-kb DNA ladder  (described as “M-dsDNA” in the figures) was constantly used as a molecular size marker for nucleic acids.2. Specific dsRNA bands were detected with cDNA probes labeled with digoxigenin-11-dUTP (DIG) and the antibody for DIG  according to the manufacturer’s instruction . The DIG-labeled cDNA probes were prepared by PCR DIG Labeling Mix  with the primers listed in Northern hybridization of the ssRNA-enriched fractions (10 μg per lane) was performed via the MOPS-formaldehyde denaturing system as previously described . Northerhttps://imagej.nih.gov/nih-image/manual/tech.html).The intensity of dsRNA bands was quantified by ImageJ version 1.52a according to the provider’s instructions  using the R package “multcomp” version 1.4.8 (https://cran.r-project.org). The mean and standard deviation (SD) were calculated by “ggpubr.”The statistical differences among the virus-free and -inoculated fungal strains were analyzed by Tukey’s test  with the accumulation of partner dsRNA viruses under the presence or absence of yadokariviruses  was analyzed by Spearman’s rank correlation test by “ggpubr.”


In order to determine the selectivity of the developed anti-H1-based electrochemical influenza A immunosensor, the response of this system towards the H3N2 virus was investigated. The matrix effect was also investigated by using synthetic saliva supplemented with H1N1 virus.H1N1 is a kind of influenza A virus that causes serious health issues throughout the world. Its symptoms are more serious than seasonal flu and can sometimes be lethal. For this reason, rapid, accurate, and effective diagnostic tests are needed. In this study, an electrochemical immunosensor for the sensitive, selective, and practical detection of the H1N1 virus was developed. The sensor platform included multi-walled carbon nanotube gold-platinum (MWCNT-Au-Pt) hybrid nanomaterial and anti-hemagglutinin (anti-H1) monoclonal antibody. For the construction of this biosensor, a gold screen-printed electrode (AuSPE) was used as a transducer. Firstly, AuSPE was modified with MWCNT-Au-Pt hybrid nanomaterial via drop casting. Anti-H1 antibody was immobilized onto the electrode surface after the modification process with cysteamine was applied. Then, the effect of the interaction time with cysteamine for surface modification was investigated. Following that, the experimental parameters, such as the amount of hybrid nanomaterial and the concentration of anti-H1 were optimized. Under the optimized conditions, the analytical characteristics of the developed electrochemical immunosensor were investigated for the H1N1 virus by using electrochemical impedance spectroscopy. As a result, a linear range was obtained between 2.5–25.0 µg/mL with a limit of the detection value of 3.54 µg/mL. The relative standard deviation value for 20 µg/mL of the H1N1 virus was also calculated and found as 0.45% ( Concerns about the reemergence of an influenza pandemic have been increased since low rates of COVID-19 were observed. It has been reported that the rate of seasonal influenza has dropped since April 2020 based on the COVID-19 precautions such as quarantine, travel restrictions, the closures of companies and schools, the usage of masks, etc. ,4,5. HowOrthomyxoviridae family 4−/[Fe(CN)6]3, graphite powder, ethylene glycol (EG), NaHCO3, NaCl, and KCl were obtained from Sigma-Aldrich, St. Louis, Missouri, ABD. KH2PO4, NaOH, H2SO4, KMnO4, and H2O2 were purchased from Merck. In addition, influenza A hemagglutinin H3 monoclonal antibody (anti-H3), inactive influenza A/New Caledonia/20/99 (H1N1), and inactive influenza A/Brisbane/10/07 (H3N2) virus were purchased from HyTest, Turku, Finland. Virus solutions of different concentrations were prepared in pH 7.4 phosphate buffer with saline. All aqueous solutions were prepared using pure water. All the reagents were of analytical grade and were used without further purification.MWCNT, HAuCl6]−3/−4 in pH 7.4, 50 mM phosphate buffer. The EIS working frequency range was set between 0.1 Hz–10 kHz at a potential value of 0.10 V. Throughout the entire study, a commercial 220 AT model Methrohm branded screen-printed gold electrode (AuSPE) was used, which contained the gold working electrode (diameter = 4 mm), gold auxiliary electrode, and silver reference electrode on a single platform.EIS measurements were performed with a FRA-modeled μ-AUTOLAB potentiostat–galvanostat device that was supported by NOVA Software, in the presence of 10 mM 4−/[Fe(CN)6]3− as a redox probe indicator in the frequency range of 0.1 Hz–10 kHz and with a potential of 0.10 V. Each experiment was carried out in triplicate, and the results were presented with error bars.Different concentrations of 10 µL of H1N1 virus solutions were incubated with developed influenza A immunosensor at 25 °C for 1 h. For the investigation of interactions between H1N1 virus and anti-H1 antigen which was immobilized onto the working electrode, EIS measurements were carried out in 50 mM pH 7.4 phosphate buffer including 10 mM 4−/[Fe(CN)6]3− as a redox probe in the frequency range of 0.1 Hz–10 kHz and with a potential of 0.10 V. In order to select the most convenient hybrid nanomaterial, experiments by using plain AuSPE, AuSPE/Grafen-Au, AuSPE/MWCNT-Au, AuSPE/Grafen-Pt, and AuSPE/MWCNT-Au-Pt, were conducted via EIS in 50 mM pH 7.4 phosphate buffer including 10 mM [Fe(CN)SEM and EDX analyses were used to provide information regarding the morphological and structural composition of the synthesized MWCNT-Au-Pt hybrid nanomaterial. In EIS was used for the layer-by-layer electrochemical characterization of developed influenza A immunosensor. The Nyquist plots consist of two parts which are a semicircle and a straight line. Whereas the straight line demonstrates the Warburg impedance, which is due to diffusion at low frequencies, the semicircle diameter represents the charge transfer resistance on the electrode surface. As shown in 2 functional groups in its structure, cysteamine plays a significant role in binding the biological materials onto the gold surface. Without any pre-thiolation, cysteamine can bind AuNPs from -SH ends and biological materials from -NH2 ends [2 and -SO3 in a long waiting time. In addition, there is a possibility that -COOH ends in the MWCNT-Au-Pt structure on the electrode surface may have an additional interaction with cysteamine [Because of the presence of –SH and –NHNH2 ends ,41. As msteamine ,43.Moreover, MWCNT-Au-Pt hybrid nanomaterial amount in the developed immunosensor structure was also optimized. For this purpose, five electrochemical influenza A immunosensors that contain different amounts of MWCNT-Au-Pt hybrid nanomaterials  and 60 µg/mL of anti-H1 antibody were prepared, and then the responses against 7.5 µg/mL of H1N1 virus detection were investigated . As can The detection ability of the electrochemical biosensor depends on bioactive layer preparation procedure and interaction conditions between that layer and the biological analyte ,46,47. T2 = 0.99) with the linear regression equation of y = 20.424x + 159.01. In addition, limit of detection  and limit of quantification  values were calculated as 3.54 µg/mL and 11.80 µg/mL, respectively. Relative standard deviation (RSD) value was found as 0.45% for 20 µg/mL concentration of the H1N1 virus (n = 3).After the experimental parameters were optimized, analytical characteristics of the developed system towards different concentrations  of H1N1 were examined with EIS technique . AccordiSelectivity study was performed by using 10 µL of 5 µg/mL H1N1 and H3N2 viruses. When the developed MWCNT-Au-Pt hybrid nanomaterial-modified anti-H1 antibody-based electrochemical immunosensor for influenza A detection was incubated with H3N2 virus for 1 h, no change was observed in the resistance value, whereas a remarkable increase in the resistance value was determined after the interaction with H1N1 virus. The obtained results demonstrates specific binding of H1 protein on H1N1 virus to anti-H1 antibody. Moreover, as expected, since H3 proteins on H3N2 virus do not react with anti-H1 antibody, no response was observed for this type of virus .The matrix effect was examined using synthetic saliva supplemented with H1N1 virus. For this purpose, EIS measurements were performed with developed influenza A immunosensor after the incubation with a synthetic saliva solution containing 5 µg/mL of H1N1 influenza A virus for 1 h. According to the obtained resistance values, recovery value was calculated as 99.18% ± 0.17.In conclusion, an MWCNT-Au-Pt hybrid nanomaterial-modified anti-H1-based electrochemical immunosensor was produced for influenza A detection. Throughout the entire study, inactive H1N1 influenza A virus was used as a model virus. This developed platform has a potential application to diagnose different types of influenza viruses that contain the H1 protein on their surfaces. Besides being selective, sensitive, and practical, due to SPE usage, very small volumes of samples were analyzed with the developed immunosensor. In addition, we believe that developed system is of great importance because it can be adapted to all kinds of influenza virus determinations for the future just by changing the antibody type.


