Blood virosphere in febrile Tanzanian children.

Viral infections are the leading cause of childhood acute febrile illnesses motivating consultation in sub-Saharan Africa. The majority of causal viruses are never identified in low-resource clinical settings as such testing is either not part of routine screening or available diagnostic tools have limited ability to detect new/unexpected viral variants. An in-depth exploration of the blood virome is therefore necessary to clarify the potential viral origin of fever in children. Metagenomic next-generation sequencing is a powerful tool for such broad investigations, allowing the detection of RNA and DNA viral genomes. Here, we describe the blood virome of 816 febrile children (<5 years) presenting at outpatient departments in Dar es Salaam over one-year. We show that half of the patients (394/816) had at least one detected virus recognized as causes of human infection/disease (13.8% enteroviruses (enterovirus A, B, C, and rhinovirus A and C), 12% rotaviruses, 11% human herpesvirus type 6). Additionally, we report the detection of a large number of viruses (related to arthropod, vertebrate or mammalian viral species) not yet known to cause human infection/disease, highlighting those who should be on the radar, deserve specific attention in the febrile paediatric population and, more broadly, for surveillance of emerging pathogens.Trial registration: ClinicalTrials.gov identifier: NCT02225769.

Fecal Components Modulate Human Astrovirus Infectivity in Cells and Reconstituted Intestinal Tissues.

Human astroviruses (HAstV) are among the most common causative agents of viral gastroenteritis, especially in children, and extraintestinal manifestations have also been described. These viruses are transmitted by the fecal-oral route, implying that stool composition and the gut microbiota may impact their ability to remain infectious. For some enteric viruses, individual bacterial envelope components and other polysaccharide-containing molecules, which are abundant in stools, have been shown to enhance capsid stability. However, the role of the complex stool environment and, most importantly, the role of interindividual differences have been poorly studied. We used HAstV as a model to investigate how the stool environment in itself, its interindividual variability, and some specific stool components could affect HAstV stability and infectivity. Using two different HAstV genotypes, we found that stools as a whole modulate astrovirus infectivity not only in an individual-dependent manner but also in a manner that depends on the viral genotype. A virus-protective effect was observed after incubation with various Gram-positive and Gram-negative bacteria as well as with bacterial components, such as lipopolysaccharide and peptidoglycan. These results were further confirmed in human intestinal tissues, a more physiologically relevant system. Astrovirus infectivity was also preserved by mucin, a major component of intestinal mucus. We further confirmed that these components stabilize the viral capsid. These results show that although HAstV benefits from the stabilizing effect of fecal components, the complexity and variability of the stool composition and the multiple potential interactions may explain the interindividual differences in viral transmission observed in real life.IMPORTANCE To ensure transmission, enteric viruses must maintain their infectivity during the various environmental challenges that they face in transit within and between hosts. Increased knowledge of the factors affecting enteric virus survival may help to control their transmission. This study reveals that specific fecal bacterial components preserve classic human astrovirus infectivity by stabilizing viral particles. However, the outcomes of stool-virus interactions are very variable, ranging from protection to a reduction of viral infectivity, depending on the viral genotype and the individual from whom the stool has been collected. We show that the transmissibility of enteric viruses is dependent on the intestinal contents of the infected individual and highlight the complex multiple interactions that could explain the stochastic nature of enteric virus transmission in humans.

Nearly Complete Genome Sequence of a Novel Phlebovirus-Like Virus Detected in a Human Plasma Sample by High-Throughput Sequencing.

Here, we report a novel phlebovirus-like virus sequence detected in a plasma sample from a febrile adult patient collected in the United Republic of Tanzania in 2014. A nearly complete RNA sequence was generated by high-throughput sequencing on a HiSeq 2500 instrument and further confirmed after repeating the analysis, starting from the initial sample.

Detection of dicistroviruses RNA in blood of febrile Tanzanian children.

