The persistence of biological systems requires evolved mechanisms which promote stability. Cohesive primate social groups are one example of stable biological systems, which persist in spite of regular conflict. We suggest that genetic relatedness and its associated kinship structure are a potential source of stability in primate social groups as kinship structure is an important organizing principle in many animal societies. We investigated the effect of average genetic relatedness per matrilineal family on the stability of matrilineal grooming and agonistic interactions in 48 matrilines from seven captive groups of rhesus macaques. Matrilines with low average genetic relatedness show increased family-level instability such as: more sub-grouping in their matrilineal groom network, more frequent fighting with kin, and higher rates of wounding. Family-level instability in multiple matrilines within a group is further associated with group-level instability such as increased wounding. Stability appears to arise from the presence of clear matrilineal structure in the rhesus macaque group hierarchy, which is derived from cohesion among kin in their affiliative and agonistic interactions with each other. We conclude that genetic relatedness and kinship structure are an important source of group stability in animal societies, particularly when dominance and/or affilative interactions are typically governed by kinship. Stability in biological systems has been described as the persistence of regularities, and evolved mechanisms are necessary to promote or maintain this stability Stable social groups are those that persist through time in spite of competition that regularly arises among group members. Among wild primate groups, a reduction in group stability may lead to group fission Macaca nemestrina). Conflict management by third parties, also called policing, involves impartial intervention upon others' conflicts resulting in termination of the conflict. Flack and colleagues found that temporary removal of conflict managers resulted in an increase in the rate of biting and intensity of aggression, thereby destabilizing the group The mechanisms that contribute to group stability may depend on a number of different factors, such as conflict resolution or reconciliation The mechanisms which promote group stability have yet to be identified for most animal societies, including the study species, rhesus macaques. Although conflict management appears to be a stabilizing mechanism in pigtailed macaque society, the highly skewed social power structure required to give some individuals sufficiently high social power with which to police the rest of the group may not be present in most societies. We suggest an alternative source of stability: kinship.Kinship has long been thought to contribute to group and matriline cohesion in primate groups, although this assumption has rarely been demonstrated empirically. According to kin selection theory, most altruism and cooperation occurs between close genetic relatives because the cost to the actor is offset by the fitness benefit gained through genes shared with the recipient by common descent, thereby maximizing the actor's inclusive fitness Stability in a biological system is a higher-level outcome which arises from the interactions among lower-level components within the system. Social network theory (SNT) is therefore an ideal method for investigating the emergence of stability from interactions among group members. In recent years, biologists have increasingly used SNT to detect higher-level properties of biological systems from dyadic interactions among components Grooming has long been known to serve a social function in primate societies, thus social grooming is one potential way for individuals to cope with competition and preserve group stability. First, grooming reduces tension and stress by lowering heart rate among those being groomed and lowering cortisol levels among both groomers and those being groomed Social groups of rhesus macaques consist of clusters of maternally related females called matrilines, and females show a preference for associating with kin. Grooming and aid in fights are patterned by degree of kinship, creating a matrilineal structure to agonistic and affiliative relationships Rhesus macaques are classified as the most despotic of the macaques same matriline. Both types of instability could originate from a low average relatedness coefficient among members of a matriline. First, a low average relatedness coefficient within a matriline indicates that the matriline is composed of a greater proportion of distant kin dyads than close kin dyads. Since agonistic alliances among kin are patterned upon degree of relatedness Macaca fuscata), lower-ranking matriline members given the opportunity to outrank a more dominant female  will sometimes ally with the dominant female to outrank her own kin Matrilineal ranks might become destabilized if there is instability regarding the relative ranks among matrilines or among members of the The goal of this study was to investigate the factors that influence the persistence of social groups, using rhesus macaques as a model species, by identifying those factors or circumstances which result in the opposite: instability. We calculated measures of instability at the matriline-level in seven groups of captive rhesus macaques. We investigated whether increased fragmentation within a matrilineal pedigree  is associated with a greater number of communities per matrilineal grooming network, which may be an indication of instability within the matriline. We further investigated whether fragmentation within a matrilineal pedigree is associated with instability of matrilineal dominance using four measures of aggressive behavior at the matriline level: (1) the proportion of aggressive dyadic interactions initiated by a matriline using intense aggression, (2) the proportion of fighting events in which a matriline participates where the initiator and recipient are kin (3) the proportion of fighting events in which the matriline participates and in which intense aggression is involved where the initiator directs intense aggression at members of her own matriline, and (4) the frequency of wounding/injury received per matriline. Finally, in order to assess whether matrilineal-level instability influences group-level instability, we investigated whether a group-level average of matrilineal fragmentation is associated with the average wounding rate per group and the age of the group. In total, we used these analyses to evaluate the potential role of kinship structure, via genetic relationships, as a mechanism of stability in rhesus macaque social groups.All research reported in this manuscript adhered to the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, the laws of the United States government, and the recommendations of the Weatherall report, “The use of non-human primates in research”. All research subjects were housed in large social groups in half-acre outdoor enclosures to provide for their psychological well-being. The methodological approach was purely observational and involved no experimental or invasive treatment of the animals. All occurrences of illness or injury among study subjects were immediately reported to and treated by CNPRC veterinary staff, and all efforts were made to ameliorate suffering. This project was approved by the University of California, Davis Institutional Animal Care and Use Committee, protocol #11843.The study was conducted at the California National Primate Research Center (CNPRC) in Davis, CA from June 2008 through November 2009. The subjects of this study were 48 matrilines from seven groups  of rhesus macaques housed in 0.2 ha enclosures . MinimumAll enclosures were similar in having ten A-frame houses, multiple suspended barrels, swings and several perches. Groups were fed a standard monkey chow diet twice per day at approximately 0700 hours and again between 1430 and 1530 hours. Fresh fruit or vegetables were provided twice per week.Rhesus macaques in this outdoor colony were managed with a minimal level of disturbance, and individuals of each group were free to interact with one another as they chose. Disturbances within the enclosure were typically limited to daily morning health checks, two round-ups per year to conduct health examinations on all animals and removal of injured or sick animals for medical treatment.Two observers (primary observers: BAB and MEJ) recorded both affiliative and aggressive interactions among members of 48 matrilines in seven groups to evaluate the degree of fragmentation in matrilineal grooming networks, measured as the number of communities per groom network An event sampling design was used to collect data on agonistic interactions for approximately 6 hours per day. Agonistic interactions were recorded as an ordered series of dyadic interactions. Both aggressive and submissive behaviors were categorized in increasing levels of severity. Aggression included threat, vocal threat or threat and follow, lunge or mild slap, chase <3 meters, chase >3 meters or grapple, bite <5 seconds, chase and bite <5 seconds, and bite >5 seconds. Submission included silent bared teeth display (SBT), turn away, turn away with SBT, move out of arms' reach, move out of arms' reach with SBT, run away <3 meters, run away <3 meters with SBT, run away >3 meters, run away >3 meters with SBT, prolonged scream, crouch , and crouch with SBT. Intense aggressive interactions included bite <5 seconds, chase and bite <5 seconds, and bite >5 seconds.Scan samples of dyadic interactions of grooming and contact-sitting were