To prospectively monitor Zika viral loads in semen from Belgian travellers with confirmed Zika virus infection, who returned from the Americas during the 2016 Zika virus epidemic.We recruited symptomatic travellers consulting our clinic and we confirmed infection with either reverse-transcriptase (RT) polymerase chain reaction (PCR) assay or virus neutralization test. The participants produced semen samples weekly, either at the clinic or at home. For the initial sample, the laboratory staff did a microscopy analysis if they received the sample within an hour of production. Using RT–PCR, we monitored Zika virus ribonucleic acid (RNA) loads in semen until we obtained two negative results.We detected Zika virus RNA in nine of 15 participants’ semen, one of whom was vasectomized. The median time to loss of RNA detection in semen was 83 days after symptom onset . The longest duration of viral shedding in semen before obtaining the first negative RT–PCR result was 144 days after symptom onset. All of the 11 participants, for whom we microscopically analysed their semen, had presence of leukocytes, 10 showed haematospermia and six showed oligospermia. These abnormalities occurred irrespective of Zika virus detection in semen.The majority of men in our study had detectable Zika virus RNA in their semen. We recommend that semen from Zika virus-infected men should be analysed with RT–PCR and that health professionals should advise infected men, even if they are vasectomized, about current recommendations for prevention of sexual transmission of the virus. Therefore, knowledge of the incidence and kinetics of Zika virus in semen is important for assessing the probability of viral sexual transmission. Here we present the results from a cohort study that prospectively monitored viral loads in semen from Belgian travellers with confirmed Zika virus infection returning from the Americas.From February 2016 to May 2017, we tested all travellers who consulted the Institute of Tropical Medicine Antwerp, Belgium, with symptoms matching the European Centers for Disease Control clinical case definition for Zika virus infection – that is, maculopapular rash with or without fever, and painful joints or muscles or non-purulent conjunctivitist-values), because a reference method for RNA quantification is not available. Any Ct-value below 45 is defined as positive. We defined confirmed infection as a positive virus neutralization test resultTo diagnose Zika virus infection, we first used the anti-Zika virus Immunoglobulin (Ig) M and IgG enzyme linked immunosorbent assay  according to the manufacturers’ exact instructions. We confirmed positive or equivocal ELISA results the same week with a non-commercial Zika virus neutralization test on refrigerated serum. To detect viral RNA, we performed commercial available Zika virus-specific RT–PCR  according to the manufacturers’ instructions on the LightCycler 96 . We tested serum from participants who presented within 7 days after symptom onset or urine within 14 days after symptom onset.Men who were 18 years or older, with a laboratory-confirmed symptomatic Zika virus infection and no history of immunosuppression were eligible to participate in the study. Staff members of the Institute of Tropical Medicine recruited men and recorded clinical and epidemiological data in a standardized case record form . Two autIf the laboratory staff received the first sample within an hour after production, they analysed the sperm, leukocyte and erythrocyte counts. Oligospermia was defined as a count of less than 15 million spermatozoa/mL.Because of the exploratory character of the study, we arbitrarily set the sample size at 20 participants. For each participant, we calculated the time from symptom onset until Zika virus RNA could no longer be detected in semen. We assessed the duration of Zika virus persistence in semen using the Kaplan–Meier estimator and the parametric Weibull regression models. All analyses were done in R, version 3.3.2 .We obtained ethical approval from the institutional review board at Institute of Tropical Medicine and the ethics committee of the Antwerp University Hospital, Belgium. We registered the protocol at ClinicalTrials.gov (NCT 02733796).From February 2016 to May 2017, the institute recruited and followed-up 15 Caucasian men, who had travelled to countries with active vector-borne transmission of Zika virus . We did RT–PCR analyses confirmed Zika virus infection in 10 men, while virus neutralization tests confirmed infection in five men. All men consented to participate and 13 men completed the study; two patients did not provide enough numbers of semen samples for obtaining two consecutive negative results. Patient 9 provided only two semen samples (at 23 and 100 days after symptom onset) and patient 12 provided only one sample at 30 days after symptom onset, after which he withdrew (no reason given).We detected Zika virus-RNA in the semen from nine men (60%), of which one man has had a successful vasectomy. For patients with RNA in semen, the median number of samples analysed was 6 (range: 2–18). For men with positive RT–PCR result, the median time to collection of the first semen sample was shorter than for men with a negative result, though not significantly . The longest duration before obtaining the first negative semen RT–PCR result was 144 days after symptom onset, with the last sample testing positive at 137 days after symptom onset . For patThe Weibull distribution curve showed that the median loss of RNA detection in semen occurred at 83 days after symptom onset (95% CI: 57−108). At 149 days after symptom onset (95% CI: 104−194) RNA could no longer be detected in the semen of 95% of the men .t-value of 19.6. Cytopathic effect appeared only in Vero cells after passage on the C6/36 culture.We managed to isolate the Zika virus from one semen sample, collected on 11 days after symptom onset and with a RT–PCR CFor 11 patients, the first collected semen sample was available for microscopic analysis. We detected leukocytes in all samples and erythrocytes in nine samples; these included five Zika virus positive and four negative samples . For one,,Here we report on the frequency and persistence of Zika virus in semen after acute symptomatic infection in Belgian men who had travelled to the Americas during the 2016 Zika virus outbreak. In 60% of the men with confirmed Zika virus infection, we also detected Zika virus RNA in the semen. Several studies have reported a similar proportion of detection, both in endemic settings,,,,,,,Persistence of Zika virus RNA in semen after acute infection appears common. In our study, sequential semen samples showed decreasing level of RNA until the virus became undetectable, confirming previous findings.The decline of Zika virus-RNA levels in semen observed in our patients suggests elimination of the virus. In only one patient, we could detect viral RNA after one negative RT–PCR result, but who subsequently had two consecutive negative samples that concluded follow-up. This finding demonstrates the need for two consecutive negative results, since the possibility of viral recurrence cannot be excluded.,,,Almost all men who had their semen analysed showed macroscopic or microscopic haematospermia. Other studies have also investigated haematospermia following Zika virus infection, but the symptom has not been consistently found.Some of the participants had oligospermia, which could result from inflammatory destruction of the seminiferous epithelium directly, from the fluctuation of reproductive hormone concentrations or from the febrile state.,As reported previously, presence of viral RNA in the ejaculate of the vasectomized participant suggests that the Zika virus does not only infect spermatogonia, but also tissues distal of the vasectomy site.,Isolation of the virus is regarded as a proxy for infectivity. However, we were only able to isolate the Zika virus from one of our patients’ semen. The failure to isolate the virus from the other patients could be an indication of viral degradation, although others have demonstrated Zika virus replication competence in semen up to 69 days after symptom onset.,Zika virus in semen may become the major transmission route in areas where arthropod vectors do not thrive. However, mathematical modelling suggests that sexual transmission alone is not likely to drive or sustain a Zika virus outbreak in absence of a suitable vector population.Our study has at least three limitations. First, we had to discontinue the recruitment of participants before reaching the projected sample size, because incidence of Zika virus infection had declined by May 2017.,The findings presented here emphasize that further studies are needed to increase our understanding of the host-pathogen relationship and implications of Zika virus infection for reproductive function. Pending new evidence, we recommend the use of RNA detection assays for semen of returning travellers with confirmed Zika virus infection, especially for couples planning a pregnancy. To reduce the risk of sexual transmission of the virus, our findings highlight that health professionals should advice patients, even vasectomized men, about the current recommendations from the World Health Organization and United States Centers for Disease Control and Prevention.


