To describe how diabetes nurses in primary care experience the process of learning to practise the person‐centred counselling approach Guided Self‐Determination among adults with type 2 diabetes.A descriptive qualitative design.Data were collected in 2014–2015 by means of individual interviews with four diabetes nurses at two points in time. The data were analysed using qualitative content analysis.Three themes that reflect nurses’ processes in learning to use the Guided Self‐Determination approach were identified: (1) from an unfamiliar interaction to “cracking the code”; (2) from an unspecific approach to a structured, reflective, but demanding approach; and (3) from a nurse‐centred to a patient‐centred approach. The overall findings indicate that the process of learning to practise Guided Self‐Determination increased the nurses’ counselling competence. Moreover, the nurses perceived the approach to be generally helpful, as it stimulated reflections about diabetes management and about their own counselling practices. At pres1.1Guided Self‐Determination is a theory‐driven counselling approach founded in a synthesis of grounded theories  disseminated information about the study during a professional meeting for DNs and by telephone to GPs and invited them to participate. As many GPs in Norway did not have employed Registered Nurses, potential recruits for this study were few. However, the inclusion criteria were as follows: Registered Nurse employed in a GP, more than 1 year of experience in diabetes care and a willingness to participate in GSD training and to use the GSD method when counselling people with T2DM. The head of the GP approved their participation in the study. In total, four DNs working at different GPs consented to participate; all were women of ages 34–54 who had experience in diabetes care ranging from 2 to 20 years (mean = 8 years). Three of the four DNs had formal postgraduate education in diabetes care (60 ECTS).2.3The four DNs included in this study attended the Steno Diabetes Centre for training in the original GSD in 2014. The programme consists of four course days (24–32 hr) over 9 months and delivers competences in using GSD. It includes lectures in the theoretical foundation and application of the reflection sheets, workshops, discussions, supervisions and practising the use of GSD in own clinical practice. The participants were trained in three advanced professional communication skills: mirroring, active listening and values‐clarification response. As part of the clinical practice, each DN recruited two adults with T2DM from their GPs to participate in seven GSD consultations. At the end of the course, the DNs completed a test regarding the GSD approach. A validity assessment tool evaluated whether the DNs’ performances with GSD were congruent with its theoretical foundation. To acquire more experience in practising in GSD, each DN recruited two additional patients with T2DM from their GPs to participate in the GSD method adapted to T2DM, which consisted of four consultations and fewer reflection sheets. As part of this training, each DN participated in three group counselling guided by two GSD supervisors (V.Z. and J.M.).2.4Data were collected over 5 months in 2014–2015 and individual interviews were performed at two points in time: (i) after the DNs had received training and gained some experience using the GSD during consultations with two adults with T2DM; and (ii) after the DNs had used the adapted GSD for two additional adults with T2DM. The rationale to conduct interviews at two points in time was to provide insights and understanding of the process of learning to practice GSD. Each interview took place at the university and each was 40–60 min in length. A semi‐structured interview guide consisting of open‐ended questions about DNs’ experiences of learning to practice the GSD was used in both interview sessions. The first session began with questions related to the expectations of the GSD training and progressed to questions specific to their practice: “Could you please tell me about your experiences in practising the method”? “What challenges did you experience”? and “Can you describe situations where you mastered the method”? The participants were asked to give concrete examples of their experiences. These questions were followed up in the interview guide during the second interview session.2.5The individual interviews from the two points of time were combined for analysis to explore the process of learning over time. The individual interviews were analysed using the qualitative content analysis described by Graneheim and Lundman . Qualita2.6We used the criteria of credibility, dependability and transferability to ensure the rigour of the research . All respondents provided informed written consent before the individual interviews and were guaranteed confidentiality and the right to withdraw from the study at any time.3The analysis resulted in the identification of three themes: (i) from an unfamiliar interaction to “cracking the code”; (ii) from an unspecific approach to a structured, reflective, but demanding approach; and (iii) from a nurse‐centred to patient‐centred approach. We used quotes from all nurses in the result section; however to ensure confidentiality, the quotations from each participants are not labelled in the text.3.1This theme demonstrates that all DNs were initially unfamiliar with GSD, particularly with the communication skills, but frequent repetition and feedback from patients promoted confidence and belief in their ability to “crack the GSD code”.3.1.1I feel this way of communicating is awkward. I feel mirroring is unnatural. It can perhaps become internalised, as it should be, but the method requires experience.All DNs emphasized that GSD training at the Steno Diabetes Centre facilitated their understanding of how to use the reflection sheets and practise advanced professional communication skills. However, although the DNs had some training, they initially experienced using the method as “groping in the dark” and felt unfamiliar with the communication techniques as described by one nurse:Working with the reflection sheets feels quite awkward. Especially in the beginning, I fumbled a bit and it took a lot of focus from the talk with the patient. I thought that was difficult and I did not know quite how to explain the forms [to the patient].In addition to the reported difficulty in incorporating the communication skills, all DNs also experienced challenges in learning to use the reflection sheets. One nurses stated:3.1.2I experience that the patients feel cared for and that they are very happy when we talk. I feel that something is achieved. There is a feeling of mastery when they leave and are happy.The DNs reported that it became gradually easier to use GSD; however, they emphasized that they needed frequent repetition before becoming comfortable with it. In addition, patient feedback was an important factor in influencing their self‐confidence in practising the method, as they perceived this as confirmation that they were on their way to “cracking the code”. Moreover, feedback from the patients stimulated the nurses’ willingness to persevere in the process of learning to practise the method. One nurse reported:3.2This theme reflects that DNs experienced a shift from a diabetes approach developed from various sources and concepts to a structured approach characterized as a tangible tool, but time‐ and energy‐consuming. Overall, they perceived GSD as a good investment.3.2.1I picked out what I think are good ideas and then I have done it my way, which sometimes works and sometimes does not.The DNs reported that before they were introduced to GSD, their approach to diabetes was inspired by advice and ideas acquired from diabetes courses, seminars and conferences. One nurse reported:However, although the DNs were not dissatisfied with their approach, they said that they sometimes failed or were unable to stimulate patients to achieve adequate diabetes management. They were, therefore, interested in learning a new way of counselling. Several DNs explained that they “wanted a tool to use” in the consultations.It led to an awareness in me of a totally different way of communicating with the patient.Another reason for participating was that the DNs wanted to develop themselves as supervisors in the field of diabetes. Succeeding as counsellors was important for all DNs and throughout the training in and applying the GSD, they experienced improved communication skills and increased awareness of and reflections on their own counselling practices as exemplified by the following quotes:3.2.2Normally, we are not used to having anything defined or written in advance. Now you have something in writing—something concrete, which works as a starting point for each consultation. I think that is good.All DNs experienced the GSD as a tangible tool that allowed the dialogues with patients to be more focused, directed and structured. In particular, they perceived the reflection sheets as useful in achieving a more “to the point” conversation with patients. One DN explained:Actually having something concrete [a sheet] to fill in for every consultation—and have your patient reflect on this at home; the various questions, their turning up prepared having thought some questions through and their being followed up and seen by the same person [is good].The DNs reported that they saw the benefits of each individual working with the reflection sheets at home, as they came prepared to the consultations and had developed their own thoughts and reflections about their diabetes management:Consequently, when patients came to consultations prepared and presented their own thoughts about their actual problems in diabetes management, DNs had better opportunities to respond to the challenges and to guide patients to find solutions that matched their own values.3.2.3It takes time. You have to make time for it. And it requires that you are really “there” yourself. In order for me to feel that I provide a good GSD consultation, I have to be absolutely, one hundred per cent present.It emerged from the first point in time that all DNs experienced the GSD, with seven consultations, as time‐ and energy‐consuming. This was also emphasized in the second occasion, when the DNs had used the modified GSD, which consisted of four consultations and fewer reflection sheets. Indeed, the DNs did not perceive the modified version as reduced, merely as more condensed, as it resulted in a more intensive consultation session. Accordingly, all DNs reported that the GSD method was still demanding and energy‐consuming and that they had to be completely aware in each consultation:I enjoy when you see that the patients think it is fun—when you see them offer up in their reflection sheets and you see that they have spent time on it. So I enjoy spending time on this [method].However, despite perceiving the method as time‐ and energy‐consuming, all DNs emphasized that using GSD in their consultations was a good investment, as they felt they succeeded in stimulating patients’ reflections on and motivations for diabetes management:3.3This theme demonstrates shifting from an approach where DNs direct the consultation and “do things to” patients to patients directing their own care and working together with DNs to develop appropriate solutions. The sub‐themes describe what the nurses experienced and characterize their interaction with patients when using the GSD approach.3.3.1We used to give advice all the time. If they said “I wish I could get more exercise”, I would say “you can do this or that”. Now, it is a more open way [of communicating], giving the patients room to figure out for themselves what is good for them. It sort of turns it around.The DNs became aware that they had reoriented their support from the “compliance‐expecting” approach to a “user‐focused” approach to facilitate patients’ reflections, decision‐making, choice and autonomy:It is exciting to observe that it is a process—that the patient reaches a decision regarding changes—they become aware—the patient taking responsibility himself.The DNs realized that using GSD changed the nature of interactions with patients, as GSD encouraged them to focus on understanding patients’ perspectives and priorities rather than quickly prescribing a standard treatment pathway. The DNs characterized the new GSD interaction as a positive and exciting process. One DN reported:That is the real goal—the patient sort of finding his own solution and gradually the solution to a problem as defined by himself. Then it is easier to make changes than if we only tell them how things should be done.The DNs moved from feeling completely responsible for delivering adequate diabetes advice and information to focusing on stimulating patients’ responsibility for their own health. They said that it was easier to stimulate change in diabetes management when patients defined their own problems and solutions. Indeed, they perceived that it was essential to guide patients to identify problems and develop solutions in their own diabetes management:3.3.2I'm the one in charge at annual check‐ups while, in these consultations, it is the patient who is in charge.Another aspect that characterized the GSD interaction was that DNs said that they lost control as patients set the agenda in consultations. Consequently, one DN reported that she had to be “prepared for the unexpected”:In a way, I'm intruding in their lives—their most intimate and vulnerable parts of themselves. It is quite personal. It isn't difficult, but it is still very sensitive. You have to tread carefully. But it isn't hard, it is just that I have to feel my way.The DNs emphasized that they did not experience losing control as difficult. Rather, they explained this situation as a sensitive meeting with the patients, which gave them deeper insight into patients’ thoughts and experiences, as exemplified by one DN:I think about how much the patient actually gives of him‐/herself when they respond to these sheets. They are supposed to draw metaphors and a picture. One patient wrote a long story, which was really touching to read. Just think of what it cost him to share so openly on paper. It made a deep impact really. It was amazing.The willingness of many patients to share their vulnerable and deepest thoughts and narratives with the DNs affected the nurses strongly. Understanding how difficult it was to tell these stories, they were impressed by patients’ openness and honesty:I don't think the patient could have shared this directly in words, how he experienced living with diabetes. You get a bit deeper when they have to think and reflect—and he opened up for a lot of topics that I might not have thought of asking him about.The DNs thought that without the GSD method, they were not sure that they would be able to capture these narratives, nor that the patients would have the opportunity to share these perspectives with them:4The aim of this study was to describe how diabetes nurses experience the process of learning to practise the Guided Self‐Determination counselling approach among adults with T2DM. Three themes were identified from the analysis of the individual interviews, all of which reflect the DNs’ process of learning to practise the method. According to Jarvis , learninNot surprisingly, the first theme, ‘from an unfamiliar interaction to initially “cracking the code”’, indicates that the learning process was initially difficult, as it required new ways of thinking, acting and communicating. The DNs were unfamiliar with communication skills such as active listening and mirroring. Person‐centred approaches such as GSD are typically not easy to master . All respondents provided informed written consent before the individual interviews and were guaranteed confidentiality and the right to withdraw from the study at any time.