conducted every half hour. Affiliation scan samples were discontinued when aggressive events occurred. A total of 5767 grooming interactions were recorded (427–1290 per group) and 12,250 fighting events (1521–2175 per group) involving 29,849 aggressive dyadic interactions. Each fighting event consisted of one or more sequential aggressive dyadic interactions, involving two or more individuals. Affiliative and aggressive interactions were recorded for both males and females 3 years and older.The heart of the matter of matrilineal cohesion is the definition of a matriline. The boundary of matriline membership is not necessarily defined by the presence of a matriarch, as two halves of a matriline whose matriarch is gone may still regard one another as kin, particularly if sisters have a strong relationship or the matriarch has not been gone long. However, the precise number of connecting females (such as the matriarch) that must be absent before separate branches of a matriline no longer regard one another as kin is not known. Among our study groups, defining matriline boundaries solely by descent from a matriarch that is present results in little variation in the average coefficients of relatedness, and actual kinship relationships appear to be discounted. Therefore, individuals were considered part of the same matriline if they could be traced back to the same female genetic common ancestor at the time of group formation. The oldest groups (10 and 16) were formed in 1976 (matrilines span 5–7 generations) and the youngest group (1) was formed in 1995 (matrilines span 3–5 generations). Males in these captive groups cannot disperse, and were therefore available to interact with their maternal kin and contribute to matrilineal cohesion. As natal males in these study groups do regularly interact  with their maternal kin All study subjects were born in captivity and all subsequent births recorded, thus all maternal kin relationships were known. Paternal kin relationships, whereby paternal siblings or half-siblings form special relationships, were not taken into account. Although paternal kinship has been found to influence social behavior in rhesus macaque societies Relative matrilineal ranks were determined from behavioral management staff records of weekly observations of displacements and aggressive interactions and were supplemented by observations from this study. All members of the same matriline generally held the same rank. However, in cases where some matriline members held a different rank from the rest of their family, the matrilineal rank assigned was the rank held by the majority of the matriline members.Social networks of grooming interactions were visualized for each matriline  using UCINET 6.247To determine whether random processes could have created the observed community structure, we constructed random networks for each matriline and compared their community structure with the observed networks. Each random network was created by simulating a set of edges for all possible dyads; the probability of edge presence was equal to the proportion of all possible edges observed in the real networks. One thousand simulations were run for each matriline. Inspection of the simulated random networks revealed that few of the observed matrilineal networks had a community structure that could have been produced by random processes. The proportion of simulated networks having the same community structure as the observed network was less than 5% for 31 matrilines, 5–9% for 11 matrilines, and greater than 10%  for six matrilines. We conclude from these comparisons that the observed network structure is not better explained by random processes.We analysed the data using linear and generalized linear mixed-effects regression models A generalized linear mixed-effects regression model (Poisson distribution) was fit to the counts of communities per matrilineal groom network using a robust estimator (aka Huber variance) of the covariance matrix Linear mixed-effects regression models (Gaussian distribution) were fit to the three measures of matriline aggression: proportion of aggressive dyadic interactions initiated per matriline using intense aggression, proportion of fighting events in which a matriline participates where the initiator and recipient are kin, and the proportion of fighting events in which the matriline participates and in which intense aggression is involved where the initiator directs intense aggression at kin. Fixed effects included adult matriline size, average coefficient of relatedness within a matriline, matrilineal rank, and presence of the matriline's matriarch. A generalized linear mixed-effects regression model (Poisson distribution) was fit to the counts of wounds/injuries received per matriline. Fixed effects included adult matriline size, the number of communities per matrilineal groom network, number of nodes per matrilineal groom network, matriline rank, and presence of the matriarch. Finally, simple linear regressions were run on the group-level dependent variables and each analysis was limited to a single variable due to small sample size (N = 7 groups). All analyses were performed using Stata  and the R statistical computing program The best fit model included fixed effects for average coefficient of relatedness per matriline and matriline size. However, there were four other models with ΔAIC ≤2, indicating that all five models are equally good at explaining the observed variation in number of communities per matrilineal groom network. Three of these five models included average relatedness as a significant predictor, and four of the five included matriline size as a significant predictor .In the best fit model, matrilines having a higher coefficient of relatedness showed significantly fewer communities in their grooming networks  matriline size , (3rd) matriline size and number of nodes per network , (4th) average matrilineal coefficient of relatedness , matriline size , and number of nodes per network , and (5th) average matrilineal coefficient of relatedness  and nodes per network . These models indicate that, in addition to the average matrilineal coefficient of relatedness and matriline size, the networks with a greater number of nodes had significantly more communities than those with fewer nodes . The direction and magnitude of the effect of all variables is similar among all five best fit models.The remaining four best fit models included the following fixed effects: . As expected, higher ranking matrilines initiate a significantly greater proportion of fights using intense aggression than lower ranking matrilines .The best fit model included a fixed effect for average coefficient of relatedness per matriline . Matrilines with a lower average coefficient of relatedness participated in a higher proportion of fighting-events in which both combatants were members of that matriline . The predicted proportion of fighting-events between kin, calculated for minimum, mean, and maximum values of average coefficients of relatedness observed in the study groups, are 12.2%, 7.9%, and 1%, respectively.The best fit model included a fixed effect for average coefficient of relatedness per matriline . As predicted, matrilines with a lower average coefficient of relatedness participated in a higher proportion of fighting-events involving intense aggression in which both combatants were members of that matriline and directed intense aggression at one another . Matrilines having a greater number of communities in their groom networks received wounds and injuries requiring hospitalization more frequently than matrilines having fewer groom communities . Matrilineal groom networks having fewer nodes  received wounds and injuries more frequently than those with more nodes . Larger matrilines received more wounds and injuries than smaller matrilines . Finally, matrilines whose matriarch was present received fewer wounds and injuries than matrilines whose matriarch was absent . The expected number of injuries received per matriline, assuming the matriarch is present and average values for matriline size and number of nodes, is 4.8, 6.4, and 9.7 for networks with 1, 3, and 6 communities, respectively. The expected number of injuries received per matriline, assuming average values for matriline size, number of communities and number of nodes, is 7.3 and 12.9 for matrilines with their matriarch present and absent, respectively.2 = 0.69).We fit a linear regression model to the average value per group (N = 7 groups) of communities per matrilineal groom network using a single predictor: the group-mean average coefficient of relatedness per matriline . This analysis revealed the same relationship between matriline fragmentation and community structure on the group-level as was found for the matriline-level: groups having a higher average value of the average coefficient of relatedness per matriline have a lower average value of communities per matriline .We fit a linear regression model to the rate of wounding per group (N = 7 groups) using a single predictor: the group-mean average coefficient of relatedness per matriline. Groups with a higher group-mean average coefficient of relatedness per matriline showed lower rates of wounding . The age range observed for the seven study groups is 13.5–32.2 years (mean  = 22.1 years).We fit a linear regression model to the group-mean average coefficient of relatedness per matriline using a single predictor: cage age (measured in years). Older cages had significantly lower group-mean average coefficient of relatedness per matriline than younger cages  further supports this relationship between matriline fragmentation