Monkeypox virus is a zoonotic DNA virus related to the virus that causes smallpox. It was first isolated and identified in 1958 but its first confirmed human case was in 1970 when the virus was isolated from a child in the Democratic Republic of Congo. Since then, several cases have been reported within the African continent and globally. Despite its spread, Monkey pox disease has continued to suffer neglect in terms of research and funding due to its reported inefficiency in transmitting from Man to man as its transmission was reportedly limited to its endemic regions in Africa. Poor health data management, inadequate capacities in terms of testing infrastructure and health care workers and weak surveillance systems are some of the challenges faced by African countries. Multisectoral collaboration in breaking the transmission cycle of monkeypox infection and other preventive measures must be improved upon. Community advocacies and education play integral roles in infection spread preparedness, as well as in strengthening the healthcare system. •Monkeypox virus infection remains a challenging health threat in sub-Saharan Africa, and the world at large.•Research, monitoring, supporting, and sharing information among countries affected by monkeypox should be improved.•Research priorities towards monkeypox virus vaccine production should be prioritised.•Sensitization toward the populations at risk must be invested into. While it was first isolated and identified in 1958 . Since tFurthermore, several studies have reported coextensive immunity to monkeypox virus through vaccination against smallpox. However, eradicating smallpox and subsequent lack of vaccination efforts have paved the way for monkeypox to gain clinical relevance . Also, tHowever, more than 3000 monkeypox virus infections have been reported in over 50 countries across five regions, since May 2022. This has prompted the World Health Organization to declare monkeypox an “evolving threat of moderate public health concern” .With an increasing number of cases being reported across regions, it is important to update knowledge of this re-emerging Public health threat, including its diagnosis prevention and clinical management in a bid to prepare health systems for the present reality.2Monkeypox virus is a viral zoonotic disease, that chiefly occurs in regions of central and Western Africa, while being exported occasionally to other parts of the world. It can be classified under the genus orthopoxviruses in the family Poxviridae, and is a double-stranded DNA virus according to the Baltimore classification of viruses  Like mos●Isolation and euthanization of animals who have been suspected to be the reservoirs of the monkeypox virus .●Isolation of infected patients in a negative pressure room to prevent any form of human-to-human outflow and spread of the virus .●Eliminating contact with infected material that may have been in contact with the infected human or animal●Use of proper protective equipment for front-line medical workers when managing infected patients and other high-risk individuals .●Avoiding direct contact with infected individuals, animals, and suspected animals of monkeypox virus in geographical regions having high monkeypox prevalence .For many viral infections, vaccination is a great measure of prevention. Seeing as monkeypox virus has many genetic similarities with smallpox, data further suggest that vaccination against smallpox infection is believed to confer about 85% cross-protection against monkeypox, and may improve the clinical manifestation of infected individuals . However3Clinical and epidemiological data must be correlated with diagnostic assays in order to confirm monkeypox infection. This data may include the date of onset of fever, date of rash onset, current state of individual, as well as age. The data collection helps in the identification of people that have a higher risk of false positive results .The confirmation of monkeypox infection is based on the sum total of patient history, clinical presentations, and confirmatory laboratory testing - these tests include Polymerase chain reaction (PCR), Enzyme-linked immunosorbent assay (ELISA), Immunohistochemistry, and Western blot .Isolation of the viral nucleic acid is also done using lesion exudates, dry crusts, and biopsy to isolate further and confirm the diagnosis. This confirmatory diagnosis plays a critical role in eliminating the possibility of other possible infections like smallpox . While WIn most cases, monkeypox usually presents with mild to moderate symptoms, and patients may not necessarily need therapy for recovery. There are no specific treatments for monkeypox virus infections, however, symptom alleviation, complication management, and prevention of long-term sequelae should be optimal during the clinical care for monkeypox infection .For specific care, an antiviral, tecovirimat, which was developed for smallpox, has been approved for the treatment of monkeypox in 2022, based on recovery data derived from animal and human clinical studies. Although it is not widely available yet, if necessary, tecovirimat must be used under excellent clinical supervision .4Much is still yet to be known regarding monkeypox virus. However, as of June 22, 2022, only one death out of 3413 confirmed cases of monkeypox has been recorded. Various public health agencies like the World Health Organization and Centre for Disease Control (CDC) have played critical role in monitoring, supporting, and sharing information of monkeypox virus among member states. The overall risk of monkeypox virus globally has been determined to be moderate, according to the resolution of the International Health Regulations Emergency Committee in June 2023. Because of the existing concurrent covid-19 health precautions, case finding, contact tracing, laboratory investigations, and various forms of clinical management have been more seamless in the control of monkeypox infection.While genomic sequencing of the monkeypox virus DNA is still ongoing, immunization against monkeypox is encouraged. Since the 1980s, the smallpox vaccination, which offer some protection against monkeypox, has been discontinued, leaving a huge portion of the population exposed to the monkeypox virus. In recent years, only a very limited number of military personnel, frontline medical personnel, and laboratory personnel have received the smallpox vaccine, and still possess immunity against monkeypox.With the world just recovering from the global Covid 19 Pandemic, there has been some comparison between the MPXV and the Coronavirus 19. However, unlike coronavirus 19, the MPVX is not a novel virus as there are current experienced expertise in prevention and management gathered from previous outbreaks. The challenges in Africa regarding containment of Monkey Pox disease is quite similar to that encountered during Covid 19 outbreak. The challenges faced by Africa include poor health data management, inadequate capacities in terms of testing infrastructure and health care workers complexed with the fact that the surveillance systems in many African countries are weak.Several studies in sub-Saharan Africa identified weaknesses and deficiencies in the quality of data collected for the surveillance of infectious diseases. The reported deficiencies have ranged from incomplete data to inconsistent data at different levels of the surveillance system. Without a doubt, several factors have contributed to the continuous persistence of these reported weaknesses. They include but are not limited to low motivation, inadequate health management information systems–related resources, and lack of skills among healthcare workers owing to the lack of training.A lack of MPX knowledge among health care workers and communities will also increase cases of underreporting, increased spread and improper management of cases, and poor prevention and control strategies, leading to the further spread of the disease.If the past outbreaks of infectious diseases in Africa are anything to go by, there's an urgent need to strengthen Africa's preparedness in tackling these issues. These strengthening need to bring together concerted efforts targeted at increasing research funding to bridge gaps in knowledge and introduce new perspectives to current issues. Strengthening of surveillance systems should be done through active, real-time digitalization of health care data with secure data sharing for research and policy decisions. The place of multisectoral collaboration can not be under estimated in controlling the current realities. There is an urgent need for collaboration with the veterinary communities in understanding the disease reservoir and trends of transmission. Community advocacies should be geared towards increasing preparedness and the need for active preventive measures. High-risk individuals such as sex workers, immunocompromised individuals, children and other key populations should be well-sensitized about risks and prevention.5As newer cases unfold, we will gain new perspectives to the impending threats posed by the Monkeypox virus. Africa as a continent, needs to act fast and effectively in curbing the spread and casualties from the infection spread. Although there have been a lot of challenges inherent to Africa, lessons learned from previous outbreaks need to be applied in winning the current fight against monkeypox infection, and preventing another full-blown pandemic.No ethical approval was required or obtained for this study.This study was self-fundedThis review was conceptualized by Damilola Quazeem Olaoye and Ernesto Dibia. Ernesto Oluwafemi Dibia and Damilola Quazeem Olaoye contributed to writing the manuscript. All the authors read and approved the final manuscript.There are no conflicts of interest.1.Name of the registry:2.Unique Identifying number or registration ID:3.Hyperlink to your specific registration (must be publicly accessible and will be checked):DIBIA, Ernesto Oluwafemi.OLAOYE, Oluwadamilola Quazeem.Not applicable.Not Applicable.


