The sodium-glucose cotransporter (SGLT) inhibitors represent a new alternative for treating patients with diabetes mellitus. They act primarily by inhibiting glucose reabsorption in the renal tubule and therefore, decreasing blood glucose levels. While little is yet known about SGLT subtype 1, SGLT2 inhibitors have demonstrated to significantly reduce cardiovascular mortality and heart failure hospitalizations. This cardioprotective benefit seems to be independent of their glucose-lowering properties; however, the underlying mechanism(s) remains still unclear and numerous hypotheses have been postulated to date. Moreover, preclinical research has suggested an important role of SGLT1 receptors on myocardial ischemia. Following acute phase of cardiac injury there is an increased activity of SGLT1 cotransport that ensures adequate energy supply to the cardiac cells. Nonetheless, a long-term upregulation of this receptor may not be that beneficial and whether its inhibition is positive or not should be further addressed. This review aims to present the most cutting-edge insights into SGLT receptors. Optimal myocardial performance relies upon incessant generation and supply of ATP molecules to the contractile apparatus. To generate this energy, the heart utilizes a variety of different metabolites including glucose, fatty acids (FA), ketone bodies (KB), and lactate among others. Interestingly, cardiac cells are capable of shifting energy dependence among these substrates depending on their availability and myocardial workload changes  . NumerouThe novel antidiabetic drug family of sodium-glucose cotransporter (SGLT) inhibitors has been found to significantly reduce cardiac mortality in patients with type 2 diabetes mellitus (T2DM) ,6,7. FurHuman hearts obtain energy from two sources, adenosine triphosphate (ATP) (~5 μmol/g wet weight) the primary molecule to act as fuel in all cellular processes, and phosphocreatine (PCr) (~8 μmol/g wet weight) which acts more as an energy reserve . CardiomThe myocardium requires high amounts of energy to meet the constant demand of sarcomere proteins. In order to maintain this, the cardiac cells rely on substrate flexibility which allows them to switch from one source of energy to another one depending on cardiac workload and metabolite availability . During When the heart fails to supply enough energy (ATP molecules) to meet energy demands , which enhances FA oxidation . TherefoIn addition, another proposed pathophysiological mechanism for myocardial dysfunction in diabetes leading to the development of diabetic cardiomyopathy is myocardial stiffness, resulting from cellular and extracellular matrix stiffness as well as cell-matrix interactions. Being the intrinsic cardiomyocyte stiffness its major contributor as a result of the impairment in its cytoskeleton. Several mechanisms are also involved in myocardial stiffness: inflammation, oxidative stress and SGLT-2 mediated effects, the reason why SGLT2i are emerging as a potential treatment option .In summary, there seem to be two different metabolic disturbances that account for the failing heart: HF (which increased glucose metabolism and reduced FA utilization) and diabetic cardiomyopathy (with greater metabolic reliability on FA oxidation but lower glucose consumption). These two mechanisms suggest the existence of a very precise balance of different substrate consumption to ensure adequate cardiac function.Following acute myocardial ischemia, a wound repair process of the left ventricle is triggered. This phenomenon, also termed “adverse remodeling”, involves changes such as myocyte death and hypertrophy, inflammatory response and connective tissue alterations. The extent of these changes is a major determinant of mortality and morbidity in patients with acute myocardial infarction ,21. In aMyocardial ischemia triggers a local and systemic inflammatory response in which both cytokines and cardiac-monomeric C-reactive protein seem to play a crucial role . In addiIt is also important to highlight the difference between acute and chronic alterations after cardiac ischemia. Following acute ischemia, there is an upregulation of carbohydrates metabolism and glucose intake, which may be beneficial in early stages since glucose is a more oxygen efficient metabolite than FA . HoweverGiven its hydrophilic nature, glucose molecules are unable to pass the lipid bilayer of cardiac cells. Two families of transporters are involved in facilitate the transport of glucose from the bloodstream into the myocyte cytosol: 1) the glucose transporters 1 and 4 (GLUT1 and GLUT4), which transport exclusively glucose molecules via facilitated-diffusion and 2) the sodium-glucose cotransporter 1 (SGLT1), which transports various substrates into the cell  against chemical gradient using the energy related in the simultaneous transport of sodium in favor of electrochemistry ,27. WhilSLC5A1 and SLC5A2, respectively, are of essential importance for glucose homeostasis [The SLGT family is the subgroup solute carrier 5 (SLC5) of the solute carrier’s group. Among them, SGLT1 and SGLT2, encoded by the genes eostasis . Therefoeostasis . Its expeostasis . While leostasis . Functional SGLT1 is an oligomer, resulting from dimerization. At least 3 bands have been observed in human hearts: 2 bands of 70 and 140 kDa (that could reflect dimerization), and also an intermediate band, which its significance is unknown and could be related to post-translational modifications  . The expIntestinal SGLT1 is modulated by dietary carbohydrate consumption. Following a high-glucose diet in animal models, SGLT1 activity and expression increase. Glucose appears to be a local rather than a systemic modulator since oral but not intravenous glucose administration enhances its expression . Cardiac ischemia results in 2- to 3-fold increased glucose uptake and utilization , that seWhether SGLT1 receptors exert protective or deleterious effects has not been yet determined. On one site, SGLT1 expression may be beneficial following acute myocardial ischemic injury since they facilitate glucose uptake, which is the only source of ATP through anaerobic glycolysis during ischemia. On the other side, chronic SGLT1 overexpression has been demonstrated to cause a phenotype similar to glycogen-storage cardiomyopathy ,34. ImpoIn summary, the metabolic switch towards carbohydrate over FA consumption, following acute myocardial ischemia, seems to be essential to maintain adequate ATP supply to the cardiac contractile apparatus. SGLT1 receptors favor this glucose utilization, and SGLT1 inhibition may not be beneficial in the acute scenario. However, a long-term cardiac metabolism mainly based on glucose consumption has also been associated with cardiac impairment and thus, SGLT1 inhibitors may play a more decisive role in this setting. Due to poor control of diabetes even with the high number of medications available, new therapeutic interventions are still being sought to try and control this fearsome epidemic. Type 2 sodium glucose co-transporter receptor inhibitors (SGLT2i) are a new class of drug with a promising future. SLC5A2, have been associated with glycosuria suggesting that by blocking this cotransporter the reabsorption of glucose from the renal tubules may be impaired and therefore reducing hyperglycemia [This receptor plays a key role in the tubular reabsorption of the urinary glucose. Kidneys filter glucose molecules that are subsequently reabsorbed in the proximal tubules back to the bloodstream. When the amount of glucose in the renal tubules surpasses 160–180 mg/dL , the kidneys are unable to reabsorb such a high quantity and glucose is eliminated through urine  . Approxiglycemia . This observation offers the nature response and settles the basics for an alternative treatment of DM patients. In terms of side effects, the most frequent and relevant is mild-moderate GI infections, more commonly in women with prior history of infections and post – menopause that resolved with standard treatment. Given that its effect is not related to increased insulin release, hypoglycemia was not seen in patients with T2DM or nondiabetic, presumably due to an increase in endogenous glucose production in the liver. In addition, the is a mild diuretic effect that may increase the risk of orthostatic hypotension, postural dizziness and dehydration specially in older patients and with combination of loop diuretics   , we post2+ concentration, via promoting Na+ influx. As a consequence, calcineurin signaling is enhanced resulting in cell death and myocardial damage [Additionally, SGLT2i inhibit the sodium-hydrogen exchanger (NHE) isoform 1, which is expressed in the myocardium and modulates cardiomyocyte pH. There is an upregulation of NHE1 activity in the failing heart, which leads to an increase in intracellular Cal damage . Long-tel damage . Moreovel damage . Importal damage ,54 whichCardiac fibrosis, an important final common pathway of HF, is also ameliorated by SGLT2 inhibition. On DM murine models after 8 weeks of empagliflozin myocardial structure and function improved showing a reduction on myocardial oxidative stress in addition of ameliorated fibrosis . DapagliAnother interesting postulated mechanism by which SGLT2i may exert their cardiac benefits is via AMPK activation. This protein-kinase promotes FA metabolism and oxidation and therefore, improving cardiac bioenergetics . MoreoveA tight glycemic control takes years to display changes in CV outcomes and this is unlikely to be the reason behind the previously mentioned benefits. Interestingly, SGLT2 receptors are not expressed in the heart and that suggests an indirect effect on cardiac metabolism that remains still unknown . Therefo2+ metabolism. Overexpression and Ca2+ dependent activation of Ca2+/calmodulin dependent kinase II CaMKII are hallmarks of HF. Empagliflozin reduces CaMKII activity and CaMKII dependent Ca2+ leak from the sarcoplasmic reticulum, which may contribute to the beneficial effect in HF [+. Elevated cardiac cytoplasmic Na+ concentration is a driver of HF. By inhibiting NHE, SGLT2i directly lower cytoplasmic Na+ concentration in the cardiomyocyte [SGLT2 inhibitors additionally affect intra cellular Cact in HF . SGLT2 iomyocyte ,66. A Significant number of effective drugs for long-term control of high-glucose levels in T2DM patients have demonstrated their effectiveness in reducing microvascular complications. Unfortunately, these drugs have not shown clear benefits in reducing CV adverse events, even few of them may be harmful when administered to patients with concomitant cardiac disease. The SGLT2i represent a new class of drug with a promising future. Recent large randomized controlled trials investigating safety outcomes with these agents have showed surprising cardiovascular benefits and even a significant mortality rate reduction. Furthermore, these drugs have also gained great attention due to their unclear mechanism of action that seems to be independently of their hypoglycemic effects, and whether is a drug-specific or generalizable to the drug class . As a consequence of all the data reviewed in this article, SGLT2 inhibitors have evolved from antidiabetic treatments towards being studied also for HF, both in diabetic and in non-diabetic patients. The EMPEROR clinical trial is an event-driven trial that is studying this potential benefits. Building up on this hypothesis, we have initiated the EMPA-TROPISM (Are the “Cardiac Benefits” of Empagliflozin Independent of Its Hypoglycemic Activity?) (NCT03485222) clinical trial to investigate the effects of empagliflozin specifically in nondiabetic HF , while oAdditional clinical benefits have been observed in renal patients. Canagliflozin for instance, has demonstrated to mitigate kidney dysfunction and to attenuate eGFR decline and albuminuria, that suggests also a nephroprotective effect of this drug . (Table The SGLT receptors have drawn a major interest following the surprising results of the EMPA-REG OUTCOME trial . Its rolIn addition, SLGT1 but not SGLT2 have demonstrated to exert pro-inflammatory properties that may intensify myocardial infarct size and contractile dysfunction. Whether SGLT1 inhibition contributes to reduce this phenomenon and provide better outcomes after I/R injury remains still unclear.Interestingly, SGLT2 inhibitors but not SGLT1 have demonstrated to promote KB metabolism and therefore, to enhance cardiac bioenergetics. This circumstance is of particular interest in patients with chronic damaged hearts. Whether this effect could be strengthened by a dual SGTL1 and SGLT2 inhibition should also be further investigated.


