Arboviruses are capable to establish long-term persistent infections in mosquitoes that do not affect significantly the physiology of the insect vectors. Arbovirus infections are controlled by the RNAi machinery via the production of viral siRNAs and the formation of RISC complexes targeting viral genomes and mRNAs. Engineered arboviruses that contain cellular gene sequences can therefore be transformed to “viral silencing vectors” for studies of gene function in reverse genetics approaches. More specifically, “ideal” viral silencing vectors must be competent to induce robust RNAi effects while other interactions with the host immune system should be kept at a minimum to reduce non-specific effects. Because of their inconspicuous nature, arboviruses may approach the “ideal” viral silencing vectors in insects and it is therefore worthwhile to study the mechanisms by which the interactions with the RNAi machinery occur. In this review, an analysis is presented of the antiviral RNAi response in mosquito vectors with respect to the major types of arboviruses . With respect to antiviral defense, the exo-RNAi pathway constitutes the major mechanism while the contribution of both miRNAs and viral piRNAs remains a contentious issue. However, additional mechanisms exist in mosquitoes that are capable to enhance or restrict the efficiency of viral silencing vectors such as the amplification of RNAi effects by DNA forms, the existence of incorporated viral elements in the genome and the induction of a non-specific systemic response by Dicer-2. Of significance is the observation that no major “viral suppressors of RNAi” (VSRs) seem to be encoded by arboviral genomes, indicating that relatively tight control of the activity of the RNA-dependent RNA polymerase (RdRp) may be sufficient to maintain the persistent character of arbovirus infections. Major strategies for improvement of viral silencing vectors therefore are proposed to involve engineering of VSRs and modifying of the properties of the RdRp. Because of safety issues (pathogen status), however, arbovirus-based silencing vectors are not well suited for practical applications, such as RNAi-based mosquito control. In that case, related mosquito-specific viruses that also establish persistent infections and may cause similar RNAi responses may represent a valuable alternative solution. RNA interference (RNAi) has become an important tool to analyze gene function in eukaryotes, including insects. RNAi technology is based on the administration of dsRNA that will trigger the degradation of homologous cellular mRNAs. Because of the specificity of gene silencing effects, RNAi has become a powerful reverse genetics tool for analysis of gene function, especially in non-model organisms, which include many insects . HoweverArbovirus infections of mosquito vectors represent an interesting system of how persistent and systemic virus infections become established and maintained in insect hosts. As such, studies of arbovirus infections can provide important insights to inspire the optimization of viral silencing vectors in other insects. As a background, this review will investigate the process of RNAi as an antiviral response during arbovirus infections and to what extent arboviruses (in their persistent state) can function as RNAi transduction vectors in mosquitoes. While RNAi is considered the most important factor that modulates arbovirus–mosquito interactions, it should be noted that other pathways and processes  and ticks . In this review, most attention is focused on mosquitoes because of the wealth of information that is available, while other insect or tick vectors are occasionally also mentioned if relevant information is available for discussion.Togaviridae (genus Alphavirus), Flaviviridae], monopartite linear (-) ssRNA genome (Rhabdoviridae), segmented linear (-) or ambisense ssRNA genome  and segmented linear dsRNA genome (Reoviridae). While arboviruses can cause some of the most devastating diseases in humans , a distinguishing feature of arbovirus infection in mosquitoes is the establishment of a non-pathogenic, persistent state (Most arboviruses are RNA viruses and characterized by monopartite linear (+) ssRNA genome  (Drosophila consists of two branches: (1) the primary (intermediate) pathway in which (single-stranded) transcripts from discrete genomic loci termed piRNA clusters (consisting of remnants of transposable elements) are processed by the nuclease Zucchini, followed by loading of piRNAs in Piwi-containing effector complexes to perform transcriptional gene silencing in the nucleus; and (2) the secondary (mature) pathway that occurs in the cytoplasm and is characterized by the ping-pong amplification loop. In the ping-pong mechanism, transposon (sense) transcripts are cleaved by Aub (loaded with antisense piRNAs) to generate complementary piRNAs (sense orientation) that are loaded in Ago-3-complexes. Because sense piRNA-Ago-3 complexes will subsequently target again transposon sequences in the antisense orientation, an amplification loop is created of production of both mature antisense and sense piRNAs that expedite transposon silencing. In the ping-pong mechanism, the 5′-end of the guide piRNA is located at exactly 10 nt from the 5′-end of the cleavage site. Because the 5′-ends of piRNAs associated with Piwi or Aub have a characteristic U-bias, products are generated with A at the 10th position, that become regularly associated with Ago-3. The U1/A10 bias and the 10 nt overlap between the 5′-ends in complementary small RNAs are therefore regarded as hallmarks of the ping-pong mechanism in the piRNA pathway  pathway, was initially identified as a defense mechanism against transposition of mobile elements in the germline of osophila . Biogenesophila] . The piR pathway .Drosophila, research in insects and other arthropods revealed an ancestral role for piRNAs in defense against transposable elements in both somatic and germline tissues  in Drosophila reflects the duplication of an ancestral gene (“piwi/aub”) at the base of the Brachycera. In aedine and culicine mosquitoes, on the other hand, the hypothetical “piwi/aub” ancestral gene has undergone a remarkable expansion   encodes one ago-3 and two “aub/piwi” genes  . By contpiwi/aub . Also thi” genes .Ae. aegypti and Ae. albopictus. Of the seven Piwi-related genes and one ago-3 gene that are identified in the genome of Ae. aegypti, piwi-4, piwi-5, piwi-6, and ago-3 are clearly expressed in the somatic tissues of the mosquito  and three homologs of ago-5 of An. gambiae   . In Ae. Flaviviridae)  (Ae. aegypti and Ae. triseriatus) as well as in C6/36 cells that are derived from Ae. albopictus   as well virales) . Pathogevirales) . This phbopictus .Ae. aegypti mosquitoes that expressed an RNA hairpin targeting the prM region of DENV-2 and that were shown to be refractory to DENV-2 infection , confirming the specificity of the antiviral RNAi response. Introgression of the RNA hairpin transgene into mosquitoes of another genetic background resulted in a change from the susceptible to the refractory phenotype  the production of viral siRNAs (vsiRNAs); (2) increase in viral replication and mortality in ago-2 and dcr-2 mutants; (3) the presence of genes in the viral genome that encode VSRs  arboviruses in general are considered to be lacking VSR genes; although a few convincing candidates were proposed, conclusive evidence within the native viral context or in the natural vector was not achieved , obtained after deep sequencing analysis, are mapped to the viral genomes, cold and hot spot regions of under- and over-representation, respectively, are typically observed. The occurrence of these regions may result from preferential processing by Dcr-2 but other causes, such as differential stability of small RNAs and preferential targeting of regions for production of viral DNA forms  need to be considered. Moreover, the preferential detection may be the result of technical issues that occur in deep sequencing experiments, such as library preparation . IdeallyBelow follows a detailed analysis of the function of the exo-RNAi pathway as an antiviral defense mechanism in mosquitoes. Production of vsiRNAs is summarized in Togaviridae) are Sindbis virus (SINV), Semliki Forest virus (SFV), Chikungunya virus (CHIKV) and o’nyong-nyong virus (ONNV). Alphaviruses are characterized by a single linear (+) ssRNA genome (11–12 kb) that also serves as the mRNA for the non-structural polyprotein while a subgenomic RNA produced during infection functions as the source for the structural proteins. Under natural conditions, SINV circulates between Culex mosquitoes and birds, while humans act as “dead-end” hosts. Both SFV and CHIKV are transmitted to humans by Aedes species while the primary vectors of ONNV are anopheline mosquitoes , resulted in enhancement of viral infection by feeding but did not affect mortality. Increased replication of recombinant SINV vectors was also observed in dcr-2 null mutant Ae. aegypti mosquitoes  although in this case only increased dissemination of SINV from midgut to other tissues was observed  and ago-3  but not ago-1 (miRNA pathway) or the Piwi/Aub-related ago-4 and ago-5 (piRNA pathway)   resultedpathway) .A. gambiae mosquitoes resulted in the production of 21 nt vsiRNAs that show similar properties to that observed following injection of SINV in Ae. aegypti mosquitoes  and Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways; However, in contrast to what is observed during midgut infection of Ae. aegypti and Ae. albopictus mosquitoes were injected with CHIKV, vsiRNAs (21 nt) were produced but also viral small RNAs of piRNA size (23–30 nt)    are DENV (present as four serotypes), West Nile virus (WNV) and Zika virus (ZIKV). Flaviviruses are characterized by a single linear (+) ssRNA genome (10–11 kb) that also functions as mRNA and encodes a single polyprotein that is processed to both structural and non-structural proteins by viral and host proteases. Both DENV and ZIKV  are transmitted to humans by Ae. aegypti and Ae. albopictus mosquitoes. The primary vectors for transmission of WNV are Culex mosquitoes with birds as preferential and humans as “dead-end” hosts, respectively  were detected predominantly in whole body samples, in contrast to other classes of viral small RNAs that were more prevalent in midgut  mosquitoes show a clear RNAi response upon oral infection with WNV  compared with wild-type at comparable time points (Replication of YFV was demonstrated to be significantly higher in e points . In addiPhenuiviridae family), La Crosse virus, Schmallenberg virus and the type species Bunyamwera virus . Bunyaviruses are characterized by a tripartite linear (-) or ambisense ssRNA genome that is organized in three segments, L (6–7 kb), M (3.0–4.5 kb), and S (1.0–1.7 kb). Several different species of mosquito  are able to act as vectors for transmission of RVFV  species  resulted in the production