For the past 20 years, the Ontario child welfare sector has made significant legislative and policy changes. Changes to legislation and policy can impact the public and sector’s response to child maltreatment and inform identified trends. Using an investigative taxonomy of urgent protection and chronic need this paper examines the shift in the nature of investigated maltreatment over time.Data from five cycles of the Ontario Incidence Studies of Reported Child Abuse and Neglect  were used. Provincial incidence rates were calculated by dividing the weighted estimates by the child population 15 years of age and under and then multiplying by 1000 in order to produce an annual incidence rate per 1000 children. Investigations were divided into urgent  and chronic . Tests of statistical significance were calculated to assess changes in subtypes between cycles.Between 1993 and 2013, the rate of child maltreatment related investigations completed in Ontario has increased from 20.48 per 1000 children to 53.27 per 1000 children. Overall there has been a decline in the incidence of urgent investigations from 9.31 per 1000 child maltreatment investigations in 1993 to 5.94 per 1000 maltreatment investigations in 2013. There has been a fourfold increase in the incidence of chronic investigations from 11.18 per 1000 child maltreatment investigations in 1993 to 47.33 per 1000 maltreatment investigations in 2013.The nature of child protection work using the urgent-chronic taxonomy shows a dramatic shift in the types of concerns identified without a corresponding shift in the way families are assessed for need. The provision of a forensic investigation to all families does not distinguish between urgent safety concerns and needs that may require prolonged engagement. Effective service provision requires more precision in our response to these diverse concerns. Three children, ages 7, 9 and 11 are observed alone on public transit by a concerned citizen and a child welfare authority is contacted with an allegation that the children are not being provided with appropriate supervision. The next day, the same child welfare authority is contacted by a cousin of a woman who has been assaulted by her husband, worried that their teenager has witnessed the assault. The following day a teacher calls with a concern that a 2 year old sibling of one of her students is being left home alone when the mother walks her pupil to school in the morning. Each of these referrals to the child protection agency is judged to meet the threshold for investigation and while the number of “collaterals” interviewed may vary in each case, each family will receive a visit and an interview, the worker will complete a risk assessment and assess the child’s safety. There will be a determination about whether a child has been maltreated and whether the family needs ongoing child welfare services. The assessment of maltreatment varies across provinces and is determined by the clinical judgement of the investigating worker based on the balance of probabilities of whether the child has been maltreated.Similar to other Canadian Provincial and Territorial statutes, safety and well-being are central and equal considerations within Ontario child welfare legislation . TypicalThe Ontario Incidence Study of Reported Child Abuse and Neglect (OIS) is the only source of aggregated provincial data on reported child maltreatment and provides an opportunity to explore possible changes in the incidence of reported child abuse and neglect over time. Reported child abuse and neglect in the province of Ontario doubled between 1998 and 2003; from a rate of an estimated 27.42 investigations per 1000 children to a rate of 53.56 per 1000 children in 2003. Since 2003, the rate of investigated maltreatment has remained consistent with the latest estimate in 2013 indicating that 53.27 investigations per 1000 children were conducted [Changes to legislation and policy can impact the public and sector’s response to alleged child maltreatment and inform identified trends. The increase in the incidence of investigations in Ontario is believed to be driven by the broadening of the child welfare mandate and the inclusion of children’s exposure to intimate partner violence, as well as those who were at future risk of maltreatment , 9. GreaIn 2006, further policy reform was initiated through the Ontario Child Welfare Transformation Agenda (Transformation Agenda), which included a more balanced approach to practice that protected children while also promoting their well-being and supporting families . The TraIn 2009, the Commission to Promote Sustainable Child Welfare (the Commission) was established to better understand the impact of the Transformation Agenda, and to develop and implement further changes to improve the child welfare sector , 15. A sDespite significant policy and legislative changes that have occurred in Ontario over the last 20 years, there are concerns with traditional child welfare service models that emphasize child safety, and the ability of the sector to meet the complex, chronic needs of children and families served , 11, 19.The objective of this research is to determine rates at which child welfare authorities investigate maltreatment using the urgent-chronic taxonomy in Ontario to identify trends over time. A detailed analysis of investigative trends in Ontario by applying the urgent-chronic taxonomy can help to increase our understanding of how the child welfare system has been responding to population, policy and practice changes, and to its dual mandate of promoting safety and well-being.Data from the five OIS cycles were analyzed to explore trends in the investigative taxonomy of urgent-chronic need. Each of the five cycles of the Ontario Incidence Study of Reported Child Abuse and Neglect (OIS) utilized a multi-stage sampling design . The firThe final stage of sampling includes identifying children investigated as a result of maltreatment concerns. In each of the five OIS cycles, estimates of the provincial annual rates of maltreatment investigations in Ontario were derived by applying a regionalization and annualization weights. Estimates for each of the cycles do not include incidents not reported to Ontario child welfare authorities, cases that were screened out and were not fully investigated, new reports on cases that were opened, and cases that were only investigated by police . For grerisk only investigations.Data for each of the OIS cycles is collected directly from investigating child welfare workers in each of the sampled organizations using a three-page standardized data collection instrument, the maltreatment assessment form. This instrument is completed at the conclusion of the investigation and collects clinical information that is routinely gathered by child welfare workers during the course conducting the investigation, including caregiver, child, case, and short-term service dispositions. In 2008, the maltreatment assessment form was amended to include investigations that were conducted which focused not on an event of maltreatment that alleged or suspected but rather assessed the risk to the investigated child of future maltreatment or SPSS Statistic version 23 was used to conduct the analyses. Provincial incidence rates were calculated by dividing the weighted estimates by the child population 15 years of age and under and then multiplying by 1000 in order to produce an annual incidence rate per 1000 children.The population for Ontario children is based upon the appropriate Census data for the study cycle. The Census is conducted by Statistics Canada every 5 years.First, the overall investigation rate for investigations in Ontario in each of the five OIS cycles  was compared over time. Next, we examined the change in the rates of investigations using the urgent-chronic taxonomy across cycles of the OIS. Investigations were classified as urgent or other maltreatment-related investigations or assessments (i.e. chronic need) by using the taxonomy developed by Trocmé and colleagues [Statistical significance was calculated to examine whether there had been a change in the incidence from the previous OIS cycle. Tests of significance were produced using WesVar 5.1 software.Figure Table As shown in Table Overall there has been a decline in the incidence of urgent investigations from 9.31 per 1000 child maltreatment investigations in 1993 to 5.94 per 1000 maltreatment investigations in 2013. As a proportion of all investigations, those of an urgent nature have declined from 45% of all investigations in the OIS-1993 to 11% of all investigations in the OIS-2013.The nearly doubling of the rate of child maltreatment investigations in Ontario between 1998 and 2003 is reflected in some of the trends in the subtypes of chronic investigations. Physical abuse investigations for children 4 years of age and older went from a rate of 8.15 per 1000 investigations in 1998 to 13.29 per 1000 investigations in 2003, returning to 8.46 per 1000 investigations in 2008 and 9.28 per 1000 investigations in 2013. A very similar pattern is seen in the rate of neglect investigations for children 4 years of age and older.In 1993, emotional maltreatment was investigated in only .91 per 1000 investigations. In 1998 there was a twofold increase to 2.15 per 1000 investigations and the rate of investigated emotional maltreatment continued to increase in 2003 to 7.70 per 1000 investigations. In 2008, there was a statistically significant decline in investigated emotional maltreatment to 3.37 per 1000 investigations. In 2008 one in three investigations focused on risk of future maltreatment (17.52 per 1000 investigations). In 2013 this proportion declined to one in five investigations (11.63 per 1000 investigations), although this decline was not statistically significant. Children exposed to intimate partner violence were not identified in 1993; in 2013 it had the highest incidence of investigations, 13.28 per 1000 investigations.Overall there has been a fourfold increase in the incidence of chronic investigations from 11.18 per 1000 child maltreatment investigations in 1993 to 47.33 per 1000 maltreatment investigations in 2013. As a proportion of all investigations, those of a chronic nature have increased from 55% of all investigations to 89% of all investigations.The rate of child maltreatment related investigations completed in Ontario since 1993 has gone from 20.48 per 1000 children to 53.27 per 1000 children. Two decades of policy and legislative changes have resulted in a dramatic change in the type of situation that child protection workers are routinely faced with. The overall increase in child maltreatment investigations in Ontario is difficult to interpret; for instance, the rate of child homicides has remained fairly consistent in Ontario for several decades . OntarioUrgent cases are investigations where the child has sustained harm serious enough to require medical treatment; there is an allegation of sexual abuse or there is a concern of physical abuse or neglect for a child under the age of four. The rate of urgent cases has both declined by nearly half (from 9.31 per 1000 investigations in 1993 to 5.94 investigations in 2013) and in proportion to the overall composition of investigations. The findings in this paper of a decline in reported sexual abuse investigations is consistent with a steady decline in sexual abuse since the 1990s in the United States , 30 and The Ontario child welfare legislation specifically includes situations where a child has been harmed or is at risk of being harmed, consistent with an emerging body of research that show chronic, unaddressed maltreatment results in behavioural, emotional, cognitive and health issues , 32, 33.Despite the changing nature of the type of maltreatment reported and investigated in Ontario, the response by the system is nearly identical to the investigation procedures. The concerns with a traditional or child protection response to maltreatment often considered adversarial and intrusive has led some jurisdictions in the United States and Canada to develop and implement a formal differential or alternative response to children and their families . This rerisk only in 2008 makes comparisons across cycles challenging. For example, the variation in reported emotional maltreatment investigations is likely to have been impacted by the inclusion of risk only investigations in 2008 as the chronic nature of these situations may be similar.The OIS collects information directly from the investigating worker and the data collected are not independently verified. The study only examines the case until the point of initial assessment—the data are not able to describe the longer term impact of the events described. The data do not include children who are only reported to the police, known to a community member or never disclose their abuse or neglect. There have been procedural and study definitional changes over time that reflect changes in legislation and procedures, in particular allowing workers to describe investigations as Data from the Ontario Incidence Studies of Reported Child Abuse and Neglect describe a child protection system that expanded rapidly between 1998 and 2003 and since then has consistently investigated five and a half percent of children 15 years of age and under for a child maltreatment-related concern. The nature of child protection work using the taxonomy developed by Trocmé and colleagues [


