Household food purchasing decision is a complex process influenced by factors such as marketing, cost, children food preference and parental choices. Most food products targeted toward children are unhealthy and are aggressively marketed to increase desirability among parents and children making healthier food selection even harder. The warning label (WL) is identified as a simple front-of-package labeling format that assist consumers to easily identify unhealthy foods and reduce their purchasing. This was a qualitative study that aimed to investigate the perceived effect of the warning label (WL) on parental food purchasing and drivers of food selection among parents. The study was conducted in a mainly rural part of South Africa, in Limpopo Province. Data were collected from 44 adult participants, all parents with children aged below 16 years selected using the snowball sampling method. Seven focus groups diversified according to age, literacy, income and urbanicity were utilized for data collection. Using a focus group discussion guide, parents were shown images of six products  superimposed with the WL and questions asked were based on those images. Thematic analysis revealed that although some parents felt undeterred by the WL, some felt they would alter their food purchasing in the presence of the WL. Other parents felt they would reduce the frequency or the amount purchased or completely stop purchasing labeled products for their children. Motives behind perceived behavior modification included children's health being perceived as a priority and labeled products being viewed as unhealthy. Factors such as pressure from children, taste, poor nutrition knowledge and affordability seemed to influence parental food selection. These findings have important policy implications by providing evidence to policymakers that the WL may alter parental food purchasing and also provide insight into drivers of food selection among South African parents. Non-communicable diseases account for more than 51% of all deaths in South Africa . The linUnhealthy diets are one of the major modifiable risk factors responsible for NCDs , 4. CurrAlthough mostly experienced later in life evidence suggests that diet-related NCDs start early in childhood and adolescence  ChildhooParents are primary household food purchasers and although influenced by other external factors such as time constraints , pressurInternational organizations recommend provision of nutritional information as a strategy to assist consumers identify healthier food options , 21. EviExisting research reveals that in the presence of the WL, an example of an interpretive FOPL, consumers are better able to understand the nutrient quality of food and select healthier food options , 26. WL Two conceptual framework models were adapted to explain pathways through which the WL influences purchasing decisions and to secondly explain drivers of food selection  31, 32), 3231, 3Warning labels have been reported to positively impact consumer behavior by shifting the desire away from unhealthy products , 43. AnoA previous study evaluating the opinion of South African adult consumers on WLs revealed that consumers found the labels attention-grabbing, easy to understand and effective in warning against unhealthy food . FindingThe latter study did not however investigate the perceived effect of the WL on parental food purchasing. Parents seem to select food differently for their children , 17 and We collected data from seven focus groups consisting of 44 participants residing in Limpopo Province, South Africa. All participants were parents with children below the age of 16 years. Focus groups varied according to age (18–29 years and 30–50 years), income (low and middle-high), literacy level (low literacy and literate) and urban or rural residency .Low income was defined as an income below or equal to R1600.00 (approximately 94 USD) and income above R1600.00 was categorized as middle-high. Low literacy was defined as educational attainment at or below Grade 6 and a participant with Grade 7 and above was considered literate. The purpose of diversifying the groups was to capture potential differences in perceptions according to different ages, educational and socioeconomic status and urbanicity. The sample consisted of parents primarily responsible for either purchasing or preparing food within the households and having children below the age of 16 years. MB, one of the researchers, recruited participants both face-to-face and telephonically through the snowball sampling method. Ethical approval was obtained from Biomedical Research Ethics Committee of the University of the Western Cape. The materials and methods followed in this study are presented according to the Consolidated Criteria for Reporting Qualitative Research (COREQ) .Discussions were based on 2D images of mock-up products  superimposed with the WL (referred to as labeled products in this study) . The nutAll discussions were conducted by MB using a focus group discussion guide  developeBefore the commencement of the study, the moderator explained the aim of the study which was to explore the views of the participants on the images to be displayed during discussions. Once the purpose of the study was explained, participants were then requested to sign the focus group confidentiality binding form. Participants were shown different images and responded to questions based on those images. The images were first rotated within the focus group to ensure each participant had a closer view of the images together with all the graphics. Participants were requested to view the images in silence. Once all participants had viewed the images, the moderator presented the images again, one at a time, without providing any explanation, to ensure all participants were aware of all the images. Once completed, the moderator started the discussions with one image, chosen at random, and led the discussions until all responses were pointing to the warning label and not the product. Once participants' focus was on the warning label, the moderator then continued to ask questions based on the focus group discussion guide. Focus group discussions lasted between 40 min to 45 min and were conducted until data saturation was reached was for the questions. Discussions were held in Sepedi, the language that participants understood. MB moderated the discussions transcribed the recordings verbatim and translated the data to English.The researchers developed a focus group discussion guide that was used during focus group discussions. The guide was based on the adapted conceptual framework , 32 Fig which suData were analyzed following inductive thematic analysis . AlthougTrustworthiness of the study was ensured throughout the study process , 48. To We extracted six themes with several subthemes from the data.WL cautions against nutrients in excess, WL promotes informedfood choices and WL reminds of health consequences.During discussions all parents were requested to share their views about the WL and the responses ranged from: There is too much fat and too much salt in that package” . Another parent said: “And this is high in salt. This tells you that this product contains too much salt” . This implies that some parents were able to correctly understand the message conveyed by the WL.A number of parents' remarked that the WL alerts them to nutrients that are contained in excessive amounts, to which one parent said: “. This is what one parent said: “As a parent, I will be the one going to the shops then I will know which products to buy for my child. I will first check the label and then know how my child will be eating. I will be aware of what I am feeding him ”. Another parent added: “So if we have inherited diseases in the family and we are diagnosed with certain diseases or hypertension in the family, I'm going to check the label first. If the label says this product contains too much fat or too much sugar I'm going to stop buying it .”One other view from some of the parents was that the WL would enable them to make informed food choicesBut if you think carefully, you will remember that eating too much sugar and too much salt causes diseases and then you will not buy them” . “I cannot buy a product that will make me sick at the end .”From the discussions it was evident that the presence of the WL made a number of parents think about the health consequences related to overconsumption of the labeled products. “It will scare us” . Another parent in the same group added: “We will no longer buy as usual, we will start to be afraid” . In response to the question, another parent said: “I will be scared”  s:When asked how they would react if the WL was implemented and put on products in the supermarkets, the perceived emotional reaction from several parents was fear as illustrated by these responses: “reduce the amountand frequency of purchasing, stop buying labeled products, continue buying labeledproducts and switch to a different product.Parents were asked about their perceived reaction if products they usually purchased for their children would contain a WL. The following subthemes emerged: It's not going to be easy to buy products with labels for them”  ' indicating some discomfort in buying products should they contain the WL in future. One parent said: “This label is going to be helpful as we will be able to see that we actually were not feeding our children well and things would have to change . Even myself that's what I normally buy for my kids. So starting today I'm going to start paying attention to what I buy for them .It was evident that the WL affected a number of parents as noted by quotes such as: “Because mostly what we saw in those pictures is what we normally put in their (children) lunchboxes, which means we are going to cut' .” Another parent said: “Let me give an example, like with crisps, there are those that come in strips of seven individual packets. I will buy one strip of seven small packets for my child and a big packet for myself, not for my child” . Similarly another parent offered: “Firstly maybe I'm going to buy the smaller amount of the pack. So I'm not going to buy the big bag” . This implied that parents viewed smaller packets of labeled products as better than the big ones.Although some parents felt they would continue buying labeled products, others felt they would reduce the amount and frequency of buying and consuming such products. One parent said: “Ya from my side I will completely stop. I'm not going to compromise the life of my children because of the nice time for only a short term” . Another parent added: “I will not buy them for my children. We also want them to grow well. If we do not want fat and salt for ourselves, we also do not want it for them” .When asked how the labels would affect food selection for their children, one parent said: “I will continue buying products with labels” . Another parent added: “Personally, I will buy the one with a warning label. A child will not eat one without the label as it will not be containing sugar” . This implied that parents were willing to accommodate their children's food preferences.Some parents acknowledged they would continue purchasing labeled products especially when shopping with their children. “One parents said: “I will check for other products without the warning signs” . When asked what they would pack for children's lunch if they stopped buying labeled products parents in the current study said they would pack non-labeled products, fruit and water. One parent said: “We will pack the one without the warning label” . Another parent said this in response: “Fruit, they are the healthy option. They do not have any negative consequences” . When asked what would happen if the child does not enjoy the non-labeled product one parent said: “They will get used to it” .Other parents' opinion was that they would instead consider other alternatives to labeled products. health as a priority and labeled products viewed as unhealthy.Some parents further offered their reasons for intention to modify purchasing behavior that included: A number of parents alluded to the importance of children being healthy from a young age: “Because children should also be healthy from childhood. We should take care of them while they are still small and not only start giving them healthy