Schizophrenia imposes significant economic burden on patients, families, caregivers, and society. To our knowledge, place of care and associated costs of acute schizophrenia episodes have not been well characterized.To describe the care settings and costs associated with likely acute episodes and untreated remission periods among patients with schizophrenia.Adults with schizophrenia were identified using the IBM MarketScan Commercial and Medicare Supplemental databases (2009-2018); claims for capitated benefits plans were excluded. Acute episode index date was defined as at least 1 inpatient schizophrenia claim or outpatient schizophrenia claim . Mental health–related medical costs  associated with acute episodes were collected over a 2-month follow-up period and stratified by setting (inpatient vs outpatient); acute episode data were reported in subgroups of patients without or with prior clozapine use, as an indication of disease severity. Remission index date was defined as at least 1 outpatient claim with a schizophrenia diagnosis with no acute episode and no oral or injectable antipsychotic therapy. Remission costs were assessed over a 3-month period. All data were analyzed descriptively.A total of 14,824 patients with schizophrenia met criteria for an acute episode . Most acute episodes were treated in an outpatient setting . When treated inpatient, mean (SD) episode medical costs were $17,045  for all patients, $16,060  for those without prior clozapine use, and $22,827  for those with prior clozapine use. When treated outpatient, mean (SD) medical costs for acute episodes were $2,478  for all patients, $2,609  for those without prior clozapine use, and $1,770  for those with prior clozapine use. For all patients with acute episodes, regardless of clozapine use, patient-incurred out-of-pocket costs were approximately 30% of total medical costs. For an untreated period of remission, 6,950 patients with schizophrenia met criteria. Total medical costs were $2,399 for these patients over a 3-month period.The majority of acute schizophrenia episodes were treated in the outpatient setting. For episodes that required inpatient care, inpatient episodes were approximately 7 times more costly than episodes treated in outpatient-only settings. For acute episodes and remission periods, health plans covered most costs; however, there were additional patient-incurred out-of-pocket costs. This study looked at the care setting and costs during acute schizophrenia episodes and periods with no symptoms. The results showed that episodes are mostly treated in outpatient settings. Inpatient costs were 7 times higher than outpatient costs. Pharmacy costs were lower than medical costs in both inpatient and outpatient settings. Health insurance plans covered most costs. Implications for managed care pharmacyIn this claims-based study, we evaluated the place of care and associated costs of acute schizophrenia episodes and periods of remission. Although most acute episodes were managed in the outpatient setting, episodes with inpatient care were approximately 7 times more costly than outpatient-only episodes. Regardless of setting, pharmacy costs were considerably lower than medical costs. These findings should inform managed care policy by encouraging access to effective schizophrenia medications to potentially avoid costly inpatient services.1 For most patients with schizophrenia, the course of illness is characterized by multiple episodes of relapse,2 which can worsen long-term disease outcomes.3 In addition, schizophrenia places significant economic burden on patients, families, caregivers, payers, and society.4 In 2013, the annual health care costs associated with schizophrenia exceeded $155 billion in the United States alone.5 Approximately one-quarter of these costs were attributed to direct health care costs, which included inpatient and outpatient services, emergency department (ED) visits, long-term care, medications, and other medical services. Inpatient services accounted for $15.2 billion of the total excess costs, which was over twice those of outpatient services ($7.4 billion). Moreover, patients with schizophrenia incur higher health care costs than those without schizophrenia. For example, in a study that compared the costs of care between Medicare beneficiaries with and without schizophrenia, those with schizophrenia had 80% higher per-patient-per-year costs.6 In an analysis of commercially insured patients, the average total claim cost per patient with schizophrenia was over 4 times that of a demographically adjusted population without schizophrenia.7Schizophrenia is a severe, chronic, and heterogenous psychiatric disorder that affects approximately 3.5 million people in the United States.3 For both acute and maintenance treatment of schizophrenia, antipsychotic medications have demonstrated efficacy vs placebo and are considered the mainstay of treatment.10 Per the American Psychiatric Association treatment guidelines, which recommend antipsychotic medication use as first-line treatment in patients with schizophrenia,11 patients should be monitored for efficacy and side effects during antipsychotic use and should continue their medication if symptoms improve. Adverse effects (AEs) associated with antipsychotics include weight gain,12 hyperprolactinemia (elevated prolactin levels),13 metabolic changes,14 and extrapyramidal symptoms.8 Atypical antipsychotics are often preferred because they tend to cause fewer extrapyramidal symptoms than conventional, typical antipsychotics.16 According to the American Psychiatric Association guidelines,11 clozapine should be reserved for more severe illness, such as cases of treatment resistance or if the patient remains at substantial suicide risk despite other treatments17; consequently, clozapine use is generally infrequent among patients with schizophrenia (< 5%)18 and likely signals greater schizophrenia severity.The clinical and economic consequences of relapse underscore the importance of achieving the long-term goals of schizophrenia management, which include remission and relapse prevention.20 particularly if they are exhibiting aggressive behavior or are at risk of harming themselves or others.22 Gaps in antipsychotic medication use is also a predictor of inpatient admission.23 Patients with a recent relapse  incur approximately 3 times greater annual direct mental health costs and significantly greater annual hospitalization costs following the relapse than patients without a recent relapse.24 Overall rates of schizophrenia-related hospitalizations significantly increased from 2005 to 2014 in the United States despite decreases in the average length of stay.25 Setting aside inflation as a contributing factor, authors hypothesized that the increase was due to poor medication adherence among younger patients with schizophrenia that led to more inpatient visits and subsequently higher inpatient costs.Inpatient services account for a substantial portion of the costs associated with schizophrenia care. During acute schizophrenia episodes, patients may experience active psychosis that requires ED visits and subsequent hospitalizations,Although it is known that there are considerable direct costs associated with schizophrenia, there are limited published data reporting the setting (inpatient vs outpatient) in which patients are treated for an acute episode and the associated costs. To address this knowledge gap, the objective of this study was to describe the place of care and estimate health-related costs associated with acute episodes, as well as untreated remission periods among patients with schizophrenia.Adults (aged ≥ 18 years) with schizophrenia were identified using the IBM MarketScan Commercial and Medicare Supplemental databases . The Commercial database included medical and pharmacy claims for more than 225 million patients from 300 contributing employers and 40 contributed health plans across the United States. The Medicare database contained enrollment and health care claims of 6.4 million retirees and people with disabilities with Medicare insurance. All included patients were continuously enrolled for at least 12 months prior to and 6 months after the index date. The study was divided into 2 cohorts: patients in an acute treatment phase (acute episodes) and patients in a stable, no treatment phase (remission).Supplementary Table 1, available in online article), place of service, and frequency of service within a short duration (2 weeks). An acute episode index date was defined as the first claim date in patients who met any of the following criteria: at least 1 inpatient schizophrenia claim or at least 1 outpatient claim at an urgent care facility, outpatient hospital (on campus), ED , skilled nursing facility, nursing facility, ambulance (land), or comprehensive inpatient or outpatient rehab facility or at least 3 outpatient claims on 3 distinct days within a 2-week period for services associated with pharmacy, telehealth, office, patient or group home, assisted living facility, outpatient hospital (off campus), custodial care facility, independent clinic, federally qualified health center, psychiatric facility partial hospitalization, community mental health center, intermediate care/intellectual disability, residential or nonresidential substance use facility, psychiatric residential treatment center, state/local public health clinic, rural health clinic, or outpatient (not elsewhere classified). A schizophrenia claim was defined as any claim with an International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9) or ICD-10 code for schizophrenia in any position.To identify acute episodes of schizophrenia, a novel, claims-based approach was used. Guided by expert opinion, a combination of the following factors and criteria were determined to be suggestive of acute episodes: schizophrenia diagnosis codes  in the baseline period. For acute episodes, the main cohort  was evaluated as well as subgroups of patients with or without prior clozapine use (defined as the presence of ≥ 2 prescriptions for clozapine in the baseline period on separate days), with clozapine use as an indication of greater disease severity.Supplementary Table 2). Patients in this remission cohort were also required to have at least 1 inpatient or outpatient claim with a schizophrenia diagnosis (excluding schizophreniform) in any position during the baseline period.The remission index date was defined as the first claim date in patients who had at least 1 outpatient claim with a schizophrenia remission diagnosis (ICD-9) or a schizophrenia stable, no treatment diagnosis (ICD-10) with no acute episode. Patients also could not have any oral or injectable antipsychotic therapy claims in the 6-month follow-up period  and health plan–only payments. For analysis of pharmacy costs, the pharmacy was considered the provider. For acute episodes, cost outcomes