N-isopropylacrylamide (NIPAAm)-co-2-acrylamido-2-methylpropanesulfonic acid (AMPS)-co-N-phenyl acrylamide (PhAAm)), P(NIPAAm-co-AMPS-co-n-butyl methacrylate (BMA)), and P(NIPAAm-co-AMPS-co-tert-butylacrylamide (tBAAm)), were modified on silica beads through atom transfer radical polymerisation. Rituximab elution profiles were observed using the prepared beads-packed column. Rituximab adsorption at high temperature and elution at low temperature from the column were observed, as a result of the temperature-modulated electrostatic and hydrophobic interactions. Using the column, rituximab purification from contaminants was performed simply by changing the temperature. Additionally, three types of antibody drugs were separated using the column through temperature-modulated hydrophobic and electrostatic interactions. These results demonstrate that the temperature-responsive column can be applied for the separation and analysis of biopharmaceuticals through a simple control of the column temperature.Antibody drugs play an important role in biopharmaceuticals, because of the specificity for target biomolecules and reduction of side effects. Thus, separation and analysis techniques for these antibody drugs have increased in importance. In the present study, we develop functional chromatography matrices for antibody drug separation and analysis. Three types of polymers, poly( Antibody drugs play an important role in biopharmaceuticals, because of the specificity for target biomolecules and reduction of side effects3. Thus, separation and analysis techniques for these antibody drugs have increased in importance. Various types of separation and analysis techniques have been developed. Affinity chromatography using protein A ligand is a powerful tool for this purpose, because of the effective affinity between protein A and antibodies5. However, antibody elution from the columns is performed using a lower pH aqueous solution. The elution process includes the possibility of losing the activity of the antibody drugs and contamination owing to the elution of protein A. Thus, an antibody drug separation column that uses different retention and elution mechanisms from protein A is demanded.Recently, biopharmaceuticals have become effective medical treatments for refractory disease9. The chromatography column uses a thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAAm). This polymer exhibits temperature-dependent hydrophilic and hydrophobic changes across the lower critical solution temperature (LCST) of 32 °C, owing to hydration and dehydration10. This temperature responsive property has been widely used in biomedical fields13, such as drug and gene delivery systems15, biosensors and diagnostic devices18, and cell culture dishes for tissue engineering and regenerative medicine23. In the application of PNIPAAm to chromatography for temperature-responsive chromatography, PNIPAAm-modified beads are used as the chromatography column27. The chromatography column can modulate the retention of analytes by changing the column temperature, because the surface hydrophobicity of the beads is changed with the hydration and dehydration of the modified PNIPAAm, which leads to the modulation of the hydrophobic interaction with analytes.Temperature-responsive chromatography is one of the candidates as an antibody separation technique, because the chromatography system can modulate the retention and elution of analytes by changing the temperature34, because bioactive compounds and proteins have an ionic property and the use of electrostatic interactions is an effective approach for separation. Temperature-responsive ion exchange chromatography uses thermoresponsive ionic copolymer modified beads as the packing material. The electrostatic interaction can be modulated by changing the column temperature. The thermoresponsive ionic copolymer is prepared by copolymerisation with an ionic monomer and NIPAAm. Furthermore, a hydrophobic monomer is incorporated to adjust the lower critical solution temperature, because the ionic monomer provides hydrophilicity to the polymer, which leads to an increase of the LCST of the polymer37. The main driving force of temperature-responsive ion exchange chromatography is the electrostatic interaction. Thus, various types of thermoresponsive ionic polymers have been investigated as ligands in thermoresponsive ion exchange chromatography by changing the incorporated ionic monomer39. Using the silica beads modified with the thermoresponsive ionic polymer P(NIPAAm-co-acrylic acid (AAc)-co-n-butyl methacrylate (BMA)) as the packing material of the solid phase extraction column, the antibody drug rituximab was separated from a contaminant40. However, in this attempt, AAc was used as the ionic monomer. AAc has a carboxyl group, which is a weakly acidic group that leads to a weak electrostatic interaction with the targeted analytes. Thus, if a thermoresponsive polymer with a strongly acidic group is used in the packing material, a more effective antibody drug separation column would be developed.Additionally, temperature-responsive ion exchange chromatography has been investigated for the separation and analysis of ionic biomolecules and pharmaceutical proteins through temperature-modulated electrostatic interaction with analytes41. However, depending on the property of the analytes, a hydrophobic interaction with the analytes can also contribute to the retention of the analyte37. Thus, if the hydrophobic interaction in addition to the electrostatic interaction with analytes are optimised, the separation efficiency of the temperature-responsive ion exchange column will increase remarkably, which will lead to the development of an effective antibody drug separation column.Among the strongly acidic monomers, 2-acrylamido-2-methylpropanesulfonic acid (AMPS) has been shown to be an effective ionic co-monomer owing to its strong acidic propertytert-butylacrylamide (tBAAm), n-butyl methacrylate (BMA), and N-phenylacrylamide (PhAAm), for the effective separation of an antibody  and P(NIPAAm-co-AMPS-co-BMA) have not been investigated as a ligand for temperature-responsive ion exchange chromatography. Although P(NIPAAm-co-AMPS-co-tBAAm) has already been investigated as a temperature responsive chromatography material for the adsorption of lysozyme and α-chymotrypsinogen A, antibody separation has not been investigated using polymer modified beads39.In the present study, to optimise the electrostatic and hydrophobic interactions with the antibody, we have prepared three types of thermoresponsive anionic polymer modified silica beads having the acidic AMPS monomer and three types of hydrophobic monomers, ody Fig. . Among tThe electrostatic and hydrophobic interactions between the prepared beads and antibody drug are investigated by the elution behaviour of the antibody drug from the prepared beads-packed column. From the results, an effective temperature-modulated antibody drug separation column is developed. The developed column is able to separate the antibody without losing activity or using protein A ligand, which cause deactivation of the antibody and contamination of the products.39. The compositions of BMA and tBAAm were set at 5 mol% and 20 mol%, respectively, to modulate the proper phase transition temperature according to the previous reports42. The composition of PhAAm was not determined, since the copolymer was not investigated. Thus, we set the composition of PhAAm to 1 mol% in the first preparation. The hydrophobicity, logP, of the hydrophobic monomers, PhAAm, BMA, and tBAAm, were calculated using Crippen’s fragmentation43, and these values were 1.63 for PhAAm, 2.23 for BMA, and 0.84 for tBAAm (Supplementary Table co-AMPS-co-PhAAm) was relatively high. Thus, we prepared P(NIPAAm-co-AMPS-co-PhAAm) including 2 mol% and 3 mol% of PhAAm to lower the phase transition temperature. However, these polymers were insoluble in a phosphate buffer solution even at low temperature  prevented the hydration of the polymer. The results indicated that the composition of PhAAm in the copolymer could not be increased to above 1 mol%.Unbound thermoresponsive polymers with various hydrophobic co-monomers and the same composition of polymers for modifying the silica beads were prepared to investigate the phase transition property Fig. A. The mo2, which was almost the same value as for silane modification on silica beads44. The amount of polymer modification ranged from 1.83 to 2.47 mg/m2, which indicated that a relatively large amount of polymer was in the form of successfully-modified silica beads, which contributed to a densely packed polymer brush conformation45. P(NIPAAm-co-AMPS-co-tBAAm)-modified beads exhibited a relatively small amount of polymer modification compared with those of P(NIPAAm-co-AMPS-co-PhAAm)-modified beads and P(NIPAAm-co-AMPS-co-BMA)-modified beads. This was proposed to arise from the reactivity of each hydrophobic monomer and the difference in monomer composition. Acrylamide monomers, such as PhAAm and tBAAm, have a low reactivity in the ATRP reaction compared with methacrylate monomers, such as BMA. Additionally, the reactivity of tBAAm would be low compared with NIPAAm, because of the larger monomer size of tBAAm compared with NIPAAm. Furthermore, the tBAAm composition was 20 mol%, which was quite high. Thus, the amount of P(NIPAAm-co-AMPS-co-tBAAm)-modified beads was relatively small compared with the other beads.Atom transfer radical polymerisation (ATRP)-initiator-modified silica beads were prepared by a silanization reaction of [(chloromethyl)phenylethyl] trimethoxysilane (CPTMS), and the polymer was modified on silica beads through ATRP Fig. A. The pr46. Thus, to investigate