Enterovirus A71 (EVA71) infection can result in paralysis and may be fatal. In common with other picornaviruses, empty capsids are produced alongside infectious virions during the viral lifecycle. These empty capsids are antigenically indistinguishable from infectious virus, but at moderate temperatures they are converted to an expanded conformation. In the closely related poliovirus, native and expanded antigenic forms of particle have different long-term protective efficacies when used as vaccines. The native form provides long-lived protective immunity, while expanded capsids fail to generate immunological protection. Whether this is true for EVA71 remains to be determined. Here, we selected an antigenically stable EVA71 virus population using successive rounds of heating and passage and characterized the antigenic conversion of both virions and empty capsids. The mutations identified within the heated passaged virus were dispersed across the capsid, including at key sites associated with particle expansion. The data presented here indicate that the mutant sequence may be a useful resource to address the importance of antigenic conformation in EVA71 vaccines. Enteroviruses (EVs) are globally distributed and are associated with a range of pathologies ranging from the common cold  to lethal neurological disease . Enterovirus A 71 (EVA71) is the primary causative agent of hand, foot and mouth disease (HFMD), a generally mild, self-limiting condition affecting young children. In severe cases the disease can present with neurological complications reminiscent of poliomyelitis . The finWhile the ability of inactivated virus vaccines to control the spread of EVA71 is extremely promising, there remains a significant biosafety concern associated with the large-scale production of infectious virus required for the generation of inactivated virus vaccines. There have been several instances where the production of PV vaccine material has resulted in the accidental release of infectious virus. In 1995 the incomplete inactivation of PV in ≈120 000 vaccine doses resulted in ≈40 000 cases of muscle weakness in recipients, 51 individuals were paralysed and 5 died. Additionally, spread of the virus to close contacts of the vaccine recipients resulted in an additional 113 cases and 5 deaths . BetweenEV VLPs are composed of three viral structural proteins, VP0, VP3 and VP1, assembled as icosahedral capsids that lack infectious genetic material, rendering these particles inherently safe . EV viriHere, we selected an antigenically stable EVA71 virus population using successive rounds of heating and passage and characterized the antigenic conversion of the virion and ECs. The mutant described will be a useful resource to address the importance of antigenic conformation in EVA71 vaccines and could inform the design of stabilized EVA71 VLPs for future vaccine production.EVA71 strain MS/7423/87 was a gift from SingVax (Singapore); an EVA71 reverse genetics system was generated from this strain as previously described . HeLa ce6 p.f.u. ml−1 virus was incubated at 52.5 °C for 30 min and an aliquot stored on ice as a non-heated control. After incubation at a range of elevated temperatures the samples were directly titrated by plaque assay and inoculated into T25 flasks of confluent Vero cells. These were incubated at 37 °C until full cytopathic effect (CPE) was observed. Supernatants were clarified and used for subsequent cycles of heating and passage until the titres obtained from the unheated and heated virus populations were similar for at least three consecutive passages. This resulted in a total of nine cycles of heating and passaging. Samples from passages 4, 5, 7, 8 and 9 were analysed by next-generation sequencing (NGS). The virus isolated after nine passages with heating was subjected to five further passages in the absence of heating to assess the genetic stability of the thermally resistant virus population.To select thermally resistant virus, and similar to previously described protocols , 100 µl The genetic sequence of stabilized virus populations was determined by next-generation sequencing using previously described protocols . BrieflyAatII and BamHI within the infectious clone plasmid. The ligated plasmid was transformed in Escherichia coli DH5α and plasmids isolated for Sanger sequencing.RNA was extracted from clarified supernatant of passage 9 virus using Trizol (Qiagen) and a Direct-zol purification kit (Zymogen), per the manufacturer’s instructions. The RNA was reverse transcribed and the sequence encompassing residues 784–3888 was amplified using Q5 polymerase (NEB) per the manufacturer’s instructions. Overlap PCR was used to combine the recombinant stabilized virus amplicon with the 5′ UTR derived from our EVA71 infectious clone . The resin vitro-transcribed RNA into HeLa cells. Briefly, the EVA71 infectious clone plasmid was linearized with XhoI followed by phenol–chloroform extraction. RNA was synthesized using the RiboMAX T7 express large-scale RNA production system (Promega) and purified using RNA clean and concentrator columns (Zymo Research). RNA was electroporated into HeLa cells within 0.4 mm cuvettes at 260 V for a single pulse of 25 ms using square wave. Cells were transferred to plates and incubated at 37 °C, 5 % CO2 in a humidified chamber overnight. Plates were freeze-thawed to enhance viral release from cells, and cellular debris was pelleted at 17 000 r.c.f. for 10 min. The supernatants were collected and samples titred using median tissue culture infectious dose (TCID50).Virus was recovered as previously described , followi50, 1×104 Vero cells were seeded into each well of a 96-well plate in 100 µl of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 2 % FBS. A series of 10-fold serial dilutions were made from viral supernatants in DMEM supplemented with 2 % FBS and 100 µl of each dilution (10−2 to 10−7) was added to five replicate wells. Plates were returned to a 37 °C, 5 % CO2 humidified incubator for 5 days before being fixed and inactivated by the addition of 100 µl per well of 4 % formaldehyde for 30 min. The contents of wells were discarded and residual cells stained with crystal violet solution. Titres were determined using the Reed–Muench method and are expressed as TCID50 ml−1 . Co. Co35]. The antigenic stabilities of the ECs and virions were investigated by incubating the particles at a range of temperatures and assessing their NAg and HAg reactivity by ELISA. The WT ECs maintained HAg reactivity up to 60 °C, but this decreased at higher temperatures; consistent with this, WT ECs were not reactive in the NAg-specific ELISA . As expeSurprisingly, the recombinant stabilized ECs lost reactivity with mAb 979 at a lower temperature (55 °C) than the equivalent WT sample , suggestThus, although the WT and recombinant stabilized virions have similar NAg reactivity , the WT We have selected for a combination of mutations that provides NAg stability to ECs. We propose that capsid proteins incorporating this combination of mutations will be ideal candidates for expressing stabilized VLPs in heterologous systems. We further suggest that these particles will facilitate future research to address the NAg/HAg paradigm surrounding EVA71 vaccine efficacy.Click here for additional data file.