Mansonia uniformis collected in July 2017, in the Kitgum District of northern Uganda. Sequence analysis determined that the virus is Yata virus . The only previous reported isolation of YATAV was in 1969 in Birao, Central African Republic, also from Ma. uniformis mosquitoes. The current sequence is over 99% identical at the nucleotide level to the original isolate, indicating a high level of YATAV genomic stability.As a part of a systematic study of mosquitoes and associated viruses in Uganda, a virus was isolated from a pool of  Rhabdoviridae is a globally distributed, diverse family of viruses comprising 275 species in 45 genera [5 genera . Rhabdov5 genera . Due to 5 genera . Most vi5 genera . Many alEphemerovirus genus includes some of the largest viruses in the Rhabdoviridae family due to their relatively complex genomes [Ephemerovirus genus is named, is the most well-characterized of the ephemeroviruses. It is found extensively in Africa, Asia and Australia, and it causes severe, debilitating, febrile illness in cattle and water buffalo [The  genomes . In addi genomes . This ge genomes ,6. These genomes . Bovine  buffalo . Outbrea buffalo ,8. Due tEphemerovirus yata; YATAV) occurred in 1969 from a pool of Mansonia uniformis mosquitoes collected near Birao, Central African Republic . DNase-treated RNA was reverse-transcribed  into cDNA, amplified, and sequenced using a MinION Mk1C and Ligation Sequencing Kit . Reads were identified using integrated ONT software, Epi2Me, and confirmed by NCBI BLAST search.Using the YATAV sequence of Blasdell et al.  (GenBankA de novo and referenced assembly of reads into contigs was performed using CLC Genomics Workbench ver. 20 (Qiagen). The phylogenetic relationship of YATAV UG12209 to other ephemeroviruses was determined by a maximum likelihood analysis of the nucleic acid sequence using ClustalW alignment and the Tamura–Nei model with 1000 iterations on MEGAX software .Ma. uniformis collected using CDC light traps in July 2017 from Ogwapoke village, Kitgum District in north-central Uganda  [−4 s/n/y. The majority of the changes were silent, resulting in only 24 amino acid differences. Such similarity suggests that the virus likely persists in a relatively stable transmission cycle. Additional sequences, particularly those that may be obtained from other vectors and/or hosts, will help refine the YATAV rate of evolution. Further study of the effect of the nucleotide and amino acid differences is also needed to understand what, if any, functional and antigenic effects these changes might have on the viral life cycle.We report the isolation of a little studied ephemerovirus, YATAV. As only the second known isolation of this virus, 48 years removed from the original, this isolation offers an opportunity to examine the change over time of the YATAV genome. Rhabdoviruses are known to mutate quickly due to an error-prone RNA-dependent RNA polymerase and adaptation resulting from selective pressures ,16. Whil (s/n/y) . The curIxodes scapularis ticks in the United States and is known only by sequence. Koolpinyah virus was isolated from cattle in Australia, and an arthropod vector has not yet been identified [Culicoides spp. in Nigeria but may be primarily vectored by mosquitoes [Ma. uniformis mosquitoes but has yet to be associated with the infection of vertebrates. The virus was isolated and amplified in Vero cells, indicating that it is not an insect-only virus. Further, based on blood meal analyses and bait preferences, Ma. uniformis is known to feed on vertebrates, particularly mammals, possibly preferring humans [Phylogenetic analysis demonstrates that ephemeroviruses can be separated into two clades based on the nucleotide sequence . One claentified . Kotonkasquitoes . Serosursquitoes . Kotonkasquitoes ,20. Yatag humans ,24,25,26g humans , evidencThe results presented herein are part of a larger study with the aim of understanding the circulation of arboviruses throughout Uganda by collecting mosquitoes in various ecological niches at different times of the year over multiple years and analyzing them for the presence of replication-competent viruses. The past decade has seen numerous large viral epidemics and pandemics, several of which have been caused by arboviruses: Zika virus, chikungunya virus, and yellow fever virus, to name a few. The results of surveillance studies such as this will increase our understanding of the viral emergence potential and possibly suggest lines of effort toward diagnostic development and control measures for these viruses.


