MVC) of the elbow flexors prior to, immediately, and 5 min following a submaximal contraction protocol. During rest or 5% MVC, transcranial magnetic stimulation, transmastoid electrical stimulation, electrical stimulation of biceps brachii motor point and Erb's point were elicited to induce motor‐evoked potentials (MEPs), cervicomedullary MEPs (CMEPs), potentiated twitch (PT) force, and maximal muscle compound action potential (Mmax), respectively prior to, immediately, and 5 min postcontraction protocol. MEP amplitudes increased  only at 1 and 6s postcontraction protocol, respectively during rest but not 5% MVC. CMEP amplitudes decreased during rest and 5% MVC  for up to 81 sec postcontraction protocol. Peak twitch force increased immediately postcontraction protocol and remained elevated for 90 sec . There was a significant positive correlation between MEP and PT force during rest  and a negative correlation between CMEP and PT force during rest  immediately postcontraction protocol. In conclusion, the change in corticospinal and spinal excitability was state‐ and time‐dependent whereas spinal excitability and postactivation potentiation were time‐dependent following the contraction protocol. Changes in corticospinal excitability and postactivation potentiation correlated and were also state‐dependent.We examined the effects of a submaximal voluntary elbow flexor contraction protocol on measures of corticospinal excitability and postactivation potentiation of evoked muscle forces and if these measures were state‐dependent (rest vs. voluntary muscle contraction). Participants completed four experimental sessions where they rested or performed a 5% maximum voluntary contraction ( The malleability of central nervous system excitability and evoked muscle twitch force is dependent on muscle contractile history. For example, immediately following and well beyond the cessation of a voluntary contraction(s) of a given muscle there is altered excitability of the corticospinal tract projecting to that muscle  of corticospinal excitability occurs in fresh and fatigued muscles including the biceps brachii  to the muscle during voluntary contraction have shown that motor unit discharge rates were decreased in the presence of postactivation potentiation in the triceps brachii  to assess the effects of repeated, submaximal voluntary contractions of the elbow flexors on corticospinal excitability to the biceps brachii and postactivation potentiation of the elbow flexors during rest and a slight contraction (5% MVC) and (2) determine if a state‐dependent relationship existed between corticospinal excitability and postactivation potentiation. We hypothesized that (1) there would be postexercise facilitation of corticospinal excitability , postexercise depression of spinal excitability  and postactivation potentiation of the elbow flexors postcontraction protocol and (2) there would be a negative relationship between spinal excitability and postactivation potentiation during 5% MVC.n = 9 for experiment A and n = 6 for experiment B). We chose to recruit only resistance‐trained males because corticospinal excitability is training‐dependent  were recruited for the study  activity was recorded from the dominant biceps brachii muscle using surface EMG recording electrodes . Electrodes were placed 2 cm apart over the midpoint of the muscle belly. A ground electrode was placed over the lateral epicondyle of the dominant knee. Skin preparation for all recording electrodes included shaving to remove excess hair and cleaning with an isopropyl alcohol swab to remove dry epithelial cells. EMG signals were amplified (×1000) (CED 1902) and filtered using a 3‐pole Butterworth filter with cut‐off frequencies of 10–1000 Hz. All signals were analog‐digitally converted at a sampling rate of 5 kHz using a CED 1401 interface.μV, in 50% of the trials  in the biceps brachii during rest and an identifiable response during 5% MVC. The mean stimulator output was then increased by 20% above that used to determine threshold for the remainder of the experiment ‐evoked MEPs were used to measure corticospinal excitability from the relaxed dominant biceps brachii muscle as well as during a 5% contraction, using a transcranial magnetic stimulator  with a circular coil (13 cm outside diameter) directly placed over the vertex . The latencies of all CMEPs were monitored because evoked stimulation to the mastoid processes can activate axons near the ventral roots which subsequently decreases the onset latency of the CMEP by ~2 msec (Taylor Stimulation was applied via surface electrodes placed over the mastoid processes and current was passed between them (100 c Taylor . Based oMmax). Erb's point was electrically stimulated via a cathode on the skin in the supraclavicular fossa and an anode on the acromion process. Current pulses were delivered as a singlet . The electrical current was gradually increased until Mmax of the dominant biceps brachii was observed. Stimulator intensity was then increased by 20% above that used to determine Mmax for the remainder of the experiment  was used to measure maximal compound muscle action potential . The electrical current was gradually increased until there was no longer an increase in the twitch force of the dominant elbow flexors. Stimulator intensity was then increased by 10% above that used to determine maximal twitch force for the remainder of the experiment , Erb's point and motor point).Mmax and PT force were determined. The experimental procedures then began. Pre and postcontraction protocol measurements consisted of three sets of 30 sec duration trials, with each 30 sec‐trial consisting of a twice repeated sequence of stimulations in the same order . Each stimulus was separated by 5s. Erb's Point stimulation occurred at 28 sec within each 30 sec trial. Each series of neuromuscular testing lasted 90 sec and included 12 transcranial, 6 motor point and 3 Erb's point stimulations. This sequence was performed pre, immediately, and 5‐ min postcontraction protocol. Approximately 6.5‐min following the contraction protocol another elbow flexor MVC was performed to assess maximum force production capability.Participants performed a MVC of the elbow flexors prior to the precontraction protocol. Approximately 15‐ min of rest was provided following the initial MVC and the stimulation intensities for MEP, Participants performed the exact same protocol as experimental session 1, however, participants continuously held a contraction with no rest at 5% of their MVC during each 90 sec block  of testing, as well as during determination of stimulation intensities.experiment A completed two experimental sessions in experiment B. Experimental sessions 1 and 2 in experiment B were identical to those outlined in experiment A, with one major difference. In experiment B, TMS was replaced with TMES to assess spinal excitability. All other portions of the protocol remained the same. See Figure Six participants from Mmax responses throughout each testing block. All MEPs and CMEPs were normalized to the recorded Mmax within the same 30 sec trial and all MEP and CMEP data reported in the results section are expressed as a percentage of Mmax. Peak twitch (PT) force of the elbow flexors was defined as the peak amplitude of the twitch force. Figure Mmax (middle panel) and PT force (bottom panel) recorded precontraction protocol and the first (black line) MEP or CMEP, Mmax and PT force recorded immediately postcontraction protocol. To determine if central drive to the biceps brachii was similar within each experimental session, mean biceps brachii root mean square (RMS) EMG amplitude was measured for 100 msec prior to each stimulus. RMS EMG was also calculated over a 1 sec epoch during each 50% MVC within each experimental session. Because there was no difference between MEP and CMEP amplitudes and PT forces precontraction protocol, they were averaged and each MEP, CMEP, and PT response recorded postcontraction protocol was compared with the average as previously done in our lab  from the precontraction protocol average.P < 0.05 and a Bonferroni post hoc test was performed to test for significant differences between time points. Pearson product‐moment correlation coefficients between percent changes in corticospinal excitability and PT force were also performed. Absolute values are reported in text and mean percentage change from the averaged precontraction protocol value in Figures . Assumptions of sphericity (Mauchley test) and normality (Shapiro‐Wilk test) were tested for all dependent variables. If the assumption of sphericity was violated, the corrected value for non‐sphericity with Greenhouse‐Geisser epsilon was reported. All data were normally distributed. We did not compare corticospinal excitability responses recorded during rest and 5% MVC because corticospinal excitability is different between resting and muscle contraction conditions, nor did we compare MEPs to CMEPs because their amplitudes were not matched , CMEP  or PT force  amplitudes during rest and 5% MVC, respectively. There was no significant main effect of time on prestimulus EMG, thus overall central drive was similar within each experimental session during rest  and 5% MVC .There was no significant main effect of time on precontraction protocol MEP (P < 0.01) on individual MEP amplitudes  and 6 sec  postcontraction protocol compared to the averaged precontraction protocol MEP amplitude (4.7 ± 1.2% Mmax).There was a significant main effect for time  on individual MEP amplitudes  lower at 51s (15.3 ± 4.0% Mmax) and significantly (P < 0.05) higher at 361 sec (29.2 ± 8.7% Mmax) postcontraction protocol compared to the averaged precontraction protocol MEP amplitude (16.9 ± 3.1% Mmax).There was a significant main effect for time  on individual CMEP amplitudes  postcontraction protocol were significantly (P < 0.05) lower than the averaged precontraction protocol CMEP amplitude (22.5 ± 6.2% Mmax).There was a significant main effect for time  on individual CMEP amplitudes  postcontraction protocol were significantly (P < 0.03) lower than the averaged precontraction protocol CMEP amplitude (20.6 ± 2.5% Mmax).There was a significant main effect for time  of time on individual PT forces  postcontraction protocol were significantly (P < 0.04) higher than all other PT forces (range: 64.5 ± 6.0–77.1 ± 7.0 N)  were significantly (P < 0.05) higher than all other PT forces (range: 69.6 ± 6.7–74.7 ± 5.4 N)  Fig. C. In expexperiments A and B, P < 0.01) of time on individual PT forces  postcontraction protocol were significantly (P < 0.01) higher than all other PT forces (range: 71.0 ± 5.7–76.1 ± 6.5 N)  were significantly (P < 0.05) higher than all other PT forces (range: 70.9 ± 4.4–78.4 ± 4.2 N)  for the contraction protocol on Mmax amplitudes  during rest and 5% MVC, respectively.There was no significant main effect      Fig. A but not22) Fig. B. In expexperiment A, P = 0.13 and P = 0.25 and experiment A, P = 0.36 and P = 0.44) for the contraction protocol on MVC force pre and postcontraction protocol  or 5% MVC , respectively.There was no significant main effect  for contraction number during the contraction protocol on EMG between 50% MVC 1 and 5 during rest  or 5% MVC , respectively.There