the difference in the molecular weight of the grafted thermoresponsive polymers on beads, unbound thermoresponsive polymers were prepared by the same polymerization condition, except that an unbound ATRP initiator, α-chloro-p-xylene, was used in place of the ATRP-initiator, CPTMS, modified beads. The molecular weight of the synthesized polymer was measured by a gel permeation chromatography (GPC) system. There was no significant difference in the molecular weight of the three polymers, P(NIPAAm-co-AMPS-co-PhAAm), P(NIPAAm-co-AMPS-co-BMA), and P(NIPAAm-co-AMPS-co-tBAAm)  measurements Fig. B. An addThe morphology of the beads was observed by field-emission scanning electron microscopy , the values were decreased after the thermoresponsive anionic polymer modification. This is because the thermoresponsive polymer was modified inside the pore of the beads through the ATRP reaction, and the polymer filled the pore, which led to a reduction of the surface area.The surface area of the beads was measured by nitrogen adsorption , and dexamethasone (logP = 1.83). This indicated that the retention of steroids on the column resulted from a hydrophobic interaction. The retention time increased with increasing the column temperature, and separation was performed at higher temperatures. This was because the thermoresponsive anionic polymers on the beads became dehydrated and interacted with the steroids through a hydrophobic interaction as the temperature was increased. Among the three types of beads, the P(NIPAAm-co-AMPS-co-BMA) beads-packed column exhibited the largest retention time and relatively good separation compared with the other columns. This indicated that the P(NIPAAm-co-AMPS-co-BMA) beads exhibited a relatively strong hydrophobicity compared with those columns packed with the other polymers. BMA exhibited the greatest hydrophobicity (logP = 2.23) compared with PhAAm (logP = 1.63) and tBAAm (logP = 0.84). Thus, the P(NIPAAm-co-AMPS-co-BMA) beads-packed column exhibited longer retention time and better separation of the steroids mixture compared with those obtained with the P(NIPAAm-co-AMPS-co-PhAAm)- and P(NIPAAm-co-AMPS-co-tBAAm)-modified beads, because of the strong hydrophobicity of BMA and the hydrophobic interaction with analytes. In the comparison of the P(NIPAAm-co-AMPS-co-BMA) beads with the P(NIPAAm-co-AMPS-co-PhAAm) beads, P(NIPAAm-co-AMPS-co-BMA) contains a larger hydrophobic monomer unit, BMA 5 mol%, compared with PhAAm 1 mol% of P(NIPAAm-co-AMPS-co-PhAAm). Also, BMA has higher hydrophobicity compared with PhAAm. Thus, P(NIPAAm-co-AMPS-co-BMA) has a higher hydrophobicity compared with P(NIPAAm-co-AMPS-co-PhAAm). In contrast, by comparison of P(NIPAAm-co-AMPS-co-BMA) and P(NIPAAm-co-AMPS-co-tBAAm), P(NIPAAm-co-AMPS-co-tBAAm) has quite a large hydrophobic monomer composition, 20 mol% of tBAAm, compared with P(NIPAAm-co-AMPS-co-BMA), 5 mol% of BMA. However, P(NIPAAm-co-AMPS-co-BMA)-modified beads exhibited a strong retention of hydrophobic steroids. This was proposed to arise from the difference in the basic monomer structures between BMA and tBAAm. BMA and tBAAm have a methacrylate and acrylamide monomer structure, respectively. The methacrylate monomer has a more hydrophobic structure compared with acrylamide. Thus, P(NIPAAm-co-AMPS-co-BMA) exhibited a strong hydrophobic interaction with steroids, which led to a longer retention time.The prepared polymer-modified beads were packed into a stainless steel column, and the packed column was connected to a high performance liquid chromatography (HPLC) system. To investigate the hydrophobicity of the prepared beads, the elution behaviour of hydrophobic steroids from the columns was observed at various temperatures Fig. . The proL-alanine (DOPA) and adrenalin  and adrenalin (pKa = 9.8–9.9)). Thus, the retention behaviour of these catecholamines predicted the electrostatic interaction between the basic analytes and the modified thermoresponsive anionic polymers on the beads28. The catecholamines eluted from the column in the order of the basicity, DOPA (pKa = 8.72) and adrenalin (pKa = 9.8–9.9). This indicated that these analytes were mainly retained through the electrostatic interaction between the sulfonic acid of AMPS and the analytes. Also, the two catecholamines were separated on all columns, which indicated that all columns exhibited a sufficient electrostatic interaction for the separation of the analytes. The retention time of adrenaline was slightly increased with an increase of the temperature. This was attributed to the increased anionic property with increasing temperature, as predicted from the zeta potential of the beads -co-tBAAm)-modified beads-packed column, the catecholamines were retained on the column through electrostatic and hydrophobic interactions, although the electrostatic interaction was the main driving force37. Thus, in the case of the columns used in this study, the catecholamines were also proposed to be retained on the column through electrostatic and hydrophobic interactions. Thus, the retention time of adrenaline increased with an increase of the temperature, because the modified polymers on the beads became hydrophobic upon increasing the temperature. Additionally, the increase in the retention time of adrenaline was most obvious with P(NIPAAm-co-AMPS-co-BMA). This was proposed to arise from the BMA monomer unit included in the polymer inducing a strong hydrophobic interaction with the analytes, the same as the retention of hydrophobic steroids.Additionally, the anionic property of the prepared beads was investigated by observing the elution behaviour of catecholamines, 3--50. Thus, rituximab was adsorbed through electrostatic interactions with the thermoresponsive anionic polymers on the beads in the column. Additionally, rituximab was adsorbed only at high temperature, which indicated that the hydrophobic interaction attributed to the dehydration of polymers at higher temperature also contributed to the adsorption of rituximab. Thus, rituximab was adsorbed on the thermoresponsive anionic polymer on the beads through both electrostatic and hydrophobic interactions. We investigated the effect of the ionic strength of the mobile phase on the adsorption of rituximab -modified bead-packed column exhibited a clear temperature-modulated adsorption and elution of rituximab. This was attributed to the effective hydrophobic interaction with the BMA-incorporated polymer similar to the retention of steroids and catecholamines. Thus, P(NIPAAm-co-AMPS-co-BMA)-modified beads are suitable for the retention of rituximab.Additionally, by comparing the three columns, the P(NIPAAm-co-AMPS-co-BMA) copolymer are important factors, because there is an optimal balance between the electrostatic and hydrophobic interactions for an effective interaction with an antibody that are attributed to AMPS and BMA, respectively. In a previous report, the optimal AMPS composition in a thermoresponsive polymer was investigated by changing the AMPS composition in the thermoresponsive polymer from 0 to 10 mol%39. As a result, 5 mol% of AMPS was a suitable composition for an effective interaction with proteins, because a higher AMPS composition above 5 mol% reduced the dehydration property of the thermoresponsive polymer with increasing temperature, which was attributed to the strong hydrophilic property of the copolymer39. Additionally, the optimal BMA composition in the thermoresponsive polymer for temperature-responsive chromatography was investigated in a previous study52. In these investigations, the retention of analytes increased with increasing the composition of BMA52. However, a BMA composition above 5 mol% reduced the hydration property of the thermoresponsive polymer, because of the excessive hydrophobicity of the incorporated BMA in the polymer42. These previous reports suggested that the composition of 5 mol% AMPS and 5 mol% BMA in the copolymer, which was used in this study, is suitable for the effective interaction with proteins.The composition of AMPS and BMA in the P(NIPAAm-co-AMPS-co-BMA)-modified beads-packed column, rituximab separation was performed by a step temperature gradient  analysis indicated that the elution fractions at 40 °C and 10 °C contained albumin and rituximab, respectively. Two bands were observed in the lane of the rituximab, which arose from the degradation of rituximab into a light chain and heavy chain through the SDS-PAGE analysis. This indicated that the mixture sample of rituximab and albumin can be separated simply by changing the column temperature.Furthermore, for the mixture of rituximab and hybridoma cell culture medium, the mixture sample was injected into the column at 40 °C, and rituximab was adsorbed and the component of the hydridoma cell culture medium was eluted. Then, the column-temperature was reduced to 10 °C, and the adsorbed rituximab was eluted from the column. SDS-PAGE analysis indicated that the fraction eluted at 40 °C did not contain rituximab and that at 10 °C contained rituximab. The results indicated that rituximab purification from the cell culture medium can be performed simply by changing the column temperature.In these experiments, albumin and the component of hybrydoma cell culture medium were eluted at probably the same retention time, which indicates that albumin and the component of hybrydoma cell culture medium would not be separated. However, the separation of albumin and the component of hybrydoma cell culture medium is not necessary for the actual antibody purification process. In the production process, the antibody and albumin are dissolved in hybrydoma cell culture medium. In the purification process, the antibody should be separated from the albumin