and stability of dominance relationships. Although wounding is rarely witnessed during observations, there is a strong implication that fragmented matrilines are targeted with more severe aggression because their social position is not stable, and is therefore contestable.Our results further support the relationship between social instability and frequency of aggression found in pigtailed macaques. Induction of group instability increased the frequency and intensity of aggression and was associated with smaller, less diverse affiliative networks Our matriline-level results indicate that a high degree of matrilineal genetic relatedness results in cohesive matrilineal relationships that are characterized by less aggression and wounding and integrated grooming ties. Cohesive relationships among kin may serve two functions: (1) to unite kin against non-kin during conflict, which reduces the likelihood that other, lower-ranking matrilines will perceive an opportunity to improve its current social position, and (2) to unite kin such that their unity reduces the likelihood of fighting amongst themselves for higher social rank within the family.The mechanism by which genetic relationships influence the stability of dominance ranks is through alliances. Inter-matriline ranks as well as individual ranks are maintained by complex networks of alliances among kin as well as among nonkin Matriline-level instability appears to translate into group-level instability when multiple matrilines within the group are fragmented. Groups with a lower group-mean matrilineal coefficient of relatedness had higher rates of wounding and more communities per matrilineal groom network, suggesting that there is a cumulative effect of matriline-level instability on group-level instability. Furthermore, high levels of genetic fragmentation within a matriline appear to be related to the age of the group. Among our study groups, those that have been together longer had lower values of group-mean matrilineal coefficient of relatedness. Thus, older groups will have more matrilines which have lost their matriarchs or other adult females that had served as connections between dyads of more distant kin. Genetic fragmentation within a matriline (and within a group) may be an inevitable outcome for an aging group, particularly when group membership cannot be adjusted to new circumstances via fission or dispersal. In human social networks, for example, groups persist for longer when group membership is fluid and individuals have the option of joining or leaving a given group Social groups at their maximum degree of instability are expected to respond to the instability via group fission or social overthrow. In captivity, maximal instability results in a social overthrow, whereby the social hierarchy is disregarded by group members and severe aggression erupts Group stability may be robust to the absence of a key adult female or the presence of a single fragmented matriline. However, we suggest that a perturbation to an already fragmented high-ranking matriline or to a group consisting of multiple fragmented matrilines may be the final push toward maximal instability. The social overthrows observed in two CNPRC groups (16 and 12) support this conclusion. In group 16, the alpha matriline average coefficient of relatedness and group-mean matrilineal coefficient of relatedness were the lowest values recorded for all study groups , and social overthrow occurred 4 days following the removal of the alpha female (for treatment of conjunctivitis). In group 12, the alpha matriline average coefficient of relatedness was 0.14 (study group mean: r = 0.18), and social overthrow occurred 10 days following the removal of the alpha female (for pregnancy complications). Thus, the sudden absence of a key adult female within an already unstable matriline or group may be a necessary perturbation to precipitate the sufficient degradation of alliance networks or the matrilineal hierarchy such that a social overthrow occurs.The persistence of stable social groups in primate societies, like the persistence of other complex biological systems, appears to be dependent upon the presence of a number of factors that promote stability. Genetic relationships create a higher-level structure within the group, and this kinship structure has a significant impact on both the affiliative and hierarchical relationships that govern interactions among group members. In general, our results further support that stability in biological systems may stem in large part from the nature of the underlying structures of the system, structures which are characterized by the pattern of relationships among the individual agents of the biological system


Micronutrient deficiencies (MNDs) are a chronic lack of vitamins and minerals and constitute a huge public health problem. MNDs have severe health consequences and are particularly harmful during early childhood due to their impact on the physical and cognitive development. We estimate the costs of illness due to iron deficiency (IDA), vitamin A deficiency (VAD) and zinc deficiency (ZnD) in 2 age groups (6–23 and 24–59 months) of Filipino children by socio-economic strata in 2008.We build a health economic model simulating the consequences of MNDs in childhood over the entire lifetime. The model is based on a health survey and a nutrition survey carried out in 2008. The sample populations are first structured into 10 socio-economic strata (SES) and 2 age groups. Health consequences of MNDs are modelled based on information extracted from literature. Direct medical costs, production losses and intangible costs are computed and long term costs are discounted to present value.Total lifetime costs of IDA, VAD and ZnD amounted to direct medical costs of 30 million dollars, production losses of 618 million dollars and intangible costs of 122,138 disability adjusted life years (DALYs). These costs can be interpreted as the lifetime costs of a 1-year cohort affected by MNDs between the age of 6–59 months. Direct medical costs are dominated by costs due to ZnD , production losses by losses in future lifetime  and intangible costs by premature death  and losses in future lifetime (43%). Costs of MNDs differ considerably between SES as costs in the poorest third of the households are 5 times higher than in the wealthiest third.MNDs lead to substantial costs in 6-59-month-old children in the Philippines. Costs are highly concentrated in the lower SES and in children 6–23 months old. These results may have important implications for the design, evaluation and choice of the most effective and cost-effective policies aimed at the reduction of MNDs. Micronutrient deficiencies (MNDs) are a chronic lack of vitamins and minerals and constitute a huge public health problem. According to WHO, worldwide nearly one in two preschool children suffer from anemia, many of them because of iron deficiency ,2, and nPrevious studies have shown that the impact of MNDs on public health can be substantial for countries with high prevalence rates -11. StudThis study is part of a more extensive project on the cost-effectiveness of food fortification for 6-59-month-old children. In a first step we examined whether food fortification is effective in controlled trials . In a seWe chose the Philippines for our cost-of-illness study because it is a large country in which MNDs are still considered to be a public health issue. Furthermore, detailed data on the nutritional status of the population is available.In this study we estimate the economic and human costs of illness due to iron, vitamin A and zinc deficiencies in 6-59-month-old Filipino children by socio-economic strata with a model simulating the lifetime consequences of MNDs.The datasets used in this study were obtained from MEASURE DHS  and fromThis study aims to quantify the cost of MNDs in the Philippines by comparing the current nutritional situation with a hypothetical scenario in which all children have a sufficient MN intake, either through their diet or through MN supplementation. We build a health economic model simulating the consequences of MNDs in childhood over the entire lifetime. Costs of MNDs in adulthood are only considered if they are the consequences of childhood micronutrient malnutrition. The model is referenced to the year 2008 for which we have the results of a major health survey, the 2008 National Demographic and Health Survey (DHS) , and of Bottom-up means that costs are first calculated on the data collected from single households contacted in the 2008 DHS and NNS surveys and then extrapolated to the total population. Incidence-based means that the model computes the lifetime costs for a one-year cohort of children 6–17 months old in 2008. Societal perspective means that all the costs occurring in the society are considered. These costs include disease related health care expenditures , current and future income losses of individuals of working age due to absence from work and reduced productivity (production losses) and quality and quantity of life years lost (intangible costs)   and two age groups (6–23 months and 24–59 months). Levels of MNDs as well as their consequences are particularly severe in these age groups as the body stores of micronutrients tend to be progressively exhausted at the age of 6 months and the child’s diet is often not adequate to meet the great need for micronutrient intake in periods of rapid growth. Although children below this age range may also suffer from MNDs  they areBoth the DHS , carriedsample population consists of the 6–59 months old children from the DHS and NNS. The model population is the sample population