There is growing evidence that plant viruses manipulate their hosts and vectors in ways that increase transmission. However, to date only few viral components underlying these phenomena have been identified. Here we show that cauliflower mosaic virus (CaMV) protein P2 modifies the feeding behavior of its aphid vector. P2 is necessary for CaMV transmission because it mediates binding of virus particles to the aphid mouthparts. We compared aphid feeding behavior on plants infected with the wild-type CaMV strain Cabb B-JI or with a deletion mutant strain, Cabb B-JIΔP2, which does not produce P2. Only aphids probing Cabb B-JI infected plants doubled the number of test punctures during the first contact with the plant, indicating a role of P2. Membrane feeding assays with purified P2 and virus particles confirmed that these viral products alone are sufficient to cause the changes in aphid probing. The behavior modifications were not observed on plants infected with a CaMV mutant expressing P2Rev5, unable to bind to the mouthparts. These results are in favor of a virus manipulation, where attachment of P2 to a specific region in the aphid stylets–the acrostyle–exercises a direct effect on vector behavior at a crucial moment, the first vector contact with the infected plant, which is essential for virus acquisition. Some pathogens including plant viruses manipulate vectors to optimize transmission. The manipulations can be indirect meaning that pathogens alter host traits such as color or odor that attract or deter vectors. Other modifications are direct, i.e. uptake of virus compounds changes vector behaviors. Direct effects have been reported for viruses that are internalized by their vectors and interact strongly with the vector from within, for example with the nervous system. Here we show that contact of a virus protein with the vector’s exterior mouthparts suffices to induce a direct effect: binding of the non-structural cauliflower mosaic virus protein P2 to aphid stylets during test punctures modifies probing activity instantly, thereby facilitating virus transmission. The fact that here no intimate virus-vector interactions are required for vector manipulation and transmission, could explain the broad vector range of CaMV and other non-circulative viruses. Plant viruses are economically important pathogens and most of them require a vector for transmission . InsectsA growing corpus of theoretical modelling and empirical research shows that viruses and other parasites modify hosts and vectors to optimize their transmission –7. TheseViruses can–after acquisition by vectors–also alter vector behavior directly. This has been studied in particular for plant viruses relying on the circulative transmission mode. Such viruses traverse the intestine, cycle through the hemocoel and accumulate in the salivary glands, before inoculation as a saliva component into new plant hosts. During their passage, they can interact with various host organs, for example the brain, the salivary glands or antenna and modify vector behavior in ways that are conducive to virus transmission –20. SimiCaulimovirus) is transmitted by aphids using the non-circulative mode. CaMV virions are retained in the aphid mouthparts (stylets) by attaching to cuticular proteins (stylins) located at the stylet tip in a zone called the acrostyle  A letter containing a detailed list of your responses to all 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.Thank you again for your submission to our journal. 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,John P CarrGuest EditorPLOS PathogensShou-Wei DingSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064***********************Reviewer Comments :Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: This is a well researched and written manuscript. The authors provide evidence that the helper component (P2 protein) of a plant virus (CaMV) can directly modify the probing behaviour of its aphid vector. The authors make use of electrical penetration graph (EPG), wild type virus and a mutant form that doesn’t produce the P2 protein to show that aphid probing and feeding actions associated with virus acquisition and successful inoculation are enhanced in the presence of P2 protein. These findings are significant in that they demonstrate that initial contact between an aphid vector and the virus results in behavioural changes in the aphid and that these changes can be caused by the direct binding of P2 to receptors in the aphid stylet. Further, the authors demonstrate the power of EPG as a valuable tool in deciphering how viruses manipulate the behaviour of their insect vectors. These results are important in modelling acquisition and dispersal of viruses by aphid vectors. These findings will be valuable to the scholarship of molecular plant virology and plant-virus-insect interactions whose significance will increase as novel ways to control aphids and the viruses they spread are sought.Reviewer #2: In regard of novelty, the study addresses a less well studied area where the authors investigated if a virus factor can modify vector fitness or behaviour. They chose CaMV P2 protein and Myzus persicae or the green peach aphid as CaMV vector. CaMV is a semi-persistently transmitted plant virus whereby it is not known if CaMV can have a direct involvement in modifying vector (aphid) behaviour, and its implication on onward transmission of the virus.The strength of the study lies in the structured way they conducted their studies.The group did prior work on CaMV , i.e.: Chesnais et al., 2020, 2021; Drucker et al., 2002 as referenced in the manuscripts. They discovered that P6-TAV, a multifunctional protein of CaMV, led to altered feeding behaviour of the aphids on Arabidopsis and this is likely to be mediated by indirect host-mediated effect as P6-TAV is involved mainly in modification of host plant physiology. CaMV P2 protein is a helper protein which has been shown to form a protein bridge between the aphid’s stylet and the virions. Hence the authors chose to investigate P2 protein in this study as a candidate for a virus-encoded factor which may modify vector behaviour.Overall this paper is well laid out, where the authors investigated whether P2 protein of CaMV are involved in modifying aphid feeding behaviour as CaMV vector. The authors utilised wild-type CaMV virus (JI), mutant virus with abolished P2 (JIdP2), and a mutant virus whereby P2 has lost the ability to bind to the aphid mouthparts (JI-P2Rev5). The authors further tested whether the effect of P2 on aphid feeding behaviour is independent from other plant factors by using membrane feeding set up to allow aphids to feed directly on food solution with P2 virion, or P2:P3 virion complexes. The results did add new knowledge in the field.The weakness of this study lies in the sentence structure and language, as well as there are additional experiments which can help clarify the results. There were several occasions where the choice of word and sentence structure need editing. The titles for the subsection in the results part of the manuscript also needs re-phrasing to increase its accuracy in reflecting the experimental result, and to avoid over assumption. Several statements are seemingly contradictory to one another, which can be due to poor sentence structuring and will need to be re-written to improve its clarity. The labelling on Figure 1 needs to be corrected (further details below). The suggested experiments are described in the following section.Reviewer #3: This manuscript presents convincing evidence for a role of CaMV P2 in influencing aphid vector behaviour directly. The implication that a protein of a non-circulative virus manipulates the vector is highly significant and will be of broad interest to researchers in plant-pathogen-vector interactions. The experimental approaches are rigorous and nicely combine EPG recording with use of mutant viruses and an artificial membrane feeding system to provide consistent and significant results. The findings are novel and the work is suitable for publication following some minor changes.**********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: n/aReviewer #2: Fig 1b : can the authors check if the labelling is correct on the westen blot? If anti P2 is an antibody specific for P2 protein, then why does bands appear on JIΔP2 samples whereby P2 were not supposed to be present? Please verify.Have the authors tested whether the aphids were able to acquire the mutant CaMV JI-P2Rev5 virus?The authors did demonstrate that turnip plants were infected with CaMV JI-P2Rev5 virus, but with P2 being unable to bind to the aphid stylet – have the authors checked whether aphids can acquire and subsequently transmit CaMV JI-P2Rev5 mutant virus? Figure 4 did show that aphids fed on JI-infected plants performed significantly more intracellular punctures than aphids fed on CaMV JI-P2Rev5 infected plants. Whilst Figure S4 showed no significant difference between aphid feeding behaviour when different parameters were measured than Fig 4 (sap ingestion and time to first ingestion) using EPG on JI-infected and JI-P2Rev5 plants.For L. 230  : Did the authors detect P2 and/or P3:virions titer or verify its presence in the individual aphids after they were fed on the artificial membrane containing P2 and P3:virions? this is to verify that the effects observedon the aphids were genuinely due to the P2 and/or P3 being taken up by the aphids and not just due to aphids experiencing feeding difficulties on the membrane for example? . Figure S2 showed viral components used for aphid feeding assay on the artificial medium, but is this is measured directly from the artificial medium itself and not from the individual aphids feeding on it? Figure S5(c) showed SDS PAGE gel of Sf9 crude extracts from the artificial medium, has any been done from the aphids itself?L. 391 and L. 403: This paragraph gives out two seemingly contradictory statements which reduces clarity. In L. 391, the authors states “Our membrane feeding assay support the direct effect of P2”, whilst L. 403 reads as follows “Therefore, although the membrane feeding experiments do show a direct effect of P2 on aphids, interpretation of the results from the membrane feeding assays are difficult”. I prefer the more cautious way the authors framed their results here in L. 403, therefore can L. 391 please be re-phrased or rewritten?Reviewer #3: The correlation analysis  provides a tantalising hint that P2 not only modifies aphid behaviour on virus-infected plants, but may also be involved in manipulating behaviour on healthy plants that are subsequently encountered and probed. Have the authors also considered investigating a correlation between number of punctures on healthy plant (the same data plotted here) against number of intracellular punctures on the virus-infected acquisition plant? Since P2 is acquired during intracellular punctures , this approach may reveal a more biologically meaningful effect of P2 through the comparison between the three treatment groups. I therefore recommend that this additional analysis is carried out and included in the manuscript.