and the component of hybridoma cell culture medium. Thus, the separation of albumin and the component of hybrydoma cell culture medium is not important for the application of antibody purification.The activity of the eluted rituximab from the column was evaluated by observing the reactivity with CD20-positive human B cell leukaemia cells, because rituximab is an anticancer drug that recognizes CD20-positive cells. The rituximab activity with B cell leukaemia cells was maintained after column elution compared with that before column injection  modified beads-packed column exhibited an effective retention of analytes, which was attributed to the strong hydrophobic interaction. To investigate the suitability of the prepared column to a biopharmaceuticals application, the elution behaviour of rituximab was observed. Rituximab was adsorbed on the column at high temperature, while rituximab was eluted from the column at low temperature. The P(NIPAAm-co-AMPS-co-BMA) modified beads-packed column exhibited the most effective adsorption and elution of rituximab among the three types of columns. Using the temperature-dependent adsorption and desorption properties, rituximab was purified from contaminants. Furthermore, three types of antibody drugs were separated using the column simply by changing the temperature. These results demonstrate that the prepared thermoresponsive anionic copolymer modified beads-packed column will be useful for bioseparation and bioanalysis for biopharmaceutics simply by changing the column temperature.Three types of thermoresponsive anionic copolymer brush modified beads were developed as packing materials for functional temperature-responsive chromatography columns for functional antibody drug separation. To develop the thermoresponsive anionic copolymer brush modified beads, AMPS was used as a strong anionic monomer, and the hydrophobic monomers, PhAAm, BMA, and tBAAm, were used as a hydrophobic monomer. The electrostatic and hydrophobic properties of the prepared beads were investigated by observing the elution behaviour of hydrophobic steroids and catecholamines from the beads-packed column. Among the three types of columns, the P, and Me6TREN  were added into the solution with continuous argon gas bubbling. The flask was sealed after filling with argon gas. The CPTMS beads (1.0 g) were put into a glass vessel. The ATRP reaction solution in a flask and the CPTMS beads in a glass vessel were placed into a glove bag. Oxygen in the glove bag was removed by a cycle of vacuuming and flowing argon. The ATRP reaction solution was added to the CPTMS beads in the glass vessel. The glass vessel was sealed. The ATRP reaction proceeded with shaking of the glass vessel for 16 h at 25 °C. After the reaction, the beads were washed with acetone, EDTA aqueous solution, methanol, and water, followed by drying in reduced pressure.The polymer-modified beads were prepared by ATRP of the thermoresponsive monomer, anionic monomer, and three types of hydrophobic monomers. The anionic monomer AMPS  was dissolved in 20 mL of water. Then, the AMPS solution was neutralised by adding NaOH aqueous solution, and the total volume was adjusted to 50 mL. To prepare the monomer solution containing PhAAm as a hydrophobic monomer, NIPAAm  and PhAAm  were dissolved in 2-propanol (38.5 mL) in a flask. The prepared AMPS solution (2.14 mL) and water (2.14 mL) were added to the solution. To prepare the BMA-containing monomer solution, NIPAAm  and BMA  were used. To prepare the tBAAm-containing monomer solution, NIPAAm  and tBAAm  were used. The prepared monomer solutions were deoxygenated by flowing argon for 15 min. Then, CuCl , CuClp-xylene  was used in place of the CPTMS-modified silica beads. The synthesised polymer was purified through dialysis using a cellulose dialysis tube (1 kDa MWCO).To characterise the thermoresponsive polymer, an unbounded polymer was synthesised using the same procedure except that α-chloro-The prepared polymer-modified silica beads were characterised by CHN elemental analysis, FTIR, scanning electron microscopy (SEM) observation, zeta potential observation, and observation of the transmittance change of the polymer solution.CI is the increase in the percentage of carbon in the ATRP-initiator modified beads compared with the unmodified beads following a silane coupling reaction; %CI  is the calculated percentage of carbon in the initiator, and S is the surface area of the beads (100 m2 g−1). The amount of polymer on the silica beads was obtained using the equation:Cp is the increase in the percentage of carbon in the polymer-modified beads compared with the initiator-modified beads. %CP  is the calculated percentage of carbon in the initiator.The amount of ATRP-initiator and polymers on the silica beads was estimated by the carbon content obtained from CHN elemental analysis . The immobilised initiator was obtained from the equation:The morphology of the beads at each reaction step was observed by FE-SEM .Polymer modification onto the silica beads was confirmed using an FTIR spectrometer .Surface area and pore diameter of the beads were measured by nitrogen adsorption using a surface area analyser .The zeta potential and diameter of the polymer-modified beads were measured using a zeta potential analyser . The polymer-modified beads were suspended in a 5 mM potassium chloride solution. The zeta potential of the beads was observed from 10 to 50 °C.The polymer phase transition was observed by the temperature-dependent optical transmittance change of the polymer solution. A polymer solution (0.5 wt%/v) was prepared at pH 7.0, using a 33.3 mM phosphate buffer solution.−2 for 1 h. The elution behaviour of the analyte from the beads-packed column was observed on an HPLC system . The properties of the analytes are summarised in Supplementary Tables −1. Elution of steroids and catecholamines was detected at 254 nm. Elution of rituximab was detected at 280 nm. Column temperature was controlled by an equipped column oven in the HPLC system. The eluted protein samples at each fraction were analysed by SDS-PAGE.Prepared beads were packed into stainless steel columns (4.6 mm in diameter and 50 mm in length). For three types of antibody separation, a column that was 4.6 mm in diameter and 150 mm in length was used. Prepared beads were dispersed into a water and methanol (1:1) mixture solution. The dispersion was added into a column packer connected to a column. Packing of the beads was performed by flowing a water and methanol (1:1) mixture solution into the packer at a constant pressure of 350 kg cmThe eluted rituximab activity was evaluated by flow cytometry. Rituximab eluted from the column was incubated with CD20-positive BALL-1 cells. Then, PE mouse anti-human IgG was reacted with the rituximab on CD20-positive cells as a secondary antibody. The amount of CD20-positive cells with PE was evaluated by a flow cytometer .Supplementary Information.


Culex sp. mosquitoes in 2018. The virus was successfully isolated, and complete genome sequence was acquired. The whole genome of the WNV was also sequenced from the patient’s urine sample. The whole genome sequences of the WNV virus detected in Slovenian patient and mosquito indicate the virus most likely spread from the north, because of the geographic proximity and because the sequences cluster with the Austrian and Hungarian sequences. A sentinel study was performed on dog sera samples, and we were able to confirm IgG antibodies in 1.8% and 4.3% of the samples in 2017 and 2018, respectively. Though Slovenia is not a highly endemic country for WNV, we have established that the virus circulates in Slovenia.West Nile virus (WNV) is a flavivirus transmitted by mosquitoes. Birds are the reservoir for the virus; humans, horses and other mammals are dead-end hosts. Infections caused by WNV in humans can vary from asymptomatic infections to West Nile fever (WNF) or West Nile neuroinvasive disease (WNND). In 1995, a serosurvey was performed in Slovenia on forest workers, and WNV specific IgG antibodies were confirmed in 6.8% of the screened samples, indicating that WNV is circulating in Slovenia. No human disease cases were detected in Slovenia until 2013, when the first case of WNV infection was confirmed in a retrospective study in a 79-year old man with meningitis. In 2018, three patients with WNND were confirmed by laboratory tests, with detection of IgM antibodies in the cerebrospinal fluid of the patients. In one of the patients, WNV RNA was detected in the urine sample. In 2017, 2018 and 2019, a mosquito study was performed in Slovenia. Mosquitoes were sampled on 14 control locations and 35 additional locations in 2019. No WNV was detected in mosquitoes in 2017 and 2019, but we confirmed the virus in a pool of  Culex pipiens mosquitoes [West Nile virus (WNV) is a flavivirus transmitted by mosquitoes. Birds are reservoir for the virus; humans, horses and other mammals are dead-end hosts and do not contribute to circulation and further spread of the virus in nature. The main vector of WNV in Europe are the squitoes . Infectisquitoes .The first outbreak, caused by WNV in Europe was reported in Romania in 1996, and since then, cases have been reported in several European countries: Albania, Bosnia, Bulgaria, Croatia, Cyprus, France, Greece, Germany, Hungary, Italy, Kosovo, Montenegro, North Macedonia, Portugal, Romania, Russia, Serbia, Spain, Turkey and Ukraine ,4. EntomFor almost a decade after the first evidence of virus circulation in neighboring countries, no human cases were diagnosed in Slovenia. Only in 2013, the first human case was confirmed in a retrospective study.The goal of the study was to detect the local virus circulation in