Robotic-assisted surgery research has grown dramatically in the past twodecades and the advantages over traditional videolaparoscopy have beenextensively debated. For hernias, the robotic system can increaseintraoperative strategies, especially in complex hernias or incisionalhernias.This study aimed to compare the direct cost differences between robotic andlaparoscopic hernia repair and determine each source of expenditure that maybe related to the increased costs in a robotic program from the perspectiveof a Brazilian public institution.This study investigated the differences in direct costs from the datagenerated from a trial protocol (ReBEC: RBR-5s6mnrf). Patients withincisional hernia were randomly assigned to receive laparoscopic ventralincisional hernia repair (LVIHR) or robotic ventral incisional hernia repair(RVIHR). The direct medical costs of hernia treatment were described in theBrazilian currency (R$).A total of 19 patients submitted to LVIHR were compared with 18 submitted toRVIHR. The amount spent on operation room time , inhaled medical gases in operating room, human resources inoperating room ,material resources , and medications  for RVIHR was higher than that for LVIHR, implying a highertotal cost to RVIHR . No significant difference was noted in costs related to thehospital stay, human resources in intensive care unit and ward, diagnostictests, and meshes.Robotic system adds a significant overall cost to traditional laparoscopichernia repair. The cost of the medical and robotic devices and longeroperative times are the main factors driving the difference in costs. A bibliometric analysis demonstrated the growth of 573% of robotic surgeryarticles published in the past decade. The advantages of robotic platform surgeries over traditionalvideolaparoscopy have been extensively debated. For hernias, the robotic system can increase intraoperative strategies,especially in complex hernias or incisional hernias. The benefits comprise high-quality 3D visualization of the abdominal cavity,gain in movement allowing easier dissection of multiple adhesions, the release ofthe rectus muscle, intraperitoneal mesh suturing, and complex reconstruction of theabdominal wall.Robotic-assisted surgery research has grown dramatically in the past two decades,leveraged by top Gross Domestic Product (GDP) countries. Both robotic and laparoscopic hernia repair approaches are equally effectivefor postoperative recovery compared with open surgery. In the first randomized controlled trial comparing robotic versuslaparoscopic incisional ventral hernia repair in Brazil, we also found no evidenceof differences in hospitalization, surgical complications, and recurrence rate.However, robotic surgery has nearly doubled operating room (OR) time.Several comparative studies of robotic-assisted primary, inguinal, or incisionalventral hernia repair versus laparoscopy found no significant differences inpostoperative outcomes. The Brazilian Public Health System (SUS) is the result of the Health ReformMovement, which culminated in the creation of the Unified Health System (inPortuguese: Sistema Único de Saúde — SUS) based on the principlesof universality, integrality, and equality. Despite its implementation, its processis considered unfinished, and with deviations. The relationship between funding and the system’s care model is one of thebiggest obstacles to the deployment of expensive new treatments, such as the roboticsystem. In this aspect, it is essential to know better the cost impacts of a publichealth system robotic program. Consequently, this study aimed to compare the directcost differences between robotic and laparoscopic hernia repair surgery anddetermine each source of expenditure that may be related to the increased costs in arobotic program from the perspective of a Brazilian public institution.The first robotic systems acquired in Brazil date back to 2007, covering privateinstitutions. In 2012, public hospitals in Brazil acquired this system, makingsurgical technology available to patients treated in the public health system.This study investigated the differences in direct costs from the data generatedfrom a randomized trial protocol . The clinical outcomes of this protocol were previously reportedInstituto do Câncer do Estado de SãoPaulo (ICESP), where the robot-assisted program was implemented in2015. ICESP is a public Brazilian cancer institute located in Sao Paulo and issupported by the National Healthcare System (SUS). Recruitment was performed in2015, and patients were followed up for 2 years. All patients were treated forincisional hernia following open oncologic surgery.All hernia repairs took place at The local Ethics Committee approved the study protocol (CAAE:40789014.3.0000.0065), and all patients signed a written informed consentform.The direct medical costs of hernia treatment are described from the Institute’sperspective. A mixed methodology of micro-costing and apportionment of themacro-costing was used. The estimate for daily costs related to hospitalizationand surgery  werevalued by apportioning fixed and variable costs  to assess the respective unit values of theInstitute health care service costs used. Drugs, medical devices, nutrition,blood, laboratory, and imaging studies were valued by micro-costing calculationaccording to individual patient consumption multiplied by the respectiveacquisition cost. The OR time was measured from entry until the patient’sdeparture from the OR, including the anesthesia and surgery. The mean cost oflaparoscopic ventral incisional hernia repair (LVIHR) and robotic ventralincisional hernia repair (RVIHR) was calculated by averaging patient costs foreach group. Costs were expressed in the Brazilian currency . In May2015, R$1.00=US$ 0.33.Patients with an incisional hernia were randomly assigned to receive anyinterventions: LVIHR or RVIHR, with either an intraperitoneal onlay mesh (IPOM)or a Rives-Stoppa procedure. All robotic-assisted procedures performed in thisstudy used the da Vinci Si platforms. When possible, any defect was closed usinga unidirectional suture. An intraperitoneal-coated macroporous multifilamentpolyester mesh was placed at least 5 cm overlap in all directions to cover theoriginal hernia size.Qualitative variables were described as counts and percentages. Continuousvariables were described as mean, median, standard deviation, and 95% confidenceintervals (95%CIs). Differences between groups were assessed by the Mann-WhitneyU test for continuous variables and Fisher’s exact test or chi-square test forcategorical variables. Statistical analyses were performed with the STATAsoftware, version 16.0 (StataCorp LLC).In the study, 19 patients submitted to LVIHR were compared with 18 patients submittedto RVIHR.