Naja haje haje venom. The purified N. haje PLA2 showed a specific activity of 22 units/mg protein against 6 units/mg protein for the whole crude venom with 3.67-fold purification. The antiviral activity of purified N. haje PLA2 has been investigated in vitro against bovine coronavirus (BCoV) and simian rotavirus (RV SA-11). Our results showed that the CC50 of PLA2 were 33.6 and 29 µg/ml against MDBK and MA104 cell lines, respectively. Antiviral analysis of N. haje PLA2 showed an inhibition of BCoV and RV SA-11 infections with a therapeutic index equal to 33.6 and 16, respectively. Moreover, N. haje PLA2 decreased the BCoV and RV SA-11 titers by 4.25 log10 TCID50 and 2.5 log10 TCID50, respectively. Thus, this research suggests the potential antiviral activity of purified N. haje PLA2 against BCoV and RV SA-11 infections in vitro.Viral infections are linked to a variety of human diseases. Despite the achievements made in drug and vaccine development, several viruses still lack preventive vaccines and efficient antiviral compounds. Thus, developing novel antiviral agents is of great concern, particularly the natural products that are promising candidates for such discoveries. In this study, we have purified an approximately 15 kDa basic phospholipase A2 (PLA2) enzyme from the Egyptian cobra  Rotaviruses (RV) and bovine coronaviruses (BCoV) are identified globally as the most important enteropathogens responsible for acute viral gastroenteritis leading to diarrhea in infants, young children and animal species. Both types of viruses are important in global health and also in animal industries. At present, no approved antiviral medications are effective in treating infections caused by the two viruses. Therefore, the recovery is primarily dependent on fluid and electrolyte replacement until the infection is resolved , Chikungunya virus (CHIKV), Dengue virus (DENV-2) and Yellow fever virus (YFV) compared to native PLA2. Thus, the necessity of exploring venom and its components as potential alternative therapeutics against viral infections  that catalyze the hydrolysis of phospholipids at sn-2 position producing lysophospholipids and free fatty acids, particularly arachidonic polyunsaturated fatty acid. According to the classification of phospholipases based on their structure, catalytic action and localization, the elapids  and hydrophids (sea snakes) PLA2s belong to secreted PLA2 group IA. On the other hand, vipers and cortalids PLA2s belong to group IIA, except many sPLA2s in Bitis sp. are listed in group IIB . Currently, several studies have demonstrated the potential therapeutic properties of venom enzymes purified from various species of snakes, which are progressively acquiring an attractive interest in their use in the biomedical field ; Moroccan cobra (Naja haje legionis) has lower PLA2 content (3–4%) compared to Nigerian Naja haje (26%)  is still unpublished.Phospholipases A2 are mostly abundant in elapids, particularly Naja haje haje) belongs to Elapidae family and is located in the cultivated areas around the Nile, Delta and Western Mediterranean Coastal Desert. It is one of the most medically important snakes, and its envenomation causes severe neurotoxic and myotoxic symptoms and may lead to death. In a previous study, the crude N. haje venom and its fractions showed moderate virucidal properties against herpes simplex viruses type I and type II (HSV-I and HSV-II)  on rotavirus gastroenteritis and mammalian coronavirus in vitro.The Egyptian cobra . The pooled venom was centrifuged at 10,000×g for 10 min at 4 ℃ to remove debris prior to lyophilization and storage at ‒ 20 ℃ until use. The chromatographic resins and substrates for the biochemical and enzymatic assays were purchased from GE Healthcare, Thermo Scientific and Sigma-Aldrich companies. The reagents were of analytical grade.2 humidified incubator .The Madin-Darby bovine kidney (MDBK) and African Green monkey fetal kidney (MA104) cells were purchased from VACSERA. The Mebus strain of bovine coronavirus (BCoV) as a surrogate model for SARS-CoV-2 was kindly obtained from the Department of Virology, Faculty of Veterinary Medicine, Cairo University. A simian rotavirus SA-11 (RV SA-11) as a surrogate model for human rotavirus was obtained from the Department of Virology, National Institute for Cholera and Enteric Diseases (NICED), Kolkata, India. Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% of heat inactivated fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin under 5% CON. haje venom using two consecutive chromatographic steps on sephacryl S-200 molecular exclusion chromatography followed by CM-sepharose ion exchange chromatography. First, N. haje crude venom (120 mg) dissolved in 1 ml of 50 mM Tris–Hcl buffer, pH 7, was applied to a sephacryl S-200 column (1.6 × 90 cm) previously equilibrated with the dissolving buffer. The elution was carried out with the same buffer, collecting fractions of 3 ml/tube at a flow rate of 20 ml/h at room temperature. All fractions were monitored at 280 nm for protein, and phospholipolytic activity was determined at 900 nm using egg yolk suspension as a substrate according to the Marinetti (The PLA2 enzyme was purified from arinetti  method. SDS-PAGE of the purified PLA2 (Nh-PLA2) was performed according to Laemmli  using 152 dissolved in 50 mM PBS, pH 7.4. After overnight incubation at 37 ℃, the clear zone indicated the presence of PLA2.The purified Nh-PLA2 was run under non-reducing conditions on either a 15% SDS-PAGE or a 7% IEF gel followed by protein transfer onto a nitrocellulose membrane according to the method by Towbin . Blood aIsoelectric focusing gel were performed as described by Garfin , using aThe phospholipolytic activity of the chromatographic fractions was determined turbidimetrically according to Marinetti  method. Protein content was determined according to Bradford  method, 50/0.1 ml) were estimated as described previously by the Karber . After an additional 48 h at 37 ℃ under a humidified 5% CO2 atmosphere, the PLA2 was discarded and 100 μl of MTT solution (5 mg/ml) was added to all wells. After 4 h at 37 ℃, the MTT was carefully removed from the wells and replaced with 50 μl of dimethyl sulfoxide (DMSO). The plates were further incubated for 30 min at 37 ℃. The optical density was read on a multi-well ELISA reader at 540 nm. The 50% cytotoxic concentration (CC50) was calculated as (A‒B/A) × 100, where A and B are equal to the mean of three OD540 of untreated and treated cells, respectively. All experiments were repeated three times and the results were expressed as a percentage of cell viability in comparison to the untreated control cells.The cytotoxicity of the PLA2 was investigated using the MTT assay after 4 days of cell culture according to the previously described protocol by Abid et al. . Briefly6.5 log10TCID50/0.1 ml or RV SA-11 at 106 log10TCID50/0.1 ml were incubated separately with the same volume of three non-toxic concentrations  of PLA2 for 1 h at 37 ℃. One hundred microliters of the above mixture was added to a sub-confluent monolayer cells. After 1 h of incubation, the mixed solution was removed. The cell lines were washed twice with PBS, and then incubated with 200 μl of FBS free DMEM. Cell control (test medium without samples) and virus control (the virus suspension) were included. All plates were incubated for 3 days at 37 ℃ in CO2 incubator or until typical CPE was visible. The 50% inhibitory concentration (IC50) was defined as the PLA2 protein concentration that can protect 50% of viable cells from the cytopathic effect caused by virus and it was calculated by the MTT method as stated above in the cytotoxicity assay by multi-well ELISA reader at 450 nm as [(A‒B)/(C‒B) × 100], where A, B and C indicate the mean three absorbance of the test protein with virus infected cells, positive virus control, and negative cell control, respectively. Then the therapeutic index (TI) was estimated as the ratio of CC50/IC50. For determination of the yield reduction assay, tenfold dilutions of the virus were prepared in FBS free growth medium. One hundred microliters of viral dilutions 10–4–10–9 was mixed and incubated with 100 μl of tested PLA2 at a concentration of 3.75 μg/ml for 1 h. Microscopic examination for CPE was performed after 3 days post-infection. The virus titer as 50% tissue culture infection dose (TCID50) was calculated using the method by Karber (One hundred microliters of BCoV at 10y Karber . The redn = 3). Data were analyzed using the Student’s t test using GraphPad Prism 8.0 software.The results were expressed as Means ± S.D.  and phospholipase A2 activity was monitored in protein peak number 4 as shown in Fig. N. haje PLA2 had a molecular mass of approximately 15 kDa as determined by SDS-PAGE gel   was purified from je venom 20 mg andje venom 20 mg andje venom 20 mg andje venom 20 mg andCrotalus durissus terrificus, inhibited the entry of Dengue virus (DENV), Yellow fever virus (YFV), Hepatitis C virus (HCV), and Chikungunya virus (CHIKV) via direct degradation of viral envelope . Similarly, the Bothrops asper venom has catalytically active sPLA 2 (Mt-I) and the enzymatically inactive sPLA 2 (Mt-II). However, the Mt-I is markedly more potent than Mt-II isoform  against DENV-2  purified from the venom of the Mozambique spitting cobra, Naja mossambica mossambica, exhibited higher antiviral activity against HCV, DENV and Japanese encephalitis virus (JEV) (IC50 in ng/ml) than against Sindbis virus (SINV), influenza A virus (IAV), Sendai virus (SV) and Herpes simplex virus (HSV) (IC50 in µg/ml)  has virucidal activity against the rabies virus. To our knowledge, our study is the first investigation highlighting the virucidal effect of basic Naja haje haje PLA2 against rotavirus (RV) and mammalian coronavirus (BCoV).Currently, specific antiviral agents and vaccines are still insufficient to prevent or control emerging and re-emerging viral illnesses  PLA2, we have the same calcium loop. while the D99 of the catalytic network is absent (data not shown). On the contrary, despite their high catalytic activity, the acidic PLA2 enzymes have less biological effects. Two interesting hypotheses were considered to explain the lack of acidic PLA2 toxicity. The first was offered by Fernandez et al. , Influenza A (IAV), Coxsackie virus B3 (CVB3), HIV, Dengue virus (DENV), Hepatitis C virus (HCV), Herpes simplex virus (HSV), Japanese encephalitis (JEV), Mayaro virus (MAYV), Middle East respiratory syndrome coronavirus (MERS), SARS-CoV-2 virus, as well as they illustrated the mechanisms of inhibition action against several viruses , which spread out in several tissues in the human body, leading to systemic damage. Similarly, other studies demonstrated the pathways where PLA2 interferes with the various stages of virus during replication cycle. sPLA2s have been shown to have the ability to interact with viral capsid , interfere in viral adsorption , interfere in internalization , and interfere in host cell components   enzyme from the Egyptian cobra


