Bunyaviridae, are thrips borne and cause severe agricultural losses worldwide. Based on the serological relationships of the structural nucleocapsid protein (NP), the current tospoviruses are divided into six serogroups. The use of NP-antisera is convenient for virus detection, but it is insufficient to identify virus species grouped in a serogroup due to the serological cross-reaction. Alternatively, virus species can be identified by the N gene amplification using specific primers. Tomato spotted wilt virus (TSWV) is the type species of the genus Tospovirus and one of the most destructive plant viruses. Eight known tospoviruses, Alstroemeria necrotic streak virus (ANSV), Chrysanthemum stem necrosis virus (CSNV), Groundnut ringspot virus (GRSV), Impatiens necrotic spot virus (INSV), Melon severe mosaic virus (MeSMV), Pepper necrotic spot virus (PNSV), Tomato chlorotic spot virus (TCSV) and Zucchini lethal chlorosis virus (ZLCV), sharing serological relatedness with TSWV in NP, are grouped in the TSWV serogroup. Most of the TSWV-serogroup viruses prevail in Europe and America. An efficient diagnostic method is necessary for inspecting these tospoviruses in Asia, including Taiwan.Tospoviruses, the plant-infecting genus in the family Chenopodium quinoa leaves separately inoculated with ANSV, CSNV, GRSV, INSV, TCSV and TSWV were used for testing purposes. The 5’-biotinylated degenerate forward and reverse primers were designed from the consensus sequences of N genes of TSWV-serogroup tospoviruses for reverse transcription-polymerase chain reaction (RT-PCR) amplification. Virus-specific oligonucleotide probes were spotted on the surface of polyvinyl chloride (PVC) chips to hybridize with PCR products. The hybridization signals were visualized by hydrolysis of NBT/BCIP with streptavidine-conjugated alkaline phosphatase. The microarray was further applied to diagnose virus infection in field crop samples.A microarray platform was developed for simultaneous detection and identification of TSWV-serogroup tospoviruses. Total RNAs extracted from Amplicons of approximately 0.46 kb were amplified from all tested TSWV-serogroup tospoviruses by RT-PCR using the degenerate primer pair Pr-dTS-f/Pr-dTS-r. Virus species were identified on chips by hybridization of PCR products with respective virus-specific probes. The microarray was successfully used to diagnose TSWV infection in field pepper samples.In this study, a rapid, sensitive and precise microarray method has been developed to simultaneously detect and identify six TSWV-serogroup tospoviruses. The microarray platform provides a great potential to explore tospoviruses that can help researchers and quarantine staff to prevent invasions of tospoviruses.The online version of this article (doi:10.1186/s12985-016-0669-1) contains supplementary material, which is available to authorized users. Bunyaviridae, are destructive plant viruses with a very broad host range, infecting more than 1090 plant species, causing lethal necrotic lesions, wilting and dieback of infected plants [Tomato spotted wilt virus (TSWV) is the first tospovirus found in Australia in 1915 [Tospoviruses, classified in the family d plants . Tomato  in 1915 , 5. To d in 1915 .Virions of tospoviruses are enveloped quasi-spherical particles measuring 80–120 nm in diameter, possessing a tripartite segmented ssRNA genome, named large (L), medium (M) and small (S). The L RNA is of negative sense, whereas the M and S RNAs are ambisense . L RNA cWatermelon silver mottle virus (WSMoV) and Iris yellow spot virus (IYSV) due to the serological relatedness of their NPs [Groundnut ringspot virus (GRSV), Impatiens necrotic spot virus (INSV), Melon severe mosaic virus (MeSMV), Pepper necrotic spot virus (PNSV), Tomato chlorotic spot virus (TCSV) and Zucchini lethal chlorosis virus (ZLCV) were grouped in the TSWV serogroup [NP is abundant in infected plant cells . The ideheir NPs . The memheir NPs . The toserogroup –28, and Prompt and accurate methods for detection and identification of tospoviruses are crucial for preventing epidemics of domestic and invading species. The serological enzyme-linked immunosorbent assay (ELISA) is the most convenient method for virus detection. Antisera against the NPs of tospoviruses are commonly used to detect tospoviruses. Due to the cross reactions of different species in the same serogroup, the use of NP antisera for virus identification could be limited , 29. TheChenopodium quinoa leaves separately inoculated with ANSV, CSNV, GRSV, INSV, TCSV and TSWV in RT-PCR. Virus species were further identified by hybridization of the PCR products with the virus-specific oligonucleotide probes that were pre-spotted on the surface of polyvinyl chloride (PVC) microchips. The signals were visualized by hydrolysis of NBT/BCIP with streptavidine-conjugated alkaline phosphatase. Furthermore, the microarray was used to diagnose TSWV infections in field pepper samples. The developed microarray has a great potential to prompt the diagnosis of TSWV-serogroup tospoviruses.In this study, an oligonucleotide-based microarray method was developed for detection and identification of tospoviruses, which are serologically indistinguishable with TSWV. The degenerate primer pair Pr-dTS-f/Pr-dTS-r was designed from the consensus sequences of N genes of TSWV-serogroup tospoviruses and successfully used to amplify a certain cDNA fragment from the total RNAs of Alstroemeria sp. in Colombia [Chenopodium quinoa Willd. by mechanical transmission in an isolated temperature-controlled (25–28 °C) greenhouse according to quarantine regulations. A buffer consisting of 0.01 M potassium phosphate (pH 7.0) and 0.01 M sodium sulfite was used for inoculation.The NY isolate of TSWV was collected from tomato in New York, the United States . The oriColombia . All virwww.ncbi.nlm.nih.gov)  (v) Table  for desiv) Table . One hunv) Table  using thThe Lasergene 7 software package  was used to analyze sequences. Multiple alignments of nucleotide sequences of N genes listed in Table PCR was conducted to evaluate the degenerate primers using the constructed plasmids. The reaction mixture of 25 μl is comprised of 100 ng of plasmid DNA, 2.5 μl of 10× Taq buffer  , 1 μl of 10 mM dNTPs (Protech), 1 μl of 100 μM forward primer, 1 μl of 100 μM reverse primer and 0.1 μl of Pro Plus Taq DNA polymerase (5 U/μl) (Protech). The amplification was carried out by Applied Biosystems GeneAMP PCR System 9700  under the setting of a hot start at 95 °C for 5 min; 35 cycles of 95 °C for 30 s, 50 °C for 40 s and 72 °C for 30 s; and a final reaction at 72 °C for 6 min. The amplicons were visualized by 2% agarose gel electrophoresis and ethidium bromide (EtBr) staining. For virus detection, the mixture of 25 μl consisting of 200 ng of plant total RNA, 0.2 μl of M-MuLV reverse transcriptase (5 U/μl) (Protech), 2.5 μl of 10× Taq buffer (Protech), 1 μl of 10 mM dNTPs (Protech), 0.1 μl of 100 μM forward primer, 0.1 μl of 100 μM reverse primer and 0.1 μl of Pro Plus Taq DNA polymerase (5 U/μl) (Protech) was used in RT-PCR amplification. Complementary DNA was synthesized at 45 °C for 30 min then termination at 95 °C for 5 min. The PCR amplification was set as a hot start at 95 °C for 5 min; 35 cycles of 95 °C for 30 s, 50 °C for 40 s and 72 °C for 30 s; and a final reaction at 72 °C for 6 min. The amplicons were observed by 2% agarose gel electrophoresis and EtBr staining.Twenty micromolar of each probe were spotted on the surface of PVC microchips with an automatic spotting machine  and immobilized by an ultraviolet crosslinker  with 1.2 J for 5 min following the previous report . In addiThe PCR product amplified from pTOPO-TSWV-N  using th3, pH 9.8) were used for colour development. The absorbance at 405 nm (A405) was determined by Model 680 microplate reader (Bio-Rad) after 10 to 60 min of substrate addition.Indirect ELISA was conducted to detect TSWV in diseased plant samples according to the previous report . The cru20) at the 5’ terminus for fixing on microchips , exhibiting chlorotic spots or ringspots on leaves and fruits, were collected from Sinyi Township, Nantou County, Taiwan in December 2015 for detecting tospovirus infection by indirect ELISA using the monoclonal antibody against the TSWV NSs protein . SY-1 anAn efficient detection method is necessary for inspection of viral diseases in quarantine. The previous studies reported that the tospoviruses ANSV, CSNV, GRSV, INSV, TCSV and TSWV share a serological relationship in their NPs, so that they can be detected, but not be differentiated, by antisera against the NP in serological assays , 40. On Groundnut bud necrosis virus (GBNV), Groundnut chlorotic fan-spot virus (GCFSV), IYSV, Melon yellow spot virus (MYSV), Soybean vein necrosis-associated virus (SVNaV), Tomato necrotic spot-associated virus (TNSaV), Tomato yellow ring virus (TYRV), Tomato zonate spot virus (TZSV), Watermelon bud necrosis virus (WBNV) and WSMoV, were also used to test the developed microarray. No unexpected signals were observed to demonstrate the excellent specificity of the microarray in TSWV serogroup only (data not shown). Using the same approach, microarrays for detecting tospoviruses in other serogroups will be further developed.Consensus nucleotide sequences can also be found within the N genes of tospoviruses belonging to a phylogenetical clade. According to this finding, the degenerate primer pair Pr-dTS-f/Pr-dTS-r were designed from the N genes of all nine TSWV-serogroup tospoviruses, and can successfully detect all six tested viruses, ANSV, CSNV, GRSV, INSV, TCSV and TSWV, in one-step RT-PCR Fig. . UnfortuCymbidium mosaic virus, Odontoglossum ringspot virus and CaCV for orchid inspection , and the identification of insect species [The DNA microarray is basically developed as a combination technique of nucleic acid amplification and hybridization for high-throughput assay and has a wide application including use in analyses of gene expression, transcription factor binding and genotyping . It also species  and the  species .The developed microarray was used to inspect virus infection in the fields of Sinyi Township in central Taiwan, and indirect ELISA using MAb-TNSs-82D3B4 was performed for comparison. MAb-TNSs-82D3B4 reacted with TSWV but not reacted with GRSV, INSV and TCSV was mentioned in our previous report , and itsTospoviruses are vectored by thrips for oversea distribution. The identification of the thrips category and the inspection of viruliferous thrips are necessary for preventing an invasion of foreign tospoviruses. The microarray is sufficient for multiplex detections. The identifications of tospoviruses and thrips will be integrated into a biochip to strengthen inspection for quarantine.In this study, a degenerate primer pair Pr-dTS-f/Pr-dTS-r, designed from the consensus sequences of N genes, was used to amplify a DNA fragment from total RNAs of plant tissues infected by six tested tospoviruses, ANSV, CSNV, GRSV, INSV, TCSV and TSWV. The virus species were further differentiated by hybridization with specific probes spotted on PVC biochips. The microarray platform was used to diagnose TSWV infections in field peppers. Our results showed that the microarray method can be used to simultaneously detect and identify TSWV-serogroup tospoviruses that provides a great applicable potential in inspection of tospoviruses for quarantine.