of vsiRNAs that were derived from all three segments with abundance gradient M > S > L and corresponded to genome and antigenome strands in approximately equal ratios (Culex mosquitoes) could be caused by hybridization of complementary N and NSs mRNAs  of three different mosquito species  and during oral infections of mosquitoes with SINV-B2 (An. gambiae with recombinant ONNV expressing B2 from Nodamura virus (Nodaviridae) by both injection and feeding also resulted in increases in mortality and viral titers  persistent infections in mosquito vectors such that transmission to the next vertebrate host can be accomplished. The importance to replicate at low levels for maintenance of the persistent state was dramatically demonstrated in experiments using recombinant SINV that expresses the strong RNAi inhibitor protein B2 of FHV ( SINV-B2 . Altoget SINV-B2 . Infectil titers .Flaviviridae) possesses strong VSR activity. VSR activity could also be demonstrated for capsid proteins of other flaviviruses infecting different hosts  isolated from bats but with unknown vector)   . Furthert siRNA) . While nWhile alphaviruses  are generally not considered to encode VSRs , a recent study nevertheless showed that the non-structural proteins nsP2 and nsP3 of CHIKV can inhibit dsRNA- and siRNA-mediated silencing in insect, mammalian and plant cells . VSR actDuring flaviviral infections, accumulation of sfRNA, an abundant non-coding subgenomic sfRNA representing the last 525 nt of the 3′UTR, is observed as a result of incomplete degradation of the flaviviral genome by the 5′–3′ exoribonuclease XRN1 located in processing bodies (PBs) in mammalian cells, where also the RNAi machinery is located . IncomplAe. albopictus U4.4 cells and Drosophila S2 cells. Engineering of the alphavirus SFV with sfRNA of WNV increased its replication in mosquito cells  while reporter assays established that sfRNA could interfere with both siRNA- and miRNA-mediated silencing in insect cells  or a mutant form that is defective for production of sfRNA  while no effect was observed in RNAi-deficient cells  . Furthermore, persistent infections of wild-type LACV and LACV deficient for NSs showed similar growth properties in C6/36 cells, indicating that NSs did not have a function as a suppressor of innate immunity (while a role as inhibitor of the exo-RNAi pathway could not be addressed in this experiment)    . In the eriment) . RVFV ineriment) .Evidence has been presented for selection of genomes with mutations in regions highly targeted by RNAi as a mechanism of evasion , 2015a,bCulex strains  to test candidate VSRs in infected mosquitoes may result in the identification of false positives. While recombinant SFV expressing capsid protein of ZIKV could replicate to higher levels than control SFV as observed before for recombinant SINV , this efDrosophila, where expression is predominantly in the germline , such information is not available in many studies that describe the generation of “viral small RNAs of piRNA size” or “vpiRNA-like small RNAs.” Using a strict definition, typical hallmarks of piRNAs are: (1) sizes of 25–29 nt; (2) ping-pong amplification signature ; (3) enrichment of the separation of the 5′-ends of complementary viral piRNAs by 10 nt; and (4) resistance to β-elimination indicating 2′-cleotide . HoweverBelow follows a detailed analysis of the occurrence of the piRNA pathway as a potential antiviral defense mechanism in mosquitoes. Production of vpiRNAs is summarized in Ae. aegypti and Ae. albopictus mosquitoes were injected with CHIKV, not only vsiRNAs (21 nt) were produced but also viral small RNAs of piRNA size (23–30 nt)  . VpiRNAsth CHIKV . During th CHIKV .Ae. aegypti mosquitoes with DENV-2, the presence of piRNA-sized viral small RNAs (24–30 nt) was revealed preferentially during early infection along with the presence of canonical vsiRNAs and viral small RNAs of unusually short length (<20 nt)  . After a(<20 nt) . It is n(<20 nt) .Ae. albopictus mosquitoes with DENV-2, three classes of viral small RNAs could be identified: unusually small (ultrashort) RNAs , vsiRNAs (20–23 nt) and candidate vpiRNAs (24–30 nt)  . Both vs4–30 nt) .Ae. albopictus, expression of piwi-1-4 is increased in adult females after blood feeding. However, no increase in expression of ago-3 and Piwi-related genes is observed following DENV-2 infection of adult females  resulted in the production of both vsiRNAs and vpiRNAs . Clear strand enrichment was observed for RVFV-derived vpiRNAs but differed according to the different segments . For the S segment, the majority of  vpiRNAs were derived from the region of the mRNA encoding the nucleocapsid (N) protein (the other gene of the ambisense S segment (NSs) being transcribed from the antigenome strand; Rift Valley Fever virus infection  of three different mosquito species  (Flaviviridae) infection   while thnfection  or SINV nfection . AlthougAe. albopictus cell lines (C6/36 and C7-10) that are deficient in the production of vsiRNAs and that show higher production of viral titers in comparison with mosquito cell lines that are competent for vsiRNA production  ] . Another defense . In thesToxorhynchites amboinensis (Culicidae) infected with SFV (Togaviridae) (Cu. sonorensis with Bluetongue virus (Reoviridae), SBV or BUNV   . By contvirales) .Below follows a detailed analysis of the occurrence of the piRNA pathway as a potential antiviral defense mechanism in mosquito cell lines. Comparison of the significance of the piRNA pathway with the exo-RNAi pathway regarding antiviral defense is provided. Production of vpiRNAs and vsiRNAs in cell lines is summarized in Ae. albopictus, non-canonical patterns of viral small RNAs are observed that are likely to be caused by its inability of dicing dsRNA substrates and a non-functional Dcr-2 enzyme  and U4.4 .During RNA virus infection of the C6/36 cell line from 2 enzyme . Genotypop codon . Anotherof Dcr-2 . In seveDuring SINV infections of C6/36 cells mainly viral small RNAs of piRNA size (23–28 nt) were produced that were distributed unevenly between genomic (70%) and antigenomic (30%) strands . Hot spoDuring DENV-2 infections of C6/36 cells, viral small RNAs were generated that corresponded almost exclusively from the sense strand and were derived from a few specific regions of the genome . MoreoveAnalysis of viral small RNAs produced during infections of C6/36 cells with LACV also revealed the predominant presence of viral small RNAs of piRNA-like size (24–28 nt) . DespiteA comparative study was carried out among Dicer-2-competent U4.4 and Aag-2 cell lines and the Dcr-2-defective C6/36 cell line with respect to RVFV infection     . The impviridae) .Infections of Aag-2 cells with alphavirus  did not only result in production of vsiRNAs but also of viral small RNAs that have the characteristics of piRNAs . VpiRNAspiwi-4 affected accumulation of piRNAs derived from transposable elements, no piRNAs corresponding to transposon sequences were detected in Piwi-4 immunoprecipitates, leading to the proposal that Piwi-4 indirectly regulates the function of the piRNA pathway , possibly by modulating the activity of the other piRNA factors or regulating the amount of substrate that enters the piRNA pathway. The diversification of Piwi-related factors in Ae. aegypti mosquitoes likely reflects their specialization in the control of different classes of parasitic nucleic acids (mainly transposons), with Piwi-5 and Ago-3 involved more specifically in production of piRNAs during viral infections . Knock-down of all expressed Piwi-related proteins  resulted in a strong decrease in production of vpiRNAs during SFV infection. However, knock-down of piwi-4 had no effect on vpiRNA accumulation, indicating an effector function of Piwi-4 to target and silence the virus independent of vpiRNAs . Despite differences in vpiRNA levels, the properties of SFV-derived vpiRNAs were very similar between Dcr-2-deficient AF319 and Dcr-2-competent AF5 cells such as distribution along the viral genome and the occurrence of the ping-pong signature. Re-introduction of Dcr-2 by expression plasmids also did not alter the properties of vpiRNAs in AF319 cells , vpiRNAs with ping-pong signature (but without positive strand bias as is commonly observed) were detected in association with both Ago-2 and Piwi-4. However, binding may be indirect since Piwi-4 is found in complexes with proteins of both siRNA  and piRNA  pathways  was used to engineer a Dcr-2-defective cell line (AF319) representative of ineering . When in19 cells . Knock-dago-2 resulted in increased expression of a CHIKV reporter replicon  since its knock-down did not affect BUNV  or ZIKV (Flaviviridae) replication , that was identified as a cofactor in the ping-pong amplification loop in osophila . No antilication .O-methylated at their 3′ termini), show an almost exclusively sense orientation and derive from a very limited number of hotspots  piwi-6. However, none of the knock-downs of ago-3 and Piwi-related genes (as well as ago-2) resulted in an increase in DENV replication levels in Aag-2 cells  in Aag-2 cells, implicating the involvement of both the siRNA and the piRNA pathway in antiviral defense  caused production of both vsiRNAs and vpiRNAs in non-host Aag-2 cells (Also infections of SBV (with -2 cells . As for -2 cells .Infections of Aag-2 cells (and U4.4 cells) with BUNV resulted in production of both vsiRNAs and vpiRNAs . VsiRNAsago-2 provided antiviral activity to all infections, piwi-4 only protected against mosquito-borne bunyaviruses  SBV infections. The analysis therefore supports the adaptation of the piRNA pathway in Aag-2 cells to specific vector-virus combinations. The miRNA pathway also has a proviral function during infections with mosquito-specific bunyaviruses, while silencing of ago-1 increases replication of midge-specific viruses ] and midge-borne bunyaviruses [SBV and Sathuperi virus (SATV)]. While aviruses . InteresThere are several indications of interactions between siRNA and piRNA pathways during antiviral defense in mosquitoes which were revealed in experiments with recombinant viruses expressing the B2 VSR that sequesters dsRNA and also by the detection of protein complexes that contain factors of both RNAi pathways and their respective small RNAs. During viral infection temporal patterns of vpiRNA and vsiRNA accumulation were also observed suggesting the predominance of a particular pathway at different stages of infection.Ae. albopictus mosquitoes with recombinant CHIKV that expresses the B2 VSR protein (CHIKV-B2) resulted in higher virulence/mortality and decreased production of vsiRNAs while levels of vpiRNAs were increased . During oral infection of nfection . In Aag-nfection . Also due genome . ProductAe. aegypti, abundant small RNAs were detected that mapped to transposon sequences  and on their association with particular Piwi proteins  in immunoprecipitates. Since the genome of Ae. aegypti is very