**********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: There is clarification required in the statistical analyses. For example, the authors used Tukey for pairwise comparisons. However, I did not find any p values for this in the supplementary files, especially where differences were significant.Figure 2 Panel ‘b’. It seems implausible that the pairwise comparisons for ‘Intracellular punctures during first penetration’ are not significant for Mock vs the mutant JIDP2. What was the p-value?Figure 3 ‘penetration before first ingestion’ and ‘intracellular punctures’ What are the p values for the pairwise comparisons between the 3 and 5 treatments, respectively?Figure 4: Panel ‘c’ ‘intracellular punctures’ What are the p values for the pairwise comparisons between the five treatments?Line 328 – do you mean ‘less’ in place of ‘smaller’?Reviewer #2: Please see more detailed comments below.The title can be edited and made more concise? Current title is “The transmission helper protein of a plant virus….”. I would suggest to mention the name of the helper protein and the virus on the title directly.L. 35: ‘first plant contact’ ? First contact with the plantL. 38: ‘virus’ would be a more fitting choice of word here rather than ‘parasite’L. 48: ‘instantly’ might be going too far here, as it was not shown here whether it is ‘instantenous’ or not. Please re-phraseL.49: ‘prepares’ here personified CaMV, can this sentence be re-phrased? It is not accurate to assume that ‘CaMV prepares for..’ as what this sentence seems to be implying.L. 50-51: This sentence is not clear. What does the authors mean by ‘minimizes vector implication’? Were the authors trying to say that it minimises the risk of non-transmission by the vector? Please re-phrase.IntroductionThe introduction started with introduction to plant viruses and their mode of transmission. Followed by examples of research on virus factors which mediates changes in vector fitness. Some relevant references in this subject area are missing and should be included. Certain references displayed here are not the most accurate references available to support the statement made and ideally should be edited. Please see comments below.L. 58: ‘their’ morphology should be ‘the stylet’s morphology’. Not the morphology of the entire aphid as an insect as what the sentence reads as at the moment.L. 70-74: Ray and Casteel 2022 is a review paper which focused more in depth about the role of effectors to mediate plant-virus-vector interaction. The statement on L. 70 whereby “the viral factors responsible for changes in vector fitness are still largely unknown” is not entirely accurate as more has been known about viral factors affecting vector fitness. Other references are more fitting to support the statement whereby particular viral factors have been identified which are responsible to change sin vector fitness , for example in Ziebell et al., 2011; Westwood et al., 2013; Shi et al., 2016; Wu et al., 2017; Tungadi et al., 2020; Arinaitwe et al., 2022.L. 76: Donelly et al. 2019 is a paper on epidemiological modelling. Their work is still very much relevant to the theme of this manuscript. However in this manuscript, it was cited as though as it was a lab experiment paper. Donelly et al 2019 generated a modelling framework on non-persistent virus transmission where it includes aphid feeding behaviour, wingless or winged aphids, and inhibition of aphid settling on plants as parameters.L. 87: ‘or not a’ ? unfinished sentence?L. 102: will exploreL. 104: ‘ingest actively some cytosol’ does not sound right, please re-phrase this sentence.L. 104: ‘if the plant is accepted’ , please re-phrase this sentence as well.L.106-107: ingest phloem sap passively and continuously.L. 102-111 : This paragraph can be shortened. I understand that the authors are trying to describe the sequence of events that occurred after the aphid lands on the plant which is relevant to the paper.The authors then settled to choose P2 to test their hypothesis whether a non-circulative virus like CaMV can encode factors having a direct effect on the vector.L. 121-122 : Why only P2 and not P3 as well?ResultsL.124: The title of this subsection does not really make sense, particularly in the part of “P2 probing”. Please re-phrase.Fig 1a : can the authors add labelling on the figure itself please?Fig 1b : can the authors check if the labelling is correct on the westen blot? If anti P2 is an antibody specific for P2 protein, then why does bands appear on JIΔP2 samples?The main take home message is number of intercellular punctures made by the aphids increased significantly during JI (wild-type) virus infection but not on plants infected by JIΔP2 virus.L. 148 : The authors then went on to test the effect of P2 on aphids using wild-type CaMV and P2 deletion mutant, JIΔP2. Deletion of P2 does not affect infectivity of the virus.They did EPG and found that deletion of P2 also does not affect on phloem sap ingestion by the aphids.However the number of intracellular punctures were a lot higher on JI-infected plants than JIΔP2 or mock inoculated ones. This section was summarised nicely on L.179-182 where they stated that CaMV infection (with and without P2) did increase phloem feeding and reduce the time taken by the aphids to reach the phloem.L. 178-182: They also stated that the number of intercellular punctures were higher on aphids fed on wild-type CaMV Ji virus but not on the mutant virus or mock inoculated plants. The closing statement from this section where the authors indicated that ‘P2 was responsible for this’ sounds premature from the conclusion of this work to date. This work here does not exclude the possibility that the observed effects can be the due to alteration in the host plant, thus an indirect effect of CaMV.Have the authors tested whether aphids were able to acquire the mutant CaMV JI-P2Rev5 virus? The authors did demonstrate that turnip plants were infected with CaMV JI-P2Rev5 virus, but with P2 being unable to bind to the aphid stylet – have the authors confirmed that aphids can acquire CaMV JI-P2Rev5 ? iL. 183: The title for this subsection is not entirely accurate. Please re-phrase and be more specific, i.e: mention P2 protein on the title?L. 227 – 228: The title for this subsection on “Direct interaction between P2 and Myzus persicae modifies the probing behaviour of the aphid vector” needs to be re-phrased as the authors have not shown protein-protein interaction between P2 and aphid stylet here in their results. It would be more accurate to re-name the subsection as “Acquisition of P2 virions (and/or P3 virions) xxx by M. persicae modifies xxx ”.L. 265-266: Similar to my previous comment. The title for this subsection on ‘The interaction between P2 and the aphid stylets is required for changing probing behaviour’ needs to be re-phrased as the authors have not shown a direct protein-protein interaction here between P2 and the aphid stylets. It would be more accurate to re-name the subsection as “Acquisition or feeding from xxx “.. or “Aphids feeding on mutant virus with abolished function of P2 interaction to xxxx “.L. 287: ‘interacting with intestine’? please re-phrase. Did the authors meant to say interacting with host factors in the aphid gut/intestine?Figure 2 : has the authors showed here that aphids can acquire JIΔ2P?L. 301: performed more than twiceL. 315: the authors did acknowledge that ‘the acrostyle-binding capacity for P2 is important for this’. Hence I think it is important that the authors demonstrate whether aphids can acquire and subsequently able to transmit CaMV JI-P2Rev5 from a CaMV JI-P2Rev5 infected plants or not. This is important as otherwise, the observed aphid behaviour when feeding on CaMV JI-P2Rev5 infected plants will be due to an indirect host-mediated effect whether the aphids were responding to virus-mediated effect to the plant hosts.L. 340-341: Is there any reference that can be cited which shows that phloem contained higher virus titer than the surrounding single cells?L. 338-345: This paragraph is confusing, does the authors meant to say that P2 is not involved in the alterations of aphid behaviour (i.e: lower number of cell punctures) as they observed a similar effect on aphids fed on both wild-type CaMV inoculated plants and CaMV P2 deletion mutant inoculates plants? This somewhat contradicts the results from Fig.1 where on L. 172-175, it states that ‘we observed P2-speficic alterations for the number of intracellular punctures during the first probe which was twice as high on JI-infected plants than on JI P2-deletion mutant infected plants’. This is also re-iterated again on L. 347-348. Please edit this paragraph.L. 347: Is there more than one CaMV isolate used in this study?L 357 : Has there been studies to show whether the level or titer of P2 protein on the stylet remained the same throughout ? whether aphids for example had higher titer of P2 protein on their stylet tips during the initial acquisition, which then increased and tapered off?L.364-367 contradicts L. 338-339, please check and edit.L. 389: The opening sentence where “We show here that P2 changes aphid probing behaviour..” again