Slovenia by analyzing the presence of WNV in mosquitoes in Slovenia and by establishing the presence of the diseases in patients in Slovenia. A study of mosquito populations was performed in the past three years to evaluate the WNV presence as well as retrospective analysis of human meningitis cases. Additionally, to aid this objective, a WNV serosurvey was performed on dog sera samples to estimate their potential role as sentinels. The data obtained in these studies are described in this article with the aim to explain the virus circulation in Slovenia.From July 2017 to October 2019, mosquitoes were sampled throughout Slovenia . We samp®Fast Virus 1-Step Master Mix , 1 µM of each primer and 0.3 µM of probe. Cycling conditions were as follows: 95 °C for 20 s, 40 cycles of 95 °C for 15 s and 60 °C for 30 s. The prevalence of infection was calculated using the program PooledInfRate version 3.0 .Nucleic acid was extracted from the aliquotes of mosquito homogenate and human whole blood samples with the BioRobot EZ1-XL Advanced (Qiagen) using the EZ1 Virus Mini Kit v2.0 (Qiagen) and eluted in 60 µL. For RNA extraction from human urine samples MagNA Pure Compact System MagNA Pure Compact Nucleic Acid Isolation Kit I-Large Volume was used. WNV was detected by real-time RT-PCR . BrieflyPossible cases of WNND and WNF occurring in Slovenia were referred to the Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana. Molecular diagnostics was performed on patients’ whole blood, urine and cerebrospinal fluid samples with real-time RT-PCR as described above. Detection of WNV IgM and IgG antibodies in serum and cerebrospinal fluid samples was done by ELISA .Since 2011, we have retrospectively investigated WNV infection in all patients with meningitis/meningoencephalitis that were TBEV IgM and IgG negative or were only IgG positive (due to previous TBE vaccination or past in apparent TBE infection) and were >65 years of age. Enzygnost Anti-TBE/FSME Virus assay  was used for detection of TBEV IgM and IgG antibodies. The stored samples were tested at the end of season for the presence of WNV IgM and IgG antibodies in serum, and cerebrospinal fluid samples were done by ELISA .Anaplasma phagocytophilum. The samples were sent from different regions in Slovenia. For screening sera were diluted 1:40 and tested using indirect immunofluorescence assay on spot slides containing Vero E6 cells infected with WNV (strain: Eg101). As conjugate anti-Dog IgG-FITC  in the dilution 1:32 was used. The sera from seropositive dogs were further diluted to establish the end-point titer.Dog sera were referred to the Institute of Microbiology and Immunology for antibody screening against To confirm the presence of WNV specific neutralizing antibodies in dog sera we performed virus neutralization assay. Two-fold serial dilutions (starting dilution was 1:10) were mixed with 100 TCID50 of WNV (strain: Eg101) and incubated for 1 h at 37 °C. Subsequently, 50 µL virus-antibody mix were inoculated on 96-well plate containing 80% confluent Vero E6 monolayer. After 1.5 h 50 µL of fresh DMEM containing 4% fetal bovine serum (FBS) was added to each well. Plates were incubated for 4 days at 37 °C, before they were fixed with 50 µL of 4% formaldehyde. Afterwards, plates were checked for cytopathic effect (CPE) under the microscope, and cell monolayer was additionally visualized using crystal-violet staining. The antibody titer was defined as the reciprocal of the highest dilution of the serum that showed 100% neutralization. Positive and negative control sera were included in each plate. Sera with a titer of 1:20 were considered positive.Each 100 µL pool aliquot of mosquitoes was inoculated onto 6-well plate containing 80% confluent monolayer of Vero E6 or C6/36 cells. Briefly, after inoculation, Vero E6 cells were incubated 1 h at 37 °C; then, 2 mL of fresh DMEM containing 4% FBS was added, and plates were incubated at 37 °C in a 5% CO2 atmosphere for 7 days. C6/36 cells were first incubated for 1 h at room temperature, and subsequently, 2 mL of L-15 media containing 10% FBS was added to each well, and plates were further on incubated for 7 days at 28 °C. All plates were daily examined for presence of CPE. The cells were blindly passaged 3 times.Additionally, the homogenate of the mosquito pool Ko169/19, where WNV RNA was detected, was inoculated intracerebrally into suckling mice BALB/c. Six days after infection, 1 of the 4 infected mice showed symptoms of infection . The mice were euthanized with CO2, and brains were removed. WNV RNA was detected in the brain of the infected rodent, and the tissue was inoculated on Vero E6 cells as described above.For sequencing, RNA was isolated with Direct-zol RNA Kit , following manufacturer’s instructions. From the mosquito sample, Ko169/19 cDNA was transcribed with Maxima H Minus Double-Stranded cDNA Synthesis Kit (Thermo Scientific) and further amplified using SISPA protocol to acquire sufficient quantities. For construction of NGS library, we used 2 different Nextera kits , according to manufacturer’s instructions . From the human sample, WNV genome was reverse transcribed and amplified using overlapping genome specific primers kindly provided by Victor Corman . PCR amplicons ranging from 486 to 806 bp were excised from UltraPure™ Agarose gel  and purified using QIAEX II Gel Extraction Kit (Qiagen). Concentrations of PCR amplicons were then determined with Qubit™ dsDNA HS Assay Kit . For preparation of NGS library, equimolar concentrations of all 20 PCR amplicons were prepared, and NGS library was constructed with Nextera XT kit and labelled with Nextera XT Index Kit according to manufacturer’s instructions . All NGS libraries were sequenced with Miseq Reagent Kit V3 on a MiSeq system  in two separate runs.Data from the first run was filtered, and adapter sequences were removed with Trimmomatic  , and genHuman and animal samples were sent to the Institute of Microbiology and Immunology, Faculty of Medicine for diagnostic purposes. No samples were specifically collected for this study. All procedures involving animals were approved by the National Ethical Committee and the Administration of the Republic of Slovenia for Food Safety, Veterinary and Plant Protection . Animal care and treatment were conducted in accordance with the institutional guidelines and international laws and policies .Aedes, Ochlerotatus, Culex, Anopheles, Culiseta and Coquillettidia. The species distribution is described in Culex pipiens mosquitoes captured in August 2018 in a village in north-eastern Slovenia, close to the Austrian border .Dog sera samples were screened for the presence of WNV specific antibodies. In 2017, we detected antibodies in 3 out of 209 sera samples (1.4%), and in 2018, we confirmed WNV infection in 8 out of 216 (3.7%) tested dogs. All dog sera had neutralizing antibodies with titers ranging from 1:40 to 1:320. Complete genome sequence was successfully obtained from mosquito pool and a patient’s urine sample. From first library, prepared from mosquito pool, we were able to de-novo assemble the complete WNV sequence, and the data from the second run was used to generate better coverage. The complete genome sequence was 10,317 bp long and had one open reading frame  translating the genome into one polyprotein. The average coverage was 7.3. Since WNV RNA in a patient’s urine sample was detected at very low concentration , we decided to use amplicon sequencing to obtain complete WNV cds. The complete sequence was 11,017 bp long, and the average coverage was 6846. Nucleotide alignment of Slovenian WNV sequences showed 92.9% identity on the nucleotide level. Both newly obtained Slovenian WNV sequences belonged to the Lineage 2 and clustered well within the sequences obtained in the neighboring countries. The WNV sequence obtained from the first acute Slovenian patient  was most closely related (99.6% on the nt level) to the WNV sequence obtained from Belgian patient that contracted WNV in Hungary in 2017 . The patSlovenia is a very heterogenous country located on the transient area between the Balkans and Central Europe. The influence of the Mediterranean and the Alps is seen in the country and affects both fauna and flora. Slovenia is a stopover for migratory birds, and therefore, we see significant biodiversity in bird population . In SlovCulex pipiens mosquitoes captured in August 2018. The minimum infection rate, 2,4 × 10−4, was significantly lower compared to those detected in neighboring Austria  in non-epidemic years [One hundred ninety-nine, 3054 and 7009 mosquitoes were tested in 2017, 2018 and 2019, respectively. No WNV RNA was detected in mosquito pools in 2017 and 2019, but we confirmed WNV RNA in a single pool of ic years . Thus, oIn 1995, a serosurvey study of various arboviruses was performed on sera samples obtained from forest workers in Slovenia in order to estimate the extent of exposure of these viruses in population at risk. WNV specific IgG antibodies were confirmed in 6.8% of the screened samples indicating that WNV is circulating in Slovenia . The firWNV was detected in Slovenia decades ago, but the virus caused no notable disease in humans, birds or horses. With the introduction of a new lineage to Europe and the changes that we see in our environment, the transmission of the virus has become increasingly likely, especially in years when the climate supports the increase in the abundance of mosquitoes. Therefore, monitoring of mosquitoes as well as the knowledge of clinicians and public health officials regarding the virus’ presence in our environment are necessary to introduce the preventive measures, for example screening of blood donations and real-time diagnosis of patients.


