CCR5 gene. CCR5Δ32 originated in Europe, but the time of origin as well as the selective pressures that allowed the maintenance of this variant and the establishment of its current frequencies in the different human populations is still a field of debates. Due to its origin, the CCR5Δ32 allele frequency is high in European-derived populations (~10%) and low in Asian and African native human populations. In Brazil, the CCR5Δ32 allele frequency is intermediate (4-6%) and varies on the Brazilian States, depending on the migratory history of each region. CCR5 is a protein that regulates the activity of several immune cells, also acting as the main HIV-1 co-receptor. The CCR5 expression is influenced by CCR5Δ32 genotypes. No CCR5 expression is observed in CCR5Δ32 homozygous individuals. Thus, the CCR5Δ32 has particular effects on different diseases. At the population level, the effect that CCR5Δ32 has on European populations may be different than that observed in highly admixed populations. Besides less evident due to its low frequency in admixed groups, the effect of the CCR5Δ32 variant may be affected by other genetic traits. Understanding the effects of CCR5Δ32 on Brazilians is essential to predict the potential use of pharmacological CCR5 modulators in Brazil. Therefore, this study reviews the impacts of the CCR5Δ32 on the Brazilian population, considering infectious diseases, inflammatory conditions, and cancer. Finally, this article provides a general discussion concerning the impacts of a European-derived variant, the CCR5Δ32, on a highly admixed population.The genetic background of Brazilians encompasses Amerindian, African, and European components as a result of the colonization of an already Amerindian inhabited region by Europeans, associated to a massive influx of Africans. Other migratory flows introduced into the Brazilian population genetic components from Asia and the Middle East. Currently, Brazil has a highly admixed population and, therefore, the study of genetic factors in the context of health or disease in Brazil is a challenging and remarkably interesting subject. This phenomenon is exemplified by the genetic variant CCR5Δ32, a 32 base-pair deletion in the  Until the year 1500 CE, Brazil was inhabited only by Native Americans belonging to different linguistic groups, distributed along the coast and hinterland of the country. This scenario changed dramatically after the arrival of the Portuguese explorers in the Brazilian territory that year, affecting many cultural and biological aspects of the native populations. The European colonization of Brazil and the associated influx of Africans had a strong influence on the genetic makeup of the Brazilian population. In Brazil, as well as in other countries colonized by the Europeans, the Native American population deeply declined after colonization (contracted around 90% in the Americas) . The remIn general terms, the genetic background of current Brazilians has Amerindian, African, and European components in different proportions , 3, 5–7,Throughout history, Brazil also received migrants from other countries beyond those from Europe and Africa, including countries from Asia and Middle East , 9. The Y-chromosome haplogroup analysis corroborates the high genetic miscegenation observed in the Brazilian population. Abe-Sandes et al.  investigIn the same direction, evaluation of mitochondrial DNA in different populations of Brazil showed the presence of diverse haplogroups characteristic of African, European, Native American and Asian populations, again evidencing the high level of miscegenation in the Brazilian population , 29–37. CCR5 gene , a fundamental component of the immune system responsible for encoding the CCR5 protein, which acts mainly in the regulation of inflammatory cell migration. It is unclear what selective pressures (considering positive selection) were responsible for fixing CCR5Δ32 in the human genome. Smallpox, bubonic plague, and other infectious diseases have already been suggested, but there is no consensus on this aspect (The CCR5Δ32 polymorphism (reference SNP ID number: rs333) is a genetic variant that originated in the European population , and thes aspect . Neutrals aspect . What iss aspect , 44, pots aspect , 46, andThe CCR5Δ32 allele presents a higher frequency in northern Europe , being less frequent in countries located in the south of the European continent. For example, the frequency of the CCR5Δ32 allele is 8.1% in Spain, 6.9% in Portugal, 6.2% in Italy, and 5.1% in Greece. The allele frequency is very low or even absent in most Asian and African countries: for example, 0.4% in China, 2.2% in Korea, 0.7% in Cameroon, 0.26% in Eritrea, and 2.9% in Egypt . A recenThe main function of the CCR5 is coordinating leukocyte migration during inflammatory reactions through interaction with different chemokines, especially CCL3, CCL4, and CCL5 . Of noteCCR5 gene have CCR5 expression constitutively, with some variation between individuals. CCR5Δ32 causes important phenotypic effects, affecting the interaction of the CCR5 with chemokines. Due to the induction of a change in the CCR5 gene reading frame, the CCR5Δ32 produces a truncated protein that is not expressed on the cell surface, presenting a gene-dosage effect. In brief, the presence of the CCR5Δ32 allele in heterozygous causes a reduction in the expression of CCR5 at the membrane. The presence of the CCR5Δ32 allele in homozygosis culminate in virtually no expression of CCR5 molecules on the cell surface  with the virus on the cell surface, thus avoiding infection of the host , 76. As Another major achievement involving CCR5Δ32, and HIV infection was the sustained remission of the infection in the ‘Berlin Patient’, reported in 2009  and confCurrently, it is known that the influence of CCR5 and CCR5Δ32 goes beyond protection against HIV infection and is much broader than previously believed, influencing the susceptibility and outcome of different conditions, such as other different viral, bacterial, and parasitic diseases , 92, as Although Brazilians form a population of more than 210 million individuals, genetic studies in this population are still limited, with most genetic studies focusing on populations with European ancestry , 9. The Considering that (I) the frequency of CCR5Δ32 is quite varied among Brazilians from different country’s regions and that (II) the role of CCR5Δ32 in various pathological conditions is an emerging topic with several knowledge gaps, the primary objective of this article is to review the effects of the genetic variant CCR5Δ32 on the Brazilian population, considering several diseases and clinical conditions. The secondary objective of this article is to discuss the impacts of a European-derived variant, the CCR5Δ32, on a highly mixed population.https://pubmed.ncbi.nlm.nih.gov/). Subsequently, the same search strategy was used on Scientific Electronic Library Online - SciELO (https://scielo.org/). The articles were initially selected based on the title and abstract. Only articles addressing CCR5Δ32 in Brazilian populations were included in this review. Articles published in English and Portuguese were considered in the evaluation, without restriction concerning the date of publication. On some specific occasions, the reference list of selected articles was also used as an additional source of published works involving CCR5Δ32 in the Brazilian population. Additional unstructured searches were performed on PubMed to select the articles cited in the introduction section and additional points of the review.For the initial selection of articles, the terms “CCR5”, “CCR5 delta 32”, “CCR5Δ32” and “rs333”, used in combination with “Brazil” or “Brazilian”, were searched on PubMed  is based on skin color, and for this reason many Brazilian studies classify individuals using this criterion. Alternatively, ‘white’ individuals can be classified as Caucasians, and ‘brown’ and ‘black’ can be classified as non-Caucasians.Vargas et al.  investigFerreira-Fernandes et al.  analyzedCarvalho et al.  evaluateFinally, we summarized in Candida albicans infection, acting as a fundamental molecule for a proper immune response. The absence of CCR5 expression resulted in uncontrolled inflammation and increased renal damage in face of C. albicans infection (Schistosoma spp.), the poor recruitment of Treg cells to the inflammation sites due to CCR5 absence causes uncontrolled inflammation and related tissue damage  and T-regulatory (Treg) cells] towards inflammation sites. For instance, a recent study with mice showed that CCR5 has a pivotal role in the recruitment of NK cells to the kidney allowing an adequate neutrophil activity during systemic nfection . Also, Te damage , 115. One damage . Taking e damage . In thisCCR5 gene   , 63 and chanism) , 66. Manchanism) –120, altchanism) , 122. ThExperimental evidence indicated that the course of HTLV (type 1 and 2) infection and HIV/HTLV co-infection may be affected by CCR5 expression patterns, which can be modulated by such viruses , 139. ThHCV and HBV are associated with the development of hepatocarcinoma and other liver diseases . CCR5 coMore recently, we evaluated the influence of CCR5Δ32 on susceptibility to HBV infection and HBV/HIV co-infection in a study involving 1113 individuals. We found no significant effect of CCR5Δ32 on susceptibility to HBV mono-infection. On the other hand, the CCR5Δ32 allele exerted a protective influence on HBV/HIV co-infection. Of note, this result was potentially due to the known protective effect of CCR5Δ32 on HIV infection . In a stHPV is strongly associated with the development of cervical cancer  and it wTrypanosoma cruzi infection. CCR5 mediates the control of acute infection, assuming a favorable role for the host. In opposition, the increased expression of CCR5 during Chagas disease is associated with exacerbated inflammation and related cardiac complications  was associated with increased risk of ocular toxoplasmosis, potentially due to the persistent CCR5-mediated inflammation in individuals with normal CCR5 expression  and intensity of inflammation. In this topic, we review the role of CCR5Δ32 on the following inflammatory diseases or inflammation-related clinical conditions: multiple sclerosis, systemic lupus erythematosus, preeclampsia, rheumatoid arthritis, juvenile idiopathic arthritis, periodontitis, osteomyelitis, transplant rejection, and sickle cell disease. Details of each study are described in Multiple sclerosis is an autoimmune, chronic, and inflammatory disease showing heterogeneity in clinical findings. Chemokines and chemokine receptors are molecules involved in the pathogenesis of multiple sclerosis , 194, anSystemic lupus erythematosus is a chronic inflammatory autoimmune disease characterized by the large production of autoantibodies, triggering generalized tissue damage. This disease has different clinical manifestations and a complex genetic