does not sit comfortably with me as the authors have not shown yet whether the effect was direct or indirect. Rather than addressing the ‘direct or indirect’ effect in the second sentence, it would be better if the authors opens this paragraph by stating the fact directly “We show here that P2 may directly or indirectly changes…….”L. 391 and L. 403: This paragraph gives out two seemingly contradictory statement which reduces clarity. In L. 391, the authors states “Our membrane feeding assay support the direct effect of P2”, whilst L. 403 reads as follows “Therefore, although the membrane feeding experiments do show a direct effect of P2 on aphids, interpretation of the results from the membrane feeding assays are difficult”. I prefer the more cautious way the authors framed their results here in L. 403, therefore can L. 391 please be re-phrased. Did the authors measure the success rate of subsequent virus inoculation onto healthy plants by the aphids after being fed on the membrane? Did the authors also detect and measure P2 and/or P3 protein presence on the aphids after they were fed on the membrane to verify that the effects observed were genuinely due to the P2 and/or P3 proteins being taken up by the aphids and not just due to aphids experiencing feeding difficulties on the membrane.L. 175 – 176 and L. 339-342 : Both set of sentences also seems to convey contradictory message whereby on L. 175-176, the authors stated that the number of intracellular punctures during the first probe was twice as high on JI-infected plants than on JIdP2 infected ones or mock-inoculated ones. This seemingly contradict the statement on L. 339-344 where the authors stated that P2 plays no role in this as the observed the same behaviour on plants infected with wild-type virus and on plants infected with JIdP2.. whereby “this” was mentioned in L. 339 as “lower number of test punctures combined with the longer phloem ingestion on infected plants”. Implying that on L. 175 : aphids made more punctures on JI-infected than on JIdP2 infected plants, however on L. 339-342 : aphids showed similar number of test punctures on both JI- and JIdP2 infected plants.This is either a grammatical error or the sentences need to be restructured as to avoid confusion to readers.Reviewer #3: I have a general issue with the term ‘feeding behavior’ (e.g. in the heading line 124 and numerous other places in the text) to indicate all behaviours displayed by aphids during stylet penetration. On plants, only E2 (sustained phloem sap ingestion) is really “feeding”. Better to use more generic (‘stylet penetration’ / ‘probing’) alternatives throughout the manuscript.The authors seem to over-state the evidence for causative links in some parts of the text. I have suggested more cautious language in parts, and made some other specific comments with line numbers indicated as follows.Line Comment50 The meaning of ‘minimises vector implication’ is not clear, what is the significance?56 ‘Hemiptera’ is an order 94 ‘fixation’ implies a non-reversible link - change to ‘adherence’108, 110 ‘tissue cells’ – can a more useful and specific term be used? Peripheral? Phloem sieve elements are part of a (phloem) tissue, so ‘tissue and from phloem sap’ does not make sense125, 158 The statement does not agree with Figure 1 b), which indicates that P2 is not detected in WT plants – has the figure been mislabelled?125 Figure 1 c) & d) and subsequent figures need full axis labels – presumably duration (hours) and occurrence (number of events per recording)? Note spelling of ‘occurrence’ (two r’s). What is being plotted – means and standard errors? This also needs clarifying in subsequent figures.160 Change ‘taking’ to ‘taken’167 Change ‘1a’ to ‘1c’178 Change to ‘….without P2) was associated with an increase in the duration….’ .179 ‘reduce the time required to reach it’ is not supported by the data and should be deleted, unless it can be evidenced. Figure 1c) shows no significant difference (time to first sap ingestion).182 Change to ‘… was associated with this.’ .187 Change ‘lists’ to ‘presents’.215 Change ‘was’ to ‘may be’.287 I suggest changing ‘intestine’ to ‘more posterior parts of the alimentary canal’ (or something similar). There seem to be lots of other possibilities for potential interaction sites (other than the intestine).306 Change ‘only on the rim of significance’ to ‘marginally not significant’.315 Change ‘this’ to ‘the observed changes in vector behavior’ (or similarly expand this rather brief last sentence).316 Delete ‘a’326 Change ‘behavior’ to ‘behavioral’.328-330 Can these statements  be supported by reference to specific parameters and statistical outcomes? Time to first E1 / E2?352 Change ‘is’ to ‘may be’.362 Change ‘did’ to ‘exhibited’.393 Change to ‘altered subsequent probing…’433 Suggest changing to ‘is not definitive evidence’. Otherwise this sentence does not make sense, particularly when the next sentence allows for other hypotheses.434 Again, it seems strange to highlight particular regions of the alimentary canal  when many other potential binding sites are possible.454 Change ‘inversed’ to ‘distinctly different’.502 Change ‘depression’ to ‘negative air pressure’.509 Change aphids to aphids’.556, 566, 586 Change ‘in’ or ‘by’ to ‘previously’.**********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. 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Insurers manage the cost of specialty medicines via rebates, however it is unclear if the savings are passed on to patients, and whether reducing rebates may lead to changes in patient out-of-pocket (OOP) costs and medication adherence. This study examined two drug classes to understand the impact of reducing list prices to net prices, via lower-priced national drug codes (NDCs) or authorized generics, on patient OOP costs and adherence.This retrospective analysis assessed IQVIA PharMetrics ® Plus adjudicated medical and pharmacy claims for commercially insured patients. Patient OOP costs per prescription and payer drug costs were assessed for evolocumab or alirocumab (proprotein convertase subtilisin/kexin type 9 inhibitors [PCSK9is]) or velpatasvir/sofosbuvir or ledipasvir/sofosbuvir (hepatitis C virus [HCV] medications). For PCSK9is and HCV medications, the original and lower-priced versions were compared. Adherence was estimated based on proportion of days covered (PDC) (PCSK9is) and receipt of full treatment regimen (HCV medications).p < 0.01; alirocumab: 51%, p < 0.01; velpatasvir/sofosbuvir: 30%, p < 0.01; ledipasvir/sofosbuvir: 14%, p = 0.03). Patients with coinsurance as the largest contributor to their OOP costs had the largest reductions in OOP costs, ranging from adjusted, mean values of US$135 to US$379 (>60% reductions). Six-month PDC for PCSK9is and proportion receiving full HCV treatment regimen were high with the original versions and did not substantially differ with the new, lower-priced versions.In total, 10,640 patients were included . After list price reductions, mean payer drug costs decreased by over 60%, while patient OOP cost reductions ranged from 14% to 55%  resulted in lower patient OOP costs, particularly for those with coinsurance. Our findings suggest that future reduction of rebates may assist in patient affordability, although additional transparency is needed. The list prices for brand name drugs have been under scrutiny in the US in recent years –6. This One way insurers are able to manage the cost of specialty drugs is through discounts and rebates, offered by pharmaceutical manufacturers, in exchange for access to their formularies . While sWhile rebates may get larger over time due to competition, typically list prices do not decline prior to patent expiry , 10, 11.Pricing changes in PCSK9is and HCV medications provide a rare opportunity to examine how reducing list prices to approximate net prices (thereby reducing rebates) may potentially have an impact on patient OOP costs and outcomes, further informing policy decision making. Furthermore, little is known about the impact of such changes on commercially insured patients, who comprise approximately half of PCSK9i users  and abou® Plus database, which comprises of fully adjudicated medical and pharmacy claims data of mostly commercially insured patients. The database contains health plan claims of over 218 million enrollees, 61 million of whom have at least 3 years of continuous enrollment. The PharMetrics Plus data contains patient demographic , primary care and specialist visits, hospitalization, pharmacy use, and costs associated with healthcare services. Cost variables included in the database are charge (amount billed for services), allowed , paid amount , coordination of benefit (the reduction in the amount paid to the provider to reflect adjustments as a secondary payer), deductible , copayment (the fixed amount paid by patients for covered services/products after a deductible is met), and coinsurance (a percentage of OOP amount paid by patients for covered services/products after a deductible is met). Data were de-identified according to Health Insurance Portability and Accountability Act patient confidentiality requirements, and institutional review board approval was not required. Data from January 1, 2015 through June 30, 2020 were used. Fig 1 provides an overview of the study concept.This was a retrospective analysis using the IQVIA PharMetricsS1 Table.Four medications which underwent price policy changes, reflected by new NDCs or authorized generics with lower allowed amounts, during the study period were examined: two PCSK9is  and two HCV drug combinations (velpatasvir/sofosbuvir and ledipasvir/sofosbuvir). The list of NDCs used to identify claims can be found in For the PCSK9is, the original NDC was compared with the new NDC (lower priced version). The lower priced version for evolocumab was introduced in October 2018; however, not all formulations were available at the new lower price until January 2019 . The staThe selection window for alirocumab was March 4, 2019 (date of introduction of the lower priced version) to December 31, 2019. The date of the first alirocumab or evolocumab claim identified during