The antiviral activity of different mutagens against single-stranded RNA viruses is well documented; however, their activity on the replication of double-stranded RNA viruses remains unexplored. This study aims to investigate the effect of different antivirals on the replication of a chicken embryo fibroblast-adapted Infectious Bursal Disease virus, FVSKG2. This study further explores the antiviral mechanism utilized by the most effective anti-IBDV agent.The cytotoxicity and anti-FVSKG2 activity of different antiviral agents  were evaluated. The virus was serially passaged in chicken embryo fibroblasts 11 times at sub-cytotoxic concentrations of ribavirin, 5-fluorouracil or amiloride. Further, the possible mutagenic and non-mutagenic mechanisms utilized by the most effective anti-FVSKG2 agent were explored.Ribavirin was the least cytotoxic on chicken embryo fibroblasts, followed by 5-fluorouracil, amiloride and 5-azacytidine. Ribavirin inhibited the replication of FVSKG2 in chicken embryo fibroblasts significantly at concentrations as low as 0.05 mM. The extinction of FVSKG2 was achieved during serial passage of the virus in chicken embryo fibroblasts at ≥0.05 mM ribavirin; however, the emergence of a mutagen-resistant virus was not observed until the eleventh passage. Further, no mutation was observed in 1,898 nucleotides of the FVSKG2 following its five passages in chicken embryo fibroblasts in the presence of 0.025 mM ribavirin. Ribavarin inhibited the FVSKG2 replication in chicken embryo fibroblasts primarily through IMPDH-mediated depletion of the Guanosine Triphosphate pool of cells. However, other mechanisms like ribavirin-mediated cytokine induction or possible inhibition of viral RNA-dependent RNA polymerase through its interaction with the enzyme’s active sites enhance the anti-IBDV effect. Ribavirin inhibits ds- RNA viruses, likely through IMPDH inhibition and not mutagenesis. The inhibitory effect may, however, be augmented by other non-mutagenic mechanisms, like induction of antiviral cytokines in chicken embryo fibroblasts or interaction of ribavirin with the active sites of RNA-dependent RNA polymerase of the virus. The growing human-animal contact can lead to the evolution of certain viruses and generate diseases with the potential to spread between species and cause pandemics , 2. NumeInfectious Bursal Disease Virus (IBDV) is a non-enveloped, double-stranded (ds)-RNA virus belonging to the genus Avibirnavirus and family Birnaviridae, that causes a substantial economic impact on the poultry industries globally . The virin vitro and comprehensive in silico approaches.Ribavirin  is a classic example of base-analogue mutagens clinically used against various viruses affecting human health. Lethal mutagenesis is a primary antiviral mechanism utilized by ribavirin against ssRNA viruses through an accumulation of mutations in the virus beyond a threshold . However2.2.1.viz.; ribavirin, 5-fluorouracil, 5-azacytidine, and amiloride  were used in this study. Each mutagen was dissolved separately in Dulbecco’s modified Eagle media  at stock concentrations of 15 mM (Ribavirin), 20 mM  and 5 mM (5-azacytidine) which were further sterile-filtered using a 0.22-μm syringe filter . The filtrate was aliquoted and stored at −20°C until use, as described in previous studies (FVSKG2 (Accession number: OP161172), a local IBDV isolate, was used in the study. Four mutagens  studies , 36.2.2.The chicken embryo fibroblasts (CEFs) were isolated from 9 days old embryonated chicken eggs and maintained in growth media containing DMEM, 5% Fetal Bovine Serum  and 1% Penicillin–Streptomycin Solution with 10,000 U penicillin and 10,000 μg streptomycin/mL , at 37°C and 5% CO2 in a humidified chamber as described in our previous studies .2.3.™ 3, Winooski, VT, United States) -2,5-diphenyltetrazolium bromide) assay . Confluent CEFs were prepared in 96-well plates . Cells were rinsed and replenished with 100 μL growth medium containing one of the four concentrations of mutagens  in three replicates. To determine cell viability at specific time point, cells were incubated at 37°C and 5% CO2, as described in the previous study , 39. At  States) , 41. Con States) .2.4.2 flasks prior to virus inoculation. At the time of virus inoculation, cell number was determined and multiplicity of infection (MOI) of 0.1 was used. The anti-IBDV activity of ribavirin was evaluated against IBDV isolate FVSKG2. Confluent cultures of CEFs were infected with FVSKG2 and incubated for 2 h in a humidified incubator at 37°C and 5% CO2 following which the virus inoculum was discarded, and the cells were replenished with 5 mL maintenance media, each containing a specific concentration of mutagens. Different sub-cytotoxic concentrations of mutagens, ribavirin, 5-Azacytidine, and Amiloride at 0.0, 0.05, 0.1, 0.2, and 0.3 mM, 5-Florouracil at 0.0, 0.1, 0.2, 0.3, and 0.5 mM were tested against FVSKG2 based on previous studies  of viruses in DMEM were prepared and inoculated in triplicates with 100 μL media per well and incubated for 2 h under cultural conditions. After incubation, the inoculum was discarded and cells were replenished with growth media. The cells were then incubated for 96 h and monitored for cytopathic effects (CPE). The wells were scored for CPE after 96 h. Virus titer was expressed as 50% tissue culture infective dose (TCID50/mL) and calculated using the method of Reed and Muench  were used to evaluate the titer of FVSKG2 samples. Eight-fold serial dilution .2.7.® SV Gel and PCR Clean-Up System, Promega Madison, Wisconsin, United States) and sent for sequencing to Macrogen .According to the manufacturer’s instructions , 300 μL of the supernatant of the stock virus of FVSKG2 was used to isolate the RNA, with the following changes. The VL buffer was used for 30 min of incubation with the viral sample. The Takara one-step RT-PCR kit was used to perform the one-step RT-PCR . Amplification was performed according to the manufacturer’s instructions, following some modifications suited for amplifying a dsRNA virus. In a 1:4 ratio, DMSO  was added to the RNA template with 0.5 μL RNAse inhibitor  and incubated at 99°C for 3 min and snap-chilled on ice following which primers, forward A_124F:CGCAGCGATGACAAACCT; reverse A_1100R: GATCCCCCGCCTGACCACCACTT for 976 bp region of segment A and forward B_1080F: CTGAAAGGTACGACAAAAGCACAT; reverse B_2002R: TACCAACCTCAACGCCTCATACCT for 922 bp region in segment B were added separately. Then 12.5 μL of 2X RT-PCR buffer and 1 μL enzyme mix were added to set up a 25 μL reaction in thermo-cycler . PCR products of size 976 bp and 922 bp, respectively, were amplified by using the following thermal cycling conditions 50°C for 30 min, 95°C for 2 min, followed by 35 cycles at 95°C for 30 s, 62°C and 61°C, respectively, for 1.5 min, 72°C for 1.5 min, and final extension of 72°C for 5 min. PCR products were purified by DNA purification kit  to determine viral titers as described in previous studies , 45.2.9.Confluent CEFs in 6-well plates were pre-treated with ribavirin at the concentration of 0, 10 and 40 μM in the presence or absence of a fixed concentration of 40 μM guanosine. Separately, confluent CEFs in 6-well plates were pre-treated with mycophenolic acid at 0, 1, 5, and 10 μM in the presence or absence of fixed concentration of 40 μM guanosine. FVSKG2 at MOI of 0.01 was added to the cells and incubated for 2 h. Inoculum was then removed and the cells were replenished with the same concentrations of the ribavirin, guanosine and mycophenolic acid as in pre-treatment, incubated at 37°C and 5% CO2. The viral titer was determined every 48 h post-infection.2.10.Ribavirin was added at 0, 0.05 and 0.1 mM concentrations in CEFs cultured in 24-well plates. RNA was isolated from the ribavirin-treated CEFs 24-h post-treatment using an RNA isolation kit  following the manufacturer instructions. RNA was reverse-transcribed into complementary DNA (cDNA) using a high-capacity cDNA reverse transcription kit  as per the following conditions: 25°C for 10 min, 37°C for 120 min and 85°C for 5 min and holding at 4°C. Real-time PCR was performed on Analytik Jena, qRT-PCR  system using various cytokine-specific primers , followi2.11.Two proteins, RdRp (Uniprot ID: Q9Q6Q5) and IMPDH2 (Uniprot ID: Q5F4A4), and two ligands ribavirin (PubChem ID:37542) and mycophenolic acid (Pubchem ID: 446541) were used in this study. The Crystal Structure of RdRp (PDB ID: 2PGG)  was retr2.12.viz. ASN402, ASP403, and ASN416 in the catalytic palm of RdRp. Site 2 was built around SER166, a self-guanylation site present in RdRp. For IMPDH2_GG, the potential binding sites were identified based on the structural comparison with human IMPDH2. The crystal structure of IMPDH2 with mycophenolic acid (1JR1) was used as the template to define the binding pocket for IMPDH_GG. Ribavirin and mycophenolic acid were docked into this binding pocket. All the complexes generated were subjected to the molecular dynamics simulation run for further analysis.AutoDock Tool  was used2.13.viz. ribavirin, and mycophenolic acid were processed in the CHARMM General Force Field (CGenFF) programGROMACS 2021 series version-2  molecula2.14.The effect of mutagens on CEFs was analysed by Mann–Whitney U test while the effect of mutagen on FVSKG2 replication, serial passages and multi-step growth curve was analyzed by Repeated measures analysis of variance (ANOVA) followed by Dunnet’s post-test using GraphPad Prism 8 . Nucleotide sequences were aligned and analyzed using DNASTAR . Cytokine expression was analyzed by the Mann–Whitney U test (SPSS Version 20) , 54. Thi3.3.1.The cytotoxicity of the four mutagens was evaluated and is summarized in the 3.2.p < 0.0001). When FVSKG2 was treated with 0.3 mM ribavirin, there was about a 3.5-log10 reduction in virus titer at 72 h post treatment, followed by a 4.5-log10 reduction at 120 hpt. Although similar activity was observed with 0.2 mM ribavirin, a 4-log10 reduction and 2.5-log10 reduction were observed in the presence of 0.1 mM or 0.05 mM ribavirin, respectively. Significant suppression of FVSKG2 replication was observed at 0.5 mM of 5-fluorouracil with 2-log10 reduction in virus titer (p < 0.0001). However, no significant antiviral activity was observed with 0.1, 0.2, and 0.3 mM 5-fluorouracil. Treatment with 0.2 and 0.3 mM 5-azacytidine significantly achieved up to 2-log10 reduction in FVSKG2 replication (p = 0.0019). Similarly, high concentrations (1 and 2 mM) of amiloride also caused significant suppression of FVSKG2 replication with up to 2.5-log10 reduction in virus titer (p = 0.0266). Further, no significant antiviral activity was measured at low concentrations of 5-azacytidine (<0.1 mM) and amiloride (<0.05 mM).The antiviral effect of ribavirin, 5- fluorouracil, 5-azacytidine and amiloride on replication of FVSKG2 in CEFs was evaluated using a multi-step growth curve. This was done by determining the titer (TCID50/mL) of mutagen-treated viral populations at indicated concentrations relative to the control concentration (0 mM) of mutagens, summarized in 3.3.10 reduction at p1 and p2. However, the virus disappeared in the third passage and did not emerge until the eleventh passage. Furthermore, FVSKG2 replication was completely suppressed at higher ribavirin concentrations (>0.1 mM). The virus appeared to be less sensitive to 5-fluorouracil and amiloride at