=0.023), humanresources (OR) ,material resources ,medications  for RVIHR wassignificantly higher than that for LVIHR, implying a much higher total cost to thepatient in RVIHR than in LVIHR  , inhaled medical gases (ICU), inhaled medical gases (ward) , human resources (ICU) , human resources (ward) , diagnostic tests , and prosthesis, meshes, and special devices . Meantime, the amount spent on operation room time, inhaled medical gases(OR)  , Table 1To investigate the medications costs difference between RVIHR and LVIHR, we analyzedthe drug vials consumed per patient .Regarding drug vials consumed per patient, no significant difference was noted inroutine medications ,intravenous fluids ,electrolyte replacement ,neuromuscular blockade reversal agents , vasoactive drugs , antibiotics ,analgesics , antiemetics, and other . On the other hand, there was a significantdifference in the consume of anesthetics , local anesthetic agents , and neuromuscular blocking agents .The outcome of the present study showed that comparing RVIHR and LVIHR, there is asignificantly higher average cost in robotic-assisted use. These costs are mainlyassociated with prolonged surgical time, higher consumption of anesthetics, highmobilization of human resources, and, evidently, material devices related to therobotic machine. Knowing the costs of each variable related to the surgicalintervention is essential to better allocate resources and to depict a detailed andprecise budget impact analysis before implementing a robotic program in a publichealth institution.. However, the Brazilian National Medical Board imposes that any surgery(robot-assisted or not) should be performed by at least two surgeons. Consequently,in Brazilian robotic surgeries, a physician assistant is not an alternative toreduce costs in robotics, and LVIHR and RVIHR shall have similar personal costsrelated to surgeons.Beyond material resources, the robotic system demands higher costs related to humanresources. In theory, the robotic system demands only one surgeon for each surgery,decreasing the need for other surgeons during the procedures. In the United States,the physician assistant, a mid-level health care provider, may act as a bedsideassistant and helps position the patient and docking, decreasing the costs relatedto robot-assisted procedures. Consequently, the complexity of the robotic OR requires comprehensive andcontinuous training of the robotic nurses and, frequently, more professionals thanthe traditional laparoscopic approach. Robotic machine failure or malfunction can result in delay and prolongedoperating times, and robotic nurses should correct and prompt identify the systemfailure, report, and take quick and suitable measures. Robotic system-related material resources are expensive, and carelesshandling may lower the life span of robotic machine apparatus, increasing thematerial costs. In this setting, robotic nurses’ training can reduce the demand foran inflated number of professionals and devices in the OR, reduce operating time,and consequently lower costs. All robotic teams, including surgeons, bedside assistants, nurses, and theengineer team, should be continuously trained to improve surgical outcomes andmitigate costs.Due to the prolonged OR time and the complexity of the procedure, there is a demandfor more number and more qualified professionals during robotic surgery. Nurses inrobot-assisted surgery need to have high technical proficiency and active attitudes.Their roles include scheduling, checking for supplies, system operating,administration of circulating nurses, patient and console positioning, placingrobotic arms, robotic arm sterile draping, configuring equipment and instruments,docking, and undocking. However, even being a low cost compared to the human and material resources,the drug expenses in robotic system impose a significant additional cost comparedwith laparoscopic hernia repair. Besides, some technical difficulties during LVIHR,such as field visualization and structures mobilization, frequently can be easilymanaged by adjusting bed inclination or lateralization. Nonetheless, the difficultyin changing the patient’s position after docking may compel a continuous deepneuromuscular blockage for suitable field visualization and contribute to the higherconsumption of neuromuscular blockade agents in the RVIHR group.Prolonged surgical time implicates higher demand for more professionals in OR andexpenditure with the OR time. Besides, prolonged surgical time implies more use ofanesthetics and neuromuscular blockade agents. Anesthesia costs usually represent aminority proportion of the perioperative costs showed that RVIHR has higher costs than LVIHR and open hernia repair. Khorakiet al., in a retrospective study, showed that the added cost related to the roboticsystem was $3,106 per patient. Olavarria et al., in a multicenter controlled trial comparing RVIHR with LVIHR, the cost ratiowas 1.21 (95%CI 1.07–1.38). These previously quoted studies evaluated only theglobal health care costs in North-American robotic centers. Abdelmoaty et al. did a more detailed cost analysis and grouped costs into fixed, personnel,medical device, and variable costs. Each of these costs was significantly higher forrobot-assisted surgery than for laparoscopic. However, the authors evaluated onlyinguinal hernia repair. Zayan et al. included both inguinal and ventral hernia repair and grouped costs in directand indirect. The authors showed that only direct costs were significantly higherfor robotic surgery than laparoscopic, yet their methodology for cost estimation wasless depicted.Recent studies demonstrated the higher costs of the RVIHR compared with LVIHR.Nationwide American studiesAll the studies quoted in the last paragraph were North American, and worldwideextrapolations are questionable. The present study gives a picture of a roboticprogram from a public institution in a middle-income country. Nonetheless, thisstudy has several limitations. First, it was conducted in a single center, lackingexternal validity from other middle and low-income countries. In addition, onlydirect costs were accounted for, and costs related to the rehabilitation facility,days off work after surgery, and their impact on quality of life were notconsidered. Another inherent issue with any cost-analysis study is due to thefluctuation of the exchange rate over time, making it difficult to obtain adefinitive analysis of the costs involved in any longitudinal study. Well-designedfuture cost-effectiveness and cost-utility studies can answer whether the high costsof robot-assisted approach are justifiable for all countries. Economic studiesevaluating the robotic systems’ budgetary impact on health systems are crucial fordetermining their utility in public programs in developing or underdevelopedcountries. defined “surgeons and nurses” robotic training as the main modifying factorsof the cost equation in robotics. Medical residency and robotics fellowship programsmust be prepared and well-trained for this new world of robotic surgery and its evolution. The benefits of robotic surgery, including visualization, increased degreesof freedom, and ergonomics, must be incorporated worldwide, including in low- and middle-incomecountries, and the cost differences between robotic and laparoscopic proceduresshould be overcome. Identifying each source of expenditure that may be related tothe increased costs in a robotic program from the perspective of a publicinstitution in a middle-income country, such as Brazil, may help propose newstrategies to facilitate robotic program dissemination worldwide.The present study’s findings raise the question: “Is there a role for the RVIHR in apublic health system?” The answer is “yes.” If a robotic program does not root inassistance, there will be a significant delay in the dissemination of trainedrobotic teams and experienced surgeons and professionals worldwide, andconsequently, expenses lowering tend to linger. Nayeemuddin et al.A robotic system adds a significant overall cost to traditional laparoscopic herniarepair. The cost of the medical and robotic devices and longer operative times arethe main factors driving the difference in costs. These costs should be well knownbefore starting any robotic public program.