Viruses are major aetiological agents of acute respiratory infection in young children. Although many studies have reported detection and analysis of respiratory viruses in sporadic cases, there have been few follow-up studies of individuals. The purpose of this study was to investigate the frequency of respiratory viral infections in a young child and to examine the duration of viral genome detection in clinical specimens.A total of 284 nasal swabs were collected during symptomatic (196 specimens) and asymptomatic (88 specimens) periods of respiratory symptoms from a young female child . Multiplex real-time PCR for 19 respiratory viruses or subtypes was performed. One hundred and ninety-eight of the tested specimens were virus positive (69.7 %) . Rhinovirus was the most frequently detected (26 times). Long durations of detection were observed for human coronavirus NL63 (30 days), rhinovirus (28 days) and human bocavirus 1 (22 days).Young children living in a group context have a high risk of respiratory virus infections, especially rhinovirus. In some instances, viral genomes were detectable for about 1 month by PCR. Over 200 serologically or genetically different viruses have been identified as major aetiological agents of acute respiratory infections . AlthougMany studies have reported and identified respiratory viruses in sporadic cases of acute respiratory infection, but there have been few follow-up studies of individuals with multiple respiratory virus infections  in the same household in a city. The child was 4 months of age at the start of the study and was admitted to a nursery school at 9 months of age. The brother was also admitted to the same nursery school during the study period. About 15 children were present in the same room, and about 140 children (0–6 years old) were present in the nursery school.One hundred and ninety-six swabs were taken during symptomatic periods when the child showed some clinical signs such as rhinorrhoea, phlegm, cough and/or fever. The remaining 88 swabs were collected during asymptomatic periods. Informed consent was given by the parents for collection of specimens and analysis of viruses present in the specimens. Specimens were collected by the parents in the home and stored at −20 °C before testing. During the symptomatic period, at least two specimens were collected each week. When no respiratory symptoms were observed, a specimen was collected every 1 or 2 weeks. When specimens collected within 7 days of each other were consecutively virus positive, the child was judged to be virus positive for the entire period between sampling.Viral RNA was extracted using a QIAamp Viral RNA Mini kit (Qiagen), and cDNA synthesis was performed using SuperScript III (Life Technologies) as described previously (P<0.001). Of the 198 virus-positive specimens, 132 (66.7 %) were positive for a single virus, whereas the remaining 66 (33.3 %) were positive for more than one virus type. A slightly higher ratio of multiple/total virus-positive specimens was collected during the symptomatic period  than the asymptomatic period  .A total of 198 (69.7 %) specimens were found to be virus positive. The number of virus-positive samples was higher during symptomatic periods  compared with during asymptomatic periods  .A total of 13 viruses and subtypes were detected during the study period. The total number of detections (co-detections) of each virus were: HRV, 131(58); HAdV, 36 (24); HBoV1, 32 (24); HEV, 14 (four); HCoV-NL63, 13 (nine); HCoV-OC43, eight (two); hMPV, eight (four); FLUCV, seven (five); HPIV-3, seven (two); HCoV-HKU1, six (two); RSV A, four (one); FLUAV, three (two); and HPIV-4, three (three). The combinations of viruses co-detected in 66 samples are shown in The distribution and duration of virus detection and clinical signs are shown in Although some follow-up studies of respiratory viral infections have been reported, there are no reports describing the detection and analysis of 19 different respiratory viruses from numerous specimens collected from one person (P<0.001). These results indicate a high risk of respiratory virus infection in young children in a group context. It has been reported that young children (<1.5 years of age) in daycare have a higher risk of upper respiratory virus infections compared with homecare children  than before she was admitted  , analyses of clinical symptoms and a consideration of bacterial detection, would be helpful to understand respiratory infections in young children. Additional epidemiological data may also help to prevent severe respiratory virus infections and facilitate the development of a vaccine(s).


Cruise tourism has become more popular. Long-term personal contact, complex population flows, a lack of medical care facilities, and defective infrastructure aboard most cruise ships is likely to result in the ship becoming an incubator for infectious diseases. In this paper, we use a cruise ship as a research scenario. Taking into consideration personal behavior, the nature and transfer route of the virus across different surfaces, virus reproduction, and disinfection, we studied contact infection of infectious disease on a cruise ship. Using gastroenteritis caused by the norovirus as an example, we analyzed the characteristics of infectious disease propagation based on simulation results under different conditions. We found hand washing are the most important factors affecting virus propagation and passenger infection. It also decides either the total number of virus microorganisms or the virus distribution in different functional areas. The transfer rate between different surfaces is a key factor influencing the concentricity of the virus. A high transfer rate leads to high concentricity. In addition, the risk of getting infected is effectively reduced when the disinfection frequency is above a certain threshold. The efficiency of disinfection of functional areas is determined by total virus number and total contact times of surfaces. With rapid population growth and economic development, convenient public transport, and high pressure of people, the number of tourists is increasing. China has the biggest population in the world and this populace takes more than 2 billion domestic tourism trips every yearIn recent years, because tourists preferred natural resources, uncrowded areas, easy travel, and new places, cruise tourism has become more popularestionnaireMost infectious diseases propagate via air-borne, water-borne, food-borne, vector-borne routes, as well as contact borne routes9In this paper, we analyzed the impacts of different functional areas such as restrooms, aisles, seats, retail counters, restaurants, stair rails, and the handrails around the sightseeing deck, on norovirus gastroenteritis propagation. Taking into consideration personal behavior, the nature and transfer route of the virus across different surfaces, virus reproduction, disinfection by crews, and influences resulting from the different position of functional areas, we analyzed norovirus propagation on a cruise ship. From this analysis we provide some optimized suggestions focusing on functional area distribution, characteristics of contact surfaces, personal behavior, and disinfection on board to reduce the risk of norovirus infection.A cruise ship typically has many functional areas such as restrooms, seats, restaurants, etc. Passengers will go to these areas and touch surfaces at different frequencies. In this paper, we considered 6 different functional areas and 8 types of contact surfaces. The 7 functional areas are seats, restrooms, retail counters, aisle, restaurant, stair rails, and handrails around the sightseeing deck. The 8 contact surfaces include the top surface of seats, the inner and outer doorknobs of restrooms, retail counters, handrails of stairs and the sightseeing deck, and surfaces of dining tables and chairs. All surfaces are divided into multiple grids of 0.4 m × 0.4 m. Every grid is regarded as an independent contact surface. The 7 functional areas are introduced below:Seats: on a short journey aboard a cruise ship, all passengers have a seat. Because of the nature of a ship’s motion, passengers have to steady themselves by holding the top of the seats when they move from a branch aisle to the main aisle and on to other functional areas. Therefore, the top surfaces of seats are prone to be touched by passengers, especially those near the main aisle.Restroom: norovirus easily appears in restrooms because infected passengers usually spread the virus accidentally. In this paper, a restroom is treated as the source of infection. Passengers might be unintentionally in contact with the norovirus from vomit or stools in the restroom. The restroom is the critical area for norovirus propagation on a cruise ship. There are many public facilities inside the restroom such as door handles, toilet seat, and washbasins. Passengers usually use these facilities in a regular sequence. In this paper, we regard all facilities inside a restroom as one facility which is represented by the inner door handle. For outside the restroom, the outer door handle is considered to be a contact surface.Retail counter: the retail section is a necessary functional area on cruise shipsAisle: aisles exist everywhere on cruise ships. As shown in Stairs: on a cruise ship, different functional areas are often connected by stairs. In Restaurant: passengers frequently eat meals in the restaurant during meal times. Sightseeing deck: passengers usually enjoy the scenery on the sightseeing deck. Handrails surround the deck for safety. As shown in There is a total of 844 grids (independent contact surfaces) in the study area .Passenger behavior directly affects the distribution and transmission of a virus on board cruise ships. In this paper we define passenger behavior in different functional areas including using the toilet, eating, sightseeing, and buying. As shown in Passengers & seats: the majority of tourists travel in groups. An investigation of the tourism market from one Chinese city showed that touring with a family accounted for 54 percent of all tourists while touring with friends accounted for 31 percent1. Because practical difficulties of contact monitoring of passengers on cruise shipH2. Three members are regarded as a family group and 2 to 4 members as a group of friends.Hhttp://worldtoilet.org/). The probability of a passenger going to the restroom is therefore considered to be 1/160 every minute. According to the statistical data from different scenarios, the probability of a person washing their hands is anything between 70 and 90 percent2021Passengers & restrooms: restroom is a critical place for passengers not only in virus infection, but also in virus disinfection. Handwashing is one of the most efficient ways to remove the norovirus on the hand183. One out of every seven passengers who visits the restroom will have a bowel movement.H4. Defecation time is