A focus group interview with clinicians, coordinators, and leaders involved in stroke survivors' rehabilitation trajectories was conducted. The group consisted of a strategic selection of participants with diverse professional backgrounds from specialist and primary healthcare services. The transcribed material was analyzed thematically using systematic text condensation based on an inductive, interpretive approach.  We found that patients with mild and moderate cognitive impairment after stroke were characterized as a neglected group in rehabilitation services and that neglect was related to both structural and professional issues. First, neglect seemed partly related to the availability of existing healthcare services, which mainly followed up on physical challenges after stroke. Second, cognitive rehabilitation seemed to be less prioritized than other health services, and the established interdisciplinary municipality teams did not seem prepared to follow-up on cognitive issues. Finally, at a professional level, the study reveals the need for building competence in cognitive rehabilitation and having services available in the long run.  The study demonstrates the need to increase knowledge concerning cognitive rehabilitation and how rehabilitation trajectories and services should be organized to fulfil stroke survivors' and carers' long-term needs. Cognitive impairment is a frequent consequence following stroke. Nearly half of stroke survivors have been reported to display a varied level of poststroke cognitive impairment (PSCI) during the first year . CognitiMany stroke survivors and their next of kin need help and support from health professionals, placing special demands for appropriate services in different phases of the rehabilitation process , 12. A gIn Norway, rehabilitation usually takes place in hospitals , where mIt is well known that patients with mild or moderate impairment after stroke in most cases benefit the most from rehabilitation in their own environment, if possible, combined with day-rehabilitation services . Access The aim of this study is to investigate how multidisciplinary health professionals experience long-term services provided to patients with mild and moderate PSCI in a North Norwegian context. We explore how organizational and individual structures contribute to shaping the service provision for this specific patient group in a given geographical area.This research employed a qualitative design, which allowed for an in-depth exploration of cognitive rehabilitation practices after stroke. To answer the research question, we conducted a multidisciplinary focus group interview with both specialist and community healthcare service providers. Focus group interview was considered suitable because it facilitates interaction that may elicit experiences and ideas and elaborate research participants' perspectives through debate within the group , 32. TheA purposeful sampling strategy was used , throughThree of the participants worked at a patient-centred healthcare team for elderly individuals with complex and chronic diseases, aiming at strengthening transitions between hospital and municipalities. One participant represented an ambulatory rehabilitation team providing prolonged advisory follow-up from hospital to home communities to patients of working age with complex, long-term rehabilitation needs. One participant represented a municipal-based interdisciplinary reablement team that offer time-limited home training to patients with mild rehabilitation needs. Their aim is to provide support in reoccupying activities that are important to people in their everyday life. Also, one therapist worked with community-based, institutional rehabilitation that provided interdisciplinary rehabilitation, either prior to, -after, -or instead of hospitalization. Last, one participant worked administratively at a coordinating municipal team aiming to coordinate the flow in services between specialist and primary healthcare to adult patients with somatic, complex rehabilitation needs.The interview guide was jointly developed in cooperation with all authors. Moreover, in the preparation phase, the content of the interviews in the project were discussed with a reference group that consisted of patients and next of kin from a stroke organization, which aided in developing the guide. The main theme for the focus group was discussions about the multidisciplinary professionals' experiences concerning service support for patients with mild and moderate cognitive impairments. Key points were patients' needs and changing needs, professional content and support, collaboration, and challenges and ideas for improvements.The focus group interview took place in a meeting room at the first author's workplace. The first author (AB) led the interview, presenting topics for discussion and encouraging the participants to share their experiences and discuss within the group. The interviewer and PhD candidate had a background as an occupational therapist with primary knowledge and experience working with neurorehabilitation. The coauthor (AG) followed up with supplementary questions and took notes to help summarize the interview. The focus group interview lasted two hours and was audio-recorded and subsequently transcribed verbatim by the first author.The material was analysed using systematic text condensation (STC) , which iThe authors received approval for the study from the Norwegian Centre for Research Data (reference number 60366), according to personal data protection. The multidisciplinary professionals signed an informed consent form to participate and were informed that they could withdraw at a given time without specifying the reason. The data were treated confidentially, and information about the participants was anonymized and presented with caution to avoid being traceable to individual contributions.Through the thematic analysis, we identified three main themes. The initial two reflect experiences of services available and healthcare priorities at a structural level in relation to patients with PSCI. The last theme concerns the experiences of health professionals' needs in terms of building competence and providing access to cognitive rehabilitation competencies, where available, in the long-term follow-up see .An overall finding was that the participants described patients with mild and moderate cognitive impairments after stroke as being a neglected patient group throughout the rehabilitation trajectory. According to the participants, the inattention to this group during the early inpatient stay could be due to a lack of investigation and therefore a missed opportunity to reveal the patient's cognitive challenges. In the phase of transitioning home, the inattention could be due to not identifying patterns in cognitive consequences over time and discussing a follow-up plan. The negligence in the long run could be related to not having services available in the municipality when cognitive impairments make everyday life for both the stroke survivor and their family members difficult. Overall, patients with mild and moderate PSCI can be characterized as a neglected patient group because it seems that the system is not capable of identifying their needs and having trained personnel available for cognitive rehabilitation, particularly in the long-term rehabilitation trajectory.A recurring issue in the material was that the participants described that this group of patients did not fit into the traditional rehabilitation services. One participant explained,"If the main concern is cognition, then the experience is that there are often challenges because who is there to follow-up, then? It has something to do with attaching it [cognitive rehabilitation needs] to some services, because they do not belong in any of the traditional services."The participants used the term “traditional services” when referring to the healthcare services that were usually provided at hospitals, community centres, and outpatient clinics to patients in need of rehabilitation in general. Common features were that follow-up begins as early as possible, that health services provide intensive follow-up on specific functional impairments and over a limited period. When talking about the traditional services in the municipalities, the participants often referred to speech therapy, OT, PT, and different interdisciplinary teams. However, the participants experienced that the patients in this group “do not fit into the ways in which the traditional services are organized.”Several participants described that from the early inpatient rehabilitation phase, a main concern often involved providing intensive follow-up on physical functioning, compared to assessing and supporting cognitive and psychosocial needs. However, different viewpoints were discussed. One participant expressed that in regard to stroke patients in general, cognitive needs and recommendations for further priorities seem readily described in patient reports and handover meetings. Another participant, however, expressed that for patients with especially mild and moderate PSCI, cognitive concerns were “perhaps not most commonly described.” The consequences when cognitive and psychosocial issues were not being identified and addressed by professionals initially were discussed. One participant said,"… we know that many have been discharged when they are physically fine. Maybe there is a mild impairment. But… you do not know for certain, and… maybe those who know him well, are starting to notice it after some weeks, and… where they are sent home after a few days at the hospital. Without a further plan, then."However, when cognitive impairments were assessed and followed up and when referrals were made before discharge, the participants experienced that the services in the municipalities were not adjusted to the patients' cognitive needs for support. A lack of services that could address these needs in the municipality was one aspect. One participant said, “Like we've talked about, having an overview at a municipal level, we don't have sufficient health care services available to this patient group.” For example, several participants described follow-up in returning to work life being “somewhat neglected,” as such follow-up requires close cooperation between the patients, interdisciplinary health professionals, and different sectoral services, although no one was appointed to follow-up in this domain. Several participants also talked about not having designated coordinators or health professionals appointed to a liaison role for stroke service provision in the community that could prioritize cognitive rehabilitation. A participant who worked in one of the interdisciplinary teams described overall challenges with the availability of health professionals who could assist patients with cognitive concerns:"…it depends on having someone to cooperate with, whether they [the municipalities] have someone they can give that task to… if there are any personnel available, if they [the patients] do not need anything else. If home care service is there already or there are occupational therapists available, if they are a part of follow-up or who receives counselling. Then, there is someone for our part at least, who we can collaborate with."The participants described that to support recovery after stroke, it was essential to offer early, intensive multiprofessional rehabilitating support. Simultaneously, they discussed how in many circumstances cognitive challenges often first become apparent for patients and relatives after living at home for some time, further highlighting how cognitive rehabilitation requires quite different organization and content for follow-up. One participant described, “Cognitive rehabilitation takes time. In a way, you need the services after you have finished the physical part, or you need help with care. You still need time... for the cognitive rehabilitation.” Another participant said,"Many fall, as you mentioned, in between two chairs. As the cognitive impairments become perhaps more visible…then there is no one there guiding them further. They sort of do not fit into any boxes in the municipalities."The participants further discussed the importance of carrying out cognitive rehabilitation in settings and through activities that are familiar and important to the patient. One participant expressed, “The world outside the hospital could be totally different for the patient. Their focus and needs may have changed completely.” Except for reablement teams that provided home training, traditional rehabilitation services rarely took place in the patients' natural everyday life surroundings. One participant said, “That's where it's missing; we do have day services, but we must have services available for those who need it at home.”In summary, the descriptions revealed a gap between how the services were organized and what patients' cognitive rehabilitation needs were. Patients with mild and moderate PSCI were reported to have needs that required following another timeline and possessing another understanding of their recovery process. Overall, the participants acknowledged that this patient group was being overlooked in different stages throughout the rehabilitation trajectory and increasingly overtime.Priorities in healthcare services also seem to influence why this patient group appears to be systematically neglected. An interesting finding was that cognitive rehabilitation may suffer a triple burden: first, rehabilitation does not seem to be a priority service compared to other service needs; second, cognitive rehabilitation seems to be a deprioritized area within the rehabilitation field; and third, established interdisciplinary teams in the municipalities