rich in transposon sequences   . Recentler (51%) . In Aag-er (51%) . As is ter (51%) , cellula vpiRNAs . It can Ae. albopictus mosquitoes by CHIKV. While inhibition of RT activity increased mortality in infected mosquitoes, viral replication levels remained unaffected leading to the proposal that production of vDNA forms increased the tolerance (but not the resistance) of mosquitoes to CHIKV infections  in the inoculum, could decrease the infection rate of CHIKV during blood feeding of Ae. aegypti mosquitoes  infections of mosquitoes and/or mosquito cell lines, viral DNA forms (vDNAs) are produced by cellular retrotransposon-derived reverse transcriptase (RT) activity . Synthesfections . During produced . In extesquitoes .Culex-derived cell lines) showed that production of vDNA forms did not involve the entire viral genome but rather discrete patches that indicate separate events of template switching of RT activity between template retrotransposon RNA and nearby arboviral genomic RNA or mRNA  but may be the result of a combination of the expansion of the piRNA pathway (in Culex and both Aedes species but not in Anopheles) and the load of retrotransposons in the genome   . Infectiopheles) .Aedes mosquitoes, particularly with long terminal repeat (LTR) retrotransposons of the Gypsy and Pao Bell families  during viral infections. The DExD/H helicase domains of Dcr-2 enzymes in insects and the RIG-I-like receptors (RLRs), that trigger interferon I responses in mammals, were shown to be phylogenetically related and could be grouped together in the family of duplex RNA-activated ATPases (DRAs) family . A recenize vDNA . Evidencize vDNA .Cx. quinquefasciatus (Hsu) induced the expression of the secreted peptide CxVago in a Dcr-2-dependent manner  and after infection of the Ae. albopictus RML12 cell line with DENV  but not the classical CxDome receptor for the unpaired ligands, leading to the speculation of the antiviral response occurring through an alternative JAK receptor  spread of infection was stimulated rather than viral replication in initially infected cells .Drosophila species showed the accelerated evolution of genes in the exo-RNAi pathway with respect to miRNA genes, which was contributed to the molecular arms race between host antiviral response and pathogenic virus infection  and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis reveals the identification of immune genes among different predicted cellular targets   . In anotsferase) . Also aan factor . Regardisquitoes .Ae. albopictus mosquitoes through the injection of miRNA mimics or antagomirs revealed a positive role for the midgut-specific aae-miR-281 to regulate DENV replication  transmission was the most frequent pathway for viral dispersion in the mosquito population . Thus, mNodaviridae; Culex Y virus , the production of vsiRNAs is dwarfed by the presence of vpiRNAs that show a clear ping-pong pattern  (In at 21 nt . During igenome) . As alsoigenome) , hot spoCulex-derived cell lines, different patterns of viral small RNA production are observed following infection with the mosquito-specific flavivirus Calbertado virus  and the mosquito-specific rhabdovirus Meridavirus  (Flaviviridae and Rhabdoviridae) while it is expected that infections with arboviruses and mosquito-specific viruses of the same family will generate similar patterns of viral small RNAs in similar hosts. Mosquito-specific (CLBOV) and arboviral (WNV) flaviviral infection of Culex tarsalis-derived CT cells indeed results in the generation of very comparable profiles of viral small RNAs (In gnature) . These dall RNAs .Wolbachia co-infection. While both mosquito-specific and arboviral flavivirus (with (+) ssRNA genome) infections are effectively cleared by Wolbachia, bunyaviruses [with segmented (-) or ambisense ssRNA genome] are resistant to the endosymbiont and the resistant phenotype applies to both mosquito-specific and arboviral species  , it was reported that miRNA expression was very little affected during infection of us (PCV) . FurtherThe above observations of viral small RNA accumulation indicate that mosquito-specific viruses can activate both the exo-RNAi (siRNA) and piRNA mechanism while other unknown RNA degradation pathways are also revealed. In conclusion, there is no evidence for significant differences between infections of arboviruses and mosquito-specific viruses belonging to the same virus family with respect to the RNAi response and other resistance mechanisms, although higher amounts of viral small RNAs may accumulate in infections with mosquito-specific viruses because of their higher replication level, potentially leading to pathogenicity.The ability of (arbo)viruses to induce RNAi effects and achieve VIGS is mainly determined by two properties, i.e., the encoding of VSR genes and the characteristics of the RdRp enzymes with respect to kinetics and replication capacity . Both pr-4 mutations per nucleotide copied  of CHIKV exhibited lower infection and dissemination titers which was attributed to reduced genetic diversity  effects of both antimutator and mutator variants were initially thought to be mainly caused by the reduction and augmentation of genetic diversity leading to the proposal that RdRp enzymes of RNA viruses have become optimized during evolution to be neither too accurate nor too erroneous. However, in mammalian cells it was also demonstrated that mutator variants of SINV show an increased recombination rate leading to defective interfering (DI) particle production . WhetherArbovirus infections of mosquitoes are characterized by the absence of pathogenic effects that allow their spread within the mosquito body to the salivary glands from where infection of new vertebrate hosts can be initiated. Despite the persistent character of the infections, functional viral small RNAs were produced by recombinant viruses that could confer resistance to secondary infections by unrelated arboviruses . HoweverBecause of the persistent character of their infections, engineered arboviruses or related mosquito-specific viruses could be developed as effective gene silencing vectors in mosquitoes. RNAi is a very powerful tool to carry out reverse genetics studies but in many cases the delivery of the dsRNA trigger is limiting . EngineeUnderstanding of the parameters that determine the persistent character of arbovirus infections may have important implications for the design of silencing vectors in insects in general. In this review, an extensive discussion is presented regarding the interaction of arbovirus infections with the mosquito RNAi machinery that can serve as a background for the improvement of the capacity of arbovirus vectors to induce specific gene silencing. As already mentioned in other studies , two impIt remains under debate whether arboviruses encode canonical VSR genes. However, other strategies for evading the RNAi response can exist such as the shielding of viral replication complexes in membrane structures  or the aBesides activation of the exo-RNAi pathway, which is thought to carry out the gene silencing effects, arbovirus infections in mosquitoes also can result in the abundant production of vpiRNAs. Since their involvement in antiviral defense was not clearly demonstrated , the funDrosophila and mosquitoes and may be related to the abundance of retrotransposons encoding active RT enzymes in the insect genomes (An interesting observation is the production of viral DNA forms during infection which is thought to be related to antiviral defense . This ph genomes . Viral D genomes . Integra genomes .Besides its role in the exo-RNAi pathway, Dcr-2 was also found to have a function in the systemic antiviral response. The helicase domain of Dcr-2 functions as a dsRNA sensor and initiates a signaling cascade for the production of secreted antiviral signaling peptides , 2014, sWhile arboviruses establish persistent infections and trigger an RNAi response in mosquitoes, their development as silencing vectors may not be practical because of their capacity to cause disease in humans and livestock. Fortunately, mosquito-specific viruses related to arboviruses were described for which reverse genetics systems are available . In addiJL and LS conceived the idea, and designed and wrote the manuscript. AK designed the figure and contributed to the revision of the text. GS revised and contributed to improve the final version of the manuscript. All authors read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The specific uptake size index (SUSI) of striatal FP-CIT uptake is independent of spatial resolution in the SPECT image, in contrast to the specific binding ratio (SBR). This suggests that the SUSI is particularly appropriate for multi-site/multi-camera settings in which camera-specific effects increase inter-subject variability of spatial resolution. However, the SUSI is sensitive to inter-subject variability of striatum size. Furthermore, it might be more sensitive to errors of the estimate of non-displaceable FP-CIT binding. This study compared SUSI and SBR in the multi-site/multi-camera (MULTI) setting of a prospective multi-center study and in a mono-site/mono-camera (MONO) setting representative of clinical routine.n = 438) and healthy controls (n = 207) from the Parkinson Progression Marker Initiative. The MONO setting included 122 patients from routine clinical patient care in whom FP-CIT SPECT had been performed with the same double-head SPECT system according to the same acquisition and reconstruction protocol. Patients were categorized as “neurodegenerative” (n = 84) or “non-neurodegenerative” (n = 38) based on follow-up data. FP-CIT SPECTs were stereotactically normalized to MNI space. SUSI and SBR were computed for caudate, putamen, and whole striatum using unilateral ROIs predefined in MNI space. SUSI analysis was repeated in native patient space in the MONO setting. The area (AUC) under the ROC curve for identification of PD/“neurodegenerative” cases was used as performance measure.The MULTI setting included patients with Parkinson’s disease . The SUSI (computed in MNI space) performed slightly better than the SBR in the MULTI setting  and slightly worse in the MONO setting . There was a trend toward larger AUC difference between SUSI and SBR in the MULTI setting compared to the MONO setting (p = 0.073). Variability of voxel intensity in the reference region was larger in misclassified cases compared to correctly classified cases for both SUSI and SBR .In both settings, the highest AUC was achieved by the putamen (minimum over both hemispheres), independent of the semi-quantitative method (SUSI or SBR). The putaminal SUSI provided slightly better performance with ROI analysis in MNI space compared to patient space (AUC = 0.969 vs. 0.961, The SUSI is particularly useful in MULTI settings. SPECT images should be stereotactically normalized prior to SUSI analysis. The putaminal SUSI provides better diagnostic performance than