Creative exploration is central to science, art and cognitive development. However, research on creative exploration is limited by a lack of high-resolution automated paradigms. To address this, we present such an automated paradigm, the creative foraging game, in which people search for novel and valuable solutions in a large and well-defined space made of all possible shapes made of ten connected squares. Players discovered shape categories such as digits, letters, and airplanes as well as more abstract categories. They exploited each category, then dropped it to explore once again, and so on. Aligned with a prediction of optimal foraging theory (OFT), during exploration phases, people moved along meandering paths that are about three times longer than the shortest paths between shapes; when exploiting a category of related shapes, they moved along the shortest paths. The moment of discovery of a new category was usually done at a non-prototypical and ambiguous shape, which can serve as an experimental proxy for creative leaps. People showed individual differences in their search patterns, along a continuum between two strategies: a mercurial quick-to-discover/quick-to-drop strategy and a thorough slow-to-discover/slow-to-drop strategy. Contrary to optimal foraging theory, players leave exploitation to explore again far before categories are depleted. This paradigm opens the way for automated high-resolution study of creative exploration. Creative exploration and discovery processes are defined as the search for novel and valuable elements within a set of constraints. Creative exploration is central for many fields, including art , design One obstacle for understanding creative exploration is the lack of automated and high-resolution experimental paradigms. For example, one of the most commonly used tests for creativity is the alternate usage task (AUT) . In the More fundamentally, AUT and other common creativity tests record only the discovered solutions and not the intermediate steps leading from one solution to another. Thus, they do not allow insight into the process of exploration. Several studies attempted to map the processes leading to creative solutions by recording verbal transcripts of thought processes—the ‘think aloud’ method ,17. HoweHere, we introduce a new paradigm for creative exploration that allows automated tracking of the search process in a space of possibilities that is large yet well-defined. To achieve this, we borrow techniques from the study of human foraging.In a series of studies Hills, Todd and colleagues used a spatial foraging task and a letter puzzle task to experimentally study human foraging behavior –21. ThesIn order to develop a similar task for creative search, we need to address one key difference between foraging and creative search. In the foraging tasks, the goal is to find as many solutions as possible; in creativity tests the goal is two-fold: (i) to find many solutions (fluency) (ii) of high quality . In other words, whereas the value of the different solutions in the foraging tasks is constant , the distribution of solution values in creativity tests is skewed, with many low-quality solutions (boring or non-feasible) and only a handful of high-quality creative solutions.creative foraging game enables high-resolution measurement of creative, intrinsically motivated search in the framework of visual exploration and discovery [We present a paradigm for creative foraging in which players seek solutions that they find interesting and beautiful, embedded within a well-defined search space. The iscovery –28.The creative foraging game is a computer game in which players can move ten adjacent squares to create different shapes . There aWe present data on 100 participants and interpret the results in the context of OFT according to the following hypotheses:1) Participants’ trajectories in shape space are composed of distinguishable phases of exploration and exploitation: exploration for new shape categories and exploitation of similar shapes within a category.(H2) In phases of exploitation people move along optimal (shortest) paths. In creative foraging participants leave exploitation phases sooner than predicted by naïve OFT, far before patch depletion. Shapes at the entry points to phases of exploitation are more ambiguous, in the sense that they can lead different players to different categories. People show individual differences in their search strategies and patterns. and 46 shapes chosen to the gallery . We 6,324) an1).We noticed that the gallery shapes gathered in a given exploitation phase were similar to each other. To test this, we performed an ‘odd-shape-out’ test with a new set of participants who did not play the game . We findNext, we considered a prediction of optimal foraging theory (OFT) that within exploitation phases, players move along direct  paths between discoveries . To test2).We find that during exploration phases players did not take the shortest path between gallery shapes; their paths averaged three times longer than the shortest possible path  . In contWe noticed that different players rediscovered the same categories of shapes- for example numerical digits and shapes resembling airplanes. To study this, we developed an automated definition of shape categories. We defined a shape category as a set of shapes often rediscovered by different players. To do so, we used a network representation of shapes found by different players and used a community-finding algorithm to automatically detect shape categories see . Each caThe algorithm revealed 14 categories that were found again and again by different players. These 14 shape categories contain a total of 653 shapes, 34% of all gallery shapes collected by participants.Each category includes shapes with a shared theme: familiar objects such as animals or airplanes, or symbols such as digits or letters. Other categories were quite abstract but seemed to share a visual similarity. Examples of shape categories are shown in We tested whether people can reliably distinguish between these categories using an ‘odd-shape-out’ test, similar to the one reported above. We asked 86 people who did not play the game to match a randomly chosen triplet of shapes from a single category either to a set of six shapes from the same category or to a set of six shapes from another randomly chosen category .We find that players cover on average only 6.8%  of the aggregated shapes of each category (see Section I in 5). Players varied considerably in the average duration of their exploitation phases To adjust for possible differences in individual move speeds, we plotted the mean number of moves in exploration and exploitation for each player . The aveWe conclude that people in our sample vary along a continuum between two strategies: a fast strategy of short exploration/exploitation and a slow strategy of long exploration/exploitation. Those quick to discover are quick to drop, and those slow to discover are slow to drop and are thorough in their exploitation of a category.The creative foraging game allowed us to focus on the moments of discovery of new categories of shapes. These moments occur at the transition between exploration and exploitation. We call the shape that marks the beginning of a new exploitation phase the ‘transition shape’. Different players enter exploitation of a given category through different transition shapes. For example, in 4): they belong to multiple meaning categories more often than other gallery shapes  of apes Fig   . The AUTWe find that both fluency and originality in AUT showed a trend in correlation with their respective measures in the CFG . To assess the total matching between AUT and CFG, we defined a composite creativity score that averages over the two measures; we averaged the Z-transformed fluency and originality score of each player. We find that the composite creativity score in the CFG shows medium correlation with the composite creativity score in AUT .We presented the creative foraging game as a paradigm to study intrinsically motivated creative exploration. In the creative foraging game, people search a defined metric space of shapes for interesting and beautiful shapes. Multi-dimensional information about the players search trajectories is automatically recorded at high resolution.1). Different people rediscover the same shape categories, including letters, digits, airplanes and other more abstract categories with shared visual properties. Thus, the creative foraging game induces meaning categories on the space of shapes. We find that these categories are interleaved rather than segregated in this space, such that shapes from a given category have neighbors from different categories. Hence, participants need only change their focus of attention in order to discover a new category. This contrasts with a common metaphor in which creative search is like seeking an oasis of novel and valuable solutions in the desert of ordinary. Search in an interleaved landscape might be relevant for exploring creative solutions in information-rich digital environments.We find that participants’ trajectories are composed of alternating phases of meandering exploration, punctuated by transitions to exploitation of patches of similar shapes (H2). This finding of meandering paths in exploration versus direct paths in exploitation can ground the metaphor from creativity research of ‘fogginess’ in exploration vs. ‘visibility’ in exploitation [The existence of exploration and exploitation phases suggests a link to studies on human foraging behavior. Human foraging studies supported the predictions of OFT ,20. In loitation ,32.3). This difference might be the result of the goals of naïve OFT and creative foraging. OFT assumes that resources have a defined and time-invariant value, and hence foraging aims to maximize the amount of discovered solutions, where all solutions have the same quality. In contrast, in creative foraging the intrinsically evaluated ‘quality’ of solutions varies considerably—some solutions are novel and interesting while others are more dull. The process of evaluating solution quality may be dynamic: the value of the first exemplar from a category is higher than the value of the nth exemplar, suggesting that creative foraging is guided by a mechanism related to novelty seeking [Our results differ from OFT regarding the point of departure from an exploitation phase, highlighting the different nature of the goals of naïve OFT and creative foraging. Naïve OFT predicts that foragers leave a patch due to diminishing returns caused by resource depletion. In contrast, we find that participants exit exploitation far before their patch of shapes is depleted , other explore longer and exploit longer . This correlation is preserved also when we adjusted for the speed in which people make the moves in the game. Other possible strategies such as short exploration and long exploitation (quick to discover and thorough exploitation) seem to be missing in our sample.The quick-to-discover/quick-to-drop versus the slow-to-discover/slow-to-drop behavioral continuum may hint that a common mechanism with a single time-scale governs both exploration and exploitation phases in the creative search process. A similar connection between the two phases was shown in previous studies. For example, in decisions from experience-sampling paradigms, people who switch more rapidly between options tend to sample less before making a decision . SimilarMore subtle differences in exploration and exploitation strategies between individuals might be observed with a larger pool of participants. Future research can explore possible mechanisms in terms of different personality traits  or atten4) than other gallery shapes. We suggest that these transition shapes can serve as an experimental proxy for moments of insight [Finally, we characterized the moments of discovery of new categories using the shapes at which participants enter a new exploitation phase. We find that the transition between exploration to exploitation occurs through shapes that are more ambiguous  = 25(±4)), took part in the experiment either for credit or payment equivalent to 5$. This study was approved by the IRB committee of the Hebrew University of Jerusalem.“Your goal is to explore the world of shifting shapes and discover those that you consider interesting and beautiful”. At each point in the game players could store the current shape to a gallery by pressing a gray square at the top-right side of the screen. The gray square showed the last gallery shape chosen. The gallery had no limit on the number of shapes. Games lasted 15 minutes. After the game, players performed another task—choosing the five most creative shapes from their gallery. This task is not analyzed in the current study.The creative foraging game was run on a PC with on-screen instructions. Players created shapes by moving at each step one of ten identical squares, keeping the squares connected by an edge . Error bars were computed by bootstrapping 10,000 times. See Section D in We tested the similarity of shapes within the same exploitation phase by means of an odd-one-out test. Participants who did not play the creative foraging game  = 36 (±12)) observed one of four blocks of 50 quartets of shapes. Sample size was calculated assuming medium effect size, and was sufficient for statistical power exceeding 0.95 assuming μrder see . A totalWe defined categories of shapes found by different players in exploitation phases using a network community approach. We constructed an undirected network in which each node is a group of shapes found in a single exploitation phase, hereafter termed patch. Two patches were connected in the network if they share at least two shapes. The network had a giant component of 334 patches and 17 smaller connected components . We defined shape categories by finding communities (modules) in the giant component using the Girvan–Newman algorithm. We find 14 shape categories of varying sizes (mean number of shapes (±std) = 70 (±38)). See Table A in 0 = 0.5, μ1 = 0.7, σ = 0.05, α = 0.05. We created for each of the largest nine categories a set of 40 such questions for a total of 360 unique questions. Groups of at least 10 participants responded to one of eight blocks of 45 questions posed in random order. See Section G in We tested the perceptual grouping of the categories by a grouping experiment. 86 participants who did not play the game  = 41.6 (±15.2)) were asked to match a triplet of randomly chosen shapes from the same category to one of two groups–six random shapes from the same category or six random shapes from another randomly chosen category . See Section H in In creativity tests such as AUT and TTCT, experimenters have access to the submitted solutions provided by the participants verbally or in drawn form, but not to intermediate steps or solutions. These submitted solutions carry a clear conceptual meaning, which in the CFG corresponds to the gallery shapes found in exploitation phases. We therefore scored exploitation gallery shapes rather than all gallery shapes in CFG. We note that considering all gallery shapes slightly improves all correlations: fluency (CFG)-fluency (AUT)- r = 0.2, p = 0.13, originality (CFG)-originality (AUT)- r = 0.21, p = 0.12, and the correlation of the CFG and AUT composite scores is- r = 0.29, p = 0.02.S1 FileSupporting Information for methods and data analysis.(DOCX)Click here for additional data file.