do not seem to be attuned to supporting the needs of this patient group.The quote below illustrates that rehabilitation in general is not given a very high priority and seems to be a nonpriority service compared with supporting care and nursing needs."Rehabilitation is… far out in the rank of priorities. Number one is what we must do first, and that is to make sure the patient receives, for instance, nursing and care, according to their needs. That is a high level of attention. While the rehabilitation part – it can wait."Another participant described how cognitive rehabilitation appeared to not be prioritized within the rehabilitation field: “There are few municipalities that have someone dedicated to work with cognitive functioning.” Several participants described excessive attention to physical functioning in both specialist and primary healthcare services. A “bodily focus” was apparent in that the visible, physical, and impaired body was reported to be initially at the forefront of health personnel's attention. It was also the physical body that patients first became aware of and requested help for recovery. One participant said, “There is often a bodily focus, and then the cognitive part comes… somewhat on the sideline.” Attention to bodily functioning from an early, postacute phase was reported to be necessary to minimize long-term medical and physical consequences. Even so, this was reflected as having implications for the rehabilitation process in the sense that health professionals were not always prompted to look for, attend to, and work with cognitive functioning.Furthermore, the participants described their engagement in different interdisciplinary teams that were aimed at follow-up either in the transition between hospital and home or that were established in some of the municipalities. For example, clinicians who worked at a patient-centred healthcare team stated that they addressed overall issues related to physical functioning and facility adjustments in the home environment in general, although they rarely attended to rehabilitation towards stroke patients. One participant said, “…it is not like specifically, that we come in and rehabilitate someone. At least not cognitively.” Another participant who worked in a reablement service described providing intensive rehabilitation for a limited period, elaborating as follows:"…it is a general rehabilitation form, where we should go in for four weeks and it should be intensive, at least five days a week. And we do have quite a few stroke patients, but then, there are some that need longer follow-up than we can provide. And they fall a bit “in between chairs.”"The participants acknowledged that the teams were not tailored to meet these patients' long-term needs and that cognitive rehabilitation was not considered a prioritized area. The exception was an ambulatory rehabilitation team. A participant engaged in this team described that many patients and next of kin who attended their self-management program one year after a mild or moderate stroke reported no prior follow-up after discharge from the hospital in terms of addressing cognitive impairments. In general, the participants described a lack of a formal structured system available in each municipality for stroke service provision that prioritized cognitive rehabilitation."We need to have a professional emphasis on cognitive rehabilitation in the municipalities. In a larger municipality, you can have an interdisciplinary team, right? That is harder in smaller municipalities, where multiple tasks must be done at once, though... that there is someone who gets good at it and that you have some guidelines on what to do."All in all, a healthcare system ranking physical functioning and physical needs first and foremost was portrayed. Moreover, rehabilitation in general, especially cognitive rehabilitation, was reported to be deprioritized among other healthcare service needs. Although several different interdisciplinary teams were established, cognitive rehabilitation was not a prioritized area. Thus, cognitive rehabilitation had to break through several layers of barriers to address these needs when necessary.The deprioritization of cognitive rehabilitation at a structural level seemed to have a direct impact on professional support and how health services were put to practice. One participant said, “It's about having it [cognitive rehabilitation] as a devoted part, that it is something one should work with and become good at – I mean, in terms of competence.”The participants described a need for developing competence in cognitive rehabilitation and for having services available. For instance, an absence of interdisciplinary rehabilitation teams and OTs with diagnosis-specific competence were described, especially in some of the smaller municipalities. While some municipalities had health professionals who worked with this patient group, limited time for competence building was reported:"Occupational therapists in the municipalities do follow-up over time. However, we have challenges when it comes to waiting lists. Nevertheless, that is where patients are referred because that's the right health service. But they have had… a lack of time, because of the pressure on… it becomes a great deal of fixing assistive devices and less time left for building competence or working on rehabilitation."The participants also expressed a need for increased resources and time for competence development in home care services. However, although playing an important part in different patient group rehabilitation trajectories, home care was reported as being more often provided to stroke survivors if they had physical impairment as well. One of the clinicians elaborated as follows:"There is no room for home care services to follow-up interventions they [stroke survivors] have received on these skills at the health centre. I think they could reach a higher functional level if they were given more time… for cognitive rehabilitation at home."In addition, the participants highlighted how addressing patients' cognitive rehabilitation needs also seemed to rely on health professionals' opportunities to utilize their professional competence. One participant expressed that OTs and others had competence working with cognitive rehabilitation but that “they do not get in position to use it.” Another indicated that there are health professionals that “want, can, and work with cognitive matters in the municipalities.” Nevertheless, few had cognitive rehabilitation as a dedicated and explicit task in their job descriptions. Consequently, other requested assignments were prioritized.Last, competencies that were available, although less known and therefore not sufficiently utilized, were discussed. For example, several participants described a lack of awareness of day centre services and therapists who had competence in cognitive rehabilitation and could follow-up patients of working age overtime. While every municipality should have a coordinating unit to turn to for information, it seemed undefined who was to obtain and provide information and when about the poststroke situation and competence available to support patients' recovery."… so, the problem lies with us, not the ones [the patients or caregivers] trying to get in hold of [services]. I mean we are “off the hook.” Instead, we are the ones that should provide information about the professional support available."All things considered, professional competencies in general appeared not adjusted to meet these patients' needs in the long term. Although some services that provide relevant competence were described, health professionals with cognitive rehabilitation skills in this study region were reported to either not be in a position to use inherent resources or not be sufficiently utilized.The aim of this study was to explore multidisciplinary health professionals' experiences with rehabilitation trajectories and factors affecting healthcare services to patients with PCSI in a North Norwegian context. The results show that the participants found that the long-term rehabilitation services after discharge from hospital in general were not adjusted to patients' most prominent needs. Recurring issues in the empirical material were that patients with mild and moderate PSCI were at risk of receiving few services, receiving follow-ups that were not tailored their needs or being left with no services at all.when during the stroke survivors' trajectories follow-up was needed. A review based on self-reported cognitive disorders summarized cognitive difficulties as a prominent phenomenon after stroke, where the consequences seem to intensify overtime through increasing demands for activity and participation [Although cognitive impairments in many cases have a profound impact on everyday life for patients and their families, there is a lack of services that address cognitive rehabilitation needs in municipalities. However, our study showed that if services were available, emphasis was not given to cipation . Thus, ccipation  that wascipation . These scipation , 39. OptThe results further reveal that training is rarely given in patients' own environments. Initially, during postacute inpatient rehabilitation, interdisciplinary professionals may adjust interventions that align with the patient's stage of recovery. At the same time, these interventions are not performed in ideal surroundings, whereby the activities are out of context and apart from the familiar environment. After discharge to home, different needs often occur. These needs may be reflected in more advanced instrumental activities of daily living, where access to the patients' everyday life and habitual surroundings is called for . NeverthIn line with previous research , 22, 26,Furthermore, our study found that there is often a lack of multidisciplinary teams in municipalities: where teams are established, they are generic in nature, and PSCIs are seldom part of the teams' assignment. Within the field of multidisciplinary healthcare, a systematic review pointed out that to deliver effective care across stroke trajectories, collaborating teams, especially when adopting an interdisciplinary approach, are a key contributor to quality in stroke services . HoweverThe participants acknowledged various factors impacting health professionals' competence and discussed how clinical competencies were put into practice. Some of the obstacles in providing long-term management were described as a lack of resources in terms of interdisciplinary stroke-specific teams, coordinators, and health professionals addressing cognitive impairments and to overcome the workload for competence development. The participants reported no designated public health professionals specialized in supporting cognitive rehabilitation in the municipalities, except services at a day centre, which were reported as not being sufficiently utilized. In rural areas and small municipalities, the availability of professionals with stroke specific competence are difficult to obtain . EnsurinThe Norwegian Stroke Guidelines recommend that stroke survivors with mild to moderate impairments are discharged directly from hospital with an equivalent program of rehabilitation provided in their own home settings , as it iAs discussed by Heiberg et al. , in the Since our main focus was to explore follow-up in long-term trajectories, participants with experience with the transition home and further follow-up in the municipalities were included. Multidisciplinary professionals from both specialist and primary healthcare were strategically recruited for this study, representing a broad range of experiences and years working in the neurorehabilitation field. Thus, participants working in stroke units or inpatient rehabilitation units within specialist healthcare were not represented, which may have contributed to the exclusion of some perspectives in the material. The study was based on a small sample size. Although we strove for heterogeneity in the sample, coincidentally, only one man was represented due to the composition among employees at the different departments at the time. However, the group represented a diverse selection of professionals engaged in different interdisciplinary teams, coordination services, and a municipal rehabilitation unit. Their experiences added to our understanding of vital perspectives on different factors impacting healthcare services and stroke rehabilitation trajectories.The study demonstrates that stroke rehabilitation services focused on early, intensive time-limited support must be supplied with long-term support to help stroke survivors manage cognitive impairment in daily living. Greater efforts are needed to ensure a systematic development of the cognitive rehabilitation field in the North Norwegian area, where occupational therapists, speech therapists, and neuropsychologists in particular play an important role. The study shows that the availability of personnel and competence in cognitive impairments after stroke can be demanding in rural areas. To ensure good, personalized services, available ambulatory specialized rehabilitation services are necessary. Continued efforts are needed to further develop interdisciplinary, cross-sectoral, and digitally supported rehabilitation models to strengthen the rehabilitation service for this patient group.A key policy priority should be to promote cognitive rehabilitation and further research addressing how long-term follow-up of these patients and their families should be organized to provide optimal rehabilitation services. This information can be used to further develop targeted interventions aimed at ensuring good follow-up for stroke patients with cognitive challenges in North Norway.