An acute respiratory disease caused by a novel coronavirus , the coronavirus disease 2019 (COVID-19) was first detected in Wuhan, China. Since then, the virus has spread rapidly worldwide leading to a global public health crisis. Due to its devastating effect on public health, it is crucial to identify a viable therapeutic option to mitigate the damage the disease causes. In spite of various governments implementing aggressive global lock-down and quarantine protocols, the number of cases continues to follow an upward trend. At present, the therapeutic strategies are supportive or preventative, focusing on reducing transmission. Given the gravity of the situation, we aim to explore the drugs that have been tried so far and their efficacy when applied in clinical trials. Since newer interventions would take months to years to develop, by looking at the pool of existing therapeutic options, including remdesivir (RDV), plasma exchange or cytapheresis, hydroxychloroquine, baricitinib, and lopinavir (LPV), we have tried to detail the principles behind their use to treat COVID-19, current application, and adverse effects. Many coronaviruses have a highly mutable single-stranded RNA genome and hence discovering new drugs against the virus is going to be challenging owing to the possible viral genetic recombination. Extensive research is still needed to safely advocate the efficacy of the currently available therapeutic options. Today, the entire world has come to a standstill, grappling with a common enemy. Although we have had numerous outbreaks of diseases in the past, including Ebola, cholera, Middle East respiratory syndrome (MERS), the novel coronavirus pandemic has undoubtedly proven to be a tougher challenge leading to millions of cases and a staggering number of deaths. An outbreak that was presumably first detected in a province in China in late December has found its way into almost every part of the world, leading to a global health crisis. Having a better understanding of the virus through various studies will give us an opportunity to hopefully mitigate the severity of this crisis.Orthocoronavirinae subfamily, which also includes severe acute respiratory syndrome coronavirus (SARS-CoV) that caused the severe acute respiratory syndrome (SARS) outbreak in 2002 and the Middle East respiratory syndrome coronavirus (MERS CoV), which caused the MERS outbreak in 2012. The severity of the SARS-CoV-2 infection, when compared to that of SARS and MERS, shows the potential of these viruses to adapt and evolve according to the changing environment  and the structural protein (spike protein) play an important role in viral pathogenesis and hence they are being recognized as potential targets for drug therapy . The knoWith the rapidly escalating situation worldwide, there are multiple approaches to finding potential therapeutic options as rightly explained by Lu et al. . The firAnother approach involves looking through the existing compounds and testing their efficacy for antiviral properties including information about transcription characteristics in different cell lines . These cA third approach is to redevelop new drugs to act specifically against individual coronaviruses. This would require a thorough understanding of the genomics and structural characteristics of the virus. This includes small interfering RNA (siRNA) molecules or enzymes that will target specific viral enzymes, inhibition of host cell protease, or host viral endocytosis. Although this seems an effective option because of these drugs' specific anti-coronavirus properties, there is limited data regarding the safety profile as well as the pharmacodynamic and pharmacokinetic properties of these drugs. Hence, it would take a long time to prove the efficacy and reliability of these drugs in patients.A combination of these approaches can help us to evolve treatment options that we can broadly classify into being virus-based or host-based. In this review article, we aim to explore the drugs that have been tried so far and their efficacy when applied in clinical trials. Also, we attempt to explore emerging drug options that look promising.Genetic characteristicsCoronavirus is classified into four types based on the genomic structure: Alphacoronavirus (AlphaCoV), Betacoronavirus (BetaCoV), Deltacoronavirus (DeltaCoV), and Gammacoronavirus (GammaCoV). As of today, seven human CoVs (HCoV) infecting human species have been identified. BetaCoVs of A lineage (HCoV-OC43 and HCoV-HKU1) and AlphaCoVs (HCoV-229E and HCoV-NL63) can cause the common cold and upper respiratory infections in immunocompetent individuals while lower respiratory infections can occur in immunocompromised people. Other human CoVs, BetaCoVs of the B and C lineage  cause epidemics involving the respiratory and extra-respiratory systems As mentioned under pathogenesis, SARS-CoV-2 enters the cells of our body through ACE2. Some have suggested that the use of ACE inhibitor impairs (ACEI) the ACE/angiotensin-II/angiotensin-1 receptor pathway. which further impedes the integrity of the ACE2/angiotensin 1-7/MAS (MAS-related G protein-coupled receptor). This ultimately could result in decreased production of ACE2, thereby reducing the chances of SARS-CoV-2 entering the cell. However, it has also been argued that ACEI inhibits ACE and decreases the level of angiotensin-1. This in turn through possible negative feedback, upregulates more ACE2 receptors, leading to increased binding sites for SARS-CoV-2. A recent article published in The Lancet mentions that drugs causing an increase in ACE2 receptors are at a higher risk for severe COVID-19 infection . TherefoClinicalTrials.gov database [In Table database .Emerging optionsThere are many emerging options being tried for the treatment of COVID-19. We have listed a few important ones. Stem Cell Therapy Stem cell therapy has sparked interest in many researchers as they are resistant to tissue damage, promote tissue repair, and have immunomodulatory effects. Treatment with mesenchymal stem cells (MSCs) has been found to inhibit the overactivation of the immune system and, by improving the lung microenvironment, they promote endogenous repair . More thNeutrophil Extracellular Trap (NET) TherapeuticsClinicalTrials.gov identifiers: NCT04324021, NCT04330638, NCT02735707, NCT04326790, NCT04328480, NCT04322565, NCT04322682, NCT04366232, NCT04424056, NCT04412291, NCT04362943, NCT04362111, NCT04360980, NCT04363437, NCT04350320, NCT04355143, NCT04367168, NCT04326790,NCT04359654) [NETs are web-like DNA proteins expelled from the neutrophils that trap pathogens. Excessive NET formations can trigger a cascade of inflammatory reactions that destroy surrounding tissues, facilitating microthrombosis leading to permanent damage to pulmonary and other systems. The coagulation abnormality seen in infected patients is most likely due to the widespread inflammatory response as the virus does not appear to have intrinsic procoagulant effects. Early reports from the first 99 patients hospitalized in China showed that 5% had elevated prothrombin time (PT), 6% had elevated activated partial thromboplastin time (aPTT), 36% had elevated D-dimer, with increased biomarkers of inflammation such as IL-6, Erythrocyte sedimentation rate (ESR) and c-reactive protein (CRP). Drugs like neutrophil elastase inhibitors, protein arginine deiminase 4 (PAD4) inhibitors, gasdermin D inhibitors, deoxyribonuclease (DNase), colchicine, and IL-1β are some drugs targeting NETs while some are still under development. Of these, colchicine and anakinra are under study . (Clinic4359654) .ArbidolClinicalTrials.gov identifiers: NCT04273763) [Arbidol is an antiviral that has been approved in China and Russia for treating influenza, SARS, and Lassa viruses. It blocks virus replication by inhibiting the fusion of the lipid membrane of the virus with the host cells. A study with Arbidol treatment showed it to be possibly superior to LPV/ritonavir in treating COVID-19 because the viral load was undetectable after administration of Arbidol in patients . (Clinic4273763) .FavipiravirClinicalTrials.gov identifiers: NCT04351295, NCT04346628, NCT04392973, NCT04387760, NCT04425460, NCT04359615, NCT04445467, NCT04358549, NCT04349241, NCT04310228, NCT04373733, NCT04333589) [Favipiravir is an antiviral drug approved for the treatment of influenza in Japan due to its activity against a wide spectrum of RNA viruses including coronaviruses . This dr4333589) .ClinicalTrials.gov database [Table database .The COVID-19 pandemic has given rise to a public health crisis resulting in an extensive search for curative treatments. Research findings and recommendations are constantly evolving as various aspects of the virus and its transmission are being studied. Apart from the recently approved RDV, there are no other approved specific drugs that can cure patients infected by the virus. It has also been suggested that there is a possibility of a large number of undocumented asymptomatic infections, which facilitates the rapid spread of the virus. This is why the containment of the virus will be a particularly challenging task. Social distancing, contact tracing, and self-isolation are important strategies to avoid the spread of COVID-19 infection as mentioned by the Centers for Disease Control and Prevention (CDC). Many drugs listed in this review are encouraging and the benefits of these most likely outweigh the risk of adverse events and could potentially be life-saving. In addition, many coronaviruses have a highly mutable single-stranded RNA genome. Discovering new drugs against the virus is going to be challenging owing to the possible viral genetic recombinations. Extensive research is still needed to safely advocate the efficacy of the currently available therapeutic options.


