In the development of end-to-end large-scale live virus vaccine (LVV) manufacturing, process analytical technology (PAT) tools enable timely monitoring of critical process parameters (CPP) and significantly guide process development and characterization. In a commercial setting, these very same tools can enable real time monitoring of CPPs on the shop floor and inform harvest decisions, predict peak potency, and serve as surrogates for release potency assays. Here we introduce the development of four advanced PAT tools for upstream and downstream process monitoring in LVV manufacturing. The first tool explores the application of capacitance probes for real time monitoring of viable cell density in bioreactors. The second tool utilizes high content imaging to determine optimum time of infection in a microcarrier process. The third tool uses flow virometry  to monitor total virus particle counts across upstream and downstream process steps and establishes a robust correlation to virus potency. The fourth and final tool explores the use of nucleic acid dye staining to discriminate between “good” and “damaged” virus particles and uses this strategy to also monitor virus aggregates generated sometimes during downstream processing. Collectively, these tools provide a comprehensive monitoring toolbox and represent a significantly enhanced control strategy for the manufacturing of LVVs. The traditional control strategy to monitor LVVs during manufacturing by measurements of cell viability and cell metabolism cannot achieve continuously monitoring cellular “fingerprints” during the viral production cycles and de-risk in a timely fashion. The assessment of the parameter optimization relies on classical cell-based potency assays, like tissue culture infectious dose 50% (TCID50) and plaque assays, which take at least 1–2 weeks and are difficult to adapt to high-throughput workflows7. The lack of tools to directly monitor and measure LVV potency rapidly results in longer iteration time for process development and process characterization of these products, and this is evidenced by the significantly longer times to launch LVVs against infectious diseases. PAT development to better track LVVs in real time and ensure their quantity as well as quality has thus become the focus of many big pharmaceutical manufacturers and are being increasingly recommended by regulatory agencies9.The race to vaccinate the world against SARS-CoV2 has led to significant efforts to compress the time to manufacture vaccines by improving control strategies around large-scale manufacturing operations. Unlike some of the newer mRNA vaccines that have well understood ways for degradation of their critical quality attributes (CQAs) and analytical assays that can reliably track and correct these changes during manufacturing10. However, these “bulk” analytical methods cannot provide direct information on single virus particles and virus aggregation7. As viruses are sub-visible particles in the nanoscale range, reliable tools to directly measure individual virus particles are not many. Microscopy-based techniques, such as electron microscopy (EM) and atomic force microscopy (AFM) could provide high resolution single-particle analysis for size, morphology, and concentration11. However, these techniques are time-consuming, labor-intensive, low-throughput and expensive, which limit their widespread utilization in LVV manufacturing7. Methods like Nanoparticle tracking analysis (NTA) are developed for nanoparticle size, concentration, and aggregation state characterization based on a laser-illuminated microscopy technique13 The Brownian motion of individual nanoparticles, i.e., virus particles in suspension are tracked and analyzed in real time to calculate their hydrodynamic size using the Stokes–Einstein equation. However, NTA only works when samples are suspended in a clear matrix and a relatively small concentration range is required for accurate analysis, which may be a challenge for samples with unknown concentration7 Flow virometry, the method described here, depends on light scattering of short wavelength (UV) light by virus particles14 and therefore, can only be reliably used for virions that have the size and the density to generate side scatter that can be discriminated above electronic noise. Appropriate and rigorous positive and negative controls are thus needed prior to establishing this as a tool to monitor viruses, but once established, can provide a highly quantitative and accurate measure of total virus count. Combined with protein, nucleic acid, and/or lipid dye staining, flow virometry can not only provide quantitative but also qualitative information about individual virions18.Several methods are available for direct virus quantification, such as detection of virus proteins via enzyme-linked immunosorbent assays (ELISA), quantification of virus nucleic acid by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), and both measuring viral protein or nucleic acid content by capillary electrophoresis (CE)20 and highlight their application as PAT in large scale manufacturing.In this report, we introduce Biocapacitance as an on-line PAT tool for continuously monitoring viral vectored vaccines production in cell culture processes. In addition, we propose high throughput potency assays for infectivity assessment of LVVs that can further speed up the process development of these lifesaving products. Moreover, we show applications of flow virometry to the near real time monitoring of two virus vaccine candidates21, and used genetic engineering to replace VSV surface glycoprotein with the target protein  used a VSV virus vector  calculations for virus seeding and subsequently bioreactor monitoring of cells for viability and metabolites , biocapacitance has been used for real-time monitoring of biomass or biovolume in cell culture processes23. For both the C1 and C2 viral vector processes, the capacitance measured at a frequency of 580 kHz models the cellular state in a scale independent manner  based on optimal cell density on each individual microcarrier and media exchange parameters for LVV programs using microcarrier cultures. Briefly, microcarrier cultures that had grown for 4 or 5 days (4- or 5-days post plant (DPP)) were infected with C1, and cells were fixed directly on the microcarriers 24 h post infection. In addition, 5DPP cells infected with C1 were also compared between bioreactors that had undergone a media exchange (MX) or had not had a media replacement (NMX) prior to infection. Fixed samples were stained with a primary anti-Target protein (expressed on surface of C1) specific monoclonal antibody conjugated to Alexa Fluor 488, anti-VSV primary antibody with Alexa Fluor 568 secondary antibody, and Hoechst 33342 nucleic acid dye. We then used automated high content imaging to analyze ~ 100 microcarriers per condition.We have previously established the utility of high content imaging as a robust tool for potency measurements with a faster turnaround time than traditional potency assaysWe detected both VSV and target proteins for all conditions studied, as evidenced by orange and green fluorescent expression, respectively Fig. A. Using 17. To evaluate if this tool can be used for the two new virus-vectored vaccine candidates, we first assessed if C1 and C2 can be detected using violet laser side scatter (VSSC).We have previously established that flow virometry can be used to directly measure and count virus particles and can thus, be effectively utilized as an at-line process monitoring tool for LVV manufacturing24. To further confirm identity of the viruses being detected by flow virometry, we performed antibody staining. For C1, we stained samples with anti-virus surface protein specific monoclonal antibody and the viral entry receptor specific fusion protein, using secondary single stained samples as controls . Data from upstream 50L and 2000L bioreactors was collected for the full length of production time and plotted for total particles and TCID 50 potency Fig. A–D. Once17. Here we wanted to explore a similar application in monitoring virus quality using the C2 virus vaccine. As part of process development, we had generated two downstream intermediate samples, both of which had undergone identical upstream production and downstream clarification steps. The only differences in generation of the two intermediates was an increased flow rate (39 L/min vs 19.5 L/min) and virus sample concentration factor being targeted (26.8 × vs 14.5×) during tangential flow filtration (TFF), which resulted in increased shear stress. The sample generated at 19.5 L/min had a TCID50 potency value within expected range, but the sample generated at 39 L/min had no detectable potency using the same assay. Total particle counts measured by CytoFLEX were not significantly different between the two samples . Cells were cultured in OptiPRO™ Serum Free medium (Thermo Fisher Scientific) supplemented with 4 mM L-glutamine (Thermo Fisher Scientific) and propagated in a humidified incubator (Thermo Fisher Scientific) at 37 °C with 5% COThe vesicular stomatitis virus (VSV) vectored virus and Measles vectored virus used in this study were obtained from MSD laboratories.The Apogee A60 is configured with 405 nm violet laser and forward, side, and green fluorescence detectors. Apogee refers to traditional forward and side scatter as small-angle light scatter (SALS) and large-angle light scatter (LALS) respectively. The CytoFLEX S is in standard configuration with three color lasers and assorted detectors; for the purposes of this study, we are concerned primarily with violet side scatter . No customization was required of the equipment after initial set up..Highly filtered sheath fluid: either 0.1 µm phosphate buffered saline (PBS), 0.12 µm Milli-Q water, or purchased Beckman Coulter sheath fluid, were used for all of these studies. Both cytometers were confirmed to be capable of visualizing and enumerating control beads and viral particles with control samples prior to using for experimental conditionsAll samples were diluted in 0.1 µm filtered PBS and run on Apogee and CytoFLEX Flow Virometers, dilutions are noted in the sample names on Figures. For Apogee, a flow rate of 1.5 µL/min was selected and for CytoFLEX a flow rate of 10 µL/min was used. Total particles count for samples was measured by multiplying events/second recorded with dilution of the sample and total volume collected during each run. The profile of each sample was recorded and analyzed using FlowJo software.Control polystyrene beads for confirming operation of flow cytometers were sourced through Apogee and utilized per manufacturer recommendations. The Apogee A60 utilizes a traditional photomultiplier tube (PMT) with variable voltage, settings were identical for all profiles which we compared to one another.For antibody staining of virus particles, samples were directly stained with primary and secondary antibodies of choice in PBS and purified between staining to remove unbound antibody using a Capto Core 700 column. Stained samples were detected on the Y-axis of the Apogee and CytoFLEX flow cytometers using the relevant fluorescence detection channel.Uninfected and C1 infected Vero cells on microcarriers were sampled from 3L bioreactors of varying experimental conditions at 4 h pre-infection and 24 h post infection, respectively. Samples were fixed and permeabilized using BD Biosciences Cytofix/Cytoperm™ kit and blocked with 4% goat serum. Infected cells were stained sequentially with anti-VSV primary antibody, Alexa Fluor 568 secondary antibody, anti-Spike primary antibody conjugated to Alexa Fluor 488, and Hoechst 33342 nuclear dye in 1X BD Perm/Wash™ Buffer. Uninfected cells were stained using the same sequence to act as negative controls. Secondary antibody controls were prepared by staining infected cells with Alexa Fluor 568 or Alexa Fluor 488 and Hoechst 33342 only. After staining, samples were transferred to a Cell Carrier Ultra 96-well Microplate (Perkin Elmer). High content imaging was performed using the 20× water objective on the Operetta CLS™ in confocal mode with Digital Phase Contrast, Hoechst 33342, Alexa 568 and Alexa 488 imaging channels. ~ 20,000–30,000 cells (~ 150–250 microcarriers) were analyzed in each condition. Cells were determined to be “positive” for VSV or Target protein expression if their mean A568 or A488 fluorescent intensity was greater than 500 based on the fluorescent intensities of unstained, secondary alone and uninfected controls. Cells with a mean A568 or A488 fluorescent intensity less than 500 were determined to be “negative” for VSV or Target protein expression.Biocapacitance data was collected at multiple