A proof of concept for a targeted enrichment approach to the simultaneous detection and characterization of rickettsial pathogens from clinical specimens.

Infection with either Rickettsia prowazekii or Orientia tsutsugamushi is common, yet diagnostic capabilities are limited due to the short window for positive identification. Until now, although targeted enrichment had been applied to increase sensitivity of sequencing-based detection for various microorganisms, it had not been applied to sequencing of R. prowazekii in clinical samples. Additionally, hybridization-based targeted enrichment strategies had only scarcely been applied to qPCR of any pathogens in clinical samples. Therefore, we tested a targeted enrichment technique as a proof of concept and found that it dramatically reduced the limits of detection of these organisms by both qPCR and high throughput sequencing. The enrichment methodology was first tested in contrived clinical samples with known spiked-in concentrations of R. prowazekii and O. tsutsugamushi DNA. This method was also evaluated using clinical samples, resulting in the simultaneous identification and characterization of O. tsutsugamushi directly from clinical specimens taken from sepsis patients. We demonstrated that the targeted enrichment technique is helpful by lowering the limit of detection, not only when applied to sequencing, but also when applied to qPCR, suggesting the technique could be applied more broadly to include other assays and/or microbes for which there are limited diagnostic or detection modalities.

Immune and behavioral correlates of protection against symptomatic post-vaccination SARS-CoV-2 infection.

Introduction: We sought to determine pre-infection correlates of protection against SARS-CoV-2 post-vaccine inzfections (PVI) acquired during the first Omicron wave in the United States. Methods: Serum and saliva samples from 176 vaccinated adults were collected from October to December of 2021, immediately before the Omicron wave, and assessed for SARS-CoV-2 Spike-specific IgG and IgA binding antibodies (bAb). Sera were also assessed for bAb using commercial assays, and for neutralization activity against several SARS-CoV-2 variants. PVI duration and severity, as well as risk and precautionary behaviors, were assessed by questionnaires. Results: Serum anti-Spike IgG levels assessed by research assay, neutralization titers against Omicron subvariants, and low home risk scores correlated with protection against PVIs after multivariable regression analysis. Commercial assays did not perform as well as research assay, likely due to their lower dynamic range. Discussion: In the 32 participants that developed PVI, anti-Spike IgG bAbs correlated with lower disease severity and shorter duration of illness.

Peripheral immune responses to filoviruses in a reservoir versus spillover hosts reveal transcriptional correlates of disease.

Several filoviruses, including Marburg virus (MARV), cause severe disease in humans and nonhuman primates (NHPs). However, the Egyptian rousette bat (ERB, Rousettus aegyptiacus), the only known MARV reservoir, shows no overt illness upon natural or experimental infection, which, like other bat hosts of zoonoses, is due to well-adapted, likely species-specific immune features. Despite advances in understanding reservoir immune responses to filoviruses, ERB peripheral blood responses to MARV and how they compare to those of diseased filovirus-infected spillover hosts remain ill-defined. We thus conducted a longitudinal analysis of ERB blood gene responses during acute MARV infection. These data were then contrasted with a compilation of published primate blood response studies to elucidate gene correlates of filovirus protection versus disease. Our work expands on previous findings in MARV-infected ERBs by supporting both host resistance and disease tolerance mechanisms, offers insight into the peripheral immunocellular repertoire during infection, and provides the most direct known cross-examination between reservoir and spillover hosts of the most prevalently-regulated response genes, pathways and activities associated with differences in filovirus pathogenesis and pathogenicity.

SARS-CoV-2 Infections in Vaccinated and Unvaccinated Populations in Camp Lemonnier, Djibouti, from April 2020 to January 2022.

The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the disparity between developed and developing countries for infectious disease surveillance and the sequencing of pathogen genomes. The majority of SARS-CoV-2 sequences published are from Europe, North America, and Asia. Between April 2020 and January 2022, 795 SARS-CoV-2-positive nares swabs from individuals in the U.S. Navy installation Camp Lemonnier, Djibouti, were collected, sequenced, and analyzed. In this study, we described the results of genomic sequencing and analysis for 589 samples, the first published viral sequences for Djibouti, including 196 cases of vaccine breakthrough infections. This study contributes to the knowledge base of circulating SARS-CoV-2 lineages in the under-sampled country of Djibouti, where only 716 total genome sequences are available at time of publication. Our analysis resulted in the detection of circulating variants of concern, mutations of interest in lineages in which those mutations are not common, and emerging spike mutations.

