Favipiravir is a nucleoside analogue which has been licensed to treat influenza in the event of a new pandemic. We previously described a favipiravir resistant influenza A virus generated by in vitro passage in presence of drug with two mutations: K229R in PB1, which conferred resistance at a cost to polymerase activity, and P653L in PA, which compensated for the cost of polymerase activity. However, the clinical relevance of these mutations is unclear as the mutations have not been found in natural isolates and it is unknown whether viruses harbouring these mutations would replicate or transmit in vivo. Here, we infected ferrets with a mix of wild type p(H1N1) 2009 and corresponding favipiravir-resistant virus and tested for replication and transmission in the absence of drug. Favipiravir-resistant virus successfully infected ferrets and was transmitted by both contact transmission and respiratory droplet routes. However, sequencing revealed the mutation that conferred resistance, K229R, decreased in frequency over time within ferrets. Modelling revealed that due to a fitness advantage for the PA P653L mutant, reassortment with the wild-type virus to gain wild-type PB1 segment in vivo resulted in the loss of the PB1 resistance mutation K229R. We demonstrated that this fitness advantage of PA P653L in the background of our starting virus A/England/195/2009 was due to a maladapted PA in first wave isolates from the 2009 pandemic. We show there is no fitness advantage of P653L in more recent pH1N1 influenza A viruses. Therefore, whilst favipiravir-resistant virus can transmit in vivo, the likelihood that the resistance mutation is retained in the absence of drug pressure may vary depending on the genetic background of the starting viral strain. In the event of a new influenza pandemic, drugs will be our first line of defence against the virus. However, drug resistance has proven to be particularly problematic to drugs against influenza. Favipiravir is a novel drug which might be used against influenza virus in the event of a new pandemic. Is resistance likely to be a problem for the use of favipiravir? Our previous work has shown that resistance to favipiravir can be generated in cell culture but we don’t know whether there will be a cost preventing the spread of resistance in whole organisms. Here, we used a mix of wild-type and resistant influenza viruses from early in the 2009 pandemic to test whether viruses resistant to favipiravir could transmit between ferrets. We found that the resistant viruses could transmit but that the resistance mutation was selected against within some ferrets. Using modelling and in vitro experiments, we found that the resistant mutation was selected against in the influenza strain from our experiment but not in more recently evolved strains. Our results show that favipiravir resistant viruses could spread if resistance is generated but the probability will depend on the genetic background of the virus. Influenza virus is a negative strand virus that causes significant morbidity and mortality worldwide. Like most other RNA viruses, influenza virus has a fast rate of evolution which allows it to evolve in response to immune pressure as well as antiviral drugs –3. AntivFavipiravir is a novel antiviral drug licensed in Japan for the treatment of influenza in the event of a new pandemic –12. FaviTransmission studies in animal models such as the ferret have revealed whether drug resistant influenza viruses have fitness costs and can help inform about the likelihood of the emergence of resistance –20. EarlFavipiravir resistance is only likely to develop in an individual treated with favipiravir . HoweverTo test whether favipiravir resistant virus could transmit by direct or indirect contact, we inoculated ferrets with a mix of resistant virus bearing PB1 K229R + PA P653L and the corresponding wild-type virus, Eng195, a prototypical first wave pH1N1 2009 virus. By inoculating with a mix of virus, we could investigate whether there was a detectable fitness difference between the favipiravir resistant and the wild-type virus. We used a low percentage of wild-type virus to maximize the probability of resistant virus transmitting in the event that there was a fitness cost to resistance in the ferrets. Four donor ferrets were inoculated with K229R + P653L and Eng195 viruses in the ratio of 95:5. After 24 hours, each donor ferret was housed with a direct contact sentinel ferret to measure contact transmission. In addition, an indirect contact sentinel animal was housed in a separate adjacent cage to measure airborne transmission.All 4 donor ferrets were successfully infected and shed virus in the nasal wash with a peak viral titre on day 2 and a secondary peak for most donors on day 4 or 5 as has been seen previously for ferrets infected with this dose of pH1N1 virus  Fig 1)..Fig 1). Two time points were selected from the daily nasal washes collected from each ferret to sequence virus shed in the nasal washes by both whole genome sequencing and more targeted sequencing of the PB1 and PA segments . The firViruses shed in nasal wash underwent whole genome sequencing to search for additional mutations which might be required for efficient transmission or to further compensate for K229R or P653L. We found no additional mutations in PB1 or PA in any of the donor ferrets or the sentinel ferrets occurring above 5%. This confirmed that the K229R + P653L virus was able to productively infect and transmit between ferrets. There were a low number of non-synonymous mutations at >25% in direct contact (5 mutations) and respiratory sentinel ferrets (4 mutations) which were likely due to bottlenecking occurring during transmission. These mutations were not observed in multiple ferrets, which could have indicated parallel evolution and positive selection. The one exception was a mutation in M2 (T60A) which was present in both the direct and indirect contact from the same donor but in no other ferrets. This sequencing provided evidence that favipiravir resistant virus could transmit without additional compensatory mutations.Next, we sought to understand how the K229R and P653L mutations might change over time within an individual ferret. In all four donor ferrets, the proportion of the PB1 wild-type amino acid, K229 increased compared to R229 from the inoculum at the earliest time point and further increased at the later time point . From 5%f) of 1 to wild-type virus and K229R + P653L mutant, 1.25 for the P653L mutant and 0.01 for the K229R mutant. As the actual fitness values in ferrets remain unknown, the fitness values were chosen to qualitatively reflect the fitness advantage for P653L and the fitness cost of K229R seen in MDCKs. The effect of varying these fitness values can be seen in the appendix  which allowed viruses to coinfect cells and for segments to reassort. Previous studies have shown that reassortment is common in experimental infections ,33. We nndix see .In the baseline model , the inif = 1). We kept other parameters the same including the fitness of K229R (0.01). Without a fitness advantage to the P653L single mutant, the WT virus was still lost after 20 generations but the frequency of P653L did not rise above 5%, the starting frequency in the inoculum . We maintained a fitness advantage for P653L (1.25). Without a fitness cost to the K229R mutant, there was an increase in the P653L single mutant and a loss of WT virus. There was also a small proportion of K229R single mutant viruses which did not rise above 5%. Taken with the previous result, our model implies that when the WT virus reassorts the P653 allele is lost if there is a cost to the K229R mutant. However, the loss of the R229 allele was caused by the fitness advantage of the PA P653L single mutant not the fitness cost of the K229R mutant.Next, we tested whether the fitness advantage of the P653L single mutant was sufficient to cause the observed increase of the K229 allele . Here, wNext, we tested whether reassortment or mutation was more important for driving the dynamics of our model . We remoA sensitivity analysis showed that our model was robust to changes in values of the initial variables as well as changes in the model assumptions see . We showGiven the large fitness advantage of the P653L mutant virus in ferrets, it is surprising that the mutation has not been observed in pH1N1 isolates. We hypothesized that one reason the mutation might not be present is that more recent mutations in the pH1N1 virus polymerase also confer a fitness advantage, achieving the same increase in polymerase activity as P653L. Our previous work showed that the polymerase of Eng195 was less well adapted to human cells compared to later pandemic isolates and identified the N321K PA mutation as being a key mutation that led to improved polymerase activity in second and third wave pH1N1 virus isolates ,35. To trd wave pandemic H1N1 virus, Eng687, which already contained N321K. As had been observed in the background of Eng195, Eng687 K229R resulted in low but appreciable polymerase activity which was fully compensated by the presence of P653L  or by outgrowth of low levels of K229, which were initially present but were not detected by sequencing. Our modelling supports that the virus with single PA mutation L653 coupled with wild-type PB1 K229 was generated by reassortment in vivo before increasing in frequency due to positive selection as seen in the schematic of the ferrets from Group 1 . The higDespite the large fitness advantage of L653, this mutation is not found in currently circulating pH1N1 viruses. We showed that one potential explanation for this observation is that other PA mutations have evolved that ameliorate the maladapted PA of the early pH1N1 isolates compared to more recent pH1N1 isolates. We have previously shown that later isolates from the third wave could outcompete first wave isolates in vitro due to the N321K mutation in PA . Here weA recent study examined the transmission of baloxavir resistant virus in ferrets in the absence of drug . Here a Our results have interesting implications for transmission dynamics of favipiravir resistant viruses in the absence of drug. We have demonstrated that the virus can transmit between ferrets that are untreated with drug, which means that a localised epidemic of drug resistant virus would be possible. However, we also found that the K229R mutation was lost over time within a single ferret which implies that if the virus can be outcompeted, resistance is less likely to spread in the absence of drug. The exact probability of resistance emerging and resistance then spreading will likely depend on the precise genetic background of the virus. This has been seen in the case of oseltamivir resistance in pH1N1(2009) where, despite outbreaks of resistant virus, oseltamivir resistance remains at low levels globally ,38. ThisAll animal research described in this study was approved and carried out under a United Kingdom Home Office License, PPL 70/7501 in accordance