Anx-A1 knockout, Anx-A1 knockdown, or treatments with the FPR2 antagonist reduced the mortality and tissue viral loads of infected mice. Our results show that Anx-A1 is a novel enhancing factor of HSV-1 infection. Anx-A1-deficient mice displayed no evident physiology and behavior changes. Hence, targeting Anx-A1 and FPR2 could be a promising prophylaxis or adjuvant therapy to decrease HSV-1 lethality.Herpes simplex virus 1 (HSV-1)-induced encephalitis is the most common cause of sporadic, fatal encephalitis in humans. HSV-1 has at least 10 different envelope glycoproteins, which can promote virus infection. The ligands for most of the envelope glycoproteins and the significance of these ligands in virus-induced encephalitis remain elusive. Here, we show that glycoprotein E (gE) binds to the cellular protein, annexin A1 (Anx-A1) to enhance infection. Anx-A1 can be detected on the surface of cells permissive for HSV-1 before infection and on virions. Suppression of Anx-A1 or its receptor, formyl peptide receptor 2 (FPR2), on the cell surface and gE or Anx-A1 on HSV-1 envelopes reduced virus binding to cells. Importantly,  Herpes simplex virus 1 (HSV-1)-induced encephalitis is the most devastating consequence of HSV-1 infection, even in patients treated with anti-HSV-1 drugs. Moreover, encephalitis induced by drug-resistant HSV-1 has been reported in immunocompromised patients. Identifying the cellular factors in promoting HSV-1 replication, especially those increasing virus attachment and entry, could facilitate the development of alternative or adjuvant therapy. Here, we identified annexin A1 (Anx-A1) and its receptor, formyl peptide receptor 2 (FPR2), facilitating HSV-1 attachment to the cell surface. Suppression of Anx-A1 or blockage of FPR2 impaired HSV-1 attachment to cells, viral yields in cells, and HSV-1 lethality in mice. Moreover, blocking FPR2 decreased the replication of drug-resistant HSV-1 in BABL/c nude mice. Hence, targeting Anx-A1 and FPR2 could be alternative or adjuvant therapy for HSV-1 infection. Herpes simplex virus (HSV)-induced encephalitis is the most common cause of sporadic fatal encephalitis, with an incidence of approximately 1 in 250,000 persons per year . There aDuring virus entry, viral envelope glycoproteins interact with cellular proteins on the cell surface. HSV-1 has at least ten different envelope glycoproteins . Among tannexin A1 (Anx-A1) gene generated by insertion of a retrovirus vector reduces HSV-1 replication in a rat epithelial cell line  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,Neal A. DeLuca, Ph.D.Guest EditorPLOS PathogensShou-Jiang GaoSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogens  orcid.org/0000-0002-7699-2064  ***********************Please address all the comments of the reviewers. The points raised in the "Major Issues" should be addressed experimentally.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 provide evidence for their claim that HSV-1 glycoprotein gE binds to Anx-A1 on cell surface to promote infection of cells. Their in vivo results show that suppression of Anx-A1 reduces the mortality and viral loads in infected mice. Multiple in vitro assays as well as in vivo assays were performed to validate the claim. The manuscript is well written and the results well described.Reviewer #2: Wang and colleagues provide evidence that the cell protein annexin A1 (Anx-A1) enhances attachment of HSV-1 to cells via an interaction with gE. Furthermore, that blocking this interaction reduces the lethality of the virus for mice after infection via the cornea.The experiments with mice consist of comparing wildtype and Anx-A1-negative mice, of treating mice with an oligonucleotide that reduces Anx-A1 expression, and of treating mice with a peptide that blocks binding of the soluble Anx-A1 to its cell surface receptor FPR2. In all cases, the data support a role for Anx-A1.The results of experiments with tissue culture are less straightforward, in part because some of the methods used are not described adequately. Details are below.There is novelty and potential significance to the study, and the Discussion is written with appropriate references to prior work.Comments on the execution and interpretation of the experiments using tissue culture systems are as follows.(1) In the first experiment, cells were incubated with virus at an MOI of 5 for 1 h at 4C, then incubated at 37C for various times before measurement of cell surface Anx-A1. There was an initial increase, but then the level went back down. The conclusion is that infection raised the level of Anx-A1 on the cell surface, and that UV-inactivated virus had a smaller effect. However, in a later experiment it was shown that Anx-A1 is actually present on virions, which suggests that the increased cell surface level of Anx-A1 was actually due to Anx-A1 on virions. This would be consistent with an initial increase, followed by a decrease, because there is no indication that the inoculum was removed after the 4C step. More virus could have bound after raising the temperature, followed by entry into cells resulting in a later decrease of virions at the cell surface. The UV virus was only tested after 1 h at 37C, at which point the level of Anx-A1 is not greatly different from that seen without the UV treatment.(2) A virus binding assay is described:Cells were "infected with HSV-1 for 1 h, washed, and harvested at 4C to determine the number of virus binding to cells by plaque assay."What is the significance of the plaque assay? This is not usually the way that virus binding is measured, and it is entirely unclear if the procedure is actually measuring binding or a combination of binding and a later step or steps.(3) In Figs 1C and 1D, immunofluorescence is used to detect increased Anx-A1 at the cell surface after infection. Quantitation shows an approximately 2-fold overall increase. The number of positive cells increases from 32% to 78%. This is sufficient to account for the overall 2-fold increase, so it is difficult to accept the report that fluorescence intensity also increased, i.e., that positive cells became more positive. The images do not support this. And again, is the overall increase the result of Anx-A1 being brought to cells on virions?(4) In Fig 2A, a virus overlay protein blot assay is used to demonstrate that virions bind to Anx-A1. Details of sample preparation are not given, but presumably this is a denaturing gel, in which case virions are binding to denatured Anx-A1, which is an interesting and surprising observation.(5) An interaction between gE and Anx-A1 is demonstrated, and said to be independent of other virus proteins . Nevertheless, quantitation of the gel would be useful, comparing the fraction of total Anx-A1 that is co-IP'd with gE; is it the same when only gE is present versus when all virus glycoproteins are present?(6) Anx-A1 interacts with the extracellular domain of gE. The experiment to demonstrate that it does not interact with the cytoplasmic domain  suffers from the problem that the hydrophobic transmembrane region is also included in the construct. This would likely result in aggregation of the cytoplasmic domain, preventing any meaningful conclusion.(7) Figs. 3A and 3C are concerned with the effect of anti-Anx-A1 serum on binding of virus to cells. The effect is measured via plaque assay. As with comment (2), this is unclear. How does a plaque assay measure virus binding? Also, how can antibody binding to the Anx-A1 on virions be eliminated as a major contributor to the result?(8) Yields of virus are measured in Figs 3B and 3D. Time points are 24 h and 36 h. A greater effect of anti-Anx-A1 antibody is seen at 36 h. The problem with this protocol is that by 36 h, there will have been two cycles of infection. Therefore, the effect of the antibody on virus spread from cell to cell is also a factor. Curiously, in the next experiments, with Anx-A1-negative cells, the results suggest that spread is NOT affected. To eliminate this complication, it would be preferable to use a higher MOI for the initial infection in all of these experiments, and to measure the virus yield at no later than 24 h.(9) There is an experiment to investigate a role for Anx-A1 in virus entry, i.e., a post-binding step, but the experimental protocol is unusual. Virus was incubated with cells for 1 h at room temperature, followed immediately by a low pH treatment to inactivate virions still on the cell surface, and then incubation at 37C to allow plaque formation. The assay therefore measures a combination of virus binding and entry into cells at room temperature, which is not very informative.The conventional way to assess entry is to begin with an initial virus attachment step at 4C. Next to raise the temperature to 37C to allow virus entry, with low pH treatment at various times after temperature increase. This allows the rate of virus entry to be determined.(10) A virus lacking gE is used in Fig 3I. Do the authors have an explanation for why the level of binding (if that is what is being measured) when gE is absent but Anx-A1 is present is only half that of when gE is present but Anx-A1 is absent?