randomly distributed between 8 and 12 minutes, and urination duration is set to be 2 minutes.HPassengers & retail: the procedure followed by passengers going to a retail area is very similar to that of going to the restroom. There are 2 hypotheses for this activity:5. The probability of passengers going to a retail point per minute is set to 1/180.H6. The time that passengers stay at the retail point is set to be 1 minute.HPassengers & restaurants: in a restaurant, passengers in the same group will eat at the same time7. Because the majority of restaurants on a cruise ship sell fast food, the time taken by passengers to have a meal is assumed to be 25 minutes.H8. According to the data set in general simulation, lunch time is usually set from 11 a.m. to 1 p.m.HPassengers & sightseeing deck: big cruise ships always have a big sightseeing deck. Passengers who want to enjoy the scenery can reach this deck via some stairs. In this study we assume the sightseeing deck is rectangular and obey these three hypotheses:9. The average frequency for passengers going to the sightseeing deck is once per 2 hours, and the probability of tourists going to the deck is 1/120 times per minute.H10. Passengers will enjoy the scenery in all four directions and they will touch the four contact surfaces from four different sides.H11. They will remain on deck for 12 minutes (3 minutes per side).H1 to H11. Based all the hypotheses, the results can only reflect the norovirus propagation in the cruise ship whose structure is the same with setting in our study.In this study, some value of parameters we mentioned above came from the references and other value we hypothesized from HIn this paper we have used norovirus gastroenteritis as an example to simulate and analyze infectious disease propagation through contact12. Because contact duration is limited we assume a virus transfer rate of 0.1. In other words, 10 percent of the virus will be transferred from the higher-density surface to the lower-density surface .where H4 to 108 under 1 atmosphere of pressure at 20 °C. When the temperature rises to 30 °C, after 490 minutes, the colony of Escherichia coli can increase from 105 to 109 Virus not only can transfer among different surfaces but also reproduce on the same surface following the reproduction of cells they board. Because the nature of Escherichia coli is similar to the host cell of norovirus, we used Escherichia coli as a substitute to study the reproduction. Research has shown that after 1459 minutes, a colony of Escherichia coli can increase from 10where R(20), R(25), and R(30) expresses the virus reproduction rate at a temperature of 20, 25 and 30 degrees respectively.Combining the two previous studies mentioned above, the virus reproduction rate under standard conditions could be calculated using The norovirus is very infectious, usually entering the body with food or water. According to previous research, people may be infected if they ingest more than 100 norovirus microorganismsAt the beginning of the simulation, we assume one person is infected with the norovirus. After going to the toilet, we assume that this person’s hand is unintentionally tainted with 3000 virus microorganisms. This time is set to be the initial time from which the norovirus started to spread on board the cruise ship.In addition to the functional areas, personal behavior, and reproduction and transmission of the virus, we also studied the impact of disinfection on virus propagation. Disinfection is a critical method of controlling or even preventing contact infection. Thorough disinfection of restroom facilities such as toilet seats, flush buttons, and door handles is very important in the prevention of infectious disease propagation on board cruise shipsIn order to analyze virus propagation under different conditions, we assume standard values to act as the control group. Considering all the influencing factors mentioned above, the value of each factor in the control group is shown in nd and 3rd day, respectivelyIn general, comparing Comparing the virus distribution on stair railings on cruise ship, the volume of virus on stair railings to the sightseeing deck is always higher than it is on stair railings to the restaurant because the restaurant is visited less frequently. In the restaurant, the relative volume of the virus on dining tables increases with rising P and E. Restrooms have a strong disinfection effect when P and E are high. The volume of the virus on the main floor is reduced, so the relative volume of the virus in the restaurant increased. However, in all situations, since they have a low contact frequency, dining chairs are the only area that always has the lowest volume of virus. The relative volume of the virus on the sightseeing deck increases with increasing P and E.T) between surfaces. With an increasing transfer rate, the area with high-density virus moves to the right. When RT = 0.1, the volume of virus on the seats near the main aisle gradually decreases from left to right. But when RT = 0.4, the middle seats closer to the main aisle have the most virus. Moreover, with increasing RT, the concentricity of virus distribution becomes more obvious. As shown in T is less than 0.2, the percentage of infected passengers dramatically decreases with increasing transfer rate. If RT is too low, the high volume of virus carried by some passengers is difficult to dilute via surface contact. However, looking at the red square in T when RT is greater than 0.25. For this value of RT the disinfection effect of restrooms is small, and passengers will be rapidly contaminated by the virus from other surfaces. From these results we infer that controlling the transfer rate can effectively prevent passengers from being infected. For example, in this case, the infection risk will reach a minimum when the transfer rate is equal to 0.25. At this time, the infection rate reached to 0.0058, and about 2.32 passengers would be norovirus-positive. When transfer rate reduce to 0.10 from 0.25, the number of norovirus-positive passengers will increase to 8.84.On the main floor, the functional areas including restrooms, retail counters, and the stairs to the restaurants and the sightseeing deck are disinfected. Because many disinfected surfaces can dilute the virus, when the disinfection frequency is high, the virus is distributed uniformly on the seats that are nonadjacent to the main aisle. As shown in D = 1, the infection rate reached 0.0048, on overage, 1.93 passengers would be norovirus-positive after 8 hours journey on the cruise ship. While ID = 8, the infection rate will increase to 0.023. In D = 4 hours is the inflection point. When the interval between disinfections is greater than the inflection point, the efficiency in reducing the number of infected passengers is reduced. On the other hand, efficiency is higher when the interval is shorter than that at the inflection point. From this study, taking into account the layout of the cruise ship and the virus propagation simulation for different disinfection frequencies, some suggestions can be made regarding infectious disease prevention.5 to reflect both total virus number and contact times). With a decreasing sensitivity, R is monotonously decreasing. Therefore, the disinfection effect of surfaces of the different functional areas is related to the product of total virus number and contact times. We should therefore prioritize the disinfection of the surfaces with a high R-value. In this study, the seats near the main aisle and the handrails on the sightseeing deck should be given the most attention.We considered 7 areas for disinfection as described in 1 to H11 are given in the section of passenger behaviors and the results could be referred in these conditions. Based on the computational simulation, the results can partially reflected the conclusion on relationship among norovirus infection and other influencing factors.There are also some limitations of this study. Our study focuses in the computer modelling of the probability of contamination through personal contacts so as to identify potential methods to control infection in actual context (without laboratory support) by the frontline workers (e.g. cruise ship workers) to avoid outbreak. Meanwhile, we will explore the importance of how the use of laboratory investigations (and their indicators) might further support infection control in future studies. In addition, limited passenger’s movement regulation and influencing factors in this study are also constrained to develop effective norovirus propagation measures in cruise ship. The total number on the cruise ship is 400 and simulation time is 8 hours. The results are only suitable for the norovirus propagation in this scale. In addition, all hypotheses from HThis study focuses on the potential transmission dynamics of contact based infectious diseases. For future investigation, similar models should be built to examine air-borne, food borne as well as vector-borne diseases. In addition, to ensure effective infection control in public locations, other important public health measures such as public risk communication and education of the use of face masks, hand hygiene, proper food safety and sanitation maintenance would be important. Future studies should include how these important dynamics might affect infection control in various settings.In this paper, using norovirus gastroenteritis as an example, we study the contact infection of an infectious disease on a cruise ship. We considered six functional areas: seats; restrooms; retail counters; stair handrails; restaurant; and sightseeing deck railings, as well as eight contact surfaces. Combining the influencing factors of personal behavior, virus transfer between surfaces, virus reproduction, passenger infection, and disinfection, we simulated infectious disease propagation on a cruise ship.In the control group, passengers far away from the restrooms have a higher infection rate than those near the restrooms. In this case, virus distribution across the seating area is in the shape of a V, with the seats nearer the main aisle being cleaner than those further away. The volume of virus microorganisms in the restaurant is low because of infrequent contact, and dining chairs are cleaner than dining tables. There are more viruses on the sightseeing deck, with the average number of viruses on the shorter handrails being higher than found on the longer handrails.The probability and efficiency of hand washing is the most important factor in determining the number and distribution of the virus. On the main floor, the left side is cleaner than the right side and the seats nearer the main aisle have more viruses. With an increasing probability and efficiency of hand washing, the distribution of the virus changes, and volume of virus microorganisms in the aisle and on the right side decreases. Through quantitative analysis we find that the percentage of infected passengers