CD73, CD90, and CD105 were highly expressed, and CD14, CD19, CD34, CD45, and HLA-DR were barely expressed in rPBMSCs. rPBMSCs possessed the potential to differentiate into chondrocytes, adipocytes, and osteoblasts under their respective induction conditions. Cell growth curve and viability experiments were performed under hypoxic conditions: 19% O2, 5% O2, and 1% O2. Specifically, 5% O2 accelerated the proliferation and expression of the stemness of PBMSCs. Cycle experiments proved that hypoxia promoted the cell transition from the G1 phase to the S phase. Molecular experiments confirmed that 5% O2 hypoxia significantly elevated the expressions of hypoxia-inducible factor 1α and β-catenin and simultaneously the expressions of cycle-related genes including CyclinE/CDK2 and stemness-related genes including Nanog and SOX2. The appropriate concentration of hypoxia  enhanced the proliferation and stemness of rPBMSCs and increased the multidirectional differentiation potential of stromal cells. The proposed culture method could improve the viability and maintain the phenotype of rPBMSCs in cartilage or bone tissue engineering.This study aimed to address the dilemma of low peripheral blood-derived mesenchymal stromal cell (PBMSC) activity and reduced phenotype in bone or cartilage tissue engineering. Rat PBMSCs (rPBMSCs) were obtained by density gradient centrifugation, and stromal cell characteristics were confirmed by flow cytometry (FCM) and multi-differentiation potential induction experiments. Cell growth curve, viability experiments, and clone formation experiments were performed by [3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) and cell counting, and the cell cycle was confirmed by cell FCM. The proliferation signal pathway and stemness-related proteins were detected by molecular methods including Western blot and real-time polymerase chain reaction.  In recent years, mesenchymal stromal cells (MSCs) derived from adults have been widely used not only for bioregenerative tissue engineering but also for pathophysiological research and cell and gene therapy of bone diseases –4. Many CD105, CD73, and CD90 and negatively express CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA-DR. MSCs also have the potential to differentiate into chondrocytes, osteoblasts, and adipocytes   , transfor (bFGF) , and oxyr (bFGF) –14. Hypor (bFGF) . To enhar (bFGF) . In the red MSCs .2  were seeded onto the T-25 flask with 10 mL of complete Dulbecco’s Modified Eagle Medium . The complete medium contained 1% penicillin/streptomycin (Gibco), 20 ng/mL bFGF , and 20% fetal bovine serum (Gibco). With 21 days of culture, the cell convergence was 80%, and the third-generation cells digested by 0.25% trypsin were used for subsequent experiments. Representative bright-field images were captured by an inverted phase-contrast microscope .The animal ethics committee of Guangzhou Red Cross Hospital approved the research. Following previously published methods , a 3 cm CD73, CD105, and CD90  were selected as positive markers of rPBMSCs, whereas CD14, CD19, CD34, CD45, and HLA-DR  were chosen as negative markers of rPBMSCs. rPBMSCs (2×105 cells/mL) were resuspended in PBS and mixed in CD14, CD19, CD34, CD45, CD73, CD90, CD105, and HLA-DR antibody solutions for 30 min, and the cell samples were then loaded on the machine for analysis.The cell immune phenotypes of third-generation PBMSCs (P3 PBMSCs) were identified by flow cytometry (FCM). 2) conditions for 24 h were collected, and rPBMSCs were then fixed with 70% (V/V) ethanol overnight. Moreover, 50 μg/mL propidium iodide (PI)  was diluted by PBS solution containing 1% Triton X-100. Cells were fully infiltrated in the freshly prepared PI solution for 30 min and were analyzed by a BD FACScan flow cytometer .rPBMSCs treated under normoxic and hypoxic . The differentiation was evaluated by alcian blue staining. For osteogenesis, the cultures were induced with an osteogenesis induction medium kit . After culture for 21 days, alizarin red staining was performed to evaluate the osteogenic products. For adipogenesis, cells were induced for 21 days in an adipogenesis induction medium kit . The formation of lipid vacuoles was assessed by Oil Red O staining. All images were captured under an inverted phase-contrast microscope .P3 rPBMSCs were seeded into a 24-well plate at a density of 2 × 104/well and cultured at 37°C in a 21% O2 and 5% CO2 incubator or a 5% O2 and 5% CO2 incubator. The induction medium and experimental procedures performed in the subsequent experiments were the same as the methods described in “Multilineage Differentiation Potential Assay.”P3 rPBMSCs were seeded into a 24-well plate at a density of 2 × 103/well) were inoculated in microplates (24-well) in 5% CO2 incubators with a gradient concentration of oxygen at 37°C. The experiments were set up as the control group (21% O2 and 5% CO2), 19% O2 and 5% CO2 hypoxia group, 5% O2 and 5% CO2 hypoxia group, and 1% O2 and 5% CO2 hypoxia group, with three replicate wells in each group. Starting from the next day, each group of cells was digested and counted accurately with a cell counter at each time point (Days 1–8). The growth curves of each cell group were made according to the number of cells. For the MTS assay, the above-mentioned groups of cells were planted on the well plate after Day 8, and the absorbance was measured at 450 nm by a multifunctional microplate reader .P3, P5, and P6 rPBMSCs  value using the following formula :CPD was plotted against time in culture and performed in triplicate for each counting procedure.Moreover, 500 rPBMSCs were cultured in 6-well plates in an incubator capable of adjusting oxygen concentration for 14 days. After fixation with paraformaldehyde for 15 min, 1 mL of crystal violet staining solution was added to the culture plate for staining clones for 30 min. Under an inverted phase-contrast microscope , the number of clones containing more than 50 cells was counted.2) treatments for 5 days, and whole-cell lysates were prepared for Western blotting in radioimmunoprecipitation assay buffer. Then, 30 μg of protein was loaded into the sample well, dispersed in the gel according to the molecular weight, and directly transferred to the poly(vinylidene fluoride) membrane  in a band-to-band manner through the semi-dry transfer method. The membranes were immersed in a square dish filled with primary antibody diluent. These antibodies  were purchased from Cell Signaling Technology (MA), and when used, the dilution ratio was 1:1000. On the next day, membranes were incubated with secondary anti-rabbit/mouse IgG, HRP-linked antibody . The electrochemiluminescence detection mixture was used to detect the protein on the membranes. ChemiDoc XRS imaging system with Image Lab software (Bio-Rad) was used to analyze the graphs.rPBMSCs were collected after normoxic and hypoxic (5% O4/well rPBMSCs were seeded in glass slides placed in plates treated under normoxia and hypoxia (5% O2) for 5 days. After sequential fixation, blocking, incubation of primary  and secondary  antibodies, a fluorescence microscope  was used to observe and capture pictures of interest.Furthermore, 10-ΔΔCt formula . With reference to the instructions, SYBR-Green reagent (Takara Bio) was used to perform real-time PCR in triplicate in a fluorescence quantitative PCR instrument . GAPDH was used as a control to analyze relative gene expression in the 2 formula . Primer Data in three replicates are presented as mean ± standard deviation. Student’s t-test or one-way analysis of variance was used to analyze differences between the two groups and among multiple groups; P < 0.05 was used to mark significant differences.In this study, rPBMSCs were successfully isolated and cultured. Stromal cell characteristics were proved by FCM and multi-differentiation potential induction experiments. The cell growth curves of P3, P5, and P6 rPBMSCs cultured under different oxygen concentrations were drawn based on the number counted at each time point. Then, the 5% hypoxia condition that significantly promoted cell growth was used for subsequent experiments. Hypoxia (5%) significantly increased the number of stromal cell clones and the proportion of S phase cells. Real-time RCR and Western blot results revealed that hypoxia (5%) significantly promoted the expressions of HIF-1α, β-catenin, and proliferation-related and stemness-related genes.CD73, CD90, and CD105, extremely low expressions of CD14, CD19, CD34, CD45, and HLA-DR , fifth-generation PBMSCs (P5 rPBMSCs), and sixth-generation PBMSCs (P6 rPBMSCs). After 3 days, hypoxia (5% O2) significantly increased the number of cells and proliferation rate of P3, P5, and P6 rPBMSCs. After 8 days, the cells approached the plateau stage. At this time, the number of P3 rPBMSCs in the control group, 19% O2 hypoxia, 5% O2 hypoxia, and 1% O2 hypoxia groups were 55 × 103, 70 × 103, 96 × 103, and 71 × 103, respectively. Statistical analysis showed that compared with the number of P3 rPBMSCs in the control group, those in the 19% O2 hypoxia, 5% O2 hypoxia, and 1% O2 hypoxia groups were increased significantly (P < 0.05). Compared with 19% O2 hypoxia and 5% O2 hypoxia, 1% O2 hypoxia further increased the number of P3 rPBMSCs (P < 0.05). Similar to the growth curve, 5% O2 hypoxia significantly promoted the absorbance of P3 rPBMSCs seeded at Day 8. The CPD curve of P6 PBMSCs proved that the CPD value of PBMSCs in the 5% hypoxia group was significantly higher than that in the normoxia group. Compared with the normoxia group, the CPD values for P6 PBMSCs in the 19% O2 hypoxia and 1% O2 hypoxia groups did not change significantly on Day 7. The shape of the growth curve and viability of P5 and P6 rPBMSCs were similar with those of P3 rPBMSCs, but the amounts of rPBMSCs and optical density values of P5 and P6 on Day 8 were lower than those of P3 rPBMSCs. Based on the cell growth curve and MTT assay results, 5% O2 hypoxia was selected for subsequent experiments. As presented in 2 hypoxia significantly promoted the formation of rPBMSC colonies. The number of rPBMSC colonies in the 5% O2 hypoxia group was increased by 53% compared with that in the control group (P < 0.05).The results of the experiments are displayed in 2). Hypoxia exerted a significant increase and decrease in the number of S phase and G1 phase cells, respectively (2 hypoxia increased the percentage of rPBMSCs in the S phase from 27.26% to 46.32% (P < 0.05) and reduced the percentage of rPBMSCs in the G1 phase from 58.24% to 47.19% (P < 0.05). These data indicated that 5% O2 hypoxia increased the DNA synthesis and cell cycle of rPBMSC progression at the S phase. After 21 days of culture with a differentiation agent under hypoxia or normoxia, the effect of hypoxia on the pluripotency of rPBMSCs was investigated. FCM was used to investigate the cell cycle transition of rPBMSCs treated under normoxia and hypoxia (5% O2 hypoxia significantly increased the HIF-1α (red) nuclei expression in rPBMSCs, compared with the control rPBMSCs. Simultaneously, the nuclei expression for β-catenin (green) in rPBMSCs was also upregulated significantly with 5% O2 hypoxia. Immunohistochemistry results  successful isolation, culture, and identification of rPBMSCs; (2) proliferation, phenotype maintenance, and differentiation potential of hypoxia-treated rPBMSCs; and (3) proliferation- and phenotype-related gene expressions of hypoxia-treated PBMSCs. This study proposes a culture method that is conducive to maintaining self-renewal and proliferation capabilities to ensure cell yield and long-term expansion.As a typical representative of adult pluripotent stromal cells, PBMSCs provide various possibilities for clinical application and transformation in the field of regenerative medicine . PBMSCs n vitro . Thus, tCD45, CD34, CD14, CD19, and HLA II, but highly expressed CD73, CD90, and CD105, indicating that the rPBMSCs had stromal cell performance, without other surface antigen markers  culture can be used as a convenient strategy to maintain the function of rPBMSCs. The general process and conclusions of this study are clearly illustrated in The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.This animal study was reviewed and approved by animal ethics committee of Guangzhou Red Cross Hospital.Study design: PW; Data collection: PZ; Data analysis: CY and JW; Interpretation of data: PW; Draft manuscript: PW; Review manuscript: PW. All authors contributed to the article and approved the submitted version.This work was supported by the Medical Science and Technology Research Foundation of Guangdong , Traditional Chinese Medicine Bureau of Guangdong Province , and Guangdong Provincial Basic and Applied Basic Regional Joint Fund .