food when they are older. He should get used to them from an early childhood” .: “My child's health is a priority. My child's health cannot be compared to any amount” .When asked what they would do should unlabelled products be more expensive, some parents maintained that they would still not buy labeled products for their children. One parent remarkedParents viewed products containing high amounts of nutrients of concern as unhealthy and that seemed to serve as motivation for intention to modify purchasing behavior. There was a general concern about the poor nutritional value of the labeled products. One parent said: “Food high in fat is unhealthy. They can cause diseases” . In addition, another parent said: as you can see, crisps contain salt and; fat is also written there (pointing at the pack). in the body they will just create a mess. We need to be selective with the type of snacks we eat, (choose) healthy ones .pressureexerted by children, taste, poor nutrition knowledge and affordability. Regarding pressureexerted by children one parent said: “Most of the time we go with them and it's not easy. Because that kid will scream in the shop like you stole him whereas he is yours. even if you agree at home that you are not going to cry for these and that. .” Another parent felt taste play a role: “A child will not eat one without the label .” Another parent said: “Lite ones do not taste nice. Children are controlled by sugary stuff, even us. It will stay in the fridge for a long time” .During discussions, drivers of food selection mentioned by parents included: Poor nutrition knowledge also emerged as one of the reasons for food selection. One of the parents believed that children are not at health risks due to their young age: “I don't think these will affect children that much. Because they are still young they might not get very sick . On the other hand other parents viewed food high in sugar as harmless.” “I don't think there is anything wrong with food high in sugar. For example if cereals are high in sugar, I can eat them with milk and then add no additional sugar” . Another parent said: “It is not possible to drink a hot drink (referring to fizzy drink). But you will not even feel its sweetness when it is cold, you just drink, no problem at all” . This implied there were compensatory measures one could take to balance the amount of sugar in food.affordability. One parent said: “I will buy labeled products if unlabelled products are more expensive” . Another one said: “We give them whatever is available. If it's a month where you managed to buy cheaper ones that's what they will take to school, if you managed to buy expensive ones, that's what they will carry” .Other parents felt compelled to purchase certain foods due to Once a week — because some of the products are needed in our bodies, even salt must not be a lot, but it is needed. Even sugar and alcohol. Not too much alcohol but just a little bit. We have to balance it like that” . Another parent said: “So we still need snacks but not too often or every day” and when asked how often this parent said: “Maybe three times a week” .When asked how frequently they felt labeled products should be consumed, their responses ranged from one to three times per week. One parent said: “One parent said: “It's only adults and literate people who will be able to read the label. Children and the illiterate will not be able to read it” . Another parent added: “They cannot understand what is happening there” . Right, it will take time because they will not understand. Children are just a children, they will not understand what is happening. But in future, that thing will build up .As a possible determinant of food selection, parents were asked whether they thought children would understand the WL or not. Parents were divided, with some believing the label would be confusing for children. Strategies to maximize WL effectiveness: Following were parents' recommendations to improve awareness of the WL: parental education of children at home, education ofchildren at school and education through mass media.parental education of children at home one parent said: “But the best thing it will need us as parents or whoever is staying with the child to educate them” . Another parent held a similar view: Like if she buys the product and comes home with it, we can inform her that she can eat it but it has negative consequences' .Regarding Because if I keep on buying, my children will think these products are okay. If these products are not available at home, sometimes they might not have money to buy them” .Other parents also emphasized the importance of modeling healthy eating habits at home. One parent said: “education of children byteachers at school would be better. Their view was that children were more receptive to teachers than their parents. One parent said: “When you tell children not to buy biscuits or other stuff they sometimes think that you just don't want them to eat biscuits, but if they are taught about the label at school…. Sometimes children become moody when you tell them to do school work, but when they are at school they listen and do what teachers instruct them to do. So, if schools could be the ones promoting the label, teach them that such products are not good, they do this to the body… they would be hearing that in their classrooms and they listen to their teachers” .On the other hand some parents were of the opinion that education through mass media. Parents recommended several avenues such as health education at the clinics, broadcasting over the radio and TV and address by the Ministry of Health. Some parents likened the implementation of the WL with the introduction of face masks for prevention of the spread of the Corona Virus. One parent said: “But the issue about whether children would understand the label or not, if this could be addressed nationally, by the Minister of Health for example, and it's broadcasted live, same as when the president warns us to be careful, it will not be difficult, just like with masks, we are used to then now. It will not be difficult” .Another strategy that emerged during discussions is This study revealed that a number of parents felt the WL would discourage selection of labeled products for their children. Motives for perceived behavior modification were child health being viewed as a priority and labeled products being viewed as unhealthy. In addition the current study revealed diverse drivers of food selection that included pressure exerted by children, taste, poor nutrition knowledge and affordability.Some parents in the current study felt the WL enabled them to identify products that were high in nutrients of concern. This finding is supported by other experimental studies where the WL performed better in assisting consumers identify products with high amounts of risk nutrients , 52. TheThe WL seemed to have made a number of parents think about the negative health effects of indulging in products bearing the WL. Some parents indicated that the presence of the WL would trigger feelings of fear toward products bearing the label. Similar reactions of fear evoked by the WL and thinking about health harms have previously been reported . WLs flaRegarding the perceived effect of the WL, although some parents felt they would continue purchasing labeled products, should the WL be implemented, others felt they would alter their purchasing behavior. A number of parents felt they would reduce the amount and frequency of purchasing labeled products, others felt they would stop purchasing labeled products while some planned to switch to other alternatives. One of the goals of the WL is to discourage purchasing of unhealthy products  and pareParents could imagine using the WLs to help guide purchases for children's lunches. This remark was made upon the realization that products shown during the discussions were those they usually include in their children's lunchboxes. Nathan et al.  found thEnablers of the WL in this study included health being viewed as a priority and labeled products being viewed as unhealthy. Health and nutrition were also previously reported as motivators for parental food choices . ParentsPoor nutrition knowledge surfaced as one of the influencers of parental food selection. One view held by parents was that sugar does not pose any health problems for younger children. Others felt that sweetened beverages if taken cold would not have any health repercussions as they lose their sweetness when chilled. Such misconceptions could potentially fuel excess sweetened beverage consumption and obesity among children. Any implementation of WL regulations should therefore be linked to strong health education campaigns to improve label understanding and broader nutrition knowledge. This calls for the need to strengthen nutrition literacy initiatives by the health sector, academia and other non-governmental organizations.Affordability was mentioned as another factor generally affecting parental food selection similar to findings in other studies , 24. LowRegarding parental perception on label understanding, a number of parents felt the WL would be meaningless to children. Parents recommended strategies to improve its effectiveness and that included education of children at home, education of children at school, education through mass media and demonstration of healthy eating habits at home. Similar strategies have previously been recommended , 62 and The strength of this study is that discussions were based on a variety of products commonly classified as unhealthy  and those usually perceived as healthy . Another strength was that all parents had children below the age of 16 years and were suitable to give views as parents. Understanding parental view on the effect of the WL is an important policy consideration as parents play an important role in shaping children's eating habits. The limitation of this study is that data were collected in only one Province and the results may not be generalizable to the entire population. However, the focus groups were diversified to capture opinions from diverse groups to improve the richness of the data. Another limitation inherent in qualitative studies is that focus group discussions can be easily swayed by one vocal group member. Quantitative studies with a representative sample size could be conducted to understand the widespread perceptions of parents in South Africa. This study was experimental and may not translate directly into actual purchasing behavior. The actual effect can only be determined once WLs are implemented. A potential bias for this type of study is demand effects. To deal with this effect, participants were invited to participate freely and were informed that there were no correct or incorrect responses. Additionally participants were only informed that the study was about their perceptions of the images (pictures) to be displayed during the discussions.Based on our results parents believed they would reduce the quantity and frequency of consuming labeled products, stop purchasing labeled products and switch to non-labeled products. Some parents felt they would continue purchasing labeled products. Motives to switch to non-labeled products included health being a priority and labeled products being perceived as unhealthy. Drivers of food selection included pressure exerted by children, taste, poor knowledge and affordability. This study provides more clarity on factors influencing food selection by parents and how policy efforts may influence purchasing behavior of South African parents. These results strengthen the importance of implementing WLs in South Africa to benefit children health.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Biomedical Research Ethics Committee; University of the Western Cape. The patients/participants provided their written informed consent to participate in this study.MB, LT, and RS conceptualized the study and contributed to the study design. MB collected and analyzed the data and drafted the original manuscript. RS acquired funding for the study and supervised the project. LT and RS reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.This study was received from Bloomberg Philanthropies, Subcontract # 5108311 with the University of the Western Cape.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.