This article presents the results of a multidisciplinary study which investigated the effects of a period with floorball training on health status, psychological health and social capital of older men. Thirty-nine untrained men aged 69.9 ± 0.6 (range: 65–76) were randomized into a group playing floorball (n = 22) or a group playing petanque (n = 17) one hour twice a week for 12 weeks. Both groups filled out the Health Survey Short Form (SF-12) and the Hospital Anxiety and Depression Scale (HADS) before and after the 12-week intervention. Linear regression analyses with bootstrapping showed that the men in the floorball group improved in the SF-12 composite score for mental health, as well as the HADS subscales anxiety and depression, compared to the men in the petanque group. In addition, 21 interviews were conducted with a sample of the men engaged in floorball. According to the statements in the interviews, the men in the floorball group experienced a high degree of solidarity and group cohesion which seemed to have increased their social capital during the intervention. In particular, the fun and joyful experiences of playing led to a high degree of social connectedness, which were mentioned by many of the men as the main reason for their participation throughout the 12-week period. The statistical results and the interview findings suggest that participation in a ball game such as floorball has several benefits regarding health status, psychological health and social capital and in addition that playing floorball is experienced as enjoyable amongst older men. Thus, it can be concluded that floorball is an activity that benefits older men and should be provided in relevant contexts, such as e.g. sport clubs or centres for seniors. Among these consequences is physiological decline, such as accumulation of body fat However, there are possibilities to prevent or diminish the negative consequences of aging. Participation in physical activity seems to be a possible way to alleviate the physical decline that is often connected aging The decline in psychological health can be alleviated as well. It is commonly accepted that physical activity has positive effects on psychological health e.g. . FurtherTeam games may be an additional remedy for the decline in physical and psychological health. Regular small-sided football and floorball training among untrained men and women seems to bring about important health benefits The above mentioned benefits of participation in team games are of particular importance to older men. Although participation in sports and physical activity is relatively high among Danes aged 60 years or older, men in this age group are less physically active than women  To find the answers to this question, the overall purpose of the study was to investigate the effects of a team game, namely floorball training, on health status and psychological health compared to a control group participating in an a team game with little or no physical activity, namely petanque, in a group of untrained men aged 65 years or older. In addition, we wanted to investigate possible reasons for the hypothesized difference between the effects of participation in floorball and petanque.2.This study was an integrated part of a multidisciplinary intervention, which also included physiological investigations. For more details on the study and the results, see Vorup et al. 2.1.Thirty-seven untrained men aged 69.84  years were recruited , but had not been involved in any type of regular (<2 weekly sessions) intense physical training for at least 10 years. Over two intervention periods, the men were randomized into a floorball group (n = 22) or a petanque group (n = 15). The reason for the unequal group sizes is that in the first intervention period, the participants were randomized 1:1, but in the second, they were randomized 2:1. This was a decision made by Vorup et al. 2.2.Both groups met twice per week to participate in floorball or petanque, respectively, and adherence was 64.64% (SD = 10.49) for floorball and 64.74% (SD = 12.88) for petanque.2.2.1.Floorball is a team sport similar to hockey, but is played with plastic sticks . The training was arranged as small-sided games, e.g. 3 vs. 3 or 4 vs. 4, on in- or outdoor courts sized 10–15 × 20–30 meter. The training sessions were organized as intervals with 4 min of playing followed by 4 min of rest with 3 intervals during the first four weeks (=12 min “active play” in each session), 4 intervals during week five to eight (= 16 min “active play” in each session), and 6 intervals in week nine to twelve (= 24 min “active play” in each session). Each training session was preceded by 10 min warm-up, which included mobility, stretching and technical exercises.2.2.2.Petanque is a traditional team sport game, in which two teams compete to throw their balls as close to a smaller ball as possible (petanque.org). It offers little physical activity or contact. The training was organized as small-sided games, typical 2 vs. 2 or 3 vs. 3, and was performed outside in a park area. Each session has a total duration of 60 minutes, and in case of bad weather conditions, the men played billiard inside.2.3.2.3.1.To measure health status and psychological health, the following questionnaires were distributed to the men before and after the intervention.The Danish version of the Health Survey Short Form SF-12  was usedA Danish version of the Hospital Anxiety and Depression Scale HADS  was also2.3.2.To test whether the groups had the same vantage point before the intervention, group differences in age, as well as all subscales of SF-12 and HADS before the intervention were tested with a Mann-Whitney test.To test effects of the training intervention, linear regression analyses with bootstrapping were used, in which measurements of the SF-12 composite scores for physical and mental health and the HADS subscales anxiety and depression at the end of the intervention were used as dependent variables, and measurements before the intervention and group affiliation  were used as independent variables.2.4.To gain insight into the men's subjective experiences of participating in floorball training, 21 individual interviews were conducted with the men in the floorball group one to three weeks after the finalisation of the intervention. To avoid bias, for example from socially desirable answers, these interviews were conducted by a researcher who had no prior involvement with the participants.2.4.1.Prior to the interviews, participant observations were conducted with the purpose of establishing positive relationships to the men in an informal manner and gaining insight on how the floorball sessions were conducted. The observations and reflections were written down as field notes and influenced the development of themes in the interview guide as a supplement to the theoretical framework 2.4.2.The interview guide was influenced by the participant observations of the training sessions during the intervention. As a theoretical framework, Bourdieu's concept of habitus and social capital, which provides insight and understanding of the social relationships between individuals 2.4.3.The interviews were based on a semi-structured interview guide 2.4.4.A list of themes was developed inspired by the reviewed literature 3.3.1.3.1.1.The Mann-Whitney test revealed no significant differences between groups for age or the subscales in SF-12 and HADS before the intervention. The results are presented in 3.1.2.The linear regression revealed that there were significant differences between the floorball and the petanque group in development of the SF-12 mental health composite score, as well as the HADS subscales anxiety and depression, from before to after the intervention. It seems that the floorball group developed better in these three subscales .3.2.3.2.1.When the men were asked about their experiences with the floorball training, they compared their experience with previous attempts to engage in a physical active lifestyle. As a part of the men's sports biographies many of the men have previously had negative experiences with training in a fitness centre or in individual physical activity. In a Bourdieusian sense, an individual's experiences with sport can be characterised as habitus. Bourdieu describes habitus as a set of”…dispositions, reflexes and forms of behaviour people acquire through acting in society. It reflects the different positions people have in society” “At some point, you want to do something with a little more ‘power’, something that is a bit more exciting, a bit more masculine, I must say (...). I was tired of that old woman gymnastics” .In contrast to these earlier experiences of exclusion, the interviewees mentioned several things that made them feel included in the team and attracted to floorball. One element was the fact that the group only consisted of men age 65+, an aspect that the interviewees emphasized as positive. One interviewee expressed his experience of cohesion by comparing it with relationships that were established in their youth: “It's a bit like being army buddies, but just in a much, much later age” . The men found it very beneficial to train in a group with participants of the same gender and age. The importance of being “at the same stage in life” was highlighted by several interviewees. One phrased it like this:“I felt that it was like meeting some old school friends (...), it was very encouraging really that we almost had some common background, although we have lived many different lives, then we have after all lived in the same time period (...). There was something that seemed very alluring from the start. I hadn't thought of that as a possibility before” .The fact that the men shared a common background was perceived as more important than the differences in their subjective habitus.The interviewees also emphasized the importance of floorball being an activity that none of the men had prior knowledge of, meaning that the men hadn't negatively experienced a significant difference in the level, competences or technical skills:“We start from scratch all together (...). There is none of us who are world champions in this game, because there is no one who has played much before. Whereas, if you come in a football club, then there is always somebody who is better than others. And I think it has in any case been the contributory factor to why it has been so easy” .The challenge of creating an inclusive and enjoyable environment when skills and competences differ, was—as the quote above shows—perceived as a barrier that have kept some of the men from starting or resuming previous sport and exercise programmes. The fact that none of the men were skilled in floorball was an inclusive element that positively influenced the interviewees' perception of floorball.Several men have expressed that it has been motivating to experience a development in skills, both personally and as a team. It is particularly the competitive element, inherent in the nature of team games, which has been mentioned as a motivating aspect of floorball. This is highlighted as a difference between floorball and individual physical activities:“You show up to win. You can't change that. No one wants to walk around and think it is fun to lose on purpose. It bothers you. They will win. Every time. And it gives a little extra. ‘Okay, now that damned guy has scored twice, he shall not be allowed any more.’ So you keep an extra eye out for him, and you start to push a little. And one cannot do that in the gym, I can't go and push the guy next to me off his bicycle” .It seems that the competitive element inherent in team games is a motivational factor, not commonly found in the physical activity opportunities available to older men.The interviewees of this study consider floorball as a form of play that is fun and generates a sense of well-being:“I must admit, I am incredibly surprised. I had thought that I would be glad to have moved my body, but that it would be so good mentally, I did not expect that in any way (...). I had pulled myself together to go to the gym, I know I would. That's not it. But it hadn't been fun. I do not believe that (...). You'd just go [and exercise], so you can drink a little bit of red wine and eat some good food (…). Whereas here [floorball], it's been a joy. It's almost like if you broke your leg, you would come here anyway and just stand and shout ‘Come on!’” .It seems that the men found floorball to be enjoyable and exciting, in contrast to earlier physical activities, that were seen as boring:“You can't go to gymnastics, it gives me nothing, even though I am an old gymnast. You can't find a place where there is gymnastics that is suitable for old age” .They describe physical activity as exhausting or boring work, which some of the men mention as a barrier to their engagement in physical activity. Contrary to these experiences, the interviewees describe floorball as fun and pleasurable, and something that they were looking forward to with enthusiasm, excitement and joy.3.2.2.The majority of the interviewees named the aim of improving fitness as their main motive to begin floorball training. The physiological effect of the training was tested during a health check conducted prior to and after the 12-week intervention. The fact that the men could “follow” their own physical development seemed to have been the initial gain of attending floorball training.Although the interviewees did not know of their personal results from the health check at the time where the interviews were conducted, they expressed that they looked forward to hearing about their performance. Aside from the outcome of the health check, the interviewees talked about the physical and mental improvements they had experienced in their everyday lives as a result of the training. It was especially an increased sense of well-being and quality of life as well as a feeling of improved physical health that were expressed as the main result of the intervention:“I haven't really had that experience since I was young, that you've used your body. You feel fantastic afterwards (...). I had not thought that it would give that kind of energy. So in that way, I will certainly continue, one way or another, even if I have to arrange it myself” .The fact that the men experienced fewer physical and mental limitations was stated by the interviewees as a factor that their significant others also have noted and appreciate: “My wife says it too, she thinks she can feel that I am in a little bit better mood”  or as Benny describes his son to have noted: “I have become much more... – it sounds intense, but my youngest son, he says ‘it is almost like you are a socially minded human after all’” .As previously underlined, the men experienced the atmosphere on the team as unique because they were of the same gender and age. This created a kind of cohesion between the players and the social capital was created as an implication of that:“It's actually fun, for like a start, you could say that we were obviously very heterogeneous in the way of [working in] different industries, different lives and in general. But anyway, then there is, all of the sudden, these things that make you have a lot of common denominators” .According to the statements in the interviews, the men experienced a high degree of solidarity and group cohesion, in particular through the fun and joyful experiences on the pitch: “We have great fun together. You could not know that in advance. I think I'm, in general, a little bit suspicious of the social, so it's such a positive surprise that it has been so lovely” .The social relations seem to not only develop on the pitch during the interaction in the floorball training, but the social relationships have eventually grown to exist off the training pitch as well:“I think as we get further in the process, we get more and more socially involved and we are also beginning to talk about what one actually did [workwise] (...). The unity that actually has happened between us. That we have now, after these 12 weeks, begun to talk about many other things to each other than when we started up, where we were more cautious (...). It seems to me, that it [the floorball] has given us something good. You could say it's a better unity that has come out of it” .It appears that the men established new social relations, and hence has increased their social capital, during the floorball intervention. The social environment established beyond the training contributes to the team spirit on the team, and it seems that the group of floorball players, because of their “membership” on the team and development of social networks, share a set of values, norms and certain practices in the training setting. Many of the interviewees appreciated that some of the most engaged team members took initiative for a Christmas dinner, for example, which added to the feeling of “being part of something” and belonging to the group.Consequently, the floorball training was an important source of social capital for the men. It was by many of the interviewees highlighted as an anchor contributing to a meaningful everyday life and several of them compared the team with the community of colleagues before their retirement:“The difference from being retired and being at work is that you are not part of a community anymore. I could feel that going to the training had a positive effect similar to going to work because then there were three days a week when I returned to the same group” .This “anchor” seemed to exist because of the combination of the enjoyable exercise and the social networks that had become crucial to the players' continued participation throughout the 12 weeks.When the interviewees were asked if these networks had grown into personal relations with meeting outside of the weekly training sessions, the majority of the interviewees expressed that the friendships were restricted to the training sessions:“They are surprisingly sweet and lovely guys all together. For the time being, there is no one I would like to meet with privately, but I am looking forward to being with them every time. They are damn nice and positive, and we are in the same boat with respect to age and weight class many of us, so I think that's fine” .Taken together, it seems that being part of the floorball group created social capital despite differences in habitus, but through the similarities in gender and age, as well as through the enjoyment of and interaction during the floorball activities. It also seems that this social capital is closely tied to the floorball context.4.In the present study, the two purposes were to (1) investigate the differences between floorball and petanque in effect on health status and psychological health in untrained men aged 65 or older; and (2) investigate possible reasons for the differences. Overall, we found that (1) floorball, compared to petanque, produced desirable effects for health status and psychological health; and (2) the homogeneous group of men above age 65 created social relationships on and off the floorball pitch, which was expressed as a key factor for the men's positive experiences in the 12-week floorball intervention.In the SF-12 mental health composite score, the floorball group improved to a larger extent than the petanque group. It seems that the combination of social interaction and physical activity is more beneficial to mental health than social interaction only. This is not surprising, as research suggest that physical activity is beneficial to mental health However, it is also possible that the nature of the game floorball yields higher quantity or quality of social interaction between the men, and this is what contributed to the better development in psychological health. Nielsen et al. Turning to the interview findings, it seems that it was important for the men that they had common background and shared the fact that they are retired. Scholars have argued that it is especially the transition from working life to retirement that is vital in terms of promoting a physical active lifestyle Some interviewees expressed that the floorball training was an “anchor” that fill in a gap they have experienced as retiree, as the training create a daily routine similar to work-life and a social community comparable with having colleagues. In line with this, Barnett, Guell, and Ogilvie From a Bourdieusian perspective, interacting with one another in itself may not purely be positive. It is the fact that one experiences a feeling of group membership In addition, the interactive and inclusive nature of floorball contributed to the development of social capital, a result similar to the findings of Nielsen et al. On another note, it is crucial to remember that a “dark side” to social capital and social relations may also exist, where individuals who are not part of a group feel excluded from certain norms and values that are practiced in a particular setting Lastly, another cause of adherence is enjoyment. According to Nielsen et al. All in all, it seems that floorball is an activity well suited to older men for a number of reasons: (1) it increases physiological health and functional capacity 4.1.This study has a few limitations that should be mentioned. First, the two participant groups were quite small, making our results prone to random error, and to type II errors. However, since our quantitative results and qualitative findings are in congruence, we consider our conclusions to be valid. Second, although floorball and petanque are easy to compare in terms of intensity level, since petanque is an activity with a negligible level of physical activity, the difference is not as clear in terms of social interaction. Petanque also includes social interaction, making it harder to attribute the better development in the floorball group to the amount of social interaction. Furthermore, we argue that there are more social interactions during floorball, but do not substantiate this claim. Again, leaning on earlier research, it seems fair to speculate that the amount of social interactions is higher in floorball because of the amount of physical “interaction” that exists on the pitch as an inherent aspect of team games. Third, we chose to only perform interviews with the floorball players because the team dynamic and group cohesion of floorball were an interest of ours, and hence, we have no grounds for comparing experiences of the two groups. However, we believe that the interview findings can shed some light on why the floorball group developed better than the petanque group in the questionnaire variables. Fourth, no follow-up data collection has been conducted, giving us no possibility to investigate if our findings change over time, or if there is a group difference in adherence over time.4.2.The findings in this study have several implications for future physical activity interventions whose aim is long-term commitment to physical activity for older men. Floorball appears to be a suitable activity for older men, as it seems beneficial for both physical and psychological health, and seems motivating for older men, a target group that in Denmark has trouble with participation in physical activity. First it is important to remember that older men prefer a homogenous group in terms of gender, age, and point in life to ensure a fertile ground for development of social capital. Second, it is advisable to introduce activities that are based on playful team activities such as ball games