decreases rapidly when the probability and efficiency of hand washing increases from 0.3 to 0.7. Therefore, keeping the probability and efficiency of hand washing over 70 percent is very important to prevent infection.The virus transfer rate between surfaces decides the distribution and concentration of the virus. High transfer rate led to high concentricity. With a low transfer rate, virus distribution was shaped like a V, and the number of virus microorganisms in the high-density areas was greater than 900 per grid. When the transfer rate rose to 0.4, the viruses were densely distributed near the restrooms and on the walls. The number of virus microorganisms in the high-density areas was approximately 500 per grid. Through quantitative analysis we found that the infection rate was lowest when the transfer rate was 0.25. If contact surfaces of the densely populated areas on board ship were designed to reduce roughness and control relative factors to keep the transfer rate at 0.25, this would help prevent virus propagation and reduce the infection rate.Through simulation we can see that regular disinfection of seats near the main aisle, door handles of the restrooms, handrails of stairs and on the sightseeing deck can effectively reduce the total number of virus microorganisms. The average number of virus microorganisms per grid on the sightseeing deck and in the restaurant is around 100 when the disinfection frequency is once every 2 hours. When the disinfection frequency increased to once per hour, the grid with the highest risk had only 600 virus microorganisms. Comparing this with the condition where there is no disinfection (majority of seats have 800 virus microorganisms), the total number of virus microorganisms is dramatically reduced. Based on quantitative analysis, a disinfection frequency of once every 4 hours is an inflection point. Continuing to increase the disinfection frequency will reduce the efficiency of its impact on infection prevention. Finally, we found that disinfecting the surfaces with the highest total virus number and longest contact times is more useful in reducing the percentage of infected passengers.All obtained results can provide some scientific basis for contact infection of infectious diseases in densely-populated places. The results also provide the basis for suggestions on how to control and prevent infectious disease.How to cite this article: Zhang, N. et al. Contact infection of infectious disease onboard a cruise ship. Sci. Rep.6, 38790; doi: 10.1038/srep38790 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Because crop plants vary in their tolerance to acidity and plant nutrients have different optimal pH ranges, target soil pH values in the UK are set at 6.5 (5.8 in peaty soils) for cropped land and 6.0 (5.3 in peaty soils) for grassland. Agricultural lime products can be sold as ‘EC Fertiliser Liming Materials’ but, although vital for soil quality and agricultural production, liming tends to be strongly influenced by the economics of farming. Consequently, much less lime is being applied in the UK than required, and many arable and grassland soils are below optimum pH.Soil acidification is caused by a number of factors including acidic precipitation and the deposition from the atmosphere of acidifying gases or particles, such as sulphur dioxide, ammonia and nitric acid. The most important causes of soil acidification on agricultural land, however, are the application of ammonium‐based fertilizers and urea, elemental S fertilizer and the growth of legumes. Acidification causes the loss of base cations, an increase in aluminium saturation and a decline in crop yields; severe acidification can cause nonreversible clay mineral dissolution and a reduction in cation exchange capacity, accompanied by structural deterioration. Soil acidity is ameliorated by applying lime or other acid‐neutralizing materials. ‘Liming’ also reduces N A soil exposed to such rain, but no other acidifying inputs and receiving no lime, would attain the same equilibrium pH as that of the rain. There are, however, very strong localized effects because human activity has increased the acidity of precipitation through emissions of acidifying compounds such as SO2 and nitrogen oxides (NOx) from industry and motor vehicles, and NH3 volatilized from manures and fertilizers  from approximately 85 kg/ha/year in 1970 to approximately 15 kg/ha/year in 1995  is taken up by the crop, there is no net acidification because the NO3− takes up protons with it , magnesium (Mg2+), potassium (K+) and sodium (Na+)] by H+ and aluminium (Al3+) through the cation exchange (CEC) buffer and then (iii) the dissolution of Al‐bearing and manganese minerals, if manganiferous minerals proliferate, and finally (iv) the dissolution of iron‐bearing minerals. These processes buffer the pH at approximately 7–8, 5–6, 4 and 3, respectively. Thus soil acidification results in periods of constant or slowly decreasing pH while one process buffers inputs, followed by a relatively rapid decrease in pH when that process is exhausted and the next takes over. Examples of field soil buffer curves from the Long‐Term Experiments at Rothamsted can be seen in Figure Soils are ‘buffered’ against acidification by a series of chemical processes: (i) firstly, the dissolution of carbonates and other basic rocks, the ‘carbonate/bicarbonate’ buffer, then (ii) the replacement of exchangeable base cations [calcium (Ca2 and Ca2+ (or Mg2+ in dolomitic limestone areas). Once cation exchange becomes the main buffer, essential nutrient cations  are leached, base saturation decreases together with nutrient availability, Al3+ saturation increases and crop yields begin to decrease. Figure There are no observable effects of acidification on soil while lime or chalk buffers the system , accompanied by structural deterioration. Such weathering is not reversible except over geological timescales and so represents a serious and costly degradation of soil quality , chloride (Cl−), NO3− and SO42−, and Ca2+ removed in farm products  of the soil. Thus, for the same required change in pH, soils with a low BC, such as sands, require less lime than those with a higher BC, such as clays.This produces double the lime requirement to that calculated for Caet al., For many years, in England and Wales, the standard method for measuring the lime required to adjust the pH of a soil was the buffer method of Woodruff . Soil pHhttp://www.rothamsted.ac.uk/rothlime; Goulding et al., http://www.aglime.org.uk/lime_calculator.php_calculator.php) also has an online Lime Calculator. Recommendations from these calculators correspond well to those in the Fertiliser Manual  sieve’. The seller must also declare the neutralizing value (NV) and the amount of material as a percentage by weight that will pass through a 150‐micron sieve. The NV of the material defines the amount of acidity that it will neutralize and is based on a reaction with HCl in a laboratory. Typical NVs of the three most commonly used materials are as follows:The most commonly used liming materials are ground limestone, dolomitic ground limestone, chalk, ground chalk, burnt lime and hydrated lime; almost 70% of the material currently used in the UK is ground limestone. Their definitions or ‘Meaning’, specifications and ‘Declarations’ (what the buyer must be told about them) must comply with the Fertiliser Regulations 1991 , NV = 50–55% depending on the geological strata;Limestone (CaCO3)2, usually 42% CaCO3 and 53% MgCO3), NV = 56%;Dolomitic limestone (CaMg(CO3), more readily broken down and absorbed into the soil solution than limestone, NV = 48–54%.Chalk , the grade of product, the NV and the Caet al., et al., 2O5, 5–7 kg MgO and 4–6 kg SO3 per tonne of lime and has a fine particle size, so is fast‐acting.A number of ‘waste products’ are available that neutralize acidity: sugar factory lime, basic slag, wood ash, coal combustion products such as fly ash and bottom ash, calcium humates and fulvates from oxidized brown coal and by‐products of the paper and pulp industry  of pelletized lime is being applied to the fields of the North Wyke Farm Platform  but, in CO2‐C equivalents, the GHG emissions from liming were about four times those saved by reducing N2O emissions.One area of greatly increased interest in recent years has been the impacts of lime and liming on C sequestration by soils and thus on climate change. Paradelo et al.  reviewed (et al.  used datEquation , which m, et al.  looked a, et al. , reducedLiming to the pH recommended in the Fertiliser Manual Defra,  is essen3, estimated by Goulding & Annis (Data compiled from the Agricultural Lime Producers Council, the Ministry of Agriculture, Fisheries and Food to 2001 and the Department for Environment, Food and Rural Affairs thereafter and the British Survey of Fertiliser Practice show that insufficient lime is being applied: annual amounts applied have declined from c 6000–7000 kt product/year to c 2500 kt product/year today. This is much less than the calculated average annual lime loss for the UK of 4 250 000 t CaCO & Annis .2 when lime reacts with soil acidity. Despite the significant reduction in acidic atmospheric deposition in the UK, the acidification from it together with ammonium‐N and elemental S fertilizers, the use of legumes for N fixation and crop growth and nutrient removals will continue to require significant amounts of lime or other acid‐neutralizing materials. However, the economics of farming continues to override agronomy in decision making for liming, and areas limed and amounts applied are well below what is necessary for maintaining recommended soil pH values.Although a short‐term saving, reducing or omitting the application of lime to correct acidity risks significant economic loss through unachieved crop yield and wasted fertilizer, plus an increased risk of the pollution of water and air by N and P fertilizers. Liming to recommended soil pH values increases productivity, benefits soil structure, improves degraded soils and, when used with other appropriate management practices, can benefit grassland biodiversity. It also reduces some greenhouse gas emissions, but this has to be set against the emissions of CO