with the approved guidelines.labtech.com), 1% penicillin-streptomycin (Invitrogen) and 1% non-essential amino acids (Gibco) at 37°C and 5% CO2.Madin-Darby canine kidney  and HEK293T (293T) were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  is a first-wave isolate from the 2009 A(H1N1) pandemic grown from a reverse genetic virus . FavipirMap to Reference) to an Eng195 reference (GenBank ID: GQ166654) and all mutants at location 653 were analysed by eye.11,455 pH1N1 (2009) viruses with full length PA segments were downloaded from GISAID on 12/03/2017. They were aligned in Geneious v11  weighing 750–1000 g were acclimatized for 14 days before inoculation. Donor ferrets were lightly anaesthetized with ketamine (22 mg/kg) and xylazine (0.9 mg/kg) and then inoculated intranasally with virus diluted in phosphate buffered saline (PBS) (0.1 ml per nostril). The virus inoculum was ~10,000 plaque forming units consisting of a mix of K229R + P653L virus and Eng195 in the ratio 95:5. Sentinel ferrets were introduced day 1 post infection and remained for the duration of the experiment. Ferret body weight was measured daily to check for significant weight loss due to sickness. Ferrets were nasal washed daily, while conscious, by instilling 2 ml PBS into the nostrils, and the expectorate was collected in 250 ml centrifuge tubes. Virus titre in the nasal wash expectorate was calculated by plaque assay. The nasal wash was stored with 4% Bovine Serum Albumin Factor V (Gibco) at -80°C prior to RNA extraction. All sentinel ferrets were handled before donor ferrets to prevent accidental transmission of virus.https://www.ebi.ac.uk/ena (project number PRJEB39934.)Samples were chosen at multiple time points for each ferret  and sequ6 virions, a mix of K229R + P653L virus and Eng195 in the ratio 95:5, as in the inoculum, and assumes a well-mixed population with virions infecting cells randomly. Evolution of the virus was tracked over 20 generations of replication, with 106 cells available for infection during each generation giving an average MOI of 1. Both the number of virions and the population of cells stayed constant between generations. We allowed for varying amounts of reassortment by adjusting the ratio of viruses to cells. During each replication cycle, each virion enters a random cell. The burst size from each cell was Poisson distributed, with mean proportional to the summed fitnesses of the virion(s) infecting that cell. As default, the mean burst size for a cell infected by 1 virion was 10  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,Seema Lakdawala, PhDReviews EditorPLOS PathogensSara CherrySection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064***********************Based on the three reviews, there are at least a few additional in vitro studies needed to address the issue of Favipiravir-resistance in different genetic strain backgrounds and to correlate polymerase activity with replication fitness. In addition, clarity on the description and interpretation of the computational model should be added to the manuscript.Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: Goldhill et al., performed transmission studies with a favipiravir-resistant strain of 2009 pandemic H1N1. The resistant virus was previously generated on the A/England/195/2009 (H1N1) background and contains the PB1 K229R and PA P653L mutations. Herein the authors evaluated the in vivo fitness effects of these resistance mutations. When the A/England virus carrying the PB1 K229R+ PA P653L mutations was inoculated into ferrets at a ratio of 95% mutant virus to 5% wild-type, the resistant virus was capable of both direct contact and airborne transmission. However, reassortant viruses carrying the PA P653L mutation with the wild-type PB1 also transmitted and eventually became the dominant strain. Subsequently, polymerase assays were performed to determine the fitness effect of the PA 653L mutation. These studies show that the PA mutation enhances polymerase activity in the A/England/195/2009 (H1N1) strain. When this mutation was introduced into a more recent pandemic H1N1 isolate, the PA P653L mutation did not confer enhanced polymerase activity. This was due to an additional mutation, N321K, that enhances polymerase activity in more recent pandemic H1N1 strains.Reviewer #2: Goldhill et al. use an elegant combination of approaches to examine the within- and between-host dynamics of an IAV favipiravir escape mutant during mixed infection with wild type virus. In particular, a double mutant Eng195 (pH1N1) virus is examined that contains both a PB1 K229R escape mutation and a PA P653L compensatory mutation. Mixed infection with wild type virus is performed and relative proportions of the wild type and mutant segments are monitored by deep sequencing of nasal swabs from inoculated, direct contact and aerosol contact ferrets. The results reveal transmission of the escape mutant virus, but also a decline in its prevalence over time within multiple ferrets. The particular dynamics observed suggest that the PA P653L (compensatory) mutation is uncoupled from PB1 K229R by reassortment, and that this PA mutation undergoes positive selection. This model is tested in a simple but robust computational simulation; the results of which support the model given. Since the PA P653L mutation appears to be driving the dynamics observed but is not seen in circulating pH1N1 viruses, the authors then extend their thinking to recent pH1N1 viruses. Recent strains have higher baseline polymerase activity owing to a PA N321K polymorphism. In the context of PA N321K, or a full Eng687 strain background, PB1 K229R and PA P653L mutations have lesser and no effect, respectively, on pol activity. The authors conclude that the fate of 229R favipiravir resistance mutations will depend in part on the particular strain background and that resistance may arise more easily in recent pH1N1 viruses compared to 2009 pandemic isolates.This is an interesting and elegant study, which not only gives insight into the potential for favipiravir resistance to spread but also reveals the factors governing within- and between-host dynamics in mixed IAV infection.Reviewer #3: In this study, the authors build on their previous work, in which they showed in vitro that the favipiravir resistance mutation PB1 K229R, which conferred a fitness cost, could be compensated for by a PA P653L mutation. Here, they use a ferret model to ask whether this compensated resistance phenotype could spread in vivo, and hence whether favipiravir resistance could spread in human influenza viruses.The paper is well conceived and for the most part well-written . The experimental work is well-performed and the overall aims of the paper have significant importance. However, in my opinion the paper as written has three key weaknesses (at least two of which could be addressed without the need for further experiments).**********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) The authors utilize viral polymerase activity assays to infer fitness effects of mutations in different viral strains. While these results are valid, they do not directly measure viral fitness, and the standard in the field is to perform viral growth kinetics studies to complement polymerase assays. Thus, additional in vitro growth kinetic studies of the mutant viruses are required. Moreover, the authors do not indicate the temperature at which the polymerase assays were performed. From review of the methods, this is most likely at 37ºC. Richard et al.,  demonstrated that during transmission between ferrets, viruses from the upper respiratory tract are transmitted. Therefore, both polymerase and growth kinetics studies should be performed at 33ºC.Reviewer #2: noneReviewer #3: (1) Discussion of mutations. The authors detected nucleotide-level changes but only express them as amino-acid level changes. This makes sense for the modelling sections, but when handling experimental data it means that they ignore key information that would be useful for identifying selection vs contamination or reinfection, an issue that comes up several times in the paper. Specifically, where multiple synonymous variants could give the same aa, the detection of a novel nt sequence encoding the original aa would suggest selection for that aa, in a way that detection of the original nt sequence would not. And a separate point: when describing changes in genotype frequency and detection of mutations, a statement of the depth of coverage would be helpful, as would an assessment of the intrinsic error rate of the NGS used.(2) Which virus is interesting? The paper focusses most of its content on discussing work on A/England/195/2009 before concluding that subsequent changes in genotype render the fitness compensation constellation that had been studied irrelevant to more recent pH1N1 viruses . This is an important point, and the authors have my sympathy as it was probably very frustrating to discover. The reader is currently left wondering: ‘now that they’ve found this effect in a virus that is no longer circulating, what might it mean for viruses that are still out there?’ How best to address this is a balance of effort vs impact that should probably be decided in discussion with the editor. I can see that it could be ethically and financially difficult to justify repeating the ferret studies with other viruses. However, it would presumably be entirely feasible to consider the effects of fitness compensation on different backgrounds using a version of the model in Fig 3. It would also be feasible to repeat Fig 4 on a wider range of genetic backgrounds – indeed, the authors have already done much of this work in Fig 4 of their earlier study . One way or another, the paper needs to find a way to say more about viruses that people ‘care about,’ even if only indirectly.(3) What is the model saying? I am not a specialist in modelling but I found a number of aspects of the model troubling and would like to know the author’s reasoning; I suspect most readers would too.To begin with, it would be helpful to have a bit more background about the scenarios under which favipiravir resistance would be expected to spread ‘in the wild’ – perhaps by expanding the introductory section on the spread of resistance to previous anti-influenza drugs. Influenza is an acute infection, and favipiravir would be expected to be administered to severely ill patients only in a hospital setting. Even if the drug was used more widely, there are no reasonable circumstances in which the majority of human-to-human influenza transmission events in a human population would occur between favipiravir-treated individuals (in this sense it is very different from evasion of adaptive immunity). There are therefore a limited set of scenarios in which resistance could spread , for example if the resistant constellation had little or no fitness cost in the absence of treatment, or if prophylactic treatment was excessively used in e.g. susceptible livestock. A brief but clear articulation of the selective pressures under which favipiravir resistance might be expected to spread is key context for the entire paper, but is currently missing.Moving on to the model itself, we have Figure 3, which presents four scenarios all with starting composition 95% (K229R + P653L) to 5% WT:(a) Relative fitness: P653L (1.25) > WT (1) = K229R+P653L (1) >> K229R (0.01). Reassortants generating K229R (653P) are lost rapidly. Reassortants generating P653L (229K) have a fitness advantage and slowly outcompete the starting WT and K229R+P653L forms. At generation 20 nearly all the population carries 653L, and 229R has declined from 95% to c.60%.