(11) Fig 4 shows that Anx-A1 is present on virions, as mentioned earlier. The statement is also made that Anx-A1 is "adjacent to gE". This statement cannot be justified without comparing the proximity of Anx-A1 to other virus glycoproteins, which was not done.(12) For Figs 4D-4F, with cells lacking Anx-A1, the level of binding is said to be "slightly higher" for virus containing Anx-A1 than for virus lacking Anx-A1 at an MOI of 1, and with a more pronounced difference at lower MOIs. It is difficult to appreciate how this effect could be influenced by the MOI. Is it caused by the assay measuring something other than, or additional to, binding? Is it influenced by the unavoidable presence of non-infectious virions in the inoculum?(13) In Fig 7, a peptide that blocks Anx-A1 binding to its cell surface receptor FPR2 reduces virus binding to cells. There are two negative controls that would strengthen the conclusion. One is to show that the peptide has no effect on virus binding to Anx-A1-negative cells. The other is to show that the peptide has no effect on gE-negative virus binding to Anx-A1-positive cells.**********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: Anx-A1 is mostly cytosolic. Could it reduce cell-to-cell spread by neutralizing gE intracellularly? Similar things could happen when neighboring cells secrete the protein. This alternate possibility should be analyzed.Why did the authors choose not to study cell-to-cell spread when gE is a known player there? A simple cell mixing (infected to uninfected) assay can be done to demonstrate effect on spread of fluorescent virions.Figs. 1/3, Results (and interpretations) are confusing. Anx-A1 expression goes up 1-2h later, by that time most virions should have been internalized. Several experts believe that virus entry happens within 5 min.Fig. 2. Results shown in this figure are very important. However, it is not clear whether a loading or input protein control was used.Fig. 4, results are confusing. Normally the presence of gE next to Anx-A1 on viral envelope should reduce the ability of gE to interact with Anx-A1 on the cell membrane.Fig. 5, Could the results shown here be due to stronger compensatory mechanisms that takeover the normal functions of Anx-A1?Reviewer #2: Key modifications/new experiments:(1) Experimental protocols must be changed so that the relevance of Anx-A1 to individual aspects of virus attachment, entry, and virus yield is measured, rather than a combination of them.Binding should be measured by an assay that does not allow for subsequent entry, and not by plaque assay.Entry, or more informatively, rate of entry should be measured by an assay that begins with attachment under conditions where entry cannot occur, and then proceeds to different lengths of time at 37C such that entry can occur.Virus yield should be measured up to, but not exceeding, 24 h post-infection, and with an appropriately high MOI, such that only one cycle of replication is possible. This avoids the complication of effects on virus spread, in addition to attachment and entry.(2) The effect of Anx-A1 on virions must be addressed. If this is the source of the increase in cell surface Anx-A1 after virus binding/infection, then one conclusion of the manuscript is invalidated. This conclusion is that infection results in exit of intracellular Anx-A1 from cells, such that it is then available to bind to the cell surface.(3) Negative controls are needed for experiments with FPR2. Does the peptide have an effect on virus binding to Anx-A1-negative cells, and does it have an effect on gE-negative virus binding to Anx-A1-positive cells?If it does, then a different interpretation of the results already in the manuscript would be needed.**********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)Reviewer #2: None.**********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 Jun 2022Attachment20220615 Responses to reviewers.docxSubmitted filename: Click here for additional data file. 20 Jun 2022Dear Dr. Chen,We are pleased to inform you that your manuscript 'Suppression of annexin A1 and its receptor reduces herpes simplex virus 1 lethality in mice' 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,Neal A. DeLuca, Ph.D.Guest EditorPLOS PathogensShou-Jiang GaoSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogens  orcid.org/0000-0002-7699-2064  ***********************************************************All the concerns have been addressed.Reviewer Comments : 24 Jul 2022Dear Dr. Chen,We are delighted to inform you that your manuscript, "Suppression of annexin A1 and its receptor reduces herpes simplex virus 1 lethality in mice," has been formally accepted for publication in PLOS Pathogens.We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles  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


Being aware of global pandemics, this research focused on the potential infection routes in building drainage systems. Case studies have found that dysfunctional building drainage systems not only failed to block contaminants but also potentially became a route for the spreading of viruses. Several fluid simulations in pipelines were conducted in this research using COMSOL Multiphysics. In particular, virus transmission from one patient’s room to other uninfected residential units through pipelines was visualized. A 12-story building, which is commonly seen in the local area, was designed as a simulation model to visualize the transmission and analyze its hazards. Furthermore, five environmental factors were filtered out for discussion: distance, time span, pressure, initial concentration, and environment temperature. By manipulating these factors, the relationship between the factors and the behavior of the contaminant could be explored. In addition, a simulation with a different pipeline arrangement was included to observe the virus diffusion behavior under different scenarios. The visualized simulation concluded that the contaminant would spread through the drainage system and arrive at the neighboring four floors within an hour under the circumstances of a 12-story building with broken seals and constant pressure and contaminant supply on the seventh floor. Meanwhile, the whole building would be exposed to infection risks by the continuous virus spreading through a drainage system. Distance, time span, and pressure were considered critical factors that affected indoor contamination in the system. On the other hand, initial concentration and environmental temperature did not have significant roles. Visualizing the behavior of viruses provides a glimpse of what happens behind walls, paving the way for recognizing the importance of maintaining functional drainage systems for individuals’ health. Building drainage systems are well recognized as one of the essential facilities in building service engineering. However, many substantial issues regarding health and indoor contamination in many existing buildings should be highlighted. The gravity drainage system is commonly used worldwide, and the appliance trap seal plays a vital role in safeguarding occupied spaces from stench and vermin spreading from the drainage network . FurtherA couple of years have passed since the COVID-19 outbreak, taking countless lives and reshaping human behaviors. As a response, studies have been conducted in different scopes and research fields to prevent further virus dispersion. From the architectural perspective, this research focused on visualizing virus diffusion in building drainage systems while treating dysfunctional water traps through simulation using Computational Fluid Dynamics. In addition, five environmental factors were examined concerning their influence on the system, and an additional simulation with different pipeline arrangements is also included in this article. Furthermore, several fluid simulations for pipelines were conducted in this research using COMSOL Multiphysics. In particular, the virus transmission from one patient’s room to other uninfected residential units through pipelines was visualized. This research mainly visualized the movement within the pipelines through computer simulation and observed how the contaminant diffused through the drainage system. On the basis of related literature, five environmental factors—distance, spreading time, pressure, contaminant concentration, and room temperature—were examined in this article concerning their influences on the system. Moreover, the simulation results in this study demonstrate that contamination may occur when water traps are left impaired.Conventional construction often hides building drainage systems behind walls. Hence, the quality of drainage systems is easily neglected. Rarely maintained pipelines could lead to water trap abnormalities, leakage, drying, or blockage. More issues can also arise due to dysfunctional drainage systems. For instance, during the SARS outbreak, the Amoy Gardens in Hong Kong experienced a massive