In order to meet the domestic urgent needs of evaluating the immunogenicity of vaccines and the potency testing of therapeutic antibody products against coronavirus disease 2019 (COVID-19), the first Chinese national standards for SARS-CoV-2 neutralizing antibody were established. The potency and stability of the candidate standards were determined by neutralization assay and accelerated degradation study. The stability studies showed that the standards were stable in the short-term. The collaborative study showed that the candidate standards could reduce the variations in neutralization titers between labs and thus improve comparability of neutralizing antibody measurements. Sample 22 has been approved by the Biological Product Reference Standards Sub-Committee of the National Drug Reference Standards Committee as the first Chinese National Standard for Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) neutralizing antibody, with an assigned potency of 1,000 units per milliliter (U/ml). This standard will contribute to the standardized assessment of the quality and efficacy of vaccines and therapeutics for COVID-19 in China. Written informed consents were obtained from all the volunteers.2.2Two plasma samples from convalescent patients were first tested for hepatitis B virus deoxyribonucleic acid (HBV DNA), hepatitis C virus ribonucleic acid (HCV RNA), human immunodeficiency virus (HIV) RNA, and SARS-CoV-2 RNA, as well as the corresponding Hepatitis B surface antigen (HBsAg), HIV-1 / HIV-2 antibody, HCV antibody, and syphilis antibody. Meanwhile, the plasma was inactivated at 56 °C for 30 min.4 cells) were added to each well. Following 24 h of incubation in a 5% carbon dioxide (CO2) environment at 37 °C, the relative light unit (RLU) was detected according to the instruction manual provided by PerkinElmer . The neutralization titers were defined as 50% maximal inhibitory dilutions and calculated with the Reed-Muench method. Inhibition percentage was calculated as the following: [1− (RLU in test samples − RLU in cell controls) / (RLU in virus controls − RLU in cell controls)] × 100%.The neutralizing antibody titer of convalescent COVID-19 patient plasma was detected by a pseudo virus neutralization assay as described previously − 30 °C.The plasma with higher titer neutralizing antibodies was selected to prepare candidate standards for this study. After being inactivated, the plasma was placed in the biosafety cabinet until it reached room temperature, and then kept at 4 °C overnight. The next day, the plasma samples were centrifuged at 5,000 revolutions per minute (rpm) for 15 min. The supernatant was separated and packed into 0.5 ml (ml) screw cap tubes, with each tube containing 100 Âµl (ul), which was then sealed and stored at 2.3•Sample 22 was convalescent COVID-19 patient plasma provided by Boya Bio-pharmaceutical Group Co., Ltd.•Sample 77 was convalescent COVID-19 patient plasma provided by Sinopharm Wuhan Plasma-derived Biotherapies Co., Ltd.•Sample 44 was prepared by mixing SPF rabbit antiserum  with plasma from healthy donors.•Sample 99 was prepared by mixing rabbit antiserum  protein) with plasma from healthy donors.•Sample 55 was human plasma, which was derived from a large mixture of plasma from healthy donors.A total of five samples were provided to participants:2.4The following eleven labs engaged in the collaborative study: the Institute of Microbiology, Chinese Academy of Sciences; the Institute of Medical Biology, Chinese Academy of Medical Sciences ; the Sinocelltech Ltd.; the Wuhan Institute of Biological Products Co., Ltd.; the Institute of Biotechnology, Academy of Military Medical Sciences; the Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences; the Sinovac Biotech Ltd.; the Beijing Institute of Biological Products Co., Ltd.; the Division of Arbovirus Vaccines, National Institutes for Food and Drug Control (NIFDC); the Division of HIV/acquired immunodeficiency syndrome (AIDS) and Sexually Transmitted Virus Vaccines, NIFDC ; and the Division of Blood Products, NIFDC. All labs are assigned code numbers and not reflected in the order presented in 2.5et al.The NIFDC organized this collaborative study. The eleven labs mentioned above participated in the study. Live virus- and pseudo virus-based neutralization assays were used to detect the neutralization titer against SARS-CoV-2 . The pse2.6Due to the inadequate time and plasma with high titer neutralizing antibody against SARS-CoV-2, we did not conduct long-term stability studies on Sample 22, and only did partial temperature acceleration tests in this study. Sample 22 was separately stored at − 30 °C, 4 °C, and 25 °C, and the titers of neutralizing antibody were determined on days 7, 11, 18, 27, and 33 by pseudo virus neutralization assay.2.7et al.50 (median effective dilution) was calculated using the Reed-Muench method The pseudotyped viruses corresponding to the SARS-CoV-2 variants B.1.1.7, B.1.351 and P.1 were constructed using the methods reported in our previous papers 2.8The neutralization titers in the collaborative study samples were assessed by neutralization assays based on pseudo virus or live virus. For the pseudo virus-based neutralization assay, the neutralization titer was calculated by Reed-Muench method based on the results of the neutralization inhibition rate Minitab 17 was used for statistical analysis. GraphPad Prism 8  was used for correlation analysis. Based on the results of the pseudo virus neutralization assay, parallel-line analysis (PLA 3.0 software) was used to trace the first Chinese national standard for SARS-CoV-2 neutralizing antibody (Sample 22) to the WHO IS (Sample G).33.1To screen plasma with high neutralizing potency, we detected the neutralizing antibody against SARS-CoV-2 in two plasma samples from Boya Bio-pharmaceutical Group Co., Ltd. and Sinopharm Wuhan Plasma-derived Biotherapies Co., Ltd. The geometric mean values of the neutralizing antibody titers in the two samples were 1307 and 136, respectively. The BOYA sample (No. 22), with a higher neutralizing antibody titer, was selected as the candidate national standard for SARS-CoV-2 neutralization antibodies.3.2Ten of the eleven labs returned the results of the pseudo virus neutralization assay, which are listed in the Table S1. Four labs returned live virus neutralization assay results, which are shown in the Table S2. Among them, both Lab JS and Lab ZY returned one set of pseudo virus neutralization assay results and one set of live virus neutralization assay results. Lab WS returned one set of pseudo virus neutralization assay results and two sets of results from two different live virus neutralization assays. Sample 55 was a negative control; the results of different labs were consistent and will not be analyzed in the future study.3.3The geometric mean potencies for samples in the pseudo virus neutralization assay are shown in 3.4The geometric mean estimates for samples in the live virus-based neutralization assay are listed in 3.52 = 0.9781)  .Fig. 2Co3.6Our previous results showed that the geometric mean of the neutralizing antibody titer of Sample 22 was 1,460 . The results of this accelerated test showed that within one month, whether Sample 22 was stored at − 30 °C, 4 °C, or 25 °C, the changes in the titer of the neutralizing antibodies were within the 95% confidence interval. There was no significant loss of neutralization activity at 4 °C and 25 °C relative to the − 30 °C baseline for Sample 22 . These r4Due to the high pathogenicity and infectivity of SARS-CoV-2, COVID-19 has become a global pandemic, causing tremendous devastation to the world. Currently, there are no approved effective anti-viral products or vaccines against SARS-CoV-2. Accurate determination of neutralizing antibodies against SARS-CoV-2 is critical for evaluating the immunogenicity of vaccines, assessing the potency of antibody products, and establishing quality control standards for vaccines and antibody products. Both the live virus-based neutralization assay, such as PRNT and CPE, and pseudo virus-based neutralization assays were used to detect neutralization antibody titers et al. reported a strong correlation between the neutralization antibody titers generated by pseudo virus and live virus systems To assess the suitability of candidate national standard virus neutralization assays, the collaborative calibration of five samples was conducted in eleven labs across China. In this collaborative study, although the method of pseudo virus neutralization assay was unified, there were differences in the testing conditions, cell status, and personal operations in various labs, which affected the consistency of results between labs . In the 5Sample 22 was approved by the Biological Product Reference Standards Sub-Committee of the National Drug Reference Standards Committee as the first Chinese National Standard for SARS-CoV-2 neutralizing antibody in September 2020, with an assigned potency of 1,000 U/ml (Lot: 280034–202001). The WHO IS for anti-SARS-CoV-2 antibody was established in December 2020, with an arbitrary assigned unitage of 250 IU/ampule for neutralizing activity 6The collaborative study of the first Chinese national standard for SARS-CoV-2 neutralizing antibody only used the methods of detecting the neutralizing antibody titer, and did not include the methods of detecting the binding antibody such as ELISA. The neutralization potency of the first Chinese national standard for SARS-CoV-2 neutralizing antibody was affected by spike substitutions found in variants of concern (VOC). Compared with the Wuhan-1 reference strain (Wild Type), the neutralizing titer of the national standard against B.1.1.7 decreased by 1.9-fold, and the neutralizing titer against B.1.351 and P.1 reduced by more than 10-fold . In the The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Viruses, and in particular the deformed wing virus (DWV), are considered as one of the main antagonists of honey bee health. The ‘suppressed in ovo virus infection’ trait (SOV) described for the first time that control of a virus infection can be achieved from genetically inherited traits and that the virus state of the eggs is indicative for this. This research aims to explore the effect of the SOV trait on DWV infections in queens descending from both SOV-positive (QDS+) and SOV-negative (QDS–) queens. Twenty QDS+ and QDS– were reared from each time four queens in the same starter–finisher colony. From each queen the head, thorax, ovaries, spermatheca, guts and eviscerated abdomen were dissected and screened for the presence of the DWV-A and DWV-B genotype using qRT-PCR. Queens descending from SOV-positive queens showed significant lower infection loads for DWV-A and DWV-B as well as a lower number of infected tissues for DWV-A. Surprisingly, differences were less expressed in the reproductive tissues, the ovaries and spermatheca. These results confirm that selection on the SOV trait is associated with increased virus resistance across viral genotypes and that this selection drives DWV towards an increased tissue specificity for the reproductive tissues. Further research is needed to explore the mechanisms underlying the interaction between the antiviral response and DWV. Varroa destructor [Over the last decades, honey bee health has been severely affected by the presence of the ectoparasitic mite structor ,2,3,4,5.structor , virus tstructor . During structor . This nestructor ,10,11. Astructor . Infectistructor ,14. Notwstructor ,16,17,18structor . Being rstructor ,16 and ostructor , DWV canVarroa destructor virus-1 [The name ‘deformed wing virus’ groups multiple genetically similar viruses of the Iflaviridae family . At pres virus-1 ) is show virus-1 ,9,19. Th virus-1 .Transmission of viruses within and between colonies can be divided in vertical and horizontal transmission. Horizontal transmission is the spread of a virus between individuals of the same generation through either bodily contact, trophallaxis, feeding or common flower visits. Vertical transmission is the spread of a virus from parents to their offspring and can occur via eggs or semen . Where hIn 2012, Belgian beekeepers started screening breeding queens for the presence of viruses in eggs deposited in worker brood cells and found 75% to be infected with at least one virus and 40% to be infected with DWV . These hSo far, the SOV trait has not yet been described from the perspective of the queen and has been focused on the DWV-A genotype. In this paper, the infection patterns of DWV in the different tissues  of queens descending from SOV positive (QDS+) and SOV negative (QDS–) queens are presented. In addition, DWV is quantified for both the DWV-A and DWV-B genotype to gain insights in the effects of the increased virus resistance associated with the SOV trait on different genotypes. As viruses are one of the most important threats for honey bee health, a better understanding of the interaction between virus resistance and the virulence and transmission characteristics of viruses is of paramount importance to increase honey bee health globally.Four SOV-positive and four SOV-negative honey bee colonies were randomly selected from the breeding stock of Ghent University. All colonies were healthy, did not show signs of disease and were either free of viruses or infected with DWV only  according to the manufacturer’s instructions and eluted in 30 µL elution buffer. RNA was reverse-transcribed with the RevertAid H Minus First Strand cDNA Synthesis Kit  using random hexamer primers starting from 5 µL RNA.