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Psychosocial care centers for alcohol and drug users (CAPS-ad) are reference services for treatment of drug users within the Brazilian National Health System. Knowledge of their total costs within the evidence-based decision-making process for public-resource allocation is essential. The aims here were to estimate the total costs of a CAPS-ad and the costs of packages of care (according to intensity of care); to ascertain the ratio between total CAPS-ad costs and the federal funding allocated; and to describe the methods for estimating unit costs for each CAPS-ad cost component. Retrospective study conducted in a public community mental health service. This was a retrospective cost description study on a CAPS-ad located in a city in the state of São Paulo, using a public healthcare provider perspective and a top-down approach, conducted over a 180-day period from March 1 to August 30, 2015. The total mean monthly costs of the CAPS-ad were BRL 64,017.54. Healthcare staff accounted for 56.5% of total costs. The mean costs per capita and per month for intensive and non-intensive care packages were, respectively, BRL 668.34 and BRL 37.12. The federal budget allocation covered 62.1% of the CAPS-ad costs and the remaining 37.9% end up funded by the municipal government. The cost of the intensive package of care was 18 times greater than the non-intensive package. Developing criteria for using services and different packages of care based on patients’ needs, and optimizing human resources according to specific actions, may improve people’s mental health and avoid wasted resources. The additional objectives were to ascertain the ratio between total CAPS costs and the federal funding allocated to this service and to describe the methods for estimating the unit costs for each CAPS cost component.This was a cross-sectional study that was conducted to estimate the total costs of a CAPS-ad located in the city of Rio Claro, state of São Paulo, covering a 180-day period from March 1 to August 30, 2015. Data on costs were extracted from the municipal accounting database and a top-down approach was applied for the cost estimation. The CAPS-ad costs were estimated for a 180-day period in the year 2015 and then the ratio between costs funded by the federal and municipal governments was examined. The 180-day period was chosen because this provided the best data quality in the administrative database that was made available by the municipal government. This study adopted the perspective of the public healthcare provider because there are no national or regional published data on the unit costs and cost components of this kind of service in Brazil.This study was approved by the Research Ethics Committee of the Federal University of São Paulo , under protocol number 0296/15.A CAPS-ad is a community-based mental health service that promotes public comprehensive care for people with substance-related disorder. The CAPS-ad that was studied here covers a population of approximately 215,960 people. It welcomes spontaneous and referred demand, is integrated with primary care and psychiatric and general hospitals for acute inpatient hospitalizations. This service is one of five that, together, make up the municipal mental health setting: another two CAPS, of which one deals with all mental disorders and is open 24 hours per day and the other deals with children and adolescents; and two outpatient services. The service that is the focus of the present study offers treatment through three types of package of care: intensive, semi-intensive and non-intensive.Individuals with severe use of drugs are generally directed towards the intensive package of care. The semi-intensive package of care is for individuals who seek the service presenting moderate use of drugs that puts them at risk of greater worsening of their functioning; or those who migrate to an intermediate treatment after showing improvements through intensive treatment. The non-intensive package of care offers support for users who have good social and family ties, those who make moderate or severe use of drugs but who work and can only attend the service at specific times, or those who are in the process of leaving the service because they have presented improvements.The following CAPS-ad cost component categories were considered:Healthcare staff costs: These were the costs of healthcare professionals working at this center, including two psychiatrists, one general practitioner, one nurse, two nursing technicians, two psychologists, two occupational therapists and one social worker. Firstly, their total costs over a 180-day period were estimated. Secondly, unit costs were determined. Lastly, the costs for each healthcare staff intervention within the care packages were estimated.Medication costs: These were categorized as the costs of psychotropic and non-psychotropic medications. Firstly, the per capita monthly costs of medication consumption were obtained. Secondly, the per capita monthly costs were extrapolated to the 180-day period, considering the mean number of patients assisted by the service over this period (810 patients). Then, the unit costs were defined.Revenue costs: These were the costs of support services (diet service: lunch and snacks), utilities , consumables , non-healthcare human resources  and overheads . Their costs were estimated considering the number of working hours and salaries.Capital costs: These consisted of rent, equipment and building repair. Equipment costs were adjusted according to the consumer price index by using the market price for 2015 (presented as Supplementary .Medication funding package (arrow 1) and the Medium and High-Complexity healthcare funding package (arrow 2).Medication funding package is firstly allocated to the state government for drug purchasing.It is expected that CAPS-ad will be funded from federal, state and municipal public healthcare budgets . The fedMedium and High Complexity healthcare funding is allocated directly to the municipal healthcare budget to fund CAPS-ad and other healthcare services. In 2015, the federal government released Brazilian reais (BRL) 39,780.00 per month to fund each CAPS-ad within the national territory, and the municipal healthcare budget funded the remaining costs, which were not publicly known until the analysis of the present study. In The intensive package of care (IPC) offered support from Monday to Friday, from 8 am to 5 pm in this CAP-ad. This one-month package included: one visit to a psychiatrist, one visit to a general practitioner, 12 group sessions with an occupational therapist, 12 group sessions with a psychologist, 10 group sessions with a social worker, six group sessions with a nurse, 20 individual routine nursing care sessions and eight individual sessions with a health case manager, approximately. One visit to a psychiatrist or general practitioner was estimated to last for 30 minutes. The group sessions were considered to last 90 minutes and each session was expected to cater for a mean of 10 patients per session. An individual session was estimated to last for 60 minutes, and an individual routine care session with an assistant nurse care was estimated to last for 15 minutes. The health case manager could be an occupational therapist, a nurse, a social worker or a psychologist, and their individual sessions were estimated to last for 60 minutes.The semi-intensive package of care (SIPC) was delivered on three days a week. This one-month package included: one visit to a psychiatrist, one visit to a general practitioner, eight group sessions with an occupational therapist, eight group sessions with a psychologist, four group sessions with a social worker, four group sessions with a nurse, 12 individual routine care sessions with a nurse and four individual sessions with a health case manager, following the same patterns of length as mentioned previously.The Patients attending the IPC and the SIPC sessions received three meals per day during the treatment: breakfast, lunch and afternoon snacks.non-intensive package of care (NIPC) included one visit to a psychiatrist and one weekly group session with a health case manager. Meals were not included in this package.The one-month Descriptive analyses on cost distributions were carried out for each cost component. Unit costs were calculated by means of a top-down approach, and unit costs for each mental health intervention per individual were estimated, considering the average length of one visit and one-tenth of the costs of a group session. The ratio between funding sources was obtained by calculating the cost difference between the total CAPS costs and the federal government funding. Sensitivity analysis was carried out both on the total costs of the CAPS-ad and on the total costs of the packages of care, in order to enable transferability of the study results to other Brazilian regions. Regarding the total costs of the CAPS-ad, a 15% cost variation in healthcare staff costs The mental health treatments offered by the CAPS-ad and their respective costs are described in Regarding capital costs over the 180-day period, the rent cost was BRL 6,826.68, or BRL 37.92 per day, and the maintenance/repair cost was BRL 125.50, or BRL 0.70 per day. The total cost of equipment was BRL 6,500.00, or BRL 750.67 over the 180-day period and BRL 4.17 per day. The total capital cost over the 180-day period was BRL 7,702.85 .Considering healthcare staff, revenue, medications and capital costs, the CAPS-ad costs over the 180-day period were BRL 384,105.27, i.e. an average of BRL 64,017.54 or USD 20,131.54 (using PPP) per month. Healthcare staff accounted for 56.5% of the total CAPS-ad costs, revenue costs 24.3%, medication costs 17.2% and capital costs 2%. In the same