Oryza sativa) protoplasts suitable to reconstitute CK signaling branches using the CK reporter construct TCSn:fLUC, consisting of a synthetic CK-responsive promoter and the firefly luciferase gene, as a sensitive readout of signaling output. We used this system to systematically test the contributions of CK signaling components, either alone or in various combinations, with or without CK treatment. The type-B response regulators (RRs) OsRR16, OsRR17, OsRR18, and OsRR19 all activated TCSn:fLUC strongly, with OsRR18 and OsRR19 showing the strongest induction by CK. Cotransfecting the reporter with OsHP01, OsHP02, OsHP05, or OsHK03 alone resulted in much weaker effects relative to those of the type-B OsRRs. When we tested combinations of OsHK03, OsHPs, and OsRRs, each combination exhibited distinct CK signaling activities. This system thus allows the rapid and high-throughput exploration of CK signaling in rice.The major components of the cytokinin (CK) signaling pathway have been identified from the receptors to their downstream transcription factors. However, since signaling proteins are encoded by multigene families, characterizing and quantifying the contribution of each component or their combinations to the signaling cascade have been challenging. Here, we describe a transient gene expression system in rice ( Plants interact with their environments via the perception of physical and chemical signals, such as light, temperature, and nutrients. Among the multiple mechanisms employed by plants to respond to their surroundings, phytohormones are endogenous chemical signals that take on a prominent role to modulate and coordinate plant growth and development by integrating the external environment and intracellular programs ,2. Each Phytohormone signaling cascades are complex due to redundancy among signaling components, which are typically encoded by multigene families, as well as multilayered effects among phytohormones . Thus, aSaccharomyces cerevisiae), and homologous systems for transient gene expression systems, such as protoplasts and Agrobacterium-mediated infiltrations, have been used to reconstitute a phytohormone, ABA signaling pathways, and quantitatively measure the function of each signaling component [Transient gene expression systems are powerful methods, as they allow rapid and large-scale screening of signaling components, despite the potential limitations mentioned above. Accordingly, heterologous systems, such as yeast  gene acted as a sensitive CK reporter of type-B RR transcriptional activity [TCSn , which provided an attractive and easy readout of CK signaling in rice and maize (Zea mays) when driving the expression of the Green Fluorescent Protein (GFP) gene in the TCSn::GFP reporter [TCSn promoter was also used to drive the expression of β-GLUCURONIDASE (GUS) to reveal CK signaling in the root after treatment with 6-benzylaminopurine (BA) [TCSn promoter is therefore an efficient CK sensor that can be combined with different reporter genes to monitor CK signaling in diverse tissues. Recently, a new version of a CK-responsive promoter was also developed, referred to as TCSv2 [The phytohormone cytokinin (CK) modulates cell division, senescence, and chloroplast development ,13. The activity . This CKreporter . The synine (BA) . The TCSas TCSv2 .TCSn::LUC in various combinations. We monitored the activation of CK signaling, as determined by luciferase activity. Our results illustrate the applicability and usefulness of this system to discriminate the contribution of each CK signaling protein in relation to its signaling partners within networks and will be instrumental in deciphering the role of other components of the CK signaling cascade.In this study, we attempted to reconstitute the CK signaling cascade and identify the effects of each signaling component in rice protoplasts. We transiently cotransfected protoplasts with constructs overexpressing the three CK signaling components  in addition to the CK sensor TCSn promoter consists of 16 copies of the ARR1A motif (5′-NGATT-3′), which is recognized by the type-B RR binding site, and induces the expression of reporter genes in a CK-dependent manner in both Arabidopsis and rice [TCSn promoter into a vector harboring the firefly luciferase gene (fLUC) to generate a versatile reporter for CK signaling in rice protoplasts. First, we tested whether this promoter induced luciferase activity specifically in response to CK treatment in rice protoplasts. To this end, we treated transiently transfected rice protoplasts expressing TCSn::fLUC and pUbi10::rLUC with major phytohormones individually at a final concentration of 5 µM. Only 5 µM BA resulted in a fivefold increase in luciferase activity over mock controls. Other phytohormones failed to raise LUC activity significantly over mock controls, demonstrating the specificity of the TCSn::fLUC reporter for CK  and trans-zeatin (tZ), together with BA; all three CKs induced LUC activity to the same extent, although BA appeared to be slightly less potent at lower concentrations  are the MYB transcription factors that bind to the am genes . The TCSt manner A. Of allcontrols A. We detcontrols B.OsHP01 or OsHP02 alone in rice protoplasts weakly induced fLUC activity, depending on DNA concentration irrespective of CK. By contrast, overexpression of OsHP05 failed to significantly raise fLUC activity over levels measured for TCSn:fLUC alone up to 5 µM DNA concentration  [Histidine phosphotransfer proteins (HPs) relay the phosphoryl group from the CK receptor to type-B OsRRs, leading to their activation. The overexpression of ntration A. To monreatment B. Notabln (BiFC) . All theArabidopsis genomes both encode eight histidine kinases (HKs). However, three of them  are actually CK receptors in Arabidopsis, and four of them are functional CK receptors in rice [OsHK03 and cotransfected the resulting construct with the TCSn::fLUC reporter into rice protoplasts. OsHK03 overexpression only weakly raised fLUC activity over that measured from the reporter alone in an effector DNA concentration- and BA-dependent manner. We then monitored the effect of cotransfecting OsHPs and OsHK03 in protoplasts on reporter activity. Basal transcriptional activation of the reporter significantly increased when OsHK03 was co-transfected with OsHP01 or OsHP02, but not with OsHP05, when compared with fLUC activity from OsHK03 transfected alone. Upon treatment with BA, the OsHK03/OsHP01 and OsHK03/OsHP02 pairs further increased reporter activity, in sharp contrast to the OsHK03/OsHP05 pair in which reporter activity showed no significant difference with or without BA , and 16 type-B RRs. While  and HKs , rice isbidopsis .Arabidopsis and rice [TCSn promoter was previously developed and applied to research on CK signaling in Arabidopsis, maize, and rice. In maize protoplasts, the TCSn promoter conferred a 15-fold induction of the reporter gene when treated with tZ; in rice roots, GUS staining from TCSn::GUS reporter lines showed induction about sixfold when exposed to various CKs [TCSn::fLUC reporter vector alone displayed an induction around fivefold upon treatment with CK, comparable to the TCSn::GUS results in rice roots. Coexpression of the type-B RR genes OsRR17, OsRR18, and OsRR19 induced the TCSn:fLUC reporter over 100-fold, thus providing a sensitive system in which to monitor the transcriptional activities of type-B RRs and reconstitute the CK signaling cascade. In this transient system, four type-B RRs exhibited distinct activities in terms of CK specificities and transcriptional activation of the reporter. Indeed, OsRR16 and OsRR17 did not activate the reporter in response to CK, whereas OsRR18 and OsRR19 strongly activated the reporter in response to CK. These results suggest that OsRR18 and OsRR19 might have specific functions at various developmental stages or affect tissues or phytohormone levels, which will await experimental validation.While many genetic studies have characterized the functions of CK-related genes in and rice ,23,31, mand rice . The TCSious CKs ,25. In oOsHP or OsHK genes alone failed to significantly induce the activity of the TCSn::fLUC reporter. However, the cotransfection of all three components, OsHKs, OsHPs, and type-B OsRRs, significantly increased the reporter activity, supporting the notion that all three components are required for full activation of CK signaling. Endogenous CK signaling components are present in rice protoplasts and activate a CK-responsive reporter to interfere in the dissection of the activity of each signaling component. Heterologous systems, such as yeast or animal cell, can be used to avoid the background noises.The overexpression of TCSn::fLUC reporter by OsRR17 in the absence of CK treatment. We interpret this observation as an indication that OsHP increases the CK-specific response of OsRR17. However, other type-B RRs did not show this behavior in relation to OsHPs, possibly because the reconstituted signaling cascade failed to deliver their cognate OsHPs; our conditions may also not have supplied each OsHP with its cognate type-B OsRR. Sun et al. 2014 showed that single knockdown lines for OsHP01 and OsHP02 exhibited significant developmental phenotypes during abiotic stress, supporting the notion that these HPs are functional [Arabidopsis or rice protoplasts [OsRR, OsHP, and OsHKs significantly increased the transcriptional activation of the TCSn::fLUC reporter already in the absence of exogenous CK treatment, while CK treatment only increased LUC activity two- to threefold further, suggesting that the endogenous CK content of protoplasts is sufficient to activate CK signaling in our conditions.Of the three OsHPs tested here, OsHP05 had very low activity toward the reporter, or even suppressed CK responses. OsHP05 was reported to carry a Glu residue instead of His in the phosphotransfer domain, which might make OsHP5 a pseudo HP  Supplem. This hynctional . HoweverOsHP01 and OsHK03 weakly activated the reporter construct.Based on the signaling activities observed here, we hypothesize that the OsHK03/OsHP01 and OsHP02/OsRR17 or OsHP02/OsRR18 pairs work alongside the same signaling network because their combination during transfection generated similar signaling