frequencies from Aber 220 mm Annular probes (Aber:Futura Probes:6532-52/PG) using the provided software (FuturaTool V3.0.5.0) in accordance with the manufacturer’s recommended setup instructions. Briefly, the probe was connected to the head amplifier (Aber:Futura Hardware:2330-00) which was connected to the data hub (Aber:Futura Hardware:2801-00). Data hub was connected to a laptop running the Aber software (FuturaTool V3.0.5.0) via USB. Multiple frequency data was outputted as a CSV file with post run analysis in Microsoft Excel (version: Microsoft 365 Apps for enterprise).t-test and a p value < 0.05 was considered statistically significant. Microsoft Excel was used to generate graphs and perform statistical analysis.Operetta CLS high content imaging data was analyzed using Perkin Elmer Harmony 4.9 analysis software. Each experimental condition includes data from three unique 3L bioreactors tested in triplicate (N = 9) and are expressed as means ± standard deviation. Conditions were compared using a two-tailed Student’s Flow cytometry data was analyzed using Flowjo 10.6.2. Graphs were generated using GraphPad Prism 8 (GraphPad Software). The correlation was analyzed by linear regression between CytoFLEX total particle count and potency. Pearson’s correlation coefficient and the coefficient of determination (R2) were analyzed with Microsoft Excel and GraphPad Prism 8.27, here we look at a more mainstream application of these tools on two different virus vectors and show that both can be quantitatively and qualitatively monitored using these methods.The large-scale commercial manufacturing of LVVs consists of a series of steps starting from the culture and expansion of mammalian cells, followed by infection with virus of interest and the subsequent purification of the viral material in large quantities to finally be vialed and distributed. Throughout this process, a series of analytical measurements are implemented to ensure the quantity and quality of the final product and exert suitable control over the manufacturing process. Such controls notwithstanding, the risk of a low potency investigation in LVV manufacturing remains a pertinent risk and a challenge for pharma companies. The need for more real time monitoring of biopharmaceutical processes using PAT tools has been discussed by regulatory agencies in recent years and is increasingly becoming a regulatory expectation (ICH guidelines). The Covid-19 pandemic has further highlighted the need for efficient and fast large-scale manufacturing of vaccines with a quick turnaround time and minimal waste, which most traditional LVV processes are not yet equipped for. A typical LVV process can take years of process development and many iterations of troubleshooting to establish the final method. One of the bottle necks for process development work in the LVV space is the long turnaround time for potency assays. While several unified and platform technologies are being used for the commercial upstream and downstream manufacturing of vaccines such as the use of Vero and CHO cell lines, such a unified approach to the application of PAT tools is still missing. Here we showcase a set of PAT tools that can be effectively used for process monitoring and potency prediction of many different LVVs. Advancing prior work on application of PAT tools to support investigations around low potency33. Our model confirms that in-line real-time monitoring by probes can be used to replace the more traditional ways to measure cell viability. In addition, we present here the intriguing possibility of directly monitoring virus particle biomass using capacitance at a higher frequency, which, if confirmed by more rigorous future studies, would present a PAT tool that could offer a way to obtain potency results in real time and would result in significantly enhanced process control and time saving.Measurement of cell viability and monitoring of the expected initial increase followed by the post infection decline is one of the most critical process parameters for LVV manufacturing. Most established methods are “at line” which means they require manual sampling and tend to be labor intensive and error prone. With the application of capacitance probes to measure biomass of adherent cells growing on microcarrier cultures in real time, we were able to advance monitoring of cell viability significantly for LVV cultures, whereby, past applications of capacitance probes have mostly been limited to applications in suspension mammalian and microbial cell culturesVirus particle counting by flow virometry is a biophysical measurement of the virus and does not directly measure virus infectivity. However, unlike true potency measurements, these methods are usually easier and significantly faster. To use this tool as a proxy for potency though, first an optimal volume of data needs to be generated under a relevant range of manufacturing conditions and design of experiment (DOE) studies for parameters like time, temperature, pH, MOI and cell density to establish the correlation. The assumption in using such a measure as a surrogate method for potency is that for a consistent and repeatable process, the total to infectious particle ratio will remain constant. Conversely, any changes in total particle counts as measured by these means will raise concerns around virus potency and will be a suitable in-process red-flag meant to offset large scale batch failures due to OOS potencies. In addition to generating a critical mass of data, direct antibody staining studies are essential to confirm identify of particles being detected by these methods and establish that these can be discriminated from cellular vesicles of similar size. As it has been shown by others, bioreactors can also produce non-viral particles of similar size which could be a mixture of extracellular vesicles (EVs), exosomes, etc. Thus, it is not enough to confirm virus identity by staining with an extracellular antibody that can also be present in EVs, but one must also try to confirm the presence of an intra-viral protein to ensure that the particles being tracked by this method is largely the virus of interest. It is expected and not unusual that some non-viral particles as well as cellular components will be retained in this mix, especially in upstream processes, and are gradually removed upon downstream purification. Here we confirm identity of the particles using virus specific antibodies as well as rely on strong correlation to established potency assay prior to utilizing these tools for routine process monitoring. Once established, such confirmatory staining no longer needs to be performed to prevent these methods from becoming long and tedious.Finally, high content imaging offers an attractive PAT for at-line monitoring of cells and the off-line measurement of potency. With advanced image analysis software that can measure multiple parameters within seconds and the potential to visualize them in centralized data repositories like SIPAT and Delta V, such advanced applications are now well within the reach of routine manufacturing.Supplementary Figures.


Arabidopsis thaliana, and to some extent in other plants, such as Solanum lycopersicum, Nicotiana benthamiana, Brassica napus, Oryza sativa, Triticum aestivum, Hordeum vulgare, Brachypodium distachyon, Medicago truncatula, Gossypium barbadense, Phaseolus vulgaris, Solanum tuberosum, and Malus robusta. Most LRR-RLKs tend to form different combinations of LRR-RLKs-complexes , and some of them were observed as important receptors in immune responses, cell death, and plant development processes. However, less is known about the function(s) of LRR-RLKs in response to abiotic and biotic stresses. Here, we give recent updates about LRR-RLK receptors, specifically focusing on their involvement in biotic and abiotic stresses in the model plant, A. thaliana. Furthermore, the recent studies on LRR-RLKs that are homologous in other plants is also reviewed in relation to their role in triggering stress response processes against biotic and abiotic stimuli and/or in exploring their additional function(s). Furthermore, we present the interactions and combinations among LRR-RLK receptors that have been confirmed through experiments. Moreover, based on GENEINVESTIGATOR microarray database analysis, we predict some potential LRR-RLK genes involved in certain biotic and abiotic stresses whose function and mechanism may be explored.Plants live under different biotic and abiotic stress conditions, and, to cope with the adversity and severity, plants have well-developed resistance mechanisms. The mechanism starts with perception of the stimuli followed by molecular, biochemical, and physiological adaptive measures. The family of LRR-RLKs (leucine-rich repeat receptor-like kinases) is one such group that perceives biotic and abiotic stimuli and also plays important roles in different biological processes of development. This has been mostly studied in the model plant,  LRR-RLK mutant lines by inserting tDNA fragments into their exon [Arabidopsis thaliana, and thereafter in tomato (Solanum lycopersicum) and tobacco (Nicotiana benthamiana). The ubiquitous presence of LRR-RLK with varying sequences across the plant genera underscores the need for its investigation in other plants in order to expand the knowledge and thereby to understand their role more specifically. RLKs were classified into different groups based on their functions, such as growth and development processes and biotic and abiotic stress responses  or SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3 (SERK3); SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1, 2, and 4 (SERK1/2/4); BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1); BAK1-INTERACTING RECEPTOR-LIKE KINASE1 (BIR1); and SUPPRESSOR OF BIR1-1 (SOBIR1), ELONGATION FACTOR-Tu (EF-Tu) RECEPTOR (EFR) were discussed as being essential for immune responses [RECEPTOR-LIKE PROTEIN KINASE1 (RPK1) and RECEPTOR-LIKE KINASE 7 (RLK7 or other name LRR XI-23), are involved in water stress [PHLOEM INTERCALATED WITH XYLEM-LIKE 1 (PXL1) in cold stress [LRR-RLKs, such as BAK1/SERK3; SERK1,-2, and -4; and BRI1, share the same signaling pathways in Mitogen-Activated Protein Kinase (MAPK) activation, Ca2+ influx, and the production of reactive oxygen species (ROS) in order to initiate plant responses to biotic or abiotic stresses and/or developmental cues  have evolved, and they are classified based on their kinase domain and extracellular domain sequences ,2,3. Amoeir exon ,13,14, ieir exon ,15,16 anarticles ). Some oesponses ,18. Credd stress . Notablye review ). Among A. thaliana.The in silico studies have shown that LRR-RLK harbors a transmembrane domain (TM), intracellular kinase domain (KD), and LRR-containing extracellular domain (ECD) . This exA. thaliana, and in other plants as well, such as N. benthamiana, S. lycopersicum, Oryza sativa, Triticum aestivum, Hordeum vulgare, Brachypodium distachyon, Brassica napus, Medicago truncatula, Gossypium barbadense, Phaseolus vulgaris, Solanum tuberosum, and Malus robusta. Additionally, we compiled the information on genetic tools used in the investigation of LRR-RLK’s role in biotic and abiotic stress response. The Microarray data analysis  of the LRR-RLK genes, which responded to biotic and abiotic stimuli, suggested the possibility of additional functions and their cross-link in triggering different signaling processes. Furthermore, we have presented the information on stress-related LRR-RLKs as dimer, trimer, and tetramer complexes, which were experimentally achieved in A. thaliana. Furthermore, an ATTED databases  analysis for protein interactions pointed towards additional possible interactions among the LRR-RLKs, however, these need to be validated experimentally [Here, we update the functioning of LRR-RLKs in response to biotic and abiotic stresses in mentally .LRR-RLK in different plants, such as annual and perennial plants, crops, trees, herbs, etc.   to form new genes may have occurred after the divergence of land plants from green algae [LRR-RLKs is a result of tandem and segmental duplication events  See . It was n events . Addition events .LRR-RLKs were mainly conducted in A. thaliana due to the availability of a large dataset. The increase in DNA and RNA sequencing in different crops and A. thaliana allowed for the identification of the numbers of stress-related LRR-RLKs  [LRR-RLKs is BAK1/SERK3, which regulates multiple processes such as Brassinosteroid (BR) signaling, growth and development, and stomatal patterning, and also activates the expression of antimicrobial proteins [bak1 mutants showed more loss of water as compared to wild-type. The ABA increased the formation