extrapolated to the total population representing all 6-59-month-old Filipino children in 2008. The model cohort is the 1-year-cohort of 6-17-month-old children from the model population for which lifetime cost consequences of MNDs are calculated. Costs are calculated separately for MNDs at the age of 6–23 months and 24–59 months.We use the following terminology for the populations considered: The The MNDs considered in this study are those of iron, vitamin A and zinc, as the health consequences of these deficiencies are well documented. We exclude iodine deficiency, although its health consequences are also well documented, because iodine fortification is focused on salt as a carrier  and not Information on the prevalence of MNDs was extracted from the 2008 NNS for the 2 age groups over the 10 SES. Prevalence rates are calculated by assuming a normal distribution defined by the mean and the standard deviations (SD) of the blood values provided by FNRI and applying the cut-off levels reported in Table Information on blood values were obtained from FNRI as mean and SD for each of the 20 groups of children. Although this information is mostly based on reasonable sample sizes of up to 250 children per group, some groups have a small sample size with only 15 observations in the smallest group. In order to be able to extrapolate the sample data to the whole population, without magnifying uncertainty due to limited sample size, we smoothed the distribution of these group means. The levels of MNDs in the 20 groups correspond to the average group specific deficiency levels measured in the NNS.Whenever possible, the attribution of health consequences to MNDs is based on systematic reviews of the effects of MN supplementation trials for children aged 6–59 months. We assume that MN supplementation trials were designed to fill the gap of MN intake (e.g. in terms of adequate intake) due to the insufficient diet of the children and thus eliminate all the adverse health consequences of MNDs, an approach also applied in previous research . The heaSome of the health effects of MNDs are calculated by attributing a fraction of the current prevalence of diarrhea and lower respiratory diseases to a specific micronutrient. We calculate the population attributable fractions (PAF) based on relative risks (RR) extracted from the literature by applying Levin’s classical formula . These PAttribution of costs to the health consequences of MNDs is based on information extracted from the DHS, literature and other sources. These costs include costs in the immediate occurrence of the disease as well as long-term costs due to the lifetime consequences of impaired physical and mental development. Intangible costs are measured in DALYs, while monetary costs are measured in US Dollars (USD). Quantities originally measured in Philippine Pesos (PHP) were converted into USD using an average 2008 exchange rate.direct medical costs is mainly based on the DHS [Calculation of  the DHS . The hou the DHS ,32.Production losses are calculated according to the human capital approach and thus correspond to the current and future gross monetary income losses. They include income losses of parents who cannot work because they have to take care of their sick children and the lifetime income losses due to impaired physical and cognitive development and premature death of children. We assume that income losses of parents only occur in households in which both parents work. Whereas current production losses are valued with SES specific incomes, future losses are all valued with an average income. In order to account for future economic growth, we assume a yearly increase in wages of 1.66%, corresponding to the average growth rate of real GDP per capita in the years 1990–2011 [990–2011 .Intangible costs are calculated as Disability Adjusted Life Years (DALYs) lost without applying age weights [ weights . DALYs a weights .Production losses and intangible costs can be considered as two separate and non-overlapping dimensions of the costs of MNDs as recent research has shown that double counting of losses considered in both cost dimensions is negligible ,37.Future production losses have to be discounted to their present value because a dollar today is worth more than a dollar in 20 or 30 years . The model population consists of all Filipino children aged 6–59 months stratified by 10 SES and 2 age groups. This population is modeled on the basis of the DHS 2008 population  and extrapolated to the total population of 90,354,753 individuals . Table 3Table The prevalence of IDA, VAD and ZnD in the model population is calculated on the basis of hemoglobin, serum retinol and serum zinc values measured in the NNS  Table 5. OverallThe epidemiological frameworks of adverse health consequences due to IDA, VAD and ZnD Figures , and 6 aTables IDA leads to impaired cognitive development, low physical activity and, in case of severe IDA, to increased all-cause mortality Figure . 60% of VAD leads to a rise in the number of cases of diarrhea and measles and increased all-cause mortality . We exclZnD leads to a rise in the number of cases of diarrhea and respiratory disease, stunting and increased all-cause mortality. Cost consequences of diarrhea and respiratory disease are calculated as for VAD. The effect of ZnD on mortality is unclear. An extensive review finds a significant reduction in mortality risk when zinc is given alone but no significant reduction when given in combination with other nutrients . We thusor between 24–59 months.Total lifetime costs of iron, vitamin A and zinc deficiencies in 6-59-month-old Filipino children amounted to direct medical costs of 30 million dollars, production losses of 618 million dollars and intangible costs of 122,138 DALYs . Production losses are dominated by losses in future lifetime , due to impaired mental and physical development, and premature death (10%) while current losses due to the absence of parents from work when caring for their sick child appear to be of minor importance (<1%). Intangible costs are mainly due to premature death (47%) followed by future lifetime losses  and losses in the current year (10%).Costs of IDA are mainly due to its impact on mental development in 6-23-month-old children (100% of production and 84% of DALY losses). The additional effect of IDA in 24-59-month-old children is limited to DALY losses due to reduced physical activity. As IDA has no relevant direct effect on other diseases, it does not lead to medical costs . Costs of VAD are predominately caused by increased mortality of children in both age groups (99% of production and 99% of DALY losses). The main cost-effect of ZnD is on future production losses due to stunting which amounts to 28% of total production losses.Costs of MNDs differ considerably between SES Table  as costsIn the deterministic sensitivity analysis (SA) we first record how results change as single model parameters are increased or diminished by 20% and then summarize these results in tornado diagrams, by ordering them from the largest to the smallest effect. Figure Future production losses are most affected by the discount rate applied to future losses. Changes in the factors influencing labor market participation and wage level have an almost one-to-one effect on production losses. Further important factors are the parameters with an impact on impaired cognitive development induced by IDA.Intangible costs are most affected by a change of the discount rate applied to future DALY losses, as the present value of DALYs far away in time is strongly reduced by discounting. For example 70 life years in full health lost, equal to 70 DALYs lost, correspond to 29 DALYs at present value when discounted with a 3% rate. Without discounting DALYs lost in future lifetime due to impaired development and premature death would increase by 138% and total DALYs by 125%. Intangible costs are also strongly affected by changes in the parameters influencing the lifetime costs of IDA and the relative risk of mortality due to VAD.Due to the high uncertainty regarding future wages, we chose to evaluate two alternative scenarios: 1) Future earnings corresponding to current SES specific earnings. 2) Future earning corresponding to median income (median income is lower than mean income as distribution of earnings is highly skewed with 53% of income in the upper 20% of households). In both scenarios productivity losses are considerably reduced. Overall productivity losses decrease by 37% in scenario 1 and by 36% in scenario 2.The systematic review that is the source of the effects of ZnD on health provides two estimates of relative risks for mortality. In the base case we use the estimate from supplementation trials providing zinc in conjunction with other micronutrients. For the sensitivity analysis we also run our model using information on the RR derived from supplementation trials where only zinc is tested against a placebo. Based on these studies, the authors find a RR for mortality of 1.22 . When apProbabilistic SA evaluates the overall variability of the results by randomly varying all model parameters in 10,000 model runs. Figure We present a health economic model simulating the lifetime cost consequences of iron, vitamin A and zinc deficiencies in 6-59-month-old children in the Philippines stratified in 10 SES and 2 age groups. The model includes a detailed representation of the consequences of MNDs on health and physical and mental development. Total lifetime costs amount to medical costs of 30 million dollars, production losses of 618 million dollars and intangible costs of 122,138 DALYs. Costs are dominated by future lifetime