indicated concentrations, with no significant change in the virus titer across the 11 passages relative to the control (0 mM). The only exception was the 0.1 mM 5-fluorouracil, which exhibited significant fluctuations in virus titers (p < 0.05). In the presence of 5-fluorouracil at a concentration of 0.1 mM, the virus initially experienced a 1-log10 reduction in titer at passage 2, reaching an equilibrium from passage 4 to passage 6 with 105.5 TCID50/mL virus titer. By passage 8, the virus titer increased again to 105.5 TCID50/mL and reached a new equilibrium by passage 9 with a 1.5 log10 reduction in virus titer. The virus titer persisted at 105 TCID50/mL from passage 9 to passage 11.Based on the cytotoxicity and antiviral activity of the selected antiviral mutagens, serial passages of FVSKG2 in CEF cells were carried out in the presence of an indicated concentration of each mutagen . We obse3.4.FVSKG2 replication was inhibited to an undetectable level at 0.05 mM of ribavirin in CEFs. However, at 0.025 mM ribavirin, FVSKG2 was replicated at a low titer. No mutation was observed in 1,898 nucleotide bases in FVSKG2 passaged five times in the presence of 0.025 mM ribavirin in CEFs when compared to FVSKG2 passaged for the same number of times in the absence of ribavirin.3.5.2 TCID50/mL and a 5.5-log10 reduction in viral titer compared to 0 mM ribavirin. However, when 0.025 mM guanosine was added in addition to ribavirin, the viral titer increased to 106 TCID50/mL at 48 h. Therefore, Ribavirin inhibited FVSKG2 replication, while adding guanosine to the culture revived the virus replication . The addition of guanosine resulted in a 4.5 log10 increase in virus titer at 1 μM mycophenolic acid, which persisted to higher concentrations of mycophenolic acid. The virus achieved a 3.5-log10 increase in titer at 10 μM mycophenolic acid, reaching about 103.5 TCID50/mL at 48 h post-infection compared to non-guanosine treated mycophenolic acid control . While site-1, formed of ASP 402, ASN 403, and ASP 416, constitutes the conserved catalytic active site, site-2, formed by SER 166, constitutes the RdRp self-guanylation site . Ribavir4.In the current study, ribavirin was found to be the least cytotoxic on CEFs among the tested mutagens, which agrees with the previous studies that used different cells , 35, 55.viz. FMDV, HCV, polio virus, hepatitis E virus and mumps virus was found to be due to mutagenic action of ribavirin  Seqin-silico studies. This is in agreement with previous studies where the antiviral effect of ribavirin on flaviviruses, paramyxoviruses, dengue virus and influenza A virus was found to be due to depletion of cellular guanosine pool via IMPDH inhibition , accession number OP161172.1.The data presented in the study are deposited in the NCBI repository , Faculty of Veterinary Sciences and Animal Husbandry, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir (Registration no: 1809/GO/ReBi/15/CPCSEA).in-vitro and in silico study and drafted the manuscript. W-IK and AK conducted thorough reviews and editing of the manuscript. AH performed additional in vitro experiments suggested by the reviewers. RAS, SMA, and NAG contributed to the reading and revision of the manuscript. NS and NC contributed to the conception and design of the study, the data analyses, and manuscript revisions. All authors contributed to the article and approved the submitted version.TA and MP performed the in vitro experiments and drafted the manuscript. IG performed the part of This work was supported by a grant from the Science and Engineering Research Board, Department of Science and Technology, Government of India, under the early career research award scheme (No: ECR/2016/001172).NC is employed by Daskdan Innovations Pvt. Ltd.The remaining 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.


The space between the outside of the MF membrane and the module inside was filled with the PES beads. UV at a wavelength of 352 nm was irradiated from outside of the acryl module. A quantity of humic acid and kaolin was dissolved in distilled water for synthetic water. Water or air intermittent backwashing was performed outside to inside. The membrane fouling resistance after 3 h process  was minimum at 30 g/L of the PES beads for water backwashing, and at 40 g/L for air backwashing when increasing the PES beads from 0 to 50 g/L. The irreversible membrane fouling resistance after physical cleaning (Rif) was at the bottom at 5 g/L of the PES beads for water backwashing, which was 3.43 times higher than minimal at 40 g/L of the PES beads for air backwashing. The treatment effectiveness of turbidity increased when increasing the PES beads’ concentration from 0 to 50 g/L; however, it reached a maximum at 98.1% at 40 g/L and 99.2% at 50 g/L for water and air backwashing, respectively. The treatment effectiveness of UV254 absorbance, which was dissolved organic matter (DOM), increased dramatically when increasing the PES beads; however, it reached a peak of 83.0% at 40 g/L and 86.0% at 50 g/L for water and air backwashing, respectively. Finally, the best PES beads’ concentration was 20~30 g/L to minimize the membrane fouling; however, it was 50 g/L to remove pollutants effectively. The water backwashing was better than the air at treating DOM; however, the air backwashing was more effective than the water at removing turbid matter and reducing membrane fouling.The membrane process has a limit to the decay of various pollutants in water. To improve the problem, the roles of backwashing media and titanium dioxide (TiO In a membrane water treatment system, membrane fouling generally occurs by the adsorption–precipitation of inorganic and organic composites on the membrane inside and surface and leads to a decline in the treated water flux, to a rise in membrane cleaning prices, and to a decrease in membrane life. Approaches for dropping membrane fouling stay insufficient, which is the chief interruption in the prosperous application of a membrane separation system; however, significant improvement has been made in membrane fouling regulation ,2. A pri2, which was utilized in this research, is superior, because it is non-toxic, photochemically stable, highly active, and inexpensive [2 has been applied to various membrane water treatment research. Cu2+/ZnO/TiO2 nanocomposites were prepared by the sol-gel method and applied to decay benzene, toluene, and xylene in oilfield-produced water [2 mesocrystals were manufactured on sanded glass and tested by the photo-degradation of various dyes [2 on glass beads was tried to eliminate seven pharmaceuticals [Among a number of photocatalysts, TiOxpensive ,6. TiO2 ed water . Immobilous dyes . A packeeuticals .Backwashing/backpulsing occasionally can be installed to get rid of reversible fouling for membrane fouling control ,11. Chem2-immobilized PES beads with UV radiation was utilized for excellent water quality.Nowadays, the combined system of membrane separation and photo-oxidation by UV radiation can successfully solve the membrane fouling limitation ,14. The Ceramic membranes applied in this study usually have a triple upper price than polymeric membranes containing comparable membrane surface area. Nevertheless, those have numerous advantages, which include a long lifetime, chemical, thermal, and mechanical resistance. The ceramic membranes were cost-effective, paralleled with polymeric membranes, owing to greater permeating flux, and essentially a long-lasting lifetime . PresentPhoto-oxidation has various benefits which are a comprehensive kind of utilization, small energy consumption, and great effectiveness. Especially, for non-biodegradable organic contaminants, the mineralization of organic composites to small inorganic molecules by oxidization of most of them can be explained by the mechanism of the photo-oxidation process. It is also one of the outstanding technologies of advanced water treatment systems. For these inspirations, the photo-oxidation method, which was applied in this study, has been utilized broadly ,25,26,272 coated-PP beads and multi-channel ceramic MF or tubular carbon fiber UF with UV radiation were researched by our group [The influence of water backwashing in the combined water treatment system of TiOur group ,30. Alsour group . 2) photocatalyst-immobilized PES beads’ concentration were investigated in a combined system of tubular ceramic MF and PES beads for advanced drinking water treatment. Replacing DOM and turbid substances, a persistent quantity of humic acid (HA) and kaolin was dissolved in distilled water. This research was the unique application of TiO2-immobilized PES beads [2-coated-PP beads [2-immobilized PES beads and the ceramic MF. The PES beads were packed between the space of the acryl module case and the ceramic MF.In this research, the roles of backwashing media  and titanium dioxide  MF membrane, manufactured by Nanopore Inc. , was utilized in this work. The characteristics of the tubular ceramic MF are summarized in rch team ,33; theiReplacing soluble natural organic substances and very small inorganic particles in natural water sources such as a river or lake, a persistent quantity of HA and kaolin was dissolved in water prepared by distillation equipment. In this study, it was applied as artificial feed water. Two UV lamps  irradiated UV with 352 nm, which was the most effective wavelength to decompose DOM from the acryl module outside and to reduce DOM by photo-oxidation. 2-immobilized PES beads were packed in the gap between the ceramic MF outside and the module inside. Additionally, for protection of the PES beads loss out of the module, a 100 mesh (0.150 mm) sieve, which was much less than the 4–6 mm size of the PES beads used in this study, was attached to the outlet of the combined module. To remove the turbid matters and DOM, the TiO2-immobilized PES beads (7) instead of PP beads, which was applied in former research [The combined water treatment system (6) of the ceramic MF membrane and the TiOresearch , is pres2-immobilized PES beads was lower than that of water, and the PES beads were gathered at the top of module.The filtration time (FT), which was the water backwashing period, was fixed at 10 min, and the backwashing time (BT) at 10 s. Kaolin and HA were fixed at 30 mg/L and 10 mg/L in all of the research, individually. The treated water flux (J) was tested continuously during each research condition during the total 3 h process. In the combined system of tubular ceramic MF, the water or air backwashing pressure was fixed at 2.5 bar, transmembrane pressure (TMP) at 1.8 bar, and the feed flow rate at 1.0 L/min. To maintain a constant viscosity of water in all experiments, the feed water temperature was set at 20 °C. The PES beads’ concentration was changed from 0 to 50 g/L in the combined module space.254 absorbance was examined by a UV spectrophotometer  for determining turbid matters and DOM. The detection limits of the turbidimeter and UV spectrophotometer were 0~4000 NTU (±0.001 NTU) and −0.1~3.0 cm−1 (±0.001 cm−1), respectively. Before testing UV254 absorbance, each sample was filtered by a 0.2 μm syringe filter to reject turbid matters.The quality of feed and treated water was analyzed every 30 min during each experiment for calculating the treatment efficiencies of turbid matters and DOM. Turbidity was checked by a turbidimeter , and UV3) of 15% for 24 