Seasonal influenza viruses account for 1 billion infections worldwide every year, including 3–5 million cases of severe illness and up to 650,000 deaths. The effectiveness of current influenza virus vaccines is variable and relies on the immunodominant hemagglutinin (HA) and to a lesser extent on the neuraminidase (NA), the viral surface glycoproteins. Efficient vaccines that refocus the immune response to conserved epitopes on the HA are needed to tackle infections by influenza virus variants. Sequential vaccination with chimeric HA (cHA) and mosaic HA (mHA) constructs has proven to induce immune responses to the HA stalk domain and conserved epitopes on the HA head. In this study, we developed a bioprocess to manufacture cHA and mHA inactivated split vaccines and a method to quantify HA with a prefusion stalk based on a sandwich enzyme-linked immunosorbent assay. Virus inactivation with beta-propiolactone (βPL) and splitting with Triton X-100 yielded the highest amount of prefusion HA and enzymatically active NA. In addition, the quantity of residual Triton X-100 and ovalbumin (OVA) was reduced to very low levels in the final vaccine preparations. The bioprocess shown here provides the basis to manufacture inactivated split cHA and mHA vaccines for pre-clinical research and future clinical trials in humans, and can also be applied to produce vaccines based on other influenza viruses. Current seasonal influenza virus vaccines are effective when they are well matched to circulating strains . The imm®, Fluad®, Fluarix®, FluLaval®, Fluzone®)  . By the luzone®) . Formaldluzone®) . DetergeTrichoplusia ni medium-formulation Hink insect cell medium  supplemented with 10% v/v fetal bovine serum , penicillin (100 U/mL) -streptomycin (100 μg/mL) solution (Gibco), and 0.1% v/v Pluronic F-68 (Gibco). High Five cells  were used for recombinant hemagglutinin (rHA) production and were grown in Express Five SFM (Gibco) supplemented with 16 mM L-glutamine (Gibco)  for baculovirus generation and amplification were grown in  (Gibco) .Cal09N1PR8 virus was derived from the HA of A/mallard/Sweden/24/2002 H8N4 virus, and the H5 head domain of the cH5/1Cal09N1Cal09 virus from A/Vietnam/1203/2004 (H5N1) virus. The HA stalk domain of both viruses was derived from A/California/04/2009 (H1N1) virus, with the NA of the cH5/1Cal09N1Cal09 virus derived from this same virus, and the NA of the cH8/1Cal09N1PR8 virus derived from the high-yielding donor vaccine strain A/Puerto Rico/8/1934 (H1N1) virus. As for group 2 cHA viruses, the H15 head domain of the cH15/3HK14N2HK14 virus was derived from the HA of A/wedge-tailed shearwater/Western Australia/2576/1979 (H15N9) virus, and the H4 head domain of the cH4/3HK14N2HK14 virus from A/duck/Czechoslovakia/1956 (H4N6) virus. The HA stalk domain and NA of both viruses was derived from A/Hong Kong/4801/2014 (H3N2) virus. The internal genes of group 1 and 2 cHA viruses were derived from A/Puerto Rico/8/1934 (H1N1) virus. In the case of mHA influenza B viruses, the major HA head antigenic sites of B/Brisbane/60/2008 were replaced by the putative antigenic sites of A/black-headed gull/Sweden/1/1999 (H13N6) virus, resulting in the mH13/BBrisbane virus. For the mH5/BPhuket virus, the major antigenic sites of B/Phuket/3073/2013 were replaced by those from A/Vietnam/1203/2004 (H5N1) virus. For both mHA influenza B viruses, the NA and internal genes were derived from the mouse-adapted B/Malaysia/2506/2004 virus. The sequences of the HA genes of the vaccine strains have been submitted to Genbank under accession numbers listed in the end of the methods section. Work with live virus was performed at appropriate biosafety level in a biosafety cabinet.Viruses expressing different chimeric hemagglutinin (cHA) proteins were generated by reverse genetics as previously reported . For groThe six cHA and mHA viruses were grown in 10-day-old embryonated chicken eggs (Charles River) for 48 h at 37°C or for 72 h at 33°C, respectively. Eggs were then cooled overnight (O/N) at 4°C before collection of the allantoic fluid. The harvested allantoic fluid was clarified at 2000 ×g for 10 min at 4°C to remove debris, aliquoted and stored at −80°C, and titrated by the plaque assay method on Madin Darby canine kidney (MDCK) cells .Murine monoclonal antibodies (mAbs) 1A7 (directed against a linear epitope on the H8 head) , 1H4 di, 1G4 di, 6G8, 3GStalk-reactive mAbs were biotinylated for HA quantification via sandwich enzyme-linked immunosorbent assay  on ice. 25–30 mL of the concentrated virus was loaded on centrifuge tubes (Seton Scientific) containing 5 mL of 30% w/v sucrose cushion prepared in 1X NTE buffer  and with the pH adjusted to 7.4. Loaded centrifuge tubes were ultracentrifuged at 25000 rpm, 4 °C for 2 h, and the pelleted virus was resuspended in phosphate-buffered saline . The resuspended virus was inactivated with beta-propiolactone  or methanol-free formaldehyde  prepared in ice-cold water for injection after pH buffering with 0.01 M disodium hydrogen phosphate (Millipore). βPL activity was stopped by incubation at 37 °C for 2 h, whereas FA was removed by diafiltration with 100 kDa centrifugal filters (Millipore). To assess the efficiency of the inactivation step, the highly concentrated virus samples were diluted 1:1,000 in fresh PBS and injected into eggs, incubated at 37 °C for 48 or 72 h, and a hemagglutination (HA) assay was conducted to detect viral replication. After inactivation, the virus sample was centrifuged at 4000 rpm, 4 °C for 30 min. The virus was split with Triton X-100  or sodium deoxycholate , and the detergent was removed by incubation with 0.2–0.3 g of bio-beads SM-2 (BioRad) per mL of inactivated split virus at 4 °C O/N. As for DCO treated viruses, a pre-incubation step of the bio-beads with DCO split viruses at 37 °C was conducted to avoid sample solidification. The supernatant was collected, and the total protein concentration was adjusted to 0.5–1 mg/mL with PBS (pH = 7.4) using the Bradford assay (BioRad). Samples were aliquoted to 50–100 µL and stored at −80 °C until analysis.The presence of virus in the clarified allantoic fluid was assessed by the HA assay method. Briefly, 50 µL of sample were added to 96-well V-bottom plates (Nunc) and serially diluted 1:2. Then, 50 µL of 0.5% v/v turkey or chicken red blood cells (RBCs) in PBS were added to each well, and plates were incubated on ice or at 4°C for 45 min. The HA titer was calculated as the highest dilution showing an RBC tear drop after tilting the plate to 90° for 10–20 s.O-phenylenediamine dihydrochloride (OPD) substrate  was added to each well. After 10 min incubation, the reaction was stopped by adding 50 µL of 3 M hydrochloric acid (HCl) to each well. The optical density was measured at 490 nm (OD490) on a Synergy H1 microplate reader (BioTek). The endpoint titer or minimum binding concentration was defined as the final dilution of the mAb at which the signal was greater than the mean plus three times the standard deviation of blank wells on a given plate using the GraphPad Prism 9 software. Cutoff values were calculated independently for each plate.Immulon 4HBX plates (Thermo Fisher Scientific) were coated with 5 μg/mL of inactivated split virus (50 µL per well) in PBS (pH = 7.4) O/N at 4°C. The next day, plates were washed three times with PBS containing 0.1% v/v Tween 20 (PBS-T) and blocked in blocking solution  for 1 h at RT. After blocking, mAbs were added to the first well at a final concentration of 30 μg/mL in blocking solution (150 µL/well). mAbs were then serially diluted 1:3 (100 µL/well) in blocking solution and incubated for 2 h at 20°C. Afterwards, plates were washed three times with PBS-T before adding sheep anti-mouse peroxidase conjugated IgG  in blocking solution (100 µL/well). Plates were incubated for 1 h at 20°C and then washed four times with PBS-T while shaking. To develop plates, 100 µL of 490 was measured on a Synergy H1 microplate reader. The data were analyzed using GraphPad Prism 9 and values were expressed as area under the curve (AUC). The cutoff was defined as the average of all blank wells plus three times their standard deviation.Immulon 4HBX plates were coated with 5 μg/mL of inactivated split virus (50 µL per well) in PBS (pH = 7.4) O/N at 4°C. Plates were washed three times with PBS-T and blocked in blocking solution for 1.5 h at RT. After blocking, competing mAbs at a concentration of 20 μg/mL (100 µL/well) were added and incubated for 2 h at 20°C. Afterwards, plates were washed three times with PBS-T, and the target biotinylated mAb was added. The biotinylated mAb was added to the first row at a concentration of 30 μg/mL (150 µL/well), serially diluted 1:3 in blocking solution, and incubated for 2 h at 20°C. Plates were washed three times with PBS-T and subsequently incubated with streptavidin conjugated to horseradish peroxidase (Thermo Fisher Scientific) in blocking solution (100 µL/well). After 1 h incubation at 20°C, plates were washed four times with PBS-T with shaking and then developed with 100 µL of OPD substrate per well. After 10 min incubation, the reaction was stopped by adding 50 µL of 3 M HCl to each well. The OD50) values were calculated, and the absolute HA concentrations in the test samples were obtained with relation to the recombinant HA protein standards by parallel line analysis/EC50 comparison using GraphPad Prism 9 (The sandwich ELISA method was adapted and further optimized from . Immulon Prism 9 .The enzymatic activity of the NA from cHA and mHA viruses was determined by using the NA-Star™ Influenza NA Inhibitor Resistance Detection kit (Applied Biosystems) according to manufacturer’s instructions. A total protein concentration in the range of 5–50 μg/mL was used as the starting dilution to measure the NA activity of live, inactivated, and inactivated split viruses. Samples were serially diluted 1:2 across the plate. The read-out was based on the luminescence signal measured in a Synergy H1 hybrid multimode microplate reader. The NA activity was measured as the AUC using GraphPad Prism 9.Residual Triton X-100 content in inactivated split virus samples was measured by absorbance at 280 nm. Test samples were mixed 1:1 with pure methanol and centrifuged at 25,000 ×g for 30 min at RT. A standard curve of known concentrations of Triton X-100 in pure methanol was also included for absolute Triton X-100 quantification. 