Two-stage revision arthroplasty is the gold standard for treating chronic prosthetic joint infection (PJI), but there has been limited analysis of the costs incurred beyond the index procedure and how additional complications and/or surgeries impact the cost of care.The electronic health record was queried for patients who underwent a total hip arthroplasty complicated by PJI and then underwent removal of the prosthesis with implantation of an antibiotic-impregnated articulating cement spacer. Patient demographics, surgical variables, hospital readmissions, emergency department (ED) visits, and postoperative complications were recorded. Data on total costs were collected with an internal accounting database. The average follow-up duration was 3.35 years.P = .007). Patients with a Girdlestone resection arthroplasty had higher total costs at 1 year  and higher costs attributable to ED visits at 2 years than the reimplantation group .Univariate analyses showed statistically significant differences between outcome groups  in total overall costs, ED visit costs, and postoperative costs at 1 and 2 years after the initial spacer placement. The median total cost at 2 years for each group was $38,865  (reimplantation), $79,223  (reimplantation with revision), $54,096  (retained spacer), $62,134  (Girdlestone). Patients who underwent successful reimplantation requiring no further surgery had significantly lower total costs than patients who needed revision surgeries after reimplantation ($38,865  vs $79,223 , A significant variation exists among total costs for the 2-stage treatment of hip PJI when stratified by the final outcome. Prosthetic joint infection (PJI) is a devastating complication following primary total joint arthroplasty (TJA). The incidence of PJI has been estimated to range from 1% to 3%  and accoTreatment algorithms for PJI typically depend on the timing of symptom onset and the mechanism of infection. Acute PJI is often treated with debridement, antibiotics, and implant retention while patients with chronic PJI undergo 2-stage revision arthroplasty, the current gold-standard in North America . SuccessPJI significantly impacts not only the affected individual but also the broader health care system, especially as the push to control health care spending and shift to value-based reimbursement models accelerates. The economic burden associated with PJI was estimated to be around $1B in 2017 and is projected to reach $1.85B by 2030 . With inThe aim of this study is (1) to quantify direct costs of 2-stage exchange arthroplasty with the placement of an articulating spacer for hip PJI in the 90-day, 1-year, and 2-year period; costs attributable to ED visits; and costs related to postoperative care and (2) to analyze the costs of PJI treatment stratified by the final outcome .After receiving institutional review board approval, an institutional electronic health record database was retrospectively queried from January 2009 through December 2020 for patients who developed a PJI after THA with subsequent removal of the hip prosthesis and placement of an antibiotic-impregnated articulating cement spacer. All procedures were performed by 1 of 7 fellowship-trained surgeons at an academic tertiary referral center. Data were gathered through retrospective chart reviews and algorithmic queries of the institutional electronic health record database.Patients aged 18 years or older with a diagnosis of PJI per the Musculoskeletal Infection Society criteria (score >6) who were subsequently treated at any time with an articulating spacer were included in the study . PatientDemographics of the patients were collected, including age, gender, race, ethnicity, and body mass index. Substance-use history  and Elixhauser Comorbidity Index score were also collected using their respective International Classification of Diseases (ICD)-10 codes.Data on hospital readmissions, emergency department (ED) visits, and complications following the initial surgery were collected. The primary outcome of this study was to determine the direct costs of 2-stage exchange arthroplasty with the placement of an articulating spacer for hip PJI in the 90-day, 1-year, and 2-year periods; costs attributable to ED visits; and costs related to postoperative care. These costs were then stratified by the patient’s final outcome . Reimplantation was defined as reimplantation after spacer placement with no additional septic revision procedures. Reimplantation with revision was defined as reimplantation after spacer placement with additional septic revision procedures. Spacer was defined as the failure to complete the second-stage reimplantation at the time of final follow-up. Girdlestone/resection arthroplasty was defined as spacer explantation and subsequent removal of a portion of the hip joint.A.Intermediate service (nursing)B.Intensive service (nursing)C.Pharmacy service D.Surgery services E.Physical therapist, occupational therapist, and speech servicesF.Radiology servicesG.Labs (testing)H.Emergency Department (ED) transportI.Outpatient clinic Total costs from the initial surgery, additional subsequent surgeries, hospital readmissions, and ED visits through 2 years were captured through an internal accounting database (EPSi). EPSi prospectively tracks expenses at various phases of care throughout the hospital encounter . The time of encounter was defined as the time a patient was admitted to the hospital until the time they were discharged. All costs accrued from the initial surgical encounter through 2 years postoperatively were recorded. Costs for each patient were aggregated according to the service group. These were broken down as follows:Costs not related to orthopedic care were excluded.Descriptive statistics of the cohort included both continuous and categorical variables that were represented as means or medians with standard deviations or interquartile ranges (IQRs) and counts and percentages, respectively.P values were corrected for multiple comparisons using the Bonferroni method.Univariate analyses were performed to compare patient demographics and comorbidity variables across the final outcome groups. Costs were compared using nonparametric Mann-Whitney U tests. Other continuous or categorical variables were compared using t-tests, chi-square test, or Fisher’s exact test as appropriate. Post-hoc pairwise comparisons were performed between each final-outcome group to compare costs at each time point . P = .003). The remainder of patient demographics are recorded in In total, 102 hip PJIs were identified, with 47 excluded due to incomplete follow-up or cost data. A total of 55 hip PJIs were included in the final cohort. The mean age of the overall cohort was 62.5 years (SD: 11.4), and 54.5% of the cohort were female, with 85% reporting their race as Caucasian/White, 5.5% as African American, and 9.1% as other/unknown. The mean Elixhauser Comorbidity score was 6.78 (SD: 3.62), and the mean Elixhauser score of the Girdlestone/resection arthroplasty group was significantly higher than that of the other final outcome groups  and second year  postoperatively. This is followed by nursing services  and pharmacy services  for the first year postoperatively. For the second year postoperatively, the next largest contributors to cost after surgical services are again nursing services  and pharmacy services (median: $856 [IQR: $0-$3340]).P = .007). In addition, the total costs of postoperative care for patients who underwent reimplantation with more than 1 revision surgery were significantly higher at both 1 and 2 years . Patients whose final outcome was a Girdlestone resection arthroplasty had higher total costs at 1 year  than the reimplantation group . These patients also had higher costs attributable to ED visits through the 2 years of follow-up than the reimplantation group .Costs stratified by the final outcome at 1 and 2 years after the initial spacer placement are provided in As reimbursement models continue to evolve and more focus is placed on quality of care and patient outcomes, health-care