This article addresses the tradeoff between the losses caused by a new virus and the size of the team for developing an antivirus against the virus. First, an individual-level virus spreading model is proposed to capture the spreading process of the virus before the appearance of its natural enemy. On this basis, the tradeoff problem is modeled as a discrete optimization problem. Next, the influences of different factors, including the infection force, the infection function, the available manpower, the alarm threshold, the antivirus development effort and the network topology, on the optimal team size are examined through computer simulations. This work takes the first step toward the tradeoff problem, and the findings are instructive to the decision makers of network security companies. Computer networks and online social networks provide us with a fast channel of acquiring information and communicating ideas. Meanwhile, computer viruses can also spread rapidly through these networks, inflicting enormous economic losses . For theConsider a network security company that is dedicated to developing antiviruses. Suppose that when the fraction of the victims of a new virus exceeds a presupposed alarm threshold, the company will initiate a project of developing an antivirus against the virus. First, the amount of effort needed for the project, which is typically measured by persons years or persons months, is estimated –10. WhenThe key to solving the tradeoff problem is to accurately estimate the speed and extent of virus infections. Computer virus spreading dynamics as an emerging interdiscipline is devoted to gaining insight into the consequence of computer viruses through modeling and analyzing their spreading process. Since the seminal work by Kephart and White , 12, larThis article addresses the above-mentioned tradeoff problem. First, an individual-level virus spreading model, which is known as the individual-level SI model, is proposed to capture the spreading process of the virus before the appearance of its natural enemy, which is then utilized to assess the expected losses caused by the virus during the development period of an antivirus aiming at the virus. Then, the tradeoff problem is modeled as a discrete optimization problem. On this basis, the influences of different factors, including the infection force, the infection function, the available manpower, the alarm threshold, the antivirus development effort and the network topology, on the optimal team size are examined through computer simulations. This work takes the first step toward the tradeoff problem, and the findings are instructive to the decision makers of network security companies.The subsequent materials of this work are organized as follows. Section 2 presents the individual-level SI model, and models the tradeoff problem. Section 3 experimentally examines the influences of different factors on the optimal team size. Finally, this work is summarized by Section 4.Imagine that a network security company prepares to develop the antivirus aiming at a new computer virus. From the company’s perspective, the losses inflicted by the virus should be minimized, and the manpower allocated for the development project should be minimized so that there is enough manpower to undertake other projects. Therefore, the decision maker of the company must make a tradeoff between the two conflicting demands. This section is dedicated to modeling the tradeoff problem. For this purpose, the virus spreading process must first be modeled.t = 0 and then spreads through a network G =  connecting N hosts labelled 1, 2, ⋯N. Let A = (aij)N×N denote the adjacency matrix of the network. Before the release of the relevant antivirus, the virus is able to spread freely through the network, and every host in the network is either susceptible or infected. Let Xi(t) = 0 and 1 denote the event that at time t, host i is susceptible and infected, respectively. Let Si(t) and Ii(t) denote the probability of host i being susceptible and infected at time t, respectively.Suppose the new virus appears at time θ denote the presupposed alarm threshold for the virus, τ the time at which the expected fraction of the infected hosts in the network exceeds θ.W denote the effort of the project, n the number of the team members assigned to the project. Then the development period for the project is Let i gets infected at rate β > 0 is referred to as the infection force, the function f is referred to as the infection function, which is strictly increasing and concave, f(0) = 0, f(x) ≤ x, x ≥ 0. For the rationality of the assumption, see Ref. [individual-level SI model.It is assumed that due to the infections by neighboring infected hosts, at time see Ref. . Accordin should be maximized. However, with the increase of n, the company will take a higher risk of having no enough manpower to undertake other projects. To reduce the risk, n should be minimized. To the extreme, it is best to assign only a single person for the project. Therefore, the decision maker of the company must make a deliberate tradeoff between the two conflicting demands. Let k > 0 stands for the relative weight of the two parts in the tradeoff; a larger k value means an emphasis on the reduction of the risk of manpower shortage, whereas a smaller k value implies that a lower loss is pursued. The tradeoff problem is then reduced to solving the following discrete optimization problem.Suppose the losses per unit time led by an infected host are one unit. Then the overall losses caused by the virus in the time interval G, the infection force β, the infection function f, the alarm threshold θ, the antivirus development effort W, and the available manpower The optimal team size, i.e., the optimal solution to the optimization problem θ can be set flexibly by the company, the development effort can be estimated with the software cost estimation techniques given in Refs. [G is obtainable using the network crawler described in Ref. [The available manpower in Refs. –10, and  in Ref. .f can be approached by applying the deep learning techniques presented in Refs. [The infection function in Refs. , 35 to tβ can be estimated by applying the time series analysis techniques exhibited in Ref. [The infection force  in Ref.  to the s in Ref. , 38. ThiWhen these factors are all determined, the optimization problem can be solved numerically.k ∈ {1, 3, 5}, β = 0.001, θ = 0.01, W = 100, G0. The team size vs. the tradeoff is shown in k = 1, 3 and 5.Consider three instances of the optimization problem  in Ref.  is takenβ, the infection function f, the available manpower θ, the development effort W, and the network G. This section is devoted to exploring the influence of each of these factors on the optimal team size.The optimal team size is dependent upon six factors: the infection force G = G0, the infection function In the following five experiments, To understand the influence of the infection force on the optimal team size, we present It is concluded from the figure that the optimal team size is increasing with the infection force. This phenomenon can be explained as follows. The loss part in the tradeoff is increasing with the infection force. To better balance the two parts in the tradeoff, the team size must be increased properly.f on the optimal team size, we present To understand the influence of the infection function It is concluded from the figure that the optimal team size is increasing with the infection function. The explanation of this phenomenon is similar to that of the previous one.To understand the influence of the available manpower on the optimal team size, we present It is concluded from the figure that the optimal team size is increasing and tends to saturation with the available manpower. This phenomenon can be explained as follows. When there is a small available manpower, the balance between the two parts in the tradeoff would lead to an optimal team size that is equal to the available manpower. With the increase of the available manpower, the balance would lead to an optimal team size that is increasing less rapidly than the available manpower and finally tends to saturation.To understand the influence of the alarm threshold on the optimal team size, we present It is concluded from this figure that the optimal team size is increasing with the alarm threshold. This phenomenon can be explained as follows. The loss part in the tradeoff is increasing with the alarm threshold. To better balance the two parts in the tradeoff, the team size must be increased properly.To understand the influence of the antivirus development effort on the optimal team size, we present It is concluded from this figure that the optimal team size is increasing with the antivirus development effort. This phenomenon can be explained as follows. The loss part in the tradeoff is increasing with the effort. To better balance the two parts in the tradeoff, the team size must be increased properly.G ∈ {Gi: 1 ≤ i ≤ 5}, where Gi are scale-free networks with 100 nodes, 109 edges, and a power exponent of 2.7, 2.8, 2.9, 3.0, and 3.1, respectively [To understand the influence of the network heterogeneity on the optimal team size, the following experiment assumes ectively . See FigWe present It is concluded from this figure that the optimal team size is increasing with the network heterogeneity. This phenomenon can be explained as follows. The loss part in the tradeoff is increasing with the effort, because malware spreads more rapidly in a more heterogeneous network than in a more homogeneous network. To better balance the two parts in the tradeoff, the team size must be increased properly.This article has addressed the tradeoff between the losses caused by a new virus and the size of the team for developing an antivirus against the virus. First, an individual-level virus spreading model has been proposed to capture the spreading process of the virus before the appearance of its natural enemy. Then, the tradeoff problem is modeled as an optimization problem. Next, the influences of different factors, including the infection force, the infection function, the available manpower, the alarm threshold, the antivirus development effort and the network topology, on the optimal team size have been examined through computer simulations. The findings are instructive to the decision makers of network security companies.Towards this direction, there are a number of problems that are worth study. As was indicated previously, the infection force and the infection function must be determined. The model should be extended to more sophisticated virus spreading models such as the impulsive spreading models –43, the


The provision of safely managed sanitation in informal settlements is a challenge, especially in schools that require durable, clean, sex-segregated facilities for a large number of children. In informal settlements in Nairobi, school sanitation facilities demand considerable capital costs, yet are prone to breakage and often unhygienic. The private sector may be able to provide quality facilities and services to schools at lower costs as an alternative to the sanitation that is traditionally provided by the government. We conducted a randomized trial comparing private sector service delivery (PSSD) of urine-diverting dry latrines with routine waste collection and maintenance and government standard delivery (GSD) of cistern-flush toilets or ventilated improved pit latrines. The primary outcomes were facility maintenance, use, exposure to fecal contamination, and cost. Schools were followed for one school year. There were few differences in maintenance and pathogen exposure between PSSD and GSD toilets. Use of the PSSD toilets was 128% higher than GSD toilets, as measured with electronic motion detectors. The initial cost of private sector service delivery was USD 2053  per school, which was lower than the average cost of rehabilitating the government standard flush-type toilets ) and constructing new facilities ). The private sector delivery of dry sanitation provided a feasible alternative to the delivery of sewage sanitation in Nairobi informal settlements and might elsewhere in sub-Saharan Africa. More than 800 million people live in urban informal settlements, a number that is expected to double by 2030 . This raFor the millions of individuals living in informal settlements in sub-Saharan Africa, inadequate funding for urban sanitation and a lack of sanitation regulations exacerbate poor sanitation provision. Over 65% of those living in informal settlements in Nairobi, Kenya, have inadequate sanitation . As a reSafely managing sanitation needs in informal urban settlements is challenging, especially in schools, where facilities must be sex-segregated, hygienic, durable, and used by a large number of students. Further, despite substantial capital costs, school sanitation facilities are prone to disrepair and are often not well maintained. Schools experience many of the same obstacles as communities in informal settlements when providing WASH services. Space is limited to build WASH facilities, and certain sanitation technologies can be inappropriate when schools have inconsistent access