were assessed for 2 mutually exclusive time periods: (1) acute episode  and (2) 2-month follow-up, which began the day after discharge for inpatient acute episodes or after the 2 weeks following the first outpatient claim for outpatient acute episodes. Costs for these periods were stratified by setting of the acute episode (inpatient vs outpatient). For acute episodes, mental health–related pharmacy costs were additionally classified by the following medication subgroups: oral atypical antipsychotics, nonoral atypical antipsychotics, typical antipsychotics, and other mental health–related medications . Multiple episodes per patient were included in the analysis. Additionally, for acute inpatient episodes, pharmacy costs observed between the dates of hospital admission and discharge were included in addition to the inpatient admission costs. Further, mental health–related costs were assessed during remission periods, which had a duration of 3 months. Demographics and baseline characteristics were assessed descriptively. Place of care and cost data described herein were analyzed descriptively and presented as mean mental health–related costs.Supplementary Figure 1). A total of 6,950 patients met the criteria for remission.A total of 14,824 patients with schizophrenia met the criteria for an acute episode, of whom 12,896 (87.0%) were included in the “without prior clozapine use” cohort and 1,427 (9.6%) in the “with prior clozapine use” cohort  were treated in an outpatient setting, regardless of clozapine use . For thoFor acute episodes occurring in the inpatient setting, total mean (SD) mental health–related medical costs were $17,045  for all patients, $16,060  for patients without prior clozapine use, and $22,827  for patients with prior clozapine use . Mean S. Mean . Oral atypical antipsychotic costs were between 60% and 70% of total pharmacy costs for all patients or patients without prior clozapine use and more than 70% of total pharmacy costs for patients with prior clozapine use. For all patients, regardless of clozapine use, health plans covered more than 85% of pharmacy costs. The percentage of patient-incurred out-of-pocket total pharmacy costs was approximately 12% during both acute episodes and follow-up.Total mental health–related pharmacy costs for all patients during the acute episode and follow-up period were $197 and $806, respectively ; costs wSupplementary Figure 3). The bulk of these mental health–related costs (≥95%) were for medical visits.The 3-month outpatient mental health–related total costs for patients with an untreated period of remission were $2,501 and health plan–only costs were $1,602  associated with acute schizophrenia episodes, updating our understanding of the location of care and its associated costs. Using a novel, claims-based methodological approach, we analyzed claims data to define acute schizophrenia episodes. Key factors  were intentionally selected to identify likely acute episodes.Overall, the majority of acute episodes (approximately 75%) in adults with schizophrenia were treated in the outpatient setting. Interestingly, approximately 90% of acute episodes in patients with prior clozapine use, which represented a more likely severe disease state, were treated in an outpatient setting. Regardless of setting, mental health–related pharmacy costs associated with acute episodes were considerably lower than medical costs. The majority of pharmacy costs were for oral atypical antipsychotics, which would be expected because they are the primary treatment prescribed to patients with schizophrenia. During periods of remission, medical visits accounted for the majority of 3-month outpatient mental health–related costs. Health plans covered most medical and pharmacy costs both for acute episodes and for untreated periods of remission; however, patients also incurred out-of-pocket costs, which were approximately 30% of total medical costs and 12% of total pharmacy costs for patients with acute episodes.26 Given this high likelihood of schizophrenia relapse, the relatively long follow-up period (2 months in duration) used in this study was designed to capture the full extent of costs of care associated with acute schizophrenia episodes. Results indicated that, in both inpatient and outpatient settings, the service costs for managing acute episodes were distributed throughout the follow-up period. Approximately half of the total costs of each inpatient episode were incurred during the 2-month follow-up period after an inpatient stay, which suggests that calculating event costs only during inpatient hospitalization could underestimate true episodic costs by approximately 50%. In addition, follow-up outpatient costs were greater than costs incurred during the acute episode  for all patients, regardless of clozapine use. These results suggest that the costs and utilization of services associated with acute schizophrenia episodes are incurred over an extended period of time, regardless of the place of care. Therefore, study designs that exclude or use a shorter follow-up period may substantially underestimate the costs associated with acute schizophrenia episodes, especially for outpatient care. These findings may be relevant to future policy assessments, such as cost-effectiveness analysis modeling; in future studies, researchers should accurately reflect the place of service (inpatient vs outpatient) and account for follow-up costs to fully capture the true impact of schizophrenia relapse.Schizophrenia is associated with high rates of relapse, particularly following hospitalizations. For example, schizophrenia and other psychotic disorders are the leading cause of 7-day readmission and the second leading cause of 30-day readmission after an initial hospital stay.7 Similarly, in a study of Medicare beneficiaries with schizophrenia, more than half of the per-patient-per-year costs were attributed to psychiatric and medical hospitalizations, which occurred in approximately 30% of those with schizophrenia6Inpatient care services represent a considerable portion of the economic burden of schizophrenia in the United States. For example, Fitch et al reported that inpatient resources accounted for more than 40% of the per-patient-per-month costs in patients newly diagnosed with schizophrenia and that these costs were highest during the month following diagnosis, primarily because of high costs associated with inpatient care.In this study, mental health–related inpatient costs were approximately 7 times greater than outpatient costs among patients who required inpatient care for acute schizophrenia episodes.27 which threaten symptomatic stability and functioning28 and have been shown to be a predictor of hospitalization.23 It has been estimated that approximately half of patients with schizophrenia are treatment-nonadherent to some degree.30 Further, treatment nonadherence has been associated with increased use of emergency psychiatric services, hospitalizations, and higher inpatient care costs.31 In cases of partial adherence, medications with longer half-lives may prolong drug exposure, which can bridge gaps in medication intake. Nonadherence in schizophrenia is also multidimensional and can vary between patients, but the AEs associated with antipsychotic treatment are a potential barrier to adherence.33 Particularly distressing AEs that may contribute to nonadherence include sexual dysfunction due to hyperprolactinemia34 and weight gain.35 Given these issues, newer antipsychotic medications with better tolerability profiles may improve adherence and potentially reduce the significant costs associated with inpatient care. Future research is needed to identify effective strategies to reduce schizophrenia-related hospitalizations and minimize costly inpatient services.One potential strategy for decreasing costly inpatient services is to reduce medication nonadherence and partial adherence,The methods used for defining likely acute schizophrenia episodes represent a new approach that may overcome some of the methodological challenges associated with using claims data, which provide valuable information for evaluating care settings and costs. However, there are inherent limitations associated with claims database analyses, and the results of this study should be interpreted within these limitations. As with all claims data, there was potential for data coding and/or entry errors. Analysis was limited to individuals with commercial or Medicare coverage, and out-of-network treatments for schizophrenia or capitated costs were not captured. Therefore, the results may not be generalizable to patients with other insurance , no insurance coverage, or patients in fully capitated health plans. A minimum coverage period was required, which may have contributed to selection bias. Further, the claims database analysis did not include baseline comorbid conditions or adherence rates, both of which could contribute to patients being treated inpatient vs outpatient.In addition, episode and remission costs cannot be compared directly given the different follow-up periods . Acute episodes and untreated periods of remission were both identified using proxy criteria based on claims data via a novel algorithm that is not yet validated. Psychiatric diagnosis and visits were used as proxies for acute schizophrenia episodes, but we could not be certain that these patients actually experienced an acute episode. In addition, the ICD-10 lacks a code for remission so the “stable, no treatment” code was used, and the ICD-9 schizophrenia remission code may be infrequently used. Therefore, we can only assume that untreated periods of remission were adequately captured. Although both sets of proxy criteria were discussed with and approved by all investigators prior to the start of the study, it is possible that the criteria did not capture the full nature of schizophrenia episodes and remission. Further, length of diagnosis was not considered in the analysis, a factor which could have impacted outcomes. Finally, the relatively small number of patients with prior clozapine use  should be noted as a potential limitation. Because adjusted analyses were not performed, comparisons between the cohorts  should be interpreted with caution.The results of this study indicate that most acute schizophrenia episodes are treated in the outpatient setting. Although episodes that required inpatient care were less frequent, inpatient costs were approximately 7 times higher than outpatient costs. Pharmacy costs were considerably lower than medical costs, regardless of setting. Although health plans covered the majority of medical and pharmacy costs, there were additional patient-incurred out-of-pocket costs. This study is the first to estimate costs and setting of acute schizophrenia episodes, advancing our understanding of the location of schizophrenia care and the associated costs to the health care system.