influence, and chemokines and their receptors, such as CCR5, are implicated in the pathogenesis of lupus , 204. ThSchauren et al.  investigBaltus et al.  evaluatePreeclampsia is a hypertensive gestational complication and an important cause of maternal-fetal mortality in Brazil. Relevant clinical findings of the disease, such as edema and proteinuria after the 20th week of pregnancy, are intricate with an excessive inflammatory process and endothelial dysfunction. In preeclampsia, increased systemic production of pro-inflammatory chemokines was observed, highlighting the role of the chemokine-ligand system in this condition , 209. TwRheumatoid arthritis is a systemic autoimmune disease characterized by progressive damage to the joints caused by chronic inflammation in the synovial fluid. Given the intense migration of immune cells to the inflammation sites, the role of CCR5 in rheumatoid arthritis appears to be of great importance , 180. InJuvenile idiopathic arthritis is a chronic inflammatory condition characterized in the synovial joints of young people up to 16 years of age , 212. ScPeriodontitis is an oral disease characterized by a chronic infection accompanied by inflammatory processes, causing irreversible and progressive destruction of dental support structures. The CCR5-mediated immune responses affect multiple aspects of periodontitis. For instance, not only CCR5 and its ligands are important in the context of disease protection, but also influence periodontal destruction and bone resorption , 214–217Staphylococcus aureus infection  being one of the best-described immunostimulators , 234. Macarriers . Zambra carriers  also evacarriers .p53 genotypes, a known tumor suppressor gene, together with the CCR5Δ32 genotypes, was also evaluated revealing a higher frequency of individuals with the p53 Arg homozygous genotype and the CCR5Δ32 wild-type genotype amongst controls as compared to patients  and one polymorphism located in the CCR2 gene , but high in others . In Native American populations, the allele is absent or occurs at low frequencies. In Brazil, CCR5Δ32 is not uncommon in non-Caucasian populations, because of the miscegenation that has occurred in the country.Many studies corroborated the protective effect of the CCR5Δ32 on susceptibility or clinical aspects of HIV infection in the Brazilian population. On the other hand, there is no evidence pointing to a relevant role for CCR5Δ32 on Cutaneous leishmaniasis, Chagas disease, HTLV-1, Dengue virus, Influenza A, HPV, HBV and HCV infections, or HCV-HIV co-infection in Brazilians. Limited evidence indicates a potential involvement of CCR5Δ32 wild-type genotype in ocular toxoplasmosis and a protective effect of the variant on HBV/HIV co-infection.Considering inflammatory conditions, the CCR5Δ32 can influence both the susceptibility and the clinical outcome of multiple sclerosis. Of note, CCR5Δ32 reduces the risk of preeclampsia and periodontitis development, potentially due to the CCR5Δ32-associated reduced inflammation. Moreover, CCR5Δ32 can reduce the risk of rheumatoid arthritis, but contributes to the progression and clinical status of juvenile idiopathic arthritis patients. CCR5Δ32 can also influence sickle cell anemia-related immune conditions. However, the impact of CCR5Δ32 on systemic lupus erythematosus is controversial. Concerning tumoral development, the CCR5Δ32 has varying influences on the development of different types of cancer, including prostate cancer and breast cancer. It is not possible to generalize the impact of the variant on cancer development, especially in the Brazilian population.Understanding the real impact of the CCR5Δ32 variant in different conditions is essential to indicate in which diseases the use of CCR5 modulators may be relevant. This knowledge is fundamental for the advancement of CCR5-based therapies, especially in populations with a complex genetic structure. Finally, CCR5Δ32 influences should be assessed within the context of each population, since genetic admixture and interactions with other alleles may alter the expected phenotypic effects attributed to CCR5Δ32.BK-L and JE wrote the first version of the manuscript. JC revised and edited the text. All authors contributed to the article and approved the submitted version.BK-L receives a fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico . JE receives a postdoctoral fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior . JC receives a research fellowship from CNPq  and has research projects funded by Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul  and CAPES (Brazil).JE is Guest Associate Editor of Frontiers in Immunology but he was not involved in editing this article.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Our aim was to examine attitudes of the general population towards reasonableness of these costs, as well as the degree to which these costs are shared across society (solidarity financing) and to determine the factors associated with them.Repeated cross-sectional data from a nationally representative online-survey. More precisely, data from wave 8 (21–22 April 2020) and wave 16 (7–8 July 2020) were used , ranging from 18 to 74 years, 51.8% female; in wave 16: analytical sample with n = 978, average age was 46.1 years (SD: 15.9 years), ranging from 18 to 74 years, 50.9% female). After a short introduction emphasizing considerable economic costs associated with the measures against the spread of the coronavirus, individuals were asked to rate the following statements (outcome measures), in each case from 1 = strongly disagree to 7 = strongly agree: “These economic costs are currently reasonable in relation to the objective pursued” (reasonableness of costs), “These economic costs should be borne jointly by all citizens and depending on income” (solidarity financing).In wave 8 (wave 16 in parentheses), the average rating for the attitude towards reasonableness of costs was 4.3, SD: 1.8  and the average rating for the attitude towards solidarity financing was 3.7, SD: 1.9 . In wave 8, more positive attitudes towards the reasonableness of costs and solidarity financing were associated with being male, higher education, not being in a partnership/being unmarried, higher affect regarding COVID-19 and higher presumed severity with respect to COVID-19. Furthermore, more positive attitudes towards the reasonableness of costs were associated with having a migration background. More positive attitudes towards solidarity financing was associated with higher age groups. Mainly similar findings were observed in wave 16.Agreement with reasonableness of costs of preventative measures as well as solidarity financing was moderately high. Knowledge of these attitudes is important to ensure social cohesion during the fight against COVID-19. The adequacy of measures to protect health during the COVID-19 pandemic will ultimately be assessed based on their direct positive, but also negative effects on health, social life and the economy . SpecifiKnowledge about how the general population perceives the activities implemented to prevent the spread of COVID-19 is important to prepare for communication about costs and hardships.It is also important to understand the population’s perception on how these activities should be financed, namely whether the costs should be shared across society.In Germany, like in other countries, discussion about the economic costs of the lock-down policies has been part of the public debate from the start on. According to the ifo Institute the German economy will probably shrink by 6.6 percent in 2020 . If thesThus far, there is a general lack of studies focusing on the attitudes of the general population towards reasonableness of these costs, as well as the degree to which these costs are shared across society (solidarity financing). One recent study  focused It should be repeated that this knowledge is important as it provides a rough approximation of the acceptance of such activities in society in April (two days after end of first lockdown) and July 2020 . Ultimately, this is important to maintain social cohesion within countries.th March 2020. These measures covered, among other things, closure of schools and day-care centers. These measures were intensified on 22nd March 2020 by implementing travel bans or contact restrictions in public. In the upcoming weeks, prolongations of these measures took place. On 20th April 2020, some COVID-19 measures were loosened such as shops under a certain size were allowed to reopen. By early May (4th May), schools slowly started to open again. Further loosening of the restrictions took place in May such as loosening the contact ban or reopening museums or playgrounds. Restrictions were further eased in June and remained relatively stable in July 2020. However, in case the number of infections increases, an “emergency brake” was implemented .In Germany, nationwide COVID-19-measures were implemented on 16Repeated cross-sectional data were derived from wave 8 and wave 16 of the COVID-19 Snapshot Monitoring (COSMO) [rd to 4th March 2020) with weekly cross-sectional online-surveys (15–20 minutes) on the COVID-19-situation for the general public. In each wave around 1,000 respondents complete an online questionnaire. The eighth wave took place from 21st to 22nd April 2020 (wave 16: 7th to 8th July 2020). The individuals were recruited from a market research company named Respondi (https://www.respondi.com/ - ISO 26362 certified online sample provider).The COSMO study started in March 2020  in such a way that it corresponds to the distribution of age, gender (crossed-quota: gender x age) and federal state (uncrossed) in the German population . Both, wRepeated cross-sectional data were used to display potential trends over time  in the outcome measures. However, it should be emphasized that it is impossible to examine individual trajectories over time when using repeated cross-sectional data.Informed consent was obtained from all individual participants included in the study. Ethical approval for COSMO was obtained by University of Erfurt’s IRB (#202000302). All procedures performed in the COSMO studies involving human participants were in accordance with the ethical standards of the University of Erfurt institutional research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.https://doi.org/10.23668/psycharchives.2776) and are made available to scientists upon request. Requests should be submitted to the COSMO consortium by contacting Prof. Dr. Cornelia Betsch, University of Erfurt, Nordhäuser Str. 63, 99089 Erfurt, Germany (cornelia.betsch@unierfurt.de).The data from the COVID-19 Snapshot Monitoring (COSMO) are stored in the repository PsychArchives  will cause considerable economic costs”), individuals were asked to rate the following statements (our outcome measures), in each case from 1 = strongly disagree to 7 = strongly agree :“These economic costs are currently