activity. By contrast, OsRR19 does not appear to engage in a significant interaction with OsHP01, as its cotransfection with Plant signaling is very complex and relies on multilayered interactions. A better understanding can be obtained via bioinformatics analysis of gene families and gene expression profiles in response to genetic perturbations, from physical interaction networks, yeast two-hybrid assays, and pull-downs, as well as from proteomics and metabolomics, which require thorough, time-consuming integration. In this work, we aimed to reconstruct individual branches of CK signaling to dissect the effects of each component. The protoplast system allows high-throughput analysis in planta and shows low noise compared with whole plants. Luciferase activity can be easily monitored from transfected protoplasts and constitutes a very powerful tool to study CK signaling and inform candidate gene identification and analysis.Oryza sativa) cultivar Dongjin was used in this study. Hulled rice seeds were surface-sterilized with 70% ethanol for 30 s, followed by 50% NaClO containing a drop of Tween-20 for 15 min with constant agitation and five washes with distilled water. The NaClO sterilization step was repeated once but without Tween-20. The surface-sterilized seeds were thoroughly dried using sterilized dry filter paper. The seeds were then sown on half-strength Murashige and Skoog (1/2 MS) medium (supplemented with 0.4% Phytagel and adjusted to pH 5.8) and allowed to germinate for 10 d in the dark and 2 d in the light at 28 °C before protoplast isolation.The rice . The diluted cell suspension was centrifuged at 100× g for 10 min at room temperature. The protoplast pellet was then resuspended in 3 mL of W5 solution and placed on top of 23% sucrose and centrifuged at 100× g for 10 min to remove remaining debris. The volume of separated protoplasts was then adjusted to 10 mL with W5 solution, and the cell density was determined on a hemocytometer. The protoplasts were centrifuged at 100× g for 7 min at room temperature and resuspended in MaMg solution  for transfection.To isolate protoplasts from young rice seedling, shoots were sliced into 1 mm strips and incubated in enzyme solution , 0.75% Macerozyme R-10 , 0.6 M mannitol, and 10 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.7 adjusting with KOH) that had been activated at 65 °C for 20 min. The mixtures containing rice tissue and enzymes were incubated by shaking for 4 h at 28 °C, filtered on a 145 μm mesh, and diluted with five volumes of W5 solution  were suspended in 300 µL MaMg solution and mixed with up to 30 μg of DNA and 40% PEG solution 2, and 40% PEG 6000). The solution was mixed thoroughly using a fixed-angle rotating wheel for 10 min and incubated for 30 min at room temperature. After incubation, 630 µL, 1.25 mL, and 2.5 mL of W5 solution were added every 10 min over the course of 30 min. The mixtures were incubated for another 25 min and centrifuged at 100× g for 5 min to remove the PEG solution. The protoplast pellets were resuspended in 3 mL of W5 solution and incubated at 28 °C overnight (18 h) with or without CKs in the dark.Protoplasts (4–5 × 10TCSn) promoter as a reporter in rice, as it specifically responds to CK [TCSn promoter was cloned upstream of the firefly luciferase (fLUC) gene to generate a CK reporter. The coding sequences for various CK signaling components (type-B response regulator (RR), histidine phosphotransfer protein (HP), and histidine kinase (HK)) were PCR-amplified, cloned into the pENTRY D-Topo vector , and then recombined into destination vectors for signaling effectors, subcellular localization, and bimolecular fluorescence complementation (BiFC) assays by LR recombination  as previously described [We selected the synthetic Two Component signaling Sensor new , and effector constructs were introduced into rice protoplasts by PEG-mediated transfection. After 18 h of incubation, the protoplasts were centrifuged at 300 g for 30 s and the supernatants removed. Pelleted cells were resuspended in 50 µL 1× passive lysis buffer (PLB). Renilla and firefly luciferase activities were measured with 10 µL lysate according to the manufacturer’s protocol ; firefly luciferase activity was then normalized to Renilla luciferase activity to account for transfection efficiency.For dual luciferase assays, OsRR–GFP fusion constructs. Red fluorescent protein (RFP) with a nuclear localization sequence (NLS) was used as nucleus-localized control. GFP fluorescence (493–546 nm) was observed and imaged using a Leica TCS SP8 confocal microscope . For BiFC assays, OsRR17 was fused to the N-terminus of the Venus variant of GFP (OsRR17-VN), while three OsHPs were fused to the C-terminus of Venus (OsHP-VC). The corresponding constructs were transfected in rice protoplasts as above. OsbZIP10-VC was used as a negative control, and NLS-RFP was used as a nucleus-localized control. RFP fluorescence (581–652 nm) was observed and imaged using a Leica TCS SP8 confocal microscope.To determine the localization of type-B OsRRs in plant cells, we transfected rice protoplasts with


Human T-lymphotropic virus type 1 (HTLV-1) infection may cause serious disease, while pathogenicity of HTLV-2 is less certain. There are no screening or surveillance programs for HTLV-1/-2 infection in Brazil. By performing this systematic review, we aimed to estimate the prevalence of HTLV-1/-2 infections in pregnant women in Brazil. This review included cohort and cross-sectional studies that assessed the presence of either HTLV-1/-2 infection in pregnant women in Brazil. We searched BVS/LILACS, Cochrane Library/CENTRAL, EMBASE, PubMed/MEDLINE, Scopus, Web of Science and gray literature from inception to August 2020. We identified 246 records in total. Twenty-six of those were included in the qualitative synthesis, while 17 of them were included in the meta-analysis. The prevalence of HTLV-1 in Brazilian pregnant women, as diagnosed by a positive screening test and a subsequent positive confirmatory test, was 0.32% (95% CI 0.19–1.54), while of HTLV-2 was 0.04% (95% CI 0.02–0.08). Subgroup analysis by region showed the highest prevalence in the Northeast region  for HTLV-1 and in the South region  for HTLV-2. The prevalence of HTLV-1 is much higher than HTLV-2 infection in pregnant Brazilian women with important differences between regions. The prevalence of both HTLV-1/-2 are higher in the Northeast compared to Center-West region. It is mainly transmitted through sexual contact, sharing of syringes and needles, blood transfusion and vertical transmission from mother to child through breastfeeding5. The virus geographical distribution follows a clustering pattern—areas with a high prevalence of endemic infection are adjacent to areas in which the disease is extremely rare6. While prevalence seems to be decreasing in endemic areas7, such as in Japan that has several government measures and actions have been implemented to prevent new HTLV-1 infection8, it remains stable in areas in which infection rates are relatively low9.The human T-lymphotropic virus type 1 (HTLV-1), also known as human T-cell leukemia virus, was the first human retrovirus to be discovered and isolated10. Adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) are the conditions most commonly associated with HTLV-1 infection; while development of these conditions is relatively rare, their unfavorable prognosis makes them particularly important. In addition to these major diseases, HTLV-1 infection has also been associated with uveitis and dermatological lesions, and the appearance of these conditions may indicate the future development of ATL or HAM/TSP11. A recent meta-analysis showed that HTLV-1 is associated with increased all-cause mortality, inflammatory conditions , infectious conditions , and other types of cancer (eg Lymphoma other than ATL)12.HTLV-1 infection leads to serious disease in approximately 10% of infected individuals13. The pathogenicity of HTLV-2 is less certain, also has been linked to HAM/TSP, besides a connection to other neurological and pulmonary disorders14. On the other hand, HTLV-3 and HTLV-4, were more recently described in isolated forest areas of Cameroon4, and not yet associated with clinical manifestations16.The HTLV-2 is considered an ancestral infection and this subtype can be used as a marker of human migration17. This makes cases hard to detect and creates silent networks of transmission in restricted areas with high endemicity, contributing to the HTLV-1 distribution in somewhat well-delimited clusters. Its main modes of transmission tend to vary by study area; sexual transmission is predominant in some areas18, while some authors report that transmission is common through breastfeeding21.Despite significant morbidity and mortality in some cases, persons with HTLV-1 infection are otherwise mostly asymptomatic23. Also, the HTLV-1/-2 infection is not included in the list of conditions that require compulsory notification to the Brazilian Ministry of Health24. In this scenario, more comprehensive screening programs could be an alternative, considering that they are currently restricted to blood donors25 or restricted in geographical scope26. Although there is no treatment for HTLV-1/-2 infection and progression to active disease is rare, a focus on pregnant women could be helpful in reducing vertical transmission of the virus. It has been demanded of the Brazilian government to include HTLV screening in the standard range of tests offered during pre-natal care in the public health system. Therefore, obtaining a national estimate of HTLV-1/-2 prevalence in pregnant women is needed to assess the possible benefits of implementing such a wide screening program. An estimate of prevalence generated through meta-analysis could be an adequate way to approximate national prevalence figures. As such, this systematic review aimed to estimate the prevalence of HTLV-1/-2 infection in pregnant women in Brazil.Although HTLV-1/-2 during pregnancy is within the scope of surveillance program in Brazil, the screening test for HTLV-1/-2 among pregnant women is not implemented as a universal screening program in the Public Health System until now27. The protocol for this review has