the selection window was the index date. Given the known access hurdles for PCSK9is,  the cohoInclusion criteria for patients in the PCSK9is included: continuous enrollment in a health plan for at least 12 months before the index date and 6 months after the index date; having at least one filled prescription of PCSK9i prior to index date; no switching of NDCs or drugs during the 6-month post-index period; aged 18 years or older; and having no missing age, payer type, plan type or region of residence data.For the HCV medications, branded  versions of the drugs were compared with the authorized generics (lower priced version). The selection window for velpatasvir/sofosbuvir and ledipasvir/sofosbuvir was the date the authorized generic was introduced, with different NDCs  until March 31, 2020. Similarly, the date of the first claim for velpatasvir/sofosbuvir or ledipasvir/sofosbuvir was defined as an index date.Inclusion criteria for patients in the HCV medication cohorts included: continuous enrollment in a health plan for 6 months before the index date and 3 months after the index date; no switching of NDCs or drugs during the 3-month post-index period; aged 18 years or older; no HCV treatment before the index date; and having no missing age, payer type, plan type or region of residence data. Additional details on the patient attrition can be found in S2 Fig). Patient OOP costs were calculated as the sum of copayments and coinsurance, excluding deductibles, because list price changes were not anticipated to have an impact on the total deductible required for a given patient . The total amount paid by both the health plan and the patient to the pharmacy for a given medication was estimated based on the allowed amount. If a single OOP cost component  was missing , OOP costs were calculated as the difference between the allowed amount and the actual amount paid to service providers by the health plan, as well as any coordination of benefits amount. OOP costs were reported as OOP costs per prescription, calculated by dividing patient total OOP costs by the total number of prescriptions filled during follow-up. The driver of OOP costs for each patient was estimated by categorizing individuals based on the largest contributor to their total OOP costs . As a sensitivity analysis, OOP costs including deductibles were estimated.We estimated rebates from list price in the quarter prior to introduction of the lower price versions of the drugs using data from SSR Health . These wMedication adherence was calculated using the proportion of days covered (PDC), defined as the number of days covered with the drug of interest divided by 6 months. PDC was chosen instead of medication possession ratio because it may provide a more accurate estimate in a situation where patients refilled the medication before finishing the current fill of the medication. In such cases, overlapping days supplies were shifted forward . For HCVt approximation for continuous variables.Descriptive statistics, including means and proportions, were used to describe baseline patient characteristics, unadjusted PDC values, and HCV drug use, with comparisons made using Chi-squared tests for categorical variables and Wilcoxon two-sample tests with Adjusted analyses were performed for OOP costs per prescription for PCSK9is and HCV medications, mean PDC for PCSK9is, and completion of the full regimen for HCV medications. Mean OOP costs per prescription and mean allowed amounts were estimated using a log-link gamma generalized linear model adjusted for age, sex, region, payer type, plan type, polypharmacy (defined as three or more medications with medication supplies overlapping the index date), Charlson Comorbidity Index and index month for all medications in the study. The log-link gamma generalized linear model was selected to provide more precise estimates of the population means as it accounts for high positive skewness commonly observed in healthcare cost .Mean PDC for PCSK9is was also estimated using a generalized linear model with log-link and gamma distribution to address the distribution of PDC values due to truncation. Adjustment variables for both models  were based on results from a previous study examining factors associated with PCSK9i treatment interruption  and inclAll analyses were performed using SAS Studio Release 3.7 (Enterprise Edition), 2012–2017 .Table 1. Across all treatments, age, sex and the number of comorbidities were similar. Among the PCSK9is, baseline mean PDC was statistically significantly different between the original and the lower priced version ; however, values were numerically similar . Although the mean number of months from first ever PCSK9i use (prior to the index PCSK9i) was similar for the lower priced version and original NDC for evolocumab (p > 0.05), the time was significantly longer for the lower priced version of alirocumab than for the original NDC .In total, 10,640 patients were included in the analysis . Patient characteristics are shown in Fig 2A), mean allowed amounts were reduced by more than 60% with the lower priced version for all treatments . Percentage and absolute reductions in mean OOP costs per prescription  were less than those observed in allowed amounts , with greater percentage reductions ], % difference) for the lower priced versions of the PCSK9i  than for the HCV lower priced versions .In adjusted analyses . In contrast, in the subgroup with coinsurance as the largest proportion of OOP costs, absolute mean adjusted OOP costs differences between the original and the new lower priced versions ranged from US$135 to US$379, with percentage reductions over 60% .Copayment was the largest contributor to OOP costs in the majority of patients , while coinsurance was the largest contributor of OOP costs in less than 10% of patients . Absolute differences in adjusted mean OOP costs between the original and lower priced versions for those with copayments as the largest contributor to OOP costs were less than US$65, with percentage reductions ranging from 48% to 7% . When including deductibles in the OOP costs, the subgroup of patients for whom deductibles were the largest contributor accounted for approximately 10–15% of patients . For those with deductibles as the largest contributor to OOP costs, absolute differences in adjusted mean OOP costs between the original PCSK9i NDC and lower priced version differed from that of the original HCV medicines compared with their lower priced versions . Among the subgroup with deductibles as the largest contributor to OOP costs, the absolute differences between the original NDC and the new lower priced version for the PCSK9i (US$155–305) were larger than the subgroup with copayments, as the largest contributor (US$52–74) and similar in magnitude to the coinsurance subgroup (US$124–279). Conversely, the absolute differences between the original and lower priced versions for the HCV medicines (among those with deductibles as the largest contributor) was not significantly different (p > 0.05), and in fact more similar to that of the subgroup with copayments as the largest contributor to OOP costs (US$70–73), and smaller in magnitude compared with the coinsurance as the largest contributor to OOP costs subgroup (velpatasvir/sofosbuvir: US$483).Percentage changes in adjusted mean OOP costs when OOP costs included deductibles were similar to when these costs were not included, although slightly attenuated (Table 2), the mean 6-month PDC for the lower priced version of evolocumab was statistically significantly higher than that of the original, but values were numerically similar . Mean 6-month PDCs for the lower priced version compared with the original for alirocumab were similar .After adjusting for patient characteristics (S2 Table). After adjusting for patient characteristics (Table 2), the odds (odds ratio [OR] [95% CI]) of completing the HCV regimen were not significantly greater with the lower priced version velpatasvir/sofosbuvir  or ledipasvir/sofosbuvir  than with the original NDC.The proportion of patients completing their original NDC HCV medication full regimen was 92% and 96% for velpatasvir/sofosbuvir and ledipasvir/sofosbuvir, respectively , suggesting that patients with high deductible plans may not benefit from reduction of rebates for higher cost drugs. This may be due to the fact that the cost of the HCV medications were likely well over most patient deductible amounts while those patients using PCSK9i whom only filled a single or two prescriptions may still be under their deductible amount. While those utilizing PCSK9i used less of their deductible on the cost of the drug at the new lower priced NDC, it is unknown whether they would still use their full deductible amount owing to the use of other medical services. Additional research is needed to further understand if OOP costs due to overall utilization of healthcare services would differ, despite using less of the deductible on drug costs.Previous studies have demonstrated that small changes in patient OOP costs may result in substantial changes in adherence to therapy. A prior study of medication adherence in veterans at US Veterans Affairs Medical Centers found that even a US$5 copayment increase (from US$2 to US$7) led to poorer treatment adherence for diabetes, hypertension and hyperglycemic medications . Among aThis study has a number of limitations to consider. First, limited information was available on actual rebate amounts for individual payers, so public statements from manufacturers , 19 and List price reductions to that of net prices resulted in lower OOP costs, suggesting that future insurance benefit design reform that involves reduction of rebates and/or changes in benefit designs may also potentially result in lower OOP costs for patients; however, further transparency across the drug supply chain is needed to determine the extent to which additional savings could be realized.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 FigPatient attrition for A) PCSK9is  and B) HCV medications (velpatasvir/sofosbuvir and ledipasvir/sofosbuvir).(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.