to water and/or sewage . The comThere is limited evidence on the effect of different sanitation and hygiene provision modalities to overcome the challenges among schools in informal settlements. In close collaboration with Ministry of Education, Science and Technology of Kenya and the Nairobi City Council, we conducted a randomized controlled trial comparing a private sector sanitation service delivery (PSSD) model of urine-diverting dry sanitation facilities with routine waste collection and maintenance to the government standard delivery (GSD) of ventilated improved pit latrines or sewered cistern-flush toilets in four informal settlements in Nairobi, Kenya. The study was conducted within the urban informal settlements in Nairobi City County, including the areas of Mukuru, Mathare, Kamukunji, and Makadara. The overall population of Nairobi City County is over three million inhabitants, and it is estimated that 60% of this population lives in informal settlements . The priThis study was part of SWASH+, an applied research project designed to address policy-relevant, school-based challenges and to provide evidence to ensure that decision-makers within the GoK have access to high quality data to inform investment decisions on the provision of school WASH services. SWASH+ has included several studies, including large randomized trials to assess the health and educational benefits and sustainability of school WASH to inform an increase in recurrent payments to improve school conditions ,13,18,23Twenty schools were randomly selected from a list provided by the Nairobi City County. Sample size was determined by the maximum number of schools that could be included given the study budget. Primary schools were considered eligible for inclusion if they had poor sanitation conditions, the willingness to pay for a private sector sanitation service delivery model or receive government standard flush-type sanitation facilities, sufficient space to accommodate the new facilities, and an adequate population to support the use of five sanitation facilities. Selected schools were visited to confirm eligibility. Schools were then randomly allocated to one of two intervention arms (10 schools in each arm), stratifying the group by private (4 schools in each arm) and public (6 schools in each arm). Randomized selection and allocation was conducted using a random number generator in Microsoft Excel  by Emory University research staff.Schools allocated to the PSSD arm received five Fresh Life Toilets (FLTs) per school and a hygiene curriculum to promote behavior change provided by Sanergy, a private sector enterprise based in Mukuru, Nairobi. Sanergy adapted their business model  in order to provide FLTs, handwashing facilities, maintenance support, and a comprehensive hygiene promotion curriculum for schools in the informal settlements. The FLTs are urine-diverting dry toilets, which have cartridges that collect the waste. A waste collection team removes and replaces the cartridges to dispose of the waste on a daily basis. In collaboration with WASH United, a non-profit behavior change communication organization, Sanergy organized a two-day training of teachers, where Sanergy provided them with the knowledge, resources, and instructional tactics they needed to educate their pupils about WASH and to encourage adoption of healthy behaviors. Soap was not provided as part of the intervention.The GSD arm received sanitation packages typical of GoK provision of school sanitation—five stances of cistern-flush toilets connected to the municipal septic system designed by an architect at Nairobi City County (NCC). Implementation of this arm by local contractors was facilitated by Cooperative for Assistance and Relief Everywhere (CARE), an international non-governmental organization (NGO) focused on serving individuals in the poorest communities in the world. These designs were derived from approved GoK blueprints and conformed to GoK policies. Some schools had toilet facilities that were in disrepair and consequently were not in use; rehabilitation of existing facilities was completed in four schools in the GSD arm. One school received five stances of ventilated improved pit (VIP) latrines rather than cistern-flush toilets due to lack of space to build a septic tank. A table comparing PSSD and GSD components can be found in Installation of FLTs in schools in the PSSD arm was completed by December 2014 . Eight of the ten GSD arm schools had completed construction by July 2015. The remaining two schools completed construction by November 2015. A timeline of intervention delivery is shown in Since construction of the flush-toilets in the GSD arm was completed at different times in Term 2 (May–July), we opted to report data from Term 1 (January–March) and Term 3 (September–November) only. Given these construction delays in the GSD arm, data from Term 1 assess the impact of FLTs in the PSSD arm versus no new delivery in the GSD arm, whereas Term 3 compares the two different interventions.We collected data from January 2015 to November 2015. Each school was visited twice per term, for a total of six visits throughout the school year, with the exception of one school in the GSD arm, which was visited once in Term 1. All visits were unannounced. Two trained enumerators collected the following data at each visit: school-wide roll call, head teacher interview, structured facility observations, hand washing and toilet use observations, and environmental swabs or hand rinses.One of the enumerators interviewed the head teacher or school director. The survey included questions about enrollment, activities influencing enrollment, the number and functionality of sanitation facilities, and maintenance of toilets. That enumerator also conducted visual inspections of drinking water containers, rainwater harvesting tanks, toilet compartments, and hand washing stations (if available). Observations were made for safety, privacy, functionality, and cleanliness of the sanitation facilities. The head teacher interview and structured facility observations were recorded in the Open Data Kit (ODK) mobile application .Data on toilet maintenance was collected through the head teacher interview and structured facility observations. A scale score was constructed by assigning values to five binary (yes/no) toilet conditions observed during the structured facility observation: availability of cleaning materials, absence of flies, no odor, no visible feces, and absence of urine/stagnant water. Affirmative responses were assigned one point, and values were summed to create a scale of toilet maintenance ranging from 0 (very dirty) to 5 (very clean). In addition, head teachers reported the weekly frequency of toilet cleaning to quantify toilet maintenance.We calculated toilet use using three measures: observed signs of use from the structured facility observation, direct observation of toilet use, and detected use from toilet sensors. For observed signs of use, the enumerator noted whether or not a toilet appeared to be in use. For direct observation, the enumerators were seated near the toilet area and recorded the number of pupils entering/exiting each toilet compartment or toilet block from 08:00 a.m. to 04:30 p.m. They also observed handwashing behaviors from where they were positioned. Handwashing behaviors of pupils utilizing the toilet were recorded as handwashing with water only, handwashing with soap or ash, or no handwashing. We deployed Passive Latrine Use Monitors (PLUMs)  in toilet blocks to measure usage in an unbiased manner . In the E. coli, a commonly used fecal indicator bacteria [E. coli exposure throughout the day in toilets, at the last visit we sampled six random toilets in each school at three fixed time points  in a day . Swabs were kept in a cooler with ice packs for a maximum of 6 h until they were transported to the lab, where they were processed immediately.Exposure to fecal contamination was measured using environmental swabs and hand rinses that were analyzed for bacteria . At fourEnumerators collected hand rinses from 20 randomly selected pupils in each school at two different visits during Terms 2 and 3 . Each selected pupil placed his or her right hand in a 500-mL Whirl-Pak bag  containing 250 mL phosphate buffered saline (PBS) solution . The enuE. coli using standard membrane filtration and m-ColiBlue24® broth  [E. coli directly by membrane filtering 100 mL of the rinse solution without dilution and plating on mColiBlue24 broth media. Plates were incubated at 37.5 ± 0.5 °C. After 24 h, blue E. coli colonies were counted. The data are reported as colony-forming units (CFUs) per swab or hands, after adjusting for dilutions and filtered volume. Plates with ≥200 CFU were recorded as too numerous to count (TNTC) and were set to 200 CFU for the analyses. For handrinses, data are reported as presence of any E. coli on the pupils’ hands (<100 CFU/hands) versus high contamination of E. coli (≥100 CFU/hands). The swabs and hand rinses were processed for CO, USA) . Seven mCO, USA) . The swaCO, USA) . HandrinThe cost of both interventions was tracked throughout the study. Cost in Kenyan shillings and person hours were recorded in an Excel spreadsheet and compared side by side. Costs were tracked by CARE and Sanergy for the two respective study arms using mutually agreed upon parameters and compiled into a table for comparison.All data were cleaned and analyzed in Stata v12 . For each outcome, three comparisons were analyzed: (1) FLTs in the PSSD arm compared to existing toilets in the GSD arm in Term 1, representing a comparison of private sector service delivery versus no sanitation improvement; (2) FLTs in the PSSD arm compared to newly constructed facilities in the GSD arm in Term 3, representing private sector service delivery versus government standard sanitation provision; and (3) FLTs over all three terms. For comparisons 1 and 2, random effects linear (continuous outcomes) and logistic (binary outcomes) regression models were utilized with the data clustered at the school level to analyze differences in primary outcomes between interventions. For comparison 3, data were tested for trend across all three terms using the nptrend command.Mixed-effects negative binomial regression was used to compare daily rates of use measured by the motion detector enabled sensor PLUMs between FLTs and government standard toilets. We fit a three-level, random intercept model to adjust for the clustering of days within toilets (level 2) and schools (level 3). The model contained an offset term in order to account for the number of individual toilets in the toilet block and robust standard errors were calculated for model coefficients . The coeThe study was conducted in accordance with the Declaration of Helsinki. Ethics approval was granted by the Institutional Review Board at Emory University  and the Ethical Review Committee at the Great Lakes University of Kisumu . A waiver of parental consent was granted and head teachers of each school signed an in loco parentis form. For hand rinses, informed oral assent was obtained from all participants. The study adhered to CONSORT guidelines for randomized trials .p = 0.001). In Term 3, the FLTs were significantly cleaner (4.0) than the new toilets in the GSD arm . However, the maintenance score for FLTs decreased each term, indicating decreased maintenance over time. There was no difference in the average number of days per week the FLTs were cleaned (5.0) compared to the existing toilets in the GSD arm (4.7) in Term 1 (p = 0.16). In Term 3, the average number of days for toilet cleaning was similar per week for the FLTs and the new toilets in the GSD arm . There was no statistical difference in the average number of days that toilets were cleaned across terms for FLTs in the PSSD arm . Toilet maintenance characteristics are shown in p = 0.37). Of these unlocked toilets, 93% of FLTs and 100% of existing GSD toilets appeared to be in use. Results were similar in Term 3 comparing FLTs to new toilets in the GSD arm. All of the unlocked FLTs and new GSD toilets appeared to be in use in Term 3. Use among FLTs in the PSSD arm across all three terms was similar.Toilet use characteristics are shown in p = 0.5).The percentage of pupils that used an FLT in one day’s time in Term 1 was 12% compared to 14% of pupils using existing toilets in the GSD arm. The percentage of pupils that used an FLT in Term 3 was 15% compared to 20% of pupils using new toilets in the GSD arm. There was no statistical difference in FLT usage across terms in the PSSD arm (p = 0.003), while the percent of students observed not washing their hands was higher in the GSD arm (p = 0.004). There was no difference in the percent of students who washed their hands with water only in Term 1 (p = 0.43). There were no differences in observed handwashing practices between the PSSD and GSD arms in Term 3. The percentage of students practicing handwashing with soap following FLT use was not different across all three terms (p = 0.54).In Term 1, the percent of students observed handwashing with soap was higher in the PSSD arm compared to the GSD arm  and eight were new cistern-flush toilets (GSD