the SUSI of the whole striatum. Errors of the estimate of non-displaceable count density in the reference region can cause misclassification by both SUSI and SBR, particularly in borderline cases. These cases might be identified by visual checking FP-CIT uptake in the reference region for particularly high variability. Single-photon emission computed tomography (SPECT) with the I-123 labeled dopamine transporter (DAT) ligand FP-CIT is widely used for detection (or exclusion) of nigrostriatal degeneration in patients with clinically uncertain parkinsonian syndrome (PS) [Semi-quantitative analysis in radionuclide imaging often aims at estimating the binding potential (BP), first defined by Mintun and co-workers as BP = Bmax / KD, where Bmax is the density of available binding sites (here DAT) and 1/KD is the affinity of the tracer (here FP-CIT) for the binding site . The binwhere Cs and Cnd are the equilibrium concentration of specifically bound and non-displaceable tracer in the ROI, respectively. Determination of BPnd requires dynamic imaging and arterial blood sampling for full tracer kinetic modeling. This is not feasible in clinical routine so that a number of methods have been developed to estimate BPnd from a single static scan during equilibrium  void of DAT. The SBR according to formula (2) is based on the assumption that CR approximates the count concentration originating from non-displaceable FP-CIT binding in the striatal ROI . The striatal ROI for computation of the SBR usually anatomically delineates the striatum or the striatal subregion of interest such as caudate nucleus or putamen , 9. A macount density in the striatal ROI by a measure of total counts in the ROI. More precisely, the SUSI is defined asThe specific uptake size index (SUSI) has been proposed to improve accuracy of semi-quantitative analysis in FP-CIT SPECT in practice , 15, 16.where T is the total number of counts originating from a large striatal ROI, V is the volume of the striatal ROI, and CR is the count concentration in the reference region.The striatal ROI for the computation of the SUSI is chosen sufficiently large around the striatum to guarantee that all counts originating from the striatum are detected within this ROI. Assuming that image reconstruction is “activity conserving” , summing the activity over all voxels within the large striatum ROI collects all counts originating from the striatum. This holds true independently of the spatial resolution in the reconstructed SPECT image as long as the ROI is large enough to encompass all counts originating from the striatum. Thus, the SUSI is independent of spatial resolution . The SUSThe SUSI has been used successfully for the quantitative characterization of age effects on DAT availability in healthy subjects from the European normal control database of FP-CIT SPECT (ENC-DAT) . A totalThe SUSI is typically computed in the original SPECT images in patient space after manual reorientation , 15, 16.A recent study suggested the SUSI to be more sensitive than the SBR to errors of the estimate of non-displaceable count density in the striatum by the count density CR in the reference region . In bothAim of the present study was to compare the SUSI and the SBR with respect to detection of nigrostriatal degeneration in (i) a sample of patients with PS in whom FP-CIT SPECT has been performed with the same SPECT camera using the same acquisition protocol with radius of rotation within very tight limits  and the n = 84, 39 females, 67.3 ± 10.0 years) comprised (i) the Lewy body disease spectrum (n = 60) including Parkinson’s disease , PD dementia , and dementia with Lewy bodies , and (ii) atypical parkinsonian syndromes  including multiple systems atrophy , progressive supranuclear palsy , and corticobasal degeneration . The non-neurodegenerative group  comprised (i) essential tremor (n = 2), (ii) vascular parkinsonism , (iii) drug-induced parkinsonism (n = 11), (iv) psychogenic parkinsonism (n = 1), (v) possible Alzheimer’s disease , and (vi) normal pressure hydrocephalus . The clinical diagnoses were established by a movement disorder specialist based on current consensus criteria and review of all relevant medical charts and clinical follow-up data (mean follow-up 26.8 ± 14.5 months). Patients with other than the listed diagnosis were excluded.One hundred twenty-two patients in whom FP-CIT SPECT had been performed for detection (or exclusion) of nigrostriatal degeneration as part of routine clinical work-up were recruited retrospectively from the patient database of the Department of Nuclear Medicine of the University Medical Center Freiburg. The patients were categorized into two groups: “neurodegenerative PS” and “non-neurodegenerative PS.” The neurodegenerative group  [www.ppmi-info.org. The PPMI is a longitudinal, multi-center study that aims to assess the progression of clinical features, imaging, and biologic markers in patients with (idiopathic) PD and healthy control (HC) subjects. All PD patients were in an early stage of the disease (diagnosis of PD within the last 2 years prior to screening). Details of the eligibility criteria are given at http://www.ppmi-info.org/wp-content/uploads/2014/01/PPMI-AM7-Protocol.pdf.Data were obtained from the Parkinson’s Progression Markers Initiative (PPMI) database (d-data/) . Up-to-dn = 1710). The analyses of the present study included the first FP-CIT SPECT of all HC subjects and all PD patients, that is, the analyses included also FP-CIT SPECT at an “unscheduled” visit if FP-CIT SPECT at the screening visit was not available. Furthermore, the analyses included not only “regular” HC subjects and “regular” PD patients but also subjects who (i) declined participation in the PPMI study after the screening visit but before inclusion in the study (n = 20), or (ii) withdrew agreement after inclusion in the study (n = 78), or (iii) were excluded from participation in the study due to a reason not related to FP-CIT SPECT (n = 23). This resulted in 656 FP-CIT scans. Visual inspection resulted in exclusion of 11 of these scans: three HC scans were excluded because of clearly reduced striatal FP-CIT uptake ; eight PD scans were excluded because of clearly normal FP-CIT uptake in the striatum . This resulted in the inclusion of a total of 645 FP-CIT SPECTs: 438 of PD patients and 207 of HC subjects.All FP-CIT scans available from the PPMI database on 22nd November 2017 were downloaded in DICOM format, independent of subgroup and visit (http://www.ppmi-info.org/study-design/research-documents-and-sops/). Raw projection data had been transferred to the PPMI imaging core lab for central image reconstruction using an OSEM algorithm on a HERMES workstation  [FP-CIT SPECT data had been acquired at 24 different centers using different SPECT camera models. All centers had been qualified for participation in the study by an image center qualification process including a technical set up visit to optimize the acquisition and reconstruction protocol for the specific SPECT system to be used in the study . The tar Sweden) . PMOD  with ROI analysis in MNI space. For this purpose, individual FP-CIT SPECTs were transformed  into MNI space using the Statistical Parametric Mapping software package (version SPM12) and a custom-made FP-CIT template  with ROI analysis in MNI space. It was computed separately for unilateral caudate and unilateral putamen using large three-dimensional unilateral ROIs predefined in MNI space Fig. c. The unSUSI and SBR were tested for identification of “neurodegenerative PS” in the MONO setting and for identification of PD patients in the MULTI setting using the minimum over both hemispheres for both semi-quantitative measures. The area (AUC) under the receiver operating characteristic (ROC) curve was employed as performance measure. Total accuracy, sensitivity, and specificity were computed with the cut-off fixed by Youden’s criterion .In order to test for potential impact of individual putamen size on the diagnostic performance of the putaminal SUSI computed in patient space, the volume of the AAL putamen ROI after transformation into patient space was used as measure of individual putamen size (mean of both hemispheres). Putamen size was compared between true positive, true negative, false positive, and false negative cases as classified by the SUSI computed in patient space. Univariate analysis of variance was used for this purpose.t tests. This was done separately for SUSI and SBR. Second, SUSI and SBR were compared with respect to the difference of the relative IQR between correctly classified scans and incorrectly classified scans using univariate analysis of variance of relative IQR with both, correctness of the SUSI-based classification and correctness of the SBR-based classification as fixed factors. Cut-offs fixed by Youden’s criterion were used to categorize scans as correctly or incorrectly classified.In order to test the secondary hypothesis, that is, the impact of the uncertainty of the estimate of non-displaceable count density by the 75th percentile of voxel intensities in the reference region on the diagnostic performance of SUSI and SBR, the interquartile range (IQR) of voxel intensities in the reference region relative to the 75th percentile of voxel intensities in the reference region was used as uncertainty measure (relative IQR). First, relative IQR was compared between correctly classified scans (true positive or true negative) and incorrectly classified scans  using unpaired SUSI – AUCSBR) (MULTI) > (AUCSUSI – AUCSBR) (MONO), where AUCSUSI is the AUC of the putaminal SUSI and AUCSBR is the AUC of the putaminal SBR. Bootstrapping with 100,000-fold resampling was used to estimate the distribution of (AUCSUSI – AUCSBR), separately for the MULTI and the MONO setting. Then, the distribution of (AUCSUSI – AUCSBR) was compared between the MULTI and the MONO setting by generating 10,000 random pairs of (AUCSUSI – AUCSBR) (MULTI) and (AUCSUSI – AUCSBR) (MONO) from their respective distributions and counting the number of random pairs that fulfilled the alternative hypothesis (AUCSUSI – AUCSBR) (MULTI) ≤ (AUCSUSI – AUCSBR) (MONO).In quantitative terms, the primary hypothesis of the study (the diagnostic performance of the SUSI relative to the SBR is better in the MULTI setting than in the MONO setting) states that . All these discrepant cases had “neurodegenerative PS” and presented with borderline findings in FP-CIT SPECT Fig. . Classifp = 0.314) and correctness of patient space SUSI  as fixed factors (standard of truth * correctness interaction p = 0.170).Putamen volume was smaller in the two false positive cases compared to the 36 true negative cases based on putaminal SUSI in patient space; it was larger in the ten false negative cases compared to the 74 true positive cases .The relative IQR of voxel intensities in the reference region was not different between the 12 patients with incorrect classification based