The incidence of stroke and the burden on health care and society are expected to increase significantly in the coming years, due to the increasing aging of the population. Various sensory, motor, cognitive and psychological disorders may remain in the patient after survival from a stroke. In hemiplegic patients with movement disorders, the impairment of upper limb function, especially hand function, dramatically limits the ability of patients to perform activities of daily living (ADL). Therefore, one of the essential goals of post-stroke rehabilitation is to restore hand function. The recovery of motor function is achieved chiefly through compensatory strategies, such as hand rehabilitation robots, which have been available since the end of the last century. This paper reviews the current research status of hand function rehabilitation devices based on various types of hand motion recognition technologies and analyzes their advantages and disadvantages, reviews the application of artificial intelligence in hand rehabilitation robots, and summarizes the current research limitations and discusses future research directions. A stroke is one of the most common causes of adult labor loss. It significantly affects people’s quality of life. A patient may endure certain movement disorders after a stroke, such as paralysis of the face, arm, and leg on one side of the body. This is known as hemiplegia . ReducedEffective hand function rehabilitation training based on rehabilitation systems should, at least, meet the following three principles ,6: firstIn the development of hand rehabilitation robots, software systems are equally important in the design and production of hardware devices. With the development of artificial intelligence, big data, cloud computing, and 5G technology, the requirements for the software system of hand rehabilitation robots have also increased. It should not only fulfill the basic hardware control function and the human–machine interaction function, but also be more intelligent, diverse, and personalized . ArtificThe outline of this paper is as follows: Hand gesture recognition based on computer vision technology mainly uses external devices to collect image data of gestures, such as cameras, and then processes the images with vision techniques, such as deep convolutional networks, to complete recognition and classification. This method is non-invasive, does not require wearing extra equipment, the user is not easily fatigued, and the calibration procedure is simple and convenient to use. It can be applied to fine hand rehabilitation training and hand function assessment tasks. However, this method also has some shortcomings. First, it has high requirements for the external environment. Second, the recognition speed and accuracy of it are relatively low. Third, sometimes, it needs to affix specific markers. The devices that collect gestures usually include color cameras, depth cameras, etc. These hand gesture recognition techniques based on computer vision have been applied to hand rehabilitation robots and independent hand function rehabilitation game systems.First of all, some cameras and computer vision techniques can be used to locate the hand’s position and recognize some basic hand gestures, which will provide patients with some more exciting games through augmented reality and virtual reality to enhance their interest in training. Studies have shown that video games are beneficial for improving cognitive dysfunction in patients . Other sFor example, Hondori et al.  developeIn addition to webcams, similar hand modeling, tracking, and recognition functions can be accomplished using depth cameras and color cameras without markers. The primary devices currently used are Leap Motion, Microsoft Kinect V1 and V2, etc.The Leap Motion uses infrared LED and a gray-scale camera, which is cheaper and can handle hand models only. Wang et al.  demonstrMukai et al.  developeThe Kinect uses depth cameras, infrared emitters, color sensors, infrared depth sensors, turnover motors, RGB cameras, etc., all of which are needed to generate a full-body or half-body model, resulting in the latter being less efficient and accurate in operation than the former.For example, Cipresso et al.  developeHowever, these systems lack tactile feedback, game angle, and time-based safety constraints based on the patient’s motor ability. Saini et al.  solved tAvola et al.  proposedIn addition to the above applications, computer vision technology can also be used simply for gesture recognition. The hand rehabilitation robot can be controlled by recognized gestures to carry out the corresponding motion. It can also identify the current motion state of the rehabilitation robot and provide feedback to the system for further adjustment of the robot or evaluation of the training situation.Cordella et al.  developeFarulla et al.  proposedNam et al.  developeThere are also many related types of research and achievements in China. For example, Hefei University of technology ,29,30,31Nanchang University  developeLiu Hongmei et al.  from ShaGesture recognition based on wearable devices requires users to wear devices, such as gloves, rings, bracelets, wristbands, armbands, etc., and collect the motion data or physiological signal data of the user’s hand movements through the sensors on the devices for recognition. This method can recognize more gestures with small input data and high precision. It can identify hand motion and three-dimensional information in space in real-time. It is not easily disturbed by external interference and has good robustness, but it is usually expensive. Wearing equipment may interfere with the therapist’s motion, and the accuracy may be affected when the user is sweating. It is also easy to make the patient tired, and calibration is usually required before each use. According to the types of data collected, wearable devices can be divided into physiological signal sensors, kinematic signal sensors, optical signal sensors, etc. Multiple transmissions are often selected simultaneously as multi-mode fusion data inputs in actual use. Gestures recognized by wearable devices can provide control targets for hand function rehabilitation robots and complete hand function training in the active mode. They can also be used for the evaluation of a patient’s rehabilitation.The bioelectric signals that can be used for hand function rehabilitation robots usually include electroencephalogram (EEG), electro-oculogram (EoG), electromyography (EMG), etc. Generally, the acquisition system collects the required bioelectric signals from the patient’s body surface, processes and analyzes the signals to obtain the patient’s motion intention, which is equivalent to synchronously identifying the patient’s hand action, and then transmits the corresponding motion instructions to the hand rehabilitation robot to drive the patient’s hand to make related motions.Wang Jing et al.  from Xi’Soekadar et al.  from GerZhang et al.  from ChaBioelectrical signals to control hand rehabilitation robots have considerable application prospects. This mode is more in line with the physiological function of the human body; that is, there are physiological signals first, and then muscle activities to produce hand movements. However, the balance and optimization between comfort, diversity, stability, accuracy, timeliness, and timeliness need to be further studied, and the safety of unsupervised training is also worthy of attention.The kinematic parameters of hand motion include fingers and joints’ coordinates, displacement, angular displacement, motion speed, angular velocity, acceleration, inertia, etc. The kinematic sensors used to measure these parameters in a hand rehabilitation robot mainly include flexible angle sensors, accelerometers, gyroscopes, pressure sensors, etc. Compared with rehabilitation robots based on physiological signals, most rehabilitation robots with integrated kinematic sensors can provide a signal of superior quality and better tolerance with regard to placement of electrodes. They can recognize finer gestures and complete finer-grained training tasks, which is very helpful for the treatment of patients in the later stages of stroke rehabilitation.Most of the kinematic sensors of hand rehabilitation robots choose the angle sensors, because they can obtain the angle information of each finger joint more directly and then recognize more fine gestures, which can be used for hand function rehabilitation and evaluation. For example, Hong et al.  from theWhen the patient has a certain degree of hand function, the tactile sensor and pressure sensor can also be used to identify the motion intention of the affected hand when grasping the object and judge the required force. For example, Nilsson et al.  from SweMany devices simultaneously use angle and pressure sensors to obtain more comprehensive kinematic information. For example, Chen et al.  proposedThe angular information during a hand motion is not only the finger joint bending angle mentioned above, but also the opening angle and the degree of overlap between each finger. Sometimes, it is necessary to use an opening angle sensor to measure the degree of the spread between fingers, which can better identify different types of hand gestures and analyze the patient’s hand function rehabilitation more comprehensively. For example, Li Nan et al.  designedFor the recognition of dynamic hand motions, in addition to static motion information, some dynamic posture information, such as velocity and acceleration, and spatial information, such as relative coordinates, are required. At this time, accelerometers, gyroscopes, and other sensors are needed. These applications are often combined with inertial sensors or posture sensors in practice. For example, Zhengzhou University  designedConventional sensors are usually directly mounted on hand rehabilitation robots, which are difficult to integrate with actuators, complex in structures, and challenging to install on flexible actuators. Sensors made of elastomers or conductive liquids will deform when subjected to surface pressure, resulting in non-linear output and compromising accuracy. Optical sensors applied to hand rehabilitation robots mainly include optical fiber sensors and photoelectric sensors. The fiber optic sensor has high sensitivity, a compact structure, and strong anti-interference ability, which means it cannot affect the hand motion when applied to the hand rehabilitation robot. Optical sensors generally work by measuring the deviation of a light beam incident on a photosensitive surface.He et al.  proposedDiez et al.  proposedLiu Chenglong et al.  from HuaIn addition to the above-mentioned research on hardware devices for hand movement recognition, hand rehabilitation robots also require research in the field of software, combining hardware and software to achieve intelligence and wisdom in the devices. With the development of artificial intelligence technology, many new fields and products have emerged. Artificial intelligence can be applied to various modules of hand rehabilitation robots, such as hand movement recognition, control of hand rehabilitation robots, human-machine intelligent collaboration, interactive game design, training program design and result evaluation, cloud platform, structural design and optimization of hand rehabilitation robots, and so on. However, in practice, the research and application of artificial intelligence in hand rehabilitation robots are still not significant or not deep enough due to technical limitations and insufficient deployment of related medical facilities .In hand rehabilitation robots, gesture recognition mainly requires real-time isolated dynamic gesture recognition, and artificial intelligence algorithms can be used for this recognition. The core idea is mainly to build a matching model from the data training set and then use the model to predict the recognized gestures. The more commonly used methods are mainly linear discriminant analysis (LDA), support vector machines (SVM), convolutional neural networks (CNN), long short-term memory (LSTM) and recurrent neural networks (RNN). For example, Zhang Guangxing  from QinSafety issues need to be addressed in the control strategy or algorithm of the hand rehabilitation robot. More precise control strategies are needed, as well as balancing the ratio between the degree of control the robot has over the hand and the risk of miscalculation. For example, Jun Wu  designedArtificial intelligence can also be applied to the human-robot collaboration of hand rehabilitation robots, mainly in the assisted training mode. For example, Wang Xiangyu  from HarIn order to increase patients’ interest and motivation during rehabilitation training, the hand rehabilitation robot can be equipped with some interactive games, such as interactive control based on voice recognition, virtual reality games based on visual recognition, and games based on brain–computer interface, etc. Artificial intelligence is also applied here. For example, Zhu Xikun  from ZheTraditional rehabilitation training programs