The ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR) are apical kinases that orchestrate the multifaceted DNA damage response (DDR) to a variety of genotoxic insults and regulate genomic stability. Whether RNA virus also manipulates the host’s DDR machine to facilitate replication is largely unknown. In this study, we revealed that single-stranded RNA virus replication specifically elicits host ATM- and ATR-mediated pathway activation and boosts their expression. The activated ATM and ATR are hijacked to the virus replication factory in the cytoplasm and facilitate viral gene expression and replication. Specific inhibitors targeting ATM and ATR strikingly block the viral proliferation and replication and inhibit expression of virus proteins. Our results reveal a novel, or otherwise noncanonical, conserved function of ATM/ATR outside DDR in promoting the replication of single-stranded RNA virus and provide an important mechanism of host–pathogen interactions.The online version contains supplementary material available at 10.1007/s42764-022-00064-3. Dear Editor,A variety of exogenous and endogenous factors have constantly caused extensive DNA damage and genomic instability. DNA damage response (DDR) is a complex protein modification regulatory network, which detects DNA damage, signals its presence and mediates its repair . It was found that ATM inhibitor (KU55933) and ATR inhibitor (VE821) can significantly inhibit the formation of progeny and reduce the viral titer, which is more evident at the late replication state , themselves (pATM and pATR), to regulate cell cycle progression, replication fork stability, and DNA repair , we observed the expression and localization of ATM and ATR kinase underwent significant changes during virus replication Below is the link to the electronic supplementary material.


Background and Purpose: Studies on the regional differences in hospital costs of acute ischemic stroke (AIS) are scarce in China. We aimed to explore the regional differences in hospital costs and identify the determinants of hospital costs in each region.Methods: Data were collected from the Chinese Acute Ischemic Stroke Treatment Outcome Registry (CASTOR), a multicenter prospective study on patients diagnosed with AIS and hospitalized from 2015 to 2017. Univariate and multivariate analyses were undertaken to identify the determinants of hospital costs of AIS.Results: A total of 8,547 patients were included in the study, of whom 3,700 were from the eastern area, 2,534 were from the northeastern area, 1,819 were from the central area, and 494 were from the western area. The median hospital costs presented a significant difference among each region, which were 2175.9, 2175.1, 2477.7, and 2282.4 dollars in each area, respectively. Each region showed a similar hospital cost proportion size order of cost components, which was Western medicine costs, other costs, diagnostic costs, and traditional medicine costs, in descending order. Male sex, diabetes mellitus, severe stroke symptoms, longer length of stay, admission to the intensive care unit, in-hospital complications of hemorrhage, and thrombectomy were independently associated with hospital costs in most regions.Conclusion: Hospital costs in different regions showed a similar proportion size order of components in China. Each region had different determinants of hospital costs, which reflected its current medical conditions and provided potential determinants for increasing medical efficiency according to each region's situation. As the second leading cause of death and third leading cause of disability globally, stroke places an enormous burden on current health care and social costs . IschemiData were obtained from the Chinese Acute Ischemic Stroke Treatment Outcome Registry (CASTOR), a multicenter prospective study that included 10,002 cases of AIS from 80 hospitals in 44 cities across mainland China. The trial design and protocol have been described previously . The hosThe covariates included demographic variables, medical history, and clinical features of the stroke index, including age and sex; payment type; medical history of prior stroke (including ischemic stroke and hemorrhagic stroke), hypertension, diabetes mellitus (DM), coronary heart disease , or atrial fibrillation (AF); geographic region; hospital level; National Institutes of Health Stroke Scale (NIHSS) score on admission ; length of stay (LOS); use of the intensive care unit (ICU); in-hospital complications including infection, deep venous thrombosis (DVT), cardiac events, hemorrhage, and serious falls; use of thrombolysis; and thrombectomy. Hypertension was defined as a history of elevated blood pressure >140/90 mmHg on two independent occasions or the use of antihypertensive medication. DM was defined as a history of elevated blood glucose on 2 independent occasions or the use of antidiabetic medication. AF was defined as a history of AF or characteristics of AF identified by ECG during hospitalization. The participating hospitals were divided into four groups according to their geographical region: the eastern area, the northeastern area, the central area, and the western area . Data frHospital costs were divided into four parts: diagnostic costs, Western medicine costs, traditional medicine costs, and other costs. Diagnostic costs included the laboratory and examination costs for pathological examinations, laboratory tests, and imaging examinations. Western medicine costs included the costs of various Western medicines. Traditional medicine costs included the costs of traditional herbs, traditional patent medicine, and traditional operation treatment such as acupuncture and Chinese massage. Other costs mainly included labor costs, such as medical service costs, nursing costs, and surgery costs. A mean exchange rate of US $ 1 = 6.75 CNY was used for 2017. The functional outcome was evaluated with the modified Rankin Scale (mRS) at discharge (poor outcome: mRS > 2) .t-test or Wilcoxon rank-sum test as appropriate according to whether they were normally distributed. Categorical variables were presented as frequencies (percentages) and were analyzed with the chi-squared test. A multivariate analysis was used with multiple linear regression models with the “backward” method, and the levels of significance were a P < 0.05 for inclusion and a P > 0.1 for exclusion in the stepwise procedure, which included all covariates mentioned above   was considered indicative of significance. All statistical analyses were performed with the IBM SPSS 22.0 software package .Continuous variables were presented as mean ± standard error or median [interquartile range (IQR)] and were analyzed with Student's  stroke) . Subgroun = 48) and patients with missing data . A total of 8,547 patients were included in the final analysis. The median age of the overall cohort was 64.0 years (IQR: 56.0–73.0), and 5,604 (65.6%) participants were male. 1,497 (17.5%) patients paid at their own expense, and 7,050 (82.5%) patients had medical insurance, in which 154 (1.8%) patients had private insurance and 6,896 (80.7%) patients had public insurance. The median hospital cost was 2248.0 dollars (IQR: 1507.3–3339.2). Five (0.1%) patients died at discharge in this study. Patients from areas with higher per capita disposable income, such as the eastern area and northeastern area, tended to have DM, AF, milder stroke severity, and shorter LOS. Regarding hospital costs, patients from areas with higher per capita disposable income tended to have lower total hospital costs, lower Western medicine costs, and lower other costs with higher diagnostic costs  classification, which have been proven to be related to hospital costs in young patients, and this limited the further exploration of hospital costs from the aspect of etiology  and Peking University Medicine Seed Fund for Interdisciplinary Research (No. BMU2018MX020). The CASTOR study was funded by Techpool Bio-Pharma Co., Ltd. All funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


N-isopropylacrylamide) (PNIPAAm)-modified bead-packed columns for effective and safe drug monitoring. PNIPAAm-modified silica beads were prepared as packing materials using atom transfer radical polymerization of NIPAAm. The increase in the retention times of the drugs requiring TDM with increasing temperature, was attributed to enhanced hydrophobic interactions at elevated temperatures. The drugs and serum proteins were separated on the prepared column at 40 °C using an all-aqueous mobile phase. Differences in the hydrophobic interactions accounted for the elution of the serum proteins and drugs at short and long retention times, respectively, and a primary column was employed to separate the serum proteins and drugs. After eluting the serum proteins from the column, the drug was introduced into the secondary column, leading to a peak of its purified form and enabling determination of the drug concentration. Two-dimensional temperature-responsive chromatography can benefit TDM by allowing the drug concentration in the serum to be measured in all-aqueous mobile phases without sample preparation.Therapeutic drug monitoring (TDM) is an effective pharmacological approach for controlling drug concentration in a patient’s serum. Herein, a new two-dimensional chromatography system was developed using two poly( Thus, the dosage must be adjusted based on the drug concentration in the patient’s serum; a low drug concentration may be ineffective, whereas an excessive concentration may be toxic3. Various methods including immunological and chromatographic methods have been developed for TDM4. Liquid chromatography (LC) is an effective and versatile method for monitoring drug concentrations in the serum because it does not require a specific drug ligand5. In contrast, ordinary chromatography requires an organic solvent in the mobile phase to adjust drug retention in the column. However, it is challenging to use organic solvents in hospitals because of exposure risks. Additionally, ordinary chromatography requires sample preparation (pretreatment) to remove serum proteins. This time-consuming sample preparation process, which generally requires organic solvents for serum protein denaturation, delays the monitoring of drug concentrations in a patient.Therapeutic drug monitoring (TDM), that is, the monitoring of drug concentrations in patients, is essential for effective pharmacological therapy, as the dosages of specific types of drugs differ among patients because of their varying absorption, distribution, and elimination levelsN-isopropylacrylamide) (PNIPAAm)-modified bead-packed columns and two-dimensional chromatography. Temperature-responsive chromatography employing a PNIPAAm-modified stationary phase can modulate analyte retention by changing the column temperature through the intrinsic properties of PNIPAAm8. Various types of thermoresponsive polymers, including polys, poly copolymers, and polys, have been investigated9. Among these, PNIPAAm is the most widely utilized because of its sharp phase-transition properties12. PNIPAAm exhibits temperature-dependent hydrophilic and hydrophobic changes, which can be attributed to hydration and dehydration processes across its phase-transition temperature of 32 °C13. In addition, PNIPAAm exhibits expansion and shrinkage at low and high temperatures, respectively. Thus, different biomedical applications, such as temperature-modulated drug and gene delivery systems19, biosensor and bioimaging systems based on the phase-transition of PNIPAAm25, nano-actuators induced by chemical oscillations28, cell separation systems exploiting the difference between the adhesive properties of cells to