h to melt out inorganic or organic pollutants lingering inside the membrane. For washing and eliminating air inside the membrane pore, it was retained in distilled water for 24 h. Before performing the next study, the water-treated water flux (Jw) was checked for valuing of the membrane recovery when a cleaning process without any backwashing was accomplished with distilled water. The membrane was recovered enough if the Jw was obtained in 95–105% of the new membrane, and it was used for another study. To decrease the effect of the membrane condition on the treatment effectiveness, the recovered membrane was utilized in all of the research. After finishing each experiment, all of the synthetic solution was discharged from the combined water treatment system, and distilled water was circulated in the line of the system for 15 min for cleaning. The PES beads were recovered, and the ceramic membrane was separated from the module. The physical cleaning was performed by brushing inside the membrane, and the treated water flux of pure water was checked to decide the irreversible membrane fouling. The combustion at a 550 °C furnace for 30 min could eliminate most of the fouling constituents inside the ceramic membrane. After dropping the membrane temperature, it was dipped in a sodium hydroxide (NaOH) solution of 0.25 N for 3 h, and in a nitric acid  ceramic MF membrane and PES beads with UV radiation and intermittent water or air backwashing. Applying the resistance-in-series filtration theory (J = ΔP/(Rm + Rb + Rf)) as the equivalent means utilized in the former results [m, Rb, Rf) were calculated from treated water flux (J) data. For a fresh membrane, the theory was shortened to J = ΔP/Rm because the Rm, Rb, and Rf were zero. Finally, the Rm could be decided from the J value for a fresh membrane. The theory was reformed to J = ΔP/(Rm + Rb) at the starting time for the solution prepared with HA and kaolin, and Rb could be decided from starting the J (J0) and Rm data. Furthermore, resistances of the irreversible and reversible membrane fouling  could be estimated from the J data, before and after physical cleaning, by applying a brush inside the membrane tube.The roles of backwashing media  and TiO results , where Δ2-immobilized PES beads on membrane fouling and treatment effectiveness with air backwashing was investigated by changing the PES beads’ concentration from 0 to 50 g/L for the solution of HA 10 mg/L and kaolin 30 mg/L. The resistances of membrane fouling (Rf) maintained the bottom data at 50 g/L of the PES beads until 45 min of the process and suddenly increased at 60 min; however, it kept the peak continuously at 0 g/L of PES beads throughout all of the process, and the bottom at 30 g/L of the PES beads after 90 min, as shown in 2-immobilized PES beads could be coated rapidly by the humic acid and kaolin until 60 min into the process, and then did not have the role of TiO2 anymore after 60 min. Finally, the optimal PES bead condition could be 30 g/L for a long time during the process with air backwashing.The role of TiOf obtained the peak at 0 g/L of pure PP beads, and the bottom at 50 g/L for 3 h. This means that the more PP beads could repress the membrane fouling, the more remarkable for this combined water treatment system. It verified that the best media concentration was dependent on the materials of the beads in the combined water treatment system with air backwashing.In the former work  regardin0), where J0 was the starting treated water flux expected by utilizing the starting two values by an extrapolation means, was paralleled to inspect the role of PES beads for relative decline of treated water flux. The J/J0 data overlapped at almost every PES bead concentration; however, those obtained the peak at 50 g/L of the PES bead until the 45 min process; however, it was the peak at 30 g/L of the PES bead after 90 min which detailed experimental data. In the former results [0 obtained the maximum at 50 g/L of the PP beads during the 3 h process, and the minimum at 0~10 g/L.As shown in  results  regardin180) was at the peak at 20 and 30 g/L of the PES beads. It verified that the treated water flux could maintain the highest data at the best with 20 and 30 g/L PES beads, for the reason that the membrane fouling was repressed effectively at 20 and 30 g/L in the combined water treatment system with intermittent air backwashing. In conclusion, the J180/J0 after the 3 h process at 20 and 30 g/L of the PES beads obtained the peak 0.361, which was 1.10 times higher than 0.328 at NBW condition. Nevertheless, the total treated water volume (VT) acquired the maximal at 7.06 L at 40 g/L of the PES bead, for the reason that J was conserved higher during the 2 to 60 min process than that of 20 and 30 PES bead concentrations, as displayed in T in this PES bead concentrations. As summarized in T was the maximum at 2.09 L at 30 and 50 g/L of the PP beads because J was greater preserved during 3 h process at 30 g/L of the PP beads. It verified that the best condition of PP beads could be 30 g/L, owing to the maximum J180/J0 and VT. The best condition of PES beads and PP beads did not agree with each other, owing to dissimilar material beads.In the former study  regardinb), that was made by concentration polarization on the surface of the membrane, was the maximum at 20 g/L of the PES beads in the combined system of tubular ceramic MF and TiO2-immobilized PES beads. It verified that 20 g/L of the PES bead could diminish the concentration polarization on the surface of the membrane the most powerfully. The Rf,180 after the 3 h process at 50 g/L of the PES bead obtained the maximum at 1.443 × 109 kg/m2s, which was 1.16 times higher than the minimum at 1.245 × 109 kg/m2s at 20 and 30 g/L of the PES bead with air backwashing. As paralleled in m, Rb, Rf,180, Rif, Rrf) are visibly paralleled as bar graphs. As the Rrf obtained a dominant portion of Rf,180, it could become a main membrane fouling in this combined water treatment system with air backwashing. This means that a major part of membrane fouling could be easily recovered by physical washing such as a bushing. In addition, the Rrf and Rif were the minimum at 20 and 40 g/L of the PES beads, respectively. It verified that the 20 and 40 g/L PES beads could diminish the reversible and irreversible membrane fouling effectively in this PES bead scope, respectively.In 2-coated PES beads could remove turbid matters, such as kaolin, more effectively in the combined water treatment system of tubular ceramic MF and PES beads with air backwashing. In the former results [The treatment efficiencies of turbidity obtained a diminishing tendency when reducing the PES beads from 50 to 0 g/L, as displayed in  results  for a co254 absorbance, which could replace the DOM concentration, decreased dramatically when declining the PES bead from 50 to 0 g/L. The high input concentration of titanium dioxide-immobilized PES beads in this hybrid module could decompose the humic acid more efficiently by photo-oxidation of UV radiation in the combined water treatment system. It verified that the best PES bead condition could be 50 g/L for DOM treatment in the experimental scope of PES beads. In the former research [As paralleled in research  for the 2-immobilized PES beads on membrane fouling and treatment effectiveness, the Rf was compared depending on the PES bead concentration with water backwashing in the combined system of tubular ceramic MF (NCMT-7231) and TiO2-immobilized PES beads with water backwashing, as presented in f data overlapped almost during the whole 3 h process. As compared in f,180 obtained the peak at 0 g/L, and the bottom at 40 g/L PES bead, respectively. Finally, the 40 g/L of the PES bead could be the best condition in the combined system of tubular ceramic MF and TiO2-immobilized PES beads with water backwashing. It was a bit of a dissimilar tendency, paralleled with the optimal PES bead with air backwashing which could be 30 g/L for a long process, owing to a dissimilar backwashing medium, as presented in To examine the role of TiOf obtained the peak at 50 g/L of the PP beads and the bottom at 5 g/L during the 3 h process. This means that the best PP beads’ concentration could be 5 g/L to control the membrane fouling and high treated water flux in this combined system of 7-channel ceramic MF and PP beads.In our former research  for a co2-immobilized PES beads in relative treated water flux, the J/J0 data overlapped almost during all of the 3 h process, which was similar with the tendency of Rf in 180/J0 after the 3 h process at 40 g/L of the PES bead obtained the maximum at 0.0927, which was 1.04 times higher than the minimum 0.0888 at 50 g/L of the PES beads. It verified that the best PES bead concentration could be 40 g/L to preserve the peak treated water flux because too many PES beads could not effectively reduce the membrane fouling by blocking UV radiation to the PES bead positioned at the module inside. However, the peak J180/J0 0.361 at air backwashing was 3.89 times higher than the maximum at 0.0927 at water backwashing. It verified that air backwashing could be more effective in reducing the membrane fouling than the water backwashing in this combined water treatment. As displayed in 0 continued greater until 90 min at PP beads 5 g/L than the data at other PP beads’ condition, and obtained the bottom at 50 g/L of the PP beads after 60 min. The values of J0 and J180 declined at 40 g/L and 50 g/L, respectively, when increasing PP beads’ concentration, because the Rb and Rf amplified at 40 g/L and 50 g/L of the PP beads, respectively. Lastly, the J180/J0 after 3 h of the process at 0 g/L of the PP beads was the peak. Nevertheless, the VT was the peak at 5 g/L of PP beads because J continued greater throughout the process than the data of other PP bead conditions.In the former study  regardinT was 2.67 L at 40 g/L of the PES beads, because the treated water flux could be maintained highly throughout the 3 h process. The maximum VT 7.06 L at air backwashing was 2.64 times higher than the peak of VT 2.67 L at water backwashing. This means that the air backwashing system could acquire more treated water than the water backwashing system. As arranged in f,180 obtained the peak of 5.151 × 109 kg/m2s at 0 g/L of the PES beads, which was 1.06 times higher than the bottom at 4.862 × 109 kg/m2s at 40 g/L of the PES beads. This verified that the best PES beads could be 40 g/L because the PES beads could reduce the membrane fouling by UV photo-oxidation; nevertheless, too many PES beads blocked UV radiation to the PES beads positioned at the module inside.In conclusion, the peak of Vrf presented the main resistance of total membrane fouling; however, the Rif was a trivial one, as it had an analogous tendency with the consequences of air backwashing as shown in f,180, Rif, and Rrf was little, depending on the PES bead concentration; however, the data regarding Rf,180, Rif, and Rrf obtained much higher results than those of air backwashing. The Rif was at the bottom at 5 g/L of the PES beads for water backwashing, which was 3.43 times higher than the minimum at 40 g/L of the PES beads for air backwashing. This verified that the air backwashing could be more efficient than water backwashing in this combined water treatment system.As paralleled in As displayed in In the former work  regardinIn 