80–90 μL of the supernatant was transferred to 96-well UV plates (Corning), and the absorbance at 280 nm was measured in a Synergy H1 hybrid multimode microplate reader. The amount of Triton X-100 in test samples was calculated using Excel (Microsoft) according to the Triton X-100 standard curve and plotted with GraphPad Prism 9.6 in diluent I (sample buffer and distilled water at a ratio of 1:20). An ovalbumin protein standard (egg standard) was also included for absolute ovalbumin protein quantification. 100 μL of sample or standard were dispensed per well of the antibody-coated microplate module and incubated at 20°C for 1 h. Plates were washed six times with washing solution (wash buffer and distilled water at a ratio of 1:20), and 100 μL of enzyme-conjugated antibody was added per well and incubated at 20°C for 30 min. Afterwards, plates were washed six times with washing solution, and 100 μL of enzyme substrate was added per well and incubated at 20°C for 30 min. The colorimetric reaction was stopped by the addition of 100 μL of stop solution. Plate read-out was measured at an absorbance of 450 nm on a Synergy H1 hybrid multimode microplate reader. The amount of ovalbumin in test samples was calculated in Excel according to the ovalbumin standard curve and plotted with GraphPad Prism 9.The quantity of ovalbumin in live, inactivated, and inactivated split virus samples was measured by ELISA according to the manufacturer’s instructions (Morinaga). Briefly, 40 μL of sample was mixed in 760 μL of sample extraction solution , vortexed for 30 s, and heated at 90°C–95°C for 10 min. Then, samples were vortexed for 30 s, and centrifuged at 3,000 ×g at RT for 20 min. The supernatant was diluted depending on the sample in the range of 1:1,000 to 1:10SDS-PAGE was performed to characterize live, inactivated, and inactivated split virus samples. Before running the SDS-PAGE, samples were deglycosylated with rapid peptide:N-glycosidase (PNGase) F (New England Biolabs) according to the manufacturer’s instructions for a better resolution of the bands. After deglycosylation, 20 μL of sample was mixed with 2X Laemmli buffer (BioRad) containing 50 mM NuPAGE sample reducing agent . Samples were then incubated for 10–15 min at 90°C–95°C and run on a 4%–20% precast polyacrylamide Mini-PROTEAN TGX gel (BioRad) for 1 h at 120 V (30 μL/well). The gel was stained with SimplyBlue SafeStain (Novex) O/N, and destained in distilled water. Images were taken in a Chemidoc MP Imaging System using the Image Lab software (BioRad).The average size of live, inactivated, and inactivated split viruses was assessed in a Litesizer 500 dynamic light scattering (DLS) instrument (Anton Paar) at an angle of 175° with 10 × 12 × 45 mm polystyrene cuvettes (Sarstedt) and 1 mL of sample. Triplicate measurements were performed per sample, each with 11 runs, and processed with the Kalliope software (Anton Paar). Live and inactivated virus samples were diluted 1:1,000 in 0.22 µm filtered PBS (pH = 7.4), while inactivated split virus samples were diluted 1:100.Samples for transmission electron microscopy (TEM) were prepared by the negative staining method using a 2% w/v solution of uranyl acetate (Electron Microscopy Sciences) in distilled water. Virus samples were diluted to approximately 20–100 μg/mL protein in Tris-buffered saline . Briefly, 3 μL of diluted virus sample was applied to glow discharged  formvar/copper support film TEM grids (Electron Microscopy Sciences). Following a brief incubation of 30 s, the sample was blotted away from the grid using filter paper (Whatman). Sample-laden grids were washed twice by contact with two successive droplets of distilled water, and subsequently dabbed twice into droplets of uranyl acetate solution. Micrographs were obtained using a Hitachi 7,500 (Hitachi) TEM at the Icahn School of Medicine at Mount Sinai, and a Tecnai F20 (Field Electron and Ion company) and JEOL JEM-1230 (Gatan) TEMs at the New York Structural Biology Center.Cal09N1PR8 and cH5/1Cal09N1Cal09) was previously determined (HK14N2HK14 and cH4/3HK14N2HK14) and mHA influenza B inactivated split viruses (mH13/BBrisbane and mH5/BPhuket). No competition for binding to inactivated split viruses was measured in any of the combinations of anti-HA head and stalk mAbs tested, indicating different possibilities of mAb pairs for the sandwich ELISA  and cH5/1Cal09N1Cal09 (1H4 and CR9114) viruses captured prefusion HA in inactivated split virus samples as compared with the recombinant HA (rHA) protein standards used in the assay .Different monoclonal antibodies (mAbs) targeting the HA head or stalk domains of diverse cHA and mHA constructs were initially screened as candidates for prefusion HA quantification by ELISA. Some of these mAbs had already been previously characterized . The bastermined , but it termined . The samch ELISA . For prehe assay . Indeed,ch ELISA . A simil results . As for Cal09N1PR8, cH15/3HK14N2HK14, and mH13/BBrisbane respectively, were selected to evaluate the impact of different methods of virus inactivation and splitting with the aim to increase the amount of prefusion HA and neuraminidase (NA) activity in the final vaccine preparations. The main steps of the bioprocess followed are summarized in One influenza virus from each group, cH8/1An increase in the concentration of prefusion HA was observed after inactivation and splitting for some of the viruses , 3A, sugTriton X-100 was selected as the detergent for splitting over DCO since it proved to be the best option to retain HA conformation and NA activity. The amount of residual Triton X-100 in the three splitting conditions tested for each virus after detergent removal with hydrophobic beads was below the 5% of the initial amount of Triton X-100 used for splitting. The ratio of residual Triton X-100 to HA content was also below 2 μg/μg HA . The conCal09N1PR8 and cH15/3HK14N2HK14 viruses, and from 130–230 nm for the mH13/BBrisbane virus. Virus splitting with 0.5% v/v Triton X-100 resulted in a less decrease in particle size in comparison with 1% v/v Triton X-100 splitting, possibly indicating a lower level of virus disruption achieved in the former. Virus splitting with 1% v/v Triton X-100 at RT or 37°C yielded similar levels of virus disruption. Therefore, splitting with 1% v/v Triton X-100 at RT for 1h was the condition selected for the final bioprocess. Characterization of the three cHA and mHA influenza viruses after UC (live virus), after 0.05% v/v βPL inactivation (inactivated virus), and after 1% v/v Triton X-100 splitting and detergent removal with bio-beads (inactivated split virus) was performed by TEM , the matrix protein 1 (M1), and some faint bands that might represent the NA and the polymerase proteins  after virus inactivation (HK14N2HK14 (3G11/CR9114) and cH4/3HK14N2HK14 (1G4/CR9114), and influenza B mHA viruses mH13/BBrisbane and mH5/BPhuket (CR8033/4C10) were also determined, proving that these mAb pairs can be used to quantify prefusion HA in live, inactivated, and inactivated split virus samples by sandwich ELISA. However, changes in binding of the mAb pairs to these different forms of the virus were detected, especially for the mH13/BBrisbane virus. We hypothesized that the accessibility of the mAb pair to their HA epitopes in the inactivated and inactivated split samples might be enhanced after the inactivation and splitting step. However, increasing the quantity of zwittergent 3–14 detergent, a mild ionic agent used in the sandwich ELISA for membrane permeabilization and disruption of aggregated HA molecules, did not further improve the binding of these mAbs. Changes in HA structure that alter the binding to the epitopes targeted by these mAbs might explain the increase in HA concentration after inactivation and splitting. In contrast to the influenza A virus HA stalk mAbs used, which are selective for the prefusion conformation of the HA, mAb 4C10 also binds well to the postfusion conformation. Therefore, the sandwich ELISA might be further improved for influenza B mHA viruses in the future with mAbs targeting prefusion-only conformational epitopes in the stalk domain. Additionally, we envision the use of the sandwich ELISA to measure HA conformation in stability studies in combination with reversed-phase high-performance liquid chromatography for a more accurate HA quantification (One important aspect to consider with our cHA and mHA constructs is that the HA stalk should be in the prefusion conformation. The single radial immunodiffusion assay, which is considered as the gold standard to measure HA concentration, is not able to distinguish between HAs with a native or denatured stalk domain . To thistivation . In thisfication .Inactivated split influenza virus vaccine production is mainly conducted in embryonated chicken eggs , though The presence of the specific contaminants OVA and Triton X-100 was also addressed. No specific guidelines on the amount of residual Triton X-100 in the final influenza virus vaccine preparation are available, but we wanted to keep the detergent to a low level  while noIn summary, a bioprocess to produce inactivated split cHA and mHA virus vaccines in eggs has been developed. A method based on a sandwich ELISA for prefusion HA quantification in cHA and mHA viruses has also been defined. Virus inactivation with βPL and splitting with Triton X-100 provides the highest yields of prefusion HA and active NA. In addition, the amount of residual Triton X-100 and OVA is reduced to low levels. This study provides the basis for a bioprocess to produce a cHA and mHA trivalent universal influenza virus vaccine for testing in future clinical trials in humans.Cal09N1Cal09: OQ349657;cH5/1Cal09N1PR8: OQ349625;cH8/1HK14N2HK14: OQ349617;cH4/3HK14N2HK14: OQ349633;cH15/3Brisbane: OQ349641;mH13/BPhuket: OQ349649.mH5/BHA gene sequences have been submitted to Genbank and will become available under the following identifiers:


The use of electric scooters (e-scooters) is increasing in Australia and internationally. The increasing availability of e-scooters has led to a rise in the number of injuries, with most patients sustaining orthopaedic injuries. This retrospective case series describes the incidence, management, and hospital costs of the orthopaedic injuries, which presented to the emergency department (ED) of the major trauma center in Western Australia. Data on demographics, ED dispatch destination, management, follow-up clinics, and hospital costs were collected between 2017 and 2022. Since June 2020, there have been 61 e-scooter crashes, which resulted in orthopaedic injuries, with more than half of the crashes occurring after the introduction of regional e-scooter sharing schemes. Thirty-two patients (52%) were admitted to the hospital. The most common orthopaedic fracture was to the upper limb (44%), followed by the lower limb (41%) and the axial skeleton (15%). Fourteen (23%) patients sustained more than one fracture. Twenty-two patients (36%) required operative management. The median number of outpatient clinic attendances per patient was 3 (interquartile range (IQR): 1–5), with inpatients requiring twice the number of clinics as compared to those discharged from the ED. The median cost per presentation was AU$5880.60  with inpatient costs exceeding those discharged from the ED. The range of the total costs was AU$413.80 to AU$100,239.80. The rise in the accessibility of e-scooters in Western Australia has led to a rise in ED presentations with orthopaedic injuries. Considering the recent implementation of e-scooter sharing schemes in metropolitan areas, ongoing surveillance of e-scooter injuries by clinicians and policy makers is warranted to inform harm minimization strategies. Marketed as a cost effective, time efficient, and environmentally friendly form of personal transportation, electric scooters (e-scooters) have gained widespread popularity since first appearing in 2017 . In certThe most common injuries sustained during helmeted e-scooter crashes are orthopaedic injuries ,15. A reWith the increasing use of e-scooters and continual introduction of trials across metropolitan areas, it is important to understand the harm associated with their use and the burden they place on the healthcare system. The primary aim of this study is to assess the incidence of patients attending the ED with e-scooter-related orthopaedic injuries, their dispatch destination, and management at a level one major trauma center in Australia. The secondary aims of this study included a description of patient demographics and to characterize the orthopaedic injury patterns. Hospital costs were calculated to establish the financial burden to the healthcare system. This study builds on previous work that utilized the trauma registry database, which captured admitted major trauma patients only and included all forms of injury .We performed a retrospective review of prospectively collected data using the ED Information System (EDIS). All e-scooter-related ED presentations to a major trauma center  in Western Australia between 1 January 2017 and 14 June 2022 were identified.The EDIS was queried for all ED presentations that included the words “scooter”, “e-scooter”, and “electric scooter”. Two datasets were created with the first containing the words “e-scooter” or “electric scooter” with the second containing all remaining scooter presentations. The datasets were merged to include all e-scooter presentations. The data were manually reviewed (KS and KR) to remove inappropriate presentations that were not related to e-scooter crashes, such as injuries involving motorized scooters, electronic bikes (e-Bikes), unpowered scooters, pedal bicycles, electronic skateboards, wheelchairs, or other forms of wheeled transport. Inclusion criteria for the present study included:Any ED presentation during the study period that involved an e-scooter, where the injuries were managed by the orthopaedic department.Upper limb fractures and dislocations;Lower limb fractures and dislocations;Pelvic fractures;Spinal fractures.Orthopaedic injuries included:Patients were identified using three techniques. First, ICD-10 codes were screened for fractures and dislocations. Second, the ED diagnoses were screened for inclusion of the words “fracture” and “dislocation”. Finally, all patients’ triage notes, clinical notes, and radiology reports were reviewed and assessed to see if a fracture or dislocation was noted during the ED presentation. If any of the 3 techniques were positive for fracture or dislocation, the patient was considered to have a fracture/dislocation. The investigator assessment served as a quality control step with misdiagnoses from the ICD-10 codes, ED diagnosis, or no radiological evidence of orthopaedic injury being excluded from the study. Exclusion criteria aimed to ensure relevance of the burden to orthopaedic services. Therefore, maxillofacial fractures, skull fractures, hand fractures, and rib fractures were excluded from the dataset, as these fractures are managed by the maxillofacial, neurosurgical, plastic and reconstructive surgical, and trauma surgical specialties, respectively. If a patient sustained any of the aforementioned fractures concurrently with orthopaedic injuries, only the non-orthopaedic injuries were excluded.This study primarily aimed to determine the incidence, disposition, and management of patients who presented to the ED after having suffered orthopaedic injuries related to e-scooters. Outcomes included the number of presentations per year, disposition from the ED, and management (operative vs. non-operative). Secondary outcomes included injury patterns  and costs associated with orthopaedic injuries sustained following e-scooter use. All orthopaedic injuries that were sustained were counted in both isolated orthopaedic injuries and in patients with injuries involving other body systems (such as in polytrauma patients). Days to theatre and days to first orthopaedic clinic were calculated from time of hospital presentation. Number of outpatient appointments and “did-not-attend” clinics were recorded. For patients who met inclusion criteria, outcomes were obtained from the hospital information system, iSOFT Clinical Manager (i.CM) , which included management type, time to operation, and time to orthopaedic outpatient clinic. Hospital cost data were captured by the hospital Business Intelligence Unit (BIU). All patient presentations were linked by their hospital unit medical record number to the BIU database. Direct costs were defined as all measurable and identifiable activities involved in patient care. Staff salaries  and treatment costs were included in the direct costs. Indirect costs are costs that are not directly related to patient activity but still provide some form of benefit to the patients. Examples of these costs include information technology, human resources, finance department, and executive director expenses. Radiology-specific costs were provided and included in the cost calculations. The BIU uses price weights for each patient and the National Efficient Price determined by the Independent Hospital Pricing Authority to determine the cost of health services. Cost calculations from index admission to last recorded outpatient appointment were collected between 1 January 2020 and 1 May 2023. Dates for hospital costs were determined by the first year that an orthopaedic e-scooter injury was recorded in the ED dataset, and up to the date at which the hospital cost calculations were requested to capture as many outpatient appointments as possible. Index admission was defined as the date of the emergency department presentation for e-scooter injury. From January 2020 to June 2021, ED costs were classified using the Urgency Related Group classification. From July 2021, ED presentations were classified and priced using the Australian Emergency Care Classification, which replaced the Urgency Related Group classification for activity-based funding purposes. The sum of direct costs, indirect costs, and radiology costs for a given ED presentation were defined as ED costs. Inpatient costs were classified according to the Australian Refined Diagnostic Related Group for activity-based funding purposes. Inpatient costs included the sum of direct and indirect costs per admission stratified by department, operation, and radiology costs. Outpatient costs were defined as direct and indirect costs billed to the orthopaedic department and radiology costs associated with outpatient appointments. Cost calculations included median cost with interquartile range (IQR) for ED costs, inpatient costs, outpatient costs, and total costs from index admission to last captured outpatient appointment. All costs were calculated in Australian dollars (AU$) in 2023. At the time of submission of this manuscript, the exchange rate of AU$ to United States dollars (US$) was 1 AUD to 0.64 US$ and 1 AU$ to 0.61 Euros. Outpatient “did-not-attend” appointments were determined by reviewing administrative costs associated with outpatient appointments that were not associated with a surgical team review billing.All data were housed on a hospital-based information system that required 2-factor authentication for access with password encryption