arm). All of the FLTs were individual units. In contrast, all of the cistern-flush toilets contained multiple individual toilets per toilet block (range 2–11). Not counting installation and removal days, the sensor was in place and functional for an average of 11.6 days (standard deviation (SD) = 4.4) in the FLTs and 10.4 days (SD = 5.7) in the GSD toilets. The proportion of these days that were weekdays was roughly equivalent in both groups (67.5% in the FLTs vs. 68.5% in the GSD toilets).Because the PLUM installation locations varied between the PSSD arm and the GSD arm, structured observations combined with PLUM monitoring were conducted in both arms to estimate a scaling factor for estimating usage events to PLUM clicks—a recording of movement with a resolution of 3 s. A total of 1.741 unique observations at FLT locations corresponded to recorded PLUM data, with a median rate of 8.6 clicks per use. In the case of the GSD toilets, only 164 observations with corresponding PLUM data were available, indicating a median rate of 8.2 clicks per use. The mean number of clicks among individual toilets in the FLTs was 220.0 (SD = 177.5) and in the GSD toilets was 76.1 (SD = 84.7). The median click count was 247.0 (interquartile range (IQR) = 344.0) in the FLTs and 39.5 (IQR = 124.5) in the GSD toilets. These rates were scaled by the median clicks per use in each arm, identified through structured observations. In the case of the GSD toilets, the PLUM recorded events were then further scaled by the number of toilet stalls observed per PLUM.p < 0.001) when compared with cistern-flush toilets in the GSD arm. Comparable results were found in sensitivity analyses examining usage rates on weekdays only  or when recorded use on the days of sensor install and removal was included .Statistical analysis of sensor data revealed significantly higher daily use among the FLTs in the PSSD arm .FLT use in Term 1 was 19.85 uses per toilet per day (SD = 23.21); in Term 2 was 31.79 (SD = 30.04) and in Term 3 was 25.52 (SD = 20.59). Applying an ANOVA adjusted for clustering, there was no difference in mean FLT use between the terms . The percent of pupils with high levels of E coli (≥100 CFU/hand) on their hands was 20% and 16% in the PSSD and GSD arms, respectively (p = 0.59).Results from the handrinses collected in Term 3 indicate that the percent of pupils with any E. coli in Term 1, whereas 22 (14%) of the existing toilets in the GSD arm tested positive (p = 0.31). In Term 3, E. coli was detected in 21 (50%) of FLTs compared to 53 (59%) of new toilets in the GSD arm (p = 0.46). Over the three terms, the percentage of FLTs with detectable E. coli levels decreased .In Term 1, 35 (35%) environmental swabs from FLTs in the PSSD arm tested positive for E. coli was measured longitudinally over the course of one day. E. coli colonies was highest at the beginning of the day and decreased throughout the day (p = 0.01). The new toilets in the GSD arm followed the opposite trend (p = 0.25), in which the mean number of E. coli colonies was lowest in the morning and increased as the day progressed.In Term 3, The initial cost of the hardware and one year of field maintenance for private sector service delivery was USD 2053  per school. This was lower than the average cost of rehabilitating flush-type toilets in the GSD arm ) and the average cost of constructing new flush-type toilets ), which did not include field maintenance support. Fewer visits (five visits) and less total personnel time (40 h) were used to install the FLTs than the toilets in the GSD arm (11 visits and 92 h). Additionally, the PSSD arm provided a comprehensive WASH training for teachers that cost USD 193 . From the head teacher survey, schools in the PSSD arm reported having, on average, a larger budget for WASH facilities ) than those in the GSD arm ). The average intervention costs per school for both arms is shown in E. coli, handwashing behaviors), toilet use as measured by infrared sensor was higher among FLTs (PSSD arm) compared to cistern-flush toilets (GSD arm), and toilet maintenance was better in the PSSD arm compared to the GSD arm. Additionally, the overall cost for private sector delivery and service of urine-diverting dry latrines was less expensive than the government standard of flush-type toilet provision. Further, flush-type toilets incur additional costs with water and sewage connections, which is not always sustainable in urban settings [We conducted a randomized trial to evaluate two modalities of sanitation delivery to schools in informal settlements. Our study demonstrates the potential for the private sector to deliver sanitation services to schools that is financially viable, sustainable, and scalable, and which could be achieved through public–private service delivery partnerships between the Ministry of Education and private sector enterprises. Feasibility of private sector service delivery of sanitation was defined as the ability to provide sustained sanitation services that were similar or superior to standard government sanitation approaches. Although there were no significant differences between the two approaches for some key indicators  at an extra cost; the total cost of the PSSD including the provision of consumables would still be far cheaper than that of the government standard of flush-type toilets.E. coli contamination in the swabs of toilets. The increase in E. coli throughout the day in the toilets of the GSD arm suggests that the surfaces are becoming contaminated with more usage, despite reported cleaning of the toilets. The difference in technologies between the two study arms would help support the conclusion that flush-type toilets result in the aerosolization of fecal contamination [E. coli on surfaces.Despite the lack of statistical significance, we noted a trend in the environmental mination . FurtherE. coli recovery with swabs, which could bias the microbial data. Third, there was a nationwide teachers’ strike that closed all public schools for five weeks between September 2015 and October 2015. As a result, data collection was suspended in the 12 public schools enrolled in our study. In Term 1, three schools in the PSSD arm allocated the FLTs to girls only; therefore, the usage did not reflect that of the entire school. Lastly, and perhaps most importantly, the study was designed so that that installation of the interventions in all 20 schools would be completed by the beginning of the school year. However, construction in eight of the GSD schools was not complete until Term 2 and construction was never completed in two schools in the GSD arm. The delay in construction was, in part, because of unforeseen issues with access to a sewer connection, weak ground soil formation, and a high water table, representing a further challenge to the standard provision of these types of facilities. These delays created a challenge for us to conduct a head-to-head comparison of the performance of these toilets over the full school year. A follow-up study is planned to look at the sustained performance of these toilets over time.There were several limitations to this study. First, we were constrained in our sample size based on the budget of the trial. Second, the difference in toilet materials  likely caused variances in In the case of the PLUM data, several limitations were observed. Firstly, the installation location of the PLUM varied between the arms. Each FLT is single-user, so one PLUM was positioned to detect use in one toilet; GSD toilets were constructed in blocks and it was not possible to position the PLUM over a single stance. As such, the PLUM was positioned to detect use between two and 10 toilets. Consequently, the measures were not equivalent and we therefore scaled the data by the median number of PLUM “clicks” per user, and then by the number of toilets observed per PLUM. There were considerably fewer observations available from GSD toilets compared to the FLTs.Secondly, in previous PLUM studies, an event algorithm was derived from, and validated with, structured observations. This algorithm assumed gaps between users, as normally observed in households. In this study, this algorithm was not applied as the structured observations identified that many users were often present one after another. Consequently, the event algorithm in this study was necessarily simplified to be based on median “clicks” per user.This study explored the feasibility of private sector sanitation provision for school children in informal settlements. We found few differences in observed use and exposure to fecal contamination between private sector service delivery and government standard delivery. However, sensor detected toilet use and toilet maintenance was superior in the PSSD arm compared to the GSD arm. Additionally, given the cost and time needed for installation, the private sector service delivery was approximately five times cheaper and a more expedient solution in the short term. A follow-up study will allow us to assess sustainability of the hardware and capture the life cycle costs of these two approaches. Our results support the provision of a private sector service delivery for school sanitation as an option in informal settlements as a complement to the existing provision options. A one-size-fits all approach to toilet designs is not sustainable in the informal settlements. Public–private service delivery partnerships could allow for the Ministry of Education to outsource sanitation responsibilities to private service providers to meet the demand for safely managed sanitation. Future research should explore the life cycle costs associated with the ability of schools to continue to pay for private sector services and sustained maintenance.


We evaluated performance of 5 case definitions for Zika virus disease surveillance in a human cohort during an outbreak in Singapore, August 26–September 5, 2016. Because laboratory tests are largely inaccessible, use of case definitions that include rash as a required clinical feature are useful in identifying this disease. Zika virus infections in humans were first reported in Nigeria, Uganda, and Tanganyika (now Tanzania) in 1951–1952 (Unlike dengue virus (a related flavivirus), Zika virus was not considered to be a major pathogen until recent reports of its association with Guillain-Barré syndrome and microcephaly , arthralgia, myalgia, and periarticular edema  to approve an interim definition in February 2016 and a final case definition in June 2016 for noncongenital Zika virus disease as >1 of the following signs or symptoms: acute onset of fever, maculopapular rash, arthralgia, and conjunctivitis  . Infected and noninfected patients were similar in age and sex. No female patients were pregnant. Among Zika virus–infected patients, rash (93.3%) was the most common symptom, followed by fever (79.2%) and myalgia (42.3%). Headache, arthralgia, and conjunctivitis were reported in <25% patients with Zika virus disease. Pruritus (11.4%) and gastrointestinal symptoms (6.7%) were relatively uncommon. For patients not infected with Zika virus, fever (86.2%) was the most common symptom, followed by myalgia (59.1%) and rash (44.8%).A total of 42.0% of the cohort had Zika virus infection confirmed in blood (4%), urine (36%), or both (60%) samples . Most (8The case definition recommended by CSTE for use in the United States (US definition) had a sensitivity of 100% and a specificity of 2% in detecting Zika virus in the cohort . The WHODespite increasing incidence of Zika virus disease and its spread across the Americas and Asia, there is no internationally adopted common clinical criteria for the surveillance of this disease. We report a large outbreak cohort of patients with suspected Zika virus infection and comprehensive documentation of clinical symptoms and parallel RT-PCR conducted on blood and urine samples for these patients by 2 laboratories. Evaluation of the performance of surveillance case definitions in such a cohort would provide useful findings that would contribute to development of guidance for Zika virus disease surveillance.Diagnosis of Zika virus disease remains suboptimal because of limited availability of confirmatory testing by RT-PCR during acute illness and cross-reactivity of serologic tests for Zika virus with other co-circulating flaviviruses , although ≈50% (range 44%–51%) of cases of Zika virus disease could be missed.The main limitation of this study is that it included only adults. However, the small number of children infected with Zika virus during the containment phase of the outbreak in Singapore had symptoms similar to those for adults . Some Zika virus infections could have been misclassified as noninfections because RT-PCR could have missed infections late in the illness course or after development of antibodies against Zika virus.In conclusion, we evaluated the performance of 5 case definitions for Zika virus disease surveillance. In the current effort to halt transmission of this virus worldwide, and with laboratory tests being largely inaccessible, use of surveillance case definitions that include rash as a required clinical criteria would provide a high yield in identifying Zika virus disease.