was no significant main effect for  change in corticospinal excitability to the biceps brachii but not spinal excitability or postactivation potentiation of the elbow flexors and (2) relationship between corticospinal excitability and postactivation potentiation but not spinal excitability and postactivation potentiation. There was no change in MVC force output from pre to postcontraction protocol or EMG during the contraction protocol. Also, prestimulus EMG was similar throughout each experimental session. Thus, the changes in postexercise facilitation, postexercise depression and postactivation potentiation and their relationship were not due to fatigue or changes in central drive to the muscle following the contraction protocol.During rest, the MEP amplitude facilitation (215 and 165%) was transient as they returned to baseline within 8–16 sec postcontraction protocol, which is a similar percentage change and time frame as previously reported  than passive  movements  that lasted up to 81 sec postcontraction protocol, which is similar to previously reported findings  immediately postcontraction protocol when recorded at rest (~58%) compared to 5% MVC (~46%). If postexercise depression of CMEPs is partially due to a reduced neurotransmitter release from the corticospinal tract onto the spinal motoneurone , the descending and/or ascending (afferent) activity may be sufficient to facilitate the release of additional neurotransmitter and thus increase the motoneuronal response. This effect would not be present when CMEPs were assessed at rest , was not altered the mechanisms for postactivation potentiation likely include a combination of changes in: (1) calcium kinetics  overall corticospinal excitability increased at rest, but not during a 5% MVC and (2) spinal excitability decreased at rest and during a 5% MVC. Whether these relationships between corticospinal excitability and postactivation potentiation are of functional significance is currently unknown. The relationship between CMEP and postactivation potentiation may have functional consequence. Postactivation potentiation may act to offset postexercise depression of spinal excitability. This relationship may exist due to a reduction in motoneurone output. Motor unit discharge rates have been shown to decrease as postactivation potentiation increases in the tibialis anterior and triceps brachii (Klein et al. Because MEPs and CMEPs were not recorded in the same session we could not make a ratio of MEP/CMEP as we (Aboodarda et al. The interaction between and changes in corticospinal excitability, spinal excitability and postactivation potentiation following submaximal contractions depended on the state in which they were measured. Postexercise depression of spinal excitability and evoked twitch force postactivation potentiation have longer lasting changes following submaximal contractions than postexercise facilitation of corticospinal excitability. Postactivation potentiation of the muscle may allow for postexercise depression of spinal excitability.None declared.


Lentiviruses are a unique class of retroviruses that infect a subset of mammalian species, including humans. Human immunodeficiency virus type 1 and 2 (HIV-1 and HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), maedi-visna virus (MVV), and caprine arthritis encephalitis virus (CAEV) infections result in immunodeficiency syndromes, neurological diseases, and other conditions. Disease is chronic, lifelong, and often fatal. Pathology is due in part to a capacity to immortalize by integrating into a host’s genomic DNA and a remarkable genetic plasticity that enables escape from potent adaptive immune responses without compromising vital viral functions. Lentiviruses encode three universal retroviral proteins , one lentivirus-specific protein called Vif (virus infectivity factor), and a handful of other less conserved accessory factors.Vif is required to protect lentiviruses from restriction by several members of the APOBEC3 family of DNA cytosine deaminases by forming a cellular polyubiquitin ligase complex to degrade these enzymes . Virusesvif-deficient virus replication . The. The7]. APOBEC3 genes themselves is driven by RUNX/CBF-β in CD4+ T cells (the primary reservoir for HIV-1 replication) . Th. Th21]. APOBEC3 gene duplication or gene conversion event creates a mimicry scenario such as for present-day human A3D and A3F.Importantly, after cross-species transmission, adaptation to each of the new host’s restrictive APOBEC3 enzymes will occur independently, as each exerts its own unique selective pressure. For example, an amino acid change at one position (a wobble) may alter the interaction with one host APOBEC3 enzyme but not others. Therefore, in an iterative fashion, a single Vif-APOBEC3 interaction surface will begin to diverge from the others. Over an evolutionary time scale, fueled by adaptive immune pressure, it is easy to imagine how an ancestral Vif-APOBEC3 interaction may have undergone many stepwise wobbles to present-day configurations. One prediction of this model is the likelihood that Vif must maintain at least a partial interaction with each restrictive host APOBEC3 enzyme during the adaption period (or face extinction). This helps explain why all known Vif-APOBEC3 interactions occur on discrete solvent exposed surfaces  driven by adaptive immune responses within infected individuals. More extreme pathogen adaptations, such as those required for cross-species transmission of ancestral lentiviruses and neutralization of more complex repertoires of restriction factors, are mediated by rare events such as the hijacking of cellular CBF-β.


Low and middle income countries face many challenges in meeting their populations’ mental health care needs. Though family caregiving is crucial to the management of severe mental health disabilities, such as schizophrenia, the economic costs borne by family caregivers often go unnoticed. In this study, we estimated the household economic costs of schizophrenia and quality of life of family caregivers in Ghana.We used a cost of illness analysis approach. Quality of life (QoL) was assessed using the abridged WHO Quality of Life (WHOQOL-BREF) tool. Cross-sectional data were collected from 442 caregivers of patients diagnosed with schizophrenia at least six months prior to the study and who received consultation in any of the three psychiatric hospitals in Ghana. Economic costs were categorized as direct costs , indirect costs (productivity losses to caregivers) and intangible costs (non-monetary costs such as stigma and pain). Direct costs included costs of medical supplies, consultations, and travel. Indirect costs were estimated as value of productive time lost (in hours) to primary caregivers. Intangible costs were assessed using the Zarit Burden Interview (ZBI). We employed multiple regression models to assess the covariates of costs, caregiver burden, and QoL.Total monthly cost to caregivers was US$ 273.28, on average. Key drivers of direct costs were medications (50%) and transportation (27%). Direct costs per caregiver represented 31% of the reported monthly earnings. Mean caregiver burden (measured by the ZBI) was 16.95 on a scale of 0–48, with 49% of caregivers reporting high burden. Mean QoL of caregivers was 28.2 (range: 19.6–34.8) out of 100. Better educated caregivers reported lower indirect costs and better QoL. Caregivers with higher severity of depression, anxiety and stress reported higher caregiver burden and lower QoL. Males reported better QoL.These findings highlight the high household burden of caregiving for people living with schizophrenia in low income settings. Results underscore the need for policies and programs to support caregivers. Mental illness accounts for about 32% of years lived with disability (YLD) and about 13% of disability-adjusted life-years (DALYs), a percentage that equals that of cardiovascular and circulatory diseases . It is eCaregiving plays a significant role in the management of schizophrenia. For example, the WHO indicates that globally up to 90% of schizophrenic patients live with their families . In manyA key provision of Ghana’s 2012 Mental Health Act Act 846) is to de-institutionalize mental health care  in order to decongest the three psychiatric hospitals. The role of caregivers in this community-based approach is crucial since family interactions influence treatment outcomes and relapse rates . However46 is to Although Ghana has a high burden of mental illness, few studies have examined the burden of mental illness in general—and schizophrenia in particular—on unpaid family caregivers. In this study, we sought to address this gap by estimating the economic burden and quality of life of family caregivers of schizophrenic patients in Ghana.The study was undertaken at the three psychiatric hospitals in Ghana: Accra, Pantang and Ankaful. Accra Psychiatric Hospital is a 600-bed hospital commissioned in 1906 as the first psychiatric hospital in Ghana and is located in the Greater Accra Region. Established in 1975 with a bed capacity of 500, the Pantang Psychiatric Hospital is situated in Pantang, also in the Greater Accra Region. Ankaful Psychiatric Hospital, which was built in 1965, is a 350-bed hospital located near the coastal town of Cape Coast in the Central Region of Ghana that serves patients from the Central, Western and Ashanti regions of Ghana and some neighbouring countries. All the hospitals are located in the southern part of the country and serve the country’s population of approximately 25 million .The study population comprised family (primary) caregivers of schizophrenic patients reporting to the outpatient department at each study site. Based on Cochran , the samn0 is the minimum required sample size; Z2 is an abscissa of the curve that cuts off an area ∝ at the tail ; e is the desired level of precision; p is the estimated proportion of schizophrenic patients that is present in the population, which was assumed to be 50% since the current proportion is unknown; and Deff is the design effect. For a 95% confidence interval, e (margin error for the study)±5%. Assuming a design effect of 1.03, we computed a minimum sample size of.Where Assuming a 90% response rate, we estimated a minimum sample size of 440 patients. We computed the sample size required in each facility using probability proportionate to size (PPS) of number of schizophrenic patients Table .Table 1Sth revision of the International Statistical Classification of Diseases and Related Health Problems (ICD-10), and primary caregivers who attended the hospital with the patients identified and interviewed. Accompanying relatives who were not the primary caregivers were excluded. Given that many patients presenting for outpatient services were unaccompanied, accompanying primary caregivers were recruited until the required sample size for the particular facility was met.In each hospital, folders of patients diagnosed with schizophrenia at least six months preceding the study were sorted and reviewed using criteria outlined in the 10Data were collected using a structured, interviewer-administered questionnaire with closed- and open-ended questions. The questionnaire elicited information on caregivers’ sociodemographic characteristics, direct health care costs  and indirect costs (i.e. productivity losses) to caregivers.We used a cost of illness analysis approach. Costs were analysed from the caregiver perspective and included costs incurred during the month preceding the data collection. All costs were computed in United States Dollars (US$) (3.9 Ghanaian Cedi (GHS) ≈ 1 US$). Costs were not adjusted for inflation. Costs were categorized into direct, indirect and intangible costs. Direct costs were further grouped into medical and non-medical costs. Medical costs included the costs of drugs, consultations, laboratory investigations and diagnostics and other therapies. Non-medical costs included the cost of travel to and from hospital, food costs incurred by the caregiver during the treatment period, accommodation costs if the caregiver travelled with the patient away from home, and miscellaneous costs such as telephone costs related to medical care. The sum of medical and non-medical costs constituted the direct costs.Indirect costs were estimated using the national daily minimum wage of US$ 2.0 per day (May 2016) for caregivers in the formal sector and a local daily agricultural wage rate of US$ 4.50 was used for caregivers working in the informal sector. Unemployed caregivers were considered part of the informal sector. Travel time was calculated by adding the total number of hours spent travelling to and from the hospital. Waiting time constituted the total time spent waiting for and receiving treatment . The time used for other caregiving activities, such as household activities and leisure, was also calculated and included in the indirect costs. Total indirect cost was calculated as the product of the sum of total time spent on personal care for patient, transport and waiting for health care, other household activities for the patient (in hours) and the respective (hourly) wage rate. Total economic cost constituted the sum of direct and indirect costs.Intangible costs (sometimes referred to as caregiver burden) were assessed using the 12-item Zarit Burden Interview (ZBI) tool developed by Bedard et al. , which cQuality of life was assessed using the abridged WHO Quality of Life (WHOQOL-BREF) tool. The WHOQOL-BREF comprises 26 items: two items assessing the overall quality of life and general health; and 24 assessing satisfaction in four main domains – seven items on physical health, six items on psychological health, three items on social relationships, and eight items on environmental health. Each item was rated on a scale of 1 to 5 with 1 being the lowest and 5 being the highest QoL. The mean score of items within each domain was used to calculate the domain mean. The mean score for each domain was then multiplied by 4 (four) to make it comparable with the full WHOQOL tool (WHOQOL-100) . The scoAge (in years) as at last birthday; Sex measured as a dummy, 1 for male and 0 for female; Marital status: Married (1), single (0); Proximity of residence from health facility visited; 1 for far  and 0 for close; Caregiver’s highest level of education: no education (0), primary/junior high school  (1), secondary (2) and tertiary (3); Caregiver’s employment status: unemployed (0), self-employed (1), private sector (2), public sector (3), student/apprentice (4); Caregivers were asked whether other members of the family spent time taking care of the patient (other family support): yes (1), no (0); and Caregiver’s mental health status . Mental health status was assessed using the Depression Anxiety Stress Scale (DASS-21), which comprises of three subscales: Depression (DASS-D), Anxiety (DASS-A), and Stress (DASS-S). Responses on each item ranged from 0  to 3 (applied to me very much).Other variables used in the analysis included Reliability of the QoL and Zarit burden scale was assessed using Cronbach’s alpha. With the exception of the depression sub-scale of the DASS-21 and the social sub-scale of the WHOQOL, the Cronbach alpha values fell within the acceptable range of alpha values (0.70 to 0.95) –24  of study variables are presented in Table Caregivers’ sociodemographic characteristics are summarized in Table The average caregiving cost per month was US$273.28, with indirect costs being $242.95 and direct costs being $30.36 (Table The mean caregiver burden score was 16.95 (SD 8.82) out of a maximum of 48. About 51% of the caregivers reported low burden . The analysis also revealed that the burden in caregiving was significantly higher for females (61%) than for males (39%) among caregivers reporting high burden.p < 0.05). Caregivers who were married had higher QoL scores than those who were unmarried. The mean score across all domains increased as the caregiver’s educational level increased.The mean quality of life scores by domain and background characteristics are summarized in Table p-value = 0.0007). A unit increase in age, resulted in a GHS 3.3 increase in indirect cost . Indirect cost for males was GHS 61.1 lower compared to females .The multivariate linear multiple regression results for covariates of economic cost are shown in Table p = 0.0022) higher than those with no anxiety disorders, controlling for other covariates.With respect to predictors of ZBI score, anxiety, depression and stress were related to Zarit burden score, as presented in Table p = 0.0001). Similar results were obtained for the psychological QoL domains. The coefficients for the Zarit burden score, taken for all the four QoL domains together, were statistically significant . There was no statistically significant relationship between the direct and indirect cost and the four QoL domains. The QoL in relation to the physical and psychological domains was higher among caregivers who reported family support. The QoL with reference to the psychological domain was about 3%  higher for participants who reported family support compared to those that did not. Depression was associated with all the four domains of QoL . The QoL was 17%  lower for participants with severe forms of depression compared to those with no depression. The results further showed that more highly educated caregivers reported higher QoL (7–15% more) than those who were uneducated, and male caregivers reported higher QoL (2–6% more) than female caregivers.Table p = 0.0030), controlling for other covariates in the model. Overall QoL was 17% lower for caregivers with depression compared to those with no depression.The results from the multiple linear regression analysis in Table The aim of this study was to estimate the economic costs and QoL of family caregivers of schizophrenic patients attending three psychiatric hospitals in Ghana and to assess the factors associated with these two outcomes. The average costs incurred by caregivers amounted to approximately US$273 per month, with about 82% of these costs being indirect costs (in terms of lost productivity). The mean caregiver burden (measured by the ZBI) was 16.95 on a scale of 0–48, with 49% of caregivers reporting high burden. Overall, caregivers reported low QoL (28.2 out of 100).The estimated mean monthly direct cost per month US$30) represented about 18% of the total cost involved in caregiving. This proportion is lower than that obtained by Addo et al.  (21%) in0 represeSimilar to Knock et al. , the curThere are limitations to the study that are worth noting. First, it was not possible to determine whether the caregiver received any assistance, in cash or in kind from other members of the family or the patient; thus, the cost burden may not be entirely attributed to the main caregiver. Second, it was not possible to conduct more nuanced analysis to assess possible underlying reasons  for some of the differences observed. Nonetheless, the current study adds to the sparse literature on the economic and social costs of mental disorders in sub-Saharan Africa.The study findings highlight the significant burden of caring for people with schizophrenia on family caregivers in Ghana. As the country considers deinstitutionalizing mental health care, it is important that measures to alleviate the direct costs on caregivers are taken into account. Currently, the National Health Insurance Scheme (NHIS) does not cover services for schizophrenia or other mental disorder, deepening the burden of families. One possible avenue to alleviate the costs incurred by caregivers of people with mental illness would be to extend social protection programs, such as the Livelihood Empowerment Against Poverty (LEAP) program (which provides cash transfers to vulnerable groups), to caregivers to cushion the effects of the shocks of the direct costs. Study findings also underscore the significant non-quantifiable burdens, such as emotional stress, that are borne by caregivers and that affect their quality of life. The quality of life of caregivers of patients with mental disorders should be considered in health policies related to mental illness.


Vespa velutina nigrithorax) in Belgium. This constitutes an unexpected report of this iflavirus outside Hawaii, USA, where it was recently described in social wasps. Although virulence of Moku virus is unknown, its potential spread raises concern for European honeybee populations.We report the detection of Moku virus in invasive Asian hornets ( Apis mellifera) being by far the major player in this process (Varroa destructor) has been shown to have a critical effect on honeybee populations, both by its direct parasitic effects and through the transmission of pathogenic viruses such as deformed wing virus , a natural predator of honeybees, has a native range spanning from India through China and as far as Indonesia  alignment to GenBank viral sequences enabled the assignment of most viral sequences to phages (not represented) and viruses of the Partitiviridae and Parvoviridae (Densovirinae) families  and has a mean nucleotide identity of 96.0% to the Hawaiian Moku virus strain (accession no. KU645789) (is virus .Vespula pensylvanica), honeybees, and Varroa mites in Hawaii (Varroa mites in areas of Europe where the Asian hornet has become endemic, such as several regions in France. As highlighted by Mordecai et al. (V. destructor mites in Hawaii is of great concern given the role played by this mite in the maintenance and transmission of viruses, including the deformed wing iflavirus, to honeybees. Furthermore, although Moku virus was shown to be highly dominant among viral species infecting V. pensylvanica wasps (V. velutina, in which Partitiviridae were much more abundant. It remains to be determined whether Moku virus is a virus of Vespulidae or, more likely given the relatively low number of reads detected, could have been picked up by these hornets from their prey, such as honeybees. Further studies are needed to establish the origin, host range, and transmission route of Moku virus; its virulence; and the risks it may represent for European honeybee populations.Our results show a large diversity of viruses in invasive Asian hornets collected in Belgium in 2016. Among these, we detected an iflavirus with high identity to the recently described Moku virus found in social wasps (Discussion of the methods used and results of the investigation of Moku virus in Asian hornets, Belgium, 2016.


