Phenotypic and genetic characterization of MERS coronaviruses from Africa to understand their zoonotic potential.

Coronaviruses are pathogens of pandemic potential. Middle East respiratory syndrome coronavirus (MERS-CoV) causes a zoonotic respiratory disease of global public health concern, and dromedary camels are the only proven source of zoonotic infection. More than 70% of MERS-CoV-infected dromedaries are found in East, North, and West Africa, but zoonotic MERS disease is only reported from the Arabian Peninsula. We compared viral replication competence of clade A and B viruses from the Arabian Peninsula with genetically diverse clade C viruses found in East (Egypt, Kenya, and Ethiopia), North (Morocco), and West (Nigeria and Burkina Faso) Africa. Viruses from Africa had lower replication competence in ex vivo cultures of the human lung and in lungs of experimentally infected human-DPP4 (hDPP4) knockin mice. We used lentivirus pseudotypes expressing MERS-CoV spike from Saudi Arabian clade A prototype strain (EMC) or African clade C1.1 viruses and demonstrated that clade C1.1 spike was associated with reduced virus entry into the respiratory epithelial cell line Calu-3. Isogenic EMC viruses with spike protein from EMC or clade C1.1 generated by reverse genetics showed that the clade C1.1 spike was associated with reduced virus replication competence in Calu-3 cells in vitro, in ex vivo human bronchus, and in lungs of hDPP4 knockin mice in vivo. These findings may explain why zoonotic MERS disease has not been reported from Africa so far, despite exposure to and infection with MERS-CoV.

Uncovering the endemic circulation of rabies in Cambodia.

In epidemiology, endemicity characterizes sustained pathogen circulation in a geographical area, which involves a circulation that is not being maintained by external introductions. Because it could potentially shape the design of public health interventions, there is an interest in fully uncovering the endemic pattern of a disease. Here, we use a phylogeographic approach to investigate the endemic signature of rabies virus (RABV) circulation in Cambodia. Cambodia is located in one of the most affected regions by rabies in the world, but RABV circulation between and within Southeast Asian countries remains understudied. Our analyses are based on a new comprehensive data set of 199 RABV genomes collected between 2014 and 2017 as well as previously published Southeast Asian RABV sequences. We show that most Cambodian sequences belong to a distinct clade that has been circulating almost exclusively in Cambodia. Our results thus point towards rabies circulation in Cambodia that does not rely on external introductions. We further characterize within-Cambodia RABV circulation by estimating lineage dispersal metrics that appear to be similar to other settings, and by performing landscape phylogeographic analyses to investigate environmental factors impacting the dispersal dynamic of viral lineages. The latter analyses do not lead to the identification of environmental variables that would be associated with the heterogeneity of viral lineage dispersal velocities, which calls for a better understanding of local dog ecology and further investigations of the potential drivers of RABV spread in the region. Overall, our study illustrates how phylogeographic investigations can be performed to assess and characterize viral endemicity in a context of relatively limited data.

MERS coronaviruses from camels in Africa exhibit region-dependent genetic diversity.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes a zoonotic respiratory disease of global public health concern, and dromedary camels are the only proven source of zoonotic infection. Although MERS-CoV infection is ubiquitous in dromedaries across Africa as well as in the Arabian Peninsula, zoonotic disease appears confined to the Arabian Peninsula. MERS-CoVs from Africa have hitherto been poorly studied. We genetically and phenotypically characterized MERS-CoV from dromedaries sampled in Morocco, Burkina Faso, Nigeria, and Ethiopia. Viruses from Africa (clade C) are phylogenetically distinct from contemporary viruses from the Arabian Peninsula (clades A and B) but remain antigenically similar in microneutralization tests. Viruses from West (Nigeria, Burkina Faso) and North (Morocco) Africa form a subclade, C1, that shares clade-defining genetic signatures including deletions in the accessory gene ORF4b Compared with human and camel MERS-CoV from Saudi Arabia, virus isolates from Burkina Faso (BF785) and Nigeria (Nig1657) had lower virus replication competence in Calu-3 cells and in ex vivo cultures of human bronchus and lung. BF785 replicated to lower titer in lungs of human DPP4-transduced mice. A reverse genetics-derived recombinant MERS-CoV (EMC) lacking ORF4b elicited higher type I and III IFN responses than the isogenic EMC virus in Calu-3 cells. However, ORF4b deletions may not be the major determinant of the reduced replication competence of BF785 and Nig1657. Genetic and phenotypic differences in West African viruses may be relevant to zoonotic potential. There is an urgent need for studies of MERS-CoV at the animal-human interface.