A panel of national experts was convened by the Brazilian Infectious Diseases Society in order to organize the national recommendations for the management of zika virus infection. The focus of this document is the diagnosis, both clinical and laboratorial, and appropriate treatment of the diverse manifestations of this infection, ranging from acute mild disease to Guillain-Barré syndrome and also microcephaly and congenital malformations. Flavivirus, in the family Flaviviridae, which was first identified in 1947, in the Zika Forest in Uganda during a monitoring program on wild yellow fever [The Zika virus is an arbovirus of the genus Outbreaks of the disease were first notified in the Pacific region in 2007 and 2013, respectively in the Yap islands and in French Polynesia, and then in the Americas (Brazil and Colombia) and in Africa (Cape Verde) in 2015 . Rapid gThrough occurrences of Zika outbreaks, the central nervous system and autoimmune complications that were previously reported in French Polynesia have also come to be observed in the Americas.Zika infection during pregnancy has been correlated with congenital microcephaly, fetal malformations and fetal losses. This led the Brazilian Ministry of Health to declare a state of public health emergency of national importance in November 2015, after observation of changes to the epidemiological pattern of occurrences of microcephaly in Pernambuco and other states in northeastern Brazil , 9.In the light of the significant increase in the incidence of neurological syndromes and cases of microcephaly that were potentially related to the Zika virus, the World Health Organization (WHO) declared an international public health state of emergency in February 2016. To put the importance of this event into context, this was the fourth time that WHO has ever declared a worldwide state of emergency in relation to a viral epidemic. The previous decisions were made in relation to H1N1 (2009), poliomyelitis (2014) and Ebola (2014).On February 18, 2016, the Brazilian Ministry of Health issued an ordinance that made it compulsory to notify suspected cases of Zika throughout the country. This disease has thus been added to other arboviruses, such as dengue, yellow fever, West Nile fever and chikungunya, which were already on the national list of diseases with compulsory notification .In humans, the Zika virus was first identified in Uganda and Tanzania, in 1952 . BetweenThe first epidemic outside of Africa and Asia occurred in 2007, in the Yap islands of Micronesia. It was estimated that more than 70 % of the population over the age of 3 years became infected [In 2014, cases of Zika virus infection were reported on Easter Island, which is Chilean territory . In May Worldwide, the virus is now circulating in 65 countries and territories, mostly in the Americas .Aedes, especially Aedes aegypti and Aedes albopictus [Zika is transmitted primarily through the bites of infected mosquitos of the genus bopictus .In humans, except for pregnant women, the period of viremia is short and it is most frequently identified by the 5th day after the symptoms start. The RNA of the Zika virus has been identified in blood as early as on the 1st day, and also only up to 11 days after the disease begins. Prolonged Zika virus RNA was detected in serum of four symptomatic pregnant women in up to 46 days after symptoms onset and in one asymptomatic pregnant woman 53 days after infection . In pregIn addition to vector transmission, other forms of transmission that so far are just theoretical or anecdotal have started to receive greater attention. The RNA of the Zika virus has now been detected in blood, urine, semen, saliva, female genital tract secretions, cerebrospinal fluid, amniotic fluid and breastmilk , 19– 25.After two likely cases of transmission through blood transfusion in Campinas, São Paulo, were identified, there has been much discussion about the importance of this transmission route .Reports of detection of Zika in urine through PCR up to 20 days after the start of symptoms, in a study conducted in French Polynesia, even without any confirmation of infectiveness, have given rise to discussion on the need for better comprehension of the importance of this biological material as an infecting agent .Zika has been detected in semen for periods of up to 10 weeks after recovery from the symptoms of the infection, and likely cases of sexual transmission from men to women have been described. Eleven countries have reported evidence of person-to-person transmission of Zika virus, probably via a sexual route .Tests conducted on the amniotic fluid of pregnant women with possible Zika virus infection whose fetuses had been diagnosed as presenting microcephaly have been found to be positive for the Zika virus . This shPresence of the virus has already been shown through detection of viral RNA in the breastmilk of mothers with conditions of acute infection. It is expected a higher viral load of Zika virus in maternal milk for women infected near delivery, and not expected to occur with first trimester infections. In New Caledonia it has been reported the presence of infectious Zika virus particles in breast milk with substantial viral loads . HoweverIt has been estimated that clinical manifestations occur in around 20 % of infected individuals. Hence, asymptomatic infection occurs more frequently. These estimates have been based on a single study that was conducted through a household serological survey involving serological tests for Zika (IgM) [The duration of incubation in humans is unknown, but it has been estimated to be 2–14 days after the bite of the vector mosquito .The clinical condition typically includes a maculopapular rash, frequently accompanied by pruritus, low fever (37.8–38.5 °C), arthralgia  and non-purulent conjunctivitis. Other manifestations that have commonly been reported include myalgia, headache, retro-orbital pain and asthenia. Periarticular edema, oral lymphadenopathy, oral ulcers, abdominal pain, nausea and diarrhea may also occur , 13, 14.In most patients, the symptoms are usually mild and present spontaneous resolution after 2–7 days. However, in some patients, arthralgia may persist for around 1 month.So far, the length of the immunity conferred through natural infection with the Zika virus remains unknown.Brazilian ministry of health’s definition of suspected zika casesFeverConjunctival hyperemia without secretion and pruritusPolyarthralgiaPeriarticular edema”Suspected case: “Patients who present pruriginous maculopapular exanthema accompanied by two or more of the following signs and symptoms:Brazilian ministry of health’s definition of confirmed zika casesIsolation of the virusDetection of viral RNA through the reverse transcriptase reaction (RT-PCR)IgM serological test ”For epidemiological control after confirmation of autochthonous circulation, other acute cases of Zika should be confirmed through clinical-epidemiological criteria, except when in pregnant women or in situations of neurological manifestations and death.Confirmed case: “Suspected case in which one of the following tests is positive or shows a specific reaction for a diagnosis of Zika:Congenital malformations, including microcephaly, generally have complex multifactorial etiology and may have been caused through infection during pregnancy or through chromosomal disorders, exposure to environmental toxins or metabolic diseases, as shown on Table This relationship has become increasingly consistent, consequent to detection of viral RNA using the PCR technique for Zika in amniotic fluid, placenta, umbilical cord blood and cerebral tissue. The capacity of the virus to infect and cross the placental barrier such that it might then affect the nerve tissue during its formation has also been demonstrated , 33.In Brazil, since the confirmation of the Zika outbreak, the incidence of microcephaly has become more than 20 times higher than what would otherwise have been expected .During an investigation conducted in relation to 35 children with microcephaly, 74 % of the mothers in northeastern Brazil who were suspected of having had Zika during their pregnancies reported that they had had a skin rash during the first and second trimesters [Retrospectively, after Brazil’s notification of cases to WHO, cases of microcephaly were also identified in French Polynesia. These cases have recently been reported in the literature .According to guidance from the Ministry of Health, it is compulsory to notify cases of fetuses that are identified as presenting central nervous system abnormalities during pregnancy.In April 2016, the Centers for Diseases Control and Prevention  concluded that there was a causal relationship between prenatal infection by the Zika virus and microcephaly other cerebral abnormalities. This conclusion was based on evidence regarding Zika virus infection during prenatal development that was consistent with the defects observed, with occurrence of a rare and specific phenotype involving microcephaly and cerebral abnormalities in fetuses or newborns with confirmed or presumed congenital infection due to the Zika virus, and on data that strongly supported biological plausibility, including identification of the Zika virus in the cerebral tissue of the fetuses and babies affected .Presence of cerebral calcifications AND/ORPresence of ventricular abnormalities AND/ORAt least two of the following signs of abnormalities of the posterior fossa: hypoplasia of the cerebellum, hypoplasia of the cerebellar vermis, widening of the posterior fossa greater than 10 mm and agenesis/hypoplasia of the corpus callosum.”“A fetus that presents at least one of the following criteria relating to abnormalities of the central nervous system, as identified through ultrasound examination :PresenceThe findings from transfontanelle ultrasound and/or cranial tomography include [Cerebral calcifications, especially periventricular, in the parenchyma, thalamic areas and basal ganglion.Ventriculomegaly.Lissencephaly.Hypoplasia of the brain stem and cerebellumAbnormality of white matter attenuation. include , 38, 39:Other ultrasound findingsArthrogryposis.Intrauterine growth retardation.Arterial flow abnormalities in the cerebral or umbilical arteries.Oligohydramnios or anhydramnios.