Despite the increased use of vaccination in several Asian countries, Japanese Encephalitis (JE) remains the most important cause of viral encephalitis in Asia in humans with an estimated 68,000 cases annually. Considered a rural disease occurring mainly in paddy-field dominated landscapes where pigs are amplifying hosts, JE may nevertheless circulate in a wider range of environment given the diversity of its potential hosts and vectors. The main objective of this study was to assess the intensity of JE transmission to pigs in a peri-urban environment in the outskirt of Phnom Penh, Cambodia. We estimated the force of JE infection in two cohorts of 15 sentinel pigs by fitting a generalised linear model on seroprevalence monitoring data observed during two four-month periods in 2014. Our results provide evidence for intensive circulation of JE virus in a periurban area near Phnom Penh, the capital and most populated city of Cambodia. Understanding JE virus transmission in different environments is important for planning JE virus control in the long term and is also an interesting model to study the complexity of vector-borne diseases. Collecting quantitative data such as the force of infection will help calibrate epidemiological model that can be used to better understand complex vector-borne disease epidemiological cycles.

Vascular permeability and plasma leakage are immune-pathologies of severe dengue virus (DENV) infection, but the mechanisms underlying the exacerbated inflammation during DENV pathogenesis are unclear. Here, we demonstrate that TLR2, together with its coreceptors CD14 and TLR6, is an innate sensor of DENV particles inducing inflammatory cytokine expression and impairing vascular integrity in vitro. Blocking TLR2 prior to DENV infection in vitro abrogates NF-κB activation while CD14 and TLR6 block has a moderate effect. Moreover, TLR2 block prior to DENV infection of peripheral blood mononuclear cells prevents activation of human vascular endothelium, suggesting a potential role of the TLR2responses in vascular integrity. TLR2 expression on CD14 + + classical monocytes isolated in an acute phase from DENV-infected pediatric patients correlates with severe disease development. Altogether, these data identify a role for TLR2 in DENV infection and provide insights into the complex interaction between the virus and innate receptors that may underlie disease pathogenesis.

Japanese encephalitis is mainly considered a rural disease, but there is growing evidence of a peri-urban and urban transmission in several countries, including Cambodia. We, therefore, compared the epidemiologic dynamic of Japanese encephalitis between a rural and a peri-urban setting in Cambodia. We monitored two cohorts of 15 pigs and determined the force of infection–rate at which seronegative pigs become positive–in two study farms located in a peri-urban and rural area, respectively. We also studied the mosquito abundance and diversity in proximity of the pigs, as well as the host densities in both areas. All the pigs seroconverted before the age of 6 months. The force of infection was 0.061 per day (95% confidence interval = 0.034–0.098) in the peri-urban cohort and 0.069 per day (95% confidence interval = 0.047–0.099) in the rural cohort. Several differences in the epidemiologic dynamic of Japanese encephalitis between both study sites were highlighted. The later virus amplification in the rural cohort may be linked to the later waning of maternal antibodies, but also to the higher pig density in direct proximity of the studied pigs, which could have led to a dilution of mosquito bites at the farm level. The force of infection was almost identical in both the peri-urban and the rural farms studied, which shifts the classic epidemiologic cycle of the virus. This study is a first step in improving our understanding of Japanese encephalitis virus ecology in different environments with distinct landscapes, human and animal densities.

The diagnosis of dog-mediated rabies in humans and animals has greatly benefited from technical advances in the laboratory setting. Approaches to diagnosis now include the detection of rabies virus (RABV), RABV RNA, or RABV antigens. These assays are important tools in the current efforts aimed at the global elimination of dog-mediated rabies. The assays available for use in laboratories are reviewed herein, as well as their strengths and weaknesses, which vary with the types of sample analyzed. Depending on the setting, however, the public health objectives and use of RABV diagnosis in the field will also vary. In non-endemic settings, the detection of all introduced or emergent animal or human cases justifies exhaustive testing. In dog RABV-endemic settings, such as rural areas of developing countries where most cases occur, the availability of or access to testing may be severely constrained. Thus, these issues are also discussed along with a proposed strategy to prioritize testing while access to rabies testing in the resource-poor, highly endemic setting is improved. As the epidemiological situation of rabies in a country evolves, the strategy should shift from that of an endemic setting to one more suitable for a decreased rabies incidence following the implementation of efficient control measures and when nearing the target of dog-mediated rabies elimination.

Background The World Health Organization (WHO) proposed guidelines on dengue clinical classification in 1997 and more recently in 2009 for the clinical management of patients. The WHO 1997 classification defines three categories of dengue infection according to severity: dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). Alternative WHO 2009 guidelines provide a cross-sectional classification aiming to discriminate dengue fever from dengue with warning signs (DWWS) and severe dengue (SD). The primary objective of this study was to perform a comparison of two dengue classifications. The secondary objective was to describe the changes of hematological and biochemical parameters occurring in patients presenting with different degrees of severity during the course of the disease, since progression to more severe clinical forms is unpredictable. Methodology/Principal findings We performed a prospective, monocentric, cross-sectional study of hospitalized children in Cambodia, aged from 2 to 15 years old with severe and non-severe dengue. We enrolled 243 patients with acute dengue-like illness: 71.2% were dengue infections confirmed using quantitative reverse transcription PCR or NS1 antigen capture ELISA, of which 87.2% and 9.0% of DF cases were respectively classified DWWS and SD, and 35.9% of DHF were PLOS NEGLECTED TROPICAL DISEASES

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.

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Dengue is a mosquito-borne viral disease caused by dengue virus (DENV). The disease is endemic to more than 100 countries with 390 million dengue infections per year. Humoral immune responses during primary and secondary DENV infections are well-investigated. However, the impact of DENV infection on B cell subsets and their antibody-independent functions are not well-documented. Through this study, we aimed to define the distribution of B cell subsets in the acute phase of DENV infection and characterize the effect of DENV infection on B cell functions such as differentiation into memory and plasma cells and cytokine production. In our cohort of Cambodian children, we observed decreased percentages of CD24hiCD38hi B cells and CD27− naïve B cells within the CD19 population and increased percentages of CD27+CD38hiCD138+ plasma cells as early as 4 days post appearance of fever in patients with severe dengue compared to patients with mild disease. Lower percentages of CD19+CD24hiCD38hi B cells in DENV-infected patients were associated with decreased concentrations of soluble CD40L in patient plasma and decreased platelet counts in these patients. In addition, CD19+CD24hiCD38hi and CD19+CD27− B cells from DENV-infected patients did not produce IL-10 or TNF-α upon stimulation in vitro, suggesting their contribution to an altered immune response during DENV infection. In addition, CD19+CD27− naïve B cells isolated from dengue patients were refractory to TLR/anti-IgM stimulation in vitro, which correlated to the increased expression of inhibitory Fcγ receptors (FcγR) CD32 and LILRB1 on CD19+CD27− naïve B cells from DENV-infected patients. Collectively, our results indicate that a defective B cell response in dengue patients may contribute to the pathogenesis of dengue during the early phase of infection.