−1). Each plate included a no template control (NTC). Absolute quantification was based on a standard curve obtained through an eightfold 5× serial dilution of viral plasmid control that ranged between 104–1010 copies/µL. All data were analyzed using CFX Manager 3.1 software . Baseline correction and threshold setting were automatically calculated by the software. Maximum accepted quantification cycle (Ct) difference between replicates was set to one Ct. The successful amplification by RT-PCR of the reference gene β-actin following the protocol described by de Graaf and colleagues [Determining the DWV load in all samples was done by uniplex qRT-PCR using Platinum SYBR Green qPCR SuperMix-UDG . Each reaction mix consisted of 0.4 µM of each primer see , 11.45 µlleagues  was usedt-Tests depending on the sample size and data type. All tests were checked for and complied with the required assumptions.Viral loads for each sample were log10-transformed to improve data visualization. RStudio version 3.6.1  was used for data analysis and visualization. Analyses of the differences in number of infections and infection load were conducted by chi-squared tests, Wilcoxon Rank Sum tests or Student’s t-test t(237.74) = 4.85, p < 0.01).All queens were infected in at least one tissue for both DWV-A and DWV-B. p < 0.05), thorax  = 6.25, p < 0.05) and eviscerated abdomen  = 6.25, p < 0.05; p = 0.87). Significantly lower number of infections were found in QDS+ compared to QDS– for DWV-A in the head  = 6.23, t-test t(102.17) = 2.04, p < 0.05). In addition, infection loads were significantly lower in QDS+ compared to QDS– for DWV-A in the ovaries (Independent sample t-test t(22.69) = 2.85, p < 0.01), spermatheca (Independent sample t-test t(16.88) = 4.66, p < 0.01), guts (Independent sample t-test t(15.25)= 4.63, p < 0.01) and eviscerated abdomen (Independent sample t-test t(13.58)= 4.44, p < 0.01; t-test t(25.93)= 2.95, p < 0.01) and eviscerated abdomen (Independent sample t-test t(29.98)= 3.89, p < 0.01; The average infection load for DWV-A was significantly lower in QDS+ compared to QDS– over all tissues  between QDS+ and QDS–, albeit being lower in QDS+ for DWV-A. As a response to the reduced vertical transmission via eggs expressed by the SOV trait the DWV quasispecies appears to be selected towards reduced virulence (lower infection loads) and increased tissue specificity for the reproductive tissues (lower effect size in the ovaries and spermatheca). These patterns confirm the role of vertical transmission as a key strategy for DWV-A  and the The screening protocol for the SOV trait initially focused on DWV-A . With thPossible infections occurring during larval feeding might be responsible for the high number of infected queens. Although larvae have a marked resistance to viral infection via oral transmission , transmiThus far, only a limited number of studies have focused on breeding for virus resistance ,32 despi


Citrus aurantifolia) is a rich source of flavonoids  and limonoids . Manufacturing of lime products  yields a considerable amount of lime peel as food waste that should be comprehensively exploited. The aim of this study was to develop a green and simple extraction method to acquire the highest yield of both limonin and hesperidin from the lime peel. The study method included ethanolic-aqueous extraction and variable factors, i.e., ethanol concentrations, pH values of solvent, and extraction temperature. The response surface methodology was used to optimize extraction conditions. The concentrations of limonin and hesperidin were determined by using UHPLC-MS/MS. Results showed that the yields of limonin and hesperidin significantly depended on ethanol concentrations and extraction temperature, while pH value had the least effect. The optimal extraction condition with the highest amounts of limonin and hesperidin was 80% ethanol at pH 7, 50 °C, which yields 2.072 and 3.353 mg/g of limonin and hesperidin, respectively. This study illustrates a green extraction process using food waste, e.g., lime peel, as an energy-saving source and ethanol as a bio-solvent to achieve the highest amount of double bioactive compounds.Green extraction is aimed at reducing energy consumption by using renewable plant sources and environmentally friendly bio-solvents. Lime ( Plant extract is one of the main ingredients in functional food, dietary supplements and herbal medicine. Production of plant extract usually requires growing a large amount of plant as raw materials and utilizing toxic solvents for extraction. Thus, a green extraction concept has been proposed as a desirable approach to save energy, reduce environmental problems, and promote sustainable development . Chemat Citrus aurantifolia) is a citrus fruit that has been cultivated around the world. Its annual world production is approximately 20,050,000 metric tons [Lime (ric tons . To promric tons .2) have been developed to solve this problem [2 extraction reduces the organic solvent for extraction, resulting in a more environmentally friendly process. Nevertheless, this process requires high costs, sophisticated devices, and special expertise [Previous studies have shown that lime is a rich source of phytochemical compounds, especially flavonoid and limonoid groups. These compounds can be found in all parts of the lime, mostly abundant in the outer and inner layers of the peel  ,5,6. The problem . SC-CO2 xpertise ,25. Thusm/z 471  are shown in m/z 471 A. The quectively B. The pr, respectively.As shown in The results of this study showed that ethanol concentration, pH value of solvent, and extraction temperature were key factors affecting the yields of limonin and hesperidin. When analyzed with ANOVA, the interactions between these factors were found. Therefore, response surface methodology (RSM) was used to demonstrate the relationship between these factors and was used to identify the most influential factor. The extraction yield was calculated as mg of limonin or hesperidin per gram of dry peel. As shown in A number of scientific evidence supports the use of plant extracts to promote good health and wellbeing . HoweverPrevious studies mostly used toxic organic solvents such as dimethyl sulfoxide (DMSO), dichloromethane, ethyl acetate, and toluene for plant extraction of limonin and hesperidin ,24. InstIn general, the efficiency of plant extraction mainly depends on type of solvent, duration, pH value, and temperature . TherefoIn this study, the extraction temperature and ethanol concentration were shown to play an important role in the extraction efficiency of limonin and hesperidin from lime peel. For ethanol concentration, we found that the use of mixed solvents (ethanol and DI water) provided higher yields of limonin and hesperidin than those of pure ethanol. Consistently, a previous report by Gertenbach demonstrated that the presence of water in the solvent mixture causes the plant cells to swell, allowing easier diffusion of the solvent into the plant cells . For extConsidering the interaction between several factors affecting extraction efficiency, our response surface methodology data is consistent with previous studies. Qin et al. showed that extraction temperature had a profound effect on the limonin concentration in the extract of pummelo seeds . AnotherThe strength of this work includes the detailed design of varied conditions for ethanol concentration, temperature, and pH to elucidate the interactions among these extraction factors. In addition, the use of LC-MS/MS allows for accurate and specific measurements of the bioactive compounds. Nevertheless, the limitations of this work include the fact that only temperatures of 50 °C and above and pH of 7 and above were tested. Furthermore, the lime peel powder used for extraction was randomly collected from the same batch of the industrial food waste. Future studies are warranted to compare the extraction yields of limonin and hesperidin from various batches of food waste. Calculation of the coefficient of variations (CV) is required to determine the consistency. Using industrial food waste, such as lime peel, from a standardized lime juice factory not only saves energy in growing new lime trees, but it also benefits from the good manufacturing practice (GMP) system to control the consistency of raw materials, i.e., the source of lime. Another limitation of this study is that we only used ethanol as an extraction solvent. The aim of this work was to develop a green extraction process. Therefore, ethanol was chosen based on its low cost, solubility, and environmentally friendly properties. This study used ethanol for extraction and LC-MS-MS for quantitation of extraction yields, while previous studies used more harmful solvents for extraction and HPLC for analysis. Therefore, the extraction yields obtained in this study cannot be fairly compared with previous works. Future studies are warranted to compare the extraction yields of ethanol with other types of solvents in parallel experiments and using similar analytical methods such as LC-MS/MS.This study discovered an optimized green extraction method to achieve the highest yields of limonin and hesperidin by using 80% ethanol in water to extract lime peel at pH 7 and 50 °C. The limonin–hesperidin-rich ethanolic-aqueous extracts of lime peel are expected to be highly effective owing to the dual bioactive compounds. Future in vitro and in vivo studies are warranted to investigate its health benefits such as anti-cancer, anti-inflammatory and anti-oxidative properties.Lime peel powder was obtained from Chiangmai Bioveggie Co., Ltd. . The powder was produced by vacuum drying fresh lime peel, which is the food waste from the GMP-certified lime juice factory. Analytical standard of limonin (≥95.0% purity) and hesperidin (≥97.0% purity) were purchased from Supelco, Inc. . HPLC-grade ethanol (≥99.8% purity), sodium hydroxide (≥98% purity), citric acid (≥99.5% purity) was purchased from Sigma-Aldrich . Phosphate-buffered saline (pH 7.4) was purchased from Invitrogen Life Science Technologies, Thermo Fisher Scientific, Inc. .Based on the chemical properties of limonin  and hespm/z) of limonin precursor and quantified product mass were 471 and 425, respectively. The collision energy of 20 V was used for the transition. The confirmation product mass for limonin was m/z 367 with the collision energy of 19 V for the transition. For hesperidin, the precursor and quantified product mass were m/z 611 and 303, respectively. The collision energy of 20 V was used for the transition. The confirmation product mass for hesperidin was m/z 465 with the collision energy of 12 V for the transition.Measurement of limonin and hesperidin was performed with a method adapted from a previous study ,44. The Standard curves were generated from standard solution of single compound (limonin or hesperidin) at the concentrations of 5, 10, 25, 50, 100 µg/ mL in ethanol. The areas under the curve (AUC) of MS chromatograms for the quantified product mass and the corresponding concentrations of limonin and hesperidin were used to create linear standard curves.p value < 0.05 indicated a statistically significant difference.Statistical analyses were performed using PASW Statistics 18 (formerly SPSS Statistics) or GraphPad prism V.9. Data were expressed as means ± SD of at least 3 independent experiments (three separate extractions). One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons or Ryan–Einot–Gabriel–Welsh F (REGWF) post hoc test was performed to compare the mean of each experimental condition. Multi-factorial ANOVA was used to determine the interaction effect of independent variables. A This study demonstrated that lime peel obtained as food waste from a lime juice factory could be a potential renewable plant source for limonin and hesperidin—a rich extract. Using LC-MS/MS for determination of the bioactive compounds together with response surface methodology, we discovered that solvent concentration and extraction temperature were crucial factors that influenced extraction yields. The optimum condition to obtain the highest yields of limonin and hesperidin from lime peel was extraction with 80% ethanol at a solid-to-solvent ratio of 0.01 g/mL and pH 7 at 50 °C for 100 min. This condition provided the yields of limonin and hesperidin at 2.072 and 3.353 mg/g, respectively. The method used ethanol and water as the extraction solvent, which is safer for extractors and more environmentally friendly than other previously used organic solvents. Therefore, this process offers a good alternative that may be applied for implementation on an industrial scale as a green extraction process of limonin and hesperidin.