In this paper an  There has been a recent interest in adaptive algorithms to handle sparsity in various signals and systems  that seeks to minimize the sum of squares of residuals on all of the variables in the equation instead of minimizing the sum of squares of residuals on only the response variable. Golub and Van Loan introduced the TLS problem to the field of numerical analysis  and the adaptive filter n(k) is the measurement noise. In this context, the goal of an adaptive algorithm is to identify the system by minimizing the cost function defined byIn the sparse system identification problem of interest, the system observes a signal represented by an M-th order M-th order sparse system, most estimation algorithms exploit non-zero coefficients of the system to obtain performance benefits and/or a computational complexity reduction (Gu et al. Especially, we call In TLS, we assume a given unknown system that both the input and output are corrupted by noise. The system should be estimated from the noisy observation of the input and the output Fig. . In thisFor TLS solution, the augmented data vector is considered as:n Davila  and Choin Davila , the TLSk-th time step. The subgradient of We adopt the modified cost function by the addition of a penalty function. This penalty function can be chosen to reflect a priori knowledge about the true sparsity system.and Tanc . We adopmentclasspt{minimaand Tanc .17\documThe proposed algorithm needs a solution in  as well Stirling . The req-RTLS in –17, \docdures in –17 need In this experiment, we follow the experiment scenario in Eksioglu and Tanc . The truγk using . The \dovalue in  is takenand Tanc , that isand Tanc  with theThe proposed algorithm needs the inversion of the covariance matrix as  in orderentclass1pt{minimaIn Fig. Table In this paper, we propose an


Metastatic rectal cancer requires a multidisciplinary and individualized approach.The authors describe a case report of a 48-year-old man with recurrence of rectal adenocarcinoma that underwent multimodal treatment, which included chemotherapy with biologic agents, cytoreduction surgery with hyperthermic intraperitoneal chemotherapy, and radiotherapy with improvement in progression-free survival and overall survival. Peritoneal carcinomatosis from colorectal cancer (CRC) represents a group of patients with metastatic disease associated with poor prognosis.1In metastatic setting, chemotherapy (CTX) eventually with association of biologic agents is the standard of care, with improvement in progression-free survival and overall survival (OS). More recently, there is evidence that cytoreductive surgery combined with hyperthermic intraperitoneal CTX can prolong patients’ life with CRC and peritoneal carcinomatosis.An otherwise healthy, 43-year-old male, with an Eastern Cooperative Oncology Group performance status (ECOG PS) of 0, was admitted in our institution in September 2012 with a recurrence of rectal adenocarcinoma.Out of our institution, in October 2011, he underwent laparoscopic anterior resection for rectal adenocarcinoma; pathologic stage was pT3N0M0 (6 nodes isolated without disease); no neo or adjuvant therapy was made.One year later, the patient underwent urgent exploratory laparotomy for intestinal occlusion; a colostomy was made and the diagnosis of peritoneal metastasis was confirmed. Mutational status of the RAS complex was wildtype.In our institution, after re-staging with computed tomography (CT) and positron emission tomography–computed tomography (PET–CT), both suggesting local recurrence and pelvic tumor implants Fig. , the casHe received 12 cycles of 5-fluorouracil (5-FU), plus irinotecan, plus leucovorin (FOLFIRI) scheme with bevacizumab, without relevant toxicity and with partial response on CT scan and PET–CT .In June 2013, CS/HIPEC (mitomycin 80 mg) was performed. The peritoneal cancer index was 11, with apparent complete cytoreduction. There were no postoperative complications. PET–CT was performed with no evidence of disease, and therefore, no complementary CTX was done.Six months later, patient was admitted to the hospital with lumbar pain. An ultrasound and a CT scan only presented right hydronephrosis with no apparent disease recurrence; a percutaneous nephrostomy was made. PET–CT was carried out and revealed increased uptake on the presacral level, suggesting local recurrence Fig. .After discussion on an MDT, it was decided preoperative concomitant chemoradiation therapy (CRT) with subsequent exploratory laparotomy associated with intraoperative radiotherapy (RT). Patient received 5-FU as radiosensitizer till March 2014.Eight weeks later, on the exploratory laparotomy, it was identified presacral recurrence and peritoneal carcinomatosis; it was made the resection of the presacral lesion and the reimplantation of the ureter into the bladder, but no intraoperative RT was performed, due to peritoneal disease. The histopathological report confirmed the pelvic recurrence, with positive margins (R1), and the presence of adenocarcinoma in a parietocolic recess.Patient restarted CTX with the same scheme, 12 cycles, until March 2015, with complete response on CT and PET–CT. MTD suggested a second-look laparotomy, but since he was completely asymptomatic without evidence of disease, it was decided to keep close vigilance.In June 2015, he repeated PET–CT Fig. , which rPatient started second-line treatment with 5-FU, oxaliplatin, and leucovorin (25% reduction of 5-FU) with bevacizumab. CT scan after 6 cycles demonstrated new disease progression and started third-line CTX with FOLFIRI  plus cetuximab. On April of the current year, patient had new peritoneal disease progression and was admitted in our center with acute bowel occlusion and treated with conservative measures, but unfortunately he get worse and died 3 weeks later.3About half of the patients with CRC will have disease recurrence as peritoneal metastasis, and 10% of these will have isolated peritoneal disease.This case represents an example of metachronous peritoneal metastasis from primary rectal cancer and describes a multimodal and individual approach.4 One thing is for sure: with the insufficient lymph nodes samples, at least adjuvant treatment should have been discussed.Regarding the treatment options undertaken, some considerations should be made. First, was this patient a candidate for neoadjuvant treatment at diagnosis instead of upfront surgery? If the tumor was a clinical T3 and/or positive for nodal disease, it should have; with neoadjuvant CRT the rates of local recurrence can be <6%.5 Our patient was selected for its young age, excellent PS, no progression under CTX, and disease evaluated for complete resection, which also meet Goéré criteria.Second, this patient with peritoneal disease at recurrence was selected for CS/HIPEC. But which patients should be candidates to undergo a CS/HIPEC approach? We don’t know; Goéré and colleagues published possible eligible criteria: ECOG (≤1), low peritoneal cancer index, absence of progression under CTX, no extra peritoneal metastasis , and no evidence of biliary or ureteral obstruction.Moreover, peritoneal carcinomatosis is found in advanced stage in most of the patients, due to absence of symptoms and because it is difficult to detect by imaging in early stage, which also make more challenging to achieve a complete resection.6Currently, there are phase II trials that support second-look surgery in patients with colon cancer at high risk for local recurrence or peritoneal metastases.7Based on this rationale, Ripley et al designed a trial to evaluate if mandatory second-look surgery with CS/HIPEC would prolong OS compared with the standard of care (surveillance) in patients who have undergone curative surgery and show no evidence of disease, but who were at high risk for developing peritoneal carcinomatosis from CRC.These high-risk patients were described as having limited and synchronous peritoneal disease completely resected with the primary tumor, ovarian metastases, tumor perforation, T4 lesions, and emergency presentation with bleeding and obstruction.The results of this study are still awaited, and at present, we do not have strong evidence-based data, so the authors do not recommend this proactive treatment strategy in rectal cancer.8 This pressure application improves tumor drug uptake, and positive results with PIPAC with low-dose cisplatin and doxorubicin for patients with platin-resistant recurrent ovarian and gastric cancer have been published.10 In addition, a retrospective analysis presented the results of PIPAC with oxaliplatin in colorectal peritoneal metastasis.11 Objective tumor responses were observed in 71% of the patients (12 of the 17 patients), with minimal adverse effects, and mean survival after first PIPAC of 15.7 months. Further studies are necessary to evaluate the possible role of this strategy in CRC with peritoneal carcinomatosis.Another promising approach in management of peritoneal carcinomatosis is delivering CTX into the peritoneal cavity as a pressurized normothermic aerosol via laparoscopy, known as PIPAC .12We would like to emphasize the patient long survival, despite the poor prognosis and life expectancy <6 months estimated, in the presence of recurrence as peritoneal disease.13Nowadays, the standard of care in the management of patients with metastatic CRC is the addition of biologic agents to CTX backbones as it is associated with improved OS that ranges to 25 to 28 months.14Our patient received FOLFIRI/bevacizumab with significant imagiologic response on PET-fluorodeoxyglucose (FDG), so it was decided to restart the same CTX after second relapse. Several CT scans were negative for presence of disease so we chose PET-FDG for disease evaluation, mostly for 2 reasons: it provides a good overall accuracy in detecting peritoneal carcinomatosis, and helps in the selection of patients for surgery; however, our experience is important only when the implants are of great dimension.In conclusion, we highlight that patients treatment should be performed in experienced centers, in a multidisciplinary setting, in order to carefully select the mostly adequate approach for each patient.None.The authors declare no conflicts of interest.