Combined and sequential liver–kidney transplantation (CLKT and SLKT) is a definitive treatment in children with end-stage organ failure. There are two major indications: - terminal insufficiency of both organs, or - need for transplanting new liver as a source of lacking enzyme or specific regulator of the immune system in a patient with renal failure. A third (uncommon) option is secondary end-stage renal failure in liver transplant recipients. These three clinical settings use distinct qualification algorithms. The most common indications include primary hyperoxaluria type 1 (PH1) and autosomal recessive polycystic kidney disease (ARPKD), followed by liver diseases associated with occasional kidney failure. Availability of anti-C5a antibody (eculizumab) has limited the validity of CLKT in genetic atypical hemolytic uremic syndrome (aHUS). The liver coming from the same donor as renal graft (in CLKT) is immunologically protective for the kidney and this provides long-term rejection-free follow-up. No such protection is observed in SLKT, when both organs come from different donors, except uncommon cases of living donation of both organs. Overall long-term outcome in CLKT in terms of graft survival is good and not different from isolated liver or kidney transplantation, however patient survival is inferior due to complexity of this procedure. Combined liver–kidney transplantation (CLKT) is a procedure in which liver  and kidney allografts, coming from the same (deceased in most cases) donor, are transplanted to the pediatric recipient during a single surgical procedure. Sequential liver–kidney transplantation (SLKT) is divided into two distinct stages: isolated transplantation of the liver (or kidney) and  – transplantation of the kidney (or liver) allograft, coming from different (deceased) or the same living-related donors. This review describes aspects of surgical and post-transplant management, immunosuppression-related issues, several specific indications for CLKT and SLKT, as well as the outcomes of both procedures.Combined liver and kidney transplantation (CLKT) is a very uncommon procedure  usually does not exceed 30 cases), therefore should be performed by experienced surgical and multidisciplinary teams, as its complexity may be the reason of early failure with fatal consequences, especially in young children of low body weight . CLKT isIn anuric patients, e.g., in children with ARPKD after bilateral native nephrectomy performed early in life , CLKT isIn cases of CLKT performed in patients with hyperoxaluria type 1 (PH1), the intensive hemodiafiltration should be continued after transplantation to remove the excess oxalate, intensively mobilized from the body deposits to plasma and urine. This approach is mandatory in cases of delayed renal graft function, however it might also be used in cases of immediate renal graft function in selected patients with severe systemic oxalosis and high oxalate burden. The aim is to protect the transplanted kidney against rapid recurrence of nephrocalcinosis. The optimal duration of post-transplant hemodiafiltration is not defined, however it is reasonable to monitor plasma oxalate concentration and maintain extracorporeal removal of oxalate until stable normalization below the saturation level (< 30 μmol/l) is achieved with good renal function –6.During the whole CLKT surgery, particular attention should be given to achieving good surgical hemostasis and controlling coagulation. In patients with liver-related coagulopathy, the major disturbances should be corrected before surgery with fresh frozen plasma (FFP), cryoprecipitate, or platelets, if necessary. The infusion of tranexamic acid may be given to control fibrinolysis during surgery, particularly in patients with a history of previous abdominal surgeries or advanced portal hypertension with multiple intra-abdominal varices and hypersplenism, as in these cases native hepatectomy is difficult and associated with marked blood loss. One the other hand it is important not to overcorrect coagulation, as it may lead to vascular thrombotic complications after reperfusion of both organs, thus administration of FFP, platelets, cryoprecipitate, fibrinogen formulations, and other procoagulants during surgery should be limited to the minimum necessary. Apart from routine laboratory tests, the best intraoperative monitoring of coagulation during CLKT is provided by repeated thromboelastometry, which shows real clinical coagulation status and guides adequate treatment. In some cases the infusion of catecholamines (dopamine and norepinephrine) should be given to maintain adequate blood pressure without fluid overload..H2O by supplementation of 100% of abdominal drainage volumes and hourly diuresis. Hematocrit is kept between 25 and 30% during the whole perioperative period and coagulation should not be corrected unless there is clinically symptomatic bleeding or the international normalized ratio (INR) exceeds 3–3.5. Administration of blood-driven products  should be kept to a minimum and related to the current needs of the recipient, measured losses, and results of laboratory tests. Our center uses anticoagulants (fractionated or low molecular weight heparin) postoperatively for 7–14 days as routine antithrombotic prophylaxis.Doppler ultrasound examination should be done in the operating room before wounds are closed to ensure adequate blood flow through vascular anastomoses. Early post-operation care requires staying in the pediatric intensive care unit as mechanical ventilation may be necessary for 1–2 days. Very close monitoring of the patient and both transplanted organs is mandatory in the early postoperative period. Very detailed fluid balance (both water-electrolyte and volume), including all drained losses and hourly urine output should be kept to promote perfusion of the grafts and maintain adequate diuresis from the transplanted kidney . CentralPatency of vascular anastomoses should be checked twice daily with Doppler ultrasound during the first 7 days after transplantation and then less frequently until discharge.In most clinical situations of sequential procedure (SLKT), the liver transplantation precedes kidney transplantation, with the exception of a few patients with renal polycystic disease and liver fibrosis. The transplantation surgery of each individual graft in SLKT is not different from combined transplantation and is just performed as two separate procedures. In SLKT, most often one of the organs may be procured from a living donor and another one from a deceased donor, however using the same living-related donor has also been reported .There are data suggesting a protective effect of the liver to the kidney when procured from the same donor and transplanted during one surgical procedure (CLKT), which translates to a low rate of acute rejection compared to isolated kidney or liver transplantation, and results in improved renal allograft survival , 8. TherP = 0.002) and overall kidney graft survival (P = 0.015) were significantly diminished among sensitized patients. Differences in patient survival translated to an estimated half-life of 10.3 years among nonsensitized recipients, versus 7.8 years among sensitized recipients. Therefore, presensitization may need to be considered in a risk stratification of CLKT   . The lat as well , 25. Theive CLKT .Recent advances in developing RNA interference (RNAi)-based therapeutics, targeting glycolate oxidase and hydroxyproline dehydrogenase, aimed at treating liver-related diseases , may change the policy of final treatment of PH1. The new drugs  are able to modify oxalate turnover to the extent that they probably will replace other therapies and limit the indications for performing CLKT/SLKT in the future .GRHPR). Despite the generally milder clinical course (compared to PH1) and fact that expression of this enzyme is not limited to the liver, cases (both pediatric and adult) of renal allograft failure due to recurrence of oxaluria and presence of oxalate deposits in transplanted kidneys have been described [There are reports on failure of isolated kidney transplantation in hyperoxaluria type 2 (PH2), associated with genetic mutations resulting in decreased activity of glyoxylate reductase/hydroxypyruvate reductase  presents in early infancy with severe renal disease, congestive heart failure, and pulmonary hypoplasia. The second group  presents predominantly progressive renal failure and Caroli syndrome in the liver, whereas the two remaining groups—infantile and juvenile—present milder renal disease and more severe hepatic complications, mostly expressed as hepatic fibrosis [2) with successful outcome, showing that the rule of qualification may be individual. Moreover, successful CLKT was associated with significant improvement of life quality and improvement of growth velocity in this series [Autosomal recessive polycystic kidney disease (ARPKD) is one of the ciliopathies and is caused by mutations of the fibrosis , 30. Manfibrosis –33. Therfibrosis –36. Therfibrosis . Howevers series . The avaTMEM67 gene) has been identified [Up to 5% of patients with nephronophthisis present hepatic fibrosis and this clinical pattern is known as Boichis syndrome. The relevant genetic background . The defect of this enzyme leads to multiorgan failure and disability caused by chronic intoxication with methylmalonic acid and creating megamitochondria in affected organs [Methylmalonic acidemia (MMA) is heterogenous inborn error of metabolism most typically caused by mutations in the vitamin-B12-dependent enzyme methylmalonyl- CoA – mutase  is a genetic disease caused by mutations in the gene antation . Membranantation . The preantation . Isolateantation . Successantation .Type 1a glycogen storage disease (GSD1) is an autosomal recessive disorder with deficiency of glucose-6 phosphatase and related multiorgan accumulation of glycogen resulting in multiple dysfunctions, including hepatomegaly and liver failure, development of hepatocellular adenomas, and renal failure in the course of secondary glomerulosclerosis. Two successful case reports of CKLT in GSD1 have been published , 51.Combined liver–kidney transplantation was used as a therapeutic option in patients with genetic aHUS at high risk of recurrence before the availability of anti-C5 monoclonal antibody (eculizumab), and the relevant recommendations concerning eligibility of patients for CLKT or isolated liver transplantation have been released by the consensus group. Successful CLKT may be curative in aHUS, however this procedure is not free from significant complications and peri-transplant overall mortality rate is about 15% –54. Curr2 at 5–10 years after liver transplantation is about 30% [2). However, conversion to mTORi was associated with a higher risk of acute rejection  and study discontinuation due to adverse events  up to 1 year after randomization [There are two distinct subgroups of patients who develop renal failure after liver transplantation. One includes patients who received liver transplant having developed CKD at stage 4/5 or developing chronic post-acute renal failure in the course of hepatorenal syndrome (HRS). The second group includes patients with normal renal function at the moment of liver transplantation, but who develop chronic kidney injury several years thereafter, mainly due to nephrotoxicity of calcineurin inhibitors (CNI). There are no specific pediatric recommendations