been registered in the PROSPERO International Prospective Register of Systematic Reviews under registration number CRD42019147362 (https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42019147362).This systematic review was prepared in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statementWe conducted searches in the following databases: the Cochrane Library/CENTRAL, PubMed/MEDLINE, EMBASE, Scopus, BVS/LILACS and Web of Knowledge (ISI). The search was not restricted by date, language or publication type. Search terms included relevant terms in the title, abstract and text, such as Human T-lymphotropic Virus, Pregnant Women and Brazil. The search strategy is presented in online Supplementary File http://www.opengrey.eu/). Furthermore, we contacted the technical staff of the Department of Chronic Conditions and Sexually Transmitted Infections of the Brazilian Ministry of Health and experts in the area to gather information from ongoing or unpublished studies. Although this strategy was adopted, no study of this nature (ongoing or unpublished) was found and used in the systematic review. Data from conference proceedings were included. Authors were contacted wherever necessary to assess inconsistent or missing data on papers.We searched the reference lists of the studies included in our review to identify additional publications. Additionally, we searched gray literature employing multiple strategies. We searched the website “bancodeteses.capes.gov.br”, a repository of Brazilian theses and dissertations, and the “Open Grey” (This systematic review of the literature included studies that met the following criteria: (1) cohort studies or cross-sectional studies; (2) studies assessing HTLV infection in pregnant women in Brazil; and (3) studies with confirmatory tests for HTLV-1/-2. No age, language or date restrictions were applied.Two reviewers (WJD and BAV) assessed the titles and abstracts of all the studies identified in our search. They were blinded to author and journal names. These researchers then independently performed full-text reviews and data extraction using standardized forms on those studies that fulfilled the eligibility criteria. Discrepancies between the reviewers were resolved by consensus; whenever needed, the opinion of a third reviewer (EMW) was sought. Records were managed with a reference management software.The following data were extracted from the full text documents: title, authors, publication year, study design, number of participants, characteristics of the population , number of women infected with HTLV-1 and HTLV-2, number of women not infected with HTLV, HTLV testing method, HTLV risk factors and comorbidities (such as HIV or syphilis coinfection), time of data collection (pre- or postpartum) and study setting. In cases of duplicate reporting, the study that provided more information was included.28.The quality of the studies included in this review was assessed with an adapted version of the NIH “Quality Assessment Tool for Observational Cohort and Cross-sectional Studies”. The overall strength of the body of evidence was assessed using the “Grading of Recommendations Assessment, Development and Evaluation” (GRADE) tool, using the principles and domains applied in the quality assessment for prognostic studiesThe narrative synthesis of the studies included in this review was structured around the prevalence of HTLV-1/-2 in pregnant women. We considered for meta-analysis only studies with confirmatory tests with typing for HTLV-1/-2. We used a random effects model where the studies weights were assigned by the inverse of their variance (inverse variance method) and the summary measure was obtained by logit transformation. This model was used to pool prevalence data and estimate the prevalence with 95% confidence intervals (CIs). In addition, prediction interval was presented.2 statistic. Meta-regression was performed to investigate the effect of age on the prevalence estimates. Cumulative meta-analysis was conducted to assess the influence of time on the prevalence of HTLV-1/-2. All analyses were performed using R 3.6.1 and the packages meta 4.9-729 and metafor 2.1-030.We performed a subgroup analysis by geographical region  into account. Heterogeneity was assessed by means of the chi-square test and the I54. Of these 26 studies evaluated that used confirmatory tests, only 17 reported which types of HTLV were studied and used for quantitative analysis . Fifteen articles presents data for HTLV-1 and -254 and two about HTLV-144. None report data just for HTLV-2.We identified 233 abstracts from the selected databases, and 13 additional studies were included from the listed references of the identified manuscripts as well as other sources. From the 100 unique citations, 60 were excluded, and the full texts of the remaining 40 publications were screened. We excluded 14 articles in the subsequent full-text assessments for reasons such as same populations of articles already included, incomplete data, study designs not included in the inclusion criteria and studies do not present confirmatory test . The prediction interval for HTLV-1 prevalence ranged from 0.04 to 2.75%, with 95% confidence . The prediction interval for HTLV-2 prevalence ranged from 0.01 to 0.29%, with 95% confidence  and 2008 (k = 7), respectively , followed by the Center-West n = 4) and Southeast (n = 2) regions; the North and South regions were represented by one study in each region. Studies for HTLV-2 have the same distribution, unless the Northeast region (n = 7). The forest plots with the estimates for each region are shown in Figs.  and Sout2 = 83%), moderate in the Southeast (I2 = 59%) and low in the Center-West (I2 = 12%), for HTLV-1 and moderate for HTLV-2, in Northeast (I2 = 44%). There was no heterogeneity in other regions. It was not possible to do subgroup analysis for North and South regions because there was just one study in each region, with very limited sample size, 618 and 643, respectively and the southern study evaluated high-risk pregnant women. Thus, the analysis results for the North and South should be interpreted with caution. The Northeast region heterogeneity was the only statistically significant (p < 0.01) for HTLV-1.The heterogeneity was high in the Northeast region .Furthermore, a meta-regression was performed with age as a covariate if the studies described this data. Few studies reported the mean age of their sample (5 of them of HTLV-1 and 4 of them of HTLV-2). This model showed no statistically significant association between HTLV infection and age in pregnant Brazilian women  to 0.663% in Kyushu (southwest area)65. The prevalence of this infection in pregnant Japanese women seems to be similar to that in pregnant women in Brazil. However, it seems our estimates are lower than those in pregnant women in other locations. In general, studies from Africa73 reported prevalence higher than our estimate, ranging around 2.0%, with the highest prevalence of HTLV-1, at 5.5%, in pregnant women who attended an antenatal clinic in Nigeria72. Our estimates of HTLV-1 were also lower than those in pregnant women who emigrated from endemic areas, such as the Caribbean (1.42%)74. In other endemic countries in Latin America, studies showed prevalence rates of HTLV-1 close to 2.0% in Peru and Jamaica77 and approximately 3.4% in French Guiana (with significant variations between ethnic groups)78.Due to the highly localized, endemic nature of HTLV infection foci, Brazilian pregnant women must be compared to women from other endemic, high-prevalence countries. Japan is a country that presents one of the areas with the highest prevalence of HTLV-1 in the world80. African studies show prevalence of this virus rates between 0.08 and 3.8% in pregnant women73. An Argentine study show a prevalence of 0.12% in this population81. Although our HTLV-2 estimates seem to be higher than native and immigrant pregnant women who live in Spain, we found lower estimates than other countries.Among pregnant women who live in Spain, prevalence estimates of HTLV-2 infection range from 0.01 to 0.03%54, and the range of mean ages was very small (23.9 to 27.0). According to the meta-regression model, age was not associated with the prevalence of HTLV infection, in contrast with the literature. The prevalence increases gradually with age83 and is significantly higher in pregnant women older than 3067 and 40 years old70. Over the years, sexual activity among women of childbearing age increases, suggesting male-to-female transmission67.Of all studies included in this systematic review, only 5 of HTLV-1 and 4 of HTLV-2 reported the mean age of the participantsThis study has some limitations that must be acknowledged. While we tried to explore and account for the differences between regions in Brazil, it is very likely that our overall estimates are not representative of the pregnant population across all of Brazil. The included studies did not cover all the states in the country, offering limited estimates of HTLV-1/-2 for each region. In addition to real differences in the prevalence of infection, regions also differed in the number of studies, type of population included in these studies and typical sample size of studies, leading to high heterogeneity due to studies intrinsic factors. Additionally, few studies were population based.84 and hepatitis B (0.4%)85. While there is an established national surveillance program for the last two, there is no national screening for HTLV-1. The similarity in prevalence could reflect either true differences in estimates or lack of nationwide information. Since 1993 serologic tests for HTLV-1/-2 became mandatory in Brazilian blood banks86, with prevalence ranging between 0.03 and 0.988. Those data could suggest a meaning to start a screening program during pregnancy, as was done in Japan. Our study highlights a lack of data on prevalence in pregnant women and the need for nationally and regionally representative measures of burden of HTLV-1/-2 infection in Brazil to support decision-making about screening to prevent vertical transmission.Compared to other infections among pregnant women, HTLV-1 appears to have a similar prevalence to HIV (0.38%)Supplementary Information 1.