Lymphatic filariasis (LF), a neglected tropical disease (NTD) preventable through mass drug administration (MDA), is one of six diseases deemed possibly eradicable. Previously we developed one LF elimination scenario, which assumes MDA scale-up to continue in all countries that have previously undertaken MDA. In contrast, our three previously developed eradication scenarios assume all LF endemic countries will undertake MDA at an average (eradication I), fast (eradication II), or instantaneous (eradication III) rate of scale-up. In this analysis we use a micro-costing model to project the financial and economic costs of each of these scenarios in order to provide evidence to decision makers about the investment required to eliminate and eradicate LF.Costing was undertaken from a health system perspective, with all results expressed in 2012 US dollars (USD). A discount rate of 3% was applied to calculate the net present value of future costs. Prospective NTD budgets from LF endemic countries were reviewed to preliminarily determine activities and resources necessary to undertake a program to eliminate LF at a country level. In consultation with LF program experts, activities and resources were further reviewed and a refined list of activities and necessary resources, along with their associated quantities and costs, were determined and grouped into the following activities: advocacy and communication, capacity strengthening, coordination and strengthening partnerships, data management, ongoing surveillance, monitoring and supervision, drug delivery, and administration. The costs of mapping and undertaking transmission assessment surveys and the value of donated drugs and volunteer time were also accounted for. Using previously developed scenarios and deterministic estimates of MDA duration, the financial and economic costs of interrupting LF transmission under varying rates of MDA scale-up were then modelled using a micro-costing approach. The elimination scenario, which includes countries that previously undertook MDA, is estimated to cost 929 million USD . Proceeding to eradication is anticipated to require a higher financial investment, estimated at 1.24 billion USD (1.17bn-1.30bn) in the eradication III scenario , with eradication II  projected at 1.27 billion USD (1.21bn-1.33bn), and eradication I  estimated at 1.29 billion USD (1.23bn-1.34bn). The economic costs of the eradication III scenario are estimated at approximately 7.57 billion USD (7.12bn-7.94bn), while the elimination scenario is projected to have an economic cost of 5.21 billion USD (4.91bn-5.45bn). Countries in the AFRO region will require the greatest investment to reach elimination or eradication, but also stand to gain the most in cost savings. Across all scenarios, capacity strengthening and advocacy and communication represent the greatest financial costs, whereas mapping, post-MDA surveillance, and administration comprise the least.Though challenging to implement, our results indicate that financial and economic savings are greatest under the eradication III scenario. Thus, if eradication for LF is the objective, accelerated scale-up is projected to be the best investment. Lymphatic filariasis (LF) is a neglected tropical disease (NTD) that is targeted for elimination and is thought to be potentially eradicable through once yearly mass drug administration (MDA) using drugs that are currently donated to LF endemic countries by the pharmaceutical companies that manufacture them. MDA has proven to be a cost-effective and efficient method of disease control, both for LF and other NTDs. Previously, we developed scale-up scenarios of varying magnitude to reach elimination  and eradication  and estimated the number of associated treatments that would be necessary in each country under each scenario. Here we project the costs—both financial and economic—of each of these scenarios. We use data from a myriad of sources to estimate the cost of various activities, and found that training and advocacy and communication activities comprise the bulk of the costs of the program. Among all scenarios, elimination requires the least total investment. However, in terms of LF eradication, faster rates of MDA scale-up are associated with decreased overall costs. Neglected tropical diseases (NTDs) are a heterogeneous group of helminthic, bacterial, viral, fungal and protozoan infections that cause chronic and debilitating disability . HoweverLymphatic filariasis (LF), an NTD, can result in irreversible disability, most often manifested as elephantiasis, lymphedema, and hydrocele . With moHowever, while LF can result in profound morbidity, it is inefficiently transmitted, with an estimated 15,500 infective mosquito bites thought necessary to generate one transmittable infection . LF is aSuccessfully eradicating a disease has innumerable long-term health benefits, and is also a classic example of a global public good –16. EradWe previously developed scenarios to reach elimination (elimination of LF transmission in countries that have previously undertaken MDA) and eradication  of LF, taking into account previous progress made under the GPELF, pre-intervention prevalence levels, and possible delays in program implementation. The elimination scenario maintains the current geographic expansion and rate of MDA scale-up as seen under the GPELF, and thus serves as the comparator scenario. The eradication scenarios were developed to assess the impact of expanding MDA to all endemic countries at an average rate of MDA scale-up (eradication I), intensifying efforts against LF (eradication II), and treating all endemic populations immediately (eradication III). Key components inherent in each scenario are outlined in Assumptions about L. loa endemic areas), we then determined the number of future treatments needed for each country under each scenario, accounting for the number of people at-risk, country-specific growth rates, duration of MDA necessary, historical rates of scale-up, and previous progress towards local elimination. By assuming that the populations at-risk for LF increase exponentially with population growth rates, scenarios with longer durations were also assumed to require more treatments  has been completed in each country under consideration. All results are listed in 2012 US dollars (USD) and future costs were discounted at 3%. One-way sensitivity on discount rates, variability rates, and probabilistic sensitivity analyses for all costing and quantity parameters were also explored. Two-way sensitivity analyses were also employed to explore the level of interdependence between key parameters and activities.NTD Master Plan Costing Tool in the African Region for Benin, Cameroon, Democratic Republic of Congo, Eritrea, Guinea, Madagascar, Niger, Senegal, and Sierra Leone ranging from January 2011 to April 2012 were reviewed to preliminarily determine essential activities and associated resources necessary for a country to successfully undertake a program to eliminate lymphatic filariasis (PELF). Subsequently, in consultation with key LF implementers from the PELF in Uganda, which has successfully been carrying out the GPELF MDA strategy since 2002 [Line items from USAID’s nce 2002 , all actnce 2002 .Retail prices from established vendors were used for tradable goods, including laboratory supplies and capital items. The WHO CHOICE database was used for unit costs which were unable to be determined elsewhere. While the percentage of time needed for LF personnel was informed by the Ugandan program, the country-specific salary estimates were taken from the WHO CHOICE database for the NTD director , LF program manager (1.0 FTE), administrative assistant (0.5 FTE), finance officer (0.3 FTE), data manager (0.3 FTE), and supplies manager (0.5 FTE). As the African Program for Onchocercaisis Control (APOC) uses a strategy similar to that employed by the GPELF , line itL. loa prevalence, as well as the added cost of biannual MDA in hyper loaisis areas were also accounted for.We took into account the cost of advocacy and communication; capacity strengthening; coordination and strengthening partnerships; mapping; data management; administration; ongoing surveillance; monitoring, evaluation, and supervision (M&E); drug delivery; and post-MDA transmission assessment surveys (TAS). As described below, the costs of increased surveillance in areas with meso Advocacy and communication was assumed to include the development and distribution of educational messages, as well as community meetings and sensitization activities with district and community leaders, sub-county and parish supervisors, and community drug distributors (CDDs). Capacity strengthening comprised trainings on MDA procedures for national trainers, district trainer of trainers, sub-county and parish supervisors, community leaders, CDDs and teachers. Trainings for monitoring sentinel and spot check sites as well as trainings for M&E officers were also considered under capacity strengthening. Conference attendance and international exchanges, cross-border meetings for regional strategies towards controlling NTDs, NTD secretariat meetings, and technical committee meetings were assumed under coordination and strengthening partnerships. Data management included all activities involved with the acquisition and distribution of MDA data, including cleaning, entering, and analyzing. The maintenance of sentinel sites—including equipment for administering microfilaria (mf) surveys and internal quality control tests—as well as the administration of sentinel and control site impact assessment surveys, associated data collection, and survey feedback meetings were grouped under ongoing surveillance. M&E included the supervision of MDA activities, monitoring for severe adverse events (SAEs), and regular feedback meetings at the district and national level. Drug delivery involved drug transport from the central stores to district stores and then onward to parish supervisors. Supplies to CDDs, including t-shirts and stationery, were also accounted for under drug delivery. Administration included overhead costs, the maintenance of office space, salaries to LF staff, and the procurement of necessary equipment.Determination of resource quantities for assumptions about district size) after the final estimated round of MDA (as determined through earlier modeling exercises) and twice thereafter at three year intervals. The number of TAS conducted has thus been assumed to vary by the number of districts achieving the specified number of MDA rounds, though the quantities of resources required for each individual TAS was assumed to remain constant.Mapping and TAS were assumed to include a preliminary visit, immunochromatographic card test (ICT) testing, data collection, and feedback meetings. The cost of mapping was included for any country that, as of 2012, had not yet completed mapping nor started MDA. In order to determine the costs of post-MDA surveillance on a global level, all TAS have been assumed to be school-based. Moreover, TAS have been assumed to occur in each district (see L. loa microfilaria per milliliter of blood are put at unacceptably high risk of developing severe adverse events (SAEs) if administered ivermectin or diethylcarbamazine citrate (DEC) [L. loa prevalence within a community has been shown to have a close correlation with individual L. loa mf density, provisional GPELF guidelines recommend communities endemic with LF that also have a L. loa prevalence greater than 40% be treated with bi-annual MDA using albendazole monotherapy coupled with vector control [Previous studies indicate that individuals harboring more than 30,000 te (DEC) –26. As L control .L. loa, however, are not yet complete. While we recognize that not all populations at-risk for L. loa are also at-risk for LF, for the purposes of this study, we make the assumption that the percentage of mapped areas from RAPLOA studies that were found to have 20–39.9% (meso) or >40% (hyper) L. loa prevalence corresponds directly to the percentage of the population in these countries also at risk for LF [L. loa, once yearly albendazole and ivermectin is still presumed. In areas of both hyper and meso L. loa, the costs associated with monitoring for SAEs are assumed to increase two-fold.Mapping studies to determine areas of co-endemicity between LF and k for LF , 28. In Timeframe and number of treatments required). In the baseline analysis, we have assumed that the number of resources required to carry out the PELF for a certain population in Uganda is relatively similar to the number of resources required to carry out the program for a population of similar size in other LF endemic countries. As MDA in Uganda is implemented at a community level, the amount of resources and duration of activities required to successfully complete the PELF in Uganda are generally organized by district, sub-county, and village units. In order to standardize the at-risk population falling into the different administrative levels  across all LF endemic countries, the average number of people at-risk for LF in each district, sub-county, and village were determined for Uganda and then assumed for all LF-endemic countries.In line with the approach for assessing necessary activities, quantities and duration of use for each required component were established through consultation with key members from the Ugandan PELF team. Aside from program activities with inherently fixed costs—such as the creation and dissemination of health messages, coordination and strengthening of partnerships, and administrative costs—budgeted line items were assumed to vary linearly by the size of the population to be treated  involving all financial unit costs and quantities assuming 10% uncertainty and gamma distributions for all parameters in order to avoid negative values . As the The total financial investment to implement the elimination scenario is projected at 929 million USD (891m-965m). To expand the campaign to all endemic countries at an average rate of scale-up (eradication I scenario) would require 1.29 billion USD (1.24bn-1.34bn), an increase of about 360 million USD (346m-374m) over the elimination scenario . The decProviding MDA to the entire at-risk population immediately, as assumed under the eradication III scenario, requires a significant initial investment, but within 10 years’ time, the annual cost of implementing the scenario becomes less than the alternatives . The shaThe average unit financial cost for undertaking each of the scenarios ranges from 0.27 USD in the elimination scenario to 0.31 USD in both the eradication II and III scenarios. However, as the scenarios progress, the unit costs increase substantially. This is due to the fact that the number of people to be treated (the denominator of the estimate) decreases, though the cost associated with some of the core activities—including coordination and strengthening partnerships, administration, and data management—are assumed to remain relatively constant. As an example of this, Capacity strengthening proves to be the most costly activity, representing between 53–55% of the overall financial costs, while advocacy and communication (22–24%); ongoing surveillance (6%); monitoring, evaluation, and supervision (8%); and drug delivery (9%) account for most of the remaining costs .L. loa coendemicity in the Democratic Republic of Congo indicates that areas of meso L. loa are anticipated to only result in an increase in monitoring and evaluation, thereby having little effect on the overall costs. In comparison to a population of comparable size without L. loa, hyper L. loa endemicity is associated with an increase of approximately 15% in the overall costs of the program due to the increase in data management, drug delivery, and monitoring and evaluation activities.Our assessment of the financial costs of treating a population of 1 million at-risk for LF with and without When the economic costs are considered, the costs of all scenarios are substantially higher (5.2 billion USD for the elimination scenario). Extending the coverage to all endemic countries is estimated to require around 7.9 billion USD (7.5bn–8.0bn), or 45% more than under the elimination scenario . DependiThis study used a micro-costing approach to provide estimates on the financial and economic investments required to eliminate and eradicate lymphatic filariasis. Across the eradication scenarios, faster rates of MDA scale-up are associated with decreased costs (1.2 billion USD in the eradication III scenario versus 1.3 billion USD in the eradication I scenario). The projected economic costs of eradication range from 7.5 billion to 8.0 billion USD, about half of which is due to the value of the donated drugs. These results serve to further highlight both the importance of higher rates of MDA scale-up as well as the crucial partnership between the GPELF and the drug donation programs. These projections may also be important in order to convince pharmaceutical companies to continue donating the drugs necessary to eliminate LF. While the cost to eliminate LF is less than that to eradicate, it must be recognized that deciding to pursue elimination rather than eradication signifies the continuation of LF-related costs indefinitely, and comes at a health burden to populations that remain untreated.With a dearth of evidence on the costs of implementing morbidity management programs , and givOur cost estimates for reaching LF elimination and eradication inherently assume that the GPELF strategy, if carried out sufficiently, will lead to the interruption of LF transmission in all areas. In our analysis we have also assumed that accelerating the rate of MDA scale-up will result in decreased marginal costs  by assuming there to be spare capacity among fixed costs . However, in some areas, additional capital may be required and, therefore, marginal costs would increase. In such a case, accelerating eradication might not be the most cost-saving option in the short term.Additionally, our analysis does not take into account the cost for certifying elimination on a country level nor the activities involved in globally assessing whether eradication has been achieved. As currently experienced by the Global Polio Eradication Initiative, the costs for finding and ascertaining the last cases to reach eradication are substantial , 41. WhiOur analysis found a 15% increase in financial costs to treat an area of hyper L. loa endemicity in the DRC. This estimate does not take into account increases in advocacy and communication in these areas, which may be necessary to achieve the targeted levels of coverage. Further, the costs for medical transport and additional medications that might be needed to treat patients suffering from severe adverse events have not been incorporated in this analysis. Costing studies for such post-MDA response activities have not previously been carried out, though such costs are likely to vary by the incidence of SAEs, the geographic location, and the intensity of response required. If substantial and significant response is required in many areas, implementing the eradication scenarios would certainly result in higher costs than projected in this baseline study.A number of methodological uncertainties in our study must be mentioned. Country-specific cost data was mostly unavailable and, consequently, was largely imputed from Ugandan data. By extrapolating cost data across countries and regions, we inherently made the assumption that each LF endemic country implements the GPELF strategy as in Uganda . Moreover, we assumed that the number of resources required to carry out the PELF for a certain population in Uganda remains relatively constant (varying by +/-10%) both across time and across countries. As described above, the variation assumed in our model was included probabilistically in both unit costs and quantities.We chose to have modest uncertainty assumptions, assuming a variation of 10% probabilistically for both costs and quantity estimates. Though 10% variation has a large impact on the individual unit costs, the impact on total costs was less than expected. As such, we chose to increase the variability to 30% during additional uncertainty analyses. However, this resulted in only a modest increase in the uncertainty intervals see . It is, Additionally, our MDA round estimates are considerably longer than the 4–6 years currently specified by the GPELF, instead ranging from 6 rounds of MDA in areas where Anopheles spp are the primary vector, treatment is through DEC+ALB, and the baseline MF prevalence is 5–10%, to 15 rounds in areas where Culex spp are the primary vector, IVM+ALB is the treatment provided and the baseline mf prevalence is 15–20% , 45. TheIdeally, LF-specific expenditure data would have been collected in all 72 LF-endemic countries. Having such data would have also allowed for an important assessment on the variance of costs from the beginning stages of implementation towards the ‘end game’ of reaching surveillance. However, 14 of these endemic countries have never carried out MDA for LF . MoreoveWe conducted a probabilistic sensitivity analysis in order to overcome some of the limitations inherent in our costing parameters. In order to provide more robust estimates on the costs of achieving elimination and eradication, costing studies in areas with the highest burden of LF could be undertaken in order to reduce the level of uncertainty in costs. While improved cost data could be used to inform policy and improve planning, the cost of acquiring additional data should be weighed against the value of such data . An addiOur findings on the costs to eliminate and eradicate LF represent very achievable investments. Our cost estimates would have likely been even lower, though, if we assumed some level of integration or cost-sharing between other disease initiatives. Many countries have, in fact, integrated similar activities across vertical programs, including onchocerciasis, trachoma, and schistosomiasis, and others have paired drug delivery for MDA with other community distribution campaigns, including insecticide treated nets (ITNs) for malaria and ongoing vaccine programs. In so doing, the overall costs per program, indeed, generally decreased and efficiency reportedly improved , 48–51. l. loa endemic areas, previous studies have indicated that including vector control could decrease the number of MDA rounds needed to interrupt transmission, thereby reducing the overall duration of the program [We further did not include the cost of vector control in this costing study, assuming instead the cost to be covered by other disease control programs . While vector control is not a core strategy of the GPELF in non  program , and potA comparison of our costs against other costs is important for validation, though challenging due to differing methodologies. Though not inclusive of overhead costs, a study from two states in Nigeria found the cost associated with conducting MDA for the prevention of LF to be between 0.02 USD and 0.12 USD per treatment delivered . A multiKnowing the global costs of the program will help decision makers assess the feasibility and rationale of investing in LF eradication, while simultaneously helping to facilitate planning and the development of strategies and policies. However, successfully eradicating LF depends on more than the monetary investment. Political will, continued community ownership, and the feasibility of the campaign all need to be taken into account . HoweverS1 File(DOCX)Click here for additional data file.S1 Fig(DOC)Click here for additional data file.S2 Fig(DOC)Click here for additional data file.S3 Fig(DOC)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S5 Table(DOC)Click here for additional data file.