resource utilization will remain under scrutiny. In orthopaedics, bundled payments have become a common alternative payment model for various procedures, including TJA. While this incentivizes both increased cost-savings and high-quality care, providers and payors must be able to accurately estimate the cost of an episode of care to ensure sustainability of the model. Our study focused on the treatment of PJI after THA and quantified costs of 2-stage revision arthroplasty at the time of initial spacer placement through the 90-day, 1-year, and 2-year periods.The present study demonstrates median total costs at 1 and 2 years after spacer placement to be $39,678  and $44,518 , respectively. This is comparatively less than what is reported elsewhere in the literature for American studies. A large retrospective study by Kurtz et al. . Bozic eAn Australian study by Peel et al. provided a cost-analysis most similar to that of this study and arrived at a base cost for hip PJI treatment of AU$30,915, translating to ∼$26,000 USD in 2021 . HoweverP = .007). Patients who underwent a Girdlestone resection arthroplasty after 2-stage reimplantation had higher total costs at 1 year than the reimplantation group and had higher costs attributable to ED visits through 2 years of follow-up. This may highlight the need for improved discharge planning and postoperative rehabilitation as this procedure substantially impacts the quality of life and ability to carry out activities of daily living.Although 2-stage revision arthroplasty is currently the gold standard for treating chronic PJI, the rate of infection clearance ranges from 70% to 90% . TherefoMany studies that report on costs for 2-stage revision arthroplasty do not include cost data that account for additional treatment for persistent infection and subsequent variation in final outcome. However, Kurtz et al.  did noteThere are several limitations to this study. Retrospective studies are subject to several biases that limit generalizability, including misclassification and recall bias. In addition, all cost data were derived from a single institution in the Southeast, and there has been reported variability in the cost of PJI revisions based on geography. However, the additional data that could be gathered from this institutional cohort provide a much more detailed picture of an episode of care for the treatment of hip PJI. The current cost data only include reported direct costs and not charges to payors, but in this case, this cost data may provide a more accurate picture of health-care resource utilization and overall economic burden. The cohort of the study was impacted significantly by incomplete follow-up and cost data, decreasing the sample size by almost half. This may be due in part to the nature of being treated at a tertiary referral center where patients often travel far for the surgical procedure but seek follow-up care closer to home.This study identified the median total 1- and 2-year costs for patients undergoing 2-stage revision arthroplasty for hip PJI to be $39,678 and $44,518, respectively. Furthermore, findings demonstrated that undergoing additional treatment for persistent infection and/or other complications can have important implications for the total cost of the episode of care. When stratified by the final outcome group , there were significant differences in all cost groupings , except for initial spacer placement. Overall, the treatment of hip PJI with 2-stage revision and antibiotic spacer can be associated with substantial costs, especially when complicated by additional treatments and procedures.Dr. Seyler receives royalties from Total Joint Orthopedics, Inc.; Pattern Health, and Restor3D; is a paid consultant for Smith & Nephew, Total Joint Orthopedics, Inc.; and Heraeus; receives research support from Next Science and Zimmer; receives publishing royalties or financial or material support from Lippincott Williams and Wilkins; and is a member of the American Association of Hip and Knee Surgeons and Musculoskeletal Infection Society. All other authors declare no potential conflicts of interest.https://doi.org/10.1016/j.artd.2022.10.011.For full disclosure statements refer to


Pinus rigida) for the removal of Cu2+ and benzene from model pollutant solutions. The properties of the alginate/biochar hydrogel beads were evaluated using scanning electron microscopy, Fourier transform infrared spectroscopy, and Brunauer–Emmet–Teller analyses. Adsorption behavior of alginate/biochar hydrogel beads indicated that the adsorption capacities for Cu2+ (28.6–72.7 mg/g) were enhanced with increasing alginate content, whereas the adsorption capacities for benzene (20.0–52.8 mg/g) were improved with increasing biochar content. The alginate/biochar hydrogel beads exhibited similar adsorption capacities for Cu2+ and benzene in the concurrent system with Cu2+ and benzene compared to those in a single pollutant system. Adsorption kinetics and isotherm studies of the alginate/biochar hydrogel beads followed the pseudo-second-order model , and Langmuir model . In addition, alginate/biochar hydrogel beads (containing 1 and 4% biochar) exhibited high reusability (>80%). Therefore, alginate/biochar hydrogel beads can be applied as adsorbents for the removal of multiple pollutants with different properties from wastewater.In this study, we prepared alginate composite hydrogel beads containing various compositions of biochar produced from pitch pine ( Rapid industrialization in primary industries and increasing industrial activities owing to exponential population growth have exacerbated environmental contamination by the emission of various organic and inorganic pollutants such as heavy metals, dyes, pharmaceuticals, antibiotics, herbicides, and volatile organic compounds . These sRecently, biochar has attracted attention as an alternative adsorbent owing to its low cost, eco-friendly properties, and physicochemical characteristics with various functional groups. Biochar is a carbonic material produced through the thermochemical conversion of sustainable biomass such as weeds, leaves, vermicompost, algae, sludge, sawdust, and manure ,13,14,15Alginate, a biopolymer extracted from brown algae, is a linear polysaccharide composed of two monomeric units: a-L-guluronate (G) and b-D-mannuronate (M). Based on the ratio of the G and M blocks, alginate forms a water-insoluble and thermo-irreversible gel via ionic crosslinking with polyvalent cations or acid treatment ,24. In pAlginate composites incorporating carbonic materials, such as activated carbon, carbon nanotubes, graphene oxide, and biochar, exhibited enhanced specific surface area, porosity, and mechanical strength compared to calcium alginate beads. Moreover, alginate/biochar composites provide a network structure for leakage prevention of fine-sized carbon-rich solids and can be easily separated from liquid treatment systems ,23,25. T2+ and benzene was investigated. The adsorptive behaviors of Cu2+ and benzene onto the alginate/biochar hydrogel beads were also analyzed using various kinetic and isotherm models. Furthermore, the Cu2+ and benzene adsorption capacities of the alginate/biochar hydrogel beads in a single contaminant solution were compared with those of the binary solution. Moreover, the reusability of the alginate/biochar hydrogel beads as adsorbents was evaluated in this study.In this study, alginate/biochar composite hydrogel beads were prepared using pitch pine-based biochar obtained through pyrolysis. The physical and chemical properties of the alginate/biochar hydrogel beads were characterized. The correlation between the compositions  of the beads and their adsorption capacities for CuPinus rigida) was obtained from G-Biotech . All the other chemicals used in this study were of analytical grade and used without further purification.Sodium alginate, calcium chloride, benzene, copper sulfate, nitric acid, ethanol, and HPLC-grade water were purchased from Samchun Pure Chemical Co., Ltd. . Sodium citrate was purchased from Deajung Chemical & Metals