infection resulting from its dysfunctional drainage system. With its incomplete water traps, virus stacking inside the drainage system reached other rooms without warning. Consequently, the virus infected over 300 people without face-to-face interaction.Drainage systems refer to the pipelines transporting domestic water within the building. The main driving power source of drainage systems in most countries is gravity. Moreover, water traps are common devices consisting of a single stack of water that remains within the pipes; their main purpose is to act as an obstacle between the clean and contaminated water. A breaking water seal is a situation in which air from two sides of the trap connects, resulting in indoor contamination. Such an incident may be caused by poor drainage system design, lack of maintenance, sudden pressure shifts ,6,7,8, oThe Amoy Garden incident occurred as a result of a lack of maintenance and long-term misuse. When mistreated seals are broken, viruses diffuse through the drainage system and spread from one room to another. Furthermore, exhaust systems, which are an increasingly common installation in modern society, accelerate the process by pumping the contaminated air out of the pipes ,10. TherCFD is a scientific research method achieved through the computational ability of computers. By dividing fluid into a grid, the computation of each point provides information about the movement of the fluid ,14,15,16This study was a combination of fluid behavior and concentration distribution. The simulation software calculated the information mathematically by employing physics formulas and controlled boundary conditions. For the fluid behavior, the Navier–Stokes Equation and the Conservation of Mass were used as the base simulation principles.Theorem 1.Navier–Stokes Equation.Theorem 2.Conservation of Mass.The Convection Diffusion Equation and Mass Transfer Equation were introduced to the system for the concentration distribution.Theorem 3. Convection Diffusion Equation.Theorem 4.Mass Transfer Equation.In this study, COMSOL Multiphysics was the software used to construct the simulation. The CFD software is verified as a trustworthy simulation tool because of the notable amount of research based on it ,31. In tWhen it comes to building drainage systems, a vast number of methods with different pipeline designs are being used depending on local conditions. One system worth mentioning is the double stack system, a drainage system design in which a ventilation pipe is connected to a soiled-water pipe or a wastewater pipe, which is widely used internationally because of its ability to balance pressure differences. In addition, various variations of the double stack system were designed to fulfill different needs of all kinds of buildings. Thus, it is not practical to simulate every scenario in a single study. In this research, we focused on a typical fitting with a domestic drainage system in residential buildings.A 12-story building community, commonly seen locally, with two households on each floor was designed. Each household had one bathroom with dimensions of 3 m × 2 m × 3 m, including two floor openings: a wastewater pipe and a soiled pipe, as shown in Additionally, another simulation was conducted under an assumed circumstance in which the two sides shared one set of vent pipes see b, which Environmental factors were considered independent variables. A series of inputs was set up. Then, the adjustment of the system was observed, and the influence of the factors on the system was studied. In this study, five factors were included and examined through simulation and discussion.(1)DistanceThe distance between the source and the target unit was considered crucial. Therefore, this paper focuses on the differences between each floor. The floor is introduced as a distance unit in the discussion.(2)Time span (t)The time span was also considered a critical factor. A three-day simulation was conducted, and the changes in the system on an hourly basis were observed.(3)0)Pressure 0)Initial concentration Temperature (T)Temperature was expected to influence fluid density. Thus, the environmental temperature was adjusted, and then the reaction of the model system was observed.0) on the seventh floor. In addition, simulation result screenshots were collected at the end of every day. The results are shown in The simulation results revealed how the virus diffused into each unit and how far it influenced the system. In order to make a comparison among the different circumstances, the contaminants’ concentration distribution is expressed in concentration ratio (C ratio), that is, the local concentration divided by the initial concentration (C0 caused the virus to spread more slowly and narrowly, while an increasing P0 worsened the contamination of the whole system.After adjusting the pressure applied to the source, a handful of data was obtained concerning the pressure’s influence on the system. The results on the third day under different pressures are shown in 0 did not have a significant direct difference on the system, as shown in Furthermore, the change in the initial concentration was addressed. However, contrary to the study’s assumptions, the adjustment of CFurthermore, the contamination mainly occupied side A and had less effect on side B. In order to examine the risk of being contaminated by horizontal units, a scenario was introduced in which both sides shared one vent pipe. The results are shown in The simulation results showed how the contaminant spread in such a short period. The virus reached the units three floors away from the source within an hour. In addition, it reached six floors within three days and raised the concentration in each unit simultaneously. With the visualized simulation as evidence, we can confirm the high risk of broken water seals, resulting in contaminant diffusion.Environmental factors may influence the system in different ways. By manipulating the parameters, the impact of each factor could be studied. First, distance was critical to virus transportation. The farther away a unit was, the farther the virus had to travel to reach it; hence, the contamination was slower and less severe. Second, time was found to be a crucial parameter in the system. Once the spreading began, the neighboring units started to become infected; thus, the longer the time span, the higher the concentration was within units until it reached its terminal concentration, which was as high as the initial concentration. Third, pressure played an important role as the driving force in the spread of the virus. A higher pressure reinforced faster virus spreading. The central concentration data in each unit was collected from side A on the third day and analyzed, as shown in Fourth, the results showed that the factor of the initial concentration did not perform as expected. There were no radical changes when the number was adjusted. This phenomenon can be explained through the Convection Diffusion Equation, as we assumed no reaction in the system. Therefore, the driving force landed on the diffusion from the concentration difference or the transportation from pressure. According to the data, transportation overpowered diffusion; the latter could not make a significant difference. Therefore, it was concluded that the system could be driven by pressure.Fifth, the environmental temperature did not significantly affect the system. Despite adjusting the atmospheric temperature, the system did not indicate different fluid behaviors.Therefore, an alternative pipeline was designed in which both sides of the building shared the same set of venting pipes. The results showed that the contaminated target gas could reach side B quickly without going through the main horizontal pipe. It was observed that the virus spread to the side B units in the first hour of the simulation, indicating the possibility of horizontal virus spread.This article sought an alternative method in which a virtual drainage system was reconstructed through computational simulation to inspect the risk of a particular scenario. The simulation results not only indicate the high possibility of potential risks but also show how a virus can spread in such a short time. Under certain circumstances, the contaminant could spread three floors away within an hour. In addition, pipelines made it possible for the virus to reach every connected unit in the building. Environmental factors, including time, distance, and pressure, played a crucial role in influencing the system. On the other hand, the initial contaminant concentration has less of an impact than other concentrations. Notably, pressure tended to be the main driving force in the spread of the virus. Additionally, a broken water trap posed a risk of a virus breach in the residential area, which could cause community infection. Visualizing the behavior of viruses provides a glimpse of what happens behind walls, paving the way for recognizing the importance of maintaining functional drainage systems for individuals’ health.