PNIPAAm32, and cell culture substrates for tissue engineering39, have been developed based on the thermoresponsive properties of PNIPAAm. A PNIPAAm-modified stationary phase is generally used for chromatography. Because the hydrophobicity of PNIPAAm changes with changing column temperature, the hydrophobic interactions between PNIPAAm and analytes can be modulated to control analyte retention in the columns. Thus, chromatography does not require addition of an organic solvent to the mobile phase to control analyte retention. The column performance of the analytes in the PNIPAAm-modified stationary phase was determined from the amount of PNIPAAm in the stationary phase8. Studies have reported that a PNIPAAm brush-modified stationary phase, which was prepared via atom transfer radical polymerization (ATRP), exhibited good performance because of the precisely controlled graft density and chain length of PNIPAAm40.To overcome these issues, we developed a method based on temperature-responsive chromatography using poly separated the analyte, and the effluent from the primary column containing the measuring object was introduced into the second column (secondary column) by switching the valve at an appropriate time. In the two-dimensional chromatography system, the primary column is employed for sample preparation, and the secondary column is employed for subsequent measurements.Two-dimensional chromatography is an effective approach for LCIn this study, a two-dimensional chromatography system consisting of two PNIPAAm brush-modified bead-packed columns was developed Fig. . PNIPAAm43; this long retention time was not suitable for the short analysis time required for TDM.The PNIPAAm brush-modified silica beads were prepared in a silane coupling reaction of ((chloromethyl)phenylethyl)trimethoxysilane (CPTMS) with glycidyloxypropyltrimethoxysilane (GPTMS) to form silica beads, followed by ATRP of NIPAAm Fig. A. We dev2, which is relatively small compared to that of beads solely modified with CPTMS, as previously reported (~ 5.0 μmol/m2)44; this is because the molecular size of GPTMS is slightly larger than that of CPTMS, thus lowering the immobilized density of the silane layer. The PNIPAAm brush-modified beads (PN-1000 and PN-1500) exhibited larger carbon compositions compared to the silane layer-modified silica beads, indicating successful modification of PNIPAAm on the silica beads via ATRP under the conditions employed in this study. The amounts of PNIPAAm, which were modified, were 2.39 and 2.65 mg/m2 for PN-1000 and PN-1500, respectively. A relatively larger amount of PNIPAAm was observed in PN-1500 than in PN-1000 because the high monomer concentration (1500 mM) in ATRP increased the polymerization rate compared to the lower monomer concentration (1000 mM).The prepared beads were characterized using elemental analysis, attenuated total reflection Fourier-transform infrared spectroscopy (ATR/FT–IR), and scanning electron microscopy (SEM). Elemental analysis of the prepared beads at each reaction step was performed to investigate the amounts of immobilized silane and modified PNIPAAm Table . The car−1, which was attributed to the Si–O bond of the silica beads. Two peaks were observed at 1550 and 1645 cm−1 for PN-1000 and PN-1500, respectively, whereas no peaks were observed at these wavenumbers for unmodified silica and silane-modified beads. These peaks were attributed to the N–H and C=O bonds of the amido group of PNIPAAm. Thus, these results indicate the modification of PNIPAAm on silica beads via ATRP. In addition, a previous report showed that the peak intensity of the amide bond represents the amount of modified PNIPAAm46. Because the amount of PNIPAAm on PN-1500 was larger than that on PN-1000, the intensity of the amide bond of PN-1500 was expected be larger than that of PN-1000. However, the heights of these peaks were approximately the same. This is likely attributed to measurement error of the ATR/FT–IR. In the measurement, the absorbance of the samples changed slightly depending on their contact with the ATR crystal. Thus, no differences in the peak height were observed between PN-1000 and PN-1500.At each reaction step, the prepared beads were characterized using ATR/FT–IR spectroscopy Fig. A. The baSEM performed at each reaction step Fig. B revealeThe prepared PNIPAAm brush-modified silica beads were packed into a stainless steel column . To investigate column performance, the elution behavior of steroids from the column was observed at various temperatures  column to prevent the adsorption of serum proteins, which can cause column clogging, onto the beads in the column. In contrast, previous reports revealed that PNIPAAm copolymers without ionic group-modified bead-packed columns eluted proteins without column clogging because non-ionic PNIPAAm copolymers did not show adsorption of  the proteinsumn Fig. . The colThe average retention time and relative standard deviation of each drug were observed via three repeated measurements  was developed. The system can benefit TDM in medical settings because it does not require sample preparation (deproteinization) or the utilization of organic solvents as the mobile phase.A two-dimensional temperature-responsive chromatography system was developed to determine the drug concentration in a serum sample. PNIPAAm brush-modified silica beads were prepared using surface-initiated ATRP. These beads were packed into a stainless steel column, and the drug retention profiles of these columns were observed. The retention time increased with increasing column temperatures because of the enhanced hydrophobic interactions between PNIPAAm and the drugs. Drugs in the serum proteins were injected into the column, and the serum proteins and drugs were separated in the column based on differences in their hydrophobic interactions. A two-dimensional temperature-responsive chromatography system was developed using two PNIPAAm brush-modified bead-packed columns. The primary column was used to separate the serum proteins and drugs, after which the serum proteins were allowed to flow into the waste. Thereafter, the purified drug was introduced into the secondary column, in which only peaks of the purified drug were obtained. From the chromatogram, a calibration curve exhibiting a high correlation coefficient was obtained, ensuring accurate determination of the drug concentration of serum proteins. The results further indicate that the two-dimensional temperature-responsive chromatography system can benefit TDM by determining the drug concentration in serum without sample preparation (deproteination) and the utilization of organic solvents as the mobile phase.2/g) were washed with hydrochloride for 3 h at 90 °C. The beads were then filtered, rinsed with pure water, and vacuum-dried for 15 h at 150 °C. The silica beads (6.2 g) were placed in a flask and humidified at relative humidity at 25 °C for 4 h. Two silane coupling reagents  and GPTMS ), were added to toluene (124 mL), and the solution was poured into a flask containing the humidified silica beads. The silane coupling reaction was performed for 16 h at 25 °C, after which the beads were washed with toluene and acetone and vacuum-dried for 3 h at 110 °C.As shown in Fig. 2 , and Me6TREN  were dissolved in NIPAAm monomer solution in an Ar atmosphere with stirring for 5 min, after which the flask was sealed and placed in a glove bag. Silane layer-modified silica beads (0.95 g) were placed in a glass vessel (50 mL), and the glass vessel containing the silica beads was placed in the glove bag. The oxygen in the glove bag containing the silica beads and monomer solution was removed by vacuuming and flowing Ar gas three times. Next, the monomer solution was poured into the silica beads in a glass vessel, and the glass vessel was sealed. This vessel was removed from the glove bag, and ATRP was performed for 16 h at 25 °C with continuous shaking of the reaction solution. Subsequently, the bead suspension was poured into two centrifuge tubes, to which acetone was added. The suspension containing the beads was centrifuged for 3 min at 1500 rpm, after which the supernatant was removed. A mixture of 50 mM ethylenediaminetetraacetic acid aqueous solution and methanol (v/v = 1:1) was added to the beads in the centrifuge tubes, and the suspension was sonicated for 30 min and centrifuged for 3 min at 1500 rpm. This procedure was repeated twice. The beads were then filtered, rinsed with pure water and acetone, and vacuum-dried for 2 h at 50 °C. All materials employed in the experiments are summarized in the Supplementary Material.PNIPAAm was modified onto the silica beads via ATRP as follows: NIPAAm  was dissolved in 42.8 mL of 2-propanol in a flask at 1000 mM. To obtain a 1500-mM NIPAAm solution, NIPAAm  was dissolved in 42.8 mL of 2-propanol. The NIPAAm solution was deoxygenated by bubbling with Ar gas for 30 min. CuCl , CuClThe prepared beads were characterized by CHN elemental analysis, FT–IR, and SEM to confirm immobilization of the silane layer and modification of PNIPAAm onto the silica beads.%CS is the increase in the carbon content of the beads after the silane coupling reaction, %CS is the calculated carbon percentage of the mixed silane coupling reagent (CPTMS:GPTMS = 3:1), and S is the surface area of the silica beads (100 m2/g).The carbon composition of the silica beads was determined using an elemental analysis apparatus , and the amount of silane layer was determined from the carbon composition of the beads, as shown in Eq. :1\docume%CN is the increase in the carbon content of the beads after ATRP and %CN is the calculated carbon percentage of NIPAAm.Further, the amount of PNIPAAm brush on the silica beads was obtained as follows:Modification of the polymer via ATRP was confirmed by observing the IR spectrum of the beads via ATR/FT–IR .The morphology of the silica beads at each reaction step was observed using SEM .The prepared beads were packed into two types of stainless steel columns . The prepared PNIPAAm-modified beads were suspended in a methanol/water mixture . This suspension was poured into the reservoir of a column packer connected to an empty stainless steel column. The beads were packed by flowing a methanol/water mixture as the solvent at a constant pressure of 35 MPa for 60 min using an HPLC pump. After packing the beads, the column was connected to an HPLC system , after which it was rinsed for 5 h at 40 °C under continuous flow of pure water at a flow rate of 0.2 mL/min.Column performance was evaluated using hydrophobic steroids (the properties of the steroids are summarized in Supplementary Table 3COONH4 solution (pH 6.75) was employed as the mobile phase at a flow rate of 0.2 mL/min, and the elution behavior of these analytes was observed at a predetermined wavelength for each drug  was used as the mobile phase. The elution behavior of the analytes was observed at predetermined wavelengths for each drug . The serum was prepared by dissolving freeze-dried serum in water (3 mL). For theophylline, sotalol, and phenobarbital, the drugs were dissolved in pure water at 1.0 mg/mL, after which the solution was filtered with a syringe filter (pore diameter: 0.2 μm). The serum solution was added to the drug solution, and the drug concentration in the serum was adjusted to 100 μg/mL. The other drugs were dissolved in tetrahydrofuran at a concentration of 1.0 mg/mL, filtered using a syringe filter, and 0.10 mL of the drug solution was collected into a microtube. The tetrahydrofuran was evaporated by flowing nitrogen gas into the solution. Next, 1.0 mL of serum was added to the drug to adjust the drug concentration to 100 μg/mL. CHSupplementary Information.