Recent advances have shed light on the importance of early therapeutic intervention for neurodegenerative diseases. Primary prevention trials present a potential disease-modifying strategy for pre-symptomatic patients of autosomal dominant neurodegenerative diseases (ADND), such as early onset familial Alzheimer’s disease (AD) and Huntington’s disease (HD). As trials target earlier disease stages, however, prospective participants face new ethical and logistical challenges, namely childbearing and reproductive health decisions. Since pregnancy is an exclusion criteria for such trials, participants of reproductive age must choose between participating in research and having a family. Such decisions carry significant burdens for ADND patients that if left unaddressed could impact patient well-being and the field as whole. We use our perspective as scientists, advocates, and ADND family members to highlight current shortcomings in the field regarding trial participation and family planning issues for ADND patients and call for the establishment of a normative standard to address these concerns. Extensive study of ADND has led to the discovery of the genes and specific mutations that give rise to these disorders , 2. DespThere are significant gaps in the field regarding the effect of family planning on ADND primary prevention trial participation for both men and women. These include: (1) the paucity of information and study on participant perspectives regarding family planning issues, (2) the lack of guidelines and implementation for family planning communication in trials, (3) the need for ethical consideration regarding informed consent on participant’s fertility-associated physical and emotional well-being, and (4) the need for an assessment of potential risks incurred by trials as well as the ADND and dementia field if these issues go unaddressed. For each of these issues related to childbearing in the ADND population, we highlight the gap within the ADND field, detail findings and strategies employed by the field of oncology to address these issues, and lastly suggest how the approach taken by the field of oncology could serve as a template to guide recommendations for ADND investigators . We acknIt remains unclear whether experimental treatments for ADND would reduce or eliminate fertility, however, committing to clinical trial participation during critical reproductive years and/or for the remainder of life may cause the loss of optimal childbearing years or push some patients out of their fertility window altogether, impacting patients’ emotional well-being. Due to the lack of data on the effects of potential fertility loss in the ADND community, we have turned to oncology—a field with experience in developing strategies for young patients concerned about their fertility future. Researchers in the cancer field have stressed that understanding the emotional aspects of infertility after cancer as well as the factors that influence a survivor’s decision about having children is of great importance . Cancer For the field of ADND, no data exists that captures the attitudes of potential trial participants on family planning issues. To gain a better understanding of patient perspectives as they relate to trial participation and family planning, we suggest that the ADND field undertake such studies. Particularly as the field moves toward treating younger pre-symptomatic and prodromal patients, investigating the experiences and perspectives of ADND patients of reproductive age will identify unmet family planning needs and provide necessary evidence for improved patient-centered care. Perspectives and questions to address Fig.  include:No guidelines are currently in place to facilitate discussions with ADND patients about fertility-related concerns regarding their disease or clinical trial participation. Guidelines established by the American Society of Clinical Oncology call for an individualized fertility consultation, including a discussion of fertility preservation options as early as possible and at every stage of treatment for oncology patients that are interested in or uncertain about their future fertility plans prior to cancer treatment . Prior tGiven the importance of this issue and the potential impact that loss of fertility can have on quality of life, there is a critical need to provide effective fertility counseling services to ADND patients of reproductive age, particularly before participation in primary prevention trials that require a fertility hold. When establishing future investigational trials, we implore investigators to facilitate conversations between patients, genetic counselors who often have experience with hereditary disorders and associated reproductive questions, and fertility specialists that can help guide family planning decisions . We postulate that taking these measures will improve psychological health, decision satisfaction, and quality of life for trial participants. We endorse the adoption of the recommendations set forth by the American Society of Clinical Oncology to facilitate clinician interactions with ADND patients both as part of trial enrollment and on an ongoing basis during trial participation as patient childbearing concerns evolve and disease-modifying treatments, hopefully, become the standard of care.There are four basic ethical principles in medicine: autonomy, beneficence, nonmaleficence, and justice. To protect human subjects during clinical research, informed consent is crucial to achieving these principles, which involves informing subjects of their rights, alternative treatment options, risks, and benefits prior to their trial participation. In the ADND field, we believe there is a current gap regarding ethical considerations and patient fertility, particularly informed consent and the measures taken to adequately address and acknowledge the potential risk to trial participants’ fertility during their participation in trials testing investigational treatments with unknown risk. In oncology, studies show that providing information to patients about treatment-associated reproductive risks is integral to helping them make informed decisions . FertiliIn ADND prevention trials, there is limited information on whether investigational drugs undergo testing for fertility-related effects, and whether this data is disclosed to patients. Although these experimental drugs are primarily brain-directed, some prevention trials are currently exploring systemic interventions given orally or intravenously. Thus, it is critically important, and necessary for adequate informed consent, for investigators to provide patients with data on the impact of these experimental drugs on egg supply, sperm health, teratogenicity, and fetal neurodevelopment, or the lack thereof. While an argument could be made that investigational trials should be held until the effects of these drugs on fertility are known, the desire for disease-modifying treatments within the ADND field cannot be overstated. Clinical trial participation forces patients to confront and weigh the importance of many major life situations and decisions: their desire to have biological children, their disease age of onset, their remaining natural fertility window, and the promise of an investigational drug to modify disease pathology Fig. . In addiInsight gained from the oncology field may shed light on potential risks that the ADND field may face for investigational treatments. While the effect of fertility concerns on trial participation for the ADND field is currently unknown, 29% of breast cancer patients report that infertility concerns affected their treatment decisions [Once a patient has decided to pursue fertility preservation, both intrinsic  and extrinsic  factors influence the patient’s ability to undergo fertility preservation. Additionally, sex can also be a factor as men and women face different barriers to fertility preservation due to ease/difficulty of sample retrieval and biological ineligibility criteria for women . However, financial burden remains the most significant deterrent against fertility preservation , cost foThe distress reported by oncology patients who lost fertility  and thei


A combination process of completely non-thermal processing methods involving high hydrostatic pressure (HHP) and vacuum-freeze drying (VFD) for producing a new snack from fruit and vegetable blends was developed, and the effect of the process on flavor quality was investigated. The HHP–VFD treatment did not significantly reduce volatile compound contents compared to single HHP or VFD. Gas chromatography–olfactometry showed that HHP–VFD raised the contents of floral-like volatile compounds  compared to the untreated sample. Sensory evaluation analysis confirmed that the overall liking was unchanged after the HHP–VFD treatment. The HHP–VFD combined treatment is effective in maintaining the flavor and extending shelf life, and is convenient for the portability and transportation of ready-to-drink juice. Flavor quality is a composite of oronasal sensory responses to aromas and taste, and is the main attribute controlling consumers’ choices of fruit and vegetable juices . Ready-tHigh hydrostatic pressure (HHP) is becoming a first-choice sterilization method to fulfill the requirements of nonthermal processing, since its limited effects on covalent bonds improve the retention of flavor quality in comparison with thermal processes ,5. As reVacuum-freeze drying (VFD) is extensively employed as one non-thermal drying tool because VFD almost reserves the same color, flavor, and nutrition as fresh fruits and vegetables ,14. VFD Herein, three common fruit and vegetable raw materials in China, including pumpkin, mango, and jujube, were selected. The study aimed to (1) identify and quantify volatiles through headspace solid-phase microextraction (HS-SPME) with gas chromatography–mass spectrometry (GC–MS); (2) discriminate the major aroma-active compounds with detection frequency and odor activity value analyses; (3) characterize the changes in volatiles; (4) validate the sensory differences by quantitative descriptive analysis, in the fruit/vegetable blended juices treated by HHP or HHP–VFD.Ziziphus jujuba Mill. cv. Hetian), pumpkins (Cucurbita moschata Duch. cv. Beibei), and mangoes (Mangifera indica L. cv. Qingmang) without physical injuries or disease infection were purchased from a local retail market in January 2021 in Beijing, China.Jujubes (N-Alkanes (C7-C30) for qualitative tests were bought from Sigma . The internal standard, 2-octanol, for quantitative tests was bought from Aladdin Biochemical . Other analytical reagents  were made by Beijing Chemical Reagent Company .Juices were prepared as per Xu et al.  with somTo carry out HHP, fresh blended juices in 100 mL bottles were pressurized using a CQC30L-600 HHP pressurization unit  as per Zhang et al. . PressurAfter preliminary freezing at −20 °C for 24 h, each cell for 5 g of fresh or HHP3-treated blended juices was injected into silastic molds . VFD was run at −40 °C (cold trap) and 100 Pa (absolute pressure) for 48 h on an LGJ-25C freeze dryer . The products were named VFD and HHP–VFD blended cubes B, respec0 (hue angle) were calculated as follows:0, a*0, and b*0 were the control values for fresh blended juice (for juices) or VFD cubes without HHP (for cubes).Colors of the samples were measured at ambient temperature using a color difference meter  in the transmission (for juices) or reflectance (for cubes) mode immediately after HHP or VFD. The meter was calibrated using a white color standard before measurements. Colors were recorded in units of L*, a*, and b* uniform color space, where L* means lightness, a* ranges from negative values for green to positive values for red, and b* ranges from negative values for blue to positive values for yellow. Numerical ΔE , C* (chroma), and hThe microstructure of the VFD- or HHP–VFD-treated blended cubes was observed by a field emission scanning electron microscope , under 100× and 500× magnification.TM/polydimethylsiloxane SPME fiber  was exposed to the headspace of the juices for 40 min at 45 °C without agitation. Finally, the fiber was removed and placed into the GC injector at 250 °C for 5 min.The volatile compounds were extracted using SPME according to Pang et al.  with minm/z.An Agilent 7890 GC system coupled with an Agilent 5975C MS meter  was operated as per a reported method  with minhttp://www.odour.org.uk/ (accessed on 14 September 2021)) less than 20 is acceptable. The concrete equation is:Ra is the retention time of the compound, tRn and tR(N+n) are the retention time of n-alkanes on the left and right sides of the compound, respectively.The volatile compounds in blended juices and rehydrated juices were identified by comparing the mass spectra with the NIST10 database, and by comparing the calculated linear retention index (LRI) with the open data of the NIST WebBook. LRI was computed using the retention time of n-alkanes  detected under the same GC–MS temperature program. A difference between the calculated LRI and those from published data or the LRI and Odour Database  linked to the GC–MS system. After the volatile compounds were isolated by the capillary column, the same volume of the effluent was removed to both the sniffing port and the MS meter. In the GC–O sniffing port, the transfer line was kept at 230 °C and moist air was continually added at 60 mL/min to maintain the sniffing sensitivity. The same temperature program for GC–MS was adopted. Detection frequency (DF) was analyzed by four trained panelists with good and stable sensation, and each panelist repeated twice. The retention time and odor descriptions were recorded, and the