This study used a propensity score matching (PSM) analysis to explore the risk factors of post-operative complications and compared the differences in clinical data between them following spinal tuberculosis surgery.The clinical data of patients with spinal tuberculosis were collected in our hospital from June 2012 to June 2021, including general information, laboratory results, surgical information, and hospitalization costs. The data were divided into two groups: complication and without complication groups. The baseline data of the two groups were obtained using the PSM analysis. Univariate and multivariate logistic analyses were used to analyze the differences between the two groups.P < 0.05).A total of 292 patients were included in the PSM analysis: 146 patients with complications and 146 patients without complications. The operation time, incision length, hospital stay, and albumin quantity in the complications group were 162 ± 74.1, 11.2 ± 4.76, 14.7 ± 9.34, and 1.71 ± 2.82, respectively, and those in the without complication group were 138 ± 60.5, 10.2 ± 3.56, 11.7 ± 7.44, and 0.740 ± 2.44, respectively. The laboratory costs, examination costs, guardianship costs, oxygen costs, and total costs in the complications group were higher than those in the without complication group. A significant difference was observed in the albumin quantity by logistic regression analysis (Several costs in the complication group were higher than in the without complication group. The albumin quantity may be an independent factor to predict post-operative complications of spinal tuberculosis by logistic regression analysis. Spinal tuberculosis is the most common form of extrapulmonary tuberculosis . It has The global tuberculosis situation is particularly tense in developing countries. The incidence of pulmonary or spinal tuberculosis is low in developed countries, however, the incidence is high in developing countries. The incidence of tuberculosis is high in the African region, which accounted for 71% of global tuberculosis and AIDS cases in 2018 . InsuffiIn the past, our team's research had found that there were various complications of spine surgery. There was a potential risk of cerebrospinal fluid leakage and reduced albumin after surgery for spinal tuberculosis , 11. In Although PSM analysis has been favored in spinal surgery, including cervical spine surgery , thoraciThe clinical data of patients with spinal tuberculosis were collected in our hospital from June 2012 to June 2021, including general information, laboratory results, surgical information, post-operative complications, and hospitalization costs. A total of 495 patients were enrolled in the study, including 298 males and 197 females. All data were divided into two groups: complication group with 146 patients –3 and wiThe general information of patients who underwent spinal tuberculosis surgery in our hospital was collected, including gender, body mass index (BMI), age, hypertension, diabetes, lesion segment, medical history, marriage, systolic blood pressure, diastolic blood pressure, pain, lower limb pain, number of lower limb pain, fatigue, fever, night sweats, appetite, weight loss, Oswestry Disability Index (ODI), Japanese Orthopedic Association (JOA) scores, visual analog scale (VAS), American Spinal Injury Association (ASIA) impairment scale, occupation, race, days before surgery, smoking, and drinking. The laboratory results of patients were collected, including blood glucose, blood type, C-reactive protein (CRP), hepatitis B surface antigen, white blood cells, hemoglobin, platelets, percentage of neutrophils, percentage of lymphocytes, absolute monocytes, percentage of monocytes, total bilirubin, direct bilirubin, indirect bilirubin, total protein, albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), AST/ALT, blood urea, blood creatinine, blood uric acid, and erythrocyte sedimentation rate. The surgical information of patients was collected, including titanium cage, internal fixation, abscess, albumin quantity, recurrence, surgical approach, hospital stay, transfusion, local streptomycin, operation time, bleeding volume, red blood cell transfusion, drainage, kyphosis, post-operative complications, and incision length. The hospitalization costs of patients were collected, including antibacterial costs, western medicine costs, surgical costs, anesthesia costs, laboratory costs, examination costs, guardianship costs, transfusion costs, oxygen costs, high consumable costs, care costs, consultation fees, and total costs. Antibacterial costs were defined as the cost of treatment with antibiotics such as penicillin, streptomycin and cefuroxime etc. Western medicine costs were defined as the cost of oral medication such as painkillers, omeprazole and glutathione etc. Surgical costs were defined as the cost of lesion removal, bone graft fusion, reconstruction of spinal stability, etc., but did not include the cost of purchasing internal fixation devices. High consumable costs were defined as the purchase of internal fixation devices such as pedicle screws, titanium cages. Anesthesia costs were defined as the cost of anesthesia during surgery. Laboratory costs were defined as the cost of blood tests before and after surgery. Examine costs were defined as the cost of pre-operative and post-operative imaging examinations, such as X-ray, CT, MRI, etc. Guardianship costs were defined as the cost of monitoring vital signs after surgery. Transfusion costs were defined as the cost of blood transfusions during hospitalization. Oxygen costs were defined as the cost of oxygen therapy during hospitalization. Care costs were defined as the costs of nurses serving patients during hospital stays. Consultation costs were defined as the cost of asking other departments for consultation and treatment during hospitalization. Total costs were defined as the total cost of hospitalization.The propensity score usually includes four methods, namely covariate adjustment using the propensity score, matching on the propensity score, stratification on the propensity score, and inverse probability of treatment weighting using the propensity score. The most popular of these in medical research is matching on the propensity score , 18. KhaUnivariate and multivariate analyses were usually applied to the analysis of related factors in the study of spinal tuberculosis. A study by Surachai et al. used logistic regression analysis and found that signal cord change and notable Cobb angle could be used as predictive factors for the neurological deficit in spinal tuberculosis . MoreoveP <0.1 in the univariate analysis were included in the multivariate logistic regression analysis. The validity of the logistic regression model was evaluated by the receiver operating characteristic (ROC) curve. The statistical difference was considered as P-value <0.05.The RStudio software (version 1.4.1717) and R software (version 4.1.0) were used for data analysis. To eliminate the confounding factors that affected the results, we selected certain parameters for the PSM analysis, including gender, BMI, age, hypertension, diabetes, abscess, titanium cage, internal fixation, percentage of lymphocytes, albumin, and lesion segment. The balance of the matched data was tested using the classical method; the alpha was set to 5. In addition, the balance of the matched data was tested using the standard difference method; the alpha was set to 15. The data following the PSM analysis were included in univariate analysis. We refer to previous published articles , 27, andThere were differences in baseline data before propensity matching, but none were statistically significant after propensity matching . The hisP-value of appetite and occupation was <0.1, and that of other parameters was more than 0.1 (P = 0.067), and there were no significant differences in other parameters (P < 0.05). In addition, there were statistical differences in the surgical approach between the two groups (P < 0.1). The laboratory costs (P < 0.05). There were no significant differences in other costs, including antibacterial costs, western medicine costs, surgical costs, anesthesia costs, transfusion costs, high consumable costs, care costs, and consultation costs, between the two groups (P > 0.05).The general data between the two groups using the PSM analysis revealed that the than 0.1 . The labrameters . The albrameters , operatirameters , incisiorameters , and hosrameters  in the crameters . There wry costs , examinary costs , guardiary costs , oxygen ry costs , and totry costs  in the cry costs . These cP-values <0.1 were included in the logistic regression analysis, including appetite, occupation, AST/ALT, albumin quantity, surgical approach, operation time, and incision length. There was a significant difference in the albumin quantity using the logistic regression model analysis (P < 0.05). However, there were no statistical differences in other parameters, including appetite, occupation, AST/ALT, surgical approach, operation time, and incision length (P > 0.05).We constructed a logistic regression model to further explore the risk factors that affected the complications. The parameters with analysis . FurtherP-values were 4.534 and 0.806, respectively. The confidence interval of the odds ratio , which implied no difference between the logistic regression model and the actual application. The value of the area under the ROC curve was 0.75, implying that this regression model can be used in clinical applications.The logistic regression model was tested through the HL test method, and the PSM analysis strategies, such as tumor chemotherapy , cervicaHowever, this study had certain limitations. (1) Some data were lost after the PSM analysis. (2) More sample data should be collected to obtain more pairs. (3) This subject was a single-center study, and the results need to be verified by a multi-center study.In this study, the costs in the complication group were generally higher than in the without complication group. Multivariate logistic regression analysis found that albumin quantity could be an independent factor for predicting post-operative complications of spinal tuberculosis. Therefore, surgeons should pay more attention to the patient's economy and the occurrence of post-operative complications in clinical work.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by Ethics Committee of the First Affiliated Hospital of Guangxi Medical University. The patients/participants provided their written informed consent to participate in this study.LC wrote the article and prepared This work was sponsored by the National Natural Science Foundation of China (81560359) and National Natural Science Foundation of China (81860393). Funding bodies had no role in the study design, collection, analysis, and interpretation of the data or in writing the manuscript.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.