Callosciurus prevostii). The links of the animal trade between private breeders and zoos, the likely introduction pathway of VSBV-1 into Germany, and the role of a primary animal distributor were elucidated. In addition, a seroprevalence study was performed among zoo animal caretakers from VSBV-1 affected zoos. No seropositive healthy zoo animal caretakers were found, underlining a probable high-case fatality rate of human VSBV-1 infections. This study illustrates the network and health consequences of uncontrolled wild pet trading as well as the benefits of molecular epidemiology for elucidation and future prevention of infection chains by zoonotic viruses. To respond to emerging zoonotic diseases rapidly, improved regulation and control strategies are urgently needed.The variegated squirrel bornavirus 1 (VSBV-1) is a recently discovered emerging viral pathogen which causes severe and eventually fatal encephalitis in humans after contact to exotic squirrels in private holdings and zoological gardens. Understanding the VSBV-1 epidemiology is crucial to develop, implement, and maintain surveillance strategies for the detection and control of animal and human infections. Based on a newly detected human encephalitis case in a zoological garden, epidemiological squirrel trade investigations and molecular phylogeny analyses of VSBV-1 with temporal and spatial resolution were conducted. Phylogenetic analyses indicated a recent emergence of VSBV-1 in European squirrel holdings and several animal–animal and animal–human spill-over infections. Virus phylogeny linked to squirrel trade analysis showed the introduction of a common ancestor of the known current VSBV-1 isolates into captive exotic squirrels in Germany, most likely by Prevost’s squirrels ( Bornaviridae, species Mammalian 2 orthobornavirus) is such a recently discovered emerging viral pathogen, initially detected by NGS-based metagenomics. VSBV-1 was retrospectively detected in 2015 as the cause of fatal encephalitis in three male private breeders of exotic variegated squirrels (Sciurus variegatoides) in the East of Germany . The three breeders had died in 2011–2013 [S. variegatoides is a species of the Sciurinae subfamily of squirrels originating from Central America. In 2018, VSBV-1 was retrospectively found to be responsible for the fatal limbic encephalitis of a female zoo animal caretaker in the North of Germany who had died in 2013 (case D.2). She had previously cared for Southeast Asian Prevost’s squirrels  in zoo D [Emerging viral pathogens, many of which emerge in clusters or outbreak scenarios and sometimes even as pandemics, are major threats to public and animal health. Owing to advances in next-generation sequencing (NGS) technology, the discovery of novel pathogens in human clinical cases or reservoir animal species has become more feasible. The variegated squirrel bornavirus 1  brain biopsy of a 41-year-old male zoo animal caretaker (case D.1) with encephalitis was retrieved from a pathology archive, after the detection of bornavirus-reactive antibodies in the patient’s serum by an indirect immunofluorescence antibody test (IFAT) using a persistently infected cell line and a line blot assay with recombinant bornavirus phosphoprotein (P) antigen  in earlyThe patient had developed encephalitic symptoms in 2007 and underwent a brain biopsy in the same year. He had been suffering from chronic encephalitis/encephalopathy of unknown origin ever since. He had worked from 1996 to 2007 in the same zoo in Northern Germany (zoo D) where a female animal caretaker (case D.2) had died of VSBV-1 encephalitis in 2013 [For complete VSBV-1 genome reconstruction from three VSBV-1 RT-qPCR-positive Prevost’s squirrels kept in zoos J and L in the Southwest of Germany, Sanger dideoxy-chain termination sequencing or NGS using the Ion S5 XL System with a 530 chip  was performed from squirrel brain tissue ,9. All oIn order to investigate the phylogenetic relationships and the spread of VSBV-1 in captive exotic squirrels in German holdings, two approaches were chosen:8 generations (with 10% burn-in) with subsampling every 104 iterations to achieve convergence as assessed by Tracer v1.5 [http://tree.bio.ed.ac.uk/software/figtree/). Given that the time of sampling of all VSBV-1 sequences included in this study was available, the temporal dynamics and the time to the most recent common ancestor (tMRCA) were estimated.First, a Bayesian maximum clade credibility (MCC) tree was reconstructed for the four newly determined VSBV-1 sequences together with all 22 complete coding VSBV-1 sequences with known time (year) and geographical origin available in GenBank using a Bayesian Markov Chain Monte Carlo (MCMC) approach available in BEAST v1.8 ,11. The cer v1.5 . The MCCSecond, a phylogenetic tree of the VSBV-1 nucleotide sequences was inferred using the ML method implemented in PhyML  with 100Focusing on zoo D in Northern Germany, where human cases D.1 and D.2 had been found infected in 2007 and 2013, respectively, all exotic squirrel trade movements were analysed in cooperation with the respective zoo veterinarians to determine the source of the VSBV-1 infection. Where possible, oral swabs, faecal or serum samples were obtained from the squirrels in these holdings and tested for VSBV-1 RNA or bornavirus-reactive antibodies as described .Within both the private and the zoo sector, information was gathered about the history and the structure of exotic squirrel holdings in Germany and other European countries. Private holders of exotic squirrels were informed in media for small mammal enthusiasts and asked to provide the numbers and species of squirrels kept in their holdings. Concurrently, possible holders and breeders of squirrels in Germany were identified by internet searches on social media or selling platforms and private websites. If contact attempts were successful, holders were interviewed about the origin and whereabouts of their squirrels. Diagnostic molecular testing of squirrels and serological testing for owners were offered. Likewise, European zoos were informed about VSBV-1 via different zoo organizations . Simultaneously, zoos keeping squirrels were identified by consulting zoo inventory databases  to contact them directly for interviews, and free testing of squirrels and staff was offered.If available in the private owners’ records, those of the zoos, or in the European monitoring studbook for Prevost's squirrels compiled by the EAZA, the datasets for each variegated squirrel, Prevost's squirrel and other susceptible species on birth, entry, transfer, and exit dates were aligned to a temporal network and combined with testing results.All present and past animal caretakers of zoo D, who had contact with exotic squirrels, and animal caretakers from other zoos that had exotic squirrel trading connections with zoo D , as well as a participating breeder, were serologically screened for bornavirus-reactive IgG antibodies by IFAT and the line blot assay.The IFAT uses Crandell-Rees Feline Kidney (CRFK) cells persistently infected with BoDV-1 strain V and uninfected cells of the same cell line as controls. Due to high antigenic cross-reactivity within the genus Orthobornavirus, the IFAT also detects antibodies against VSBV-1 . All serPresent, but not past animal caretakers of zoo D had already been serologically tested for VSBV-1 infection in 2018 [In addition, all participating animal caretakers and the breeder were interviewed for possible encephalitic episodes of themselves or fellow co-workers if known. Informed consent was obtained from all participants.Tamiops swinhoei; subfamily Callosciurinae), was sampled on a regular basis. Oral swabs or faecal samples were collected and tested in February 2016, April 2018, July–August 2019 and November 2020. Complete carcasses of deceased Swinhoei’s striped squirrels were submitted for VSBV-1 RT-qPCR analysis in July 2017, April 2019 and November 2020.The current group of remaining exotic squirrels in zoo D, Swinhoei’s striped squirrels .Complete VSBV-1 coding sequences were also generated from brain tissue of two Prevost’s squirrels kept in zoo J  and one Prevost’s squirrel in zoo L .The phylogenetic trees inferred by both Bayesian MCC  and ML aThese Prevost’s squirrel-to-human, variegated squirrel-to-human, Prevost’s squirrel-to-variegated squirrel, Prevost’s squirrel-to-Finlayson's squirrel and Prevost’s squirrel-to-Swinhoei's striped squirrel transitions were supported by a posterior probability (pp.) of 95%. The most likely host species of the common ancestor of all VSBV-1 strains in Germany was the Prevost’s squirrel (pp. 92%).The tMRCA was probably around 2003, with the 95% highest posterior density (HPD) interval for 1998–2007 .Based on information of squirrel owners and contact persons within small mammal specialist groups of private and zoo organizations, the formerly unknown German breeder , who hadThe interviews with the owner of holding X, a 55-year-old male German, revealed that he had started keeping and breeding exotic squirrels in the late 1970s in Western Germany at the age of 12 years. Thereafter, he bought his first Prevost’s squirrels from Indonesia (Sumatra and Borneo) imported by a Dutch animal broker, from whom he obtained additional Prevost’s squirrels in the following decades, lastly in 1997. In 1990, variegated squirrels imported from Nicaragua by another animal distributor were included in his husbandry, which consisted then of about 150–200 animals of more than ten species. He bred and sold the animals and their offspring during the whole period mainly to German and Dutch private holders as well as to few German and Swiss zoos. In 1998, he emigrated to Costa Rica, where he ran a squirrel breeding farm with variegated squirrels and Prevost’s squirrels both introduced from his former German holding. Throughout the years in Germany and Costa Rica, he collaborated closely with the owner of holding F, a 65-year-old male German squirrel breeder, who died of encephalitis of unknown cause in 2005 . After Based on trading information, the likeliest time period of introduction of VSBV-1 into Germany via holding X is in or before 1997. The slow spread of the virus into various other holdings is attributed to transfers of single infected animals, followed by spill-over infections to other captive exotic squirrel species.n = 26), zoo J (n = 5), zoo P (n = 6) and zoo O (n = 15), who had had contact with exotic squirrels, participated in the study. Testing for bornavirus-reactive antibodies was negative for all participants. With the exception of the already known cases D.1 and D.2, no encephalitic episodes of the study participants or of fellow coworkers were reported.A total of 52 present and past animal caretakers of zoo D , thus qualifying them as potential natural reservoirs . HoweverThe striking genetic homogeneity of all currently known VSBV-1 genomes suggests a single introduction of the virus into the known captive exotic squirrel populations in Europe, which is supported by consolidation of phylogenetic analysis and trade network investigations in this study. The Bayesian analysis estimates the most recent common ancestor of the VSBV-1 strains in Europe to have occurred in 1998–2007 (probably around 2003), which is in line with the recorded segregation of the different arms of the trade network in 1997 from holding X. Based on these observations, the introduction of VSBV-1 into German holdings is likely to have occurred prior to 1997 in holding X. Phylogenetic data indicate that the initially infected animals in the here identified network were Prevost´s squirrels, suggesting a possible Southeast Asian origin of VSBV-1 and an introduction via the import of this species.Sciurus vulgaris) from Germany and the UK [Callosciurus erythraeus; n = 35) from Italy and Eastern grey squirrels  from the UK [Examinations of wild squirrel populations and investigations of human encephalitis cases of unknown aetiology for VSBV-1 infection in Southeast Asia are currently under way and may shed more light on the epidemiology and origin of VSBV-1. However, we cannot exclude that Prevost's squirrels do not represent the original host, but have become infected by transmission from another species, either in holding X, in an unidentified holding in Europe or elsewhere prior to its import. Furthermore, the virus may have originated from a single introduction from an unknown