Zika virus disease is caused by infection with a flavivirus with broad geographic distribution and is most frequently transmitted by the bite of an infected mosquito. The disease was first identified in the World Health Organization’s Region of the Americas in 2015 and was followed by a surge in reported cases of congenital microcephaly in Brazil; Zika virus disease rapidly spread to the rest of the region and the Caribbean  conducted active investigations and collected serum specimens from 19 patients with suspected Zika virus disease during October 2015–January 2016. The National LaboratoryStandard case definitionsEpidemiologic data and laboratory testing results for persons with suspected Zika virus disease and GBS in Haiti presented in this report were obtained from this surveillance system. MSPP personnel were interviewed to describe the Zika virus disease module and response.Among 3,036 suspected cases of Zika virus disease  with suspected congenital microcephaly, and cases of GBS), the highest number originated from Ouest , Nord (583), and Centre (421) departments. Two peaks occurred in 2016 between epidemiologic weeks 5–7 and weeks 21–27 . CommuneAmong 294 (9.7%) patients with suspected Zika virus disease, congenital microcephaly, or GBS who underwent testing, 19 cases (6.5%) were confirmed by RT-PCR from serum specimens . Two cerebrospinal fluid specimens and eight urine specimens from other patients tested negative. The median age of patients with confirmed infection was 34 years  and 44.4% were male. Nine of the confirmed cases were investigated; among these, eight patients reported symptoms including nonpurulent conjunctivitis (seven cases); maculopapular rash (six); temperature >37.2°C (99.0°F) (four); arthritis (three); headache (three); general pain (three); and digestive pain (one). A male fetus (24 weeks’ gestation) had spinal and limb malformations. Blood and urine specimens from the mother were negative, but the umbilical cord blood specimen was positive for Zika virus RNA by RT-PCR.DELR completed 148 case investigations; the highest percentage of investigations (90.9%) occurred among pregnant women . The mosTwenty-nine suspected cases of Zika virus–associated congenital microcephaly . On February 24, 2016, two of the 18 women were confirmed to be infected with Zika virus by RT-PCR; they reported rashes, nonpurulent conjunctivitis, and mosquitoes present at home and work.Thirteen suspected cases of Zika-associated GBS were detected, and 11 patients were hospitalized. The median age was 31 years . Zika-related symptoms included elevated temperature (nine patients) and headache (five). Seven of the 13 GBS cases occurred in females. Among 11 serum specimens submitted to the National Laboratory for testing, two were rejected as inadequate on arrival at the lab; the remaining nine tested negative by RT-PCR. Specimens from these patients might have tested positive for Zika virus by serology and plaque reduction neutralization test, both testing methods that are unavailable in Haiti.MSPP implemented a response plan that included epidemiologic monitoring of Zika virus disease and complications, laboratory testing, vector control, social mobilization, and clinical care. MSPP released statements about modes of transmission, testing, strategies to prevent mosquito bites, and potential Zika virus disease complications through their website, fliers, and radio announcements  were positive by reverse transcription–polymerase chain reaction testing.Improving reporting of cases of suspected Zika virus disease to public health authorities by health care workers in Haiti and by providers evaluating patients with recent travel to Haiti is important to ongoing surveillance initiatives. Trained epidemiologists and health care workers are important in ensuring reporting. Although laboratory-confirmed Zika virus disease cases were detected in only three of Haiti’s 10 departments (geographic administrative units), testing should continue throughout the country according to Haiti’s guidelines. Integrated mosquito control strategies can mitigate disease spread in Haiti. Residents of and visitors to Haiti should follow recommended precautions to protect against Zika virus infection.


Cicer arietinum L. (chickpea) is the world's fourth most widely grown pulse. Chickpea seeds are a primary source of dietary protein for humans, and chickpea cultivation contributes to biological nitrogen fixation in the soil, given its symbiotic relationship with rhizobia. Therefore, chickpea cultivation plays a pivotal role in innovative sustainable models of agro-ecosystems inserted in crop rotation in arid and semi-arid environments for soil improvement and the reduction of chemical inputs. Indeed, the arid and semi-arid tropical zones of Africa and Asia have been primary areas of cultivation and diversification. Yet, nowadays, chickpea is gaining prominence in Canada, Australia, and South America where it constitutes a main ingredient in vegetarian and vegan diets. Viruses and plant parasitic nematodes (PPNs) have been considered to be of minor and local impact in primary areas of cultivation. However, the introduction of chickpea in new environments exposes the crop to these biotic stresses, compromising its yields. The adoption of high-throughput genomic technologies, including genome and transcriptome sequencing projects by the chickpea research community, has provided major insights into genome evolution as well as genomic architecture and domestication. This review summarizes the major viruses and PPNs that affect chickpea cultivation worldwide. We also present an overview of the current state of chickpea genomics. Accordingly, we explore the opportunities that genomics, post-genomics and novel editing biotechnologies are offering in order to understand chickpea diseases and stress tolerance and to design innovative control strategies. In many developing countries, grain legumes have gained much importance in view of acute shortages in the production of animal proteins and the prevalence of protein malnutrition. Conversely, they are a valid alternative as a source of protein for specific (vegetarian or vegan) or balanced diets worldwide, particularly in developed countries.6 tons, grain legumes  rank third after cereals and oilseeds   and kabuli-type (large-seeded). Desi-type accounts for about 85% of the world's production and is mainly grown in India, Pakistan, Iran, Afghanistan, and Ethiopia. kabuli-type, instead, is grown in the Middle East, India, Mexico, North and South America, Australia, Spain, and Italy. A third type is characterized by a medium-to-small size and cream-colored seed, and it is designated as “pea-shaped”  , Cucumber mosaic virus , Bean leafroll virus (BLRV) and Beet western yellows virus (BWYV) , Pea enation mosaic virus complex  and , Chickpea stunt disease-associated virus , and a number of geminiviruses of the genus Mastrevirus, the most important being Chickpea chlorotic dwarf virus (CpCDV). Faba bean necrotic yellows virus  has also been reported  Table  is suppohttps://cropgenebank.sgrp.cgiar.org/index.php/management-mainmenu-433/stogs-mainmenu-238/chickpea/guidelines/viruses). They are: Pea seed-borne mosaic virus, Bean yellow mosaic virus , AMV and CMV  approaches have opened the door to reconstructing viral populations in a high-throughput and cost-effective manner. Nowadays, NGS can be employed in environmental studies in the agro-ecosystem to either analyze known plant viruses by means of a reference-guided approach or to discover novel plant viruses using a C. arietinum L. based on short (s)RNA analysis have been carried out in Apulia, Southern Italy, during the 2013–2017 time period. The surveys revealed that a large number of known viral species co-infect chickpea plants without causing any symptoms. Surprisingly, among the viruses found were Tomato mottle mosaic virus , which had not yet been observed in chickpea or reported in Europe, and one viroid referring to Hop stunt viroid   was found, though never reported before in chickpea , reniform nematodes (Rotylenchus reniformis), cyst-forming nematodes (CNs) (Heterodera spp.) and root-knot nematodes (RKNs) (Meloidogyne spp.) have been found pathogenic for chickpea and studies were carried out to characterized chickpea-nematode interactions, to describe geographical distributions, general symptoms even at histopathological levels  Chitwood; four promising nematode-tolerant genotypes were found and conserved in the chickpea ICRISAT genebank  RNAs of the miR2118/482 superfamily members has been associated with nodulation events (plant-rhizobium interactions) and not to better specified plant pathogen defense strategies . PAMPs are evolutionarily conserved molecules across kingdoms; in plants they carry out critical functions against several microbial attacks   Figure . By contH. glycines interactions. Wu et al. , confers resistance to CNs  to enhance chickpea resistance to pests and other biotic and abiotic stress, and (ii) to achieve more sustainable food production in developing countries, such as in the semi-arid tropics where agrochemicals are inaccessible to low-income farmers , aphids (Aphis craccivora), bruchids, fungal diseases (Fusarium oxysporium/F. udum), and abiotic  stress, as well as the nutritional quality by increasing the sulfur-containing amino acid content. The first transformation studies with chickpea were performed by Srinivasan et al.  and insecticidal cry1Ac. Fontana and colleagues   cases of transformation efficiency higher that 25% have been reported -associated protein 9 (Cas9) DNA editing system has recently been developed as a new method for genome engineering. It is based on the type II CRISPR-associated immune system that protects bacteria against invading DNA viruses and/or plasmids  cloning of resistance genes from natural resources and transfer to other plant species; (ii) overexpression of different protease inhibitors; and (iii) suppression of nematode effectors in plants using RNAi (Ali et al., All the authors have contributed to the review with their proper specific expertise in plant science, plant pathology, and plant biotechnology.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