and assessment of rehabilitation results are mostly conducted by doctors themselves, but this requires a lot of medical resources and sometimes has a certain subjective component. Theoretically, the application of artificial intelligence can improve this problem by digitizing and standardizing the assessment report and training program, which can identify the patient’s current hand movement status and physiological data in real time, combine it with the relevant medical program data, assess the patient’s current muscle strength level and rehabilitation training effect, and intelligently design the training program based on the assessment results. However, the actual effectiveness of these applications has yet to be clinically validated. For example, Neofect  has deveThe cloud platform can be used for one-stop implementation of training program design and evaluation of training results, as well as remote rehabilitation and real-time interaction. The cloud platform can monitor equipment parameter data in real time and provide safe and reliable rehabilitation training for patients. It can also apply digital twin technology to break the limitation of time and space and share big data sets of patients’ medical records, intelligent rehabilitation assessment reports, and training reports. Through these data, training programs are intelligently designed and pushed to rehabilitation robots, with interactive games, real-time remote training, and full cloud health management. For example, Xinyu Tang  from SouIn addition to the software applications mentioned above, artificial intelligence can be applied to the hardware structure of the rehabilitation robot. For example, AI can optimize the mechanical mechanism based on human body data to make the machinery more ergonomic and better fit the biological curve of the human body to enhance the use experience.The previous context introduces the hardware and software of existing research on hand function rehabilitation robots. However, problems also exist that require further attention in future research. The following list summarizes the existing problems and limitations of the employed techniques in the current studies.(1)Hand function rehabilitation equipment based on computer vision techniques is sometimes cumbersome, requires affixing markers, and is not convenient or portable enough for the external environment. Some aspects of the equipment will even make users feel dizzy. When using monocular vision, the recognition accuracy is affected by the light and color of the environment, skin color, etc. Although these problems can be improved to some extent when using binocular vision, it requires more complicated calibration and correction, which increases the computational cost and creates other interference factors that affect the image quality. Therefore, it is necessary to continue to improve the equipment and optimize the algorithm.(2)When using computer vision technology for bare hand gesture recognition and training patients’ hand function with games in the virtual environment, the tactile feedback of the affected hand is often not available, ignoring the motion angle and time that needs to be strictly set according to the patient’s motion ability to ensure the safety of the game. The feedback is mandatory during sensorimotor rehabilitation. Therefore, more attention should be paid to the design by adding some threshold limits.(3)When using computer vision technology only for gesture recognition and rehabilitation training, the type of data is relatively simple. When recognizing complex gestures, it is inevitable to encounter a situation where the acquisition cannot be collected or where the acquisition and recognition are inaccurate. Some other information collection methods, such as physiological signals and so on, can be used to achieve multi-information fusion.(4)Most hand rehabilitation robots based on wearable device gesture recognition need to wear additional sensors, which increases the cost and at the same time is inconvenient to use and has certain restrictions for patients. The recognition algorithm and the central processor of the wearable device have higher requirements, so there may be some delays. The algorithm also needs to be retrained if the number of sensor positions changes. Therefore, the balance and optimization between comfort, diversity, stability, accuracy, and timeliness in acquisition need to be further studied, and the safety of unsupervised training also deserves attention.(5)The hand rehabilitation robots based on physiological signal gesture recognition are prone to instability in signal acquisition and transmission, and the amount of data that can be recognized is limited, making it difficult in the application to fine finger motion rehabilitation and the recognition of continuous motion. To address this aspect, multi-mode control strategies can also be used, in conjunction with kinematic signals, optical signals, etc.(6)Regardless of the techniques used, the rehabilitation robot should be designed with a closed-loop control network, which can, for example, provide force feedback, angular feedback, position feedback, haptic feedback, etc. through sensors to provide timely feedback and adjust parameters according to the real-time status of patients.(7)Most hand function rehabilitation robots are still in the research stage, and those based on computer vision technologies require far more attention than those found on wearable sensors. Evaluated using the “Technology Readiness Level (TRL) system” by Mankins, most hand function rehabilitation systems based on computer vision technology are still at TRL5 or 6 (technology demonstration). A few of them are at TRL7 or 8 (system/subsystem development), and few reach TRL9  . On the Besides the above existing problems, the clinical utility of hand function rehabilitation robots has been evaluated in terms of appropriateness, acceptability, and practicability . Most esIn the future, we expect that a hand function rehabilitation robot should first be able to fully simulate a human therapist’s approach and achieve complete “human-machine integration”. Secondly, it can be upgraded under human guidance to assess the patient’s condition, give rehabilitation training plans and evaluate the training effect. Moreover, it can realize a more convenient and intelligent human–machine interaction mode. Finally, it can also assist in optimizing the allocation of medical resources to achieve efficient and interesting telemedicine.


Glaucoma disease is characterized by an increased intraocular pressure (IOP), glaucomatous alterations of the optic disc and corresponding visual field defects. Even lowering the main risk factor IOP until an individual target level does not prevent this neurodegenerative disorder from proceeding. Several autoimmune mechanisms were discovered, partly showing a functionality. One of these autoimmune phenomena targets the ß2-adrenergic receptor  and is linked to an elevated IOP and an impaired retinal microcirculation. As neurodegenerative disorder, Alzheimer’s Disease (AD) is postulated to share a common molecular mechanism with glaucoma. In the present study we investigated autoimmune phenomena targeting the ß2-AR in patients with AD. Sera of the patients were analyzed in a rat cardiomyocyte bioassay for the presence of functional autoantibodies against ß2-AR. In addition, different species of amyloid beta (Aß) monomers were tested . Our results demonstrate that none of the short-chain Aß  showed any agonistic or inhibitory effect on ß2-AR. Contrary, long-chain Aß-[Pyr]3–43, representing a major neurogenic plaque component, exerted an activation that after blocking by the ß2-AR antagonist ICI118.551, could be identified as that the effect was realized via the ß2-AR. Moreover, the long chain Aß1-40, Aβ1–42, and Aβ10–37, yet not the short-chain Aß peptides prevented the clenbuterol induced desensitization of the ß2-AR. In addition, we identified functional autoantibodies in the sera of AD patients, activating the ß2-AR, like the ß2-agAAb found in patients with glaucoma. As autoimmune mechanisms were reportedly involved in the pathogenesis of glaucoma and Alzheimer’s Disease, we postulate that overstimulation of the ß2-AR pathway can induce an adrenergic overdrive, that may play an important role in the multifactorial interplay of neurodegenerative disorders. In vivo data suggested a link of the ß2-agAAb to the increased IOP and impaired retinal microcirculation  3–43). Our results demonstrate that only the Aß peptide Aß-[Pyr]3–43, but not the other Aß peptides or Aß fragments are able to activate the β2 adrenoceptor similar as the classical β-adrenergic agonists. The long-chain Aß-[Pyr]3–43, representing a major neurogenic plaque component, exerted an activation of the ß2-AR blocked by the ß2-AR antagonist ICI 118.551 but not by the β1-AR antagonists bisoprolol or the α1- AR blocker urapidil .A long-term incubation of the cardiomyocytes with adrenergic agonists leads to desensitization of the adrenoceptors and to a resistant to the used drug. The incubation of the cardiomyocytes with the β2-adrenergic agonist clenbuterol caused a time dependent reduction of the response to this agonist. A washing procedure removed this agonist completely and a new stimulation with 1μM clenbuterol generate only a strong reduced response . Yet thiGlaucoma disease is one of the leading causes of irreversible blindness worldwide . Its comThe autonomic system is a main actor in human body. It regulates important function and is the basis of human life. Any alterations in the sympathetic and/or parasympathetic pathways results in an impairment or overaction of the autonomic system. Adrenergic receptors are also present on pericytes thus it can be hypothesized that any alterations in adrenergic activity might influence and alter microcirculation. It is commonly accepted that an impaired microcirculation can induced amyloid plaques in AD –49 and iIn patients with AD disease the ß2-adrenergic dysregulation might be mediated in three different ways. First is the direct activation of the ß2-AR by its classical natural agonist adrenaline or noradrenaline. Both compounds activate the ß1-, the ß2- and the ß3-AR. The stimulation with adrenaline, isoprenaline or the more ß2-AR specific agonist clenbuterol induced a positive chronotropic response in the cardiomyocyte bioassay, which could be blocked by the specific ß2-AR antagonist ICI118.551. It was shown that patients with AD develop functional active autoantibodies directed against the ß2-AR. These ß2-agAAb activate the ß2-AR in a similar manner as the ß2-adrenergic agonists , but in Secondly, it was shown that the activation of the ß2-AR may play a role in the formation of amyloid β (Aβ). ß2-AR agonists activate the gamma-secretase, forming Aβ from the amyloid precursor proteins (APP), resulting in an elevated accumulation of Aβ . MoreoveWang and coworker observed in prefrontal cortical neurons that Aβ1–40 and Aβ1–42 can bind to the β2-AR, and activate the protein kinase A (PKA) in these ,65. ThisA third possibility to influence the β2-AR activity can be seen in fact that the long- chain Aβ peptides Aβ1–42, Aβ1–40, and Aβ10–37, yet not the short breaking products of the amyloid Aβ1–14, Aβ25–35 and Aβ28–40 prevent desensitization of the β2-adrenergic agonist clenbuterol. The combination of the long chain Aβ peptides and clenbuterol induced a permanent activation of the β2-signal cascade similar to the response seen for the agonist-like β2-AAbs. The β2-AAbs induced their long-lasting effect via a cross-link between the two arms of the antibody molecule and two receptors. This cross-link stabilized the active conformation of the receptor and led to a permanent stimulation (as described above). Why the long-chain Aβ-peptides can prevent the desensitization of the adrenoceptors is an open question and under investigation. Yet, we were able to show that other proteins for example the C-reactive protein (CRP) can also prevent the desensitization of different GPCRs . MoreoveWe hypothesize that a dysregulated β2-mediated signal cascade might be a molecular mechanism of an autonomic dysregulation, both observed in neurodegenerative disorders, e.g. glaucoma and AD. A seropositivity for β2-AAb and the β-adrenergic agonists itself can enhance β2-AR activation. In addition to β2-AAb, the Aβ peptide Aβ-[Pyr]3–43 was also able to activate the β2-AR mediated signal cascade directly. Furthermore, the long chain Aβ peptides induced like the β2-AAb a permanent stimulation and deactivate the protection mechanism of the cells against an adrenergic overstimulation. All these mechanisms may play an important role in the pathogenesis of glaucoma and AD.Glaucoma and AD are known to be neurodegenerative disorders with an involvement of autoimmune mechanisms and impaired microcirculation. As both disease entities showed a seropositivity for β2-AAb we postulate that an overstimulated ß2-AR pathway can induce an adrenergic overdrive, which can be enhanced by amyloid-β peptides in patients with AD.S1 File(DOCX)Click here for additional data file.