local squirrel or non-squirrel reservoir in Europe. However, recent studies failed to detect VSBV-1 in 77 wild Eurasian red squirrels  occurred, followed by a possible human case in 2005 (holder F), and the first proven case (D.1) in 2007. Both human occupation-related fatal encephalitis cases (D.1 and D.2) had independently occurred in the same rather small zoological garden in Northern Germany six years apart. However, the two different animals suspected as the respective sources of infection had originated from two different zoos. The Prevost’s squirrel suspected to be responsible for the spill-over infection to case D.1 in 2007 originated directly from zoo O. It had died untested in 2011, but was identified as the only possible source of infection of case D.1. The squirrel assumed to be responsible for the transmission event to patient D.2 in 2013 originated from zoo P, but its (possibly VSBV-1-infected) ancestors could be traced back via zoo K also to zoo O . This isInterestingly, the sequences of the strains originating from the two different squirrels from zoo J sampled in 2016 and 2019 also suggest two independent virus introductions from German holdings. Again, this is corroborated by the trade analysis, as zoo J received animals from at least three sources with confirmed or suspected VSBV-1 infections . The relative heterogeneity of the seven strains from Prevost’s squirrels originating from holding E  may haveHolder X and all zoo animal caretakers from affected zoos, who participated in the study tested negative for bornavirus-reactive antibodies. Although the presence of antibodies below the detection limit cannot be completely ruled out, there was no anamnestic evidence of a past neurological disease. The only so far detected living seropositive person is breeder E, who reported to have recovered after having suffered from a chronic neurological disease in 2004/05 .In conclusion, exotic pet trade is an important driver of the spread of exotic pathogens to new environments promoting contact with susceptible new hosts. This study demonstrates the spread of VSBV-1 as an emerging pathogen in captive squirrels and spill-over infections to humans. Understanding the epidemiology is crucial to develop and implement surveillance strategies for control. Our report highlights the risk of VSBV-1 transmission in private and occupational settings and the need for monitoring of all exotic squirrel holdings. Annual testing of oral swabs and faecal samples by RT-qPCR is currently recommended  in order


Background: Hospital staffing shortages are again (mid-year 2021) becoming a significant problem as the number of positive COVID-19 cases continues to increase worldwide. Objective: To assess the costs of sending HCW into quarantine (Scenario 1) from the hospital’s and the taxpayer’s perspective versus the costs arising from implementing point-of-care COVID-19 antigen testing (POCT) for those staff members who, despite learning that they have been exposed to hospital patients later found to be infected with COVID-19, continue to report to work (Scenario 2). Methods: A mathematical model was built to calculate the costs of a sample-and-stay strategy for exposed healthcare workers (HCW) in Germany by utilizing a high-quality antigen fluorescent immunoassay (FIA), compared to the costs of quarantine. Direct costs and wage costs were evaluated from the hospital as well as from the taxpayer perspective assuming a SARS-CoV-2 infection prevalence of 10%. Results: Serial POCT testing of exposed HCW in Germany (Scenario 2) who do not go into quarantine but continue to work during a post-exposure period of 14 days at their working place raises costs of EUR 289  per HCW at the expense of the employing hospital while the extra-costs to the taxpayer per exposed HCW are limited to EUR 16 (±20%: EUR 13 to EUR 19). In contrast, sending HCW into quarantine (Scenario 1) would result in costs of EUR 111 (±20%: EUR 89 to EUR 133) per exposed HCW for the hospital but EUR 2235 (±20%: EUR 1744 to EUR 2727) per HCW at the expense of the taxpayer. Conclusions: Monitoring exposed HCW who continued working by sequential POCT may considerably reduce costs from the perspective of the taxpayer and help mitigate personnel shortages in hospitals during pandemic COVID-19 waves. The severe acute respiratory syndrome COVID-19, caused by coronavirus 2 (SARS-CoV-2), first appeared in December 2019 in Wuhan, China, with an accumulation of pneumonia and has since spread across the globe .The rapid escalation of the situation caused the World Health Organization to declare a pandemic on 11 March 2020 . Since tThe current reference test used to establish SARS-CoV-2 infection worldwide is the real-time reverse transcriptase polymerase chain reaction (RT-PCR). However, in Germany, currently 71.5% of all hospitals have eliminated their in-house laboratories . To ensuIn contrast, lateral flow assay (LFA) SARS-CoV-2 antigen tests, which are based on lateral flow technology that uses monoclonal antibodies, can be performed at point of care, i.e., in the hospital itself, provide results within 15–30 min and are inexpensive. Numerous SARS-CoV-2 POC antigen tests are currently available, whose use offers the potential for rapid identification of those individuals who are not only infected, but infectious and are therefore at greatest risk of spreading the infection . AlthougIn our model, we separately calculate the economic consequences of a sample-and-stay strategy, which uses periodic POCT at least once every 48 h together with the wearing of a FFP2 mask compared to the costs of sending these HCW into quarantine during a period of 14-days. We did not include the healthcare costs arising from symptomatically infected HCW requiring treatment; such costs have to be covered by the German Public Statutory insurance and thus lie beyond the perspective of the hospital as employer and that of the taxpayer. Also, additional costs arising through quarantine caused by food suppliers or other services could not be considered.With respect to exposed HCW, two scenarios are considered: In the first one, as usually performed in Germany, the HCW are sampled for a RT-PCR test immediately at the hospital in order not to overlook a very early infection during the gap of 1–2 days when the increasing viral load may still be below the antigen tests’ limit of detection. In case of a negative result, the HCW will then go home into quarantine for 14 days. If the HCW does not have symptoms, a final antigen test must be done after 14 days, the negative result of which ends the quarantine .In the second scenario, by way of derogation from the first one , exposedRelying on recently published figures, we assumed a sensitivity of 80% and a specificity of 98.9% for POC antigen-testing in symptomatically infected COVID-19 subjects . Due to The cost parameters used in our calculation are all derived from fixed or pre-weighted costs. Our calculation does not take into consideration outcomes from clinical trials or different size samples. Therefore, we always use mean costs. Confidence intervals are not provided, as their application to this non-probabilistic model would have been inappropriate. However, we performed a sensitivity analysis by varying all costs by ±20% of the base-case value according to international practice.The incubation period for COVID-19 has a median time of 5–6 days from exposure to onset of symptoms and is thought to extend to 14 days  thereaftPersons fully vaccinated against COVID-19 are exempt from quarantine measures after exposure to a confirmed COVID-19 case  as they Absence due to quarantine, which is usually ordered and checked by the locally responsible health department according to para 28 Section 1 of the German Law on Protection against Infection (IfSG), is fully reimbursed to the hospital employing the HCW by the responsible authority, i.e., by the taxpayer, upon request as long as the HCW does not develop disease—in which case the HCW are put on sick leave and the hospital has to absorb the costs of the sick leave days from that date. That means that during the 14-day period of quarantine, the taxpayer must bear the wage costs as long as no symptoms due to SARS-CoV-2 infections appear.® SARS Antigen FIA, the final result of which is available in 15 min  minus EUR 12.83 [Scenario 2]) per exposed HCW, whereas the net cost of the hospital rises to EUR 231.62.® SARS Antigen FIA, which can claim specificity of nearly 99%, come close to laboratory RT-PCR testing in their ability to very rapidly and reliably exclude the presence in a patient of transmissible COVID-19. In the meantime, asymptomatic staff testing by POC antigen tests has been a core component of some national infection control testing strategies, such as those in the UK, the US and Germany [Newer real-time POC tests, such as the Sofia Germany ,28,29. B Germany , it coulDue to the potentially increasing pressure on hospitals to develop strategies to mitigate healthcare personnel staffing shortages, the CDC —and in tThe infection prevalence in exposed HCW under the strict security standards now in place in German hospitals can generally be considered low, with concerns mostly centered around casual attitudes towards personal protection held by a minority in a given hospital ward. Hence one may assume limited exposure risk, under which a high negative predictive value of POCT in such contacts can be expected. We assumed a moderate test sensitivity of 80% for POC antigen-testing for the 80%-symptomatically infected COVID-19 subjects and a 58.1% sensitivity for the remaining 20% infected persons in our model. As symptomatic patients will always be retested with RT-PCR even if the POCT is negative, no infections of symptomatic patients remain unrecognized. Of note, the number of false-negative results in asymptomatically infected HCW infections during the 14-day incubation period is low (0.84 persons out of 100 HCW). Given the fact that wearing FFP2 masks reduces the risk of contagion by 67% , the risAs the working wages for contact persons of COVID-19 patients in Germany who must remain in quarantine for 14 days are paid by the taxpayer, a total of EUR 2235 would have been transferred to the hospital as employer per exposed HCW. In contrast, serial POCT testing at their workplace (every other day during the 14-day post-exposure period) of exposed HCW who don´t go into quarantine but who volunteer to continue coming to work under a regulatory derogation, results in extra costs at the expense of the employing hospital of only EUR 178 per HCW. Even reducing all costs of the taxpayer by 20% in sensitivity analysis and simultaneously increasing the hospital’s costs by the same percentage shall not change this discrepancy. Although it might seem counterintuitive for a hospital to not accept pay-off, the latter approach avoids a possibly threatening thinning out of the workforce, and thus can be considered a win-win situation.Our model has several limitations: A general limitation of our study that must be kept in mind when interpreting its results is that, as always, a single-center economic model cannot depict the true local SARS-CoV-2 infection prevalence among exposed HCW. Furthermore, we do not know how many HCW would in fact follow the offer to return to work due to missing data. Finally, the probabilities of becoming infected on different days during the quarantine period could only be derived as point estimators from McAloon´s log-normal distribution. Since that model cannot be varied, a more comprehensive sensitivity analysis demonstrating the robustness of our findings was not possible. Therefore, to validate our estimates, prospective cost studies are required.High-quality POC antigen tests, used for serial testing of healthcare workers exposed towards COVID-19 patients in their hospital, can lessen the burden of quarantine on hospital staff. They allow asymptomatic HCW to continue working (or to return to work earlier) on a voluntary basis, while wearing FFP2 masks and maintaining careful self-observation for signs of developing symptoms, and thus help to avoid unnecessary shortage of urgently needed employees. As such, POCT can also reduce costs from the taxpayer perspective and allow resources to be allocated for other precautions. Prospective clinical studies should be undertaken to further evaluate its economic advantages in the immediate future.