COVID-19 is a fast-spreading pandemic that is caused by SARS-CoV-2 viral pathogen. Combination therapy of the antiviral favipiravir and the anticoagulant apixaban is one of the efficient treatment regimens. Therefore, development of novel and sensitive methods for simultaneous analysis of such combination is highly advantageous. Herein, two eco-friendly, simple, rapid, and cost-effective spectrofluorometric methods were evolved for the estimation of favipiravir and apixaban in pharmaceutical and biological matrices. Method I was based on analysis of favipiravir and apixaban by the first-order derivative of the conventional fluorescence spectra obtained after excitation at 300 nm, where favipiravir and apixaban were detected at 468.8 and 432.0 nm, respectively. Method II relied on dual scan synchronous spectrofluorometry, in which favipiravir was determined at 364 nm using Δλ = 60 nm while apixaban was analyzed at 274 nm using Δλ = 200 nm. Method optimization was performed for selecting the optimum conditions at which maximum sensitivity and selectivity were obtained. This report is the first one that describes simultaneous analysis of favipiravir and apixaban by synchronous spectrofluorometry. The developed methods were successfully applied to evaluate favipiravir and apixaban in spiked human plasma and in pharmaceutical dosages with high %recoveries and low RSD. The disease is caused by novel infectious positive single-stranded RNA (SARS‐CoV‐2) virus, and it is usually accompanied by multiple atypical pneumonia. Moreover, it shows several other symptoms such as cough, fever, fatigue, diarrhea and progressing to acute respiratory distress syndrome (ARDS). COVID-19 patients may also report neurological symptoms, including dizziness, hypogeusia headache, myalgia, ataxia, hyposmia, and seizures2.COVID-19 pandemic emerged in Wuhan, China at the end of 2019. Up to date, more than 6 million people have lost their lives because of this epidemic out of approximately 492 million who were primarily infected worldwide3. Among these effective antivirals is favipiravir (FAV) which was recently used for treatment of COVID-19 in some countries.Notwithstanding, COVID-19 treatment protocols are still under investigation and may differ from a country to another relying to severity and spread factors, however, the main treatment guidelines are followed. Because of the high reproductive number of SARS-CoV-2, antivirals were tested for efficacy to control or even stop the viral transmission. Antivirals that inhibit reverse transcription are among the most potent agents available to combat the SARS-CoV-2 infection4.Favipiravir is a nucleoside analogue (purine base) that is converted intracellularly to the active form, FAV-ribofuranosyl-5B-triphosphate by phosphoribosylation process. It inhibits RNA-dependent RNA polymerase (a core enzyme in SARS-CoV-2 replication process) selectively and potently in SARS-CoV-2. FAV was the antiviral of choice for the current study as it showed rapid viral clearance in comparison to ritonavir and lopinavir in addition to superior rate of recovery compared to umifenovir. Additionally, FAV is incorporated recently in COVID-19 treatment protocols in some countries such as India5, elevated D-dimer levels and prothrombotic parameters. Then, anticoagulants were included as prophylactic treatment in patients with high D-dimer values to reduce venous thromboembolism rate or mortality. Reported clinical trials assured the claim by significantly decreasing mortality upon using anticoagulant therapy among COVID-19 protocol6. Different anticoagulants were tested for decreasing mortality, enoxaparin and heparin therapeutic doses have not shown to downside mortality rates while apixaban (APX) prophylaxis and therapeutic doses have done. Then, APX was the preferred anticoagulant regimen6.The mortality rate increased in COVID-19 patients who suffered from multi-organ dysfunction accompanying ARDSH-pyrazine-3-carboxamide and 1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxopiperidin-1-yl)phenyl]-4,5-dihydropyrazolopyridine-3-carboxamide, respectively.Favipiravir and apixaban Fig.  have the7, two conventional spectrofluorometric methods9, electrochemical methods12, and HPLC methods13 were reported. On the other hand, spectrophotometric17, spectrofluorometric18, electrochemical19, and HPLC24 methods were reported for the assay of APX. As discussed, antivirals besides anticoagulants are co-administered during COVID-19 treatment in mild to severe cases of infection. The analytical study of such coadministration besides few reported spectrofluorimetric techniques were the motivation to develop this work. It is noteworthy that this study is the first to be performed targeting simultaneous determination of FAV and APX followed by application to human biological samples.The literature was screened for analytical procedures that were applied for determination of FAV and APX. For FAV, a spectrophotometric methodBased on the native fluorescence of FAV and APX, trials for separation of FAV and APX’s overlapped spectra were performed. Then, the two current methods were developed and applied to local market tablets having FAV or APX as the main active ingredient besides determination of both drugs simultaneously in human plasma samples.All spectrofluorometric measurements were performed by an Agilent® Cary Eclipse spectrofluorometer to which a xenon flash lamp is equipped. A smoothing factor of 20, a 5 nm slit width, and an applied voltage of 800 V were used during all experimental trials. Relative fluorescence intensities (RFI) of the estimated analytes were measured using excitation wavelength of 300 nm in method (I) and dual scan in method (II), where in first scan FAV was determined using Δλ of 60 nm and calibrated at 364 nm and in the second scan Δλ of 200 was set to detect APX and then calibration was performed at 274 nm.Favipiravir raw material was kindly supplied by EIPICO pharmaceutical company, Egypt. Avipiravir® tablets  were supplied by EVA Pharma Company, Cairo, Egypt. Apixaban raw material was supplied by Multi Apex company, Egypt. Artixiban® commercial tablets, labelled to contain 2.5 mg APX per tablet, were purchased from a local pharmacy.HPLC grade iso-propanol, acetonitrile, ethanol, methanol, and n-propanol were all obtained from Sigma-Aldrich, Egypt. Analytical grade of surfactants: tween 80, sodium dodecyl sulfate, and carboxymethyl cellulose were supplied by laboratories of faculty of pharmacy, Mansoura University, Egypt.The frozen plasma samples were supplied by Hospitals of Mansoura University, Dakahleya, Egypt. All experiments were performed according to the Institutional Ethics Approval of the relevant University Committee.−1 of both drugs, were prepared by accurately weighing 10 mg of the raw materials, quantitatively transferring each powder to a 100 mL volumetric flask, and completing the volume to 100 mL using ethanol, followed by a 10-min sonication process.Stock solutions, having the concentration of 100 μg mLSets of 10.0 mL volumetric flasks were arranged, to which aliquots of FAV or APX or both (as in synthetic mixture) were transferred. All flasks were completed to 10.0 mL using distilled water and mixed well. Against blank distilled water, RFI of flasks were measured setting excitation wavelength of 300 nm. The recorded emission spectra were converted to their first order derivatives using filter size of 20 and interval of 5. FAV was measured at 468.8 nm which represented a clear zero crossing point for APX, meanwhile, APX measurements were recorded at 432 nm, that was a clear zero crossing point for FAV. Then, calibration curves were attained by plotting ∆F of each flask against its corresponding concentration.