2-immobilized PES beads for air and water backwashing was studied in the combined water treatment system of tubular ceramic MF and PES beads. The results of TiO2-immobilized PES beads were associated with those of the former study [In this research, the role of TiOer study ,33 in th(1)f) sustained the bottom data at 50 g/L PES beads up until 45 min of the process, and suddenly increased at 60 min; however, it was the bottom at 30 g/L of the PES beads after 90 min. Too many TiO2-immobilized PES beads could be coated rapidly by the humic acid and kaolin up until 60 min of the process, and then did not have the role of TiO2 anymore after 60 min. Finally, the optimal PES bead condition could be 30 g/L for long time during the process. In our former work [f obtained the bottom at 50 g/L PP beads. This means that more PP beads could repress the membrane fouling the more remarkably in this combined water treatment system. This also verified that the best media condition was dependent on the material of the beads in the combined water treatment system with air backwashing.For air backwashing, the resistances of membrane fouling (Rmer work  regardin(2)180/J0) after the 3 h process at 20 and 30 g/L of the PES beads obtained the peak; nevertheless, the total treated water volume (VT) had the peak at 40 g/L of the PES beads. Finally, the best PES bead concentration could be 40 g/L in the combined water treatment system with air backwashing. In the former research [T was the maximum at 30 and 50 g/L of the PP beads. This means that the best condition of PP beads could be 30 g/L. The best condition of PES and PP beads did not agree with each other, owing to different material beads.The dimensionless treated water flux f,180 obtained the bottom at 40 g/L PES beads. Finally, the 40 g/L PES beads could be the best condition. It was a little dissimilar tendency, when compared to the optimal PES beads with air backwashing could be 30 g/L for long process, owing to a different backwashing medium. In our former results [f was at the bottom at 5 g/L of the PP beads during the 3 h process. This verified that the best PP beads’ concentration could be 5 g/L to reduce the membrane fouling and a lot of treated water volume.For water backwashing, the R results  regardin(4)T was 2.67 L at 40 g/L PES beads because the treated water flux could be maintained extremely throughout the 3 h process. The maximum VT 7.06 L at air backwashing was 2.64 times higher than the peak VT 2.67 L at water backwashing. This means that the air backwashing system could acquire more treated water than the water backwashing system.The peak V(5)The effectiveness of turbidity for air backwashing obtained an increasing tendency when increasing PES beads; however, for water backwashing, it obtained almost constant, independent of PES beads. The effectiveness of DOM for air backwashing increased dramatically when increasing the beads; however, for water backwashing, it increased slowly and obtained a maximum at 40 g/L PES beads.


Circoviridae. It is an emerging virus described for the first time in 2011; since then, it has been detected in different countries and can be defined as worldwide distribution virus. CanineCV infects domestic and wild canids and is mainly related to hemorrhagic enteritis in canines. However, it has been identified in fecal samples from apparently healthy animals, where in most cases it is found in coinfection with other viral agents such as the canine parvovirus type-2 (CPV). The estimated prevalence/frequency of CanineCV has been variable in the populations and countries where it has been evaluated, reaching from 1 to 30%, and there are still many concepts to define the epidemiological characteristics of the virus. The molecular characterization and phylo-evolutive analyses that allow to postulate the wild origin and intercontinental distribution of the virus. This review focuses on the importance on continuing research and establish surveillance systems for this emerging virus.Canine Circovirus (CanineCV) belongs to the family  According to the international committee on virus taxonomy . The latter is a thermodynamically stable conserved stem–loop structure with a characteristic sequence of 9 nt for the initiation of replication (TAGTATTAC), a palindromic sequence of 12 nt pairs and an open loop of 10 nt (CATAGTATTA) . These hGeminiviridae and Nanoviridae. For circoviruses, the study model has been mainly that of porcine circovirus (PCV). Due to the reduced size of the viral genome that limits its coding capacity, this type of replication depends largely on host cell factors and requires cellular enzymes expressed during the S phase of the cell cycle  and, subsequently, have adapted to domestic and wild canids .4.In 2013, Li et al. characterized the complete genome of CanineCV from the liver of a dog with severe hemorrhagic gastroenteritis, granulomatous lymphadenitis, and vasculitis. Since then, CanineCV infection has been associated with clinical profiles of acute gastroenteritis, hemorrhagic diarrhea, signs of vasculitis, lymphadenitis, thrombocytopenia, neutropenia, and lymphopenia, which is related to immunosuppressive activity due to lymphoid tissue damage; the latter has also been described in infection by PCVs in pigs and in birds suffering Beak and Feather Disease Virus (BFDV) , 28. OthDifferent forms of infection could be suggested, such as subclinical, clinical or coinfection with other infectious agents, and the fecal route as the main route for transmission , 6, 28. The prevalence of CanineCV in different countries, both in samples of patients with compatible signs and asymptomatic animals, is variable. Li et al. in 2013 in the United States  found a Sun W et al. in 2019, in the southern region of Guangxi established a molecular prevalence of CanineCV in serum samples of dogs with diarrhea of 8.75% and in healthy dogs of 9.25% . In GermIn middle east region, the presence of CanineCv has been studied in Turkey and Iran. In Turkey, from a total of 150 rectal swab samples collected from animals with manifestations of gastrointestinal disease, nine positive samples were obtained (6%) with coinfection with the other two viruses between 2 to 5% . To inveThe only study reported to date on seroprevalence of CanineCV was conducted with samples taken between 2016 and 2017 in northeast China . AuthorsThe prevalence of CanineCV varies over a wide range. The differences between the studies may be due to multiple factors as mentioned before. In addition, it is not yet possible to define whether the virus is found more frequently in dogs with diarrhea than in healthy animals, as shown by the variations in the results of the reports published to date. Therefore, it is important to conduct molecular and research that clearly defines the epidemiological characteristics of the virus and its role as an infectious agent in gastrointestinal clinical disease in domestic dogs.5.Necropsy and immunohistochemistry of retrospective cases of dogs with signs compatible with CanineCV has shown critical lesions, such as necrotizing vasculitis in the intestine and spleen, granulomatous lymphadenitis and histiocytic drainage in Peyer’s patches and lymph nodes, and microscopic lesions in the kidney . Clinicoin situ hybridization or immunohistochemistry the presence of this viral agent in compatible lesions are still lacking  from Italy by using molecular and phylogenetic approaches, CanineCV DNA was detected in one of 32 processed stool samples; confirming that dogs and foxes could share phylogenetic related circoviruses  from the Svalbard archipelago, Norway and 59 red foxes (Vulpes vulpes) from Northern Norway (mainland) sampled from 1996 to 2018, CanineCV DNA was detected in 10 red foxes and 11 arctic foxes. Positive DNA were found even in samples from Artic foxes sampled in 1996, fifteen years before the first report in domestic dogs in USA, suggesting that wild carnivores could have harbored an ancestor of CanineCV that could later be transmitted to domestic canids, thus highlighting the role of wild carnivores in the transmission of pathogens to the domestic dogs populations , canine coronavirus and canine distemper has been found , porcine reproductive and respiratory syndrome virus (PRRSV) and porcine parvovirus (PPV). Coinfection has even been described between different PCV2 genotypes. Also, it has been suggested that coinfections are associated with a greater severity of infection ; however, after multiple investigations, the association of PCV2 with other clinical conditions, such as hepatitis, reproductive failure, respiratory disease and nephropathy (PDNS), was established. It has also been associated with porcine respiratory complex disease (PRDC) , 38. Curnfection , 44.The adverse effect of PCV2 coinfections with the aforementioned agents could be explained by the repercussions on the immune system of pigs since the virus targets lymphoid tissues. The replication of PCV2 destroys the normal architecture of the lymphoid follicles, causing lymphoid depletion that is then replaced by histiocytes. Replication of the virus has been evidenced in organs such as the spleen, tonsils, lungs, kidney, liver and thymus; in the latter, PCV2 alters the selection of T lymphocytes, particularly in younger animals. It also infects dendritic cells, limiting their function; such effects generate severe lymphoid depletion and severe immunosuppression in pigs, increasing the probability of contracting other infections , 44.There are also reports of the detection of PCV2 and PCV3 in dogs. To date, PCV2 has been discovered in other reservoirs, such as calves, goats, mice, and in carnivores, such as mink and foxes, indicating that transmission to nonporcine hosts is possible. Close contact between animals is probably the main route of transmission, particularly in multispecies farms and sites accessible to wildlife , 46. TheSimilar to PCV2 infection, the pathogenesis of CanineCV and its participation as a coinfecting agent is not yet clearly defined. Current research suggests is that its role as a pathogen may be to exacerbate clinical conditions, like PCV2 infection, weakening the immune system and favoring the entry and dissemination of other viruses and bacteria. This is because, in the majority of the CanineCV detections performed, the virus has been found to be accompanied by other infectious agents, such as CPV with coinfection rates of up to 100% . To dateBased on the findings of the genetic material of CanineCV in lymphoid tissues, it has been proposed different hypotheses regarding the clinical manifestation of the CanineCV infection , 28. FirIt has been also evaluated the possible role of CanineCV in acute hemorrhagic diarrhea syndrome. According to the results, it has been reported a low probability that CanineCV acts as a putative agent of the syndrome, but CanineCV may be a predisposing element for bacterial or viral infections. The prevalence of CanineCV in the study was 4.6%, indicating that it could be only an incidental finding where the virus would be part of the normal intestinal microbiota of canines ; this ha8.The discovery of emerging viruses such as CanineCV raises an important research topic in domestic and wild animals. The evidence of multiple gaps in the knowledge of this virus in aspects such as pathogenic and epidemiological characteristics