❖ Ultrasound findings can be detected from the 18th to 20th week of pregnancy onwards.There may be ocular involvement, especially pigment abnormality and macular atrophy, and also abnormalities of the optic nerve , 41.Guillain-Barré syndrome (GBS) is an autoimmune disease characterized by acute inflammatory demyelinating polyradiculoneuropathy. Motor function is usually affected, starting distally and progressing proximally over a 4-week period. The patients present generalized weakness, areflexia and varying degrees of sensory disorders and disorders of cranial nerve involvement. Its forms range from those with both motor and sensory impairment to those solely with sensory impairment. The risk increases with age and it occurs more frequently among men than among women. Approximately 25 % of the patients require admission to an intensive care unit and 3–5 % die. The expected annual incidence is one case per 100,000 inhabitants .Higher incidence of GBS was observed concomitantly with the Zika outbreaks in French Polynesia, Brazil, El Salvador, Colombia, Suriname and Venezuela .A retrospective case–control study conducted in French Polynesia and published in February 2016 reported on 42 cases of GBS that occurred during the outbreak of 2013–2014, with detection of IgM or IgG for Zika in 98 % of the patients and neutralizing antibodies for Zika in 100 % of the cases, compared with 56 % of the control group. The most common symptoms were generalized muscle weakness (74 %) and facial paralysis (64 %) .Bilateral facial paralysis was also frequently observed. Most of the patients (88 %) reported that, on average 6 days before the start of neurological symptoms, they had a condition that was compatible with Zika virus .In Venezuela, 252 cases of GBS were notified between January 1 and 31, 2016, with PCR positive for Zika in three cases, including one fatal case .In Brazil between January and November 2015, 1708 cases of GBS were notified. This total reflected very significant increases in the numbers of cases in some states, especially Alagoas (516.7 %), Bahia (196.1 %), Rio Grande do Norte (108.7 %), Piauí (108.3 %), Espirito Santo (78.6 %) and Rio de Janeiro (60.9 %) .So far, the determinants relating to the increased incidence of GBS in Brazil, Colombia, El Salvador and Suriname have not been definitively established, especially with regard to the scenario of simultaneous circulation of dengue, chikungunya and Zika, which are all potentially related to occurrences of neurological syndromes .Other arbovirus diseases such as dengue, chikungunya, Japanese encephalitis and West Nile fever have already been incriminated as agents related to occurrences of GBS.Just like other flaviviruses, the Zika virus may cause other neurological syndromes such as meningitis, meningoencephalitis and myelitis, as described in the outbreak in French Polynesia .The laboratory abnormalities are nonspecific. There have been reports of mild to moderate leukopenia and thrombocytopenia, and slight elevation of assayed concentrations of serum lactic dehydrogenase, gamma glutamyl transferase and markers of inflammatory activity  .Because of the scarcity of clinical studies, what has been described are slight abnormalities in hematological parameters and even smaller changes in liver enzymes. In general, there are no significant abnormalities in these parameters.Dengue: higher fever and greater severity of myalgia and asthenia. There may be complications due to hemorrhage and hemodynamic abnormalities, including shock. Does not usually cause conjunctivitis. Significant abnormalities in laboratory tests, with hemoconcentration, low platelet count and liver enzyme abnormalities.Chikungunya: high fever as observed in dengue, but with the major differential that the polyarthralgia/polyarthritis starts suddenly in a severe and debilitating manner. This may be associated with articular/periarticular edema from the outset of the condition. Pruriginous skin rash may be present, with a duration that is usually shorter than in Zika. It may follow a course that includes mild conjunctivitis.Parvovirus: may cause acute symmetrical arthritis or arthralgia, most frequently in the small joints of the hands and feet, and in the wrists and knees. Skin rash is frequently observed.Rubella: generally causes low fever and coryza. Presence of skin rash initially of the face and then spreading to the trunk. There may be arthritis and lymphadenopathy.Measles: presence of fever, coughing, sore throat, coryza, conjunctivitis and lymphadenitis. Koplik spots may precede the generalized rash.Rickettsiosis: characterized by occurrence of fever, headache, myalgia and centripetal non-pruriginous maculopapular exanthema. The complications have included hemorrhagic suffusion, hemorrhagic, respiratory insufficiency, kidney failure, neurological abnormalities and shock.Malaria: periodic fever, paroxysm, kidney failure, jaundice, altered levels of consciousness, hepatomegaly or splenomegaly and history of exposure in transmission areas.Leptospirosis: severe myalgia, ocular suffusion, rubinic jaundice, oliguria and subconjunctival hemorrhage. History of exposure to contaminated water.The main differential features of dengue, chikungunya and zika virus infection are shown on Table Many recent publications have emphasized the occurrence of dual infections, including Zika and Dengue, and Zika and Chikungunya co-infections –51. WithThe specific laboratory diagnosis is based mainly on detection of viral RNA from clinical specimens. In blood samples, detection is possible for a period of 1–5 days after the start of symptoms , 53. NegBecause of the greater persistence of the virus in urine, patients seen after the fifth day of the disease should undergo RT-PCR on urine. This analysis is indicated up to the 15th day after the start of symptoms , 53.It is generally considered that serological tests are able to detect IgM from the 4th day and IgG from the 12th day.Serological tests for Zika in populations with simultaneous or previous circulation of other flaviviruses may be imprecise because of the risk of cross-reactions, thereby leading to false positive results. For this reason, positive results should be analyzed cautiously since they might represent previous exposure to other flaviviruses (such as the dengue virus) or vaccination in the past against yellow fever or Japanese encephalitis , 53.Negative serological results (non-reactive IgM and IgG) suggest that infection did not occur, if the test was done between 2 and 12 weeks after the exposure . Figure Brazilian Ministry of Health’s guidelines determines to collect samples from the first cases in an area without laboratory confirmation of acute Zika virus disease, from 100 %  of the pregnant women with suspected acute Zika virus disease, from 100 %  of the deaths suspected to be due to acute Zika virus disease and from 100 %  of the patients hospitalized in sentinel units, with neurological manifestations and with suspected previous viral infection .A positive molecular test confirms the diagnosis of Zika.A negative molecular test does not rule out the possibility of Zika.A reactive serological test for Zika may be due to acute infection by the virus, a cross-reaction with other flaviviruses or a result from yellow fever vaccination.There is no specific antiviral treatment.The treatment consists of rest, oral hydration and use of medications for symptoms.Analgesics and antipyretics such as dipyrone and paracetamol.Antihistamine medications to control itching.Non-steroidal anti-inflammatory drugs (NSAIDs) should not be used until the diagnosis of dengue has been ruled out . Avoid tAvoid use of aspirin in children under the age of 12 years because of the risk of Reye syndrome.It is important to evaluate differential diagnoses, especially in relation to dengue, because of the greater risk of evolution to severe cases. Treat all cases as dengue until this diagnosis has been ruled out.Currently, only limited data on pregnant women infected with the Zika virus are available. The data suggest that pregnant women may become infected with the Zika virus in any trimester. However, the incidence of Zika virus infection among pregnant women is unknown. There is no evidence to suggest that pregnant women are more susceptible to Zika or that they present disease of greater severity than do other individuals .Regarding transmission of the Zika virus, there is evidence of virus transmission from the mother to the fetus during pregnancy and also close to the time of delivery. Since no vaccines or prophylactic medications for avoiding Zika virus infection exist, the Brazilian Society of Infectology (SBI) recommends  that women in any trimester of pregnancy should consider postponing journeys to areas with transmission of this virus. If a pregnant woman lives in or travels to an area with transmission of the Zika virus, she should protect herself to avoid mosquito bites.Pregnant women who present symptoms compatible with Zika  need to be given priority for laboratory investigation in order to diagnose Zika virus infection.Routine prenatal use of serological tests for Zika among pregnant women living in areas with the Zika epidemic is not recommended by the Brazilian Ministry of Health.If a laboratory test is positive for Zika or inconclusive, use of serial ultrasound examinations should be considered.For pregnant women whose fetus has a confirmed diagnosis of microcephaly, the possibility of amniocentesis should be evaluated from the 15th week of pregnancy onwards.Pregnant women in a transmission area should undergo serological tests for Zika at the start of their prenatal care.Serological tests should be offered to women in areas without autochthonous transmission if they have a history of travel to an area with known Zika virus transmission and are asymptomatic (i.e. without reports of sickness consistent with Zika). These tests should be performed 2–12 weeks after the trip.The Brazilian Federation of Gynecology and Obstetrics Associations (FEBRASGO) recommends the following in cases of pregnant women with clinical manifestations of Zika:Ultrasound examination should be scheduled every month until delivery The American Society of Gynecology and Obstetrics recommends the following in cases of pregnant women with serological findings that are positive for Zika or inconclusive, and/or who show symptoms of Zika infection:If, at the time of the symptoms or the serological tests, the pregnant woman has not reached her 20th week of pregnancy, an ultrasound examination should be scheduled every 3 to 4 weeks, starting at the 18th week.If, at the time of the symptoms or the serological tests, the pregnant woman is already beyond her 20th week of pregnancy, an ultrasound examination should be scheduled every 3–4 weeks, from the time of diagnosis onwards.The summary of current reccomendations for diagnostic of zika virus infection in pregnant women in shown on Fig. It is very important to record the gestational age at birth when assessing the size of the skull in newborns .The INTERGROWTH-21st allows measuring the head circumference, at birth, across many different populations and it has been obtained through rigorous methodology and standardised procedures. This is very important in the context of the Zika virus outbreak, as reliable information on the head circumference of newborns according to their gestational age is required so as to screen for microcephaly .Criteria for notification of newborn with microcephaly according to the Ministry of Health’s recommendations :Newborns with gestational age of less than 37 weeks that present a head circumference measurement of less than −2 standard deviations for the gestational age and sex, as described in the InterGrowth table.Newborns with gestational age of 37 weeks or more that present a head circumference measurement of less than or equal to 31.5 cm for girls and 31.9 for boys, which is equivalent to less than −2 standard deviations for the neonate’s age and sex, as described in the WHO table.ndations :NewbornsAccording to WHO and the international literature, microcephaly is defined as a head circumference of less than minus two standard deviations of the reference for the sex, age or length of gestation. The head circumference must be measured using standardized technique and equipment between 24 h and 6 days 23 h after birth (i.e. within the 1st week of life).Severe microcephaly is defined as a head circumference of less than −3 standard deviations, i.e. more than three standard deviations below the mean for the gestational age and sex.According to WHO, newborns with microcephaly who