−1) were transferred. Flasks were completed with distilled water to the mark and mixed well. RFI of flasks were recorded at Δλ = 60 nm and calibrated at 364 nm against blank. Calibration curves were attained by plotting ∆F of each flask against its corresponding concentration.FAV calibration curves. A set of 10.0 mL volumetric flasks were arranged, to which aliquots of a diluted stock  and (II), respectively. The nominal contents were calculated using corresponding regression equations or already plotted calibration graphs for each method.The donated plasma was kept frozen at − 5 °C then gently thawed in a water bath at 37 °C before using. Into a set of 6 centrifugation tubes, 15-mL each, 1 mL of thawed human plasma was transferred to each tube, followed by spiking with elevating concentrations of FAV and APX, simultaneously. The 6-tubed set examined a one-minute vortex mixing and after that, completed to 5 mL with ethanol that allowed one step precipitation of plasma proteins. The vortexed set examined a 20-min 5500 rotations per minute centrifugation to efficiently separate the precipitated plasma proteins while maintaining FAV and APX in the supernatant layer. Then, supernatants were filtered by 0.2 µm disc filters to remove possible suspended particles. A specified volume (1.0 mL) of the supernatant solutions, containing FAV and APX, was transferred to a series of 10.0 mL volumetric flasks, then the procedures under “FAV-APX synthetic mixtures” section were followed and the %recoveries were measured.Plasma application was licensed by Research Ethics Committee, Faculty of Pharmacy, Mansoura University (Code number: 2022-128). The Research Ethics Committee, Faculty of Pharmacy, Mansoura University (Code number 2022-128), waived the need for informed consent to be obtained. Also, all experiments followed the guidelines and regulations by Research Ethics Committee, Faculty of Pharmacy, Mansoura University.COVID-19 or corona disease is still attacking all countries worldwide with variable strengths. Treatment protocols passed through several updates and are still under study and optimization by clinical researchers. Then, we had the eager to develop the presented methods which manifested the selectivity and sensitivity of spectrofluorometric technique. Our methods estimated the recently approved COVID-19 antiviral FAV and co-administered APX anticoagulant in their dosage forms. Additionally, both drugs were determined simultaneously in human plasma samples, and this would be further applied in forensic medicine and clinical studies.18. Upon spectrofluorometric scanning of FAV and APX, a complete overlap was observed as represented in Fig. Spectrofluorometry was the technique of choice as it is featured by high sensitivity, selectivity, simplicity, and low cost. This technique allowed utilization of reported conventional fluorescence spectra of the analytes in questionex of 323 nm and 248 nm, respectively, an overlap was observed hindering simultaneous measurement of the analytes in question Fig. 26. In conventional spectrofluorometry, recording emission at excitation of 300 nm allowed detection of FAV and APX simultaneously at 468.8 nm and 432 nm, respectively after implementing first order derivatization as in Fig. Following conventional emission of FAV and APX at their reported λ28.SFS is advantaged by simplicity and sharpness of peaks when compared to conventional spectra. Beside minimization of complex matrix interference, high selectivity is guaranteed by synchronous technique, then plasma applications are more simple−1) and APX (1.0 µg mL−1) were used for the following three parameters. Conventional spectrofluorometry was adopted to adjust optimum conditions using emission 432 nm after 323 nm excitation for FAV samples and emission at 475 nm after 280 nm excitation for APX samples.Spectrofluorometry is characterized by high sensitivity, then study conditions were cautiously examined. Where constant concentrations of FAV , FAV: ΔF = − 2.79 − 0.48C, APX: ΔF = 0.53 + 30.1C , and method (II) FAV: ΔF = 1.10 + 65.29 C, APX: ΔF = 7.88 + 313.22 C, where C: drug concentration and ΔF : relative fluorescence intensity.Following ICH guidelinesa is the standard deviation of intercept. Table r) of 0.9999 for both drugs assured linearity of methods for both drugs in the selected range of analysis.Detection and quantitation limits values were both calculated by data obtained in calibration graphs following using the reported equations: LOD = mentclass2pt{minimmentclass2pt{minimt-test and variance ratio F-test when compared to comparison procedures. Obtained data and analysis values are expressed in Table Accuracy of the proposed spectrofluorometric procedures could be expressed by accepted values of Student −19. Additionally, the reported comparison method for APX analysis relied on conventional spectrofluorometric measurement at 450 nm following excitation at 284 nm using distilled water with linearity range of 0.2–6.0 μg mL−118. The present SFS method is superior in sensitivity for FAV (1–14 ng mL−1) and APX (0.1–2.0 μg mL−1).FAV comparison method was based on direct analysis at 436 nm in Britton-Robinson buffer of pH 4 after excitation at 323 nm with linearity range of 20–350 ng mLTable max in each individual scan. The methods were applied successfully to assay the analytes of interest in their pharmaceutical dosages. Additionally, FAV and APX were analyzed in human plasma with % mean recoveries of 98.54% and 101.76% in conventional spectrofluorometric method and 98.63% and 99.95% in dual scan synchronous spectrofluorometry for FAV and APX, respectively.Two rapid, simple, direct, green, and sensitive spectrofluorometric methods were evolved for the simultaneous analysis of COVID-19 medication regimen FAV and APX in different matrices. The assay methods utilized both conventional and dual scan synchronous fluorescence spectroscopy in order to assay each analyte in presence of the other one without any interference. Dual scan technique has overcome the sensitivity and selectivity problems by measuring each analyte at its λ


New-onset atrial fibrillation (NOAF) complicating with ST-elevated myocardial infarction (STEMI) patients following percutaneous coronary intervention (PCI) is associated with worse prognosis. The systemic inflammatory response index (SIRI), serves as a novel inflammatory indicator, is found to be predictive of adverse outcomes. The aim of this study is to explore the association between NOAF and SIRI.A retrospective data included 616 STEMI participants treated with PCI in our cardiology department had been analyzed in present investigation, of which being divided into a NOAF or sinus rhythm (SR) group based on the presence or absence of atrial fibrillation. The predictive role of SIRI for in detecting NOAF had been evaluated by the logistic regression analyses and receiver operating characteristic (ROC) curve. Additionally, long-term all-cause mortality between both groups was compared using the Kaplan–Meier test.P = 0.001). In the ROC curve analysis, SIRI with a cut-off value of 4.86 was calculated to predict NOAF, with 4.86, with a sensitivity of 80.85% and a specificity of 75.57%, respectively (area under the curve (AUC) = 0.826, P < 0.001). Furthermore, pairwise compassion of ROC curves displayed the superiority of SIRI in the prediction of NOAF in comparison with that of neutrophil/lymphocyte or monocyte/lymphocyte (P < 0.05). In addition, the participants in NOAF group had a significantly higher incidence of all-cause death compared to those in SR group after a median of 40-month follow-up .NOAF during hospitalization developed in 7.6% of PCI-treated individuals. After multivariate regression analyses, SIRI remains to be an independently predictor of NOAF (odds ratio 1.782, 95% confidence interval 1.675–1.906, SIRI can independently predict NOAF in patients with STEMI after PCI, with being positively correlated to worsened outcomes. New-onset atrial fibrillation (NOAF) is one of the most common arrhythmic complications in the setting of ST-elevated myocardial infarction (STEMI) following percutaneous coronary intervention (PCI), with a reported incidence ranging from 3.0 to 13.7% . Thus, iInflammatory response and immune system cells, such as monocyte, neutrophil and lymphocyte, have been implicated in the initiation and development of atrial fibrillation (AF) –10. FurtA total of consecutive STEMI participants admitted to the Department of Cardiology between February 2016 and December 2021 had been recruited in this retrospective study. All STEMI patients aged over 18 years were treated invasively within the first 24 h after symptoms onset. Exclusion criteria contained individuals with previously paroxysmal/non-paroxysmal AF, history of PCI, heart valve disease, inflammatory-related diseases, hematological diseases, malignancy, chronic obstructive pulmonary disease, hyperthyroidism or hypothyroidism, and severe hepatic or renal dysfunction. Finally, the study cohort was comprised of 616 eligible participants  or hypertension, drinking or smoking status, were obtained from a questionnaire by trained doctors. Routine blood analyses, such as, the levels of neutrophil counts, lymphocyte counts, monocyte counts, were performed before PCI and measured with an automated blood count analyzer . Biochemical markers, including glucose, triglyceride, total cholesterol, high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c), creatinine, creatine kinase (CK), and albumin were additionally analyzed for all patients after fasting for overnight by using an automated chemistry analyzer . The value of NLR and MLR was defined as the counts of neutrophil and monocyte to the counts of lymphocyte, respectively. SIRI was calculated as follows, (N × M)/L, where N, M, and L was presented as the counts of neutrophil, monocyte, and