of a complex between BAK1 and OPEN STOMATA1 (OST1) near the plasma membrane [bak1–5 mutants revealed that the post-invasive resistance of A. thaliana to Alternaria brassicicola is independent of pathogen-triggered indole-3-carboxylic acid and its derivatives (ICAs) and camalexin biosynthesis [Phytophthora infestans) and bacterial and nematode infection [SERKs, such as SERK1 and SERK2, were also important genes for conferring resistance against bacterial leaf blight and fungal infection, respectively [OsSERK2 was shown as a positive regulator of immunity, interacting with the rice immune receptor kinases (XA21 and XA3) [SERK2 could be a target for sRNAs of Sclerotinia sclerotiorum and the resulting action may contribute to the silencing of immune components in plants [serk2 mutant lines, it was observed that SERK2 is a component of BR signaling and it regulates BR signaling and salt resistance in rice [SERK4 together with SERK3 were shown to trigger a series of defense responses [SERK4, it was observed that SERK4 negatively regulates the leaves senescence process [From a pool of 223 thaliana , some welopment) ,69,70,71proteins ,72. Recemembrane . In addiynthesis . Notablynfection ,76. Otheectively ,77,78. In plants . Recentl in rice . Previouesponses , but in  process .BRI1, which interacts with SERK3 by mediating BR signaling through the BRI1/BAK1 complex [BRI1 shows pleiotropic effects on disease resistance along with plant development regulation [B. napus, S. lycopersicum, and B. distachyon, the role of BRI1 in BR signaling was confirmed [BRI1 was involved in BR signaling through MAPK and Ca2+-dependent protein kinases in rice [SlBRI1 is related with systemin-mediated systemic defense response [NbBRI1 was involved in BR-mediated systemic defense signaling by regulating H2O2 and NO production [BRI1 manipulation in different cereals resulted in drought tolerance [TaBRI1 in A. thaliana revealed early flowering and enhancement of seed production [S. tuberosum), BRI1 was involved in the regulation of tuberization, thus suggesting other possible roles of BRI1 [SOBIR1 activated the immune response in tomato against fungal infection [NbSOBIR1 was involved in the immunity of N. benthamiana through monitoring the production of ROS [GbSOBIR1 in cotton (G. barbadense) plants resulted in resistance to Verticillium [A. thaliana efr mutants lacking EF-Tu perception are more susceptible to transformation by Agrobacterium tumefaciens [EFR in tobacco and in tomato were also involved in pathogen-associated molecular patterns (PAMP)-triggered immunity, and suggesting transgenic expression of EFR could be used as an engineering tool against broad-spectrum bacterial infections [AtEFR in different crops shows resistance to bacterial infection and/or symptoms. In wheat and apple, the overexpression of AtEFR enhanced resistance against bacterial disease, fire, and blight [M. truncatula, it reduced the bacterium infection [Another well-studied LRR-RLK gene is  complex , and it  complex ,17,82. Mgulation , and it gulation ,85. In Bonfirmed ,87,88. F in rice . Additioresponse , howeveroduction . Recentlolerance , and disolerance . Additiooduction , while ooduction . In pota of BRI1 . BIR1 is of BRI1 . The LRR of BRI1 . Notablynfection , but notnfection . In toban of ROS . Furthericillium . While Eicillium , it was efaciens . This refections . In ricefections . The oved blight , respectnfection  and in pnfection .PSKR1 was also identified as an important component of plant defense [PSKR1 suppresses salicylate-dependent defense responses, where the pskr1 mutants exhibit early senescence, a salicylate (SA)-associated response, and are impaired in wound healing, a jasmonate (JA)-associated response [OsPSKR1 was involved in the immune response against bacterial leaf streak in rice through salicylic acid (SA) pathway signaling [PEP1 RECEPTOR 1 (PEPR1) and PEPR2 were involved in immune responses through interaction with SERK3 through AtPep1-triggered ROS production and ethylene signaling [PEPR1/2 ORTHOLOG RECEPTOR-LIKE KINASE1 from S. lycopersicum was shown to regulate responses to systemin, necrotrophic fungi, and insect herbivory [SERKs also form complexes with PHYTOSULFOKINE (PSK) RECEPTOR 1 (PSKR1), which is known to regulate root growth and hypocotyl elongation . Moreove defense . PSKR1 sresponse . Moreoveignaling . PEP1 REignaling . Furtherignaling . Furtherignaling . PEPR1/2erbivory . Previouerbivory ,109.2-mediated stomatal closure [Fusarium by disbalancing salicylic acid signaling [LRR1 in rice in the XA21 genetic background  compromises resistance to bacterial leaf blight, indicating involvement in XA21-mediated immune response [LRR-RLK, RECEPTOR-LIKE KINASE 902 (RLK902) previously showed importance in resistance to Hyaloperonospora arabidopsidis (downy mildew) in A. thaliana [At-FEI2  plays a positive role in Arabidopsis and in tomato defense against Botrytis cinerea based on the study using knockout mutants of the Bc-siR37 [The LEUCINE-RICH REPEAT PROTEIN 1 (LRR1) forms a complex with pathogenesis-related protein10 (PR10), and it leads to cell-death-mediated defense signaling . Additio closure . Recentlignaling . The silresponse . Other Lthaliana . RLK902 thaliana . Not mucthaliana . It was Bc-siR37 .LRR-RLK genes have been shown to play important roles in response to abiotic stresses. In previous studies, ERECTA-LIKE1 (ERL1), a member of the gene family closely related to LRR-RLKs, was shown to synergistically regulate plant development and morphogenesis and functions in response to abiotic stresses, especially for heat response and drought [LRR-RLK genes like PXY-Like 2 (PXL2) have been proven to be essential in vascular development through recognizing small signaling peptides, and they play a role in ABA signaling [PHLOEM INTERCALATED WITH XYLEM-LIKE 1 (PXL1) is essential in cold and heat stress (through the ROS), the PXL1 regulates HISTIDINE-RICH DEHYDRIN1 (AtHIRD1) and LIGHT-HARVESTING PROTEIN COMPLEX I (AtLHCA1) by phosphorylation [LRR-RLK gene, such as SCHENGEN 3 (SGN3), is related with developmental processes, particularly with microdomain organization and enhanced suberization in the endodermis. Additionally, it was shown that the sgn3 mutant was extremely sensitive to environmental conditions, such as different temperatures and nutrient deficiency [BRL3 resulted in drought stress tolerance in A. thaliana through the accumulation of osmoprotectant metabolites, such as proline and sugars [OsBRL1 and OsBRL3 were shown to be partly involved in BR perception in the roots [RLK7/LRR XI-23, was involved in the regulation of seed germination and oxidative stress [RPK2 and CLAVATA1 (CLV1) and CLAVATA2 (CLV2) showed importance in nematode parasitism [SARK was shown to be a positive regulator of senescence through hormone imbalances [PpSARK in moss was shown as a positive regulator of senescence in salt stress responses and was also suggested to be a negative regulator of senescence [The  drought ,119. Rec drought . Furthemignaling . While trylation . Anotherficiency . Moreoveficiency . Additiod sugars . In ricehe roots . Anothere stress . Furtherrasitism . Furtherrasitism  and anthrasitism . SARK wabalances . Interesnescence . In commnescence .LRR-RLK gene(s), which were tested in different biotic or abiotic stress/senescence processes, shared similar signaling pathways, such as reactive oxygen species (ROS) production, Ca2+ influx, activation of MAPK, regulation of defense genes, regulation of stomatal patterning, hormonal regulation, and regulation of senescence-related gene(s), to trigger the response of plants  , which aM. truncatula [A. thaliana and other plants and the generation of new double or triple mutants could help in functional and interactional analyses of LRR-RLKs.Additionally, the ATTED database showed different interactions among the proteins based on various experiments and data . The anauncatula , and wheuncatula  . The double, triple, and quadruple mutant generation for LRR-RLKs in A. thaliana and/or crop plants is a powerful tool for identifying the function role of LRR-RLKs.Plants must evolve to adapt and tolerate harsh environments. The perception of biotic and abiotic stimuli is crucial for the survival of plants. Among the large numbers of receptors, the LRR-RLK are not only involved in different development biological processes but also in stress response processes. The rapid development of genome and RNA sequence analyses have allowed for the identification of the many LRR-RLKs genes in different monocot and dicot plants. As compared to


Short-term researches have studied the change of the center of rotation (COR) after the Bryan Cervical disc arthroplasty (CDA). But there is a lack of long-term studies focusing on the location of COR and its influence after surgery.Clinical and radiographic materials of patients who received CDA were retrospectively reviewed. Written informed consents were obtained. Clinical outcome was accessed by Japanese Orthopaedic Association (JOA), Neck Disability Index (NDI), and Odom’s scale. Radiographic evaluation underwent before surgery, at early (3 months) follow-up and final  follow-up. The ROM of the global cervical spine and index level, the functional spine unit (FSU) angle and C2-C7 angle were measured. COR was identified and its coordinates were calculated. The absolute change of COR-x and COR-y were compared in subgroup analysis.p = 0.03). The ROM at the index level remained unchanged from early follow-up to the final follow-up . In subgroup analysis, larger changes of both COR-x and COR-y were related with decreased ROM.Sixty patients were included, with an average age of 55.9 ± 8.1 years old. The mean duration of follow up was 135.1 ± 16.1 (120–155) months. JOA, NDI and Odom’s scale showed significant improvements at 10 years after surgery. The COR of index level was located in the posterior superior half of the caudal vertebral body. Following the implant of Bryan Disc, the COR shifted forward and downward. During the 10-year follow-up, the location of COR remained stable. ROM at the index level decreased from 10.6 ± 4.0° preoperatively to 9.3 ± 4.0° at the early follow-up (Our study illustrated that Bryan CDA could achieved favorable clinical and radiographic outcome over a minimal 10-year follow-up. The reduction of the flexion-extension ROM may be correlated with a more deviated postoperative COR. More attention should be paid to preoperative design and intraoperative technique to obtain a more native COR. Symptomatic cervical degenerative disc disease (CDDD) is a common condition affecting the population in various age groups –4. AlthoThe principal goal of CDA is to restore the physiologic kinematics at the index level and the ideal CDA prosthesis should achieve this goal by restoring physiologic quantity and quality of motion. And Bryan disc was the most widely used and researched CDA prosthesis in the world. In previous literature about CDA, many studies have proven that the clinical outcomes were satisfactory, and the ROM, the quantity of motion, was maintained –10. And We retrospectively enrolled 83 consecutive cases who underwent single-level cervical arthroplasty for cervical disc degenerative disease between January 2008 and January 2012. Written informed consents were obtained before surgery for the possibility of using their charts and radiographs. All surgeries were accomplished by a single surgeon (WT).The inclusion criteria were as follows: 1) diagnosis of single-level cervical disc degenerative disease, presented with either radiculopathy or myelopathy, or both; 2) age > 18 years; 3) no response to no-operative management for more than 6 weeks; 4) complete follow-up data; and 5) a preoperative ROM at the index level > 3°. The exclusion criteria involved: 1) patients with traumatic or infectious pathology, 2) previous cervical spine surgery, 3) diagnosis of multilevel cervical disc degenerative disease, 4) radiographically instability or loss of ROM (< 3°), severe facet degeneration, potential infection, poor bone quality or neoplasia, and 5) high level (grade III or IV ) heterotAfter general anesthesia, the patient was placed in a supine position through a standard anterior cervical approach according to Smith–Robinson. After confirming the index cervical level by intraoperative fluoroscopy, a complete discectomy was performed. An accurate decompression of the spinal cord and nerve roots was obtained. After the vertebral endplate was polished, the proper size Bryan artificial disc  was implanted.Before surgery and at last follow-up, clinical outcome was accessed by Japanese Orthopaedic Association (JOA) score for functional recovery, Neck Disability Index (NDI) for neck pain/disability, and