SARS-CoV-2 Outbreak Dynamics in an Isolated US Military Recruit Training Center With Rigorous Prevention Measures.

BACKGROUND: Marine recruits training at Parris Island experienced an unexpectedly high rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, despite preventive measures including a supervised, 2-week, pre-entry quarantine. We characterize SARS-CoV-2 transmission in this cohort. METHODS: Between May and November 2020, we monitored 2,469 unvaccinated, mostly male, Marine recruits prospectively during basic training. If participants tested negative for SARS-CoV-2 by quantitative polymerase chain reaction (qPCR) at the end of quarantine, they were transferred to the training site in segregated companies and underwent biweekly testing for 6 weeks. We assessed the effects of coronavirus disease 2019 (COVID-19) prevention measures on other respiratory infections with passive surveillance data, performed phylogenetic analysis, and modeled transmission dynamics and testing regimens. RESULTS: Preventive measures were associated with drastically lower rates of other respiratory illnesses. However, among the trainees, 1,107 (44.8%) tested SARS-CoV-2-positive, with either mild or no symptoms. Phylogenetic analysis of viral genomes from 580 participants revealed that all cases but one were linked to five independent introductions, each characterized by accumulation of mutations across and within companies, and similar viral isolates in individuals from the same company. Variation in company transmission rates (mean reproduction number R 0 ; 5.5 [95% confidence interval [CI], 5.0, 6.1]) could be accounted for by multiple initial cases within a company and superspreader events. Simulations indicate that frequent rapid-report testing with case isolation may minimize outbreaks. CONCLUSIONS: Transmission of wild-type SARS-CoV-2 among Marine recruits was approximately twice that seen in the community. Insights from SARS-CoV-2 outbreak dynamics and mutations spread in a remote, congregate setting may inform effective mitigation strategies.

Prospective Assessment of Symptoms to Evaluate Asymptomatic SARS-CoV-2 Infections in a Cohort of Health Care Workers.

BACKGROUND: The frequency of asymptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is unclear and may be influenced by how symptoms are evaluated. In this study, we sought to determine the frequency of asymptomatic SARS-CoV-2 infections in a prospective cohort of health care workers (HCWs). METHODS: A prospective cohort of HCWs, confirmed negative for SARS-CoV-2 exposure upon enrollment, were evaluated for SARS-CoV-2 infection by monthly analysis of SARS-CoV-2 antibodies as well as referral for polymerase chain reaction testing whenever they exhibited symptoms of coronavirus disease 2019 (COVID-19). Participants completed the standardized and validated FLU-PRO Plus symptom questionnaire scoring viral respiratory disease symptom intensity and frequency at least twice monthly during baseline periods of health and each day they had any symptoms that were different from their baseline. RESULTS: Two hundred sixty-three participants were enrolled between August 25 and December 31, 2020. Through February 28, 2021, 12 participants were diagnosed with SARS-CoV-2 infection. Symptom analysis demonstrated that all 12 had at least mild symptoms of COVID-19, compared with baseline health, near or at time of infection. CONCLUSIONS: These results suggest that asymptomatic SARS-CoV-2 infection in unvaccinated, immunocompetent adults is less common than previously reported. While infectious inoculum doses and patient factors may have played a role in the clinical manifestations of SARS-CoV-2 infections in this cohort, we suspect that the high rate of symptomatic disease was due primarily to participant attentiveness to symptoms and collection of symptoms in a standardized, prospective fashion. These results have implications for studies that estimate SARS-CoV-2 infection prevalence and for public health measures to control the spread of this virus.

Immunological and Genetic Investigation of SARS-CoV-2 Reinfection in an Otherwise Healthy, Young Marine Recruit.

We used epidemiologic and viral genetic information to identify a case of likely reinfection in an otherwise healthy, young Marine recruit enrolled in the prospective, longitudinal COVID-19 Health Action Response for Marines (CHARM) study, and we paired these findings with serological studies. This participant had a positive RT-PCR to SARS-CoV-2 upon routine sampling on study day 7, although he was asymptomatic at that time. He cleared the infection within seven days. On study day 46, he had developed symptoms consistent with COVID-19 and tested positive by RT-PCR for SARS-CoV-2 again. Viral whole genome sequencing was conducted from nares swabs at multiple time points. The day 7 sample was determined to be lineage B.1.340, whereas both the day 46 and day 49 samples were B.1.1. The first positive result for anti-SARS-CoV-2 IgM serology was collected on day 49 and for IgG on day 91. This case appears most consistent with a reinfection event. Our investigation into this case is unique in that we compared sequence data from more than just paired specimens, and we also assayed for immune response after both the initial infection and the later reinfection. These data demonstrate that individuals who have experienced an infection with SARS-CoV-2 may fail to generate effective or long-lasting immunity, similar to endemic human beta coronaviruses.