Fever is the leading cause of paediatric outpatient consultations in Sub-Saharan Africa. Although most are suspected to be of viral origin, a putative causative pathogen is not identified in over a quarter of these febrile episodes. Using a de novo assembly sequencing approach, we report the detection (15.4%) of dicistroviruses (DicV) RNA in sera collected from 692 febrile Tanzanian children. In contrast, DicV RNA was only detected in 1/77 (1.3%) plasma samples from febrile Tanzanian adults, suggesting that children could represent the primary susceptible population. Estimated viral load by specific quantitative real-time RT-PCR assay ranged from < 1.32E3 to 1.44E7 viral RNA copies/mL serum. Three DicV full-length genomes were obtained, and a phylogenetic analyse on the capsid region showed the presence of two clusters representing tentative novel genus. Although DicV-positive cases were detected throughout the year, a significantly higher positivity rate was observed during the rainy season. This study reveals that novel DicV RNA is frequently detected in the blood of Tanzanian children, paving the way for further investigations to determine if DicV possibly represent a new agent in humans.

Usutu virus in cerebrospinal fluid: A 2-year survey in a Tertiary Care Hospital, Geneva, Switzerland.

In 2009, the Usutu virus (USUV) was first reported as a cause of human neuroinvasive disorders. In Switzerland, USUV has been detected in wild birds with a seroprevalence of up to 6.59% in captive specimens sampled from zoo enclosures. This study investigates the clinical prevalence of USUV in human acute neuroinvasive disorders in Switzerland. Two hundred and fifty-eight cerebrospinal fluid samples collected between 2015 and 2017 for routine clinical care in a tertiary level hospital (Geneva) were tested for USUV by rRT-PCR. No samples were found positive, suggesting the absence, or the extremely low circulation of USUV in Western Switzerland.

Ebola virus disease diagnosis by real-time RT-PCR: A comparative study of 11 different procedures.

BACKGROUND: The diagnosis of Ebola virus disease relies on the detection of viral RNA in blood by real-time reverse-transcription PCR. While several of these assays were developed during the unprecedented 2013-2015 Ebola virus disease outbreak in West Africa, few were applied in the field. OBJECTIVES: To compare technical performances and practical aspects of 11 Ebola virus real-time reverse-transcription PCR procedures. STUDY DESIGN: We selected the most promising assays using serial dilutions of culture-derived Ebola virus RNA and determined their analytical sensitivity and potential range of quantification using quantified in vitro transcribed RNA; viral load values in the serum of an Ebola virus disease patient obtained with these assays were reported. Finally, ease of use and turnaround times of these kits were evaluated. RESULTS: Commercial assays were at least as sensitive as in-house tests. Five of the former (Altona, Roche, Fast-track Diagnostics, and Life Technologies) were selected for further evaluation. Despite differences in analytical sensitivity and limits of quantification, all of them were suitable for Ebola virus diagnosis and viral load estimation. The Lifetech Lyophilized Ebola Virus (Zaire 2014) assay (Life Technologies) appeared particularly promising, displaying the highest analytical sensitivity and shortest turnaround time, in addition to requiring no reagent freezing. CONCLUSIONS: Commercial kits were at least as sensitive as in-house tests and potentially easier to use in the field than the latter. This qualitative comparison of real-time reverse transcription PCR assays may serve as a basis for the design of future Ebola virus disease diagnostics.

Molecular Epidemiology of Human Rhinoviruses and Enteroviruses Highlights Their Diversity in Sub-Saharan Africa.