N-isopropylacrylamide) (PNIPAAm) hydrogel-modified silica beads were prepared via a condensation reaction and radical polymerization. The temperature-dependent elution behavior of the drugs was observed using a PNIPAAm-modified silica-bead packed column and an all-aqueous mobile phase. Sharp peaks with reproducible retention times were observed at temperatures of 30 °C or 40 °C because the PNIPAAm hydrogel on the silica beads shrinks at these temperatures, limiting drug diffusion into the PNIPAAm hydrogel layer. The elution behavior of the sample from the prepared column was examined using a mixture of serum and model drugs. The serum and drugs were separated on the column at 30 °C or 40 °C, and the concentration of the eluted drug was obtained using the calibration curve. The results show that the prepared chromatography column would be useful for therapeutic drug monitoring because the drug concentration in serum can be measured without using organic solvents in the mobile phase and without any need for sample preparation.Therapeutic drug monitoring is a key technology for effective pharmacological treatment. In the present study, a temperature-responsive chromatography column was developed for safe and simple therapeutic drug monitoring without the use of organic solvents. Poly( The dosage of particular types of drugs must be adjusted based on monitoring the drug concentration in serum because a drug at a low concentration is ineffective and an excessively high drug concentration can cause toxicity. Therapeutic drug monitoring that measures drug concentration in serum is an effective approach to monitor the therapeutic levels required for specific types of drugs3. Various measurement methods have been developed to determine drug concentration in serum including immunological and chromatographic methods4. Liquid chromatography is effective for determining drug concentration in serum because of its versatility and lack of requirement of a specific ligand for a particular drug5. However, liquid chromatography requires the use of the organic-solvent mobile phase to move drugs along the chromatography column. In addition, the blood sample must be deproteinated using organic solvents before measuring the drug concentration in the serum by chromatography. The use of organic solvents is often undesirable in hospitals because of the associated hazards of exposure. Thus, an analytical method that does not use organic solvents is required for therapeutic drug monitoring. Temperature-responsive chromatography using poly(N-isopropylacrylamide) (PNIPAAm) is one such measurement method that does not use organic solvents. PNIPAAm exhibits temperature-dependent hydrophilic and hydrophobic changes across its phase transition temperature of 32°C9. Since the phase transition temperature is close to the body temperature, PNIPAAm has been used in various biomedical applications such as temperature-modulated drug and gene delivery systems15, biosensors and bioimaging systems utilizing the phase transition properties of PNIPAAm21, cell separation systems using the difference in cell adhesive properties among cells on PNIPAAm30, and cell culture substrates for tissue engineering using temperature-modulated cell adhesion and detachment38. When applied to chromatography, PNIPAAm-modified silica beads are used as the stationary phase44. Because the hydrophobicity of PNIPAAm changes according to the external temperature, the hydrophobic interaction between PNIPAAm and the analyte can be modulated by changing the column temperature. The retention time of the analyte is modulated by changing the column temperature without changing the mobile phase composition; aqueous mobile phases can be used, thus alleviating the need for organic solvents.In pharmacological therapy, the optimal drug dosage is different among patients because of the individual variation in adsorption, distribution, and eliminationIn the present study, we developed PNIPAAm-modified silica beads as temperature-responsive chromatographic matrices for therapeutic drug monitoring Fig. . Ten difN,N-methylenebisacrylamide (BIS)  V-501) modification of silica beads, followed by radical polymerization of NIPAAm and  modifica2. This value is similar to that reported previously46. The PNIPAAm-hydrogel-modified beads exhibited a higher carbon composition compared to that of the V-501-immobilized silica beads. The results indicated that the PNIPAAm hydrogel was modified on the silica beads via polymerization of NIPAAm and BIS. PNIPAAm-modified silica beads prepared via either 5 h or 18 h polymerization exhibited different amounts of PNIPAAm, 0.098 mg/m2 or 0.215 mg/m2, respectively. A longer polymerization reaction resulted in a larger amount of PNIPAAm on the silica beads. This also indicated that the larger molecular weight of PNIPAAm was present on silica beads prepared by polymerization for 18 h compared to 5 h.The silica beads were characterized at each reaction step by elemental analysis Table . A highe−1 were observed in the spectra of the PNIPAAm-modified beads, while no peaks were observed in the spectra of the aminopropyl silica beads. The peaks were attributed to the N–H bending vibration and C=O stretching vibrations of the N–H and C=O bonds, respectively, of the modified PNIPAAm hydrogel on the silica beads. Thus, the results further confirmed that PNIPAAm was successfully generated on silica beads via a polymerization reaction.The prepared beads were characterized by ATR/FT-IR at each reaction step Fig. A. Two peThe morphology of the prepared beads was observed at each step using scanning electron microscopy (SEM) Fig. B. A simiThe prepared PNIPAAm-hydrogel-modified beads were packed into a stainless-steel column .48. However, wider peaks were observed on the column using the beads prepared via the 18 h polymerization compared to that using the beads prepared via the 5 h polymerization because of the longer diffusion time of the steroid molecules into the thicker PNIPAAm hydrogel layer. The PNIPAAm hydrogel layer coated on the beads during the 18 h polymerization was thicker than that coated on the beads during the 5 h polymerization. Thus, the diffusion of the steroids into the thicker PNIPAAm hydrogel layer resulted in a slower elution from the beads and wider peaks in the chromatogram. Previous studies have suggested that small molecular analytes tend to diffuse into a thick PNIPAAm layer composed of large molecular weight PNIPAAm on silica beads at a higher temperature50.To investigate the column performance of the prepared beads, the elution behavior of five hydrophobic steroids—hydrocortisone, prednisolone, dexamethasone, hydrocortisone acetate, and testosterone, was observed Fig. . The proTherefore, the column incorporating the beads prepared via the 5 h polymerization was used for these investigations because of the shorter retention times and narrower peaks in the chromatogram. A short analysis time is required for therapeutic drug monitoring because the serum drug concentration of patients should be measured promptly. In addition, wide peaks in the chromatograms reduced the accuracy of the determination.The suitability of the prepared column was investigated for therapeutic drug monitoring using ten types of drugs that required therapeutic drug monitoring in therapy Table .3COONH4 (pH 6.70) as the mobile phase  and vancomycin  in the prepared column is shown in Fig. A, B. DigThe chromatograms of mycophenolic acid (an immune-suppressing drug) and methotrexate (an anti-cancer drug) are shown in Fig. 3COONH4 buffer solution as the mobile phase. A pH 6.75 CH3COONH4 buffer solution was used as the mobile phase for the other drugs. Most drugs could be analyzed within a short analysis time (8 min), although mycophenolic acid and disopyramide exhibited a slightly longer retention time. Various changes in the retention time with changing column temperature were observed. The retention times of disopyramide, phenytoin, and digoxin increased, whereas those of mycophenolic acid, methotrexate, and vancomycin decreased with increasing temperature. The retention times of the other drugs, carbamazepine, lamotrigine, propafenone, and quinidine were almost independent of the column temperature. The difference in retention time with temperature is attributed to the differences in the analyte properties such as hydrophobicity and ionic properties. PNIPAAm on the column silica beads becomes hydrophobic with increasing temperature due to dehydration. Thus, the hydrophobic interaction between PNIPAAm and the analyte increase, leading to an increase in the retention time of these analytes. In contrast, the solubility of the analytes in the mobile phase increase with increasing temperature, leading to a shortening of the retention time of the analytes. The retention of the analyte is dependent on the balance between these two factors. In the case of disopyramide, phenytoin, and digoxin, the effect of increasing the hydrophobic interaction between PNIPAAm and the analyte with increasing temperature was large compared to the effect of increasing analyte solubility in the mobile phase. In contrast, for mycophenolic acid, methotrexate, and vancomycin, the effect of increasing analyte solubility in the mobile phase was large compared to the effect of increasing hydrophobic interactions between PNIPAAm and the analyte. In the case of carbamazepine, lamotrigine, propafenone, and quinidine, both effects were balanced at all temperatures, leading to the retention time being independent of temperature.The temperature-dependent retention time changes of all the analytes were summarized to compare the retention profiles with changing column temperature Fig. . The retThe reproducibility was investigated by observing three separate measurements of the retention time and expressed as the RSD value Table . The RSD58. In contrast, the PNIPAAm homopolymer modified-bead packed column scarcely adsorbs proteins even at high column temperatures52. Thus, the PNIPAAm homopolymer column prepared in the present study could be used for serum samples without deproteination because serum proteins do not adsorb onto the column beads. The suitability of the prepared column for determining drug concentration in serum was investigated using a mixture of serum and model drugs  is required before HPLC column injection to avoid serum protein adsorption onto the beads in the column resulting in the column clogging. Thermoresponsive ionic copolymers composed of NIPAAm and ionic monomers have been shown to adsorb proteins via multiple hydrophobic and electrostatic interactionsgs Figs.  and 8. TThe elution behavior of antiepileptic drugs in the serum was observed Fig. A–C. The For the determination of anti-arrhythmic drugs in the serum Fig. D–F, the The separation of four types of drugs, digoxin, vancomycin, mycophenolic acid, and methotrexate from serum was investigated at 30 °C Fig. . The elu46; we reported that drug binding to serum proteins did not in this case affect separation between drug and serum proteins46.There is a possibility for drug binding to serum proteins, and the bound drug being eluted with serum proteins. However, in a previous study, we investigated the effect of drug