A novel pestivirus species was discovered in a piglet-producing farm in Austriaduring virologic examinations of congenital tremor cases. The emergence of thisnovel pestivirus species, provisionally termed Linda virus, in domestic pigs mayhave implications for classical swine fever virus surveillance and porcinehealth management. Pestivirus consists of 4 approved species within the familyFlaviviridae , was discovered. These viruses were identified in North America  resulted in “nosignificant similarity found,” but the translated sequence of 270 aa alignedwell with different pestiviruses. The viral RNA was detected in all samples  of CT-affectedpiglets and their littermates from the farm.We tested various samples from the farm using an APPV-specific TaqMan probe-basedreverse transcription PCR (RT-PCR)  toavoid confusion with other pestiviruses. We determined the full genome of Lindavirus (LV) using the standard primer walking RT-PCR approach together with rapidamplification of cRNA ends PCR to identify the ultimate 5′- and 3′-termini using the cultured virus. We determined the length of the LV genome (GenBankaccession no. KY436034) to be 12,614 nt, with 381 nt 5′-nontranslated region,an open reading frame of 11,772 nt, and 461 nt of 3′-nontranslated region. 2354/S2355 motive in LV; the intramolecular NS3 cleavagesite (L1834/A1835) was conserved in LV , which demonstrated LV’s divergence from other pestiviruses , panel Bed in LV , even if7 50% tissue culture infectiousdose/mL). Using monoclonal antibody 6A5 in immunohistochemical analysis, we detectedpestiviral antigen in CT-affected piglets in regions with histological lesions.Specific stains in neurons of the nucleus of the trigeminal nerve  and sequence homology to otherpestiviruses (A previously unknown pestiviral agent was found in CT-affected piglets on a farm inAustria. We observed a severe hypomyelination in the entire white matter of thespinal cord and detected pestivirus antigen in the brain of the CT-affected piglets,suggesting a causal relationship between infection and lesions. Analyses of theassembled genome allowed an unambiguous assignment of LV within the genusAfter its description ≈10 years ago, BV was intensively sought worldwide, butthe virus has never been detected outside Australia. The broad cell culture tropismof BV led to the hypothesis that the virus recently jumped from another species toporcine hosts (


Zika virus, a mosquito-borne flavivirus that can cause rash with fever, emerged in the Region of the Americas on Easter Island, Chile, in 2014 and in northeast Brazil in 2015  of which were classified as laboratory-confirmed. Autochthonous Zika virus cases had been identified in two countries (Brazil and Colombia) by October 2015 . Zika viFrom May 15, 2015, to December 15, 2016, rates of Zika virus disease peaked at different times in different subregions of the Americas . In both the Southern Cone and Andean subregions, rates increased in January, peaked in February, and progressively declined. In Central America, rates peaked in January, followed by a more modest peak in June. In the non-Latin Caribbean, incidence peaks of comparable intensity were reported in February and June. In the Latin Caribbean subregion, where the highest rates of reported Zika virus disease cases were observed, rates began to increase in January 2016, and continued at high levels through July. Reported rates remained relatively low in North America.As of December 15, 2016, increases in the number of GBS cases had been reported in 13 countries and territories with documented Zika virus transmission, compared with baseline data.In December 2015, PAHO activated an incident management system to coordinate the regional Zika virus response and developed a framework for action with four pillars: 1) detection of Zika virus and its complications, 2) prevention of new infections, 3) provision of care and support for affected persons and families, and 4) implementation of research to understand the disease and its consequences .Zika virus, a flavivirus that is primarily transmitted by During May 15, 2015–December 15, 2016, autochthonous Zika virus transmission was confirmed in 48 countries and territories in the Region of the Americas. Rates of Zika virus disease peaked at different times in different subregions. During this period, the trend in reported GBS cases paralleled that of reported Zika virus disease cases.Aedes mosquitoes are present should continue surveillance for Zika virus disease, GBS, and congenital abnormalities; strengthen capacity for laboratory diagnosis of Zika virus and other arboviruses; and continue the implementation of vector control measures and other prevention activities.Because of ongoing Zika virus transmission, the occurrence of associated complications, and the risk for recurrence of large outbreaks, countries where


Mimiviridae proposed from metagenomic data. BsV infects an ecologically important microzooplankton, the kinetoplastid Bodo saltans. Its 1.39 Mb genome encodes 1227 predicted ORFs, including a complex replication machinery. Yet, much of its translational apparatus has been lost, including all tRNAs. Essential genes are invaded by homing endonuclease-encoding self-splicing introns that may defend against competing viruses. Putative anti-host factors show extensive gene duplication via a genomic accordion indicating an ongoing evolutionary arms race and highlighting the rapid evolution and genomic plasticity that has led to genome gigantism and the enigma that is giant viruses.Giant viruses are ecologically important players in aquatic ecosystems that have challenged concepts of what constitutes a virus. Herein, we present the giant Bodo saltans virus (BsV), the first characterized representative of the most abundant group of giant viruses in ocean metagenomes, and the first isolate of a klosneuvirus, a subgroup of the  In oceans, rivers and lakes, there are about a million viruses in every milliliter of water. Most of these viruses are tiny, often 10 or 100 times smaller than bacteria. However, a few reach a similar size and complexity to bacteria, and so stand out as relative giants.Relative to other viruses, Giant Viruses have much more DNA in their genome, which in turn provides the genetic template to produce the proteins that allow viruses to reproduce largely independently of its host. Typically, more than half of the genes encoded by Giant Viruses have no evident similarity to genes from other viruses or cellular life. Sequencing DNA from ocean water suggests that Giant Viruses are abundant and ecologically important; yet, few have been isolated from the microbes that they infect. Without being able to study Giant Viruses in the laboratory, little can be known about their biology, the way they infect their hosts, and their broader influence on aquatic life.Deeg et al. have now isolated and characterized the giant Bodo saltans virus (BsV), a Giant Virus that infects an ecologically important microbe commonly found in aquatic environments. Sequencing the genome of BsV revealed many previously unknown genes, as well as several unusual features. For example, the genome contains movable genetic elements that might help to fend off other giant viruses by cutting their genomes. In addition, the set of genes used by BsV to translate mRNA templates into proteins differs from those found in other giant viruses, implying that they are not derived from a more complex common ancestor.The size of the genome appears to have grown rapidly by the duplication of genes at the end of the genome – a feature known as a genomic accordion. The identity of the duplicated genes suggests that there is an evolutionary arms race with its host that forces genome expansion. Further studies of the BsV genome could help researchers to understand the origin of gigantism in the genomes of giant viruses. Viruses are the most abundant biological entities on the planet and there are typically millions of virus particles in each milliliter of marine or fresh waters that are estimated to kill about 20% of the living biomass each day in surface marine waters . This haMimiviridae, infects a marine heterotrophic flagellate that was initially identified as Bodo sp. , a giant virus that infects the ecologically important kinetoplastid microzooplankter e oceans . Kinetope oceans , and aree oceans . At 1.39mic data . The BsVBodo saltans . The addition of BsV to a culture of Bodo saltans (~2.5 × 105 cells ml−1) at a virus particle to cell ratio of two, measured by flow cytometry, resulted in free virus particles 18 hr later. Viral concentrations peaked at 2.5 × 107 particles ml−1, while host cell density dropped to 25% of uninfected control cultures  that infects an isolate of the widely occurring kinetoplastid, cultures . The cloTransmission electron microscopy (TEM) revealed that BsV is an icosahedral particle approximately 300 nm in diameter . The parBodo saltans cell presents intricate intracellular structures, including the characteristic kinetoplast and a pronounced cytostome and cytopharynx  were most similar to viruses in the nucleocytoplasmic large DNA viruses (NCLDV) group, while the remaining ORFs were most similar to bacterial (9%) or archaeal (1%) sequences. In gene cluster analysis, only 45% of protein-coding gene clusters are shared with related viruses such as CroV and klosneuviruses, highlighting the low number of conserved core genes amongst these viruses , making the BsV genome one of the largest complete viral genomes described to date, surpassing those of mimiviruses infecting is 25.3%  and, as is 25.3% , much loodo spp. ; this suodo spp. . Unlike odo spp. . The genodo spp. . A poorlodo spp. . Severalodo spp. . Most pr viruses . Essenti viruses .While no function could be attributed to 54% of ORFs, the largest identifiable fraction of annotations are involved in DNA replication and repair . Coding There were 47 sequences identified that matched enzymes involved in protein and signal modification, with the majority being serine/threonine kinases/phosphatases. These are potentially involved in host cell takeover.The genome of BsV is rich in coding sequences involved in transcription. An early transcription factor putatively recognizing the highly conserved AAAAATTGA motif and a late transcription factor putatively targeting TGCG were identified, whereas the target sequence of a third transcription factor is unknown. Further, a TATA-binding protein, a