Vaccine hesitancy has been deemed one of the top 10 threats to global health. Antivaccine information on social media is a major barrier to addressing vaccine hesitancy. Understanding how vaccine proponents and opponents interact with each other on social media may help address vaccine hesitancy.We aimed to examine conversations between vaccine proponents and opponents on Reddit to understand whether homophily in web-based conversations impedes opinion exchange, whether people are able to accommodate their languages to each other in web-based conversations, and whether engaging with opposing viewpoints stimulates higher levels of analytical thinking.t tests, and multilevel modeling to test 3 questions of interest.We analyzed large-scale conversational text data about human vaccines on Reddit from 2016 to 2018. Using deep neural network language models and computer-assisted conversational analyses, we obtained each Redditor’s stance on vaccines, each post’s stance on vaccines, each Redditor’s language coordination score, and each post or comment’s analytical thinking score. We then performed chi-square tests, 2-tailed P<.001). When Redditors interact with others who hold opposing views on vaccines, both provaccine and antivaccine Redditors accommodate their language to out-group members  and show no difference in analytical thinking compared with interacting with congruent views (P=.63), suggesting that Redditors do not engage in motivated reasoning. Antivaccine Redditors, on average, showed higher analytical thinking in their posts and comments than provaccine Redditors (P<.001).The results show that both provaccine and antivaccine Redditors are more likely to selectively respond to Redditors who indicate similar views on vaccines (This study shows that although vaccine proponents and opponents selectively communicate with their in-group members on Reddit, they accommodate their language and do not engage in motivated reasoning when communicating with out-group members. These findings may have implications for the design of provaccine campaigns on social media. The COVID-19 pandemic thrust vaccine hesitancy that has existed long before the pandemic entered the global spotlight. Vaccine hesitancy, the “delay in acceptance or refusal of vaccine despite of vaccine availability” , understWeb-based conversations, such as those about vaccines examined in this study, belong to a broader category of sociable conversations in which individuals discuss everyday interest- or leisure-related issues . SociablAlthough previous research has used different approaches to examine web-based conversations, including their frequency, valence, diffusion size, and speed, the complex nature of web-based conversations requires innovative theory and methods, particularly to understand conversations in a specific context, such as in public health . We lackTo fill the gaps in the extant literature on web-based conversations, particularly in the health context, we proposed a 3-facet approach to understanding web-based health conversations: homophily of the conversational networks, relational dynamics between parties in the conversation, and cognitive information processing in the conversation. Homophily is the tendency of people to interact with others who share a preexisting characteristic or attitude. In our study, homophily is the tendency of people who share similar attitudes about vaccines to interact more frequently with each other than with people who hold differing views. Thus, first, homophily of the conversational networks on social media potentially inhibits the formation of bridging social capital, which is crucial for obtaining information from different perspectives and seeking advice on complex health-related issues ,10. SecoTechnological affordances of social media have provided a myriad of messages and channels from which people may select. Decades of research have found that individuals tend to be consistent in their choices. Selectivity in a high-choice media environment and the tendency to remain consistent may lead to homophily; that is, people select attitude-congruent information, transmit attitude-consistent messages, and interact with like-minded individuals. Homophily is more likely to emerge as the intellectual specificity on which a community is built increases . Reddit echo chamber effect, a closed system in which people tend to seek out and disseminate information that reinforces their preexisting beliefs, attitudes, and behaviors, which is typically devoid of counterattitudinal perspectives [Homophily may lead to an pectives . Empiricpectives , includipectives . For exapectives  found ecpectives  found thpectives . Thus, wHypothesis 1: Redditors are more likely to selectively reply to others who share similar stances on vaccines.cultural bridges [power dynamics, through language coordination in web-based conversations, to examine conversations about vaccines on Reddit.Although homophily leads us to expect that both pro- and antivaccine Redditors are more likely to interact with those who share their opinions , they also interact with those who hold opposing views . We define Redditors who connect the provaccine and antivaccine groups by replying to posts within both groups as  bridges . Compari bridges . We focuaccommodation. Among different accommodative strategies, convergence is a specific accommodation strategy in which individuals adapt their communicative behaviors in linguistic , paralinguistic , and nonverbal features  to become more like the individual or group they interact with.The CAT predicts and explains the adjustments people make to manage social interactions . The conupward or downward convergence. Upward convergence describes people in subordinate positions accommodating their communicative behaviors to people in superordinate positions; downward convergence reverses the direction of the flow. The power distance between people primarily originates from 2 sources [Convergence is a function of the social power between 2 parties  and gene sources ,23: statThe CAT has been applied in the health domain. For instance, a laboratory experiment on accommodation behaviors in instant messaging conversations found a general tendency for convergence for both the length and duration of utterances about the human papillomavirus (HPV) vaccine . Althouglanguage coordination, a specific form of accommodation, in conversations about vaccines on social media. Empirical studies have shown evidence of persuasive intent on both the provaccine and antivaccine sides. A study analyzing antivaccine websites found that antivaccine websites are persuasive because they mix scientifically proximate misinformation  with value-based beliefs  [In this study, we examined duality) . Provaccduality) ,28. Thusduality) . TherefoHypothesis 2: Redditors who post about vaccines accommodate their language more when interacting with out-group members who hold different views on vaccines than when interacting with in-group members.analytical thinking to refer to the intentional and resource-demanding processing and intuitive thinking to refer to effortless and instinctive processing, following Epstein [Complex social and scientific controversies, such as the debate over vaccination, require critical thinking to evaluate scientific evidence and potentially reconsider one’s perspective . However Epstein . In addi Epstein . Individ Epstein . WhetherThe flip side of this phenomenon can be seen when people encounter arguments that are consistent with their preexisting beliefs, which they are more likely to accept uncritically ; that isIn the context of health, risk, and science communication, motivated reasoning potentially explains why people are susceptible to misinformation. Susceptibility to misinformation can ultimately be traced to people’s propensity to confirm previously held value-based beliefs and protect their social identities . This goScholars have called for more research that incorporates cognitive models into vaccination education  and examHypothesis 3: Analytical thinking is higher when people interact with out-group members who hold opposing views than when they interact with in-group members who hold similar views.We collected all posts and comments from the archive of Reddit hosted by Google’s BigQuery from January 1, 2016, to December 31, 2018. We then filtered the posts and their corresponding comments that are related to vaccines using a group of keywords  antivaccine message if the post contains an idea that would be useful to someone making an argument against vaccines, (3) provaccine author if we can tell the author of the post is provaccine, and (4) antivaccine author if we can tell the author of the post is antivaccine. We coded the message stance and the author stance separately because occasionally a post contains both provaccine and antivaccine information, but the author’s stance is clear despite the mixed information  of the posts for expert coders to code into four categories of content using four dummy variables: (1) provaccine The coded posts served as training data for the 4 supervised machine learning models corresponding to the 4 categories. We used the pretrained language