on patient space SUSI compared to the remaining 110 patients .In the harmonized MULTI setting, too, the putamen achieved the highest performance with both SUSI and SBR Table . The AUCp = 0.207). Classification of subjects (as PD or HC) was incorrect in 30 (4.7%) and 23 (3.6%) of the 645 subjects when based on putaminal SBR or putaminal SUSI, respectively , followed by the SUSI (AUC = 0.993) and the SBR of the whole putamen (AUC = 0.991) Fig. b. The AUt test p = 0.007). The same was true for the 30 subjects with incorrect SBR-based classification compared to the remaining 615 subjects .The relative IQR of voxel intensities in the reference region was higher in the 23 subjects with incorrect SUSI-based classification compared to the remaining 622 subjects with correct SUSI-based classification . SUSI-based classification was correct and SBR-based classification was incorrect in two HC subjects and 13 PD patients. SUSI-based classification was incorrect and SBR-based classification was correct in three HC subjects and 5 PD patients. The difference of relative IQR between incorrectly classified scans and correctly classified scans based on the SUSI was larger for scans with correct SBR-based classification  compared to scans with incorrect SBR-based classification  .Classification based on putaminal SUSI or on putaminal SBR was discrepant in 23 of the 645 patients (3.6%). The relative IQR of voxel intensities in the reference region was higher in the 23 subjects with discrepant classification compared to the remaining 622 subjects with concordant classification  and slightly better in the MULTI setting .The putaminal SUSI performed slightly worse than the putaminal SBR in the MONO setting  in the MULTI setting and in the MONO setting estimated by bootstrapping is shown in Fig. SUSI − AUCSBR) was 0.002 (95% CI -0.001–0.005) in the MULTI setting and -0.007 (-0.021–0.004) in the MONO setting. The sign test revealed a trend toward larger (AUCSUSI – AUCSBR) in the MULTI setting compared to the MONO setting (p = 0.073).The distribution of the difference . However, direct statistical comparison of the AUC difference between the SUSI and the SBR between the MULTI setting and the MONO setting showed a trend toward larger AUC difference in the MULTI setting compared to the MONO setting , the putaminal SUSI (computed in MNI space) performed slightly worse than the putaminal SBR in the MONO setting (AUC = 0.969 versus 0.976) and slightly better in the MULTI setting (AUC = 0.993 versus 0.991). This is in line with the primary hypothesis, although neither the difference in the MONO setting nor the difference in the MULTI setting reached statistical significance  in the PPMI by careful adaption of the acquisition protocol for each single camera and central image reconstruction at a core imaging center. Second, the relative IQR of the voxel intensity in the reference region was significantly larger in cases that were incorrectly classified by the putaminal SUSI compared to those that were correctly classified. The same was observed for the putaminal SBR. This demonstrates that the diagnostic performance of both, SUSI and SBR, is affected by uncertainty of the estimate of non-displaceable striatal FP-CIT count density by the count density in the reference region. The difference of the relative IQR between cases that were incorrectly classified by the putaminal SUSI and those that were correctly classified by the putaminal SUSI was larger in cases correctly classified by the putamen SBR compared to those incorrectly classified by the putamen SBR Fig. . This isGoethals and co-workers compared the SUSI computed in patient space and SBR computed in template space for detection of nigrostriatal degeneration in a mono-camera setting very similar to the mono-camera setting in the present study . The AUCThe following limitations of the present study should be noted. First, the classification performance of SUSI and SBR in FP-CIT SPECT not only depends on the setting (multi-site/multi-camera versus mono-site/mono-camera). Classification performance of both semi-quantitative measures also depends on the subjects referred to FP-CIT SPECT.The PPMI PD sample differs from the group of patients with neurodegenerative PS in the clinical mono-site/mono-camera sample in several respects, particularly by inclusion of patients with atypical neurodegenerative PS  in the clinical mono-site/mono-camera sample and the exclusion of subjects without evidence of dopaminergic deficit (SWEDD) from the PPMI PD sample . There iSecond, the excellent performance of both SUSI and SBR in the PPMI sample limits the power to detect performance differences (ceiling effect). Good diagnostic performance of FP-CIT SPECT independent of the analysis method in general makes it difficult to demonstrate differences in the diagnostic performance between methods and to reliably identify factors that might affect performance .Third, classification performance of semi-quantitative measures also depends on the SPECT cameras, the acquisition protocol (including radius of rotation and delay of acquisition after tracer administration), the reconstruction algorithm, and, in the multi-site/multi-camera setting, the extent of harmonization of acquisition protocol and reconstruction algorithm. These factors not only affect the absolute classification performance of both SUSI and SBR, they also might affect the performance difference between SUSI and SBR. This may affect the generalizability of the results of the present study. In particular, it is to be expected that the SUSI more clearly outperforms the SBR in multi-site/multi-camera settings with lower degree of harmonization than in the PPMI.The specific uptake size index (SUSI) of striatal FP-CIT uptake is particularly appropriate for semi-quantitative analysis in multi-site/multi-camera settings in which camera-specific effects increase inter-subject variability of spatial resolution. SPECT images should be stereotactically normalized prior to SUSI analysis in order to reduce the impact of inter-subject variability of striatum size. The SUSI of the putamen provides better diagnostic performance than the SUSI of the whole striatum. In mono-camera settings without camera-specific inter-subject variability of spatial resolution, the specific binding ratio (SBR) provides similar performance as the SUSI. Errors of the estimate of non-displaceable count density can cause misclassification by both, SUSI and SBR, particularly in borderline cases. We recommend to check for high uncertainty associated with high variability of voxel intensity in the reference ROI in addition to verification of proper placement of the ROI.


Despite the importance of placental function in embryonic development, it remains poorly understood and challenging to characterize, primarily due to the lack of non-invasive imaging tools capable of monitoring placental and foetal oxygenation and perfusion parameters during pregnancy. We developed an optoacoustic tomography approach for real-time imaging through entire ~4 cm cross-sections of pregnant mice. Functional changes in both maternal and embryo regions were studied at different gestation days when subjected to an oxygen breathing challenge and perfusion with indocyanine green. Structural phenotyping of the cross-sectional scans highlighted different internal organs, whereas multi-wavelength acquisitions enabled non-invasive label-free spectroscopic assessment of blood-oxygenation parameters in foeto-placental regions, rendering a strong correlation with the amount of oxygen administered. Likewise, the placental function in protecting the embryo from extrinsically administered agents was substantiated. The proposed methodology may potentially further serve as a probing mechanism to appraise embryo development during pregnancy in the clinical setting. A non-invasive imaging tool that reveals blood oxygen levels and perfusion in mice placentas and fetuses has potential applications for monitoring preeclampsia and other pregnancy disorders. Optoacoustic (OA) imaging involves sending laser pulses into biological tissue. The light absorption results in emission of ultrasonic waves, which are detected and used to build up images representing physiological factors such as oxygen levels. Daniel Razansky at the University of Zurich and ETH Zurich, Switzerland, and co-workers used OA imaging to non-invasively monitor placental and fetal function in pregnant mice. The team administered near-infrared contrast agent and alternate doses of 20 and 100% oxygen to the mice via breathing apparatus and monitored the resulting blood oxygenation and perfusion parameters. The images provided unprecedented detail showing how the placenta protects the embryo from excessive oxygen and extrinsically administered agents. Moreover, placental insufficiency and maternal chronic hypoxia hinder oxygen delivery to the foetus, leading to several consequences, including intrauterine growth restriction (IUGR) and foetal death2. However, despite the importance of the placental function in embryonic development, it is still not fully understood and is challenging to characterize in vivo. Therefore, the emergence of new insights in placental insufficiency and oxygenation along with changes in maternal and foetal functional parameters are of high importance and can help in the prevention of pathological cases via periodic monitoring of anatomical and functional attributes during development.Medical history has witnessed multiple complications during pregnancy and childbirth due to impaired oxygen transfer across the foeto-maternal vascular interface, resulting in pregnancy-induced hypertensive disorders (PIHD), including preeclampsia, with possible fatal maternal and/or foetal effects. Anomalous placental development and lack of oxygen perfusion through the placental-vascular interface can lead to hypoxic and ischaemic attacks. According to a survey by the World Health Organization, ~830 women die every day across the world due to complications related to pregnancy and childbirth, among which ~14% are attributable to PIHD3, X-ray computed tomography (CT)4, and blood oxygen level-dependent magnetic resonance imaging (BOLD MRI)6, have been shown to have considerable potential for better understanding of embryonic development in pre-clinical research and in clinical practice. Doppler-US provides high spatial resolution for structural phenotyping but lacks the capability of measuring placental oxygen saturation. X-ray CT provides a weak soft-tissue contrast, and its radiation exposure limits longitudinal studies, particularly in obstetrics. Additional limitations stem from the need for exogenous contrast agents and the incapability of real-time imaging of the embryo. BOLD MRI can provide high-resolution anatomical images along with estimates of changes in placental oxygen saturation. However, its clinical application is limited by the high instrumentation