Co., Ltd. . Wood powder from pitch pine  using a pyrolysis reactor . The obtained biochar was washed several times with distilled water (DW) to remove ash and impurities and then dried at 60 °C overnight.w/w) sodium alginate and various contents of biochar  were mixed in DW using a mortar and pestle. The alginate solution containing homogeneously dispersed biochar was added dropwise to a 2% CaCl2 solution at a rate of 50 mL/min with a syringe pump  using an 18-gauge needle to obtain millimeter-sized hydrogel beads. The resulting hydrogel beads were cured in a CaCl2 solution for 1 h and then washed with DW three times. The prepared alginate (Alg) and alginate/biochar (Alg/BC) hydrogel beads were stored in DW at 4 °C until further use.For the preparation of adsorbents in the hydrogel state, 4% . The Brunauer–Emmett–Teller (BET) specific surface area was investigated by N2 sorption using TriStar II . Fourier transform infrared spectroscopy (FTIR) measurements were performed by averaging 16 scans per spectrum in the range of 4000–700 cm−1  using an FT/IR-4100 type A spectrometer . The surfaces of the samples were examined using scanning electron microscopy . The functional groups and morphology of the Alg/BC hydrogel beads were analyzed by FTIR and SEM after being frozen at −70 °C and dried at −80 °C under vacuum. The biochar content of the Alg/BC hydrogel beads was measured by dissolving alginate in 100 mM sodium citrate at 60 °C for 24 h. The remaining biochar was weighed after washing with DW and drying at 60 °C.Thermogravimetric analysis (TGA) was conducted from 25 to 900 °C at a rate of 20 °C/min under N4 solution (100 mg/L as Cu2+). For benzene adsorption experiments, 10 mg (dry weight) of adsorbent was placed in a 5 mL glass vial and then 4.5 mL of benzene solution (300 mg/L) was added. Batch adsorption experiments were conducted by shaking at 120 rpm for 24 h at 25 °C. After each adsorption reaction, for Cu2+ measurement, aliquots (200 μL) were periodically collected and centrifuged before dilution with 800 μL of DW. To determine the benzene concentration, aliquots (1 mL) were filtrated using PVDF syringe filter (0.22 μm). Then, the remaining Cu2+ and benzene concentration in samples were analyzed. The amounts of adsorbed Cu2+ and benzene were measured using a UV/Vis spectrophotometer. The Cu2+ content in the solution was measured using Micro BCATM Protein Assay Kits  according to a modified protocol. BSA (Bovine Serum Albumin) solution (10 mg/mL) was added into BCA reagents instead of CuSO4 solution. The prepared BCA working reagents were mixed with the sample and incubated at 60 °C for 1 h. The reacted samples were measured at 562 nm. The benzene concentration was quantified using standard curve  at 254 nm. The adsorption efficiency (qe) of the adsorbent was calculated as:0 and Ce (mg/L) denote the initial and equilibrium concentrations of the adsorption solution, respectively; V (L) denotes the volume of the adsorption solution; and m (g) denotes the weight of the dried adsorbent.10 mg (dry weight) of the adsorbent, that is, biochar or Alg/BC hydrogel beads, was added to 10 mL of CuSO2+ and benzene adsorption onto the Alg/BC hydrogel beads, the pH of the solution was adjusted between 3 and 6 with 0.1 M HCl or 0.1 M NaOH. The co-adsorption of Cu2+ (100 mg/L) and benzene (300 mg/L) onto the Alg/BC hydrogel beads was performed by shaking at 120 rpm for 24 h at 25 °C and pH 6.To evaluate the effect of solution pH on Cu2+ and benzene on Alg/BC hydrogel beads were implemented in Cu2+ (100 mg/L) and benzene (300 mg/L) solutions with 10 mg (dry weight) of Alg/BC hydrogel beads at 120 rpm and 25 °C. The adsorption isotherm studies were conducted in Cu2+ (10–800 mg/L) and benzene (50–800 mg/L) solution for 24 h. Adsorption kinetics and isotherm models were applied to elucidate the results from the adsorption and isotherm experiments using the equations shown in Adsorption kinetics studies for Cu2+ was desorbed from the adsorbents using 0.1 M HCl as the desorption solvent at 120 rpm for 2 h at 25 °C. After the desorption process, the Alg/BC hydrogel beads were regenerated with 2% CaCl2 for 2 h and then washed with DW. Benzene desorption from the adsorbents was conducted using ethanol at 120 rpm for 24 h at 25 °C. The adsorbents with desorbed benzene were washed three times with DW.The adsorbed Cut-test or one-way ANOVA with Tukey’s HSD test using Minitab 16.0 software . Statistical significance was set at a 95% confidence interval.The triplicate data were analyzed by An increase in the pyrolysis temperature enhanced the formation of pores on the surface of the biochar . The sur32−), 1049 (C-O stretching), 1270 (C-OH phenolics), 1430 (C-H bending), 1640 , 1724 (C=O stretching of carboxyl), 2930 , and 3200–3400 cm−1 (-OH band) [OH band) ,19,20. H2+, and benzene were selected as adsorbate models. The adsorption capacity of Cu2+ on biochar decreased with increasing pyrolysis temperature (p < 0.05), whereas the amount of adsorbed benzene on biochar exhibited the highest value (60.4 mg/g) when using biochar obtained through pyrolysis at 550 °C (p < 0.05)  and the higher specific surface area (SSA) of biochar  (2+ (<3 mg/g) compared to that of benzene (>25 mg/g). Thus, the biochar generated at 550 °C was only appropriate for the adsorption of benzene. Hence, the biochar prepared in this study was unsuitable for adsorbing heavy metals such as Cu2+ without chemical activation or modification processes. Therefore, in the following experiments, alginate (a negatively charged biopolymer) was blended with pitch pine-based biochar obtained at 550 °C to improve its heavy metal adsorption capacity.To evaluate the adsorption capacities of the pitch pine-based biochars produced at 350, 550, and 750 °C for heavy metals, BTEX, Cu < 0.05) . These r.4 m2/g) . All bioThe 4% alginate solutions containing various amounts of biochar were made into hydrogel beads of regular spherical shape by the extrusion method using an 18-gauge needle; however, the Alg/BC solution containing more than 10% biochar formed irregular beads. The alginate beads were pale yellow, whereas the addition of biochar to the alginate solution changed them to black . Further2+ and benzene based on the added biochar content. The adsorption capacity of the alginate hydrogel beads for Cu2+ was 54 times higher than that of the biochar generated at 550 °C. The increase in the added biochar content caused a decrease in the Cu2+ adsorption capacity, whereas the adsorption capacity of benzene increased with an increase in biochar. Furthermore, good linear correlations were observed between added biochar content and adsorption capacity . Thus, these results indicated that the adsorption capacities of the Alg/BC hydrogel beads for Cu2+ and benzene were highly dependent on the ratios of alginate and biochar content in the Alg/BC hydrogel beads. Sigdel et al. (2017) showed that powdered activated carbon-impregnated alginate beads exhibited a similar phenomenon for the adsorption of heavy metal ions and aromatic compounds owing to the physicochemical characteristics of adsorbents. Hence, the ratios of alginate and biochar contents can be selectively applied depending on the concentration and types of contaminants in the binary solution.