Among various post-translational modifications (PTMs), acetylation is an essential PTM recognized in AR and is governed by the regulated actions of acetyltransferases and deacetyltransferases. Acetylation of AR has been identified as a critical step for its activation and depending on the site of acetylation, the intracellular dynamics and activity of the AR can be modulated. Various acetyltransferases such as CBP, p300, PCAF, TIP60, and ARD1 that are known to acetylate AR, may directly coactivate the AR transcriptional function or help to recruit additional coactivators to functionally regulate the transcriptional activity of the AR. Aberrant expression of acetyltransferases and their deregulated activities have been found to interfere with AR signaling and play a key role in development and progression of prostatic diseases, including prostate cancer (PCa). In this review, we summarized recent research advances aimed at understanding the role of various lysine acetyltransferases (KATs) in the regulation of AR activity at the level of post-translational modifications in normal prostate physiology, as well as in development and progression of PCa. Considering the critical importance of KATs in modulating AR activity in physiological and patho-physiological context, we further discussed the potential of targeting these enzymes as a therapeutic option to treat AR-related pathology in combination with hormonal therapy.The development and growth of a normal prostate gland, as well as its physiological functions, are regulated by the actions of androgens through androgen receptor (AR) signaling which drives multiple cellular processes including transcription, cellular proliferation, and apoptosis in prostate cells. Post-translational regulation of AR plays a vital role in directing its cellular activities  It is a serine protease secreted by both normal and neoplastic prostate epithelium and is mainly responsible for liquefaction of the seminal fluid coagulum by proteolytic cleavage of semenogelin I and II, major gel forming proteins present in the semen coagulum. High serum levels of PSA are strongly correlated with advanced stage of PCa and therefore, measurement of PSA levels serve as an important biomarker for clinical detection and evaluation of PCa progression, as well as in the assessment of the patient’s response to PCa drugs. However, several other non-cancerous prostate conditions, such as benign prostatic hyperplasia (BPH) or inflammation or infection of the prostate or recent ejaculation, can also elevate or modify PSA levels, hence, the use of the PSA test alone for diagnosis of PCa is considered controversial by urologists as it can lead to over-diagnosis and over-treatment as part of screening (Prostate cancer (PCa) is one of the most frequently diagnosed solid tumors in men and remains the second leading cause of cancer-related death after lung cancer in men, according to the latest data provided by Cancer Statistics 2022 . The procreening . Alternacreening .A wide range of AR antagonists and antiandrogens that block androgen-induced stimulation of the prostate or suppress androgen levels, are frequently used for androgen-dependent PCa therapy . In most617RKCYEAGMTLGARKLKKL634), crucial for mediating AR translocation into the nucleus , a DNA binding domain (DBD), and a ligand binding domain (LBD), each serving specific functions Figure 1 nucleus . Besides nucleus , 17. Pos nucleus . AR LBD  nucleus . AF-2 re nucleus . In addi nucleus . The cla nucleus , however. KATs are an important category of AR coregulator proteins and possess intrinsic acetyltransferase (KAT) activity. KAT-induced acetylation of lysine residues in histone tails neutralizes its positive charge which weakens its interaction with DNA and thus, alters the structure of chromatin and favors the binding of effector proteins to the chromatin, thereby facilitating the process of transcription , estrogen receptor (ER), retinoid X receptor (RXR), farnesoid X receptor (FXR), progesterone receptor (PR), androgen receptor, and steroidogenic factor 1 (SF-1) have shown to be direct substrates for p300 lysine acetyltransferase activity   located within the hinge domain of AR, which are targeted for acetylation by p300 , that facilitate PCa progression poptosis –48. Morepoptosis . Similarpoptosis . Clinicapoptosis . Later, poptosis . Altogetvia acetylation and modification of nucleosomes in cooperation with other proteins  (PCAF was identified as a p300/CBP interacting protein and belongs to the superfamily of GCN-5 related N-acetyltransferases (GNATs) . The PCAproteins . Direct proteins , 58. PCAteins  . Fu and teins  . Interesteins  . An inveteins  . A recenteins  . AltogetGcn5-/-) die as early as day 10.5 post-coitum , a member of the MYST family of lysine acetyltransferases, was originally identified as an interacting partner of ORC1, the largest subunit of the origin recognition complex, and like other members of MYST family, HBO1 contains a highly-conserved catalytic MYST domain and a unique N-terminal serine rich domain  Figure, 101. AlSharma et al. demonstrated, for the first time, direct interaction of HBO1 with the AR using a yeast two hybrid system, pull-down and immunoprecipitation assays . Androge). Important roles for ARD1 have been detected in a wide variety of cellular activities like cell division, proliferation, hypoxia, apoptosis, and autophagy (Also known as N-α-acetyltransferase 10 (NAA10), is an N-acetyltransferase (NAT) enzyme that belongs to the GCN5-related N-acetyltransfererase (GNAT) superfamily that is widely-known to catalyze the transfer of an acetyl group from acetyl coenzyme A to the target protein’s N-terminal amino acid . Althougutophagy . Not surutophagy , 110. Thutophagy , 112. Liutophagy , 117–119utophagy . The firutophagy . They shutophagy . Lysine utophagy . Interesutophagy , 121. Toutophagy , 121. Siutophagy . These fAlthough there is no definitive cure available for advanced and metastatic prostate cancer, several therapeutic options are available that can prolong survival and improve the patient’s quality of life for quite some time. A number of factors, such as the patient’s age, health, type of symptoms, PSA level, grade of tumor, extent of tumor spread, and response to the therapy, can help in making treatment plan decisions and predicting outcomes. As over 80% of PCa shows dependence on androgens, most treatments are directed towards inhibiting the AR activity either by using antiandrogens, lowering the secretion of testosterone by surgical castration, or by using luteinizing hormone-releasing hormone (LHRH) agonists and antagonists , 123. Cy). Indeed, treatment with LHRH agonists are also used as a preferred alternative to surgical castration. Leuprolide, goserelin, and triptorelin are clinically approved LHRH agonists to treat PCa, but can cause testosterone flare as a noted side effect observed during the first few weeks of treatment which is usually countered by coadministration of antiandrogen drugs  or an LHRH (gonadotropin-releasing hormone (GnRH)) analogs to achieve combined androgen blockade or total androgen blockade . Althougen drugs . Whereasen drugs Table 1.via obstructing AR nuclear translocation, downregulating the AR levels, and inhibiting the expression of AR-dependent genes such as PSA and TMPRSS2 , which were obtained as secondary metabolites by culturing a production strain belonging to Penicilliumsp. and examined their effect on the viability of DHT-dependent LNCaP cell line and DHT-independent PC-3 cell line  on histone acetylation and expression of AR target genes and found a significant decrease in H3K27ac at AR/p300 overlapping peaks, which inhibited AR-mediated transcription, although AR–p300 interaction or binding of the AR or p300 to the target gene promoter was not found to be inhibited by this compound -ethan; an isothiazole), a highly-selective and potent inhibitor against TIP60 catalytic activity, and showed that NU9056 treatment could inhibit cell growth and colony formation in several PCa cells including androgen sensitive LNCaP and insensitive PC-3 cells . Furtherinst PCa .Garcinia indica, shows non-specific inhibitory activity against both p300 and PCAF, while anacardic acid extracted from the cashew nut shell, is a broad-spectrum KAT inhibitor that can non-selectively inhibit p300, PCAF, and TIP60 activity , India BMS/ADHOC/CMB/ 2015-0817/JUN-16/1 /UP/PVT to AG and DST grant (SR/WOS-A/LS/228/2017) to BJ. AA acknowledge financial support from the DST-INSPIRE fellowship (DST/INSPIRE Fellowship/2019/IF190432).