lymphocyte, respectively . Body maThe treatment of PCI was performed for all STEMI patients according to the contemporary guidelines . The degAll STEMI patients treated with PCI were monitored by telemetry electrocardiogram (ECG) during hospitalization at the coronary unit. Subsequently, any suspected symptom of arrhythmia was assessed by a 12-lead ECG at the ward. AF was defined as irregular RR intervals with the absence of discrete P waves, complicating by fibrillatory waves. NOAF was identified as the occurrence of an AF episode lasting 30 s during hospital period. A total of 40 NOAF patients had been treated with either electrical or chemical cardioversion, of note, amiodarone intravenously is regarded as the pharmacologic treatment of AF. Ventricular tachycardia (VT) was presented as any VT event lasting ≥ 16 consecutive beats with a rate more than 125 beat/min .The primary adverse outcomes during hospitalization were comprised of cerebrovascular events, pulmonary edema, cardiogenic shock, VT, mortality. The long-term endpoint with respect to all-cause death was collected from medical records or obtained by telephonic interviews. Participants were followed-up from the enrollment to June 15, 2022.P < 0.05), were used to determine the independent predictors of NOAF. Receiver operating characteristic (ROC) curve and area under the curve (AUC) were generated to define the predictability of potential factors. The comparison of ROC curves for different risk factors was conducted by the DeLong method. Kaplan–Meier curves were performed to estimate time-to-event data, while the comparison of different groups was evaluated using a log-rank test. All data were analyzed by IBM SPSS 23.0, with P < 0.05 of statistical significance.Kolmogorov–Smirnov test was performed to evaluate the distribution patterns of the continuous variables. Continuous data were defined as mean ± standard deviation or median (interquartile range) based on whether variables are normally distributed or not, whereas the categorical variables were expressed as numbers (percentages). The comparison of parametric variables was conducted by the Student’s t or Mann–Whitney U-test, as appropriate, while non-parametric data was compared using the Chi-squared or Fisher’s exact test. Multivariable logistic regression analyses, which incorporated potential risk factors originated from univariable regression analysis . Further multivariate analyses demonstrated that the values of SIRI, albumin, glucose and eGFR were the independent predictors of NOAF in STEMI treated with PCI  experienced all-cause death after hospital discharge. Kaplan–Meier analysis showed that the all-cause mortality in NOAF group is significantly higher than that in SR group -6, IL-1β, tumor necrosis factor (TNF)-α, thus leading to both structure and electrical atrial remodeling, which can provide the occurrence of AF with necessary substance –37. AddiSeveral clinical implications have been stated according to the present results. First, SRI is a relatively common and easily obtained biomarker in clinical practice. Further, it might be beneficial for clinicians to efficiently identify the patients with high risk of NOAF by using the pre-procedural SIRI, thus providing them with better individualized treatment and care. Additionally, our results may broaden current understanding of underlying pathological mechanisms for the NOAF in STEMI patients, thus implying new targets for the prevention of NOAF.There are several limitations in our study. First, it is a single-center study based solely on Chinese individuals, thus limiting the generality of the present result. Second, due to the nature of retrospective analysis and limited samples, the result is inherently subject to selection bias and only provide an association between SIRI and NOAF instead of causality. Third, Several inflammatory indicators, such as, CRP, interleukins, were not assessed. Additionally, the level of SIRI was evaluated on admission for one-time and the fluctuation of SIRI had not been measured during hospitalization. Future large-scale prospective investigation is thus necessary to validate our result.In conclusion, SIRI is independently associated with NOAF in STEMI patients treated with PCI, with NOAF patients experiencing higher risks of in-hospital complications and long-term all-cause death as compared to those with SR.


Although comorbidity of major depressive disorder (MDD) and chronic pain (CP) has been well-studied, their association with pain catastrophizing is largely elusive. This study aimed to investigate the potential effects of pain catastrophizing in patients with a comorbidity.n = 45; depression group: patients with depression without chronic pain, n = 47; and healthy controls: n = 48). The Hamilton Depression Rating Scale (HAMD)-24 and Hamilton Anxiety Rating Scale (HAMA)-14 were used by professional psychiatrists to evaluate the severity of depression and anxiety. Beck Depression Inventory-II (BDI-II) and Beck Anxiety Inventory (BAI) were conducted by patients' self-report to assess the symptom severity. The pain intensity numerical rating scale (PI-NRS) was used to assess the pain intensity. Pain Catastrophizing Scale (PCS) and Pain Anxiety Symptoms Scale (PASS) were used to estimate pain-related negative thinking.In total, 140 participants were included in this study and divided into three groups according to the Diagnostic and Statistical Manual of Mental Disorders and the International Association for the study of pain . A stepwise regression analysis further demonstrated that the total PCS score, high monthly income level, and BDI score had positive impacts on PASS (P < 0.05). We also found that the total BDI score, disease course ≥1 year, and pain intensity had positive effects on PCS (P < 0.05), whereas years of education (≤ 12 years) had a negative effect on PCS (P = 0.012). In all, we have clearly demonstrated that PCS and PASS could serve as potentially predictive factors in patients suffering from comorbidity of MDD and CP.The results showed that PASS and PCS scores were significantly different among the three groups. Particularly, the scores in the comorbidity group were the highest. The Pearson correlation analysis revealed a positive correlation between PCS  and the severity of depression symptoms, anxiety symptoms, and pain intensity (Our results suggested that the pain-related catastrophic thinking and anxiety were more severe in the comorbidity group than in MDD-only group and healthy group. Pain-related catastrophizing thoughts and anxiety may have potentially effects on the comorbidity of depression and chronic pain. Empirical research has shown a strong correlation between major depressive disorder (MDD) and chronic pain (CP). Epidemiological studies have explored that the average prevalence of pain in MDD is as high as 65%, and the average prevalence of MDD in patients with CP is 52% , 2; the Psychosocial factors, such as pain-related anxiety and catastrophic thoughts, are critical determinants of differences in the development of MDD or CP , 13. PaiFrom a clinical perspective, the majority of studies addressing the relationship between MDD and CP have limitations . First, n = 45), depression group , and control group . The International Association for the Study of Pain (IASP) defines the CP as an unpleasant sensory and emotional experience, when it lasts or recurs for longer than 3 months  between January 2019 and July 2021. According to trial standards, two professional doctors used the Mini-International Neuropsychiatric Interview (MINI) 6.0.0. to evaluate all participants. A total of 162 participants were initially evaluated, among which 12 participants could not complete the scale assessment and 10 individuals refused to sign the informed consent. Hence, 22 subjects who did not meet the experimental criteria were excluded from this experiment, and the remaining 140 participants were included. Ultimately, 140 participants were included in this study and divided into the following three groups according to the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5), namely, comorbidity group  fulfillment of the DSM-5 criteria and IASP for patients with depression and CP by two independent experienced psychiatrists; (2) aged between 16 and 60 years; (3) CP is not caused by physical trauma injury or serious somatic disease or inflammatory disease; (4) pain intensity numerical rating scale (PI-NRS) ≥3. The inclusion criteria for the depression group were as follows: (1) fulfillment of the DSM-5 criteria for depression by two independent experienced psychiatrists; (2) aged between 16 and 60 years; and (3) PI-NRS <3. All the participants did not have any of the following exclusion criteria: (1) history of craniocerebral trauma or