model in natural language processing, Decoding-Enhanced Bidirectional Encoder Representations From Transformers With Disentangled Attention (DeBERTa). BERT is a pretrained language model developed by Google. Specifically, for each category, we fine-tuned the 4 DeBERTa models with the same hyperparameters based on DeBERTaV3   provaccine message and (2) antivaccine message. If a post only contains provaccine information , its post stance is a provaccine stance. Similarly, if a post only contains antivaccine information , its post stance is an antivaccine stance. If a post contains both provaccine and antivaccine information , its post stance is a 2-sided stance. If a post contains no opinions about vaccines or readers could not clearly identify its point of view , its post stance is neutral , we define the language coordination of author b toward author a as: defines the probability of author b uses marker m in the comment 2u directly replying to author a’s post or comment 1u that uses marker m, and  defines the probability of author b’s normal use of marker m in conversations with author a.where author b toward a group of authors A with the same stance on vaccines. Specifically, given a set of exchanges A,bS between author b and a group of authors aA. The set A,bS includes the posts or comments 1u of various authors aA and the comment 2u of author b. The language coordination of author b to the group A is as follows:The linguistic style markers include 8 categories of function words generated by the Linguistic Inquiry and Word Count (LIWC) dictionary , includiA,bS.where the probabilities in the generalized equation are estimated over ConvoKit (version 2.5.3) [We used the coordination feature of the Python package n 2.5.3)  to calcuAnalytical thinking of each post or comment was measured by the analytical score using LIWC , we focused on authors who posted at least once about vaccines and left at least 1 comment about vaccines.  We then filtered the comment authors and posts whose stance is either provaccine or antivaccine to test hypothesis 1. We obtained 13,899 posts and 89,347 comments to use to test hypothesis 1. We then matched the corresponding posts’ stance to that of the authors of the comments that replied to the posts. Finally, we performed a chi-square test of independence to test the relationship between the 2 variables: t tests: one compared how provaccine and antivaccine Redditors coordinate their language toward antivaccine Redditors, and the other compared how provaccine and antivaccine Redditors coordinate their language toward provaccine Redditors. We further filtered out provaccine Redditors and antivaccine Redditors who replied to both their in-group members and out-group members  and performed 2-tailed paired-sample t tests to examine whether the patterns of linguistic styles differed for the same Redditor when they replied to in-group or out-group members.To test whether Redditors with different opinions on vaccines are more likely to coordinate language toward their in-group members or out-group members (hypothesis 2), we filtered posts and comments whose stance is either provaccine or antivaccine and whose count of function words is not equal to 0, as function words are integral to our measure of language coordination. We obtained 14,865 posts and 84,544 comments that met the criteria for further analyses. After obtaining the language coordination score(s) for each author depending on whether they replied to their in-group members, out-group members, or both, we then performed 2 separate 2-tailed independent sample To test the interaction effect of post stance and comment authors’ stance on analytical thinking in the comments , we filtered the posts and comments whose authors are either provaccine or antivaccine and whose count of function words is not equal to 0. Similar to our analysis of language coordination scores, we only used posts and comments with nonzero function words because analytical thinking was measured based on function words. We further selected the comments that directly replied to posts but not to other comments to avoid the confounding effects of other comments on the target comments. We obtained 7777 posts and 14,978 comments for further analysis. We then used multilevel modeling to control for the unmeasured interdependence between comments nested within the same authors, the same posts, or the same post authors . Both provaccine and antivaccine Redditors are more likely to reply to their in-group members than to their out-group members. Hypothesis 1, which is about whether provaccine and antivaccine Redditors selectively reply to their in-group members than to their out-group members, is supported. The results of the chi-square test of independence show that there was a significant relationship between post stance and comment authors’ stance . Antivaccine Redditors are more likely to coordinate their language toward provaccine Redditors  than provaccine Redditors coordinating their language toward provaccine Redditors . Hypothesis 2, which is about whether provaccine and antivaccine Redditors are more likely to coordinate language toward out-group members than toward in-group members, is supported. Provaccine Redditors are more likely to coordinate their language toward antivaccine Redditors  than antivaccine Redditors coordinating their language toward antivaccine Redditors . In contrast, provaccine cultural bridges tend to coordinate their language more toward antivaccine Redditors  than toward provaccine Redditors . The difference in language coordination scores  is statistically significant . For Redditors who serve as cultural bridges, antivaccine cultural bridges do not show significant differences between replying to provaccine Redditors  and antivaccine Redditors  in terms of language coordination. The difference in language coordination scores  is not statistically significant (tP<.001). Analytical thinking in comments replying to provaccine posts was significantly lower than that in comments replying to antivaccine posts . We did not observe a significant interaction effect between post stance and comment author’s stance on analytical thinking in the comments ; that is, the difference in analytical thinking in the comments from provaccine Redditors and antivaccine Redditors does not depend on the stance of the posts that they reply to.Hypothesis 3, which considers whether Redditors with different opinions on vaccines are more likely to engage in analytical thinking when they reply to out-group members, is not supported. The results of the multilevel model also show that analytical thinking in the posts about vaccines is a significant predictor of analytical thinking in the comments. We visualized the trend of analytical thinking in the posts from 2016 to 2018 in In this study, we examined the content and linguistic patterns of posts and comments about vaccines on Reddit. The results show that provaccine and antivaccine Redditors are more likely to selectively reply to Redditors who share similar views on vaccines. However, when Redditors interact with others who hold opposing views on vaccines, both provaccine and antivaccine Redditors accommodate their language to out-group members. However, for cultural bridges who interact with both in-group and out-group members, only provaccine cultural bridges accommodate their language toward out-group members. In addition, provaccine and antivaccine Redditors do not show greater analytical thinking when interacting with out-group members, suggesting that Redditors do not engage in motivated reasoning when interacting with people who hold different stances on vaccines. Antivaccine Redditors, on average, have higher analytical thinking in posts and comments than provaccine Redditors. These findings help further our understanding of web-based conversations in the public health domain and have implications for future provaccine campaign design on social media.First, this study shows that both provaccine and antivaccine Redditors are more likely to comment on posts that share the same or similar views on vaccines. The results are consistent with previous research that shows that people are likely to seek health-related information on the internet that confirms prior beliefs  and thatSecond, our results show that provaccine and antivaccine Redditors accommodate their language when they interact with out-group members; that is, when both provaccine and antivaccine Redditors interact with those who hold opposing views, they tend to accommodate their linguistic styles accordingly, compared with the conversations with in-group members. This is a promising and optimistic finding, as listening to one another is the first step in any kind of effective communication. This indicates that Redditors with different opinions on vaccines display positive and healthy relational dynamics, which provides an open and connected environment to exchange arguments. However, when we filter cultural bridges who communicate with both in-group and out-group members, the results show a slightly different story. Compared with communication with in-group members, only provaccine cultural bridges accommodate language