costs due to impaired mental and physical development (90% of production and 43% of DALY losses) and costs of premature deaths (10% of production and 47% of DALY losses) over current losses. Burdens of MNDs differ substantially between SES as the costs in the poorest third of the households are 5 times higher than in the wealthiest third. Intangible costs are considerably higher for VAD and IDA than for ZnD while direct medical costs are dominated by ZnD. Production losses on the other hand are mainly determined by IDA, followed by ZnD. Nearly all the costs of MNDs in the Philippines are incurred by households, as they sustain a large share of medical costs and of the income lost by parents looking after their sick children and, most importantly, the huge income and DALY losses borne by children in their future lifetime.It may be helpful to examine the magnitude of these results. The 122,138 DALYs lost are driven by the premature death of 1,951 child deaths corresponding to 57,569 DALYs lost. If we compare total DALYs lost with DALYs lost due to premature death, we may conclude that the equivalent of 4,000 complete lifespans are lost each year because of MNDs. Comparing our undiscounted results with the total undiscounted 22.2 million DALYs lost in the Philippines in 2010 according to the GBD . AlthougTo our knowledge, this is the first study combining the information on health and nutritional status of 6-59-month-old children in the Philippines collected in two large and representative surveys in 2008. We build a detailed and transparent health economic model describing the lifetime health and cost consequences of iron, vitamin A and zinc deficiencies in these children. Prevalence of MNDs and health and cost consequences are differentiated by 10 SES and 2 age groups. The results are an important contribution to the design, evaluation and choice of the most effective and cost-effective policies aiming at the reduction of MNDs. We carry out a comprehensive sensitivity analysis in order to identify the parameters with the largest effect on the results and to test the overall robustness of the model. The model may in future be adapted to other countries.First, our approach in identifying the health consequences of MNDs via the results of micronutrient supplementation trials has two potential caveats: The supplementation of vitamin A and zinc in high doses may have a “pharmacological effect” which may lead to an overestimation of the adverse health consequences. High doses of vitamin A are for example employed in the treatment of measles, malaria, diarrhea and respiratory diseases and high doses of zinc are employed in the treatment of diarrhea in children. However, some children with severe deficiencies and consequent health impairments are excluded from supplementation trials as it would be unethical to give these children a placebo supplement. This may lead to an underestimation of the health consequences of MNDs. These uncertainties are at least in part explored in the probabilistic SA. Second, the consequences of some MNDs can only be quantified with single studies and not with systematic reviews. This is particularly the case for the long-term consequences of IDA on cognitive scores and the consequent reduction of adult wages. Third, we compute future production losses with an average future wage based on the current average wage and the assumption of a constant productivity growth rate. We thus assume that future wages of children will converge to the average wage. We believe that this assumption can be supported in face of the uncertainty of the long-term development of a rapidly evolving emerging country and the circumstance that lower current wages in lower SES are in part due to the long term consequences of malnutrition in childhood. In the sensitivity analysis we show that dropping this assumption and assuming productivity growth rate adjusted future wages equal to the current SES specific wages would reduce production losses by 37%.Some limitations have to be mentioned. Our results appear in line with most results of previous studies although a comparison is often difficult because of differences in population, cost-categories, micronutrients and countries considered. A direct comparison can be made with 2 studies which include the calculation of the costs of MNDs in the Philippines: Zimmermann and Qaim  examinedAn apparent difference between our results and those of previous studies is the lower magnitude of DALY losses due to ZnD  in comparison to those of VAD 47%) and IDA (46%). In the important study by Black et al. 7% and ID, which sOur results show that the costs of MNDs are highly concentrated in the lower SES and the younger age group of children. Costs are 5 times higher in the poorest third of households than in the wealthiest third. The difference in cost per child between the 6-23-month age group and the 24-59-month age group is even larger with a 4.4-fold difference in production losses and a 6.5-fold difference in intangible costs. These results are important because interventions focusing on the SES and the age classes with most severe deficiencies and highest costs are likely to have a higher health impact and to be more cost-effective.invisible or hidden nature of the costs of MNDs is particularly apparent for IDA, as 84% of DALY losses and nearly all production losses occurring in future lifetime are triggered by IDA between 6 and 23 months of age. Without any specific knowledge of the links between nutrition and future cognitive development of their child, the parents will have no reason to improve the diet of the child. The reasons why parents do not invest sufficiently in the future health of their children although the cost of prevention appears small relative to its future benefits have, for example, been investigated in the research on the use of mosquito nets for malaria prevention [The evention  or vaccievention . PolicieThe much higher prevalence of MNDs in lower SES may be one of the factors leading to a health-based poverty trap . BecauseMNDs lead to substantial costs in 6-59-month-old children in the Philippines. These costs are dominated by future production losses and intangible costs. Costs are highly concentrated in the lower SES and in 6-23-month-old children. These results may have important implications for the design, evaluation and choice of the most effective and cost-effective policies aimed at the reduction of MNDs.DALY: Disability adjusted life year; DHS: Demographic and Health Survey; FNRI: Food and Nutrition Research Institute; IDA: Iron deficiency anemia; MNDs: Micronutrient deficiencies; NNS: National Nutrition Survey; PAF: Population attributable fraction; RR: Relative Risk; SA: Sensitivity analysis; SD: Standard deviation; SES: Socio-economic strata; USD: United States Dollar; VAD: Vitamin A deficiency; ZnD: Zinc deficiency.The authors declare that they have no competing interests.SW & UB: Designed the study. SW: designed the cost of illness model and prepared the first draft of the manuscript. RP: Was responsible for data analysis, made major contributions to the cost of illness model and provided comments on the manuscript KE: Made major contributions to the health economic model and provided comments on the manuscript. OM: Analyzed literature and participated in the data analysis. MC: Provided important inputs to the cost of illness model and supervised the analysis of nutrition data. IA: Was responsible for the analysis of nutrition data. All authors revised the manuscript critically for important intellectual content and approved the final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/13/1167/prepubDetails of the economic model.Click here for file


Overall, primary viruses collected from patients at first visit were resistant to neutralization by all anti-HIV-1 mAbs with the exception of one virus sensitive to IgG1b12. Four of the five patients' viruses evolved increased sensitivity to neutralization by anti-V3 mAbs. Virus collected from a patient obtained 31 months later, evolved increased sensitivity to anti-V2, anti-V3, and anti-CD4bd mAbs. Furthermore, the anti-V2 and anti-CD4bd mAbs also exhibited increased neutralization capacities against virus collected from a patient 29 months later. Of the seven anti-V3 mAbs, five showed increased potency to neutralize the evolved virus from a patient collected after 11 months, and three exhibited increased potency against viruses from two patients collected 29 and 36 months later. Anti-V3 mAbs exhibited the most breadth and potency in neutralizing the evolving viruses. Sequence analysis of the envelope regions revealed amino acid conservation within the V3 loop, while most of the changes identified occurred outside the core epitopes and in particular within the C3 region; these may account for increased neutralization sensitivity. These studies demonstrate that  To date, almost all studies that have examined the sensitivity of human immunodeficiency virus type 1 (HIV-1) isolates to anti-HIV-1 human monoclonal antibodies (mAbs) have been cross-sectional and they show that only a few mAbs neutralize HIV-1 primary isolates. The lack of neutralization by most antibodies has been attributed to a variety of reasons such as the occlusion of the neutralization sensitive epitopes on primary HIV-1 isolates by carbohydrate moieties, the absence of the specific epitope on the intact HIV-1 virion and the differences in conformational structures on the virions in-vitro cultured isolates (e.g SF162) are sensitive to neutralization by mAbs, many of these antibodies do not neutralize HIV-1 primary isolates when tested in cross sectional