In this study, emulsion gel beads for loading quercetin were prepared through an emulsification/gelation process using whey protein isolate (WPI) and pectin. Emulsion gel beads’ properties were investigated by different pectin content. Additionally, the physicochemical properties, morphology and quercetin release properties from beads were explored. Firstly, electrical characteristics and the rheology of bead-forming solutions were measured, revealing that all systems had strong negative charge and exhibited shear-thinning behavior. The textural results demonstrated that the properties of emulsion gel beads were improved with increasing the content of pectin. It was also confirmed that crosslinking was formed between WPI emulsion and pectin by Fourier Transform Infrared (FTIR) analysis and thermogravimetric analysis (TGA). In addition, the shape of the beads was spherical or ellipses with smooth surfaces and they had a tight gel network of internal structures, which was visualized by using electron microscopy (SEM). Finally, the amount of quercetin released in vitro was gradually decreased with increasing pectin content; it was as low as 0.59%. These results revealed that WPI emulsion–pectin gel beads might be an effective delivery system for quercetin as a colon target and are worth exploring further. Quercetin , a plant flavonoid, exists in a variety of vegetables, fruit and grains. Recently, quercetin has attracted considerable attention due to its beneficial biological activities, such as antioxidative, anti-inflammatory, antibacterial and anticancer effects ,2. HowevRecently, several delivery systems have exhibited that the bioaccessibility of quercetin can be improved, including emulsions and hydrogels, etc. ,5,6. EmuHydrogels can absorb a great deal of water due to their three-dimensional network structure, making them ideal materials for functional food or pharmaceutical applications when they are swelled under physiological environments . Among tIn fact, proteins ) and polysaccharides  have been widely investigated as gelling agents. WPI contains more positively charged amino acids (267.9 mg/g) at pH 7.0 ), which are easy to react with anionic polysaccharides through electrostatic attractions. Moreover, WPI has higher solubility and surface hydrophobicity. Several reports have shown that protein solubility affects the interactions between protein and water, and surface hydrophobicity has an impact on protein–oil interactions on droplet surfaces . Simultaneously, pectin can improve the stability of emulsion through slowing down the movement of droplets, decreasing the collision frequency and coalescence rate, which are attributed to increasing the viscosity or forming a gel network in continuous phase. Therefore, the purpose of this study was to investigate the properties of emulsion gel beads loaded with quercetin. WPI and pectin were used as emulsifiers and structural strengthening agents, respectively. The encapsulation of quercetin in WPI emulsion–pectin gel beads was characterized and the release behavior of quercetin in the simulated gastrointestinal tract was estimated. The developed emulsion gel beads will have broad application prospects for delivering and controlling the release of active substances in the food and pharmaceutical industries.− when dissolved in water [The electrical characteristics of WPI emulsion–pectin complex were tested because these determine the nature of the electrostatic interactions between biopolymers . All beain water .In order to assess the rheological properties of the WPI emulsion–pectin complex solutions whether impacted the formation of beads or not, their apparent viscosity were measured b. There The texture characteristics of beads were measured. Hardness is regarded as the force required to attain a certain deformation, and it serves as a measure of food firmness . As show−1, which represented the carbonyl (C=O) stretching of the secondary amide (amide I band), the bending vibrations of the N-H (amide II band) and the N–H stretching (amide III band), respectively. The absorption bands of pectin was detected at 3394, 2926, 1746, 1411 and 1048 cm−1 corresponding to the stretching vibrations of hydroxyl groups, the stretching vibrations of –CH bonds of methyl esters groups or pyranoid ring carbons, the carbonyl (C=O) stretching of the methyl esterified carboxyl groups, asymmetric stretching vibrations of –C–O–C– bonds and −CH groups or methyl esters groups and skeletal C–O and C–C vibration bands of glycosidic bonds and a pyranoid ring, respectively [−1, which were stood for –OH stretching, aromatic ketonic carbonyl stretching, aromatic bending, C=C stretching of the aromatic ring, –OH phenolic bending, and aromatic stretching, respectively [As shown in ectively ,26. As fectively ,28.−1, suggesting that the cross-linking existed between WPI and pectin. In addition, the peaks at 1633 and 1411 cm−1 of pectin were shifted to around 1650 and 1460 cm−1, respectively, indicating that carboxyl groups (–COO−) were cross-linked with Ca2+ [−1 which was a characteristic peak of quercetin . However, it was interested that the peak of 80% pectin beads shifted to 1327 cm−1 because the fraction of WPI emulsion decreased.Furthermore, it was clearly shown that a new peak of 20%, 40%, 50%, 60% and 80% pectin beads was appeared around at 2850 cmith Ca2+ . The encA thermogravimetric analysis of all beads as shown in the The swelling degree is a crucial factor in the release of drugs from delivery systems . Gel beaThe visual appearances, surface and tangent plane morphologies of all beads were determined. The WPI emulsion–pectin gel beads with different concentrations of pectin  for the quercetin delivery system were prepared by an ionic gelation method. The particle sizes of freshly prepared beads depended on the pectin content, and their diameter was significantly increased when the pectin content increased from 20% to 80% . ResearcThe differences in the surface morphology and internal structure of quercetin containing beads (after they were lyophilized) from observing SEM micrographs exhibited in The content of quercetin in WPI emulsion–pectin beads was measured. Compared with WPI emulsion, the encapsulation efficiency of all beads was improved a. The enThe in-vitro release study was aimed to reveal that quercetin could be released slowly from the beads. It was found that the release of quercetin from all samples was below 10% after exposure to simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) in the In this study, a core-shell bioactive delivery system using natural food grade materials was successfully constructed. Beads were fabricated by mixing WPI emulsion with pectin under the ionotropic gelation method. FTIR, TGA and texture methods were used to investigate the effect of pectin content on the properties of beads the gel beads, confirming that incorporation of high pectin content into gel beads affect their texture, mesh-like structure and thermodynamics. These findings are important for producing emulsion gel beads during gelation because the properties of emulsion gel beads may affect encapsulation, stability, and release of hydrophobic functional ingredients embed in emulsion gel beads. As for in vitro digestion, it was found that beads loading quercetin were stable in SGF and SIF, indicating indirectly that it might be a promising delivery system to colon target for quercetin release. Nonetheless, the delivery systems for colon target should be further explored deeply. To sum up, these results might provide valuable information for nutraceutical delivery systems in functional food industry.2) were bought from Sinopharm Chemical Reagent Co., Ltd. . All chemicals were of analytical grade and the water used in this study was deionized.Whey protein isolate (WPI) was purchased from American Davisco chemical co., LTD. The product contained 97.6% protein (dry basis), as determined by the supplier’s standard proximate analysis procedures. Medium-chain triglyceride (MCT) oil and low-methoxy pectin were bought from Yuanye Bio-Technology Co., Ltd.  and Solarbio Co. Ltd. , respectively. Quercetin, pepsin, lipase, pancrelipase and bile salts were purchased from Sigma-Aldrich . Sodium chloride (NaCl), hydrochloric acid (HCl), sodium hydroxide (NaOH) and calcium chloride (CaClThe dispersions of WPI (10 wt%) and pectin (2 wt%) were stirred at room temperature for 2 h using a magnetic stirrer, respectively, and then pH values of dispersions were adjusted to 7.0 with 1 M HCl and NaOH. These solutions were kept overnight to ensure complete dispersion and dissolution.For production of quercetin emulsion: WPI stabilized quercetin emulsion (10 wt% protein) was prepared with 10 wt% medium chain triacylglycerol (MCT) oil containing quercetin  as the dispersed phase and 90 wt% aqueous phase solution at room temperature. The quercetin emulsion was prepared using an Ultra-Turrax  at a speed of 10,000 rpm for 3 min to form coarse emulsions, which were subsequently homogenized by using an ultrasonic cell disruptor  at ultrasonic power was 300 W for 10 min.w/w) calcium chloride solutions by using 5 mL measuring pipets and the distance between the tip of pipet and the surface of CaCl2 solutions was fixed at 10 cm, respectively. The samples were allowed to gel in CaCl2 solutions for 30 min with mild magnetic stirring, and then beads were rinsed with distilled water.For production of emulsion gel beads: the above emulsions mixed with pectin solution  at room temperature for 1 h, and then they were dropped into 2% . All stock solutions were diluted to 500 folds using distilled water (the resistivity was 15 MΩ cm) to avoid multiple scattering effects. After loading the samples into the instrument, they were equilibrated for about 120 s before particle charge data was collected over 11 continuous readings.−1.The apparent viscosity of stock solutions was measured by using a dynamic shear rheometer  with a parallel plate geometry  at 25 °C. The samples were placed on the plates and equilibrated for 2 min before testing. The apparent viscosity of sample was calculated when shear rates were ranging from 2 to 200 sThe texture profile analysis (TPA) tests were performed by using a texture analyzer  equipped with a cylindric shaped probe . The freshly prepared spherical beads (about 2 g) were placed on a fixed bottom plate under the probe at 50% compression. The pre-test, test and post-test speeds were set at 1.0 mm/s, 0.5 mm/s and 1 mm/s, respectively. Furthermore, trigger force was 1 g, and the time between two loadings was 5 s. The texture parameters including hardness, springiness, cohesiveness, resilience, gumminess and chewiness values of beads are calculated by the software Texture Expert Version 1.22.The water content of beads with different pectin content was detected with Moisture Analyzer . The fresh gel beads (about 0.8 g) were placed on the plate made by aluminized paper for measuring.−1 and 60 scans at a resolution of 4 cm−1, and all spectra of the prepared disks were recorded.Fourier transform infrared spectroscopy (FT-IR) spectra was used for detecting WPI, quercetin, pectin and freeze-dried beads. All freeze-dried samples were examined on an FT-IR spectrometer . Firstly, all samples should be mixed with KBr reagent (a mass ratio of 1:50) and were ground into powders. Next, the powders were compressed into disks by using Hydraulic press  at 1.7 tons, which are used for FTIR detection. The wavenumber of FT-IR spectrum was between 4000 and 400 cmThe thermal stability of freeze-dried beads was measured by using Thermogravimetry Analyzer . The sample was placed on a platinum pan and the temperature was raised from 30 °C to 500 °C and the heating temperature was scanned at the rate of 10 °C/min. The weight loss was monitored with respect to time and temperature.s and Wi are the weight of the swollen beads and initial beads, respectively.The swelling behavior of the freshly prepared beads was detected according to the methods previously described ,37. BrieThe morphologies of freeze-dried gel beads were analyzed by a scanning electron microscope  using an accelerating voltage of 5 kV under low vacuum conditions. The surface and internal (cut with a blade) morphologies of freeze-dried gel beads were examined. The beads were affixed to conductive carbon tapes and coated with a thin layer of gold by a sputter coater before observation. Moreover, gel beads diameters were measured using Leica Application Suite EZ software 3.4.0.d is the quercetin concentration that remained in the sodium citrate solution and Ci is the initial quercetin concentration.The amount of quercetin present in beads was quantified using an Ultraviolet spectrophotometer . Before measuring, all beads were added into 2% sodium citrate solution until they were dissolved completely. Subsequently, referring previous study to measure the amount of quercetin . BrieflyThe quercetin-loaded gel beads were used to explore the release behavior in vitro digestion. The method was according to previous research . Briefly