use when communicating with out-group members. This is consistent with what the CAT predicts would occur when people attempt to convince out-group members, which creates a power distance between those with opposing views ,23, and Third, the results of analytical thinking in posts and comments do not support our hypothesis that provaccine and antivaccine Redditors engage in motivated reasoning when they interact with others who hold different opinions about vaccines. These findings are partially consistent with the findings of Pennycook and Rand , who fouIt is important to note that the antivaccine Redditors’ comments and the comments replying to antivaccine posts, on average, involve more analytical thinking than their provaccine counterparts. In other words, web-based conversations about vaccines consistently stimulated more cognitive information processing among vaccine opponents than among vaccine proponents from 2016 to 2018. These findings are consistent with those of Faasse et al , who fouThis study contributes to the theoretical understanding of web-based conversations between people with opposing views through the lens of homophily, the CAT, and motivated reasoning. The 3 theories help deepen our understanding of web-based conversations between people with opposing views on controversial public health issues, as discussed earlier. This study also extends the CAT by testing whether people accommodate a particular type of communicative symbol  on social media, in which the many-to-many mode of communication changes the nature of communication. In addition, this study extends the literature on motivated reasoning by testing it in a real-world setting using social media data.This study has several important practical implications. First, the findings deepen our understanding of how vaccine proponents and opponents interact on social media. Although previous research has explored this at the macro level, there is a lack of deep understanding of micro level interactions between vaccine proponents and opponents. Our study fills this gap through the lens of language coordination and analytic thinking in posts and comments on vaccines on social media. Our findings provide an optimistic view that vaccine proponents and opponents accommodate their language and do not engage in motivated reasoning when interacting with people with opposing views. Public health practitioners can build on our research by designing campaigns that use social media to facilitate communication and mutual understanding between the 2 sides.Second, the findings offer potential explanations for why some social media–based provaccine campaigns do not achieve the expected effect and may have implications for the future design of strategic campaigns. We found that when communicating with others who hold opposing views on vaccines, provaccine cultural bridges accommodate language use, whereas antivaccine cultural bridges do not. This indicates that although antivaccine cultural bridges play a crucial role in the success of provaccine campaigns, they are not as proactive as they could be, as reflected by their lack of language accommodation. Future social media–based provaccine campaigns may want to target these cultural bridges to facilitate communication between the 2 sides. In addition, our findings suggest that provaccine posts have not successfully stimulated analytical thinking on either side. Low analytical thinking may hinder the understanding of information in provaccine campaigns. Future social media–based provaccine campaigns may want to design messages and corresponding visuals to increase analytical thinking among audiences. Third, we found that provaccine cultural bridges and general vaccine proponents accommodate their languages when interacting with vaccine opponents on social media. Future provaccine campaigns may include moderators or bots to proactively send provaccine messages in languages accommodated toward vaccine opponents.This study has several limitations that future research should examine. First, this study relies on a single item from the LIWC dictionary to measure analytical thinking. Although previous studies have used the analytical thinking score from the LIWC to answer various research questions -47, the This study examined the content and linguistic patterns of conversations about vaccines in Reddit from 2016 to 2018 using deep learning and computer-assisted conversational analyses. This study found that although people are more likely to reply to posts that share similar views, people accommodate their language styles and do not engage in motivated reasoning when they interact with people with opposing views. Theoretically, this study deepens our understanding of the nature of web-based conversations, particularly web-based conversations about vaccines. Practically, this study has implications for future social media–based provaccine campaign design, such as increasing analytic thinking in campaign messages and targeting cultural bridges to facilitate conversations between those who support and oppose vaccination.


Aedes (Stegomyia), other transmission routes have been reported: between sexual partners and from mother to fetus, as well as via the transfusion of blood and blood products [Zika virus (ZIKV) is a mosquito-borne flavivirus that was first identified in Uganda in 1947, then was essentially neglected for six decades. However, in 2007, a ZIKV outbreak emerged in the Western Pacific Ocean, and between 2013 and 2014, a larger outbreak spread in the South Pacific, reaching South America in 2015 [ZIKV infection usually causes a self-limited disease presenting acute febrile illness, but recent studies link the infection to a rising number of severe neurologic complications in adults, such as Guillain–Barré syndrome, myelitis, and meningoencephalitis, as well as congenital ZIKV infection that can result in Zika congenital syndrome including fetal damage and microcephaly in newborns . Despite efforts to find one, there is currently no commercially available vaccine or treatment to prevent or fight against this viral pathogen ,5, resulAs we have learnt from the recent pandemic caused by SARS-CoV-2, we cannot predict what might come next or when that might be, but it is clear that viruses can emerge unexpectedly and cause global human disease. All the challenges in, and possible contributions to, unveiling the pathogenic mechanism of ZIKV are welcome for the current Special Issue entitled “Mechanisms of Infection in Zika Virus”, which aims to analyze and discuss the development of diagnostic methods, prevention and control strategies, and genetic mechanism of the virus, provide solutions, and expand the knowledge covering all aspects of viral pathogenesis and host–virus interactions, essential steps in creating new strategies to fight the virus.


Aedes aegypti, several strains of the intracellular bacterium Wolbachia have been shown to inhibit the transmission of dengue, Zika, and chikungunya viruses, important human pathogens that cause significant morbidity and mortality worldwide. In addition to pathogen inhibition, many Wolbachia strains manipulate host reproduction, resulting in an invasive capacity of the bacterium in insect populations. This has led to the deployment of Wolbachia as a dengue control tool, and trials have reported significant reductions in transmission in release areas. Here, we discuss the possible mechanisms of Wolbachia-virus inhibition and the implications for long-term success of dengue control. We also consider the evidence presented in several reports that Wolbachia may cause an enhancement of replication of certain viruses under particular conditions, and conclude that these should not cause any concerns with respect to the application of Wolbachia to arbovirus control.Following transfer into the primary arbovirus vector  Wolbachia pipientis are alphaproteobacteria found in many arthropod species and some nematodes [Wolbachia are transmitted from mother to offspring in the egg cytoplasm during oogenesis. Wolbachia-host interactions often combine elements of parasitism and mutualism: in some instances, they provision a host with nutrients [Wolbachia-carrying male mates with a Wolbachia-free female or with a female carrying a reciprocally incompatible Wolbachia strain, but can be rescued by a female carrying the same or otherwise compatible Wolbachia strain. Because CI results in a relative reproductive advantage for Wolbachia-carrying females, CI-inducing Wolbachia strains can often maintain very high population infection frequencies. The fitness advantage for Wolbachia-carriers generated by CI is frequency dependent and is highest when a Wolbachia strain is close to fixation, while at very low frequencies the population-level effects of CI are negligible. This results in a threshold frequency above which there is spread and below which Wolbachia is lost; the threshold depends on CI penetrance, efficiency of maternal transmission, and fitness effects. Phenotypes that improve the spread of a Wolbachia strain are expected to be selected for; mathematical models suggest that pathogen protection can significantly lower the frequency thresholds required for