Diversity of Dromedary Camel Coronavirus HKU23 in African Camels Revealed Multiple Recombination Events among Closely Related Betacoronaviruses of the Subgenus Embecovirus.

Genetic recombination has frequently been observed in coronaviruses. Here, we sequenced multiple complete genomes of dromedary camel coronavirus HKU23 (DcCoV-HKU23) from Nigeria, Morocco, and Ethiopia and identified several genomic positions indicative of cross-species virus recombination events among other betacoronaviruses of the subgenus Embecovirus (clade A beta-CoVs). Recombinant fragments of a rabbit coronavirus (RbCoV-HKU14) were identified at the hemagglutinin esterase gene position. Homolog fragments of a rodent CoV were also observed at 8.9-kDa open reading frame 4a at the 3' end of the spike gene. The patterns of recombination differed geographically across the African region, highlighting a mosaic structure of DcCoV-HKU23 genomes circulating in dromedaries. Our results highlighted active recombination of coronaviruses circulating in dromedaries and are also relevant to the emergence and evolution of other betacoronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV).IMPORTANCE Genetic recombination is often demonstrated in coronaviruses and can result in host range expansion or alteration in tissue tropism. Here, we showed interspecies events of recombination of an endemic dromedary camel coronavirus, HKU23, with other clade A betacoronaviruses. Our results supported the possibility that the zoonotic pathogen MERS-CoV, which also cocirculates in the same camel species, may have undergone similar recombination events facilitating its emergence or may do so in its future evolution.

Isolation and full-genome sequences of Japanese encephalitis virus genotype I strains from Cambodian human patients, mosquitoes and pigs.

Japanese encephalitis remains the most important cause of viral encephalitis in humans in several southeast Asian countries, including Cambodia, causing at least 65 000 cases of encephalitis per year. This vector-borne viral zoonosis - caused by Japanese encephalitis virus (JEV) - is considered to be a rural disease and is transmitted by mosquitoes, with birds and pigs being the natural reservoirs, while humans are accidental hosts. In this study we report the first two JEV isolations in Cambodia from human encephalitis cases from two studies on the aetiology of central nervous system disease, conducted at the two major paediatric hospitals in the country. We also report JEV isolation from Culextritaeniorhynchus mosquitoes and from pig samples collected in two farms, located in peri-urban and rural areas. Out of 11 reverse-transcription polymerase chain reaction-positive original samples, we generated full-genome sequences from 5 JEV isolates. Five additional partial sequences of the JEV NS3 gene from viruses detected in five pigs and one complete coding sequence of the envelope gene of a strain identified in a pig were generated. Phylogenetic analyses revealed that JEV detected in Cambodia belonged to genotype I and clustered in two clades: genotype I-a, mainly comprising strains from Thailand, and genotype I-b, comprising strains from Vietnam that dispersed northwards to China. Finally, in this study, we provide proof that the sequenced JEV strains circulate between pigs, Culex tritaeniorhynchus and humans in the Phnom Penh vicinity.

Risk factors for MERS coronavirus infection in dromedary camels in Burkina Faso, Ethiopia, and Morocco, 2015.

Understanding Middle East respiratory syndrome coronavirus (MERS-CoV) transmission in dromedary camels is important, as they consitute a source of zoonotic infection to humans. To identify risk factors for MERS-CoV infection in camels bred in diverse conditions in Burkina Faso, Ethiopia and Morocco, blood samples and nasal swabs were sampled in February-March 2015. A relatively high MERS-CoV RNA rate was detected in Ethiopia (up to 15.7%; 95% confidence interval (CI): 8.2-28.0), followed by Burkina Faso (up to 12.2%; 95% CI: 7-20.4) and Morocco (up to 7.6%; 95% CI: 1.9-26.1). The RNA detection rate was higher in camels bred for milk or meat than in camels for transport (p = 0.01) as well as in younger camels (p = 0.06). High seropositivity rates (up to 100%; 95% CI: 100-100 and 99.4%; 95% CI: 95.4-99.9) were found in Morocco and Ethiopia, followed by Burkina Faso (up to 84.6%; 95% CI: 77.2-89.9). Seropositivity rates were higher in large/medium herds (≥51 camels) than small herds (p = 0.061), in camels raised for meat or milk than for transport (p = 0.01), and in nomadic or sedentary herds than in herds with a mix of these lifestyles (p < 0.005).