Previously, we reported that Zika virus (ZIKV) causes ocular complications such as chorioretinal atrophy, by infecting cells lining the blood-retinal barrier, including the retinal pigment epithelium (RPE). To understand the molecular basis of ZIKV-induced retinal pathology, we performed a meta-analysis of transcriptome profiles of ZIKV-infected human primary RPE and other cell types infected with either ZIKV or other related flaviviruses . This led to identification of a unique ZIKV infection signature comprising 43 genes (35 upregulated and 8 downregulated). The major biological processes perturbed include SH3/SH2 adaptor activity, lipid and ceramide metabolism, and embryonic organ development. Further, a comparative analysis of some differentially regulated genes  revealed that ZIKV induced their expression relatively more than dengue virus did in RPE. Importantly, the pharmacological inhibition of ABCG1, a membrane transporter of cholesterol, resulted in reduced ZIKV infectivity. Interestingly, the ZIKV infection signature revealed the downregulation of ALDH5A1 and CHML, genes implicated in neurological  and ophthalmic (choroideremia) disorders, respectively. Collectively, our study revealed that ZIKV induces differential gene expression in RPE cells, and the identified genes/pathways  could potentially contribute to ZIKV-associated ocular pathologies. It is increasingly clear that ZIKV infection has broad implications beyond microcephaly, because infants born with congenital ZIKV have pathology in their eyes, ears and limbs5. Consequently, there has been significant interest in understanding the pathogenesis of ZIKV in various diseases. Because of clinical studies linking ZIKV to ocular abnormalities, primarily in the retina of infants and adults (uveitis)6, and our laboratory interests in innate retinal defense to microbial infections, we initiated host-pathogen interaction studies of ZIKV in the eye. In our recent study7, we reported for the first time that (1) ZIKV causes retinal lesions in mouse eyes with a clinical presentation  resembling some of the features of ZIKV-associated ocular pathology described in humans; (2) cells lining the blood-retinal barrier (BRB), the retinal vascular endothelium and RPE were permissive to ZIKV replication and expressed receptors for its entry; and (3) ZIKV induces retinal cell death and evokes innate retinal inflammatory and antiviral responses both in vitro and in vivo. These findings led us to postulate that being a blood-borne pathogen, ZIKV must overcome the BRB to gain access to the eye and cause retinal abnormalities, a key manifestation of ZIKV infection in the eye6. Moreover, three recent studies also support our findings by demonstrating the permissive nature of retinal endothelium and RPE to ZIKV infection10.The emergence of Zika virus (ZIKV) in both endemic and non-endemic regions of the world has been accompanied by an unprecedented rise in the spectra of ZIKV-associated diseases11. We hypothesized that ZIKV might have a specific infection signature that distinguishes it from other flaviviruses. In this study, we generated a transcriptome profile of ZIKV-infected human primary RPE cells and performed a meta-analysis12 of transcriptome profiles of cells infected with other flaviviruses to gain insights in understanding the molecular mechanisms of ZIKV pathogenesis. Given the non-availability of vaccines against ZIKV, the results of this study will lead to the identification of novel pathways and/or drug targets to prevent ZIKV infection and its associated ocular complications.Since RPE constitutes the outer BRB, the specific contribution of RPE in ZIKV-induced retinal pathology such as chorioretinal atrophy remains to be elucidated. Similarly, upon infection, how ZIKV modulates innate antiviral responses and cell signaling in RPE for its own survival (replication) is currently unknown. Because ZIKV is closely related to other members of the Flaviviridae family such as dengue virus (DENV), West Nile virus (WNV), and Japanese encephalitis virus (JEV), an important question is why only ZIKV, but not other pathogenic flaviviruses, causes congenital diseases and associated complicationshttps://www.ncbi.nlm.nih.gov/geo/)  Table  and normTranscriptome profiling was performed on uninfected control and ZIKV-infected (48 and 96 hrs) human primary RPE cells Fig.  using ulThe comparison of ZIKV-infected and uninfected samples at 48 h and 96 h identified 633 and 1,198 significantly differentially expressed genes respectively, with a P value < 0.01 and at least a 2-fold change, with 452 genes being commonly associated with ZIKV infection at both time points Fig. . Among tNext, we compared the transcriptome profiles of ZIKV-infected RPE with ZIKV-infected human neural progenitor cells (hNPCs). This led to the identification of a signature comprising 601 genes (484 upregulated and 117 downregulated) that were specifically dysregulated in ZIKV-infected RPE cells Fig. . These dFurther pathway analysis of genes that are specifically downregulated only in ZIKV-infected RPE cells revealed significant enrichment in EIF2 signaling, antiproliferative role of TOB in T cell signaling, mTOR signaling, paxillin signaling and FAK signaling pathways Fig. . On the To gain further insight into system-level perturbations and to identify key molecules associated with ZIKV Infection in RPE cells, we performed a systems biology-oriented analysis on the genes that are differentially expressed in RPE cells after ZIKV infection. The regulatory analysis revealed strong activation of immune and inflammatory responses, mediated through activation of TNFSF10, IFNB1, IL6, STAT2, TLR2, DDX54 (RIG-I), NLRC5, and IRF7, indicating that the RPE response to ZIKA infection is dominated by genes involved in inducing cytokines, chemokines, and type I interferons Fig. . On the 7, further validating systems biology prediction.The comparative analysis of ZIKV-infected RPE and hNPC signatures identified 115 common genes that were dysregulated consistently across both cell types . Out of 115 genes, 99 were upregulated, and 16 were downregulated Fig. . FurtherThe meta-analysis of ZIKV signatures from different cell types (RPE versus hNPCs) was not sufficient to identify genes or pathways that are specifically linked to ZIKV infection or pathogenesis, because this analysis only revealed genes and pathways linked to general antiviral responses. To identify genes specifically dysregulated by ZIKV in addition to the generalized antiviral response, we performed a comparison of the ZIKV meta-signature with transcriptome signatures from other flaviviruses closely related to ZIKV, i.e., JEV, WNV, and DENV. Of the ZIKV meta-signature 115 genes Fig. , 72 geneGene ontology enrichment analysis on ZIKV core signature genes revealed multiple biological processes related to SH3/SH2 adaptor activity, lipid and ceramide metabolism (P < 0.05) Fig. . FurtherFurther interactive network analysis was performed on core ZIKV dysregulated genes to identify key regulators that are crucial for ZIKV pathophysiology. The interactive network was generated using the co-expression information of genes based on a Pearson correlation from the transcriptome profile of ZIKV and other viruses that cause neurological diseases. The network was constructed using highly correlative interactions of genes . Since these networks are scale-free and undirected in nature, their topological properties reveal the impact of weak and strong connections measured based on “degree of centrality.” Our results showed significant enrichment of networks controlling immune, inflammation, and metabolic functions, mirroring the result of the GO analysis. Key nodes were isolated based on the degree of centrality ranking, and the top 10 key nodes included ALDH5A1, SIX4, ABCG1, TNFSF10B and CHML from the ZIKV core signature Fig. . To furtAfter meta-analysis and systems biology-led predictions of the ZIKV infection signature, we sought to determine whether other flaviviruses also modulate the expression of identified key molecules. First, we show that similar to ZIKV, human primary RPE cells are permissive to DENV Fig. . Second,Among the genes predicted to be downregulated, qRT-PCR validation revealed that both ZIKV and DENV reduced the expression of ALDH5A1 and CHML in infected cells. While no significant difference was observed in downregulation of the CHML gene in ZIKV- versus DENV-infected cells, ALDH5A1 levels were drastically reduced by ZIKV compared to DENV at both time points 48 and 96 hrs post-viral challenge Fig. . These r15. In addition to mRNA  because its expression has been demonstrated in the retina, including RPE cellsRNA Fig. , we confRNA Fig. . Our resRPE Fig. . In cont19. This prompted the WHO to initially declare ZIKV as a public health emergency of international concern. Our interest in studying ZIKV pathogenesis emanated from clinical reports linking ZIKV with ocular complications such as uveitis21, acute maculopathy22, pigmentary retinopathy, and chorioretinal atrophy26. These clinical findings highlight that macular and chorioretinal disease can significantly impact visual outcomes in ZIKV-infected infants and adults. However, it is not yet clear whether congenital ocular complications are directly caused by ZIKV or are secondary to microcephaly27. To investigate whether ZIKV can directly affect the health of the retina, the primary target of ZIKV in the eye, we recently developed a mouse model of ZIKV-induced chorioretinal atrophy and demonstrated that cells lining the BRB and the retinal pigment epithelium (RPE) are highly permissive to ZIKV7.The most recent outbreak of ZIKV in Brazil and other South American countries featured unexpectedly severe neurological manifestations that were not observed in prior ZIKV or other related flavivirus outbreaks7 and other studies10 could create a portal for ZIKV entry to the eye from the fenestrated choroidal capillaries. To investigate the molecular mechanisms of ZIKV pathogenesis in RPE cells, here we performed transcriptome analysis of ZIKV-infected human primary RPE cells using a highly sensitive RNA sequencing approach. Consistent with the previous study of WNV-infected RPE cells28, our data also suggest the predominance of pathways regulating innate antiviral responses. These include interferon signaling, as indicated by upregulation of type-I IFNs, and the production of antiviral molecules, such as MX1, ISG15, OAS2, and CXCL10. Other pathways significantly upregulated in ZIKV-infected RPE were cytosolic pathogen recognition receptors like RIG-I, and activation of IRFs by cytosolic pattern recognition receptors and Toll-like receptors (TLRs), which play an essential role in recognition of viral RNAs, resulting in the modulation of IFN signaling. Indeed, our recent study showing ZIKV-induced antiviral innate responses in retinal endothelial cells and RPE, including induced expression of RIG-I and TLR37, validates the transcriptome data obtained here. In addition to TLR3, unexpectedly, we observed the induction of TLR2 and TLR9, suggesting that these innate receptors might also be involved in recognition of ZIKV. The pathway analysis of genes that are specifically down-regulated in ZIKV-infected RPE showed significant enrichment in EIF2 signaling, mTOR signaling, paxillin signaling, and FAK signaling pathways, indicating the induction of cellular stress-related signals.Because RPE maintains the outer BRB and shields the neuroretina from blood-borne pathogens, RPE cell death as reported by our30 and current studies, meta-analysis after uniform normalization and preprocessing can provide a valid comparative analysis to identify most robust genes associated with pathophysiology. We normalized all the datasets and performed outlier analysis independently on all the studies to ensure that the studies are at the same normalized values and contain no outliers. Moreover, we have confirmed our meta-analysis findings using qRT-PCR analysis of identified key genes.To identify the universal set of genes associated with ZIKV pathogenesis, we performed a comparative analysis of our transcriptome data with publicly available transcriptome data of other cell types infected by ZIKV. First, we compared our data with transcriptome data of ZIKV-infected human neural progenitor cells (hNPCs) from two other studies after uniform pre-processing and analysis. This led to the identification of 115 common genes that are dysregulated  consistently across both cell types (the ZIKV meta-signature). The most dysregulated pathways were linked to interferon production, ER stress, and defense response to viruses. This meta-analysis of ZIKV signatures from different cell types  was unable to distinguish genes or pathways specifically linked to ZIKV pathogenesis from those of the common antiviral response. To further identify genes