binding on serum proteins on separation using PNIPAAm-modified silica beadsThe separated peaks from serum were observed for each of the types of drugs using the prepared column. Thus, the developed column can determine the drug concentration in serum using all aqueous mobile phases and without sample deproteination, whereas the conventional method using a reversed-phase chromatography system requires an organic mobile phase and deproteination using an organic eluent.The calibration curves of seven drugs—phenytoin, lamotrigine, carbamazepine, disopyramide, quinidine, propafenone, mycophenolic acid, digoxin, and methotrexate in the effective drug concentration range for therapeutic drug monitoring were quantified using the method reported here Fig. . The effThese results indicate that the drug concentration in serum proteins can be measured using the developed PNIPAAm-hydrogel-modified silica bead packed column with an all-aqueous mobile phase and without pre-deproteination of samples. The developed chromatography column is useful for therapeutic drug monitoring, and the chromatography system is safe in a medical setting because of the absence of organic solvents in the mobile phase; further, it does not require deproteination as a sample preparation step.A temperature-responsive chromatography column was developed for safe and simple therapeutic drug monitoring. PNIPAAm hydrogel-modified beads were prepared via the immobilization of V-501 on aminopropyl silica beads using a condensation reaction and subsequent radical polymerization with a polymerization time of either 5 h or 18 h. Silica beads prepared via the 18 h radical polymerization exhibited a larger concentration of PNIPAAm on the silica beads compared to silica beads prepared via the 5 h radical polymerization. Shorter retention times and sharper peaks were observed for the column using the beads prepared via the 5 h polymerization compared to using the beads prepared via the 18 h polymerization because a thinner PNIPAAm hydrogel layer was deposited on the silica beads via the 5 h polymerization and the thin PNIPAAm hydrogel layer reduced analyte diffusion into the PNIPAAm layer and hence the retention time. The elution behavior of ten types of drugs was examined to determine the feasibility of using the silica-bead packed column for therapeutic drug monitoring. All the drugs were retained on the prepared column and exhibited sharp peaks at high temperatures because the PNIPAAm hydrogel shrunk and prevented analyte diffusion into the hydrogel layer. The elution behavior from the column was determined using a mixture of serum and drugs as model samples. At 30 °C or 40 °C, the serum and drugs were separated, and the drug concentration was estimated using the calibration curve. The results indicated that the prepared temperature-responsive chromatography column would be useful for therapeutic drug monitoring because the chromatography column enables the determination of drug concentration in serum without using organic solvents in the mobile phase and without requiring sample deproteination.N,N-dimethylformamide (DMF) (50 mL) in a flask. Aminopropyl silica beads (5.0 g) were added to the solution. The bead suspension was deoxygenated by bubbling with nitrogen gas for 30 min. The flask was sealed, and the condensation reaction was carried out for 6 h at 25 °C. After the reaction, the beads were filtered, rinsed with ethanol, and dried at 25 °C in vacuo.PNIPAAm hydrogel-modified silica beads were prepared using the following procedure Fig. A. All maN,N′-methylene-bisacrylamide (BIS)  were dissolved in ethanol (100 mL) in a flask. V-501 modified silica beads (2.0 g) were added to the solution. The bead suspension was deoxygenated for 30 min. The flask was then sealed, and radical polymerization proceeded with continuous shaking at 70 °C for either 5 or 18 h. Different amounts of PNIPAAm were generated on the bead surface by the different duration of polymerization. After the polymerization reaction, the beads were rinsed with methanol and dried at 25 °C in vacuo.NIPAAm  and The prepared beads were characterized by CHN elemental analysis, FT-IR, and SEM at each reaction step.CI is the increase in carbon content after V-501 modification, %CI  is the calculated carbon percentage of the V-501 molecule, and S is the surface area of the silica beads.The carbon composition of the prepared beads was measured using a CHN elemental analyzer . The quantity of modified V-501 and PNIPAAm on the silica bead surfaces was obtained using the carbon composition of the beads  is the calculated carbon percentage of PNIPAAm.The quantity of PNIPAAm hydrogel on the silica bead surfaces was obtained using Eq. :2\documePolymer modification of the silica beads surface was also confirmed by attenuated total reflection Fourier-transform infrared spectroscopy .The bead morphologies were observed by SEM  for each polymer modification step.The prepared beads were packed into a stainless-steel column (4.6 mm diameter × 50 mm length). The PNIPAAm-modified beads (0.8 g) were suspended in a methanol/water (1:1) mixture. The suspension was then poured into a column packer connected to a stainless-steel column. Beads were packed by pouring the methanol/water (1:1) suspension into the column at a constant pressure of 35 MPa for 90 min. After packing, pure water was used to rinse the column for 5 h.) in methanol was prepared. Ten types of drugs required for therapeutic drug monitoring were used as samples. The properties of the drugs are summarized in Table 3COONH4 solution (pH 6.75 and pH 4.8) was used as the mobile phase. The elution behavior of these analytes was observed at a predetermined wavelength for each drug . The column performance was evaluated by HPLC by examining the elution behavior of steroid analytes. The results are summarized in Table ug Table  using a Supplementary Information 1.


Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine virus A (GVA), Grapevine leafroll-associated virus-1 (GLRaV-1), Grapevine leafroll-associated virus-2 (GLRaV-2), Grapevine leafroll-associated virus-3 (GLRaV-3), Grapevine fanleaf virus (GFLV), and Grapevine fleck virus (GFkV) using RT-PCR. Out of all samples tested, 54.5% were positive for at least one of the viruses  in the Stavropol region, 49.8% in the Krasnodar region and 49.5% in the Republic of Crimea. Some plants were found to be infected with several viruses simultaneously. In the Republic of Crimea, for instance, a number of plants were infected with five viruses. In the Krasnodar region and the Republic of Crimea, 4.7% and 3.3% of the samples were predominantly infected with both GFkV and GRSPaV, whereas in the Stavropol region, 6% of the selected samples had both GLRaV-1 and GVA infections. We carried out a phylogenetic analysis of the coat protein genes of the detected viruses and identified the presence of GVA of groups I and IV, GRSPaV of groups BS and SG1, GLRaV-1 of group III, GLRaV-2 of groups PN and H4, GLRaV-3 of groups I and III. The results obtained make it possible to assess the viral load and the distribution of the main grapevine viruses on plantations in the viticultural zones of Russia, emphasizing the urgent need to develop and implement long-term strategies for the control of viral diseases of grapes.Viral diseases can seriously damage the vineyard productivity and the quality of grape and wine products. Therefore, the study of the species composition and range of grapevine viruses is important for the development and implementation of strategies and tactics to limit their spread and increase the economic benefits of viticulture. In 2014–2019, we carried out a large-scale phytosanitary monitoring of Russian commercial vineyards in the Krasnodar region, Stavropol region and Republic of Crimea. A total of 1857 samples were collected and tested for the presence of  A large variety of fruits and berries provide humans with beneficial substances necessary for the vital activities of the body; moreover, some cultures, including grapes which is one of the most ancient and valuable fruit crops , are theClosteroviridae, Betaflexiviridae, Caulimoviridae, Geminiviridae, and Secoviridae. About half of viral phytopathogens are associated with four main disease complexes known as infectious degeneration and decline (fanleaf), leafroll, rugose wood, and fleck [Grapevine fanleaf virus (GFLV) and grapevine leafroll disease, the most significant infectious agents of which are Grapevine leafroll-associated virus-1 (GLRaV-1), Grapevine leafroll-associated virus-2 (GLRaV-2) and Grapevine leafroll-associated virus-3 (GLRaV-3) [Among woody plants worldwide, grapevine is the culture which is most affected by viral phytopathogens . To datend fleck ,7. The mGLRaV-3) ,9,10. ThGLRaV-3) . It is kGLRaV-3) ,12,13.Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine virus D (GVD), Grapevine virus E (GVE), and Grapevine virus F (GVF). They are characterized by the presence of rupestris stem pitting, kober stem grooving, corky bark, and grooving and pitting of the woody cylinder [Viruses that cause rugose wood include cylinder . Infectecylinder .Grapevine fleck virus (GFkV) is one of four representatives of the fleck complex, along with Grapevine asteroid mosaic-associated virus (GAMaV), Grapevine rupestris vein feathering virus (GRVFV), and Grapevine red globe virus (GRGV) [s (GRGV) . The pats (GRGV) ,15.Since fighting against viral phytopathogens is difficult and the ways to completely destroy viruses directly in the vineyard are unknown, it is necessary to use healthy planting material tested for viruses at the stage of seedling production in nurseries .Early detection of foci of viral infection of grapevines allows to prevent their large-scale spreading and take measures to reduce the economic damage to the industry . In RussThe aim of this work was to study the distribution and genetic diversity of grapevine viruses in Russia. Among them, we selected 7 pathogens that have the most significant economic effect and carried out a large-scale phytosanitary study of grapevine plantations in three southern regions of Russia. Five viruses we previously detected were included to understand how widespread they are, as well as GFLV and GFkV, which are usually included in the monitoring of vineyards for viruses.During the studies, we carried out phytosanitary monitoring of 335 grape plantations in various agricultural landscapes, including commercial, small farms, and abandoned plantations. In total, we examined grape plantations of 83 cultivars and hybrids, as well as a number of unspecified cultivars.An analysis of the infection symptoms in the plants of the surveyed territories revealed that over 56% of the samples collected in the Crimea had various types of chlorotic leaf yellowing and spots, which may be a manifestation of infection with various viruses and viroids. The second most common symptom (about 17%) was reddening of the interveinal areas of the leaf blade with main veins remaining green . Often wDiagnostics of collected samples for the presence of 7 grapevine viruses showed that in the Crimea 610 plants (49.5%) were infected with at least one of the viruses under study, in the Krasnodar region—295 plants (49.8%) and in the Stavropol region—18 plants (54.5%) .In the Krasnodar region and Crimea, we detected all viruses under study, in the Stavropol region—5 out of 7 (GFLV and GFkV were not detected) . This reIn the Stavropol region, GLRaV-1 was more widespread; it was identified in 33% of the collected samples, while GLRaV-2 and GLRaV-3 were detected in 6% of the plants. This was the highest percentage of GLRaV-2 infections in all surveyed areas. The highest percentage of GLRaV-3 was observed in the Republic of Crimea, where it was found in 10% of the samples. In general, leafroll viruses were less prevalent than viruses belonging to the stem pitting complex.GRSPaV associated with grapevine rupestris stem pitting disease was identified in 15% of the samples in the Crimea, in 10% of the plants in the Krasnodar region, and in 6% of the samples in the Stavropol region. GVA was more prevalent in the Stavropol region and the Republic of Crimea, where it was detected in 12% of the plants, while in the Krasnodar region the virus was detected only in 4% of the tested samples.Some of the plants were infected with two or more viruses at the same time . In the Since phylogenetic reconstruction can be affected by recombination , a recomAccording to phylogenetic analysis of the coat protein gene, GVA isolates are usually classified into 4 groups . AlignmeIn recent decades, a large number of studies on the genetic diversity of GRSPaV have been carried out, which made it possible to identify several molecular groups of GRSPaV ,24,25,26A BLASTn analysis for a fragment of the GFkV coat protein gene showed that the identity of the nucleotide sequences of the five Russian isolates with isolates from the GenBank database varied from 88.38% to 100% ; the cloA comparison of fragments of the GFLV coat protein gene using BLASTn demonstrated that the sequences of the Russian isolates are 86.77–90.12% identical with the sequences of the GFLV coat protein gene available in the GenBank. According to previous studies in the field of GLRaV-1 phylogeny, all isolates belong to 3 groups . We analCurrently, 6 phylogenetic groups of GLRaV-3 are distinguished . In our According to previous studies, there are 5  or 6 molFour GLRaV-2 isolates from commercial grape cultivars Cabernet Sauvignon and Muscat Golodrigi in the Crimea, as well as from table grape cultivars Libya and Moldova in the Krasnodar region and Stavropol region, respectively, belonged to the H4 group. Two isolates found in the vineyards of the Cabernet Sauvignon cultivar and an unknown grape cultivar in the Yalta district of the Republic of Crimea belonged to the PN group. A BLASTn analysis showed the identity of our isolates with isolates from the GenBank at the level of 88.63–99.66%.An RFLP analysis of two amplicons of the coat protein gene produced different restriction profiles that are presented in Previously, we have determined that the isolate 3 belongs to group 3 of GLRaV-2 (GenBank ID MH074870) by digestion of PCR product amplified with primers V2dCPf2/V2dCPrl . In thisTaqI restriction profile of the V2dCPf2/V2dCPrl amplicon corresponds to the profile of groups 3 and 4, while the RsaI restriction profile—to the profile of the RG group isolates. The data obtained made it possible to suggest the presence of an isolate of a new group in the analyzed sample or the presence of a mixed infection of isolates belonging to several groups of GLRaV-2. At the same time, phylogenetic analysis shows that isolate 346 belongs to the H4 group which is separate from the RG isolates.As a result of digestion of two fragments of the GLRaV-2 coat protein gene of isolate 346, we obtained restriction profiles which on the whole did not correspond to any of the previously described groups. For instance, the restriction profile of the GLR2CP1/GLR2CP2 amplicon was the same as the profile of the group 3 isolates. The RsaI and TaqI.PCR with primers V2dCPf2/V2dCPrl did not result in amplification of a fragment of the coat protein gene of isolate 1853 collected from grapevine samples in the Stavropol region. Therefore, we were not able to identify the exact RFLP group by the digestion of two PCR products with the enzymes Here we describe the results of a large-scale study of the distribution of 7 economically significant grapevine viruses conducted in the southern regions of Russia. For the first time in Russia, we have earlier detected a number of grapevine viruses in individual vineyards of the Crimea and Krasnodar region ,18,19,20Grapevine Pinot gris virus (GPGV), Arabis mosaic virus (ArMV), Grapevine virus B (GVB), Grapevine red blotch-associated virus (GRBaV), etc.The diversity of symptoms of viral diseases that we observed in the process of phytosanitary examination of grapevine plantations is likely to be associated with the difference in action of the same species of viruses on different cultivars and hybrids of grapes. In addition, the size of the territory and the location of vineyards in different zones that we studied indicate the influence of climatic conditions on the symptoms. Infection of the grapevine with several viruses as well as infection with other phytopathogens  can result in unique symptoms and an increase in their manifestation ,30,31. FIn this work, we expanded the species composition of viruses for the first time detected in the Krasnodar region; such are GVA, GRSPaV, GLRaV-1, GLRaV-3, GFLV and GFkV. In the Stavropol region, we for the first time detected GVA, GRSPaV, GLRaV-1, GLRaV-2, and GLRaV-3. In the Crimea, we showed the distribution of 7 grapevine viruses: GVA, GRSPaV, GLRaV-1, GLRaV-2, GLRaV-3, GFLV and GFkV.The data obtained on the prevalence of GRSPaV  indicate its ubiquitous presence in the vineyards of Russia. A phylogenetic analysis showed that most of the studied isolates belong to the BS group and one isolate belongs to the SG1 molecular group. Such clustering did not depend on the geographical location of the plants from which the samples were collected. The SG1 and BS groups and the GRSPaV-1 group have previously been described in Brazil . IsolateThe prevalence of GVA varied from 4% in the Krasnodar region to 12% in the vineyards of the Crimea and Stavropol region. Phylogenetic typing confirmed the prevalence of molecular group IV in the vineyards of southern Russia, whereas group I was less prevalent. For comparison, the absolute majority of isolates in Tunisia (70%) belonged to group I, while group IV was the second most prevalent . The preIn this work, we studied the distribution of three main pathogens of grapevine leafroll disease: GLRaV-1, GLRaV-2, and GLRaV-3. In general, from 2 to about 10% of the analyzed samples were infected with one or more leafroll viruses at the same time. The data on the distribution of these viruses obtained in this study are in line with the results of our previous studies in the Crimea, when out of 689 samples, 4.9% were found to be infected with GLRaV-1 and 5.4%—with GLRaV-3 .As a result of the study of evolutionary relationships, Russian GLRaV-1 isolates were found to belong to molecular group III. A similar situation was observed in Slovakia, where most of the samples belonged to group III, while several isolates belonged to molecular group I . IsolateVitis vinifera allowed to identify GLRaV-2 in about 15% of the samples collected in the Krasnodar region [Our earlier studies of five table cultivars of r region . As a rer region .GLRaV-3 is the predominant species in the vineyards of the Crimea, regardless of the cultivar and age of the grapevine, while GLRaV-1 is predominant in the Stavropol region and Krasnodar region. The overall infection level of GLRaV-3 in the Crimea was about 10%, which is higher than in our previous studies on a smaller selection of samples . The GLROur studies showed that GFkV is widespread almost everywhere; it was identified in 27% of the grape samples collected in the Crimea and in 36% of the samples from the Krasnodar region. The virus has not yet been detected in the Stavropol region. Our results indicating a widespread infection of grapevines with GFkV are supported by data obtained by colleagues from other countries . In CanaGFLV is considered one of the most widespread nepoviruses in the world . In our Hop stunt viroid, Grapevine yellow speckle viroid 1 and -2), improving detection methods are an integral part of the further development of viticulture in Russia.The results of phytosanitary monitoring show a wide distribution of viruses in the vineyards of Russia and emphasize the urgent need for a comprehensive approach in the fight against the spread of viral infections. This requires the development and implementation of certification programs for planting material produced in Russia, as well as the control of imported planting material for the presence of key viruses. Creation of nurseries for healthy planting material and implementation of agrotechnical and carrier control measures will make it possible to limit the spread of harmful viruses in Russia. Regular phytosanitary monitoring of main viticulture zones of the south of Russia, including new regions and vineyards, expanding the list of harmful viruses by including recently discovered ones  and commonly known  and viroids , as well as the Stavropol region, where the sum of active temperatures is insufficient for growing grapes without sheltering them for the winter.At selected vineyards, symptomatic samples presumably infected with viruses were collected. Taking into account that the symptoms of infection and the intensity of their manifestation can vary depending on the cultivar, climatic conditions, pathogen titer in the plant, and coinfection with one or several other viruses, we collected samples with any visual deviations from healthy plants.Samples of grapevines and leaves were collected from various layers, stored in a refrigerator at +4 °C, and transported to the laboratory for analysis. In the laboratory, samples were stored at −20 °C.Total RNA was extracted from 0.3 g of the combined sample which included fragments of grapevines and leaves, according to a slight modified method described in . RNA quaFor each sample, a control amplification of 18S rRNA was performed with the primers listed in Taq Buffer with (NH4)2SO4, 0.2 mM each dNTP, 1 μM each primer, 2.5 mM MgCl2, 0.375 U Taq polymerase . The primers used for detection are listed in Viral detection was conducted by PCR using 1 μL of cDNA as a template in a reaction mixture containing The PCR amplification consisted of an initial denaturation at 94 °C for 3 min, followed by 35 cycles of 94 °C for 45 s, 56 °C for 45 s, 72 °C for 1 min. The final elongation stage was carried out at 72 °C for 7 min. The resulting PCR products were separated in 1% agarose gel and the corresponding bands were excised. DNA was purified using a Cleanup Standard Kit for DNA purification from agarose gel according to the manufacturer’s protocol . A virus detected in a single vine was considered as a single isolate. To confirm the presence of virus, the obtained amplicons were sequenced by the Sanger method with forward and reverse primers using Big Dye Terminator v.3.1 chemistry reagents on an ABI PRIZM 3730 automatic sequencer according to the manufacturer’s instructions. The complete sequence was assembled using the BioEdit Sequence Alignment Editor v.7.2.0 software. Then, using the BLASTn algorithm, we compared the sequences that we obtained with those available in the NCBI database. The obtained sequences were deposited in the GenBank, access numbers are listed in Positive DNA samples were used for sequencing the gene encoding the coat protein and studying the phylogeny of GRSPaV, GVA, GLRaV-1, GLRaV-2, GLRaV-3, GFLV, and GFkV. The list of isolates used for phylogenetic studies is presented in Multiple alignment was performed using ClustalW in the BioEdit Sequence Alignment Editor v.7.2.0 program. Phylogenetic analysis was performed in the MegaX program using the maximum likelihood algorithm ,56. The RsaI and TaqI using tools of the Clone Manager 6N program. The second pair of primers GLR2CP1/GLR2CP2 was used for more specific detection of the molecular group GLRaV-2RG; comparison of the obtained isolate profiles with those published earlier [For detection of the GLRaV-2 group, we also used RFLP . PCR pro earlier  helped up ≤ 0.05 were considered significant.To search for recombination sites in the genomes of the isolates that we found, we used nucleotide sequences of amplified coat protein genes of viruses GVA, GRSPaV, GLRaV-1, GLRaV-2, GLRaV-3, GFLV, and GFkV, as well as nucleotide sequences of coat protein genes of all viruses submitted to GenBank as of March 2021 . The sea