Adana phlebovirus, differed in the M segment from the Ponticelli I and Ponticelli II viruses. Analysis performed on the genomic segments of the newly isolated virus compared with other phleboviruses highlighted a strong purifying selection in the L segments, and different substitution saturation, highest in the M segments. Future research should address the ecological processes driving the occurrence of these novel phleboviruses and their possible impact on public health.The number of newly described sandfly-borne phleboviruses has been steadily growing in recent years. Some phleboviruses are human pathogens, but their health relevance is largely uncharacterized. We aimed to investigate the circulation of these viruses in the Emilia-Romagna region where several have already been described. A total of 482 sandflies were collected in a site in Reggio Emilia in 2019 and 2020. Sandflies collected in 2020 were grouped in 21 pools with a maximum of 25 sandflies per pool, submitted to real time PCR, and isolated in Vero cell culture. Complete genome sequencing showed the isolation of a strain of a Ponticelli III virus. This virus, which belongs to the species  Phenuiviridae includes 20 genera, the members of three of which are largely transmitted by arthropods: Phlebovirus, Uukuvirus and Ixovirus [Phlebovirus comprises about 70 named viruses widely distributed in Europe, Africa, Central Asia and the Americas. As provided by the International Committee on Taxonomy of Viruses (ICTV), these viruses are currently classified into species according to the threshold of 95% identity in the amino acid sequence of the RNA-dependent RNA polymerase (RdRp) protein [The family Ixovirus ,2. The g protein . Some of protein .Phleboviruses are single-stranded negative-sense RNA with a genomic organization consisting of three segments, designated S , M (medium), and L (large). The S segment bears two genes encoding, by an ambisense strategy, the nucleoprotein (Nc) and non-structural (Ns) protein; the M segment encodes for enveloping the glycoproteins; and the L segment for the RdRp ,6. PhlebPhlebovirus infections [In the Old World, at least 250 million people have been exposed to fections ,10. One fections .Sandfly-borne phleboviruses’ distribution and circulation peaks are closely related to vector presence and activity . SandfliPhlebovirus circulation with newly discovered viruses, many of which were detected in sandflies [Phlebotomus (Ph.) tobbi, Ph. perfiliewi, and Ph. papatasi [Recently, several studies conducted in Mediterranean countries showed an increase in andflies ,16. One papatasi . The genpapatasi . Conspecpapatasi , in Croapapatasi .In our current study, we report a new isolation of a Ponticelli III virus (PONTV III) strain in Emilia-Romagna at about 80 km from the site of the first report of this virus in the region .Samples were collected in June 2019 and September 2020 in Casalgrande locality , Reggio Emilia province. The site was located in a hilly area of the Emilia-Romagna region at 257 m altitude . Sandflyg for 15 min. Samples were inoculated in a confluent monolayer of VERO cells (African green monkey kidney cells) at passage 170 , incubated at 37 °C with 5% CO2 and observed daily for 7 days to observe the development of cytopathic effects (CPE) [−8). One of the obtained duplicates was recultured on VERO cells and subjected to sequencing.Female specimens pools were homogenized in minimal essential medium, supplemented with penicillin and streptomycin, using a sterilized glass potter and clarified by centrifugation at 3000× ts (CPE) . To exclhttps://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 28 January 2023).Viral RNA was extracted from supernatant cultures that displayed CPE using TRIzol LS Reagent ; cDNA synthesis was performed using random hexamers  and SuperScript™ II reverse transcriptase . Complementary DNA was submitted to PCR for the detection of phleboviruses (Pan-Phlebo PCR) using one reverse and two forward primers and targeting a 370-nucleotide region of the S segment . The obtThe selected viruses isolated from the cell culture supernatant were sequenced on a MiSeq Instrument . The preparation DNA libraries for whole-genome sequencing and viral RNA amplification were performed following the Kampmann et al. (2011) protocol .p value of 0.05) was used, FEL infers nonsynoymous (dN) and synonymous (dS) substitution rates using a maximum-likelihood (ML) approach for a given coding alignment [The complete genome of the segments was aligned with the homologous sequences of other phleboviruses available in GB. The nucleotide alignments were obtained using the translated amino acidic sequence with the Mega11 software   in Casalgrande locality, Emilia-Romagna region, Italy. Only some specimens collected in 2019 were identified using morphological characteristics; of these, 72 were niciosus . The 135niciosus . CPE obsniciosus . This saAccording to the previously obtained sequences, and particularly the M sequence, the new isolate was classified as PONTV III, strain 270315. Obtained sequences were deposited in GB by the accession numbers OP293791 for the L segment, OP293792 for the M segment, and OP293793 for the S segment. These sequences showed high identity for the PONTV II 238134-4/2016 (MG911981) in the L segment (99.13%), PONTV III 238134-3/2016 (MG911977) in the M segment (99.46%), and PONTV III 195684-2/2016 (MG911986) for the S segment (98.33%).Sequences of the three segments were used with homologous sequences of sandfly- borne phleboviruses from the Mediterranean and Middle East that were available in GB for phylogenetic analysis. Two alignments were obtained with L and M segments  and one None of the putative recombination events were detected by all three methods selected by RDP5 software in the alignments obtained by sequences of L and M segments.Adana phlebovirus species, according to the 95% identity threshold. The phylogenetic tree obtained for M segments , 63% in the S segment (293 on 464 non invariant sites tested), and 73% in the M segment (947 on 1295 non invariant sites tested). The FEL analysis identified only one of the sites under diversifying positive selection in segment M. Plotting nucleotide p-distance against aminoacid distance, we observed that the different segments seemed subject to a differential dynamic of evolution, with an increasing amino acidic and nucleotide diversity in M segments, while L segments showed less aminoacid diversity for the same amount of nucleotide diversity to the other segments . The anaThe saturation plot showed a higher relative number of transversions in the M segment, which exceeded that of transitions with the increase in GTR distance. This situation strongly suggested a substation saturation in the M segments .Pipistrellus kuhlii) in regions of Italy where several TOSV strains are circulating. Recently, positive sera of TOSV and SFSV measured using a seroneutralization test were detected in European bat colonies (free-tailed bats (Tadarida teniotis) and serovar bats (Eptesicus serotinus)) located in the Aragon and the Balearic Islands regions [Bregalaka virus (BREV) neutralizing antibodies were detected in 3.3% of human sera in North Macedonia, demonstrating that BREV frequently infects humans [Detection of phleboviruses in hosts is made difficult by the overall diversity and genetic complexity of clinically relevant strains, their essentially undefined clinical associations, and the resulting lack of diagnostic tools . A parad regions . The pos regions . Moreoves humans . Interesphlebovirus [Orthobunyavirus genus, in which reassortant viruses are common, but with the relevant difference that the donor virus of the M segment is often known [In phleboviruses, the same L and S segments could be associated with different M segments. The presence of viruses differing in M segments was already recorded in two group of phleboviruses beyond the PONTV, Massilia, Arrábida and Granada viruses ,33,34,35ebovirus  in Southen known . The greResults obtained by the analysis of the PONTV indicated that the three segments were differentially saturated, with the M segment more variable and the L segment more preserved. More work is needed to understand if different saturations of the segments could be caused by a different level of purifying selection and consequent extinction of deleterious genotype, or if other genetic mechanisms are potentially creating this different evolutionary dynamic.In the M segment, transversions occur more frequently than transitions, indicating a saturation of substitutions. The characterization of the mechanism at the base of this condition was hampered by the variability in M segment. While it is difficult to reconstruct recombination events that could have happened in the saturated condition of the M segment, we failed to detect a recombination signal between sequences of phleboviruses in this study. Even if it is not possible to completely exclude recombination events, this result argues in favor of the rarity of these events, as already highlighted in negative senses RNA viruses ,39. ThisThe M segment, which encodes the envelope proteins, was notably more saturated. We hypothesize that this increased variability can favor the escape of the virus from the hosts’ immunity and allow its occupation of different ecological niches. This appears to be confirmed by the different serological reaction in humans and animals of the three PONTV bearing different M segments ,40.Adana phlebovirus species. This is a new isolation of PONTV III from sandflies. Further studies should examine the host and epidemiological range of the virus in the field, especially whether the virus can infect and be pathogenic for humans, livestock, and other animals.In this study, PONTV III was isolated from sandflies collected in the field. The genomic structure, nucleotide and amino acid sequence homology, and molecular genetics evolution analyses of the virus confirmed that it is a virus of the