studies. However, several immunochemical studies have revealed that many of these antibodies, especially anti-V3 mAbs, bind to peptides, soluble proteins, recombinant proteins and intact virions, suggesting that the epitopes are present but in different forms HIV-1 viral envelope regions, which have been identified to be immunogenic and to which mAbs were developed include the V1V2, V3, CD4-binding domain (CD4bd), CD4 induced antigen (CD4i) and C5 regions of the gp120 Information is sparse on the evolution of sensitivity to neutralization of HIV-1 primary isolates by antibodies that either do or don't neutralize viruses tested in cross sectional studies. It is well known that viruses in infected individuals evolve to escape from neutralization by autologous antibodies over time The current study examines the neutralization sensitivity to anti-HIV-1 mAbs of viruses from 3 patients  3, while the CD4 counts of two study subjects (ITM60 and 3506) was 1031 and 993 cells/mm3. The CD4 counts of ITM60 and NYU104 declined to 671 and 253 cells/mm3, while the CD4 counts of two study subjects (ITM39 and 3506) stayed relatively stable over time (499 and 750 cells/mm3), respectively. It was noted that the CD4 T cell count of subject ITM27 increased from 415 to 767 cells/mm3. Viruses isolated from a portion of the blood sample that was used for CD4 count determination were used for the neutralization studies described below.A portion of the sequential blood samples were collected from the five HIV-1-infected subjects and used to determine the CD4 cell counts by FACScan. Their CD4 profiles are shown in early virus from these patients above 50% (50) of the later time point virus was 0.3 µg/ml, as shown by mAb 447-52D and 2191 , three anti-CD4bd mAbs  and one anti-carbohydrate mAb (2G12), with the sequential viruses from all five patients; ITM60 , NYU104 50 and IC90 were achieved at concentrations of <0.3 µg/ml and 41.7 µg/ml, respectively. With mAb 654-D, an IC50 was also achieved at a concentration of <0.3 µg/ml  tested against the late virus from patient NYU104, mAbs 559/64-D and IgG1b12 neutralized the virus with ICectively , Table 150 µg/ml , Table 1.7 µg/ml , Table 150 and IC90 titers for mAb IgG1b12 were achieved at 6.0 µg/ml and 24.9 µg/ml respectively for the first time point virus  and the epitopes targeted by each mAb are shown in The V3 sequence used to define the mAb core epitope was derived from HIV-1 MN strain  whose sequential viruses exhibited increased sensitivity to neutralization by anti-V3 mAbs, those from patient ITM27 exhibited the weakest increase in neutralization sensitivity . The seqThe V2 sequences of the sequential viruses from patient ITM60 and NYU104 that evolved increase sensitivity to anti-V2 mAbs were aligned and changes in the entire V2 loop sequence were examined. Several changes in the V2 sequence from the late time point virus as compared to early time point virus were identified. Unlike the constant nature of the V3 loop sequences described above, more changes were found to occur in the V2 region for the late time point virus . These cWe examined the changes in the amino acid sequence critical for IgG1b12 binding in sequential viruses obtained from patients ITM60, NYU104 and 3506. We found that although there were changes in the in the C3 region sequence, the amino acids critical for IgG1b12 binding had only one change for the late time point virus from patient 3506 that included a change from threonine (T) to alanine (A) at position 44 . No chanOur present study demonstrates that both subtype B and CRF02_AG viruses in some HIV-1 chronically-infected individuals evolve to become more sensitive to neutralization by antibodies directed at epitopes in the V2, V3 and CD4bd  and 3. TStudies that have examined virus evolution and autologous neutralization in HIV-1 infected patients during acute and chronic infection have demonstrated that over time, viruses in these individuals undergo diversification and escape from neutralization 50 concentrations needed to yield 50% neutralization were low (0.3–2 µg/ml), in particular, for the anti-V3 mAbs for the late time point virus from patient ITM60, while higher concentrations of the same mAbs were needed to neutralize the later time point viruses from patient NYU104 (4.4–6.1 µg/ml) and 3506 (1.5–35.5 µg/ml). Such variations in mAb concentrations needed to achieve 50% neutralization for different viruses have also been reported for these and other mAbs in other studies In general, HIV-1 primary isolates are often resistant to neutralization by plasma from HIV-1 infected patients and by many anti-HIV-1 mAbs. Of the mAbs tested in this study, the anti-V3 and anti-CD4bd mAbs have been documented in other studies to neutralize primary isolates at concentrations similar to those seen in our study with the later time point viruses from the patients The V3 loop sequences as well as the amino acids critical for mAb IgG1b12 binding remained relatively constant for the sequential viruses; yet, the viruses became more sensitive to neutralization. This suggests that changes outside the core epitope contribute to increased neutralization sensitivity. Because several changes were identified outside the core epitopes, especially in C3 region, the possibility exists that these mutations may have caused changes in the structural conformation exposing the V3 loop and increasing its accessibility to the antibodies. In addition to changes that occurred outside the core epitope, which might have resulted in better exposure and increased neutralization sensitivity, point mutations  might haThe late time point virus of NYU104 evolved increased neutralization sensitivity to the anti-carbohydrate mAb, 2G12. The 2G12 epitope is centered on the high-mannose and/or hybrid glycans of asparagines residues 295, 332, and 392, with peripheral glycans from 386 and 448 on either flank Viral evolution toward or away from neutralization sensitivity is likely to be an ongoing process. It now remains to be seen whether these viruses that evolve increase neutralization sensitivity will continue to be sensitive or will become resistant over time. This will require studies to examine sequential viruses from several time points from such individuals. Furthermore, our studies have examined viruses isolated from patient PBMC which might not represent the whole viral quasispecies present in the patients. However, to avoid the possibility that the neutralization sensitive viruses detected in the later time point from patients was not due to a preferential selection, adaptation and possibly infectivity differences by such variants that may be present even in the early time point, if different donor PBMCs were used for virus isolation and amplification in different donor PBMCs, the same PBMC donor pool were used in all virus isolation and amplification. Because different variants within a quasispecies can exhibit different neutralization patterns, thus, the often lack of 100% neutralization sensitivity; different variants within the patients will also need to be examined for their neutralization sensitivity over time and the proportion of variants that are susceptible to neutralization be determined.It is important to begin to understand whether viruses that evolve increased neutralization sensitivity to heterologous antibodies also induce neutralizing antibodies in their hosts that would neutralize their autologous or other heterologous viruses. In initial experiments in which we tested the sequential plasma with their contemporaneous viruses (early and late) from patient NYU104 and ITM60 whose viruses evolved increased neutralization sensitivity we observed that these viruses were resistant to neutralization by their autologous and contemporaneous plasma samples (data not show). The results of our studies therefore reveal that while viruses mutate to escape neutralization by autologous antibodies In conclusion, the results of this study are counter intuitive revealing that viruses in patients can evolve increase neutralization sensitivity. However, the antibodies tested are heterologous to the viruses, which should be the basis for a protective vaccine because the infecting virus will almost always be heterologous to the antibodies induced by the vaccine strain. Understanding how viruses evolve and how their evolution affects their sensitivity to different antibodies should improve our knowledge of the relationship between genetic evolution, immunity, and vaccine design.The sequential viruses tested were isolated from the PBMCs of four HIV-1 subtype B and one CRF02_AG chronically infected drug naïve patients. Three of the four HIV-1 subtype B infected patients  from whom sequential viruses were obtained have been described in our previous studies 6 patient PBMCs with of 5×106 of the pooled HIV negative donor PBMCs. To prepare stocks of these viruses for the neutralization studies, 1 ml aliquot of culture supernatant containing the virus isolated from patients' PBMCs was used to infect the PBMCs of 3-day phytohemaglutinin-stimulated HIV-negative pooled donors Virus isolation and virus stock preparation from all the five patients studied were performed using two donor's PBMCs that were pooled. This approach using the same donor PBMCs was to avoid differences that may exist in virus selection, adaptation, and infectivity when using different donor PBMCs . First, Thirteen mAbs were used to test the neutralization sensitivity of sequential viruses from the HIV-1 infected individuals. The mAbs were directed at epitopes in the V3  50 of the virus in the TZM-bl neutralization assay. Thus, culture supernatant containing PBMC derived viruses was pre-incubated