Flaviviridae family anda close relative of a group of important tick-borne viruses that cause humanencephalitis. RNA-dependent RNA polymerase is a significant component of thereplication mechanism of the Flaviviridae viral family. In thepresent work, a three-dimensional model of the Langat virus RNA-dependent RNApolymerase was designed through homology modeling. The experimentally determinedstructure of the RNA-dependent RNA polymerase of Dengue virus type II, anothermember of the same viral family, was employed as template for the homologymodeling process. The resulting model underwent a series of optimisations andits quality was verified using the Verify3D algorithm. Important functionalcharacteristics of the family of viral RNA-dependent RNA polymerases wereidentified in the generated model, thus affirming the potential for its use inthe possible design of anti-viral agents for Langat virus.Langat virus is a member of the  Flaviviridae viral family is asingle-stranded, non-segmented RNA, usually 9.5–12.3 kilobases long. Itcontains a large open reading frame (ORF), with untranslated regions (UTRs) at the5’ and 3’ ends. The 3’ end of the RNA molecule isnon-polyadenylated, while the 5’ region presents variability among thevarious genera  domain , along with several other important pathogensthat cause human disease, such as Tick-borne Encephalitis virus, Powassan virus andOmsk hemorrhagic fever virus . Protein-protein BLAST algorithm . SecondaThe 3D modeling of the Langat virus RdRp was carried out using MOE and the incorporatedstructure-based homology modeling module , to removegeometrical strain. Additional evaluation of the model’s quality wascarried out using Verify3D. This method investigates the similarity of a 3Dmodel with its own amino acid sequence. Considering the environment of everyresidue, an assortment of known, reliable structures is used to assign a scoreto each of the twenty amino acids, resulting in a 1D-3D profile  is the most conserved protein across theflavivirus genus, and its RNA-dependent RNA polymerase domain is of paramountimportance for the successful replication of viruses in theFlaviviridae viralfamily as the root cause continue to pose a threat to human health on a globalscale. Viruses in the Tick-borne Encephalitis virus serocomplex are responsible fora rising number of cases around the world, a fact that calls for attention from thescientific and medical community. Langat virus, a member of the serocomplex, is apathogen which while thus far naturally attenuated when it comes to humans, cannotbe excluded as a potential future threat. From that perspective, the study of Langatvirus’ life cycle is an essential step, in order to be proactive and preparedin case of such outcome. Replication mechanisms are at the core of the virus’life and transmission, which is why the availability of a structure of the Langatvirus RNA-dependent RNA polymerase is of great significance. In the absence of anexperimentally determined structure, the 3D model of the enzyme designed throughhomology modeling can facilitate the analysis of its structure and function in areliable manner. The extraction of this kind of information can pave the way forfurther research approaches. For example, the model of the Langat virus RdRp mayassist in the rational design of anti-viral agents. These agents, such as potentialinhibitors, can be employed in the possible event that Langat virus manifests theability to cause disease in the human population, reinforcing the level ofpreparedness of the medical and scientific community.Viral infections with members of the


Odocoileus virginianus), a species which is highly susceptible to the causative agent of EHD, epizootic hemorrhagic disease virus (EHDV). EHDV has been detected globally across tropical and temperate regions, largely corresponding to the presence of Culicoides spp. biting midges which transmit the virus between ruminant hosts. It regularly causes high morbidity and mortality in wild and captive deer populations in endemic areas during epizootics. Although cattle historically have been less susceptible to EHDV, reports of clinical disease in cattle have increased in the past two decades. There is a pressing need to identify new methods to prevent and mitigate outbreaks and reduce the considerable impacts of EHDV on livestock and wildlife. This review discusses recent research advancements towards the control of EHDV, including the development of new investigative tools and progress in basic and applied research focused on virus detection, disease mitigation, and vector control. The potential impacts and implications of these advancements on EHD management are also discussed.Epizootic hemorrhagic disease (EHD) is an insect-transmitted viral disease of wild and domestic ruminants. It was first described following a 1955 epizootic in North American white-tailed deer ( Orbivirus of the family Reoviridae. It is the causative agent of epizootic hemorrhagic disease (EHD), a disease of wild and domestic ruminants transmitted by Culicoides spp. biting midges  represent the species most severely impacted by EHDV, but several ruminant species, including domestic cattle, can also be infected and develop clinical disease (reviewed in [Epizootic hemorrhagic disease virus (EHDV) belongs to the genus gonidae; ). North iewed in ). EHDV iiewed in ,4,5,6, iiewed in ,8.EHDV was first identified following a 1955 United States (U.S.) outbreak in WTD, with deer mortality so remarkable that it was initially investigated as a mass intoxication event . DescripIn 2015, the state of the EHDV research field was explored through several comprehensive reviews as well as the published outcomes of a 2013 gap analysis; topics included vector management, diagnostic tools, vaccines, transmission, and other knowledge gaps ,22,23. SThe changing patterns and distribution of EHDV provide relevant context in approaching effective virus control strategies, as well as highlight the inherent challenges that these strategies face . Seven sC. sonorensis fed on EHDV-infected deer coincided with the level of viremia. The authors concluded that WTD are most infectious to midges during peak viremia which occurs shortly after EHDV infection [A well-defined animal model is a quintessential research tool for advancing knowledge of pathogen transmission and pathogenesis, and an absolute requirement for countermeasure discovery and development. Despite the inherent challenges of working with WTD in research settings, experimental infections in deer have provided insights into host susceptibility, pathogenesis, and immune responses since the initial determination that EHD was a viral disease ,71,72,73nfection . Unexpecnfection . A recennfection , are undAn apparent recent increase in reported clinical disease among cattle has highlighted the need for additional studies of EHDV in domestic bovids. Some of the recent WTD studies which are discussed above also provided insights into cattle responses to EHDV. An EHDV-6 pathogenesis study in cattle found that viremia and seroconversion were more variable in cattle challenged with the same virus as WTD, and none of the cattle demonstrated clinical, hematologic, or pathologic abnormalities . These fC. sonorensis midges to naïve ECEs, the latter observed with EHDV-1 and -2 but not EHDV-6. In addition to identifying a method to investigate vector competence, the ECE study identified the potential need for further study of C. sonorensis competency for EHDV-6.Not all EHDV researchers have the resources and facilities to perform experiments in target animals, particularly in nontraditional research species such as WTD. Therefore, mammalian cell lines such as baby hamster kidney (BHK) cells, African green monkey kidney (Vero) cells, and calf pulmonary aortal endothelial (CPAE) cells are commonly used for in vitro studies of virus growth kinetics as well as for virus isolation and propagation . The higRecent advances in molecular biology will afford researchers the ability to investigate the influence of EHDV genetics on virus replication, pathogenesis, virulence, and immunity, as well as to develop rationally designed candidate vaccines. Virus-like particles (VLPs), which previously have been used to develop vaccines for, and study the biology of, BTV, have been generated using recombinant structural proteins of EHDV. Four viral capsid proteins  of a field isolate of EHDV-6 were shown to assemble into VLPs with correct morphology and size , thus prNew molecular tools have also been developed to help diagnose EHDV more efficiently, and with increased sensitivity and specificity. TaqMan real-time RT-PCR (RT-qPCR) assays were recently described for both pan-reactive and serotype-specific detection of EHDV-specific RNA, with the ability to detect as few as two copies of viral RNA per reaction . A methoIn the absence of safe and efficacious vaccines, biting midge vector management remains the best available method to reduce EHDV transmission for wild cervids and domesticated animal populations such as pastured beef and dairy cattle, sheep, or captive cervids. Insect vector management (IVM) reduces biting midge populations and limits contact (feeding) events between vector insects and host animals; this will decrease virus transmission during outbreaks if the vector species are known ,95. FurtWolbachia infections may be possible after finding natural populations of midges infected with Wolbachia [C. sonorensis with exotic Wolbachia [To reduce pesticide exposure and non-target impact, new insect-specific management methods are being developed as next-generation pesticides; this technology is based on successes in mosquito management . Sterileolbachia , and alsolbachia . Additioolbachia ,104 may olbachia ,106,107,olbachia ,95,108. olbachia , but theThe advent of safe, effective, and fully licensed, commercially available vaccines for EHDV will provide the potential to significantly change the landscape of EHDV epidemiology and the damaging impacts that EHD historically has had on farmed, and other captive, North American WTD populations. The financial effects of EHD on the deer farming industry are difficult to determine, in part because the disease is clinically indistinguishable from a similar disease caused by BTV infection; however, reports by farmers of devastating animal losses are common during EHDV outbreaks, with mortality rates of up to 80% . Ideal sIdeally, EHDV vaccination would protect animals against the different EHDV serotypes that are present in a given region. However, the ability of the vaccines under development to generate durable universal immunity that can cross-protect between different EHDV serotypes remains uncertain. Observations from the cattle study mentioned above suggested that there is no or only limited cross-neutralization between EHDV serotypes 1, 2 and 6, but stronger cross-reactivity between EHDV-2 and -7, and EHDV-6 and -8 . This isThe emerging patterns exhibited by the ever-evolving epidemiology of EHDV in recent years make the prospect of virus control somewhat daunting. The expanding geographic distribution of various EHDV serotypes and their vectors, and the increasing severity and frequency of outbreaks in both deer and cattle, provide new challenges. However, the growing body of countermeasure tools, resources, and information available to scientists, diagnosticians, clinicians, and producers is poised to yield new, more effective approaches to meet these challenges in the near future. There are ample reasons to believe that the EHDV control landscape, while formidable, looks increasingly promising.