somatic trauma; (2) history of severe inflammatory diseases, neurological diseases, or tumor-related diseases; (3) history of alcohol or other substance use or other mental disorders; (4) history of diabetes, hypertension, and endocrine disease; (5) pregnant or lactating women; and (6) administration of electroconvulsive therapy without convulsions 3 months before enrollment.MINI 6.0.0, which is a concise diagnostic interview for psychiatric disorders used by psychiatrists in the United States and Europe, was used to verify the preliminary clinical diagnosis. All patients underwent MINI to confirm the clinical diagnoses of MDD with CP and MDD without CP .A self-reported questionnaire was used to collect information concerning age, gender, marital status, income status, employment status, educational level attained, duration of disease course, and health service accessibility.Hamilton Depression Rating Scale-24 (HDRS-24) is the most commonly used depression scale worldwide because it has a high specificity to assess the severity of depression symptoms. The Cronbach's α score of HAMD-24 is 0.88, and the κ-score is 0.92. The HAMD-24 score may be used to define clinically relevant symptom levels as follows: <8 = no depression, 8–19 = mild depression, 20–34 = moderate depression and ≥35 = severe depression  and excellent criterion validity (κ = 0.94) compared with other depression measures .Beck Anxiety Inventory is a 21-item self-report instrument that assesses the grade of anxiety symptoms. Each item is rated from 0 to 3 with general guidelines; its total score ranges from 0 to 63, and higher scores indicate more severe anxiety symptoms. BAI has excellent internal consistency  and good discriminant validity for anxiety disorders .Pain Catastrophizing Scale is a 13-item self-report instrument that assesses catastrophizing in the context of actual or anticipated pain. PCS measures catastrophizing as a multifaceted construct with three subscales, namely, rumination, magnification, and helplessness. PCS estimates how individuals respond to painful circumstances and indicates the degree to which they experience each thought or feeling when experiencing pain. Each item is rated from 0  to 4  with general guidelines, its total score ranges from 0 to 52, and higher scores indicate more serious pain-related thoughts .Pain Anxiety Symptom Scale was translated into the Chinese version by Xiao-Yi Zhou. Chinese PASS is a 20-item questionnaire that comprises four subscales, namely, cognitive anxiety, escape/avoidance, fearful appraisal, and physiological anxiety. All items were rated on a scale ranging from 0  to 5 . Its total score ranges from 0 to 100, and higher scores indicate more severe pain-related anxiety.Pain intensity numerical rating scale is a self-report questionnaire that assesses pain intensity and demonstrates strong construct validity and stability. In consideration of its simplicity and easy operation, it is widely used in the evaluation of clinical CP disease. This scale frequently measured on an 11-point pain intensity PI-NRS, where 0 indicates no pain and 10 indicates worst possible pain .t-test, chi-squared test, and ANOVA were performed to compare the differences in continuous or categorical parameters among the three groups. Symptoms related to pain anxiety and pain catastrophizing were analyzed by repeated-measure ANOVA and Tukey post-hoc analyses. Pearson product-moment correlation coefficients were calculated to examine the relationships between severe degree of depression, pain, or anxiety and pain catastrophizing thoughts. A stepwise regression analysis was conducted to analyze the confirmatory factor analysis. P < 0.05 was considered statistically significant. Finally, the mediation analysis was performed to further test whether the relationship between the potentially risk factors and depressive symptoms was mediated by pain-related anxiety and pain catastrophizing thoughts. This analysis was carried out by quantifying the direct and indirect relationships among the independent variables, mediator , and dependent variable . In the mediation models, all paths were reported as unstandardized ordinary least squares regression coefficients. A significance analysis was based on 5,000 bootstrap realizations, and a significant indirect effect was indicated when the bootstrap 95% confidence interval (CI) was not zero.Statistical Package for the Social Sciences version 22.0  was used to analyze the data. The P > 0.05). Psychometric properties and the clinical characteristics of depression and anxiety also showed no differences between the comorbidity and depression groups. However, pain intensity was more severe in the comorbidity group compared with the depression group (P = 0.025).The demographic data, anxiety psychometric properties, and clinical characteristics of the three groups are shown in F = 21.29, P < 0.001; F = 26.62, P < 0.001; F = 14.46, P < 0.001, respectively). The comorbidity group had significantly higher total PASS scores, fear of pain score, and physiological anxiety score  than the MDD and healthy groups. The three factors in the PCS scale, namely, magnification, rumination, and helplessness, were also assessed. Total PCS score, magnification score, rumination score, and helplessness score were significantly different among the three groups . Together, these results . Meanwhile, self-reported anxiety symptoms had positive correlations with fear of pain, physiological anxiety, and total PASS score . Interestingly, from the perspective of professional psychiatrists, depression symptoms only had a positive relationship with physiologic anxiety , and somatic anxiety symptoms had a positive relationship with fear of pain . In addition, we found that the course of disease positively correlated with fear of pain and the total PASS score . On the contrary, we discovered that years of education and monthly income negatively correlated with fear of pain . Except for the above, we also found the pain intensity had positive related to the fear of pain and physiologic anxiety .Remarkable correlations were found between the components of pain-related anxiety and depression symptoms. Specifically, self-reported depression symptoms had positive correlations with fear of pain, physiologic anxiety, and total PASS score . Similar to the previous results, self-reported anxiety symptoms had positive correlations with the helplessness, magnification, rumination, and total PCS scores . In addition, depression symptoms were also positively associated with the helplessness score, magnification score, rumination score, and total PCS score . Psychological anxiety and somatic anxiety had positive relations with the helplessness score . Furthermore, the pain intensity also had a strong correlation with helplessness, magnification, rumination, and total of PCS scores . Above-mentioned results are shown in We evaluated the relationship between pain catastrophizing thoughts and depression symptoms. Self-reported depression symptoms had positive correlations with the helplessness score, magnification score, rumination score, and total PCS score , whereas a significant negative impact was found between men and PASS score . Furthermore, the regression model accounted for 53.1% of the variation in total PCS scores. A positive effect was explored between PCS with total BDI score, disease course ≥ 1 year, and pain intensity , and a negative relationship was found between education >12 years and PCS score . Finally, an examination of the mediator pathways revealed the indirect role of pain-related anxiety, pain catastrophizing thoughts, and pain intensity in depression symptom. Boot strapping results indicated that the indirect effect was significant (P < 0.05). The 95% CI did not contain zero, confirming the significant mediating effect of pain-related anxiety and pain catastrophizing thoughts for depression symptoms prediction , Hospital Project of Hefei Fourth People's Hospital (Grant Number: 2019023), Fund Project of Anhui Medical University (Grant Number: 2019xkj206), Shanghai Key Laboratory of Psychotic Disorders Open Grant (Grant Number: 13dz2260500), Natural Science Research Projects in Anhui Universities (Grant Number: KJ2020A0218), Applied Medicine Research Project of Hefei Health Committee (Grant Number: Hwk2020zd0016), Applied Medicine Research Project of Anhui Health Committee (Grant Number: AHWJ2021a036), and Natural Science Foundation of Minhang District (Grant Number: 2020MHZ063). The funding sources had no involvement in the study design, collection, analysis, and writing of this article and 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. The handling editor declared a past collaboration with one of the author's, CZ.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.