regarding this, however there is ongoing discussion on optimal therapy in the first setting, conducted among transplant physicians and surgeons treating adult patients. The consensus guidelines divide (adult) candidates for CLKT into two renal categories: (1) persistent acute kidney injury (AKI) ≥4 weeks, or eGFR ≤35 ml/min (by MDRD-6 formula) or eGFR ≤25 ml/min , and (2) CKD for 3 months before qualification, defined as eGFR ≤40 ml/min (by MDRD-6 formula) or eGFR ≤30 ml/min  or proteinuria ≥2 g/day or chronic pathology in renal biopsy  or metabolic disease involving kidneys . These cbout 30% , howeverbout 30% . There about 30%  and the bout 30% . There hmization . A similmization . ClinicaP = 0.005) [P = 0.4), which is in contrast to the opinion of a protective effect of the liver on the renal graft. Overall, the early post-transplant period in CLKT is associated with high risk for patients , while long-term graft survival is not inferior compared to isolated transplantations. Data from the Scientific Registry of Transplant Recipients (SRTR), including 152 primary cases of CLKT (1987–2011), shows that patient survival was 86.8, 82.1, and 78.9%, liver graft survival was 81.9, 76.5, and 72.6% and renal graft survival 83.4, 76.5, and 66.8% at 1, 2, and 10 years, respectively. Similar survival rates for isolated liver transplantations were 86.7, 81.2, and 77.4%, and for isolated kidney transplantations 98.2, 95.4, and 90%, respectively. In patients undergoing CLKT, PH1 as the underlying disease  was associated with reduced patient, liver, and kidney graft survival . The study also showed that during one decade, graft outcome after CLKT significantly improved (p = 0.04 for liver and p = 0.02 for kidney transplantation), however this did not translate into better patient survival (p = 0.2). The study demonstrated the center effect on overall patient survival, with inferior outcomes in children receiving CLKT in centers with minor experience (defined as 1–2 CLKTs performed) [p = 0.002), however (death-censored) graft survival was similar compared to isolated kidney recipients  [There are variable data on long-term outcomes of CLKT, depending on the series of patients and specific indications (when compared to outcomes of isolated organ transplantation). The major difference is also related to the primary parameter of outcome: - incidence of acute rejection, or - patient and graft survival. Overall, the incidence of acute rejection in CKLT is lower compared to isolated liver or kidney transplantation (reports from UNOS and CTS databases) , 14, whirformed) . Overallrformed) . The UNO• Indications for combined or sequential liver–kidney transplantation (CLKT/SLKT) include parallel or sequential failure of both organs, the need to replace the liver as the source of a specific missing enzyme in a patient with renal failure, and the need for renal transplantation in the recipient of liver graft due to renal failure.• The selection of optimal approach (CLKT vs. SLKT) is individual depending on specific clinical indications, current status of both organs, general condition of the recipient, and experience of the center.• In CLKT, the liver exerts a protective immunological effect upon the kidney, which translates to low acute rejection rates and good long-term graft survival, however this effect seems to be limited to non-sensitized patients.• In SLKT, there is no protective effect, as in most cases liver and kidney come from different donors, however the HLA matching between sequential organs (liver and kidney) is important for future outcome.• Overall outcome of CLKT/SLKT is good in terms of long-term graft survival, however it is not superior to an isolated liver or kidney transplantation setting in terms of patient survival, which is related to the complexity of transplantation procedures, especially in young children and the high risk during the early perioperative period.Qualification for organ transplantation in children with primary type 1 hyperoxaluria (PH1) is based on:Basic rule: ‘kidney first’ in all casesBasic rule ‘liver first’ in all casesEvaluation of GFR and adjusting strategy to the stage of CKDDegree of liver failure2.Transplanted liver protects renal graft against acute rejection:AlwaysWhen both grafts come from the same donor and the recipient is not hyperimmunizedWhen the liver comes from living-related donor and the kidney from a deceased donorIf the time interval between the sequential transplantations exceeds 1 year3.During CLKT:Liver is transplanted first and then the kidneyBiliary anastomosis is performed before kidney reperfusionKidney reperfusion should precede liver reperfusionOptimal cold ischemia time for both organs should be not longer than 16 h4.When patients with ARPKD receive an isolated renal transplant:Liver function improvesThere is no effect of this procedure on native liverLiver volume decreases due to better fluid turnoverThere is a risk of recurrent cholangitis in the native liver, exacerbated by post-transplant immunosuppression5.CLKT in atypical hemolytic uremic syndrome (aHUS):Is a single effective curative procedureIs still widely performed, as monoclonal anti-C5 is not effective in most cases of isolated kidney transplantationsIs indicated only when a living-related donor is availableIs not currently regarded as first-line option, as repeated administration of monoclonal anti-C5 is effective in isolated kidney transplantation for prevention against recurrence of aHUS6.Imediate post-CLKT extracorporeal removal of oxalate in patients with primary type 1 hyperoxaluria (PH1):Is unnecessary, as well functioning liver transplanted easily eliminates whole pool of oxalateIs indicated in cases of  delayed graft functionIs contraindicated in patients plasma oxalate concentration > 100 μmol/l and good urine outputShould never be done due to high risk of bleeding


Although more than 95% of viral sequences found in skin tumors typically belong to human papillomaviruses (HPVs), HPV transcription has so far not been detected. As current technology allows very deep transcriptome sequencing, we examined skin tumors and precursor lesions for HPV transcription.Fresh frozen biopsies from 12 skin specimens (11/12 were positive for HPV DNA) were subjected to total RNA sequencing. The cervical cancer cell line CaSki was included as a positive control for HPV transcription.HPV RNA was detected and confirmed in 1/12 skin lesions at a median depth of 66 million reads per sample. One specimen was positive for HPV 110 transcripts mapping to E6, E7, E2/E4 and L2 open reading frames, as well as to a spliced E1^E4 transcript.In conclusion, the study revealed that a minority of skin lesions contains HPV transcripts and that HPV DNA detection does not predict HPV transcriptional activity. Human papillomaviruses (HPVs) infect keratinocytes in both mucosa and skin and can cause several diseases from benign lesions to malignant tumors , 2. CutaL1 gene which is the most conserved gene within the HPV genome [The traditional methods to identify HPV-related lesions/cancer are based on PCR, targeting usually the V genome . PCR metV genome , 16. UnkV genome . Most ofV genome , 18, 19.V genome .Detection of HPV DNA does not necessarily mean that an HPV infection has been detected, as it may represent a viral deposition on the skin surface . Simple Metagenomic sequencing without prior PCR allows detection of all HPVs present in a sample, without previous knowledge on which types might be present , 18, 25.Written informed consent was obtained from all participants in the clinical study. The study was approved by the Ethical review Committee of Karolinska Institutet and of Lund University (Sweden) . We included a set of 12 fresh frozen biopsies from immunocompetent patients  prepared for RNA analysis. Samples had been whole genome sequenced using MiSeq or HiSeq Illumina platforms in a previous study . The RNARNA was extracted using RNeasy Lipid Tissue Mini kit (Qiagen), following the manufacturer´s instructions, including the DNase digestion, and kept at -70°C until further analysis.A cervical cancer cell line (CaSki (ATCC CRM-CRL-1550)) known to contain approximately 600 copies of HPV type 16 genome integrated into the human genome, was used as a positive control for virus presence and transcription. The cell line was cultured and harvested cells were treated with RNAlater and RNA was extracted using the same RNeasy Lipid Tissue Mini kit (Qiagen), following the manufacturer´s instructions.The RNA extracted samples  together with two negative controls containing lab-grade water, were rRNA depleted, reverse transcribed and ligated to individual indexed adapters using the TruSeq Stranded Total RNA kit according to the user´s guide, Revision E . All 16 individual libraries were validated, normalized to 2 nM and pooled accordingly  before sequencing. Sequencing was performed in the NextSeq500 system  at 151 paired-end cycles. A total of 4 runs were carried out. The first 2 runs contained a pool of 6 skin lesions, 1 negative control (lab-grade water) and 1 positive control (CaSki cell line) each. In the third run, only two lesions were sequenced. The fourth run was performed as a validation of the HPV 16 RNA-positive specimen in the second run, now sequenced without the CaSki cell line positive control to discard possible index-hopping.Indices, included in the Illumina adapters, were used to assign raw sequence reads obtained from the NextSeq500 (Illumina) platform to the originating samples. Reads were quality checked and adapter trimmed with Trimmomatic . High quOnce human reads were filtered from the data set, reads from each sample were queried against all HPV protein sequences included in the PaVE database , using the open source software Diamond blastx with default parameters and—top 1  . SamplesSpecimen adequacy was confirmed for all samples, except for the negative controls, through coverage analysis of human reference genes known to be expressed in skin (ACTB and GAPDH) . All samhttps://www.ncbi.nlm.nih.gov/sra/PRJNA493709).The Illumina sequencing generated high quality sequencing data with a median of 66 million reads per sample . Most ofWhen the skin lesions were sequenced with multiplexing for 8 samples, only 2 specimens (both actinic keratosis) were positive for HPV RNA transcripts, one positive for HPV 16 (53 reads) and one positive for HPV 110 (10 reads) . The HPVIn order to investigate if increased sequencing depth would detect more HPV transcripts, we increased the depth 4 times by multiplexing only two lesions  . In addiHPV 16 RNA-reads were detected only in one actinic keratosis lesion (specimen LH130). No other sample nor water controls generated any HPV 16 reads. As HPV 16 is a mucosal high risk type for cervical cancer it would be interesting if an HPV 16 transcript was detected in a pre-malignant skin lesion, suggesting an active HPV 16-infection. We considered whether the HPV 16 reads found in LH130 might have resulted from contamination from the HPV 16-positive control (CaSki cell line) used in the experiment, most likely due to a phenomenon known as “index hopping”, where similar indexed adapters are mis-read during multiplexed sequencing. LH130 index was AR013 (AGTCAA) and the CaSki index was AR015 (ATGTCA). These indices only differ in an extra T at the second position in AR015 and an extra A in the last position in AR013.Comparing sequences of HPV 16 reads from LH130 and CaSki