Environmental predictors of West Nile fever risk in Europe.

BACKGROUND: West Nile virus (WNV) is a mosquito-borne pathogen of global public health importance. Transmission of WNV is determined by abiotic and biotic factors. The objective of this study was to examine environmental variables as predictors of WNV risk in Europe and neighboring countries, considering the anomalies of remotely sensed water and vegetation indices and of temperature at the locations of West Nile fever (WNF) outbreaks reported in humans between 2002 and 2013. METHODS: The status of infection by WNV in relationship to environmental and climatic risk factors was analyzed at the district level using logistic regression models. Temperature, remotely sensed Normalized Difference Vegetation Index (NDVI) and Modified Normalized Difference Water Index (MNDWI) anomalies, as well as population, birds' migratory routes, and presence of wetlands were considered as explanatory variables. RESULTS: The anomalies of temperature in July, of MNDWI in early June, the presence of wetlands, the location under migratory routes, and the occurrence of a WNF outbreak the previous year were identified as risk factors. The best statistical model according to the Akaike Information Criterion was used to map WNF risk areas in 2012 and 2013. Model validations showed a good level of prediction: area under Receiver Operator Characteristic curve = 0.854 (95% Confidence Interval 0.850-0.856) for internal validation and 0.819 (95% Confidence Interval 0.814-0.823) (2012) and 0.853 (95% Confidence Interval 0.850-0.855) (2013) for external validations, respectively. CONCLUSIONS: WNF incidence is increasing in Europe and WNV is expanding into new areas where it had never been observed before. Our model can be used to direct surveillance activities and public health interventions for the upcoming WNF season.

Bayesian modeling of post-vaccination serological data suggests that yearly vaccination of dog aged <2 years old is efficient to stop rabies circulation in Cambodia.

Rabies control remains challenging in low and middle-income countries, mostly due to lack of financial resources, rapid turnover of dog populations and poor accessibility to dogs. Rabies is endemic in Cambodia, where no national rabies vaccination program is implemented. The objective of this study was to assess the short and long-term vaccination-induced immunity in Cambodian dogs under field conditions, and to propose optimized vaccination strategies. A cohort of 351 dogs was followed at regular time points following primary vaccination only (PV) or PV plus single booster (BV). Fluorescent antibody virus neutralization test (FAVNT) was implemented to determine the neutralizing antibody titer against rabies and an individual titer ≥0·5 IU/mL indicated protection. Bayesian modeling was used to evaluate the individual duration of protection against rabies and the efficacy of two different vaccination strategies. Overall, 61% of dogs had a protective immunity one year after PV. In dogs receiving a BV, this protective immunity remained for up to one year after the BV in 95% of dogs. According to the best Bayesian model, a PV conferred a protective immunity in 82% of dogs (95% CI: 75-91%) for a mean duration of 4.7 years, and BV induced a lifelong protective immunity. Annual PV of dogs less than one year old and systematic BV solely of dogs vaccinated the year before would allow to achieve the 70% World Health Organization recommended threshold to control rabies circulation in a dog population in three to five years of implementation depending on dog population dynamics. This vaccination strategy would save up to about a third of vaccine doses, reducing cost and time efforts of mass dog vaccination campaigns. These results can contribute to optimize rabies control measures in Cambodia moving towards the global goal of ending human death from dog-mediated rabies by 2030.

Towards a better understanding of Rift Valley fever epidemiology in the south-west of the Indian Ocean.

Rift Valley fever virus (Phlebovirus, Bunyaviridae) is an arbovirus causing intermittent epizootics and sporadic epidemics primarily in East Africa. Infection causes severe and often fatal illness in young sheep, goats and cattle. Domestic animals and humans can be contaminated by close contact with infectious tissues or through mosquito infectious bites. Rift Valley fever virus was historically restricted to sub-Saharan countries. The probability of Rift Valley fever emerging in virgin areas is likely to be increasing. Its geographical range has extended over the past years. As a recent example, autochthonous cases of Rift Valley fever were recorded in 2007-2008 in Mayotte in the Indian Ocean. It has been proposed that a single infected animal that enters a naive country is sufficient to initiate a major outbreak before Rift Valley fever virus would ever be detected. Unless vaccines are available and widely used to limit its expansion, Rift Valley fever will continue to be a critical issue for human and animal health in the region of the Indian Ocean.