reasonable in relation to the objective pursued” (reasonableness of costs)“These economic costs should be borne jointly by all citizens and depending on income” (solidarity financing)To reduce information bias, a small pretest was conducted (with n = 15) which confirmed the comprehensibility and face validity of our dependent variables.In regression analysis, we included sociodemographic factors and factors related to COVID-19 because we were interested in the fact whether such factors are associated with both outcome measures. More precisely, the following sociodemographic independent variables were included: sex , age category , children under 18 years , relationship/marriage , living situation , education ; 10 years and more ), migration background , self-employed , chronic diseases , region ; East Germany ), town size ; medium sized town (20.001–100.000); small city (100.001–500.000); big city (> 500.000)) and the COVID-19 cases/100,000 population .Furthermore, affect (such as fear or concerns) regarding COVID-19  and presumed severity of COVID-19 infection  were used as independent variables. Exemplary items for the tool quantifying affect regarding COVID-19  to assess the size of potential differences in the outcomes from wave 8 to wave 16. Subsequently, four multiple linear regressions were used to determine the factors associated with both outcome measures (both waves separately).We performed a White test for heteroscedasticity in the error distribution. According to the test statistics , residuals are heteroscedastic. Therefore, robust standard errors were calculated in this study. Additionally, standardized normal probability plots were used to check the normality of residuals. According to these plots, the residuals have an approximately normal distribution. We also checked for multicollinearity. The variance inflation factors (VIFs) were very low. This indicates that multicollinearity is not a threat.The statistical significance was defined as p value of ≤ .05. Statistical analyses were performed using Stata 16.1 .Sample characteristics for our analytical sample  are displayed in The effect size ) were in the first case Cohen’s d = -0.04 and in the second case Cohen’s d = -0.21.Multiple linear regressions with attitudes towards reasonableness of costs  and solidarity financing  are displayed in Second column: With attitudes towards reasonableness of costs as outcome measure (wave 8)Third column: With attitudes towards solidarity financing as outcome measure (wave 8)Fourth column: With attitudes towards reasonableness of costs as outcome measure (wave 16)Fifth column: With attitudes towards solidarity financing as outcome measure (wave 16)With regard to wave 8, regressions showed that stronger agreement with the statements that the economic costs are reasonable and that these costs should be borne by all citizens was associated with being male , higher education , not being in a partnership/being unmarried , higher affect regarding COVID-19  and higher presumed severity of COVID-19 . Furthermore, more positive attitudes towards the reasonableness of costs were associated with having a migration background . More positive attitudes towards solidarity financing were associated with higher age groups .While various associations remained remarkably similar in wave 16, some differences are worth noting: In wave 16, the link between higher age groups, and attitudes towards solidarity financing disappeared. Furthermore, more negative attitudes towards solidarity financing were present in East Germany . Moreover, the link between migration background and attitudes towards reasonableness of costs vanished. Additionally, the association between chronic diseases and attitudes towards reasonableness of costs became significant . Furthermore, the association between relationship status and both outcome measures disappeared.Our aim was to descriptively analyze attitudes towards the economic costs associated with measures against the spread of COVID-19, and to determine the factors associated with them. Agreement with reasonableness of costs as well as solidarity financing was moderately high. We could not identify a substantial change over time in both outcome measures. Several sociodemographic and COVID-19-related correlates have been identified.It should be noted that there is a general lack of studies investigating the attitudes towards reasonableness of these costs, as well as the degree to which these costs are shared across society. Therefore, our findings are difficult to compare with other studies. Nevertheless, it appears plausible that attitudes towards the reasonableness of costs and solidarity financing were positively associated with higher affect regarding COVID-19 and higher presumed severity of COVID-19. Potential individual health consequences may increase the desire that society as a whole covers the costs associated with the COVID-19 pandemic. Furthermore, when individuals fear the severity of a COVID-19 infection, they may be more willing to accept the restrictions in daily life, and ultimately may appreciate the actions taken to address this pandemic. Similarly, the link between higher age and more positive attitudes towards solidarity financing can be explained by potential individual health consequences. However, it should be noted that this link disappeared in wave 16.Higher education may reflect a higher ability to pay and may also reflect the increased ability to critically reflect on the pandemic and its economic consequences . MoreoveSurprisingly, unmarried/single and male individuals reported more positive attitudes towards the reasonableness of costs and solidarity financing. However, the association between marital status and the outcomes vanished in wave 16. In addition, more positive attitudes towards the reasonableness of the costs were associated with having a migration background. Again, this association vanished in wave 16. Future research is required to clarify this association in further detail.Interestingly, the agreement with reasonableness of costs as well as solidarity financing was moderately high in both waves . This means that individuals reported similar agreements when the partial lockdown with restrictions of social gatherings, closing of schools, universities, cultural events and restaurants was first eased in April and in July (when various restrictions were further eased). Future research in the next months is required to better understand these agreements. Moreover, qualitative studies are desirable to gain deeper insights into these agreements.It should be repeated that we could not determine a considerable change over time in both outcomes. This appears somewhat surprising given the fact that wave 8 took place two days after the first lockdown and wave 16 took place in a time with few restrictions. A possible explanation may be that our outcomes reflect more stable constructs rather than quickly changing attitudes. However, future research is required to investigate these outcomes in later stages of the COVID-19 pandemic.Some strengths and limitations of this study are worth highlighting. To the best of our knowledge, this is the first study identifying the factors associated with perceptions on the reasonableness of costs associated with activities implemented to prevent the spread of COVID-19, and the degree to which these costs should be borne by society. Data were taken from a nationally representative online sample. However, future research is required to investigate the attitudes of older individuals aged 75 years and older. Additionally, due to the nature of an online sample, a response rate could not be calculated. Therefore, the possibility of a sample selection bias cannot be entirely dismissed. Moreover, a pretest was conducted showing that, among others, the outcome measures had a high face validity. However, future studies with more sophisticated tools are required to validate our findings. Additionally, only the endpoints were labeled in the dependent variables. However, the middle of the scale was the most frequently chosen response option. It remains unclear whether the respondents selected this option to express neutrality, indifference, did not have an opinion or did not know. In these questions, terms such as “adequacy”, “the objective pursued” or “depending on income”  were not further defined. Furthermore, this is a cross-sectional study, and therefore has well-known limitations .This study identified factors associated with the attitudes of the general population towards the reasonableness of economic costs associated with interventions implemented to prevent the spread of COVID-19, as well as towards bearing these costs as a society. This in turn gives us a rough approximation of the acceptance within society of implemented measures to prevent the spread of COVID-19. This study may ultimately assist in maintaining social cohesion in the general population. Differences in population subgroup perceptions have to be confirmed and should be acknowledged when communicating about costs and cost sharing. Future research in other countries is required to make cross-country-comparisons.S1 Table(DOCX)Click here for additional data file.


A side-by-side electrospinning process characterized by a home-made eccentric spinneret was established to produce the Janus beads-on-a-string products. In this study, ketoprofen (KET) and methylene blue (MB) were used as model drugs, which loaded in Janus beads-on-a-string products, in which polyvinylpyrrolidone K90 (PVP K90) and ethyl cellulose (EC) were exploited as the polymer matrices. From SEM images, distinct nanofibers and microparticles in the Janus beads-on-a-string structures could be observed clearly. X-ray diffraction demonstrated that all crystalline drugs loaded in Janus beads-on-a-string products were transferred into the amorphous state. ATR-FTIR revealed that the components of prepared Janus nanostructures were compatibility. In vitro dissolution tests showed that Janus beads-on-a-string products could provide typical double drugs controlled-release profiles, which provided a faster immediate release of MB and a slower sustained release of KET than the electrospun Janus nanofibers. Drug releases from the Janus beads-on-a-string products were controlled through a combination of erosion mechanism (linear MB-PVP sides) and a typical Fickian diffusion mechanism (bead KET-EC sides). This work developed a brand-new approach for the preparation of the Janus beads-on-a-string nanostructures using side-by-side electrospinning, and also provided a fresh idea for double drugs controlled release and the potential combined therapy. Nanotechnology has been the most widely studied and multidisciplinary technology field for the past few years. Nanotechnology can control the structure of materials at the nanoscale, so as to obtain significant changes in material properties, outstanding advantages