Hepatitis E virus (HEV) infection is the most common cause of acute viral hepatitis worldwide. Hepatitis E is usually asymptomatic and self-limiting but it can become chronic in immunocompromised patients and is associated with increased fulminant hepatic failure and mortality rates in pregnant women. HEV genome encodes three proteins including the ORF2 protein that is the viral capsid protein. Interestingly, HEV produces 3 isoforms of the ORF2 capsid protein which are partitioned in different subcellular compartments and perform distinct functions in the HEV lifecycle. Notably, the infectious ORF2 (ORF2i) protein is the structural component of virions, whereas the genome-free secreted and glycosylated ORF2 proteins likely act as a humoral immune decoy. Here, by using a series of ORF2 capsid protein mutants expressed in the infectious genotype 3 p6 HEV strain as well as chimeras between ORF2 and the CD4 glycoprotein, we demonstrated how an Arginine-Rich Motif (ARM) located in the ORF2 N-terminal region controls the fate and functions of ORF2 isoforms. We showed that the ARM controls ORF2 nuclear translocation likely to promote regulation of host antiviral responses. This motif also regulates the dual topology and functionality of ORF2 signal peptide, leading to the production of either cytosolic infectious ORF2i or reticular non-infectious glycosylated ORF2 forms. It serves as maturation site of glycosylated ORF2 by furin, and promotes ORF2-host cell membrane interactions. The identification of ORF2 ARM as a unique central regulator of the HEV lifecycle uncovers how viruses settle strategies to condense their genetic information and hijack cellular processes. Hepatitis E virus (HEV) is the major cause of acute viral hepatitis worldwide. Although infection with HEV is usually self-resolving, it can cause up to 30% mortality in pregnant women in the third trimester. There is no specific treatment nor universal vaccine to fight against HEV. In our study, we focused on the HEV ORF2 capsid protein which is produced as different forms that perform distinct functions in the HEV lifecycle. The infectious ORF2i form is the structural component of virions, while the other forms likely act as an immune decoy. Herein, we deciphered the molecular determinants of ORF2 multifunctionality. We identified an Arginine-Rich Motif (ARM) located in the ORF2 N-terminal region that controls the subcellular localization, the fate and functions of ORF2 forms. It also promotes ORF2-host cell interactions. Our observations highlight the ORF2 ARM as a unique central regulator of ORF2 addressing that finely controls the HEV lifecycle. Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide and is an emerging problem in industrialized countries. This virus causes about 20 million infections annually . While Het al. in naturally infected human hepatocytes  A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Alexander Ploss, Ph.D.Guest EditorPLOS PathogensGuangxiang LuoSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogens  orcid.org/0000-0002-7699-2064  ***********************Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: The life cycle of HEV is poorly understood due to lack of robust cell culture model. ORF2 is the viral capsid protein, which could package viral genome and mediate viral entry by engagement with the putative cellular receptor. Interestingly, ORF2 has multiple forms and they play different roles in HEV life cycle. In this manuscript, Hervouet et al constructed a panel of ORF2 mutants in Arginine-Rich Motif (ARM) to delineate the function of ARM motif in regulating ORF2s subcellular localization and host antiviral response. ARM regulates the production of different forms of ORF2 and serves as maturation site of glycosylated ORF2. This study could further deep our understanding of HEV life cycle and its interaction with cell host, however, this manuscript is not well organized, not necessarily long and the experiments are not well-control, other explanations or conclusions can be drawn. In addition, the authors performed lots of experiments by electroporation of viral RNA, instead of viral infection, which could potentially lead to some artifacts of the observation. The specific comments could be found as below.Reviewer #2: In their manuscript entitled “An Arginine-Rich Motif in the ORF2 Capsid Protein Regulates the Hepatitis E Virus Lifecycle and Interactions with the Host Cell” Hervouet et al. use an HEV cell culture model to elucidate the role of ARM on multiple steps of HEV propagation.Utilizing a series of ORF2 mutants expressed either in the p6 HEV strain or as ORF2 capsid protein they delineate a crucial role of the ARM as functional nuclear localization signal which seems to be important for viral production. With the help of specific inhibitors and co-localization studies, the authors identified a CRM1-dependent egress from the nucleus and 3 important nuclear export signals within the ORF2 protein.The main body of the manuscript focusses on the maturation and addressing of ORF2. The authors found evidence for a role of a furin-dependent secretion pathway in the maturation process. With the generation and evaluation of additional mutants including the signal and/or the encoding sequence of CD4 by state-of the art techniques the authors delineate a tight interplay of the ARM with the respective signal peptide for addressing, topology and egress of the capsid proteins. Bringing this data set together the authors finally delineate a model for the generation of the infectious or non-infectious forms of the HEV particles.The manuscript is well written, the experiments are appropriate and on a technically high level. Following on the previous reports from the group this manuscript sheds light into the so far under investigated HEV life-cycle and the generation of the different capsid forms.Overall, this manuscript describes several novelties in the field and sets the ground for several future scientific perspectives concerning HEV.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: 1) P6 L114 why the authors choose electroporation of viral RNA genome to observe the ORF2 localization, instead of viral infection? A large amount of viral RNA delivered into the cells by electroporation could produce lots of ORF2, which is not an appropriate model to observe ORF2 localization.2) The authors constructed series of ARM mutants to observe the alterations of ORF2 mutants localization to demonstrate ARM’s NLS function. Also they engineered the mutations accordingly to demonstrate the critical role of ORF2 nuclei localization in HEV life cycle . It has been well known that ORF2 is also responsible to package viral genome to form infectious particles, and it is very possible that the ARM mutation impaired ORF2 function to package viral genome, which is not related to its NLS function. I suspect these mutations affect ORF2 fold or interaction with genomic RNA to cause the phenotype.3) In addition, the authors also need to check the total ORF2 amount in Fig 2C, excluding the possibility that the mutations in ARM affect ORF2 stability. The cellular markers in the WB were required to make sure the quality of isolation of each fraction. For example, there is not nuclei maker (histone protein) in the nuclei extract, which is very important.4) Data presented in Figure 2D show the ORF2 mutants produce less infectious viral particles than WT. As the ORF2 and ORF3 are overlapped, in ORF2 mutants, the amino acid of ORF3 also changed, therefore, more data should be presented to exclude the possibility that this phenotype is caused by ORF3 mutation.5) The authors used multiple inhibitors to block the interested pathway and then analyze the phenotype , and always have the concerns about the specificity of the phenotype in the manuscript. More solid data should be used to support the conclusions.Reviewer #2: I have the following major comments.Major:All experiments have been performed with the (sub-)genotype Kernow p6. As the authors also stated in their introduction, several other (sub-)genotypes exist with marked differences in their clinical presentation. Therefore, this study would largely benefit from expanding the range of viruses used. In this regard it would be helpful toI) show by phylogenetic analysis of for example published strains the abundance of ARM as well as NESII) show a similar nuclear pattern for a different GT3 subgenotype as well as another genotype (the published data so far only investigated GT3 infections).The main body of the manuscript is focussing on virus addressing. Some parts are comparable little studied and therefore do not allow as strong statements as have been made in the manuscript. This in particular is the case for the following:I) Page 7, Lines 140-143: “In addition, high molecular weight formsof ORF2intra in the soluble fraction as well as an increase of ORF2g/c secretion were observed , suggesting a higher translocation into the secretory pathway associated to an improved functionality of ORF2 SP”This statement seems only to be true for 3R/3A and 5R/5A, not for the 2R/2A construct. The authors should either repeat the experiments to identify significant results or be gentle with the description.II) Page 7, Lines 166-168:”Intracellular titers were alsolowered in SP mutants , indicating that the ORF2 SP likely plays an important role in the assembly of infectious particles.”The intracellular HEV RNA is already significantly lower for the SP mutants intracellularly and therefore argue for a reduced replication. Overall, this figure would benefit from a positive control (such as ribavirin) and possibly a time-course to ensure a decent measuring window.III) Page 8, Lines 177-179 “Interestingly, ORF2 and Importin-α1 co-localized in the nucleus of infected cells with a Pearson correlation coefficient (PCC) of 0.670, and the mutation of arginine residues drastically reduced this colocalization. Taken together, our results indicate that ORF2 colocalizes with Importin-α1 thanks to its ARM that serves as a functional NLS.”This seems to be only a moderate co-localization. More work needs to be done to support this assumption such as performing IP experiments to reveal direct interaction.IV) Page 13, Lines 203-207: “Altogether our results suggest that the ORF2 ARM, which notably regulates ORF2 nuclear translocation, is a pivotal viral determinant for the modulation of host pathways and, especially, genes of the NF-κB-induced signalling upon infection. Further studies will be required to define precisely the impact of this HEV-driven host regulation on immune cell responses.”Although it seems to be an interesting observation, I agree that the displayed data is too preliminary and should be left out or expanded significantly . Additionally, the time-course of viral replication might be significantly affected depending on whether the construct enters the nucleus or not. Therefore, the analysis at one specific time-point (18h) is difficult to compare. Additionally, mutant delORF3 has not been investigated in the previous experiments in terms of translocation to the nucleus.V) Fig 7/8. Have the authors analysed an additional SP from a different source in terms of its phenotype?