Maternal mental health conditions (MMHCs), which include depression and anxiety disorders during pregnancy and through five years postpartum, are among the most common obstetric complications in the United States overall and in Texas in particular. In the context of potential expansion of postpartum Medicaid coverage from 60 days to one year, we sought to capture the societal, financial burden of untreated MMHCs.We estimated the economic impact of untreated maternal mental health conditions (MMHCs) among births in Texas in 2019 using a cost-of-illness model.We found that MMHCs affected 13.2% of mothers and, when left untreated, cost $2.2 billion among mothers and children born in Texas in 2019 when following the birth cohort from conception through five years postpartum. We found that MMHCs affected 17.2% of mothers enrolled in Texas’ Medicaid for Pregnant Women and cost $962 million. In addition, the prevalence of MMHCs and resulting costs varied considerably among women of different races and ethnicities. Employers and health care payers, including Medicaid, bore most of these costs.The Texas Health and Human Services Commission’s (HHSC) efforts to increase awareness about MMHCs and increase access to care represent an important step toward improving maternal and child health and maximizing benefits to Texas HHSC, employers, and insurers.The online version contains supplementary material available at 10.1186/s12884-022-05001-6. Maternal mental health conditions (MMHCs), which include depression and anxiety disorders during pregnancy and through five years postpartum, affect at least 1 in 8 pregnant and postpartum women in the U.S. and are among the most common obstetric complications in Texas , 3. In fIn Texas, legislators and health insurers have paid increasing attention to MMHCs, as evidenced by the Texas Health and Human Services Commission’s (HHSC) September 2020 release of a plan to address postpartum depression . To suppIn Luca et al. (2020), we developed a conceptual framework to quantify the economic impact of untreated MMHCs nationally Fig.  6]. In . In 6]. Our mathematical model accounts for costs incurred by the mother–child pair from conception through five years post-delivery to highlight the immediate associations most relevant to the public and to policymakers. Since exposure to MMHCs can also impact a child’s well-being in the longer term, our estimates should be interpreted as a lower bound on the true cost of untreated MMHCs in Texas.To build our cost-of-illness model, we focused on Texas-specific maternal and child outcomes linked to MMHCs in the literature and recognized by subject matter experts. We estimated societal costs incurred by mothers and their children born in 2019 and projected these costs from conception through the child’s fifth birthday. We accounted for three types of direct and indirect costs: (1) maternal income loss due to lower labor force participation and productivity losses; (2) increased use of public assistance, including Supplemental Nutrition Assistance Program (SNAP), Special Supplemental Nutrition Program for Women, Infants and Children (WIC), and Medicaid; and (3) higher health care costs due to worse maternal and child health.We synthesized evidence on the associations between untreated MMHCs and maternal and child outcomes. We compiled data from peer-reviewed literature and secondary data sources to determine model inputs, which included: (1) the prevalence of MMHCs in Texas; (2) measures of association to measure the incremental effect of exposure to untreated MMHCs relative to no exposure; and (3) costs and baseline rates for each outcome.We conducted a systematic literature search following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines to obtain measures of association on the relationship between exposure to untreated MMHCs and maternal and child outcomes . We applWe identified 111 relevant articles that (1) controlled for confounding variables or used a matched comparison group design, 2) focused on outcomes occurring between conception and the child’s fifth birthday, and (3) estimated associations in numeric or monetary terms. Supplementary Figure  focused We estimated the prevalence of MMHCs in Texas and the prevalence or incidence of each outcome in the Texas population. In selecting estimates, we prioritized recency and relevance to pregnant and postpartum women and children ages 0 to 5. For example, the baseline rates of absenteeism, presenteeism, and unemployment apply to reproductive-age women or women with young children. Supplementary Table To calculate the total cost of MMHCs in Texas, we estimated the incremental cost of each outcome attributable to exposure to untreated MMHCs. We estimated baseline costs using peer-reviewed literature and data from the Centers for Disease Control and Prevention (CDC), Bureau of Labor Statistics (BLS), and other government agencies. We collected granular estimates of direct and indirect costs from the literature, accounting for both immediate and downstream costs, and excluded overlapping cost components to avoid overstating costs. We standardized costs to annual units and converted costs to 2019 dollars using the medical component of the consumer price index (CPI) for medical costs and the CPI for all urban consumers, less food and energy, for non-medical costs. Supplementary Table Using Microsoft Excel, we collated estimates and developed a cost-of-illness model to quantify the costs of MMHCs among Texas mothers giving birth in 2019 and their children , and suboptimal breastfeeding are potential outcomes that occur once, in Year 0 (from conception through the first year postpartum).Costs of productivity loss, suicide, non-obstetric healthcare, child injury, emergency department visits, and missed well-child care visits are incurred annually until the mother achieves remission, and then fall to $0.Two-thirds of women, whether treated or untreated, achieve remission at the end of Year 0. Based on a meta-analysis by Vliegen et al. (2014), the proportion achieving remission each year is constant across years .The association between untreated MMHCs and child behavioral and developmental disorders, asthma, and obesity remains constant regardless of whether the mother achieves remission.We projected costs through five years postpartum by assuming that:We then projected costs through five years postpartum by calculating the percentage change between the December 2018 and December 2019 medical CPIs. Based on the percentage change, we assumed that medical costs increased by 4.53% annually beyond Year 0 . We summTo understand the sensitivity of our cost estimates to variation in prevalence, cost, and measures of association, we conducted one-way deterministic sensitivity analyses, calculating differences from the main model as we varied each parameter from the lowest to highest value identified in the literature. .The total estimated cost of exposure to untreated MMHCs in Texas was $2.2 billion for the 2019 birth cohort, accounting for costs incurred in the six years between conception and five years post-delivery , non-obstetric health expenditures ($445.1 million), and obstetric-specific health expenditures for excess cases of preeclampsia ($36.8 million), cesarean section ($55.2 million), and long peripartum inpatient stays ($31.3 million). The remaining 45% of costs are attributable to child outcomes. The highest-cost child outcomes over the six model years were child behavioral and developmental disorders ($556 million), preterm births ($372 million), and asthma ($33 million). Although missed well-child care visits could partially offset these costs in the short term (-$11.2 million), they are likely to lead to higher costs in