on all documents with patient datapoints. Once data were collected, all patients were de-identified prior to statistical analysis to protect patient confidentiality. This project received governance approval from the local Clinical Safety and Quality Unit .n < 5) were used to compare differences in demographics between admitted and discharged patients. Statistical significance was set at p < 0.05. Statistical analyses and visualizations were completed using R Statistical Package  [Descriptive statistics were provided for the entire cohort and two subsets of patients based on ED disposition . All data were reviewed using histograms, and the Kolmogorov–Smirnov test was used to determine if data were normally distributed. For continuous variables, a Wilcoxon ranked-sum test was used to compare the 2 independent samples. For categorical variables, chi-square tests or Fisher Exact tests (if Austria) .Between 1 January 2017 and 14 June 2022, 61 patients sustained orthopaedic injuries . There wInjury patterns are detailed in All costs are reported in This study has demonstrated a large increase in e-scooter-related ED presentations with orthopaedic injuries to the Western Australian state trauma center since 2020. In the present study, fifty-two percent of ED presentations with orthopaedic injuries were admitted. Observations regarding disposition from the ED highlight the healthcare burden imposed by e-scooter injuries. Whilst other studies have considered ED presentations of e-scooter-related orthopaedic injuries, this study considers disposition, follow-up, and the hospital costs (including outpatient appointments) from the index presentation.The increase in ED presentations with orthopaedic injuries following e-scooter crashes may reflect the widespread introduction of e-scooters in the Western Australian community. The rise of e-scooter use has been driven by the public perception of their numerous benefits ,32: theyA proposed benefit of e-scooter usage is their environmental sustainability . Due to Over half of the current cohort were treated and discharged from the ED, and over a third of all patients required surgery. Unsurprisingly, more admitted patients underwent surgery as compared to those discharged from the ED. This result is expected as more severely injured patients are more likely to be admitted than less severely injured patients. The differences in the current project as compared to other Australian studies is likely indicative of the orthopaedic focus of this project, whereas other studies considered all injuries ,6,9,14. Admitted patients had a significantly higher financial cost to the healthcare system for outpatient costs, ED costs, and total costs. Whilst ED costs were comparable to those seen in both Brisbane and the Northern Territory, inpatient costs were markedly higher in the present study as compared to those seen in the Northern Territory ,9. The rInjury patterns of this study were consistent with those found in the literature ,15. ShorWe recognize limitations in the present study. First, the dataset used for the data captured was a combination of administrative data and clinical medical records, neither of which are designed for research. However, our three-tier fracture/dislocation identification strategy and dual-investigator manual record review minimizes the risk of missed injuries or incorrect patient inclusion. The present study focused on ED presentations and therefore did not include a direct measurement of injury severity. Whilst the length of hospital stay, hospital costs, and the type of injuries may give an indication of injury severity, we acknowledge that no direct measurement of injury severity was conducted in this study, and the results should be interpreted accordingly. The retrospective nature of this study did not account for the ownership type of e-scooter used (private vs. rideshare e-scooter). Whilst the results of the current study may suggest an association between the introduction of the first e-scooter rideshare program in Western Australia and an increase in the number of orthopaedic injuries sustained in e-scooter crashes, retrospective observational data cannot provide a causal link between these two factors. Furthermore, the state trauma center receives only adult patients (over 16 years of age). Pediatric patients who sustained e-scooter-related orthopaedic injuries are diverted to a nearby tertiary pediatric center. Finally, this was a single-center study. Future studies that include data from all tertiary centers within the state may provide a more holistic perspective of the orthopaedic burden of disease imposed by e-scooter use. Despite these limitations, this study provides a summary of the current orthopaedic burden posed by the emergence of a new form of transport. Taken together, the observations of ED disposition, management, and subsequent hospital costs summarize the orthopaedic burden on the Western Australian state trauma center posed by e-scooter-related orthopaedic trauma since 2017. Orthopaedic injuries associated with e-scooter usage are on the rise in Western Australia. Whilst many ED presentations were discharged, over half of the patients with e-scooter-associated orthopaedic injuries were admitted to the hospital. Admitted patients were more likely to undergo surgery and have higher ED, outpatient, and total costs. The introduction of e-scooter sharing schemes brings with them an increased risk of injury that policy makers and clinicians must be conscious of to reduce harm and financial costs to the community.


Crassostrea gigas). Both viruses were experimentally bioaccumulated in oysters, which were thereafter depurated at 12 °C and 17 °C for 4 weeks. Infectious TuV and viral RNA were monitored weekly for 28 days by TCID50 and (PMAxx-) RT-qPCR, respectively. TuV RNA was more persistent than NoV and decreased by < 0.5 log10 after 14 days, while NoV reductions were already > 1.0 log10 at this time. For RT-qPCR there was no detectable benefit of elevated water temperatures or PMAxx for either virus (p > 0.05). TuV TCID50 decreased steadily, and reductions were significantly different between the two temperatures (p < 0.001). This was most evident on days 14 and 21 when reductions at 17 °C were 1.3–1.7 log10 higher than at 12 °C. After 3 weeks, reductions > 3.0 log10 were observed at 17 °C, while at 12 °C reductions did not exceed 1.9 log10. The length of depuration also had an influence on virus numbers. TuV reductions increased from < 1.0 log10 after seven days to > 4.0 log10 after 4 weeks. This implies that an extension of the depuration period to more than seven days, possibly in combination with elevated water temperatures, may be beneficial for the inactivation and removal of viral pathogens.Raw oysters are considered a culinary delicacy but are frequently the culprit in food-borne norovirus (NoV) infections. As commercial depuration procedures are currently unable to efficiently eliminate NoV from oysters, an optimisation of the process should be considered. This study addresses the ability of elevated water temperatures to enhance the elimination of NoV and Tulane virus (TuV) from Pacific oysters (The online version contains supplementary material available at 10.1007/s12560-022-09547-8. Shellfish are commonly viewed as culinary delicacies, giving high retail prices. This is especially true for bivalve molluscs. Among those, raw oysters are the pinnacle of fine dining. Still, this high-class product is often associated with food-borne illness. In this context, norovirus (NoV) is of special relevance, as it is perceived as the most common cause of non-bacterial gastroenteritis globally. Even though NoV is known to spread directly from person to person, transmission through food is common. Shellfish, predominantly oysters, are repeatedly listed as the culprit in food-borne NoV infections  have been bioaccumulated in the Pacific oyster, 2. Cells were infected with TuV at a multiplicity of infection of 1 in M199 Earle's salts/GlutaMAX without additional supplements (maintenance medium). When a cytopathic effect was observed, TuV was harvested by 3× freeze-thawing. Debris was removed by centrifugation at 2500×g for 5 min. The supernatant was collected, and the titre of virus stock determined (3.1 × 107 TCID50/mL). The NoV GII.2 faecal sample was acquired from a child with acute gastroenteritis. The genotype was determined based on ORF2 sequencing and the Norovirus Automated Genotyping Tool  were grown in cell culture flasks with filter cap in Medium 199/Earle's salts/GlutaMAX  with 10% foetal bovine serum (Thermo Scientific/Gibco) and 2% antibiotic/antimycotic solution (Thermo Scientific/Gibco) at 37 °C and 5% COOysters harvested near Nøtterøy, Vestfold and Telemark County, Norway, were provided by a local supplier. Oysters were size graded by the supplier and weighed 120 g on average.4 TCID50/mL and 4.0 × 103 genome copies per millilitre (GC/mL), respectively. Two trays, each containing ~ 75 oysters were placed in tanks with sea water so that all were completely submerged. Bioaccumulation lasted for 20 h at a water temperature of ~ 12 °C and under constant aeration of the water. There was no feed added to the bioaccumulation tank. Oysters were rinsed in clean sea water and 15 oysters were collected to determine the initial virus concentration. The remaining oysters were divided equally and placed into two depuration tanks that differed in water temperature. Low temperature depuration was conducted at 12 ± 0.9 °C using incoming sea water routinely used in the depuration facility. Elevated temperature depuration was at 17 ± 0.2 °C. The water temperature was held stable by a swimming pool heat pump . Depuration lasted for 4 weeks in a flow-through system without water recirculation.Bioaccumulation was conducted in late June 2021 in natural sea water that was