that are specifically dysregulated by ZIKV in addition to the generalized antiviral response, we performed a comparison of the ZIKV meta-signature of 115 genes with transcriptome signatures from DENV, JEV, and WNV. This meta-analysis led to the identification of a unique signature of 43 genes referred to as the ZIKV core signature, which is dysregulated upon ZIKV infection but not by the other flaviviruses tested. While the meta-analysis significantly reduces experimental efforts and assists in identifying the genes associated with a specific infection signature, the availability of only limited transcriptome datasets from similar cell types and/or time points  validated ACBG1 expression both at mRNA and protein levels in ZIKV-infected RPE cells, and (2) that pharmacological inhibition of ACBG117 resulted in reduced ZIKV infectivity, whereas cholesterol supplementation increased ZIKV infectivity in RPE cells. Viruses subvert cholesterol homeostasis using multiple mechanisms34. For example, DENV increases intracellular cholesterol levels by LDL uptake and promoting the activity of HMG-CoA reductase35, while herpes simplex virus 1 alters cholesterol trafficking36. Because host cell lipids and cholesterol play an essential role in various stages of viral replication, including entry, uncoating, genome replication, assembly, and release37, further studies should investigate how ZIKV infection of RPE alters chorioretinal cholesterol homeostasis38 and whether cholesterol transporters contribute to retinal pathology in vivo, using ABCG140 or ABCA113 (another cholesterol efflux transporter) knockout mice.The gene ontology (GO) analysis of upregulated genes in the ZIKV core signature identified enrichment in multiple biological processes related to SH3/SH2 adaptor activity, lipid metabolism, and ceramide metabolism. This is consistent with the studies showing that WNV and DENV use ceramide differentially during their replication cycles41. Under inflammatory conditions, SH2B3/LNK counteracts leukocyte adhesion to endothelial cells by inhibiting VCAM-1 expression42. Endothelial cells lining the retinal blood vessels are the major regulatory interface for the trafficking of hematopoietic cells into the retina and provide an innate barrier to viral pathogens44, including ZIKV9. In response to inflammatory stimuli such as TNFA, activated endothelial cells highly express SH2B345, which negatively regulates integrin signaling via inhibition of the integrin-linked kinase, thereby restricting endothelial cell adhesion and migration. SH2B3 also regulates integrins and cell motility in other cell types, such as platelets and megakaryocytes47. Our data showing induced expression of SH2B3 in ZIKV-infected retina/retinal cells indicates that ZIKV may employ SH2B3 to evade the immune system by attenuating the inflammatory response and infiltration of innate immune cells to the site of infection. The mechanism of SH2B3 modulation of the innate antiviral response to promote ZIKV replication needs further investigation, e.g., by inhibiting the SH2B3 activity or the use of Lnk−/− knockout mice.Another key regulator gene identified was SH2B3, originally characterized as LNK, a negative regulator of multiple cytokine signaling pathways, and which is associated with an increased risk of myocardial infarction48. Epilepsy is present in about half of affected individuals49. Studies in Aldh5a1−/− mice revealed that multiple metabolites associated with gamma-aminobutyric acid (GABA) metabolism , 4,5-dihydro hexanoate) and oxidative stress are significantly increased in multiple organs/tissues50. Myelin and lipid abnormalities in the cortex and hippocampus are also implicated in the pathophysiology of Aldh5a1−/− mice51. Based on our findings demonstrating ZIKV-induced downregulation of the ALDH5A1 gene, we postulate that a reduction in ALDH5A1 enzyme activity would increase endogenous GHB and GABA levels, and therefore contribute to neurological manifestations of ZIKV infection such as microcephaly and Guillain-Barré syndrome in adults52. Further studies should address whether ZIKV indeed triggers SSADH and determine the underlying mechanisms. Another gene downregulated by ZIKV was CHML, which is linked to an ocular disorder called Choroideremia (CHM)53, a slowly progressing X-linked retinal disease characterized by degeneration of the choroid, the RPE, and the neural retina54. In both human and mouse eye, CHML and Rab escort-protein-1 (REP-1) are expressed throughout the retina in multiple cell types including RPE54. The CHML gene encodes REP-1, an essential player in the geranylgeranylation of Ras-related GTPases, which are Rab proteins involved in intracellular trafficking of vesicles in endocytic and exocytic pathways55. To determine whether ZIKV-infected RPE has impaired phagocytic function, we performed phagocytic uptake of fluorescent beads and observed no significant difference in infected versus uninfected control cells (data not shown). Silencing of REP-1 in RPE cells does not affect outer photoreceptor segment (POS) internalization but delays POS protein clearance and increases the secretion of inflammatory mediators56. The alteration of the activity of Rab proteins, including REP-1, in regulating endocytosis could be an important factor in the pathogenesis of diseases caused by intracellular pathogens55. Further studies are warranted to elucidate the function of the role of CHML in ZIKV and other flavivirus infections. The fact that ALDH5A1 and CHML, two key molecules significantly associated with the stability of the ZIKV network, are found in both ZIKV core and extended set of genes, indicates their importance in ZIKV-induced pathophysiology  -dependent succinic semialdehyde dehydrogenase (SSADH) enzyme. In humans, SSADH deficiency is a rare autosomal recessive metabolic disorder affecting γ-aminobutyric acid degradation and is characterized by developmental delay, cognitive impairment, expressive language deficit, and mild ataxiaIn summary, to our knowledge, this study is the first to reveal the molecular signature of ZIKV infection, contributing to better understanding of ZIKV pathogenesis. By performing RNA sequencing on ZIKV infected human primary RPE cells and performing innovative comparative analysis of ZIKV transcriptome profiles with other flaviviruses, we identified the involvement of novel pathways unknown to play a crucial role in ZIKV pathogenesis. Moreover, in the wake of rapidly spreading ZIKV infection and lack of treatment options, the identified genes/pathways specifically perturbed by ZIKV infection could allow the rational design of therapeutic strategies to treat or prevent ZIKV infection and its complications.™Bulletkit® media as per manufacturer’s instruction (Lonza). Vero cells (ATCC CCL-81) and Aedes albopictus clone C6/36 cells (ATCC CRL-1660) were cultured in DMEM, and EMEM media supplemented with 10% FBS, 10 µg/ml L-glutamine, and 1% penicillin and streptomycin solution as per manufacturer’s recommendation. ZIKV Virus (ZIKV) strain PRVABC59, NR-50240, originally isolated from human blood in Puerto Rico in December 2015, and DENV type 2 strain NR12216 were obtained through BEI Resources, NIAID, NIH. This ZIKV strain is 97–100% genetically similar to the current ZIKV strain circulating in Brazil57. ZIKV and DENV were propagated in ATCC CCL-81 Vero cells and ATCC CRL-1660 C6/36 cells respectively, and titers were determined by plaque assay. RPE cells were infected with ZIKV and DENV at MOI of 1 for the indicated time points as described previously7. For ABCG1 functional studies, Pr. RPE cells were treated with a pharmacological inhibitor of ABCG1, Benzamil (50 µM) , and cholesterol-water soluble (10 µM)  1 h before ZIKV challenge.Human primary retinal pigmented epithelial (RPE) cells  were cultured in RtEGM7. Briefly, cells were infected with ZIKV PRVABC59 and DENV NR12216 for the indicated time points. Following infection, ZIKV/DENV infected, and uninfected control cells were fixed overnight with 4% paraformaldehyde in PBS at 4 °C. Following rinse with PBS cells were incubated with primary mouse monoclonal antibody 4G2 (1:100)  overnight at 4 °C. Following removal of the primary antibody, the cells were washed extensively with PBS and incubated for 1 h with anti-mouse Alexa Fluor 594-conjugated secondary antibody (1:200) at room temperature. Finally, the cells were extensively washed with PBS, and the slides were mounted in Vectashield anti-fade mounting medium containing DAPI  and visualized using fluorescence microscope .For immunostaining procedures, primary RPE cells were cultured in a four-well chamber slide  and infected with ZIKV and DENV at MOI of 1 and immunostained for anti-flavivirus group antigen 4G2 as described previouslyTo understand the molecular mechanism of ZIKV infection, RNA derived from ZIKV-infected, and uninfected human primary RPE cells were subjected to next-generation sequencing to generate deep coverage RNA-Seq data. For each treatment group, sequencing was performed on at least two biological replicates. Sequencing libraries of Poly(A)-selected mRNA were generated from the double-stranded cDNA using the Illumina TruSeq kit, as per the manufacturer’s protocol. Library quality control was checked using the Agilent DNA High Sensitivity Chip and qRT-PCR. High-quality libraries were sequenced on an Illumina HiSeq. 2500. To achieve comprehensive coverage for each sample, we generated ~30 million paired-end reads from each sample.58. The quality of the reads with checked using FastQC program that provides a very comprehensive estimate of sample quality based on read quality, read length, GC content, uncalled based, the ratio of bases called, sequence duplication, adaptor and PCR primer contamination59. These high quality clean reads were aligned against human genome using Tophat2 algorithm that uses bowtie2 aligner (http://tophat.cbcb.umd.edu/)60. We used hg19 human genome assembly as a reference genome for alignment. Gene expression measurement was performed from aligned reads by counting the unique reads using the HTSeq-count function in HTSeq algorithm61. The features with low read counts were filtered out using counts per million approach. The filtered read count-based gene expression data was normalized using voom approach, which estimates the mean-variance relationship of the log-transformed gene count data and generates a precision weight for each gene expression62.The raw sequencing data were processed to remove any adaptors, PCR primers and low-quality transcripts  using the Trimmomatic program63. LIMMA estimates the differences between uninfected and infected samples by fitting a linear model and determining the significance of differences in gene expression by applying empirical Bayes moderated-t-statistics. The differentially expressed genes were identified based on absolute fold change and raw P value. Genes were considered significantly differentially expressed if the P value was <0.01 and absolute fold change >1.5. The unsupervised analysis was performed using principal component analysis (PCA) using prcomp library in R, which projects multivariate data objects onto a lower dimensional space while retaining as much of the original variance as possible.The differentially expressed genes were identified from normalized sequencing data using a linear model microarray analysis software package )64. The read count-based gene expression data were normalized on the basis of library complexity and gene variation using the R package EdgeR65. The normalized count data were compared among groups using a negative binomial model to identify differentially expressed genes. The differentially expressed genes were identified based on multiple tests corrected P value and fold change. Genes were considered significantly differentially expressed if the P-value was <5% false discovery rate (FDR) and absolute fold change >1.5. Heatmaps of differentially expressed genes were generated using heatmap.2 or complex heatmap packages in R66. In heatmaps, clustering of genes was performed using Hclust function in R using Euclidean distance, and complete linkage parameters on gene-wise scaled data. The scaled data was generated using “normalize” function of SOM package in R.To further establish the transcriptome alterations associated with ZIKV infection in different cell types and model systems, we performed a meta-analysis. For the meta-analysis, RNA sequencing or microarray data were obtained from public repositories, namely the GEO database Table . The rawThe meta-signature of ZIKV was generated by comparing the results of our study with other two published studies Table . The com67. The normalized data were generated using original Affymetrix CDF files. After rigorous quality control analysis and log (base 2) transformation of normalized data, the differential analysis was performed using