Coronavirus disease 2019 (COVID‐19) is a zoonosis like most of the great plagues sculpting human history, from smallpox to pandemic influenza and human immunodeficiency virus. When viruses jump into a new species the outcome of infection ranges from asymptomatic to lethal, historically ascribed to “genetic resistance to viral disease.” People have exploited these differences for good and bad, for developing vaccines from cowpox and horsepox virus, controlling rabbit plagues with myxoma virus and introducing smallpox during colonization of America and Australia. Differences in resistance to viral disease are at the core of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) crisis, yet our understanding of the mechanisms in any interspecies leap falls short of the mark. Here I review how the two key parameters of viral disease are countered by fundamentally different genetic mechanisms for resistance: (1) virus transmission, countered primarily by activation of innate and adaptive immune responses; and (2) pathology, countered primarily by tolerance checkpoints to limit innate and adaptive immune responses. I discuss tolerance thresholds and the role of CD8 T cells to limit pathological immune responses, the problems posed by tolerant superspreaders and the signature coronavirus evasion strategy of eliciting only short‐lived neutralizing antibody responses. Pinpointing and targeting the mechanisms responsible for varying pathology and short‐lived antibody were beyond reach in previous zoonoses, but this time we are armed with genomic technologies and more knowledge of immune checkpoint genes. These known unknowns must now be tackled to solve the current COVID‐19 crisis and the inevitable zoonoses to follow. Coronavirus disease 2019 (COVID‐19) results from a virus making an interspecies leap that tests genetic resistance to viral disease, like most of the great plagues sculpting human history. Here I set out known unknowns for tackling COVID‐19, taking advantage of progress understanding how viral disease is countered by fundamentally different genetic mechanisms for resistance: resistance to virus transmission primarily by activation of immune responses; and resistance to pathology primarily by tolerance checkpoints to limit immune responses. This adaptation clearly occurred in late 2002 and it may happen again in the future. But given the present understanding and awareness about SARS, we expect that such re‐emergence is unlikely to lead to a global outbreak on the scale of 2003.”The 2003 severe acute respiratory syndrome (SARS) outbreak infected a cumulative total of 8098 people worldwide, of whom 774 died. As history now shows, our understanding of SARS and the cause and treatment of zoonotic coronavirus disease falls short of the mark. Coronavirus disease 2019 (COVID‐19) is a new human disease closely related to the SARS pandemic of 2003. Like SARS, COVID‐19 is caused by a zoonotic jump of a bat coronavirus to humans followed by adaptation for high human‐to‐human transmission, yielding a new virus called severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). As of 17 November 2020, SARS‐CoV‐2 has infected more than 55 million people globally, of whom more than 1.3 million people have died.Public health responses quickly countered and eradicated the two successive zoonotic outbreaks of SARS in 2003 and 2004 because there were very few asymptomatic virus shedders. The great majority of SARS‐infected people required medical attention, including 20–30% requiring intensive care, and symptoms and fever almost always preceded onset of virus shedding.SARS‐CoV‐2 has proved much harder to contain because of a wider spread in clinical presentation: 80% of people have mild or asymptomatic infections, but an unmanageably high percentage develop severe disease requiring hospitalization including 5–10% requiring intensive care. Severe COVID‐19 disease is broad based and unpredictable. While age is a risk factor as it was for SARS (discussed later), 30% of hospitalized cases in New South Wales at the end of March 2020 were people under 60 years. In the Chinese analysis of 72 314 cases “14.8% of confirmed cases among health workers were classified as severe or critical.”In Australia, these unique epidemiological parameters have forced Federal and State governments to deviate from well‐prepared influenza pandemic response plans.What explains the range of clinical outcomes when viruses leap into a new species, from asymptomatic to lethal? Historically it has been ascribed to differing levels of “genetic resistance to viral disease” in the new host. To clarify, in this review the word “genetic” is used in the sense of “encoded in the genome,” even if the relevant gene and trait is not known to be variable within a species. The nature of resistance is a major gap in the knowledge needed to reduce the rates of intensive care unit admissions for COVID‐19 and to promote durable immunity to eradicate the virus with a vaccine.It is valuable to split apart virus transmission and host pathology as separate parameters of infectious disease Figure  because,A range of genetically encoded mechanisms counter transmission of a new virus within the population Figure , includiCCR5 gene deletion. Significantly, the prevalence of this CCR5 allele in Europeans may have been driven by conferring lower mortality to smallpox.ACE2 receptor gene.Genetic resistance conferred by absence of virus receptors on host cells is best illustrated by “elite resistors” of human immunodeficiency virus infection who inherit two alleles of a The central role of adaptive antibody responses in blocking virus transmission is highlighted by Sabin’s attenuated oral poliovirus strains, which have been given to millions of children to eradicate polio from most countries around the world. These cause an asymptomatic infection and transient virus shedding from the gut, while triggering durable neutralizing antibodies that block infection and transmission of wild poliovirus. However, in people with genetic deficiencies in antibody formation, such as X‐linked agammaglobulinemia, the attenuated virus causes a persistent asymptomatic infection with intestinal virus shedding for up to 18 years.Genetic resistance to communicable disease by inhibiting microbe transmission, whether by loss of receptors or by gaining immune responses, is nevertheless unstable over time because variant microbes are rapidly selected that evade resistance and transmit better, as plant breeders have known for more than half a century.Coronaviruses have the largest genomes among RNA viruses and, unlike influenza virus or retroviruses, replicate their genome with much higher fidelity.Instead of blocking transmission, an alternative and more evolutionarily stable strategy involves genetic mechanisms to diminish pathological host responses to infection Figure , referreSalmonella enterica causing many severe or lethal cases of typhoid.Disease resistance by tolerating microbe replication is evolutionarily more stable because it can be achieved without decreasing microbe transmission and therefore without selecting for resistance escape variants.Contrary to popular view, CD8 T lymphocyte responses to viruses may primarily represent a genetic mechanism to tolerate infection. Unlike neutralizing antibodies to influenza, the strong CD8 T‐cell responses to conserved influenza epitopes do not appear to diminish virus transmission as evidenced by the need to update the seasonal influenza vaccine annually. However, these T cells may account for the diminished morbidity after a new influenza zoonosis becomes established in the population. Transmission of ectromelia virus in mice, a close relative of variola virus , is primarily blocked by neutralizing antibody but not by CD8 T cells: however, in the absence of CD8 T cells infected mice do not tolerate infection and rapidly succumb to lethal inflammatory disease.SH2D1A, which encodes SLAM‐associated protein, an intracellular adaptor for SLAM‐family receptors on CD8 T cells.SH2D1A‐deficient people, Epstein‐Barr virus provokes morbid pathological immune responses resulting in severe mononucleosis and, in some, a lethal “cytokine storm” terminating in the macrophage hyperactivation syndrome of hemophagocytic lymphohistiocytosis.Epstein‐Barr virus is widely transmitted and establishes a lifelong infection in most people, which we nevertheless tolerate asymptomatically by maintaining very high numbers of circulating CD8 T cells against the virus. These CD8 T‐cell responses do not develop in people who inherit a genetic defect in The importance of considering resistance to transmission and resistance to viral pathology separately is as important for SARS‐CoV‐2 as it was for polio. The first‐generation inactivated Salk vaccine did not protect against poliovirus infection and fecal–oral transmission but did confer resistance to viral infection of the central nervous system and paralyzing poliomyelitis.In experimental challenge studies in nonhuman primates, a nonreplicating adenovirus vector encoding the SARS‐CoV‐2 spike elicited virus‐reactive CD8 T cells but only low (less than 1/100) titers of neutralizing antibodies, which were insufficient to protect any of the animals from virus infection in the upper respiratory tract but diminished virus replication and pathology in the lung.Oryctolagus rabbit populations.Sylvilagus rabbits, but caused over 99% lethal systemic disease when introduced to Oryctolagus rabbits. After seven successive epizootic outbreaks of myxomatosis, the recovering Australian rabbit population was genetically more resistant to lethal disease, despite the virus replicating equally well in cells from genetically resistant or susceptible Oryctolagus rabbits.Oryctolagus rabbits revealed convergent selection for chromosomal segments containing pre‐existing gene variants in immune regulators including IFNA, CD96, FCRL3 and MHC‐I.+ regulatory T cells with common inherited variants associated with susceptibility to multiple autoimmune diseases.The paradigm‐setting example of varying genetic resistance when viruses leap between species was the deliberate release of myxoma virus in Australia to control European Myxoma virus release was, very regrettably, the second history‐shaping exploitation of differing host genetic tolerance to viruses in Australia. The first was an unexplained release of variola virus into the indigenous Sydney aboriginal community by the first fleet of European colonists. The likely source was ship surgeons’ stocks of preserved smallpox scabs used for variolation.Like COVID‐19, smallpox started as a zoonosis approximately 3000 years ago when variola virus evolved selective replication in the human population after leaping from rodents. Around the same time and location, camels were introduced to the Horn of Africa and closely related viruses evolved to replicate selectively in camels (camelpox virus) and in African naked‐sole gerbils (taterapox virus).In contrast to the clinical severity of human variola infection, its predecessor approximately 10 000 years ago is represented today by cowpox virus,Ironically, cowpox is not the vaccine strain employed to eradicate smallpox from the globe. Vaccinia virus—the strain used for eradication—was revealed by genome sequencing to have diverged from cowpox virus approximately 8000 years ago, around the same time as variola virus, but diverged only in the last 300 years from horsepox virus—a now extinct virus that caused pocks and pustules on the legs of horses referred to as “grease heel” in the 18th century.Like cowpox and horsepox infections, almost all humans are genetically resistant to systemic disease caused by vaccinia virus infection. Inoculation causes only a mild, localized virus replication in a skin pock but nevertheless induces high titers of neutralizing antibodies that continue to be produced for many decades,Poxviruses, along with herpesviruses such as Epstein‐Barr virus, have the largest genomes of all DNA viruses and carry