(b) Relative fitness: WT (1) = P653L (1) = K229R+P653L (1) >> K229R (0.01). Reassortants generating K229R (653P) are lost rapidly. Reassortants generating P653L (229K) are in equilibrium with K229R+P653L. WT would be in equilibrium too without reassortment but, due to the large starting amount of K229R + P653L, in any reassortant event WT is likely to mix with a double mutant and so be lost. At generation 20 nearly all the population carries 653L, and 229R remains at 95% but is now largely on a PA 653L background.(c) Relative fitness: P653L (1.25) > WT (1) = K229R (1) = K229R+P653L (1). Reassortants generating K229R (653P) do not have a fitness disadvantage but are rare events due the starting composition; they behave much as WT in (b). Reassortants generating P653L (229K) have a fitness advantage and exponentially outcompete all other species much as in (a). At generation 20 nearly all the population carries PA 653L (slightly less than in (a)), and PB1 229R, now largely on a PA 653L background has declined from 95% to c.60%.(d) Relative fitness: P653L (1.25) > WT (1) = K229R+P653L (1) >> K229R (0.01); no reassortment. Here the starting composition of K229R+P653L and WT is in equilibrium, and the single mutants can only be generated by spontaneous mutation at a much lower rate. Where this happens K229R (653P) is rapidly lost, and P653L (229K) once it appears begins to outcompete the other species with similar eventual kinetics as in (a) and (c) but from a much lower starting position. At generation 20 the starting composition is only slightly changed, but if you ran this out to 60 generations the eventual increase of P543L (229K) would presumably end up displacing 229R from the population.I wouldn’t normally write this out in this much detail in a review, but I want to do so here for three reasons.First, it took a great deal of work for me to work all of this out (most of an evening), and until I had done so I was entirely unable to make any sense of Figure 3. It should have been evident from the way the paper was written - the explanation needs to be clearer.Second, note that in the summary above the fitness effects of K229R and P653L are strictly independent. The conclusion that ‘the fitness advantage of the PA P653L single mutant was necessary and sufficient to explain the loss of the R229 PB1 resistance mutation’ (lines 211-3) is not obvious to me. Rather, the models suggest to me that (as is intuitively obvious) K229R is lost because it carries a fitness cost, and that if a large proportion of double mutants are present in the initial mix, they will eventually dominate all reassortment events.Third, the outputs of these models are clearly dependent on arbitrary parameters, as any model would be. The authors claim that their sensitivity analysis shows that the model outputs are ‘robust to changes in values of the initial variables’ (line 221). This is simply incorrect . The variable fWM, currently 1.25 (a 1.25-fold increase in fitness), behaves radically differently if increased by just 20% to 1.5. The variable fMW, currently 0.01 (a 100-fold decrease in fitness) is not tested at all, and nor is pMM  both of which I would argue have a major effect on the model outcomes. A much more thorough and explicit sensitivity analysis is needed, exploring model outcomes over a range of plausible values. Indeed, this could add to the value of the paper, as it would allow some consideration of what might happen in a favipiravir-treated patient where the relative fitness of WT PB1 will be, by definition, depressed, as well as considering other scenarios (see above).**********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) In Figure 3, the authors provide a mathematical model of their findings. The model is consistent with the authors results; however, caution needs to be taken when interpreting this model. The model is derived from a single set of parameters in which ferrets were inoculated with a single mixture of viruses at a ratio of 95% mutant viruses to 5% wild-type. The model would benefit from an additional set of data inputs, as this would validate the findings. This is beyond the scope of the manuscript, but the authors should discuss the limitations of using a single set of parameters to develop the model or provide justification for why a single set of parameters is sufficient.Reviewer #2: 1. Line 156 refers to a low number of mutations in recipient ferrets and surmises that their prevalence may be due to transmission bottlenecks. Please elaborate. Were these few mutations present at high frequencies? The prior sentence states that no mutations were found in the pol genes. Were the mutations referred to in other genes? Did their frequency increase over time in the recipient ferrets (evidence of positive selection)?2. In the description of the baseline model (ca. line 196), a fitness value is given for only one of the four viruses. Please indicate parameters used for all viruses here. Did the values used correspond quantitatively with the data cited in ref 17? At this location in the text, it would also be helpful include basic information on how MOI was determined and what assumptions were used to govern reassortment rates - or at least refer to the appendix/methods here.3. In the appendix:A. Please add references indicating the sources of default parametersB. Please include results obtained with the default parameters for comparison.C. The default parameter for viral load seems low. Is the model sensitive to changes in this parameter?D. How would a higher MOI impact results? 4. This is not absolutely necessary in my opinion, but to really round out the paper, it would be great to see a second  ferret experiment done in the Eng687 background to test the prediction that the evolutionary dynamics of the K229R resistance mutation depends on strain background.5. The discussion nicely discusses the results of the present study but could do more to put these results in context relative to the literature.Reviewer #3: The arguments in the paper can often be quite hard to follow, something which would be helped considerably by a clear concluding statement at the end of each results section . The discussion of Figure 3 is currently ‘upside down’ (the text refers to the frequency of K at PB1 229 while the figure shows the frequency of R).Missing references: Line 100-2: ‘In humans so far…’ Line 302 ‘not being structurally close to the active site’ .Line 103-4: ‘equivalent mutation’ please explainLine 140: ‘shortly after infection and a later time point’ – please explain more clearly. It appears to be after the first increase in titre and at the last time point before the final fall in titre, but this isn’t made clear.Line 143: ‘high levels’; line 156 ‘low number’, line 197 ‘a fitness advantage’ – please quantify (at least approximately)Line 144 -6: ‘indicating that the K229R + P653L virus … was efficiently transmitted…’ given the (reasonable) choice to stack the deck in favour of the mutant virus the fact that it was transmitted does not actually say much about how efficient this was.Line 156-9: if ‘bottlenecking’ could have removed low-frequency mutations, why is this not also a reasonable explanation for the loss of the WT genome segments?Line 159: ‘pattern of repeated mutations’ meaning unclear (to me)Line 197 (and elsewhere): I believe that the term ‘fitness’ here should more properly be ‘relative fitness’Why is Figure 5 in the discussion rather than the results and why is only group 1 shown?Line 303-5: ‘This might imply… cost.’ Meaning unclear (to me). Due to compensation?Lines 322-4: ‘Given that… mutations.’ Reasoning unclear (to me).Line 340: date of download and number of sequences downloaded requiredLine 341: please briefly explain how the alignment was performed and give the accession number (or other source) of the reference sequence.Line 385-6: ‘mean proportional to the summed fitness’ – summed (as opposed to mean) fitness implies that a cell infection with, say, 5 viruses should produce 5 times more progeny than a cell infected with one virus. Is this what was meant? If it is – given that high MOIs of IAV tend in practice to increase the rate of virus production rather than the maximum yield – does this not skew the model excessively to considering reassortant events? Line 393: units for the mutation rate are missing, and there appears to be an additional superscripted term. Line 544: how many cells for this amount of DNA?Figure 4 legend: What is N? What measure of central tendency and variance has been plotted?Line 557-8: Suggest removing interpretation from a figure legend.Figure 1: the black outlines are hard to distinguishAppendix: Keys are missing for the colours in the figures.**********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.. 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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,Seema Lakdawala, PhDReviews EditorPLOS PathogensSara CherrySection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064***********************************************************The authors have successfully address the critiques raised in the first submission. In particular the expansion of the appendix to describe the sensitivity of the model was an extensive addition as was the additional experiments on polymerase activity. We are pleased to accept the revised submission with these additions.Reviewer Comments : 26 May 2021Dear Dr Goldhill,We are delighted to inform you that your manuscript, "Favipiravir-resistant influenza A virus shows potential for transmission," 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. 