odorant with DF ≥ 6 was regarded a potent aroma-active compound in a total of eight tests .The OAV was calculated as follows:® (QDA) [According to relevant requirements of Quantitative Descriptive Analysis® (QDA) ,29, the p < 0.05 were detected via one-way and two-way analysis of variance (ANOVA) and on SPSS 25.0 . Differences in volatile profiles among the samples were compared by principal component analysis (PCA) and cluster analysis with MetaboAnalyst online tools ). Plotting was finished on Graphpad prism 8.0  and Origin 2019 .Data are expressed as mean ± standard deviation of three repetitions. Differences between samples at the significance level 0, and ΔE of HHP-treated or HHP–VFD-treated blended juices. No significant differences in color parameters of the blended juice samples were observed at all pressures of HHP (p > 0.05), which was consistent with previous research in juices where HHP was shown to better preserve the color of raw materials [p < 0.05) after rehydrating from VFD-treated cubes compared to fresh blended juice, indicating a brighter and more saturated color after VFD and rehydration. VFD has been reported to retain the color characteristics of raw materials relative to other drying methods [aterials . On the  methods ,32,33. T methods . This maMicrostructure has an important effect on the quality of dried food. The microstructure of VFD-treated cubes changed after HHP treatment . IrregulA total of 64 volatile compounds were detected in blended juices and rehydrated juices, including 10 ketones, 19 aldehydes, 8 terpenes, 8 alcohols, 9 acids, and 10 esters . Most ofE)-2-hexenal, benzaldehyde), terpenes , acids , esters , and some other ketones, alcohols, and benzene derivatives were predominant in the volatile compounds of blended juices and rehydrated juices. As reported, HHP can alter the compositions and quantities of volatile compounds in many fruit and vegetable juices and wines [Hydrocarbon aldehydes  with the pressure of HHP. Pressure levels of HHP may enhance or retard enzymatic and chemical reactions and thereby indirectly influence the increase or decrease in volatile compounds [p < 0.05) the contents of total ketones, whereas HHP–VFD led to a twofold rise (p < 0.05). In addition, the proportion of total ketones was low in HHP-treated blended juices and was less than 2% of total volatiles, but was higher in HHP–VFD-treated rehydrated juices  from 22.66 μg/kg in fresh blended juice to 77.88 μg/kg at 400 MPa and 102.59 μg/kg at 600 MPa. This result is similar to a previous study on the effect of HHP on kiwifruit pulp beverages [E)-2-nonenal, and -2,6-nonadienal in HHP-treated blended juice significantly declined (p < 0.05) in comparison with fresh blended juice. These aldehydes resulted from the peroxidation of polyunsaturated fatty acids under catalysis by LOX, which were completely inactivated in tomatoes at 550 MPa [p < 0.05) the contents of -2,4-heptadienal and β-cyclocitral, which may be due to the decomposition of unsaturated fatty acids during drying. Linolenic acid degradation leads to the formation of 2,4-heptadienal isomers, which is the same as in mango peels [Nineteen aldehydes were detected, which were the most representative volatile compounds in blended juices. Most of these aldehydes belong to C6–C9 aldehydes, which originate from the oxidative breakdown and cleavage of fatty acids by lipoxygenase (LOX) and hydroperoxide lyase (HPL) . The hexeverages . HPL waseverages , and waseverages . On the  550 MPa . Similar 550 MPa  after HHgo peels ,58. β-Cyp < 0.05) the content of total terpenes in blended juices, which is similar to the results in different varieties of orange juices [p < 0.05) the content of total terpenes, some new terpenes appeared , which is different from previous studies on freeze-dried cabbages and carrot slices [Terpenes account for the typical aroma profiles of many fruits, particularly tropical fruits such as mangoes . One of e juices ,60. Intet slices ,62. Thist slices . Additiot slices  observedp < 0.05) after HHP treatments. Moreover, the level of 1-octen-3-ol in HHP-treated kiwifruit pulp beverage and black truffle significantly increased or appeared after the treatment [In total, eight alcohols were identified, which all possessed only one hydroxyl group. The proportion of total alcohols rose after processing , which mreatment ,66. Thisreatment . Carveolreatment , and is reatment .p < 0.05) after HHP treatment. However, this decrease was not linear with a high-pressure increase. The most acid retention was observed for blended juices pressure treated at 400 MPa, which is consistent with the study of Zabetakis et al. [p < 0.05) than in the juices. As reported, acids disappeared and decreased after freeze drying in jujubes, which was much more pronounced than other dehydration methods such as air drying and microwave drying, which evidenced the improvement in flavor by freeze drying [Nine acids were detected in blended juices, which occupied the second-largest proportion of all volatile compounds. The content of all acids in the samples decreased  the concentrations of esters in samples, which is consistent with the changes in most matrices, such as mango juice, red plum puree, and kiwifruit pulp beverage [p < 0.05) the contents of esters, except for ethyl acetate, though VFD can extremely retain the ester content due to the lower temperature compared with other drying methods [Esters are pivotal to the aroma of most fruits and comprise the important proportion of volatiles in jujubes and mangoes ,41,72. Nbeverage ,54,73. Mbeverage ,75. More methods .p = 0.155), isovaleraldehyde (p = 0.165), valeraldehyde (p = 0.053), -2,4-heptadienal (p = 0.821), (E)-2-nonenal (p = 0.515), 4-carene (p = 0.394), α, p-dimethylstyrene (p = 0.479), 1-pentanol (p = 0.223), (E)-3-Hexen-1-ol (p = 0.221), p-cymen-8-ol (p = 0.758), the interaction and main effect between HHP and VFD in terms of their effects on the contents of volatile compounds were significant (p < 0.05). These results further confirmed the interaction of HHP and VFD, rather than a one-way independent casual effect.In addition, a two-way ANOVA was applied to four fresh, HHP3, VFD, and HHP3–VFD samples to analyze the effect of combined HHP and VFD treatments on the contents of volatile compounds . The resE)-2-octenal, -2,6-nonadienal, (E)-3-hexen-1-ol, 1-octen-3-ol, propionic acid, butanoic acid, 2-methyl butanoic acid, hexanoic acid, and octanoic acid, were detected by assessors eight times at least in one sample, meaning that these 13 compounds were potentially critical contributors to the overall aroma of blended juices and rehydrated juices. Of them, 6-methyl-5-hepten-2-one, hexanal, 1-nonanal, -2,6-nonadienal, butanoic acid, and 2-methyl butanoic acid were reported as aroma-active compounds contributing to the aroma profile of mangoes of different varieties [E)-2-octanal were recognized as the most powerful odor-active compounds in jujube fruits from three cultivars, on the basis of both GC–O and OAV [E)-3-hexen-1-ol, and 1-octen-3-ol were described as the most characteristic compounds of the variety of pumpkin [arieties ,78. Hexa and OAV . In anot and OAV . Further pumpkin . HoweverE) and (Z) isomer conformations -2-hexenal, (E)-2-nonenal, (E)-2-octenal, and -2,6-nonadienal herein) have higher odor thresholds and are usually compounds that affect characteristics. The shorter-chain analogs provide green aromas. It is worth mentioning that the odor thresholds of both saturated aliphatic and unsaturated aldehydes seemingly decrease gradually with the prolonging of chain length. Floral-like β-ionone in VFD-treated or HHP–VFD-treated rehydrated juices had the highest OAV , followed by cucumber-like -2,6-nonadienal in fresh juice (1076.69). Generally, HHP does not considerably alter the OAV of most volatiles [E,Z)-2,6-nonadienal, which had the largest OAV in fresh juice, was undetected in HHP1 and HHP2 juices. -2,6-Nonadienal is formed from linoleic acid, which is the substrate of LOX, and the activity of LOX may change under different pressures and in different matrices [OAV is a reasonable tool for aroma potency assessment based on the equilibrium between air and the food matrix. olatiles , but -2-hexenal, and 1-octen-3-ol were found in all six samples, indicating they were potent and major aroma contributors and unaffected by processing. β-Ionone and β-cyclocitral were only detected in VFD-treated and HHP–VFD-treated samples and were screened as aroma-active compounds, and therefore, they can be used as indicators to identify whether a sample was treated with VFD or not. β-Ionone was derived from the degradation of β-carotenene under the enzymatic formation of carotenoid cleavage dioxygenase [E)-3-hexen-1-ol, propionic acid, butanoic acid, 2-methyl butanoic acid, hexanoic acid, and octanoic acid had OAVs < 1, suggesting they made limited contributions to the overall aroma. The differences between the two assessments were mainly due to the different principles applied [After comparison of the aroma-active compounds detected by DF and OAV , the joixygenase ,85, and xygenase . As repoxygenase ,88. Simixygenase  compared applied . OAV was applied ,91. ThusHeat map, hierarchical clustering, and PCA of the concentrations of the 13 major aroma-active compounds were performed to characterize the aroma profiles of blended juices with different treatments . Heat map < 0.05). Noticeably, HHP-treated blended juices showed a stronger grassy aroma, the intensity of which was strengthened as pressure increased. Reportedly, HHP-treated kiwifruit pulp can cause more intense grassy and fresh notes, which may be contributed by hexanal [Quantitative descriptive analysis (QDA) including six aroma notes and overall liking was used to distinguish the sensory quality with different processing methods . Six aro hexanal . HHP–VFD hexanal . As for  hexanal . Thus, tCompared to HHP or VFD alone, the combination of HHP–VFD resulted in a better retention of flavor quality in blended juices. HHP treatment at 200, 400, and 600 MPa did not lead to significant changes in the color of blended juices but decreases in the total volatiles and changes in sensory quality. The HHP–VFD treatment changed the color, microstructure and caused a decrease in total volatiles. Moreover, HHP–VFD resulted in the highest terpene and ketone contents of blended juices among the different treatments, especially the aroma-active compounds β-ionone and β-cyclocitral, so they can be recognized as discriminant indicators of HHP–VFD treatment. Rehydrated juice from HHP–VFD-treated rehydrated juices can better retain the flavor quality of fresh juice, indicating a possible application for the non-thermal processing combination of HHP and VFD in the fruit and vegetable juice industry. Although we maximized flavor retention based on the extended shelf life, further research in this field is required to apply this non-thermal processing combination into food and other matrices.


An incomplete interlobar fissure makes thoracoscopic lobectomy difficult and is predictive of morbidity after thoracoscopic lobectomy. This report demonstrates the robot-assisted thoracoscopic (RATS) lobectomy technique for patients with severe incomplete interlobar fissures. A fissureless approach was chosen for pulmonary resection. Near-infrared fluorescence imaging with intravenous indocyanine green (ICG) was used to detect the interlobar line after transection of the bronchus, pulmonary artery and vein. Interlobar fissure was identified and divided by robotic staplers. This combined technique using ICG and fissureless lobectomy made RATS lobectomy safe for patients with severe incomplete interlobar fissures. Incomplete interlobar fissures increase the surgical difficulty of video-assisted thoracoscopic surgery (VATS) lobectomy. Furthermore, the degree of pulmonary fissure completeness is a categorical predictor of both major and minor morbidity after VATS lobectomy . TherefoThis is the case of a 38-year-old man with cStage IB adenocarcinoma. Computed tomography showed a 33-mm mass in the right upper lobe , and no RATS right upper lobectomy with ND2a-2 was performed via four port incisions and one assist incision. One 12-mm robotic port was placed in the eighth intercostal space on the anterior axillary line, and three 8-mm robotic ports were placed in the eighth intercostal space. The assist incision was made in the fourth intercostal space on the anterior axillary line. The Da Vinci Xi surgical system  was docked. A vessel sealer extended, fenestrated bipolar forceps and long bipolar grasper were inserted. Then the upper bronchus, V1 + 2 + 3, and A1 + 2 + 3 were exposed and transected by a robotic stapler. To identify the minor fissure, indocyanine green (ICG) (7.5 mg) was injected through the peripheral venous catheter, followed by a 10-ml flush of sterile normal saline. After injecting, the surgical field was visualized with the integrated fluorescence imaging capability of the Firefly Fluorescence Imaging camera  . The intTo the best of our knowledge, this was the first report on RATS lobectomy using the combined ICG and fissureless technique for patients with severe incomplete interlobar fissure. We used this technique in three cases, including the present case. All operations were completed without complications, and the postoperative courses were uneventful.Fissureless VATS lobectomy has been reported as a superior alternative to conventional lobectomy in preventing prolonged air leak, and shortening hospital stay and chest tube duration , 3. AlthCompared to VATS, RATS was associated with a lower conversion rate in meta-analyses . HoweverRATS_incomplete_fissure_rjab336Click here for additional data file.