and wide applications. Nanomaterials include nanotubes , nanosheWith the rapid development of preparation and characterization of nanofibers, probable applications of nanofibers have been explored, including biomedicine ,12,13, fAccording to their chemical composition and function, nanostructures prepared by electrospinning can be designed into two independent parts, which have raised wide concern over the years ,49,50. TJanus is the ancient Roman “double faced God”, also known as the double-sided particle. Double-sided particles refer to a class of anisotropic particles with different physical or chemical properties in two different directions, and these two parts are separated in space that each part can be designed, respectively, ,52. MoreJanus particles and Janus nanofibers have been widely reported, but the study of Janus beads-on-a-string nanostructures  is still largely ignored. It is generally considered that beads-on-a-string are undesirable “by-products” that must be removed to collect smooth nanofibers and uniform particles . TherefoIn this work, Janus beads-on-a-string was prepared by a side-by-side electrospinning process with a home-made eccentric spinneret. One side of the Janus beads-on-a-string used a hydrophilic polymer  as the nanofiber forming polymer and loaded methylene blue (MB)as a model drug, while the other side used a hydrophobic polymer  as particle forming polymer and carries ketoprofen (KET) as a model drug. Two drug release behaviors were different, because the different polymer matrices and structures in Janus nanocomposite. In addition, the relationship between structures and properties of material could be established, which would instruct to control the release of double drugs. Ethyl cellulose  was bought from Shanghai Aladdin Chemistry Ltd. . Polyvinylpyrrolidone K90 , which were used for the crescent side of electrospinning. In this study, four Janus structures (Janus beads-on-a-string and Janus nanofibers) were manufactured. The specific parameters of preparing side-by-side electrospun nanostructures are shown in −1, and the round side was 1.0 mL·h−1. For optimization, the applied voltage between the eccentric spinneret and the grounded collector was fixed at 12.0 kV, and the collection distance was 16 cm. Relative humidity and Working environmental temperature were 55 ± 5% and 25 ± 5 °C, respectively, through all electrospinning processes.Working fluids were poured into 10 mL plastic syringe, and syringes were settled on two pumps, respectively, and led to connect a home-made eccentric side-by-side spinneret. Two working fluids were driven simultaneously through KSD100 and KSD200 syringe pumps . The electrostatic power is obtained from a high-voltage power supply . The fluid flow rate of the crescent side was 0.8 mL·hThe prepared Janus nanostructures were covered with gold for 120 s under a nitrogen atmosphere to give them electrical conductivity. The morphology of Janus nanostructures was evaluated using scanning electron microscopy . The sizes of 100 nanofibers and microparticles were measured from scanning electron microscopy images, and the average diameters of nanofibers and particles were reckoned employing Image J .Using Bruker-AXS diffractometer , XRD analysis of the simple, including raw KET/PVP/EC/MB powders and Janus nanostructures, were analyzed over the 2θ angle range of 10° to 60°. The voltage applied was 40 kV and the electric current was 30 mA.−1 and within the scope of 500 cm−1 to 4000 cm−1. The raw powders and Janus nanostructures were evaluated by FTIR Spectrometer . The spectrum was recorded with a resolution of 2 cmmax = 260 nm and 664 nm using a UV-vis spectrophotometer . Referring to the Pharmacopoeia of Chinese (2020 Ed.), in vitro drug dissolution tests were implemented successfully. Here, 44.4 mg Janus nanostructures were immersed in 200 mL phosphate buffer saline  at 37 ± 1 °C, and that is a method using constant temperature shaker device (set the speed to 50 rpm). At predetermined time points, 4.0 mL aliquot of the samples was taken out of the solution and replaced with fresh PBS to hold the volume constant. The amounts of KET and MB released were analyzed at λ−1), and −1), t, h), and the experiments were implemented 6 times.The accumulative percentage of KET and MB released from the Janus nanostructures was figured according to Equation (1) :(1)P(%)=g , and then were dried in a DHG-101-OA Electric Oven  to constant weight for SEM assessments. After the dissolution tests, the residues of F2 in the bulk dissolution media were separated through a TL-5.0W Table Centrifuge at 5000× A diagram of the side-by-side electrospinning process are exhibited in A digital image of the home-made side-by-side electrospinning system appeares in −1 for EC/PVP side), all fluids could work continuously and stably in side-by-side electrospinning. A typical Taylor cone was shown in Under the pre-optimized working parameters  and their average diameters and size distributions are displayed in w/v), the nanostructures have distinct beads that include spindle-shaped and concave-shaped beads (w/v), the nanostructures have been mostly concave beads and the latter has fewer microparticles than the former (w/v) (w/v), the 5% (w/v) concentration of nanofibers performs well linear morphology clear of spindles or beads phenomenon. As a result, the minimum spinnable PVP concentration (5% (w/v)) can be combined with EC solution to prepare Janus beads-on-a-string, and most of the microparticles are EC particles. When the concentration of PVP increased to 8% (w/v), the spinnable PVP can drive the unspinnable EC solution to form Janus nanofibers.As expected, the SEM images of the Janus nanostructures (includes beads-on-a-string and nanofibers) were obtained by changing PVP concentration. When PVP concentration was 4%  d. From Fw/v) PVP concentration). By comparison to the smooth surfaces of Janus nanofibers F4 (or core/sheath nanofibers), beads-on-a-string has ultrafine nanofibers to release drugs rapidly. Moreover, it also has larger microparticles as a storehouse to conduct sustained release.The Janus nanocomposites F1, F2, F3 (beads-on-a-string) and F4 (nanofibers) prepared through side-by-side electrospinning have average nanofiber diameters of 0.12 ± 0.004 µm e, 0.17 ±The XRD patterns of raw powders  and the Janus nanostructures  are shown in −1. Then, the characteristic peak at 1668 cm−1 was aroused by C=O stretching vibration and the peak at 1424 cm−1 was produced by -CH bending vibration, and the peak at 1292 cm−1 was raised by C-N stretching vibration. With regards to EC, the characteristic peaks at 1378 cm−1 for -CH bending, 3486 cm−1 for -OH stretching vibration and 1056 cm−1 for C-O stretching are shown in its spectrum. In the spectrum of KET, 1698 cm−1 and 1655 cm−1 for stretching vibrations of the carbonyl group and ketone group were observed. The typical peak at 1592 cm−1 for benzene ring skeletal stretching vibration, 1322 cm−1 and 1136 cm−1 for C-N and C-S stretching vibrations were shown in the spectrum of MB. The peaks at 2955 cm−1, 2978 cm−1, 2979 cm−1, 2925 cm−1 were the stretching vibrations of C-H in the spectrum of PVP, EC, KET and MB, respectively. Whereas, the peak at 1698 cm−1 of KET was vanished in the spectrum of F2 and F4, proving that hydrogen bonds were formed between the proton donor of EC and the receptors of KET. As was known to all, methylene blue had strong adsorption for polymers and no concern about their compatibility. The results of ATR-FTIR demonstrated that the components had well compatibility and stability.The compatibility between the drugs and polymers was investigated by FTIR analysis, which is necessary for preparing steady and high-quality Janus nanostructures. The secondary interactions, such as hydrophobic interaction, electrostatic attraction and hydrogen bonding enhanced the compatibility among the components . The ATR−1) and KET and MB have maximum UV absorbance peaks at 260 nm and 664 nm, respectively. According to the predetermined calibration curve The in vitro drug controlled release profiles of the Janus beads-on-a-string F2 and Janus nanofibers F4 are concluded and compared in Meanwhile, as shown in the inset of In contrast, KET release from the EC sides is controlled by the typical diffusion mechanism because of the insolubility of EC and the homogeneous distribution of KET molecules in the EC matrices. Shown in The KET release profiles from Janus beads-on-a-string F2 and Janus nanofibers F4 were analyzed employing the Peppas Equations (2) and (3) :(2)Q=MtMn is smaller than the critical value of 0.45, which indicates that drugs controlled release from side-by-side electrospinning products can be controlled through a typical mechanism of Fickian diffusion regardless of Janus nanofibers or Janus beads-on-a-string products. These results suggested that the structure of the product had no significant effect on the drugs diffusion mechanism.The equation of regression for F2 are The above study demonstrated that drug release could be controlled through adjusted the polymer concentration, and alter the nanomaterials structure. The Janus beads-on-a-string F2, prepared by the process, is more advantageous than the Janus nanofibers F4 in the aspects of controlled release profiles of double drugs and the potential combined therapy. The ultrafine hydrophilic nanofibers of the Janus beads-on-a-string released the first drug (MB) more rapidly than Janus nanofibers, while the larger hydrophobic microparticles of the Janus beads-on-a-string released the second drug (KET) more slowly than Janus nanofibers. Compared with Janus nanofibers, it provided more accurate control over the release of double drugs.A schematic diagram of drugs release mechanisms is given in Janus beads-on-a-string composite structures composed of PVP-MB//EC-KET were successfully achieved by a side-by-side electrospinning process. SEM images showed the fibers and particles of the Janus beads-on-a-string clearly. Through changing the polymer concentration, Janus beads-on-a-string with different particle distribution and diameter could be obtained. XRD patterns proved that all the components were present in the Janus nanostructures with amorphous states. ATR-FTIR spectrum testified that the compatibility and stability of the components were very well due to hydrogen bonding. In vitro drug dissolution tests demonstrated that the Janus beads-on-a-string could provide typical double drugs controlled release profiles, which had the faster immediate release and slower sustained release than Janus nanofibers. The drugs released from the Janus beads-on-a-string were controlled through a representative mechanism of Fickian diffusion. The current work showed a new preparation method of the Janus beads-on-a-string structures using side-by-side electrospinning, which also provided a fresh idea for double drugs controlled release and the combined medication.


