TRAF6 Plays a Proviral Role in Tick-Borne Flavivirus Infection through Interaction with the NS3 Protease.

Tick-borne flaviviruses (TBFVs) can cause life-threatening encephalitis and hemorrhagic fever. To identify virus-host interactions that may be exploited as therapeutic targets, we analyzed the TBFV polyprotein in silico for antiviral protein-binding motifs. We obtained two putative tumor necrosis factor receptor-associated factor 6 (TRAF6)-binding motifs (TBMs) within the protease domain of the viral nonstructural 3 (NS3) protein. Here, we show that TBFV NS3 interacted with TRAF6 during infection and that TRAF6 supports TBFV replication. The proviral role of TRAF6 was not seen with mosquito-borne flaviviruses, consistent with the lack of conserved TBMs. Mutation of the second TBM within NS3 disrupted TRAF6 binding, coincident with reduced abundance of mature, autocatalytically derived form of the NS3 protease and significant virus attenuation in vitro. Our studies reveal insights into how flaviviruses exploit innate immunity for the purpose of viral replication and identify a potential target for therapeutic design.

Morbidity and mortality due to shigella and enterotoxigenic Escherichia coli diarrhoea: the Global Burden of Disease Study 1990-2016.

BACKGROUND: Shigella and enterotoxigenic Escherichia coli (ETEC) are bacterial pathogens that are frequently associated with diarrhoeal disease, and are a significant cause of mortality and morbidity worldwide. The Global Burden of Diseases, Injuries, and Risk Factors study 2016 (GBD 2016) is a systematic, scientific effort to quantify the morbidity and mortality due to over 300 causes of death and disability. We aimed to analyse the global burden of shigella and ETEC diarrhoea according to age, sex, geography, and year from 1990 to 2016. METHODS: We modelled shigella and ETEC-related mortality using a Bayesian hierarchical modelling platform that evaluates a wide range of covariates and model types on the basis of vital registration and verbal autopsy data. We used a compartmental meta-regression tool to model the incidence of shigella and ETEC, which enforces an association between incidence, prevalence, and remission on the basis of scientific literature, population representative surveys, and health-care data. We calculated 95% uncertainty intervals (UIs) for the point estimates. FINDINGS: Shigella was the second leading cause of diarrhoeal mortality in 2016 among all ages, accounting for 212 438 deaths (95% UI 136 979-326 913) and about 13·2% (9·2-17·4) of all diarrhoea deaths. Shigella was responsible for 63 713 deaths (41 191-93 611) among children younger than 5 years and was frequently associated with diarrhoea across all adult age groups, increasing in elderly people, with broad geographical distribution. ETEC was the eighth leading cause of diarrhoea mortality in 2016 among all age groups, accounting for 51 186 deaths (26 757-83 064) and about 3·2% (1·8-4·7) of diarrhoea deaths. ETEC was responsible for about 4·2% (2·2-6·8) of diarrhoea deaths in children younger than 5 years. INTERPRETATION: The health burden of bacterial diarrhoeal pathogens is difficult to estimate. Despite existing prevention and treatment options, they remain a major cause of morbidity and mortality globally. Additional emphasis by public health officials is needed on a reduction in disease due to shigella and ETEC to reduce disease burden. FUNDING: Bill & Melinda Gates Foundation.

Rotavirus Vaccination and the Global Burden of Rotavirus Diarrhea Among Children Younger Than 5 Years.