numerous genes encoding proteins that manipulate host innate and adaptive antiviral immune responses. Variola virus has lost parts of the genome present in its more benign cowpox virus ancestor.IFNA genes,RNASEL and OAS genes,AIM2/PYHIN genes,NLRP3 inflammasome expression and activityTMEM173 Ser358 substitutions causing partial loss of function in stimulator of interferon genes .The coronavirus precursors of SARS‐CoV, SARS‐CoV‐2 and MERS‐CoV appear to be carried and shed for prolonged periods by many bats without causing disease, indicating high tolerance to coronavirus infection in these species.Genetic resistance to microbe transmission conferred by innate or adaptive immune responses imposes a fitness cost in the form of collateral damage to host tissues and extra energy demands.The innate immune response of pyroptosis highlights how the host cell’s response to virus or bacterial replication can be much more cytopathic than the microbe itself. A web of intracellular sensors of viruses, bacteria or host cell damage converge to activate caspase cleavage of a host cell protein, gasdermin D (GSDMD). The cleaved N‐terminal GSDMD fragment oligomerizes into plasma membrane pores causing inflammatory host cell death—pyroptosis.GSDMD illustrate how differences in genetic tolerance to infectious disease can arise. Phenotypic screening of mouse populations for genetic resistance to pyroptosis revealed a Gsdmd missense variant, I105N, that preserves GSDMD expression, cleavage and binding to membranes, but slows the rate of membrane pore assembly.IRF2) resulting in low transcription of Gsdmd messenger RNA,Gsdmd genetic deficiency have 50‐fold greater rotavirus replication and more severe diarrhea because infected intestinal epithelial cells are unable to activate pyroptosis upon sensing viral RNA through NLRP9B.Gsdmd genetic deficiency is beneficial in sepsis models where it confers resistance to lethal diseaseInherited variants in SAMD9 and SAMD9L, against which both viruses have evolved unique evasion proteins that bind with high affinity to the SAMD9/SAMD9L proteins and block their antiviral effect.de novo truncating mutations in SAMD9L that appear to remove negative regulatory domains, causing a severe autoinflammatory disorderApparently intrinsic traits of genetic resistance to virus replication turn out to reflect carefully regulated innate immune responses within the infected cells. Replication of myxoma and vaccinia viruses in cells from most mammalian species is aborted by “intrinsic” host cell restriction genes, TMEM173), which in turn activates TAK1, IRF3, interferon and inflammatory responses. As with the aforesaid SAMD9L example, people who inherit TMEM173 point mutations that lower the threshold for STING activation develop a severe autoinflammatory disease.The simplest demonstration of genetically tuned thresholds to tolerate virus replication comes from another intracellular virus sensing protein, cyclic GMP–AMP synthase (cGAS). cGAS binds and is activated by DNA in the cytoplasm, which could either be replicating viral DNA or short fragments of self‐DNA that have escaped from the cell’s nucleus or mitochondria.TNFAIP3 exemplifies a disease caused not by microbes per se but by loss of host tolerance to these microbes. When innate or adaptive immune receptors are stimulated by microbes or self‐constituents, they initiate signaling pathways to activate the transcription factor nuclear factor kappa B, which initiates diverse immune responses but also induces TNFAIP3 messenger RNA. TNFAIP3 protein, also called A20, serves as a potent inhibitory feedback to dampen nuclear factor kappa B activation. Without this checkpoint in intestinal epithelial cells and macrophages, commensal bacteria in the gut stimulate an overexuberant immune response through a vicious cycle that progressively thins the mucin layer and epithelial layer that normally serve as barriers physically separating commensal gut bacteria from inflammatory immune cells.Devastating mucosal inflammatory disease in peopleTNFAIP3 gene variants illustrate the goldilocks problem of genetically varying immune tolerance.TNFAIP3 variant in mice that lessens the protein’s inhibitory activity decreased immune tolerance to self‐tissues and caused subclinical colitis, but also increased resistance to virus replication and otherwise lethal disease caused by Coxsackievirus B4. However, the same gene variant caused a fatally overexuberant immune response when the mice were infected with ectromelia virus. The right amount of immune response to one virus is too much for another.Subtle inherited TNFAIP3 variant from extinct Denisovan hominins similarly decreases the activity of the TNFAIP3 checkpoint.An ancient human in utero, mice acquire the ability to tolerate viral replication throughout postnatal life without the brain disease and convulsions that follow adult‐onset infection. Traub did not use the word “tolerance” but noted the similarities with plant immunity to tobacco ring spot virus: “Immune plants are always carriers of virus, although they show no signs of disease.”Cellular immunologists sometimes protest against applying the word “tolerance” to describe asymptomatic viral infections, but that condition is the origin of the concept. Lifelong, asymptomatic replication and shedding of lymphocytic choriomeningitis virus (LCMV) in mice was revealed by Traub.in utero in dizygotic cattle twins,Traub's observations, together with Owen's finding that antibodies were not made against foreign erythrocyte alloantigens encountered Medawar and colleagues experimentally demonstrated Burnet and Fenner's idea by showing that neonatal inoculation with allogeneic hematopoietic stem cells resulted in “actively acquired immunological tolerance” to foreign skin grafts that would normally have been swiftly rejected.in utero by clonal deletion of cells with self‐reactive antibodies (before these antibodies could be tested for their utility in binding foreign microbes), immunological self‐tolerance is actively acquired throughout life by sequential checkpoints that balance tolerance with immunity.Contrary to Burnet and Fenner's idea that tolerance could only be acquired Tolerance checkpoints in B and T lymphocytes balance activating and inhibitory receptors and intracellular signals, switching an individual receptor between stimulating immunity and stimulating tolerance. This was first revealed by surface immunoglobulin antigen receptors on B cells where differences in the tempo and strength of stimulation, together with costimuli from other inhibitory  or activating  receptors, change the pattern of intracellular signals and genetic responses: either to trigger clonal proliferation and antibody secretion or to inhibit the maturation and survival of the B cell and actively establish tolerance.IGHV4‐34 gene in adult volunteers following vaccinia virus infection.IGHV4‐34 B cells were released from some checkpoints, proliferated and acquired somatic mutations in their antibody genes. Daughter cells were selected with antibody mutations that removed self‐reactivity and the risk of autoimmune hemolytic anemia, while other antibody mutations were selected that improved binding to the virus.Without self‐tolerance checkpoints, the production of neutralizing antibodies could paradoxically increase pathology during an infection Figure . This isIGHV1‐69 gene, which encodes structural features that target a key site on the virus envelope E2 protein.IGHV1‐69‐encoded structural features confer polyreactivity with many other molecules, including frequent binding to the Fc domain of human immunoglobulin G as a rheumatoid factor autoantibody.IGHV1‐69 rheumatoid factors that become extremely pathogenic causing mixed cryoglobulinemic vasculitis.IGHV1‐69‐encoded antibodies normally make only transient antibody responses and fail to accumulate as memory B cells.The limits on tolerance during antiviral antibody responses are illustrated by chronic infection with hepatitis C virus. Broadly neutralizing antibodies against hepatitis C virus primarily use the in utero, a similar state arises following infection of adult mice with a subclone of LCMV (clone 13) that was initially isolated from an in utero infected animal. In adult mice infected with LCMV clone 13, CD8 T cells bearing T‐cell receptors against viral peptides initially proliferate to form large clones but when these are unable to control chronic virus replication the T cells switch into a tolerant state of “exhaustion.” The tolerant state comes in part by expressing high levels of the inhibitory receptor, PD‐1,The concept of sequential tolerance checkpoints developed for B cells was subsequently extended to T cells, with the T‐cell receptor integrating tempo and strength of antigen stimulation with costimuli from inhibitory  and stimulatory  receptors.The burden of somatic mutations in cancer cells creates neoantigens that are not “self.” While immune responses initially retard growth of tumors bearing these neoantigens,CTLA4 gene,People whose CTLA4 checkpoint is blocked by antibody immunotherapy,Upon SARS‐CoV‐2 infection, many people exhibit mild or no clinical symptoms or radiographic evidence of lung disease despite actively shedding viral RNA and transmitting the infection to others who develop severe pneumonia.The incidence of severe COVID‐19 is much higher in older people, and the same was true for SARS. This is difficult to dissect because so many things change with age, including clonal hematopoietic stem cell mutations that exaggerate inflammatory responses.Serum immunoglobulin G against SARS‐CoV appears on days 10–20 after symptom onset and may contribute to the decline in viral RNA in nasopharyngeal and fecal samples on days 15 and 20 after a peak on day 10.Antibody responses can cause pathological reactions to virus replication, either through polyreactivity and self‐reactivity as discussed earlier or by the phenomenon of “antibody‐dependent enhancement.” The latter is an insurmountable barrier to vaccine control of a widespread coronavirus disease in cats, feline infectious peritonitis, which is made much worse because of antibody responses against the virus. In feline infectious peritonitis, even potent neutralizing monoclonal antibodies paradoxically enhance virus‐induced pathology because the antibody Fc segment enables antibody‐bound virus to enter cells bearing Fc receptors.It is unclear whether a similar mechanism contributes to severe SARS and COVID‐19 disease. Experimental evidence supporting this possibility has come from vaccination of macaques with a recombinant vaccinia virus expressing the SARS‐CoV spike protein.The current COVID‐19 crisis shines a beacon on the need to answer “known unknowns” about the mechanisms responsible for varying tolerance and immune response posed by every zoonotic leap from animals into humans. Several fundamental questions need to be answered:With respect to transmission, why are neutralizing antibody titers low and short‐lived following infection with COVID‐19, SARS and other coronaviruses? What titers and against which parts of the spike protein are needed to stop community transmission, and how can these be durably induced by a vaccine if the natural infection does not?On the pathology side, is severe COVID‐19 caused by inadequate (e.g. type 1 interferon) or by exaggerated (e.g. pyroptosis) innate immune responses? Or is it caused by inadequate or unchecked CD8 T‐cell responses, or by pathological antibody responses ? Are there particular immune tolerance checkpoints that fail in people with severe COVID‐19, and why is this more common as we get older?Pinpointing and targeting the mechanisms responsible for varying pathology and inadequate antibody to stop transmission were out of reach in earlier coronavirus zoonoses. But this time around medical science is armed with the necessary genomic technologies and knowledge of immune response and tolerance checkpoint genes. It is time to deploy.I have no conflicts of interest to declare.