Pulmonary fibrosis (PF) is a serious lung disease which can result from known genetic or environmental exposures but is more commonly idiopathic (IPF). In familial PF (FPF), the majority of identified causal genes play key roles in the maintenance of telomeres, the protective end structures of chromosomes. Recent evidence suggests that short telomeres may also be implicated causally in a significant proportion of idiopathic cases. The possible involvement of herpes viruses in PF disease incidence and progression has been examined for many years, with some studies showing strong, statistically significant associations and others reporting no involvement. Evidence is thus polarized and remains inconclusive. Here we review the reported involvement of herpes viruses in PF in both animals and humans and present a summary of the evidence to date. We also present several possible mechanisms of action of the different herpes viruses in PF pathogenesis, including potential contributions to telomere attrition and cellular senescence. Evidence for antiviral treatment in PF is very limited but suggests a potential benefit. Further work is required to definitely answer the question of whether herpes viruses impact PF disease onset and progression and to enable the possible use of targeted antiviral treatments to improve clinical outcomes. A role for herpes viruses in pulmonary fibrosis remains controversial. Herpes viruses are DNA viruses and include herpes simplex (HSV) 1 & 2, Epstein Barr Virus (EBV), cytomegalovirus (CMV) and human herpes viruses . Herpes viruses are ubiquitous; HSV affects up to 95%  of EuropLytic viral expression is often subclinical but may also be responsible for typical clinical manifestations such as cold sores (HSV1) or infectious mononucleosis (EBV or CMV), or in severely immunosuppressed people necrotising retinitis, encephalitis or pneumonitis . PeriodiPulmonary fibrosis (PF) is a feature of the interstitial lung diseases, marking a progressive disorder characterized by alveolar-interstitial inflammation, accumulation of extracellular matrix, distortion of normal architecture and impairment of gas exchange. Slow deterioration in lung function is interspersed with acute exacerbations; these episodes of rapid decline in lung function, associated with high mortality, may or may not be associated with triggers such as infection. For Idiopathic PF (IPF), the most common form of PF, overall mortality is high with median survival of 3 years from diagnosis . FamiliaSome herpes viruses appear able to augment other insults to the lung epithelium in causing and exacerbating fibrosis from both the lytic and latent states. In addition to the established role of these viruses in destruction of cells, there appear to be several potential herpes virus-associated mechanisms for enhancement of telomere shortening in alveolar epithelial cells (AEC). These include: induction of a pro-fibrotic cytokine environment; virally-induced endoplasmic reticulum stress which induces inflammatory signaling via NFκB and AP-1 pathways; enhanced cell proliferation regulated by viral DNA; an influence over telomeric repeat-containing RNA (TERRA) regulation on both maintenance of telomere integrity and degradation of shelterin; and a possible impact on two of the key genes implicated in FPF via the GA binding protein GABP. Given the recognized significance of telomere shortening in IPF, described in more detail below, it would be reasonable to postulate a potential role for herpes viruses in contributing to telomere attrition.Herpes viruses may have different roles in PF, depending on whether they are latent  or lytic. Various studies have looked at possible roles for herpes viruses in the initiation, maintenance and acute exacerbation of pulmonary fibrosis. The results of studies in humans are summarized in p = 0.001), although interestingly, not with acute exacerbation (p = 0.988) where there are less data. Not all studies have shown associations between herpes viruses and PF. In particular, the evidence linking acute exacerbations with herpes viruses is limited.A large meta-analysis of case-control studies showed significant association between herpes virus infection and IPF . Presencp = 0.0001 (p = 0.047). Immunohistochemistry showed LMP protein in 9 of 29 patients with IPF, but no immunoreactivity in SSc (with PF) or healthy control biopsies. LMP detection was significantly associated with PF itself and with disease progression (p = 0.022)  IgA in serum of IPF patients compared with controls with PF of known cause . Egan an= 0.0001 . In agre= 0.022) .p < 0.0001)  contained PCR-amplifiable DNA from at least one Herpes virus, compared with 36% of controls ( 0.0001) . The domrophages .p = 0.01). Three antibody positive PF patients but none of the controls had detectable EBV DNA, confirmed by sequencing (p = 0.02) (p = 0.0005), implying a functional consequence of infection. Calabrese et al. found CMV, EBV, HHV6, 7 or 8, but not other viruses by PCR in bronchoalveolar lavage in 40% of PF vs. 7.3% of disease control, non-IPF diffuse parenchymal, lung transplant explants (p = 0.0003), and 0% of healthy lungs  in AEC in 40% of positive PF cases.Other studies again showed increased prevalence of serum EBV IgA, possibly implying a mucosal process, and EBV DNA in bronchoalveolar lavage fluid from IPF patients. Sixty percent of 17 PF patients compared with 22% of 50 healthy controls and 24% of 46 controls with other interstitial lung diseases had detectable serum EBV IgA to early, nuclear or capsid antigens ( = 0.02) . The fol = 0.02) . Herpes hy lungs . EBV wasHerpesvirus saimiri, a virus closely related to HHV8, in the regenerating epithelial cells of 21/21 IPF cases and 0/21 controls -K, the expression of which is potentiated by EBV. Since only 1 EBV-associated RNA (EBER) was sought it remains possible that this latency-associated gene is only expressed at levels below the limit of detection in infected lung cells . A very er group . LikewisPF lungs . A studyPF lungs . HHV8 anPF lungs .Since patients in the studies discussed above were stable at the time the lung material was obtained, the results cannot address any role for herpes viruses in acute exacerbation, including subclinical exacerbation, which may nevertheless contribute to functional decline. Investigation of acute exacerbation has necessarily been limited by barriers in obtaining suitable material in very sick patients. Acute exacerbations were previously defined as deterioration in the absence of infection or other known factor. The role of infection, often cryptic, in triggering acute decline is now well-accepted but the definition has made it difficult to assess the role of virus in such decline.In 2010, Huie et al. noted the presence of CMV or HSV by PCR or culture in bronchoalveolar lavage of 4 of 27 patients with IPF and other fibrotic diseases. In the patients who had biopsy material available, no CMV or other viral inclusions were seen, nor was there detectable viraemia, suggesting that the viruses were either commensals, or at least were not exerting any direct cytopathic effect .Wootton et al. investigated bronchoalveolar lavage samples from PF patients undergoing acute exacerbation and found PCR, array or deep sequencing evidence of virus presence in only 19 of 43 cases, of which 3 were herpes viruses (EBV in 2 cases and HSV in 1) . Ushiki Overall epidemiological evidence appears to be in favor of a role for herpes viruses, at least in stable disease , but a lAn extensive study of asymptomatic relatives of patients with FPF showed that patients with PF had increased prevalence of herpes virus DNA in cell-free bronchoalveolar lavage fluid and evidence of herpes virus antigen expression in AEC ; asymptoIn contrast, observations in other animals provide more compelling evidence of a role for herpes virus in PF , 21, 54.Respiratory viruses can cause damage via direct cytopathic effects, by potentiation of secondary bacterial infections or via associated inflammation , 63. PreThe microbiome is increasingly recognized as an important modulator of disease, including in pulmonary fibrosis , 73, 74 H. saimiri or HHV8  or Th17 -polarized lymphocyte response can induce epithelial-mesenchymal transition of AEC2, their differentiation to myofibroblasts, activation of a type 2 cytokine-mediated immune response, profibrotic macrophages, fibroblast-myofibroblast transformation and neutrophil influx, in a complex manner which incorporates several paracrine amplification loops. Extracellular matrix, such as collagen, hyaluran or fibronectin, laid down by myofibroblasts causes architectural distortion and impaired gas transfer of clinical pulmonary fibrosis, and will itself contribute to fibrotic progression , 78. TGFH. saimiri is known to promote expression of human IL-17. Folcik et al. demonstrated co-expression of H. Saimiri and IL-17 in regenerating AEC2 from human PF lung biopsies  mRNA and protein, as well as platelet-derived growth factor (PDGF), are expressed by alveolar epithelium in IPF but not normal human lung biopsies . CCL2 atAnimal studies demonstrate association of herpes viruses with a pro-fibrotic immunological environment. γHV-68 infection of Th2-biased mice increases secretion of TGFβ and cysteinyl leukotriene synthesis in AEC2 , hyperplAnother study showed that aged, but not younger mice developed γHV-associated pulmonary fibrosis, with TGFβ production by alveolar macrophages and AEC2 and increased sensitivity of fibrocytes to TGFβ, despite similar levels of viral DNA and Th1/Th2 cytokine ratio . Use of In a Th2-biased (IFNγ knockout) mouse model, PF was initiated by γ-herpes virus infection and was associated with high levels of lung VEGF, which were reduced when lytic infection was controlled or prevented by infection with lytic-phase-incompetent virus .Alveolar macrophages and other immune cells in γHV-68-infected mice also produce chemokines such as CCL3, 4 & 5 , and morshowed that pulmonary CMV reactivation was universal when Th2-biased mice were subject to sepsis induced by caecal puncture. CMV reactivation under these circumstances was associated with increased and prolonged secretion of TNFα, IL1β, neutrophil chemokine KC and MIP2/CCL4 with collagen deposition and pulmonary fibrosis, which did not occur in uninfected mice  mice. As well as increased AEC and macrophage CCL2/12 production, there were increased levels of the type I cytokines, IFNγ and TNFα. Type 1 cytokines are usually considered protective against PF, but, as illustrated by this model, may be associated with acute alveolar damage and be deleterious early in the course of an exacerbation, particularly since TNFα can induce production of profibrotic cytokines such as TGFβ and PDGF , associated with increased CXCL12, a fibrocyte-attracting chemokine, and TGFβ expression , 56 suggH. pylori , suggesting exposure to such infectious agents should be an important consideration in studies of telomere dynamics . A very . pylori .Latent herpes virus infection may impact telomere length by various mechanisms. In human diploid fibroblasts, HSV1 rearranges telomeres, is able to induce transcription of TERRA, a non-coding RNA essential for telomere homeostasis and degrades TPP1, a component of shelterin, a protein complex critical for stabilizing telomeres. Increased TERRA dampens the DNA damage response, and together with the enhanced telomere loss associated with TPP1 reduction, promotes cellular senescence, rather than apoptosis of aged cells, facilitating further HSV1 replication .Human herpes virus 6A and 6B, and possibly human herpes virus 7 can integrate into telomeres, enabling somatic and sometimes vertical transmission . Indeed,Stem cell transplant is known to be associated with reactivation of chromosomally-integrated HHV6, e.g., . People