Wolbachia invasion [ematodes . As obliutrients , enhanceutrients , and canutrients ,107–110.invasion .Table 1.Wolbachia as a vector control intervention. Firstly, the spread of virus-blocking Wolbachia strains through mosquito populations can reduce their competence for certain arboviral diseases [Wolbachia strains that cause CI, and therefore sterility, when mated with wild females [Wolbachia-carriers, while the latter attempts to achieve the local elimination of vector populations. Trials with artificial transinfections in the major arbovirus vector Aedes aegypti, which is not a natural Wolbachia host, have generated promising results with both approaches.Two distinct strategies are currently being deployed to utilize diseases . Secondl females ,112–114.Ae. aegypti carrying the Wolbachia strain wMelPop were unable to achieve stable and persistent population replacement, likely a result of the high fitness costs associated with this high-density strain [Ae. aegypti carrying the lower density wMel strain were released in Cairns, Australia, a city that experienced a relatively low number of locally acquired dengue cases annually. wMel spread swiftly in the city and was maintained at proportions close to fixation, with case notification data indicating a reduction in dengue incidence of 96% [wMel Ae. aegypti in the cities of Yogyakarta, Indonesia and Niteroi, Brazil met with similar success, with estimated average decreases in dengue incidence in release sites of 77% and 69%, respectively [wMel Ae. aegypti are also ongoing in sites in several additional countries including Columbia, Mexico, Fiji, and Sri Lanka. However, not all releases have resulted in the successful in establishment of wMel; in Nha Trang City in central Vietnam the initial establishment of wMel was followed by seasonal fluctuations in frequencies associated with elevated temperatures and the infection was subsequently lost from two release site areas [wMel frequencies [Releases in Northern Australia and Vietnam with y strain . Ae. aege of 96% . Releaseectively ,117. Relte areas ; furtherquencies .Wolbachia strain wAlbB has been successfully deployed for dengue control in Greater Kuala Lumpur, Malaysia [wAlbB-carrying Ae. aegypti in a variety of sites comprising different urban landscapes/building types (e.g. high-rise apartment buildings and landed houses) with intervention sites also selected for persistently high dengue incidence over preceeding years. Monitoring of the Ae. aegypti population size indicated no major release-related increases in population density, which is likely to be the result of compensatory CI-induced sterility between released wAlbB males and wild females. wAlbB invaded wild populations rapidly and reached a frequency of more than 90% in all intervention sites. After the cessation of releases, wAlbB frequencies remained stable in a majority of sites, while a minority experienced fluctuations and frequency drops that were largely overcome by additional low-level releases. The presence of wAlbB was associated with 40–85% decreases in dengue cases when comparing pre-intervention with post-inervention incidence over multiple years, although this is expected to be an underestimate of the true impact on transmission, given that dengue infection could be acquired by the routine travel of residents outside of the release areas [Malaysia , releasise areas .Wolbachia can confer on host insects RNA human arboviral pathogens in research studies), a small number of reports suggest that Wolbachia can in some circumstances enhance host susceptibility to infection with certain viruses. There is substantial divergence in insect host species and virus phylogeny examined in the various Wolbachia-virus interaction studies, as well as differences in experimental methodology, ranging from purely correlative studies to those with controlled laboratory infectious bloodmeal challenges. Among the laboratory studies, there is significant variation in the methods used to deliver virus, quantify virus, and even in the nature of the Wolbachia transinfection (e.g. stable germline or transient somatic infection). As any potential for viral enhancement is of clear public health importance for the use of Wolbachia in vector control, claims of enhancement should be closely examined and any implications carefully considered.A large body of experimental data has been published in recent years demonstrating the virus-inhibiting effect that ects see . This anWolbachia-mediated protection from infection with pathogenic viruses, conferring increased survival, was first demonstrated in Drosophila melanogaster carrying the wMel Wolbachia strain. wMel-carrying flies displayed reduced mortality following challenge with (+)RNA viruses: Drosophila C virus (DCV), Nora Virus and Flock House virus [Wolbachia could inhibit viral replication [wMel and wMel-Pop strains were also shown in to protect Drosophila against cricket paralysis virus [Drosophila simulans was subsequently used to test the protective capacity of an array of Wolbachia strains, demonstrating substantial differences in virus inhibition between Wolbachia strain variants [se virus , and whilication ,99; wMelis virus . It was is virus . Drosophvariants .Wolbachia transinfections were generated in Ae. aegypti and several showed a potent capacity to inhibit the transmission of dengue and other arboviruses that cause human disease , thr absent . Non-nat tissues ,123. Howlication ,54.Wolbachia-mediated virus blocking remains elusive, there are strong indications of which processes are likely to be involved  replication without affecting Wolbachia density [Within the host cell membrane . Sequencneration . Lipidomeramides , while peramides . Ae. aeg density .Wolbachia, which has been linked to activation of the Toll innate immune pathway and the production of antimicrobial peptides in Ae. aegypti [Wolbachia may modulate host cell autophagy [Wolbachia densities through the fusion of Wolbachia-containing endosomes with autophagic lysosomes [Wolbachia. DENV has been shown to induce autophagy in mammalian cells [Wolbachia and virus over the regulation of cellular autophagic flux.Other mechanisms that may contribute to viral inhibition include the production of reactive oxygen species (ROS) induced by the presence of  aegypti . There iutophagy ,131. Incysosomes ; it is tan cells , which ran cells . Hence, Drosophila, Dnmt2 actively binds DCV RNA and Dnmt2 mutant flies show elevated levels of DCV, suggesting an anti-viral role in this species [Drosophila carrying the wMel Wolbachia strain have increased Dnmt2 expression, and wMel-carrying flies that are Dnmt2 mutants show reduced inhibition of Sindbis virus (SINV) [Wolbachia-carrying Ae. albopictus and Ae. aegypti show reduced levels of AaDnmt2 [Ae. aegypti cells results in increased levels of DENV in Wolbachia-negative cells and AeDnmt2 has been shown to be pro-viral in both Ae. aegypti and Ae. albopictus for various Alphaviruses. Overexpression of AeDnmt2 in A. albopictus cells containing wMel resulted in reduced viral inhibition of chikungunya virus (CHIKV) and SINV. Furthermore, viral RNA produced in cells containing Wolbachia show a significant reduction in m5C methylation [Wolbachia-carrying cells produce less infectious virus.RNA-binding proteins (RBPs) play a critical role in the life cycle of RNA viruses and can have both pro and antiviral effects . One suc species . Drosophs (SINV) . In cont AaDnmt2 ,136. OveWolbachia-mediated antiviral activity [Wolbachia is present however, there is less viral replication and thus a reduced accumulation of XRN1-inhibiting sfRNAs and a more rapid degradation of viral RNAs by XRN1, likely enhancing the antiviral effects of Wolbachia.The RBP exoribonuclease 1 (×RN1) has also been implicated in activity . Part ofactivity . This leWolbachia of pathways involved in the synthesis or localization of membrane lipids. Although not currently fully understood, there are likely to be fundamental mechanistic reasons for disparities in inhibition between different virus types, with evidence suggesting that those viruses most affected will be those most sensitive to Wolbachia-induced perturbations in lipid homeostasis.A highly conserved feature of (+)RNA virus replication is a dependence on the formation of subcellular compartments through virus-induced rearrangements of organelle membranes, including those of the mitochondria, endosomes, or endoplasmic reticulum , which pWolbachia-mediated virus inhibition for the long-term prospects for the use of Wolbachia in dengue control. Firstly, the likelihood that multiple cellular perturbations contribute to virus inhibition, while making the phenotype more challenging to experimentally dissect, does reduce the possibility that viral escape mutations will arise that restore DENV replication. Indeed, no examples of DENV escape mutants have to date been reported. This apparently built-in robustness is analogous to multidrug therapy using drugs with different modes of action to reduce the likelihood of evolution of pathogen drug resistance. Nevertheless, differences between Wolbachia strains should also be considered in this context. The wMel strain in Ae. aegypti was