year, the federal funding allocated to the municipal government for financing the CAPS-ad was BRL 39,780.00 per month or BRL 238,680.00  over the 180-day period.Over the 180-day period, the CAPS-ad offered assistance to approximately 810 patients. Thus, the mean monthly CAPS-ad treatment cost per capita was BRL 474.20 . BRL 294.47 was funded by the federal government and BRL 179.73 by the municipal government.Data on the care packages are shown in The Brazilian federal government spent BRL 39,780.00 per CAPS-ad in 2015,Healthcare staff accounted for 56.5% of the total CAPS-ad cost. This result was close to the findings of Araujo et al.,The Rio Claro municipal government funded 37.9% of the monthly CAPS-ad costs, or BRL 64,017.54. In that same year, the total budget of the city council was BRL 698 million, and 30% (BRL 209 million) was invested in healthcare according to the official city council newspaper.,Although the federal government’s budget covered a large part of the CAPS-ad costs, there have been no adjustments for inflation since 2011,The monthly CAPS-ad treatment cost per capita was almost three times lower than the amount paid per month  by the São Paulo state government to provide inpatient treatment for substance users at private clinics through the “restart program” (Programa Recomeço).,The unit costs reported here may inform further economic evaluations and modelling studies within similar contexts of services in the state of São Paulo. This is especially important, considering the lack of information on unit costs within healthcare in Brazil. This is unlike the situation in some European countries, where national guidelines for unit costs reference for healthcare services and interventions are available, thus facilitating development of cost studies and economic evaluations.,-The expansion of the CAPS network in Brazil,According to the current legislation on public health financing in Brazil,It is necessary to ascertain municipal governments’ capacity to manage the costs of these services, in order to plan public investment in services that can be sustained over time. After the costs of this type of service have been established, the discussion can shift from focusing on coverage to analysis in greater depth, including in relation to the cost-effectiveness of services and interventions.The present study has three important limitations:the state government may have participated in the purchase of medications prescribed by CAPS-ad doctors, and thus its participation in the CAPS-ad funding system may have been underestimated;the study period may not have detected possible fluctuations in costs over the twelve months of the year; andthere is some uncertainty regarding inaccuracies of cost estimations, given the variations in healthcare staff salaries, revenue and medication costs according to the different regions of Brazil. These limitations impede generalizations.The federal government funded two-thirds of the CAPS-ad costs, while one-third was funded by the municipal government. These findings may enable better planning and management, both for the federal government and for municipal governments that are interested in expanding the CMHS network for people with substance-related disorders. Moreover, these findings also highlight the need for government agencies and the national academic community to focus on mental health policies, not only to expand treatment coverage, but also to attain the best allocation of resources, in terms of costs and outcomes. Careful use of packages of care based on patient needs can improve people’s mental health and avoid wasting resources.


SARS-CoV-2 is a lipid-enveloped Betacoronavirus and cause of the Covid-19 pandemic. To study the three-dimensional architecture of the virus, we perform electron cryotomography (cryo-ET) on SARS-Cov-2 virions and three variants revealing particles of regular cylindrical morphology. The ribonucleoprotein particles packaging the genome in the virion interior form a dense, double layer assembly with a cylindrical shape related to the overall particle morphology. This organisation suggests structural interactions important to virus assembly. Electron cryotomography expands our knowledge of SARS-CoV-2 virus architecture and reveals a distinct and uniform morphology for which interactions between RNP subunits within the nucleocapsid and between the nucleocapsid and the membrane are likely to be important Cryotomography of frozen-hydrated virus particles enables the visualisation of particle exterior and interior in three dimensions and thus is an important method for understanding the three-dimensional architecture of pleomorphic lipid-enveloped viruses. These previous studies have described SARS-CoV-2 virions as spherical or ellipsoidal and in addition have mainly visualised the spike protein (S) in pre-fusion and post-fusion states on the virus surface and ribonucleoprotein assemblies in the virus interior. Other virus structural components such as the membrane protein (M), the envelope protein (E) and additional non-structural proteins may play important roles in virus structure and assembly.SARS-Cov-2, the causative agent of Covid-19, has been the object of intense investigation, including structural studies by cryo-EM of individual component proteins at high-resolution, as well as cryotomography of virusesBecause a number of fundamental questions remain about the virus architecture, such as how virion components interact in three-dimensions to assemble particles and how the large positive strand genome is packaged, we apply cryotomography to study the architecture of virions of the original Wuhan strain of SARS-CoV-2 and three variants that arose during the first year of the pandemic. The tomograms show the particles are of a uniform cylindrical shape with spike proteins distributed over the whole envelope. The interior of the particle reveals an organised RNP assembly with implications for virus assembly.3 and other coronaviruses8. A gallery of Wuhan virion cross-sections is shown in Fig. We recorded tilt-series and reconstructed cryotomograms of frozen-hydrated SARS-CoV-2 virions from the original Wuhan strain and Alpha (B.1.1.7), Beta (B.1.351) and Delta (B.1.617) variants. The tomograms show that the virions are predominantly of a single and uniform morphology which is an extremely flat cylindrical shape. A slice through a tomogram of Wuhan strain virions shown in Fig. We have measured the three principal axis dimensions as shown in Fig. 3 and which we also observed for the variants . We placed copies of the pre-fusion spike map at their locations in the virion tomograms  up  that package the genome to form the nucleocapsid. The nucleocapsid has a cylindrical shape that matches the particle geometry. By comparison, particles described at budding sites in the ERGIC compartment of the ER have less compact interiors3, and are also similar to particles assembled in vitro from N (nucleocapsid) protein and RNA16. The side cross-section shows compact packing of RNP subunits within a layer and typically staggered packing between layers  as shown in Fig. ers Fig. . We makeers Fig. . By comp8. A membrane association of the nucleocapsid cylindrical circumference is also apparent in virions with more ellipsoidal shapes . In the side-view cross-section, the nucleocapsid is only contiguous with the membrane at the highly curved ends Fig. , and thepes Fig. . These apes Fig. .17.RNP-RNP interactions may themselves form a cylinder-shaped condensate that interacts with a membrane component and shapes the surrounding membrane. Alternatively, membrane-associated proteins that are organised by interaction with the RNP assembly may also play a role in creating membrane curvature and determine particle morphology. These association properties are consistent with biochemical characterisation of the nucleoprotein, N, which has been observed to interact and form condensates with RNA or M protein, potentially in annular structures, and to interact with membranes via the M proteinOur observations of a distinct and uniform morphology of the SARS-CoV-2 virion and its organised interior indicate the importance of interactions between RNP subunits within the nucleocapsid, and between the nucleocapsid and membrane components. During the assembly of new virus particles these interactions may determine the overall shape of the virion and may also play roles in particle disassembly following cell entry by membrane fusion.Four different viral strains were obtained as follows:Wuhan (hCoV-19/England/02/2020), (GISAID EpiCov™ accession EPI_ISL_407073) from The Respiratory Virus Unit, Public Health England (PHE), UK.Alpha variant B.1.1.7 (hCoV-19/England/204690005/2020) from PHE through Professor Wendy Barclay, Imperial College, London, UK.Beta variant B.1.351 (501Y.V2.HV001) from the African Health Institute, Durban, South Africa.Delta variant B.1.617.2 (GISAID accession EPI_ISL1731019) from Professor Wendy Barclay, through Genotype-to-Phenotype National Virology Consortium.All viruses were grown at the World Influenza Centre, Francis Crick Institute, London, UK under Biosafety level 3 conditions in Vero V1 cells, provided by Professor Steve Goodbourne, St George’s Hospital, University of London, UK and maintained in Dulbecco’s Modified Eagle Medium (DMEM) Gibco™ 41965039, with 100 U/ml penicillin, 100 μg/ml streptomycin (Pen-Strep) and 10% (v/v) heat-inactivated fetal calf serum (FCS).Vero V1 cells were infected at low multiplicity of infection (MOI) of 0.0001PFU per cell with viral seed stock and incubated as above for 4-5 days until about 90% cytopathic effect (CPE) was observed before the supernatant was harvested. The low MOI and long incubation time was chosen to ensure a multiple cycle infection thus maximising the proportion of infective to defective virus particles that are produced with low cycles of infection. The length of incubation time was experimentally identified to maximise the virus yield. The supernatant was harvested and clarified twice by centrifugation at 3180 g for 30 mins at 4 °C in an Allegra 12-R centrifuge, SX4750 rotor. Virus was pelleted from the supernatant through 30% (w/v) sucrose in 10 mM MOPS, 150 mM NaCl, 1 mM EDTA, pH 6.8 (MES buffer), spun at 111,063 g for 90 mins at 4 °C in an Optima XPN-90 Beckman ultracentrifuge, SW32Ti rotor. Viral pellets were resuspended overnight in MES buffer at 4 °C and then formaldehyde added to a final concentration of 4% (v/v), incubated at 20 °C for 20 mins and then stored overnight at 4 °C.See Supplementary Table All virus preparations were screened by negative stain EM.2 μl drops of sample were absorbed to a carbon-coated 400 mesh copper grid (TAAB). After 30 s the grid was floated onto water, sample side in contact with the water for 30 s, then transferred to 1% sodium silicotungstate pH 7.5 (Agar Scientific) for 30secs. The grid was air dried, viewed and imaged at a pixel size of 7 Å with a Technai Spirit TEM (FEI) operated at 120 kV, with an Eagle 4 K detector (FEI).Virus samples were mixed with 10 nm Protein A gold (BBI Solutions) in a ratio of 4:1 by volume. 