with mAb for 1 hour at 37°C and added to TZM-bl cells  in a 96-well plate. The plates were incubated for 2 days after which 150 µl of supernatant was removed and 50 µl of Bright Glo  was added and incubated for 2 min at room temperature. Following this incubation period, 80 µl of the mixture was transferred to a 96 well black plate and luminescence was measured using a Lumimark Plus System microplate reader . Reduction of infectivity was determined by comparing the relative luminescence units (RLU) in the presence of anti-HIV-1 mAbs and negative control wells in which the viruses were mixed with the human mAb 860-55D to parvovirus B19. All experiments were performed in triplicate wells. The average of the neutralization results from the triplicate wells for each virus/mAb test combination was expressed as percent neutralization. All the neutralization assays were repeated two-three times and the average of the independent neutralization assays was taken to determine the neutralization sensitivities of the various tested mAbs.TZM-bl cell lines carrying CD4 and both CCR5 and CXCR4 were used in the neutralization assays as previously described GCCCTGGAAGCATCCAGGAAGTCAGCCT 3′ (HXB2 location 5857–5884) and ENV N (reverse) 5′ CTGCCAATCAGGGAAGTAGCCTTGTGT 3′ (HXB2 location 9171–9145), and with inner primers R8003B (forward) 5′ AAAAGGCTTAGGCATCTCTATGGCAGGAAGAAGCG 3′ (HXB2 location 5950–5985) and R8004B (reverse) 5′ ATATGTCGACCTCGAGATACTGCTCCCACCCCTTCTGCTACTG 3′ (HXB2 location 8912–8870). The amplification protocol for the first- round PCR was one cycle at 94°C for 2 minutes, followed by 30 cycles at 94°C for 10 seconds, 55°C for 30 seconds and 68°C for 8 minutes, ending with a single extension cycle at 68°C for 10 minutes. For the second-round PCR, the protocol started with one cycle at 94°C for 2 minutes, followed by 10 cycles at 94°C for 10 seconds, 60°C for 30 seconds and 68°C for 8 minutes; it continued with an additional 20 cycles at 94°C for 15 seconds, 62°C for 35 seconds and 68°C for 8 minutes, ending with a single extension cycle at 68°C for 10 minutes.Viral RNA was extracted from virus culture supernatant  as described by Boom et al, E. coli cells according to manufacturer's recommendations . Between 3 and 10 colonies selected by Kanamycin (Kan) resistance and blue-white screening were grown in 3 ml of LB+Kan broth overnight. Plasmids were purified using the QIAprep Spin Miniprep kit , and analyzed by PCR to determine insert size. Positive clones were sequenced at the 5′ and 3′ ends with the universal T3 and T7 primers. Primer walking was performed to obtain the full sequence. Sequences were assembled using the Pregap and Gap programs from the Staden software package http://hiv-web.lanl.gov/content/sequence/gene-cutter/cutter.html).PCR products were cloned into the Topo TA cloning vector and plasmids were transformed into chemically competent


Crimean-Congo hemorrhagic fever (CCHF), a tick-borne disease caused by Crimean-Congo hemorrhagic fever virus (CCHFV), is a member of the genus Nairovirus in the family Bunyaviridae. Recently, CCHFV has been reported as an important emerging infectious viral pathogen in Sudan. Sporadic cases and multiple CCHF outbreaks, associated with nosocomial chain of transmission, have been reported in the Kordufan region of Sudan.To confirm CCHF in an index patient and attending physician in North Kordufan region, Sudan, and to provide some information on virus genetic lineages.Antibody captured ELISA, reverse transcription PCR, partial S segment sequences of the virus and subsequent phylogenetic analysis were used to confirm the CCHFV infection and to determine the virus genetic lineages.CCHF was confirmed by monitoring specific IgM antibody and by detection of the viral genome using RT-PCR. Treatment with oral ribavirin, replacement with fluid therapy, blood transfusion and administration of platelets concentrate resulted in rapid improvement of the health condition of the female physician. Phylogenetic analysis of the partial S segment sequences of the 2 CCHFV indicates that both strains are identical and belong to Group III virus lineage, which includes viruses from Africa including, Sudan, Mauritania, South Africa and Nigeria.Further epidemiologic studies including, CCHFV complete genome analysis and implementation of improved surveillance are urgently needed to better predict and respond to CCHF outbreaks in the Kordufan region, Sudan. Ixodid ticks or by contact with blood, bodily fluids or tissue from viraemic livestock and infected humans [Crimean-Congo hemorrhagic fever (CCHF), caused by Crimean-Congo hemorrhagic fever virus (CCHFV), is a viral zoonotic disease with a high mortality rate in humans. CCHFV can be transmitted to humans by bites of d humans . CCHFV id humans ,3. CCHF d humans -13. In rd humans ,14,15.GQ862371-2) were identified as etiologic agents of the nosocomial outbreak [In the past few years, CCHFV has been reported as an important emerging infectious viral pathogen in Sudan. We reported on nosocomial outbreak of CCHF in Alfulah Rural Hospital, West Kordufan. Two virus strains designated Al-fulah 3 and 4 . On Saturday 27/02/2010, the health condition of the physician deteriorated very rapidly and she developed massive vaginal bleeding, which lasted after 12 days. She also developed renal insufficiency. The serum urea and creatinine levels were as high as 135 mg/dL and 13 mg/dL, respectively. Blood platelets count was as low as 9 x10In the present investigation, we report on a nosocomial transmission of CCHF to an attending physician in North Kordufan as a result of medical referral of an infected patient, who is resident of Lagawa District, South Kordufan. Rapid diagnosis allows proper management of the disease during outbreak settings. In addition, molecular characterization of the virus provides information on the virus genetic lineages, which subsequently provides a more detailed understanding of the movement of virus strains in Kordufan region and in Sudan at large .The case definition used for identification of Crimean Congo hemorrhagic fever patients included hemorrhagic manifestations , and one or more of the following symptoms: Fever, severe headache, joint pain, chills, and nausea.Blood samples were collected from the two acute hemorrhagic fever (HF) patients in clean, sterile vacutainers. Samples were collected as part of routine diagnostic testing. Ethical clearance was obtained from the Ministry of Health of Sudan and informed consent from all patients was provided through an ethical clearance form, which permitted use of the samples for diagnostic and research purposes. In addition to being used for routine malaria screening, blood was allowed to clot, and sera were separated and sent to the Division of Virology, National Medical Health Laboratory, Khartoum, Sudan, for serological diagnostic screening. Extracted RNAs from serum samples were provided to the Molecular Biology Laboratory, Faculty of Veterinary Medicine, University of Khartoum for conventional RT-PCR amplification and subsequent sequencing.GQ862371), followed by direct sequencing of the PCR ampilicon. Serologic tests were conducted to screen the sera for CCHFV antibodies as determined by detection of IgM using antibody capture enzyme-linked immunosorbent assay (ELISA) kits from Biological Diagnostic Supplies Limited (South Africa).Virus isolation attempts were not conducted as the cultivation of CCHFV requires high laboratory containments (biosafety level 4), a facility which is restricted to a few laboratories in Africa. Therefore, identification of the virus was solely dependent on serology and conventional RT-PCR amplification, using primers targeting the S segment of the Al-fulah strain of CCHFV (Genbank accession number GQ862371). The primers were designed based on multiple sequence alignment of several published sequences of the gene using BioEidit software . A forward primer CCHF1 (5): CTG CTC TGG TGG AGG CAA CAA (3) and a reverse primer CCHF2 (5): TGG GTT GAA GGC CAT GAT GTA T (3) were used to amplify a 452-bp primary PCR product. An internal pair of forward primer CCHFn1 (5): AGG TTT CCG TGT CAA TGC AAA (3) and a reverse primer CCHFn2 (5): TTG ACA AAC TCC CTG CAC CAG T (3) were used to amplify a 207-bp nested PCR product.It is well documented that CCHFV small (S) RNA genome segment, which codes for viral nucleoprotein (NP) and non-structural protein, has the less variable nucleotide sequences among cognate genes of CCHFV strains . Thus, s2, 5.0 U enzyme mix, 2.0 μl of 20 picomole of each primers (CCHF1 and CCHF2), 5.0 μl of target RNA, were used. The total volume was brought to 50.0 μl using RNase free water. Target genes were amplified in low-profile 0.2 ml tube . Rift Valley fever virus (RVFV) and Dengue virus RNA templates were used as negative controls. Thermal profiles were performed on a Techne PHC-2 thermal cycler . The thermal cycling profiles were started with 30-min incubation at 50°C for reverse transcription of the CCHFV RNA templates into cDNA copies. The PCR tubes were then incubated at 95°C for 15 min to destroy the excess amount of RT enzyme and to activate the Taq DNA polymerase. The synthesized cDNA copies in the PCR tubes were subjected to 40 cycles of denaturation at 95°C for 1 min, annealing at 56°C for 30 sec and extension at 72°C for 45 sec, and a final incubation at 72°C for 10 min.A single-tube RT-PCR assay was carried out for CCHFV RNAs amplification basically as described previously . BrieflyFor nested amplification, reaction mixtures similar to those of RT-PCR were used, except that 2 μl of the primary PCR products were used as a template DNA for PCR amplification. The thermal