LIMMA approach63. The mapping of Affymetrix probes to gene symbols and entrez IDs was performed using the biomaRt package in R68. The genes that were significantly dysregulated by different viral infections were identified by comparing uninfected and infected samples using LIMMA63. The genes with absolute fold change >2 and P-value < 0.05 FDR were considered significantly differentially expressed in this analysis. The comparison of the ZIKV meta-signature with transcriptome signatures from different viruses was performed on the basis of gene symbols using Venn diagram approach. The genes that were selectivity dysregulated in ZIKV-infected samples but not in other virus-infected samples were considered as the ZIKV core transcriptome signature.To further identify molecular changes associated with only ZIKV infection, we also performed a comparative analysis of the ZIKV transcriptome with transcriptomes of other closely related flaviviruses such as DENV, WNV, and JEV. The raw transcriptome datasets for other viruses were obtained from the GEO database Table . After q69. DAVID is an online implementation of the EASE software that produces a list of over-represented categories using jackknife iterative re-sampling of the Fisher exact probabilities. P values are assigned to each category based on enrichment. Smaller P values reflect increasing confidence in over-representation. The GO categories with P values < 0.05 and comprising at least 2 genes were considered significant. The GO enrichment analysis was performed both on meta-signature and core signatures of ZIKV infection to understand the key biological processes that are affected by ZIKV infection.To identify the over-represented GO categories in host altered genes by ZIKV infection, we used the biological processes and molecular functions enrichment analysis available from the database for annotation, visualization and integrated discovery (DAVID)Ingenuity pathway analysis  was used to identify the pathways that are significantly affected by genes specifically associated with ZIKV infection. This software consists of functions, pathways and network models derived by systematically exploring the peer-reviewed the scientific literature. A detailed description of IPA analysis is available at the Ingenuity Systems’ website (http//www.ingenuity.com). It calculates a P value for each pathway according to the fit of users’ data to the IPA database using one-tailed Fisher exact test. The pathways with P values < 0.05 were considered significantly affected.70 was used to identify the cascade of upstream transcriptional regulators that can explain the observed gene expression changes due to ZIKV infection. The analysis helps in identifying first which transcription regulators are significantly affected by the infection and then determining whether they are activated or inhibited. The activation or inhibition of transcriptional regulators was determined on the basis of overlap among our data with activation or inhibition signatures of key regulators. The significance of overlap was determined using the one-tailed Fisher exact test.Regulatory module analysis71. The topological analysis was performed on the network to identify nodes critical for network stability. We performed topological analysis using the degree of the node of centrality parameter, which is a standardized measure to calculate a number of edges connected to each node72.To define master regulators that are perturbed by genes associated with ZIKV infection, we performed an interactive network analysis. Co-expression-based interactive networks were developed from normalized expression matrix data from ZIKV studies  and an extended group of viruses  causing neurological diseases. For co-expression analysis, network analysis returns pairwise gene interactions, Pearson R values (t-static), P values and Bonferroni-adjusted P values. We restricted interactions building with a P value < 0.01 to identify high confidence hub genes that regulated the network. The topological network parameters were calculated using cytoNCA package in CytoscapeTotal RNA was extracted from mock-treated and ZIKV/DENV-infected RPE cells using RNeasy Mini kit , per the manufacturer’s instructions. An aliquot of the same RNA was used for RNAseq analysis and another aliquot for gene validation studies by qRT-PCR. For qRT-PCR, cDNA was synthesized using 1 µg of total RNA using a Maxima first strand cDNA synthesis kit, as per the manufacturer’s instructions (Thermo Scientific). The cDNA was amplified using gene-specific PCR primers. qRT-PCR was conducted in a StepOnePlus™ Real-Time PCR system (Applied Biosystems). Integrated DNA Technologies synthesized all the primers. The quantification of gene expression was determined via the comparative ΔΔCT method. Gene expression data in the test samples were normalized to the endogenous reference β-actin level and were reported as fold change relative to β-actin gene expression. All assays were performed in triplicate and repeated at least twice. The data are presented as the mean ± SD, and statistical analysis was performed using Student’s t-test or one-way ANOVA followed by post hoc test using GraphPad Prism 7.02 .Supplementary Information


The Zika virus (ZIKV) is a recently emerged mosquito-borne flavivirus that, while typically asymptomatic, can cause neurological symptoms in adults and birth defects in babies born to infected mothers. The interactions of ZIKV with many different pathways in the human host ultimately determine successful virus replication and ZIKV-induced pathogenesis; however, the molecular mechanisms of such host-ZIKV interactions have just begun to be elucidated. Here, we summarize the recent advances that defined the mechanisms by which ZIKV antagonizes antiviral innate immune signaling pathways, with a particular focus on evasion of the type I interferon response in the human host. Furthermore, we describe emerging evidence that indicated the contribution of several cell-intrinsic mechanisms to an effective restriction of ZIKV infection, such as nonsense-mediated mRNA decay, stress granule formation, and “reticulophagy”, a type of selective autophagy. Finally, we summarize the recent work that identified strategies by which ZIKV modulated these intrinsic antiviral responses. Flaviviridae, which is comprised of enveloped viruses with a positive sense, single-stranded RNA genome [Aedes aegypti mosquito vector is the primary route of ZIKV circulation, its ability to transmit through a vertical and sexual route makes this virus unique amongst the other mosquito-transmitted flaviviruses that cause diseases in humans [Zika virus (ZIKV) is a member of the A genome . Other mA genome . While tn humans ,4,5.Since its discovery in 1947, ZIKV infection in humans has been historically linked to sporadic cases of self-limiting symptoms such as fever, rash, and conjunctivitis . After aZIKV has a broad cell tropism in vitro, infecting human skin cells, such as dermal fibroblasts and epidermal keratinocytes; human myeloid cells, such as dendritic cells (DCs) and macrophages; and human progenitor cells of neuronal, placental, and testicular origin . In ordeImportantly, there is no approved vaccine or specific antiviral treatment available for ZIKV infection to date. Current drug design efforts primarily aim to target ZIKV proteins with enzymatic activities that are essential for virus replication, such as NS3 and NS5, while progress towards a ZIKV vaccine has been challenging due to the potential risk of neurological side effects and antibody-dependent enhancement of infection, a phenomenon that is best-characterized for DENV infection ,24,25,26In this review, we summarize recently described roles of the mammalian antiviral type I interferon (IFN) system in restricting infection by ZIKV and related flaviviruses, as well as the molecular mechanisms by which these viruses evade the type I IFN response. Furthermore, we highlight recent work that showed that several host cell-intrinsic pathways play important roles in ZIKV restriction that, in turn, are modulated by ZIKV-encoded proteins. A detailed understanding of the mechanisms that ZIKV uses to evade or suppress host intrinsic or innate antiviral pathways might provide novel avenues for the development of antiviral drugs against ZIKV.transducer and activator of transcription factor 1 and 2 (STAT1 and STAT2) and results in the upregulation of many antiviral factors, generally known as IFN-stimulated genes (ISGs) [Early defense against viral pathogens is exerted by the innate immune system, which is comprised of pattern-recognition receptors (PRRs) that serve to detect conserved features of invading pathogens, universally called pathogen-associated molecular patterns (PAMPs) . While ms (ISGs)   mice [IFNAR as well as STAT2–/– mice were more susceptible to ZIKV infection and showed more severe ZIKV-associated pathology, compared to WT mice [IFNAR1–/– mice led to severe fetal growth restriction and fetal demise, compared to control mice [ZIKV replication has been shown to be inhibited by type I IFNs in several human cell types and in mouse models. For example, ZIKV replication was reduced in primary skin fibroblasts that were pre-treated with IFN-α or IFN-β, compared with cells that remained untreated . MoreoveWT) mice . Similar WT mice ,34. In arol mice ,36. CollIFIT1-3, RSAD2/Viperin, and OAS1 in primary human DCs [IFN-β and several ISGs , compared to mock infection [Restriction of ZIKV infection is mediated by a collection of ISGs, which are transcriptionally upregulated by IFNAR signaling in response to infection. ZIKV infection was shown to induce the expression of the ISGs uman DCs . Furthernfection . Anothernfection . TogetheIFN-α/β gene expression and subsequent upregulation of ISGs are triggered by an ensemble of PRRs following sensing of the invading pathogen. At least three major classes of PRRs contribute to the effective detection of flaviviruses—(1) toll-like receptors (in particular TLR 3 and 7), which sense viral RNA within the endosome; (2) retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) such as RIG-I itself and melanoma differentiation-associated protein 5 (MDA5), which recognize RNA species in the cytoplasm; and (3) cyclic GMP–AMP synthase (cGAS), which detects cellular dsDNA that is mislocalized during flavivirus infection [nfection ,40,41,42nfection . Upon acnfection . cGAS, wnfection . Signalinfection .Endosomal TLRs, such as TLR3 and TLR7, are critical for innate detection of flavivirus infections, evidenced by work from many groups (reviewed in detail in ). For exSimilar to other flaviviruses, ZIKV RNA is believed to be sensed by endosomal TLR3 and TLR7, which are abundantly expressed on several cells relevant for ZIKV infection, including monocytes and DCs. Stimulation of monocyte-derived macrophages with a TLR7/8 agonist restricted ZIKV replication by inducing expression of several ISGs . AdditioRIG-I–/– or MDA5–/– mouse embryonic fibroblasts (MEFs), although at differing time points after infection [MAVS–/– mice revealed enhanced WNV replication and viral-induced lethality due to increased inflammation in the brain [MAVS–/– mice were shown to be highly susceptible to DENV (serotype 2) infection and showed delayed IFN-α and IFN-β production [Sensing of flaviviruses within the cell by the cytoplasmic sensors MDA5 and RIG-I has been well-characterized in the context of WNV and DENV infection. For example, WNV infection was enhanced in both nfection . Similarnfection . Infectihe brain . Similaroduction .RIG-I and MDA5, although silencing of these sensors did not significantly affect ZIKV replication in these cells [RIG-I and MDA5 transcript and protein expression following ZIKV infection, and treatment with a RIG-I agonist derived from HCV RNA restricted ZIKV replication [RIG-I–/–, MDA5–/–, and MAVS–/– human trophoblast cells—a cell type of the placental barrier—with the most enhanced replication observed in MAVS–/– cells [IFN-β gene expression was reduced in RIG-I–/– and MDA5–/– trophoblast cells, and completely abolished in MAVS–/– cells [IFN-β and several ISGs following ZIKV infection and, accordingly, enhanced the viral titers. Furthermore, silencing of both RIG-I and MDA5 strongly enhanced ZIKV replication in this cell type [MDA5-deficient cells, reporter activation was strongly reduced in cells that lacked RIG-I or MAVS [The critical role of RLRs in ZIKV sensing and restriction has also been demonstrated by several studies. ZIKV-infected primary human skin fibroblasts transcriptionally upregulated se cells . Similarlication . Further/– cells . Accordi/– cells , suggestell type . Experimell type . Moreove or MAVS . ZIKV RN or MAVS . These sIFN-β induction in –/–cGAS THP1 (human monocytic) cells and peripheral blood mononuclear cells [–/–STING human fibroblasts and that ZIKV actively antagonizes