Importance: Rotavirus infection is the global leading cause of diarrhea-associated morbidity and mortality among children younger than 5 years. Objectives: To examine the extent of rotavirus infection among children younger than 5 years by country and the number of deaths averted because of the rotavirus vaccine. Design, Setting, and Participants: This report builds on findings from the Global Burden of Disease Study 2016, a cross-sectional study that measured diarrheal diseases and their etiologic agents. Models were used to estimate burden in data-sparse locations. Exposure: Diarrhea due to rotavirus infection. Main Outcomes and Measures: Rotavirus-associated mortality and morbidity by country and year and averted deaths attributable to the rotavirus vaccine by country. Results: Rotavirus infection was responsible for an estimated 128 500 deaths (95% uncertainty interval [UI], 104 500-155 600) among children younger than 5 years throughout the world in 2016, with 104 733 deaths occurring in sub-Saharan Africa (95% UI, 83 406-128 842). Rotavirus infection was responsible for more than 258 million episodes of diarrhea among children younger than 5 years in 2016 (95% UI, 193 million to 341 million), an incidence of 0.42 cases per child-year (95% UI, 0.30-0.53). Vaccine use is estimated to have averted more than 28 000 deaths (95% UI, 14 600-46 700) among children younger than 5 years, and expanded use of the rotavirus vaccine, particularly in sub-Saharan Africa, could have prevented approximately 20% of all deaths attributable to diarrhea among children younger than 5 years. Conclusions and Relevance: Rotavirus-associated mortality has decreased markedly over time in part because of the introduction of the rotavirus vaccine. This study suggests that prioritizing vaccine introduction and interventions to reduce diarrhea-associated morbidity and mortality is necessary in the continued global reduction of rotavirus infection.

Morbidity, mortality, and long-term consequences associated with diarrhoea from Cryptosporidium infection in children younger than 5 years: a meta-analyses study.

BACKGROUND: The protozoan Cryptosporidium is a leading cause of diarrhoea morbidity and mortality in children younger than 5 years. However, the true global burden of Cryptosporidium infection in children younger than 5 years might have been underestimated in previous quantifications because it only took account of the acute effects of diarrhoea. We aimed to demonstrate whether there is a causal relation between Cryptosporidium and childhood growth and, if so, to quantify the associated additional burden. METHODS: The Global Burden of Diseases, Injuries, and Risk Factors study (GBD) 2016 was a systematic and scientific effort to quantify the morbidity and mortality associated with more than 300 causes of death and disability, including diarrhoea caused by Cryptosporidium infection. We supplemented estimates on the burden of Cryptosporidium in GBD 2016 with findings from a systematic review of published and unpublished cohort studies and a meta-analysis of the effect of childhood diarrhoea caused by Cryptosporidium infection on physical growth. FINDINGS: In 2016, Cryptosporidium infection was the fifth leading diarrhoeal aetiology in children younger than 5 years, and acute infection caused more than 48 000 deaths (95% uncertainty interval [UI] 24 600-81 900) and more than 4·2 million disability-adjusted life-years lost (95% UI 2·2 million-7·2 million). We identified seven data sources from the scientific literature and six individual-level data sources describing the relation between Cryptosporidium and childhood growth. Each episode of diarrhoea caused by Cryptosporidium infection was associated with a decrease in height-for-age Z score (0·049, 95% CI 0·014-0·080), weight-for-age Z score (0·095, 0·055-0·134), and weight-for-height Z score (0·126, 0·057-0·194). We estimated that diarrhoea from Cryptosporidium infection caused an additional 7·85 million disability-adjusted life-years (95% UI 5·42 million-10·11 million) after we accounted for its effect on growth faltering-153% more than that estimated from acute effects alone. INTERPRETATION: Our findings show that the substantial short-term burden of diarrhoea from Cryptosporidium infection on childhood growth and wellbeing is an underestimate of the true burden. Interventions designed to prevent and effectively treat infection in children younger than 5 years will have enormous public health and social development impacts. FUNDING: The Bill & Melinda Gates Foundation.

Global disability-adjusted life-year estimates of long-term health burden and undernutrition attributable to diarrhoeal diseases in children younger than 5 years.