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Culex pipiens mosquitoes in 2010; then, it was found in several mosquito cell lines in a further study in 2018. For mammalian cells, we were able to confirm that CYV does not replicate as it was previously described. Additionally, we found that CYV does not replicate in honey bees or locusts. However, we detected replication in the Culex pipiens biotype molestus, Aedes albopictus, and Drosophila melanogaster, thus indicating dipteran specificity. We detected significantly higher mortality in Culex pipiens biotype molestus males and Drosophila melanogaster, but not in Aedes albopictus and female Culex pipiens biotype molestus. CYV could not be transmitted transovarially to offspring, but we detected venereal transmission as well as CYV in mosquitos’ saliva, indicating that an oral route of infection would also be possible. CYV’s dipteran specificity, transmission routes, and killing effect with respect to Culex males may be used as powerful tools with which to destabilize arbovirus vector populations in the future.Mosquitoes are vectors of various pathogens that cause diseases in humans and animals. To prevent the outbreak of mosquito-borne diseases, it is essential to control vector populations, as treatment or vaccination for mosquito-borne diseases are often unavailable. Insect-specific viruses (ISVs) have previously been described as being potentially helpful against arboviral disease outbreaks. In this study, we present the first in vivo characterization of the ISV Culex Y virus (CYV). CYV was first isolated from free-living  Aedes aegypti mosquitoes [Mosquitoes are widespread vectors of various pathogens. In particular, mosquito-borne arboviruses such as dengue virus (DENV), West Nile virus (WNV), Sindbis virus (SINV), and chikungunya virus (CHIKV), to name a few, regularly lead to disease outbreaks in human populations and livestock worldwide . Varioussquitoes . Thus, nAedes aegypti cells) as well as in laboratory mosquito colonies [Birnaviridae family, the Culex Y virus (CYV), was isolated from free-living Culex (Cx.) pipiens mosquitoes [Birnaviridae family includes four genera, with three genera  infecting vertebrates excluding mammals, and a fourth genus, the Entmobirnaviruses, that infects insects [Cx. pipiens is considered an important vector of WNV [Cx. pipiens complex, which comprises several biotypes. The biotypes within this complex are morphologically indistinguishable and differ in terms of their essential ecological and behavioral characteristics; however, they can hybridize, thus significantly expanding their host range [One strategy that has gained increasing attention in the past years is the use of insect-specific viruses or entomopathogenic bacteria and fungi to control vector populations. Insect-specific viruses (ISVs) are viruses from a variety of virus families that can only replicate in insect/arthropod hosts and, therefore, not in vertebrate cells . They hacolonies , but alscolonies ,17,18. T insects . In Germr of WNV ,21. Thisst range .Cx. pipiens biotype molestus mosquitoes, we characterized the virus’s replication in vivo, mosquito survival after infection, and the possible transmission routes of CYV. Furthermore, we analyzed the host specificity of this virus by conducting in vitro experiments with mammalian cells and in vivo experiments with the Cx. pipiens biotypes molestus (mosquitoes), Aedes (Ae.) albopictus (mosquitoes), Drosophila (D.) melanogaster (fruit flies), Apis (A.) mellifera (honey bees), and Locusta (L.) migratoria (locusts). All in all, we propose that CYV might be a candidate for new vector control strategies in the future.To expand our knowledge regarding the interaction of CYV with the Cx. pipiens biotype molestus  colony was established in our laboratory in Hannover, Germany, in 2016, and was derived from blood-fed gravid females collected in 2012 in Wendland (W strain), Germany, and from egg rafts collected in 2013 in Langenlehsten (LL strain), Germany [Cx. pipiens biotype molestus colonies were maintained in climate chambers at 26 °C (±1 °C), a relative humidity of 45–75%, and a 16:8 (light:dark) hr photoperiod with 1 h twilight periods at dusk and dawn. Our Cx. pipiens biotype molestus mosquito colony was naturally infected with Wolbachia bacteria. They tested negative for Alphaviruses, Orthobunyaviruses and Flaviviruses (data not shown); the primer sets and thermal conditions employed for amplification have already been published [Ae. albopictus  colony from Nice, France, was provided by Infravec2 (Laboratory of Prof. Failloux), Nice, France, and was maintained at 28 °C (±1 °C), a relative humidity of 60–75%, and a 16:8 (light:dark) hr photoperiod with 1 h twilight periods at dusk and dawn. Mosquito larvae were reared in plastic basins or bowls filled with dechlorinated tap water and fed with TetraPleco fish food tablets  until pupation and hatching. Adult mosquitoes were housed in cages  and fed ad libitum with 8% fructose solution. As a dietary supplement, 0.5 g/L of 4-aminobenzoic acid (PABA) was added to the fructose solution. Adult female mosquitoes were fed with dog blood once a week.The  Germany . In addiublished ,25,26,27Mosquitoes used for the experiments were between two and seven days old. All experiments with mosquitoes were performed in rearing cages (BugDorm-1) in a climate chamber at 26 °C (±1 °C), relative humidity of 45–75%, and a 16:8 (light:dark) hr photoperiod with 1 h twilight periods at dusk and dawn.D. melanogaster yellow white (YW) strain from Bloomington Drosophila Stock Center were reared in drosophila cultivation tubes  filled with approx. 2 cm of corn flour meal agar , 8.5 g of dry yeast , 23.5 g of glucose , 5 g of sugar beet syrup , 0.04 g of Nipagin , 4 g of agar , 1.625 mL of propionic acid , and 400 mL of tap water) to provide food and moisture. D. melanogaster specimens were held at 24 °C (±1 °C) and 45–55% RH in constant darkness, i.e., without a light:dark cycle.Flies of the Locusts were reared in a 12:12 cycle (light:dark) at 30 °C in crowded culture in the facilities of the Institute of Physiology and Cell Biology of the University of Veterinary Medicine Hannover. The animals were fed ad libitum with fresh wheatgrass and wheat bran. Adult locusts were offered tubes filled with moist sand and water-soaked vermiculite for egg laying. Egg tubes were incubated at 30 °C until eclosion.A. mellifera colonies from the apiary of the Justus-Liebig University  in the second half of August 2021. A piece of capped brood comb with late-stage pupae was incubated at 35 °C and 70% relative humidity. The newly emerged bees were transferred to a conical plastic box (18 cm × 18 cm × 7.9 cm) and fed sugar solution (1:1 sucrose and water) ad libitum.Honey bees were collected from two ® CCL-185™). Cells were cultured in Dulbecco’s Modified Eagle Medium  (A549) or Minimum Essential Medium  (BHK-21), both supplemented with (1%) penicillin–streptomycin (100 units/mL) , (1%) L-glutamine (200 mM) , and 10% FBS (A549) or 5% FBS (BHK-21)  at 37 °C and 5% CO2. Cell cultures were split twice weekly to prevent cells from reaching full confluence. To detach the cells from the bottom of the flask, cells were treated with trypsin.Mammalian cells used for infection experiments were BHK-21  and A549  for 1 min, and the homogenate was frozen at −18 °C until further processing. RNA extraction was performed using the QIAamp Viral RNA Mini Kit , according to the manufacturer’s instructions.All insect samples were homogenized before further analyses. For this purpose, insect samples were transferred in groups or individually, depending on the assay, to reaction tubes containing 500 µL of Schneider’s Drosophila Medium  or 1 mL of nuclease-free water (bees) containing two 5 mm steel balls  and were homogenized using the TissueLyser II  at 30 Hz for 30 s or 25 Hz for 3 min (bees). Samples were then centrifuged at full speed , OSM_319 (5′ CGTTGTTAAGGAAGACTC 3′), and a probe OSM_320 (FAM-CACAAGAGGATACACAAGCAGCG-BHQ1) were used. The Luna Probe One-Step RT-qPCR Kit , was used for all samples with 9 µL master mix and 1 µL template in an AriaMx Real-time PCR System . The thermal program consisted of the application of 55 °C for 10 min, followed by 1 min at 95 °C and 40 cycles of 10 s at 95 °C and 30 s at 54 °C . The resulting fragment was 114 bp long.4 RNA copies, which had Ct values between 33 and 35; thus, measurements over Ct value 35 were excluded in further analyses.A PCR standard was prepared using the SuperScript III One-Step RT-PCR Platinum Taq DNA Polymerase Kit  according to the manufacturer’s instructions, thus reducing the final volume from 50 μL to 25 μL, including 1 µL template. A fragment length of 414 bp was amplified using primers OSM_321 (5′ GGCAATGTTCGGGCTCATC 3′) and OSM_322 (5′ CGACAGTGCAGGAGTAGGGG 3′). The cycler program was set to 1 min at 60 °C and 45 min at 50 °C, followed by 2 min 95 °C for reverse transcriptase. This was followed by 40 cycles of DNA denaturation at 94 °C (15 s), annealing at 59 °C (30 s), and elongation at 68 °C (30 s). A final elongation of 7 min was carried out. The resulting PCR product was then purified using the NucleoSpin Gel and PCR Clean-up Kit  according to their instructions. For transcription, a T7 promoter was added to the previous purified PCR product. For this purpose, a T7 promoter was added to the standard OSM_321 forward primer, OSB_728 (5′ TAATACGACTCACTATAGGGGGCAATGTTC 3′). The RNA concentration was measured, and the RNA copy number/mL was calculated. The detection limit for our standard was 1 × 102-anesthetized adult mosquitoes were injected in the lateral metathorax [COtathorax ,29 usingtathorax  (first itathorax )  were sampled in equal numbers of males and females. To study viral growth and dissemination, mosquitoes were injected with 46 nL of CYV (approximately 3.32 × 105 RNA copies) and RNA copies were measured by qRT-PCR using the CYV standard described in 2 and immobilized by removing legs and wings. The immobilized mosquitoes were then rinsed with 70% ethanol to sterilize their exoskeletons and remove static charge from the mosquitoes; then, they were placed on a microscope slide in 50 μL of 1× phosphate-buffered saline , which provides necessary physiological conditions and prevents contamination of the hemolymph. The salivary glands , ovaries or testes, intestines, heads, and carcasses were dissected and washed three times in 200 µL 1× PBS and then pooled in 1.5 mL reaction tubes containing 500 µL Schneider’s Drosophila Medium and two 3 mm steel balls and stored at −18 °C until further processing.In vivo kinetics of viral growth in Drosophila tubes until hatching. After injection, males and females were placed in BugDorm-1 cages and fed ad libitum with an 8% fructose solution under conditions described in To test vertical transmission of CYV within mosquito populations, a total of 126 virgin males and 324 virgin females were infected with CYV by injection and a control group, containing 108 virgin females and 42 virgin males, was injected with Schneider’s Drosophila Medium. To ensure virginity on the day of injection and prior to assembly, pupae were placed in individual Virgin mosquitoes were injected and then incubated again separately sexed for five days. On the fifth day, CYV-infected mosquitoes were joined with virgin