Human rotaviruses (HuRVAs) are highly important causes of acute gastroenteritis in infants and young children worldwide. A lack of reliable and reproducible reverse genetics systems for HuRVAs has limited a proper understanding of HuRVA biology and also the rational design of live-attenuated vaccines. Since the development of the first reverse genetics system for RVAs  in 2006, there have been many efforts with the goal of generating infectious recombinant HuRVAs entirely from cloned cDNAs. However, the establishment of a HuRVA reverse genetics system was very challenging until 2019. This review article provides an overview of the historical background of the recent development of long-awaited HuRVA reverse genetics systems, beginning with the generation of recombinant human-simian reassortant RVAs with the aid of a helper virus in 2006 and the generation of recombinant animal (simian) RVAs in a helper virus-free manner in 2017, and culminating in the generation of recombinant HuRVAs entirely from plasmid cDNAs in 2019. Notably, the original HuRVA reverse genetics system has already been optimized to increase the efficiency of virus generation. Although the application of HuRVA reverse genetics systems has only just been initiated, these technologies will help to answer HuRVA research questions regarding viral replication and pathogenicity that could not be addressed before, and to develop next-generation vaccines and intestine-specific rotaviral vectors. Reverse genetics, a technology that enables the de novo synthesis of live (infectious) viruses from cloned cDNA(s), is a most instructive tool to understand multiple aspects of virus biology , as well as to generate vaccine seed strains and viral vectors. Since the first generation of an infectious RNA virus with a single-stranded RNA (ssRNA) genome of positive-sense orientation, bacteriophage QB, in 1978, numerous reverse genetics technologies have been developed for the majority of important viruses in the human population. Common reverse genetics approaches permit the rescue of infectious recombinant viruses upon transfection of cDNA(s) into cultured cells .Rotavirus and family Reoviridae. RVAs are ubiquitous intestinal pathogens causing severe diarrhea in the young of many mammals, including humans, and avian species worldwide. An RVA virion contains an 18.6 kb 11-segment dsRNA genome, which encodes 11 or 12 viral proteins, six structural ones , and five or six non-structural ones (NSP1-NSP5/6) [https://rega.kuleuven.be/cev/viralmetagenomics/virus-classification/rcwg) (accessed on 10 July 2021).Group A rotaviruses (RVAs) are non-enveloped, double-stranded (ds)RNA viruses belonging to the genus -NSP5/6) . All gen-NSP5/6) . An RVA -NSP5/6) . The 11-Human RVAs (HuRVAs) are primary pathogens of childhood severe diarrheal disease and are associated with high morbidity and mortality. HuRVA gastroenteritis is associated with ~128,500–215,000 deaths each year in children <5 years of age ,8. Despi6 plaque-forming unit (PFU)/mL) is still very low compared to that of animal RVAs (~108 PFU/mL) [Many animal RVAs can grow to high titers in cell lines; however, HuRVAs are more difficult to adapt to cell culture ,10,11. A PFU/mL) ,15.The other barrier in the HuRVA research field was the inability to develop a reverse genetics system for HuRVAs. The development of reverse genetics systems has been very difficult even for high-yield animal RVAs. The nucleotide sequences of all the genomic segments of simian RVA strain SA11 had been determined in 1990 ; howeverIn contrast to positive-sense ssRNA viruses, the naked genomic dsRNAs of RVAs alone do not initiate the production of infectious viruses when transfected into permissive cells. In the RVA replication cycle, a positive-sense ssRNA with a 5’ cap acts as both the mRNA and template for negative-sense ssRNA synthesis to yield genomic dsRNA . NotablyThe breakthrough in genetic engineering of live RVAs came in 2006, when Komoto et al. (2006)  developeBy employing the original partially plasmid-based reverse genetics system and modifications of it, novel infectious recombinant RVAs with a cDNA-derived modified VP4 ,22,23, NAs described above, strong selection condition(s) to isolate a recombinant RVA from the vast majority of helper viruses have to be developed for partially plasmid-based reverse genetics systems, but such conditions have been developed for only three of the 11 gene segments  over the past 15 years. Therefore, rotavirologists could not manipulate RVA genomes at will using the initial reverse genetics systems. However, several important lessons could be learnt from these primary systems, that is, intracellular synthesis of artificial RVA positive-sense ssRNAs with precise 5’ and 3’ ends from transfected rescue T7 plasmids was the key to the successful recovery of recombinant RVAs, suggesting that transfection of cultured cells with a set of 11 rescue T7 plasmids encoding a whole RVA genome could lead to the production of replicating RVAs in a helper virus-free manner. Thus, to overcome the limitation that helper virus-dependent reverse genetics systems are restricted to only three of the 11 genomic segments, rotavirologists devoted themselves to the development of a helper virus-free RVA reverse genetics system. Kanai et al. (2017)  reported5) can be obtained following introduction into BHK-T7 cells of just a set of 11 rescue T7 plasmids encoding a genome of SA11-L2 virus with 3-fold increased amounts of two rescue T7 plasmids for the NSP2 and NSP5 segments    describe system) . By usinVery recently, a further optimized 11 plasmid-only system-based reverse genetics system with increased virus generation efficiency was developed (Sánchez-Tacuba’s reverse genetics system)  Figure . SánchezAnimal RVAs, such as simian SA11-L2 and RRV viruses, and HuRVAs share many features, but they differ in such as their host range, antigenic structures on outermost capsids, VP7 and VP4, and receptor-binding specificities.50) in pup infection experiments than simian RVAs [RVAs are generally host restricted, although the interspecies transmission of RVAs with or without reassortment can occur in rare cases ,46. HuRVian RVAs ,56. Lastian RVAs ,58. TherThe development of the first HuRVA reverse genetics system was reported by Komoto et al. (2019) . As a HuTo generate infectious recombinant KU virus entirely from plasmid cDNAs, BHK-T7 cells were transfected with a set of 11 rescue T7 plasmids encoding a genome of KU virus according to the 11 plasmid-only system . HoweverBecause the functionalities of at least 10 rescue T7 plasmids for strain KU could be confirmed, the authors then modified the recovery procedure for recombinant HuRVAs for efficient multiplication of a small number of nascent recombinant KU viruses within the transfected BHK-T7 cells. As modifications, the authors employed the roller-tube culture technique and a 3-fold increased trypsin concentration in the maintenance medium for co-culture of transfected BHK-T7 cells and overlaid CV-1 cells, and a roller-tube culture with MA104 cells. These processes are beneficial for HuRVA propagation, and similar to the procedures for the isolation by cell culture of fastidious HuRVA strains from diarrheic stool specimens from RVA gastroenteritis patients. These modifications led to the successful recovery of infectious recombinant KU virus (named rKU virus). The rKU virus showed similar growth kinetics and plaque size to the native KU virus, demonstrating that the replication characteristics of rKU virus are indistinguishable from those of the native KU virus. Thus, the minor modifications raising the efficiency of isolation of a small number of recombinant HuRVAs from the transfected BHK-T7 cells were revealed to be the critical steps in the development of a HuRVA reverse genetics system.After this initial report of the development of a HuRVA reverse genetics system, the engineering of two other HuRVA strains was described ,65. SáncReoviridae appear to be nonrandom [The segmented nature of the RVA genome allows for reassortment between/among two or more viruses within a co-infected cell, and this phenomenon has been harnessed to generate mono-reassortant viruses for determining the function(s) of a gene segment and their use as live-attenuated vaccines such as worldwide-used RotaTeq . The reassortment of RVAs can occur at a high frequency both in vitro ,78,79,80onrandom ,83,84,85onrandom ,87. Reveonrandom  demonstronrandom  reportedIf a novel RVA strain possessing a new genotype emerges in humans, the VP7 and/or VP4 genes from the newly emerging RVA can be amplified by reverse transcription (RT)-PCR or quickly chemically synthesized, and then cloned into rescue T7 plasmids used for virus engineering by reverse genetics. The reverse genetics approach will speed up the preparation of possible vaccine seed viruses against newly emerging HuRVAs from 2–6 months to 2 weeks, bypassing the need to isolate and evaluate the reassortants. Moreover, compared to classical procedures for the preparation of mono-reassortants, a reverse genetics approach provides a direct and more reliable technique for the purpose.Because HuRVA disease remains a major medical problem, generation of recombinant HuRVAs to control the disease is a long-awaited achievement for the clinically important viral pathogens in humans. HuRVA reverse genetics systems are highly valuable techniques that have the potential to increase our understanding of the molecular biology of HuRVAs and provide a new way of vaccine design to ultimately control HuRVA disease. That is, recombinant HuRVAs engineered in this way can be employed to address multiple issues regarding virus-host cell interactions during HuRVA replication, such as cell entry, uncoating, genome replication, gene transcription, packaging, maturation, and viral release. Although there are no antivirals against HuRVA disease currently, the identification of any amino acid sequence that interacts with a cellular factor could lead to the development of new antivirals. Currently licensed and used live-attenuated RVA vaccines are safe and efficacious, but one of the greatest concerns about the use of the live vaccines is their genetic stability and hence their reversion to pathogenic wild-type viruses. Clearly, reverse genetics technology can also be exploited to produce live-attenuated vaccine strains with several amino acid replacements and/or deletions in gene segment(s), making reversion to the wild-type virus highly unlikely. Moreover, recombinant HuRVAs can be generated from just 11 rescue T7 plasmids for an RVA genome without the need of any helper expression plasmid in the 11 plasmid-only system, which is possibly suitable for clinical research purposes as well as basic ones. On the other hand, reverse genetics approaches with increased efficiency of virus generation requiring helper expression plasmid(s) may be more suitable for basic research purposes. In any case, the future appears to be bright for progress in understanding the molecular mechanisms that regulate HuRVA replication and pathogenesis, and in developing next-generation vaccines to prevent HuRVA disease and rotaviral vectors of intestine-specificity. HuRVA reverse genetics will serve as a useful platform for a new generation of HuRVA investigations in both basic and clinical research areas.


