Real-time, portable genome sequencing for Ebola surveillance.

The Ebola virus disease epidemic in West Africa is the largest on record, responsible for over 28,599 cases and more than 11,299 deaths. Genome sequencing in viral outbreaks is desirable to characterize the infectious agent and determine its evolutionary rate. Genome sequencing also allows the identification of signatures of host adaptation, identification and monitoring of diagnostic targets, and characterization of responses to vaccines and treatments. The Ebola virus (EBOV) genome substitution rate in the Makona strain has been estimated at between 0.87 × 10(-3) and 1.42 × 10(-3) mutations per site per year. This is equivalent to 16-27 mutations in each genome, meaning that sequences diverge rapidly enough to identify distinct sub-lineages during a prolonged epidemic. Genome sequencing provides a high-resolution view of pathogen evolution and is increasingly sought after for outbreak surveillance. Sequence data may be used to guide control measures, but only if the results are generated quickly enough to inform interventions. Genomic surveillance during the epidemic has been sporadic owing to a lack of local sequencing capacity coupled with practical difficulties transporting samples to remote sequencing facilities. To address this problem, here we devise a genomic surveillance system that utilizes a novel nanopore DNA sequencing instrument. In April 2015 this system was transported in standard airline luggage to Guinea and used for real-time genomic surveillance of the ongoing epidemic. We present sequence data and analysis of 142 EBOV samples collected during the period March to October 2015. We were able to generate results less than 24 h after receiving an Ebola-positive sample, with the sequencing process taking as little as 15-60 min. We show that real-time genomic surveillance is possible in resource-limited settings and can be established rapidly to monitor outbreaks.

Laboratory Findings, Compassionate Use of Favipiravir, and Outcome in Patients With Ebola Virus Disease, Guinea, 2015-A Retrospective Observational Study.

BACKGROUND: In 2015, the laboratory at the Ebola treatment center in Coyah, Guinea, confirmed Ebola virus disease (EVD) in 286 patients. The cycle threshold (Ct) of an Ebola virus-specific reverse transcription-polymerase chain reaction assay and 13 blood chemistry parameters were measured on admission and during hospitalization. Favipiravir treatment was offered to patients with EVD on a compassionate-use basis. METHODS: To reduce biases in the raw field data, we carefully selected 163 of 286 patients with EVD for a retrospective study to assess associations between potential risk factors, alterations in blood chemistry findings, favipiravir treatment, and outcome. RESULTS: The case-fatality rate in favipiravir-treated patients was lower than in untreated patients (42.5% [31 of 73] vs 57.8% [52 of 90]; P = .053 by univariate analysis). In multivariate regression analysis, a higher Ct and a younger age were associated with survival (P < .001), while favipiravir treatment showed no statistically significant effect (P = .11). However, Kaplan-Meier analysis indicated a longer survival time in the favipiravir-treated group (P = .015). The study also showed characteristic changes in blood chemistry findings in patients who died, compared with survivors. CONCLUSIONS: Consistent with the JIKI trial, this retrospective study revealed a trend toward improved survival in favipiravir- treated patients; however, the effect of treatment was not statistically significant, except for its influence on survival time.

Utility of primary cells to examine NPC1 receptor expression in Mops condylurus, a potential Ebola virus reservoir.

The significance of the integral membrane protein Niemann-Pick C1 (NPC1) in the ebolavirus entry process has been determined using various cell lines derived from humans, non-human primates and fruit bats. Fruit bats have long been purported as the potential reservoir host for ebolaviruses, however several studies provide evidence that Mops condylurus, an insectivorous microbat, is also an ebolavirus reservoir. NPC1 receptor expression in the context of ebolavirus replication in microbat cells remains unstudied. In order to study Ebola virus (EBOV) cellular entry and replication in M. condylurus, we derived primary and immortalized cell cultures from 12 different organs. The NPC1 receptor expression was characterized by confocal microscopy and flow cytometry comparing the expression levels of M. condylurus primary and immortalized cells, HeLa cells, human embryonic kidney cells and cells from a European microbat species. EBOV replication kinetics was studied for four representative cell cultures using qRT-PCR. The aim was to elucidate the suitability of primary and immortalized cells from different tissues for studying NPC1 receptor expression levels and their potential influence on EBOV replication. The NPC1 receptor expression level in M. condylurus primary cells differed depending on the organ they were derived from and was for most cell types significantly lower than in human cell lines. Immortalized cells showed for most cell types higher expression levels than their corresponding primary cells. Concluding from our infection experiments with EBOV we suggest a potential correlation between NPC1 receptor expression level and virus replication rate in vitro.

Different features of Vδ2 T and NK cells in fatal and non-fatal human Ebola infections.

BACKGROUND: Human Ebola infection is characterized by a paralysis of the immune system. A signature of αß T cells in fatal Ebola infection has been recently proposed, while the involvement of innate immune cells in the protection/pathogenesis of Ebola infection is unknown. Aim of this study was to analyze γδ T and NK cells in patients from the Ebola outbreak of 2014-2015 occurred in West Africa, and to assess their association with the clinical outcome. METHODOLOGY/PRINCIPAL FINDINGS: Nineteen Ebola-infected patients were enrolled at the time of admission to the Ebola Treatment Centre in Guinea. Patients were divided in two groups on the basis of the clinical outcome. The analysis was performed by using multiparametric flow cytometry established by the European Mobile Laboratory in the field. A low frequency of Vδ2 T-cells was observed during Ebola infection, independently from the clinical outcome. Moreover, Vδ2 T-cells from Ebola patients massively expressed CD95 apoptotic marker, suggesting the involvement of apoptotic mechanisms in Vδ2 T-cell loss. Interestingly, Vδ2 T-cells from survivors expressed an effector phenotype and presented a lower expression of the CTLA-4 exhaustion marker than fatalities, suggesting a role of effector Vδ2 T-cells in the protection. Furthermore, patients with fatal Ebola infection were characterized by a lower NK cell frequency than patients with non fatal infection. In particular, both CD56bright and CD56dim NK frequency were very low both in fatal and non fatal infections, while a higher frequency of CD56neg NK cells was associated to non-fatal infections. Finally, NK activation and expression of NKp46 and CD158a were independent from clinical outcome. CONCLUSIONS/SIGNIFICANCES: Altogether, the data suggest that both effector Vδ2 T-cells and NK cells may play a role in the complex network of protective response to EBOV infection. Further studies are required to characterize the protective effector functions of Vδ2 and NK cells.

Neuraminidase-Inhibiting Antibody Response to H5N1 Virus Vaccination in Chronically Ill and Immunocompromised Patients.

Neuraminidase-inhibiting (NAi) antibodies have been reported to be an independent correlate of protection from influenza disease, but the NAi antibody response to influenza vaccination has never been assessed in chronically ill or immunocompromised participants. Using an enzyme-linked lectin assay, we demonstrated that 2 immunizations with a Vero cell culture-derived, whole-virus H5N1 A/Vietnam vaccine induces NAi antibodies in 94.3% of chronically ill and 83.8% of immunocompromised participants. A booster with a heterologous A/Indonesia H5N1 vaccine induced comparable NAi antibody titers in both groups and resulted in 100% seropositivity. These data support prepandemic H5N1 vaccination strategies for these highly vulnerable risk groups.