Gorham-Stout disease (GSD) is a rare osteolytic disease with unknown etiology, varied clinical manifestations and unpredictable prognosis. This disease is characterized by progressive massive local osteolysis and resorption caused by intraosseous lymphatic vessel structure and thin-walled vascular proliferation. The diagnosis of GSD has not yet formed a uniform standard, but the combination of clinical manifestations, radiological features and unique histopathological examinations and excluding other diseases contribute to early diagnosis. Although medical therapy, radiotherapy and surgical interventions or combinations have been used for the treatment of GSD, there is currently still no recommended standardized treatment regimen.This paper presents a case of a previously healthy 70-year-old man presented with a 10-year history of severe right hip pain and progressive walking disorder of the lower limbs. Based on the patient's clear clinical presentation, unique radiological features, and histological findings, a diagnosis of GSD was made with the exclusion of other potential diseases. The patient was treated with bisphosphonates to slow the progression of the disease followed by total hip arthroplasty to help restore walking function. At the 3-year follow-up, the patient returned to normal walking and no recurrence was observed.Bisphosphonates combined with total hip arthroplasty may be an effective method for the treatment of severe GSD in the hip joint. Gorham-Stout disease (GSD), also termed idiopathic massive osteolysis, vanishing bone disease, phantom bone disease, acute spontaneous absorption of bone, and Gorham disease, is an exceedingly rare osteolytic disease characterized by progressive massive local osteolysis and resorption caused by spontaneous intraosseous lymphatic vessel structure and thin-walled vascular proliferation –3. At prIn most cases, GSD usually occurs in a single bone, and in a few cases, it occurs in multiple bones. GSD occurs in a wide range of bones and has been reported to involve the femur, ribs, jaw, humerus, tibia, fibula, and spine –9. The cLaboratory examinations, radiological examinations and histopathological examinations are crucial for the diagnosis of GSD. Laboratory blood tests can provide evidence to rule out infections, myeloma, hyperparathyroidism, and other diseases, but they are less specific for diagnosis , 11. OstA 70-year-old man presented with a 10-year history of severe right hip pain and a 2-month history of progressive walking disorder of the lower limbs. The patient was previously treated with nonsteroidal anti-inflammatory drugs elsewhere due to pain, but the pain relief was not significant until the patient was unable to walk and was admitted to the emergency department of our hospital in May 2018. The patient had no history of alcoholism, hormone use, or trauma. Initial physical examination revealed positive tenderness in the right groin, positive patrick's sign, and rotation and abduction significantly less than the left hip joint. No redness or skin temperature increase was observed in the right hip joint.3 tissues were obtained from the right femoral neck, synovial membrane, and acetabulum. Subsequent histological examinations showed that the necrotic bone tissue was accompanied by focal aggregation of osteoclasts, giant cells and plasma cells without cellular atypia, some proliferative thin-walled capillaries and lymphatic structures, and replaced by fibrous connective tissue (Preliminary anteroposterior  and latee tissue . Combinee tissue  (Table 1e tissue .GSD is a rare benign osteolytic disease characterized by trabecular destruction and resorption of bone, thin-walled capillaries and lymphatic hyperplasia, and the absence of cellular heterogeneity –3. This β, PDGF-BB, and VEGF produced by bone macrophages (GSD pathology includes osteoclast activation, angiogenesis and lymphatic angiogenesis caused by cytokines such as TGF-rophages . T cellsrophages . Clinicarophages , 9, 11. rophages . In our rophages . Relevanrophages , 4, 11. rophages . In controphages , 22. CT rophages . ECT is Medical therapy, radiotherapy, and surgical interventions or combinations are currently available for the treatment of GSD , 5, 11. GSD is a rare benign osteolytic disease, and its etiology and pathogenesis are still unclear. There are no uniform standards for diagnosis and treatment. The combination of clinical manifestations, laboratory examinations, imaging features, and histopathological characteristics is helpful for diagnosis. Medical therapy, radiotherapy, and surgical interventions or combinations are the options for the treatment of GSD. In our case, the combination of bisphosphonates and artificial prosthesis reconstruction achieved good results in the treatment of GSD involving the hip joint, and there was no recurrence at the 3-year follow-up. Medical therapy combined with artificial prosthesis reconstruction may be an effective treatment approach for GSD.


Infections of white spot syndrome virus could be a lethal disease in shrimp culture systems. The 3D structures of the virus-associated structures were obtained through electron tomography. The relationships between the mature and immature particles were connected by the localizations of viral proteins. Our study provided new structural information on the virus through 3D, which extends the limited 2D knowledge of pathogen assembling in the white spot disease, further contributing to the development of a strategy for therapy or prevention.Procambarus clarkii). The samples from various tissues were prepared on the seventh day post-infection. The serial sections of the intestinal tissue were obtained for electron tomography after the ultrastructural screening. After 3D reconstruction, the WSSV-associated structures were further visualized, and the expressions of viral proteins were confirmed with immuno-gold labeling. While the pairs of sheet-like structures with unknown functions were observed in the nucleus, the immature virions could be recognized by the core units of nucleocapsids on a piece of the envelope. The maturation of the particle could include the elongation of core units and the filling of empty nucleocapsids with electron-dense materials. Our observations may bring to light a possible order of WSSV maturation in the cell nucleus of the crayfish, while more investigations remain necessary to visualize the detailed viral–host interactions.White spot syndrome virus (WSSV) has been reported to cause severe economic loss in the shrimp industry. With WSSV being a large virus still under investigation, the 3D structure of its assembly remains unclear. The current study was planned to clarify the 3D structures of WSSV infections in the cell nucleus of red swamp crayfish ( Since its outbreak was reported in Asia in the early 1990s, white spot disease (WSD) has caused huge economic losses in the crustacean farming industry throughout the world . The disIt has been revealed in the literature that the life cycle of WSSV includes the entry of viruses through endocytosis, escaping from endosomes, viral replication in the nucleus, the inhibition of host apoptosis, and finally the disruptions of the host cells ,7. The mThe development of the techniques of electron microscopy has played a major role in the study of the structures of viruses . PreviouProcambarus clarkii) captured in the area near Taipei city were continuously maintained in water tanks for more than 2 generations in the lab under a neutral light cycle. Adult (about 20 g), intermolt male crayfish with normal behavior were selected for inoculations. The selected crayfish were isolated in water tanks of 30 cm (L) × 20 cm (W) × 25 cm (H). Food was supplied daily and 1/3 of water was changed weekly. The crayfish were acclimated in the tank for 7days before inoculation.Red swamp crayfish  paraformaldehyde (PFA) and 2.5% (w/v) glutaraldehyde (GA) in 0.1 M sodium phosphate buffer , while for immune-gold labeling, a fixative containing 4% (w/v) PFA and 0.25% (w/v) GA in 0.1 M PB (pH 7.3) was used. The virus solution was diluted in PBS  and injected intramuscularly into healthy crayfish (about 20 g) between the second and third abdominal segments . On day The procedures for perfusion were modified from the literature . Before For