CYV-naïve mosquitoes of the opposite sex. After seven days, the surviving individuals, consisting of a total of 168 CYV-injected females, 127 CYV-injected males, 70 CYV-naïve females, and 71 CYV-naïve males, were pooled into a maximum of twelve individuals per pool and sexed and processed as described for qRT-PCR analyses. The minimum infection rate (MIR) was calculated for this experiment as described by Pettersson et al. (2014) , since aCx. pipiens biotype molestus mosquitoes were injected with CYV two to three days after hatching, as described earlier, and caged until five days post-infection (dpi) to ensure viral growth. We examined saliva from 30 CYV-injected males and 30 CYV-injected females. Saliva was obtained by forced salivation of male and female mosquitoes via removal of wings and legs and placing the proboscises in 10 µL of 1× PBS using a pipette tip. Mosquitoes were allowed to feed for 1.5 h. Saliva and mosquito bodies were then tested for CYV by qRT-PCR.For the oral transmission experiments 6 RNA copies was prepared. Feeding was performed in tubes in groups containing a maximum of 12 animals each. The mosquitoes were allowed to feed on the fructose solution containing CYV via cotton swabs (200 µL per cotton swab) for 180 min. Animals with distinctly red-colored abdomens were used for growth kinetics experiments, as described in Furthermore, we performed viral growth kinetic experiments on orally CYV-infected mosquitoes with a total number of 144 CYV-infected mosquitoes comprising 72 males and 72 females. An 8% fructose solution containing 35 µL of red ink as a dye and 5 µL of CYV containing approximately 3 × 102 instar to the early L3 instar, 100 larvae were kept at a constant density of 4 cm3/larvae to prevent the crowding factor from affecting development. Resulting pupae were counted and hatched mosquitoes were frozen at −18 °C.All autogenous egg rafts laid by CYV-injected females described in Wings from 295 frozen female offspring and 286 frozen male offspring from CYV-infected mosquitoes and from 102 female and 97 male offspring from medium-infected mosquitoes were severed near the thorax with precision forceps and wing length (WL) and wing area (WA) were measured. The detached wings were placed on a white sheet of paper with a scalable grid for size reference. A glass slide was carefully placed on the wings to straighten them. Wings were photographed using a Moticam 2.0 camera on a binocular microscope at 10× magnification and measured using ImageJ 1.53c. Wing length was measured using the linear distance between the distal end of the alula and the tip of the third radial vein at the apical margin. Wing area was defined as the area on the upper surface excluding the fringe. The proximal end of the area was defined by a vertical line connecting the end of the fringe and the posterior margin of the alula . WL and Cx. pipiens biotype molestus males and 96 CYV-injected Cx. pipiens biotype molestus females. A control group injected with Schneider’s Drosophila Medium that comprised the same number of mosquitoes was also included in each replicate. Mosquitoes were injected as described in We monitored the survival of 96 CYV-injected 6 cells per well with 500 µL of the appropriate cell culture medium and 10% FBS and allowed to grow to approximately 80% confluence. Approximately 3 × 107 CYV RNA copies were added to 500 µL of Schneider’s Drosophila Medium and used for infection. Mammalian cells were inoculated at 37 °C and 5% CO2 for one hour, with plates gently shaken every 15 min. As stated above (2.4), CYV did not induce any CPE in cells, thus hindering a regular plaque-forming unit assay; therefore, we used RNA copies of the virus to standardize our experiments. After inoculation, the inoculum was removed, and the cells were washed three times with 500 µL 1× PBS (pH 7) and replenished with medium and FBS. Viral kinetics were monitored for seven days, with 200 µL of supernatant collected on day zero, day three, and day seven post-inoculation. Infection and negative controls were performed and treated in triplicate and in three independent experiments.Mammalian cells (BHK-21 and A549) were seeded in 12-well plates  at 0.1 × 10We further investigated the host specificity of CYV by injecting the virus into other insect species. For all infection experiments, each replicate included a virus-infected group and a Schneider’s Drosophila Medium control group. Individuals that died within 24 h post-injection were excluded from further analyses.Ae. albopictus mosquitoes were injected with 46 nL of CYV (approximately 3.32 × 105 RNA copies), as described in For this investigation, D. melanogaster flies of a yellow white wildtype (YW) strain, as representatives of Brachycera, were injected with 18.4 nL of CYV (approximately 7.34 × 103 RNA copies) or Drosophila Schneider’s Medium as control group, respectively. In total, 125 males and 125 females per virus and control group (three to six days old) were observed. For the experiments, two to three D. melanogaster tubes  of strain YW were completely emptied. On the third day after hatching, the newly hatched individuals were placed on fresh drosophila cultivation tubes  filled with approx. 2 cm of corn meal agar and left for three days at 24 °C (±1 °C) and 45–55% RH in constant darkness to allow the immune system of flies fully develop. After injection, 50 flies  per group were placed in fresh Drosophila tubes containing cornmeal agar and maintained under the same conditions as Cx. pipiens biotype molestus mosquitoes. The flies were checked every day for survival, except on weekends. Dead flies were removed from the tubes and stored at −18 °C. After seven days post-infection, the flies were replaced in new Drosophila cultivation tubes to ensure that generations did not mix. We performed survival experiments over 14 days. Dead individuals were removed from the tubes and tested for CYV via qRT-PCR. For viral growth, fruit flies were injected as described and individual pools of five males and five females per pool were tested for CYV at day zero, one, five, ten, and fourteen after injection.A. mellifera, the honey bee, a member of the Hymenoptera order, were injected with 1 µL of CYV (approximately 8.17 × 105 RNA copies) or Schneider’s Drosophila Medium. The bees were quickly anesthetized with CO2 and their thoraxes were injected using Nanoject II . Subsequently, the bees were distributed into the plastic experimental boxes (11.5 cm × 8.5 cm × 8 cm) with a piece of wax, provided with sucrose solution (1:1) ad libitum, and kept under the conditions described above. A total of 29 bees were injected with CYV and 51 were injected with medium for survival experiments described in Specimens of For the study of viral growth kinetics, 36 bees were injected as described above and samples were analyzed for CYV by qRT-PCR at four time points post-injection: zero, three, seven, and fourteen. Two live bees per treatment were frozen and stored at −20 °C until further analysis.L. migratoria, a member of the order Orthoptera, was also injected with CYV. For each treatment group (Schneider’s Drosophila Medium as control and the CYV-injected group), 24 first-instar locusts were injected a few hours after eclosion [5 RNA copies). Locusts were kept in groups of six in Drosophila tubes filled up to 2 cm with agar to provide adequate humidity and were maintained at ambient conditions in the laboratory . The agar-filled tubes were replaced every second day, and the animals were fed with fresh or thawed wheatgrass daily. We performed survival experiments as described in eclosion  with a vAll statistical analyses were performed using Prism 9 Version 9.4.0 . To determine the survival rates, Kaplan–Meier survival curves were generated and significance levels between curves were determined using the log-rank test.t-test with Welch’s correction for parametric data and the Mann–Whitney test for non-parametric data. Multiple t-tests were performed using Bonferroni correction. Results of tests were considered significant when p ≤ 0.05. Significance levels were displayed in four levels by different numbers of asterisks: p ≤ 0.05 is shown as *, p ≤ 0.01 is marked with **, p ≤ 0.001 with ***, and p ≤ 0.0001 is marked with ****.Data were tested for normality using the Shapiro–Wilk test. Parametric analyses of more than two groups were conducted with a one-way ANOVA test or two-way ANOVA test, while non-parametric data were tested using the Kruskal–Wallis test. Individual testing of two groups was performed using the unpaired Cx. pipiens biotype molestus mosquitoes. In total, we dissected 72 males and 72 females divided into 3 independent replicates. The results show that within two hours after its injection into the thorax, all organs were flooded with CYV, although all organs were washed three times in 1× PBS. We obtained an increase in RNA copy numbers in all the tested organs of the females at day three and day seven post-injection (t-test) , the measured RNA copies in the mosquitoes’ organs were lower after injection. On the fourteenth day, higher amounts of viral RNA were measured for all organs compared to the inoculum, namely, 1.16 × 107 in males and 1.40 × 107 in females. The data shown include measurements that passed the detection limit.To investigate viral replication and dissemination in vivo, the viral growth kinetics of CYV were examined. Accordingly, viral RNA was measured in the heads, carcasses, reproductive organs, intestines, and salivary glands  at four time points after CYV injection  in njection A. Exceptnjection B. In alm(t-test) A,B. The t-test, Bonferroni-corrected). In the carcasses, significantly lower viral RNA copy numbers were detected in both sexes at day zero compared to day seven  and day fourteen in males . In addition, a significant difference was observed between day zero and day three in males   .Cx. pipiens biotype molestus mosquitoes, namely, vertical, venereal, and oral