costs associated with microscopic level resolution and the inability to operate at the bedside. Overall, none of the clinically established imaging modalities can offer real-time readings of placental and foetal oxygenation and perfusion parameters.With technological advancements in imaging methods, several probing mechanisms, such as Doppler ultrasound (US)8. After several years of continuous technological developments, the use of OA in pre-clinical research and, more recently, in clinical studies has widely spread, with applications including characterization of cerebral hemodynamic responses upon external stimuli9, multi-scale whole-body imaging of small animals11, cell tracking13, assessments of tumour growth and progression15 and visualization of cardiovascular dynamics16. OA has been shown to complement or enhance the hemodynamic readings provided by Doppler US17 or functional MRI18. Particularly, five-dimensional OA tomography can simultaneously measure multiple hemodynamic parameters in real time19 that have remained inaccessible with other modalities. In addition, OA has successfully demonstrated its potential in delivering highly specific information from targeted and activatable probes20 as well as from genetic labels22. Moreover, OA imaging has shown significant ability to detect retinal neovascularization in larger animals23, which can be further exploited in the investigation of retrolental fibroplasia (RLF) or retinopathy of prematurity (ROP), a major cause of blindness in premature cases that originates from factors such as neovascularization, high oxygen exposure to preterm babies25 among others. OA structural phenotyping of mammalian embryos has been shown to be feasible by either imaging mice embryos ex vivo26 or using relatively slow scanning-based systems28. More recent studies have focused on monitoring placental and foetal oxygenation at different developmental stages with real-time OA systems based on linear arrays29. However, these systems are strongly affected by so-called limited-view effects and hence offer sub-optimal imaging performance and very limited quantification capabilities30. Significant limitations yet remain in terms of inadequate depth penetration and lack of a high-resolution anatomical lay-out of whole cross-sectional areas, thereby encumbering its application in the clinical domain. More importantly, the spectroscopic imaging paradigm has not been fully explored in simultaneous monitoring and comparative evaluation of changes in oxyhaemoglobin (HbO) and deoxyhaemoglobin (HbR) from maternal to foeto-placental areas.Optoacoustic (OA) imaging emerged in the early 2000s as a novel non-invasive imaging method harnessing the advantages of optical and US imaging modalities to provide high-contrast characteristic responses of functional and molecular attributes without sacrificing resolution (for depths of millimetres to centimetres) in highly optically scattering biological tissues2), in different areas  of tomographic scans. The system can provide cross-sectional images over a large area (~40 mm across) with a high (~200 µm) spatial resolution31, providing both structural and functional data from pregnant mice at gestation days E14 and E19.This work focuses on the study of the capabilities of a self-developed ring-shaped optoacoustic tomography (ROAT) system based on an array of cylindrically focused transducers for in vivo and non-invasive embryonic imaging in pregnant mice. Particular emphasis is placed on functional analysis of the spatio-temporal distribution of hemodynamic parameters, such as HbO, HbR and oxygen saturation  at different gestation days (E14 and E19) in which no such significant change in SO2 levels could be observed. The measured changes of SO2 in the maternal artery and placenta are in close accordance with the theoretical values, as previously reported7. Although a quantitative measurement of SO2 in the entire cross-sections is challenging, it appears that the maternal artery consistently exhibits higher signal changes compared with the placenta and embryo regions.A study of the changes in the functional parameters obtained from the multispectral images was carried out in two phases, namely, a breathing gas challenge Fig.  and ICG 2 Fig.  during htPA) and placenta (ΔtP), where ΔtPA<ΔtP, the changes in the OA signal are significantly higher in the peripheral artery (ΔaPA) compared to the placenta (ΔaP) due to higher ICG perfusion. The lower panel  may preserve placental oxygen for a period of time, so that relative changes in oxygen exposure may have a lower effect in the placenta and hence in the embryo. Similar studies were performed to assess placental and foetal oxygenation using BOLD MRI in different animal models, where a subsequent change in foetal blood oxygenation was reported during hypoxia of the maternal animal33. A comparison of OA hemodynamic readings with standard BOLD MRI signals was conducted in a previous study18 where a good correlation was found in the hemodynamic parameters of tumour vasculature in mice in vivo. Functional studies with respect to the change in oxygen concentration at normoxia and hypoxia have also been performed2, in which a gradual decrement in oxygen saturation in the placental and skin regions of the mother mouse was shown while changing the oxygen exposure from hyperoxia to hypoxia. Our study is the first to extend pre-clinical placental and embryonic research to the whole-body scale in pregnant mice to characterize various anatomical features and subsequently perform a functional study in a longitudinal manner. To ascertain the functional attributes and efficacy of the ROAT approach, an oxygen challenge (hyperoxia) was induced in pregnant mice. The change in the blood oxygenation parameters  while changing the oxygen exposure from 100% to 20% also depicts the nature of their alterations, substantiating the standard rule of change in blood-oxygenation parameters. Considering the established correlation between the OA response to blood oxygenation and Doppler flow34, our functional responses are in good agreement with the previously reported data, substantiating the efficacy of the ROAT imaging instrumentation for non-invasive studies with an unprecedented spatio-temporal resolution and field of view. We can therefore state that ROAT is a potentially viable imaging paradigm to monitor changes in the functional attributes of foetal and placental regions, which may help to overcome complications during gestation periods. For example, RLF and other pathological changes in the retina are known to be induced in the foetus by hyperoxic conditions35. In this context, the designed system and methodology can also provide significant input into the investigation of the pathologic conditions related to high oxygen exposure (hyperoxia) in premature infants, causing RLF in resource-limited clinical settings. Moreover, inadequate O2 perfusion through the placenta can directly cause placental insufficiency and IUGR. Early symptoms of trivial embryo development and onset of placental insufficiency can potentially be detected by in vivo monitoring of the placental oxygenation. In this context, ROAT can significantly reduce the risk of anomalous placental function leading to abnormal embryo development and often foetal death.This work demonstrates that ROAT is suitable for monitoring placental and embryonic function during pregnancy in rodents. Imaging at a wavelength of 1064 nm facilitated accurate labelling and identification of internal organs within the abdominal cavity of pregnant mice. Pulsations associated with the heartbeat of the embryo and the respiration of the maternal mouse were further observed with a high spatio-temporal resolution. More subtle anatomical structures of the embryo may be resolved by using US arrays with a broader bandwidth, thus attaining a better spatial resolution. In addition, multi-wavelength image acquisition at 700, 730, 755, 800 and 850 nm enabled the analysis of functional attributes, such as HbO, HbR and SO36, which to quantification errors in approximating the bio-distribution of ICG in deep vascular networks. Although other spectroscopic algorithms based on blind unmixing or statistical approaches have been shown to enhance the sensitivity of detecting specific chromophores37 and eigenspectra analysis has been employed to mitigate wavelength-dependent light fluence variations38, quantification inaccuracies generally remain in images. Likewise, the sensitivity to other external stimuli must be studied to obtain a detailed characteristic response of the foeto-placental function.The functionality of the placental barrier in protecting the embryo from external stimuli was observed in an additional experiment in which the perfusion of ICG through the placental barrier was dynamically monitored. Although this experiment was aimed at studying the functional behaviour of the placenta, it is important to emphasise the fact that only a qualitative assessment of the ICG distribution was performed. The linear unmixing method used in this work enables only qualitative temporal mapping of ICG at deeper locations. Quantitative estimations are challenging in this situation due to alterations in the spectral signature of OA signals owing to wavelength-dependent light attenuation in deep tissues39. It may then be feasible to assess the anatomical and functional attributes of the human placenta as well as to monitor embryonic development in women suffering from pathological placental insufficiencies. It is important to take into account that minor changes in oxygen saturation with respect to normoxia are expected in the clinical scenario, and the sensitivity of the system must be evaluated in such cases. However, monitoring the human embryo is expected to be hampered by a limited penetration of photons even for NIR laser sources. To facilitate clinical translation, the light delivery approach must first be optimized along with the frequency bandwidth, number of elements and arrangement of such transducer arrays. This optimization would allow the pregnancy stages in which the new imaging approach may be applicable in clinics to be determined. It is also important to highlight that ICG is an FDA-approved contrast agent at the concentrations injected in our study40. Therefore, assessment of the functionality of the placenta as a barrier against external agents may also be possible in humans. The reduced changes in oxygenation compared with the maternal peripheral artery, as reflected by the experiments with a breathing gas challenge, corroborate two important facts: placental insufficiency due to maternal oxygen challenge and preservation of placental oxygen even in the challenging environment. Additionally, the performed ICG perfusion experiments substantiated the functionality of the placental barrier in protecting the embryo from external stimuli. Thus, the new imaging approach can potentially