raises the need to create and continue with studies that allow expanding and defining the knowledge about CanineCV infection. Scientific evidence supports that this virus poses a risk to the health and well-being of the canine population, so work must be done to define many concepts and gaps in the understanding of this emerging pathogen. Therefore, its necessary to strength the knowledge of veterinarians and clinicians about the possible manifestations as a disease not only as a single agent but also in coinfection with other pathogens. Additionally, the role of wild canids should be highlighted, since, according to recent research, it is evident that in this population, CanineCV seems to be more pathogenic, and its participation in the transmission of the virus to domestic canines seems to be relevant, especially because human invasion of wild habitats favors contact between both populations, which contributes to the transmission of interspecies pathogens.The role of CanineCV as a primary agent or coinfecting in enteric diseases in canines remains uncertain; existing research suggests the importance of continuing with other study models that provide new information to define the concepts that remain doubtful until now. As can be observed in the development of this article, CanineCV could have different pathways of disease development in canines, either as a primary agent that leads to generating the clinical signs described or to cause subclinical infection where there are no signs but where the animal could spread the virus or contribute to the severity of the disease in coinfection with other agents. In addition, CanineCV, like the other circoviruses, could play an important role in the immune response of the animal, favoring the entry of other infectious agents or preventing its recovery .9.The pathogenic and epidemiological characteristics of CanineCV are currently poorly understood, so CanineCV represents a multidisciplinary exploration challenge both in the field of research and in the clinic. Understanding the particularities of this new virus either in healthy animals or coinfected with other agents could provide valuable information regarding prophylactic or therapeutic practices in enteric diseases in canines, contributing to the health and well-being of canine populations. Thus, it is necessary to continue carrying out research in different canine populations that allow the evaluation of the epidemiology of CanineCV in geographical sites not yet explored and its behavior in healthy or coinfected animals, in addition, to monitoring the clinical evolution of the infected animals to help to understand the pathogenesis of the CanineCV.JR-S and JJ: conceptualization, validation, resources, and data curation. DG-B and DV-B: methodology, formal analysis, and investigation. DG-B: writing—original draft preparation. DG-B, DV-B, SG-R, JJ, and JR-S: writing—review and editing. SG-R: software and visualization. JR-S: supervision, project administration, and funding acquisition. All authors contributed to the article and approved the submitted version.This research was funded by a CONADI-UCC grant to JR-S and the APC was funded by CONADI-UCC.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.


Many viruses form highly pleomorphic particles. In influenza, virion structure is of interest not only in the context of virus assembly, but also because pleomorphic variations may correlate with infectivity and pathogenicity. We have used fluorescence super-resolution microscopy combined with a rapid automated analysis pipeline, a method well-suited to the study of large numbers of pleomorphic structures, to image many thousands of individual influenza virions; gaining information on their size, morphology and the distribution of membrane-embedded and internal proteins. We observed broad phenotypic variability in filament size, and Fourier transform analysis of super-resolution images demonstrated no generalized common spatial frequency patterning of HA or NA on the virion surface, suggesting a model of virus particle assembly where the release of progeny filaments from cells occurs in a stochastic way. We also showed that viral RNP complexes are located preferentially within Archetti bodies when these were observed at filament ends, suggesting that these structures may play a role in virus transmission. Our approach therefore offers exciting new insights into influenza virus morphology and represents a powerful technique that is easily extendable to the study of pleomorphism in other pathogenic viruses. Viruses are significant human pathogens. In influenza, virus structure and morphology has been linked to pathogenicity; however commonly used methods to study virus structure are often low through-put, or lack the resolution required to resolve virus particle features. In addition, influenza filaments are frequently understudied due to their fragile nature and loss of filamentous morphology during viral passage. In order to address this, we have developed a fluorescence super-resolution microscopy and rapid automated analysis pipeline to image many thousands of individual influenza virions at a time, gaining information on their size, morphology and protein distribution. Using this method we were able to show that there is no specific alternation of the surface glycoproteins in filaments, and that neuraminidase-enriched Archetti bodies preferentially house viral ribonucleoprotein complexes. Our findings offer new insights into influenza virus particle assembly and virus transmission, and further, our versatile methods have the potential to contribute towards multiple areas of virus research. Viral disease results in significant illness and deaths in humans each year, and thus represents a large healthcare and economic burden to countries around the world. The COVID-19 pandemic has resulted in the deaths of millions of people, while annual influenza epidemics can result in up to 650,000 respiratory deaths per year [Influenza virus particles are highly pleomorphic , rangingInfluenza virus particles are surrounded by a lipid bilayer, in which two surface glycoproteins, the hemagglutinin (HA) and neuraminidase (NA), as well as the M2 protein, are embedded . The M2 Inside the M1 core lies the viral genome, which is comprised of 8 viral RNA segments, each of which is bound by multiple nucleoproteins (NP) and the RNA polymerase  to make ribonucleoprotein (RNP) complexes. Imaging work has suggested that RNP complexes cluster at one end of filamentous virus particles ,19–21,26The imaging of viruses by conventional fluorescence microscopy is limited as virus particles are generally smaller than the ~ 250 nm resolution limit of optical microscopy for visible light, making it impossible to gain detailed information on the shape, size and protein organisation of individual virions. Electron microscopy (EM), and more recently cryo-EM, are excellent methods for high-resolution imaging of virus structure, but can be comparatively low throughput, time consuming, and offer limited molecular identification. The pleomorphy of many viruses also precludes visualization by procedures that rely on averaging many identical particles, and as a result, it remains challenging to measure protein distributions and abundance at the single-virus level across an entire population. To overcome those limitations and facilitate the high-throughput acquisition of images of filamentous influenza virions, we used direct stochastic optical reconstruction microscopy (dSTORM) , a technIn this study, we have combined dSTORM imaging methodology with rapid automated analysis software to carry out a high-throughput and high-resolution analysis of thousands of virions at a time. We have used these tools to investigate the structure and organization of influenza H3N2 A/Udorn/72; a strain which exhibits both spherical and filamentous morphology. We found that length analysis provided a useful way of characterizing virions: filaments longer than ~ 230 nm formed a broad size distribution, while smaller particles formed two distinct populations, likely corresponding to spherical and elongated virions. In contrast to previous analyses , axial rIn order to establish a rapid, robust and high-throughput method of imaging virus particles we used influenza A/Udorn/72, an influenza strain with a well-characterized spherical and filamentous phenotype . We initNext, we imaged the immobilized virus particles using a widefield total internal reflection fluorescence (TIRF) microscope. A/Udorn/72 virions were dual-stained using polyclonal anti-HA and anti-NA primary antibodies and secondary antibodies labelled with Alexa546 (green) and Alexa647 (red) respectively . A singlOur initial images showed a population of elongated virus particles with large variability in length, ranging from approximately 250 nm to several microns. Due to the large size of the viral filaments (larger than the diffraction limit), we initially performed a high-throughput length analysis of multiple filaments using widefield images containing diffraction-limited signals. Multiple images of A/Udorn/72 virus labelled with an anti-HA antibody were taken and a rapid automated analysis pipeline was used to measure filament length . We adjuThis process was completed on 486 individual FOVs, allowing the lengths of 46,872 filamentous particles to be measured. The resulting histogram was fitted with a single exponential function . RepeatiHaving completed a comprehensive length analysis of long virions we next considered how to do this for smaller virus particles  categories). The small size of these viruses precluded the use of widefield images to get accurate length data, and so we turned to super-resolution images to obtain their size distribution. A/Udorn/72 virus was immobilized, stained and imaged using dSTORM as described above; the DBSCAN clustering algorithm  was usedThis process was completed on 46 individual FOVs, allowing the size of 41,754 particles to be measured. By repeating the process on virus-negative FOVs, the data was corrected for noise or background . The resEM images of virus particles have previously been categorized based on their axial ratio , as well as length. We therefore plotted the major/minor axis histogram from the measured particles fitted with a single exponential function , as wellIn order to demonstrate the versatility of our imaging and analysis pipelines, we carried out similar experiments on the SARS-CoV-2 virus, the causative agent of the COVID-19 pandemic. Fixed SARS-CoV-2 virions were dual-labelled for the spike (red) and nucleocapsid (green) proteins ; multiplThe distributions of the HA and NA glycoproteins over the virion surface are not entirely random; indeed, when we stained filaments for either HA  or NA F the resu-1 which is due to the spatial frequency of the filament as a whole (-1) may suggest that individual filaments can contain patterning at a certain spatial frequency, however these frequencies can only be seen in long filaments—the peak suggests patterning on the length scale of >300 nm—and so this is likely to be an artefact of undersampling of longer filaments. Similar analysis was conducted for the NA protein . In ordIn order to determine the position of the RNA in each filament, a line was drawn along the axis of the filaments  and the intensity profiles of the line