Identifying landscape features associated with Rift Valley fever virus transmission, Ferlo region, Senegal, using very high spatial resolution satellite imagery.

INTRODUCTION: Dynamics of most of vector-borne diseases are strongly linked to global and local environmental changes. Landscape changes are indicators of human activities or natural processes that are likely to modify the ecology of the diseases. Here, a landscape approach developed at a local scale is proposed for extracting mosquito favourable biotopes, and for testing ecological parameters when identifying risk areas of Rift Valley fever (RVF) transmission. The study was carried out around Barkedji village, Ferlo region, Senegal. METHODS: In order to test whether pond characteristics may influence the density and the dispersal behaviour of RVF vectors, and thus the spatial variation in RVFV transmission, we used a very high spatial resolution remote sensing image (2.4 m resolution) provided by the Quickbird sensor to produce a detailed land-cover map of the study area. Based on knowledge of vector and disease ecology, seven landscape attributes were defined at the pond level and computed from the land-cover map. Then, the relationships between landscape attributes and RVF serologic incidence rates in small ruminants were analyzed through a beta-binomial regression. Finally, the best statistical model according to the Akaike Information Criterion corrected for small samples (AICC), was used to map areas at risk for RVF. RESULTS: Among the derived landscape variables, the vegetation density index (VDI) computed within a 500 m buffer around ponds was positively correlated with serologic incidence (p<0.001), suggesting that the risk of RVF transmission was higher in the vicinity of ponds surrounded by a dense vegetation cover. The final risk map of RVF transmission displays a heterogeneous spatial distribution, corroborating previous findings from the same area. CONCLUSIONS: Our results highlight the potential of very high spatial resolution remote sensing data for identifying environmental risk factors and mapping RVF risk areas at a local scale.

Dosiomics-Based Prediction of Radiation-Induced Valvulopathy after Childhood Cancer.

Valvular Heart Disease (VHD) is a known late complication of radiotherapy for childhood cancer (CC), and identifying high-risk survivors correctly remains a challenge. This paper focuses on the distribution of the radiation dose absorbed by heart tissues. We propose that a dosiomics signature could provide insight into the spatial characteristics of the heart dose associated with a VHD, beyond the already-established risk induced by high doses. We analyzed data from the 7670 survivors of the French Childhood Cancer Survivors' Study (FCCSS), 3902 of whom were treated with radiotherapy. In all, 63 (1.6%) survivors that had been treated with radiotherapy experienced a VHD, and 57 of them had heterogeneous heart doses. From the heart-dose distribution of each survivor, we extracted 93 first-order and spatial dosiomics features. We trained random forest algorithms adapted for imbalanced classification and evaluated their predictive performance compared to the performance of standard mean heart dose (MHD)-based models. Sensitivity analyses were also conducted for sub-populations of survivors with spatially heterogeneous heart doses. Our results suggest that MHD and dosiomics-based models performed equally well globally in our cohort and that, when considering the sub-population having received a spatially heterogeneous dose distribution, the predictive capability of the models is significantly improved by the use of the dosiomics features. If these findings are further validated, the dosiomics signature may be incorporated into machine learning algorithms for radiation-induced VHD risk assessment and, in turn, into the personalized refinement of follow-up guidelines.

Genetic diversity and molecular epidemiology of Middle East Respiratory Syndrome Coronavirus in dromedaries in Ethiopia, 2017-2020.

Middle East respiratory syndrome coronavirus (MERS-CoV) is enzootic in dromedary camels and causes zoonotic infection and disease in humans. Although over 80% of the global population of infected dromedary camels are found in Africa, zoonotic disease had only been reported in the Arabia Peninsula and travel-associated disease has been reported elsewhere. In this study, genetic diversity and molecular epidemiology of MERS-CoV in dromedary camels in Ethiopia were investigated during 2017-2020. Of 1766 nasal swab samples collected, 61 (3.5%) were detected positive for MERS-CoV RNA. Of 484 turbinate swab samples collected, 10 (2.1%) were detected positive for MERS-CoV RNA. Twenty-five whole genome sequences were obtained from these MERS-CoV positive samples. Phylogenetically, these Ethiopian camel-originated MERS-CoV belonged to clade C2, clustering with other East African camel strains. Virus sequences from camel herds clustered geographically while in an abattoir, two distinct phylogenetic clusters of MERS-CoVs were observed in two sequential sampling collections, which indicates the greater genetic diversity of MERS-CoV in abattoirs. In contrast to clade A and B viruses from the Arabian Peninsula, clade C camel-originated MERS-CoV from Ethiopia had various nucleotide insertions and deletions in non-structural gene nsp3, accessory genes ORF3 and ORF5 and structural gene N. This study demonstrates the genetic instability of MERS-CoV in dromedaries in East Africa, which indicates that the virus is still actively adapting to its camel host. The impact of the observed nucleotide insertions and deletions on virus evolution, viral fitness, and zoonotic potential deserves further study.