morphological observations, the tissues were further subjected to a standard protocol of post-fixation (1% Osmium tetroxide in 0.1 M PB for 90 min), dehydration, and embedding (Spurr’s medium) after overnight fixation at 4 °C. Semi-thin sections (500 nm) were cut for toluidine blue O (TBO) stain and optical microscopic examinations. Ultra-thin sections were collected on 150-mesh copper grids and stained with 10% uranyl acetate (UA) in methanol (20 min) and Reynold’s lead citrate . The virus-infected nuclei were confirmed using a TEM (FEI Tecnai G2 TF20 Super TWIN) operating at 120 kV. Two-dimensional images were collected in the area without obvious artifacts. The blocks with acceptable structural preservations were further processed for electron tomography.For electron tomography, the blocks were further trimmed and the serial sections (200 nm) through the target area were obtained as previously described . Double-For immuno-gold labeling, small pieces of tissues were fixed in precooled fixative for 10 min and rinsed with cold 0.1 M PB three times (10 min each). The tissues were subjected to a standard protocol of dehydration without post-fixation. The dehydrated samples in ethanol were incubated with 0.1% UA in acetone for 8 h at −20 °C in a homemade freeze-substitution chamber. After rinsing with acetone three times (1 h each) at −20 °C, the specimens were subsequently infiltrated through an ascending gradient of Lowicryl HM20 resin  at −20 °C. The samples were further immersed overnight in 100% HM20. After the final change of 100% HM20 (1 h), the samples were embedded in a sample container the next day. The container was further sealed in a transparent air-tight box and the ultraviolet (360 nm) polymerization was set at −20 °C for 48 h. The container was then rewarmed to room temperature and ultraviolet radiation continued for another 24 h. Next, 100 nm sections of the tissues were prepared from the polymerized blocks and placed on 150-mesh nickel grids for immuno-gold labeling.For labeling procedures, the sections on nickel grids were blocked with 5 % BSA in PBS for 20 min and incubated with the rabbit anti-VP664 (500×) [Since the ultrastructure analysis at the 2D level is still a common way to visualize the virus, here we confirmed the appearance of the virus using negative stain and ultra-thin sections . The 2D Since larger numbers of virus-infected nuclei were found in the intestines, serial sections and electron tomography on the intestines were performed to generate the serial tomograms (Videos S1–S4). In serial tomograms, the linear structures in 2D images were actually shown to be sheet-like structures . Those sThe matured WSSV particles could be well arranged in the nucleus, while the area with immature particles could be found nearby. In our datasets, the immature particles could be classified into four different types . For theTo correlate the virus-associated structures with the protein assembling in matured virus particles, we checked the expressions of viral proteins with immuno-gold labeling . To confThe gold particles on virus-associated structures were then searched under TEM. Firstly, we checked protein expressions on the sheet-like structures . AlthougThen, we focused our observations on the immature particles in the cell nucleus . Based oAs the key step in the viral life cycle, the assembling of virus particles could be a series of reactions among the protein subunits in the crowded intracellular spaces. During such reactions, the ordered and reproducible particles were generated to be infectious agents. Since the viruses could play a critical role in many animal diseases, understanding the assembling procedures could aid in the development of antiviral strategies . In our Research reports that the capsids of viruses could be briefly classified into rod-like and spherical (polyhedron) structures . As prevSince there are about 515–684 open reading frames that encode about 180 functional proteins  in the WSSV genome, the majority of viral protein functions may still call for investigations ,24. As tPenaeus vannamei, P. stylirostris [Penaeus monodon [Procambarus clarkii. Although the existence of species- and tissue-specific structures after WSSV infection could not be denied, our observations suggested that the rod-shaped structures of the repeating subunits without the electron-lucent core mentioned in the literature could be an image of the paired sheets from the oblique projections [From early investigations in the WSSV-infected nucleus, a tubular structure of two electron-dense 20nm-wide longitudinal bands separated by a central 6nm-wide electron-clear band was reported in irostris ,10,27, a monodon . The str monodon ,10 or vi monodon . InconsiFor the immuno-gold labeling of VP664, it is reported that the gold particles were not detected on the matured viruses with intact viral envelopes . While tEarly 2D ultrastructural investigations have revealed the structures of immature WSSV virions in the center of the infected nucleus ,10,11,12Recently, the knockdown of the envelope protein VP19 showed a connecting disruption between envelope and nucleocapsids of the WSSV particles in crayfish, where the nucleocapsids with matured size could still be obtained . In our For the viral capsid formations, various mechanisms have been reported in the literature . As the Procambarus clarkii in this study. Our observation at the ultrastructural level revealed novel information about 3D structures after WSSV infections. The structures were also correlated to the matured virions through the expressions of viral proteins. However, more structural investigations on protein interactions and time-specific changes remain to be carried out to uncover the virus assembling procedures.The 3D structures of the WSSV-infected nucleus were reconstructed from


Essential for potexvirus Accumulation 1 (EXA1) has been identified as a susceptibility gene required for potexvirus, lolavirus, and bacterial and oomycete pathogens. In this study, EXA1 knockdown in potato (StEXA1) was found to confer novel resistance to potato virus Y  in a strain-specific manner. It significantly compromised PVYO accumulation but not PVYN:O and PVYNTN. Further analysis revealed that StEXA1 is associated with the HC-Pro of PVY through a member of eIF4Es (StnCBP). HC-ProO and HC-ProN, two HC-Pro proteins from PVYO and PVYN, exhibited strong and weak interactions with StnCBP, respectively, due to their different spatial conformation. Moreover, the accumulation of PVYO was mainly dependent on the stress granules (SGs) induced by StEXA1 and StnCBP, whereas PVYN:O and PVYNTN could induce SGs by HC-ProN independently through an unknown mechanism. These results could explain why StEXA1 or StnCBP knockdown conferred resistance to PVYO but not to PVYN:O and PVYNTN. In summary, our results for the first time demonstrate that EXA1 can act as a susceptibility gene for PVY infection. Finally, a hypothetical model was proposed for understanding the mechanism by which StEXA1 interacts with StnCBP to facilitate PVY accumulation in potato through the SG-dependent RNA regulatory pathway.Plant viruses recruit multiple host factors for translation, replication, and movement in the infection process. The loss-of-function mutation of the susceptibility genes will lead to the loss of susceptibility to viruses, which is referred to as ‘recessive resistance’.  Published by Oxford University Press on behalf of Nanjing Agricultural University. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: 13 February 2022; Revised: 27 June 2022; Accepted: 6 July 2022
 Solanum tuberosum L.) has become the third most important food crop worldwide next to rice and wheat in terms of human consumption . Then, 15-μl beads  were added to 500-μl extracted protein supernatant. After incubation at 4°C for 2 hours, the beads were washed thrice with wash buffer . Finally, the beads were added with 50-μl 5× SDS buffer containing 5% β-mercaptoethanol, and boiled at 95°C for 10 minutes to elute the proteins for Western blot assay as previously described [escribed . PrimersAll data are expressed as the mean value ± standard deviation (SD) of biological replicates. Statistical significance was determined using Student’s t-test.Web_Material_uhac159Click here for additional data file.