in both the green and red channels was measured. The averaged intensity of the HA signal from all 68 filaments analysed was uniformly distributed over the length of the filaments , howeverThe small size and pleomorphic nature of viruses precludes their visualization by single-particle averaging methods or conventional fluorescence imaging, making it difficult to measure protein distributions at the single-virus level across an entire population. In this work, we have shown that a combination of super-resolution imaging and high-throughput analysis of virus particles allows for the structural features of multiple virions to be examined at a time. Using the highly pleomorphic influenza strain A/Udorn/72 as a proof of principle, we imaged thousands of filamentous and spherical virions, allowing the features of a large population of virus particles to be ascertained at high-resolution.Large viral filaments that measured over 250nm in length were analysed using widefield microscopy; analysis of the size distribution of >40,000 filamentous virus in this way showed a broad range of filament sizes. The exact binding epitopes of the antibodies used are unknown, however for HA , we observed uninterrupted HA signal along the entire length of the imaged filaments  the use of antibodies, which can add an additional linkage error between reporter and epitope which has been estimated to be 10–15 nm , ultimatOur analysis demonstrated large variability in filament length, as well as a lack of distinctly sized sub-populations within a filamentous virus population. In influenza, the initiation of virus budding is thought to be initiated by clustering of HA and NA in lipid raft domains on the cell surface, followed by recruitment of M1 which serves as a docking site for the RNPs just below the plasma membrane . ElongatNA has been shown previously to preferentially cluster at the filament pole, either at the same  or the oIn this work, we have shown that high-resolution microscopy and rapid automated analysis software can be used to gain structural information at the single-virus level on thousands of virions at a time. Although this study introduces these methods on two well-studied viral species with available antibodies against the major viral proteins, there could be general applicability of the techniques to less-well studied virus types, and so our methods may also be useful for future studies of virus assembly and budding, cell-to-cell transmission and virus pathogenesis, or the development of viral diagnostic strategies that rely on virus morphology for identification.The influenza strain H3N2 A/Udorn/72 (Udorn) was grown in Madin-Darby Canine Kidney (MDCK) cells as described previously . To prodUnless otherwise specified, viruses were immobilized using poly-L-lysine. Coverslips  were heated in a furnace at 500°C for 1 hour to burn off any dust. A silicone gasket  was added to the glass slide and 0.01% poly-L-Lysine (Sigma) solution was added to the wells for 30 minutes. The excess was removed and the chambers were washed three times with MilliQ water and the slide allowed to dry. As an alternative chitosan was used in place of poly-L-lysine (0.015% chitosan powder (Sigma) in 0.1M ethanoic acid).For specific immobilisation of viruses via biotinylation, passivated microscope slides were prepared by washing in acetone and Vectabond solution (Vector Laboratories) before being incubated with NHS-PEG:Biotin-NHS-PEG in an 80:1 ratio. 0.5 mg/mL neutravidin was incubated for 10 minutes at room temperature on the slide shortly before virus was added. Viruses were biotinylated by incubation in a 1 mg/mL Sulfo-NHS-LC-Biotin (ThermoFisher) for 3 hours at 37°C before being fixed and immunolabelled as described below.Virus was incubated in the wells at 45°C until the well was dry. Immobilised virus was fixed with 4% formaldehyde (Thermo Scientific) in phosphate buffered saline (PBS) for 10 minutes at room temperature, before being permeabilised in 0.5% Triton-X-100  for 15 minutes. The sample was blocked to prevent non-specific binding with 10% donkey serum (Sigma) and 0.1% Tween-20 in PBS for 1 hour at room temperature. Primary antibodies were diluted in blocking buffer and incubated with the sample for 1 hour, followed by washing in PBS and labelling with secondary antibodies (Invitrogen). A final washing step was carried out and samples were stored in PBS at 4°C until imaging. For super resolution imaging, the PBS was replaced with a STORM imaging buffer comprising an enzymatic oxygen scavenging system consisting of 1 mg/mL glucose oxidase and 40 μg/mL catalase, 10% glucose and 0.05 M mercaptoethylamine (MEA) in 1x PBS. Multiple independent repeats, with different virus preparations, were taken over different days.A/Udorn/72 virions were labelled with the mouse anti-HA primary antibody Hc83x  and goat anti-NA and anti-M1 . SARS-CoV-2 virions were labelled with the human anti-spike antibody EY6A   and subsequently permeabilized with 0.5% Triton X-100 in PBS for 10 min. After washing with 2x SSC, the surface was blocked with blocking buffer  for 30 minutes at 37°C. The sample was then incubated with 1 μM FISH probes  in blocking buffer for 1-3h at 37°C. After hybridization, the sample was washed thrice with 2x SSC, 10% formamide, 1% RNasin Plus and then washed three times with 2x SSC before being imaged.2 for both the green (532 nm) and red (647 nm) lasers, and movies of between 5,000 and 15,000 frames were taken. The red channel was imaged first, followed by the green channel.All fluorescence imaging experiments were performed on a commercially available Nanoimager fluorescence microscope (Oxford Nanoimaging). The sample was imaged using total internal reflection fluorescence (TIRF) microscopy. The laser illumination was focused at an angle of 53° with respect to the default position. Images of a field of view (FOV) measuring 80 x 50 μm were taken with an exposure time of 30 ms. For super resolution imaging the laser intensities were gradually increased up to a maximum of 780 kW/cm2, y2) in the x and y coordinates of the green channel for all colocalised localisations.Drift correction was computed using the inbuilt Nanoimager software, which uses phase correlation between consecutive frames containing a minimum threshold number of localisations to find a translative offset between the two images. In the event that the minimal localization threshold isn’t reached for some later frames in very long acquisitions, there may still be the appearance of minor drift in some clusters in the image. Channel mapping was also computed using the inbuilt Nanoimager software. This uses a mapping file generated from images of a Tetraspek microsphere bead sample which fluoresces in both the red and green channels. The channel offset is found from the phase correlation shift between the two channels and then, using a maximum distance, the software finds colocalised localisations in the channels that do not colocalise with multiple localisations in the other channel. Finally, the mapping matrix is found using least squares minimisation for up to the second order  labelled with the protein of interest (HA or NA) were skeletonized as in the filament length analysis. For each pixel in the skeleton of filaments, a fit line was produced (using the MATLAB polyfit function) using the next 5 points in each direction from the pixel of interest. From this quadratic, the normal was found and the intensity of the pixels in the normal were summed. After iterating this over the skeleton, the calculated line profile of each filament were Fourier transformed using the MATLAB fft function and the resulting distributions across all filaments were summed to produce the average frequency spectrum for that surface protein .dSTORM images of viruses were simulated using a Monte Carlo method with the probability of a protein being placed equally across the virion and an exclusion distance of 20nm. Filaments were modelled as cylinders with hemispherical caps. After projecting the simulated filaments into 2D, localisations were randomly placed with a normal distribution about the protein location with a standard deviation of 7.4nm  and the image was coloured as a STORM image . AlternaSupernatant containing both floating cells and virus was harvested from MDBK cells infected with A/Udorn/72 virus was spun onto 8-well plates (Thermo Scientific) at 5000 RPM for 5 minutes to immobilise the sample onto glass. Samples were stained for HA and NA and imaged as described above. Using data from both channels as a guide, a line was drawn in FIJI from the tips of filaments to just before the filament broadened at the cell membrane, with a width slightly greater than the filament width. The lengths of the filaments and intensity profile were normalised (0–1) per filament and summed.The inbuilt Nanoimager software was used to create dual-colour images of diffraction-limited green fluorescence and super-resolution localisations of red fluorescence of the filaments. To create the intensity along the filament figures, movies recorded at 30 ms exposure with 1000 frames were projected in FIJI to make a single summation image. In FIJI, a line was taken along the axis of the filaments measuring the intensity in the green and red emission channels of the microscope. Filaments containing Archetti bodies were orientated with the Archetti bodies at one end, while filaments without were measured from left to right on the screen, assuming they randomly orientate themselves on the surface. The background intensity  was subtracted to produce the raw intensity of the fluorescence, the length in pixels was normalised (0–1), and graphs plotted for all lines.S1 FigA) A virus negative control (top) or an A/Udorn/72 virus sample (bottom), were immobilized via a specific biotin/PEG linkage. Virus particles were biotinylated by incubation with 1 mg/mL Sulfo-NHS-LC-Biotin (ThermoFisher) for 3 hours at 37°C before being immobilised on a pegylated slide. The virus was labelled with an anti-Udorn primary antibody and an Alexa647 secondary antibody and imaged on a widefield TIRF microscope. Scale bars 10 μm. B) A virus sample was incubated on a slide pre-treated with 0.01% poly-L-lysine. C) A virus sample was incubated on a slide pre-treated with 0.015 mg/mL chitosan in 0.1 M acetic acid.(JPG)Click here for additional data file.S2 FigA) A virus negative control (top) or an A/Udorn/72 virus sample (bottom), were incubated on a slide pre-treated with 0.01% poly-L-lysine at 4°C for 10 minutes. The excess sample was removed from the well and the immobilized virus was fixed and labelled with an anti-Udorn primary antibody and an Alexa647 secondary antibody before being imaged on a widefield TIRF microscope. Scale bars 10 μm. B) As in A) but the slide was not pre-treated with poly-L-lysine and the samples were dried directly onto glass coverslips by heating at 45°C for 10 minutes. C) As in A) but the samples were dried directly onto glass coverslips by heating at 45°C for 10 minutes. D) dSTORM reconstructions of Udorn-stained influenza filaments either dried directly onto glass coverslips by heating at 45°C for 10 minutes or incubated on the coverslips at room temperature for 10 minutes. Scale bars 0.5 μm. E) Plot of number of particles detected per diffraction-limited field-of-view (FOV) under different fixation and permeabilization conditions.