positive controls, one mutation was detected (substitution C3470T) between specimens. While LH130 showed 87% reads (13/15) with a thymine at position 3470, CaSki controls presented 100% cytosine .To validate this positive HPV 16 results we sequenced this specimen at 8 times greater depth (2x75 cycles) without the CaSki positive control in the same run. No HPV reads were detected for this sample at a deeper sequencing.The DNA and RNA detection of HPV 16 in the different skin lesions do not coincide as 2 skin lesions were positive for HPV 16 DNA, but negative for HPV 16 RNA . An explCutaneous HPVs are commonly detected both on healthy skin , 35 and In 11/12 skin lesions, we did not find any HPV expression. Besides true negativity for HPV expression, possible explanations for not finding HPVs might include: specimen inadequacy and detection method still not sensitive enough . SpecimeWe choose to align our sequencing reads against all HPV protein sequences present in the PaVE database, not to miss a large number of recently reported HPV types, which are not established yet, and to see if we were able to detect divergent putative novel HPVs. We did not find any novel HPV type, nor did we detect any of the HPV types previously detected by DNA sequencing in the same specimens, which may suggest that these types are only present as environmental deposition rather than active infections. DNA from HPV 197  wasIn conclusion, RNA sequencing can detect presence of HPV transcription. While HPV is actively transcribed in cervical cancers, HPV transcription in skin lesions seems to be a rare event. Using this strategy of RNA-seq in further studies on the non-human transcriptome in NMSCs and precursor lesions may give more insight into the role of viruses in these tumors. In addition, all tumors with increased incidence among immunosuppressed patients may be analyzed in the same manner in order to investigate putative viral etiologies in those cancers.S1 TableNormalization of reads (RPKM units) for ACTB (actin beta) and GAPDH  protein coding genes for skin specimens.(DOCX)Click here for additional data file.S1 FigThe height of the grey bars corresponds to the number of reads at each nucleotide position. The exons of the genes ACTB is visualized at the bottom as blue boxes and the introns as blue lines. The high coverage of most of the exons and lack of coverage of intron regions indicates that the sequences originate from RNA with no or very little DNA contamination.(TIF)Click here for additional data file.S2 FigThe height of the grey bars corresponds to the number of reads at each nucleotide position. The exons of the gene GAPDH is visualized at the bottom as blue boxes and the introns as blue lines. The high coverage of most of the exons and lack of coverage of intron regions indicates that the sequences originate from RNA with no or very little DNA contamination.(TIF)Click here for additional data file.


This article contains data related to the research article entitled “Assessing terrestrial laser scanning for developing non-destructive biomass allometry” (Stovall et al., 2018  Specifications TableValue of the data•Volumetric data are useful for studies of wood allocation in 10 dominant species on the East Coast of the United States.•The data provide information useful for estimating forest carbon storage in hardwood forests in the United States.•The data can be used as-is or combined with regionally similar measurements to create high sample size allometry including large diameter and height trees.1We present data on terrestrial LiDAR-based tree-level estimates of diameter at breast height (DBH), height, length, volume, and biomass ; Fig. 1.2We detail the terrestrial LiDAR sampling and post-processing method thoroughly in Refs. Field-based measurements of DBH were collected during the same sampling season as the TLS acquisitions and used to validate the TLS-derived estimates. The presented tree measurements were collected across 14 1/10th ha circular plots. At each plot, we collected 5–6 TLS scans with a resolution totaling 28.2 pulses per scan, positioned approximately at the center and the four cardinal directions. Scans were registered using multiple registration points with an average error of less than 1 cm.Registered point cloud data were imported into Computree software


Progress in diagnostics and immunotherapy for autoimmune encephalitis has significantly improved survival rates. However, autoimmune encephalitis remains a challenging condition, associated with a protracted recovery course and uncertain long-term outcome. Clarification of the risk factors for autoimmune encephalitis, and the development of scales to aid with prognostication, may help improve clinical management.This month’s journal club explores three papers relating to autoimmune encephalitis. The first paper examines the frequency of and risk factors for autoimmune encephalitis following herpes simplex encephalitis. The second paper describes the development of a score that predicts 1-year functional status in patients with anti-NMDA receptor encephalitis. The third paper discusses the development of a general scale for assessing severity in diverse autoimmune encephalitis syndromes.It is well established that herpes simplex encephalitis can trigger autoimmune encephalitis. This paper explores the association between herpes simplex encephalitis and consequent autoimmune encephalitis using prospective (cohort A) and retrospective (cohort B) analyses. Cohort A comprised 51 patients with newly diagnosed herpes simplex encephalitis. These patients were followed up at several stages over a 12-month period with a neurological assessment and evaluation of serum and CSF autoantibody levels. Cohort B comprised 48 patients with a clinical diagnosis of autoimmune encephalitis following successful treatment of herpes simplex encephalitis.In the prospective analysis, 14 patients (27%) developed neurological worsening consistent with probable autoimmune encephalitis. All 14 patients (100%) tested positive for IgG antibodies; 9 patients tested positive for anti-NMDA receptor antibodies, and 5 for other antigens. All patients had been negative for neuronal antibodies at onset of herpes simplex encephalitis. In contrast, of the 37 patients who did not develop clinical autoimmune encephalitis, 11 (30%) tested positive for IgG antibodies during follow up. The time period between herpes simplex encephalitis and the development of autoimmune encephalitis for cohort A patients ranged between 7 and 61 days. There were no statistically significant differences between clinical symptoms of herpes simplex encephalitis, cerebrospinal fluid parameters, radiological findings or treatment duration between patients who developed autoimmune encephalitis to those who did not.In cohort B, 44/48 (92%) had neuronal antibodies either against NMDA (34/44) (77%) or against unknown antigens. Of all 58 patients who developed autoimmune encephalitis across both cohorts, patients younger than 4 years were more likely to present with choreoathetosis, whereas patients over age 4 years more frequently demonstrated behavioural problems and psychosis.Comment: This is a robust study demonstrating a 27% probability of developing autoimmune encephalitis following herpes simplex encephalitis. Positive antibodies 3-week post-diagnosis of herpes simplex encephalitis may predict autoimmune encephalitis. Strengths include the objectivity of serum and CSF antibody testing and the follow-up of patients in the prospective analysis at a number of different time-points. However, it is unclear which autoantibodies, other than anti-NMDA, were tested for, and whether antibodies against unknown antigens found in the sera and CSF are genuinely pathogenic.Armangue et al. (2018) Lancet Neurol 17:760–72.The objective of this retrospective observational study was to construct a grading score that predicts neurological function 1 year after the diagnosis of NMDAR encephalitis.This study examined a cohort of 382 patients identified as having anti-NMDA receptor encephalitis defined by the clinical picture as well as positive serum or CSF findings. All patient CSF and serum samples were sent either to the University of Pennsylvania or the University of Barcelona from 200 centres in 35 countries. All patients within the cohort had been part of a prior observational study, with previously collected data regarding demographics, symptom onset, clinical, laboratory and radiological features, time to treatment initiation, time from treatment initiation to improvement, and functional status using the modified Rankin scale (mRS). Good functional status at 1 year was defined as a mRS ≤ 2.pThe group analysed a range of clinical factors to determine predictors of functional status at 1 year using a multivariate regression model. Five independent variables were identified including: need for ICU admission, delay of treatment initiation beyond 4 weeks of symptom onset, lack of clinical improvement 4 weeks after initiating treatment, presence of an abnormal MRI scan and CSF WBC count > 20 cells/uL. Points were assigned to each of these variables to construct the NEOS score , with a maximum possible score of 5. A strong association was found between the NEOS score and functional status at 1 year, with a NEOS score of 0 relating to good functional status and higher NEOS scores relating to greater disability  Neurology Jan 15;92(3):e244–e252.This study describes the development of a novel scale for assessing severity of disease and evaluating response to treatment in patients with diverse autoimmune encephalitis syndromes. Currently the modified Rankin Scale is used to assess neurological outcome in autoimmune encephalitis, however, as the mRS is designed to evaluate disability post stroke, a rating scale for autoimmune encephalitis is desirable.p < 0.001).Two expert panels developed the scale. Initially, 4 experts in autoimmune encephalitis generated the key clinical determinants of severity of autoimmune encephalitis. Thirteen neurologists then reviewed the key items put forward by the expert panel and answered whether the item was or was not essential for reviewing severity of autoimmune encephalitis. The final scale comprised nine items: seizure, memory dysfunction, psychiatric symptoms, consciousness, language problem, dyskinesia/dystonia, gait instability and ataxia, brainstem dysfunction and weakness, with sub-criteria for each key item giving a possible score of 0–3, and a maximum score of 27 for all nine key items. The scale was tested by three neurologists on a development cohort of 50 patients with confirmed autoimmune encephalitis, and by two neurologists on a validation cohort of 38 patients to evaluate its reliability. The scale showed excellent inter-observer and intra-observer reliability and good correlation with the modified Ranking Scale  Ann Neurol 85 (3):352–358.The first paper discussed raises awareness of the likelihood of autoimmune encephalitis following herpes simplex encephalitis. Clinicians should have a low threshold to test for autoimmune encephalitis in patients who deteriorate following viral encephalitis. The second and third papers demonstrate encouraging work in the development of assessment scales for autoimmune encephalitis. Although treatment and recovery remains an individualised process, these scales may help to identify patient subgroups who could benefit from novel therapies.