Semen evaluation is used to estimate the testicular function. In bulls, the spermatozoa present in the ejaculate are the result of a process that begun more than 2 mo earlier, bequeathing a delayed depiction of the actual function of the testis. Since testis vascularization might be critical for the gonad function, selected pulse wave Doppler ultrasound parameters were assessed in this study, for instance the peak systolic velocity, the end diastolic velocity and the resistive index of the testicular artery along the spermatic cord, the marginal portion of the testicular artery and the intratesticular branches of the testicular artery both in healthy adult and young bulls. Correlations between these parameters and characteristics of semen that was collected numerous times, before and after the Doppler ultrasound examination.P < 0.01), and between the marginal portion of the testicular artery and the intratesticular branches of the testicular artery (P < 0.05). No differences were recorded for the pulse wave Doppler ultrasound parameters in young bulls compared with adults. A significant correlation was found between the resistive index of the marginal portion of the testicular artery and total sperm in the ejaculate , the immature sperm , the teratoid sperm , and the “Dag defect” sperm . Similarly, the resistive index of the intratesticular branches of the testicular artery were found correlated with the total sperm number in the ejaculate , the immature sperm , the teratoid sperm , and the “Dag defect” sperm .The peak systolic velocity and the end diastolic velocity measured in the testicular artery along the spermatic cord (supratesticular artery – SA) were variable among the bulls and within individual bulls, likely due to the convoluted course of the vessel. The resistive index was found highly repeatable in the same bull. A reduction in the resistive index was found between the supratesticular artery and the marginal portion of the testicular artery (The data presented in this study suggest that the resistive index, measured at the marginal portion of the testicular artery, could be an easy-to-perform parameter to evaluate the spermatogenesis quality in young bulls and normal adults. In the breeding soundness evaluation of the bull, the scrotal circumference and semen analysis are widely used as reference to assess the testicular function . HoweverTesticular parenchyma is supplied by a long testicular artery, showing low oxygen tension –6 and loThe ultrasonographic evaluation of the scrotal content represents one of the central investigations that can be exploited to assess the testicular function. Several studies tested the use of ultrasonography (US) to determine the normal testicular function  or to deHigh-resolution color Doppler US was recently proposed as a tool to evaluate the testicular vasculature and, in turn, the perfusion of the testis. In human medicine color Doppler US has been employed to evaluate testicular blood flow in pathological conditions , 19. FurThere are no data concerning the application of the PWDU to evaluate the testicular function in the bull. Consequently, the aim of the present study was to assess the associations between the selected PWDU parameters  obtained from the testicular artery and its branches, and sperm characteristics. The ultimate goal of the study was to verify if these PWDU parameters could be used to evaluate the actual status of the testis in the bull.This study was performed on 18 Swiss Brown bulls, nine adult  and nine young  regularly used in artificial insemination (AI) programs. The bulls, owned by the Superbrown Consortium, were housed in the Alpenseme AI Center of the Provincial Breeders Federation of Trento . The animals were maintained according to the Italian legislation on animal care . The owner of the animals gave his consent to the performed procedure. Aliquots for semen evaluations were part of the ejaculates routinely collected in the artificial insemination centre, and no semen collections were performed specifically for the study.For the ultrasound examination, the bull was restrained in a bovine individual steel stanchion, and a posterior approach to the scrotal region was used, without the need of sedative administration. The scrotal skin was cleaned, and 70% ethyl alcohol and ultrasonographic gel  were applied to increase the quality of the ultrasound image. A portable ultrasound machine Logiq Book XP  equipped with a 10-MHz linear probe  was used. The scan depth was 10 cm for all the examinations.The images were acquired at 10 MHz, and the sample volume was set to 1 mm. All the ultrasound machine settings remained unchanged during the trial. Initially, the probe was applied on the spermatic cord with horizontal orientation to examine the SA. Subsequently, the probe was moved to the rear face of the scrotum with vertical orientation and moved equatorially with the same orientation around the scrotum, in order to identify and examine the MA and the IA. After the vessel detection, the orientation of the probe was adjusted to achieve the visualization of at least 3-mm recognizable longitudinal section, the sample volume marker was pointed on the vessel, the angle of insonation was adjusted in the window below 60 degrees to obtain a valid velocimetric measures along the course of the artery, and at least three spectral curves were followed.The peak systolic velocity , the end diastolic velocity , and the resistive index  were measured by the internal algorithm. All the measurement procedures were repeated three times.6 sperm/mL) was estimated using Accucell photometer . Total sperm/ejaculate  was calculated as: VOL × CONC.For each bull, semen collections and evaluations were performed d − 60 and d − 30 before the PWDU, the day of the PWDU (d 0), and d 30, d 60, and d 90 after the PWDU. Semen was collected using an artificial vagina in a sterile 13-mL plastic tube. The volume  was estimated by the ejaculate weight measured with a precision balance CP6201 . The concentration  was evaluated using a conventional epifluorescent double stain with propidium iodide (PI) and SYBR-14 fluorescent stain  as previously described  with somSperm morphology was evaluated using phase contrast microscopy (magnification: × 1000) after fixation with 0.9% NaCl solution with 3% glutaraldehyde . In thisThe sperm kinetic parameters were determined using a computer assisted sperm analyzer (CASA) IVOS 12.3 . Motility parameters were collected and recorded by the analysis of 12 nonconsecutive fields. The anticollision algorithm was activated. Makler chamber was used for analysis . The parP > 0.05), the PWDU parameters , measured in triplicate, were analyzed using generalized linear model (GLM) for repeated measures while the testicle (right vs. left), the age (young vs. adult), and the type of the vessel  were considered fixed factors. As per the vessel type, the post-hoc evaluation was performed using the Scheffé test. For semen characteristics, VOL, CONC, TSE, MI, sperm abnormalities, and sperm kinematics, the GLM for repeated measures was used, considering the age of the bull as a fixed factor.The data presented in this study were reported as mean ± standard deviation (SD). The data recorded for normality were tested using the Kolmogorov-Smirnov test. Since the data were normally distributed .The differences were considered significant when P > 0.05). All PWDU values , were significantly higher (P < 0.01) in SA than in MA, which was in turn significantly higher (P < 0.05) than the IA for PSV, and RI. End diastolic velocity showed similar (P < 0.05) values in the MA and IA. No differences in the crude values were recorded in the corresponding region of the artery between young and adult bulls. In all cases, the PWDU values registered in the different regions of the testicular artery, were similar in the right and left testis (Table P < 0.01) in the same bull, but the MA and the IA were similar (P < 0.05). Resistive index measured in various regions of the testicular artery was similar within the same bull.The PWDU of the testis was relatively simple in all bulls. Only the evaluation of the IA required more time for the measurement, due to the frequent movement of the bulls during the procedure. The pulse wave Doppler US parameters and the sperm characteristics were normally distributed (r P < 0.0 in SA thP < 0.01). On the other hand, sperm membrane integrity was similar in young and adult bulls. The total amount of sperm abnormalities ranged between 10 to 23% in all subjects, with mean values of 8.4 ± 3.2% in the young bulls and 14.3 ± 6.9% in the adults. However, the abnormal forms were highly variable among individuals (P < 0.01), as evidenced by the standard deviation (Table P > 0.05) were found in the percentage of the different abnormalities between adult and young bulls. Furthermore, the percentage of total sperm abnormalities, such as the different type of sperm defects, were similar (P > 0.05) within samples collected from the same bull.Semen characteristics are summarized in Table r = 0.516, P < 0.05; r = 0.568, P < 0.05, respectively), immature sperm , teratoid sperm , and sperm with “Dag defect” . The other seminal characteristics were not significantly correlated with the PWDU parameters.Significant correlations were found between the RI, measured in both the MA and the IA, and TSE (The PWUD of the testicular vasculature was previously studied in several species, such as human , 33–37, In our study, the RI recorded in the different locations of the testicular artery and its branches had similar values in different bulls, and within the same bull, while the PSV and the EDV measured in the SA were variable in the same bull. Our approach was different from those reported previously in other studies for stallions  and dogsOur data showed a progressive reduction in the PWDU parameters along the course of the testicular artery and its branches. The trend found in the present study in the bull confirmed the data reported in stallions  and dogsAs previously reported in stallions  and dogsThe blood perfusion of the testis seemed effectively measured by RI in the MA and in the IA. However, the measurement of the PWDU parameters in the IA could be tedious, time consuming, and is referred to a part of the testis. On the other hand, RI in the MA could be considered representative of the whole testicular perfusion. The relevance of RI in indirectly measuring the testicular function was supported by the evidence that a significant increase of this parameter in the MA was recorded in stallions whose the testicular dysfunction was induced pharmacologically . The freAmong the perfusion parameters, only the RI measured in the MA and in IA was significantly correlated with the TSE, the percentage of immature sperm, the teratoid sperm, and the sperm with “Dag” defect, without differences between adult and young bulls. The correlation between all these parameters and the RI corroborates that the perfusion of the testis could be related, at least partially, to the efficient function of the testis in normal bulls. The TSE could be considered a non-invasive parameter to estimate the testicular function in bulls in which the semen is routinely collected . ImmaturThe data reported in the present study suggest that the normal testicular function, resulting in normal sperm production, is correlated to the perfusion of the testis. Pulse wave Doppler ultrasound measures and indices of the blood flow in the bull testis, especially RI obtained from the marginal part of the testicular artery, can be proposed as objective parameters of testicular function occurring at the same time as the PWDU evaluation is performed.