Cellular competition for transcription factor binding can be impacted by herpes viruses, which could decrease telomere length by “micro-competition.” After establishing a latent infection, the viral N-box (a core binding sequence contained by many viruses in their promoter /enhancer regions), binds the cellular transcription factor GA binding protein (GABP) p300 transcription complex, causing production of abnormal levels of cellular proteins. Viruses with strong N-boxes in their promoters/enhancers include herpes viruses EBV, CMV, HSV, VZV, Hepatitis B & C  and Human Papillomavirus (HPV). CMV has the strongest promoter/enhancer of these. The micro-competition theory suggests that a latent CMV binds the host GABP-binding N box, decreasing the availability of GABP to the promoter of TERF2, which encodes a shelterin protein, essential for telomere maintenance . Herpes To summarize for ageing, telomere attrition and cellular senescence, there is growing evidence for the causal role of compromised telomere maintenance or short telomeres in PF pathogenesis, certainly in a substantial subset of patients. Here we present several reports of herpes virus interactions with cellular DNA that could also affect telomere maintenance.Ageing individuals have higher levels of endoplasmic reticular (ER) stress. Increased ER stress and apoptotic markers are evident in explants of idiopathic PF patients, perhaps related to generically shorter telomeres, higher cellular senescence and higher TGFβ levels , 110. CMHerpes virus antigens co-localize with ER stress markers in AEC2 in IPF lung biopsies as evidenced by markers of the unfolded protein response in the AEC  and corrAged, but not young, Th1-biased mice infected with γHV developed prolonged pulmonary inflammation and PF, compared with young mice who showed resolution without long-term fibrosis. Aged mice showed marked increase of ER stress markers and apoptosis of AEC2, prior to onset of fibrosis, despite controlling herpes virus to the same extent as young mice . Thus, aIn summary, there appear to be several plausible mechanisms by which herpes viruses could also enable or promote lung fibrosis via ER stress.Several antiviral drugs with activity against lytic herpes viruses are currently available acyclovir and ganciclovir are widely available, and well-tolerated even in long-term use for treatment and prophylaxis of herpes simplex, zoster, and cytomegalovirus disease, respectively , 115. FoDespite our inability to cure latent herpes virus, or to prevent infection by vaccination, several animal studies have demonstrated clinical benefit from antivirals. Cidofovir administered to γ-herpes virus-infected IFNγR deficient (Th2-biased) mice, reduced the detection of herpes-related protein in lung and spleen, prevented progression of fibrosis and loss of lung function, and reduced production of TGFβ, inflammatory cytokines IFNγ, TNFα, and Th2 cytokines IL5 and IL13 . TreatmeEvidence of clinical benefit has also been demonstrated in humans. A patient with pulmonary fibrosis and a family history of rapid progression experienced clearance of pulmonary EBV expression, as measured by sputum PCR viral load and stabilization of lung function over 9 months, while on valaciclovir. A second patient treated with valaciclovir experienced clinical deterioration. However, this patient continued to have detectable sputum HHV7, despite EBV clearance .In a small open-label study of 2 weeks' intravenous ganciclovir, 9 of 14 patients were classified as “responders” with improvement in a composite outcome of FVC, DTPA clearance, shuttle-walk test and reduction in prednisolone dose. Ganciclovir was well-tolerated without significant adverse events. Of those treated, 3 were alive without transplant, 2 had died and 3 had had lung transplant at 12 months follow up, compared with “non-responders” all of whom had died (3) or were post-transplant (2 subjects) .Interferon gamma also has antiviral properties. In human PF and normal lung fibroblast cultures, IFNγ downregulates proliferation and differentiation to myofibroblasts in response to TGFβ and to act in a synergistic manner with pirfenidone . AlthougApplying learning from mouse models to treatment of patients, the anti-fibrotic effect of inhibition of TGFβ-ALK5 signaling in bleomycin-induced pulmonary fibrosis in mice is attenuated in the presence of concurrent γ-herpes virus infection, suggesting that herpes virus control may be necessary in order to optimize benefit from licensed antifibrotic drugs such as Pirfenidone. Pirfenidone may act by inhibiting TGFβ, probably indirectly, and, in the absence of pharmacological virus control, could potentially enhance herpes virus burden via the immunosuppressive effect of TGFβ .Given the high community prevalence of herpes virus infections, the significance of current diagnostic tests, whether serological, antigen-based or histological, in a patient with PF, is largely unknown.Better identification of biomarkers for relevant herpes virus infection may enable targeting of treatment to those most likely to benefit, and along with other biomarker-targeted treatment could enable truly personalized treatments. Animal studies clearly show that herpes virus-associated changes occur prior to onset of clinical features and in some cases that early treatment provides more greater benefit than later . For thiPulmonary fibrosis is an end-stage manifestation of many different and interacting processes. Although human and animal data suggests that herpes viruses are implicated in initiation, progression and exacerbation of pulmonary fibrosis, other studies suggest no such effect. Direct alveolar epithelial damage, induction of a profibrotic immunological environment, endoplasmic reticular stress, telomere attrition and cellular senescence are consequences of herpes virus infection, which, in concert with other environmental and genetic factors could increase the risk of pulmonary fibrosis. Techniques such as single cell RNA sequencing will provide important answers as to whether herpes viruses are implicated in pulmonary fibrosis. Cell specific data are required to understand the mechanisms by which herpes viruses could induce, maintain or exacerbate the fibrotic process. If herpes viruses are indeed relevant to pulmonary fibrosis, anti-herpes virus treatments could play a part in modifying or slowing pulmonary fibrosis progression, especially when used at an early stage.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.AD conceived the paper and together with HL wrote the first draft. CS and JP reviewed the manuscript and contributed new sections. AD and JP compiled the tables. All authors contributed to the literature review and approve the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bornaviridae produce enveloped virions containing a linear negative-sense non-segmented RNA genome of about 9 kb. Bornaviruses are found in mammals, birds, reptiles and fish. The most-studied viruses with public health and veterinary impact are Borna disease virus 1 and variegated squirrel bornavirus 1, both of which cause fatal encephalitis in humans. Several orthobornaviruses cause neurological and intestinal disorders in birds, mostly parrots. Endogenous bornavirus-like sequences occur in the genomes of various animals. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Bornaviridae, which is available at ictv.global/report/bornaviridae.Members of the family  Where known, bornavirus virions are spherical in shape with a bimodal size distribution of larger (110–130 nm diameter) and smaller particles (70–90 nm diameter) . VirionsN-X/P-M-G-L-5′ (Orthobornavirus) or 3′-N-X/P-G-M-L-5′ (Carbovirus and Cultervirus) [The bornavirus genome consists of a linear negative-sense non-segmented RNA with six open reading frames (ORFs) in the order 3′-ervirus)  that encStudies of Borna disease virus 1 indicate that N encapsidates the genomic RNA, forming the viral nucleocapsid, and, together with L and P, forms the ribonucleoprotein (RNP) complex . The genwww.ictv.global/taxonomy. Bornaviruses form a family in the haploviricotine order Mononegavirales, most closely related to members of the families Mymonaviridae, Nyamiviridae and Xinmoviridae. Like most other mononegaviruses, bornaviruses (i) have linear negative-sense non-segmented RNA genomes, (ii) have five conserved motifs (A–E) in the amino-acid sequence of their RdRP domain and (iii) produce enveloped virions.Current taxonomy: Bornaviridae: www.ictv.global/report/bornaviridae.Current ICTV Report on the family


Background: The most common reason for hospitalization in the United States is childbirth. The costs of childbirth are substantial.Materials and Methods: This was a retrospective cohort study of hospital deliveries identified in the MarketScan® Commercial and Medicaid health insurance claim databases. Women with an inpatient birth in the calendar year 2016 were included. Severe maternal morbidity (SMM) was identified using the Centers for Disease Control and Prevention algorithm of 21 International Classification of Diseases-10 codes. Mean costs and cost ratios for women with and without SMM were reported. Generalized linear models were used to analyze demographic and clinical variables influencing delivery costs.Results: We identified 1,486 women in the Commercial population, who had a birth in 2016 and met the criteria for SMM. The total mean per-patient costs of care for women with and without SMM were $50,212 and $23,795, respectively. In the Medicaid population there were 29,763 births, of which 342 met the criteria for SMM. The total mean per-patient costs of care for women with and without SMM were $26,513 and $9,652, respectively. A multifetal gestation, a cesarean delivery, maternal age, and pregnancy-related complications were independently predictive of increased delivery costs in both Commercial and Medicaid populations.Conclusions: The occurrence of SMM was associated with an increase in maternity-related costs of 111% in the Commercial and 175% in the Medicaid population. Some of the factors associated with increased delivery hospitalization costs could be treated or avoided. The Commercial database includes paid medical and prescription drug claims for several million individuals annually from ∼300 self-insured U.S. employers and 25 health plans, while the Medicaid database contains the pooled health care experience of Medicaid enrollees from 11 states.18 Due to privacy agreements between the Medicaid state agencies and the agency that manages the coordination of benefits, the states included are not publicly identifiable. The MarketScan data were deidentified, so that Institutional Review Board approval was not required.This was a retrospective cohort study of SMM in beneficiaries enrolled in the MarketScan19 In the primary analysis, continuous enrollment (without gaps) was required during the 9 months before and 30 days following the delivery.The study population included all women with an inpatient birth in 2016, identified by ICD-10 diagnostic and procedural, Current Procedural Terminology, and Diagnosis-Related Group codes for a delivery .19 In th13 Preexisting comorbidities and obstetric-related complications were identified from the literature and the corresponding ICD-10 codes identified , gestation type , delivery method , geographic region , and race/ethnicity (Medicaid population only). Geographic regions included the four divisions defined by the U.S. Census: Northeast, Midwest, South, and West. Race/ethnicity was categorized as White non-Hispanic, Black non-Hispanic, Hispanic, and other.SMM was defined by the occurrence during delivery hospitalization of one or more of the 21 potentially life-threatening conditions or complications identified by the diagnostic and procedural ICD-10 codes identified by