be leveraged in periodic monitoring of maternal and foetal health and also in clinical applications to study and aid pregnancy-related abnormalities.The demonstrated good performance of the ROAT imaging system in providing both anatomical and functional information about maternal and foetal bodies in real-time and in a non-invasive manner further validates its possible clinical application in monitoring foeto-placental health through measurement of different blood-oxygenation parameters. In this regard, hand-held OA scanners based on the same type of concave arrays of cylindrically focused transducers have been shown to offer promising prospects for characterizing human tissues at centimetre-scale depths2 (height × width). The pressure waves for all elements were simultaneously sampled at 2032 instances with a sampling frequency of 40 MHz using a custom-made data acquisition system . The acquired signals were subsequently transferred to a host PC through a 1 Gbit Ethernet connection for reconstruction and post-processing. The imaging system can acquire images at frame rates of up to 100 Hz determined by the PRF of the laser. Faster frame rates could be achieved with higher PRF lasers by sparsely acquiring signals from a reduced number of channels41.A schematic description of the whole-body ROAT is presented in Fig. n = 4) and E19 (n = 3). Pregnancy was safely determined by palpation of the embryos.All experimental procedures including animal handling were carried out in compliance with the institutional guidelines of Helmholtz Zentrum Muenchen and with the approval from the Government of Upper Bavaria. In this study, we used pregnant mice (CD-1 (Crl: CD1 ICR)) from Charles River, Wilmington, MA of two different days of gestation: E14  were imaged under a breathing gas challenge in which the inhaled gas was altered periodically between normoxia  and hyperoxia (100% O2) every 2 min for a total duration of 14 min. This periodic alteration of gasses was controlled manually using the gas flowmeters of the anaesthesia unit  while keeping the isoflurane level constant. In a different experiment with an E14 pregnant mouse, 100 nmol of indocyanine green (ICG) diluted in 100 μl of phosphate buffer saline (PBS) was injected intravenously through a tail-vein catheter for 20 s. OA signals were acquired by tuning the laser to a wavelength of 800 nm and PRF of 25 Hz. It is also noted that all in vivo imaging experiments were terminal in the sense that all pregnant mice, including embryos, were euthanized during anaesthesia at the end of the experiments.Prior to in vivo imaging, the abdominal region of the mice was thoroughly shaved in an anaesthetic environment with 1.2–2% v/v isoflurane in O42) during each in vivo tomographic scan. The reconstruction parameters  can be adjusted in the GUI for improved quality and better signal-to-noise ratio. Prior to image reconstruction, the time-resolved OA signals were deconvolved with the frequency response of the US transducer elements, followed by band-pass filtering (BPF) in the frequency range of 0.1 to 6 MHz. Each 2D cross-sectional scan of 45 × 45 mm2 was reconstructed in a 400 × 400 pixel area (pixel size of ~113 µm). Furthermore, all raw OA signals, corresponding to each scan, were also stored for offline image reconstruction, spectral unmixing and post-processing. The offline processing is described as follows. Following the aforementioned frequency-selective filtering, an adaptive filtering method was implemented based on least mean squares adaptation to reduce the noise effect within the frequency band of interest. This algorithm is described in detail in Section 1: Supplementary Document 44. The multispectral acquisitions were further unmixed to retrieve the distribution of oxygenated (HbO) and deoxygenated (HbR) haemoglobin45. This was achieved by least squares spectral fitting of individual pixels reconstructed at the five different wavelengths to the molar extinction spectra of HbO and HbR. Subsequently, pixel-wise oxygen saturation (SO2) mapping was calculated as SO2 = HbO/(HbO + HbR). The reconstructed images at 800 nm corresponding to the ICG injection experiment were additionally time-averaged for 10 consecutive frames to increase the signal to noise ratio (SNR).All acquired data were further analysed in MATLAB , installed in a graphics processing unit-based computer. A graphical user interface (GUI) was also developed to render on-the-fly image reconstruction (using a back-projection algorithm46. The details of this processing step are presented in Section 2: Supplementary Document After the reconstruction of whole-body anatomical cross-sections and multispectral unmixing, a few image processing steps were further implemented, which enabled the production of high-contrast OA images with substantial enhancement of subtle features that clearly demonstrate both structural and functional attributes in different experiments. First, the mouse surface in the 2D cross-sectional scans was segmented using a modified snake-based active contour algorithmSupplementary MaterialSupplementary Video 1Supplementary Video 2


ClinicalTrials.gov Identifier: NCT03197467) is to assess feasibility and safety of pre-surgical anti PD-1 treatment in order to improve long term survival.Immunotherapies targeting the PD1/PD-L1 pathway have had a large impact on the treatment of advanced NSCLC. Concerning multimodality tumor therapy, only few trials until today have been performed investigating neoadjuvant treatment with anti PD-1 immunotherapy prior to curative intent surgery. Aim of the NEOMUN investigator initiated trial , tumor resection with lobectomy or bilobectomy will be performed. Primary objectives are to assess the feasibility and safety of a neoadjuvant immunotherapy and to assess antitumor activity of Pembrolizumab with regard to clinical and pathological tumor response. Secondary objective is disease free and overall survival. Exploratory objective is to analyze potential predictive biomarkers and to evaluate the therapeutic efficacy of Pembrolizumab by extended immune cell and cytokine analysis of tumor tissue.The study protocol was approved by the local ethics committee and the federal authority. Start of patient enrollment is scheduled for June 2018.The NEOMUN trial will be one of the first clinical trials investigating a multimodal treatment strategy including neoadjuvant immunotherapy using Pembrolizumab as an investigational drug. Assessing the safety and therapeutic potential of neoadjuvant immunotherapy in connection with lung surgery will be of great interest for thoracic surgeons.www.clinicaltrials.gov; NCT03197467 .Prospectively, the NEOMUN study has been registered on  Lung cancer is one of the leading causes of death worldwide with approximately 230.000 new diagnoses per year in the United States and 410.000 in Europe . In thesIn contrast to standard cisplatin based chemotherapy, novel drugs modulating the patient’s immune-system unleashing the anti-tumor immunity have been implemented into modern treatment strategies. Special interest has been raised by so-called regulatory immune-inhibitory pathways including the programmed death-receptor 1 (PD-1) and its ligand (PD-L1). Besides others, these molecules are expressed on the tumor cell surface (PD-L1) or the patients activated T-cells (PD-1). Ligation of PD1/PD-L1 suppresses activated T-cells, thus resulting in impaired anti-tumor immunity , 11.p < 0.001) and an overall survival of 80.2% at 6 months compared to 72.4% in patients receiving standard chemotherapy (p < 0.005). These findings resulted in the recent approval of Pembrolizumab as first line-treatment for patients with a PD-L1 expression > 50%. Nevertheless, the KEYNOTE study population consisted of patients with advanced or metastatic NSCLC [Antibodies blocking the ligation of PD-1/PD-L1 have been investigated in recent clinical trials in NSCLC stage IV patients . Treatment of selected patients  with a monoclonal humanized antibody against PD-1  correlated with an increased progression-free survival  will be administered in a neoadjuvant setting to patients with resectable NSCLC stage II/IIIA who are eligible for curative intent surgery. The percentage of patients reaching the surgical therapy following neoadjuvant immunotherapy should exceed 80%.Primary Objectives are to assess the feasibility and safety of a neoadjuvant application of Pembrolizumab and to evaluate the effectivity of an anti-PD1-treatment on clinical and pathologic tumor response.Secondary Objective is to assess the impact of neoadjuvant Pembrolizumab application on patient disease free- and overall survival.Exploratory objective is the translational assessment of treated and untreated tumors  in order to generate a hypothesis on potential biomarkers predicting the efficacy of Pembrolizumab.Primary endpoints are to evaluate the frequency and severity of adverse events including peri- and post-operative complications (grade 2–4 AEs according to NCI-CTCAE V4.03) in all participants. Immunotherapy-associated tumor response will be assessed byradiological change (Δ tumor size / lymph node size), according to RECIST  and iRECfunctional  andpathological response parameters (regression grading according to Junker criteria )*Immunotherapy-trials described unique patterns of tumor response (pseudoprogression). A subset of patients meeting RECIST 1.1 criteria for disease progression (in size or number of lesions) showed delayed but durable responses to therapy by time. iRECIST therefore widens RECIST to immune-related response criteria, notably iUPD (unconfirmed progressive disease). A new or growing lesion is then defined as iUPD until definite progression is confirmed over time.N = 30 and assuming that the number of events follows a binomial distribution B , events with an incidence rate p > 9.5% will be observed at least once with a 95% probability. This observation limit will exclude individual Pembrolizumab related SAEs but covers most of the historical reference events/rates . The reported frequency of these events has been taken into account to calculate the 95%-confidence interval (under a sample size of n = 30) using the Clopper-Pearson-Method [Targeted sample size is 30 patients. The rationale for the sample size is based on ethical, clinical and scientific considerations. The neoadjuvant treatment approach with an immune checkpoint inhibitor is experimental with only limited data on safety and feasibility available until today. Therefore, only a small number of patients should be subjected to this experimental treatment. The sample size should allow the generation of statistically meaningful evidence for feasibility and safety to permit the decision to further develop this treatment strategy. With a sample size of n-Method , 18.All patients  with clinically suspected lung cancer that are scheduled for surgical cancer therapy at the Thoraxklinik Heidelberg will be screened for eligibility