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Bunyavirales), is an endemic viral infection of the wrinkle-lipped free-tailed bat (Chaerephon plicatus aka Tadarida plicata plicata). Little is known about the ecology and maintenance of KKV within the bat population, nor the infection dynamics and transmission among bats or between bats and other vertebrates. Therefore, KKV was studied in Kaeng Khoi cave, Saraburi province, Thailand, during 1973–1974 with the objectives to (1) characterize the seasonal infection rates of KKV in the context of the bat population ecology, and (2) describe the infection dynamics and viral shedding by naturally- and experimentally-infected bats. To this end, the free-tailed bat population was estimated by a series of timed photographs taken during the evening exodus. The case population of 900,000 adult bats doubled at the time of weaning of the young and returned to its previous level soon thereafter. The newborn bats had neutralizing antibodies to KKV that were likely to be maternal in origin. The KKV antibody prevalence in adult bats was high (69–91%) in March–May and low (29–40%) in August and September. Kaeng Khoi virus was isolated from 75% of dead and 50% of moribund bats, but was not found in nearly 400 apparently healthy bats. Virus was present in saliva, urine and blood of most of the naturally-moribund bats tested. Consistent with observations from naturally-infected bats, experimental infection of bats with KKV revealed significant liver pathology, also suggestive that this is not a benign infection. Kaeng Khoi virus is an endemic, year-round infection maintained by the annual recruitment of a large number of immunologically-naïve juvenile bats. Moreover, it produces an acute infection in the bat, either leading to death by hepatitis, or immunity.Kaeng Khoi virus (KKV; Order:  Chaerephon plicatus; aka Tadarida plicata plicata),in Saraburi province, Thailand in 1969 [Bunyavirales, family Peribunyaviridae; it clusters phylogenetically with Mojuí dos Campos virus (MDCV) which was isolated from an unknown species of bat in Brazil in 1976 [Chaerephon plicatus) in Cambodia [Eucampsipoda sundaica) associated with rousette bats in the Yunnan Province of China [Kaeng Khoi virus (KKV) was first isolated from a wrinkle-lipped free-tailed bat  was the most numerous of the vertebrates with a population approaching one million (see Results). The population of tomb bats  was much lower and was estimated to be less than 100,000 individuals.The Kaeng Khoi cave is located 180 m above the valley floor on the steep western face of Khao Lorn Phat, a mountain which forms part of the eastern border of the valley . The cav2 (6.25 × 4.2 m2 exposure plane), with a depth of field of approximately 7.5 m to infinity. The distance of 9.l m between the camera and the bats in the exodus remained essentially constant because of the structural limitation of the cave entrance. The cross-section of the exodus at the exposure plane varied in dimensions (1–3 m) as the number of bats in the exodus increased and decreased. Exposures (Tri-X film at ASA1000 film speed) were started when the first bats left the cave and were made at 30 s intervals for the first 7–10 min and thereafter at one- or five-min intervals. Each exposure of a photographic exodus series  from rooms 1 and 2 . They weBlood from adult bats was drawn by cardiac puncture with 2.5 mL disposable syringes. To obtain blood from newborn bats, a skin pouch was made around the axillary artery. The artery was then cut and the blood collected in the pouch was withdrawn by syringe. Whole blood from bats was diluted 1:2 (sera 1:4) at the time of bleeding by drawing the blood into syringes containing appropriate volumes (0.5 mL for adult and 0.25 mL for newborn) of sterile tissue culture growth medium 199 (GM). Oral swabs from bats were collected by massaging the oral cavity with a cotton swab. Urine was collected from handheld urinating bats with Pasteur pipettes. Oral swabs and urine samples were diluted in l.0 mL GM. All specimens were stored on dry ice at the field station until their transfer to the main laboratory, where the sera were stored at −20 °C. The blood clots, oral swabs, urine samples and animal bodies were stored at −60 °C.5 cells/mL in GM. GM contains medium 199 with 1.4 g Na HCO3 per liter, and was supplemented with 10% heat-inactivated fetal calf serum, 100 units penicillin/mL and 10 μg streptomycin/mL. Tissue culture tubes and 1 oz prescription bottles were seeded with 0.9 mL and 4 mL of cell suspensions, respectively, and incubated for four days at 37 °C prior to inoculation.Vero cell cultures (African green monkey kidney) and/or Swiss mice obtained from the Southeast Asian Treaty Organization (SEATO) mouse colony were used in virus isolation attempts. Vero cells were propagated in Blake bottles. Monolayers were removed with typsin-versene, resuspended to a final concentration of 10g for 30 min at 4 °C. The supernatant was withdrawn and divided into two parts; one portion was frozen at −60 °C for re-isolation attempts, and the other one was inoculated intracerebrally (i.c.) into a litter of newborn Swiss mice (0.02 mL per mouse) and/or into 3 tubes of Vero cells (0.1 mL/tube). Urine, oral swabs and tissues from moribund bats were inoculated into Vero cell cultures only. Drained cell culture tubes were inoculated with 0.l mL of supernatant and incubated for l h at 37 °C. After adsorption, maintenance medium (1 mL) was added. Maintenance medium (MM) was GM supplemented with 5% fetal bovine serum. The inoculated cultures were incubated at 37 °C, and were examined for virus-induced cytopathic effect (CPE) over a period of 14 days. Supernatants from tubes showing CPE were sub-passaged. Maintenance medium was changed every third day. Inoculated mice were observed for 15 days. Brains of those found sick or dead were harvested and sub-passaged by intracerebral inoculation of mice. Virus isolates were identified by neutralization (N) tests, using Kaeng Khoi hyperimmune rabbit serum or mouse ascitic fluid. These tests were done with constant serum and varying virus dilutions.Individual tissues were triturated in sterile, 7 mL Ten Broeck tissue grinders containing 2 mL GM. The resultant suspension was centrifuged at 9750 3 solution (7.5% stock); 1.8 mL 100× penicillin-G and streptomycin; and 3 mL neutral red (1: 1000). Drained tubes were inoculated with 0.1 mL of serum-virus mixture. After absorption at 37 °C for 1 h, 1 mL of MM was added to each tube. Bottles and tubes were incubated at 37 °C and observed, respectively, for plaques and CPE on day five. A plaque reduction of 80% or more and no CPE in tubes were the criterion for a positive test.Neutralization tests for antibodies against KKV were performed by plaque reduction or in tube cultures using Vero cell monolayers. Sera were inactivated at 56 °C for 30 min and diluted with GM. Virus was diluted to give an effective virus dose of 100 TCID 50 (50% tissue culture infectious dose) or 50–180 p.f.u. (plaque forming units). A 0.4 mL volume of each serum dilution was mixed with an equal volume of an appropriate virus dilution. The serum–virus mixture was incubated for one hour at 37 °C. Following incubation, two drained 1 oz. prescription bottles were inoculated, each, with 0.3 mL of serum-virus mixture. After adsorption at 37 °C for 1 h, 8–10 mL of overlay medium was added to each 1 oz. bottle. The overlay medium consisted of equal volumes of sterile 2% Ionagar No.2  with an inserted hanging screen. Bats were hand fed by a 2.5 cc syringe. A diet with the following ingredients was prepared in a Waring blender with a small amount of tap water: hardboiled egg yolk, cream cheese, and banana, each one part by weight, and ants, substituted for meal-worms, one half part by weight. The diet was fed once daily, approximately 2 cc per day.The free-tailed bats used for experimental infection were collected from the Kaeng Khoi cave and were caged individually in cardboard boxes , and declined to about 900,000 in late June . These vThe recorded population doubling at the time of weaning is what would be expected, since others  have shoSera from bats of four age groups were tested for neutralizing antibodies to KKV. Sera from one-to-seven day old neonates and from reproductively active females were collected during the same time period (mid-April 1973) for a comparison of antibody titers and prevalence in the two groups. Most of the neonates had attached placentae. Further, the cohort born in mid-April was resampled in mid-May and in late June to obtain sera from 32–39 day old bats and 73–80 day old bats, respectively. Additionally, milk from ten seropositive lactating bats was obtained in late June to test for the presence of neutralizing antibodies to KKV.Tadarida is chorioallantoic [The antibody prevalence in both the neonates and the reproductively active females was 84% and the distribution of antibody titers of positive sera was also comparable in these two groups . These fllantoic  a placenllantoic . The tenSera from a total of 253 adult bats collected at different times of the year in 1973 were tested for prevalence and titers of neutralizing antibodies to KKV. The results are given in In the earlier study  Kaeng KhTissues were collected from 24 moribund bats in rooms 1, 2, and 3 during January, February and March 1974 and examined for virus by inoculation of Vero cells. Virus was recovered from pooled tissue suspensions of 12 (50%) moribund bats . Kaeng KA histological examination of the tissues of morbid bats showed that viral infection was associated with a massive destruction of the liver parenchymal cells . A grossTissues of a total of 324 bats, including 68 bats less than nine weeks old, were collected over a seven month period and tested for KKV by inoculation into both in Vero cell cultures and suckling mice . No isolVirus isolation from dead and morbid bats suggests that KKV produced a marked systemic infection and some mortality, and that KKV could disseminate from an infected bat through saliva, urine and/or by a hematophagous arthropod. The lack of isolation from the apparently healthy bats suggests that the virus typically produces an acute, rather than a chronic infection.The pathological lesions produced in bats infected with KKV were studied by three approaches. Histopathological examination was performed on (i) tissues from naturally infected morbid bats from Kaeng Khoi cave; (ii) livers from experimentally infected bats; and (iii) livers from experimentally infected suckling mice. The tissues were fixed in buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for histopathological examination.Twelve of 24 morbid bats had naturally acquired KKV infection. The highest KKV titers were consistently found in the liver and the lung . Of thesA consistent feature of the pathological process was the absence of an inflammatory infiltrate. Intranuclear and intracytoplasmic inclusion bodies were not seen. All hepatic lesions observed were well-advanced and were probably responsible for the mortality and morbidity in the infected bats. Livers of the virus-negative morbid bats were essentially normal .Lung pathology was observed regularly in KKV-positive and KKV-negative bats. These lesions varied from a mild focal hemorrhage to a severe acute interstitial pneumonia. Hyalinization of the pulmonary vascular walls was observed in ten of the 16 bats examined, and was severe in two cases. Interstitial pneumonia was more common in the virus-infected bats  than in The massive liver destruction in morbid bats naturally infected with KKV and the high titer of virus in the livers indicated that KKV produced an acute hepatitis in wrinkle-lipped free-tailed bats. An attempt was made to confirm this