On November 28, 2016, the Texas Department of State Health Services (Texas DSHS) reported its first confirmed case of local mosquitoborne Zika virus transmission in the city of Brownsville, located in south Texas along the U.S.-Mexico border. Zika virus infection during pregnancy has been linked to adverse congenital outcomes including microcephaly, neural tube defects, early brain malformations, structural eye abnormalities, congenital deafness, and limb contractures  and laboratory test results is collected and reviewed by the Texas DSHS Zoonosis Control Branch as a part of routine arboviral disease surveillance. Each week, the Zoonosis Control Branch shares a line list of pregnant women with laboratory evidence of possible Zika virus infection and their reported pregnancy outcomes with the Texas DSHS Birth Defects Epidemiology and Surveillance Branch. Among possible cases with no reported pregnancy outcome, recent birth certificate data are searched for the reported pregnant woman’s name and birth date to determine whether a live birth has occurred. Birth Defects Epidemiology and Surveillance Branch staff members review neonatal medical records of all babies born to women with possible Zika virus infection during pregnancy to identify all documented birth defects.During January 1, 2016–July 31, 2017, a total of 219 pregnant women with laboratory evidence of possible recent Zika virus infection were identified in Texas, including 49 (22%) with laboratory-confirmed Zika virus infection (Table). One woman was infected in Texas; all others were exposed outside the United States and its territories. Among the 219 pregnancies, outcomes were recorded for 185 (84%), including 182 live-born infants and three pregnancy losses that occurred at any time during gestation. Among the remaining 34 pregnant women, 20 have an estimated due date which has not yet passed, four have an estimated due date which has passed but no pregnancy outcomes have been reported, and for 10, there was no reported estimated due date. All recorded completed pregnancies were reviewed by the Zoonosis Control and Birth Defects Epidemiology and Surveillance Branches to ascertain Zika virus testing status and to identify any birth defects. Zika virus testing was completed for 80 (43%) of the 185 infants or fetuses, and Zika virus–associated birth defects were documented in 15 (8%) pregnancies , including six (17%) of the 36 infants or fetal losses delivered by women with laboratory-confirmed Zika virus infection. Ten infants or fetuses had microcephaly; five of those with microcephaly had additional birth defects, including holoprosencephaly, hydranencephaly, craniosynostosis, and clubfeet. Zika virus–associated birth defects identified in the remaining three infants included holoprosencephaly, cataracts, and ventral pons hypoplasia.Zika virus testing was not completed for 105 (57%) infants or fetuses; including three pregnancy losses and 10 live-born infants for whom only a placental or cord blood specimen was tested. In the absence of other evidence, testing of cord blood is insufficient to determine an infant’s infection status (The occurrence of travel-related Zika virus infections, combined with the threat of local transmission in Texas, indicates a need for continued surveillance for birth defects associated with Zika virus infection. This analysis found that only 43% of identified infants or fetuses for whom testing was indicated received testing. Efforts to increase the frequency of collecting and testing of specimens from infants born to mothers with laboratory evidence of possible recent Zika virus infection are needed. Physicians caring for newborn infants need to be aware of the Zika testing status of the mother, particularly in geographic locations with high potential for local mosquitoborne transmission. Serum specimens are strongly preferred to placenta or cord blood specimens for infant testing, and should be collected soon after birth (


Ipomoviruses that cause cassava brown streak disease (CBSD); Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), with up to 100% yield loss for smallholder farmers in the region. To date, vector studies have not resulted in reproducible and highly efficient transmission of CBSV and UCBSV. Most virus transmission studies have used Bemisia tabaci (whitefly), but a maximum of 41% U/CBSV transmission efficiency has been documented for this vector. With the advent of next generation sequencing, researchers are generating whole genome sequences for both CBSV and UCBSV from throughout eastern Africa. Our initial goal for this study was to characterize U/CBSV whole genomes from CBSD symptomatic cassava plants sampled in Kenya. We have generated 8 new whole genomes (3 CBSV and 5 UCBSV) from Kenya, and in the process of analyzing these genomes together with 26 previously published sequences, we uncovered the aphid transmission associated DAG motif within coat protein genes of all CBSV whole genomes at amino acid positions 52–54, but not in UCBSV. Upon further investigation, the DAG motif was also found at the same positions in two other Ipomoviruses: Squash vein yellowing virus (SqVYV), Coccinia mottle virus (CocMoV). Until this study, the highly-conserved DAG motif, which is associated with aphid transmission was only noticed once, in SqVYV but discounted as being of minimal importance. This study represents the first comprehensive look at Ipomovirus genomes to determine the extent of DAG motif presence and significance for vector relations. The presence of this motif suggests that aphids could potentially be a vector of CBSV, SqVYV and CocMov. Further transmission and ipomoviral protein evolutionary studies are needed to confirm this hypothesis.Cassava is the main staple food for over 800 million people globally. Its production in eastern Africa is being constrained by two devastating  Most plant-infecting viruses are transmitted to their host plants by vectors. This interaction between vector and virus varies in duration and specificity, but some common themes have emerged. Firstly, plant viruses encode proteins on the surface of the virion that are essential for transmission and in some cases additional non-structural helper proteins act to bridge the virion to the vector binding site. For example, current evidence suggests that successful transmission of Potyviruses by their aphid vectors depends upon the interaction of two viral proteins; the coat protein (CP) and helper component proteinase–HC-Pro , 2. PotyCucumber mosaic virus, distinct CP motifs are responsible for transmission by different aphid species. The amino acids in position 25, 129, 162, 168, and 214 of CMV CP are critical for efficient transmission by Myzus persicae. In contrast, amino acids in position 129, 162, and 168 are critical for efficient transmission by Aphis gossypii , 0.15 μl Taq DNA polymerase and 1.0 μl of cDNA. The PCR conditions were: 94°C for2 min followed by 35cycles of 94°C (30s), 51°C (30s) and 72°C (30s) for denaturation, annealing and extension, respectively. PCR products were analysed by gel electrophoresis in a x1 TAE buffer on a 1.5% agarose gel, stained with ethidium bromide, visualized under UV light and gel picture documented.The CTAB method  was usedEight (8) samples were transported to the laboratory and extracted as detailed above. Total RNA was blotted on to FTA cards and later extracted using methods previously described . Total Rde novo assembly function of CLCGW with automatic word size, automatic bubble size, minimum contig length 500, mismatch cost two, insertion cost three, deletion cost three, length fraction 0.5 and similarity fraction 0.9. Contigs were sorted by length and the longest subjected to a BLAST search (blastn and blastx). In addition, reads were also imported into Geneious 6.1.6 [For each sample, reads were first trimmed using CLC Genomics Workbench 7.5 (CLCGW) with the quality scores limit set to 0.01, maximum number of ambiguities to two and removing any reads with <30 nucleotides (nt). Contigs were assembled using the us 6.1.6  and provus 6.1.6 . Open reSweet potato mild mottle virus (SPMMV) (GenBank reference Z73124), Eggplant mild leaf mottle virus (EMLMV) (GenBank HQ840786) and Tomato mild mottle virus (ToMMov) (GenBank HE600072) were also analyzed for the presence of a DAG motif in the coat protein.Twenty-six whole genomes (12 CBSV and 14 UCBSV) were downloaded from GenBank and imported into Geneious , and theIndividual gene trees were estimated using MrBayes 3.2.1  and run After observing the DAG motif in the CBSV whole genome data, all sequences found in GenBank overlapping the DAG region found in the coat protein were obtained. Megablast implemented in Geneious was carried out using the coat protein sequence for CBSV_TZ_Ser_6_KR108830 as the query sequence and all hits matching the query were downloaded and aligned using MAFFT with the default parameters.To determine if the DAG motif of CBSV is exposed to the cytosol both I-TASSER (Iterative Threading ASSEmbly Refinement)  and ProtEight samples were subjected to sequencing on an Illumina Hiseq2000, which resulted in raw reads ranging from 13,839,232 to 22,613,080 for each sample. Upon trimming for quality using CLCGW, these reads were reduced to 12,846,217 to 16,825,692 . After dCassava brown streak virus (CBSV) sequences generated in this study and those recovered from GenBank all have the DAG motif at the N-terminus of the coat protein  or NEG (1 sample-FJ039520) at the same location in the alignment (The three (3) new whole genome lignment . In addilignment . While tDeletion in CBSV P1: Five CBSV whole genome sequences have a deletion of 4 amino acids in the P1 protein. The sequences with the deletion form a monophyletic group  and CBSV_TZ_KoR6_GU563327. This clade corresponds to clade B in Ndunguru et al. [et al. [PTK and KIC motifs: CBSV has a PTK in the coat protein , while UCBSV does not. There is a KIC motif in the CBSV P1, but no KITC motif in either CBSV or UCBSV.The UCBSV genome is longer than that of CBSV and contains other unique characteristics as described here; (i) ic group , which cu et al.  and was  [et al. . The UCBCBSV_TZ_GQ329864 and CBSV_TZ_Ser_6_Kr108830. Five new UCBSV whole genomes have been added from Kenya. K4 and K5 form a monophyletic group and are placed next to two Tanzanian UCBSV genomes from Serengeti. While K12 is grouped with another Kenyan whole genome sequence (UCBSV_KE_54_FN433933), K13 and K14 are found on branches by themselves indicating