the CDC .13 Preexentified .20–22 Pat-test. Generalized linear models were used to analyze the relationship between demographic and clinical variables and delivery costs, adjusting for preexisting conditions and pregnancy-related complications. Generalized linear model methodology is an appropriate technique for analyzing skewed cost data.23 The models used a log link and gamma error distribution function that conforms to the non-normal cost distributions typical of health care costs. Results are presented as cost ratios and mean costs with 95% confidence intervals (CIs). For the Medicaid population, a sensitivity analysis was conducted for delivery costs, in which the continuous enrollment criterion was removed. This sensitivity analysis was completed to account for potential selection bias in the Medicaid population, which was observed to have a fragmented enrollment history. All analyses were conducted using SAS Version 9.4.31.Costs are presented in 2016 U.S. dollars. All-cause costs incurred, whether obstetrics related or not, were classified into one of three time periods: the prenatal period , delivery period , and postdelivery period . Total costs were determined for all services, using the amounts paid by insurers plus out-of-pocket and third-party payments. Cost data were calculated as mean, standard deviation. Costs were also calculated as median and interquartile range. Costs were calculated by SMM status, delivery method, gestation type, maternal age category, region in the Commercial population, and race in the Medicaid population. Comparisons of mean costs between women with and without SMM were made using the p < 0.001 for women with SMM versus women without SMM  cost of $22,672 . Delivery costs were 39.3% of total costs for women without SMM, and 28.5% of total costs for women with SMM. The SMM indicator associated with the greatest delivery cost was shock  were 177% higher than for women without SMM $9,652; . Costs o$25,769; .In the generalized linear model analysis, the adjusted delivery cost was 31% greater for women with than for women without SMM . AdjusteIn the sensitivity analysis, conducted without the continuous enrollment criterion, there were 219,670 delivery hospitalizations in the Medicaid population, with a similar rate of SMM  as in the primary analysis . Total mean delivery costs with or without SMM in the sensitivity analysis were twice those in the primary analysis . NeverthIn this analysis, the occurrence of SMM during delivery hospitalization was associated with an increase in total mean health care costs of 111% and 175% in the Commercial and Medicaid populations, respectively. SMM was more strongly predictive of increased delivery hospitalization costs than a cesarean delivery, maternal age, or multifetal gestation. Pregnancy-related complications were independently associated with increased delivery costs in both the Commercial and Medicaid populations. Furthermore, racial and ethnic disparities were observed in the Medicaid population, with elevated costs associated with SMM among Black non-Hispanic women compared with White non-Hispanic and Hispanic women.24 Comparing the present 2016 cost analysis with the previous 2013 analysis,16 total pregnancy-related costs with or without SMM were considerably higher in 2016 in both the Commercial and Medicaid populations: total costs in the Commercial and Medicaid populations were 40%–60% higher without SMM and 150%–260% higher with SMM. Delivery costs with SMM were 48% higher in 2016 than in 2013 in the Commercial population, but similar (4% higher) in 2016 compared with the 2013 Medicaid populations—which suggests that the change in SMM coding algorithm did not itself increase costs.Prenatal, delivery, and postpartum costs were all considerably higher in the Commercial than in the Medicaid population in both women with or without SMM. Commercial insurance is known to reimburse for services at higher payment rates compared with government sponsored insurance.15 Direct comparison of these delivery cost estimates with estimates based on reimbursements in separate Commercial and Medicaid populations is difficult, given the different population structures and cost data sources. The earlier analyses estimated the costs expended by the hospital but excluded physician expenses. In the present study and in the previous 2013 analysis,16 populations of employer-sponsored insurance beneficiaries and Medicaid beneficiaries were analyzed separately. In contrast, the NIS and NYC studies analyzed mixed populations of commercially insured and Medicaid patients .15 In addition, the earlier studies used an algorithm for identifying SMM based on ICD-9 codes, whereas the present study was based on ICD-10 codes.14–16Two earlier studies reported delivery hospitalization costs associated with SMM, using, respectively, discharge data from the 2011 National Inpatient Sample (NIS) and a dataset of all delivery hospitalizations in New York City (NYC) from 2008 to 2012.25 Furthermore, the sensitivity and predictive value of the ICD-10 version of the CDC algorithm for SMM have not been determined.The accuracy of SMM algorithms based on either ICD-9 or ICD-10 codes depends on the correct assignment of codes in hospital discharge records. In a study set in the period 2001–2011, in which the ICD-9 codes for SMM indicators were checked against medical records, the codes were correctly assigned in 82%–86% of instances, although this varied substantially by code.Removing the requirement for continuous enrollment increased the number of delivery hospitalizations in the sensitivity analysis of the Medicaid population. The requirement for continuous enrollment especially impacts the Medicaid population due to the fragmented patterns of enrollment. Delivery costs in the sensitivity analysis were greater overall and across all demographic and delivery characteristics, although the SMM cost ratios were similar. The increased costs could be attributed to the fragmentation of care, in that complications may not be identified or treated in a timely manner. Noted differences in the demographic and clinical profile of the women in the sensitivity analysis could explain the increased costs, but further research will be necessary to understand the specific cost drivers.26 Analysis of cost data is problematic, because the data tend to be non-negative and positively skewed, with heavy tails. In this data set, as is typical, median costs were lower than means in both the Commercial and Medicaid populations  associated with higher costs are preventable. There is evidence supporting the implementation of quality improvement programs designed to reduce maternal morbidity.27 Collections of best practices, or care bundles, have been developed to address obstetric hemorrhage and severe hypertension in particular.27–29 In the postdelivery period, the American College of Obstetricians and Gynecologists has recommended that postpartum care should become an ongoing process, with services tailored to each woman's individual needs.31 In addition, the fragmentation of care in the Medicaid population could be addressed by modifying eligibility and enrollment criteria. Our data suggest that the costs of these initiatives could be offset by reductions in costs associated with SMM.The data in this study indicate that there may be financial incentives for initiatives to reduce maternal morbidity. In the predelivery period, many of the pregnancy-related complications (gestational hypertension, preeclampsia, obstetric infection, In conclusion, women experiencing SMM incurred significantly greater health care costs during the prenatal, delivery hospitalization, and 30-day postdelivery periods compared with women without SMM. Several demographic factors, preexisting comorbidities, and obstetric complications were associated with increased delivery hospitalization costs.


The transmission of SARS-CoV-2 is likely to occur through a number of routes, including contact with contaminated surfaces. Many studies have used reverse transcription-PCR (RT-PCR) analysis to detect SARS-CoV-2 RNA on surfaces, but seldom has viable virus been detected. This paper investigates the viability over time of SARS-CoV-2 dried onto a range of materials and compares viability of the virus to RNA copies recovered and whether virus viability is concentration dependent. Viable virus persisted for the longest time on surgical mask material and stainless steel, with a 99.9% reduction in viability by 122 and 114 h, respectively. Viability of SARS-CoV-2 reduced the fastest on a polyester shirt, with a 99.9% reduction within 2.5 h. Viability on the bank note was reduced second fastest, with 99.9% reduction in 75 h. RNA on all surfaces exhibited a 1-log reduction in genome copy number recovery over 21 days. The findings show that SARS-CoV-2 is most stable on nonporous hydrophobic surfaces. RNA is highly stable when dried on surfaces, with only 1-log reduction in recovery over 3 weeks. In comparison, SARS-CoV-2 viability reduced more rapidly, but this loss in viability was found to be independent of starting concentration. Expected levels of SARS-CoV-2 viable environmental surface contamination would lead to undetectable levels within 2 days. Therefore, when RNA is detected on surfaces, it does not directly indicate the presence of viable virus, even at low cycle threshold values.IMPORTANCE This study shows the impact of material type on the viability of SARS-CoV-2 on surfaces. It demonstrates that the decay rate of viable SARS-CoV-2 is independent of starting concentration. However, RNA shows high stability on surfaces over extended periods. This has implications for interpretation of surface sampling results using RT-PCR to determine the possibility of viable virus from a surface, where RT-PCR is not an appropriate technique to determine viable virus. Unless sampled immediately after contamination, it is difficult to align RNA copy numbers to quantity of viable virus on a surface. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) has spread globally, and many countries are experiencing ongoing local transmission despite various levels of control efforts. SARS-CoV-2 is primarily transmitted via respiratory droplets from an infected host 24, with SSARS-CoV-2 RNA has been detected on environmental surfaces, potentially indicating the presence of viable virus , 10. CurThe three aims of this study were (i) to measure the persistence of viable SARS-CoV-2 virus on common personal protective equipment (PPE) materials , high-touch surface materials, and commonly worn fabrics; (ii) to investigate the relationship between recoverable viable virus from these surfaces and the levels of SARS-CoV-2 RNA in the same sample; and (iii) to determine the relationship between inactivation rate and initial viral titer load on surfaces.10, with standard deviations across materials of 1.06 log10  from a high starting inoculum of approximately 4 × 105 PFU per material . Viable virus could be recovered from the surgical mask and stainless steel coupons for the longest periods of time . The recovery time for cotton t-shirt and polyester sports shirt materials was shorter (log10 reductions of 5.15 over 5 days and 3.9 within 1 day). RNA copy number was recovered at higher concentrations in all samples compared to the levels of viable virus, decreasing by ∼1.5 log10  and ∼1 log10  over the initial 7 days and then stabilized at around 107.5 copies per coupon from day 7. The ratio of viable virus recovered ranged from 103 to 108 times less than the viral RNA assayed from start to finish of the study period .The results from the comparative study involving two viral titers revealed an initial rapid decrease in the recovery of viable virus from the surface during the drying period, with the inactivation rate