transcription initiation factor (TFIIIB) and a transcription elongation factor (TFIIS) were identified that should aid transcription. As well, RNA polymerase subunits a,b,c,e,f,g and I were identified and are assisted by DNA topoisomerases Type 2 and 1B. BsV encodes amRNA specific RNase III, a poly A polymerase, several 5’ capping enzymes and methyl transferases. Transcription is presumably terminated in a manner similar to that described in ApMV, since hairpin structures were detected in the 3’ UTR of most transcripts . They arBsV also encodes several enzymes associated with nucleic-acid transport and metabolism, including several AT-specific nucleic-acid synthesis pathway components. For instance, adenylosuccinate, thymidylate and pseudouridine synthetases and kinases, as well as ribonucleoside-diphosphate reductase were evident. Other ORFs were associated with nucleotide salvaging pathways, including nucleoside kinases, phosphoribosyl transferases, and cytidine and deoxycytidylate deaminase. A mitochondrial carrier protein was identified that, similar to ApMV, likely provides dATP and dTTP directly from the kinetoplast to the virus factory, as evident from electron microscopic observations .Several genes were identified that are involved in membrane trafficking. A system based on soluble N-ethylmaleimide-sensitive factor (NSF) attachment proteins (SNAPs) and the SNAP receptors (SNAREs) appears to have been acquired from the host by horizontal gene transfer in the recent evolutionary past. In combination with several NSF homologues, including the vesicular-fusion ATPases that also seems to have been acquired from the host. Other proteins putatively involved in membrane trafficking are rab-domain containing proteins, ras-like GTPases, and kinesin motor proteins.The BsV genome encodes four major capsid proteins. One of these proteins contains several large insertions between conserved domains shared among all four capsid proteins, and with 4194 aa boasts a size almost seven times that of its paralogs. This enlarged version of the major capsid protein might be responsible for creating the halo around the virus particles observed by TEM, by producing shortened fibers similar to those observed in ApMV  Xiao et. FurtherThere were numerous ORFs that were similar to genes encoding metabolic proteins, like enzymes putatively involved in carbohydrate metabolism. However, no one continuous metabolic pathway could be assembled and therefore these enzymes likely complement host pathways. BsV also encodes coenzyme synthetases such as CoA and NADH and to meet the demand for amino acids that are rare in the host, BsV encodes the key steps in the synthesis pathways of glutamine, histidine, isoleucine, and asparagine.Another group of genes putatively mediate competitive interactions, either directly with the host, or with other viruses or intracellular pathogens. These include genes involved in the production of several toxins such as a VIP2-like protein as well as antitoxins containing BRO domains. Further, a partial bleomycin detox pathway was found, as well as multidrug export pumps and partial restriction modification systems.Mimiviridae and shows the largest turnover of these genes resulting in a net contraction  and their ribozymes differ in secondary structure . SubsequMimiviridae. Whole genome analysis based on gene cluster presence/absence of NCLDVs resulted in BsV clustering within the large mimiviruses (recently proposed ‘Megavirinae’) and separate from the small mimiviruses (‘Mesomimivirinae’)  with BsV, and CroV as the sole member of an outgroup (http://www.igs.cnrs-mrs.fr/TaraOceans/) were mapped to a maximum likelihood tree of DNA polymerase B sequences fromthe Mimiviridae, it was apparent that the ‘Megavirinae’ were dominated by klosneuviruses, with BsV as their only isolate    . BsV is iviridae . Within outgroup . Phylogeoutgroup ,2. When  isolate . Thus, BAureococcus anophagefferens virus; AcV: Anomala cumrea entomopoxvirus; AMaV: Acanthamoeba castellanii mamavirus; AMgV: Moumouvirus goulette; AMoV: Acanthamoeba polyphaga moumouvirus; ApMV: Acanthamoeba polyphaga mimivirus; ASFV: African swine fever virus BA71V; AtcV: Acanthocystis turfacea chlorella virus 1; BpV: Bathycoccus sp. RCC1105 Virus; BsV: Bodo saltans virus NG1; CatV: Catovirus; CeV: Chrysochromulina ericina virus 1B; CroV: Cafeteria roenbergensis virus BV-PW1; EhV: Emiliania huxleyi virus 86; FauV: Faustovirus E12; HokV: Hokovirus; HvV: Heliothis virescens ascovirus 3e; IiV: Invertebrate iridescent virus; IndV: Indivirus; ISKV: Infectious spleen and kidney necrosis virus; KloV: Klosneuvirus; LauV: Lausannevirus; MarV: Marseillevirus T19; MoVs: Mollivirus sibericum; MpVS: Micromoas pusillae Virus SP-1; MsV: Melanoplus sanguinipess entomopoxvirus; MVc: Megavirus chiliensis; MyxV: Myxoma virus; OLV1: Organic Lake Phycodnavirus 1; OLV2: Organic Lake Phycodnavirus 2; OtV: Ostreococcus tauri virus 1; PbCV: Paramecium bursaria chlorella virus 1; PgV: Phaeocystis globosa virus 16T; PiVs: Pithovirus sibericum P1084-T; PoV: Pyramimonas orientalis virus; PpV: Phaeocystis pouchetii virus; PVd: Pandoravirus dulcis; PVs: Pandoravirus salinus; SfV: Spodoptera frugiperda ascovirus 1a; SGV: Singapore grouper iridovirus; TnV: Trichoplusia ni ascovirus 2 c; VacV: Vaccinia virus; WiV: Wiseana iridescent virus; YLV1: Yellowstone lake phycodnavirus 1AaV: Mimiviridae, making it one of the largest completely sequenced genome of the family. BsV groups with the klosneuviruses and is separate from CroV and the Acanthamoeba infecting mimiviruses, and falls within the proposed ‘Megavirinae’  has the one of the largest sequenced genome of the Virus concentrates were collected from 11 fresh water locations in southern British Columbia, Canada . To concentrate giant viruses, 20 l water samples were prefiltered with a GF-A filter  over a 0.8 um PES membrane . Filtrates from all locations were pooled and were concentrated using a 30 kDa MW cut-off tangential flow filtration cartridge  .Bodo saltans strain NG, the host of BsV, was isolated from a water sample collected near the sediment surface of the pond in Nitobe Memorial Garden, The University of British Columbia, Canada . Clonal cultures were obtained by end-point dilution, and maintained in modified DY-V artificial fresh water media with yeast extract and a wheat grain (http://www3.botany.ubc.ca/cccm/) reference number CCCM 6296  vs. FSC on FACScalibur )). After aey, USA)). Cell nuThe similarity to the flow cytometry profile of Cafeteria roenbergensis virus suggested that the lytic agent was in deed a giant virus.Bodo saltans lysates after BsV infection were applied to the carbon side of a formvar-carbon coated 400 mesh copper grid  and incubated at 4°C in the dark overnight in the presence of high humidity. Next, the lysate was removed and the grids were stained with 1% Uranyl acetate for 30 s before observation on a Hitachi H7600 transmission electron microscope at 80 kV.B. saltans cultures were infected at a concentration of 5 × 105 cells ml−1 with BsV at a relative particle to cell ratio of ~5 to ensure synchronous infection. Cells were harvested from infected cultures at different time points  as well as from uninfected control cultures. Cells from 50 ml were pelleted in two consecutive 10 min at 5000 xg centrifugation runs in a Beckmann tabletop centrifuge. Pellets were resuspended in 10–15 µl DY-V culture medium with 20% (w/v) BSA and immediately place on ice. Cell suspensions were cryo-preserved using a Leica EM HPM100 high-pressure freezer. Vitrified samples were freeze-substituted in a Leica AFS system for 2 days at −85°C in a 0.5% glutaraldehyde 0.1% tannic acid solution in acetone, then rinsed 10 times in 100% acetone at −85°C, and transferred to 1% osmium tetroxide, 0.1% uranyl acetate in acetone and stored for an additional 2 days at −85°C. The samples were then warmed to −20°C over 10 hr, held at −20°C for 6 hr to facilitate osmication, and then warmed to 4°C over 12 hr. The samples were then rinsed in 100% acetone 3X at room temperature and gradually infiltrated with an equal part mixture of Spurr’s and Gembed embedding media. Samples were polymerized in a 60°C oven overnight. 50 nm thin sections were prepared using a Diatome ultra 45° knife  on an ultra-microtome. The sections were collected on a 40x copper grid and stained for 10 min in 2% aqueous uranyl acetate and 5 min in Reynold’s lead citrate. Image data were recorded on a Hitachi H7600 transmission electron microscope at 80 kV. Image J (RRID:SCR_003070) was used to compile all TEM images. Adjustments to contrast and brightness levels were applied equally to all parts of the image.Exponentially growing B. saltans cultures were infected at a concentration of approximately 5 × 105 cells ml−1 with BsV lysate (107 VLP ml−1) at a multiplicity of infection (MOI) of ~0.5. After 4 days, when host cell densities had dropped below 30%, cultures were centrifuged in a Sorvall SLC-6000 for 20 min, 5000 rpm, 4°C to remove remaining host cells and the supernatant was consecutively subjected to tangential flow filtration with at 30 kDa cut-off (Vivaflow PES) and concentrated approximately 100x. Viral concentrates were subjected to ultracentrifugation at 28,000 rpm, 15°C for 8 hr in a Ti90 fixed angle rotor . Pellets were resuspended and virions lysed using laurosyl acid and proteinase K subjected to pulsed-field gel electrophoresis on a CHEF II pulse field gel electrophoresis aperture (BioRad) for 25 hr at 14°C in a 0.8% LMP agarose gel with 60–180S switchtimes and 16.170 ramping factor in 0.5 TBE under 5.5 V/cm and 120°. Genomic DNA was visualized under UV light after 30 min SYBR gold  staining. The dominant PFGE band belonging to genomic BsV DNA (1.35 Mb) was cut and DNA was extracted using a GELase kit  and ethanol purified according to manufacturer’s protocol. Libraries were prepared using the Illumina Nextera XT kit  as per manufacturer’s recommendation and library quality and quantity were checked by Bioanalyzer 2100 with the HS DNA kit (Agilent Technology). 