The thermal stability of viruses in gelatin liquid formulations for medical research and application is poorly understood and this study aimed to examine the thermal stability of 4 enveloped and nonenveloped DNA and RNA viruses in hydrolyzed gelatin liquid formulations.Bovine herpesvirus (BHV) was used as a model virus to examine the molecular weight (MW), concentration and gelatin type and to optimize virus stability in liquid formulations at 25 °C and 4 °C. Using the model virus liquid formulation, the stability of multiple enveloped and nonenveloped RNA and DNA viruses, including parainfluenza virus, reovirus (RV), BHV, and adenovirus (AdV), was monitored over up to a 30-week storage period.The BHV model virus was considered stable after 3 weeks in hydrolyzed gelatin (MW: 4000) with a 0.8 LRV  at 25 °C or a 0.2 LRV at 4 °C, compared to the stabilities observed in higher MW gelatin  with an LRV above 1. Based on the gelatin type, BHV in alkaline-treated hydrolyzed gelatin samples were unexpectantly more stable than in acid-treated hydrolyzed gelatin sample. All four viruses exhibited stability at 4 °C for at least 8 weeks, BHV or AdV remained stable for over 30 weeks of storage, and at 25 °C, AdV and RV remained stable for 8 weeks.The results demonstrated that 5% of 4000 MW hydrolyzed gelatin formulation can act as a relevant stabilizer for the thermal stability of viruses in medical research and application.The online version contains supplementary material available at 10.1186/s12985-022-01819-w. Many viruses used for medical research or therapeutic purposes exhibit a lack of thermostability in ambient environments. Viruses can be placed in two main categories based on the presence or absence of a lipid envelope around their capsid, which classifies them as an enveloped or nonenveloped virus, respectively. The latter is more thermostable than the former group , 2. The There are two main strategies currently used to improve virus stability: cold chain storage and freeze-drying. However, these strategies create some issues, including the challenges of cost and extensive infrastructure and maintenance for cold chain transport, especially for deep freezing , 5. The Very few studies have reported the thermal stability of viruses in gelatin formulations. Some studies have investigated the temperature-induced aggregation of measles particles in several stabilizing excipients, including gelatin , or the The mechanism of gelatin-mediated stabilization of viral particles in liquid inoculant formulations remains broadly unknown. Gelatin is an additive that is thought to be a good stabilizer of viruses in viral transport medium . GelatinBovine herpesvirus 1 (BHV-1) as Herpesviridae, PI-3 (bovine parainfluenza type 3) as Paramyxoviridae, Reovirus 3  as Reoviridae, and human adenovirus 5  as Adenoviridae were used as test viruses to examine virus thermal stability in gelatin liquid formulations. The BHV-1, PI-3, RV-3, and AdV-5 inoculums were prepared, and infected monolayers of MDBK cells (BHV-1 and PI-3), HEK-293 cells (AdV-5), and LLC-MK2 cells (RV-3) were cultured with Dulbecco's modified Eagle’s medium  containing 5% fetal calf serum following incubation at 37 °C for 6–7 days post-infection. Each virus-infected culture was harvested and centrifuged at 2500 rpm for 15 min at 4 °C, and the supernatant was collected, transferred into SW41Ti Beckman UC tubes, and ultracentrifuged for 2 h at 28 K rpm to produce pellets of high titer of virus stocks. Pellets were dissolved in gelatin liquid formulations. Aliquots of 250 µL/tube were made and pretested to determine the initial virus titer, and the rest were stored at − 80 °C as references and at ambient temperatures, including 4 °C and 25 °C, for virus thermal stability evaluations.The gelatin samples used for formulation were of either porcine or bovine origin. They included pigskin and bovine bone gelatin resulting from acid or alkaline extraction methods. All bovine materials used for gelatin production are not from countries at risk of BSE or specified risk materials, and a process for prion inactivation is included in the gelatin manufacturing process. Specifically, the samples were beMatrix™ Gelatin series, from Nitta Gelatin Inc. . Additionally, recombinant human serum albumin (rHSA) of molecular weight 66,000 from Albumedix  was also used as a reference control for the viral stability test. Various MWs of hydrolyzed gelatin (HG) were also obtained from one of the original products (beMatrix™ Gelatin LS-H) through heat hydrolysis at different temperatures and pH values, and the samples were all finally adjusted to pH 7 prior to use in the formulations. The resulting MW of the samples was measured by gel permeation chromatography following the manufacturer’s protocol (Tosoh Bioscience). Briefly, a 0.2% gelatin solution adjusted with distilled water was passed through a 0.2 µm filter and vortexed. The samples were placed in the autosampler for analysis under the following conditions: a 1 mL/min flow rate, column temperature at 40 °C, 10 µL injection volume, and a 214 nm UV wavelength. The isoelectric point determination for each sample was performed using the zeta potential method with a ZetaSizer Nano ZS . Briefly, samples were dissolved in buffer made of a 0.1 M Tris–HCl (pH 7.7–9.0) solution and acetate solution (pH 4.0–5.50) to make 0.25% (w/v). The zeta potential was measured, and the pH variation depending on the zeta potential was plotted and extrapolated to derive the isoelectric point at zero .To select cytotoxicity-free samples for the gelatin formulation, a total of three buffers  and gelatin samples of various MWs were evaluated. Briefly, each sample was serially diluted twofold in DMEM (Dulbecco’s modified Eagle’s medium) and dispensed at 50 µL/well (triplicate) into a 96-well plate, followed by the addition of 10,000 Vero cells per well and incubation at 37 °C for 72 h. A volume of 20 µL of MTS -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) from Promega (Cat #G3580) was added and incubated at 37 °C for 3 h, followed by absorbance measurement at 490 nm on a plate reader . The survival rate was determined using the formula [Survival rate = (Sample O.D.- Blank)/(Mock OD- Blank) × 100].50/mL) following the Karber method [Virus titration was carried out before and after storage at − 80 °C, 4 °C and 25 °C for a certain number of weeks. Briefly, triplicates of aliquots for each virus sample before or after a storage period were serially diluted tenfold in cell medium; and inoculated on relevant monolayers, including MDCK cells for BHV-1 and PI-3, HEK-293 cells for AdV-5, and LLC-MK2 cells for RV-3, in 96-well plates; and incubated at 37 °C. The cytopathic effect was recorded 6 days post-infection. The virus titers were determined as the 50% tissue culture infectious dose .Data analysis, including group comparisons and graphical representations, was conducted using Prism software 8.0 , and a GTS buffer  and gelatin samples of various MWs with final concentrations ranging between 0.4% and 25% demonstrated no cytotoxicity (data not shown). In conducting the virus storage stability test, BHV-1, which had the highest decrease in stability under 5% FCS DMEM conditions at 4 °C for 4 weeks, was used as a model virus for the development of gelatin liquid formulations.In total, 5 samples with different gelatin molecular weights in the formulation in GTS buffer with BHV-1 were monitored primarily for 3 weeks at 25 °C and 4 °C. BHV-1 remained stable after 3 weeks of storage in 4000 MW hydrolyzed gelatin liquid formulation at 25 °C, in contrast to the loss of stability observed in gelatin liquid formulations with 60,000 and 160,000 MWs.  % to find the optimal concentration for virus storage stability. In addition, 0.5% rHSA in GTS buffer was also used as a control in the same evaluation. Only the concentration of 5% showed good stability after 3 weeks of storage at 25 °C, while loss of rHSA stability occurred  showed better results than the sample obtained by the acid extraction method (acid-treated) with the BHV-1 model, and an interpretation of this finding would require further investigation. In general, the pI (isoelectric point) values of the alkaline-treated and acid-treated gelatin are low (acidic) and high (basic), respectively. These HG samples with pIs around 5 or 8 were produced by alkaline and acid extraction methods, respectively. The pI of herpesvirus is reported to be approximately 9.6 . There iNevertheless, this research has some limitations. The stability profiles of other viruses, including PIV, RV, and AdV, with known gelatin parameters, such as MW, concentration, or extraction method, were not evaluated. Additionally, only the stability of BHV was evaluated in gelatin-free buffer or in freeze–thaw stability tests. Moreover, the effect of the current formulation on a vaccine model or genetically modified virus such as the oncolytic virus for potential immunogenicity retention or thermal stability were not evaluated and would be examined in a future study.In summary, this study included the examination of the stability of four relevant viruses (enveloped/nonenveloped RNA/DNA viruses) in HG liquid formulation for storage in ambient environments. All virus models remained stable for 8 weeks, and BHV and AdV showed extended stability in the liquid formulation after 30 weeks of storage at 4 °C in 5% HG. The hydrolyzed gelatin used to develop the liquid formulation for all four model viruses was of pharmaceutical grade, had very low levels of endotoxin (< 10 EU/g) and would be relevant for virus and medical research studies and applications.Additional file 1: Table S1. Eight-week storage stability of BHV in GTS buffer at 25 °C, 4 °C and -80 °C. Table S2. Stability of RV with two different virus stock titers (LRV ± STD).