Prostate cancer (PrCa) demonstrates a heterogeneous clinical presentation ranging from largely indolent to lethal. We sought to identify a signature of rare inherited variants that distinguishes between these two extreme phenotypes.We sequenced germline whole exomes from 139 aggressive  and 141 non-aggressive  PrCa cases. We conducted rare variant association analyses at gene and gene set levels using SKAT and Bayesian risk index techniques. GO term enrichment analysis was performed for genes with the highest differential burden of rare disruptive variants.BRCA2, ATM and NBN the most frequently mutated genes. Differential burden of rare variants was identified between metastatic and non-aggressive cases for several genes implicated in angiogenesis, conferring both deleterious and protective effects.Protein truncating variants (PTVs) in specific DNA repair genes were significantly overrepresented among patients with the aggressive phenotype, with Inherited PTVs in several DNA repair genes distinguish aggressive from non-aggressive PrCa cases. Furthermore, inherited variants in genes with roles in angiogenesis may be potential predictors for risk of metastases. If validated in a larger dataset, these findings have potential for future clinical application. Although common variants identified thus far explain over a quarter of the familial relative risk of PrCa,We hypothesised that additional rare germline variants exist that are predictive of poorer prognosis and could improve clinical management of the disease. However, due to the large number of neutral rare germline variants carried by each individual, detection of causative variants is challenging. In an attempt to enrich for rare variants that predispose to the aggressive disease outcome, we designed a case–case study that sampled the extremes of the PrCa phenotype. Our cohort compared young onset, metastatic patients against cases with older onset, indolent disease. We performed whole exome sequencing to identify genes and biological processes with the highest differential burden of disruptive rare variants, which may in turn represent a signature of aggressiveness.14 were obtained from whole blood and distributed on three 96-well plates for DNA library preparation and sequencing. To minimise any potential for case–case confounding caused by batch effects, samples were block randomised based on case status and DNA extraction method. DNA samples were fragmented using a Covaris E220 Ultrasonicator and exome sequences enriched using Agilent SureSelectXT2 Human All Exon V5 baits, in 36 pools (8 samples/pool) using 7 bp molecular barcodes. Pools were sequenced on an Illumina HiSeq 2500 instrument .Germline DNA samples for 144 aggressive  and 144 non-aggressive  PrCa cases from the UK Genetic Prostate Cancer Study (UKGPCS)15 and aligned to the GRCh37/hg19 reference genome using BWA-MEM 0.7.10.16 Variants were jointly called across all samples using GATK 3.5,17 following the specified best practices (https://software.broadinstitute.org/gatk/best-practices/). Analysis was restricted to the exome capture regions plus additional 100 bp padding. Variant annotation was performed using wAnnovar,18 Oncotator 1.819 and WGSA20 . Combined Annotation Dependent Depletion (CADD) scores21 were used to predict deleteriousness of single nucleotide variants and indels. Transcript annotation was taken from the Oncotator pipeline using the transcript list giving priority to known clinical protein changes (Feb 2016). GENCODE  was used as the reference transcript set.Paired end reads were adaptor-masked using Cutadapt 1.522 Samples not achieving these quality thresholds were excluded from further analysis . After exclusion of data from 8 cases due to mixed ancestry, high heterozygosity or insufficient sequencing coverage, the final sample set consisted of 139 aggressive and 141 non-aggressive cases, and 150,787 coding or splice site variants that passed QC  and low contamination  <10%), as estimated by VerifyBamID 1.1.0%, as es23 One-way ANOVA was performed to test for association of ‘missingness’ rate with sample preparation batch. Neither batch effects nor case-case confounding were detected  analysis, using a general linear model to test the association of PCs with the case and batch status. The first 10 PCs were tested, with no association found for either case or batch status. Differences in genotype call rates between cases were assessed using PLINK 1.9.27 in R and a Bonferroni adjusted significance threshold .Association of individual variants with case status was tested using logistic regression with Firth correction using the rv package21 Rare variants were defined as those with MAF <1% in the healthy non-Finnish European population, as reported by the Exome Aggregation Consortium (ExAC).28To assess the potential association between gene-level burden of rare disruptive variants with phenotype, potentially damaging variants were grouped into two categories. Tier 1 (predicted deleterious) were protein truncating variants  and Tier 2 (predicted damaging) all non-truncating variants with CADD score ≥20  or SKAT-O.31 and SKAT-O29 methods. BROCA gene set analysis was conducted based on the expanded BROCA cancer risk gene panel of 60 genes.32 DNA repair gene set analyses were performed using 177 curated DNA repair genes and pathway groupings.33 Hallmark gene set analyses were conducted according to the 50 gene sets curated to represent specific biological processes in the Molecular Signature Database (MSigDB) Hallmark Gene Set Collection (http://software.broadinstitute.org/gsea/msigdb/collections.jsp).34Gene set analyses were performed separately on Tier 1 only, or Tier1 and Tier 2 variants combined, using Bayesian Risk Index35 PANTHER Overrepresentation Test (2016-07-15 release) and GO Ontology database annotation (2016-10-27 release), applying a Bonferroni adjustment to correct for multiple testing.For GO term enrichment analysis, we selected genes with the highest differential burden of disruptive (Tier 1 and Tier 2) variants between aggressive and non-aggressive cases. Genes were ranked based on collapsing burden count odds ratios (ORs) of >2 or <0.5 and filtered with an additional criterion of a minimum count difference of 3 disruptive variants between groups. The enrichment analysis of biological processes and molecular functions was performed using AmiGO,36 of patients from the UK Genetic Prostate Cancer Study (UKGPCS), in which ~90% of patients had clinically presenting disease at diagnosis.14 We selected 144 aggressive and 144 non-aggressive cases for whole exome sequencing. All cases were of self-reported European ancestry and unrelated. The criteria for defining aggressive PrCa cases were metastatic disease combined with early age of onset ( <60 years), while the non-aggressive cases were later onset (≥60 years) and had low risk clinical presentation .To investigate the genetic signature of aggressive PrCa risk through a case–case analysis, we performed extreme phenotype sampling37After exclusion of data from 8 cases due to mixed ancestry, high heterozygosity or insufficient sequencing coverage, the final sample set consisted of 139 aggressive and 141 non-aggressive cases Table . The medWe first assessed association of all coding variants individually and collapsed at the gene level with the extreme phenotypes. Single variant tests were conducted for all variants with no MAF filter applied, whilst gene level burden tests were performed using rare (MAF<1%) variants for Tier 1 (PTVs) or Tier 1 plus Tier 2 (non-truncating variants with a CADD score ≥20) variants separately. As expected due to the modest sample size, no individual variant or gene was associated with aggressive status at a statistically significant level after adjustment for multiple testing (data not shown).32 are associated with aggressive PrCa in a familial cohort.11 In this study, we identified 22 rare Tier 1 mutations in the extended 60 gene BROCA panel , ATM  and NBN  were the most frequently mutated genes, with one metastatic patient a carrier of two Tier 1 BROCA mutations, both in NBN. 17 of the Tier 1 BROCA variants in our cohort have been previously reported in ExAC and/or ClinVar, of which 13 are classified as pathogenic and 1 as likely pathogenic. Five variants had not been reported before: p.K828fs in ATM, p.Y1527* in ATR, p.R89fs in NBN, p.Q244* in PMS2 and p.A1653fs in SLX4.We subsequently conducted analyses at the gene set level, using previously curated gene collections linked to either specific biological pathways or disease in the literature. We have previously shown that deleterious mutations in the original 22 gene BROCA panel of high and moderate risk genes involved in cancer predisposition, primarily focussed on hormone-driven breast and ovarian cancers,31 , which identified NBN and BRCA2 as the genes contributing most strongly to this enrichment  and Fisher’s exact test . This association remained with BRCA2 excluded from the panel . Carriers of BROCA gene PTVs also demonstrate reduced survival compared with non-carriers , or including both Tier 1 and Tier 2 .24 of the 60 genes in the BROCA panel are DNA repair genes; of the 22 Tier 1 BROCA panel variants identified, 21 were within DNA repair genes and only 1 in the remainder of the panel. These DNA repair gene PTVs were identified across genes participating in a number of different DNA repair pathways, with the Fanconi Anaemia pathway, homologous recombination, DNA damage response and mismatch repair all represented. We therefore analysed an expanded set of 177 curated DNA repair genes,NBN, for which a Tier 1 or 2 variant was observed in 8 metastatic patients (5.8%) and no non-aggressive cases.As no evidence for association with the metastatic phenotype was observed for the full set of curated DNA repair genes, we further stratified the analyses by the primary process or pathway each gene participates in. These analyses were performed for both Tier 1 and Tier 1 plus 2 variants separately. We observed very strong evidence for association between the homologous recombination pathway and the metastatic phenotype  and strong evidence for the HALLMARK_ANGIOGENESIS gene set . The genes contributing most strongly to these enrichments were ACVRL1 and LUM respectively  that may represent additional candidates for differential susceptibility towards metastatic PrCa . Analysis of only the genes with lowest OR (higher burden of variants in the non-aggressive cases) showed a 9.0-fold enrichment of the term “fibril organisation” . Further investigation of the 8 genes contributing to this enrichment  revealed all to be involved in the collagen metabolism and angiogenesis pathways apart from RIPK3, which is involved in amyloid fibril formation (Supplementary Table P = 0.012), a higher hierarchy term containing the “extracellular matrix organisation” category.To further interrogate wider biological processes that may contribute towards the