Myrica rubra) is a subtropical fruit crop widely grown in southern China. Twig dieback is a disease of Chinese bayberry caused by Pestalotiopsis spp. and results in great economic losses to Chinese bayberry production. A virus survey was conducted in the population of Pestalotiopsis spp. infecting M. rubra in China. We explored the viral diversity in Pestalotiopsis spp., which may provide resources for further development as biocontrol agents of twig dieback.Chinese bayberry (Pestalotiopsis spp. were isolated from diseased twigs of M. rubra, and cultured on potato dextrose agar for RNA extraction. The total RNA of each strain was extracted, mixed, and used for RNA sequencing. The resulting sequences were deduplicated, annotated, and then used for phylogenetic analysis.Strains of M. rubra collected from 14 localities in China. Based on the phylogenetic analysis, these viruses were classified into five viral families/orders, Botourmiaviridae, Mitoviridae, Partitiviridae, Tymovirales and Bunyavirales, and one virus, Pestalotiopsis negative-stranded RNA virus 1, which likely belongs to a new viral family.Seven novel viruses were characterized from 59 isolates of Pestalotiopsis spp. isolated from M. rubra in China. The genomes of eight putative viruses were identified, seven of which were nearly full-length. Some of these viruses of Pestalotiopsis spp. may have the potential for the biological control of twig dieback of M. rubra.Metatranscriptomics analysis showed the presence of various mycoviruses in The online version contains supplementary material available at 10.1186/s12985-021-01513-3. Myrica rubra) is a subtropical fruit crop with great economic value that is widely grown in southern China. The fruits of M. rubra are not only eaten fresh but also can be processed into various products, such as juice, jam, sweets, and wine [M. rubra was found in all major Chinese M. rubra producing areas [M. rubra. Many species of Pestalotiopsis, like Pestalotiopsis mangiferae, P. vismiae, P. microspora, and P. versicolor, were determined to be the causal agents of twig dieback of M. rubra in China [Pestalotiopsis spp. are also endophytes in many plant species, representing a huge and largely untapped resource of natural products [Chinese bayberry , positive single-stranded (+ ss) RNA , or negative single-stranded (-ss) RNA (Mymonaviridae). Nevertheless, many mycoviruses remain unclassified [Sclerotinia sclerotiorum [Fusarium graminearum [Mycoviruses or fungal viruses refer to viruses parasitizing or inhabiting fungi and oomycetes. They are widely discovered in all major groups of fungi. The earliest record of mycoviruses was believed to be the discovery of virus-like particles in diseased bisporus . Due to bisporus  and the bisporus –12. Mycoassified . Besidesrotiorum  and Fusaminearum .S. sclerotiorum population [Pestalotiopsis spp. Therefore, in this study, we aimed to identify the viral population in Pestalotiopsis spp. infecting M. rubra in China by analyzing the total RNA with NGS technology.Next-generation sequencing (NGS) technology is a popular way to explore mycoviral diversity, including sequencing the total RNA that is depleted of ribosomal RNA or small RNAs –11. Manypulation . HoweverPestalotiopsis spp. were isolated from diseased twigs of M. rubra collected from 14 localities  and then mixed. A mixed RNA sample (~ 120 μg) was sent to Novogene  for RNA sequencing (RNA-seq).The total RNA of each strain was extracted from 0.1 g of fungal mycelium by using a Plant RNA Extraction Kit . The total RNA was stored at -80https://www.ncbi.nlm.nih.gov/) were used for BLASTx annotation, and the viral sequences were selected. Pairwise sequence comparisons were carried out for both botourmiavirus and mitovirus related sequences, using Sequence Demarcation Tool (SDT) v.1.0 with the MUSCLE alignment option [Ribosomal RNA depletion , library preparations  and Illumina sequencing (Illumina Novaseq 6000) were performed by Novogene . The filtered data was spliced from scratch using Trinity software (version: 2.3.3), and the resulting sequences were then deduplicated with cd-hit (version: 4.6). Finally, Diamond software (version: 0.8.22) and the non-redundant protein database in the National Center for Biotechnology Information (NCBI)  and conserved domain(s) were determined by using the ORF Finder program and CD-search in NCBI. Then, the selected amino acid (aa) sequences of viral replicases or other conserved regions were used for the construction of phylogenetic trees with the neighbor-joining (NJ) method and tested with a bootstrap of 1000 replicates to ascertain the reliability of a given branch pattern in MEGA X software . MultiplPestalotiopsis strains were isolated form the diseased samples collected from 14 locations  were obtained, and a total of 164,628 contigs were assembled by using the software Trinity. A BLAST search of non-redundant protein database identified a total of 8 putative viral contigs  sequence of Colletotrichum gloeosporioides ourmia-like virus 1  with an aa sequence identity of 96%. Therefore, this fragment may represent a strain of CgOLV1. The fragment c55068_g1 was 2634 nt long, encoding a putative RdRp of 639 aa, which was 61% identical to the RdRp sequence of Plasmopara viticola associated ourmia-like virus 44 (QGY72574.1), and it was named Pestalotiopsis botourmiavirus 2 (PBV-2). The fragment c59323_g1 was 2460 nt in length encoding a putative RdRp of 636 aa, namely Pestalotiopsis botourmiavirus 3 (PBV-3). The RdRp of PBV-3 was most homologous to the RdRp sequence of Plasmopara viticola associated ourmia-like virus 81 (QGY72611.1) with aa sequence identity of 39%  and c61286_g1 (2486 nt), were predicted to encode mitoviral RdRps, namely Pestalotiopsis mitovirus 1 (PMV-1) and Pestalotiopsis mitovirus 2 (PMV-2), respectively. The RdRp of PMV-1 was most similar to Sclerotinia homoeocarpa mitovirus (AAO21337.1), with an aa sequence identity of 49%, while PMV-2 was most similar to Rhizoctonia solani mitovirus 10 (ALD89102.1), with an aa sequence identity of 44%  and a viral helicase (Hel) domain  was also identified, namely Pestalotiopsis negative-stranded RNA virus 1 (PNSV-1). The identified PNSV-1 contig was 7009 nt long and putatively encoded a polyprotein of 2300 aa in length, containing an RdRp domain and a Hel domain  of 1646 bp in length, encoding a 518 aa long putative RdRp, showing homology to partitiviruses, namely Pestalotiopsis partitivirus 1 (PPV-1)  with viral genomes of + ssRNA, and all members contain a linear genome of 5.9–9.0 nt in length [Deltaflexiviridae with 100% bootstrap support. These results suggest that PDFV-1 may be a novel member of this family, but it is more distantly related to the reported deltaflexiviruses  and mitoviral (b) sequences in Pestalotiopsis strains to other known botoumiaviruses and mitoviruses. See Table S3 for detailed information of each virus. Figure S3. Conserved amino acid sequence motifs of the putative RNA-dependent RNA polymerases of Colletotrichum gloeosporioides ourmia-like virus 1 (CgOLV1), Pestalotiopsis botourmiavirus 2 (PBV-2), Pestalotiopsis botourmiavirus 3 (PBV-3), Botrytis ourmia-like virus (BOLV), Phoma matteucciicola ourmia-like virus 1 (PmOLV1), Magnaporthe oryzae ourmia-like virus (MOLV), and Rhizoctonia solani ourmia-like virus 1 (RsOLV1). The GenBank accession number of each virus is listed in Table S3. “*” indicates identical amino acid residues; and “.” indicates low chemically similar amino acid residues. Figure S4. Conserved amino acid sequence motifs of the putative RNA-dependent RNA polymerases of Pestalotiopsis mitovirus 1 (PMV-1), Pestalotiopsis mitovirus 2 (PMV-2), Cryphonectria cubensis mitovirus 2a (CcMV2a), Ophiostoma mitovirus 1a (OnuMV1a), Sclerotinia homoeocarpa mitovirus (ShMV), Ophiostoma mitovirus 3a (OnuMV1a), and Cryphonectria parasitica mitovirus 1-NB631 (CpMV1-NB631). The GenBank accession number of each virus is listed in Table S3. “*” indicates identical amino acid residues; and “.” indicates low chemically similar amino acid residues. Figure S5. Multiple alignment of amino acid sequences of conserved domains including methyltransferase (Mtr), viral RNA Helicase (Hel), and RNA-dependent RNA polymerase (RdRp) domains of Pestalotiopsis deltaflexi-like virus 1 (PDFV-1), Sclerotinia sclerotiorum deltaflexivirus 1 (SsDFV1), Sclerotinia sclerotiorum deltaflexivirus 2 (SsDFV2), and Fusarium graminearum deltaflexivirus 1 (FgDFV1). The GenBank accession number of each virus is listed in Table S3. “*” indicates identical amino acid residues; and “.” indicates low chemically similar amino acid residues. Figure S6. Conserved amino acid sequence motifs of the putative RNA-dependent RNA polymerases of Pestalotiopsis negative-stranded RNA virus 1, rose rosette emaravirus (RRV), and fig mosaic emaravirus (FMV). The GenBank accession number of each virus is listed in Table S3. “*” indicates identical amino acid residues; and “.” indicates low chemically similar amino acid residues. Figure S7. Conserved amino acid sequence motifs of the putative RNA-dependent RNA polymerases of Pestalotiopsis partitivirus 1 (PPV-1), beet cryptic virus 1 (BcV1), beet cryptic virus 2 (BcV2), beet cryptic virus 3 (BcV3), and fig cryptic virus (FcV). The GenBank accession number of each virus is listed in Table S3. “*” indicates identical amino acid residues; and “.” indicates low chemically similar amino acid residues. Table S1. Strains used in this study. Table S2. List of PCR primers used for viral contig detection. Table S3. Viruses used for multiple sequence alignments and phylogenetic analysis.