An examination of motor unit firing rates during steady torque of maximal efforts with either an explosive or slower rate of torque development.

NEW FINDINGS: What is the central question of this study? The aim was to explore agonist and antagonist motor unit firing rates during maximal efforts performed with either an explosive or a slower rate of torque development. What is the main finding and its importance? The antagonist muscle presented a motor unit firing rate relationship similar to the agonist muscle. Additionally, the motor units of both muscles exhibited higher firing rates during explosive maximal contractions than during maximal contractions performed at a slower rate of torque development. These results could prove useful to future research analysing the effects of age, disease, resistance training and/or fatigue-related alterations to motor unit firing rates. ABSTRACT: The primary purpose of the present study was to examine motor unit (MU) firing rates in agonist and antagonist muscles during periods of steady, maximal efforts using explosive and slower rates of torque development. A secondary purpose was to analyse the MU firing rate versus action potential amplitude relationships of the agonist and antagonist muscles during maximal efforts. Thirteen subjects (mean ± SD; age, 21.2 ± 3.6 years; mass 81.1 ± 21.3 kg; and stature, 177.1±9.9 cm) performed two maximal isometric trapezoid muscle actions of the elbow flexors that included either an explosive or a slower, linearly increasing rate (ramp) of torque development. Surface EMG signals of the biceps brachii (BB) and triceps brachii (TB) muscles were collected and decomposed into their constituent MU action potential trains. The MU firing rate versus action potential amplitude relationships of the BB (agonist) and TB (antagonist) muscles were analysed. Moderate to strong relationships (|r| ≥ 0.65) were present for the explosive and ramp contractions in the agonist and antagonist muscles. Firing rates of smaller and larger MUs were higher during the explosive [mean ± SD; agonist = 18.1 ± 6.9 pulses per second (pps), antagonist = 22.0±3.9 pps] than the ramp (agonist = 14.0 ± 5.1 pps, antagonist = 18.3 ± 4.4 pps) contractions for the agonist (P = 0.013) and antagonist muscles (P = 0.007). The antagonist muscle exhibits a similar MU firing rate versus action potential amplitude relationship to the agonist muscle at maximal efforts. Future research should investigate the effects of short-term resistance training on antagonist firing rates and the involvement of peripheral feedback on firing rates during maximal efforts performed at various rates of torque development.

Host response transcriptomic analysis of Crimean-Congo hemorrhagic fever pathogenesis in the cynomolgus macaque model.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a highly pathogenic tick-borne RNA virus prevalent in Asia, Europe, and Africa, and can cause a hemorrhagic disease (CCHF) in humans with mortality rates as high as 60%. A general lack of both effective medical countermeasures and a comprehensive understanding of disease pathogenesis is partly driven by an historical lack of viable CCHF animal models. Recently, a cynomolgous macaque model of CCHF disease was developed. Here, we document the targeted transcriptomic response of non-human primates (NHP) to two different CCHFV strains; Afghan09-2990 and Kosova Hoti that both yielded a mild CCHF disease state. We utilized a targeted gene panel to elucidate the transcriptomic changes occurring in NHP whole blood during CCHFV infection; a first for any primate species. We show numerous upregulated genes starting at 1 day post-challenge through 14 days post-challenge. Early gene changes fell predominantly in the interferon stimulated gene family with later gene changes coinciding with an adaptive immune response to the virus. There are subtle differences between viral strains, namely duration of the differentially expressed gene response and biological pathways enriched. After recovery, NHPs showed no lasting transcriptomic changes at the end of sample collection.

Transcriptomic Analysis Reveals Host miRNAs Correlated with Immune Gene Dysregulation during Fatal Disease Progression in the Ebola Virus Cynomolgus Macaque Disease Model.