treated by UV irradiation. TuV and NoV stock was diluted 1:1000 in 60 L sea water to 3.1 × 10g for 15 min. The supernatant was collected and immediately used for molecular and cell culture analysis. Excess virus extract was stored at − 80 °C.Samples were collected weekly until day 28. From each depuration tank at each sampling time, 15 oysters were collected, rinsed in clean sea water, and transported chilled to the laboratory within 3 h. Oysters were placed at 4 °C upon arrival for a maximum of 2 h until analysis. Oysters were shucked, DT removed and homogenised with scalpel blades. DT from five oysters was pooled to make one sample, resulting in three replicates per depuration temperature per sampling day. Virus was extracted from oyster tissue as described in Araud et al.  with min4 cells/well) and grown to confluency. Virus extracts were diluted ten-fold in maintenance medium. Fifty microlitre of these virus dilutions was inoculated into the wells in quadruplicate for incubation at 37 °C and 5% CO2 for 2 h. Maintenance medium (200 µL) was added, and plates were incubated at 37 °C and 5% CO2 until 7 days post infection. Cytopathic effect was converted to TCID50 per mL virus extract with the Spearman & Kärber algorithm described by Hierholzer and Killington  at room temperature for 15 min in the dark. They were exposed to LED light (464–476 nm) for 15 min in the PhastBlue Photoactivation System for Tubes  at 100% intensity and placed on ice. Virus extracts that did not receive PMAxx treatment were kept on ice.PMAxx was included in the analysis to reduce PCR amplification of viruses with damaged capsids  as described in ISO 15216–1 ISO, . RNA wasThe RNA UltraSense One-Step Quantitative RT-PCR System (Thermo Scientific/Applied Biosystems) was used at a reaction volume of 20 µL. TVIF primer and probe were chosen for TuV and COG2R/QNIF2d primers and QNISP probe for GII Table . TVIF pr6 GC/mL according to the method described by Persson et al. (For quantification of genome copies (GC) per mL after RT-qPCR, NoV and TuV positive controls were quantified in RT-ddPCR. The One-Step RT-ddPCR Advanced Kit for Probes  and the QX200 Droplet Digital System (Bio-Rad) were used. The NoV stock was determined to be 4.0 × 10n et al. . QNIFSP 1 and N2 are the amount of viral RNA in the sample and in the positive control, respectively; Ct1 and Ct2 are threshold cycles for sample and control, respectively; E is the efficiency of amplification. These numbers were the basis for calculating log10 reductions as follows: log10 reduction = log10 (control virus) – log10 (inactivated virus). ANOVA and Tukey HSD post-hoc test was conducted in RStudio version 1.3.959 .RT-qPCR underestimated the reduction in infectious TuV compared to TCID50 Fig. a, b. TuV10 after week one .NoV GII decreased by 0.8 logp > 0.05, data not shown). PMAxx data is available as a supplementary file online.There was no observable benefit of applying PMAxx for differentiating between infectious and non-infectious virus for either TuV or NoV . This was most evident on days 14 and 21 .TuV titre was monitored throughout the 4-week depuration period. The initial TuV concentration was 3.0 × 10Depuration has shown a limited ability to remove NoV from oysters. One important factor influencing virus removal rates may be water temperature during depuration. Lower temperatures lead to low metabolic activity in oysters and seem to decrease virus removal rates  NoV contamination levels in oysters averaged at 337 GC/g, and only 5.5% of samples were > 1000 GC/g EFSA, . In the r et al. . If we a in EFSA . While t in EFSA  determin in EFSA . In that10 and 2.6 log10 at the low and elevated temperature, respectively. After 7 days, reductions were not noticeably different. This implies elevated water temperatures may primarily be relevant for a 2- to 3-week depuration. After 28 days, we detected reductions in TuV infectivity of > 4.0 log10, regardless of temperature. In case of suspected recent faecal contamination and high virus concentrations, an extended depuration of 3 to 4 weeks could be considered. The economic burden of prolonging the depuration period must be weighed against the burden of heating up the depuration water.Overall, we observed an advantage of elevated water temperatures for the reduction in infectious TuV. This was noticeable after 14 days when TuV was reduced by 1.0 logSupplementary file1 (XLSX 53 kb)Below is the link to the electronic supplementary material.


Progressive supranuclear palsy (PSP) is a rare and fatal neurodegenerative movement disorder with no disease modifying therapy currently available. Data on the costs associated with PSP are scarce. This study aims to assess the direct medical expenditure of patients with PSP (PwPSP) throughout disease course.This retrospective cohort study is based on the data of a large state-mandated health provider in Israel. We identified PwPSP who were initially diagnosed between 2000 and 2017. Each PwPSP was randomly matched to three health-plan members without PSP by birth-year, sex, and socioeconomic status. Healthcare resources’ utilization and related costs were assessed.We identified 88 eligible PwPSP and 264 people in the reference group; mean age at diagnosis was 72.6 years (SD = 8.4) and 53.4% were female. The annual direct costs of PwPSP have risen over time, reaching US$ 21,637 in the fifth year and US$ 36,693 in the tenth year of follow-up vs US$ 8910 in the year prior diagnosis. Compared to people without PSP, PwPSP had substantially higher medical expenditure during the years prior- and post-index date.The present study demonstrates higher economic burden, which increases with time, in PwPSP as compared to those without.The online version contains supplementary material available at 10.1007/s00415-023-11714-1. Progressive supranuclear palsy (PSP) is a fatal but rare neurodegenerative disease  with an This retrospective cohort study was performed using the comprehensive database of the Maccabi Healthcare Services (MHS), a 2.6-million-member state-mandated health plan (payer–provider) in Israel. Membership in MHS is free and open to all Israeli residents. MHS has maintained a computerized database of electronic health records since 1993, containing extensive longitudinal data on a stable population .Classification of Diseases ninth edition (ICD-9) code 333.0. Due to ICD-9 = 333.0 unspecific nature, potential cases indicated solely by an external diagnosis were verified by reviewing hospital discharge reports. The index date was defined as the earliest date of PSP diagnosis. Eligible study patients were also required to have at least 1 year of continuous enrollment in MHS prior to the index date (to ensure cases were incident) as well as 1-year post-index date. Each PwPSP was randomly matched by birth-year, sex, and socioeconomic-status (SES) categories  to MHS members without PSP in a 1:3 ratio. MHS members without PSP were given their respective matched PwPSP’s index date.We identified all MHS members aged 40 or above that had at least 1 documented PSP diagnosis anytime between 2000 and 2017 (inclusive). PSP was documented either using an internal MHS specific code for PSP or an external documented diagnosis using International All data were anonymous. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline for cohort studies.® using data on patients’ residential area. This score is highly correlated with SES measured by the Central Bureau of Statistics  n = 88, respectively). Even when comparing the cost increase in the fifth year (n = 42), there is a 2.5-fold increase of costs . While, an increase of number of hospitalizations days was observed  were calculated using GLMM for each year of follow-up from the last year prior to PSP first diagnosis (“− 1”) to the 10th year post-diagnosis. For each year, the model included only patients with at least one event. Note, each panel has a different Y axis.During 1-year prior index date Table , compareIn the year post-index date Table , PwPSP wFigure This retrospective cohort study utilized a clinical database spanning over two decades to quantify the HCRU and direct costs associated with PSP. It is well established that PSP is a progressive disease, where gait, swallowing, and speech functions deteriorate with time . To imprOur results show that PSP is associated with a substantial direct medical costs that quadrupled between the year prior diagnosis and the tenth year after diagnosis, indicating that a greater use of services that PwPSP require over time. Throughout a period of 10 years after diagnosis, there is a cumulative added cost of US$ 148,000 per patient.Data on the direct medical costs of PSP are scarce. Our findings are in line with two previous publications assessing the economic burden of PSP, demonstrating that disease severity is a significant contributor to the increase of direct costs , 20; theOverall, during 1 year prior- and post-index date, when compared with people without PSP, PwPSP had higher HCRU. Direct medical expenditure per patient and that for the whole cohort were higher in PwPSP during both years. These findings resemble those of a recent published abstract . In addi[total semiannual costs – the unpaid care]*2) is approximately €16,000, similar to our corresponding fourth year costs; US$ 18,358 [The present study has several limitations. First, the analysis was conducted from the healthcare system perspective, and therefore, we focused on direct medical expenditure. Direct costs of services provided beyond the framework of the MHS or indirect costs, such as home renovation, nursing home, loss of caregiver income, and unpaid care, were not included. However, when excluding the unpaid costs in a study reporting cross-sectional costs analysis (mean PSP duration ranges 3.9–4.4), the mean direct annual costs Below is the link to the electronic supplementary material.