STING within the cGAS pathway [In addition to RNA sensors, the cGAS-STING signaling axis has been shown to detect and restrict flaviviruses ,42,59. Mar cells . Further pathway .In summary, recent studies have demonstrated that TLRs, RLRs, and the cGAS-STING pathway play an important role in innate immune sensing and control of the ZIKV infection. These sensors likely act together within the infected cell to detect a combination of ZIKV-derived PAMPs and host-derived molecules, or act in a cell type-specific manner. Thus, determining the relative contribution of these innate immune sensors to IFN/ISG induction and ZIKV restriction in different cell types and tissues will be important. Studies in knockout mice lacking specific sensors, especially TLRs, would provide important insight into the contribution of these sensors to ZIKV restriction and pathogenesis. Along these lines, the precise PAMPs recognized by PRRs during ZIKV infection are largely unknown. For example, it has not been fully established whether cGAS senses mtDNA during ZIKV infection, or perhaps other host DNA species. Moreover, the precise mechanism(s) whereby ZIKV induces cGAS-PAMPs during infection have not yet been elucidated, which will be an exciting avenue for future research.Flaviviruses have evolved many mechanisms for antagonizing the host’s type I IFN response during infection . These mA critical step in the activation of RIG-I is its Lys63-linked ubiquitination, mediated by the ubiquitin E3 ligase TRIM25 (and other E3 enzymes), which ultimately leads to RIG-I oligomerization and its binding to the adaptor protein MAVS . The sfRGiven that the cGAS-STING axis also effectively restricts flavivirus infection ,42,59, iGiven the recent emergence of ZIKV as a human pathogen, innate immune evasion strategies mediated by this virus are not as well-understood as those utilized by other mosquito-borne flaviviruses. Whereas antagonism of the TLR sensing pathways by ZIKV remains largely elusive, recent studies uncovered some of the mechanisms by which ZIKV inhibits the RLR-MAVS and cGAS-STING pathways, or their shared downstream signaling proteins TBK1 and IRF3.Several studies have found that overexpression of specific ZIKV NS proteins inhibits IFN-β promoter activation stimulated by ectopic expression of RIG-I 2CARD, which is constitutively-active, and/or stimulated by RIG-I agonists in HEK293T cells ,77,78,79Recent work showed that ZIKV is able to counteract RIG-I- and MDA5-mediated innate immunity by disrupting the interactions of both RLRs with their respective scaffold proteins, 14-3-3ε and 14-3-3η . PreviouWith the knowledge of species-specific DENV-mediated antagonism of the cGAS-STING pathway , Ding etIFN-β expression [In addition to viral antagonism of PRRs or their adaptor proteins, ZIKV NS proteins were shown to inhibit innate immune signaling at the level of TBK1 and IRF3, as well ,79,81. Epression . NS1 andpression . LikewisDownstream of TBK1 phosphorylation and oligomerization, ZIKV NS5 was found to interact with both TBK1 and its activating binding partner TRAF6 in an overexpression context, resulting in a reduction of the TBK1–TRAF6 interaction. However, the functional consequence of this competitive binding on IFN induction was not investigated . In a seIn regards to IRF3 antagonism by ZIKV, it was found that the ectopic expression of full-length ZIKV NS5 reduced TBK1 and MAVS-mediated phosphorylation of ectopically-expressed IRF3 at Ser-396 in HEK293 cells . LikewisIn summary, several NS proteins of ZIKV have been reported to antagonize IFN production. However, many of these antagonism strategies are yet to be confirmed during native ZIKV infection, and their relevance for effective virus replication and immune evasion has to be tested in vivo. Studies investigating these IFN-antagonistic mechanisms in suitable mouse infection models might reveal cell-type- or tissue-specific roles for individual NS proteins, and provide important insight into ZIKV-mediated pathogenesis. Moreover, it will be important to evaluate the relative contribution of the proposed evasion mechanisms to IFN antagonism by ZIKV, potentially through generating mutant recombinant viruses in which specific ZIKV-host interactions are ablated.While many studies have focused on NS protein-mediated IFN antagonism, our understanding of the role of the recently identified and characterized ZIKV sfRNAs  in modulFurthermore, although type I IFNs are the primary IFNs secreted by a wide variety of cells, several cell types, including trophoblasts of the placental barrier, have been shown to secrete type III IFNs (IFN-λ) to control the ZIKV infection . Therefotransducer and activator of transcription factor 1 and 2 (STAT1 and 2), inducing their dimerization and subsequent assembly with IRF9 into the multimeric protein complex IFN-stimulated gene factor 3 (ISGF-3) [After production and secretion from the infected cell, IFN-α/β binds IFNAR1/2 to activate receptor-associated kinases Janus kinase (JAK) and tyrosine kinase 2 (TYK2) . These k(ISGF-3) . ISGF-3 (ISGF-3) . ISGs en(ISGF-3) . Other I(ISGF-3) . In turnBesides antagonizing signaling that leads to IFN gene expression, flaviviruses have evolved many mechanisms to suppress signaling downstream of IFNAR1/2 activation. At the level of entry, WNV and likely also other flaviviruses induce the expression of suppressors of cytokine signaling (SOCS) 1 and 3 after binding to and activating TAM (Tyro3/Axl/Mer) receptors on dendritic cells; this ultimately results in SOCS1/3-mediated inhibition of JAK1 and further downstream signaling ,86. WNV ISG15, IFIT1, and IFIT2 in response to IFN-β treatment, as compared to mock infection [ISG15 and OAS1 gene expression triggered by exogenous addition of IFN [Viperin/RSAD2 and IFIT1 during ZIKV infection, following poly(I:C) stimulation. Interestingly, cells of neural and placental origin also showed a reduced ISG response during ZIKV infection, compared to poly(I:C) stimulation, with the expression of Viperin/RSAD2 being particularly diminished. Since overexpression of Viperin reduced ZIKV titers and, inversely, Viperin/RSAD2–/– MEFs showed enhanced ZIKV permissiveness, the dampening effect of ZIKV on Viperin/RSAD2 gene expression is likely a mechanism of immune evasion [ZIKV infection has been shown to modulate innate immune signaling downstream of IFN production. For example, ZIKV infection of A549 cells has been associated with down-regulated expression of ISGs including nfection . Likewisn of IFN . Further evasion .More specifically, ZIKV has been shown to antagonize IFNAR-induced signaling through a variety of mechanisms that include (1) JAK1 degradation, (2) inhibition of STAT1 and STAT2 phosphorylation, and (3) STAT2 degradation. The protein abundance of JAK1 was found to be reduced during ZIKV infection of the A549 cells, both in the absence and presence of IFN stimulation . FurtherDownstream of JAK1, STAT1 and STAT2 must be phosphorylated in order to activate the expression of ISGs. In A549 cells and human DCs, ZIKV infection diminished the pools of endogenous phosphorylated STAT1 and STAT2 (p-STAT1/2), at residues Tyr-701 and Tyr-689, respectively, in the presence and absence of exogenous IFN treatment . LikewisUBR4–/– cells, suggesting a unique mechanism of STAT2 inhibition by ZIKV NS5 [PDLIM2–/– Huh7.5 cells were more resistant to HCV, DENV, and ZIKV infection than parental cells [PDLIM2 resulted in less efficient IFN-α-mediated STAT2 degradation; however, STAT2 degradation during flavivirus infection was not investigated in PDLIM2–/– cells [Similar to DENV, ZIKV has been shown to degrade human—but not mouse—STAT2 via its NS5 protein. Grant et al. found that ZIKV infection resulted in an MOI-dependent reduction in endogenous STAT2 protein abundance in Vero cells . EctopicZIKV NS5 . Of noteZIKV NS5 ,77  cells. When CHME3 cells were treated with an Axl antagonist, ZIKV infection resulted in enhanced nfection . Of notetagonist . On the  and MxA . These r and MxA . HoweverSeveral recent studies have reported on additional host pathways implicated in ZIKV restriction and, in turn, mechanisms whereby ZIKV is able to modulate these pathways. Below, we summarize the current data describing an important role for the cellular stress response, nonsense-mediated mRNA decay (NMD), and selective autophagy during ZIKV infection.The cellular stress response induces the accumulation of dynamic cytoplasmic aggregates called stress granules (SGs), which are comprised of stalled mRNA-bound translation pre-initiation complexes and several protein factors . During Recent studies have provided evidence for ZIKV-mediated modulation of the NMD machinery. NMD is a cytoplasmic RNA surveillance pathway that functions to recognize and degrade aberrant RNAs (predominantly mRNAs) by recruiting Up-frameshift Protein 1 (UPF1) to nucleate assembly of the NMD machinery on target mRNAs . SeveralWith regards to flaviviruses, HCV infection has been shown to cause accumulation of canonical NMD RNA targets, mediated by binding to and sequestering of the NMD component WIBG by the HCV core protein . SimilarAutophagy is a tightly-regulated cellular process wherein cytoplasmic materials are targeted for lysosomal degradation in a non-selective or selective manner . MechaniThe role of autophagy during flavivirus infection is also multi-faceted, playing both proviral and antiviral roles during the viral lifecycle. For example, knockdown of autophagy machinery resulted in reduced HCV replication, correlated with enhanced IFN and ISG signaling in HCV-infected hepatocytes . DENV (sAtg16L1, suggesting that ZIKV might utilize autophagy to cross the placental barrier [Like other flaviviruses, ZIKV infection has been shown to induce autophagy in a variety of permissive cell types, including human skin fibroblasts , fetal n barrier . CollectWhile non-selective autophagy has been shown to be required for certain stages of the ZIKV lifecycle ,119,120,In summary, these studies provided evidence that stress granule formation, NMD, and reticulophagy have antiviral activity against ZIKV, and as such, ZIKV antagonizes these host processes to promote its replication. While the mechanistic details of viral targeting by these intrinsic pathways as well as of viral evasion, thereof, are currently not well-understood, the recent findings should stimulate more detailed investigation into these novel ZIKV-host interactions.Since the emergence of ZIKV as an epidemic threat in 2015, impressive progress has been made in identifying and understanding ZIKV-mediated evasion of host antiviral pathways. While many viral mechanisms of innate immune antagonism have been elucidated, most studies have focused on those mediated by direct actions of ZIKV NS proteins. However, there is much to be discovered regarding potential host factors involved in these inhibitory mechanisms, as well as other means for evasion, such as ZIKV sfRNA-mediated antagonism of innate and intrinsic immunity. Interestingly, the majority of innate immune molecules antagonized by ZIKV have been previously shown to be targeted by related viruses, such as DENV and WNV. Along these lines, although the molecular mechanisms of ZIKV-mediated inhibition of the type I IFN response still need to be further elucidated, current data suggests that many of the evasion strategies are conserved among mosquito-transmitted flaviviruses. Since ZIKV infection in humans shows unique pathological features, it is expected that ZIKV has evolved unique innate evasion mechanisms, which will be an exciting avenue for future research. In particular, investigation of the combined role of type I IFN evasion and additional antiviral pathways that ZIKV is known to subvert, such as type III IFN production, might more clearly define the physiology of ZIKV infection at a molecular level. Furthermore, recent data have indicated that the activation of stress granule formation, NMD, and reticulophagy play an antiviral role during ZIKV infection. Additionally, elucidating the precise mechanisms by which these cellular pathways suppress ZIKV replication and contribute to the ZIKV-induced pathogenesis will be an exciting question in this emerging field. Importantly, whether ZIKV directly antagonizes these pathways as a means to subvert their antiviral activities, or whether ZIKV co-opts these cellular pathways to promote its replication is still not clearly understood. Moreover, it will be interesting to define whether these intrinsic pathways show cell-type- or viral strain-specific effects. Understanding the mechanisms of innate and intrinsic antiviral pathways in restricting ZIKV might identify potential molecular targets for therapeutic intervention of ZIKV infection. Elucidating novel host defense responses to ZIKV will stimulate research into the mechanisms of how ZIKV, in turn, evades or hijacks these pathways.