Vector Competence of Mediterranean Mosquitoes for Rift Valley Fever Virus: A Meta-Analysis.

Rift Valley fever (RVF) is a zoonotic disease caused by a virus mainly transmitted by Aedes and Culex mosquitoes. Infection leads to high abortion rates and considerable mortality in domestic livestock. The combination of viral circulation in Egypt and Libya and the existence of unregulated live animal trade routes through endemic areas raise concerns that the virus may spread to other Mediterranean countries, where there are mosquitoes potentially competent for RVF virus (RVFV) transmission. The competence of vectors for a given pathogen can be assessed through laboratory experiments, but results may vary greatly with the study design. This research aims to quantify the competence of five major potential RVFV vectors in the Mediterranean Basin, namely Aedes detritus, Ae. caspius, Ae. vexans, Culex pipiens and Cx. theileri, through a systematic literature review and meta-analysis. We first computed the infection rate, the dissemination rate among infected mosquitoes, the overall dissemination rate, the transmission rate among mosquitoes with a disseminated infection and the overall transmission rate for these five mosquito species. We next assessed the influence of laboratory study designs on the variability of these five parameters. According to experimental results and our analysis, Aedes caspius may be the most competent vector among the five species considered.

Modelling Japanese encephalitis virus transmission dynamics and human exposure in a Cambodian rural multi-host system.

Japanese encephalitis (JE) is a vector-borne zoonosis and the leading cause of human viral encephalitis in Asia. Its transmission cycle is usually described as involving wild birds as reservoirs and pigs as amplifying hosts. JE is endemic in Cambodia, where it circulates in areas with low pig densities (<70 pigs per km2), and could be maintained in a multi-host system composed of pigs, but also poultry as competent hosts, and dogs, cattle and humans as non-competent hosts. We used a mathematical model representing Japanese encephalitis virus (JEV) transmission in a traditional Cambodian village that we calibrated with field data collected in 3 districts of Kandal province, Cambodia. First, R0 calculations allowed us to assess the capacity of the epidemiological system to be invaded by JEV and sustain virus transmission in villages in the 3 districts, and we predicted human exposure at the epidemiological equilibrium, based on simulations. Changes in spatial density of livestock, in agricultural practices, and epizootics (e.g., African swine fever), can profoundly alter the composition of host communities, which could affect JEV transmission and its impact on human health. In a second step, we then used the model to analyse how host community composition affected R0 and the predicted human exposure. Lastly, we evaluated the potential use of dog JE seroprevalence as an indicator of human exposure to JEV. In the modeled villages, the calculated R0 ranged from 1.07 to 1.38. Once the equilibrium reached, predicted annual probability of human exposure ranged from 9% to 47%, and predicted average age at infection was low, between 2 and 11 years old, highlighting the risk of severe forms of JEV infection and the need to intensify child immunization. According to the model, increasing the proportion of competent hosts induced a decrease in age at infection. The simulations also showed that JEV could invade a multi-host system with no pigs, reinforcing the assumption of poultry acting as reservoirs. Finally, the annual human exposure probability appeared linearly correlated with dog seroprevalence, suggesting that in our specific study area, dog seroprevalence would be a good proxy for human exposure.

Mechanistic models of Rift Valley fever virus transmission: A systematic review.