Odom’s scale for overall efficacy. Each evaluation was performed by two separate doctors, and if disagreement was met, an advice from a third senior doctor would be sought.Radiographic evaluation included lateral neutral and flexion-extension cervical radiographs before surgery, at early (3-month) follow-up and final follow-up. The ROM of the overall cervical spine (C2-7) and index level were measured on lateral flexion-extension radiographs according to the Cobb method . The ROMAn orthogonal plane coordinate system is set up, the posterior inferior corner of the caudal vertebra (point O) is set as origin of coordinates, and the inferior endplate of the caudal vertebra (line OC) is set as x-axis. The y-axis was directed upward perpendicular to the x-axis. COR-x is positive in the anterior direction and COR-y is positive in the cranial direction. The coordinates of points A1, A2, B1 and B2 were measured in millimeter and expressed as , ,  and . Analytical geometry method is used to calculate the coordinates of COR , as shown below:Then the COR-x value was normalized as percentage (%) of the inferior endplate length of the caudal vertebra body, and the COR-y value was normalized as percentage (%) of the posterior height of the caudal vertebra body.Sagittal alignment included C2-C7 angle (the angle between lower endplate of C2 and the lower endplate of C7) and functional spine unit angle . The ICC values were graded using the following criteria: excellent for values in the 0.9–1.0 range, good for 0.7–0.89, fair/moderate for 0.50–0.69, low for 0.25– 0.49, and poor for 0.0–0.24.Statistical analysis was performed using IBM SPSS Statistics 20.0 . All continuous variable data were assessed for normality using Shapiro–Wilk test statistics. The continuous variable data is given as the mean ± standard deviation. The Mann–Whitney U-test was used for non-normally distributed data. The student t-test was used to compare normally distributed data, and paired t-test for paired data. One-way repeated measures analysis of variance was used for comparisons involving 3 time points on the same subject to correct P value from paired t-test. The χWe reviewed 83 patient’s charts, and finally 60 patients were included. Twelve patients were lost to follow up, 7 patients were excluded because of incomplete materials and 4 were due to the occurrence of grade III or grade IV heterotopic ossification (HO). The average age was 55.9 ± 8.1 (38-66) years old at last follow-up. It was composed of 24 females and 36 males, including 1 patient at the C3-4 index level, 13 patients at the C4-5 index level, 39 patients at the C5-6 index level and 7 patients at the C6-7 index level. The mean duration of follow up was 135.1 ± 16.1(120-155) months (Table p < 0.001). The NDI declined from a preoperative score of 27.0 ± 10.0 to a 10-year postoperative score of 12.0 ± 8.0 (P < 0.001). Using Odom’s Scale, 91.7% of patients reported good or excellent outcome. And there was no infection, revision surgery or neurologically/technically related complications occurred upon minimal 10-year follow-up.There was immediate relief of radiculopathy and/or myelopathy in all cases, with no operative or device-related complications over the follow-up. The JOA score of the overall group increased from 13.5 ± 3.3 before surgery to 16.5 ± 2.0 at the final follow-up (p=0.229), and it remained stable during the 10-year follow-up . There was no significant change in mean FSU angle from the preoperative values to the postoperative values , and at the final follow-up, it remained stable . The FSU height value was normalized as percentage (%) of the posterior height of the caudal vertebra body. The FSU height before surgery was comparable to that at early follow-up , and it was maintained to the 10-year follow-up .The C2-C7 angle before surgery was similar to that at early follow-up , while COR-y decreased significantly after surgery from 70.6 ± 12.7% to 67.2 ± 14.0% (p = 0.003). At the 10-year follow-up, COR-x and COR-y showed no significant change compared with the early postoperative values . . The ROM at the index level remained unchanged from early follow-up to the final . The mean global (C2-7) ROM before surgery was similar to that at early follow-up , and it was well preserved during the 10-year follow-up .The range of motion (ROM) at the index level decreased from 10.6 ± 4.0° preoperatively to 9.3 ± 4.0° at the early follow-up (All radiographic results were summarized in Table p < 0.001), which means that COR shifted more anterior in group D-ROM. The change of COR-y was larger in group D-ROM than that in group I-ROM, , indicating that COR in group D-ROM shifted more inferior.  , and by the decreased COR-y after surgery . The possible reasons for this deviation may be the more caudal insertion angulation and/or insufficient insertion depth of the artificial disc. Over-milling of the posterosuperior corner of the caudal vertebra during operation may result in caudal insertion angulation of the prosthesis, and this may cause COR deviation. It may warrant detailed studies on that.Pickett  and Kowap = 0.203 and p= 0.109, respectively). It means that the COR remained relatively stable during the 10-year duration after CDA, which strengthened the conclusions of the short-term studies above.Despite of the statistical significance of the anterior-inferior change of COR, the amplitude of variation was relatively small . Whether this small margin of variation would cause clinical change still needs further researches. But COR-x and COR-y showed no significant change at the last follow-up compared with the early postoperative values (In our subgroup analysis, a deviated COR was related with a decreased ROM. Previous studies found that if the simulated location of COR was close to the inherent location of 1 healthy functional spinal unit, the segmental ROM on the sagittal plane would be well preserved , 24. AndLimitation exists in our study. Firstly, we used only end flexion-extension images to calculate the mean COR and did not depict the path of the COR from full flexion to full extension. Secondly, all surgeries were performed by a single surgeon. The influence of the surgeon’s preferred technique and experience may not be fully ruled out.However, our study does have some strengths. First of all, the minimal 10-year follow-up makes our results more stable and solid. Second, we evaluated not only the quantity of the motion-ROM, but also the quality of the motion-COR. Last but not the least, it can provide clinicians some guidance in the preoperative design of cervical disc arthroplasty.Our in vivo study illustrated that Bryan cervical disc arthroplasty could achieved favorable clinical results and radiographic outcomes over time. Bryan discs preserved ROM at the index level over a ten-year follow-up. The reduction of the flexion-extension ROM may be correlated with a more deviated postoperative COR, and more attention should be paid to preoperative design and intraoperative technique to obtain a more native COR.


A monkeypox virus outbreak has spread worldwide since April 2022. We report a young woman in France positive for monkeypox virus transmitted through oral and vaginal sex. Ulceronecrotic lesions developed intravaginally and around her vulva. Health professionals should become familiar with all aspects of infection from this virus, including possible vertical transmission. A monkeypox virus (MPXV) outbreak has spread worldwide since April 2022. Although the risk to the general public was previously considered low, the World Health Organization is now responding to this outbreak as a high priority to avoid further spread  were all negative for both persons. A real-time reverse transcription PCR, designed at the French National Reference Center of Orthopoxvirus, was performed on a pharyngeal swab sample from the patient on September 7 and tested positive for MPXV on September 8.The patient had been in a relationship for 9 months with her boyfriend and had only had vaginal and oral intercourse with him; the most recent intercourse was 8 days before her first symptoms. On August 25, 2022, after a vacation in the southwest of France, her boyfriend began experiencing fever, pimples on his penis, and swollen inguinal glands. His rashes disappeared on September 1. We have no additional information for him. The main transmission hypothesis for MPXV in this case is oral and vaginal sex with the patient’s boyfriend. She was not hospitalized but placed in confinement in her home. On October 12, the patient was seen at Foch Hospital for follow up, at which time the rashes were beginning to disappear .Most current MPXV outbreaks in Europe have involved young men who have sex with men who attend festivals and other public events  is contraindicated during pregnancy because it is a vaccinia virus (live virus); other vaccines, such as third-generation vaccines , are preferred in pregnant women (To date, only 10 cases in pregnancy have been reported worldwide, predominantly in Brazil and the United States (Additional information about monkeypox virus infection in 18-year-old woman after sexual intercourse, France, September 2022


The reported recurrence rate of endometrial polyps (EPs) after hysteroscopic polypectomy varied widely, and the factors influencing the recurrence of EPs are still controversial. Furthermore, the known definite independent risk factors are almost unchangeable, such as the number of EPs and previous polypectomy history. The aim of this study was to evaluate the impact of chronic endometritis (CE) on the recurrence of EPs in premenopausal women who underwent hysteroscopic polypectomy.A retrospective study was conducted at a university-affiliated hospital. Premenopausal women who underwent hysteroscopic polypectomy were enrolled, and those with definite confounding factors for polyp recurrence were excluded, including endometriosis and previous polypectomy history. A total of 233 women were enrolled in this study, including 64 (27.5%) cases with CE and 169 (72.5%) cases without CE. CE was diagnosed via immunohistochemical detection of CD138 on the endometrial specimen. Comparison of the recurrence rate of EPs was performed in women with or without CE at each monitoring stage  after hysteroscopic polypectomy.P = 0.001). Similarly, the recurrence rate of EPs was significantly higher in women with CE than in those without CE at each monitoring stage . CE and multiple EPs were risk factors for EPs recurrence. The HR for EPs recurrence in the EPs with CE cohort compared with the EPs without CE cohort was 3.06, after adjustment for the number of EPs.The recurrence rates of EPs at one year in patients with and without CE were 26.6%  and 9.5% (95% CI 5.0–14.0%), respectively, with an overall recurrence rate of 14.2% (95% CI 9.7–18.7%). The hazard ratio (HR) for EPs recurrence in the EPs with CE cohort versus the EPs without CE cohort was 3.08 (95% CI 1.56–6.09) (CE was a harmful factor for the recurrence of EPs in premenopausal women after hysteroscopic polypectomy. Thus, routine screening for CE during hysteroscopic polypectomy was needed. Frequent monitoring was needed for multiple EPs as the number of EPs also contributed to polyp recurrence. Endometrial polyps (EPs) represent a common benign gynecological condition that manifests via excessive local hyperplasia of the interstitial and endometrial glands. The prevalence of EPs ranges from 7.8 to 34.9% across different populations . AlthougCE involves persistent local inflammation of endometrium, characterized by the presence of plasma cells infiltrating the interstitium , 6. CE iEPs are associated with AUB, infertility and recurrent reproductive failure , 2, 15. In the retrospective study, we investigated whether CE increased the recurrence of EPs in premenopausal women after hysteroscopic polypectomy.Between January 2019 and January 2020, a retrospective study was conducted at the Affiliated Hospital of North Sichuan Medical College (China) for women who underwent hysteroscopic polypectomy. 