**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: 1) Fig 1, it is required to present the nuclei staining results.2) Fig 2D needs RdRp inactive (GAD) control.3) P9 L187 whether overexpression of ORF2 can cause similar results on NF-kB-related signaling.4) P10 L230 The phenotype is not consistent among these three mutants, suggesting that there is nothing to do with nuclear localization.5) P13 L287-L289 The localization of ORF2 is also dynamic like infection condition? If not, OE system cannot reflect true functional ORF2 in infection condition.6) Figure 2C. Tubulin of ΔSP1 is much less than the others, indicating the quality of ΔSP1 soluble extract is not good.7) Figure 4A and 4D-F. At What time points post-electroporation are the cells treated with inhibitors? Some related descriptions should be added to the legend.Reviewer #2: Overall, the study would benefit from additional controls and more detailed analysis in particular in the first part.1) Supplement Figure 6: Please provide data with addition of a positive/cytotoxic control.2) Page 7 Lines 151-155: he PSG/3R mutant showeda marked nuclear localization  but was impaired inORF2g/c secretion , as observed for the SP deletion mutants,indicating that the addition of arginine residues strengthens the NLS function of ARMbut inhibits the functionality of ORF2 SP.To underline this point it would be interesting to perform an additional time-kinetic  to understand and learn where the ORF2 dose eventually reside and if it is retained in the nucleus for a longer period of time.3) Page 14, Lines 231-234, “Of note, intracellular replication wasnot altered by NES mutations , indicating that the loss of infectious particleassembly is due to the differential subcellular localization of the mutants and not to areplication defect.”This seems counterintuitive. Please provide appropriate replication inhibitor control of the assay. Additionally, this would also benefit from a time-kinetic analysis.4) In line 114 page 6: Titel: “phase of infection”: This is not correct, as the cells were transfected. Therefore, the term replication would be more appropiate.5) Fig 5C convincingly shows that nuclear ORF2 is present in the WT construct. However, for Fig5B this is difficult to judge by eye. In Fig. 2C the nuclear signal is displayed as nuclear fluorescent intensity. However, in the following figures this is delineated as nuclear/cyto intensitiy ratio, which makes a comparison difficult. Please harmonize the display of these results.**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, Data Requirements:http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here on PLOS Biology: Reproducibility:https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocolsTo enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at  19 Apr 2022AttachmentReviewers comments PPATHOGENS-D-21-01762-R1.docxSubmitted filename: Click here for additional data file. 10 Jun 2022Dear Dr. Cocquerel,Thank you very much for submitting your manuscript "An Arginine-Rich Motif in the ORF2 Capsid Protein Regulates the Hepatitis E Virus Lifecycle and Interactions with the Host Cell" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a revised version that takes into account the reviewers' comments. We would like you to focus on the major points raised by reviewer 1.Your study is of considerable interest but these remaining points need to be addressed before we can make a final decision on your submission..When you are ready to resubmit, please upload the following:[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Alexander Ploss, Ph.D.Guest EditorPLOS PathogensGuangxiang LuoSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogens  orcid.org/0000-0002-7699-2064  ***********************Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: The authors added new data in revised manuscript, but the following concerns are still not been addressed.Reviewer #2: In their manuscript entitled “An Arginine-Rich Motif in the ORF2 Capsid Protein Regulates the Hepatitis E Virus Lifecycle and Interactions with the Host Cell” Hervouet et al. use an HEV cell culture model to elucidate the role of ARM on multiple steps of HEV propagation. Utilizing a series of ORF2 mutants expressed either in the p6 HEV strain or as ORF2 capsid protein they delineate a crucial role of the ARM as functional nuclear localization signal which seems to be important for viral production. With the help of specific inhibitors and co- localization studies, the authors identified a CRM1-dependent egress from the nucleus and 3 important nuclear export signals within the ORF2 protein. The main body of the manuscript focusses on the maturation and addressing of ORF2. The authors found evidence for a role of a furin-dependent secretion pathway in the maturation process. With the generation and evaluation of additional mutants including the signal and/or the encoding sequence of CD4 by state-of the art techniques the authors delineate a tight interplay of the ARM with the respective signal peptide for addressing, topology and egress of the capsid proteins. Bringing this data set together the authors finally delineate a model for the generation of the infectious or non-infectious forms of the HEV particles.The manuscript is well written, the experiments are appropriate and on a technically high level. Following on the previous reports from the group this manuscript sheds light into the so far under investigated HEV life-cycle and the generation of the different capsid forms.Overall, this manuscript describes several novelties in the field and sets the ground for several future scientific perspectives concerning HEV.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: 1) Subcellular localization of ORF2 does not change obviously during infection condition  compared with electroporation , suggesting that it may be an artifact in electroporation condition due to ORF2 overexpression.2) Fig 2D, there are multiple rounds of infection already, so the phenotype could be due the alterations of ORF3. This concern is not been addressed. According to the result in S7 Fig, the direct conclusion is that deletion of ORF3 don’t impair ORF2 nuclear localization. Our question is how to ensure that the fewer infectious particles produced is indeed caused by ARM mutation rather than ORF3 mutation (ORF2 and ORF3 ORFs are largely overlapped)? To answer this question, I think the possible strategies are as follows: (1) When generating ARM mutation to impair ORF2 nuclear localization in the contents of genomic sequence, please make sure the ORF3 amino acids unchanged. (2) utilize the trans-complementation system to uncouple the expression of ORF2 and ORF3 protein, then investigate the ARM mutation on virus production.3) From the total extract panel in new Fig 2, it is obvious that ORF2 stability is dramatically impaired of ΔSP1, but the authors didn’t mention it in response.Firstly, in S7 Fig, a WB result is required to show that the deletion of ORF3 is successful. More importantly, this assay  can’t answer our question.Reviewer #2: This reviewer feels, that all questions have been adequately adressed by the authors in this revision.**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: 1) Please check primers to construct 5R/5A mutation. It seems to be wrong.Reviewer #2: This reviewer feels, that all questions have been adequately adressed by the authors in this revision.**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, Data Requirements:http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here on PLOS Biology: Reproducibility:https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocolsTo enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at  15 Jul 2022AttachmentPLoS-Patho-R2-Comments.pdfSubmitted filename: Click here for additional data file. 6 Aug 2022Dear Pr Cocquerel,We are pleased to inform you that your manuscript 'An Arginine-Rich Motif in the ORF2 Capsid Protein Regulates the Hepatitis E Virus Lifecycle and Interactions with the Host Cell' has been provisionally accepted for publication in PLOS Pathogens.Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.Best regards,Alexander Ploss, Ph.D.Guest EditorPLOS PathogensGuangxiang LuoSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogens  orcid.org/0000-0002-7699-2064  ***********************************************************Reviewer Comments :Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: The authors performed additional experiments to address reviewers questions. Most of the concerns and questions have been addressed.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: (No Response)**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: (No Response)**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  are generated on a different schedule and may not be made available as quickly.Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens. Best regards,Kasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064