found to be unstable when larvae were reared at elevated temperatures  [wAlbB was stable at the high rearing temperatures used. This affects the relative utility of different strains of Wolbachia for dengue control in very hot climates – an important factor for long-term robustness of the approach in the face of climate change. Furthermore, the use of strains with high-temperature stability could also ensure that any risks of the selection of virus escape mutations are minimized. This is analogous to the concept that drug dose and treatment duration affect the risk of selection of drug resistance, with a ‘mutant prevention concentration’. More research is needed on the effects of high temperatures on the Wolbachia-induced cellular perturbations relevant to virus replication. It is important to note that no virus escape mutations have been reported to date.There are some important implications of the research published to date on the mechanistic basis of ve 34°C) ,142, witve 34°C) , and subve 34°C) . In contWolbachia-mediated virus inhibition will be very useful is for assessing the implications of any Ae. aegypti-Wolbachia co-evolutionary changes that could occur over time to minimize host fitness costs. For example, virus transmission blocking could be reduced over time if mosquito-Wolbachia co-evolution results in lower Wolbachia density overall, or more restricted tissue distribution to the ovaries and testes. The most important Wolbachia-associated costs detected in Ae. aegypti are in reduced hatch of embryos following quiescence (dry storage) [Ae. aegypti and its relationship to virus inhibition. It is also important to note that no evidence for density reduction or loss of virus transmission-blocking capacity has been reported to date in Ae. aegypti field populations tested several years after Wolbachia introduction [A second area where greater knowledge of the mechanisms of storage) , and redstorage) . Thus, soduction ,48,147.Wolbachia strains into Ae. aegypti genetic backgrounds from different geographical areas does not have a major impact on Wolbachia density [In addition, there is mounting evidence that introgressing  density  or the c density ,150. Art density .Wolbachia-mediated enhancement of a (+)RNA virus is from Dodson et al [Culex tarsalis mosquitoes transiently infected with the wAlbB Wolbachia strain. Following challenge with WNV, Cx. tarsalis transiently infected with wAlbB showed a significantly higher WNV infection rate compared to non-Wolbachia controls at 7-days post infection – although there was no effect of the Wolbachia infection on rates of viral dissemination to mosquito legs or transmission in salivary secretions at this time point. A second time point (14-days post infection) found no significant differences in rates of infection, dissemination or transmission between Wolbachia-positive and Wolbachia-free controls.The first published study describing on et al , and desWolbachia infection. Although technically easier to generate, transient infections resulting from adult intrathoracic injection do not reliably recreate the Wolbachia densities or tissue distributions observed in natural germline transinfections [Wolbachia appears to be largely cell autonomous, requiring the co-localization of Wolbachia and virus in the same host cell – although there may also be a systemic contribution in some cases in the form of immune activation [Wolbachia in certain host tissues involved in viral infection and transmission, such as the midgut and salivary glands, is important for transmission blocking. While stable germ-line transinfections tend to produce stable midgut and salivary gland densities, transient infections are less consistent. Quantitative PCR analysis of Wolbachia titers in the Dodson study [wAlbB levels varied over 400-fold between individual transiently infected Cx. tarsalis mosquitoes , a level of variability far greater than that typically observed in germline infections [Wolbachia titer and probability of infection with WNV, as may be expected if Wolbachia was causing enhanced host susceptibility to virus infection. A separate study comparing WNV blocking in transient and germline wAlbB-infected Ae. aegypti found that transient somatic transinfection significantly underestimated levels of WNV inhibition compared to germline transinfection, although significant WNV inhibition was observed in both cases [Wolbachia, including the wAlbB strain in Ae. albopictus cells [wMel in Ae. aegypti cells [Wolbachia-mediated antiviral activity. While it is possible that Cx. tarsalis differs from other host species in its interactions with Wolbachia such that it causes viral enhancement instead of inhibition, the use of transient Wolbachia infections in the Dodson study [Wolbachia-infected cohort was injected with a wAlbB-containing An. gambiae cell-line extract, the Wolbachia-negative cohort was injected with clean media – rather than the preferred control of a Wolbachia-negative An. gambiae cell-line extract. It is likely that a complex mixture of An. gambiae cellular debris/mitochondrial/insect specific virus (ISV) material was co-introduced into Cu. tarsalis along with wAlbB, with unknown effect on the host. The Dodson study [Wolbachia-injected cohort had a slight but significant downregulation of Rel1, a Toll pathway transcription factor important in the Toll-mediated innate immune response. This is surprising given that Wolbachia-derived cellular components such as the Wolbachia surface protein (WSP) can act as pathogen-associated molecular patterns (PAMPs) [Wolbachia infections [Cx. tarsalis.As noted by the authors , a key cfections . Virus itivation ,74,129. on study  suggesteth cases . Severalus cells  and wMelti cells , indicaton study  makes thon study  reports  (PAMPs) , and havfections ,129, sucWolbachia in a mosquito host concerns an increased infection rate of insect-specific flaviviruses (ISFs) in field-caught Ae. aegypti mosquitoes carrying the wMel Wolbachia strain [Ae. aegypti were sampled from sites in Cairns, Australia: wMel-carrying mosquitoes were collected from two sites, while Wolbachia-free mosquitoes were sampled from a third site. Using specific primers, ISF fragments were PCR amplified and then sequenced. The authors reported a higher proportion of wMel-carrying mosquitoes positive for known ISF sequences compared with Wolbachia-free mosquitoes. While this result is consistent with an ISF enhancement effect by wMel, the limited number of sampling sites used, and the absence of both Wolbachia positive and negative samples from the same sites, leaves the possibility that differences may simply reflect geographical variation in ISF abundance and/or host background [wMel-carrying and Wolbachia-free mosquitoes. An analysis of ISF levels in mosquitoes of field and laboratory origin by qRT-PCR suggested a tendency for a stronger ISF signal in wMel carriers, although there were no instances where significant differences in putative ISFs were consistent across both field and laboratory sampled mosquitoes, and in one of the five putative ISFs, both field and laboratory mosquitoes showed significant differences between wMel-carriers and non-carriers, but with conflicting outcomes . Unexpectedly, one of the sequences that was significantly more abundant in laboratory-reared wMel-carriers showed high (99%) similarity to cell fusing agent virus (CFAV), an ISF for which strong inhition by Wolbachia has previously been shown [A further study reporting the enhancement of (+)RNA viruses by a strain . In thisckground . The auten shown ,43,44.Wolbachia on viral infection in Drosophila species. Martinez et al. 2014 [Wolbachia strains in Drosophila simulans, and challenged the lines with Drosophila C virus (DCV) and Flock House virus (FHV), (+)RNA viruses from the Picornaviridae and Nodaviridae families, respectively. For DCV, 7 out of the 19 Wolbachia lines showed a significant reduction in viral titer compared to Wolbachia-negative controls, while one showed a significant increase. For FHV 5 out of the 19 lines showed a significant reduction in viral titer, while one showed a significant increase. Interestingly, the Wolbachia strain associated with increased DCV titer was not the same as the strain associated with increased FHV titer – each strain correlated with increased titers of one virus showed no significant interaction with the other.Two studies report an enhancement effect of al. 2014  tested gWolbachia infections in Drosophila pandora: wPanMK and wPanCI. Challenge of flies with cricket paralysis virus (CrPV), a (+)RNA virus from the Family Dicistroviridae, revealed that wPanMK was associated with reduced CrPV-induced mortality, while wPanCI was associated with increased mortality compared to Wolbachia-negative controls. Measurements of viral titers showed no significant differences in CrPV levels between the fly lines , suggesting that the Wolbachia infections did not have a direct impact on CrPV replication, but rather appeared to affect the ability of D. pandora to tolerate the infection.Asselin et al. 2019  characteWolbachia infection and the frequency of deformed wing virus (DWV) in the parasitic honeybee mite Varroa destructor, with