35 patients without regular surveillance in 12 months were excluded. Finally, a total of 233 premenopausal women after hysteroscopic polypectomy were enrolled in this study, including 64 (27.5%) cases with CE and 169 (72.5%) cases without CE. The expression of CD138 in the endometrium was analyzed by immunohistochemistry to identify CE. Comparison of the recurrence rate of EPs was performed in women with and without CE at each monitoring stage .Patients were eligible for inclusion if they had enough endometrial specimen for immunohistochemical analysis of CD138 to identify CE, were premenopausal women (18–50 years) and had no severe systemic diseases. The exclusion criteria were pelvic inflammatory disease (PID) occurring in the 12 months after hysteroscopic polypectomy, previous endometrial polypectomy history, endometriosis, endometrial hyperplasia, and use of hormone therapy or antibiotics. This study was conducted in accordance with the principles of the Declaration of Helsinki.Hysteroscopic polypectomy was conducted in the follicular phase with a bipolar plasmakinetic resection system under intravenous anesthesia using a 3-mm 15° inside rigid hysteroscope and an 8.5-mm outside sheath . All operations were performed by the same physician (DC Qu). Endometrial samples were obtained visually using an unpowered plasma cutting ring from the upper uterine cavity, while hysteroscopic polypectomy was performed to remove local polyps. Samples of polyps and endometrium were detected for routine histologic analysis after hysteroscopic polypectomy, not only to confirm the diagnosis of EPs, but also to show whether there was malignant transformation or endometrial lesion.The diagnosis of CE should be made first by looking to the morphology of the field and the presence of plasma cell with or without lymphoid follicles. If the morphology is controversial, immunohistochemistry for the plasmacyte marker CD138 was needed. A histopathology consultant performed immunohistochemical detection of CD138 on the endometrial specimen slices as previously described , 21. FivWomen who underwent hysteroscopic polypectomy at our hysteroscopic center were surveilled routinely. Three dimensional colour-flow doppler transvaginal ultrasonography was performed to evaluate the uterine cavity condition at 3, 6, 9 and 12 months after hysteroscopic polypectomy. All ultrasound examinations were performed with a Voluson E8 ultrasound system and a 5 to 9 MHz endocavitary transducer .P value < 0.05 was considered to be statistically significant. After analyzing the distribution of the data and confirming that the age and body mass index (BMI) of the population were not normally distributed, we adopted the Mann–Whitney U test to analyze age and BMI expressed as median (interquartile range). Categoric variables were compared using the chi-square test. The chi-square test was used to compare the recurrence of EPs between each group at each stage. The Kaplan–Meier method with the log-rank test was used to estimate recurrence-free survival and to compare recurrence rates between the two groups. Univariate and multivariate Cox regression models were used to assess the factors associated with the recurrence of EPs. Factors for which P < 0.2 on univariate analyses were included in the subsequent multivariable model.For all statistical analyses, we used SPSS version 22.0 , and a P > 0.05).The prevalence of CE in the population was 27.5% (64/233). The demographic details and clinical features of the two groups of patients were shown in Table P = 0.001). The HR for EPs recurrence in the EPs with CE cohort versus the EPs without CE cohort was 3.08  and 9.5% (95% CI 5.0–14.0%), respectively, with an overall cure rate of 14.2% (95% CI 9.7–18.7%)  at 3 months, 15.6% (95% CI 6.8–24.4%) versus 4.1%  at 6 months, and 21.9% (95% CI 11.7–32.1%) versus 7.1%  at 9 months  versus 1.2% . Multivariate analysis confirmed that CE was a harmful factor for the recurrence of EPs . In addition, as previously mentioned [P = 0.040).Univariate and multivariate Cox regression models were used to assess the factors associated with the recurrence of EPs. Univariate analysis showed that the recurrence of EPs was not affected by age, BMI or polyp size, but significantly higher EPs recurrence rates were observed in women with multiple EPs and with CE compared with the rates in patients without the respective conditions . The HR for EPs recurrence in the multiple EPs cohort compared with the solitary EP cohort was 3.02, which is consistent with previous studies, showing a 3.45 HR with a high number of EPs [In the current study, to better evaluate the impact of CE on polyp recurrence, patients with definite external confounding factors for polyp recurrence, including endometriosis and previous polypectomy history, were excluded. Univariate analysis was used to determine the relationship between the recurrence of EPs and its potential risk factors, including age, BMI, polyp size, number of EPs and CE. According to the previous studies , 18, polr of EPs .There are some limitations in this study. For example, because this was a retrospective study, we could not take into account enough factors affecting the recurrence of EPs, and other concealed factors need to be explored. Furthermore, CE treated with antibiotics cohort was not included in our study, we do not know whether the effect of CE on the recurrence of EPs can be eliminated when treated with antibiotics.In conclusion, this study demonstrated that CE was a harmful factor for the recurrence of EPs in premenopausal women after hysteroscopic polypectomy. Thus, routine screening for CE during hysteroscopic polypectomy was needed. Frequent monitoring was needed for multiple EPs as the number of EPs also contributed to polyp recurrence. The role of CE in the recurrence of EPs and whether cured CE can provide prevention to eliminate the harmful effect need to be further confirmed by prospective trials with larger sample sizes.


Saccharomyces cerevisiae, whereas other viruses from the family Totiviridae infect a diverse group of organisms including protists, fungi, arthropods, and vertebrates. The presence of totiviruses alters the fitness of the host organisms, for example, by maintaining the killer system in yeast or increasing the virulence of Leishmania guyanensis. Despite the importance of totiviruses for their host survival, there is limited information about Totivirus structure and assembly. Here we used cryo-electron microscopy to determine the structure of L-BC virus to a resolution of 2.9 Å. The L-BC capsid is organized with icosahedral symmetry, with each asymmetric unit composed of two copies of the capsid protein. Decamers of capsid proteins are stabilized by domain swapping of the C-termini of subunits located around icosahedral fivefold axes. We show that capsids of 9% of particles in a purified L-BC sample were open and lacked one decamer of capsid proteins. The existence of the open particles together with domain swapping within a decamer provides evidence that Totiviridae capsids assemble from the decamers of capsid proteins. Furthermore, the open particles may be assembly intermediates that are prepared for the incorporation of the virus (+) strand RNA.L-BC virus persists in the budding yeast  A 2.9 Å resolution structure of the L-BC virus provides insight into the contacts between capsid proteins and the mechanism of capsid assembly. Totiviridae is a family of viruses with monopartite double-stranded RNA (dsRNA) genomes, which contain two open reading frames encoding the capsid protein and the RNA-dependent RNA-polymerase (RdRp). Members of this family infect fungi (genera Totivirus and Victorivirus) and unicellular protists . In addition, several as-yet unclassified totiviruses were identified in arthropods2 and vertebrates4. Most totiviruses belong to the ecological group of mycoviruses that persist as complete virions in the cytoplasm of infected cells and spread through cell division or contact with the cytoplasm5. In contrast, the arthropod totiviruses and Giardia lamblia virus were shown to be capable of extracellular transmission8.Saccharomyces cerevisiae virus L-A (L-A) and Saccharomyces cerevisiae virus L-BCLa (L-BC)9. Wild strains of yeasts occasionally also contain M particles, which carry the sole gene for a toxin-antitoxin system and ensure retention of the virus similarly to bacterial plasmid addiction systems11. The M-viruses do not encode capsid proteins and RdRp, but are maintained by the L-A helper virus. There are at least four types of M-viruses , each maintained by a specific L-A virus that competes with the others in a given yeast population12. This proverbial tug of war results in a co-evolution of L-A and M viruses13. The L-BC virus accompanies L-A in at least 50% of natural yeast populations and co-evolves with it14. However, it is possible to generate a L-BC-positive/L-A-negative yeast strain. In such a strain, the L-A-associated killer phenotype is lost, which indicates that L-BC cannot serve as a helper virus for the toxin-expressing M-virus15. Various stable L-A/L-BC combinations can be obtained by the cytoplasmic mixing of laboratory yeast strains, suggesting that there is no interdependency between naturally co-occurring L-A and L-BC viruses14.Two totiviruses are known to infect yeasts: Totiviridae and two arthropod totiviruses were determined23. The capsids of totiviruses are organized with icosahedral symmetry and have a diameter of 40 nm. The icosahedral asymmetric unit is formed by two copies of a capsid protein. The arrangement of a capsid with two subunits in an icosahedral asymmetric unit is a feature shared by many viruses with dsRNA genomes that infect bacteria and eukaryotes. Capsids of dsRNA viruses either consist of a single protein layer  or possess one or two additional layers with more complex structure (cystoviruses and reoviruses with the exception of a single-shelled Cypovirus)25. Structural studies revealed polymerase in the virions of several members of the families Reoviridae31 and Cystoviridae33. The incorporation of RdRp into a capsid is a crucial step in dsRNA virus assembly as it enables the replication and transcription of the viral genome inside the virion, avoiding the host antiviral response35. Open reading frames of capsids and RdRps of most totiviruses that infect protists and fungi overlap. The RdRp is expressed as a C-terminal extension of the capsid protein thanks to a ribosomal frameshifting sequence between the two genes39. In L-A virus, the efficiency of the ribosomal frameshifting is 1.9%, which results in the incorporation of two RdRps per virion on average40. A secondary structure at the 3′-end of the (+)RNA strand of the L-A virus genome is recognized by the RNA-binding domain of the RdRp43 and ensures genome encapsidation44.Capsid structures of six members of the family 45. Therefore, the virus RNA molecules are not recognized by the host translation factors and may be targeted for degradation by host cell RNA quality control systems46. Particles of L-A and L-BC were reported to de-cap cellular mRNAs, which may serve as decoys to overload cellular RNA-degradation machinery48. Furthermore, there is evidence that both L-A and L-BC have a unique cap-snatching mechanism that transfers eukaryotic cap 0 (m7G) to viral transcripts. First, m7G from a host mRNA is covalently bound to a histidine from a capsid protein and then transferred to a viral mRNA50.The RdRps of totiviruses produce RNA molecules that are not capped at the 5′-endsIn this study we describe the structure of the L-BC capsid, determined to a resolution of 2.9 Å. The putative mRNA decapping site in the capsid protein of L-BC is homologous to that of L-A virus. We describe domain swap stabilizing interactions between capsid proteins of L-BC that form a decamer. Furthermore, we identified an open particle of L-BC that lacks one decamer of capsid proteins. These observations indicate that the decamers of capsid proteins are the building blocks for the L-BC capsid. We discuss the stochastic incorporation of capsid-RdRp fusion protein into the L-BC capsid, and pinpoint the role of the −1 ribosome frameshifting site in successful virion assembly.51.Two-dimensional classification of particle images from a purified L-BC virus sample identified 87% empty particles, 9% open particles, and 4% genome-containing virions Fig. . The str21. In L-A virus, the largest interface between A and B subunits has a buried surface area of 1500 Å2, whereas the second largest interface has an area of 1400 Å221,. In contrast, in L-BC both of the corresponding interfaces have buried surface areas of 1350 Å2  of A subunits. As a result, an eight-stranded beta-sheet is formed Fig. . The conResidues 637–639 of A and B subunits form the strand βU which is the central part of an antiparallel beta-sheet that also includes strands βJ (res 387–389) and βK (res 412–414) Figs. , 5a. HowTrichomonas vaginalis virus 222. However, it is possible that decamers of capsid proteins of these viruses are stabilized by other structural features such as the “thumb” protrusions of Trichomonas vaginalis virus 222. Since L-BC virus has smaller interfaces between capsid proteins than L-A and Trichomonas vaginalis virus 2 (Supplementary Table Omono River Virus (unclassified Totivirus-like virus). Unlike in L-BC, the domain swap involves the C-terminus of the B