Several viral, bacterial, and parasitic diseases have been associated with cognitive function and neuropsychiatric outcomes in humans, including human T-cell lymphotropic virus 1 (HTLV-1). In this study, we sought to further generalize previously reported associations of cognitive function and depression with HTLV-1 seropositivity and serointensity using a community-based sample of adults aged approximately 40 to 70 years (mean = 55.3 years) from the United Kingdom. In this sample, the results of adjusted linear regression models showed no associations of HTLV-1 seropositivity or serointensity with reasoning, pairs-matching, or reaction-time cognitive tasks or with depression. In addition, neither age, sex, educational attainment, nor income moderated associations of HTLV-1 seropositivity or serointensity with cognitive function or depression. In this middle-aged to older middle-aged adult community sample, HTLV-1 seropositivity and serointensity do not appear to be associated with reasoning, pairs-matching, and reaction-time tasks or with depression. Several viral, bacterial, and parasitic diseases appear to be associated with cognitive function and dementia ,3,4,5,6 HTLV-1 is a retrovirus that infects approximately 5 to 10 million people throughout much of the world, although this likely underestimates the actual number of people infected with HTLV-1 . TransmiIn addition to its association with adult T-cell leukemia , HTLV-1 In addition to its association with HAM/TSP and other diseases including encephalomyelopathy, and its possible relation to elevations in cytokines and chemokines , emerginIn addition to its association with worse cognitive function, HTLV-1 also has been associated with depression and anxiety. A cross-sectional study from Brazil that investigated 20 patients with HAM/TSP, 38 asymptomatic HTLV-1 patients, and 340 healthy controls found that both HAM/TSP and asymptomatic HTLV-1 were associated with depression and anxiety , althougIndeed, much of the research investigating associations between HTLV-1 and cognitive function and between HTLV-1 and other neuropsychiatric outcomes has used samples from low-to-middle-income regions where HTLV-1 is endemic, with little research investigating these associations in non-endemic, high-income regions. However, evidence suggests that differences in early-life factors such as exposure to stress, pollution, and infectious diseases might affect outcomes to later infectious diseases , suggestIn adjusted models, there were no associations between HTLV-1 seropositivity, the natural-log transformed concentration of the gag antibody, the natural-log transformed concentration of the env antibody, and the mean of the natural-log transformed concentrations of the env and gag antibodies and performance on reasoning, pairs matching, or reaction-time cognitive tasks or with depression .Of the 64 interaction models , only thPrevious findings primarily from regions endemic for HTLV-1 suggest that HTLV-1 is associated with cognitive impairment and depression, consistent with associations between HTLV-1 and several types of neurological disease, including HAM/TSP ,17,19. DOur findings showing no associations between HTLV-1 and cognitive function differ from several other previous reports that have shown associations between HTLV-1 and cognitive function and depression in comparisons of HAM/TSP with healthy controls and with asymptomatic HTLV-1 and healthy controls ,11,19,21The large sample sizes we used that ranged from 2269 to 6785 in the analyses of cognitive function and 1673 in the analyses of depression suggest that our analyses were adequately powered to detect any associations between HTLV-1 and reasoning, pairs matching, and reaction time and between HTLV-1 and depression, arguing that in this sample there were indeed no associations between HTLV-1 and performance on these particular assessments of cognitive function and with depression. However, the small size (33) of the HTLV-1 seropositive group with depression and the small size of the HTLV-1 seropositive group who had data for reasoning could have lowered the power of our analyses to detect associations between HTLV-1 seropositivity and either depression or reasoning.As ours was a community sample, it is likely that most of the people in our study did not have HAM/TSP, whereas some previously reported findings were from studies that had solely investigated HAM/TSP compared to healthy controls. In this regard, spinal-cord injury itself is associated with decreased cognitive function , and theAnother factor possibly accounting for potential geographical differences in the effects of HTLV-1 on cognition and depression is that there are different subtypes of HTLV-1 , which cFurthermore, while our study was based on people living in the UK, the majority of people with HTLV-1 in the UK, however, appear to be in people who have come from a region endemic for HTLV-1, who have had sexual contact with a person from an endemic region, or who are children of or descended from someone from an endemic region , suggestDifferences in host factors other than the covariates we used in our models could also account for some of the differences between the results we found and other reports showing associations between HTLV-1 and worse cognitive function and depression, particularly because host immune factors are likely involved with differential responses to HTLV-1 . NonetheSimilarly, and of relevance to our findings showing no association between HTLV-1 and cognitive function and depression in the UK sample, evidence suggests that early-life events such as exposure to pollution, nutritional deficits, infection, and stress might be associated with persistent changes in immune function that could influence the outcomes of infectious diseases in adulthood. Adversity in early life might result in relative immune senescence that contributes to a proinflammatory state later in life that affects the immune response to subsequent infection . As suchIn addition to exposure to early-life events, differences in previous and comorbid infections in the sample we used compared to other previously reported samples also could be factors accounting for our not finding associations between HTLV-1 and either cognitive function or depression. Related to this issue are findings showing that coinfection with human immunodeficiency virus or hepatitis C virus is an independent risk factor for mortality in patients with HTLV-1 .Somewhat consistent with our findings of no associations between HTLV-1 and cognitive function are findings from one small study showing no associations between HTLV-1 and HAM/TSP with brain volume , althougFindings so far from investigating associations between HTLV-1 and cognitive function lead to several unanswered questions, including whether geographical region affects the association between HTLV-1 and worse cognition and depression. In addition, if HTLV-1 is indeed associated with worse cognitive function, an important question is whether it is associated with subsequent dementia, as cognitive deficits have been associated with later dementia , and becDespite not finding an association between HTLV-1 and cognitive function, our study has several strengths. The exposure variables are based on objective measures, as were the cognitive outcome variables. In addition, we included multiple covariates in the statistical models to account for possible confounding. The sample sizes were large, reducing the chances of finding false positives and false negatives. However, the study also has several limitations in addition to those we have already considered. For independent variables, we used measures of seropositivity and serointensity but not viral load, which could be better associated with cognitive function and depression than seropositivity and serointensity. Because of the cross-sectional data we used, we were unable to determine when the initial exposure to HTLV-1 might have occurred. It is feasible that different subgroups classified according to the time of initial exposure to HTLV-1 and to when during neural development the exposure occurred could be more or less susceptible to worse cognitive effects or to depression from HTLV-1 exposure.In conclusion, we found no associations between HTLV-1 seropositivity and serointensity with cognitive function assessed by reasoning, pairs-matching, and reaction-time tasks and between HTLV-1 seropositivity and serointensity with lifetime depression in this community-based study of adults from the UK Biobank. The potential for geographical region and other variables to influence previously reported associations between HTLV-1 and cognitive function and between HTLV-1 and depression and the large number of people seropositive for HTLV-1 worldwide requires additional research into associations between HTLV-1 and cognitive function and between HTLV-1 and depression.http://biobank.ctsu.ox.ac.uk/crystal/field.cgi?id=200, accessed on 10 September 2021). Upon application, the UK Biobank gave us approval to use deidentified data under application number 41535. The UK Biobank enrolled participants sampled from population-based registries  and accessed at 22 centers in the United Kingdom [http://biobank.ctsu.ox.ac.uk/crystal/field.cgi?id=200, accessed on 21 October 2021). Although the UK Biobank sample was not designed to be representative of the UK population and even though there was a very low response rate to the initial surveys in the study, data from the UK biobank appear valid for establishing exposure-outcome associations [For this study, we used a subset of participants of the UK Biobank Resource, a large community-based sample of adults that received ethical approval from the National Research Ethics Service Committee North West-Haydock (reference 11/NW/0382) and consent from all participants . Across the study’s timeline, the UK Biobank includes a variety of neuropsychological tasks given to selected participants to assess cognitive function. However, the combination of the relatively small subgroup of the larger UK Biobank sample that were assayed for HTLV-1 and its low prevalence in the sample limited our cognitive analyses to the reasoning, pairs-matching, and reaction-time cognitive measures . Overall, there were 9430 participants who were assayed for HTLV-1. Most of these had valid data for the pairs-matching and reaction-time cognitive variables, but there were only about 3000 who had valid data for reasoning and approximately 2000 for depression. After removing observations with missing information on the covariates, the respective analyses included 2269 participants for reasoning, 6785 for pairs matching, 6757 for reaction time, and 1673 for depression.We used different analytic samples for each outcome variable. Samples were limited to participants assayed for HTLV-1 and who had undergone cognitive testing or evaluation for depression and who had the preidentified covariates available  [https://biobank.ctsu.ox.ac.uk/crystal/field.cgi?id=23062, accessed on 21 October 2021) [The UK Biobank defined HTLV-1 seropositivity based on levels of HTLV-1 gag and env antibodies .The reasoning task the UK Biobank used assesses fluid intelligence (higher score is better). In the pairs matching task that the UK Biobank used, participants attempt to remember the position of matching pairs of cards. Both the number of correct and incorrect matches were recorded. Because all participants were allowed to complete the task, almost all reached the total number of pairs . Therefore, we only used the number of incorrect responses (lower score is better). Reaction time is the mean time of attempts to identify matching pairs of images (lower time is better). We included the natural log of reaction time in the analyses because reaction time was highly skewed. Further information about these cognitive tasks is available at https://biobank.ctsu.ox.ac.uk/showcase/showcase/docs/TouchscreenQuestionsMainFinal.pdf, accessed on 10 September 2021) to identify participants with a lifetime history of major depression.In assessing depression in the UK Biobank, we used methods outlined by Smith et al.  that inc2), smoking history , and alcohol use .We were particularly interested in including variables in our models that could potentially confound the associations of HTLV-1 with cognitive function or depression. To do so, we included covariates already associated with cognitive function  or that We explored the relationship of HTLV-1 seropositivity and serointensity with cognitive functioning and depression in a series of analyses. We used linear regression to model the relationship between HTLV-1 and cognitive functioning because the cognitive functioning dependent variables were continuous; we used logistic regression to model the association between HTLV-1 and depression as the depression variable was dichotomous. suest command [p-values smaller than 0.05 were considered statistically significant.The thorough examination of the relationship of HTLV-1 with cognitive functioning and depression presented here requires a relatively large number of statistical tests, which also presents a heightened risk of identifying false positive results. To protect against this risk, we estimated multivariate tests that examined the relationship between a single focal predictor and the four dependent variables . The tes command , which pWe used Stata 17.0  for all statistical analyses.