One of the systems of plant defense against viral infection is RNA silencing, or RNA interference (RNAi), in which small RNAs derived from viral genomic RNAs and/or mRNAs serve as guides to target an Argonaute nuclease (AGO) to virus-specific RNAs. Complementary base pairing between the small interfering RNA incorporated into the AGO-based protein complex and viral RNA results in the target cleavage or translational repression. As a counter-defensive strategy, viruses have evolved to acquire viral silencing suppressors (VSRs) to inhibit the host plant RNAi pathway. Plant virus VSR proteins use multiple mechanisms to inhibit silencing. VSRs are often multifunctional proteins that perform additional functions in the virus infection cycle, particularly, cell-to-cell movement, genome encapsidation, or replication. This paper summarizes the available data on the proteins with dual VSR/movement protein activity used by plant viruses of nine orders to override the protective silencing response and reviews the different molecular mechanisms employed by these proteins to suppress RNAi. In plant virus infections, once a virus genome enters the initial cell(s), the further systemic infection involves cell-to-cell genome movement via plasmodesmata (PD) connecting neighboring cells in plant tissues and subsequent long-distance movement via the vascular system ,5,6,7,8.MPs, in addition to VSRs, are considered to be a specialized virus-specific system for the colonization of land plants having a symplastic organization in which the PD channels serve as cytoplasmic connections between adjacent cells ,21,22,23Potato virus X (PVX) is a member of the genus Potexvirus  . Lo. LoTrivie leaves .Alphaflexiviridae and Betaflexiviridae families, viruses in the Tymoviridae family have icosahedral virions. Turnip yellow mosaic virus (TYMV) is the best-studied member of this family. Unusually, the TYMV P69 MP gene is located in the 5′-proximal region of the genomic RNA and overlaps significantly with a replicase gene .,62.61,62amovirus . Furtheramovirus ,65. In aamovirus , suggestTomato mosaic virus strain N5 (ToMV-N5), which causes systemic necrosis in certain tomato cultivars, has identified MP as a determinant of virus pathogenicity [A recent analysis of genicity . Comparegenicity . ToMV-N5genicity . At presgenicity . The latgenicity . These dgenicity . FurtherTobravirus have bipartite genomes. RNA1 encodes two components of the viral replicase complex, with the larger protein component being expressed by read-through translation of the stop codon of the smaller replicase subunit, the viral MP and a cysteine-rich VSR protein, whereas RNA2 encodes the viral CP and two or more proteins involved in the virus transmission by nematodes [Tobacco rattle virus (TRV), the type tobravirus, has been shown to suppress GFP silencing induced by single-stranded and double-stranded RNA in agrobacteria infiltration assays, to reduce the amount of GFP-specific siRNA and to cause a delay in the local and systemic spread of silencing signals [Viruses of the genus ematodes . The 16- signals ,71,72. I signals . The mec signals . 16K has signals .TRV RNA1-encoded MP has also been shown to suppress silencing under certain conditions . Indeed,Luteovirus have positive-stranded RNA genomes containing six ORFs. Two 5′-proximal ORFs encode components of the viral replicase that are translated from the genomic RNA by a mechanism involving ribosomal frameshifting, ORF3 encodes virus CP; a translational readthrough of ORF3 into ORF5 gives rise to a protein required for the virus aphid transmission; ORF4, located in a different reading frame within ORF3, encodes MP [Barley yellow dwarf virus-GAV (BYDV-GAV) P6 has been found to suppress local silencing in a GFP patch agroinfiltration assay and to inhibit systemic transport of silencing signals in transgenic plants [Luteovirus genus encode two proteins with VSR activity, MP, and P6 but that their activities vary in different viruses of the genus , the best studied dianthovirus, viral infection has been shown to be associated with silencing suppression accompanied by reduced levels of siRNAs and miRNAs; however, individual RCNMV proteins are unable to suppress silencing in a patch agroinfiltration assay [Turnip crinkle virus (TCV) genome, in which the gene for CP, the TCV VSR, has been replaced by a GFP gene. Such a TCV-GFP reporter construct is restricted to single initially infected cells unless co-expressed with a VSR, which allows TCV-GFP to be transported from cell to cell, resulting in readily detectable fluorescent infection foci [Members of the genus viral MP . For theon assay . Furtheron assay . These oon assay , the VSRion foci ,83. Co-eion foci ,83. ThusSobemovirus have five ORFs [The single-stranded RNA genomes of viruses of the genus ive ORFs ,85. ORF3ive ORFs ,86. An aive ORFs ,88,89, sive ORFs .Rice yellow mottle virus (RYMV) P1 protein is found to inhibit post-transcriptional transgene silencing when expressed either from a PVX-based viral vector in N. benthamiana [Cocksfoot mottle virus (CfMV) can suppress silencing in a GFP patch agroinfiltration assay, as manifested by increased GFP levels and decreased levels of GFP-specific 24-nucleotides-long small RNAs  P38 and the Pothos latent virus P14 which both bind long dsRNA molecules [The mechanism of silencing suppression by sobemoviral MPs is only partially understood. CfMV P1 binds ssRNA in a sequence-nonspecific manner . Howeverduplexes ,100, andolecules ,102.dcl4 mutant [Expression of RYMV P1 in transgenic rice has been shown to result in developmental abnormalities phenocopying those known for a 4 mutant . This ef4 mutant . Thus, t4 mutant . This P14 mutant , suggestRecently, the tertiary structure of the RYMV P1 protein has been resolved by X-ray crystallography and nuclear magnetic resonance spectroscopy, providing insight into the structural features of the protein relevant to its functions . The RYMFabavirus (family Secoviridae) have bipartite genomes; RNA1 encodes a polyprotein that is autocatalytically processed into proteins required for virus replication, whereas RNA2 encodes two CPs and a viral MP [Broad bean wilt virus 1 (BBWV1) [Viruses of the genus viral MP . The latviral MP . A short (BBWV1) . In addi (BBWV1) . HoweverPotyviridae have a single-stranded RNA genome of positive polarity  with a 5′-terminus covalently linked to the protein VPg and a polyadenylated 3′-terminus [Members of the family terminus . The genterminus ,108. In terminus ,110. Accterminus ,109.In infected cells, the CI protein forms pinwheel cytoplasmic inclusions that are detected at the PD entrance, can cross the cell wall through PD to form connections between two CI-inclusions in adjacent cells, and are associated with clusters of virions ,112. SimThe VPg and HC-Pro proteins, which interact with each other and with the virion-bound CI protein, are known to form a morphologically distinctive structure at one end of the virions ,119 thatPotyvirus and Rymovirus, whereas in other genera of the family Potyviridae, this function depends on the P1 protein (Sweet potato mild mottle virus (genus Ipomovirus), P1 has been shown to suppress RNA silencing by binding to AGO1 through GW/WG-motifs, inhibiting both existing and de novo formed AGO1-containing RISC complexes [Zucchini yellow mosaic virus (ZYMV) but result in the loss of this ability in the case of Tobacco etch virus (TEV) and Potato virus Y (PVY) [Turnip mosaic virus (TuMV) HC-Pro has been shown to inhibit the methylation activity of HEN1 through physical interaction via the FRNK motif in which the arginine residue plays a key role [Tobacco etch virus (TEV) HC-Pro has been reported to interfere with the self-regulation of the plant RNA-silencing pathway and increase the expression of miR168 which negatively regulates AGO1 expression by targeting AGO1 mRNA for degradation [Potato virus A (PVA) HC-Pro has been shown to interact directly with AGO1 in ribosomal complexes. It has been suggested that this interaction may be involved in the repression of potyviral translation [Arabidopsis plants expressing the P1/HC-Pro precursors of TuMV, ZYMV, and TEV have shown that TuMV P1/HC-Pro triggers post-translational degradation of AGO1, but this effect is not observed in plants expressing P1/HC-Pro ZYMV and P1/HC-Pro TEV [HC-Pro has RNA silencing suppression activity only in viruses of the genera  protein  [132,133omplexes . Interesomplexes ,136. Foromplexes . Since tomplexes . Althougomplexes ,139,140.omplexes ,139,141.omplexes ,143,144. Y (PVY) . This co Y (PVY) . More li Y (PVY) ,147,148, Y (PVY) . HC-Pro  Y (PVY) . Alternakey role . Anotherradation . In addinslation . Howevernslation . On the -Pro TEV . In viewArabidopsis thaliana are caused by the interference of HC-Pro TuMV with pathways that depend on the negative regulation involving miRNA. The Arg residue in the FRNK motif of HC-Pro, which is responsible for binding small RNA duplexes, is also responsible for suppressing the host miRNA-mediated response. It is thought that HC-Pro interferes with the assembly or activity of RISC complexes, resulting in increased accumulation of those mRNAs that are otherwise negatively regulated by miRNA-directed cleavage [Transgenic plants constitutively expressing HC-Pro have been shown to exhibit developmental defects associated with the disruption of miRNA function ,154. Appcleavage ,145,153.Papaya ringspot virus (PRSV) HC-Pro interacts with papaya calreticulin and appears to modulate calcium signaling-mediated defense response [The function of HC-Pro as a VSR appears to intersect with plant innate immunity. TEV HC-Pro has been shown to interfere with the expression of jasmonate-regulated transcripts . In addiresponse .As described above, the mechanisms by which HC-Pro can suppress RNA silencing are highly variable. Given the limited number of studies comparing the function of HC-Pro in RNA silencing suppression and its transport function, it is difficult to extrapolate these data. An apparent correlation between the involvement of TEV HC-Pro in long-distance transport and its VSR function has been demonstrated: mutant viruses defective in long-distance transport have no suppressor activity while retaining the ability to replicate and move from cell to cell, albeit less efficiently ,127. SimRecently, it has been shown that HC-Pro induces the formation of cytoplasmic potyvirus-induced RNA granules (PGs) during PVA and TuMV infections, which contain plant proteins such as acidic ribosomal protein P0, AGO1, oligouridylate-binding protein 1 (UBP1), varicose (VCS), and eukaryotic initiation factor iso4E (eIF(iso)4E). PGs are associated with the RNA silencing suppression activity of HC-Pro, as point mutations that disrupt this activity of HC-Pro also abolish its ability to generate PGs . Of partCytorhabdovirus and Nucleorhabdovirus and bipartite in the genera Dichorhavirus and Varicosavirus) have five canonical genes encoding nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and polymerase (L) [Plant rhabdoviruses are enveloped bacilliform viruses with negative-sense ssRNA genomes capable of replication in both host plants and specific arthropod vectors . The virrase (L) ,169.Orchid fleck virus (OFV-citrus) (genus Dichorhavirus), the silencing suppressor function of the virus MP has been found. This non-structural protein is encoded by a highly conserved gene in RNA-1. The OFV-citrus MP has a diffuse cytoplasmic localization and is additionally localized to PD. Importantly, MP associates with structural N and P proteins and directs these proteins from the nucleus to the cell cytoplasm, and subsequently the complex of MP and N (nucleocapsid) proteins is transported to PD [In the citrus strain of ed to PD ,171. Theed to PD ,171,172.Dichorhavirus, the structural (virion) P protein of monopartite plant rhabdoviruses has been shown to have a silencing suppressor function [Barley yellow striate mosaic virus, the structural P3 protein has been shown to have silencing suppressor activity in addition to the P protein [Rice yellow stunt virus the only identified suppressor is the virion P6 protein [In contrast to members of the genus function ,175,176.function ,175. Oth protein .Rice ragged stunt virus (RRSV) is a member of the genus Oryzavirus in the family Spinareoviridae. RRSV infects plants of the Graminae family and is transmitted in a persistent manner by the brown plant hopper (Nilaparvata lugens). The RRSV genome contains 10 double-stranded RNAs ranging in size from 1.2 to 3.9 kb [The o 3.9 kb . The RRSo 3.9 kb .N. tabacum and N. benthamiana plants [N. benthamiana. The Pns6-eGFP fusion protein transiently expressed in N. benthamiana epidermal cells is mainly localized mainly close to the cell wall as well as in punctate sites corresponding to PD [N. benthamiana. RRSV Pns6 has been shown to suppress silencing in the patch agroinfiltration assay in GFP-transgenic N. benthamiana plants [N. benthamiana with a 35S-GFP construct. Both Pns6 and Pns9 proteins inhibit local silencing, but only Pns6 affects the long-range movement of the silencing signal [The RRSV RNA binding Pns6 protein has been reported to complement the cell-to-cell movement of a movement-deficient TMV in a plants ,181. Howng to PD . Expressa plants . More spa plants . These da plants ,181,182.g signal .Rice dwarf virus (RDV) and Rice gall dwarf virus (RGDV) are members of the genus Phytoreovirus in the family Sedoreoviridae. These viruses have icosahedral double-shelled virions with an average diameter of approximately 70 nm [Nephotettix cincticeps or Resilia dorsalis) and multiply in both the plants and the invertebrate insect vectors. In RDV, six nonstructural proteins are products of genomic RNA components S4, S6, S10, S11, and S12, respectively [N. benthamiana. This MP has been observed to localize near or within the cell walls of epidermal cells [N. cincticeps during insect feeding on infected plants, RDV first infects the filter chamber epithelium. After the assembly of progeny virions, RDV spreads to adjacent organs and eventually moves to the salivary glands from where it can be introduced into rice hosts [N. cincticeps [The ly 70 nm ,184. Phyectively ,187,188.al cells . Strikince hosts . Severalncticeps . These rncticeps . It is incticeps ,191.N. benthamiana plants carrying a GFP transgene. The green fluorescence intensity remains strong in the patches co-infiltrated with 35S-GFP and 35S-S10 constructs for a nine day observation period. GFP-specific siRNAs levels are significantly reduced in leaves co-infiltrated with 35S-GFP and 35S-S10 constructs compared to 35S-GFP-infiltrated leaves [Pns10, encoded by genomic segment S10, has also been identified as a VSR ,192. Thed leaves . Pns10 hd leaves .In grafting experiments with transgenic plants expressing GFP and Pns10, the silencing signal is unable to spread beyond specific veins in systemic leaves. In the case of dsRNA-induced silencing, Pns10 appears to interfere with signal perception in the recipient tissue but does not inhibit silencing signal formation . InteresGeminiviridae belong to the recently introduced virus phylum Cressdnaviricota, which includes hundreds of discovered circular Rep-encoding ssDNA viruses [Begomovirus have bipartite ssDNA genomes, but other members of this genus and the members of thirteen other genera have monopartite genomes. These genomes typically encode six to seven proteins. In most cases, the virion DNA-sense transcripts contain two partially overlapping ORFs , whereas the complementary-sense mRNAs usually encode four proteins, namely a replication-associated protein , which is essential for the rolling-circle replication of the viral genome by the plant DNA polymerases alpha and delta: a transcription activator protein , a replication enhancer protein , and a rather small C4 protein (85–100 amino acids long). The gene for the latter protein is located within the C1 gene [Viruses of the family  viruses ,196. Mos C1 gene ,201,202.Mastrevirus performs functions of a membrane-bound MP and works in coordination with CP to enable the trafficking of virus ssDNA from cell to cell through PD [It has long been recognized that the V2 protein in the viruses of the genus rough PD ,204,205.rough PD ,209,210.rough PD ,212, whirough PD ,214,215.rough PD ,217.Tomato yellow leaf curl virus (TYLCV), and other members of the genus Begomovirus is a strong suppressor of gene silencing (PTGS) that acts as an interactor of SGS3 and suppresses secondary siRNA production [Curtovirus, V2 also functions as a strong PTGS suppressor by interfering with the RDR6/SGS3 pathway of secondary siRNA formation [In general, many studies have shown that C1 and C2 proteins encoded by different geminiviruses can suppress silencing . Being poduction ,232,233.ormation ,227.The C4 protein is known to suppress both PTGS and TGS. To counteract TGS, C4 suppresses SAM synthetase enzyme activity and interacts with AGO4 to eliminate viral genome methylation ,214,215.In the last decades, RNA silencing-based strategies have been developed for plant protection against viruses. Currently, the main schemes of this strategy include the construction of transgenic plants expressing virus-specific hairpin transcripts and artificial virus-related miRNAs, or the exogenous application of dsRNAs. Host-induced gene silencing (HIGS) is observed in transgenic plants that are genetically engineered to synthesize pathogen gene-targeting sRNAs or dsRNAs that are further processed into siRNAs. These siRNAs are then led to silencing virulence-related genes. Importantly, HIGS is effective against a wide range of plant pathogens and pests, including viruses, viroids, fungi, insects, and nematodes infesting many important crops ,235,236.Another newly developed method is CRISPR/Cas system, which shows great promise as a simple and scalable platform that can be efficiently exploited to achieve virus resistance in plants ,244,245.Turnip yellow mosaic virus (P69) [It seems reasonable that the promising targets of antiviral RNAs might be key functional regions of viral genomes, especially those encoding MPs and VSRs ,247. Indus (P69) , showed Furthermore, studies on engineered defense against animal viruses have revealed fundamentally new methods not previously used in plants. In particular, it has been shown that the inhibition of an enterovirus VSR by a specific artificial peptide that blocks the VSR dimerization leads to a significant increase in the antiviral siRNA activity in animal cells . Thus, d


Ind heart  pp. 7. H6 PFU of each virus. One day later, naïve ferrets were placed into neighboring cages that prevented direct contact between the inoculated and exposed animals but allowed virus respiratory droplet transmission through the air. Nasal swab samples collected after inoculation or exposure revealed robust virus titers in infected, but not exposed animals , by a Project Promoting Support for Drug Discovery (JP21nf0101632), by the Japan Program for Infectious Diseases Research and Infrastructure (JP23wm0125002), and by the Japan Initiative for World-leading Vaccine Research and Development Centers (JP233fa627001) from the Japan Agency for Medical Research and Development. YK has received unrelated funding support from Daiichi Sankyo Pharmaceutical, Fuji Film , Tauns Laboratories, Shionogi, Otsuka Pharmaceutical, KM Biologics, Kyoritsu Seiyaku, Shinya Corporation, and Fuji Rebio.All other authors declare no competing interests.