Human rhinoviruses (HRVs) and enteroviruses (HEVs) belong to the Enterovirus genus and are the most frequent cause of infection worldwide, but data on their molecular epidemiology in Africa are scarce. To understand HRV and HEV molecular epidemiology in this setting, we enrolled febrile pediatric patients participating in a large prospective cohort assessing the causes of fever in Tanzanian children. Naso/oropharyngeal swabs were systematically collected and tested by real-time RT-PCR for HRV and HEV. Viruses from positive samples were sequenced and phylogenetic analyses were then applied to highlight the HRV and HEV types as well as recombinant or divergent strains. Thirty-eight percent (378/1005) of the enrolled children harboured an HRV or HEV infection. Although some types were predominant, many distinct types were co-circulating, including a vaccinal poliovirus, HEV-A71 and HEV-D68. Three HRV-A recombinants were identified: HRV-A36/HRV-A67, HRV-A12/HRV-A67 and HRV-A96/HRV-A61. Four divergent HRV strains were also identified: one HRV-B strain and three HRV-C strains. This is the first prospective study focused on HRV and HEV molecular epidemiology in sub-Saharan Africa. This systematic and thorough large screening with careful clinical data management confirms the wide genomic diversity of these viruses, brings new insights about their evolution and provides data about associated symptoms.

Clinical features and viral kinetics in a rapidly cured patient with Ebola virus disease: a case report.

BACKGROUND: A detailed description of viral kinetics, duration of virus shedding, and intraviral evolution in different body sites is warranted to understand Ebola virus pathogenesis. Patients with Ebola virus infections admitted to university hospitals provide a unique opportunity to do such in-depth virological investigations. We describe the clinical, biological, and virological follow-up of a case of Ebola virus disease. METHODS: A 43-year-old medical doctor who contracted an Ebola virus infection in Sierra Leone on Nov 16, 2014 (day 1), was airlifted to Geneva University Hospitals, Geneva, Switzerland, on day 5 after disease onset. The patient received an experimental antiviral treatment of monoclonal antibodies (ZMAb) and favipiravir. We monitored daily viral load kinetics, estimated viral clearance, calculated the half-life of the virus in plasma, and analysed the viral genome via high-throughput sequencing, in addition to clinical and biological signs. FINDINGS: The patient recovered rapidly, despite an initial high viral load (about 1 × 10(7) RNA copies per mL 24 h after onset of fever). We noted a two-phase viral decay. The virus half-life decreased from about 26 h to 9·5 h after the experimental antiviral treatment. Compared with a consensus sequence of June 18, 2014, the isolate that infected this patient displayed only five synonymous nucleotide substitutions on the full genome (4901A→C, 7837C→T, 8712A→G, 9947T→C, 16201T→C) despite 5 months of human-to-human transmission. INTERPRETATION: This study emphasises the importance of virological investigations to fully understand the course of Ebola virus disease and adaptation of the virus. Whether the viral decay was caused by the effects of the immune response alone, an additional benefit from the antiviral treatment, or a combination of both is unclear. In-depth virological analysis and randomised controlled trials are needed before any conclusion on the potential effect of antiviral treatment can be drawn. FUNDING: Geneva University Hospitals, Swiss Office of Public Health, Swiss Agency for Development and Cooperation, and Swiss National Science Foundation.

Critical analysis of rhinovirus RNA load quantification by real-time reverse transcription-PCR.

Rhinoviruses are the most frequent cause of human respiratory infections, and quantitative rhinovirus diagnostic tools are needed for clinical investigations. Although results obtained by real-time reverse-transcription PCR (RT-PCR) assays are frequently converted to viral RNA loads, this presents several limitations regarding accurate virus RNA quantification, particularly given the need to reliably quantify all known rhinovirus genotypes with a single assay. Using an internal extraction control and serial dilutions of an in vitro-transcribed rhinovirus RNA reference standard, we validated a quantitative one-step real-time PCR assay. We then used chimeric rhinovirus genomes with 5'-untranslated regions (5'UTRs) originating from the three rhinovirus species and from one enterovirus to estimate the impact of the 5'UTR diversity. Respiratory specimens from infected patients were then also analyzed. The assay quantification ability ranged from 4.10 to 9.10 log RNA copies/ml, with an estimated error margin of ±10%. This variation was mainly linked to target variability and interassay variability. Taken together, our results indicate that our assay can reliably estimate rhinovirus RNA load, provided that the appropriate error margin is used. In contrast, due to the lack of a universal rhinovirus RNA standard and the variability related to sample collection procedures, accurate absolute rhinovirus RNA quantification in respiratory specimens is currently hardly feasible.