to participate in the NEOMUN-trial. Baseline inclusion criteria are non-small cell histology in clinical stages IIA to IIIA.The decision for radical surgical therapy as an integral part of the therapy must be agreed on the oncological interdisciplinary tumor board. Each possible candidate is simultaneously re-evaluated by the principal investigator or authorized medical staff. Only patients with adequate cardiopulmonary function according to current treatment guidelines will be counted eligible for trial inclusion . FurtherAll patients with histologically proven NSCLC in stages IIA to IIIA who are likely to meet all of the inclusion criteria and none of the exclusion criteria, may be included into the trial between day − 28 and 0. The treatment period will start after the first administration of Pembrolizumab (day 1). The second application is planned at day 21. During the pre-surgical period between day 42 to 50, a second PET-scan will be performed for radiological baseline assessment followed by curative intent surgery (day 50+).Adjuvant standard chemo- and/or radiotherapy will be administered according to the current guidelines. Each patient will be followed up 24 months after completion of the entire tumor therapy Fig. .Fig. 1TiA contrast enhanced thoracic computed tomography (CT) will initially be performed in all patients that are screened for the trial. Distant metastatic disease will be radiologically ruled out using a diagnostic positron-emission-tomography-scan (PET) of the whole body and magnetic resonance imaging of the brain (MRI).All patients receive rigid and flexible bronchoscopy to evaluate the endobronchial anatomy. The mediastinum is examined using endobronchial ultrasound. Enlarged  or PET positive mediastinal lymph nodes will be clarified by EBUS-guided biopsy. If malignancy cannot be ruled out, mediastinoscopy will be performed. Histological confirmation of NSCLC will be provided by either transbronchial biopsy (TBB), transbronchial needle aspiration (TBNA) or transthoracic biopsy (ultrasound-guided or computed tomography guided).Clinical staging will be proceeded in accordance to the actual TNM-classification. As defined by protocol, patients that meet the stage inclusion criteria according the 7th edition will be screened for eligibility to participate (Stages IIA - IIIA). Nevertheless, TNM-classification from the 7th and the actual valid 8th edition will both be provided , 20 .Investigational drug is intravenous Pembrolizumab at a fixed dose of 200 mg every three weeks ((q3w), 21 days) for 2 cycles .Following neoadjuvant immunotherapy, curative intent radical surgery will be performed in each patient meeting eligibility criteria. Thoracic surgery is no study procedure. Depending on individual patient factors , access into the chest cavity will be provided either by open surgery  or endoscopic surgery . The study investigators will plan the patients for at least anatomical pulmonary lobectomy including systematic complete hilar and mediastinal lymphadenectomy, as systematic nodal dissection is an integral part of the surgical procedure to accurately assess the patients correct regional nodal status .All patients will be discussed in the institutional multidisciplinary tumorboard before discharge. Depending on the individual tumor stage and postsurgical physical recovery, adjuvant chemotherapy will be recommended according to the current national and international guidelines.After completion of the initially recommended tumor-specific therapy, routine follow up will be performed every three months up to 24 months and includes thoracic and abdominal sectional imaging as well as lung function assessment. In addition, translational blood analysis will be conducted on e.g. tumor markers, peripheral blood mononuclear cells, antibody profiles and cellular as well humoral immune-related response parameters. Clinical or radiological findings suspicious for tumor recurrence will result in whole body restaging and further individual examination. The end of the entire trial is defined as the end of follow up of the last patient included.An adverse event (AE) is defined in the International Conference on Harmonization (ICH) Guideline for Good Clinical Practice as “any untoward medical occurrence in a patient or clinical investigation subject administered a pharmaceutical product and which does not necessarily have a causal relationship with this treatment” (ICH E6: section 1.2). An adverse event can therefore be any unfavorable and unintended sign , symptom, or disease temporally associated with the use of a medicinal  product, whether or not considered related to the medicinal  product.A serious adverse event (SAE) is defined as any untoward medical occurrence (adverse event) that at any dose results in death, is life-threatening, requires new or prolonged in-patient hospitalization, results in persistent or significant disability/incapacity, is a congenital anomaly/birth defect or, any other significant medical condition.Progression of the NSCLC and symptoms caused by progression of the NSCLC need not to be reported as SAE in this protocol, unless progression or symptoms of progression are assessed as causally related to study medication.Medical or ethical reasons affecting the continued performance of the studyOccurrence of AEs unknown to date in respect of their nature, severity, and duration or the unexpected incidence of known AE with a negative impact on the risk/benefit assessmentmedically unacceptable risks for the treatment of the patientsAt the interim analysis (15 patients included): if the rate of unresectability is equal or greater 20%; i.e. if 3/15 or more study subjects did not undergo surgery as planned according to the protocol (unresectable disease or excessive delay of surgery).The whole study will be stopped in the event of any of the following:All patients that are included into the trial will participate in a translational research program. The standard collection of specimen includes pre-therapeutic diagnostic biopsy, surgical specimen and sequential blood samples. Of major interest is the analysis of possible therapy-associated changes in different tumor regions , the mediastinal and hilar lymph-nodes, the healthy lung tissue and the peripheral blood. Detailed research will be conducted to further investigate the tumor microenvironment, tumor mutational burden, mutational status, cytokine expression levels and genomic alterations. Further examinations of tumor tissue taken pre- and post Pembrolizumab will include whole genome sequencing and FACS-examination of the intratumoral immune infiltrate. Polarization of tumor associated macrophages (TAMs) in response to anti PD-1 treatment will be investigated.Workup of pre- and posttreatment blood will focus on cytokine−/chemokine-profiles and antibody-profiles using multiplex assays. Specific interactions between tumor and immune-cells will be examined by further research on neo-epitope- specific T-cell responses and T-cell-receptor -clonality analysis. In order to evaluate a correlation between the mutational-load/transcriptome of the tumor and the peripheral blood, whole exome sequencing of cell free plasma DNA will be performed.Data of the patients participating in the NEOMUN trial will be compared to patient data from an institutional biobank cohort matched to age, gender, stage of disease and tumor-dependent parameters . The matched cohort will only include patients who did not receive immunomodulatory treatment.p-value of < 0.05 will be considered statistically significant.All demographic and clinical characteristics recorded at baseline will be submitted to descriptive analyses using descriptive statistics by means of listing and tables. Continuous data will be summarized with at least the following: frequency (n), median, quartiles, mean, standard deviation (standard error), minimum and maximum. Statistical test between changes of parameters over time will be performed for continuous as well as categorical variables. Wilcoxon signed rank test, Fisher’s exact test and other appropriate tests will be used where applicable. Time-to-event measures  will be analyzed by means of Kaplan-Meier curves. A For the analysis of adverse events, summary tables providing the number and percentage of patients will be generated for the incidence of AEs overall and by severity. Laboratory parameters will be reported with descriptive statistics.Patients with NSCLC and nodal N1 and/or N2 disease suffer from an overall relapse rate of 52% even when multimodality treatment including curative intent surgery with chemo- and/or radiotherapy is administered. The overall 5-year survival benefit in these patients accounts of only 4 to 5% , 23. In Recently, immunotherapies targeting the PD-1/PD-L1 pathway have had significant impact on survival in patients with advanced stages of NSCLC in comparison to standard chemotherapy . A correIrrespective of the results in metastatic stage IV patients, the predictive value of the PD-L1 status might have a different impact in patients with non-metastatic earlier-stage lung cancer with less tumor burden. This hypothesis is supported by data recently published by Forde et al. . Their tMoreover, the techniques used to quantitatively assess the PDL-1 status in small pre-treatment biopsies are under critical debate. Several factors contributing to a false-negative or a false-low PD-L1 status have recently been identified . From a In addition to the PD-L1 status analysis, EGFR-mutation and ALK-translocation have been implemented in routine diagnostics and represent important prognostic parameters in the era of targeted therapy . A correclinicaltrials.gov).The question whether checkpoint blockade should be performed before or after surgery has been addressed by Liu et al. in a preclinical breast-cancer model. The authors reported about an increased tumor specific CD8+ T-cell response in patients after neoadjuvant immunotherapy . PircherIn NSCLC, clinical data on neoadjuvant immunomodulatory treatment is scarce but few encouraging trials investigating anti-PD-1/PD-L1 checkpoint inhibition prior to surgery for NSCLC patients are currently ongoing . In a first interim analysis, Forde et al. recently published data on patients with NSCLC receiving neoadjuvant anti-PD-1 therapy (Nivolumab). They observed a major pathological response (less than 10% viable tumor cells) in 9 of 20 patients (45%) with neoadjuvant immunotherapy. These preliminary data and the results of the KEYNOTE-trials identified immunotherapy as a promising strategy in NSCLC treatment [The scope of the enrolled NEOMUN trial is to generate pilot data on the efficacy, safety and the feasibility of a neoadjuvant anti-PD-1 immunotherapy with Pembrolizumab. The research endeavor will be flanked by a translational research program to elucidate potential predictive biomarkers and to investigate the mode of action of anti PD-1 treatment in NSCLC patients.