Rift Valley fever (RVF) is a zoonotic arbovirosis which has been reported across Africa including the northernmost edge, South West Indian Ocean islands, and the Arabian Peninsula. The virus is responsible for high abortion rates and mortality in young ruminants, with economic impacts in affected countries. To date, RVF epidemiological mechanisms are not fully understood, due to the multiplicity of implicated vertebrate hosts, vectors, and ecosystems. In this context, mathematical models are useful tools to develop our understanding of complex systems, and mechanistic models are particularly suited to data-scarce settings. Here, we performed a systematic review of mechanistic models studying RVF, to explore their diversity and their contribution to the understanding of this disease epidemiology. Researching Pubmed and Scopus databases (October 2021), we eventually selected 48 papers, presenting overall 49 different models with numerical application to RVF. We categorized models as theoretical, applied, or grey, depending on whether they represented a specific geographical context or not, and whether they relied on an extensive use of data. We discussed their contributions to the understanding of RVF epidemiology, and highlighted that theoretical and applied models are used differently yet meet common objectives. Through the examination of model features, we identified research questions left unexplored across scales, such as the role of animal mobility, as well as the relative contributions of host and vector species to transmission. Importantly, we noted a substantial lack of justification when choosing a functional form for the force of infection. Overall, we showed a great diversity in RVF models, leading to important progress in our comprehension of epidemiological mechanisms. To go further, data gaps must be filled, and modelers need to improve their code accessibility.

Accessibility to rabies centers and human rabies post-exposure prophylaxis rates in Cambodia: A Bayesian spatio-temporal analysis to identify optimal locations for future centers.

Rabies is endemic in Cambodia. For exposed humans, post-exposure prophylaxis (PEP) is very effective in preventing this otherwise fatal disease. The Institut Pasteur du Cambodge (IPC) in Phnom Penh was the primary distributor of PEP in Cambodia until 2018. Since then, and to increase distribution of PEP, two new centers have been opened by IPC in the provinces of Battambang and Kampong Cham. Data on bitten patients, who sometimes bring the head of the biting animal for rabies analyses, have been recorded by IPC since 2000. However, human cases are not routinely recorded in Cambodia, making it difficult to establish a human burden of disease and generate a risk map of dog bites to inform the selection of future PEP center locations in high-risk areas. Our aim was to assess the impact of accessibility to rabies centers on the yearly rate of PEP patients in the population and generate a risk map to identify the locations where new centers would be the most beneficial to the Cambodian population. To accomplish this, we used spatio-temporal Bayesian regression models with the number of PEP patients as the outcome. The primary exposure variable considered was travel time to the nearest IPC center. Secondary exposure variables consisted of travel time to a provincial capital and urban proportion of the population. Between 2000 and 2016, a total of 293,955 PEP patient records were identified. Our results showed a significant negative association between travel time to IPC and the rate of PEP patients: an increase in one hour travel time from the living location to IPC PEP centers leads to a reduction in PEP rate of 70% to 80%. Five provinces were identified as the most efficient locations for future centers to maximize PEP accessibility: Banteay Meanchey, Siem Reap, Takeo, Kampot and Svay Rieng. Adding a PEP center in every provincial capital would increase the proportion of Cambodians living within 60 minutes of a PEP center from 26.6% to 64.9%, and living within 120 minutes from 52.8% to 93.3%, which could save hundreds of lives annually.

Childhood encephalitis in the Greater Mekong region (the SouthEast Asia Encephalitis Project): a multicentre prospective study.