development of the aggressive phenotype, we performed GO term enrichment analysis of genes with the largest OR calculated from the Tier 1 and Tier 2 variant burden. When including both genes with highest (>2.0) and lowest (<0.5) OR for metastatic disease, we observed a 1.9-fold enrichment of the term “extracellular matrix organisation”  being the most commonly mutated genes among the aggressive cohort. The findings for BRCA2 and ATM are largely in agreement with recent candidate gene studies in castration resistant metastatic PrCa cases39 and lethal PrCa cohorts13 of primarily European ancestry. The Bayesian Risk Index method that we employed ranked NBN as the gene within the BROCA panel for which PTVs contributed most strongly towards the metastatic phenotype. Furthermore, separate analyses of individual DNA repair pathways indicated a likely additional contribution by rare non-truncating NBN variants to increased risk of metastases. These findings are consistent with previous observations that carriers of a Slavic founder mutation (rs587776650 / 657del5) in NBN40 experience significantly higher PrCa mortality,41 and demonstrates that additional rare NBN variants, including missense variants, may increase the risk of aggressive PrCa in the British population.Our previous work has shown that the BROCA cancer risk gene panel, designed primarily to evaluate hereditary breast and ovarian cancer predisposition, is informative in stratifying PrCa risk among individuals with strong family history of PrCa.39 no significant association of rare truncating variants with metastatic disease was observed in our analysis of an expanded set of 177 genes curated specifically as contributing towards DNA repair.33 This suggests that only a subset of specific DNA repair genes or pathways contribute substantially to the predisposition towards the poor prognosis phenotype, and that germline PTVs within any family members for which association has not yet been established are likely to occur at extremely low frequencies in the British population. The PTVs we identified were observed across genes contributing to multiple separate DNA repair pathways, in particular homologous recombination and the Fanconi anaemia pathway. Stratification of the DNA repair gene set by pathway provided strong evidence for a role by homologous recombination in increased susceptibility to development of metastases, with the association driven primarily by NBN.Although this study therefore provides further evidence that germline variants within a handful of specific DNA repair genes increase risk of aggressive PrCa,TET2 have previously been reported to be enriched in aggressive PrCa cases of African ancestry; however this association was ethnicity specific and was not found for European ancestry individuals.42 Within our cohort, we observed truncating TET2 mutations in 3 patients, all non-aggressive, whilst Tier 2 variants were identified in one additional metastatic and one non-aggressive case. A low frequency missense variant in HOXB13 (rs138213197 / G84E) has also been robustly demonstrated to increase risk of PrCa in European ancestry populations and is associated with familial clustering and younger age of diagnosis; however no association with clinical presentation or survival has been demonstrated.45 The HOXB13 G84E variant was identified in 1 non-aggressive case only in our study and no further Tier 1 or 2 HOXB13 variants were identified within either cohort. These observations provide further support that these two genes are unlikely to contribute substantially towards increased risk of aggressive disease in European ancestry populations.Whilst we observed a high rate of germline DNA repair gene mutations among our metastatic cohort in comparison to non-aggressive PrCa cases, the absence of these mutations in the majority of the cohort implies that additional biological processes, besides faulty DNA damage response and repair, could also be instrumental in predisposition towards or protection against metastatic disease. Rare germline coding variants in 46 Interestingly, we also found damaging variants in genes associated with collagen fibril formation enriched among the non-aggressive cases, alluding to a possible protective function against tumour invasiveness of certain defects in the extracellular matrix organisation. In line with these observations, Bayesian Risk Index analysis of hallmark gene sets in MSigDB demonstrated evidence that disruptive germline variants in genes contributing towards angiogenesis can distinguish patients with metastatic and localised disease. This analysis selected two gene sets with high evidence of differential burden between the extreme phenotypes and highlighted two genes in particular driving these associations, LUM and ACVRL1. Further inclusion of genes that demonstrated a gene-level BF > 100 within any gene set analysis returns a total of 10 genes for consideration. The majority of these genes have previously been reported to be linked to angiogenesis, proliferation and poor prognosis in multiple cancer types, especially through modulation of TGF-ß signalling. Indeed, pharmacological inhibitors of two genes (ACVRL1 and AXL) are currently the subject of clinical trials in a range of tumour types as prospective anti-angiogenic therapeutics.48We conducted further gene set analyses in an attempt to identify additional genes that may influence prognosis in PrCa patients. GO term enrichment analysis on genes displaying a differential case–case burden of rare disruptive variants was performed, which highlighted genes involved in extracellular matrix organisation and remodelling, a key event in angiogenesis.LUM and AXL. LUM codes for lumican, a collagen-binding protein found in interstitial collagenous matrices throughout the body and implicated as an inhibitor of angiogenesis50; disruptive mutations within LUM would therefore be expected to facilitate tumour vasculature formation. Suppression of AXL expression has been reported to play a central role in the proliferation of disseminated PrCa cells in bone marrow into metastatic lesions from a dormant state,51 representing a plausible route through which disruptive variants in AXL could predispose individuals that develop PrCa towards a metastatic phenotype. The remainder of genes identified were enriched for mutations in non-aggressive rather than metastatic cases, with many also demonstrating biologically plausible rationale for how these variants could potentially confer a protective effect against tumour migration. ACVRL1  is a cell surface receptor in the TGF-beta signalling pathway which binds bone morphogenic protein (BMP)-9 and -10, appears to be involved in developmental and angiogenic blood vessel formation, and has been shown to be highly expressed in the vasculature and stroma in a high proportion of prostate tumours.52 Germline disruptive variants within ACVRL1 could therefore in principle inhibit development of new tumour vasculature and hinder the onset of metastases. Similarly, mTOR is a known promoter of angiogenesis through the VEGF signalling pathway and is frequently targeted for inhibition in human tumours to exert anti-angiogenic activity.53 Also of note is the CP gene, which codes for ceruloplasmin, the major copper-carrying protein in the blood. Association between angiogenesis and copper levels has been established, with elevated levels of ceruloplasmin itself linked to development of metastases in rabbit models.54 Disruptive mutations in CP could therefore feasibly be protective against angiogenesis in PrCa. Other genes enriched for mutations in patients with indolent disease include WISP2, a gene that inhibits proliferation of vascular smooth muscle cells and has been associated with invasion, metastasis and poor prognosis, although opposing effects have been reported for different cancer types,56DHRS2, which stabilises p53 by inhibiting Mdm2 and may be upregulated in breast tumour endothelium in comparison to normal breast vasculature,58 and PARK2, a gene linked to hereditary Parkinson’s disease that also demonstrates tumour suppressor functions and might confer an influence on apoptosis through modulation of mitophagy.60In keeping with the role of many of these genes in developmental or homoeostatic processes, PTVs were observed in only 2 of the 10 genes shortlisted through the Bayesian Risk Index analysis, with Tier 2 variants accounting for 81 of the 88 rare variants identified in these genes in our cohort. We also observed differential enrichment of mutations within either the metastatic or non-aggressive cohort for different genes, implying a combination of potential deleterious and protective effects towards tumour invasiveness. Two genes exhibited enrichment of disruptive mutations in metastatic cases, NBN, BRCA2 and ATM genes are associated with development of an aggressive clinical phenotype. Using gene-set enrichment analyses, we have also found strong preliminary evidence that rare, predominantly non-truncating germline variants predicted to affect protein function in specific genes contributing to angiogenesis are implicated in conferring either predisposition towards, or protection against, progression to metastatic disease in men who develop PrCa. If validated in larger independent cohorts, or meta-analyses of multiple studies, these genes would represent prognostic markers that may aid identification of PrCa patients at high or low risk of developing invasive disease, who would warrant distinct clinical management pathways. These genes could also represent exciting novel targets for targeted therapies tailored towards individual PrCa patients with aggressive phenotype. These initial findings therefore warrant follow-up in larger cohorts to validate their potential role in predisposition toward metastatic disease.The use of differential ages in the definition of our aggressive and non-aggressive cohorts enabled us to maximise our power to detect associations through the analysis of sample groups at the extremes of the phenotypic spectrum of PrCa, however could also represent a potential confounder. As 105 of the 120 deaths within our sample cohort were PrCa specific, and no cause was recurrent among the remaining 15 deaths, there would appear to be minimal evidence of any overlapping associations with large effect sizes against other disease phenotypes within our data. We cannot completely exclude however that this could represent a potential limitation of our study, whilst the use of age as a criterion in the definition of aggressiveness also limits our ability to examine the effect of mutation carrier status on age at onset; since carrier status, aggressiveness, and age are all correlated. Nonetheless, this study demonstrates that germline PTVs in specific DNA repair genes occur significantly more frequently among, and therefore can be used to distinguish, PrCa patients likely to progress to metastatic disease. In particular, mutations in the Supplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5Supplementary Table 6Supplementary Table 7