transmission, in the following set of experiments.We investigated how CYV could be transmitted within a mosquito population. For this purpose, we tested different transmission routes in 3 and 2.16 × 103 RNA copies in the testes and ovaries, respectively, on day zero to 1.67 × 105 and 2.72 × 105 on day fourteen. The peak viral RNA was measured on day seven in both sexes, with 3.03 × 105 in the ovaries and 2.06 × 105 in the testes.To investigate whether the virus could be transmitted vertically, we first examined CYV replication in the reproductive organs of both sexes A,B. We fCx. pipiens biotype molestus mosquitoes.Next, we tested the vertical transmission of the virus in the CYV-positive mosquitoes to their offspring. Therefore, 672 adult F1 generation mosquitoes were tested for viral RNA copies; consequently, it was revealed that none of the tested samples had tested positive. Therefore, it seems that the vertical transmission of CYV does not occur in 7 12 days post-injection for the females, which was calculated per animal. Their male mating partners, who were originally CYV-naïve in this experiment, had a mean RNA copy number of 3.97 × 103 per animal. Similar results were observed for the CYV-injected males 12 days after injection, with a mean RNA copy number per animal of 1.21 × 107. An average of 2.65 × 103 viral RNA copies were detected in their female, previously CYV-naïve mating partners.Another potential route of viral spread within a population might be the venereal transmission of CYV from CYV-positive to CYV-naïve mosquitoes. The RNA copy numbers were analyzed per mosquito pool; in a second step, we calculated the RNA copy number per individual animal from these data to enable better comparability between pools . To test3, while 63.3% of the males had CYV-positive saliva with a mean RNA copy number of 8.62 × 103 in the male saliva  and 3.69 × 107 .To investigate whether CYV could be transmitted orally in the mosquito populations, first, we conducted a forced salivation assay with the CYV-injected mosquitoes and additionally performed feeding experiments to determine whether the virus could be ingested in the diet and replicate in the mosquitoes. Forced saliva testing confirmed that 100% of the CYV-injected mosquitoes were positive for CYV infection after injection. Regarding the females, 86.7% also had CYV-positive saliva, with a mean RNA copy number of 7.76 × 10e saliva . RNA cop4 RNA copies. The mean RNA copy numbers of all organs combined resulted in a copy number of 4.98 × 103 in the males and 7.07 × 103 in the females on the day of oral infection.In a second step, we found that CYV can be taken up via a fructose solution and that the virus replicates after oral uptake in both sexes . All orgTo investigate whether CYV has a negative impact on mosquito survival, we monitored the CYV-infected mosquitoes in a survival assay for 14 consecutive days and counted the surviving individuals. The survival of the female mosquitoes was not significantly impaired by CYV infection in both the injected and the oral infection group A,C. In mTo investigate whether there could be effects on reproduction in terms of egg rafts, eggs, hatching, pupation and emergence rates, these traits were analyzed based on the viruses found in the reproductive organs. No significant differences were found between CYV- and medium-injected females and their offspring for all traits tested . When egAlthough we could not detect CYV in the offspring, we still considered the question of whether CYV infection of the parental generation might have an impact on the development of the offspring. For this purpose, we measured the wings of the offspring of the CYV-infected mosquitoes. Examination of the morphological characteristics of the virus-affected (CYV group) and non-virus-affected (control group) progeny revealed significant differences in terms of wing sizes, which were divided into wing length (WL) and wing area (WA) .2 (±0.3236), whereas the females of the CYV group had a significantly smaller WA of 2.699 mm2 (±0.2837). Males also showed a significant difference in WA between groups. The measured WA in the male control group averaged 1.851 mm2 (±0.1715) and was, accordingly, larger than that of the CYV group with a wing area of 1.766 mm2 (±0.1677) (With a mean length of 3.453 mm (±0.1806), the WL of the female CYV group was significantly reduced compared to the female control group (3.507 mm (±0.1934)) . Similar±0.1677) .7 viral RNA copies. For the BHK-21 cells, a decrease from the day of infection with a mean of 8.31 × 103 (±4.80 × 103) RNA copies to 6.09 × 103 (±4.50 × 103) RNA copies on the seventh day was observed (3 (±2.45 × 103) RNA copies at day zero to 1.02 × 104 (±9.35 × 104) RNA copies at day seven in the A549 cells. We observed high variances in these data, with RNA copies ranging from 7.41 × 102 to 2.43 × 104 in the A549 cells at day seven. Taken together, and with regard to the high variances we observed in this experiment, we do not assume that CYV would replicate in mammalian cells.Furthermore, in our study, we wanted to confirm that CYV does not replicate in mammalian cells. Therefore, we inoculated BHK-21 and A459 cells with CYV and examined the cell culture supernatants at days zero, three, and seven. We could not observe CPE and, therefore, measured the RNA copies via qRT-PCR with our CYV standard. The inoculum with the infection volume of 2.5 µL in the mammalian cell infection experiments contained approximately 1.82 × 10observed . The resAe. albopictus, with the highest RNA copy number on day five at 4.10 × 107  . The results of the survival tests performed on the fruit flies showed a significantly lower survival rate of the CYV-injected flies compared to the control flies (log-rank: p < 0.0001) starting from the ninth day of fly death   A. When cly death B. In thef 0.7166 C. No sigog-rank) D.Ae. aegypti mosquitoes was observed for ZIKV when co-infected with CFAV [Insect-specific viruses are found across a wide range of virus families, including virus families mainly known for their arbovirus representatives, such as the flaviviruses. Accordingly, most data on ISVs in mosquitoes have been obtained from these arbovirus-related ISVs, such as CFAV or CxFV. In the case of CxFV, the potential to suppress or at least delay arbovirus transmission when co-infected in cell culture with DENV and WNV was described. Furthermore, a lower rate of viral dissemination in ith CFAV . Similarith CFAV .Cx. pipiens mosquitoes [Culex specific virus or if it can infect a wide range of insects.In contrast to CxFV and CFAV, less is known about CYV, which was discovered in 2010 in hibernating, free-living squitoes . Followisquitoes , furthersquitoes ,35. Fransquitoes . Furthersquitoes ,35. SimiCx. pipiens biotype molestus laboratory strains originating from the north of Germany after injection and oral uptake. However, the replication rate was significantly higher after injection , and Semliki Forest virus and in Ae. australis with Whataroa virus [We found that CYV was able to replicate in njection  since three days . Our datree days . This phree days . After ty glands . The abson rates . For arbon rates . For ISVoa virus ,42. Viruoa virus . This soCx. pipiens biotype molestus leads to infection. Furthermore, we were able to demonstrate that CYV is secreted in the saliva, as all the tested saliva samples were positive for CYV after injection experiments, although the measured RNA copy numbers were low. Considering that mosquitoes salivate while feeding [Phlebotomus perniciosus [Cx. annulirostris and Cx. gelidus have shown that, following infection with various flaviviruses, the virus can be delivered to sucrose food sources, albeit in varying amounts depending on the species and virus [It can be hypothesized that one possible route of CYV transmission within mosquito populations is indirect transmission via virus-contaminated food sources, as we have shown that CYV uptake by  feeding , the virniciosus , and expnd virus . Since IDrosophila utilize this mode of transmission [Besides contaminated plant food sources, vertical transmission if often described as a primary transmission route of ISVs. Specifically, baculoviruses and polydnaviruses rely on this mode of transmission; additionally, RNA viruses such as Sigma virus in smission . Regardismission ,51,52,53Cx. pipiens biotype molestus mosquitoes  [Next, the impact of a CYV infection on mosquitoes and their survival and fertility was investigated. We detected a significantly higher mortality rate for sporidia ,72,73,74s (OGVs) . CYV infs (OGVs) . This cos (OGVs) ,78,79; ts (OGVs) ,81,82. Ts (OGVs) ,84,85. WAe. albopictus, a second tested mosquito species, seems to be unaffected by CYV, while the closely related Drosophila  is affected by this virus (reduced survival L. migratoria. The survival rates of both the honey bee and the locust were not affected by CYV infection (Finally, we analyzed the host specificity of CYV. If CYV is to be used as a tool to reduce mosquito population size or to interfere with arbovirus transmission, the environmental safety of this agent needs to be confirmed to prevent unwanted impacts of the vector control measurements on other insects. We were able to confirm that the virus could not replicate in any mammalian cell culture. This finding emphasizes the assumption that CYV may be insect-specific. By examining closely and distantly related insect species, we found that CYV is not mosquito-specific but possibly diptera-specific; nfection C,D, as cnfection C,D. ThesTaken together, due to the male-killing effect and the reduced wing size, which might be associated with the reduced reproductive success of subsequent generations, CYV may be considered for use as a tool to destabilize arbovirus vector populations. Regarding the safety of the CYV agents, we were able to show that no harmful effects on economically important organisms such as honey bees could be observed.