BACKGROUND: Diarrhoea is a leading cause of death and illness globally among children younger than 5 years. Mortality and short-term morbidity cause substantial burden of disease but probably underestimate the true effect of diarrhoea on population health. This underestimation is because diarrhoeal diseases can negatively affect early childhood growth, probably through enteric dysfunction and impaired uptake of macronutrients and micronutrients. We attempt to quantify the long-term sequelae associated with childhood growth impairment due to diarrhoea. METHODS: We used the Global Burden of Diseases, Injuries, and Risk Factors Study framework and leveraged existing estimates of diarrhoea incidence, childhood undernutrition, and infectious disease burden to estimate the effect of diarrhoeal diseases on physical growth, including weight and height, and subsequent disease among children younger than 5 years. The burden of diarrhoea was measured in disability-adjusted life-years (DALYs), a composite metric of mortality and morbidity. We hypothesised that diarrhoea is negatively associated with three common markers of growth: weight-for-age, weight-for-height, and height-for-age Z-scores. On the basis of these undernutrition exposures, we applied a counterfactual approach to quantify the relative risk of infectious disease (subsequent diarrhoea, lower respiratory infection, and measles) and protein energy malnutrition morbidity and mortality per day of diarrhoea and quantified the burden of diarrhoeal disease due to these outcomes caused by undernutrition. FINDINGS: Diarrhoea episodes are significantly associated with childhood growth faltering. We found that each day of diarrhoea was associated with height-for-age Z-score (-0·0033 [95% CI -0·0024 to -0·0041]; p=4·43 × 10-14), weight-for-age Z-score (-0·0077 [-0·0058 to -0·0097]; p=3·19â€ˆ× 10-15), and weight-for-height Z-score (-0·0096 [-0·0067 to -0·0125]; p=7·78 × 10-11). After addition of the DALYs due to the long-term sequelae as a consequence of undernutrition, the burden of diarrhoeal diseases increased by 39·0% (95% uncertainty interval [UI] 33·0-46·6) and was responsible for 55 778 000 DALYs (95% UI 49 125 400-62 396 200) among children younger than 5 years in 2016. Among the 15 652 300 DALYs (95% UI 12 951 300-18 806 100) associated with undernutrition due to diarrhoeal episodes, more than 84·7% are due to increased risk of infectious disease, whereas the remaining 15·3% of long-term DALYs are due to increased prevalence of protein energy malnutrition. The burden of diarrhoea has decreased substantially since 1990, but progress has been greater in long-term (78·7% reduction [95% UI 69·3-85·5]) than in acute (70·4% reduction [95% UI 61·7-76·5]) DALYs. INTERPRETATION: Diarrhoea represents an even larger burden of disease than was estimated in the Global Burden of Disease Study. In order to adequately address the burden of its long-term sequelae, a renewed emphasis on controlling the risk of diarrhoea incidence may be required. This renewed effort can help further prevent the potential lifelong cost on child health, growth, and overall potential. FUNDING: Bill & Melinda Gates Foundation.

Zinc deficiency in children with environmental enteropathy-development of new strategies: report from an expert workshop.

Zinc deficiency is a major cause of childhood morbidity and mortality. The WHO/UNICEF strategy for zinc supplementation as adjunctive therapy for diarrhea is poorly implemented. A conference of experts in zinc nutrition and gastrointestinal disorders was convened to consider approaches that might complement the current recommendation and what research was needed to develop these approaches. Several key points were identified. The design of novel zinc interventions would be facilitated by a better understanding of how disturbed gut function, such as environmental (or tropical) enteropathy, affects zinc absorption, losses, and homeostasis. Because only 10% of zinc stores are able to be rapidly turned over, and appear to be rapidly depleted by acute intestinal illness, they are probably best maintained by complementary regular supplementation in a primary prevention strategy rather than secondary prevention triggered by acute diarrhea. The assessment of zinc status is challenging and complex without simple, validated measures to facilitate field testing of novel interventions. Zinc bioavailability may be a crucial factor in the success of primary prevention strategies, and a range of options, all still inadequately explored, might be valuable in improving zinc nutrition. Some therapeutic actions of zinc on diarrhea seem attributable to pharmacologic effects, whereas others are related to the reversal of deficiency (ie, nutritional). The distinction between these 2 mechanisms cannot be clarified given the insensitivity of serum zinc to identify subclinical deficiency states. Why zinc seems to be less effective than expected at all ages, and ineffective for secondary prevention of diarrhea in children <12 mo of age, remains unclear. It was concluded that a reframing of the current recommendation is warranted with consideration of how to better optimize and deliver zinc and whether to provide a complementary public health primary prevention zinc strategy. This requires careful consideration of the zinc product to be used as well as strategies for its delivery.