The rapid and reliable detection of infectious agents is one of the most challenging tasks in scenarios lacking well-equipped laboratory infrastructure, like diagnostics in rural areas of developing countries. Commercially available point-of-care diagnostic tests for emerging and rare diseases are particularly scarce.Orthopoxviruses (OPV). The OPV ABICAP assay detects down to 1 × 104 plaque forming units/mL of OPV particles within 45 min. It can be applied to clinical material like skin crusts and detects all zoonotic OPV infecting humans, including Vaccinia, Cowpox, Monkeypox, and most importantly Variola virus.In this work we present a point-of-care test for the detection of Monkeypox virus infections in areas lacking proper laboratory infrastructure as well as rapid on-site testing of suspected bioterrorism samples.Given the high sensitivity and the ease of handling, the novel assay could be highly useful for on-site diagnostics of suspected The online version of this article (doi:10.1186/s12985-016-0665-5) contains supplementary material, which is available to authorized users. Poxviridae family and are large and complex DNA viruses able to infect humans, causing severe diseases [Variola virus (VARV), zoonotic infections with still circulating OPVs, namely Monkeypox virus (MPXV), Cowpox virus (CPXV), and Vaccinia virus (VACV) [Variola virus in the case of a bioterrorist attack [OPV belong to the diseases . Despites (VACV) , remain s (VACV) . Diagnoss (VACV)  are usuas (VACV) , 6 as wet attack , 8.An ideal POC diagnostic system should be affordable, sensitive, specific, simple enough to perform by untrained persons, rapid and robust, free of sophisticated equipment, and deliverable to those who need it . The ABIIn this work, we describe a rapid and sensitive detection system based on the ABICAP technology that reliably detects particles of different human-pathogenic OPV.Vaccinia virus (WR) rA27 (NR-2622) with a C-terminal histidine tag, recombinantly expressed from baculovirus.The diagnostic ABICAP is based on two monoclonal antibodies (mAbs) targeting the OPV surface protein A27: mAb A1/40 and mAb A3/710. Briefly, both antibodies bind to A27 with high affinity  and recognize non-overlapping linear epitopes located in close proximity to the N-terminal heparin-binding domain of A27 . Both antibodies have previously been selected in an antigen capture ELISA, representing the best combination to detect native VACV, CMLV, CPXV, ECTV, and MPXV. Production, purification, storage, and biotinylation of antibodies was performed as described previously . The folCamelpox virus (CMLV) strain CP-19, Ectromelia virus (ECTV) strain Nü-1, and MPXV strain MSF6 were kindly provided by Prof. Dr. Hermann Meyer . The OPV ABICAP detection system was additionally validated with two VARV strains, VARV Solaiman 1974 and VARV Niger 1969. Parapoxvirus (PPV) ORF D1701 was kindly provided by Achim Rziha  whereas herpes simplex virus 1 (HSV-1) was isolated in our lab from a patient. Propagation and titration of viruses on cell culture was done according to standard procedures [The virus panel used for development and validation included VACV strain New York City Board of Health  and CPXV strain GuWi . Camelpoocedures . Virusesocedures . All virYellow fever virus strain 17D, Ebola virus strain Zaire, Marburg virus, VACV, and MPXV in Dulbecco’s Modified Eagle Medium was kindly provided by the P.R.O.B.E. consortium. Viruses in this panel have been quantified by quantitative real-time PCR using published assays [For final validation, a panel containing inactivated (γ-irradiation) highly pathogenic viruses comprising d assays , 15 and Molluscipox virus [Parapox virus [Myxoma virus [Clinical sample material tested in this work had been sent to the Robert Koch Institute for poxvirus diagnostics. Crust material and surface swabs were delivered dry and suspended in 500 μL of phosphate buffered saline  without magnesium and calcium. Surface swabs were thoroughly mixed on a vortexer while crusts were homogenized in a Fastprep 24 homogenizer  after addition of Precellys® ceramic beads , and DNA was prepared from 200 μL of sample using the Qiagen Blood and Tissues Kit  according the manufacturer’s recommendations. Quantitative real-time PCR for OPV diagnostics was performed on 5 μL of isolated DNA, using rpo18 as target as published previously . The numox virus , Parapoxma virus  were tesThe preparation of ABICAP columns was done as described before , using m3/ Na2CO3, pH 9.5). All activating and coating steps were done at room temperature under constant stirring and vacuum. The frits were then coated with 7.5 μg/frit of mAb A1/40 in coating buffer (75 μg/mL) overnight and blocked for 25 min with PBS-T BND  + 0.2% bovine serum albumin . Finally, frits were prepared for drying by 1 h of incubation with PBS-T BND + 1% BSA + 5% saccharose and air-dried at 35 °C for 30 min in a fluid bed dryer . The columns were equipped with antibody-coated frits between two preblocked separation frits  and stored dry at room temperature until further use.To this aim, polyethylene filter frits  used for the coating of the capture antibodies were activated by successive 10-min washes with 96% ethanol, 50% ethanol, and 3 × coating buffer  is shown in Fig. To allow for maximum sensitivity of detection at low background, both SA-pHRP and biotinylated detection antibodies were titrated  in the absence of antigen. For titration of SA-pHRP, the protocol was performed as described, but instead of biotinylated detection antibody AA1-buffer only was added. For titration of biotinylated detection antibodies, SA-pHRP was added at the optimum concentration determined in the previous experiment. Low background was obtained with mAb A3/710 at 0.5 μg/mL and SA-pHRP at 0.5 μg/mL . The assay’s sensitivity was determined by measuring the LOD for recombinant A27. The assay’s specificity was determined for various OPV strains by using purified viral particles and by testing clinical crust material from CPXV-infected patients. To determine the test’s specificity, additional viruses causing similar clinical pictures were used, including HSV-1 and PPV. The LOD was determined by measuring serial dilutions of semi-purified virus particles and virus from clarified cell culture supernatant. Finally, the OPV ABICAP assay was validated with a blinded panel of PCR-quantified highly pathogenic viruses, including Yellow Fever virus, Ebola virus, Marburg virus, VACV, and MPXV.The cutoff to determine the limits of detection (LOD) and to discern positive from negative samples was calculated as the mean + 3 × standard deviation of negative control columns , 6.8 × 103 PFU/mL for CPXV, 1.3 × 103 PFU/mL for MPXV, and 6.8 × 103 PFU/mL for CMLV , which was expected since a previous study showed impaired binding of capture antibody A1/40 to ECTV [Using the presented protocol, all OPV strains tested were detected with sensitivities of 3.4 × 10MLV Fig. . ECTV sh to ECTV . As ECTVMyxoma virus as well as a surface swab sample positive for Varizella zoster virus were included to test the specificity of the ABICAP assay. Cell culture supernatants from PPV and HSV-1 infections were used as negative control. The clinical material that was tested positive for OPV by PCR could be diagnosed as positive by the OPV ABICAP assay  from two CPXV infections which had been diagnosed as CPXV positive by qPCR. Homogenized crusts of either unknown etiology (OPV negative by PCR) or those positive for T value of 30.7 corresponding to 6.4 × 104 genome equivalents (GE)/mL. The highest dilution at a CT value of 37.98 (2.3 × 103 GE/mL) could not be detected, which is in agreement with the titration experiments. In addition, to our knowledge OPV preparations contain at least 10-fold higher viral genome loads than infectious particles, meaning that the particle concentration in this sample was significantly lower. MPXV was also reliably detected, while no cross-reactivity could be seen against Ebola virus, Marburg virus, and Yellow fever virus.To evaluate further the ability of the OPV ABICAP assay to detect OPV and differentiate them from other potential bioterror agents, a panel of PCR-quantified gamma irradiation-inactivated viruses was tested Table . The pan4 GE/mL.In conclusion, the OPV ABICAP assay was able to detect OPV rapidly from clinical specimens as well as from blinded samples included in a bioterror panel. The assay was sufficiently sensitive to confirm the virus presence in all clinical samples. The observed LOD for real-world samples measured with the OPV ABICAP assay corresponded to approximately 5 × 10Molecular diagnostics of infectious diseases has undergone a rapid development over the past years. While there are numerous commercially available tests for clinically relevant infectious diseases, there is an obvious gap regarding tests for rare or emerging diseases, particularly with respect to on-site diagnostic tests. Although unrivalled in sensitivity and specificity, PCR has its drawbacks in field diagnostics, requiring a certain amount of technical equipment, practical skills, and diagnostic knowledge. In contrast, so-called rapid tests like lateral flow assays (LFA) usually circumvent these demands and are easy to perform, but lack sensitivity due to the absence of a signal amplification step. However, they present a promising approach for POC diagnostics.6 to 107 PFU/mL which is usually sufficient for the diagnosis of skin lesions containing extremely high viral loads [4 PFU/mL which allows reliable diagnosis even when viral loads in the sample are low because of inappropriate sampling or non-optimal time points in the course of infection. Similarly, an ABICAP assay for the detection of Ebola virus showed an LOD of approximately 1.2 × 104 PFU/mL, indicating that ABICAP systems generally represent sensitive platforms for particle detection [The established OPV ABICAP immunofiltration assay is the first POC detection assay covering all zoonotic OPV as well as VARV with a satisfactory sensitivity. One previously developed handheld rapid test for poxviruses, the Tetracore Orthopox Biothreat detection assay, displays an LOD of 10al loads . The newThe higher sensitivity of the ABICAP assay compared to lateral flow assays is also in agreement with previous reports with an ABICAP assay targeting botulinum neurotoxin. Furthermore, in an independent evaluation the ABICAP showed sensitivity comparable to that of an in-house ELISA whereas commercial LFAs either failed completely or lacked sensitivity, even compared to the relatively high LODs according to the manufacturers . Our stuParticularly when performing field diagnostics, adequate sampling and sample processing can be impaired dramatically by environmental conditions. Although the ABICAP assay was highly specific for all samples tested in this work, false-positive results remain a risk as in any diagnostics, especially when testing clinical sample material. Independent of the high specificity of the implemented antibodies, matrix effects might cause higher background signals. For an exhaustive validation, more samples, ideally in the field, will be required to be analyzed using this assay.Finally, as all reagents for an OPV ABICAP test can be stored stably at 4 °C or even ambient temperature, there is no need to freeze reagents to -20 °C, which is usually a limiting factor in the regions in which the diagnosed diseases are endemic. The OPV ABICAP assay is therefore a low-tech alternative, particularly when compared to PCR, and represents an optimal compromise between ease of handling and sensitivity. Most importantly, not only was the ABICAP system able to detect OPV from clinical samples, but also to differentiate OPV from other viruses that can (like OPV) cause skin lesions or potentially be used as bioterror agents.