Tissue pathogens are commonly encountered in histopathology and cytology practice, where they can present as either benign mimickers of malignancy or true malignancies. The aim of this review is to provide a timely synthesis of our understanding of these tissue pathogens, with an emphasis on pertinent diagnostic conundrums associated with the benign mimickers of malignancy that can be seen with viral infections and those which manifest as granulomas. The oncogenic pathogens, including viruses, bacteria, and parasites, are then discussed with relationship to their associated malignancies. Although not exhaustive, the epidemiology, clinical manifestations, pathogenesis, and histological findings are included, along with a short review of emerging therapies.  Cancer is the leading cause of premature death in 57 countries, along with cardiovascular diseases in 70 countries, and it is anticipated that cancer may supersede cardiovascular diseases as the leading cause of worldwide premature death in this century . Based oInfection-attributable cancers account for 15.0% to 20.0% of the worldwide cancer burden ,7. NewerThe International Agency for Research on Cancer (IARC) has classified ten infectious pathogens as carcinogenic to humans (group 1) which include H. Pylori, HBV, HCV, HPV, Epstein–Barr virus (EBV), human herpesvirus type 8 (HHV-8), human T-cell lymphotropic virus type 1 (HTLV-1), Opisthorcis viverrini (Ov), Clonorchis sinensis (Cs), and Schistosoma haematobium (Sh) . Human iThe emergence of the novel coronavirus SARS-CoV-2 (COVID-19) in late December 2019 has shifted much attention to controlling this global health crisis . There iHSV is a member of the Herpesviridae family of viruses which have enveloped double-stranded DNA genomes ,18. ThisHSV1 is typically implicated in orofacial infections  and sporadic encephalitis, while HSV2 is typically implicated in genital infections  and may be transmitted from mother to newborn . As an aIn modern medicine, microbiologic confirmation can be obtained by polymerase chain reaction (PCR) or viral isolation . HSV virHSV viral cytopathic infection can mimic malignancy on cytology. In a retrospective study of 18 cases of respiratory cytological specimens with the diagnosis of HSV infection, only 28% had classic viral cytopathic changes while 22% showed atypical squamous cells with dense keratinised cytoplasm, hyperchromatic nuclei with irregular contours, features which can be mistaken for a squamous cell carcinoma (SCC) . G. G71]. GThe presence of granulomas associated with tumours is well established, reportedly seen in 4.4% of carcinomas, 13.8% of Hodgkin’s disease, 7.3% of non-Hodgkin lymphomas , and 50.In particular, granulomatous mastitis, a granulomatous inflammation of breast parenchymal tissue, although uncommonly encountered, can be mistaken for a malignancy, both clinically and radiologically . A serieHBV and HCV are hepatotropic viruses implicated in acute hepatitis, chronic liver disease, and cirrhosis as well as hepatocellular carcinoma (HCC). Hepatitis B is a member of the Hepadnaviridae family of viruses. It bears a double-stranded DNA genome, which is able to integrate into the DNA of the host genome. There are 10 major genotypes of which genotype C and D are associated with a higher risk of progression to liver cirrhosis . In chilOn the other hand, Hepatitis C is a member of the Flaviviridae family of viruses. It bears a single-stranded RNA genome. There are seven major genotypes of which genotype 1 is associated with more aggressive disease course and reduced responsiveness to therapy. Once acquired, Hepatitis C persists as a chronic infection in approximately 80% of hosts . Clinical diagnosis of Hepatitis B or Hepatitis C infection can be achieved via serology or molecular methods. The role of histopathologic examination is primarily in the grading and staging of liver disease. Of note, the accumulation of Hepatitis B surface antigen (HBsAg) in hepatocytes gives a ground glass appearance to the cytoplasm which stains magenta and blue on interrogation with the Shikata’s orcein stain A and VicApproximately 70–85% of cases of HCC worldwide are associated with Hepatitis B and Hepatitis C infections . DespiteThe management of HCC includes surgical resection, chemoembolisation, radioembolisation, and liver transplantation . SorafenHuman papillomavirus (HPV) is a member of the Papovaviridae family. It is a non-enveloped DNA virus with a predilection for cutaneous and mucosal epithelium. More than 90% of people who acquire the virus experience transient infection and can immunologically clear the virus. In contrast, chronic persistent infection, contributed to by various factors such as immunocompromise, frequent reinfections and co-infections, is associated with progression to neoplasia . HIV staMore than 100 types of HPV virus have been isolated and sequenced to date. These can be broadly subclassified into low-risk and high-risk types, based on malignant potential. Low-risk types include HPV 6 and 11 which are associated with genital warts and respiratory papillomatosis. High-risk types include HPV 16, 18, 31, 33, 45, and 51, which are associated with malignancies of the cervix, anogenital region, and oral cavity. The central oncogenic event is the integration of the viral genome into the host genome, which results in the loss of the viral E2 repressor leading to increased expression of oncogenic proteins E6 and E7 that inhibit p53 and Rb proteins, respectively . In the cervix, the neoplastic process is represented by the progression across well-defined morphologic categories of cervical intraepithelial neoplasia (CIN) 1, 2, and 3, and finally invasive SCC. The hallmark of HPV infection is koilocytes (hollow cell), which are mature squamous cells that show binucleation, nuclear hyperchromasia, nuclear contour irregularities and enlargement, as well as the characteristic perinuclear halo  that gives the cell its name C,D 95]..95]. HisRemarkably, our understanding of the role of HPV in cancer continues to evolve, with the recognition of new entities such as HPV-related multiphenotypic sinonasal carcinoma (associated with HPV type 33) , and thePublic health measures such as cervical cancer screening programmes using Papanicolaou smears, liquid-based samples or primary HPV testing by molecular methods, and universal HPV vaccination have seen great success in developed countries which have witnessed a decline in rates of squamous cell carcinoma of the cervix over the years. These public health measures hold great promise in less developed populations that continue to suffer from relatively high rates of SCC of the cervix, an increasingly preventable cancer. The management of cervical cancers depends on the stage of disease, with options including radical surgery and radiotherapy, as well as neoadjuvant chemoradiotherapy . ImmunotEpstein–Barr Virus (EBV) is a member of the gammaherpesviruses subfamily. It is a ubiquitous virus acquired by most people by young adulthood. The virus is transmitted by saliva and infects epithelial cells and B cells of the oropharynx, causing a predominantly lytic infection in the former while establishing latency in the latter . In immunocompetent hosts, primary infection is typically asymptomatic or presents as infectious mononucleosis, a classic constellation of fever, tonsillitis/pharyngitis, lymphadenopathy, and atypical lymphocytosis. The diagnosis can usually be made on clinical grounds supported by serologic confirmation. Rarely, atypical clinical presentation leads to consideration of biopsy of the enlarged tonsil or lymph node . InterprIn the immunocompromised, such as in a patient with HIV, however, an uncontrolled B cell lymphoproliferation results, leading to acquisition of additional genomic alterations culminating in a monoclonal proliferation that amounts to lymphoma ,105. BroThe development of prophylactic EBV vaccines for young children (prior to acquisition of the virus) or in pre-transplant patients is a subject of ongoing research . TherapeHHV8, also termed Kaposi sarcoma (KS)-associated herpes virus, belongs to the gammaherpesvirus subfamily and is a large double-stranded DNA within an enveloped capsid . It is aThe incidence of KS is highest in Central Africa . The epiThe virus is transmitted primarily through sexual contact, especially through male homosexual contact, with other routes of transmission including mother-to-child, saliva, organ transplantation and other unknown routes ,112. TheThe pathogenesis and oncogenesis of HHV8 in the associated malignancies have been extensively reviewed ,117,118.Histologically, the various KS subtypes show an identical morphologic appearance. In the patch stage, there is a proliferation of vascular spaces in the upper reticular dermis, with flattened endothelial cells  Figure D,E. In tPEL is a large B-cell neoplasm which shows a spectrum of morphological appearances ranging from immunoblastic or plasmablastic to anaplastic appearance, with large nuclei and prominent nucleoli within an abundant basophilic cytoplasm . Some ceChemotherapy with anthracyclines and taxols is the mainstay of treatment for KS . MultiplHTLV-1 was the first human retrovirus to be discovered, and consists of an enveloped single-stranded RNA . HTLV-1 HTLV-1 infection is associated with adult T-cell leukemia/lymphoma (ATL) and benign entities such as HTLV-1 associated myelopathy/spastic paraparesis . There aThe current treatment options of ATL include observation, zidovudine and interferon-alfa, chemotherapy, or allogeneic haematopoietic stem cell transplantation . In a meMCPyV belongs to the family of human polyomaviruses, along with BKPyV. It is a recently identified virus implicated in the pathogenesis of merkel cell carcinoma (MCC), where the genomic sequences of MCPyV were detected in 80.0% of MCC . It is aThe pathogenesis of MCC were previously elaborated in greater details ,145,146.Histologically, the tumour is located in the dermis and/or subcutis, with cell size ranging from small to large, and the nuclei showing the characteristic salt-and-pepper chromatin seen in neuroendocrine neoplasms as well as nuclear moulding G,H 147]147]. BriThe management of MCC includes wide local excision, assessment of regional lymph nodes with a consideration for sentinel lymph node biopsy if clinically indicated, radiotherapy, chemotherapy, and immunotherapy, with promising results reported in clinical trials investigating the efficacy of anti-PD-L1 therapies such as pembrolizumab . TherapeHelicobacter pylori (H. Pylori) is a member of the Helicobacteraceae family of bacteria. It colonises the gastric epithelium of about half the global population . The bacWhile H. Pylori was first discovered through histologic evaluation of biopsies and microbiologic cultures, many non-invasive diagnostic tools are available today, for instance, urea breath test and stool antigen test . NeverthOn tissue sections, H. pylori are represented as spiral-shaped organisms, 5µm or less in length, usually located in the lumen of the superficial antral glands. Additional vigilance is required in the detection of these organisms in patients on proton pump inhibitor therapy which allows the organisms to colonise the oxyntic mucosa and the deeper glands. When present in low quantities, immunohistochemistry for H. Pylori may be used as an ancillary diagnostic tool I. AccuraChronic H. Pylori infection has been implicated in gastric adenocarcinoma J,K, gastThe management of gastric adenocarcinoma includes surgical resection, chemoradiotherapy, targeted therapy, and immunotherapy with the use of agents like pembrolizumab, trastuzumab, and ramucirumab . PD-L1 hOv and Cs are liver flukes, members of the Opisthorchiidae family . Os is pOv and Cs infections are typically asymptomatic, but mild symptoms such as abdominal pain and diarrhoea may occur, and more severe symptoms with hepatomegaly and malnutrition may be seen with chronic infections ,161. ParChronic Ov infection shows characteristic histological changes such as periportal and periductal fibrosis in the liver, and adenomatous hyperplasia and epithelial hyperplasia in the gallbladder , while CThe management of cholangiocarcinoma typically involves surgical resection, but most patients are not surgical candidates at the time of presentation and there is limited benefit of systemic chemotherapy . There aSh belongs to the Schistosomatidae family, and are blood flukes , locatedThe pathological findings of urinary schistosomiasis are related to the inflammatory reactions to parasitic egg deposition , and theFor treatment of urinary schistosomiasis, praziquantel is the primary therapy but it is ineffective against juvenile schistosomes . Non-invAt the time of writing, there are over 200 million cases of COVID-19 worldwide . PredomiPathogens are frequently encountered in the practice of anatomical pathology, with some presenting as benign mimickers of malignancies and others as more commonly seen malignancies. Considering many of the oncogenic pathogens are endemic in certain parts of the world and are often asymptomatic, there may be a greater need for early detection of these pathogens as well as identification of the pre-malignant manifestations to initiate prompt medical intervention. With increasing attention given to personalised medicine, molecular diagnostics and therapeutics, it is likely that more knowledge will be uncovered about the oncogenic molecular pathways associated with the various oncogenic pathogens. Furthermore, with an accelerated growth in the research around anti-viral and anti-cancer vaccines, it is envisioned that more will be known about these pathogens and how they may be eradicated.