decreasing after drying. Virus was recovered after 4 days for the low inoculum and up to 7 days for the high inoculum . ParalleContact with SARS-CoV-2-contaminated surfaces is thought to be a route of transmission in the current pandemic , 15. SurExisting studies examining time-based viability of SARS-CoV-2 on different surfaces have focused on a single virus titer , 13, 17.10 reduction at 28 days and Kasloff et al. at 14 days using the Australian isolate Betacoronavirus/Australia/SA01/2020 and Canadian isolate hCoV-19/Canada/ON-VIDO-01/2020, respectively (5 and 106 PFU per surface) and environmental conditions of temperature and relative humidity (RH) are similar for the two previous studies and this study, demonstrating it is likely that the differences observed in survival results primarily arise from a protective effect afforded by added serum/mucin and/or the different isolates used. A difference between the current study and the previous studies was the exposure of the coupons. During Kasloff et al.’s study, the coupons were placed in a vented box and stored within a closed cabinet once dried. In Riddell et al.’s study, the coupons, once dry, were placed in a climate chamber in the dark. The current study held the coupons in a flexible film isolator (FFI) for the exposure period  and SARS-CoV-2 (nCoV-WA1-2020) by van Doremalen et al. showed that both were similar under their experimental test conditions . Chin etDroplets on nonporous or hydrophobic surfaces dry in a beaded shape, giving a high volume-to-surface area ratio. In such an environment, these droplets can produce a core-shell structure , which c10 reduction in recovered infectivity over 7 days. Viable SARS-CoV-2 was recovered from these surface materials over longer periods of time compared to the truly porous and hydrophilic surfaces tested, cotton and woven polyester. An exception was the hydrophobic polymer bank note, from which viable virus was recovered at the limit of detection for days 5 and 7 of the study. This is different from the study of Riddell et al., where the recovery from the Australian bank note was similar to that of the other nonporous surfaces tested  was spun into fine fibers, aligned in the same orientation, and woven into fabric, it behaved like the other woven fabric tested, cotton. It is possible that the aligned polyester fibers, which are close together but not fused, causes capillary action to draw the liquid into the interstitial spaces between the fibers and trap the virus particles. Virus that was inoculated onto a polyester sports shirt was rapidly inactivated to unrecoverable levels in 1 day; there may also be interaction between the chemicals used to process/color the fabric and the virus .SARS-CoV-2 RNA has been detected on surfaces in different environments, but there have been few reports of viable virus recovery from these surfaces. The use of RT-PCR to determine the presence of SARS-CoV-2 on surfaces has advantages, i.e., increased sensitivity  and rapid high throughput of samples compared to culture-based methods. The limitation of the use of RT-PCR in such studies is its inability to distinguish between viable and nonviable virus, whereas a positive result from a culture-based method would indicate that an infectious particle was present on the surface at the time of sampling and a there was potential for transmission. Pretreatment of swabs or samples with agents such as propidium monoazide may help reduce amplification of nucleic acid from damaged or degraded viral particles; however, there are limited data available validating their utility for SARS-CoV-2 , and assTC) values from the RT-PCR assay indicate that more copy numbers of target RNA are present in that sample. Our study determined that a TC value of 18 equates to approximately 5 × 108 copy numbers of the RNA target. The initial recovery of infectious virus from the materials  lower than the copy number, showing that there is a large amount of RNA exogenous to infection-competent viral particles in the inoculum. This difference between infectious virus and RNA copy number was also reported by Kasloff et al. (The detection of SARS-CoV-2 RNA from surface samples is used to indicate that virus (viable or nonviable) was present on that surface at some point previously. Lower cycle threshold  passage 3 (P3), was propagated by the High Containment Microbiology Department at PHE, Porton Down. The virus was isolated from a clinical sample taken during acute-phase illness, using Vero E6 cells . A P2 master bank was produced using Vero E6 cells  and a P3 working bank produced in Vero/hSLAM cells . Cell lines were infected at 95% confluence at a multiplicity of infection (MOI) of 0.0005 to 0.01 and maintained in 1× minimal essential medium, GlutaMAX, 4% heat-treated fetal bovine serum (Gibco), 1× nonessential amino acids (Gibco), 25 mM HEPES buffer (Gibco). Additionally, Vero/hSLAM cells were maintained in the presence of 0.4 mg/ml Geneticin . Virus was harvested 3 to 6 days postinfection and supernatant clarified by centrifugation . Virus was aliquoted and stored at −80°C. The titer of the P3 virus stock was determined to be 2.0 × 10The surfaces used in this study are representative of nonporous hand-touch sites , a bank note (English polymer £10 note), PPE items used in the hospital and wider environments , and materials representing clothing items, a cotton t-shirt (Fruit of the Loom) and a polyester sports shirt . Coupons (1 cm by 1 cm) of each material were prepared. Once prepared, nonporous coupons (stainless steel and bank note) were cleaned with Neutracon  detergent followed by rinsing with 70% isopropyl alcohol (IPA). Additionally, stainless steel was sterilized by autoclaving. The surfaces not suitable for cleaning  were purchased new and handled aseptically. Materials were subdivided depending on their surface properties .5 PFU/ml with complete minimal essential medium (cMEM) for low loading or used neat for high loading. Only the high-concentration inoculum was used for the experiments investigating viable virus and RNA recovery on all surfaces, whereas the high and low inocula were applied only to stainless steel for viability with different concentrations. The inoculum was frozen at −80°C and assayed with the generated samples. Coupons were inoculated by the addition of two 10-μl droplets of virus culture within a negative pressure flexible film isolator (FFI) and were left uncovered in plastic petri dishes in the FFI for the duration of the study at a temperature of 21.5°C (±1°C) and an average relative humidity of 45%. Three biological replicate coupons were prepared and inoculated for each time point  and each material. Triplicate coupons exposed to but not inoculated with the virus acted as negative controls. No viable virus or viral RNA was detected in the negative controls.Viral aliquots were thawed to room temperature immediately prior to inoculation of the materials. The stock suspension was diluted to 2 × 10P = 0.345; Mann-Whitney rank sum test) was found between samples processed immediately and those freeze/thawed. Time point zero (t = 0) coupons were recovered within 5 min of being inoculated before any drying occurred. The recoveries from the zero time point coupons were used to determine the log10 reductions from the subsequent samples.Recovery of virus from the coupons at each specified time point was performed by transferring a coupon into 1 ml of cMEM in a 7-ml bijou tube with 4 glass beads (3-mm diameter), followed by vortex mixing for 1 min at maximum speed (Heidolph Multireax vortex). The resulting suspension was transferred to a cryotube for storage at −80°C before analysis. Storage of samples at −80°C was not found to significantly affect viability of the virus or denature the RNA. No significant difference (l-glutamine (Gibco), 1% nonessential amino acids, and 2.5% 1 M HEPES. A volume of 100 μl of each dilution was pipetted in duplicate  for up to four replicates for neat dilutions (400 μl) onto confluent Vero E6 cells within a 24-well plate (7.9 × 104 cells/cm2). After 1 h of incubation (±15 min) at 37°C with plate rocking every 15 to 20 min, 0.5 ml CMC overlay was added to each well, containing 1.5% CMC (3% [wt/vol] carboxymethylcellulose solution in sterile distilled water [Sigma C4888]), 1% antibiotic antimycotic solution (100×) (Sigma-Aldrich), 2× overlay medium (20% 10× MEM [Gibco]), 2% l-glutamine (200 mM) (Gibco), 2% nonessential amino acids (Gibco), 6% sodium bicarbonate solution (Gibco), 8% fetal bovine serum (Sigma-Aldrich), 5% HEPES buffer (Gibco), and 57% distilled water (Versol). After 3 days of incubation at 37°C, cells were fixed with formaldehyde and stained by addition of approximately 250 μl 0.2% crystal violet for 5 min before washing with water. The number of plaques in each well was determined and expressed as plaque-forming units.Coupon recovery liquid was assayed by thawing samples at room temperature and then serially diluted (1 in 10) with MEM (Gibco), 1% in vitro transcript  of the coupon recovery liquid using the QIAamp viral RNA minikit . RT-PCR was performed using the VIASURE SARS-CoV-2 real-time PCR detection kit  by following the methods provided. Quantification was undertaken using the N target with a standard curve generated by serial dilution of an anscript .Each time point for each material coupon had 3 biological replicates  and 2 technical replicates (plaque assay performed in duplicate). Calculations for the mean assay counts were determined from these 6 replicates and standard deviations from the biological replicates using Microsoft Excel (Office 365). Time to percent reduction values was calculated using linear regression of PFU/coupon averages in Minitab 18. High and low loading line slopes were analyzed using linear regression analysis with GraphPad Prism software, version 7.


Vulpes vulpes) cubs found in the wildwith neurologic signs in the Netherlands. The virus is related to avianinfluenza viruses found in wild birds in the same area.We detected infection with highly pathogenic avian influenza A(H5N1) virus clade2.3.4.4b in 2 red fox ( Vulpes vulpes cub (cub 1) displaying abnormalbehavior was found in Bellingwolde, the Netherlands, and taken into care of a wildliferescue center. Upon entry, the 6- to 8-week-old cub was slightly dehydrated and showedat intervals of <30 minutes lip retraction, rapid opening and closing of mouth,excessive salivation, skin twitching, head shaking, and body tremors  .Test results were negative.Although rabies lyssavirus is unlikely in the Netherlands, European bat 1 lyssavirusis endemic in serotine bats  were reported in the northern part of the Netherlands, andlater other species of waterfowl and birds of prey were also found dead. A selectednumber of dead wild birds were submitted for AI diagnostics and tested positive forHPAI H5N1 virus. We performed whole-genome sequencing of the HPAI H5N1 viruses foundin wild birds and the 2 foxes as previously described (https://www.gisaid.org] accession no. EPI_2194218) and cub 2 (GISAIDaccession no. EPI_2194219) were identical; the closest related virus was identifiedin a white-tailed eagle  near the village ofNoordlaren. We observed only 6 aa differences: mutations A152T and T521I inpolymerase basic protein 2 (PB2); M644V in polymerase basic protein 1; A336T innucleoprotein; L22S in neuraminidase protein; and D209N in nonstructural protein. Whether these changes are associated with adaptationof the avian virus to mammal species remains unknown.During April–May 2021, large numbers of dead barnacle geese  virusin wild red foxes, the Netherlands, 2021.Acknowledgment of authors and submitting laboratories of the sequences fromthe GISAID EpiFlu Database used in a study of highly pathogenic avianinfluenza A(H5N1) virus in wild red foxes, the Netherlands, 2021.