Aberrant regulation of developmental pathways plays a key role in tumorigenesis. Tumor cells differ from normal cells in their sustained proliferation, replicative immortality, resistance to cell death and growth inhibition, angiogenesis, and metastatic behavior. Often they acquire these features as a consequence of dysregulated Hedgehog, Notch, or WNT signaling pathways. Human tumor viruses affect the cancer cell hallmarks by encoding oncogenic proteins, and/or by modifying the microenvironment, as well as by conveying genomic instability to accelerate cancer development. In addition, viral immune evasion mechanisms may compromise developmental pathways to accelerate tumor growth. Viruses achieve this by influencing both coding and non-coding gene regulatory pathways. Elucidating how oncogenic viruses intersect with and modulate developmental pathways is crucial to understanding viral tumorigenesis. Many currently available antiviral therapies target viral lytic cycle replication but with low efficacy and severe side effects. A greater understanding of the cross-signaling between oncogenic viruses and developmental pathways will improve the efficacy of next-generation inhibitors and pave the way to more targeted antiviral therapies. About 15% of all human tumors have infectious etiology and yet only a handful of viruses are known to promote tumor development . ClearlyIn recent years, computational reconstruction of proteome-wide protein–protein interaction (PPI) networks between viruses and developmental pathways have enhanced our understanding of virus-induced carcinogenesis. Mei and Zhang studied PPI networks to highlight important relationships between Epstein-Barr virus (EBV) proteins and developmental pathways, including Hedgehog (HH) and Notch signaling . By explThe Notch pathway is evolutionarily conserved and participates in a plethora of physiological intercellular and intracellular signaling processes during differentiation and development of an organism. Evidence shows that virally perturbed Notch signaling may lead to cancer . OncogenThe WNT signaling developmental program is also frequently targeted by oncogenic viruses to transform the target cells. HBV, Hepatitis C virus (HCV), EBV and Human T Lymphocyte Virus-1 (HTLV-1) co-opt or modulate components of the WNT pathway to effectively subvert normal cellular processes including cell proliferation, differentiation, and survival.Thus, as our understanding of the mechanisms that regulate oncogenic transformation grows, the extent and complexity of cellular processes targeted by oncoviruses is better appreciated. The recognition that multiple developmental pathways are frequently targeted, either individually or collectively, may represent unexplored opportunities for developing unique or synergistic therapeutic strategies to treat or prevent viral tumorigenesis.In this review, we present an overview of the three developmental pathways, namely, HH, Notch, and WNT and how some oncogenic viruses interact with them. We will review immune system interactions with these viruses, and how they regulate these pathways through viral miRNAs to survive and contribute to carcinogenesis and tumor progression. We will also provide perspectives for the development of therapies that target important and common regulators of these three developmental pathways.Tissue patterning, cell differentiation and proliferation require HH signaling but aberrant HH signaling is an important cause of cancer. In humans, the HH pathway is activated by three ligands: Sonic Hedgehog (SHH), Indian Hedgehog (IHH), and Desert Hedgehog (DHH) . These lPTCH1, FOXM1, and WNT5A, a highly evolutionarily conserved non-canonical WNT ligand. Altered HH signaling is common in NPC and specifically a reduced expression of SUFU has been detected in a large number of NPC specimens , the HTLV-1 TAX transcription factor epigenetically upregulates Ellis Van Creveld (EVC) family members, EVC1 and EVC2, both of which have been associated with the cellular HH activity and thus provides the pro-survival attributes of ATL cells . AdditioInfection with oncogenic viruses may be silent for years and HH pathway reactivation later in life has been associated with tumor development . Merkel In EBV-associated tumors, the virus persists in one of the three distinct latent phases, each characterized by expression of a set of viral genes. In NPC, EBV latent gene expression is restricted to EBNA1, EBV-encoded RNA1/2 (EBER1/2), BARF1, the BamHIA transcripts (BARTs), as well as variable expression of genes encoding oncogenic membrane proteins LMP1 and LMP2. In these tumors, EBNA1, LMP1 and LMP2 all stimulate HH signaling, but only the latter two are capable of reprogramming cells toward stemness. This would suggest that each viral protein can trigger different molecular pathways involved either in tumor cell growth and survival, or in stemness-associated programs. Just like normal stem cells, cancer cells have the ability to self-renew, differentiate in different cell types and under specific tumor microenvironmental condition, dedifferentiate backward a primitive state. This feature provides tumor cells the ability to grow, metastasize and home to specific tissues. Given its ability to activate the WNT and Notch pathways, Co-expression of cancer stem/initiating and mesenchymal cell markers have been observed in peritumoral stromal tissues within nodules of Hepatocellular Carcinoma (HCC) from livers of patients with chronic hepatitis. Both HBV and HCV clearance rates are high in infected individuals, often depending on the age . Only abSHH and IHH, and target genes, PTCH and GLI2, possibly during more advanced stages of liver disease  without involving SHH. The hepatotropic HCV and HBV can cause chronic inflammation and turn on an inactive HH signaling in the healthy liver to promote fibrosis and HCC development. Another mode of action to perturb HH signaling is through alteration of Gli transcription as highlighted by HPV encoded E6 protein.Autocrine stimulation plays an indispensable role in HH-mediated viral carcinogenesis, but its effect in the tumor microenvironment is still uncovered. Future studies are required to investigate how viruses utilize and relocate HH family members in infected cells to drive host cell machinery to carcinogenesis.Drosophila. It was subsequently found to be linked to a heterozygous deletion of a gene located on the chromosome X, hence named Notch. The Notch signaling cascade is highly conserved from Drosophila to humans (2+-dependent ionic bonds: the Notch extracellular domain (NECD), consisting of multiple epidermal growth factor-like (EGF) repeats which mediate ligand binding, and the Notch intracellular domain (NICD), which is the transcriptionally active part of the molecule   . Indepennd JAG2) . One nonnd JAG2) .Ligand-receptor interactions and transcription factor activity in Notch signaling play pivotal roles in a wide variety of differentiation processes, including regulation of cell-fate determination during tissue and cell development . Notch sPerturbing the Notch pathway can lead to the onset of various diseases, including cancer . Co-optic-myc, CD21, and LMP2A, but not LMP1 or CD23  and of 1p36.11 hairy and enhancer of split-1 (HES)2/3/4/5 tumor suppressor genes belonging to the Notch pathway. Interestingly, this was associated with significantly improved overall survival (OS) of these patients. In spite of several important limitations, such as the lack of information about HPV-type associated with the aberrant amplification of the two tumor suppressor genes of the Notch pathway, this study provides new insights for the prognosis of CC patients, based on tumor cell methylation signatures.A recent study showed that in HPV-related CC, the Notch pathway is indeed affected by aberrant mutations, amplifications, and deletions . A copy JAG1 mRNA. Furthermore, in most ATL patients with high JAG1 expression, miR-124a levels were low. Interestingly, STAT3, and NFATc1 were also highly expressed. Remarkably, they are both miR-124a targets. The absence of miR-124a in ATL might enhance JAG1 and Notch1 signaling pathway sustained by constitutive expression of STAT3 and NFATc1. Therefore, inhibiting JAG1 could be a promising therapeutic strategy in ATL  cells are involved in chronic HCV pathogenesis  is essential for its replication and contributes to viral induced HCC . The Yeaogenesis . Specifiogenesis . These sSince oncogenic viruses co-opt Notch pathway components to sustain their survival leading to tumor progression, the development of inhibitors to block virus-Notch interactions could become a valuable therapeutic approach. To this end, utilizing monoclonal antibodies, drugs targeting Notch receptors, γ-secretase inhibitors (GSI), or small molecules that disrupt the interaction between Notch and RBP-Jk, RBPJ INhibitor-1 (RIN1), provide novel therapeutic avenues to pursue. For instance, monoclonal antibodies (mAbs) specifically targeting Notch1 reduced stemness of breast cancer cells , whilst With some exceptions mentioned above, such as in the case of H1N1, in which inhibiting the Notch pathway with GSI led to adverse effects in mice, the Notch inhibitors have the potential to be therapeutically implemented with necessary precautions to interrupt the Notch pathway activation by viral proteins.Immune escape by tumor cells is a fascinating phenomenon. Indeed, tumor development is, quintessentially, a failure of the immune system to recognize and eliminate the cells that have emancipated themselves from cell cycle control and have gone awry . The immA paradigmatic example of how Notch pathway activation could be central to immune suppression is provided by EBV associated Hodgkin’s lymphoma (HL). This tumor is characterized by increased infiltration of regulatory T (Treg) cells. The characteristic Reed Sternberg (RS) cells, which represent the HL tumor component, highly express Notch1 and Notch2 . InteresNon-coding RNAs, especially miRNAs, play a significant role in helping tumors escape immune control by negatively regulating critical immunomodulatory genes . MiR-346In HBV associated HCC, the metastatic event characterized by the portal vein tumor thrombus (PVTT), is common in over a third of patients. The positive