Nicotiana benthamiana for silencing. Using this toolkit, we identify the LRR receptor-like protein Response to XEG1 (RXEG1) that specifically recognizes the glycoside hydrolase 12 protein XEG1. RXEG1 associates with XEG1 via the LRR domain in the apoplast and forms a complex with the LRR receptor-like kinases BAK1 and SOBIR1 to transduce the XEG1-induced defense signal. Thus, this genome-wide silencing assay is demonstrated to be an efficient toolkit to pinpoint new immune receptors, which will contribute to developing durable disease resistance.Activation of innate immunity by membrane-localized receptors is conserved across eukaryotes. Plant genomes contain hundreds of such receptor-like genes and those encoding proteins with an extracellular leucine-rich repeat (LRR) domain represent the largest family. Here, we develop a high-throughput approach to study LRR receptor-like genes on a genome-wide scale. In total, 257 tobacco rattle virus-based constructs are generated to target 386 of the 403 identified LRR receptor-like genes in  Nicotiana benthamiana genome and identify Response to XEG1 that specifically recognizes the glycoside hydrolase 12 protein XEG1.The role of most plant leucine-rich repeat (LRR) receptors in innate immunity is unknown. Here, the authors develop virus-based constructs to silence LRR receptor-like genes in the  In general, MAMPs can be structural elements or proteins released from microbes2.Environmental microbes constantly threaten plants. Over the course of evolution, plants have developed a multifaceted innate immune system to defend against potentially harmful microbes. One of the most important determinants of plant adaptation is the capacity to perceive the evolutionarily conserved signatures of microbial pathogens, namely microbe-associated molecular patterns (MAMPs), and initiate effective defense responses accordingly3. Plant genomes encode hundreds of potential cell surface receptors including receptor-like kinases (RLKs) and receptor-like proteins (RLPs)6. RLKs contain a ligand-binding ectodomain, a transmembrane (TM) domain, and an intracellular kinase domain, while RLPs lack any known intracellular signaling domains. Cell surface receptors have diverse ectodomains4. Receptor-like genes encoding proteins with an extracellular leucine-rich repeat (LRR) domain constitute the most overrepresented family according to analyses of sequenced plant genomes8. Several reports demonstrated that these LRR receptor-like genes are crucial for plant adaptation and function in various physiological processes, including development, growth, and responses to abiotic and biotic stresses11.Plants have evolved cell surface pattern-recognition receptors (PRRs) to detect MAMPs and thereby activate immune responses12. These include RLKs, such as the bacterial flagellin receptors FLS214 and FLS315, the bacterial elongation factor Tu receptor EFR, the bacterial elicitor xup25 receptor XPS1, and the DAMP receptors PEPR1 and PEPR219. The characterized RLPs include tomato EIX2, Ve1, and Cf4, potato ELR, and Arabidopsis thaliana RLP1, RLP23, RLP30, and RLP42, which recognize fungal ethylene-inducing xylanase (EIX), fungal apoplastic effector Ave1 and Avr4, Phytophthora elicitin INF1, an unknown Xanthomonas MAMP, necrosis and ethylene-inducing peptide 1-like proteins (NLPs), Sclerotinia sclerotiorum elicitor SCFE1, and fungal endopolygalacturonases, respectively27. Interestingly, recognition of an epitope of the bacterial cold shock protein (csp22) requires two receptors including the LRR-RLP receptor CSPR and the LRR-RLK receptor CORE29. LRR receptors, such as the LRR-RLK BAK1, also function as co-receptors, by forming complexes with multiple LRR-type PRRs where they are indispensable for MAMP recognition and subsequent immune signaling10. In addition, the LRR-RLK SOBIR1 functions as an adaptor that associates with multiple LRR-RLPs to form bi-partite equivalents of LRR-RLKs30. Several other LRR-RLKs, such as ERECTA, are also involved in plant immunity31; however, their exact activation mechanisms remain unclear. In spite of this, the vast majority of this enigmatic protein family has not yet been investigated and their role in plant growth and immunity remains largely elusive.Hitherto, only a few LRR receptor-like genes have been documented encoding PRRs capable of recognizing MAMPs or receptor-like proteins (DAMPs) and function as immune receptors33. The glycoside hydrolase 12 (GH12) protein XEG1 identified from the soybean root rot pathogen Phytophthora sojae is recognized in the plant apoplast as a novel MAMP34. XEG1 contains a GH domain that can degrade xyloglucan and β-glucan. This xyloglucanase activity was shown to be essential for XEG1-mediated Phytophthora virulence, but not for plant recognition35. The GH12 proteins are widely distributed across microbial taxa and many are able to trigger cell death in plants36, indicating that the recognition system is evolutionally conserved.With the growing number of sequences of microbial genomes, multiple pathogen-secreted elicitors have been identified. A number of these elicitors represent conserved microbial patterns that can be recognized by plants, resulting in the activation of defense responsesNicotiana benthamiana is an important model plant for the study of plant biology and plant−pathogen interactions and has several advantages over other plant species37. N. benthamiana belongs to the Solanaceous plant family, with high genomic collinearity with other Solanaceous plant species such as tomato39. N. benthamiana is amenable to highly efficient protein expression and virus-induced gene silencing (VIGS). Moreover, multiple MAMPs, including elicitins, trigger defense responses or plant cell death in N. benthamiana, making it an excellent system for dissecting the recognition events.N. benthamiana as a model plant and develop a toolkit that allows high-throughput characterization of LRR receptor-like genes on a genome-wide scale. We explore the genome sequence of N. benthamiana for potential LRR receptor-like genes and perform silencing assays of the identified LRR-RLK and LRR-RLP genes using a VIGS-based approach. The silencing efficiency is evaluated and alterations in plant growth are monitored. Using this toolkit, we successfully identify the Response to XEG1 (RXEG1) protein, which is required for response to the GH12 protein XEG1 and homologs in N. benthamiana. Thus, this study has established an important toolkit for the characterization of plant immune receptors.In this study, we examine N. benthamiana using both BLAST and hidden Markov model (HMM) searches to identify fragments encoding proteins with both LRR and TM domains. These LRR receptor-like genes were used as queries for a BLAST search of the genome sequence of N. benthamiana. The retrieved sequences were validated using the publically available transcriptome data and manually corrected as needed. In total, 86 LRR-RLPs and 317 LRR-RLKs were identified that are listed in Table N. benthamiana, two copies of highly homologous LRR receptor-like genes were often identified in the genome , LRR and TM domains, as well as a cytoplasmic tail or kinase domain, although many of these proteins are missing SPs  to 494 bp  homologs, were dwarfs with short stature, dark green, and wrinkled leaves  encode a component essential for XEG1 recognition. Both constructs simultaneously silenced two genes encoding full length LRR-RLP receptors containing 31 LRRs in the extracellular domain and one encoding an RLP with 16 LRRs , RXEG1-like1 , and RXEG1-like2 , respectively. To determine which LRR receptor-like gene is responsible for XEG1 recognition, we made synthesized versions of these receptor-like genes (RXEG1(S), RXEGL1(S), and RXEGL2(S); Supplementary Data RXEG1-1- or TRV:RXEG1-2-treated N. benthamiana. All three receptors were expressed as indicated by western blotting  or a mixture of the three receptors, but not in leaves expressing the other two synthesized receptors  alone did not cause cell death in N. benthamiana , where a significant increase in Phytophthora biomass was consistently detected in the RXEG1-silenced leaves  assay and western blot. As shown in Fig. Phytophthora GH12 proteins using Co-IP assay and we found all the four tested GH12 proteins associate with RXEG1 in planta ) in the TRV:RXEG1-1 and TRV:RXEG1-2-treated plants. RXEG1SP+LRR(S) was successfully expressed in N. benthamiana, but failed to restore XEG1-induced plant cell death , but this interaction was hardly detectable in mock-treated leaves. In the case of RXEG1, the association with SOBIR1 was detected regardless of XEG1 treatment  associates with BAK1, which was confirmed by yeast two-hybrid and bimolecular fluorescence complementation assays50. Nevertheless, the interaction between RXEG1 and BAK1 was increased upon XEG1 treatment, demonstrating that XEG1 can significantly promote the RXEG1−BAK1 interaction. FLS2 is another PRR that forms a complex with BAK1 upon ligand  treatment. According to the solved structure, the FLS2LRR-flg22-BAK1LRR complex shares two interfaces14. BAK1LRR binds directly to FLS2LRR, which may account for the flg22-independent FLS2−BAK1 association detected in some cases52. The other interface is the ligand flg22-dependent bridging of FLS2LRR and BAK1LRR, which promotes the heterodimerization of FLS2LRR and BAK1LRR. Whether the RXEG1-XEG1-BAK1 complex forms in a similar manner, and the detailed molecular mechanisms thereof, remain to be uncovered.RXEG1 belongs to the LRR-RLP-type of receptors whose function is usually highly dependent on the common adaptor SOBIR154. Recently, multiple novel MAMPs were identified that require BAK1 or both BAK1 and SOBIR155, indicating that the corresponding receptors may belong to the LRR class. Moreover, the recognition of certain MAMPs is restricted in Solanaceous plants55. Therefore, the silencing of the LRR receptor candidates should be a straightforward approach to identify recognition receptors for each MAMP, which will ultimately lead to improved plant resistance. In addition, as LRR receptors belong to a conserved receptor family among plant species, functional analysis of these receptors in N. benthamiana will also improve our understanding of their function in other plant species, especially in those species in which genetic manipulation is difficult.Based on current knowledge, BAK1 and SOBIR1 participate solely in signaling mediated by LRR receptors, but not receptors with other types of ectodomains such as LysM-receptors or lectin receptor kinaseshttp://pfam.xfam.org) was performed via the Sol Genomic Network (SGN) website (http://solgenomics.net) against the genomic databases of N. benthamiana (V1.0.1) or Solanum lycopersicum (ITAG release 3.20). All non-redundant protein sequences were retrieved and analyzed with Conserved Domains (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi), SMART (http://smart.embl-heidelberg.de), Pfam (http://pfam.xfam.org/), and SignalP 3.0  for protein domain and motif prediction. Proteins with both LRR and TM domains were considered as potential LRR receptors. For N. benthamiana, both protein and cDNA sequences were retrieved and the obtained cDNA sequences were further validated using RNA-sequencing data derived from the QUT Nicotiana benthamiana Genome & Transcriptom Sequencing Consortium (http://benthgenome.qut.edu.au/)57.An HMM search by the software HMMER  using LRR . The N. benthamiana LRR-RLK candidates were classified into different subfamilies according to the reference LRR-RLKs in Arabidopsis thaliana, which represents the subfamilies I-XV6. For LRR-RLPs, phylogenetic tree was constructed using the conserved C3 and D domains59. The phylogenetic trees were displayed using iTOL (http://itol.embl.de).For LRR-RLKs, phylogenetic