Activation of innate immunity in healthy Macaca mulatta macaques by a single subcutaneous dose of GMP CpG 7909: safety data and interferon-inducible protein-10 kinetics for humans and macaques.

Following a demonstration that mouse-optimized cytosine-guanosine dinucleotide (CpG) oligodeoxynucleotides stimulated innate immune protection against intracellular pathogens, we tested the ability of CpG 7909, a primate-optimized Toll-like receptor 9 (TLR9) agonist, to stimulate rhesus macaques to produce interferon-inducible protein-10 (IP-10), a biomarker of immune activation. This study was performed prior to a similar trial with humans in order to facilitate the development of CpG 7909 as an immunomodulator for biodefense. A single subcutaneous dose of clinical-grade CpG 7909 was given to four groups of healthy adult rhesus macaques (0-mg dose [n = 5], 0.75-mg dose [n = 9], 1.5-mg dose [n = 9], and 3.0-mg dose [n = 9]). Directed physical examination findings, clinical laboratory values, and serum IP-10 concentrations were collected at scheduled intervals for 28 days. All three dose levels of CpG 7909 were safe and not associated with significant clinical or laboratory abnormality. The time to peak serum IP-10 concentration was 1.0 days at the 0.75-mg dose and 0.5 days at the 1.5- and 3.0-mg doses. A dose-dependent response was observed for the magnitude and duration of IP-10 concentrations, which remained significantly above baseline for 3 days for the 3.0-mg and 1.5-mg dose groups but above baseline for only 2 days for the 0.75-mg dose group. There were no nonresponders to CpG 7909. These rhesus macaque safety and IP-10 response data closely parallel a subsequent phase 1 human study of subcutaneously administered CpG 7909. A single dose of clinical-grade CpG 7909 induced a rapid, sustained IP-10 response, a biomarker for activation of the innate immune system. Given the similar susceptibilities of humans and rhesus macaques to infectious diseases, the rhesus macaque appears to be a suitable model to evaluate the potential of CpG 7909-mediated innate immune activation to protect humans against pathogens.

Establishment of a human hepatocyte line that supports in vitro development of the exo-erythrocytic stages of the malaria parasites Plasmodium falciparum and P. vivax.

Our understanding of the biology of malaria parasite liver stages is limited because of the lack of efficient in vitro systems that support the exo-erythrocytic (EE) development of the parasite. We report the development of a new hepatocyte line (HC-04) from normal human liver cells. The HC-04 cells have proliferated in hormone-free medium for more than 200 passages. The cells were hyperdiploid, resembled liver parenchymal cells, and synthesized major liver-specific proteins and enzymes. Using Plasmodium falciparum and P. vivax sporozoites harvested from salivary glands of infected mosquitoes, we showed that HC-04 cells supported the complete EE development of these two most prevalent human malaria parasites. The EE parasites attained full maturation as shown by their infectivity to human erythrocytes. The infection rates of the liver cells were estimated to be 0.066% and 0.041% for P. falciparum and P. vivax, respectively. As the first human hepatocyte line known to support complete EE development of both P. falciparum and P. vivax, HC-04 will provide an experimental model that can be used for studying the biology of liver stage malaria parasites.

Randomized, controlled, double-blind trial of daily oral azithromycin in adults for the prophylaxis of Plasmodium vivax malaria in Western Thailand.

We assessed the prophylactic efficacy of azithromycin (250 mg/day) against malaria in 276 adults in western Thailand in a randomized, double-blind, placebo-controlled trial. After antimalarial suppressive treatment, volunteers were randomized in a 2:1 ratio to either the azithromycin or placebo, respectively. Study medication was given for an average of 74 days. The azithromycin group (n = 179) had five endpoint parasitemias (1 Plasmodium vivax and 4 P. falciparum), and the placebo group (n = 97) had 28 endpoint parasitemias (21 P. vivax, 5 P. falciparum, and 2 mixed infections). Adverse events and compliance and withdrawal rates were similar in both groups. The protective efficacy (PE) of azithromycin was 98% for P. vivax (95% confidence interval [CI] = 88-100%). There were too few cases to reliably estimate the efficacy of azithromycin for P. falciparum (PE =71%, 95% C =-14-94%). We conclude that daily azithromycin was safe, well-tolerated, and had a high efficacy for the prevention of P. vivax malaria.