present structural abnormalities of the brain, as diagnosed through imaging examinations or observed neurological or developmental abnormalities, should be classified as having “microcephaly with brain abnormality”. All neonates with microcephaly should receive regular evaluations and follow-up during their childhood, including: head growth, the mother’s and family’s histories of pregnancies, evaluation of development and physical and neurological examinations, including evaluation of hearing and eyesight, to identify any problems. To detect structural abnormalities of the brain, WHO recommends that transfontanelle ultrasound examinations should be performed when the size of the fontanelle is sufficient for this procedure. For neonates who present severe microcephaly (−3 standard deviations), cerebral computed tomography or magnetic resonance imaging should be performed.Microcephaly may be accompanied by epilepsy, cerebral palsy, delayed cognitive, motor and speech development and hearing and eyesight problems.There is no specific treatment for microcephaly. Since each child develops complications that differ in type and severity, which may include respiratory, neurological and motor problems, the follow-up by different specialists will depend on which functions have been compromised.The summary of current reccomendations for diagnostic of Zika virus infection in newborns in shown on Fig. Because of the autoimmune nature of Guillain-Barré syndrome, its treatment in the acute phase consists of immunotherapy, such as plasmapheresis or application of human immunoglobulin. When corticosteroids are used separately, this does not accelerate the recovery or alter the long-term result .The aim of plasmapheresis is to remove the antibodies from the bloodstream and replace them with artificial plasma, usually albumin. The result is better when the procedure is started within the first 7–14 days after the onset of neurological symptoms .Human immunoglobulin accelerates recovery, as observed with plasmapheresis. It is relatively simple to administer. The best results are obtained when it is started within the first 2 weeks after the symptoms begin .Endovenous dose of human immunoglobulin: 400 mg/kg of body weight per day, for a period of 5 days.Clinical history and neurological examination.Collection of cerebrospinal fluid, in which it is expected to find an increased protein concentration, to the detriment of increased cellularity.3 should be investigated for other etiologies or for concomitant HIV infection.Patients with suspected syndrome and cellularity greater than 50 cells/mmThe cerebrospinal fluid may be normal in the hyperacute phase (first week).Electroneuromyography is the examination that confirms the diagnosis, but it may be normal in the 1st week.Imaging examinations are generally normal.Diagnosis of Guillain-Barré syndrome :ClinicalBasic sanitation.Elimination of vector foci in homes and common areas.Reduction of garbage accumulation through urban cleansing campaigns in areas where garbage collection is irregular, and through increasing the frequency of garbage collection.Implementation of vector control through physical, biological and chemical methods, with involvement of families and communities.In areas with autochthonous transmission or imported cases of transmission of dengue, chikungunya and/or Zika, blockage of cases through using pesticides targeting adult vectors, primarily through spraying, is recommended, in addition to use of larvicides.Fieldwork done by endemic disease agents for controlling both the larvae and the adult mosquitos needs to be monitored and controlled.Mosquito control is the only measure that can interrupt the transmission of arbovirus diseases such as Zika, dengue and chikungunya.Vector control measures:Aedes during the 1st week (viremic phase).In order not to infect other people, infected individuals should protect themselves from bites by There is still no vaccine for prevention of infection by the Zika virus.Steps for preventing mosquito bitesUse long-sleeved shirts and long trousers/pants.Stay in enclosed places with air conditioning or places with windows and doors that have screens to prevent the entry of mosquitos.Sleep under mosquito nets.Always follow the guidance of instruction leaflets.Avoid using products that combine repellent and sun protection in the same formulation. The sun protection factor decreases by one-third when used together with insect repellent.If sun protection is used, apply it before applying the repellent.Use registered insect repellents. When these are used as instructed, they are safe and effective, even during pregnancy or breastfeeding.Do not use repellent on children under the age of 2 months.Dress children in clothes that cover their arms and legs.Cover cots and pushchairs with mosquito nets.Do not apply repellent to children’s hands.For childrenDo not use products containing permethrin directly on the skin.Clothes impregnated with permethrin can be used.In Brazil, the National Health Surveillance Agency (ANVISA) only recommends use of repellents for children over the age of 6 months. The Centers for Disease Control and Prevention (CDC) recommends their use from the age of 2 months, except for lemon eucalyptus, which should only be used from the age of 3 years.Annals of Clinical Microbiology and Antimicrobials because it is an official document of the Brazilian Infectious Diseases Society and contains the recommendations for managing this infection based on the experience that Brazilian infectious disease specialists gained after facing this epidemic. This document also considers the most recent scientific evidence about zika transmission and clinical features. We intend to submit this article for a special supplement about Arboviruses at Annals of Clinical Microbiology and Antimicrobials.We believe that our article “Management of Infection by the Zika Virus” should be published in All authors declare no competing interests and confirm that have approved the manuscript for submission. We also confirm that the content of the manuscript has not been published, or submitted for publication elsewhere.


In plants, the chloroplast is the organelle that conducts photosynthesis. It has been known that chloroplast is involved in virus infection of plants for approximate 70 years. Recently, the subject of chloroplast-virus interplay is getting more and more attention. In this article we discuss the different aspects of chloroplast-virus interaction into three sections: the effect of virus infection on the structure and function of chloroplast, the role of chloroplast in virus infection cycle, and the function of chloroplast in host defense against viruses. In particular, we focus on the characterization of chloroplast protein-viral protein interactions that underlie the interplay between chloroplast and virus. It can be summarized that chloroplast is a common target of plant viruses for viral pathogenesis or propagation; and conversely, chloroplast and its components also can play active roles in plant defense against viruses. Chloroplast photosynthesis-related genes/proteins (CPRGs/CPRPs) are suggested to play a central role during the complex chloroplast-virus interaction. Plant viruses, as obligate biotrophic pathogens, attack a broad range of plant species utilizing host plants' cellular apparatuses for protein synthesis, genome replication and intercellular and systemic movement in order to support their propagation and proliferation. Virus infection usually causes symptoms resulting in morphological and physiological alterations of the infected plant hosts, which always incurs inferior performance such as the decreased host biomass and crop yield loss.The most common viral symptom is leaf chlorosis, reflecting altered pigmentation and structural change of chloroplasts. Viral influence on chloroplast structures and functions usually leads to depleted photosynthetic activity. Since the first half of the twentieth century, an increasing number of reports on a broad range of plant-virus combinations have revealed that virus infection inhibits host photosynthesis, which is usually associated with viral symptoms  fluctuation of chlorophyll fluorescence and reduced chlorophyll pigmentation , affect chloroplast ultrastructure and symptom development (see below).N. tabacum leaves  have been demonstrated as determinants of symptom phenotypes for a much long period  protein in tobacco plants results in a virus-like chlorosis symptom associated with the abnormal thylakoid stacks Figure , Table 2Studies on the effect of virus infection on expression of chloroplast proteins at the transcriptomic and proteomic levels provide insights into the molecular events during symptom expression. In the susceptible plant response to virus infection, the majority of significantly changed proteins are identified to be located in chloroplasts or associated with chloroplast membranes. Most of them are down-regulated and correlate with the severity of chlorosis Dardick, . During As the biosynthesis of CPRPs is a complicated process with a series of steps Seidler, , plant vflavum strain leads to a total depletion of PS II core complex and OEC, including chloroplast-encoded CPRP PsbA and nuclear-encoded CPRPs LhcB1, LhcB2  and PsbO. However, the PsbA mRNA accumulated to a higher level in the infected leaves  gene transcripts and down-regulates its expression by RNA silencing  with blocked chloroplast development and depletion of chloroplast-encoded proteins  RNA which can disturb chloroplast function and induce disease symptoms  pathway for targeting CPRGs transcripts. The tenuivirus RSV, causing a devastating disease in East Asia countries, hijacks CPRP during infection and perturbs photosynthesis  to fulfill the transport via symplastic routes within plant hosts , a gene involved in the JA signaling pathway, accelerates the development of symptoms caused by co-infection of PVX and PVY, and accumulation of viral titers at early stages of infection  in the chloroplast membranes , and the photosynthetic electron transport chain is responsible for ROS generation Asada, . Superoxs Asada, . The burs Asada, . Chlorops Asada, .N-mediated TMV resistance response. Further, a number of stromules surround nuclei during plant defense response, which is correlated with the accumulation of chloroplast-localized defense protein NRIP1 and H2O2 in the nucleus. In the absence of virus infection, suppression of chloroplast CHUP1 induces stromules and enhances programmed cell death constitutively  protein of potyviruses is required for virus replication and cell-to-cell movement. CI protein from PPV and TVMV interacts with photosystem I PSI-K protein Table , the proL88 with high RNAi suppressor activity , a rhodanese sulfurtransferase which is destined to chloroplast under normal conditions, associates with both the tobacco N receptor and 126 K replicase during TMV infection; its relocation from chloroplast to cytoplasm and nucleus is required for N-mediated resistance to TMV , the National Basic Research Program of China (2014CB138400), the Special Fund for Agro-scientific Research in the Public Interest of China (201303028), and the China Postdoctoral Science Foundation (2014M550049), the Initial Funding of Zhejiang Academy of Agricultural Sciences, and the Cultural Funding for Youth Talent of Zhejiang Academy of Agricultural Sciences (2015R21R08E03).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.