maximum-likelihood (ML) tree was constructed using the predicted kinase domain sequences and PhyML implemented in SEAVIEW software  with the primers listed in Supplementary Table 60 or the expression vectors pBIN-(c)eGFP, pBIN-(c)HA, pBIN-(c)mRFP using the ClonExpress II One Step Cloning Kit . The full length coding sequences containing the SP were amplified for INF1, NPP1, and XEG1 homologs except Avh241 without SP61. The full length coding sequence62 of EIX was synthesized in Genscript. All these fragments were subsequently cloned into vector pGR107-cHA. The resulting vectors were verified by sequencing and individually transformed into the Agrobacterium tumefaciens strain GV3101. TRV:BAK151and TRV:SOBIR163 were shown in supplementary Data Fragments used to generate silencing or overexpression constructs were amplified by PCR from cDNA or genomic DNA of Nicotiana benthamiana plants were grown in soil in a conditioned climate chamber at 19–22 °C with a 14 h photoperiod and 70–80% relative humidity. P. parasitica 025 was maintained in the dark at 25 °C on 20% (v/v) V8 juice agar64. Leaf infection assays were performed as follow65: leaves were collected from N. benthamiana with similar plant sizes at the same position. Inoculated leaves were kept in transparent plastic boxes with high humidity and placed in the climate chamber. Inoculated leaves were kept in the dark for the first 24 h. Lesion diameters were measured at 3 days post-inoculation (dpi).N. benthamiana of similar size and ground in liquid nitrogen. Total RNA was isolated from 100 mg of ground material using an RNA Kit  and used as a template for first strand cDNA synthesis using PrimeScript Reverse Transcriptase (Takara Bio Inc.). Quantitative reverse transcription PCR (qRT-PCR) was performed on an ABI 7500 Fast Real-Time PCR system  using PrimeScript RT Master Mix (Takara Bio Inc.) with translation elongation factor 1 alpha (EF-1α) as an endogenous control and the primers listed in Supplementary Data −ΔΔCT method66.Leaves were selected from the same position of Nicotiana benthamiana leaf discs (d = 4.0 cm) were collected from infection sites 3 days after P. parasitica inoculation. The leaf discs were ground in liquid nitrogen and used for DNA isolation using the genomic DNA extraction kit . The eluted products were used as templates for qPCR with N. benthamiana EF-1α as an endogenous control, while the primers PAR-F/R were used to target P. parasitica  for 3–4 h. For the TRV-mediated silencing assay, A. tumefaciens cultures expressing pTRV-RNA2 constructs were mixed in a 1:1 ratio with A. tumefaciens culture expressing pTRV1 to a final OD600 of 1.0 before infiltration into cotyledons of four-leaf-stage N. benthamiana seedlings. TRV:PDS and TRV:GFP were used as controls. For transient gene expression, A. tumefaciens suspensions were syringe-infiltrated into fully expanded leaves of six-week-old N. benthamiana with appropriate concentrations . For co-infiltration with the silencing suppressor P19, suspensions were mixed in an appropriate ratio to a final OD600 of 0.6.m NaCl, 50 mm Tris-HCl pH 7.5,10 mm ethylenediaminetetraacetic acid, 1.0% (v/v) NP-40, 1 mm phenylmethylsulfonyl fluoride, and 0.1% (v/v) protease inhibitor cocktail  for 30 min. The homogenate was centrifuged at 21,000×g for 15 min and the supernatant was incubated with GFP-trap® A beads  at 4 °C for 2 h. The beads were pelleted and washed with extraction buffer for six times. The proteins were then eluted from the beads by boiling for 10 min, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred to a polyvinylidene fluoride (PVDF) membrane . The PVDF membrane was blocked with phosphate-buffered saline (PBS) pH 7.4 with 5% skim milk for 1 h at room temperature followed by three washes with PBS with 0.1% Tween-20. Co-immunoprecipitated proteins were analyzed by incubating the membrane with 1:5000 diluted anti-GFP , anti-HA , or anti-RFP  antibodies followed by incubation with 1:10,000 diluted goat anti-mouse  or goat anti-rabbit  antibody. The blots  or KM71 (Muts). P. pastoris was cultured overnight in the YPD medium at 30 °C and subsequently grown in the BMGY (Buffered Glycerol-Complex Medium) and BMMY (Buffered Methanol-Complex Medium) (pH = 6.5) for protein expression. The recombinant protein XEG1-His or EV was purified from the supernatant of the P. pastoris culture harboring pPICZα-XEG1-His or pPICZα using the AKTA™ avant 25  through the HisTrap™ FF Ni Sepharose Columns  and HiTrap™ Desalting Prepacked Columns .pPICZα vector containing XEG1-His(ROS) production was monitored with a luminol/peroxidase-based assay on leaf discs (Ø 0.5 cm) collected from 5-week-old N. benthamiana plants and floated overnight in 200 μL of sterile H2O in a 96-well plate. H2O was replaced with the luminol (35.4 μg/mL)/peroxidase (10 μg/mL) reaction solution supplied with sterile water, 100 nm flg22 , 1 μm purified XEG1 protein, or EV. Luminescence was measured using the GLOMAX96 microplate luminometer .Reactive oxygen species N. benthamiana leaf discs (Ø 1.0 cm) were collected 3 days after agro-infiltration and floated on 4 mL of deionized water for 3 h with continuous shaking (100 rpm) at room temperature. The initial electrolyte leakage values and the final electrolyte leakage values after 15 min of boiling were measured using a conductivity meter . Relative electrolyte leakage was calculated by comparing the initial and final electrolyte leakage values.Six Sequence information can be found in GenBank with the following accession numbers: RXEG1 (MG010652), RXEGL1 (MG010653), and RXEGL2 (MG010654). All data relevant to this study are available from the corresponding author upon reasonable request.Supplementary InformationDescriptions of Additional Supplementary FilesPeer Review FileSupplementary Data 1Supplementary Data 2Supplementary Data 3


Influenza virus motility is based on cooperation between two viral spike proteins, hemagglutinin (HA) and neuraminidase (NA), and is a major determinant of virus infectivity. To translocate a virus particle on the cell surface, HA molecules exchange viral receptors and NA molecules accelerate the receptor exchange of HA. This type of virus motility was recently identified in influenza A virus (IAV). To determine if other influenza virus types have a similar receptor exchange mechanism-driven motility, we investigated influenza C virus (ICV) motility on a receptor-fixed glass surface. This system excludes receptor mobility, which makes it more desirable than a cell surface for demonstrating virus motility by receptor exchange. Like IAV, ICV was observed to move across the receptor-fixed surface. However, in contrast to the random movement of IAV, a filamentous ICV strain, Ann Arbor/1/50 (AA), moved in a straight line, in a directed manner, and at a constant rate, whereas a spherical ICV strain, Taylor/1233/47 (Taylor), moved randomly, similar to IAV. The AA and Taylor viruses each moved with a combination of gradual (crawling) and rapid (gliding) motions, but the distances of crawling and gliding for the AA virus were shorter than those of the Taylor virus. Our findings indicate that like IAV, ICV also has a motility that is driven by the receptor exchange mechanism. However, compared with IAV movement, filamentous ICV movement is highly regulated in both direction and speed. Control of ICV movement is based on its specific motility employing short crawling and gliding motions as well as its own filamentous morphology.IMPORTANCE Influenza virus enters into a host cell for infection via cellular endocytosis. Human influenza virus infects epithelial cells of the respiratory tract, the surfaces of which are hidden by abundant cilia that are inactive in endocytosis. An open question is the manner by which the virus migrates to endocytosis-active domains. In analyzing individual virus behaviors through single-virus tracking, we identified a novel function of the hemagglutinin and esterase of influenza C virus (ICV) as the motility machinery. Hemagglutinin iteratively exchanges a viral receptor, causing virus movement. Esterase degrades the receptors along the trajectory traveled by the virus and prevents the virus from moving backward, causing directional movement. We propose that ICV has a unique motile machinery directionally controlled via hemagglutinin sensing the receptor density manipulated by esterase. Virus motility is a major determinant for virus infectivity, and a novel motile mechanism was recently found in influenza A virus (IAV) . Utiliziin vivo, so virus motility is significant for virus infection. Notably, human influenza virus infects epithelial cells in the respiratory tract, the surface of which is covered by abundant cilia. Cilium membranes are inactive in endocytosis, and the endocytosis-active domains of ciliated cells are located in the cilium bases  , 10, 11.in vitro system was developed for performing imaging analyses of virus movement via the receptor exchange mechanism  (R) of the virus is equal to the product of the velocity (V) and time (t). Consequently, the MSD instead obeys the equation ⟨R2⟩ = ⟨V2⟩t2, where ⟨V2⟩ is the mean square velocity. Therefore, the MSD of viruses moving randomly is proportional to the time (t), whereas the MSD of viruses moving linearly is proportional to the square of time (t2). The log-log plots of the MSDs are suitable for a quantitative evaluation of the linearity or randomness of virus movements  from the initial viral attachment positions or from the virus positions at the time recording was begun  and B. Tseconds) , 15. In ovements . The MSDng curve  and C, wF(r) is the CCDF of r, r is the displacement of viruses per second, and P(r) is the frequency function of r. The CCDF is more suitable than the “normal” frequency function P(r) for analyses of rare events, like gliding motions (We hypothesized that the short-range motions specific for AA viruses act to generate the direction of virus movement. Previous work reported that IAV Puerto Rico/8/34 H1N1) and Aichi/2/68 (H3N2) strains moved randomly with occasional rapid (gliding) motions in addition to their ordinary gradual (crawling) motions N1 and Ai and F. T motions . HoweverShort crawling and gliding in ICV presumably generate directional movement through the degradation of receptors by viral ESs . BecauseIn the filamentous AA virus, directional movement persists over a long period of time due to the filamentous morphology of the virus  and 7. NAchatina fulica, specifically binds to Neu5,9Ac2-terminated oligosaccharide  were coated with bovine mucin , which was dissolved in phosphate-buffered saline (PBS) and filtered through a polyvinylidene fluoride membrane filter with a 0.22-μm pore  to remove insoluble mucin, and the dishes were then incubated for 1 h at room temperature. The glass surface of each dish was washed twice with PBS to remove any unbound mucin. This cycle of coating and washing was repeated twice. To examine virus movement, viruses diluted in PBS were added to the mucin-coated glass surfaces and observed by SRICM  using a 100× lens objective . Virus images were acquired every 1 s for a total duration of 15 min. Images of 50 filamentous AA, 30 spherical AA, and 30 spherical Taylor viruses were analyzed. Virions with lengths of more than 0.5 μm were regarded as filamentous, whereas virions with diameters of less than 0.3 μm were regarded as spherical.http://rsb.info.nih.gov/ij/). The positions (x and y coordinates) of viruses were determined as the centers of darkness. To test the stability and accuracy of our apparatus, IAV particles were fixed on a coverslip coated with fetuin (1 mg/ml) in the presence of an NA inhibitor , and time-lapse images of IAV particles were acquired (Images were analyzed with ImageJ software (h fetuin  mg/ml inh fetuin  mg/ml inacquired , 25.