3.5 μl drops were applied to air glow-discharged 200 mesh copper Quantifoil R2/2 grids and plunge frozen in liquid ethane using a MkIV Vitrobot (Thermo Fisher) operated at 90% humidity, 4 °C.See Supplementary Table -/Å2.The Talos microscope, fitted with a Falcon 3 camera, was operated at 200 kV. Tomograms were collected using the Thermo Fisher Tomography 5 software in linear mode, using a dose-symmetric scheme at 3° intervals to + /− 57° at a pixel size of 2.58 Å, and total dose of 94e-/Å2.The Titan Krios microscope is fitted with a Gatan GIF Quantum energy filter operated in zero-loss mode with a 20 eV slit width, and a K2 Summit detector (Gatan) operated in counting mode. Dose-symmetric tilt series were aquired using the Thermo Fisher Tomography 5 software at 3° intervals to + /−60°, using a defocus range of −2.5 to -5um, at a pixel size if 2.22 Å and 4 frames per tilt image. The total tomogram dose was 98e18 and reconstructed with SIRT, 5 iterations. Resulting tomograms were binned 4x and filtered with 10 iterations of Nonlinear Anisotropic Diffusion.Talos-acquired tilt series were fiducial aligned with the IMOD package19, tomograms were CTF corrected by phase flipping and reconstructed with novaCTF20, producing a weighted back-projection tomogram and a SIRT-like filtered tomogram. For visual analysis the SIRT-like filtered tomograms were binned 4x and subjected to 20 iterations of Nonlinear Anisotropic Diffusion filtering using the Bsoft21 program bnad with default parameters (λ = 0.1).Movie frames of Krios acquired tilt series were motion corrected, dose-weighted and fiducial-aligned using the IMOD package. The contrast transfer function was estimated with CTFFIND422. Particles were then extracted from the unbinned, CTF-corrected WBP tomograms with 4-fold downsampling  using Relion 3.123. Reference-free alignment of all subtomograms produced an initial map which was used as a reference for further 3D classification and alignment. A loose, soft mask around the outer surface of the virion envelope was used during further alignment, classification and postprocessing. The average map of 2-layered virions was estimated to have a resolution of 44 Å at the FSC = 0.143 cutoff by Relion postprocessing. After the alignment of all subtomograms, 3D classification into 5 classes without alignment was then carried out to examine the varying morphologies of the virions.268 whole virions from 20 tomograms were picked manually from the 4-fold binned SIRT/NAD-filtered tomograms using EMAN 2.9122, aligned against the Relion map and analysed using PCA-based classification, starting from 3 initial basis vectors and requesting 3 output classes.For PCA analysis, the virion subtomograms were re-imported into EMAN 2.91Spike particles were picked manually from the 4-fold binned SIRT/NAD-filtered tomograms using IMOD. 4418 particles from 251 virions in 18 tomograms were then extracted from the full-size WBP tomograms with 2-fold downsampling  for subtomogram averaging in Relion 3.1. Reference-free initial model generation in C1 produced a map with clear 3-fold features, which was symmetrised and used as a reference for further refinement with C3 symmetry applied and with a loose mask around the ectodomain. Upon convergence of refinement, the particles were reclassified with relaxation of C3 symmetry, revealing a class with the 1-RBD-up conformation. The classes were separated and the closed conformation particles were finally refined with C3 symmetry, while the 1-RBD-up particles were refined without symmetry. The final resolutions were estimated as 30 Å (closed conformation) and 28 Å (1-RBD-up conformation) at the FSC = 0.143 cutoff by Relion postprocessing.The subtomogram averaging maps were backplotted into the frame of reference of the original tomogram using in-house scripts (available from the authors). Spike particles were visually inspected and removed if they were misaligned  or duplicate particles which converged to the same position during alignment.Number of biological replicates and measurements made are given in Supplementary Table Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary DataSupplementary Data 1Reporting Summary-New


We then study the impact of these additional degrees of freedom on viral wrapping. We find that cells with an optimal density of filamentous extracellular components (ECCs) are more likely to be infected as they uptake the virus faster and use relatively less cell surface area per individual virus. At the optimal density, the cell surface folds around the virus, and folds are faster and more efficient at wrapping the virus than crumple-like wrapping. We also find that cell surface bending rigidity helps generate folds, as bending rigidity enhances force transmission across the surface. However, changing other mechanical parameters, such as the stretching stiffness of filamentous ECCs or virus spikes, can drive crumple-like formation of the cell surface. We conclude with the implications of our study on the evolutionary pressures of virus-like particles, with a particular focus on the cellular microenvironment that may include filamentous ECCs.Nanoparticles, such as viruses, can enter cells via endocytosis. During endocytosis, the cell surface wraps around the nanoparticle to effectively eat it. Prior focus has been on how nanoparticle size and shape impacts endocytosis. However, inspired by the noted presence of extracellular vimentin affecting viral and bacteria uptake, as well as the structure of coronaviruses, we construct a computational model in which  Viruses are relatively simple in structure as they consist of a container and genetic material. The container material ranges from solid-like proteins to fluid-like lipids , 2. For Since the contents of a virus are minimal, in order to replicate, viruses must enter cells to hijack their biological machinery. Viruses enter cells via multiple pathways. Two of the dominant pathways for coronaviruses are membrane fusion, and endocytosis , 13. In both the cell-like construct and the virus-like construct contain their own filamentous structures protruding from their surfaces and study the impact of these additional degrees of freedom on viral wrapping. Indeed, much research has focused on how the size and shape of the virus impact viral wrapping [outside of a cell assist in viral wrapping?Following our initial mostly experimental study of how extracellular vimentin impacts viral uptake , here, wwrapping –24 with wrapping . In addiwrapping . Filamenwrapping , 28. HowTo begin to answer the above question, we have organized the manuscript as follows. We first present the computational model with several simplifying assumptions. The first is that the cell surface is modeled as a deformable sheet with bending and stretching energy costs, representing the underlying cell cortex supporting the lipid bilayer. Second, as extracellular vimentin is our main filamentous protein candidate, vimentin self-assembles into filamentous, hierarchical structures whose tetramers are six times longer than the thickness of a cell membrane . There iTo understand the role of the filamentous structures on the outside of both a cell and a virus on viral uptake, we model viral wrapping via coarse-grained Brownian dynamics simulations. Here are the relevant players and how they are incorporated into the model and their respective interactions with the other players.rij is the distance between nearest neighbors and lo is the edge length of the equilateral triangle. Bending rigidity is introduced in the surface with a spring interaction between second nearest neighbors rik is the distance between second nearest-neighbors and We model the cell surface as a deformable sheet, as shown in Again, we are modeling the cell surface as a deformable sheet. With this assumption, we can also explore local, nontrivial shapes of the underlying cortex, which, in turn, drives the plasma membrane shape. On the other hand, since earlier work demonstrates that the head domain of vimentin can also associate with a plasma membrane , we willθlmn is the angle between three consecutive particles in co-receptors and rij is the distance between nearest neighbor and σo is the diameter of a ECC particle. Finally, we assume that these filamentous extracellular components are bound to the cell cortex just beneath the lipid bilayer [The filamentous ECCs are semiflexible polymers also modeled as particles connected with springs, as shown in  bilayer , 40, 41.A short receptor is placed in the middle of the cell surface and is the same as the ECC receptor with different values for ro is the equilibrium distance found by the triangulation. This construction of a deformable, elastic virus surface, with ultimately filamentous structures emanating from it, is a simplifying assumption as viruses with spike proteins typically consist of fluid-like containers but, nonetheless, provides a starting point for the modeling.The deformable virus surface is initialized by generating particles on a sphere. The particles are arranged in a Fibonacci sequence. We then implement a Delaunay triangulation to identify the edges between these particles. All particles on the sphere are connected with a harmonic spring potential σo is the particle’s diameter. We place the spikes on the virus’s surface such that the typical distance between two neighboring spikes is between 3–15 nm, the range found in experiments [Spikes are two particles joined by harmonic springs, as shown in eriments , 44.ro is the equilibrium distance. The receptor is made up of four particles connected via harmonic spring and has some bending rigidity.The virus is connected to the primary receptor via a harmonic spring interaction, rij is the distance between ith top spike particle and the jth top ECC particle, and σo is the diameter of the particle, orVirus spikes and extracellular components may interact via specific binding domains and charge interactions , 37, 45. domains , 46, we  domains . Thus, oKSoft−Repulsion is the strength of the soft repulsion, rij is the distance between the centers of two particles, and σo is the diameter of a particle. The repulsion force acts only when the distance between particles is lower than the diameter of the particle, orAll components of the model have volume exclusion due to soft-core repulsion given by ri is the position of an ith particle, ξ(t) is the Gaussian white noise to simulate thermal fluctuations and follows < ξ(t) >= 0 and < ξi(t)ξj(t’) >= 0. The sum of conservative forces on the ith particle ith particle coordinate We implement Brownian dynamics to quantify the dynamics of the model. The equation of motion is given by μs, and one force unit is 10−1pN. All simulation quantities are expressed in terms of these basic units. We run the total simulation with dt 10−4 for 108 simulation steps or 50 ms and recording positions at each 25 μs. Total run time is comparable to the typical viral endocytosis time[kBT. Since bending rigidity is encoded in the second nearest-neighbor springs, multiplying that spring constant with the area of the triangle made up of those springs, with side Our Brownian dynamics simulation is a coarse-grained simulation. All simulation quantities are normalized via length, time, and force scales. We can convert simulation quantities to biologically relevant quantities by the following definitions: one unit simulation length is defined as 10 nm, one unit simulation time is 1osis time, 49. To The simulation box contains up to 8624 particles. Each particle has a diameter of 10 nm. Since the typical size of the virus is 10s-100s of nanometers and the cell size is usually at the scales of 10 s of micrometers, thus virus interacts only a small part of the cell surface. So, we simulated only a small patch of the cell surface of size 550 nm×480.6 nm, which is made up of 1444 particles. The cell surface boundary is free in our simulations. Each ECC has a length of 40 nm, which is in the range of many cell surface proteins , 52 and A.