(JPG)Click here for additional data file.S3 FigA) A representative super-resolution image of a virus-negative sample virus stained with an antibody against the HA protein. Scale bar 10 μm. B) Super-resolution localisations were clustered and each cluster fitted with an ellipse to extract particle dimensions. A histogram of the major axis lengths shows that background signal is negligible. C) Zoomed in histogram of B).(PNG)Click here for additional data file.S4 FigA) Each FOV was drift corrected, and a Gaussian function was fitted to each detected localization in every frame of the acquisition, using the inbuilt Nanoimager software. The fitting error  in the x direction of each localization was exported and plotted as a histogram, providing a median error of 7.4 nm. B) Plot of the localisation precision in the y direction, providing a median error of 7.2 nm.(JPG)Click here for additional data file.S5 FigA) A plot of the major axis against minor axis for the influenza HA protein. B) A heatmap of the major axis against minor axis for the influenza HA protein. C) A plot of the major axis against minor axis for the SARS-CoV-2 spike protein. D) Histogram of the major/minor axis ratio of spike localisations shows a single distribution. E) Analysis of the envelope protein also falls into a single population centered at 83.0 nm. F) Representative super-resolution images of SARS-CoV-2 virions dual-labelled with anti-envelope and anti-nucleocapsid primary antibodies. Scale bar 100 nm.(JPG)Click here for additional data file.S6 FigA) The average structure when centering spike protein localisations on the centroid of nucleocapsid protein. B) The average structure when centering all proteins on their own centroid and aligning these centres. Scale bars 30 nm(JPG)Click here for additional data file.S7 FigInput parameters of virion size, number of localisations and localization precision were used for Monte Carlo simulations to create simulated dSTORM images of filamentous virions. Filaments were modelled as cylinders with hemispherical caps. After projecting the simulated filaments into 2D, localisations were randomly placed with a normal distribution about the protein location with a standard deviation of 7.4nm and the image was coloured as a STORM image. Scale bar 20 nm.(JPG)Click here for additional data file.S8 Fig-1; the limit of visible frequencies occurs when the length scales are on the order of half of filament lengths. B) The frequency distribution from 1000 nm simulated filaments with protein frequency patterned at 0.007 nm-1 and 0.033 nm-1 show patterning at multiple frequencies can be seen. A small peak from a harmonic of the 0.007 nm-1 frequency is also observed.A) Frequency distribution from 500 nm simulated filaments with protein frequency patterning at 0.024 nm(JPG)Click here for additional data file.S9 FigA) Diffraction limited image of A/Udorn/72 filaments stained with for HA (green) and NA (red) budding out of an infected cell. White line denotes a filament with an Archetti body (arrow) at the tip. Scale bar 1 μm. B) Intensity trace of the NA signal from the filament highlighted in A), showing higher NA signal at the Archetti body at the tip. C) Normalised averaged intensity traces of the NA signal from filaments without obvious Archetti body structures at the tip, showing an enrichment at the filament ends furthest from the cell membrane.(JPG)Click here for additional data file.S10 FigDiffraction limited images of filamentous A/Udorn/72 stained with an anti-HA antibody , super-resolution localisations from an array of FISH probes against the NA gene segment , and merged images (right panels). White boxes denote Archetti bodies. Scale bars 1 μm.(JPG)Click here for additional data file.S11 FigA) Top: Raw intensity traces (grey) of the HA signal from 14 filaments with a visible Archetti body, with the average intensity profile shown as a green line. Bottom: Normalised average RNA signal from the 14 filaments, showing peak in intensity of the HA signal in Archetti bodies. B) Same as A) but for the HA signal from 54 filaments with no visible Archetti body. C) Top: Raw intensity traces (grey) of the RNA signal from 23 filaments with intensity peaks above an arbitrary background threshold of 10, with the average intensity profile shown as a red line. Bottom: Normalised average RNA intensity from the 23 filaments. D) Same as C) but for the RNA signal from all non-Archetti filaments that have been re-orientated to line up the largest peaks on one side.(JPG)Click here for additional data file.


Current evidence suggests that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can remain suspended spread in aerosols for longer period of time under poorly ventilated indoor setting. To minimize spreading, application of antiviral filter to capture infectious aerosols and to inactivate SARS-CoV-2 can be a promising solution. This study aimed to develop a method to assess simultaneously the filtration and removal efficiency of aerosolized pseudo-type SARS-CoV-2 using a vertical-type wind tunnel with relatively high face velocity (1.3 m/s). Comparing with the untreated spunlace non-woven filter, the C-POLAR™ treated filter increased the filtration efficiency from 74.2 ± 11.5% to 97.2 ± 1.7%, with the removal efficiency of 99.4 ± 0.051%. The results provided not only solid evidence to support the effectiveness of the cationic polymeric coated filter in fighting against the SARS-CoV-2 pandemic, but also a method to test viral filtration and removal efficiency under relative fast air velocity and with a safer environment to the operators. The disease is caused by a positive-sense single-stranded RNA virus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)2 with spherical or elliptic morphology. The diameter of SARS-CoV-2 is approximately 60–140 nm, with a crown-like appearance due to the expression of spike glycoproteins on the envelope surface Some studies suggested that the spike glycoproteins are responsible for receptor binding and entering into the host cell4. It can transmit from human-to-human by multiple means, including short-range airborne transmission5 through atomization of SARS-CoV-2 in respiratory droplets (≥ 5 μm) and fine aerosols (< 5 μm) by inhalation, coughing or sneezing from an infected person6. Fears et al.7 also demonstrated that SARS-CoV-2 is persistent in aerosol suspension with the mass median aerodynamic diameter of around 2 μm.As of May 2023, the outbreak of coronavirus disease 2019 (COVID-19) has caused more than 766 million cases and more than 6.9 million deaths globally8 and high-efficiency particulate air (HEPA) for heating, ventilation, and air conditioning (HVAC) system9. Since it is difficult to quantify virus in air samples, many of filtration efficiency studies were conducted by observing any residual viral RNA contamination on-site or using aerosol containing salt solution or bacteria as model to mimic viruses. There is a lack of standard method to directly assess the removal efficiency of virus10, especially for filters used in a HVAC or air purifier with relatively high face velocity, which remains uncertainty to the effectiveness of filter materials in prevention of SARS-CoV-2 infection.In order to reduce the risk of infection, different types of filters have been thus deployed to reduce the aerosolized SARS-CoV-2, such as melt-blown polypropylene (MBPP) electret fabric11 and dielectric filter discharge12 to inactivate SARS-CoV-2 bioaerosol, these systems have their own limitations including consumption of energy, and increase the ozone concentration in the treated air. Recently, a cationic polymeric coated filter system was introduced by C-POLAR™ Technologies, Inc. (https://cpolartechnologies.com), named as C-POLAR™ treated filter, consisting of a polyamine, a cationic polymer that are widely used as a gene delivery vector with high transfection efficiencies13. The C-POLAR™ material was used as coating on spunlace filter to increase the capture of negatively polar microbials in aerosol and to inactivate microbials through penetration of membrane and envelope by its high density of positively polar groups along the backbone chain.Apart from reducing of bioaerosols through effective filtering, it is also important to inactivate the virus to prevent from fouling and secondary transmission. Some studies suggested the use of ultraviolet-C14. Pseudo-type virus is a genetical modified virus with attenuated virulence comparing with the wild-type, which get rid of the native surface protein expression and replaced with the desired surface proteins. The resulting pseudo-type virus can still infect the host cell but cannot replicate inside15. Importantly, the study provides a method to evaluate other filter system for filtration efficiency and antimicrobial properties in a relatively high air face velocity setting.In this study, a vertical-type wind tunnel was developed Fig.  to evalu17. Although the use of virulence of the pseudo-type SARS-CoV-2 virus is much lower than the wild-type, the design of the wind tunnel was made under negative pressure using vacuum pump in order to minimize the risk of accidental leaks of pseudo-type virus to the atmosphere18, where the bioaerosols together with fresh air at the entrance would pass all the way through to the test (upstream), PTFE (downstream) and vent (HEPA) filter and release at the exhaust. The whole setup together with the exhaust gas were circulated inside the biosafety cabinet to ensure the operator’s safety during the experiment. The design of wind tunnel using a vertical configuration rather than a traditional horizonal type is to minimize the effect of aerosol and droplet particles settling onto the horizontal ducting surface by gravitational acceleration and increase the recovery of virus input19. The face velocity of the wind tunnel (i.e. 1.3 m/s) was comparable to the required by the ASHRAE Standard 52.2 for uniformity testing of the standard test duct20. The mass medium aerodynamic diameter of aerosol (i.e. 1.72 ± 0.26 μm) generated by the nebulizer was found similar to other studies for viral filtration performance22. McCluskey et al.23 specified aerosols within the size range of 0.5–20 μm are more likely to retain in the respiratory tract to cause the infection. Results from the study could be benchmarked with other testing methods to mimic bioaerosol removal efficiency in heating, ventilation, and air conditioning (HVAC) systems.Compared with traditional assays, the use of pseudo-type virus assay demonstrated good correlation with wild-type SARS-CoV-2 assays, while maintaining a high throughput and requiring fewer biosafety requirements of the laboratoryn = 3) of the virus input (directly collected at the entrance of plastic tubing from the nebulizer), and virus titers retained on the upstream and downstream filters. A consistent loading of pseudo-type virus in aerosol was delivered by the nebulizer to the wind tunnel, where a total of 9.60 × 105 ± 5.17 × 104 infectious units (IU) was aerosolized in each trial. The mean diameter of aerosol generated by the nebulizer, and the total amount of virus titers filtered by upstream and downstream filters in the control group (i.e. untreated spunlace non-woven filter) were found comparable to other wind tunnel studies18, where less virus volume as well as total virus titers were inputted in