The crystal structure of DGL showed that His‐163 could serve as the proton donor. H163A mutant DGL is fully active in the steady‐state reaction, and the pre‐steady‐state kinetics are very similar to those of wild‐type DGL. However, H163A DGL accumulates a transient intermediate with λmax at 293 nm during the reaction that is not seen with wild‐type DGL. Furthermore, NMR analysis of the reaction of H163A DGL in D2O shows that the product is a mixture of deuterated diastereomers at C‐3. These results establish that His‐163 is the proton donor in the reaction mechanism of DGL. d‐gluconate‐6‐phosphate produced from d‐glucosaminate‐6‐phosphate by d‐glucosaminate‐6‐phosphate ammonia‐lyase. This protonation step is not rate determining but may be necessary to avoid the release of a potentially cytotoxic intermediate.His‐163 is the stereospecific source of the proton on C‐3 of 2‐keto‐3‐deoxy‐ KDG‐6‐P, 2‐keto‐3‐deoxyglutarate‐6‐phosphateDGL, (4.3.1.29), d‐glucosaminate‐6‐phosphate ammonia‐lyaseDGA‐6‐P, d‐glucosaminate‐6‐phosphateDGA, d‐glucosaminic acidPLP, Pyridoxal‐5′‐phosphateSVD, singular value decompositionSalmonella enterica serovar typhimurium and other related enterobacteria can utilize d‐glucosaminic acid  as a sole carbon and nitrogen source through a pathway that we have recently described (Eq. 1) [d‐glucosaminate‐6‐phosphate (DGA‐6‐P). The key step in this pathway is the deamination of DGA‐6‐P by d‐glucosaminate‐6‐phosphate ammonia‐lyase (DGL) to give 2‐keto‐3‐deoxygluconate‐6‐phosphate (KDG‐6‐P). Subsequently, an aldolase, produced by the DgaF gene, breaks down KDG‐6‐P to pyruvate and d‐glyceraldehyde‐3‐phosphate to enter the Entner–Doudoroff alternative glycolytic pathway. We previously created a homology model of DGL that predicted His‐162 or His‐163 would be possible catalytic groups, based on their proximity to the active site [2O gives a single diastereomeric 3‐deutero‐KDG‐6‐P, where the pro‐R proton of KDG‐6‐P is stereospecifically replaced by deuterium, resulting in an unusual inversion of stereochemistry in a β‐elimination reaction [d‐glucosamine hydrochloride  as described previously [DGA‐6‐P was prepared from eviously . BuffersWild‐type DGL was prepared as described previously , 2. H163GCTTGCGTACAGAAAGGCATGTTGAGC‐3′Forward: 5′‐GCGCTACTGTATGTGAAATCACACE. coli cells (Genesee Scientific). Single colonies were grown for plasmid purification to confirm the presence of the mutation by sequencing. The mutant plasmid was then used to transform competent E. coli BL21(DE3)  for expression. An overnight culture in 5 mL LB with 100 μg·mL−1 ampicillin was used to innoculate 1 L of Studier ZYP‐5052 autoinduction medium [m potassium phosphate, pH 7, 0.3 m NaCl, and 0.1 mm PLP. The cell‐free extract was obtained by sonication for 4‐min in 1‐min intervals, with cooling on ice, followed by centrifugation for 90 min. The supernatant was applied to a Ni‐NTA Sepharose  affinity column equilibrated with 0.05 m potassium phosphate, pH 7, 0.3 m NaCl, 0.1 mm PLP, and washed with the buffer containing 20 mm imidazole until the 280 nm absorbance returned to baseline. The protein was eluted with the buffer containing 200 mm imidazole and concentrated in a centrifugal concentrator , then diluted with buffer without imidazole and concentrated. After concentration, the protein was frozen in aliquots at −80 °C.Reverse: 5′‐GCGCTACTGTATGTGAAATCACACGCTTGCGTACAGAAAGGCATGTTGAGC‐3′ bases indicated in bold. The PCR reaction used Phusion DNA polymerase , with annealing for 30 s at 55 °C and extension for 3 min at 72 °C for 30 cycles. The PCR product was then used to transform competent GC‐10 m triethanolamine hydrochloride, pH 8, 0.2 mm NADH, 50 μm PLP, 16 μg·mL−1 rabbit muscle lactate dehydrogenase, 4 μL of crude DgaF, and various amounts of DGA‐6‐P. The reactions were followed on a Cary 100 UV–visible spectrophotometer at 340 nm and 37 °C. There was often a short lag (1–2 min) before the steady‐state rate was achieved. The rate was measured in the steady state, using Δε = 6.2 × 103·m−1·s−1 for the molar extinction coefficient of NADH at 340 nm. The data were fit to Eq. DGL was assayed as described previously , 2. The −1) were mixed 1:1 with 20 mm ligand solutions in 0.1 m Tris–HCl, pH 8, at ambient temperature (~ 20 °C). The data were analyzed with the Global Works program provided by OLIS [The rapid‐scanning stopped‐flow kinetics were performed in an OLIS RSM‐1000 instrument equipped with a stopped‐flow mixer. Absorbance scans were collected from 250 to 800 nm at 1000 Hz for 1–2 s, or at 62 Hz for 60 s or longer. Solutions of wild‐type or H163A DGL , and 99 μL water. It was incubated for 30 min at room temperature, then analyzed by ESI‐MS in negative‐ion mode on a Bruker Impact II Q‐TOF instrument.The sample contained 0.5 mg DGA‐6‐P, 1 mg NHm NaOD to bring the pH up to 7 and 10 μL wild‐type or H163A DGL, in 600 μL D2O. After 30 min at room temperature, the spectra were collected on a Bruker Avance instrument at 400.14 MHz with presaturation for water suppression.The samples for NMR contained 2.75 mg of DGA‐6‐P and 1.4 μL of 14.2 7LCE) manually with COOT [The model of DGA‐6‐P in an extended conformation was prepared with ChemBio3D  and docked into the structure of DGL (PDB ith COOT . The phom−1)− at m/z = 275.00679  , showing that the reaction product of the mutant enzyme is also KDG‐6‐P.H163A DGL was prepared by PCR mutagenesis, expressed, and purified by Ni‐MAC, as previously described for wild‐type DGL , 3. Althkobs = 318 ± 30 s−1, along with an increase in absorbance in the 300–360 nm region , and this peak decayed with a ion Fig. . This iss−1 Fig. , althougs−1 Fig.  with H16s−1 Fig. .m, while the enzyme concentration is 25 μm, so if it were enzyme bound, the intermediate would have a molar extinction coefficient of ≥56 000 m−1·cm−1. No PLP enzyme intermediate is known, which has an absorbance at 293 nm with such a high molar extinction coefficient. We suggest that this transient intermediate is formed in millimolar concentrations and is the substituted aminoacrylate product of the β‐elimination of water from the 3‐position from DGA‐6‐P. The spectra and time courses for the overall reaction of wild‐type DGL ‐3‐deutero‐KDG‐6‐P together with trace amounts of the epimeric (3S)‐deuterated product and protiated KDG‐6‐P . What is the function of His‐162 in DGL? A model was created PLP Fig. . The phoPLP Fig. . Thus, HHis‐163 is strictly conserved in all sequences of DGL, even those with relatively low (~ 40%) overall sequence identity . Since the protonation of C‐3 of the product is not rate limiting (Table His‐163 is the stereospecific source of the proton on C‐3 of KDG‐6‐P produced from DGA‐6‐P by DGL. This protonation step is not rate determining but may be necessary to avoid the release of a potentially cytotoxic intermediate.KLA prepared the wild‐type enzyme and created a homology model of DGL. DG created the H163A mutant enzyme, prepared the protein, and performed the NMR experiment. RSP performed the stopped‐flow experiments and wrote the paper.Fig. S1. Portion of sequence alignments of DGL from various bacteria.Fig. S2. Negative ion mode ESI‐MS of the product of H163A DGL.Click here for additional data file.