subunit, which interacts with two neighboring A subunits23.The domain swaps between A subunits are not formed in L-A and 50. His156 of L-BC is located in a pocket on the outer surface of the L-BC capsid  caps of the host mRNAsThe two-dimensional classification of L-BC particle images identified a subpopulation of 9% of particles missing segments of their capsids Fig. . Asymmet53. Thus RdRp of L-BC is probably expressed as a C-terminal extension of the capsid by means of -1 ribosomal frameshifting facilitated by a putative slippery sequence GGATTTT , which is found in the overlap between the two open reading frames , and N is the number of capsid-RdRps per particle. The Eq.  used during particle vitrification. It was shown that exposing the L-A virus to 50 mM NaCl results in a 25% decrease in transcription activity54. In addition, this tendency of fungal dsRNA viruses to lose the genome has been described previously34. The low stability of dsRNA viruses that reside in the host cytoplasm may be due to their lack of need to protect their genomes in the extracellular environment, as is the case for viruses whose virions regularly infect cells.Assuming that the efficiency of ribosomal frameshifting is the same for L-A and L-BC viruses, 10% of particles should have no polymerase or genome. However, the purified L-BC sample contained 87% empty particles and only 4% virions  the formation of asymmetric AB dimers corresponding to the icosahedral asymmetric unit, (2) the formation of decamers from five AB dimers, and (3) the assembly of a capsid from 12 decamers. The conformation of the B subunit, with a βU strand forming a βJ-βU-βK sheet within the same chain, is probably the default arrangement of the capsid protein. The release of βU from its intramolecular βJ-βU-βK sheet may be induced by the interaction of the helix(ɑ2)-turn-helix(ɑ3) motif of the future B subunit with the residues 611–618 preceding the βU strand from the future A subunit upon the formation of AB dimers. The salt bridges between Asp90, Asp84 of B, and Arg616 of the A subunit Fig.  and a hyBluetongue virus (Reoviridae) subcore, which has the same type of symmetry as Totivirus capsids56. The formation of dimers and decamers of capsid proteins of Bluetongue virus was demonstrated experimentally by deleting the “dimerization domain” from the core protein, which prevented the association of decamers into subcore particles and promoted their accumulation in the cell57. Moreover, the subcore of Bluetongue virus, devoid of middle and outer shell layers, is transcriptionally active and infectious when introduced into the cell cytoplasm58, invoking parallels with totiviruses. Owing to their much more complicated virion structure, reoviruses require viroplasm, composed of non-structural proteins, to guide their particle assembly60. On the other hand, totiviruses are not known to code for any non-structural proteins, hence, their capsid assembly is likely to be stochastic.The proposed three-step assembly pathway is similar to that of the Birnaviridae with an asymmetric inner core61), pointing to their common evolutionary origin51. Therefore, the assembly pathway of the shell may also be conserved. So far, the existence of decamer intermediates has only been experimentally verified for the Bluetongue virus57. In addition, it was also predicted for Cytoplasmic polyhedrosis virus (Reoviridae) and Penicillium chrysogenum virus (Chrysoviridae) based on capsid stabilization by protrusion proteins62 and contacts between asymmetric units63. We present the evidence for the decamer as an assembly intermediate of L-BC virus (Totiviridae). The only possible exception for the “assembly-by-decamer” rule is Penicillium stoloniferum virus F from the family Partitiviridae, which was predicted to assemble from 30 diamond-shaped tetramers52.The capsid shell immediately surrounding the genome has a unique but universally conserved arrangement among all dsRNA viruses , received from prof. Elena Servienė , was grown at 30 °C in 1% yeast extract, 2% peptone, and 2% glucose medium for 48 h. Cells were harvested by centrifugation at 5000 × g for 5 min at 4 °C, resuspended in lysis buffer , and lysed by vigorous shaking with glass beads. Cell debris was removed by centrifugation at 10,000 × g at 4 °C for 30 min. L-BC particles were precipitated from the resulting supernatant by the addition of PEG-4000 to a final concentration of 5% (v/w), 500 mM NaCl, and incubation for 24 h at 4 °C. The precipitated L-BC particles were pelleted by centrifugation at 15,000 × g at 4 °C for 10 min. The pellet was re-suspended in the lysis buffer, dialyzed against the lysis buffer containing 50% glycerol, and stored at −20 °C. A centrifugal concentrator with a 100 kDa cut-off (Corning Spin-X UF) was used to remove glycerol, change the buffer to 20 mM Tris-HCl pH 7.5, 50 mM KCl, 10 mM MgCl2, and concentrate virus particles to 1.5 mg/ml. The L-BC virus sample (3.5 µl) was pipetted on a Quantifoil R 2/2 mesh holey carbon grid, blotted, and vitrified using a Vitrobot Mark IV . Double sample application was performed to increase the amount of virus particles in holes.The yeast strain BY4741 ski3∆  parameters were estimated using Gctf 1.0665. The dose-weighted sum of aligned frames was used for automated virus particle picking by crYOLO 1.7.566 with a custom trained model and a detection threshold of 0.5, resulting in a total of 72,705 particles. Particle coordinates were passed to RELION 3.1 for image processing68. The particles were extracted with a box size of 560 pixels and subjected to reference-free two-dimensional classification. The resulting best classes corresponding to virions (1748 images), empty , and open (3812 images) particles were processed in parallel. The initial model of the L-BC virus capsid was generated de novo by stochastic gradient descent using the dataset of empty particles. Empty particle images were 3D-classified and filtered by the maximum value of probability distribution  to obtain a homogeneous set of best-aligned particles . The RELION 3dautorefine procedure was used to reconstruct the full and empty particles with imposed icosahedral symmetry. To account for varying beam tilt during data acquisition, the particle images were split into nine optic groups according to the group of the beam-shifted acquisition area of the micrograph they originated from. Estimates of magnification anisotropy, beam tilt, and optical aberrations (up to 4th order) were done for each of the nine optic groups separately. Estimations of defocus parameters and b-factors were performed for individual particle images. The refinements were done iteratively (three rounds) following the scheme: 3dautorefine -> Ewald sphere correction -> masking -> CTF/aberration estimations. The resulting icosahedral reconstruction was postprocessed using the XMIPP local deblur tool (part of Scipion 2.0 package) which performs adaptive b-factor sharpening based on the local resolution of the map69. The final map resolution was determined using the FSC0.143 threshold criterion70.The dataset was acquired on a TFS Titan Krios operated at 300 kV, equipped with a Bioquantum 967 energy filter and Gatan K2 camera operating in counting mode. Each image was recorded as a movie over 6 s of exposure, resulting in a total dose of 36 e71.The 3D reconstruction of the open particle dataset  with fivefold symmetry (C5) yielded a map with weak density on one of the capsid protein decamers positioned on the fivefold symmetry axis , suggesting that the alignment of the missing decamers was imperfect. Subsequent masked 3D classification without alignment  focused on the region of the front decamer resulted in two distinct classes: 2692 particles with a front decamer clearly present and 1120 particles without a front decamer. Further local C5 refinement of the minor class produced an open particle with no density of the front decamer. Attempts to enrich the open particle class by running classifications focused on 11 other decamers or applying an iterative refinement-classification approach focused on the missing decamer were unsuccessful. The average of two half-maps from the final reconstruction was low-pass filtered to 16 Å and multiplied by the mask representing an icosahedral particle without a front decamer. The processed map was subsequently used to rigid-body fit previously refined A and B capsid proteins. The subunits around the hole were fitted using the program Chimera 1.13The particle orientations from the final C5 reconstruction were symmetry-expanded to C1 and subjected to C1 refinement with restricted searches for tilt- and psi-angles and full search for rot-angle using RELION 3.1. The symmetry-related variants of the particle orientation were then scored based on the goodness of fit to the resulting cryo-EM density map . For each particle image, the orientation with the highest score out of the five possible variants was retained. The restricted C1 refinement was repeated, starting from the best-selected particle orientations.72. The fit of the refined coordinates to the experimental density map was assessed and corrected manually using Coot 0.973. Several rounds of real-space refinements in Phenix 1.1974 and reciprocal-space refinements in Refmac575 were performed. The quality of the model was validated using MolProbity server76. Finally, to avoid clashes of residues between icosahedral asymmetric units, reciprocal-space refinement with 60 asymmetric units in the P23 map was performed using Refmac5 with the NCS constraints option. Structure visualization was performed in UCSF Chimera (1.15)71 and UCSF ChimeraX (1.2.5)77. The interfaces between capsid proteins were analyzed using the PISA EMBL-EBI web service78.The electron density map of an empty particle was cropped, normalized, rotated into 222 orientation, the map origin set to the center of the particle, and the crystallographic P23 space group applied. The initial model for the L-BC capsid protein was obtained from RaptorX Server80. Particles were randomly assigned the two subsets that were reconstructed independently.Cryo-EM reconstructions were performed according to the “gold-standard” approach used in structure determinationFurther information on research design is available in the Supplementary InformationReporting Summary


Dear Editor,In the middle of the COVID-19 pandemic, a rapidly emerging outbreak of a formerly existent yet neglected virus, monkeypox, has alarmed the world. A member of the family Poxviridae and the genus Orthopoxvirus, monkeypox virus (MPXV), is a brick-shaped, enveloped, double-stranded DNA virus closely related to now-extinct smallpox (variola virus) that previously plagued humans .In 1970, the Democratic Republic of the Congo reported the discovery of the first human MPXV infection, where it became endemic. Ever since then, the virus has spread to other African nations, primarily Central and West Africa . In 2003The virus predominantly transmits via close contact with contaminated objects or bodily fluids, such as during sexual activity or respiratory droplets. It can also transmit to health care providers who have had close, sustained contact with a patient . FollowiMonkeypox has been endemic in the African region for the past five decades. However, the fact that new cases are recently showing up outside Africa highlights the disease's geographical growth potential and its global importance. Owing to the ongoing COVID-19 pandemic, the healthcare resources are strapped, hence the possible pandemic risk carried by the virus should not be underestimated . It is eWith the current economic instability and the toll COVID-19 took on healthcare systems, many national health systems cannot afford pharmaceutical tools like vaccinations, diagnostic kits, and antivirals. Therefore, it is imperative to consider the beneficial outcomes of various interventions such as raising awareness, surveillance including self-reporting, and quarantining measures . AlthougNone.Externally peer reviewed, not commissioned.The following information is required for submission. Please note that failure to respond to these questions/statements will mean your submission will be returned. If you have nothing to declare in any of these categories then this should be stated.No conflicts of interest to declare.No funds were granted for this work.Not required.Written informed consent was obtained from the patient to publish this case report and any accompanying images.Not applicable.1.Name of the registry: Not required.2.Unique Identifying number or registration ID: Not applicable.3.Hyperlink to your specific registration (must be publicly accessible and will be checked):Irfan Ullah.The authors declare no conflict of interest.