Flaviviridae family that has been known to circulate for decades causing mild febrile illness. The more recent ZIKV outbreaks in the Americas and the Caribbean associated with congenital malformations and Guillain-Barré syndrome in adults have placed public health officials in high alert and highlight the significant impact of ZIKV on human health. New technologies to study the biology of ZIKV and to develop more effective prevention options are highly desired. In this study we demonstrate that direct delivery in mice of an infectious ZIKV cDNA clone allows the rescue of recombinant (r)ZIKV in vivo. A bacterial artificial chromosome containing the sequence of ZIKV strain Paraiba/2015 under the control of the cytomegalovirus promoter was complexed with a commercial transfection reagent and administrated using different routes in type-I interferon receptor deficient A129 mice. Clinical signs and death associated with ZIKV viremia were observed in mice. The rZIKV recovered from these mice remained fully virulent in a second passage in mice. Interestingly, infectious rZIKV was also recovered after intraperitoneal inoculation of the rZIKV cDNA in the absence of transfection reagent. Further expanding these studies, we demonstrate that a single intraperitoneal inoculation of a cDNA clone encoding an attenuated rZIKV was safe, highly immunogenic, and provided full protection against lethal ZIKV challenge. This novel in vivo reverse genetics method is a potentially suitable delivery platform for the study of wild-type and live-attenuated ZIKV devoid of confounding factors typical associated with in vitro systems. Moreover, our results open the possibility of employing similar in vivo reverse genetic approaches for the generation of other viruses and, therefore, change the way we will use reverse genetics in the future.Zika virus (ZIKV) is a mosquito-borne member of the  Flaviviridae family, became a global public concern because of the correlation of Zika virus epidemic with fetal developmental defects, including highly publicized cases of microcephaly1. The viral genome is made of a positive sense, single-stranded RNA molecule (~10.8 kb) that contains a single open reading frame flanked by 5′ and 3′ untranslated regions4. The viral RNA is translated as a single polyprotein that is co- and post-translationally processed by viral and cellular proteases into three major structural proteins  involved in viral entry, fusion and assembly5, and seven non-structural (NS) proteins  that are involved in viral RNA replication and transcription, assembly and evasion of the host antiviral responses9.Zika virus (ZIKV), a member of the 11, and in adults with the Guillain-Barré syndrome13. ZIKV is primary transmitted to humans through the bite of infected mosquitoes11. However, non-vector ZIKV transmission can occur through sexual contact15 or vertical transmission from infected mothers17. Currently, there are no approved vaccines or antivirals to prevent or treat ZIKV infection.Although the majority of ZIKV infections are asymptomatic or associated with a mild febrile illness, infection during pregnancy has been associated with miscarriage and severe congenital malformations, including fetal microcephaly18. In 1981, Racaniello and Baltimore provided the basis of this approach with poliovirus19. Since then, numerous reverse genetics systems have been developed for positive- and negative-stranded RNA viruses24. Plasmid-based reverse genetic approaches allow the rescue of infectious recombinant viruses by the transfection of one or more plasmids encoding the components necessary for the de novo generation of infectious virus particles into cultured cells. Importantly, reverse genetic techniques are powerful platforms to modify the viral genome and they have provided critical insights into replication and pathogenesis of multiple viruses18. Importantly, viral reverse genetic approaches have also been used as platforms to develop novel and more effective live-attenuated vaccines (LAVs) against viral infections and have significantly contributed to the development of antiviral treatments20. However, there are intrinsic limitations in the in vitro recovery of recombinant viruses using reverse genetic approaches, mostly associated with cell culture-adaptive mutations that might restrict and/or change the phenotype of the virus in vivo26. In the case of LAVs, these mutations could lead to the reversion to a virulent phenotype28. Likewise, the current need of first generating recombinant viruses in cultured cells rather than directly in validated animal models of infection delays studies aimed to understand and assess viral infections in vivo. Therefore, it is of high significance to develop approaches that allow the recovery in vivo of infectious recombinant virus from cDNA infectious clones without the current need of a tissue culture step. In vivo reverse genetics or recover of virus directly from validated animal models could overcome the concern of virus adaptation to cell cultures and facilitate and simplify the study of the virus and the development of LAVs based on attenuated forms of these viruses.The establishment of plasmid-based reverse genetic systems for RNA viruses entails the rescue of recombinant viruses from cDNA clones containing the entire viral genome on a plasmid from transfected culture cells29 or Paraiba/2015 strains30 under the control of the cytomegalovirus (CMV) immediate-early promoter. Importantly, we demonstrated the feasibility of using this infectious clone to generate a virulent rZIKV with similar in vitro and in vivo characteristics to the natural Paraiba/2015 isolate30. Using the BAC infectious cDNA clone of Paraiba/2015 strain (pBAC-ZIKV), we have explored the possibility to recover infectious rZIKV directly in vivo in the type-I interferon (IFN) receptor deficient (IFNAR−/−) A129 mouse model of ZIKV infection33. The pBAC-ZIKV cDNA clone was complexed with Lipofectamine 2000 (pBAC-ZIKV/LPF) and inoculated in IFNAR−/− A129 mice using different inoculation routes, which are typically used for ZIKV infections. Recovery of rZIKV in vivo with high efficiency was observed in mice. In addition, infectious rZIKV was also recovered by direct inoculation of the infectious cDNA clone in the absence of transfection reagent. More Importantly, we demonstrate the feasibility of using this in vivo rescue approach with a BAC cDNA infectious clone encoding an attenuated rZIKV (rZIKVatt)30 that resulted in sterilizing immunity against aggressive ZIKV challenge. In vivo recovery of fully infectious and/or attenuated RNA viruses expands the potential use of reverse genetic systems and open the possibility of developing similar approaches for other viruses, which could be an innovative technology for their study or the future development of LAVs.Recently, we have described a reverse genetic approach for ZIKV based on the use of a bacterial artificial chromosome (BAC), by assembling the full-length genome of ZIKV Rio Grande do Norte Natal (RGN)et al. had previously described a method for the in vivo recovery of influenza A virus (IAV), a negative-strand RNA virus, after the inoculation in the nasal cavity of mice with cells transfected with a bacmid containing the entire IAV genome34. A similar approach was used to study the potential for reassortment between different IAV subtype strains in ferrets36. Thus, we conducted similar studies to determine whether this approach was valid for the in vivo rescue of rZIKV . Vero cells  were transfected with 5 µg of the pBAC-ZIKV cDNA clone using Lipofectamine 2000 (LPF) . At 12 h post-transfection (hpt), cells were trypsinized, washed and resuspended in PBS, and used to inoculate mice. Unless otherwise stated, animal studies were performed in 4-5-weeks old, IFNAR−/− A129 mice consisting of 6 mice/group, 3 females and 3 males  either subcutaneously (SC) in the footpad  were readily observed in samples from 3 dpi , confirming the functionality of the pBAC-ZIKV cDNA clone used for the in vivo experiments. Importantly, mice inoculated with Vero cells transfected with empty BAC plasmid did not result in any clinical signs of disease, body weight loss or death; and rZIKV were not detected at any day post-inoculation (data not shown). These data demonstrate that rZIKV can be successfully rescued in vivo after inoculation of Vero cells transfected with the pBAC-ZIKV/LPF mixture.Chen IKV Fig. . Vero celes Fig. . Mice wepad Fig.  and survpad Fig.  for 18 dpad Fig. . Mice shpad Fig. , consistmia Fig. . In genedpi Fig. , in agredpi Fig. . In addidpi Fig. . At 6 dpin vivo directly after inoculation of the pBAC-ZIKV cDNA clone, mice were inoculated with 100 µg of pBAC-ZIKV complexed with 100 µl of LPF (1:1 ratio), both diluted in Opti-MEM in a final volume of 200 µl  used to inoculate mice resulting in ~2 × 106 FFU/ml of rZIKV by 48 hpt, confirming the quality of the transfection mixture used in the in vivo studies (data not shown). Notably, mice inoculated with empty BAC plasmid did not result in the recovery of ZIKV at any day post-inoculation and no clinical signs of disease, body weight loss or death were observed (data not shown). These data demonstrate that rZIKV can be successfully recovered in vivo with 100% efficiency after inoculation in the peritoneal cavity of mice with the pBAC-ZIKV/LPF mixture.To further determine whether rZIKV could be rescued  µl Fig. . The samin vivo rZIKV rescue, we next evaluated the minimal amount of pBAC-ZIKV/LPF required for such effect . Viral titers of 2 and 4 × 106 FFU/ml were detected at 48 hpt in the tissue culture supernatants of Vero cells transfected with LPF and GenJet, respectively. As expected, no virus was recovered in vitro without the use of a transfection reagent or transfecting empty BAC with any transfection reagent (data not shown). In vivo, mice inoculated with either the pBAC-ZIKV/GenJet or the pBAC-ZIKV/LPF mixture began to lose weight by 6–7 dpi  for the successful recovery of rZIKV with 100% efficiency.GenJet (SignaGen) is a transfection reagent specifically formulated for ent Fig. . Mice weent Fig. . As intedpi Fig.  and stardpi Fig. . In agrecue Fig. . Mice weice Fig. . Howeverice Fig. . At leasice Fig.  for the in vivo reverse genetics system as a potential vaccine platform for the prevention of a typical ZIKV infection  with 100 µg/mouse of the pBAC-ZIKVatt/LPF mixture  of the in vitro generated rZIKVatt  of rZIKVatt 30. Mice in the pBAC-ZIKVatt/LPF-inoculated group and those in the control rZIKVatt group were fully protected against lethal rZIKV challenge, showing no signs of disease, no body weight loss . We systematically analyzed whether Vero cells transfected with pBAC-ZIKV, the pBAC-ZIKV/LPF mixture or naked pBAC-ZIKV without transfection reagent were suitable platforms for generation of rZIKV in vivo. We have