BACKGROUND: Encephalitis is a worldwide public health issue, with a substantially high burden among children in southeast Asia. We aimed to determine the causes of encephalitis in children admitted to hospitals across the Greater Mekong region by implementing a comprehensive state-of-the-art diagnostic procedure harmonised across all centres, and identifying clinical characteristics related to patients' conditions. METHODS: In this multicentre, observational, prospective study of childhood encephalitis, four referral hospitals in Cambodia, Vietnam, Laos, and Myanmar recruited children (aged 28 days to 16 years) who presented with altered mental status lasting more than 24 h and two of the following minor criteria: fever (within the 72 h before or after presentation), one or more generalised or partial seizures (excluding febrile seizures), a new-onset focal neurological deficit, cerebrospinal fluid (CSF) white blood cell count of 5 per mL or higher, or brain imaging (CT or MRI) suggestive of lesions of encephalitis. Comprehensive diagnostic procedures were harmonised across all centres, with first-line testing was done on samples taken at inclusion and results delivered within 24 h of inclusion for main treatable causes of disease and second-line testing was done thereafter for mostly non-treatable causes. An independent expert medical panel reviewed the charts and attribution of causes of all the included children. Using multivariate analyses, we assessed risk factors associated with unfavourable outcomes (ie, severe neurological sequelae and death) at discharge using data from baseline and day 2 after inclusion. This study is registered with ClinicalTrials.gov, NCT04089436, and is now complete. FINDINGS: Between July 28, 2014, and Dec 31, 2017, 664 children with encephalitis were enrolled. Median age was 4·3 years (1·8-8·8), 295 (44%) children were female, and 369 (56%) were male. A confirmed or probable cause of encephalitis was identified in 425 (64%) patients: 216 (33%) of 664 cases were due to Japanese encephalitis virus, 27 (4%) were due to dengue virus, 26 (4%) were due to influenza virus, 24 (4%) were due to herpes simplex virus 1, 18 (3%) were due to Mycobacterium tuberculosis, 17 (3%) were due to Streptococcus pneumoniae, 17 (3%) were due to enterovirus A71, 74 (9%) were due to other pathogens, and six (1%) were due to autoimmune encephalitis. Diagnosis was made within 24 h of admission to hospital for 83 (13%) of 664 children. 119 (18%) children had treatable conditions and 276 (42%) had conditions that could have been preventable by vaccination. At time of discharge, 153 (23%) of 664 children had severe neurological sequelae and 83 (13%) had died. In multivariate analyses, risk factors for unfavourable outcome were diagnosis of M tuberculosis infection upon admission (odds ratio 3·23 [95% CI 1·04-10·03]), coma on day 2 (2·90 [1·78-4·72]), supplementary oxygen requirement (1·89 [1·25-2·86]), and more than 1 week duration between symptom onset and admission to hospital (3·03 [1·68-5·48]). At 1 year after inclusion, of 432 children who were discharged alive from hospital with follow-up data, 24 (5%) had died, 129 (30%) had neurological sequelae, and 279 (65%) had completely recovered. INTERPRETATION: In southeast Asia, most causes of childhood encephalitis are either preventable or treatable, with Japanese encephalitis virus being the most common cause. We provide crucial information that could guide public health policy to improve diagnostic, vaccination, and early therapeutic guidelines on childhood encephalitis in the Greater Mekong region. FUNDING: Institut Pasteur, Institut Pasteur International Network, Fondation Merieux, Aviesan Sud, INSERM, Wellcome Trust, Institut de Recherche pour le Développement (IRD), and Fondation Total.

Risk assessment of the introduction of Rift Valley fever from the Horn of Africa to Yemen via legal trade of small ruminants.

Rift Valley fever (RVF) is a mosquito-borne viral zoonosis of increasing global importance. Occurring since 1930 across Africa, it was detected for the first time in Saudi Arabia and Yemen in September 2000, leading to human deaths and major losses in livestock populations. Assuming the virus has not survived in Yemen or has been circulating at a low level, authors qualitatively assessed the likelihood of "re-introduction" of RVF into Yemen through the legal importation of small ruminants from the Horn of Africa. The overall probability of introduction was assessed very low to medium, increasing during festival periods and higher when considering a direct transmission exposure as compared to a vectorial transmission exposure. The uncertainty was considered to be medium underlining important gaps in information that need to be fulfilled in the region. Options to reduce the risk are proposed and discussed, including possible improvements of the current Yemeni quarantine system.

Mosquito Vector Competence for Japanese Encephalitis Virus.

Japanese encephalitis virus (JEV) is a zoonotic pathogen mainly found in East and Southeast Asia and transmitted by mosquitoes. The objective of this review is to summarize the knowledge on the diversity of JEV mosquito vector species. Therefore, we systematically analyzed reports of JEV found in field-caught mosquitoes as well as experimental vector competence studies. Based on the investigated publications, we classified 14 species as confirmed vectors for JEV due to their documented experimental vector competence and evidence of JEV found in wild mosquitoes. Additionally, we identified 11 mosquito species, belonging to five genera, with an experimentally confirmed vector competence for JEV but lacking evidence on their JEV transmission capacity from field-caught mosquitoes. Our study highlights the diversity of confirmed and potential JEV vector species. We also emphasize the variety in the study design of vector competence investigations. To account for the diversity of the vector species and regional circumstances, JEV vector competence should be studied in the local context, using local mosquitoes with local virus strains under local climate conditions to achieve reliable data. In addition, harmonization of the design of vector competence experiments would lead to better comparable data, informing vector and disease control measures.