Randomized trial of 3-dose regimens of tafenoquine (WR238605) versus low-dose primaquine for preventing Plasmodium vivax malaria relapse.

BACKGROUND: Tafenoquine is an 8-aminoquinoline developed as a more effective replacement for primaquine. In a previous dose-ranging study in Thailand, 3 tafenoquine regimens with total doses ranging from 500 mg to 3000 mg prevented relapse of Plasmodium vivax malaria in most patients when administered 2 days after receipt of a blood schizonticidal dose of chloroquine. METHODS: To improve convenience and to begin comparison of tafenoquine with primaquine, 80 patients with P. vivax infection were randomized to receive 1 of the following 5 treatments 1 day after receiving a blood schizonticidal dose of chloroquine: (A) tafenoquine, 300 mg per day for 7 days (n=18); (B) tafenoquine, 600 mg per day for 3 days (n=19); (C) tafenoquine, 600 mg as a single dose (n=18); (D) no further treatment (n=13); or (E) primaquine base, 15 mg per day for 14 days (n=12). The minimum duration of protocol follow-up was 8 weeks, with additional follow-up to 24 weeks. RESULTS: Forty-six of 55 tafenoquine recipients, 10 of 13 recipients of chloroquine only, and 12 of 12 recipients of chloroquine plus primaquine completed at least 8 weeks of follow-up (or had relapse). There was 1 relapse among recipients of chloroquine plus tafenoquine, 8 among recipients of chloroquine only, and 3 among recipients of chloroquine plus primaquine. The rate of protective efficacy (determined on the basis of reduction in incidence density) for all recipients of chloroquine plus tafenoquine, compared with recipients of chloroquine plus primaquine, was 92.6% (95% confidence interval, 7.3%-99.9%; P=.042, by Fisher's exact test). CONCLUSIONS: Tafenoquine doses as low as a single 600-mg dose may be useful for prevention of relapse of P. vivax malaria in Thailand.

Efficacy of monthly tafenoquine for prophylaxis of Plasmodium vivax and multidrug-resistant P. falciparum malaria.

We assessed monthly doses of tafenoquine for preventing Plasmodium vivax and multidrug-resistant P. falciparum malaria. In a randomized, double-blind, placebo-controlled study, 205 Thai soldiers received either a loading dose of tafenoquine 400 mg (base) daily for 3 days, followed by single monthly 400-mg doses (n = 104), or placebo (n = 101), for up to 5 consecutive months. In volunteers completing follow-up (96 tafenoquine and 91 placebo recipients), there were 22 P. vivax, 8 P. falciparum, and 1 mixed infection. All infections except 1 P. vivax occurred in placebo recipients, giving tafenoquine a protective efficacy of 97% for all malaria (95% confidence interval [CI], 82%-99%), 96% for P. vivax malaria (95% CI, 76%-99%), and 100% for P. falciparum malaria (95% CI, 60%-100%). Monthly tafenoquine was safe, well tolerated, and highly effective in preventing P. vivax and multidrug-resistant P. falciparum malaria in Thai soldiers during 6 months of prophylaxis.

Plasmodium falciparum-based bioassay for measurement of artemisinin derivatives in plasma or serum.

Artemisinin and its derivatives, artesunate and artemether, are rapidly acting antimalarials that are used for the treatment of severe and uncomplicated multidrug-resistant falciparum malaria. To optimize treatment regimens that use this new class of antimalarials, there is a need for readily available and reproducible assays to monitor drug levels closely in patients. A sensitive and reproducible bioassay for the measurement of the concentrations of artemisinin derivatives in plasma and serum is described. By modifying the in vitro drug susceptibility test, it was found that antimalarial activity in plasma or serum containing an unknown concentration of drug could be equated to the known concentrations of dihydroartemisinin (DHA) required to inhibit parasite growth. Dose-response curves for a Plasmodium falciparum clone (clone W2) and DHA were used as a standard for each assay. Assays with plasma or serum spiked with DHA proved to be reproducible (coefficient of variation,