Malaria causes significant mortality and morbidity in sub-Saharan Africa, especially among children under five years of age and places a huge economic burden on individuals and health systems. While this burden has been assessed previously, few studies have explored how malaria comorbidities affect inpatient costs. This study in a malaria endemic area in Western Kenya, assessed the total treatment costs per malaria episode including comorbidities in children and adults.Total economic costs of malaria hospitalizations were calculated from a health system and societal perspective. Patient-level data were collected from patients admitted with a malaria diagnosis to a county-level hospital between June 2016 and May 2017. All treatment documented in medical records were included as health system costs. Patient and household costs included direct medical and non-medical expenses, and indirect costs due to productivity losses.Of the 746 patients admitted with a malaria diagnosis, 64% were female and 36% were male. The mean age was 14 years (median 7 years). The mean length of stay was three days. The mean health system cost per patient was Kenyan Shilling (KSh) 4288 (USD 42.0)  95% CI KSh 4046–4531). The total household cost per patient was KSh 1676 (USD 16.4) (95% CI KSh 1488–1864) and consisted of: KSh 161 (USD1.6) medical costs; KSh 728 (USD 7.1) non-medical costs; and KSh 787 (USD 7.7) indirect costs. The total societal cost  per patient was KSh 5964 (USD 58.4) (95% CI KSh 5534–6394). Almost a quarter of patients (24%) had a reported comorbidity. The most common malaria comorbidities were chest infections, diarrhoea, and anaemia. The inclusion of comorbidities compared to patients with-out comorbidities led to a 46% increase in societal costs .The economic burden of malaria is increased by comorbidities which are associated with longer hospital stays and higher medical costs to patients and the health system. Understanding the full economic burden of malaria is critical if future malaria control interventions are to protect access to care, especially by the poor.The online version contains supplementary material available at 10.1186/s12936-021-03958-x. While there has been a worldwide decline in the number of cases and deaths over the last decade, in 2017 there were an estimated 219 million cases of malaria, and malaria control strategies continue to place a significant economic burden on many resource-constrained health systems, especially those in sub-Saharan Africa  as well Ensuring ‘affordable’ malaria treatment is a stated priority in the malaria control strategy of the Kenyan Government . NotableIn Kenya, malaria is the most common reason for presentation at local hospitals , and togThe economic burden of malaria inpatient and outpatient care has been well-researched, and studies on household costs have found malaria imposes significant financial burden on households , 24. HowThis study used patient-level data from Iguhu hospital, a sub-county hospital in Kakamega county, in the malaria endemic-prone Western Kenya highlands. The hospital is funded by the Kakamega County Government Health serving a population of 17,860 people. It has 21 beds and 4 cots catering for children, women, and men. Critically ill patients are transferred to Kakamega County Teaching and Referral Hospital in Kakamega township 16 km away. The majority of the population work in small-scale subsistence farming keeping livestock, growing corn and tea or work in the informal sector . Water cPlasmodium-positive blood smear (or positive RDT test if laboratory testing was not available) between June 2016 and May 2017.The sample comprised all infants, children, adolescents and adults admitted to Iguhu hospital with a diagnosis of malaria, associated symptoms such as fever, malaise, headache or vomiting and a laboratory confirmed An ingredients-based approach  was usedTo estimate resource use, detailed data were collected from hospital admission records which included: patient age, sex, village of residence, sublocation, pregnancy status, type of malaria diagnostic test conducted, type of malaria treatment, any other diagnostic tests and treatments, length of stay, discharge destination; and any fees paid by patients for their inpatient stay. Key personnel from Iguhu hospital advised on specific costs for patients, including hospital charges, transport and food, provision and stock-outs of medications in the past 12 months, and the approximate costs of these medications at local village vendors.Health system cost data were extracted from patient-level medical records and included costs associated with hospitalization (bed cost), laboratory tests, medications and related overheads ” framework . These cAll inpatients had confirmed malaria and the cost of microscopic diagnosis was derived from previously published estimates for Kenya .Kenya of Government National Malaria Treatment Guidelines [for the malaria components, and for other illnesses based on recommended dosage and duration documented in the Kenya Essential Medicines List [As the hospital records only listed the names of medications, dosage calculations for malaria were based on idelines  for the nes List , Médecinnes List , and MSFnes List  based onnes List  and costThe costs of medications and equipment were estimated using the Kenya Medical Supplies Authority (KEMSA) price list . KEMSA iAll medications listed in the hospital records and administration costs  were included. For an additional diagnosis of dehydration, the cost for the insertion of an intravenous line and 24 h of IV fluids was included. For anaemia, where blood was required, it was assumed that one unit of packed cells was used , and thaDirect patient costs included: registration books, laboratory test for malaria on admission and any hospital fees documented in hospital records. Children under five years of age and pregnant women were exempted from paying a registration fee and fees for malaria laboratory tests. Hospital inpatient charges to patients were included as recorded in the patient record. Any inpatient charges were assumed to be paid at discharge and deducted from the total health system cost.Costs for medication following discharge were based on prices in the Iguhu hospital pharmacy medications price manual . HospitaTravel costs of patients and any accompanying persons were estimated for motorbike taxi transport. If the patient was a child under 6 years of age it was assumed that a caregiver travelled to and from the hospital with them and a meal was purchased each day. If a patient had to be transferred to Kakamega, an additional cost was estimated for a return trip. In the case of death, funeral costs were also included.Discounting was not necessary as all costs were estimated over one year. Any costs from the published literature were converted to local currency, Kenyan Shilling (KSh) and adjusted for inflation to 2020. Changes over time in the prices of non-medical goods and services were adjusted using the Kenya consumer price index (CPI) , and medPatient-level data were transferred securely in a Microsoft Excel format and analysis was performed using SAS 9.4 . Patient characteristics are described with chi-square tests used to calculate differences in proportions. Sensitivity analysis was conducted to examine the impact on household costs of an increase in hospital costs due to inflation (5%); and the impact of stockouts of antibiotics as this was reported to be a common issue by hospital personnel. If antibiotics were not available from the hospital, it was assumed they would be purchased from a local vendor. The percentage difference between the public sector  and private sector (pharmacies) procurement prices for locally produced and imported medicines (expressed as median price ratios)  was usedThe sample consisted of 746 inpatients, 478 (64%) females and 268 (36%) males. The mean age for patients was 14.5 years, median 7 years (IQR 2.5–21). The median age for females was 11 years (IQR 3.4–30 years) and males 4 years (IQR: 2–9). There were 41 women admitted to hospital who were pregnant. Most patients (98%) were discharged home after the hospital stay, with 7 (1%) patient deaths and 9 (1%) hospital transfers  had a documented comorbidity in addition to malaria. Younger patients (< 5 years) were more likely to have comorbidities compared to older patients (p = 0.02)  incurred some direct costs. These included 167 patients who only paid registration or laboratory costs (27%) and 229 patients (37%) who paid a hospital inpatient fee of KSh 100 (USD 1). Both of these fees were paid by 209 patients (34%) and nine patients (2%) paid higher fees between KSh 200-KSh 850.   95% CI KSh 4046–4531). Total patient and household costs averaged KSh 1676 (USD 16.4) (95% CI KSh 1488-1864) per patient, consisting of KSh 161 (USD1.6) medical and KSh 728 (USD 7.1) non-medical costs and KSh 787 (USD 7.7) indirect costs. The total societal cost  was KSh 5964 (USD 58.4) (95% CI KSh 5534–6394)  to KSh 5538 (USD 54.3), respectively. Total mean patient and household costs increased by 54% from KSh 1480 (USD 14.5) to KSh 2281 (USD 22.4), of which direct costs increased by 80% from KSh 743 (USD 7.3) to KSh 1,337 (USD 13.1), and indirect costs due to productivity losses increased by 22% from KSh 737 (USD 7.2) to KSh 945 (USD 9.3)  to KSh 6560 (USD 64.3). Total mean patient and household costs increased by 57% from KSh 1456 (USD 14.3) to KSh 2283 (USD 22.4) of which direct costs increased by 29% from KSh 825 (USD 8.1) to KSh 1062 (USD 10.4) per patient and productivity losses increased by 48% from KSh 631 (USD 6.2) to KSh 1220 (USD 12.0) per patient . Table S4. Mean health system and patient costs related to hospitalisation and antibiotic stockouts with confirmedmalaria diagnosis.