correlation between the presence of HBV and PVTT has been established . These PIt is remarkable how diverse viral proteins, such as EBNA2, LANA and Tax1, are all able to deregulate the same effector of the Notch pathway, NICD, leading to cell transformation, latency maintenance, proliferation, angiogenesis and tumor progression. HBx and NS3 viral proteins interact with other activators of the Notch pathway, such as JAG1, Dll4, and SRCAP, respectively, to favor HCC progression. Continued investigation will enable a deeper understanding of how viral proteins interact with the Notch signaling to take advantage of host cells and escape immune surveillance. The knowledge of these interactions is an important step for the development of targeted therapies in virus-associated cancer patients.WNT signaling is involved in cell proliferation, cell polarity, and cell fate determination during embryonic development and tissue homeostasis. Evolutionarily, the WNT signaling pathway is highly conserved. Under normal or homeostatic conditions, in the absence of WNT ligands, the signaling is maintained in an “off” state . Under tWNT signaling is activated when secreted WNT proteins bind to the seven-pass transmembrane receptor, Frizzled (FZD), together with its co-receptor, low-density lipoprotein receptor related protein (LRP) 6, or to the closely related, LRP5. Following WNT binding, the FZD-LRP6 receptor complex associates with the Disheveled segment polarity protein (Dvl), a scaffold protein that facilitates LRP6 phosphorylation and subsequent recruitment of Axin way from APC and GSK3b to form the FZD-WNT-LRP6-Dvl-Axin multiprotein complex . As the WNT signaling regulation is complex. Many components of this pathway are, themselves, positively or negatively regulated through the WNT pathway. Through their co-evolution with the human immune system, oncogenic viruses have devised means either to down-regulate inhibitory WNT proteins, such as Dickkopf WNT signaling pathway inhibitor1 (DKK1)  Figure  or reinfOncogenic HBV and HCV infect cells of the liver causing hepatic inflammation, fibrosis, cirrhosis, and, ultimately, HCC. The progression from initial infection to HCC is a lengthy, complex, multi-step process. HBV and HCV are vastly different – HBV is a partially double-stranded DNA virus , while HCTNNB1, the b-catenin encoding gene, is associated with HCC (AXIN1 leading to HCC (S gene), core proteins (C gene), HBV polymerase (P gene), and the HBx protein (X gene). The polymerase lacks proofreading activity, so HBV mutations occur with greater frequency than other DNA viruses. Frequently, these mutations are associated with activation of WNT signaling. Mutations in the core promoter, overlapping with the X gene, have also been described. The resulting single or combination point mutations in HBx, which is thought to act as a promiscuous transcription factor, also upregulated WNT signaling , Cyclin D1, and WISP2, in mutant HBx-expressing cells, suggesting these mutations act at multiple levels to increase WNT signaling  . Additionot FZD7 . Cyclin MYC, WNT1, and CCND1 in Huh7, but not in L02 cells, suggesting these HCV proteins increase WNT signaling to directly affect WNT-mediated cellular processes  and DKK1, leading to WNT/b-catenin activation. Studies by SFRP2 and DKK1 hypermethylation may occur early following HCV infection and may lead to HCC via a multi-step process. In this study, the authors utilized bisulfite sequencing to analyze liver biopsies from patients with chronic hepatitis, liver cirrhosis, and HCC. Compared to normal liver samples, SRFP2 and DKK1 showed significant hypermethylation in HCV-infected patients. Although no differences in DKK1 methylation was noted between the different conditions, there was a progressive increase in SFRP2 promoter methylation with normal liver < chronic hepatitis < liver cirrhosis < HCC  showed that EBV infection led to epigenetic reprogramming, CpG hypermethylation, and delayed responsiveness to differentiation cues . Using totential .DACT2, is expressed in various healthy tissues and is a methylation target in some cancers  factor, HBZ, and the Tax protein; however, they can have opposing effects on WNT signaling. For instance, HBZ and Tax, both can interact with the WNT pathway through a related protein, DAPLE (disheveled-associated protein with a high frequency of leucine residues) . In the  pathway . Knockinogenesis .in vivo data supporting this yin and yang between TCF/LEF expression and viral load using Japanese macaques as a model system. In animals infected with the closely related, Simian T Lymphocyte Virus-1 (STLV-1), a negative correlation between the STLV-1 proviral load and TCF/LEF1 expression was observed in T cells (To highlight the complex interactions between HTLV-1 and the WNT pathway, it is interesting to note that while Tax functions to promote HTLV-1 viral replication, TCF1 and LEF1 both interact with Tax to attenuate Tax-dependent viral expression and activation of NF-kB and AP-1 . In cont T cells .DKK1, which was attenuated following siRNA knock-down of p300/CBP. Forced HBZ expression in T cells uninfected with HTLV-1 caused de novo expression of DKK1, while expressing HBZ in HTLV-1-infected T cells increased its expression (Among its symptoms, patients with severe cases of ATL may exhibit hypercalcemia, bone loss, and bone lesions that are associated with osteoclast-mediated bone resorption . One keypression . These dpression .Finally, the γ-herpesvirus KSHV, can target components of the WNT signaling pathway in B cells to promote tumorigenesis . The LANCollectively, the data suggest that dysregulating WNT signaling is an effective mechanism by which oncogenic viruses target and manipulate multiple normal cellular processes to facilitate tumorigenesis. Furthermore, these viruses often employ multiple means of circumventing normal WNT signaling. HBV, HCV, EBV, HPV, and KSHV can all inhibit various components of the WNT pathway that normally keep it in its “OFF” state. These same viruses can also enhance positive regulators of WNT signaling, resulting in increased expression of WNT target genes. Thus, by “releasing the brakes and stepping on the gas” of the WNT signaling pathway, oncogenic viruses have evolved to be very efficient at driving cell proliferation, survival, and ultimately, transformation.Given the complexity of the crosstalk between viruses and the multiple members of the developmental pathways, it is of paramount importance to better understand the molecular mechanisms involved in host-pathogen interactions to develop novel therapies. Notwithstanding the multitude of signaling proteins participating in each developmental pathway, the biological outcome is the same, thus leading to proliferation, cell survival, angiogenesis, stemness, EMT, immune evasion, and maintenance of latent viral state. The ability of the same viral protein to connect cellular proteins of the different pathways, such as HPV E6 or E7 with members of the Notch, HH, and WNT pathways raises the question whether simultaneous or ordered interactions occur in enhanced cell proliferation or tumor progression. Conversely, different viral proteins can interact with signaling proteins of the same pathway. For instance, HBx and NS3/5A/5B encoded by HBV and HCV, respectively, interact with the same HH signaling proteins, leading to cell proliferation and survival. GLI1/2 is activated by HBV, HPV, HCV, KSHV, and EBV proteins bringing to cell proliferation and survival, as well as virus immune evasion . OncogenHes1 (Hes1 expression. In addition, during in vitro angiogenesis, the protein complex, NICD/RBP-Jk/β-catenin, was formed and directed the differentiation of vascular progenitor cells toward arterial endothelial cells (In addition, developmental pathways may interact with each other to form a complex intertwined network connected by common molecules. Thus, understanding the crosstalk between developmental pathways might help to reveal common, druggable targets. This could be the case with β-catenin, a transcriptional activator of the WNT pathway, which also regulates the Notch-regulated transcriptional repressor, Hes1 . Furtheral cells . In carcal cells . Crosstaal cells . Althougin vitro, including combined with three-dimensional cell models, which better recapitulate the TME, as well as in vivo experiments using robust animal models to fully evaluate how these drugs might influence anti-viral/anti-tumor immune responses in the host.Considering the complexity of signaling cascades in each developmental pathway and the existing crosstalk between them, there is a need to carefully design appropriate targeted therapies to avoid adverse, toxic, or off-target drug effects in patients. It will be mandatory to test these drugs in appropriate cellular models Developmental pathways are important mediators of the transformation potential of different oncogenic viruses. They can turn normal physiologic pathways into potent carcinogenic routes, either to promote aberrant proliferation and acquisition of stemness, and/or to evade immune surveillance. Future efforts focused on better dissecting the cross-signaling between cellular developmental pathways and viruses will further our understanding of the evolution of viral carcinogenesis, including host-pathogen communications that can also shape the cells in the tumor microenvironment and modulate anti-tumoral immune responses. Given the ability of viruses to behave as forced activators of developmental pathways, we should consider targeting their members in future experimental studies. Viruses can repress signaling pathways at early stages of carcinogenesis, as HPV does with TGFβ and Notch, but can promote later activation, as seen in malignant progression . MoreoveSKP and EM researched the literature and performed the literature review and helped in editing the figures. CM, DB, SC, and RP critically revised the manuscript. PT participated to wrote and to edit the manuscript and figures. EA, LM, and MPF put forward the idea of the manuscript, wrote and edited the manuscript, figures, and the table. All authors contributed to the article and approved the submitted 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.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.