Ebola virus is a continuing threat to human populations, causing a virulent hemorrhagic fever disease characterized by dysregulation of both the innate and adaptive host immune responses. Severe cases are distinguished by an early, elevated pro-inflammatory response followed by a pronounced lymphopenia with B and T cells unable to mount an effective anti-viral response. The precise mechanisms underlying the dysregulation of the host immune system are poorly understood. In recent years, focus on host-derived miRNAs showed these molecules to play an important role in the host gene regulation arsenal. Here, we describe an investigation of RNA biomarkers in the fatal Ebola virus disease (EVD) cynomolgus macaque model. We monitored both host mRNA and miRNA responses in whole blood longitudinally over the disease course in these non-human primates (NHPs). Analysis of the interactions between these classes of RNAs revealed several miRNA markers significantly correlated with downregulation of genes; specifically, the analysis revealed those involved in dysregulated immune pathways associated with EVD. In particular, we noted strong interactions between the miRNAs hsa-miR-122-5p and hsa-miR-125b-5p with immunological genes regulating both B and T-cell activation. This promising set of biomarkers will be useful in future studies of severe EVD pathogenesis in both NHPs and humans and may serve as potential prognostic targets.

Genomic and Virological Characterization of SARS-CoV-2 Variants in a Subset of Unvaccinated and Vaccinated U.S. Military Personnel.

The emergence of SARS-CoV-2 variants complicates efforts to control the COVID-19 pandemic. Increasing genomic surveillance of SARS-CoV-2 is imperative for early detection of emerging variants, to trace the movement of variants, and to monitor effectiveness of countermeasures. Additionally, determining the amount of viable virus present in clinical samples is helpful to better understand the impact these variants have on viral shedding. In this study, we analyzed nasal swab samples collected between March 2020 and early November 2021 from a cohort of United States (U.S.) military personnel and healthcare system beneficiaries stationed worldwide as a part of the Defense Health Agency's (DHA) Global Emerging Infections Surveillance (GEIS) program. SARS-CoV-2 quantitative real time reverse-transcription PCR (qRT-PCR) positive samples were characterized by next-generation sequencing and a subset was analyzed for isolation and quantification of viable virus. Not surprisingly, we found that the Delta variant is the predominant strain circulating among U.S. military personnel beginning in July 2021 and primarily represents cases of vaccine breakthrough infections (VBIs). Among VBIs, we found a 50-fold increase in viable virus in nasal swab samples from Delta variant cases when compared to cases involving other variants. Notably, we found a 40-fold increase in viable virus in nasal swab samples from VBIs involving Delta as compared to unvaccinated personnel infected with other variants prior to the availability of approved vaccines. This study provides important insight about the genomic and virological characterization of SARS-CoV-2 isolates from a unique study population with a global presence.

Asymptomatic Infection of Marburg Virus Reservoir Bats Is Explained by a Strategy of Immunoprotective Disease Tolerance.

Marburg virus (MARV) is among the most virulent pathogens of primates, including humans. Contributors to severe MARV disease include immune response suppression and inflammatory gene dysregulation ("cytokine storm"), leading to systemic damage and often death. Conversely, MARV causes little to no clinical disease in its reservoir host, the Egyptian rousette bat (ERB). Previous genomic and in vitro data suggest that a tolerant ERB immune response may underlie MARV avirulence, but no significant examination of this response in vivo yet exists. Here, using colony-bred ERBs inoculated with a bat isolate of MARV, we use species-specific antibodies and an immune gene probe array (NanoString) to temporally characterize the transcriptional host response at sites of MARV replication relevant to primate pathogenesis and immunity, including CD14+ monocytes/macrophages, critical immune response mediators, primary MARV targets, and skin at the inoculation site, where highest viral loads and initial engagement of antiviral defenses are expected. Our analysis shows that ERBs upregulate canonical antiviral genes typical of mammalian systems, such as ISG15, IFIT1, and OAS3, yet demonstrate a remarkable lack of significant induction of proinflammatory genes classically implicated in primate filoviral pathogenesis, including CCL8, FAS, and IL6. Together, these findings offer the first in vivo functional evidence for disease tolerance as an immunological mechanism by which the bat reservoir asymptomatically hosts MARV. More broadly, these data highlight factors determining disparate outcomes between reservoir and spillover hosts and defensive strategies likely utilized by bat hosts of other emerging pathogens, knowledge that may guide development of effective antiviral therapies.