Vespula are social insects that have become major pests and predators in their introduced range. Viruses present in these wasps have been studied in the context of spillover from honey bees, yet we lack an understanding of the endogenous virome of wasps as potential reservoirs of novel emerging infectious diseases. We describe the characterization of 68 novel and nine previously identified virus sequences found in transcriptomes of Vespula vulgaris in colonies sampled from their native range (Belgium) and an invasive range . Many viruses present in the samples were from the Picorna-like virus family (38%). We identified one Luteo-like virus, Vespula vulgaris Luteo-like virus 1, present in the three life stages examined in all colonies from both locations, suggesting this virus is a highly prevalent and persistent infection in wasp colonies. Additionally, we identified a novel Iflavirus with similarity to a recently identified Moku virus, a known wasp and honey bee pathogen. Experimental infection of honey bees with this novel Vespula vulgaris Moku-like virus resulted in an active infection. The high viral diversity present in these invasive wasps is a likely indication that their polyphagous diet is a rich source of viral infections.Wasps of the genus  Insects have been shown to harbor a remarkable viral diversity with a high abundance of novel virus families ,2. This Varroa destructor as a vector . We. WeV. pe spiders ,38,53. M spiders ,20,23.Vespula wasp populations may be achievable through the discovery of novel viruses that infect them [It will be important to determine the prevalence and pathogenicity of the novel Moku-like virus in global wasp and honey bee populations, given that the closely related Moku virus has now been widely isolated ,36,37 anect them , howeverect them .Vespula vulgaris, harbors a complex virome, as demonstrated by the 68 novel and seven previously identified virus genomes from 11 viral families described here. These viruses are likely to be shared among arthropod species, including honey bees that underpin pollination and primary industries in many countries. The invasion of wasp populations into a new range brings an obvious direct predation pressure, but also can bring a hidden and very diverse package of pathogens that may influence the population dynamics of the wider species pool.The invasive common wasp,


Marburg virus (MARV) has been a major concern since 1967, with two major outbreaks occurring in 1998 and 2004. Infection from MARV results in severe hemorrhagic fever, causing organ dysfunction and death. Exposure to fruit bats in caves and mines, and human-to-human transmission had major roles in the amplification of MARV outbreaks in African countries. The high fatality rate of up to 90% demands the broad study of MARV diseases (MVD) that correspond with MARV infection. Since large outbreaks are rare for MARV, clinical investigations are often inadequate for providing the substantial data necessary to determine the treatment of MARV disease. Therefore, an overall review may contribute to minimizing the limitations associated with future medical research and improve the clinical management of MVD. In this review, we sought to analyze and amalgamate significant information regarding MARV disease epidemics, pathophysiology, and management approaches to provide a better understanding of this deadly virus and the associated infection. Marburg virus (MARV) causes deadly outbreaks with a high fatality rate. It is responsible for several outbreaks since its concurrent discovery and characterization in 1967 in Marburg, Germany; Frankfurt, Germany; and Belgrade, Yugoslavia (now Serbia). The majority of the MARV outbreaks occurred in Africa. MARV is into the NIAID Category A Priority Pathogen list, and is the primary cause of MARV disease (MVD) ,3.Mononegavirales, the family Filoviridae, and the genus Marburgvirus. This genus only includes one species, named Marburg marburgvirus, generally known as Marburg virus [Rousettus aegyptiacus bat species as the major natural source for MARV, Hipposideros caffer and some other Chiroptera can also serve as natural sources of infection [This virus belongs to the order rg virus . Variousrg virus ,5. Theserg virus . The humrg virus , Sudan vrg virus . Due to nfection ,9. Currenfection . Laboratnfection . Howevernfection .This review elaborately describes the history of MVD outbreaks, summarizes available information regarding the viral structure and genome, and describes the known sources of MARV and the transmission methods of both natural source-to-human and human-to-human infection pathways. This review also describes the pathophysiology, cellular tropism, immune evasion, and sites of major damage within the host body to provide a better understanding of the pathogenesis of MVD. Currently available clinical findings and management approaches are also described to help researchers to make appropriate decisions in preparation for future MVD outbreaks. No methods have been approved for the control of MVD outbreaks, and numerous studies remain necessary to develop drugs or vaccines. Our review will help future researchers comprehend the background of this virus and provide a profound understanding of the mechanism of infection, which will facilitate the MARV disease management and designing of future drugs and vaccines.An organized literature search strategy was followed to find all the published articles, which reported outbreak history, genome sequence, structure, sources, pathophysiology, damaging prospects, cellular tropism, immune evasion, clinical findings, symptoms, transmission, and management of Marburg virus. To retrieve the information, we thoroughly searched for relevant literature through Google Scholar, Scopus, and PubMed between 1967 and October 2021. We have developed some specific Boolean words based on our objective, as shown in MARV is a pleomorphic virus which is observed in six, circular, U, rod-like and most commonly in filamentous shape . Usuallycis-acting element play role in replication, transcription and also in DNA packaging [It is a non-segmented negative-sense virus which contains a 19.1 kb long RNA genome that encodes seven genes in the following linear order-3'-NP-VP35-VP40-GP-VP30-VP24-L-5' . Each ofackaging ,17. All ackaging  , collected from Uganda in an attempt to produce vaccines for polio [The first outbreak of MARV was documented in Marburg, Germany, in 1967, where scientists and laboratory technicians were doing experiments with tissue derived from African green monkeys  and animals hunted for bushmeat, in addition to natural reservoirs, for example saliva, urine, and excrement of bat, are the primary vectors of MARV transmission. The transmitting paths from reservoir hosts to humans remain unknown ,79. HoweIn the early stages, MARV may spread to humans by infected intermediate animals. Besides, MARV may transmit due to sexual intercourse within humans, since the presence of virus antigens in the semen of infected males has already been substantiated . The stuMost available clinical data were obtained from a few large outbreaks, especially the 1967 outbreak of Germany and Belgrade of Yugoslavia, the 1998 to 2000 outbreak of DRC, and the 2004 to 2005 outbreak of Angola ,60,94. TMHF can be divided into 3 distinct phases; initial generalization phase, that is followed by early organ phase, and then the late organ phase or convalescence phase, according to disease progression  Table 5Table 5.TThe initial generalization phase lasts for five days after disease onset. The early symptoms that are present during this phase include generic flu-like characteristics, accompanied by high fever (39–40 °C). In addition, debilitating symptoms, including fatigue, loss of appetite, abdominal pain, severe weight loss, severe nausea, vomiting, watery diarrhea, and anorexia, have been reported by many patients. Severe headaches, myalgia, chills, and malaise are also common signs ,83,93. TA sustained high fever and other general symptoms accompany the early organ phase, which lasts from five to thirteen days after the onset of symptoms. Patients may manifest conjunctival infection, prostration, shortness of breath (dyspnea), viral exanthema, irregular vascular permeability, and edema . NeuroloThe late stage of MARV infection results in two distinct outcomes: infection either becomes fatal or patients enter a prolonged phase of restoration. Fatality generally occurs between eight and sixteen days after the onset of symptoms. Typically, shock and multi-organ failure are the primary drivers of death ,102. TheThe dissemination and replication of MARV in hosts facilitates the penetration and navigation of viral particles into several cells . MARV gaMARVs are among the most devastating and virulent pathogens that affect humans. Autopsies of MARV-infected patients have revealed swelling of the heart, brain, spleen, kidneys, and lymph nodes. Also in NHPs, hemorrhages were identified in mucous membranes and soft tissues; the most severe necrotic lesions were observed in the lymph nodes, liver, spleen, testes, ovaries, gastrointestinal tract, and endocardium . These oThe severity of MARV’s hepatic damages is apparently greater than EBOV . In MARVMARV infection in humans causes damage to lymphatic tissues, for example necrosis of the follicles and the medulla of lymph nodes, and red pulp of the spleen, in addition to lymphocyte depletion. Interestingly, lymphocytes are not infected by the virus, although bystander apoptosis occurrence results in lymphocyte depletion ,115. In The lung alveoli often showed diffuse congestion, hemorrhage, suppurative pneumonia, and bacterial co-infection ,116. SmaA large volume of plasma cells and monocytes exist in lymphatic organs and the mucous membranes of the stomach and intestines of NHPs . In humaThe kidneys were swollen, pale, and hemorrhagic; tubular necrosis and parenchymal damage cause tubular dysfunction, which leads to proteinuria in MARV patients . MultiplSkin and mucous membranes typically show hemorrhagic abnormalities in MHF, which is associated with skin lesions. Limited histopathological changes occur to skin tissues, including endothelial cell swelling, focal hemorrhage, necrosis, and dermal edema. Cutaneous effects appear regularly between the 2nd and 7th days after symptoms onset, and can occur during the recovery period as well . EpidermA study showed that following the onset of symptoms, MARV can persist in the semen for up to 7 weeks . The incMorphological changes to the bone marrow in MHF remain imprecisely defined. MARV antigen infects normocellular bone marrow, causing focal necrosis. However, thrombocytopenia can be observed without a concomitant reduction in platelet production in MARV cases, similar to EBOV cases .Pseudomonas) have been observed in the myocardium, but no viral antigen has been associated with these lesions [Morphological myocardial injuries observed in MARV autopsy cases have not been uniform. Multiple suppurative embolic foci and lesions containing gram-negative bacteria  . The mosLimited comprehensive clinical studies exist for MARV due to the rural conditions as well as severe occurrences of most of the MARV outbreaks in Africa, and the compilation of laboratory and pathological evidence from patients has traditionally been inadequate. Clinical findings to date emerge from information linked to the first epidemic in Germany, with the South African outbreak, and smaller outbreaks in