new additions to the genetic diversity of the CBSVs phylogeny.Samples from Kenya were found in every clade of the CBSVs phylogenetic tree . There aCassava brown streak virus has a DAG motif in the coat protein of all published sequences, including the 3 new whole genomes added in this study, while UCBSV does not. This observation coupled with the low transmission efficiency of CBSV by B. tabaci [Myzus persicae, three reasons can be used to explain this failure; firstly, it is possible that they worked with UCBSV, which has no DAG, other than CBSV. In 1986, species of CBSVs were not known and therefore it is unknown which virus (CBSV or UCBSV) was used for the transmission. Secondly, low virus titer of CBSVs in the experimental plants may have limited aphid acquisition and subsequent virus transmission. Thirdly, in case their transmission experiments by chance used CBSV which has DAG, it is possible that location or context of the motif was responsible for lack of transmission by aphids. For potyviruses, not only is DAG located near the surface-exposed CP N-terminus and conserved among several isolates transmitted by aphids, but specific context within which it occurs is also of considerable importance [Ugandan cassava brown streak virus sequences do not have a DAG motif in the coat protein and clearly these data further support observed biological differences between UCBSV and CBSV.. tabaci , 32 stro. tabaci  failed tportance . In contIpomovirus ToMMoV was initially reported to be experimentally transmitted by aphids [B. tabaci (MEAM1 or B Biotype) in the field. This scenario is similar to the CBSV and UCBSV hypothesis where higher incidence of the disease symptoms in the field (CBSD) is often attributed to larger populations of the most abundant vector (B. tabaci) rather than the possibility of the presence of an alternative vector [The y aphids  and subsy aphids , 68, 69.e vector , 39.B. tabaci and B. afer has been sporadic and irreproducible. Maruthi [B. tabaci. Furthermore, extremely low transmission rates, inconsistency and irreproducibility in transmission rates were also observed for CBSV [Ipomoviruses, the CBSVs are not retained in the vector for more than 24 h [B. tabaci but we now know there are at least 34 species [B. tabaci was used in the transmission studies. Also, there are now more species of CBSVs than previously thought [Transmission of CBSVs by  Maruthi  reported Maruthi  failed tfor CBSV . This mahan 24 h , 37. In han 24 h , but it  species , so it i thought , 20, so  thought .B. tabaci populations to be generally low in most cassava-growing areas in Malawi compared to what is reported in East Africa. On average the highest mean whitefly populations were recorded to be 4.8 in 2009/2010, 4.0 in 2011, 15.3 in 2013 and 5.5 in 2015 in country-wide surveys carried out in Malawi (Unpublished data). Additionally, it was confirmed through sequencing of the mitochondrial cytochrome I (mtCOI) gene that the predominant whitefly species in Malawi is B. afer and not B. tabaci . In Malawi, the spread of CBSD seems to be due mainly to planting disease affected cassava stem cuttings than vector borne infections. However, there is possibility of involvement of another insect vector (such as aphids) in the spread of CBSD. A report on outreach activities to demonstrate ‘benefits of using clean/disease-free virus indexed planting materials’, indicated a susceptible cultivar Matakolembwende which started with no CBSD, to build up high disease incidence levels when planted in Karonga district, a high CBSD pressure area in northern Malawi . In this area, whitefly populations were equally low during the study as elsewhere in the country; leaving the cause of the high rate of spread of CBSD in local susceptible cultivar in great doubt. A recent paper from Kenya also confirmed a lack of correlation between whitefly numbers and CBSD incidence [Furthermore, studies in Malawi show there being no clear correlation between whitefly populations and CBSD, despite widespread occurrence of the disease. All monitoring studies carried out by the Cassava Diagnostics Project (CDP Bill and Melinda Gates Foundation Grant no. 51466) indicate whitefly ncidence Ipomoviruses. Thirdly, several publications have mislabeled sequences as CBSV and yet they are for UCBSV. For example, Dombrovsky [There are several reasons the DAG motif in CBSV has been missed. Firstly, non-overlapping genomic regions of coat protein have been amplified in most past diagnostics. The DAG motif is 52–54 amino acids from the start of the coat protein and the diagnostic primers start at position 100 and beyond; therefore, people have missed it. Secondly, CBSV DAG could have been missed due to lack of focused scrutiny for it, as all related published literature , 72–74 cmbrovsky , 75 has mbrovsky  as CBSV Myzus persicae in Pisum sativum (field pea) non-persistently transmitting Pea seed-borne mosaic virus (PsbMV). Despite no colonization ever being recorded in field pea, winged aphids are a major driver of virus infection, along with the level of seed infection present at the time of planting [Bean yellow mosaic virus infecting Lupinus angustifolius, closure of the crop canopy as soon as possible to reduce number of aphids drawn to land on bare ground have been recommended [Non-colonizer aphid species contribute more to potyvirus spread than colonizer species , 77. Stuplanting . This scommended , 82. SimIt is well known in plant virology that continuous mechanical passage of an aphid transmitted plant virus in a greenhouse situation often ends in the virus isolate losing its ability to be transmitted by aphids . In thisAphis craccivora, A. gossypii, A. nerii, Rhopalosiphum maidis, R. rufiadominalis and Schoutedenia lutea), and Lennon et al. [Myzus persicae with no transmission success. However, they could have been using UCBSV in these transmission studies and not CBSV, because knowledge and distinguishing diagnostics of the two-virus species were not available then. The transmission studies also used the indicator plants Nicotiana debneyi or N. tabacum, and not cassava. There are many species of aphids present in East Africa [Seven species of aphids have previously been tested for transmission of CBSVs. Bock  tested sn et al.  tested Mt Africa  and sevet Africa , 20. It Ipomovirus which we have shown to contain a DAG motif in its coat protein gene . Five new UCBSV whole genomes have been added from Kenya. K4 and K5 form a monophyletic group and are placed next to two Tanzanian genomes from Serengeti. While K12 is grouped with another Kenyan whole genome sequence (UCBSV_KE_54_FN433933) K13 and K14 are found on branches by themselves indicating new additions to the genetic diversity of the CBSVs phylogeny.Our new whole genomes from Kenya are found in every clade of the CBSVs phylogenetic tree , indicatUtilizing the most advanced genetic sequencing technology, coupled with high performance computing, and user-friendly alignment visualization tools, we have uncovered patterns in CBSVs genomic data that were previously undiscovered. Translating nucleotides into amino acids and critically analyzing the resulting alignment before proceeding to downstream analyses led us to discover the DAG in CBSV genomes and not UCBSV. The importance of translating nucleotide sequences into amino acids cannot be underestimated as demonstrated here. These observations would not have been possible even ten years ago, and the pace of advancement in software and hardware capabilities ensures that this type of analysis and investigation will become commonplace. This further advances our understanding of how genomics and biological factors interact, and leads us to new routes of investigation in the field, and faster than ever before.The first actions of further investigation must be an attempt to understand the diversity of aphid species present in and around cassava fields in east Africa. This includes morphological and molecular identifications. Second will be to collect and maintain populations of common species for CBSVs aphid transmission experiments. When this occurs, it must be with isolates of CBSV whose whole genome sequence is known, and an isolate from each proposed new species group needs to be included. This is the only way to answer the most pressing questions. It is becoming clear that there are many as yet undiscovered species within the Ipomoviruses. As the genetic landscape of these viruses increases due to advances in NGS technologies we will gain a clearer picture of the virus species and be able to pin point the most recent common ancestor to these sequences. This will shine a light on the evolutionary history of the presence of the DAG motif in ipomoviruses, and their potential transmission by other vectors like aphids. In addition, a crystal structure of CBSV coat protein is needed to identify exactly where the DAG motif is located. Current predictions are based on homology to tobacco mosaic viruses, which are clearly divergent. There are opportunities to develop better diagnostic tools for both virus species confirmation and vector identification particularly in-field, to ensure researchers know exactly what they are taking back to the laboratory and greenhouse.S1 File(PDF)Click here for additional data file.


Host-pathogen interaction between Zika virus (ZIKV) and other medically relevant flaviviruses is a hot topic, in part due to the potential risk of disease exacerbation by antibody-dependent enhancement (ADE). Current data are controversial, thus care should be taken when facing this aspect of ZIKV infection, particularly during vaccine development.Mosquito-borne flaviviruses are (re-)emerging pathogens responsible for several human diseases that lately have been raising alarms both socially and in healthcare. Colonization of new geographical areas by vectors and spread of ZIKV to regions with competent vectors allows its increased settling, exemplified by the recent epidemic across new areas where other important flaviviruses, such as dengue virus (DENV), Yellow Fever virus (YFV) and West Nile virus (WNV), co-circulate. The alarm was mainly due to the rapid spread of the virus across the American continent  , ZIKV infection was linked to Guillain-Barré development independent of previous DENV infection (Cao-Lormeau et al., All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.