relationship by experimental infection of normal free-tailed bats acquired from Kaeng Khoi cave.4.0·TCID50 (per ml of blood or gram of tissue).Data on 35 experimentally infected bats are summarized in 4 TCID50 of virus per gram, while the blood had no detectable virus. Of the five bats inoculated i. c. with 2 × 102 SMLD50, livers from four were harvested for virus titer on days 4–6 post inoculation. All four livers were positive for virus and contained 6.2, 6.3, 7.2 and greater than 8.1 log10 virus per gram of liver.Nonimmune bats appeared to be more susceptible to i.c. than to i.p. inoculation. In two cases, bats which were viremic on days 2 and 3, had developed antibodies by day 7; on day 7, the tissues had over 10Pathological lesions characterized as acute multifocal hepatitis  were obsThese data suggest that KKV is hepato-trophic for bats and that naturally acquired immunity protects the immune bats against a second systemic infection.2 SMLD50 i.c. were harvested when moribund, 5–7 days post inoculation. All manifested a massive liver cell destruction similar to that observed in the virus-positive morbid bats (data not shown). The two control mice inoculated i.c. with virus diluent exhibited unremarkable livers.A litter of six suckling mice inoculated with 2 × 10Several important components of the epidemiology of KKV infection of wrinkle-lipped free-tailed bat inhabiting Kaeng Khoi cave were studied during 1973–1974. The data presented support the natural circulation of KKV in free-tailed bats at a high prevalence, driven by the integration of immunologically-naïve juvenile bats into the population after maternal antibody protection waned. Importantly, infection of these bats with KKV did not appear to be benign; isolation of KKV was only from moribund or dead bats, and experimentally-infected bats developed pathogenic lesions in the liver and lungs. Virus isolations of KKV were also only made from dead wrinkle-lipped free-tailed bats in Cambodia, with no isolations from apparently healthy bats [The April birthing period was estimated to be about 900,000 bats or an approximate doubling of the cave population. Although the accuracy of the estimates is difficult to assess without knowing the true population values, the method did record a doubling of the population in June, when these juvenile bats would have become volant . AlternaSerological results of the sampled newborns showed that 84%  of pups had neutralizing antibodies to KKV . These aPteropus hypomelanus bats vaccinated against canine distemper virus (CDV), and Hendra virus antibodies in wild-caught P. alecto bats [Eidolon helvum) exposed to Lagos bat virus or African henipa-virus was about six months, with a low point in seroprevalence at 11–12 months [The high antibody prevalence rate in the newborn bats may be expected to protect the seropositive young from systemic KKV infection. The length of time maternal antibodies would protect the newborns would depend upon the half-life of the IgG immunoglobulin of bats, the initial titer of the antibodies and perhaps the dosage of KKV that the bats are exposed to in the cave. It is not known at what time after birth the bat would become susceptible to KKV infection. The cohort of baby bats born in mid-April maintained a high antibody prevalence of 77% and 71% at 32–39 and 73–80 days of age, respectively , but witcto bats . In both2 months . The wan2 months ,21,22,23The antibody prevalence rates of the adult bats sampled from March 1973 through September 1973 are difficult to interpret because reliable methods are not available for the estimation of the age of the free-tailed bats older than about four months. Further, the movement patterns of these bats among caves and other roosting areas are unknown, so it is difficult to appreciate whether or not the samples taken at different times of year represent the same group of individuals. However, it is likely that the presence of the reproductively active females accounted for the high antibody prevalence observed in March, April and May and that the low antibody prevalence rates found in August and September reflect the loss of maternal immunity by the cohort of bats born in mid-April .Of the 136 bats initially included in the three experimental groups , 35 batsBunyavirales characteristically produce liver destruction, including many viruses in the family Peribunyaviridae: Caraparu (group C) [Phleboviridae: Rift Valley fever virus [Nairoviridae: Crimean Congo hemorrhagic fever virus [Hantaviridae: hemorrhagic fever with renal syndrome (hantaviruses) [The only characteristic lesion found in naturally infected moribund bats was an acute massive destruction of the liver parenchymal cells. All of the moribund bats found positive for KKV had hepatic lesions which were well advanced and were probably responsible for the mortality and morbidity in the infected bat . Livers group C) , Patois group C) , Guama [group C)  Oropouchgroup C) ; Phlebover virus ,29,30; NThe high isolation rate of virus from sick and dead bats and the lack of isolation from healthy bats, together with the severe hepatic lesions in the sick bats, strongly indicate that KKV infection produces mortality and that it may be responsible for as many as 50–75% of the deaths occurring in the cave . These oIn 1969 an attempt was made to estimate the number of bats dying in the cave. All dead bats found on the floor of the cave were collected three times a day, for one week of each month, for twelve months. The highest number of daily deaths for any one month did not exceed 30 bats, giving a maximal estimate of annual deaths in the cave of about 11,000, or between 1–2% of the population in the cave. This estimate seems to be very low, and suggests that a large majority of deaths are occurring outside the cave. However, the age structure and movement patterns of the bat population in the cave during this period of time were not known and, therefore, this estimate only indicates the number of deaths occurring in the cave and cannot be interpreted as a mortality rate or to indicate what proportion of this mortality outside the cave is due to KKV. The significance of mortality due to KKV on the bat population warrants further investigation.Although there is no good basis to calculate the mortality rate in bats infected with KKV, the large numbers of bats with immunity and the high proportion of newborns with maternal immunity suggest that the mortality due to this infection may be lower than indicated by the isolation rates from dead and moribund bats.The population of bats in the Kaeng Khoi cave was estimated by a timed photographic series of the evening exodus. The initial population of between 700,000 and 900,000 bats (between March and May 1973) nearly doubled in early June and declined to its previous level in late June. The increase in number was due to the recruitment of bats born in mid-April into the foraging population.Neutralizing antibodies to KKV were probably transferred from mother to the newborn. The antibody prevalence in adult bats was high in March–May 1973 and low in August and September 1973.Kaeng Khoi virus was isolated from 75% of dead, 50% of moribund and no isolations were made from healthy bats. In moribund bats, KKV was present in all tissues examined, with the highest titers in liver and lung tissue. Virus was routinely isolated from the blood, urine and saliva of infected bats.The characteristic pathology in naturally infected moribund free-tailed bats was an acute massive destruction of liver parenchymal cells. Experimentally infected bats and suckling mice also exhibited liver lesions. Bats with naturally acquired antibodies resisted the Kaeng Khoi virus challenge.This manuscript describes a series of field- and laboratory studies that collectively provide important, novel information regarding the viral ecology of KKV. Investigations into the population ecology and seasonal reproductive phenology of this bat population were highly informative to the circulation and maintenance of KKV. Importantly, bats appear to be susceptible to KKV, with significant liver pathology noted in dead and moribund bats infected with KKV. Specific key findings are summarized below:


After establishing planktonic inhibition, antifungal CS beads were introduced to fungal biofilms (n = 5) to assess biofilm formation and cell viability through a combination of standard quantitative and qualitative biofilm assays. Inoculation of a hydrogel substrate, packed with antifungal CS beads, was also used to assess diffusion through a semidry material, to mimic active infection in vivo. In general, antifungals released from loaded CS beads were all effective at inhibiting the pathogenic fungi over 7 days within standard MIC ranges for these fungi. We observed a significant reduction of pregrown fungal biofilms across key fungal pathogens following treatment, with visually observable changes in cell morphology and biofilm coverage provided by scanning electron microscopy. Assessment of biofilm inhibition also revealed reductions in total and viable cells across all organisms tested. These data show that antifungal-loaded CS beads produce a sustained antimicrobial effect that inhibits and kills clinically relevant fungal species in vitro as planktonic and biofilm cells.Calcium sulfate (CS) has been used clinically as a bone- or void-filling biomaterial, and its resorptive properties have provided the prospect for its use as a release mechanism for local antibiotics to control biofilms. Here, we aimed to test CS beads loaded with three antifungal drugs against planktonic and sessile fungal species to assess whether these antifungal beads could be harnessed to provide consistent release of antifungals at biofilm-inhibitory doses. A panel of different fungal species ( While total joint replacements are among the most successful medical procedures currently undertaken, with good survivorship and low morbidity and mortality , 2, the Calcium sulfate (CS) has been used as a biomaterial for clinical applications for many years –10. It h–15–Candida spp., other yeasts and filamentous fungi have been increasingly associated with biofilm infections  is preferentially used in the management of fungal PJIs and is distributed through oral dosing and subsequent absorption through the gastrointestinal system . Due to efficacy . Amphoteefficacy . Clinicaefficacy . Echinocefficacy , 32.In this study, we assessed the mixing of these antifungal agents with CS. In addition, we assessed whether when these antifungal agents are loaded into CS beads, they have the capacity to inhibit and kill a panel of pathogenic fungi growing as planktonic and biofilm phenotypes. Here, we show for the first time that antifungal agents loaded in CS can successfully control fungal biofilms.Candida auris and Candida tropicalis displayed little to no inhibition  after 24 h for CSP- and AMB-loaded beads compared with control beads , by an average of 76% . In C. auris, averages of 92, 99, and 74% reductions in viability