−), 1600 (asymmetric stretching vibration of COO−), and 3200–3400 cm−1 (-OH band), corresponding to the functional groups of alginate [2+ and benzene adsorption. Nevertheless, some changes (shift and/or intensity) may occur after Cu2+ adsorption as reported form the previous literature [2+, Cu2+, and Cd2+) and carboxylate group leads the ability to bind [The FTIR spectra of the alginate and Alg/BC beads  before and after adsorption are shown in alginate ,41,42,432+ and benzene on alginate and Alg/BC hydrogel beads (prepared with 1 and 4% biochar) were conducted in the pH range of 3 to 6. The adsorption capacity for Cu2+ of hydrogel beads consisting of alginate and biochar increased with increasing solution pH  . Sigdel et al. (2017) reported that the solution pH had little effect on the sorption of benzene by alginate beads containing activated carbon. These results imply that benzene adsorption onto the alginate and Alg/BC hydrogel beads progressed through non-electrostatic interactions  in addition to association with functional groups [The initial pH of the solution influences the adsorptive interactions between the adsorbates and adsorbents. Hence, adsorption experiments for Cu < 0.05) a. These l et al. 17 report2+ and benzene onto the adsorbents based on the contact time, respectively. The adsorption of Cu2+ onto all adsorbents rapidly occurred within 8 h and then plateaued . In particular, the alginate hydrogel beads exhibited 1.7 times higher adsorption capacity for Cu2+ than that of Alg/BC hydrogel beads (4% biochar) owing to the higher content of negatively charged carboxyl groups of alginate compared to Alg/BC hydrogel beads (p > 0.05), and the amount of benzene adsorbed onto the Alg/BC hydrogel beads (4% biochar) increased by up to 3.3 times compared to the pure alginate beads  for cross-linking via ion exchange between divalent metal ions and adsorbents to form an egg-box structure. Additionally, negatively charged alginate draws positively charged metal ions via electrostatic interactions [el beads a. In conte beads c. These ractions ,42. In cractions .2+ and benzene adsorption onto the Alg/BC hydrogel beads, adsorption kinetics studies were performed using pseudo-first-order, pseudo-second-order, Elovich, intraparticle diffusion, and liquid film diffusion models. The pseudo-first-order model was used to describe the pure physical adsorption. The pseudo-second-order model indicates that the rate-limiting step is the chemical or physicochemical adsorption. The Elovich model has been applied to the chemisorption of adsorbates on adsorbents [2+ and benzene onto alginate and Alg/BC hydrogel beads followed a pseudo-second-order kinetic model with high correlation values (r2 > 0.99), and the calculated qe values were close to those obtained experimentally. These results show that the adsorption of Cu2+ and benzene onto alginate and Alg/BC hydrogel beads was governed by electrostatic interactions and chemisorption [2+ adsorption using alginate beads containing biochar obtained from pine cones was fitted to the pseudo-second-order model with a high regression coefficient (r2 > 0.99), similar to the results of this study [2+ and benzene onto alginate-activated carbon beads [Typically, the adsorption process consists of multiple mass transfer steps: (1) migration of the adsorbate from the bulk solution to the boundary layer of the adsorbent; (2) external mass transfer between the boundary layer and the surface of the adsorbent (boundary layer and liquid film diffusions); (3) intraparticle diffusion within the adsorbent (pore and surface diffusions); and (4) adsorption of the adsorbate on the active site of the adsorbent via physisorption or chemisorption ,46,47,48sorbents ,45,49,50sorption . In a pris study . Gürkan is study ,43. In pon beads .2+ and benzene onto the alginate and Alg/BC hydrogel beads. It is evident that the qt vs. t0.5 plots were composed of two linear portions, indicating the boundary and intraparticle diffusions, respectively. However, the plots obtained using diffusion models did not pass through the origin. Hence, in the adsorption of alginate and Alg/BC hydrogel beads for Cu2+ and benzene, neither intraparticle diffusion nor liquid film diffusion was the sole rate-controlling step, but these may be simultaneous adsorption rate-controlling steps [The intraparticle and liquid film diffusion models suggest the rate-determining step by investigating whether their linear plots pass through the origin. ng steps ,53.2+ and benzene. Although the adsorption capacities of alginate hydrogel beads for Cu2+ and benzene were 130.9 and 21.5 mg/g, respectively, the adsorption capacities of Alg/BC hydrogel beads  for Cu2+ and benzene were 57.5 and 74.9 mg/g, respectively   indicates surface area and porosity correlated with adsorption capacity of adsorbents. Thus, the dimensionless separation factor (RL) acquired from the Langmuir model means the favorability of adsorption. The isotherm type is divided into unfavorable (RL > 1), linear (RL = 1), favorable (0 < RL < 1), and irreversible (RL = 0), depending on the RL value. In this study, the RL values for Cu2+ and benzene adsorption onto alginate and Alg/BC hydrogel beads ranged between 0 and 1, and thus the adsorption of Cu2+ and benzene on alginate and Alg/BC hydrogel beads is a favorable process.The parameters obtained from the isotherm models are listed in  > 0.99) b,d. Hencm, of alginate hydrogel beads for Cu2+ and benzene was 122.0 and 22.2 mg/g, respectively. The Alg/BC hydrogel beads  exhibited 3.7 times higher maximum adsorption capacity for benzene than that of alginate hydrogel beads. In contrast, the qm of the Alg/BC hydrogel beads  for Cu2+ decreased to 62.5 mg/g. Hence, the addition of biochar to Alg/BC hydrogel beads decreased the qm of the adsorbent for Cu2+ while increasing that for benzene adsorption.The maximum adsorption capacity, q2+ and benzene during multicomponent adsorption onto Alg/BC hydrogel beads , the adsorption reaction was conducted using a binary solution involving both contaminants (Cu2+ and benzene). 2+ and benzene in single and binary solutions. The adsorption capacity of Alg/BC hydrogel beads in single-sorbate solution for Cu2+ and benzene was 68.6 and 25.9 mg/g, respectively. In a simultaneous adsorption system, the adsorption capacity of Alg/BC hydrogel beads for Cu2+ and benzene was 70.8 and 21.8 mg/g, respectively. These results indicated that there was no significant difference in the adsorption ability (p > 0.05). Sigdel et al. (2017) reported similar results for cadmium and benzene adsorption on alginate/activated carbon beads [To confirm the interference effect between Cuon beads . In the 2+ and benzene, respectively. 2+, the residual adsorption capacity of the Alg/BC hydrogel beads was 99.1% of the initial adsorption capacity after five cycles. When a 0.1 M HCl solution was used as the desorption solvent, the desorption efficiency of Cu2+ adsorbed on the adsorbents was more than 95.5% of the adsorbed amount. These results may be due to the regeneration of active sites through ion exchange during the desorption and reconstruction processes [2+ and ranged from 79.1 to 88.4% for the adsorbed benzene. However, the residual adsorption capacity of the Alg/BC hydrogel beads after reuse was similar to or higher compared with the adsorption efficiency of Cu2+ after repeated operation. These results suggest that the Alg/BC hydrogel beads can be used repeatedly during the desorption process using an appropriate desorption solvent for Cu2+ or benzene.To analyze the reusability of Alg/BC hydrogel beads, they were prepared with 1 and 4% BC and used for the removal of Curocesses . The des2+ and benzene, based on the composition of alginate and biochar. Biochar and alginate had positive effects on the adsorption capacities of benzene and Cu2+, respectively. Furthermore, the initial solution pH influenced the adsorption of Cu2+, whereas its effect on the adsorption capacity for benzene was negligible. The pseudo-second-order and Langmuir models were the best-fitting kinetic and isotherm models for the adsorption process on the Alg/BC hydrogel beads, respectively. The Alg/BC hydrogel beads exhibited a trivial difference in adsorption capacities between the single and binary contaminant systems. Additionally, the decrease in the adsorption efficiency of Cu2+ and benzene onto the Alg/BC hydrogel beads was trivial even after four reuses. Hence, the Alg/BC hydrogel beads could be customized to be optimized for the treatment of wastewater containing a variety of contaminants, such as heavy metals, dyes, pharmaceuticals, antibiotics, and toxic organic compounds.In this study, to obtain sustainable adsorbents, pitch pine-based biochar was blended with alginate and fabricated into hydrogel beads. The Alg/BC composite hydrogel beads showed different adsorption capacities for Cu