the longer term due to worse child health.The total estimated cost of exposure to untreated MMHCs among Texas Medicaid for Pregnant Women enrollees giving birth in 2019 was $962 million, accounting for costs incurred in the six years between conception and five years postpartum  yielding the highest cost. The remaining 65% of costs are attributable to child outcomes, with child behavioral and developmental disorders ($345 million) and preterm birth , followed by Hispanic mothers (12%) and non-Hispanic White mothers (11.4%) [8.2%, folThe total estimated cost of untreated MMHCs among non-Hispanic White mother–child pairs was $599 million for the 2019 birth cohort, accounting for costs incurred over six model years. Assuming that 13,896 of the 121,899 Non-Hispanic White mothers (11.4%) who gave birth in 2019 had an untreated MMHC [The total estimated cost of exposure to untreated MMHCs among non-Hispanic Black mother–child pairs was $521 million. Assuming 8,448 of the 46,420 non-Hispanic Black mothers (18.2%) who gave birth in 2019 had an untreated MMHC, these conditions cost $61,671 per affected non-Hispanic Black mother–child pair over six years. In this population, 65% of these costs relate to maternal outcomes, with productivity losses ($189 million) and non-obstetric health expenditures ($118 million) yielding the highest costs. The remaining 35% of costs relate to child outcomes, with child behavioral and developmental disorders ($94 million) and preterm birth ($74 million) yielding the highest costs.Finally, the total estimated cost of exposure to untreated MMHCs among Hispanic mother–child pairs was $928 million. Assuming 21,421 of the 178,509 Hispanic mothers (12%) who gave birth in 2019 had an untreated MMHC, these conditions cost $43,322 per affected Hispanic mother–child pair over six years. In this population, 55% of these costs relate to maternal outcomes, with productivity losses ($229 million) and non-obstetric health expenditures ($211 million) yielding the highest costs. The remaining 45% of costs relate to child outcomes, with child behavioral and developmental disorders ($239 million) and preterm birth ($149 million) yielding the highest costs.The magnitude of the disparities in the prevalence and cost of untreated MMHCs suggests ample room for policy interventions to ensure more equitable access to screening and effective treatment options. In the long run, these interventions could reduce suffering caused by untreated MMHCs as well as reduce costs to employers, the health care system, insurers, and the government.The sensitivity analysis showed that our model estimated a range of $739 million to $4.3 billion for the 2019 Texas birth cohort through varying all prevalence and impact estimates at once . Although we focused on immediate associations in the six-year period between conception and five years post-delivery, we recognize that MMHCs can have long-term impacts on mothers and their children. For example, children who develop behavioral and developmental disorders due to exposure to MMHCs could have lower educational attainment. Our model does not account for the increased prevalence of MMHCs arising from the COVID-19 pandemic. We did not account for the impact of MMHCs on nonmaternal caregivers, though we recognize they may also have mental health conditions. In addition, most of the literature on the impacts of untreated MMHCs focused on maternal depression rather than maternal anxiety. If maternal anxiety impacts outcomes independently of maternal depression and a mother has both anxiety and depression, the model may underestimate costs of untreated MMHCs . As a reAdditionally, we did not account for treatment options and did not distinguish between untreated and inadequately treated MMHCs, so did not make the economic case for treatment, or estimate potential cost savings through treatment of MMHCs. Finally, we did not analyze primary data, but rather used inputs from the literature and secondary data sources.To inform financial and policy approaches to improving screening for and treatment of MMHCs in Texas, we estimated the total societal cost of untreated MMHCs, as well as costs among mothers enrolled in Texas Medicaid for Pregnant Women and their children. To uncover potential disparities in disease burden, we also quantified costs among non-Hispanic White, non-Hispanic Black, and Hispanic mother–child pairs.We found that untreated MMHCs generated excess costs of $2.2 billion relative to cases in which MMHCs were not experienced among Texas mothers and children in the 2019 birth cohort. This amounts to more than $44,000 per mother–child pair from conception through the child’s fifth birthday. Nearly half of the total costs are incurred before the child’s first birthday, and overall, 55% of costs are attributable to maternal outcomes and 45% of costs are attributable to child outcomes.Among births covered by Texas Medicaid for Pregnant Women, we found that the total cost of exposure to untreated MMHCs was $962 million, or $31,071 per pair, excluding costs of productivity loss, maternal suicide, and SIDS. Maternal outcomes, especially non-obstetric health issues ($276 million), accounted for 35% of total costs. Child outcomes, especially child behavioral and developmental disorders ($345 million) and preterm birth ($231 million), accounted for 65% of costs.Given termination of coverage through this program after 60 days postpartum, low income eligibility requirements, and the fact that 57% of costs are incurred beyond the birth year, Medicaid directly bears only a small portion of these costs. If a mother cannot afford to purchase health insurance and does not meet Medicaid income eligibility requirements, the health care system could bear the costs of adverse health outcomes for mother and child. If a mother can afford to purchase insurance through the Affordable Care Act Marketplace, insurers may bear these costs.We also found evidence of substantial disparities in the disease burden of untreated MMHCs among mothers of different racial and ethnic backgrounds. From conception through five years postpartum, untreated MMHCs cost $599 million among non-Hispanic White mother–child pairs , $521 million among non-Hispanic Black mother–child pairs , and $928 million among Hispanic mothers . The distribution of costs for non-Hispanic White and Hispanic mother–child pairs was comparable to the distribution among the general population, but for non-Hispanic Black mother–child pairs, the distribution was more heavily skewed toward maternal outcomes .Our study shows that failing to treat MMHCs imposes a heavy cost on society ($2.2 billion) in Texas. It also shows that employers, through reduced employee productivity, and health insurers, through worse maternal and child health, bear most of these costs.We also found substantial variation in both the prevalence and cost of MMHCs among mother–child pairs of different racial and ethnic backgrounds. These disparities in disease burden may be attributable to poorer access to screening and high-quality care among women impacted by systemic racism and socioeconomic disadvantage and highlight a need for targeted interventions. Improving access to screening and treatment for MMHCs among women of all races and ethnicities would help increase women’s productivity, reduce the need for high-cost maternal and pediatric health care, and decrease the need for public assistance. This would lead to lower costs to Texas HHSC, employers, insurers, and the health care system, and would help support the health of Texas mothers and their children for generations to come.Additional file 1: Supplementary Figure 1. Literature review: PRISMA flowchart of article selection. Supplementary Table 1. Literature Review: search terms. Supplementary Table 2. Effects of exposure to MMHCs. Supplementary Table 3. Prevalence of MMHCs. Supplementary Table 4. Studies and data sources used to inform the cost estimates used in the main model. Supplementary Table 5. Model Inputs (main model): Parameters and costs used to estimate the economic impact of untreated MMHCs among 2019 births. Supplementary Table 6. Model inputs (women enrolled in Texas Medicaid for Pregnant Women): Parameters and costs used to estimate the economic impact of untreated MMHCs among 2019 births. Supplementary Table 7. Model results for costs of untreated MMHCs (in millions of dollars) for the 2019 birth cohort: Non-Hispanic White mothers. Supplementary Table 8. Model results for costs of untreated MMHCs (in millions of dollars) for the 2019 birth cohort: Non-Hispanic Black mothers. Supplementary Table 9. Model results for costs of untreated MMHCs (in millions of dollars) for the 2019 birth cohort: Hispanic mothers. Supplementary Table 10. Model inputs : Parameters and costs used to estimate the economic impact of untreated MMHCs among 2019 Births.