−3 nm−2. ECC coverage of 100 % corresponds to the area density of 5.4 ×10−3 nm−2.We first investigate the effects of varying extracellular components (ECC) and the virus spike density on wrapping. We altered the cell surface coverage by the ECCs from 20 percent to cover it fully and varied the spike numbers from 25 to 200 spikes. In To quantify the wrapping behavior, we define virus wrapping as the ratio of the surface area covered via a cell surface divided by the whole surface area of the virus, as shown in > 20 %. Viral wrapping increases at medium percent coverage, 40 and 60 %, only to again decrease for the high ECC percent coverage of 80 and 100 %. Since we have the constraint that a spike can only adhere to one ECC at a time, getting low virus wrapping with low ECC coverage is somewhat expected. As there are few coreceptors to stick to, such viruses cannot interact with many coreceptors. On the other hand, for higher percent coverages of coreceptors, there is a shielding effect that reduces the viral wrapping. Once the coreceptors near the virus attach to the spikes, far away coreceptors cannot interact with virus spikes due to volume exclusion. Thus, having many coreceptors does not lead to higher virus wrapping. Virus wrapping is maximum at the medium coreceptors density, where virus spikes have enough coreceptors to interact with those around them and have enough space between spikes not to invoke shielding effects. Thus, we found an optimal percent coverage of cell surface coreceptors for maximal wrapping. The notion of an optimal percent coverage for maximal wrapping is a rather reasonable one if one considers effective cell surface bending rigidity that depends on such a quantity. More specifically, as the ECC percent coverage increases, the sheet stiffens such that the sheet eventually can no longer wrap around the viral. At lower ECC percent coverage, some stiffening of the sheet gives rise to a more coordinated wrapping, which we explore in more detail in the next subsection.We find non-monotonic behavior for the wrapping as a function of the percentage of coverage of extracellular components for 200 spikes, as shown in B.Next, we investigate the cell surface bending rigidity’s role in viral wrapping. Since there exists heterogeneity in the structure and mechanics of the cell cortex, as viruses invade cells, they may encounter different cellular surface rigidities, which may affect the rate of its uptake. In R, centered at the center of mass of the virus. Starting with the highest spike on the virus that is adhered to an ECC, we construct a cylinder around the virus and identify all the cell surface particles in this cylinder for a given radius. As these particles are a part of a triangulated lattice, we see the total surface area of the cell surface inside the cylinder by adding up all the triangle areas inside the cylinder. Here, the radius of cylinder R sets how close or far from the virus we find the surface area. To arrive at a dimensionless area, we divide the obtained surface area by the total area of the sheet. We cannot have a large value of R compared to the virus radius as there is a finite amount of cell surface; by taking too small R, on the other hand, we may miss some of the data. Therefore, R is 1.5 times of radius of the virus or 150 nm. For other radii, we studied the quantification, and it is robust for a range of R. Please see P as well as the cell surface area A inside the cylinder. We then define a dimensionless quantity called shape index as .399, which would be a lower bound. Larger shape indices denote less spherically symmetric wrapping and, so, less efficient wrapping in terms of the use of cell surface area.Given that we have two general types of wrapping configurations that the cell surface takes on, crumple-like wrapping and fold-like wrapping, how do we distinguish between the two types? We say “crumple-like” because the crumpling is very localized near the virus, unlike the crumpling of a sheet of paper, for example . QuantifIn Furthermore, we plotted the cell surface shape index as a function of ECC percent coverage and cell surface bending rigidity in C.We now explore the dynamics of viral wrapping. From From We also investigate the virus’s engulfing velocity to further understand the role of folds and crumples in viral uptake. We plotted the virus velocity in the z-direction, or perpendicular with respect to the cell surface, see D.−2pN/nm. The attraction between spikes and ECC with low stretching cost energy makes them more accessible to each other, leading to many attachments between them. Even though we have a significant number of spikes connected to coreceptors, the cell surface does not form a fold but a crumple. This is because these low- stretching strength coreceptors cannot transfer enough force to the cell surface to make a more coordinated fold. On the other hand, in 2pN/nm, which is enough to generate forces to fold the cell surface around the virus. Thus, we found that an optimal ECC percent coverage determined for one set of mechanical parameters does not ensure efficiency that virus wrapping can always be achieved. The stretching rigidity of spikes and filaments on the cell surface also constrains the formation of folds.In this subsection, we investigate the effects of the mechanical properties of spike and ECC on viral wrapping. Indeed, filaments on the cell surface have varied mechanical properties depending on the type of filament. Therefore, we vary the stretching strength of coreceptors and virus spikes on viral uptake. Given this additional mechanical variation, we ask the question, is the optimal percent coverage of coreceptors always guaranteed a fold? To answer this question, we maintain the percent coverage of coreceptors at 40%, which yielded maximal virus wrapping, as shown in We vary the stretching strength of spike and coreceptors up to four orders of magnitude given in −2pN/nm and vary the coreceptors stretching strength from 10−2pN/nm to 102 pN/nm. We observe that changing coreceptors stretching strength does not contribute much to the virus wrapping behavior as it just increases with time having similar trends for all coreceptor’s stretching strength values. On the other hand, from 2pN/nm, we see that low coreceptor stretching values are associated with wrapping slowly and get less virus wrapping than for high coreceptor stretching strength. This is because, with the higher stretching strength of the spike and coreceptor, the cell surface folds, leading to the faster wrapping of the virus.We also examine the time series analysis to understand how the fold and crumple formation mechanism changes due to the stretching strength of spike and coreceptors. In We demonstrate that having optimal coreceptors percent coverage is not enough for efficient wrapping via folds. Coreceptors and spikes also require a threshold stretching strength above which the cell surface folds to achieve more wrapping. We also investigate the effects of the bending rigidity of filamentous ECCs and virus spikes. Bending rigidity does not impact the cell surface morphologies as stretching. See Our study suggests that cells whose surfaces are optimally populated with filamentous protein structures acting as coreceptors are more likely to be infected as they uptake the virus faster and use relatively less cell surface area per individual virus so that more virus-like particles can be uptaken. At the optimal percent coverage, the cell surface makes folds around the virus, and folds are faster and more efficient at wrapping the virus than crumple-like wrapping. Our study also finds that cell surface bending rigidity helps generate folds, as bending rigidity enhances force transmission across the surface. We also conclude that such an optimal percent coverage does not always ensure a fold formation, as changing mechanical parameters, such as the stretching stiffness of the ECC or the virus spikes, can drive the crumple-like formation of the cell surface.There has been much work exploring the role of viruses or nanoparticle spikes and how their mechanical properties affect endocytosis. However, these studies treat receptors as sticky particles on the cell surface without any degree of freedom , 56–60. The earlier experimental finding that extracellular vimentin enhances SARS-CoV-2 uptake is intriguing , 19. It We have assumed that the virus-like particle is already attached via a small receptor to the cell surface and quantified viral wrapping. However, it would also be interesting to study how the cellular microenvironment affects the trajectory of a nanoparticle to find that initial receptor in terms of search strategy . We haveSupplement 1


Current guidelines for the control of soil-transmitted helminths (STH) recommend deworming children and other high-risk groups, primarily using school-based deworming (SBD) programmes. However, targeting individuals of all ages through community-wide mass drug administration (cMDA) may interrupt STH transmission in some settings. We compared the costs of cMDA to SBD to inform decision-making about future updates to STH policy.We conducted activity-based microcosting of cMDA and SBD for 2 years in Benin, India and Malawi within an ongoing cMDA trial.Field sites and collaborating research institutions.We calculated total financial and opportunity costs and costs per treatment administered (unit costs in 2019 USD ($)) from the service provider perspective, including costs related to community drug distributors and other volunteers.On average, cMDA unit costs were more expensive than SBD in India ($1.17 vs $0.72) and Malawi ($2.26 vs $1.69), and comparable in Benin ($2.45 vs $2.47). cMDA was more expensive than SBD in part because most costs (~60%) were ‘supportive costs’ needed to deliver treatment with high coverage, such as additional supervision and electronic data capture. A smaller fraction of cMDA costs (~30%) was routine expenditures . The remaining cMDA costs (~10%) were opportunity costs of staff and volunteer time. A larger percentage of SBD costs was opportunity costs for teachers and other government staff (between ~25% and 75%). Unit costs varied over time and were sensitive to the number of treatments administered.cMDA was generally more expensive than SBD. Accounting for local staff time  in community programmes is important and drives higher cost estimates than commonly recognised in the literature. Costs may be lower outside of a trial setting, given a reduction in supportive costs used to drive higher treatment coverage and economies of scale.NCT03014167. We used rigorous microcosting methods to collect costs associated with community-wide mass drug administration and school-based deworming and corresponding treatment data, in real time.The granularity of data collected provides rich information regarding the resource needs for deworming programmes, and how these may vary across countries and delivery modalities (school vs community-based treatment).We estimated opportunity costs of the volunteer workforce and currently employed government staff , which are often excluded from deworming costing studies.Although costs associated with research and trial administration were not included in this study, it is possible that some costs  may be higher in this research setting than what would be observed in routine deworming programmes.Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus and Trichuris trichiura) that globally affect approximately 1.5 billion individuals annually, predominantly in sub-Saharan Africa, East Asia and Latin America.2Soil-transmitted helminths (STH) are a group of intestinal parasites , where teachers and health workers deliver preventative chemotherapy to pre-school and school-aged children.5It may be possible to interrupt STH transmission by expanding deworming eligibility to individuals of all ages.This study systematically identified, measured and compared resources for implementation of 12 rounds of cMDA and 8 rounds of SBD across the DeWorm3 sites, during implementation of the trial. Determining the costs and cost drivers of expanding STH treatment to all individuals in a community will be essential for shaping future STH policy.The DeWorm3 project was implemented in Come Commune of Benin, Tamil Nadu State of India and Mangochi District of Malawi. These sites were selected because they had previously implemented lymphatic filariasis programmes over five or more rounds with albendazole coadministered with ivermectin or diethylcarbamazine.10.1136/bmjopen-2021-059565.supp1Supplementary dataIn addition to deworming, DeWorm3 also conducted an annual census to enumerate the full population in study catchment areas, annual prevalence surveys to determine STH prevalence and intensity and post-MDA coverage surveys to assess the reach of cMDA and SBD .8 These We conducted activity-based microcosting from the service provider perspective  during the first 2 years of DeWorm3 implementation in order to explore heterogeneity in costs across rounds of implementation. Across the three sites, we conducted 2 years of intensive microcosting, resulting in data from 12 rounds of cMDA and 8 rounds of SBD. Costing the first year of cMDA implementation allowed us to capture costs related to start-up, while the second year provided a more accurate portrayal of costs related to routine implementation. This analysis includes opportunity costs associated with all health worker involvement in implementation, including teachers and community volunteers engaging in drug delivery. Costs to the household were not assessed as they are assumed to be negligible.17 18et al,The methodology for costing cMDA is detailed in Galactionova Following data collection, all costing data were iteratively reviewed for quality and completeness. Costs related to DeWorm3 research only  were not included in the data collection instruments and, if identified, were removed during data cleaning.20Financial costs included actual expenditures on goods and services purchased by the DeWorm3 project or site governments. We analysed these data in Stata (V.16.1). Costs were converted to USD using the annual average exchange rate based on the year in which the costs were incurred.Opportunity costs included the costs of donated drugs, volunteer time  and time costs for currently employed government staff. We estimated costs associated with volunteer time spent delivering drugs using the DeWorm3 trial’s digital treatment forms . Supportive costs included additional activities aimed at optimising coverage and compliance. For example, electronic data were collected to monitor cMDA coverage in real-time and identify areas in need of additional sensitisation and mop-up. In general, supportive activities included: (1) start-up planning costs, (2) additional supervision from a non-governmental organisation (NGO) implementing partner, (3) additional sensitisation activities, (4) electronic data collection and (5) programme management costs associated with these supportive activities. Additional details regarding routine and supportive costs are presented in The cost per treatment administered  was determined by dividing costs per round by the total number of treatments administered. The number of treatments administered via cMDA was abstracted from MDA treatment forms (household-level forms completed by enumerators during cMDA). The number of treatments administered via SBD was estimated from paper SBD forms filled out by school and/or DeWorm3 field staff, then transferred to an electronic format. One and two-way sensitivity analyses were conducted to explore how the average cost per treatment administered would change due to variation in key costing inputs and coverage levels  and in the design and conduct of the wider DeWorm3 trial.Between February 2018 and December 2019, a total of 12 rounds of cMDA and 8 rounds of SBD were delivered across DeWorm3 sites in Benin, India and Malawi. Activity-specific unit costs for cMDA and SBD are presented in On average, SBD unit costs were $2.47 in Benin, $0.72 in India and $1.69 in Malawi. Routine financial costs, such as per-diems, fuel, and materials, were approximately 5%–30% of unit costs and were more expensive in Benin and Malawi ($0.53 and $0.48 respectively) as compared with India ($0.03). Routine opportunity costs, mainly teacher and school-level staff time, represented the largest share of costs in Benin and India ; the governments of Benin and India were the primary SBD implementers. In Malawi, where SBD was delivered by the DeWorm3 team, routine opportunity costs were only one-fourth of costs, whereas supportive activities represented half of unit costs.Across sites, average unit costs were generally higher for cMDA as compared with SBD, except for Benin. However, routine cMDA costs were consistently less expensive compared with SBD, driven in part by the high opportunity costs of SBD. Across cMDA and SBD, drug delivery followed by programme management were the most expensive activities. Drug delivery included initial drug distribution as well as mop-up activities (approximately 10%–20% of drug delivery costs). The largest resource input was staff wages and per-diems, representing 56%–91% of average unit costs, generally followed by vehicle costs . RoutineCosts of additional activities, such as an annual census, prevalence surveys and coverage surveys are not included within cMDA and SBD unit cost estimates but are detailed in In one-way and two-way sensitivity analyses , the larCosts and resource needs are important pieces of evidence for governments considering updating standards of care and related policies, such as a potential shift from SBD to cMDA. The DeWorm3 project provided a unique platform to assess and compare the costs of two deworming treatment strategies (SBD and cMDA) across heterogeneous STH-endemic settings. We found the average unit cost per treatment administered to be higher for cMDA compared with SBD in India and Malawi, and comparable in Benin.Costs of MDA for neglected tropical diseases (NTDs), including deworming, vary considerably in the literature, depending on treatment strategy, resources accounted for, and perspective. In a review of 34 studies of MDA costs, financial unit costs (excluding medicine) ranged from $0.01 to $8.50 (2015 USD).12–14This study disaggregates routine programme costs from supportive costs that are used to increase coverage . Average routine costs of cMDA were lower than SBD costs across countries. This is largely driven by salary costs for teachers and school directors who generally spend 1–3 days each year involved in SBD. Similar findings were observed in Niger, where deworming was delivered via SBD to children and via community-based treatment to children and at-risk adults (at fixed locations or their homes); unit costs of SBD were higher at $0.76 compared with $0.46 for community treatment (2005 USD). Differences in costs in Niger were attributed to CDDs treating more individuals than teachers.As it is not possible to disentangle the precise impact of supportive activities on coverage, sensitivity analyses were conducted to explore the potential impact of reducing supportive activities on unit costs. If supportive activities were removed and coverage reduced as a result, unit costs were estimated to drop between 10% and 50%. Although opportunities for electronic data collection during MDA are increasing , not all programmes may choose to proceed with more resource-intensive mobile data collection.Given the experimental nature of cMDA and the DeWorm3 platform on which it was implemented, cMDA costs may vary if launched within routine health systems. Depending on existing capacity within countries, governments could see a reduction in costs due to cost-sharing between other community-based or NTD programmes. Additionally, studies suggest that MDA costs are subject to economies of scale; according to one model, a 10-fold increase in individuals treated could reduce costs by approximately 70% in DeWorm3 countries.When examining average unit costs of cMDA and SBD across sites, we observed lowest costs in India, followed by Malawi and Benin, respectively. However, this pattern was not consistent when examining costs per round, by subactivity or by routine versus opportunity cost. For example, unit costs of cMDA were highest in Malawi during rounds 1 and 2. Our results suggest unit costs of planning, training and community sensitisation may be more similar across MDA treatment strategies and countries, while resources such as staffing and supervision for programme management and drug delivery may be more setting specific. We briefly highlight several reasons for variation in unit costs across sites and a more extensive description of drivers of variation is found in In addition to unit costs, other metrics should be considered to determine the relative value of cMDA and SBD.There are several limitations to this analysis. As described above, there were different degrees of DeWorm3 involvement in SBD across sites; data sources and some driving assumptions, thus necessarily varied. Although DeWorm3 trial costs were excluded from this costing analysis, we anticipate that programme management, planning and supervision costs may be higher than what would be observed routinely. Other assumptions are described in detail in This study provides evidence from a large microcosting study, over 12 rounds of cMDA and 8 rounds of SBD in Benin, India and Malawi DeWorm3 sites. To our knowledge, this is the first study to directly compare costs of SBD to cMDA for STH programmes.