The replication efficiencies of ZIKV-MY and ZIKV-Natal in A549 and Vero cells were similar, while ZIKV-MY replicated more efficiently in wild-type (WT) and IFNAR−/− mouse embryonic fibroblasts. Viremias in IFNAR1−/− dams were similar after infection with ZIKV-MY or ZIKV-Natal, and importantly, infection of fetal brains was also not significantly different. Thus, fetal brain infection does not appear to be a unique feature of Brazilian ZIKV isolates.The recent emergence of Zika virus (ZIKV) in Brazil was associated with an increased number of fetal brain infections that resulted in a spectrum of congenital neurological complications known as congenital Zika syndrome (CZS). Herein, we generated  Flavivirus of the Flaviviridae family [Zika virus (ZIKV) belongs to the genus e family , which ie family . ZIKV ise family .Phylogenetically, ZIKV strains are categorized into either African or Asian lineages , with moA central question associated with the ZIKV epidemic in Brazil (and in French Polynesia) is whether the virus strains involved had acquired mutations that enhanced their ability to cause CZS ,18. Seve−/− mouse model, we previously demonstrated that the Brazilian isolate  was capable of causing fetal infection and congenital malformations [−/−) dams and were both capable of causing fetal brain infections.We previously generated the contemporary Asian lineage isolate ZIKV-Natal from sequence data obtained directly from the brain tissue of an aborted ZIKV-infected human fetus during the 2015 Brazilian outbreak ,23. Usinrmations . Herein,rmations . We showThe ZIKV-MY (strain P6-740) isolate whose sequence was used in this study was reported to be passaged six times in suckling mouse brains, once in baby hamster kidney (BHK) cells, once in C6/36 cells, twice in Vero cells, and five times in Vero E6 cells (GenBank accession number KX694533). Six dsDNA fragments covering the entire viral sequence a were pu−/− MEFs (44) were infected with passage 1 of C6/36-derived stock of ZIKV-MY, ZIKV-Natal, or ZIKV-Natal/MY-prME viruses at the indicated multiplicity of infection (MOI), and 200 µL of culture supernatant was collected from each sample well at the indicated times post-infection. Three independent experiments were conducted for each cell line. Viral titers were determined by standard plaque assay on Vero cells, as previously described [Vero (CCL-81), A549 (ATCC CCL-185), wild-type mouse embryonic fibroblasts (MEFs), and IFNARescribed .−/− mice on a C57BL/6J background were provided by Dr P Hertzog  [−/− mice (between 10–20 weeks old) were infected s.c. at the base of the tail with 100 µL of medium at a dose of 104 CCID50 as described [−/− mice, infection with either ZIKA-MY or ZIKV-Natal did not result in any overt morbidity. Mice were euthanized with CO2.IFNAR1stralia)  and bredescribed . Mating All mouse work was conducted in accordance with the “Australian code for the care and use of animals for scientific purposes”, as defined by the National Health and Medical Research Council of Australia. Animal experiments and associated statistical treatments were reviewed and approved by the QIMR Berghofer Medical Research Institute animal ethics committee .50) assays for viremia and tissue titers were undertaken as described [ZIKV cell culture infectious dose, 50% endpoint  were purchased from Integrated DNA Technologies a. The fi3 pfu/mL) (−/− MEF), ZIKV-MY replicated more efficiently than ZIKV-Natal  e. Even iKV-Natal f. To ascKV-Natal . In bothKV-Natal . The bet−/− mice were infected with ZIKV-MY or ZIKV-Natal at E12.5 of gestation, and viremias were determined daily for five days post-infection. No statistically significant differences in mean viremias were observed after infection with the two viruses in either pregnant or non-pregnant mice (IFNAR1ant mice a. Viremiant mice a. The prant mice b–h.p = 0.077 Kolmogorov–Smirnov test). Placental titers were about 0.8 logs higher for ZIKV-MY than for ZIKV-Natal infection . Following ZIKV infection, placenta and fetuses often form highly deformed indistinguishable masses, as described previously [p = 0.045, Kolmogorov–Smirnov test) in ZIKV-MY- than in ZIKV-Natal-infected dams (Groups of pregnant mice were infected with ZIKV-MY or ZIKV-Natal at E12.5 of gestation and euthanized at E17.5, with fetuses and placentas collected. The viral tissue titres in fetal heads obtained from dams infected with ZIKV-MY or ZIKV-Natal were not significantly different (eviously . Viral tted dams c. These −/− mice, with similar efficiency to the 2015 Natal isolate from Brazil. Both ZIKV-MY and ZIKV-Natal also generated indistinguishable viremia and viral loads in a panel of organs tested. Thus, the propensity for congenital infection does not appear to be a unique feature of contemporary Brazilian ZIKV strains.Herein, a 1966 Malaysian isolate of ZIKV was generated de novo directly from a published sequence and was shown to cause fetal brain infection in pregnant IFNAR1in vivo characterization of ZIKV virulence. Because of the inability of ZIKV to degrade murine STAT2 [−/− C57BL/6 as a congenital infection model [The use of mice as a ZIKV animal model remains the main practical approach for ne STAT2 , IFN reson model . In otheon model ,32,33,34on model  did compA potential limitation of our study is that the Malaysian isolate generated herein was derived from the sequence of an isolate that had undergone six passages in suckling mouse brain . PotentiIn conclusion, our results argue that ZIKV has an intrinsic ability to cause congenital infection, irrespective of the sequence evolution that has been documented in recent years.


Aedes spp mosquitoes that has caused outbreaks of fever and rash on islands in the Pacific and in the Americas. These outbreaks have been associated with neurologic complications that include congenital abnormalities and Guillain-Barré syndrome (GBS). The pathogenesis of ZIKV-associated GBS, a potentially life-threatening peripheral nerve disease, remains unclear. Because Schwann cells (SCs) play a central role in peripheral nerve function and can be the target for damage in GBS, we characterized the interactions of ZIKV isolates from Africa, Asia and Brazil with human SCs in comparison with the related mosquito-transmitted flaviviruses yellow fever virus 17D (YFV) and dengue virus type 2 (DENV2). SCs supported sustained replication of ZIKV and YFV, but not DENV. ZIKV infection induced increased SC expression of IL-6, interferon (IFN)β1, IFN-λ, IFIT-1, TNFα and IL-23A mRNAs as well as IFN-λ receptors and negative regulators of IFN signaling. SCs expressed baseline mRNAs for multiple potential flavivirus receptors and levels did not change after ZIKV infection. SCs did not express detectable levels of cell surface Fcγ receptors. This study demonstrates the susceptibility and biological responses of SCs to ZIKV infection of potential importance for the pathogenesis of ZIKV-associated GBS.Zika virus (ZIKV) is a re-emerged flavivirus transmitted by  Flavivirus and is related to other mosquito-borne flaviviruses, including dengue, yellow fever, West Nile, Japanese encephalitis and Spondweni viruses1. Aedes spp. mosquitoes are the main vectors for ZIKV, yellow fever virus (YFV) and all four types of dengue virus (DENV) but ZIKV can also be transmitted through infected semen and from mother to fetus4. ZIKV was first isolated from primates in Uganda in 1947, from Aedes africanus mosquitoes in 19485 and from a febrile man working in Uganda in 19636. For the next five decades sporadic cases of benign, self-limiting acute illness characterized by fever, headache, myalgia and rash were identified in Asia and Africa9.Zika virus (ZIKV) is a plus-strand enveloped RNA virus that belongs to the genus 10. Subsequent outbreaks beginning in October 2013 and continuing throughout 2014 were recognized across Oceania in four groups of Pacific islands: French Polynesia, Easter Island, the Cook Islands, and New Caledonia with 8,750 reported cases11. In May 2015, locally acquired ZIKV disease was recognized in Brazil12 followed by rapid spread through the Americas and Caribbean islands with accompanying reports of neurological complications and congenital malformations including microcephaly15.The first recognized large outbreak of human ZIKV infection occurred in 2007 on the Pacific island of Yap, in the Federated States of Micronesia16. During the ZIKV outbreak in South America, 66 of the 68 patients with GBS in Colombia had symptoms compatible with ZIKV infection before the onset of GBS17 and in Martinique, a prospective study determined that the incidence rate of GBS during the 2016 ZIKV outbreak was 4.52 times that of prior years18. In 2016, the World Health Organization officially recognized ZIKV as a cause of GBS and microcephaly19.The first association of ZIKV infection with Guillain-Barré syndrome (GBS) was a retrospective study of the French Polynesian outbreak where there was a 20-fold increase in GBS incidence and all of the 42 patients hospitalized with GBS had anti-ZIKV antibody20. The most common subtypes of GBS are acute motor axonal neuropathy (AMAN) and acute inflammatory demyelinating polyneuropathy (AIDP), differentiated by the site of immune-mediated injury21. In AMAN, membranes of the nerve axon are the primary targets of cross-reactive anti-ganglioside antibodies while in AIDP the Schwann cell (SC)-produced myelin sheath is the target for damage but the relative roles and specificities of antibody and cellular immune responses are unclear20.GBS, a potentially life-threatening peripheral nerve disease, is characterized by a rapid onset of bilateral weakness progressing to paralysis that may be accompanied by sensory symptoms. GBS typically occurs 1–3 weeks after an infectious disease postulated to trigger a pathogen-specific immune response that cross-reacts with peripheral nervous system (PNS) antigensin silico comparison of the ZIKV protein sequence with human proteins has identified shared peptides22, there is no evidence of disease-relevant cross reactivity so the pathogenesis of ZIKV-associated GBS remains unclear. The electrophysiological findings from the French Polynesian GBS cases were reported to be compatible with the AMAN subtype of GBS, but the typical AMAN-associated anti-ganglioside antibodies were rarely detected16. In Colombia, nerve-conduction studies and electromyography were consistent with the AIDP subtype of GBS17 and the electrophysiologic findings from Martinique were also consistent with AIDP further suggesting that SCs were the target for ZIKV-associated damage18.Several studies have sought to determine the type of GBS associated with ZIKV infection. Although 23 and can initiate and regulate local immune responses25. For instance, SCs can be induced to express MHC class I and II molecules, inflammatory cytokines , chemokines  and nitric oxide synthase (iNOS) in response to damage or toll-like receptor (TLR) stimulation29. Induction of cytokines, chemokines and cell surface immunoregulatory proteins by infection of SCs could promote development of potentially damaging virus- or host cell-specific immune responses.SCs develop from neural crest cells, are the myelinating glial cells of the PNS and play a central role in peripheral nerve function, maintenance and repair. The SC myelin sheath enables saltatory conduction of action potentials by large diameter axons but SCs also ensheath and maintain axons