were recorded for CSP-, AMB-, and FLZ-loaded beads, respectively. FLZ also recorded drops in viability by an average of 64% for C. tropicalis and 16% for A. brasiliensis, despite no reduction in biomass. Despite recorded changes between control and treatments for A. fumigatus, no significance was determined.Across the 5 organisms selected for biofilm treatment testing, there was a significant reduction in biomass , a 95-fold reduction when exposed to CSP-loaded beads (P < 0.0005), and a 7-fold reduction in total cells when exposed to FLZ-loaded beads (P < 0.005). While C. tropicalis incubated with CSP-loaded beads produced a 40-fold reduction, AMB-loaded beads produced a 120-fold reduction and FLZ-loaded beads an 8-fold reduction. C. auris observed a 3-fold drop in total CFE on exposure to CSP-loaded beads (P < 0.005) and a 4.5-fold reduction when exposed to AMB-loaded beads (P < 0.001), with no significant reduction when exposed to FLZ-loaded beads. A. fumigatus also observed a 5-fold decrease in total CFE when exposed to CSP-loaded beads (P < 0.005) and a 15-fold reduction in cells when exposed to AMB-loaded beads (P < 0.001). A. brasiliensis was not significantly reduced when drug-loaded and control beads were compared. Following these analyses, we provided a comparison of the percentage of live cells compared with the total number of cells present in biofilms exposed to CS beads  values for each fungal isolate were acquired and compared with those of an unloaded control bead to observe drug differences . The greCS beads . These ded beads , the majC. albicans producing robust biofilm with clear filamentation in the unloaded control sample but obvious destruction of biofilm and malformation of cellular structure against all antifungal-loaded beads , reinforcing the atypical resistance profile seen in previous data  of biofilms exposed to antifungal beads showed that across all tested organisms, CSP- and AMB-loaded beads produce a pronounced visual effect on the developed biofilm that supports our quantitative analysis . Visualied beads . C. auriubstrate . C. tropous data . A. fumiM of biofC. albicans (P < 0.0001), 18-fold in C. tropicalis, and 17-fold in A. fumigatus, as well as a 7-fold reduction in C. auris and an 8-fold reduction for A. brasiliensis (P < 0.0005), in samples containing AMB-loaded beads compared with control beads. CSP-loaded beads also indicated significant reductions (P < 0.0005) for C. albicans (33-fold), C. tropicalis (5-fold), A. fumigatus (3-fold), and A. brasiliensis (6-fold), while C. auris observed a diminished, though still significant, 2-fold reduction (P < 0.005). For samples exposed to FLZ-loaded beads, A. brasiliensis showed no significant reduction to CFE, A. fumigatus displayed an 8-fold reduction in CFE compared with the control (P < 0.0001), while significant reductions were also recorded for all other organisms. When assessing the viability of cells present in each sample  and are found to be largely consistent with values found expressed in other literature –37. Wher–ed beads . Additio our own . Values  our own –40. Indeour own –. FurtherC. albicans biofilm susceptibility to AMB was recorded at a concentration of 8 μg/ml  for total cells present in a sample and specifically for live cells present in a sample. This provided a much clearer representation of how effective drug release from CS beads was at diminishing biofilms of each organism. Indeed, significant fold reductions were seen for almost every organism. Diminished sensitivity to treatment in sistance , 53. Addocandins , 54.in vivo growth conditions that may have developed over time, such as those of the chronic wound under conditions like diabetic foot ulceration (DFU). While this also addresses the concern of prophylactic prevention of biofilm formation under conditions such as PJI, where CS beads have been investigated clinically  agar  and refrigerated at 4°C prior to proliferation in yeast-peptone-dextrose  in a 200-rpm shaking incubator overnight at 30°C for Candida, Malassezia, Trichosporon, and Rhodotorula species. Spores of the pore-forming fungi Aspergillus, Rhizopus, and Scedosporium were harvested using a previously described method for storage at 4°C . Cells and spores were then counted using a Neubauer hemocytometer and standardized to a working concentration for further experimentation.A panel of 15 fungal species comprising e at 4°C . Spores A prepackaged kit containing 9.6 g of pharmaceutical-grade CS α-hemihydrate powder (CS-CSH)  was mixed with 3.1 ml of deionized water to create unloaded CS beads. To prepare antifungal-loaded beads, an additional 35 mg of CSP acetate powder or 80 mg of FLZ powder was added to the CS-CSH mixture prior to mixing. For AMB, 25 mg was added to a CS mixture with 3.5 ml of 0.9% saline solution. Drug concentrations were selected primarily based on daily dosage of intravenous drugs (CSP and FLZ) or the maximal concentration of drug that could be added to CS-CSH while still allowing it to fully set (AMB). These combinations were then mixed for approximately 30 s until a smooth paste had formed and then spread into a 6- or 3-mm pellet mold and allowed to harden for a minimum of 30 min, until set, under aseptic conditions. The beads were then weighed in order to determine their antifungal load. The 6-mm AMB, CSP, and FLZ beads were averaged at 0.143, 0.138, and 0.127 g, respectively. The antifungal loads per bead were determined to be 0.56 mg of AMB, 0.38 mg of CSP, and 0.79 mg of FLZ.4 CFU/ml, and fungal spores were standardized to 2 × 103 CFU/ml, as previously described, and broth microdilution testing was performed on each antifungal supernatant according to Clinical and Laboratory Standards Institute (CLSI) guidelines for yeasts and spores  over a course of 7 days, with supernatant removed daily to assess the effective concentration required to inhibit planktonic growth as a proxy measure for antifungal release. There were 7 different supernatants, representing each of the 7 days, for each antifungal agent. For time points not being immediately assessed, supernatant was changed daily to evaluate any drift in concentration across the 7 days. Fungal cells were then standardized to 2 × 10d spores . Effecti6 CFU/ml, and spore-forming organisms were standardized to 1 × 105 CFU/ml according to standardized methods  at 1-, 3-, and 5-day time points alongside 6-mm antifungal-loaded CS beads of FLZ, AMB, and CSP and an unloaded control bead. Following the incubation period, biofilms were washed 3 times with PBS and then fixed in a solution of 2% glutaraldehyde, 2% paraformaldehyde, 0.15% alcian blue powder, and 0.15 M sodium cacodylate. Next, this was counterstained with uranyl acetate for 1 h, and a gradient dehydration from 30% to 100% ethanol was performed. Finally, a critical point drying step was performed in hexamethyldisilazane (HMDS) in accordance with a previously described protocol . SamplesCTCGTAGTTGAACCTTGGGC-3′ and reverse primer 5′-GGCCTGCTTTGAACACTCTA-3′), and CFE were extrapolated from threshold cycles (TC) using the 2−ΔCT method and quantified using the 2−ΔΔCT method, outlined as described previously (3 to 1 × 108 CFU/ml by quantitative PCR (qPCR) was then undertaken , with half of the samples set aside for total cell qPCR and the other half treated with propidium monoazide , a DNA intercalating dye that renders DNA not within a membranous structure as insoluble and prevents its detection during PCR –72. In b–eviously . A standdertaken . The fol6 cells/ml, and incubated for 2 h at 30°C in a shaking incubator set to 200 rpm to allow fungal cell and spore adherence. These inoculated matrices were then placed upon a “bed” of 4- by 3-mm antifungal-loaded CS beads and unloaded control beads and incubated for 24 h. After incubation, the cellulose matrices were transferred to 5-ml Bijou bottles containing 1 ml of PBS, before being sonicated to remove adhered cells. A PCR-based quantification of adherent fungi was carried out as described above.To assess whether loaded CS beads could inhibit biofilm growth within more translatable environmental parameters than those of a traditional plate model, an alternative hydrogel model was used that was augmented from our previously published wound model . This coP < 0.01.Statistical analyses and graph production were performed using GraphPad Prism . Ordinary one-way analysis of variance (ANOVA) was applied to compare means of multiple drug effects among fungal samples. CFU and CFE/ml were log transformed before statistical analyses took place. Statistical significance was achieved at


The aim of this study was to evaluate whether high surgical volume at a single center was associated with lower healthcare costs compared to lower surgical volume in a multicenter setting.All patients had symptomatic and anatomical apical prolapse (POP-Q ≥ stage II) with or without cystocele and were operated on by a standard surgical procedure using the Uphold mesh. Data on time of resource use in terms of surgery time, hospital stay and re-interventions across 5 years were compared between the single center (97 patients) and multicenter . Unit costs for surgical time, inpatient and outpatient visits were extracted from the single-center hospital’s operation analysis program and prime production cost. Total costs were estimated for primary surgery and during 5-year follow-up.P = 0.29). Follow-up costs 5 years after primary surgery were 2.8 times higher at the multicenter than single center . Mean cost per patient over 5 years was significantly lower at the single than multicenter .Costs for primary surgery were comparable between the single and the multicenter ($13,561 ± 2688 and $13,867 ± 1177, Using a mesh kit for apical pelvic organ prolapse in a high surgical volume center was associated with reduced healthcare costs compared with a lower volume multiple-site setting. The cost reduction at the high surgical volume center increased over time because of lower surgical and medical re-intervention rates for postoperative complications and recurrence. Lite mesh kit to suspend the apical vaginal segment have shown the procedure to improve pelvic organ prolapse symptoms and have high effectiveness in restoring the vaginal anatomy , whereas general anesthesia was more commonly used at the multicenter . Operating time was longer at the single center  probably because of the higher rate of previous prolapse recurrence surgery . Hospital stay was shorter at the single center .Patients medical, socioeconomic and surgery characteristics are illustrated in detail in Table P = 0.922]. The overall time at the operation department (operation theater and recovery unit) was comparable between the single and the multicenter . Furthermore, no significant differences in the overall time at the operation department were detected when comparing spinal  to general anesthesia  for the single and the multicenter, respectively, indicating that differences in mode of anesthesia do not appear to drive differences in resource use related