specimens collected from several different apiaries. Two sets of primers were used for Wolbachia detection in these samples, with the authors reporting a significant positive correlation between infection frequency with Wolbachia and DWV with one primer set but not with the other. As there was little agreement in infection frequency between the two primer sets (in one of the hives the different PCR assays produced a disparity in Wolbachia infection frequency in mites that ranged from 0% to 100%), there are concerns over the sensitivity and/or specificity of the PCR assays used in the detection of Wolbachia. It is possible that multiple strains of Wolbachia naturally infect V. destructor, which could explain the inconsistency between primers sets; however, it is difficult to draw definitive conclusions about potential correlations with DWV without more robust characterization of the Wolbachia present.Grau et al.  investigWolbachia-virus interactions, Teixeira et al [wMel-carrying D. melanogaster were challenged with Insect Iridescent Virus 6 (IIV-6), a large double-stranded DNA virus. A small number of studies have since investigated the effect of Wolbachia on DNA viruses. In one study [Wolbachia density with titers of the ssDNA Ae. albopictus densovirus  in cultured Ae. aegypti and Ae. albopictus-derived cell lines carrying the wMelPop or wAlbB Wolbachia strains. The authors found that under normal cell culturing conditions the wMelPop and wAlbB-infected cells contained approximately 5-20-fold more AalDNV-1 genomes per host cell than tetracycline-cured controls. Using cells cultured in media containing varying concentrations of tetracycline, the authors generated a range of wMelPop and wAlbB intracellular densities and found that Wolbachia levels positively correlated with AalDNV-1 genome copies. The authors hypothesized that elevated DNA repair response pathways induced by disruption of cellular redox homeostasis triggered by Wolbachia-generated reactive oxygen species may provide the additional molecular apparatus required for increased AalDNV-1 replication. Similarly, studies investigating the interaction of Wolbachia on the load of the ssDNA Culex pipiens densovirus (CpDV) on field caught samples found a positive correlation between CpDV levels and the density of the native Culex pipiens Wolbachia strain wPip [Wolbachia-positive mosquitoes than in antibiotic-cured controls [wMelPop-CLA on viral load in the Ae. aegypti Aag2 cell line [Wolbachia compared to a Wolbachia-cured Aag2 cell line.In one of the first studies examining ra et al . reportene study , qPCR waain wPip , and labcontrols . A studyell line . A quantWolbachia strains (wExe1–3) native to the African army worm, Spodoptera exempta, were assessed for their effect on host infectivity with Spodoptera exempta nucleopolyhedrovirus (SpexNPV), a double-stranded DNA virus (family: baculovirus) that displays very strong host pathology [S. exempta which suggested a positive correlation between the three Wolbachia infections and rates of SpexNPV-induced mortality. Follow-up laboratory-based bioassays using varying doses of SpexNPV on wExe1-carrying and tetracycline-cured S. exempta larvae suggested that the Wolbachia-free group had a LD50 6-14-fold greater than that of wExe1 carriers. Interestingly however, while both the field and laboratory findings indicated increased larval mortality associated with Wolbachia infection, quantification of SpexNPV in dead larvae revealed a tendency for lower numbers of viral occlusion bodies in the Wolbachia carriers. This may be due to a faster ‘speed of kill’ of the virus in Wolbachia-carriers, resulting in less time for occluded viral forms to accumulate in moribund larvae, and may result from a lower tolerance of Wolbachia-carriers for viral infection.Three athology . The autWolbachia are being deployed in both population suppression and population replacement vector control strategies. Population replacement strategies aim to spread and maintain an introduced Wolbachia strain in a wild population; any virus enhancement is therefore of potential interest to public health. Currently, the spread of novel Wolbachia strains through wild populations has been limited to Ae. aegypti - although promising strains have also been developed for Ae. albopictus [Wolbachia-virus interactions have investigated novel transinfections in Ae. aegypti in both in vivo and cell culture systems and have used a wide variety of arboviruses, focusing on the major human pathogenic viruses belonging to the virus families Flaviviridae (genus Flavivirus) and Togaviridae (genus Alphavirus). These arboviruses contain positive-sense single-stranded RNA genomes and are responsible for the vast majority of arboviral morbidity and mortality globally [Wolbachia can have on viral replication varies depending on multiple factors including the Wolbachia strain, the host species, and the replication modality of the virus. However, only a single study out of more than 50 involving Ae. aegypti and Ae. albopictus reports evidence of enhancement of viruses known to cause pathology in humans. This study, by King et al., 2018 [wMel-carrying Ae. aegypti [wMel can increase susceptibility to mosquito dengue infection when challenge titers are low. However, the data used to generate one of the primary data sets suffers from various shortcomings, including low statistical power and the potential for false positives when challenges were performed at low viral titers, and issues with the statistical model used. For a more thorough examination of the King et al., 2018 [bopictus ,68. The globally . Studiesl., 2018 , reexami aegypti ,156, andl., 2018  study sel., 2018 .Aedes mosquitoes reports the enhancement of a pathogenic human virus, several report elevated ISV titers. ISVs do not replicate in vertebrate cells, and therefore do not pose a direct public health concern themselves, although they do have the potential to interact with and modulate the replication of human arboviruses when co-infecting the same mosquito host, primarily through mechanisms of superinfection exclusion – a process where a host cell infected with a virus has a reduced capacity to support the productive replication of a secondary viral infection. Superinfection exclusion has been observed between several ISVs and human arboviruses, including with WNV and the insect-specific Culex flavivirus (CxFV), where experimentally challenged Culex pipiens mosquitoes showed reduced WNV dissemination early in infection when superinfected with CxFV [Culex quinquefasciatus [Ae. aegypti cells infected with Wolbachia were associated with higher titers of Aedes anphevirus (AeAV), an ISV that showed a mild suppressive effect on DENV [in vitro. Interestingly, superinfection exclusion does not appear to be limited to viruses with similar genomes/replication modalities. As discussed above, Wolbachia presence was correlated with enhanced replication of the ssDNA virus, Aedes albopictus densovirus , in Ae. aegypti cells. A previous study of an Aedes albopictus densovirus  found that AalDNV could restrict the replication of DENV in Ae. albopictus cells, despite AalDNV localizing its replication to the nucleus and DENV to the cytoplasm [Wolbachia in field populations of Aedes mosquitoes, current evidence suggests that mechanisms of superinfection exclusion may actually contribute to reduced transmission competency of Wolbachia-carriers.Although only a single study in ith CxFV  – althouasciatus . Ae. aeg on DENV ,60 and Z on DENV  replicatytoplasm . AssuminWolbachia may reduce the tolerance of host insects to pathogenic ISVs [Wolbachia-carriers and therefore influence the invasiveness or population stability of a strain introduced for arbovirus control. However, there are also numerous examples of host protection against pathogenic ISVs. Protection from pathogenic ISVs appears to be the more common phenotype and has been suggested as a mechanism by which Wolbachia strains can spread from very low initial infection frequencies following natural horizontal transmission events, when the fitness advantages from frequency-dependent CI are negligible [Some studies suggest that nic ISVs ,105. A rgligible .Wolbachia can modulate the susceptibility of host cells to infection with some viruses, with the magnitude of this effect varying widely between Wolbachia strains and host species. While the altered intracellular state may be more favorable to the replication of some virus types, current evidence overwhelmingly indicates that Wolbachia has an inhibitory effect on the replication of (+)RNA viruses – which includes the vast majority of human arboviral pathogens. Although there are some rare examples of Wolbachia enhancing (+)RNA virus replication in insects, these are very much in the minority, and there are often significant caveats to the experimental design or interpretation of results in these studies. The effects of Wolbachia on viruses without (+)RNA genomes appear to be more nuanced. The vast majority of these involve ISVs, the modulation of which may influence arboviral disease transmission through mechanisms of superinfection exclusion or by altering the fitness/pathogen tolerance of mosquito hosts.