FAILED N95, fit-testPASSED N95) and personal protective equipment (PPE), and (2) determine if a portable high-efficiency particulate air (HEPA) filter can enhance the benefit of PPE.Healthcare workers (HCWs) are at risk from aerosol transmission of severe acute respiratory syndrome coronavirus 2. The aims of this study were to (1) quantify the protection provided by masks  for 40 minutes in a sealed clinical room. Virus exposure was quantified via skin swabs applied to the face, nostrils, forearms, neck, and forehead. Experiments were repeated with a HEPA filter (13.4 volume-filtrations/hour).Virus aerosol exposure experiments using bacteriophage PhiX174 were performed. An HCW wearing PPE  was exposed to nebulized viruses (10PASSED N95 resulted in lower virus counts compared to control (P = .007). Nasal swabs demonstrated high virus exposure, which was not mitigated by the surgical/fit-testFAILED N95 masks, although there was a trend for the fit-testPASSED N95 mask to reduce virus counts (P = .058). HEPA filtration reduced virus to near-zero levels when combined with fit-testPASSED N95 mask, gloves, gown, and face shield.Significant virus counts were detected on the face while the participants were wearing either surgical or N95 masks. Only the fit-testN95 masks that have passed a quantitative fit-test combined with HEPA filtration protects against high virus aerosol loads at close range and for prolonged periods of time. Only a fit-tested N95 mask provided protection against virus aerosol; surgical masks and non-fit-tested N95 masks were not superior to no-mask control. The combination of air purification and fitted N95 mask provided protection against very high virus aerosol load. Virus-laden aerosols can remain suspended in the air for prolonged periods of time and travel large distances while remaining infectious , 2. EpidThese findings led the Centers for Disease Control and Prevention (CDC) to recommend that HCWs wear an N95 (or higher-level respirator) and to optimize indoor air quality, including the deployment of portable high-efficiency particulate air (HEPA) filters when permanent air-handling systems cannot be feasibly improved . ImportaFAILED N95), and a fit-testPASSED N95 mask) in combination with face shield, gown, and disposable gloves; and second, to determine if the use of a portable HEPA filter enhances the benefit of PPE to protect the wearer against virus aerosol contamination.We aimed to examine 2 mechanistic questions regarding the effectiveness of personal PPE and air filtration to protect HCWs against virus aerosol: first, to quantify the degree of personal contamination with virus aerosol when wearing different types of masks  in lysogeny broth. The bacteriophage was purified according to the Phage-on-Tap protocol [9 plaque-forming units (PFU)/mL was obtained and diluted in 1× phosphate-buffered saline . The bacteriophage titer was determined using the standard soft agar overlay method. A 10-mL bacteriophage lysate  was aerosolized (via nebulizer) in all experiments. The choice of titer was determined by pilot sensitivity experiments [Bacteriophage PhiX174 was used as a nonhazardous model virus in all experiments. PhiX174 was propagated using bacterial host protocol . A titereriments  with the3; A nebulizer (PARI Respiratory Equipment), positioned at the head of the bed, aerosolized the bacteriophage lysate within a simulated clinical room , in 1 of 2 locations . The “beAfter exiting the room, the HCW doffed PPE according to a standardized protocol see . Skin/naSettle plates were used to quantify environmental contamination from virus aerosol , 21 ThirGiven that this research involved no human or animal participants (HCWs were researchers), the Monash University Human Research Ethics committee exempted this protocol from ethics review.Experiment 1. To assess the efficacy of PPE to protect against virus aerosol exposure, the HCW was seated at the bedside during nebulization. Virus counts from face/nasal swabs were used to assess the efficacy of the (1) surgical mask, (2) fit-testFAILED N95 mask, and (3) fit-testPASSED N95 mask. A gown, gloves, and face shield were worn in each condition. A no-PPE condition served as a control. All 4 mask conditions were tested on the same day in a randomized order using a computer-generated sequence. Each condition was replicated 5 times over 5 separate days.Experiment 2. To assess the efficacy of combining multiple control measures, the same experimental paradigm was used with constant HEPA filtration. The HCW was seated either bedside (0.85 m from aerosol source) or at a distanced location (2.70 m from source), with the HEPA filter placed at the foot of the bed (PASSED N95 mask. All 4 conditions were tested on the same day in a randomized order and replicated 3 times over 3 separate days. the bed . The HCWExperiments were performed with a single HCW (S. A. J.) wearing a gown , gloves , and face shield .Three mask variants were tested:3-ply surgical mask (OBE Premium face mask)FAILED N95 respirator Fit-testPASSED N95 respirator Fit-testQuantitative fit testing was performed via TSI PortaCount Fit Tester Model 8048, which measures the concentration of particles in the ambient air relative to within a respirator to calculate a fit factor.Before experiments, masks were individually fit checked  and form-fitted by the wearer to optimize fit. Breathing was at rest and predominantly nasal with periods of oral breathing to check/confirm mask fit. The HCW was clean shaven prior to each experiment to reduce mask/beard interactions.After aerosol exposure, PPE was doffed in a room separated from the clinical room by a corridor and 4 sealed doors. The doffing room had continuous HEPA filtration (5 air exchanges/hour). Doffing was videoed and examined independently by 2 expert nurses  to ensure doffing procedure compliance .The clinical room had a double-sealed soundproofed/insulated door. To control airflow patterns, ceiling vents were taped shut and heating/cooling appliances were switched off. The room temperature , humidity , and barometric pressure  were well controlled during experimental procedures. Individual measurements for each experiment are available via the 3/hour, which based on the room volume, achieved 13.4 volume-filtrations/hour. This device was run for 30 minutes (∼6.7 filtration exchanges) after nebulization to remove bacteriophages before repeating experiments. This was then confirmed by deployment of control plates.The IQAir HealthPro250 was used at its highest clean air delivery rate 470 mU, or Friedman tests (χ2Friedman) with post hoc comparisons (Dunn test) were used to compare virus counts between conditions.Viable viruses were quantified by counting the number of viruses from swabs and settling plates. Virus counts >200 were considered too many to count (TMTC) and rated using an ordinal visual rating scale , with TMTC++++ indicating complete lysis of the bacterial host. For graphing/analysis, TMTC ratings were given values of 200–230. Wilcoxon, Mann-Whitney Settle plates confirmed substantial virus contamination of surfaces in the clinical room across all experimental conditions .2Friedman = 9.08, P = .017; Figure 2A); however, only the fit-testPASSED N95 mask resulted in significantly lower virus counts compared to the no-mask control . Given the very high and variable virus counts recovered from the skin under the mask, skin swabs taken from within the nostril were introduced on the third experiment day . Virus counts from inside the nostril  were consistently high for control, surgical, and fit-testFAILED N95 mask conditions. There was a trend for the fit-testPASSED N95 mask to reduce virus counts on nasal swab ; however, positive virus counts were still recovered on all tests.Virus counts recovered from skin under the mask significantly differed by mask type  compared to control.There were highly variable virus counts within conditions, particularly under the mask . This vaC–E). A gown and gloves substantially reduced virus counts on the forearms/hands compared to the no-PPE control . Viruses were detectable on all neck samples and there was no significant difference between the no-PPE control . Virus counts measured from forehead swabs were significantly reduced with the face shield compared to the no-PPE control .To assess the efficacy of gloves, gown, and face shields to reduce virus counts recovered from the body, data were combined across mask conditions and compared to the no-PPE control C–E. A goSettle plates demonstrated substantial virus counts during nebulization despite the presence of the HEPA filter. Plates closest to the aerosol source .2Friedman = 7.93, P = .028; Figure 3A) and were higher at bedside with a surgical mask compared to the fit-testPASSED N95 mask at bedside (P = .027). There was trend suggesting the fit-testPASSED N95 mask at bedside outperformed the surgical mask at distance (P = .058). Notably, virus counts were near zero for the fit-testPASSED N95 mask at both distances.Virus counts were on average lower across swab locations and conditions compared to experiment 1 . Virus c2Friedman = 7.97, P = .017; Figure 3B). Nasal virus counts under a surgical mask were consistently TMTC and significantly higher than the fit-testPASSED N95 mask at both bedside (P = .017) and distanced (P = .040) positions. While nasal swab virus counts were lower with the fit-testPASSED N95 at bedside compared to the surgical mask at distance (P = .082), this was not statistically significant, similar to the results for swabs under the mask. Virus counts did not significantly differ based on distance for surgical masks , or for the fit-testPASSED N95 mask  likely to due to floor effect.Consistent with experiment 1, virus counts from nasal swabs were higher than all other swab sites and varied substantially via mask type and distance .The combination of PPE  and HEPA filtration resulted in very low virus counts on the forearms and forehead, regardless of distance from aerosol source . The neck site had low virus counts , but were consistently higher compared to forehead swabs. The difference between bedside and distanced was not significant . It is also noteworthy that the fit-testFAILED N95 was appropriately sized and had no external qualitative indicators of poor fit and was form-fitted on each application per protocol. Despite this, the fit-testFAILED N95 performed with similar efficacy as surgical mask, again highlighting the critical importance of mask fit. These findings reinforce the necessity of quantitative fit testing of N95 respirators for all forward-facing HCWs, a process that is not universal practice and that relies on available mask supply [B).Clinical evidence for the superiority of N95 respirators over other mask types in protecting against SARS-CoV-2 infection is mixed . Our datk supply . HoweverE. coli. Second, our method allows quantification of viruses settling on surfaces, infiltrating PPE, and most importantly depositing in a human upper airway.Patients with airborne infectious diseases are typically cared for in negative-pressure rooms that are designed to facilitate 12 air exchanges/hour. However, such rooms are rapidly depleted/overwhelmed in an infectious respiratory disease pandemic. An alternative method is to filter the air within a patient room with portable HEPA filters. The CDC recommends that HEPA filters could be used in lieu of negative-pressure rooms while hospital facilities are being renovated/repaired . This idPASSED N95 mask provided almost complete protection against skin under the mask and nasal contamination from virus aerosol. The level of protection offered from nasal virus deposition is critical as it directly relates to possible ports of entry of SARS-CoV-2 including via the upper respiratory tract and the lungs. Although particle deposition in the respiratory tract is complex, an extensively verified model for regional aerosol deposition shows that for molecules in the range 0.2–10 µm, deposition in the anterior nose is greater than in the bronchioles when breathing through either the mouth or nose [We demonstrated 2 significant effects of HEPA filtration on virus-laden aerosol. First, relating to HCWs, we found that a HEPA filter enhances the PPE effectiveness so that a quantitatively fit-test or nose , 33. As  or nose .8/mL) compared to aerosols generated by patients infected with coronaviruses [PASSED N95 and HEPA filter, the results indicating almost universal zero detectable virus are reassuring in this regard. Fourth, our experiments are conducted in a sealed clinical room as opposed to a standard clinical room, which should have approximately 6–12 air exchanges per hour. Therefore, no mixing between clean/external air was occurring . The lack of mixing biases toward a higher aerosol load in the experimental room and thus represents a worse-case scenario for room ventilation. This again provides reassurance about the high effectiveness of the combination of fit-testPASSED N95, PPE, and HEPA filter. Finally, although particle analysis was not performed during conditions, indicative reference recordings in our experiments demonstrate a high particle load of size distribution in the range generated by humans [There are several limitations to this study. First, this was an experimental study using high quantities of a marker virus rather than a clinical observational trial. Therefore, we would strongly caution against making direct quantitative comparisons  against published minimum infective doses for respiratory viruses. This experimental design does not allow us to directly determine whether virus counts in HCWs’ nares may result in clinical infection. Similarly, the HCW remained seated in the clinical room for the entire exposure period. While this provided good experimental control of exposure, this behavior is not directly generalizable to clinical practice. Second, we aerosolized a higher viral load (10aviruses . Howeveraviruses  and otheaviruses  using bay humans , confirmIn conclusion, the emergence of more transmissible variants of SARS-CoV-2 have highlighted the gaps in protecting HCWs that were exposed in 2020–2021. Healthcare providers must deploy a simultaneous array of mitigation strategies to optimize HCW safety. N95 masks that pass a quantitative fit-test combined with a HEPA filter can offer protection against high virus aerosol loads, at close range, for prolonged periods of time.jiac195_Supplementary_DataClick here for additional data file.