300 bp paired-end sequencing was performed on an Illumina MiSeq platform by UCLA's Genoseq center  to a nominal sequencing depth of 800x. Sequence quality was examined using FastQC  and sequence reads were quality trimmed (parameters: minlen = 50 qtrim = rl trimq = 15 ktrim = r k = 21 mink = 8 ref=$adapters hdist = 2 hdist2 = 1 tbo = t tpe = t) and cleared of human (parameters: minid = 0.95 maxindel = 3 bwr = 0.16 bw = 12 quickmatch fast minhits = 2 qtrim = lr trimq = 10) and PhiX (parameters: k = 31 hdist = 1) sequences against the whole respective genomes using BBMap v35 (http://sourceforge.net/projects/bbmap/).For Illumina sequencing, exponentially growing 2 followed by centrifugation in an SW40 rotor for 20 min at 7200 rpm and 18°C and were finally collected by centrifugation in an SW40 rotor for 30 min at 7800 rpm and 18°C. Purity of the concentrate was verified by flow cytometry  vs SSC on a FACScalibur . High-molecular-weight genomic DNA was extracted using phenol-chloroform-chloroform extraction. Length and purity were confirmed by gel electrophoresis and Bioanalyzer 2100 wit the HS DNA kit (Agilent Technology). PacBio RSII 20 kb sequencing was performed by the sequencing center of the University of Delaware. Reads were assembled using PacBio HGAP3 software with 20 kb seed reads and resulted in a single viral contig of 1,384,624 bp, 286.1x coverage, 99.99% called bases and a consensus concordance of 99.9551% . An Optiprep (Sigma) gradient was created by underlaying a 10% Optiprep solution in 50 mM Tris-Cl, pH 8.0, 2 mM MgCl2 with a 30% solution followed by a 50% solution and was equilibration over night at 4°C. One ml of viral concentrate from the 40% cushion was added atop the gradient and the concentrate was fractionated by ultracentrifugation in an SW40 rotor for 4 hr at 25,000 rpm and 18°C. The viral fraction was extracted from the gradient with a syringe and washed twice with 50 mM Tris-Cl, pH 8.0, 2 mM MgCl99.9551% .Cleaned up Illumina reads were mapped to the PacBio contig to confirm the PacBio assembly as well as extending the contig’s 5’ end by 1245 bp to a total viral genome length of 1,385,869 bp.SCR_011931, Open-reading frames were predicted using GLIMMER  on all protein-coding genes of length ≥100 aa. This resulted in the definition of 3001 distinct clusters. Gene clusters are available in source file ‘Source_data_Fig4_S3_all-Mimi_COGs’, ‘Source_data_Fig4C_translation-Mimi_COGs’, ‘Source_data_Fig6A-Fig4C_orthoMCL_groups’, and ‘Source_data_Fig6A_gene-clusters’. We computed a presence/absence matrix based on the genes clusters and calculated a distance matrix using the according to Whole genome content phylogeny was performed by OrthoMCL (RRID:SCR_010519) using MUSCLE with default parameters .Alignments of aa sequences were performed in Geneious R9 (RRID:http://www.igs.cnrs-mrs.fr/TaraOceans/) were mapped to a Mimiviridae DNA polymerase B family reference tree created as described above with pplacer . Of the 401 input sequences, 256 mapped within the Mimiviridae and are displayed in the figure.Translated environmental assemblies identified by Hingamp et al. as representing NCLDV DNA polymerase B family genes ; Eugene Koonin (Reviewer #2); Grieg Steward (Reviewer #3).Thank you for submitting your article "The kinetoplastid-infecting Bodo saltans virus (BsV), a window into the most abundant giant viruses in the sea" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.The reviewers all agree that this manuscript and work are important but feel that the inferences about its exact phylogenetic position are not sufficiently supported to justify some of the statements and that there are many small editorial fixes needed as found below in the detailed reviews.Essential revisions:1) The uncertainty with the phylogenies seems to be the main problematic issue with this manuscript. This can be resolved by simply making various statements about the abundance and phylogeny more cautious and some additional phylogeny analysis as described in reviews 2 and 3.2) Addressing the editorial revisions requested in the detailed reviews below.Reviewer #1:Mimiviridae family, ii) BsV is the first virus to be isolated and characterized in one of the 3 subclades of the Mimiviridae (the other members in this subclade only exist from metagenomic sequences), iii) this subclade of viruses appear to be the most common viruses in the ocean after bacteriophage, iv) BsV is claimed to be one of the few Mimiviridae members that does not infect amoeba , and v) the genome has some interesting features, including the absence of tRNA genes, it has several homing endonuclease-encoding self-splicing introns and many apparent gene duplications of ankyrin repeat encoding genes.This manuscript describes the isolation and characterization of a giant virus (BsV) that infects the ecologically important microzooplankton, the kinetoplastid Bodo saltans. The manuscript is important for several reasons including: i) BsV has the largest genome of viruses included in the The experiments appear to be carefully conducted by an established research group that works in the area of giant viruses and viruses in the marine environment. I only have a few comments to make about the manuscript.1) The authors claim that BsV has a stargate structure, which has been extensively studied in the prototype Mimivirus APMV. In the last paragraph of the subsection “Virus morphology and Replication Kinetics”, they refer to 2) The authors state in the first paragraph of the subsection “Virus morphology and Replication Kinetic”, that the virion has 6 layers that engulf the virion and refer to Reviewer #2:Mimiviridae from a marine protist is of major interest and value. It is particularly important that the actual virus has been obtained given that the related Klosneuviruses so far have been identified only from metagenomic sequences. This report will be of interest to all virologists, and I believe, also to a broader audience of biologists.The isolation and characterization of a novel, highly abundant giant virus of the family I do have certain concerns regarding the genome analysis described in the manuscript and the authors' interpretation.Aquavirinae". The DNAP tree (6D) does not agree, here BsV falls within the Klosneuvirus group. In this situation, I think there is little confidence in the exact position of BsV within the Mimiviridae, and also in the position of CroV at the base of the "Aquavirinae" as opposed to outside of the MImi-Klos clade as observed in the Schul et al. paper on Klosneuviruses. I find it desirable to invest some more effort into phylogenetic analysis, in particular, analyze Bayesian trees, perform tree topology tests (e.g. AU) and present trees for individual core NCLDV genes. Unless such additional analyses substantially increase the confidence in the tree topology (and I am not entirely optimistic), I think it is best to be much more circumspect regarding the phylogeny of the Mimiviridae in general and the position of BsV, in particular.My first and probably most important problem is with the phylogeny of the NCLDV shown in Second, and related to my first concern, the tree topology affects the conclusions regarding gene gain and loss. The authors indicate in the Abstract and other parts of the article that much of the translation machinery has been lost by BsV. Qualitatively, this is likely to be true but how much, critically depends on the tree topology. Furthermore, I find it highly desirable to perform a formal reconstruction of gene gain and loss using COUNT or GLUM software. Without such an analysis, claims of gene gain and loss remain poorly substantiated. Finally, on the subject of gene loss, to me, one of the most interesting findings here is the presence in BsV of pseudogenes of 3 aaRS pseudogenes and one translation factor. This finding does indeed imply that the translation system could be "on its way out", and in my opinion, should be emphasized stronger.Mimiviridae, 1227. Two of the Klosneuviruses are bigger.Third, and less important, I find it strange that the authors claim that BsV has the largest number of genes among the Aqauvirinae' but rather 'a provisional subfamily "Aquavirinae" with the family Mimiviridae'.Fourth, and least important, but still essential, the authors should be more cautious with taxonomy, especially, given the aforementioned uncertainties with the phylogeny. It is fine to propose Aquavirinae but it should not be 'a distinct evolutionary group [what's that?] Reviewer #3:Megaviridae and its genomic content. The work is noteworthy for several reasons: 1) the size of the viral genome, which is the largest yet described for an isolate within this family, 2) the unusual functional features of the genome relative to other members of the family, and 3) the lineage of the host, since it is the first DNA virus known to infect a member of the Excavata. This is a noteworthy discovery with lots of interesting tidbits including the absence of tRNAs and the presence of parasitic genetic elements (inteins and introns) that would seem to confer competitive advantage during co-infection with other viruses. The science looks sound. My main general recommendation is that the authors be more careful with the wording, specifically properly qualifying statements about the abundance and phylogenetic position of this virus and its relatives. Also, I would recommend being more circumspect in the phrasing of the headings in the Discussion (and perhaps title), many of which read as statements of fact but are inferences and hypotheses.This paper describes a new viral isolate within the  Reviewer #1:[…] 1) The authors claim that BsV has a stargate structure, which has been extensively studied in the prototype Mimivirus APMV. In the last paragraph of the subsection “Virus morphology and Replication Kinetics”, they refer to eLife guidelines. We agree that the evidence of a stargate structure in the figures is not definitive, but it is persuasive, especially in the figures in the supplemental material. We have modified the statement in the last paragraph of the subsection “Virus morphology and Replication Kinetics” to emphasize uncertainty in our statement.Supplemental figure references are formatted according to 2) The authors state in the first paragraph of the subsection “Virus morphology and Replication Kinetic”, that the virion has 6 layers that engulf the virion and refer to Arrow heads have been added to indicate these layers.Reviewer #2:[…] I do have certain concerns regarding the genome analysis described in the manuscript and the authors' interpretation.My first and probably most important problem is with the phylogeny of the NCLDV shown in Additional phylogenetic analysis was performed according to the suggestions of reviewer 2. A refined concatenated alignment of five conserved genes followed by Bayesion analysis confidently placed