to surgery time when comparing low-volume sites with the high-volume site. The only significant observation detected was the comparison of overall time at the operation department between spinal and general anesthesia at the multicenter .Operation and recovery time by type of anesthesia  at the single center is presented in Table P < 0.001). Costs for interventional anesthesia before and after surgery, including equipment and drugs used, were lower at the single center . Despite these differences, there was no significant difference between centers when the total operation theater  and recovery unit costs  were compared. Cost for hospital stay was higher at the multicenter than single center . Total costs for the primary surgery were comparable between the single and multicenter .Disaggregated total costs for primary surgery at the single and multicenter using the Uphold mesh are shown in Table P =  < 0.001)  vs. $13,805  for the multicenter and the single center, respectively (P = 0.054).Follow-up costs for direct medical and surgical re-interventions after primary surgery and up to 2 months were doubled in the multicenter compared to single center use  for the multicenter vs. the single center, leading to a significant increase of total costs at the multicenter . The follow-up cost corresponded to reoperation for prolapse recurrence, n = 7, and mesh revisions, n = 4, at the multicenter compared to mesh revision, n = 1, at the single center.The estimated follow-up costs from 2 months to the next follow-ups  were $1039 vs. $128 (P < 0.001). Additional follow-up costs at 5 years were because of reoperation for prolapse recurrence, n = 16 patients at the multicenter and n = 6 patients at the single center, and hysterectomy because of pain at the multicenter, n = 1 patient. The total follow-up cost across 5 years was $3262 at the multicenter compared to $1149 at the single center (P < 0.001). In detail, mean follow-up cost for medical and surgery interventions including out-clinic and readmission because of surgery complications and mesh related complications was $1001 at the multicenter vs. $372 at the single center (P < 0.001). For reoperation of prolapse recurrence, the mean follow-up cost was $2260 at the multicenter vs. $777 at the single center (P < 0.001). Thus, the mean total cost per patient through 5 years was significantly higher at the multicenter compared to the single center   inversely correlated to primary surgery and total costs at 5 years compared to the multicenter (difference in standardized β-coefficient -1.562).All results of multiple regression analysis on the impact of patient medical, socioeconomic and surgical characteristics on costs by type of site are shown in Table P < 0.001; difference in standardized β-coefficient of -1.883). Also, longer hospital stay in the multicenter setting was associated with an increase of costs of primary surgery and total costs at 5 years . However, shorter operation times in the multicenter setting correlated to lower primary surgery costs and total costs at 5 years . No other characteristics had a significant influence on costs.From the surgical characteristics, more common use of general anesthesia in the multicenter setting was associated with an increase of costs of primary surgery and total costs at 5 years  services have been shown to reduce costs . UndergoAccumulating evidence suggests that the Uphold vaginal mesh may be effective and safe if surgery is done at a high surgery volume unit –7. In thIn search of predictors that may have influenced costs, we performed a multiple regression and stratified analysis of covariances. Only a higher educational level at the single center predicted lower costs of primary surgery as well as total costs at 5 years. In previous studies, the educational level had an impact on patient-reported disease-specific pelvic floor outcomes and was related to improved health-related quality of life compared to lower educational levels , 19. It Strengths of the study are use of real-world data on time and costs recorded at the hospitals, capturing resource use in clinical practice. The study populations were sufficiently large to determine the impact of the most important surgical factors on the costs associated with the procedure. Furthermore, classification of the procedure was homogeneous since all operations used an identical mesh kit and were performed in a standardized surgical manner.Patient and surgeon allocation to high- and low-volume sites was not randomized, suggesting that several unmeasured effects may to some extent constitute a source of bias in estimates of costs associated to each setting. Another limitation was that the number of patients was insufficient for an analysis on the importance of individual experience and skill of the surgeons involved. Also, we did not have access to detailed socioeconomic characteristics of the patients to allow for an analysis of the influence of patient-attributed factors other than educational level. Since it has been argued that centralization should not be based solely on a minimum number of procedures, but rather on the multidisciplinary treatment of complex diseases , future In this long-term multivariate cost analysis study, we found that using a mesh kit for apical pelvic organ prolapse in a high surgical volume center was associated with reduced healthcare costs compared to a lower volume multiple-site setting. The cost reduction at the high surgical volume center increased over time because of the lower surgical and medical re-intervention rates for postoperative complication and recurrence. These results may guide future regulatory and health economic decisions when planning and implementing new healthcare routines in urogynecological surgery.


This article proposes that one should explore whether the pulmonary complications of Covid-19 can be reduced or avoided by bypassing the airway entry of the SARS-CoV-2 virus. This could possibly be achieved by injecting live SARS-CoV-2 virus intradermal (ID), subcutaneous, intra-muscular (IM) or intra-peritoneal (IP), or by targeting the virus to the digestive tract.  The effectiveness and innocuity of using those various routes could be tested very rapidly in animal models, such as Macaques, Hamsters, Ferrets or Cats. The hope is that these experiments will reveal a route of inoculation that can reliably lead to bona-fide infections, resulting in strong immune responses, with both cellular and serological components, but with much less viral replication in the lungs. This would not only hopefully reduce the incidence of pulmonary complications in the infected subjects, but would also probably reduce the amount of virus released by them via aerosols, and thus reduce the vector of contagiosity that is hardest to control, and that probably leads most effectively to viral replication in the lungs. If those experiments in animal models reveal that one or several routes can be used effectively to reduce pulmonary pathology, a clinical trial could be conducted in human volunteers with very low risk profiles. The ID route should probably be considered as a priority, since it could double-up as a skin test to reveal the immune status of the recipients towards the SARS-CoV-2 virus. The course of action proposed here may possibly provide a way of taking a step ahead of the virus, and if it works as hoped, could help to end the need for confinement within a matter of months, if not weeks. It is now clear, however, that the virus only kills a small proportion of those it infects, whilst others recover and become immune in a matter of days. The final outcome of this sanitary war is therefore a foregone conclusion, and the survival of the human race is clearly not at stake.As of 21 April, over 2 million cases of Covid-19 have been recorded worldwide and thousands are dying every day  inoculation. If resources permitted, a preferable course of action to save time would be to explore as many of these routes in parallel as possible.The ID route would, however, seem preferable to the other routes of inoculation, for the following reasons:The ID route could also double-up as a skin test, akin to the Mantoux test used to test for BCG-primed immunity. Thus, individuals previously infected by SARS-CoV-2 could be detected in a matter of days. These individuals could then be issued with a ‘sero-converted’ sanitary passport and resume a normal life .It could also boost the immunity of those who have only had mild forms of Covid-19. These people would possibly be at high risk of ADE-related complications in the form of a cytokine storm if they were to be accidentally re-infected by SARS-CoV-2 via the airway route in the course of later months or years, when their level of circulating antibodies would have decreased to sub-neutralizing levels , the virus is expected to replicate efficiently in the gut, and consequently to promote strong mucosal immunity. This is supported by the observation that digestive signs are indeed frequent in Covid-19 patients (https://www.sciencemag.org/news/2020/04/how-does-coronavirus-kill-clinicians-trace-ferocious-rampage-through-body-brain-toes#). As an elaboration, one could also conceive of using the IR route as an initial primer, followed by ID inoculation 7 to 10 days later, which would not only boost the immunity, but also provide a direct and sensitive test of the immune status of the recipients.IR inoculation may turn out to be an alternative to the ID. Indeed, for the IR route, the sanitary considerations would be minimal, and simple dilutions of viral preparations derived directly from tissue culture could conceivably be used. As experience with HIV has shown, some viruses can spread very effectively via the rectal route. Given the fact that ACE-2, the viral receptor, is expressed at very high levels in the digestive tract , and provides lifelong immunity. The major drawback of this live vaccine is that it has been found that the attenuated polio virus rapidly reverts to a wild-type, fully pathogenic virus in the intestine of the vaccinated subjects, who will then expel it in very large numbers in their faeces. This is only a very infrequent problem for the vaccinated subjects themselves , but is 7/ml) in cell lines such as Vero, Caco II, or Calu 3 cells  that will have been identified as the most promising through animal experimentation. Those volunteers would be selected from populations with very low risks of complications. The trial would also have to take place in a completely confined environment, and under close medical monitoring. I suggest that a military base would be a particularly appropriate environment for this type of project.Furthermore, it does not seem unreasonable to hope that, by then, some therapeutic approaches will have been identified to combat the virus in patients presenting complications. In this regard, passive immunisation with serum from recovered patients seems to be one of the more hopeful avenues to date  Over the coming month, completion