other areas of Africa . Experimin vitro analysis, suggesting that the hepatocyte receptor asialoglycoprotein may worsen MARV infections [At organ level, in patients with MARV infection, it seems that MARV mainly targets the adrenal glands and the liver , as wellfections . The necfections ,140. Thefections , heart afections .Figure At the cellular level, macrophage, and dendritic cell (DC) are the primary targets for MARV entrance ,141. TheMARV pathophysiology has been observed in animal models, including guinea pigs, mice, hamsters, baboon ,148, andIn the MARV-infected humanized mouse model, Marburg virus manifests malignancy in a greater range. MARV primarily infects macrophages, B cells, T cells, NK cells, and phagocytes in these mice . In non-Guinea pig shows mild feverish manifestation when infected by host-adapted variant of MARV. However, consecutive passaging the guinea pigs with MARV can cause malignant conditions among the animals. In fact, one of the first MARV guinea pig model studies showed that 100% of the MARV-inoculated guinea pigs die 7–9 days post-infection after 8 passages by whole blood . This feSyrian hamster models usually show no manifestations when infected with wild-type MARV strains. But STAT2 null immunodeficient hamster models show infections of liver and spleen, neutrophil leukocytosis, and an increase of proinflammatory cytokine . BesidesAmong all tested NHP models, rhesus and cynomolgus macaques are the best-characterized as well as widely used in MARV research due to their accurate recapitulation of human pathophysiology . MARV inIn ferrets, an experiment recently demonstrated that MARV neither causes disease nor causes viremia in them . AlthougNo established management approach for MARV currently exists. Nevertheless, a popular treatment approach is the utilization of palliatives for pain alleviating purpose. Moreover, supportive treatment is often offered, including blood volume administration and electrolyte balance . AlthougSeveral supportive methods and therapies were used pre-clinically and clinically in the past, as well as several new approaches that have been studied in animal models ,165–167.in vitro against Marburg virus [in vitro against MARV with their several pharmacological potentials [Modern researches have investigated various drugs for MARV infection. A recent study of remdesivir against Marburg virus showed that it has therapeutic efficacies in cynomolgus macaque models, which makes it an important drug to be assessed further. The study showed that it had therapeutic efficacy when given at 4 or 5 days post inoculation once daily in vehicle, for 12 days with 5 mg/kg dose, or 10 mg/kg loading dose followed by 5 mg/kg dose . Anotherrg virus . Recentlrg virus . Furthertentials . Some smtentials . Again, tentials . Also, Atentials .Numerous experiments were conducted on rodent and NHP models for testing vaccine efficacies against MARV. To date, some vaccines have been trialed for human use ,181,182.The experimental approaches have substantiated the efficacies of numerous treatments, which include anti-viral treatment, pre- and post-exposure vaccine treatment, and treatment with IFNs; these are displayed in the Rural and poor conditions, along with sporadic outbreaks in Africa, are principal reasons for unsatisfactory clinical findings of the Marburg virus. Although the African fruit bat has been identified as a potential natural reservoir for MARV and most of the outbreaks have been caused by spillover into human population from an animal reservoir, human-to-human transmission mediated by the care providers or health professionals who work in hospitals also played great role in the growth of MARV outbreaks. These outbreaks demonstrated that MARV could be malignant and able to cause severe outbreaks if not taken proper steps immediately. Although MARV strains originate only in African regions, the outbreak history suggests that other continents could also be at risk. Recent outbreaks have indicated the necessity of additional research exploring the pathophysiology and pathogenicity of MARV. Current data remain based on earlier studies of infected patients. In response to the intensity of previous outbreaks, epidemiologists have suggested MARV as a threat to global public health. Therefore, novel studies examining MARV remain crucial to provide unambiguous guidelines for the therapeutic management of patients, and for the development of vaccines. These steps could be used to minimize future outbreaks and reduces the CFRs. Vaccine studies have been ongoing for the past few years, and the virus is being studied on a variety of animal models, including NHPs, mouse models, guinea pigs, and hamsters. Vaccine studies should be perpetuated until the licensure and distribution of vaccines in humans occurs.


Giant DNA viruses of eukaryotes are notable for their extraordinary genome size and coding capacity. Once thought to be oddities in the virus world, these elusive microbes have turned out to be widely occurring in marine, freshwater, and terrestrial ecosystems and are commonly associated with diverse hosts, in particular microbial eukaryotes. This commentary discusses how new sequencing techniques and information can inform us about the interactions between giant viruses and eukaryotic hosts during the viral replication cycle and their implications for ecological and evolutionary processes across different spatiotemporal scales. Nucleocytoviricota . While NCLDVs vary along a size continuum, their relatively gigantic dimensions justify the use of giant viruses (GVs) to refer to this group of viruses with shared characteristics and genes.Viruses are traditionally defined as small infectious biological entities. However, a rapidly increasing number of viruses with large particles and genomes are associated with diverse eukaryotes, including vertebrates, invertebrates, algae, and various other protists from across the eukaryotic tree of life . These nExcept for smallpox, African swine fever, and some deadly diseases in fish, tetrapods, and insects, most of the seven or so GV families recognized to date—major clades in phylogenetic trees—infect only or predominantly microorganisms. Microbial, mostly unicellular, eukaryotes compose the vast majority of eukaryotic taxa and exhibit immense diversity in organelles, cellular structures, and life cycles. Although they make up less than 1% of the biomass on our planet , they caApproaches to studying GVs encompass culture-dependent and culture-independent methods . Since tA major disadvantage of meta-omic approaches is their bulk sampling nature that prevents tracing a particular genomic fragment back to a specific cell or organism. Cooccurrence analyses can help predict the eukaryotic taxa of GV hosts , but theDespite the relative ease of acquiring omic data, many aspects of viral biology cannot be studied without lab cultures. It is therefore important to stress the need for high-throughput protocols for systematic isolation and culture of GVs, for example, with the use of amoebae that enable the replication of a wide range of GVs  or a cusThe replication cycle of GVs involves virus entry, gene expression, DNA replication, virion assembly, and release . TypicalAcanthamoeba and Vermamoeba, are permissive to diverse GVs, although they are not necessarily the natural hosts of these GVs , likely representing the largest evolutionary radiation of GVs known so far. It is highly abundant in aquatic environments (One research focus in our lab is to elucidate GV gene content variation and evolution by taking into account the effects of different processes  and hostronments , 6, has ronments  and the ronments , 6, and ronments , 8.In summary, recent advances in understanding GV-eukaryote relationships are fundamentally changing our view of the associated ecological and evolutionary processes at all levels . Althoug


Soffritti et al.). In this Special Issue, there is a consensus: the oral microbiome is a key factor in oral and systemic health. Also, considering HOM as biomarkers for diseases is a significant emerging orientation. Saliva, the cornerstone of the HOM and systemic health relationship, has gained popularity as a readily available source of biomarkers useful for diagnosing specific oral and systemic conditions . Current findings indicate that salivary markers of oxidative stress are indicative of other clinical disease indices such as the papillary bleeding index and the caries index. Fungal species Candida dubliniensis and Candida tropicalis more abundant in the saliva of children with severe early childhood caries should play a role as caries risk markers .In oral health, biomarkers in saliva  can be used for activity determination and for periodontal disease prognosis . Oral bacteria from genera Capnocytophaga and Veillonella are apparently present in increased amounts in lung cancer patients . Also, bacterial dysbiosis plays an important role in the esophageal carcinogenesis process through microbial metabolism, inflammation and genotoxicity  and Capnocytophaga and Veillonella are reportedly present in increased amounts in lung cancer patients .Oral pathobionts are essential in the development of colorectal and pancreatic cancer with current evidence showing differences in oral microbiota composition between patients with and without digestive cancers . Immunity in saliva is, in particular, thought to have considerable impacts on the incidence and progression of respiratory viral infection. Parts of antiviral mechanisms against influenza virus and SARS-CoV-2 by immunity in saliva are similar.It is important for prevention of viral infection to draw a perspective on the role of the oral cavity in the virus infection , possessing unique niches and invasive properties, coexist with Human Papilloma Virus (HPV) within HPV-induced oral lesions in head and neck cancer patients .Saliva based biomarkers are useful in diagnosis of several viral infections such as hepatitis A virus, hepatitis B virus, hepatitis C virus, Human immunodeficiency virus (HIV) 1, etc. . The susceptibility of each individual to SARS-CoV-2 infection could therefore be characterized by HOM profile, which could facilitate virus replication and inflammation or conversely induce a protective IgA response . Several routes of SARS-CoV-2 viral entry into the saliva have been suggested. There is direct entry to the oral cavity from upper and lower respiratory tract secretions, while circulatory viruses in the blood enter the gingival crevicular fluid. Studies reported a high yield of virus particles in the gingival sulcus and crevicular fluid, which are suspected to provide favorable conditions for virus replication and maintenance (Sri Santosh et al., The fact that oral cavity is an important site for SARS-CoV-2 infection implicates saliva as a potential route of SARS-CoV-2 transmission (Overall, the 12 contributions that make up this Special Issue highlight the fundamental relationship between HOM and systemic health providing potential microbiome-based clinical applications improving prevention, diagnosis, or drug response, which is of great significance. Characterization of microbial biomarkers is of great interest for precision medicine and represents a simple method to transfer microbiome research into clinical practice (Gilbert et al., All authors listed have made a substantial, direct, and intellectual contribution to the work and approved 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.