BsV within the klosneuvirus clade. Second, and related to my first concern, the tree topology affects the conclusions regarding gene gain and loss. The authors indicate in the Abstract and other parts of the article that much of the translation machinery has been lost by BsV. Qualitatively, this is likely to be true but how much, critically depends on the tree topology. Furthermore, I find it highly desirable to perform a formal reconstruction of gene gain and loss using COUNT or GLUM software. Without such an analysis, claims of gene gain and loss remain poorly substantiated. Finally, on the subject of gene loss, to me, one of the most interesting findings here is the presence in BsV of pseudogenes of 3 aaRS pseudogenes and one translation factor. This finding does indeed imply that the translation system could be "on its way out", and in my opinion, should be emphasized stronger.COUNT analysis of conserved gene families was performed. The reconstructed evolutionary history of translational genes is now depicted in Third, and less important, I find it strange that the authors claim that BsV has the largest number of genes among the Mimiviridae, 1227. Two of the Klosneuviruses are bigger.The manuscript has been modified to indicate that we are referring to completely sequenced genomes from cultured viruses. This is in contrast to the larger, incomplete klosneuvirus genomes inferred from metagenomic analysis.Fourth, and least important, but still essential, the authors should be more cautious with taxonomy, especially, given the aforementioned uncertainties with the phylogeny. It is fine to propose Aquavirinae but it should not be 'a distinct evolutionary group [what's that?] Aqauvirinae' but rather 'a provisional subfamily "Aquavirinae" with the family Mimiviridae'.Aquavirinae” was dropped from the manuscript. The use of taxonomic names previously proposed, but not confirmed by the ICTV  throughout the manuscript has been updated as per the reviewer’s suggestion.Based on the updated phylogenetic analysis, the term “Reviewer #3:This paper describes a new viral isolate within the Megaviridae and its genomic content. The work is noteworthy for several reasons: 1) the size of the viral genome, which is the largest yet described for an isolate within this family, 2) the unusual functional features of the genome relative to other members of the family, and 3) the lineage of the host, since it is the first DNA virus known to infect a member of the Excavata. This is a noteworthy discovery with lots of interesting tidbits including the absence of tRNAs and the presence of parasitic genetic elements (inteins and introns) that would seem to confer competitive advantage during co-infection with other viruses. The science looks sound. My main general recommendation is that the authors be more careful with the wording, specifically properly qualifying statements about the abundance and phylogenetic position of this virus and its relatives. Also, I would recommend being more circumspect in the phrasing of the headings in the Discussion (and perhaps title), many of which read as statements of fact but are inferences and hypotheses.Statements have been modified throughout the manuscript as per the reviewer’s recommendations.


To estimate the timing of key events in the natural history of Zika virus infection.Zika. By pooling data, we estimated the incubation period, the time to seroconversion and the duration of viral shedding. We estimated the risk of Zika virus contaminated blood donations.In February 2016, we searched PubMed, Scopus and the Web of Science for publications containing the term We identified 20 articles on 25 patients with Zika virus infection. The median incubation period for the infection was estimated to be 5.9 days , with 95% of people who developed symptoms doing so within 11.2 days (95% CrI: 7.6–18.0) after infection. On average, seroconversion occurred 9.1 days (95% CrI: 7.0–11.6) after infection. The virus was detectable in blood for 9.9 days (95% CrI: 6.9–21.4) on average. Without screening, the estimated risk that a blood donation would come from an infected individual increased by approximately 1 in 10 000 for every 1 per 100 000 person–days increase in the incidence of Zika virus infection. Symptom-based screening may reduce this rate by 7%  and antibody screening, by 29% .Neither symptom- nor antibody-based screening for Zika virus infection substantially reduced the risk that blood donations would be contaminated by the virus. Polymerase chain reaction testing should be considered for identifying blood safe for use in pregnant women in high-incidence areas. There are concerns about potential transmission through blood transfusion because a large proportion of people infected with the virus remain asymptomatic,,,,–As temporary deferral or the banning of blood donations could result in severe shortages in blood supplies, research on the duration of viraemia and the time to antibody seroconversion is vital for quantifying the risk to blood supplies and for developing strategies for protecting them. Furthermore, better knowledge of the natural history of Zika virus infection, including the incubation period and infectious period, is essential for designing evidence-based surveillance systems and informing public health policy.The aim of this study was to better characterize the natural history of Zika virus infection and to inform disease prevention, surveillance and blood supply safety strategies by applying an extension of this analytical approach to case reports collected through a systematic review of the literature. Using pooled data, we estimated the incubation period, the time to seroconversion and the duration of viral shedding from Zika virus infection.Zika in any field. For our analysis, we included publications that provided information on: (i) the time of exposure to the Zika virus; (ii) the time of symptom onset; and (iii) the time of sample collection for, and the result (i.e. positive or negative) of, virological (e.g. by polymerase chain reaction or culture) or antibody Zika virus testing or both. Each publication was randomly assigned to two reviewers who independently screened titles and abstracts for relevance. We excluded publications that: (i) were not about the Zika virus; (ii) contained no data from humans; (iii) did not provide sufficient information to determine a bounded time of exposure; (iv) reported only perinatal viral transmission; or (v) were not in English. However, papers in French, Portuguese or Spanish that would have passed abstract screening had they been in English (i.e. 55 of 78 non-English articles) were also reviewed  for publications containing the term reviewed . Then, tWe abstracted the data necessary to estimate: (i) the incubation period of the Zika virus; (ii) the timing and duration of viral shedding; and (iii) the time to a positive serum antibody test result. In particular, we obtained information that enabled us to determine upper and lower bounds on the time of: (i) exposure to the Zika virus; (ii) symptom onset; and (iii) sample collection for virological or antibody testing. The exact timing of events was used when possible; otherwise bounds on the timing of an event were derived from the information available (e.g. the dates of travel to an endemic region). For virological and serological tests, the test result was recorded and specific immunoglobulin-M serological test results were noted, when available. We also recorded basic demographic details, the type of sample collected (e.g. blood or urine) and, when available, the mode of viral transmission.The bounds for the time of Zika virus infection were the earliest and latest possible times of exposure consistent with the case report. When no latest exposure time could be determined – for example, the patient developed symptoms in an endemic area – it was assumed to be the latest possible time of symptom onset – the time of symptom onset was specified to the nearest day in most case reports. The earliest possible time of seroconversion was considered to be immediately after the last negative serological test and the latest possible time was immediately before the first positive test. If only a positive serological test was reported, the earliest possible time of seroconversion was considered to be the same as the earliest possible time of exposure; when only a negative test was reported, seroconversion was considered to have occurred after the time of testing. Similarly, the earliest possible time of viral clearance, which was defined as no detectable virus in blood, was the time of the last positive virological test and the latest was the time of the first negative test. Missing virological test results were treated in the same way as missing serological test results.,For each observation of a time to a key event in the course of Zika virus infection , we used the observed data to derive upper and lower bounds for the time of exposure and for the event. These censored observations were then used to fit separate distributions for each key event using an adaptation of previously described techniques.,We estimated the impact of the distributions of key variables on the safety of blood supplies for a constant incidence rate. The number of blood donors with a possible Zika virus infection in every 100 000 in the absence of screening was calculated as the daily incidence of infection per 100 000 multiplied by the mean time to viral clearance. This estimate was adjusted for symptom-based screening using the mean time to symptom onset and by assuming that 80% of those infected remain asymptomatic.Zika, of which 118 were selected for full text review , with a dispersion of 1.5 days (95% CrI: 1.2–1.9). Hence, 5% of symptomatic cases would be expected to develop symptoms within 3.2 days of infection (95% CrI: 1.7–4.6), 25% within 4.6 days (95% CrI: 3.1–6.0), 75% within 7.6 days (95% CrI: 5.8–10.4) and 95% within 11.2 days  if no screening were performed. Refusing donations from people with recent symptoms of a possible infection would decrease this risk by only 7% , assuming that 80% of individuals with Zika virus infections are asymptomatic and given that those who develop symptoms are infectious but asymptomatic for an average of 5.9 days – here it is assumed that the Zika virus can be transmitted via blood from the moment of infection. Serological screening would reduce the risk by 29%  but improve the safety