of the ID inoculation experiments in macaques .2) During this time, preparations will be made for the first trial on a cohort of a few dozens of volunteers in a completely confined environment. This could take place during the coming month(s), but will obviously only go ahead if and when the results of the above experiments will have shown very promising results.3) If and when the project gets to that stage of self-inoculating by human volunteers, one of the very important aspects of this experimental stage will be to determine how contagious these inoculated volunteers will become, especially whether they excrete a lot of live infectious virus through their airways, urine, or faeces.4) If it is found that, after inoculation, the volunteers are not worryingly contagious, one could conceive of an exercise of mass immunisation, strictly on volunteers, asking the inoculated volunteers to stay confined at home during the time they generate immunity to SARS-CoV-2.5) On the other hand, if it turned out that inoculated individuals can become highly contagious, one may have to resort to performing the immunisations in confined centres, which I refer to as ‘covidodromes’. For this, I envisage that military bases could provide convenient environments, at least for the first stages of the exercise.6) If it turned out that the approach proposed here was truly efficient, and could result in the rapid sero-conversion of thousands of people with a bare minimum of complications, and even fewer casualties, one could then endeavour to step-up the process to be in a position to offer covidodromes for millions of volunteers to travel to and be inoculated with controlled stocks of virus, in a controlled environment. This could for example be achieved by using tourist places which are currently deserted, such as holiday centres and villages, ski resorts, or even some of the large islands such as those found along the French littoral.With such a large-scale approach, significant proportions of sero-converted people in the population could be achieved before this summer, ultimately reaching herd immunity, which is the only thing that will allow us to come out of confinement serenely.No data are associated with this article. The concept of using life viruses by injection in atypical location might be attractive to some extent and some other researchers had already published on it. However, I am still not encouraged to support this premise as long as safer and effective techniques can be developed. Recently, several vaccines have been approved and widely practiced worldwide, the opinion must be updated. The application of live viruses for injection of human being can be a matter of virus activation, mutation and infected or immunized persons might be seeding the virus in the surrounding community and act as a source of infection. This application requires also much care, sophisticated resources and extreme care from the medical staff to avoid contact with the live virus. Despite there are some agreements in the literature, there is no solid evidence for the success of this concept. This manuscript also needs to be updated with the recent findings and approvals in the field of COVID-19 vaccines. Since the strength of immunity might depend on the amount of virus proteins and poster doses. I am not sure that small amount of live SARS-CoV-2 virus are sufficient to give solid immunity.Is the topic of the opinion article discussed accurately in the context of the current literature?NoAre arguments sufficiently supported by evidence from the published literature?NoAre all factual statements correct and adequately supported by citations?NoAre the conclusions drawn balanced and justified on the basis of the presented arguments?NoReviewer Expertise:PharmacologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. CoV2 vaccine effectiveness.Advantages of GI immunization for possibly optimizing mucosal immunity. Possibly mention existing enteric adenovirus vaccines used for US military personnel as a relevant example.Make the obvious point that the proposed approach could be rapidly deployed for emerging CoV2 variants or future pandemic with zoonotic virus.Joly proposes immunization with live SARS-CoV-2 (CoV2) via non-respiratory routes as a rapidly employable and cost-effective vaccine. This is a brilliant proposal but compromised somewhat by the introduction of highly effective vaccines for COVID-19. I would recommend that the author update the article accordingly and possibly alter the message to incorporate:Is the topic of the opinion article discussed accurately in the context of the current literature?YesAre arguments sufficiently supported by evidence from the published literature?YesAre all factual statements correct and adequately supported by citations?YesAre the conclusions drawn balanced and justified on the basis of the presented arguments?YesReviewer Expertise:Adaptive immunity to virusesI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The article proposed the use of unattenuated wild-type SARS-CoV-2 as a live vaccine to be inoculated through routes that can bypass the lungs. Taking into purview the recent trends of the COVID-19 pandemic, the opinion seems to be justified. If we look at the present scenario of the pandemic in India, it would be true to say that the world is going to witness nature's wrath due to human complacency. To avoid further deterioration outside-the-box thinking is required, this article presents the very same. 1, which mentions the changing scenario of the pandemic with relation to age distribution. Recent reports have presented facts that differ from those stated in this article. It mentions "Since there are almost no serious cases in people under 40 years of age with no pre-existing medical condition", this can be now refuted by Abbasi (2020) To conclude, I would suggest that the opinion being presented should be experimented with in order to search all the avenues.Is the topic of the opinion article discussed accurately in the context of the current literature?YesAre arguments sufficiently supported by evidence from the published literature?PartlyAre all factual statements correct and adequately supported by citations?PartlyAre the conclusions drawn balanced and justified on the basis of the presented arguments?PartlyReviewer Expertise:Anatomy, GeneticsWe confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. 1; PMID: 325255872; PMID: 326816503). This opinion is supported by the fact that two wild-type adenoviruses have been SAFELY used as live vaccines through enterically coated capsules for 40 years in the US recruits, and reduced over 99% cases of adenovirus-associated acute respiratory diseases (PMID: 274754744). My own experiments also suggested that intra-muscular inoculation of a wild-type respiratory virus is safe and able to induce strong immunity (PMID: 326816503). This article proposed to use a wild-type SARS-CoV-2 as a live vaccine through intradermal, subcutaneous, intra-muscular, or intraperitoneal inoculation, to bypass the pathogenesis site of the fragile lungs. I published three articles discussing the same opinion . Meanwhile, the anti-αGal antibodies could greatly enhance the efficacy of the live unattenuated vaccine through immune opsonization, as the immune complexes aid the antigen capture, process and presentation of APCs (PMID: 326816503). Again, this view should be investigated using animal experiments. On the other side, some methods available readily could enhance the safety of the live unattenuated vaccine of COVID-19. For instance, if the SARS-CoV-2 is produced using PK-15 cells rather than Vero cells, it shall carry α1,3-galactose , and thus can form immune complexes with anti-αGal antibodies which are naturally abundant in human bodies. The anti-αGal antibodies could greatly enhance the safety of the live unattenuated vaccine through blocking the viral entry, aiding complements to destroy the virus, and aiding immune cells to destroy the virus  the unattenuated vaccine could be powerful to curb the COVID-19 pandemic; (2) the unattenuated vaccine is imperatively desired to be investigated using animal experiments. Meanwhile, the author should consider the relevant history, publications, and methods to enhance the safety and efficacy of the unattenuated vaccine.Is the topic of the opinion article discussed accurately in the context of the current literature?YesAre arguments sufficiently supported by evidence from the published literature?YesAre all factual statements correct and adequately supported by citations?PartlyAre the conclusions drawn balanced and justified on the basis of the presented arguments?YesReviewer Expertise:public health; veterinary medicine; control of infectious diseases; epidemiology; influenza; virology; evolutionI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Ovaliviridae is a family of enveloped viruses with a linear dsDNA genome. The virions are ellipsoidal, and contain a multi-layered spool-like capsid. The viral genome is presumably replicated through protein priming by a putative DNA polymerase encoded by the virus. Progeny virions are released through hexagonal openings resulting from the rupture of virus-associated pyramids formed on the surface of infected cells. The only known host is a hyperthermophilic archaeon of the genus Sulfolobus. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Ovaliviridae, which is available at ictv.global/report/ovaliviridae. Virions of Sulfolobus ellipsoid virus 1 are ellipsoidal, about 115×78 nm and coated with an envelope , Fig. 1.vp1, vp2, vp3 and vp4) encode structural proteins, as verified by the analysis of virus particles by SDS-PAGE. The genome is also predicted to encode a glycosyltransferase, a B-family DNA polymerase and a SAM-dependent methyltransferase. Four ORFs  are homologous to sequences in lipothrixviruses or rudiviruses, and the first three of them also to sequences in the spindle-shaped virus Sulfolobus monocaudavirus 1 (an unclassified virus related to members of the family Bicaudaviridae), implying possible horizontal gene transfer between these viruses.The Sulfolobus ellipsoid virus 1 virion contains a linear dsDNA genome of 23 219 bp with 172 bp inverted terminal repeats. The G+C content of the genome is 33%. The genome harbours 38 putative protein-encoding genes  [1]. TheAmpullaviridae) [Halspiviridae) and His2 virus (Pleolipoviridae) [Thaspiviridae) [Sulfolobus ellipsoid virus 1 encodes a putative B-type DNA polymerase belonging to the subfamily of protein-primed DNA polymerases . This poviridae) , His1 viviridae) , and Nitviridae) . Howeverictv.global/taxonomy. Sulfolobus ellipsoid virus 1 was isolated from an acidic hot spring  in Laguna Fumarolica, Costa Rica. The only known host is Sulfolobus sp. A20, which was isolated from the same hot spring [Ovaliviridae includes the genus Alphaovalivirus and the species Sulfolobus ellipsoid virus 1.Current taxonomy: Ovaliviridae: ictv.global/report/ovaliviridae.Full ICTV Report on the family