We report on the experimental observationof a Tamm plasmon statein the near-IR region characterized by an anomalously high energylevel located in the upper half of the photonic band gap. Such a “blue”Tamm plasmon was demonstrated at the interface between a conventional,completely periodic Bragg reflector and a nanostructured nonresonantthin gold grating. We study the effect of the grating period on thecharacteristics of the anomalous state and show that the anomaly resultsfrom a nontrivial topology of the nanograting’s optical nearfield, which cannot be captured by the effective medium approach andtransfer matrix method commonly employed in the analysis of Tamm plasmons. We also show thatthe appearance of such a state cannot be described in the frame ofthe commonly employed transfer matrix method and the effective mediumapproach, suggesting that the latter has limited applicability tonanostructured nonresonant metal films.EB). It comprised 11 pairs ofNb2O5 (high index) and SiO2 (lowindex) layers with a thickness of, respectively, 159 and 246 nm, whichwere deposited onto a 1 in. diameter and 1 mm thick double-side polishedfused silica substrate using reactive magnetron sputtering, as detailedin ref (2O5 layer) was coated by a 50 nm thick film of Auvia thermal evaporation (see the Supporting Information). The deposition of Au film was carried out at room temperaturewith the rate of about 12 nm/min under a working pressure of 3.5 ×10–6 Torr using high-purity Au pellets (purity 99.999%).The nanograting was milled in a 30 μm × 30 μm largerpatch of the film using a focused ion beam with the area dosage setto 7 mC/cm2 and an ion current of 26 pA  equipped with a tungsten–halogen lightsource, a broad band linear polarizer, and a cooled near-IR CCD array.Light was focused onto the sample from the Au-coated side of the mirrorto a spot of about 1.5 mm in diameter using a 15× objective withNA = 0.28 and collected upon reflection using the same objective.The spectra of the nanograting were acquired for the polarizationof incident light set parallel (||) and orthogonal (⊥) to theslits, as indicated in 20 Also, its spectral line is broadened and additionally weakenedvia angular dispersion caused by focused illumination. A similar reflectivityspectrum is displayed by the dielectric mirror capped with the nanogratingfor incident light polarized along the slits (||), featuring a slightlystronger Tamm plasmon resonance, which is shifted to lower energiesby about 8 meV , the nanograting must have effectivelybehaved as a nonresonant continuous film with metallic response somewhatweaker than that of Au film it was milled in. Hence, intuitively,the phase change acquired by light reflected off the nanograting intothe dielectric mirror should be further away from the saturation (whichoccurs for a semi-infinite Au layer)25 andresulted in a Tamm plasmon state with the energy smaller than 836meV—just like in the other case.For the orthogonal polarization (⊥),the spectrum of thenanograting also indicates the appearance of a Tamm plasmon-like statebut at a substantially higher energy, namely, 906 meV, which exceedsthe Bragg energy by about 25 meV layer, which acted as a substrate and was extended to infinity tosimplify the extraction of the effective parameters in our model.The optical constants of Au were defined by the tabulated data;29 the refractive index of Nb2O5 was assumed to be constant across the entire energy bandand equal to 2.24.24 The effective opticalconstants of the nanograting were given by the values that enabledus to reproduce, using the standard thin-film formulae,30 the simulated transmittance and reflectanceof the nanograting with the accuracy exceeding 0.3% (see the Supporting Information).To further demonstratethe anomalous character of the observedhigh-energy (“blue”) state, we resorted to the well-establishedtransfer matrix method (TMM) commonly employed in the analysis ofTamm plasmons. Using TMM, we calculated (and compare below) the reflectivityspectra of the dielectric mirror covered by the nanograting for incidentlight polarized along and across the slits. In each case, the nanogratingwas defined as a 50 nm thick continuous film with the optical constantsgiven by the effective medium approach. To avoid limitations and shortfallsof the existing analytical effective medium models DM +φnG = 2π,31 where φDM and φnG are changes of the phase caused by reflectionsoff the dielectric mirror and nanograting (into the dielectric), respectively.The energy at which this equality holds can be found graphically asthe point of intersection between [2π – φDM(E)] and φnG(E) curves. The former is given by TMM, while the latter is readilyextracted from the thin formula after plugging in the effective opticalconstants of the nanograting. As evident from The reflectivityspectra of the dielectric mirror capped by thenanograting, as calculated using TMM, are shown in 23 the latter offers little physicalinsight into the exact nature of such peculiar optical states. Tounderstand it, we decided to rigorously model the interaction of theentire sample  with polarized light using a full-wave numerical solver implementedin COMSOL Multiphysics. The reflectivity spectra of the sample obtainednumerically are shown in DM(E) and φnG(E) were extracted directly from the simulateddata. Importantly, while the φnG(E) curve obtained numerically is identical to that calculated usingthe effective optical constants for light polarized along the slits,it appears quite different from what the effective medium approachpredicted for the orthogonal polarization (compare dotted and solidblue curves in nG(E)now features a negative rather than positive slope and is offset withinthe photonic band gap by about +π/2, which reveals the trueorigin of the observed anomaly—for light polarized across theslits, the nanograting appears optically closer to the dielectricmirror than it physically is.While anomalous Tamm plasmons have been predicted using Zak phaseformalism,32 Clearly, thetopology of the near field in nanostructured metal films cannot betaken into account  by effective medium modelsand TMM, which, therefore, naturally fail to predict the appearanceof Tamm plasmon states in the “forbidden” high-energyband.The explanation of such a peculiar “mirage” phenomenonis offered by 25 or by introducing an additional low-index layer.34 In those cases, however, the anomalous Tammplasmon will be equally affected and still appear more “blue”with its energy exceeding the upper limit characteristic of the opticalstate under a semi-infinite metal layer.We note that, in principle, conventional Tamm plasmonstates canbe pushed into the upper half of the photonic band gap in a modifiedBragg reflector either by making the top high-index layer physicallythinner than λ/4Given the microscopicorigin of the high-energy Tamm plasmon, whichinvolves the appearance of virtual corrugations below the nanograting,one may conclude that the energy level of this anomalous state mustdepend on the period of the nanograting. Indeed, for a smaller period,the corrugations would appear closer to each other effectively squeezingthe electric field out of the gaps between them (and into the mirror)and, hence, should yield an even higher-energy Tamm state. Correspondingly,for a larger period, the virtual corrugations would be spaced furtherapart allowing the electric field to fill the gaps between them andextend further toward the nanograting. The energy of the resultingTamm state should, therefore, approach that of a Tamm plasmon supportedby the continuous Au film. This is exactly what we observed with furtherexperiments involving two additional Au nanogratings see 4a,c. The20 The spectral locations of the Tamm states that we observed experimentallywith the shorter- and longer-period nanogratings also agree very wellwith the predictions of our full-wave numerical model (subject to10 meV blueshift due to focused illumination), as evident from Q-factorand compare that with the case of a conventional Tamm plasmon resonance.Since the lifetimes of Tamm states observed in an experiment couldbe affected by the accuracy of employed fabrication and characterizationtechniques, we based our analysis on the simulated data presentedin Q-factor ofthe anomalous Tamm state exhibits the opposite behavior and is seento increase from about 20 to 100, with the upper limit of 137 correspondingto the continuous Au film  Tamm plasmonexcited under the nanograting when its slits are parallel to the polarizationof incident light. Its resonances, however, have a smaller linewidthand, consequently, larger Q-factor. For example,the Q-factor of a Tamm plasmon state supported bythe nanograting with a 300 nm long period appears to be almost 3 timeslarger.It is also instructive to analyzehow the observed anomalous Tammstate evolves in terms of its linewidth and  Au film 5d. In faQ-factoron the nanograting period can be readily anticipated by taking intoaccount the leakage of light through the slits as an additional mechanismof radiative losses. Correspondingly, nanograting with a larger periodhas fewer slits and will, therefore, leak light at a slower rate . Furthermore, since the leakage of lightthrough a subwavelength slit is much weaker when the latter is parallelto the electric field, Tamm plasmon resonances will generally be narrowerand characterized by a larger Q-factor for lightpolarization set parallel to the slits of the nanograting.The noted dependencies of the linewidth and We observed experimentally and analyzed numerically the appearanceof an anomalous near-IR Tamm plasmon state, which emerged at the interfacebetween a conventional, completely periodic Bragg reflector and asubwavelength nonresonant gold nanograting. The observed state hadthe energy exceeding the theoretical limit achieved in the case ofa continuous semi-infinite gold layer and could not be predicted inthe frame of the effective medium approach and transfer matrix methodroutinely employed in the analysis of Tamm plasmons. We have foundthat the anomaly stemmed from a nontrivial topology of the near fieldproduced by the nanograting inside the Bragg reflector. Our findingsillustrate how the effective medium approach may have limited applicabilityeven in the case of nonresonant nanostructured metal films featuringthe most primitive structural patterns. The demonstration of the effectintroduces a new practical degree of freedom in controlling the spatiallocalization of Tamm plasmons and enables attaining the energies ofoptical Tamm states across the entire photonic band gap with minimalstructural alterations. Our findings also offer new insights intothe design of hybrid photonic cavities, identifying an important additionaldegree of freedom in controlling their optical length.Amongthe immediate applications of the observed anomalous Tammstate is polarization discrimination in reflection. Indeed, periodic1D nanopatterning of Au film rendered the metal-coated Bragg reflectoras an efficient polarization-sensitive mirror, which operated at normalincidence and had the extinction ratio of 1:10 at the wavelength ofanomalous Tamm plasmon resonance. Carving nanogratings in the metalcoating also allows one to easily adjust the energy of Tamm statewithin a wide spectral range and, therefore, constitutes a practicallyviable approach to tuning Tamm plasmons after fabrication. Moreover,the demonstrated approach enables spatial modulation of the energyof Tamm state along a metal-coated Bragg reflector, whereby an arrayof Tamm plasmon-based sensors or light emitters can be engineeredon the same chip, all operating at different wavelengths.