of blood supplies only marginally. In settings where the risk is solely from imported Zika cases, ensuring blood supply safety is easier. We estimated that, by 23.4 days (95% CrI: 14.3–154.3) after infection, 99% of cases would no longer have detectable virus in their blood. For these estimates, we assumed that a blood donation would be safe if there was no detectable virus in the donor’s blood. However, in four reported cases, a saliva, nasal or urine sample tested positive for the virus even though it was no longer detectable in blood . Blood d,In June 2016, WHO reported that the incubation period of the Zika virus was uncertain but likely to be “a few days”.,,In practice, many countries and organizations recommend deferring blood donation until 28 days after the resolution of symptoms or after the time of a positive serological or virological test result for an asymptomatic individual, as recommended by WHOOur study highlights the need for a highly specific, simple and rapid Zika virus antigen test. As well as for screening donated blood, an antigen test could be used to monitor the incidence of Zika virus infection, to help provide advice for women considering becoming pregnant and to identify pregnant women at risk of a poor clinical outcome. In addition, saliva or urine could be used in diagnostic tests and research into the relationship between the presence of the virus in bodily fluids and the risk of disease transmission is also needed. Knowledge of the time between infection and the virus becoming detectable in various fluids is essential for ascertaining the ability of antigen testing to ensure the safety of blood and organ donations. Moreover, knowledge of the incubation period can help clinicians determine whether Zika virus infection should be considered in the differential diagnosis of febrile patients who have recently travelled abroad and a good estimate of the time to seroconversion can help optimize the timing of confirmatory testing. Knowing the time to viral clearance after a potential infection or exposure can indicate when it may again be safe to become pregnant. However, the time to clearance from seminal fluid remains unknown. Our analysis provides only a first step in determining the values of key variables in Zika virus infection as the small number of cases included means that there are substantial uncertainties and a potential for bias. Investigators should continue to collect data to refine and update our estimates and to provide information on other key variables in the disease’s natural history, such as the latent period (i.e. the time between being infected and becoming infectious). To assist that process, all our data and the analysis software we used are freely available to enable other investigators to contribute to this work or apply our methods to their own data.Our analysis necessarily involved several assumptions because the published data were not collected to assess key variables in Zika virus infection. First, we assumed that virological testing of blood or sera had a sensitivity of 100% for detecting a Zika virus infection – however there is evidence that viral shedding can continue longer in urine and other bodily fluids and the virus may persist in the blood below the limit of detection. We also assumed that the time to seropositivity for the Zika virus was independent of previous infections – however it is probable that people who have had a prior flavivirus infection may seroconvert more quickly. Consequently, it is possible we overestimated the time to seroconversion because the majority of cases in our analysis were travellers returning to countries where flaviviruses were not endemic. Another limitation is that the majority of our data were from people presumed to have been infected through mosquito bites. The timing of key events may differ for other routes of infection . Furthermore, all our cases were symptomatic – the time to seroconversion or viral clearance may be different in asymptomatic individuals and it is possible that cases reported in the literature may have been more severe than usual. Nevertheless, the principle limitation of our analysis was the small number of cases.Despite these limitations, our analysis provides the most detailed, quantitative estimates to date of the timing of key events in the natural history of Zika virus infection. Our findings can help guide disease surveillance in both endemic areas and in returning travellers and can underpin research into the basic features of this pathogen.


Healthcare interventions, and particularly those in public health may affect multiple diseases and significantly prolong life. No consensus currently exists for how to estimate comparable healthcare costs across multiple diseases for use in health and public health cost-effectiveness models. We aim to describe a method for estimating comparable disease specific English healthcare costs as well as future healthcare costs from diseases unrelated to those modelled.We use routine national datasets including programme budgeting data and cost curves from NHS England to estimate annual per person costs for diseases included in the PRIMEtime model as well as age and sex specific costs due to unrelated diseases.The 2013/14 annual cost to NHS England per prevalent case varied between £3,074 for pancreatic cancer and £314 for liver disease. Costs due to unrelated diseases increase with age except for a secondary peak at 30–34 years for women reflecting maternity resource use.The methodology described allows health and public health economic modellers to estimate comparable English healthcare costs for multiple diseases. This facilitates the direct comparison of different health and public health interventions enabling better decision making. Disease specific healthcare costs for use in health and public health economic models can be calculated using one of two different approaches: adding together the costs of all components of a patient’s care, such as staff and equipment (a micro-level or bottom-up approach), or allocating an overall healthcare budget to specific diseases (a macro-level or top-down approach).Guidelines exist for how to estimate costs for health economic cost-effectiveness models, however these do not provide specific advice on how to identify costs when evaluating the impact on multiple diseases simultaneously where the comparability of data sources for different diseases is important ,2. WithoA second challenge is how to quantify the age and sex specific future economic consequences of individuals becoming unwell from diseases unrelated to those explicitly modelled following interventions that may prolong life ,11. InclThe aim of this paper is to describe a method for estimating comparable disease specific and unrelated future healthcare costs using routinely available data in England for use in health and public health economic modelling. We present both the method and the results of applying this method to 11 diseases included in an existing multistate life table model.A case study is used to illustrate the methodology: disease specific costs are estimated for the 11 diseases simulated by the multistate life table model, PRIMEtime , and foDisease specific costs are derived from 2013/14 NHS England programme budgeting data which reports expenditure by clinical commissioning groups , and accounts for around two thirds of the total NHS England budget , an, an9], aFinally, in the absence of any UK studies estimating unrelated healthcare costs by age and sex, results are compared with Blakely et al. who estimated healthcare expenditure for diseases unrelated to tobacco consumption for the New Zealand population by age and sex using bottom-up unit patient care costs . The magIn summary, we describe a novel approach to estimating NHS England costs for multiple diseases and unrelated future healthcare costs using routinely available data. The key strength is that we use a consistent approach to estimating costs across multiple diseases meaning that the cost implications of different interventions affecting these diseases can be directly compared. This approach can be applied to other health and public health economic models that estimate the economic consequences of an intervention affecting multiple diseases in England.S1 File(DOCX)Click here for additional data file.


The ToMV genome of the Zimbabwe isolate is the second to be reported in Africa.A tomato-infecting tomato mosaic virus (ToMV) isolate was detected in Zimbabwe using lateral flow kits and double-antibody sandwich enzyme-linked immunosorbent assay. Next-generation sequencing and  Tobamovirus in the family Virgaviridae, is an important pathogen of ornamental and solanaceous crops worldwide (Tomato mosaic virus (ToMV), which belongs to the genus orldwide , 2. ToMVorldwide ; howeverDuring surveys for tomato diseases in 2015 and 2016, tomato plants exhibiting stunted growth, leaf wrinkles, and a fern-like appearance were found at four smallholder irrigation schemes in Mutoko district, Zimbabwe. Based on these symptoms, five leaf samples per site were tested on-site for ToMV using the virus-specific LoeweFast lateral flow kits . All samples were positive. Subsequently, five leaf samples per site per year were collected, preserved in RNAlater solution (Qiagen), and tested for cucumber mosaic virus (CMV), ToMV, tomato spotted wilt virus (TSWV), and tobacco mosaic virus (TMV) using double-antibody sandwich enzyme-linked immunosorbent assay kits . Only ToMV was detected in the samples.https://www.bioinformatics.babraham.ac.uk/projects/fastqc) was used to assess the quality of the reads. Adapter removal and trimming were performed using Trimmomatic version 0.33 software  was used to extract total RNA from one randomly selected ToMV-positive sample. The RNA was sequenced on an Illumina MiSeq platform at Inqaba Biotechnical Industries (Pty) Ltd. . FastQC (software  prior toon 3.9.0 .One contig, 6,383 nucleotides in size, was identified as ToMV by BLASTn analysis. It was found to be the full-length genome sequence upon further inspection. The Zimbabwe ToMV isolate was analyzed for recombination events using the RDP4 program .Tm-1 and Tm-2 resistance-breaking (The Zimbabwe ToMV isolate has a nonrecombinant genome. Furthermore, the amino acid exchanges responsible for breaking  were notKX711903.The genome sequence reported here has been deposited in DDBJ/EMBL/GenBank under the accession number