cycling profiles were started with an initial denaturation at 95°C for 5-min, and the PCR tubes were subjected to 40 cycles of denaturation at 95°C for 1 min, annealing at 56°C for 30 sec and extension at 72°C for 45 sec, and a final incubation at 72°C for 10 min. Following amplification, 10 μl from each PCR amplification product were loaded onto 2% agarose gel and electrophoresed at 80 volts for 1 h. The gels were stained with ethidium bromide, and a UV light source was used to visualize the primary and the nested PCR products.The 452-bp primary PCR products were purified using QIAquick PCR purification Kit  and sent for sequencing in a commercial company . Resulted sequences were edited using BioEdit software and the Basic Local Alignment Search Tool (BLAST) of NCBI  and used to confirm the identity of the generated sequences in the GenBank nucleotide database. The sequences were then aligned with the corresponding S-segment of known CCHFV strains from Sudan, other African countries, Asia, middle and far east, and Eastern Europe. Phylogenetic tree was constructed using PAUP version 4.0 software packgae . The name of the virus strain, country of origin and the GenBank accession numbers were given for each sequence.In the present study, serological attempts to diagnose CCHF in acute phase sera sampled during the first 3 days post-infection (pi) from both patients were largely unsuccessful. However, positive IgM antibodies to CCHFV were detected in subsequent blood samples drawn from the attending physician at day 7 pi.Application of the reverse transcriptase-polymerase chain reaction (RT-PCR) assay, to acute phase sera sampled during the first 3 days pi from both suspected HF patients resulted in amplification of a primary 452 bp PCR product Figure . The sechttp://www.ncbi.nlm.nih.gov) and phylogenetic analysis. The sequences were edited and assembled using Lasergene software 3.57 . The partial S-segment of Lagawa strain showed 99% homology with Sudan- Alfulah strain-3. Phylogenetic tree was constructed using PAUP version 4.0 software  to investigate the relationship between Lagawa CCHFV strain and other known CCHFV strains identified globally. The phylogenetic analysis placed this virus strain in Group III virus lineage, which includes virus strains from across Africa including Sudan, Mauritania, South Africa and Nigeria  variants and strains, associated with CCHF outbreaks, have recently been reported in Alfulah, Western Kordufan  and in AThe source of the infection of the index patient was considered to be the result of consumption of raw sheep liver. In certain areas of Sudan, the social habit of consumption of raw offals of cattle and raw livers of sheep with bile and spices is not uncommon, which serves as a means of transmission of various communicable diseases  and CCHFTaking into consideration the identification of multiple virus variants and strains in human cases from Alfulah (2010) and Abyei districts of Kordufan region (2011), in addition to the two CCHF cases mentioned in the present study, it is likely that these CCHFV strains or variants have been actively circulating in Western Sudan, yet undetected for decades. In fact, sporadic cases and outbreaks of acute febrile hemorrhagic illnesses compatible with CCHF have long been observed in Kordufan region for several past decades, but the virus was not identified until very recently . Given tIn Sudan, fever of unknown etiology is not uncommon, but malaria is usually considered to be the primary cause . The firGQ862371) previously described by Aradaib et al., [In the present study, we confirmed that cluster of two cases was due to CCHFV as determined by RT-PCR and serology. Clearly, the disease is becoming endemic in most areas of Kordufan region of Sudan. The two CCHFV strains involved are identical and belonged to group III virus lineage as determined by the S segment partial sequences and subsequent phylogenetic analysis . The nam et al., . It is s et al., ,21.The CCHFV is susceptible to ribavirin in vitro but there is no controlled study evaluating oral versus intravenous ribavirin in treating infected patients. However, few studies have evaluated oral ribavirin. Oral ribavirin, at a dose rate of 200 mg twice daily for 5 days, is the recommended dosage for post-exposure prophylaxis . In the In conclusion, this study highlights the need to include CCHF in the differential diagnosis, when fever with hemorrhagic tendencies is observed in clinical centers and hospitals in the Kordufan region, Sudan. The frequent occurrence of sporadic cases and multiple CCHF outbreaks, and the risks these cases pose for medical staff in resource-poor health care facilities, necessitates the need for improved surveillance programs and prevention measures for this important viral disease in Sudan.The authors declare that they have no competing interests.ATME optimized the nested polymerase chain reaction-based detection assay; MSK designed the study and prepared the draft manuscript; RME processed the clinical samples and perform the viral RNA extraction; MAH conducted examination of clinical cases and clinical presentation of CCHF cases; KHT preparation and editing of nucleic acid sequences of the virus strain; MIE designed the experiment; IEA designed the experiment and prepared the final manuscript. All authors read and approved the final manuscript.


Once inside the host cell, innate immunity may interfere with SRLV replication, but the virus develops counteraction mechanisms to escape, multiply and survive, creating a quasi-species and undergoing compartmentalization events. So far, the mechanisms of organ tropism involved in the development of different disease forms  are unknown, but different alternatives are proposed. This is an overview of the current state of knowledge on SRLV genetic variability and its implications in tropism as well as in the development of alternative diagnostic assays.Small ruminant lentiviruses (SRLV) cause a multisystemic chronic disease affecting animal production and welfare. SRLV infections are spread across the world with the exception of Iceland. Success in controlling SRLV spread depends largely on the use of appropriate diagnostic tools, but the existence of a high genetic/antigenic variability among these viruses, the fluctuant levels of antibody against them and the low viral loads found in infected individuals hamper the diagnostic efficacy. SRLV have a marked  Small ruminant lentiviruses (SRLV), which include caprine arthritis encephalitis virus (CAEV) and visna/maedi virus (VMV), also called ovine progressive pneumonia virus (OPPV), cause a progressive multisystemic chronic disease clinically characterized by wasting (visna); breathing difficulty (maedi) associated with pneumonia; encephalitis; arthritis and/or mastitis that affect considerably animal welfare and production. The economic effect of SRLV, often underestimated and still under study, depends on factors related to environment, breed, individual susceptibility, production system, farming practices and age of culling ,2. The pThe intake of infected colostrum and milk by the offspring constitutes a major route of SRLV transmission, but the virus is also transmitted via the respiratory route, especially upon close contact, particularly under intensive housing or grazing conditions ,20,21,22gag, pol and env) and regulatory  genes flanked by non-coding long terminal repeat regions (LTRs) .In SRLV infections, PCR-positive reactions are often found amongst seronegative animals   219,228228, whicgag-PCR diagnosis reveal that this method may detect infection in 88.3% of sheep and goats that are seropositive by AGID and in 11% of seronegatives  [gag-seminested-PCR procedure allows the detection of 70% of the AGID positive sheep and 11% of seronegative animals [Studies based on nverted) . In line animals . Compara animals . LTR-PCR animals . in vitro [in situ hybridization [pol-seminested-PCR on AGID-seropositive animals infected with two viral subtypes (genotypes A and B). The percentage of pol-PCR positives in this study was 62.2% and 30.5% for types A and B, respectively. In addition, the pol-PCR detected 38.2% of positive animals among AGID seronegatives [Since PCR assays may fail to detect virus when virus load is below the assay threshold, PCR sensitivity may increase upon co-cultivation of PBMC from infected animals with permissive fibroblasts in vitro ,231,232 in vitro ,219 or idization ,233 techdization  using a egatives .Although different studies indicate that in general the PCR methods tend to be less sensitive than serological tests (particularly ELISA tests)  213,220220, PCR The basic mechanisms of mutation and recombination are complex biological processes, but they may help to understand the true role of viral diversity in SRLV pathogenesis. Virus and host genetic and microenvironmental factors involved in cell, tissue/organ and host tropism of these viruses are pivotal issues in current investigations. Genetic and antigenic variations of the virus represent challenges in SRLV diagnosis. The success in avoiding SRLV infection spread depends largely on early detection and culling of infected animals in the heard/flock. Detection of specific antibodies in serum or milk by ELISA, typing the circulating strains by local strain-based ELISAs, HMA or PCR-sequencing techniques, and use of PCR for confirmatory purposes are solid technological approaches of practical use for reducing the risk of misdiagnosis in different areas. Finally, diagnostic methods suitable to both sheep and goats should be implemented in joint programs for these species for successful SRLV control and eradication strategies.