BackgroundThe copy number of the oligonucleotide 5’-purine-uridine-uridine-purine-uridine-3’ (purUUpurU) motif in a viral genome was previously shown to correlate with the severity of acute illness. This study aimed to determine whether purUUpurU content correlates with virulence in other single-strand RNA (ssRNA) viruses that vary in clinical severity.MethodologyWe determined the copy number of purUUpurU in the genomes of two subtypes of human respiratory syncytial virus (RSV), respiratory syncytial virus A (RSV-A), and respiratory syncytial virus B (RSV-B), which vary in clinical severity. In addition, we determined the purUUpurU content of the four ebolaviruses that cause human disease, dengue virus, rabies virus, human rhinovirus-A, poliovirus type 1, astrovirus, rubella, yellow fever virus, and measles virus. Viral nucleotide sequence files were downloaded from the National Center for Biotechnology Information (NCBI)/National Institutes of Health website. In addition, we determined the cumulative case fatality rate of 20 epidemics of the Ebola virus and compared it with that of the other human ebolaviruses.ResultsThe genomic purUUpurU content correlated with the severity of acute illness caused by both subtypes of RSV and human ebolaviruses. The lowest purUUpurU content was in the genome of the rubella virus, which causes mild disease.ConclusionsThe quantity of genomic purUUpurU is a virulence factor in ssRNA viruses. Blood hyperviscosity is one mechanism by which purUUpurU causes pathology. Comparative quantitative genomic analysis for purUUpurU will be helpful in estimating the risk posed by emergent ssRNA viruses. We recently reported that the number of copies of the oligonucleotide 5’-purine-uridine-uridine-purine-uridine-3’ (purUUpurU) within the genome of several single-strand RNA (ssRNA) viruses correlates with the potential severity of acute illness caused by several of those viruses . ssRNA vPurUUpurU activates innate immunity by binding to Toll-like receptor 8 (TLR8). Acting through the adaptor protein myeloid differentiation primary response 88 (MYD88) and nuclear factor kappa B (NF-κB), this causes the production of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6) . Both TNThis paper will report the findings of quantitative comparative genomic analysis for purUUpurU in several other viruses. First, we examine two subtypes of respiratory syncytial virus (RSV) that differ in the severity of the illness they cause. In our previous report, we noted a high copy number of purUUpurU in RSV, which can cause a fatal acute illness in infants and the elderly. There are two major subtypes of RSV, respiratory syncytial virus A (RSV-A) and respiratory syncytial virus B (RSV-B). These differ in the severity of illness they cause, with RSV-A causing more severe disease -9. Thus,Next, we will examine the species of ebola virus that cause disease in humans. Epidemics of these viruses, namely, Ebola virus (EBOV), Sudan virus (SUDV), Bundibugyo virus (BDBV), and Taï Forest virus (TAFV), differ in their case fatality rate. Thus, they provide another opportunity to test the pathogenicity of purUUpurU. Finally, we will also report the results of quantitative comparative genomic analysis of other viruses that cause acute illnesses of varying severity, namely, yellow fever virus (YFV), rabies virus, measles virus, human rhinovirus A (HRV-A), astrovirus, rubella, dengue virus, and poliovirus 1.https://www.ncbi.nlm.nih.gov/labs/virus/) in fasta format. All complete  nucleotide sequences cumulatively available at the time of analysis were studied. Several curations were performed to remove incomplete sequences. All data analyses were conducted in Python programming language (Python3.7) and Bio.Seq, SeqIO, motifs modules of Biopython toolbox (https://biopython.org/).Viral nucleotide sequence files were downloaded from the National Center for Biotechnology Information (NCBI)/National Institutes of Health website (The purUUpurU motifs were counted in the following manner: because purine bases are either adenine (A) or guanine (G), the possible purUUpurU motifs are AUUAU, AUUGU, GUUGU, and GUUAU. The complementary DNA sequences are ATTAT, ATTGT, GTTGT, and GTTAT. The strand present in viral particles  was used for analysis.If two motifs overlapped, both motifs were tracked. The results report the non-overlapping motif counts with the incidence rate of the overlap. For instance, AUUGUUGU would be detected as two motifs, AUUGU and GUUGU, but there is one non-overlapping motif. The following viruses were examined: RSV-A, RSV-B, BDBV, SUDV, TAFV, EBOV, measles virus, HRV-A, dengue virus, rabies virus, astrovirus, rubella, poliovirus type 1, and YFV.Definitive outcome data from the West Africa 2013-2016 EBOV epidemic were used to calculate the fatality of that epidemic. Those data were taken from a published source and collected by the World Health Organization (WHO) during the earliest phase of that epidemic, from December 2013 to September 2014 . FatalitThe mean number of purUUpurU and genome size  were calculated. The coefficient of variation (CV) of both was also calculated. Pearson’s correlation coefficient was used to determine the correlation between genome size and purUUpurU count. This statistic was performed using Microsoft Excel . The Mann-Whitney U test was used to determine the significance of the difference between the purUUpurU counts of RSV-A and RSV-B and EBOV and BDVB. A chi-square test was used to determine whether there was a significant difference between the case fatality rates of EBOV and SUDV. These calculations were performed with the Python statistics package (SciPy.stats from SciPy 1.5.2).Results of the purUUpurU counting are presented in Figure The purUUpurU count was a strong discriminant between the RSV-A and RSV-B Figure . The genFigure The mean purUUpurU count decreased in the following order: TAFV > EBOV > BDBV > SUDV. Case fatality rates were as follows: EBOV, 69.2% , SUDV, 53.7% , BDBV, 32.0% , and TAFV, 0 deaths (one case). Using these data, the case fatality rate for the disease caused by EBOV was higher than for the disease caused by SUDV .The percentages of overlapping motifs ranged from 0.1% (rubella virus) up to 9.0% (poliovirus 1)  . Based oNevertheless, very similar figures are cited in the literature -18. ThisElucidation of genomic purUUpurU content as a virulence factor provides a biological basis for a higher case fatality rate in epidemics caused by EBOV. Further, a case fatality rate of 39.5% is lower than any other epidemic caused by EBOV. Thus, we regard data from the definitive outcomes of smaller outbreaks and the beginning of the West Africa 2013-2016 outbreak to be the most reliable and that EBOV is the most virulent ebolavirus Figure .Dengue virusThe moderate copy number of purUUpurU in the genome of the dengue virus may seem surprising given its association with severe diseases such as “break-bone fever” and viral hemorrhagic disease. However, of the estimated 390 million annual dengue virus infections, 293.9 million are estimated to be clinically inapparent . Severe Nonspecific viral syndromesAt both ends of the spectrum, in many cases, the genomic purUUpurU content accounts for the potential severity of acute illness caused by the virus. SARS-CoV-2 has 247 copies of purUUpurU in its genome  and can Significance of PurUUpurUPurUUpurU can be viewed as a viral equivalent of N-formylmethionine, a product of bacterial metabolism that activates innate immunity. It is a driver of the cytokines that cause the symptoms seen in the initial nonspecific phase of many viral illnesses. If the infection progresses, continued production of cytokines driven by purUUpurU can lead to elevated blood viscosity with subsequent organ dysfunction due to reduced tissue perfusion, as seen in the disease caused by RSV and ebolaviruses. This outcome is likelier if the host interferon response is deficient. In infections caused by viruses with the greatest purUUpurU content, inflammation driven by that oligonucleotide can lead to marked elevations of cytokines, hyperviscosity syndrome, and thromboinflammation. The high copy number of purUUpurU in SARS-CoV-2 suggests that the cytokine storm seen in COVID-19 is caused by the virus, not dysregulation of the host immune system. This may also be the case in the disease caused by EBOV. Thus, the purUUpurU content of a viral genome influences the inflammatory potential of that virus.Quantitative comparative genomic analysis has contributed to the understanding of how much of the variation in a clinical outcome is determined by the virus and how much is due to variation in the host immune response. PurUUpurU data suggest that viruses can cause cytokine storm syndrome in a normally functioning immune system. Quantitative comparative genomic analysis has revealed that the genomic purUUpurU content is an important characteristic of any ssRNA virus. A 2007 publication reported that 49 of the 87 new human pathogens identified since 1980 are RNA viruses . Pri. Pri1]. A meta-analysis of inflammatory markers in COVID-19 showed that in critically ill patients, acute phase reactants and related biomarkers are elevated to a greater degree than cytokines. The authors questioned the relevance of cytokine storm in organ dysfunction in COVID-19. Instead, the degree of elevation of D-dimer, the degradation product of cross-linked fibrin, was the strongest discriminant between critically ill COVID-19 patients and those with sepsis . IncreasAlong with impaired myocardial contractility and capillary leak, blood hyperviscosity should be recognized as a severe adverse effect of marked cytokine elevations. All of these factors decrease tissue perfusion. Cytokine elevations change from being beneficial to pathologic when they cause decreased tissue perfusion in the host.The quantity of genomic purUUpurU is a virulence factor in ssRNA viruses. Blood hyperviscosity is one mechanism by which purUUpurU causes pathology. Comparative quantitative genomic analysis for purUUpurU will be helpful in estimating the risk posed by emergent ssRNA viruses.


A new virus, named Langya henipavirus (LayV), has recently been identified in Shandong and Henan provinces in China and has so far infected 35 individuals between April 2018 and August 2021. It is closely related to other known henipaviruses (Nipah and Hendra viruses) that can cause up to 70% human case fatality. Even though LayV has not been shown to be fatal in humans and does not appear to be transmitted from human-to-human, it is an RNA virus with the capacity to evolve genetically in the infected hosts (e.g. shrews) and can infect humans (e.g. farmers who have been in close contacts with shrews). It is therefore important to be vigilant about this new viral outbreak. Paramyxoviridae  released by virus-infected and/or uninfected cells ,4, if it