our experimental design. For the upstream filters, the average infectious units (IU) found on the cationic polymeric coated and untreated spunlace non-woven filters were found to contain 2.65 × 103 ± 4.29 × 103 IU and 4.47 × 105 ± 4.03 × 104 IU respectively. No detectable viable virus was found on 2 out of 3 C-POLAR™ treated filters under trial. The average and standard deviation viable counts for CPS filter were calculated with the use of detection limit (i.e. 1.03 × 102 IU/filter).Table t test, p-value = 0.036). There was also an order of magnitude decrease in titers found on downstream filter for the experiment of cationic polymeric coated filter, indicating a better trapping efficiency of the C-POLAR™ coated filter than untreated spunlace non-woven filter alone. The pH value of SARS-CoV-2 aerosol is generally alkaline and increased over time24, where the polarity of virus particles at neutral to alkaline pH would become negative as the virus’s isoelectric point is generally below 725. The cationic polarization matrix induced on the filter surface by the polymeric C-POLAR™ material coated on the spunlace non-woven filter would thus enhance the capturing efficiency of virus particles in aerosol.As shown in Fig. 21 which used MS2 bacteriophage as the model, the viral filtration efficiency for the cationic polymeric coated filter was comparable to a minimum efficiency rating values (MERV) 14 filter under similar air face velocity. For the untreated spunlace non-woven filter, the filtration efficiency was slightly poorer than a MERV 12 filter, which indicated the application of positively polar C-POLAR™ material on the filter improving the strength in arresting bioaerosol and the resulting rating value.Compared with the results from Zhang et al.12, treated filter provide comparable removal efficiency of pseudo-type SARS-CoV-2 even at a much higher face velocity (1.3 m/s versus 0.18 m/s) and no energy consumption is required. Purwar et al.22 proposed a 3-layer omni-phobic coated polypropylene/copper coated diamond-like carbon (DLC) /non-woven (Hy-Cu) filter, which caused rupture to the lipid envelope of virus by the lipophobic property of the top layer and inactivate virus by copper surface on the middle layer. From its result of virus inactivation, the Hy–Cu filter with DLC was found to have 99% inactivation of MS2 bacteriophage virus over a period of 2 h (with the face velocity of 0.01 m/s), where the treated filter in this study can inactivate with more than 99% instantaneously just after the completion of aerosol spraying in 5 min.By the use of infectious titer assay, the log reduction and removal efficiency of cationic polymeric coated filter was found to be 2.24 ± 0.038 and 99.4 ± 0.051% respectively, which imply the treated filter could inactivate pseudo-type SARS-CoV-2 effectively under relatively high face velocity. Compared with the use of dielectric filter discharge which inactivated SARS-CoV-2 by generation of ozone and other oxygen reactive species26.The wind tunnel system in this study only assessed the viable virus by means of infectious titer assay coupling with quantitative polymerase chain reaction (qPCR) technique to determine the genome DNA from infected cells through transduction rather than the use of quantitative reverse transcription polymerase chain reaction (RT-qPCR) method to determine RNA recovered from filter extracts, as the RT-qPCR technique may create false positive results by detecting longer RNA fragments released from inactivated virus retained on the filtersBy the use of the vertical-type wind tunnel system developed, the ability of cationic polymeric coated filter in capturing of bioaerosols and inactivation of pseudo-type SARS-CoV-2 under were assessed experimentally. Bioaerosols passing through the cationic polymeric coated filter under tests showed a significant reduction by its 97.2 ± 1.7% filtration efficiency and 99.4 ± 0.051% removal efficiency, providing solid evidence to support the effectiveness of the cationic polymeric coated filter in fighting against the SARS-CoV-2 pandemic. The wind tunnel system can provide a consistent virus loading, relative fast air velocity and a safer environment to the operators. In the future, studies on the application flexible design vertical-type wind tunnel to filters will be conducted by the use of other microbial strains, and further improvement of the performance of the vertical-type wind tunnel system will be considered.n = 3).In this study, both the C-POLAR™ treated filter and untreated filter (i.e. polyester based non-woven spunlace filter) were obtained from C-POLAR™ technologies , where the C-POLAR™ treated filter was prepared by applying a high-pressure stream of 6% C-POLAR™ polymer aqueous solution onto the untreated filter, followed by in-line drying the filter. Regarding the physical performance of the the C-POLAR™ treated filter, the minimum efficiency reporting values (MERV) in terms of particle filtration efficiency was increased from MERV 8 (for the untreated filter) to MERV 13 (for the C-POLAR™ treated filter), and the pressure drop of the C-POLAR™ treated filter was found to be around 31 pascals for the face velocity at 1.3 m/s. In this study, both the C-POLAR™ treated filter and untreated filter were cut into circular specimens with 25 mm diameter. Three samples for each filter were tested . 1-day post-transfection, the culture medium was removed, and the cells were washed with fresh medium twice to prevent plasmid DNA carryover. Cell media containing pseudo-typed viruses were then collected daily for 3 days. After collection, pseudo-typed virus containing media were centrifuged at 1000×g for 10 min and filtered through a 0.45-µm Polyethersulfone (PES) syringe filter  to remove cell debris. The pseudo-typed viruses in the filtrate were then concentrated with Amicon® Ultra 15 mL Centrifugal Filters, 100 kDa nominal molecular weight limit (NMWL)  before aliquoting and stored at − 80 °C before use.To reduce the safety risk of aerosolized virus, a pseudo-typed virus with the SARS-CoV-2 Spike protein expressed on the envelop surface carrying a green fluorescent protein (GFP) reporter gene was chosen as the test virus, which was produced in HEK293T cells according to the manufacturer’s instructions 20) and a constant flow rate of around 0.2 ml/min for 5 min for each test. The total virus input from the nebulizer was measured by immersing the end of plastic tubing at the wind tunnel into a test tube with 5 mL of DMEM to collect the aerosol produced, followed by quantitation using infectious titer assay. For filtration efficiency and SARS-CoV-2 inactivation study, the aerosolized pseudo-type SARS-CoV-2 virus was diverted to the vertical-type wind tunnel with internal diameter 12.7 mm and length 250 mm, where the filter being tested and a polytetrafluoroethylene (PTFE) filter (0.22 μm pore size) were fitted at the upstream and downstream positions of the wind tunnel respectively to collect the aerosol with distance 62.5 mm apart. A vacuum pump was connected in orthogonal position after the downstream filter, where the flow rate was set at 10 L/min using an in-line type flow meter  to draw the aerosol and fresh air from the opening of the wind tunnel. The face velocity inside the wind tunnel was set at around 1.3 m/s. After 5 min of running aerosolized pseudo-type SARS-CoV-2 virus through the filters in the above-mentioned conditions, both the nebulizer and the vacuum pump were switched off and both the upstream and downstream filters were extracted separately in 10 mL DMEM using vortex. Viable virus retained on the filter that extracted by DMEM was quantified by infectious titer assay. The filtration efficiency of the filters being tested was determined by the equation below, with the use of the virus titers retained on the downstream filter and the virus input:The idea was to assess the filtration and inactivation properties of the novel coated filter to aerosol with pseudo-type SARS-CoV-2 virus under certain face velocity inside a wind tunnel. The setup and the experiments were conducted in a Class II biosafety cabinet , where temperature and humidity of the environment were maintained at 23 °C and 50% relative humidity respectively. The design of the vertical-type wind tunnel is illustrated in Fig. 11:For the viral inactivation, the log reduction and removal efficiency of the C-POLAR™ treated filter was determined by comparing the titers found in the infectious titer assay between the untreated and C-POLAR™ treated filters28 with some modifications. Briefly, 1 × 104 A549 adenocarcinomic human alveolar basal epithelial cells with the expression of human angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) were cultured in a 96-well plate at 37 °C with 5% CO2 in a humidified incubator . DMEM supplied with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin  were used as cell culture media. Infections of pseudovirus were performed by adding diluted filter extract to the culture media for cell culturing. Culture medium without addition of filter extract was used as negative control. Viable pseudo-type SARS-CoV-2 virus remained in the filter extract can infect the A549 cells and integrate its genomic sequence into the A549 cell’s genome through transduction. Upon 3-day post-infection, the genomic DNA were extracted from the cultured cells using Monarch® Genomic DNA Purification Kit  according to manufacturer’s instruction. To measure the infectious titer, qPCR was performed by targeting the lentiviral-specific transgene ) and a single copy refence gene ). qPCRs were performed in a QuantStudio™ 3 Real-Time PCR System  in triplicates with the reaction mixture containing 10 μl Luna® Universal qPCR Master Mix , 800 nM for each primer (forward and reverse), 5 μl of the genomic DNA and make up the final volume to 20 μl with nuclease-free water. The thermocycling programme to amplify the lentiviral-specific fragment (WPRE) and albumin gene (Alb) was as follows: 95 °C for 5 min , followed by 40 cycles at 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 10 s (detection). A melting curve analysis (60–95 °C range) was conducted at the end of the reaction to verify specificity of the qPCR product, where the melting temperature of the WPRE gene and albumin were at Tm = 84.5 °C and Tm = 77.2 °C respectively. In each run of qPCR, ten-fold serial dilutions of the transfer plasmid for the pseudo-typed virus preparation in known concentration were prepared to create calibration curve for quantification. A no template sample was used as negative control.To quantify pseudo-type SARS-CoV-2 virus retained on each of the tested filter, adherent human ACE2 and TMPRSS2 expressing A549 cells  cells were incubated with the serially diluted DMEM extract from the tested filter. Cells infected by pseudo-type SARS-CoV-2 virus in the diluted extract was measured by quantitative polymerase chain reaction (qPCR) as described by Barczak et al.Primers used for the qPCR:ALB forward 5′-TTTGCAGATGTCAGTGAAAGAGA-3′.ALB reverse 5′-TGGGGAGGCTATAGAAAATAAGG-3′.WPRE forward 5′-GTCCTTTCCATGGCTGCTC-3′.WPRE reverse 5′-CCGAAGGGACGTAGCAGA-3′.t test in GraphPad Prism 7.1 . Data were considered significantly difference when the p-value was less than 0.05.Data are expressed as the mean ± standard error of the mean (SEM), and statistical significance was determined by student’s