previously shown that intranasal inoculation of 293T cells transfected with a reverse genetics clone of IAV in a bacmid DNA leads to generation of infectious virus34. Here we show that other routes  are also suitable for transfection-based inoculation in vivo reverse genetics, at least for the rescue of rZIKV. On the other hand, we developed an in vivo rescue approach based on the direct inoculation of the pBAC-ZIKV complexed with LPF. Direct inoculation by either the SC, IM, IP routes of pBAC-ZIKV/LPF resulted in successful and efficient rZIKV recovery, with the IP route being the most consistent and efficient. While DNA without any transfection reagent has been shown to be effective for gene transfer and expression following direct injection into skeletal muscle or skin in vivo, this is the first report of IP delivery and expression of naked DNA. Regardless of the inoculation route used, virus produced using the in vivo reverse genetics approach reproduced disease associated with ZIKV infection in the mouse model, including neurological signs, body weight loss, and death. We observed that ZIKV infection was delayed 2–3 days after pBAC-ZIKV inoculation compared to ZIKV-infected mice, which can be explained by the fact that the viral RNA has to be transcribed from the pBAC-ZIKV by the cellular RNA polymerase II in the nucleus of transfected cells and further amplified in the cytoplasm by the viral replicase. Considering the time needed to rescue viruses using reverse genetics in cultured cells, our in vivo reverse genetics also represents a significant improvement in the time that viruses can be studied in animal models of infection. The rZIKV generated after in vivo inoculation conserved the infectivity and virulence when used to infect new mice, which is important because it implies that the system recapitulates a bona fide ZIKV replication cycle. Further studies suggest that in vivo ZIKV rescue is dependent on the concentration of plasmid, but the virus can be recovered even in the absence of transfection reagent, albeit with lower efficiency. This also represent an advantage over current in vitro reverse genetic approaches that requires the use of expensive transfection reagents for successful recovery of viruses in cultured cells. Overall, these results demonstrate the feasibility of using the in vivo reverse genetics approach for the successful rescue of rZIKV in vivo. This methodology could be useful to directly test the contribution of specific mutations in the viral genome in vivo, without the need of prior recovering and amplifying the virus in vitro, to minimize potential mutations associated with passaging the virus in culture cells and overall reducing the time required to study viruses in validated animal models of infection. Moreover, this approach could be useful to rescue other RNA viruses, with poor replication properties in cultured cells41.In this report, we have expanded the potential of 45. Thus, the use of IFNAR −/− A129 immunodeficient mice represented the best suitable model to demonstrate the successful rescue of recombinant ZIKV using in vivo reverse genetics. Further studies using a more relevant animal model of ZIKV infection, currently outside the scope of this manuscript, will demonstrate the feasibility of using similar in vivo reverse genetics for the feasibility to generate recombinant ZIKV.It is important to indicate that our studies are based on the use of IFNAR −/− A129 mice that are deficient in type I IFN responses. In this animal model, infection with ZIKV using different routes  results in neurological disease and the animals succumb to viral infection with high viral load in the blood and organs , consistent with ZIKV infection and manifestation in humans. Contrary, infection of WT mice with ZIKV does not results in clinical signs of infection, mortality or detection of the virus at different times post-infection in the blood or organs of infected animalsin vitro transcribed Hepatitis C virus (HCV) viral (v)RNA was infectious after direct injection into the liver of chimpanzees43. This was an important technical advance in the field, since HCV does not replicate efficiently in cell cultures44. However, it also has some major limitations, including the need of transcribing the vRNA before in vivo delivery, which may introduce spurious mutations and is also limited to the quality, quantity, and stability of the transcribed vRNA in vivo. In that sense, our approach to recover recombinant viruses in vivo represents a major advance since plasmid DNA is easier to produce and more stable that vRNA transcripts. The plasmid DNA was inoculated IP, which allows for systemic delivery and therefore no specific organs have to be injected. We were able to obtain 100% viral rescue success when using up to 12.5 μg of plasmid DNA, alone or in combination with transfection reagents. Also, our results demonstrate that the IM route, a more validated in vivo DNA delivery, also serves as a useful method for in vivo rescue of rZIKV, although the IP route was more efficient in the recovery of rZIKV, challenging the gold standard (IM route) of plasmid DNA delivery in vivo.Previous studies have reported that 46. However, DNA vaccines have been hampered by issues related to poor immunogenicity and/or the need for inoculation of high amounts (mgs) in order to generate adequate protective responses. In contrast, LAVs have been shown to provide efficient, multidimensional protective responses by stimulating humoral and cellular protective immune responses. The major drawback of LAVs is that they may require specialized culture system substrates for production and a cold-chain to maintain the integrity of the vaccine47. The ability to combine the advantages of these two systems, like the one described in this report, opens the exciting possibility for greatly improving vaccines and vaccination strategies for a myriad of pathogens, including ZIKV. We took advantage of our previously described pBAC-ZIKVatt cDNA infectious clone30 to show that rZIKVatt was efficiently recovered and safe in vivo, which in turn produced strong humoral responses, including high levels of neutralizing antibodies capable of protecting mice against a lethal rZIKV challenge. This new in vivo viral rescue platform for vaccine development offers numerous advantages compared to traditional approaches and combines the advantages of DNA vaccines  with the strengths of LAVs 51. More importantly, our new in vivo reverse genetics approach could be extended to vaccine development for other RNA viruses that represent a concern in human health.DNA vaccines have several advantages over LAVs, including stability and cost2.African green monkey kidney epithelial Vero cells  were grown in Dulbecco’s modified Eagle’s medium  supplemented with 5% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C with 5% CO30 and obtained after transfection of Vero cells with the corresponding infectious BAC cDNA clones. The rZIKVatt contains a valine (V) to alanine (A) substitution at position 117 in the viral NS2A protein that renders the virus attenuated in mice30. Briefly, ZIKV genomes were assembled in the BAC plasmid pBeloBac11 under the control of the CMV promoter and flanked at the 3′ end by the hepatitis delta virus (HDV) ribozyme and the bovine growth hormone (BGH) termination and polyadenylation sequences52. Infectious clones were propagated in the DH10B strain of E. Coli (Gibco/BRL), and BAC were prepared using the Qiagen plasmid Giga kit, following the manufacture specifications.The rZIKV wild type Paraiba/2015 and its corresponding attenuated (att) strain were previously described6 cells/well, triplicates) were transfected with 5 μg of the ZIKV BAC cDNA clones using 15 μl of LPF (Invitrogen), as described previously29. At 6 hpt, media was removed and replaced with grown media supplemented with 2% FBS. For in vitro rescues, tissue culture supernatants were collected after 3–4 days, when cytopathic effect (CPE) was apparent (60–80%), and stored at −80 °C. Virus stocks were propagated in Vero cells and titrated by immunofocus assay in Vero cells using the pan-flavivirus envelope (E) protein monoclonal antibody (mAb) 4G2 from BEI Resources (NR-50327)27. Viral titers were expressed as focus-forming units per ml (FFU/ml)27.Vero cells grown to 90% of confluence  with a solution of ketamine (100 mg/kg) and xylazine (10 mg/kg), and then inoculated in the footpad , in the quadriceps muscle  or in the peritoneal cavity  as indicated in the main text. Morbidity  and mortality were monitored for 18 days. Mice showing severe signs of paralysis or mice that lost more than 20% of their initial body weight were humanely euthanized following AVMA guidelines. Statistical analyses were performed using PRISM software .IFNAR −/− A129 mice (Jackson Laboratory) were bred and maintained in the animal care facility of the University of Rochester Medical Center (URMC). All mice were housed under specific pathogen-free conditions. Mouse experiments were approved by the University of Rochester Committee of Animal Resources and carried out in compliance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Research Council27.Mouse sera were collected by submandibular bleeding at the indicated dpi and blood was maintained at room temperature for 30 min to allow clotting. Serum was separated by centrifugation at 5,000 rpm for 20 min and immediately stored at −80 °C. Viral titers were determined by FFU assays in Vero cells55. Briefly, 96-well ELISA plates (ThermoFisher) were coated with extracts from ZIKV-infected Vero cells, and incubated overnight at 4 °C. Coated wells were blocked with 1% BSA in PBS, and incubated with 2-fold serial dilutions of mice sera (starting dilution of 1:100) for 1 h at 37 °C, following by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG for 1 h at 37 °C. Reactions were developed with tetramethylbenzidine (TMB) substrate (BioLegend) for 10 min at room temperature, quenched with 2 N H2SO4 and read at 450 nm in a Vmax Kinetic microplate reader (Molecular Devices).To assess the levels of virus-specific antibodies present in the sera of vaccinated mice, ELISAs were performed as previously described56. Briefly, 2-fold serial dilutions of sera (starting dilution of 1:800) were incubated with 25 FFU of rZIKV for 3 h at 37 °C, and used to infect subconfluent (80%) monolayers of Vero cells in 96-well plates. After 90 min of viral absorption at 37 °C, virus-sera mixture was removed, and the cells overlaid with 100 μl of growth medium containing 2% FBS and 1% Avicel. Cells were fixed at 36 h post-infection with 4% paraformaldehyde and prepared for immunostaining assay using 1 μg/ml of mAb 4G2. Viral plaques were visualized using Vectastin ABC kit and DAB HRP substrate (Vector Laboratories Inc.), following the manufacturer’s instructions.The presence of neutralizing antibodies in the sera of vaccinated mice were determined by FRNT as previously described