that are not myelinated. In response to nerve injury SCs can trans-differentiate into a proliferating cell capable of secreting inflammatory mediators that enhance macrophage-mediated myelin removal32. To evaluate the potential for direct ZIKV-induced peripheral nerve damage, we assessed the susceptibility of immortalized primary human Schwann cells (hSCs)33 to infection with strains of ZIKV from Africa, Asia and Brazil. Infection with ZIKV was compared to infection with flaviviruses YFV 17D and DENV2 that are less commonly associated with GBS35. All strains of ZIKV and YFV, but not DENV2, replicated well in hSCs and induced innate responses and cytopathic effect.Because GBS is associated with ZIKV infection and often occurs when symptoms of ZIKV infection are still present and ZIKV RNA is detectable, it has been postulated that GBS might be directly associated with ZIKV infection of peripheral nerve cells or the immune response to viral antigens expressed by these cellse.g. S100B), transcription factors , cell surface receptors (e.g. p75NTR), chemokines (e.g. CCL2) and neurotrophic factors  and can myelinate axons in vitro36, but have not been evaluated for susceptibility or response to virus infection. To determine susceptibility of hSCs to flavivirus infection, cells were incubated (MOI = 5) with YFV 17D, DENV2 and three strains of ZIKV: 1968 Nigeria, 2014 Thailand and 2015 Brazil  chosen to represent the historical shifts of ZIKV as it moved from Africa to Asia and finally the Americas37. Supernatant fluids, collected from 0 to 120 h after infection, were analyzed by plaque assay -1 were upregulated after infection with both YFV and ZIKV with the greatest response induced by YFV. However, IFN β1 protein production was detectable only in ZIKV Fortaleza-infected cells (119 ± 18.9 pg/ml at 72 h)  protein at 24 and 36 h were similar, but at 48 and 72 h ZIKV Fortaleza-infected cells produced more IFNλ1  is an important regulator of cell susceptibility to infection. To determine the responses of hSCs to ZIKV Fortaleza and YFV 17D infection, which had similar levels of replication and similar response Fig. . Levels  h) Fig. . ZIKV inNλ1 Fig. . At 48 h40, were induced only by ZIKV. Expression of TNFα mRNA was induced in both ZIKV and YFV-infected hSCs. The NLRP3 inflammasome protein mRNA was induced by YFV, but only transiently by ZIKV. Therefore, ZIKV Fortaleza and YFV infections induced overlapping, but distinct innate immune responses in hSCs.To determine whether ZIKV or YFV upregulates hSC expression of other innate response gene mRNAs, levels of mRNAs for representative innate cytokines interleukin (IL)-6, IL-23A and tumor necrosis factor (TNF)-α as well as inflammasome protein NLRP3 were measured , CLEC5a and mannose receptor (MR) and the phosphatidyl serine receptors T-cell immunoglobulin and mucin domain (TIM)-1 and TAM receptors Tyro3 and AXL45. To begin to identify factors that may facilitate flavivirus infection of hSCs, we analyzed expression of mRNAs for protein receptor families reported to mediate entry ; MR, a receptor for DENV46 that colocalizes with MHC class II during phagocytosis by SCs47; C-type lectin CD20949; and TIM1 and TAM receptors50. We also assessed the modulation of receptor expression during culture and infection. HSPG2 and ICAM-1 mRNA expression increased during culture of hSCs, but this was not affected by ZIKV infection. MR mRNA expression showed a transient increase at 24 h in both infected and uninfected cells. There was little change and no effect of infection on expression of CD209, TIM1, AXL or Tyro3. The intercellular cell adhesion molecule (ICAM-1) a cell surface receptor that can be up regulated by IFNγ, IL1β and TNFα in SCs51 showed increased mRNA expression during culture but was not modulated by ZIKV infection.Like other flaviviruses, ZIKV probably can use multiple receptors for host cell attachment, clathrin-mediated endocytosis, pH-dependent membrane fusion and entry52, but levels of mRNA were not modulated by ZIKV infection. Several types of junctional specializations are formed in myelinating SCs between membrane lamellae, act as autotypic junctions and are disrupted during infection of retinal epithelial cells54. In addition, claudin 1 interacts with the prM protein of DENV to facilitate entry56. Therefore, we assessed expression of mRNAs for claudins (CLDN) 6 and 9, occludin (OCLN) and zona occludens (ZO)-1. CLDN9 was up regulated in ZIKV-infected hSCs, but other junctional protein mRNAs were not affected by infection.Because SC responses to injury can affect peripheral nerve function we also assessed the effect of ZIKV infection on expression of several response-relevant protein mRNAs Fig. . Galecti61. SCs in peripheral nerves have been reported to express FcγRII and FcγRIII63. To determine whether immortalized hSCs express FcγR mRNAs and whether expression is modulated by infection, we assessed the levels of FcγRIc, FcγRIIb or FcγRIIIa mRNAs before and after ZIKV infection  IFIT1 and MX1, as previously described for ZIKV infection of A549 respiratory epithelial cells and glomerular podocytes68. The IFNβ and ISG response to YFV was greater than to ZIKV, perhaps reflecting the ability of ZIKV to reduce type I IFN induction and NS5 to degrade STAT2 required for IFN signaling82.Flaviviruses can activate pathogen recognition receptors RIG-I, MDA-5, TLR3 and TLR7 to induce expression of IFN and cytokine genes83. Both ZIKV and YFV-infected cells showed significant increases in IL-29 (IFNλ1) mRNA at 48 h although larger amounts of both IFNβ and IFNλ proteins were produced by ZIKV-infected cells than YFV-infected cells perhaps because of better viability  infection86. HEV can replicate in neural cells and virus is often detectable at the onset of GBS, but tropism for SCs has not been examined87. Our study of the relationship between ZIKV and GBS, specifically examining hSCs, suggests that GBS could be mediated by pathogen-specific antibodies/T cells or a direct viral effect on SCs rather than cross-reactive antibodies to host proteins/lipids. However, pathologic evaluation of the limited tissue available has not detected ZIKV in peripheral nerves of patients dying with ZIKV-associated GBS89. The understanding that ZIKV can replicate within hSCs and induces a cellular response that this study demonstrates facilitates a better understanding of the role of ZIKV and GBS in the most recent epidemic.The mechanism(s) of ZIKV-induced GBS is not understood. Speculated mechanisms include: Antibody dependent enhancement with DENV sera, an immune mediated mechanism inducing damage through molecular mimicry, cellular mediated inflammation and demyelination induced by complement and macrophage activation, and a direct viral pathogenic effect36. hSCs were grown in Dulbecco’s modified Eagle’s medium  supplemented with 0.2% glucose, 2mM L-glutamine, 10% heat-inactivated fetal bovine serum (FBS), 2 µM forskolin, penicillin (100 U/ml) and streptomycin (100 µg/ml) at 37 °C in 5% CO2. Vero cells (American Type Culture Collection) were grown in DMEM with 10% FBS, 2mM L-glutamine, penicillin and streptomycin at 37 °C in 5% CO2. C6/36 Aedes albopictus cells (American Type Culture Collection) were grown in DMEM supplemented with 10% FBS and minimal essential amino acids, at 28 °C in 5% CO2. Cell viability was determined by trypan blue exclusion.Human SCs, a cell line derived from 60–80 day fetal sciatic nerves and immortalized with SV40 large T antigen and human telomerase reverse transcriptase have been previously described and were obtained from Ahmet Hoke 90 diluted 1:2000 in 5% milk followed by horse radish peroxidase-conjugated goat anti-mouse IgG (KPL) diluted 1:3000 in 5% milk for 1 h. Foci were developed with KPL TrueBlue Peroxidase Substrate (Sera Care). For plaque assays, cells were overlaid with 0.6% Bacto Agar in Modified Eagle Medium , incubated for 5 days, fixed with 10% formaldehyde in PBS and stained with 0.2% crystal violet in 20% ethanol.YFV (17D), DENV2 (NGC) and ZIKV strains 2014 Thailand (SCV0127/14) and 2015 Brazil  were obtained from Anna Durbin . ZIKV strain 1968 Nigeria (IBH 30656) was obtained from Andrew Pekosz . All virus stocks were grown in C6/36 mosquito cells. Stocks and supernatant fluids from infected cells (MOI = 5) were assayed on Vero cells by focus-formation for DENV and YFV and by plaque-formation for ZIKV. Samples serially diluted in DMEM plus 1% FBS were incubated with Vero cell monolayers for an hour. For focus-forming assays, cells were overlaid with 1% methylcellulose in Optimem (Gibco) with 2% FBS, 2 mM glutamine, 50μg/ml gentamicin (Sigma Aldrich) and incubated for 3 days. Cells were fixed with 80% methanol for 10 min, blocked with 5% nonfat milk for 10 min and incubated for 1 h with pan-flavivirus mouse 4G2 monoclonal antibody hSCs were infected with three strains of ZIKV, YFV and DENV (MOI = 0.1). At different times after infection the supernatant fluid was removed and 50μl of MTT reagent  in DMEM was added. After 2 h 50μl lysis buffer  was added and incubated for 4 h. Readings at 570 nm were used to calculate the percentage of viable infected cells compared to mock-infected cells.hSCs grown on poly-L-lysine-coated cover slips were infected with the three strains of ZIKV, DENV2 and YFV at a MOI of 5. At 24–96 h after infection, cells were fixed with 4% formaldehyde, permeabilized with 0.2% triton X 100 in PBS, and blocked with 5% normal goat serum in PBS. The cells were incubated with pan-flavivirus 4G2 primary antibody (1:1000 in PBS) for 1 h followed by anti-mouse IgG Alexafluor594 (Invitrogen). Nuclei were stained with DAPI. Images were captured using a Zeiss Axio Imager M2 microscope and analyzed using Volocity software. Numbers of infected and uninfected cells in three fields were counted to determine percentages of cells infected for each virus.−∆∆ct.hSCs infected with ZIKV Fortaleza and YFV (MOI = 5) were collected in RLT buffer at 0, 12, 24, 48, and 72 h after infection in triplicate. RNA was isolated from cell lysates using the RNeasy Plus Mini Kit (Qiagen) and quantified using a Nanodrop-1000 spectrophotometer. cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) on an Applied Biosystems 2720 thermal cycler with an RNA concentration of 500 ng/µl in a 20 µl total reaction volume. qPCR was performed with the Promega GoTaq qPCR Master Mix for Dye-Based Detection using gene-specific oligonucleotide primers for SYBR Green-based measurements or Applied Biosystems TaqMan probes  and IL-29 (IFNλ1) was measured using the Human IL29 Uncoated ELISA kit (Invitrogen) according to the manufacturer’s instructions. Supernatant fluids from 3 independent infections were tested and data are presented as pg per ml or OD at 450 nm. Assay range was 50 to 4000 pg/ml for IFNβ and 15.6 to 1000 pg/ml for IL-29. The limit of detection is marked as the OD value obtained for lowest concentration of assay range.hSCs were collected in ice cold PBS and live/dead staining (Invitrogen) was done on ice for 30 min in the dark. Cells were stained with FITC-conjugated mouse antihuman CD64 , CD32 , CD16  and mouse IgG1  for 1 h on ice and analyzed on a FACSCanto flow cytometer. Histograms were plotted to determine mean fluorescence intensity.Data were compared using two-way ANOVA and are presented as mean +/− standard deviation. P < 0.05 was considered significant. All statistical analyses were performed with GraphPad Prism 5.