Rousette Bat Dendritic Cells Overcome Marburg Virus-Mediated Antiviral Responses by Upregulation of Interferon-Related Genes While Downregulating Proinflammatory Disease Mediators.

Dysregulated and maladaptive immune responses are at the forefront of human diseases caused by infection with zoonotic viral hemorrhagic fever viruses. Elucidating mechanisms of how the natural animal reservoirs of these viruses coexist with these agents without overt disease, while permitting sufficient replication to allow for transmission and maintenance in a population, is important for understanding the viral ecology and spillover to humans. The Egyptian rousette bat (ERB) has been identified as a reservoir for Marburg virus (MARV), a filovirus and the etiological agent of the highly lethal Marburg virus disease. Little is known regarding how these bats immunologically respond to MARV infection. In humans, macrophages and dendritic cells (DCs) are primary targets of infection, and their dysregulation is thought to play a central role in filovirus diseases, by disturbing their normal functions as innate sensors and adaptive immune response facilitators while serving as amplification and dissemination agents for the virus. The infection status and responses to MARV in bat myeloid-lineage cells are uncharacterized and likely represent an important modulator of the bat's immune response to MARV infection. Here, we generate DCs from the bone marrow of rousette bats. Infection with a bat isolate of MARV resulted in a low level of transcription in these cells and significantly downregulated DC maturation and adaptive immune-stimulatory pathways while simultaneously upregulating interferon-related pathogen-sensing pathways. This study provides a first insight into how the bat immune response is directed toward preventing aberrant inflammatory responses while mounting an antiviral response to defend against MARV infection.IMPORTANCE Marburg viruses (MARVs) cause severe human disease resulting from aberrant immune responses. Dendritic cells (DCs) are primary targets of infection and are dysregulated by MARV. Dysregulation of DCs facilitates MARV replication and virus dissemination and influences downstream immune responses that result in immunopathology. Egyptian rousette bats (ERBs) are natural reservoirs of MARV, and infection results in virus replication and shedding, with asymptomatic control of the virus within weeks. The mechanisms that bats employ to appropriately respond to infection while avoiding disease are unknown. Because DC infection and modulation are important early events in human disease, we measured the transcriptional responses of ERB DCs to MARV. The significance of this work is in identifying cell type-specific coevolved responses between ERBs and MARV, which gives insight into how bat reservoirs are able to harbor MARV and permit viral replication, allowing transmission and maintenance in the population while simultaneously preventing immunopathogenesis.

Transcriptomics Reveal Antiviral Gene Induction in the Egyptian Rousette Bat Is Antagonized In Vitro by Marburg Virus Infection.

The Egyptian rousette bat (ERB) is the only known Marburg virus (MARV) reservoir host. ERBs develop a productive MARV infection with low viremia and shedding but no overt disease, suggesting this virus is efficiently controlled by ERB antiviral responses. This dynamic would contrast with humans, where MARV-mediated interferon (IFN) antagonism early in infection is thought to contribute to the severe, often fatal disease. The newly-annotated ERB genome and transcriptome have now enabled us to use a custom-designed NanoString nCounter ERB CodeSet in conjunction with RNA-seq to investigate responses in a MARV-infected ERB cell line. Both transcriptomic platforms correlated well and showed that MARV inhibited the antiviral program in ERB cells, while an IFN antagonism-impaired MARV was less efficient at suppressing the response gene induction, phenotypes previously reported for primate cells. Interestingly, and despite the expansion of IFN loci in the ERB genome, neither MARV showed specific induction of almost any IFN gene. However, we detected an upregulation of putative, unannotated ERB antiviral paralogs, as well as an elevated basal expression in uninfected ERB cells of key antiviral genes.

Growth-Adaptive Mutations in the Ebola Virus Makona Glycoprotein Alter Different Steps in the Virus Entry Pathway.

The Zaire ebolavirus (EBOV) glycoprotein (GP) is cleaved into two subunits (GP1 and GP2) that are both required for virus attachment and entry into cells. Sequence changes in the GP have been proposed to increase pathogenesis and to alter virus growth properties. Mutations in GP acquired during EBOV tissue culture passage have also been reported to change virus growth properties. Here, we report the isolation of six amino acid mutations in EBOV GP that spontaneously appeared during recovery and passage of an EBOV-Makona GP-pseudotyped vesicular stomatitis virus (VSV), two of which also occur during passage of EBOV clinical isolates in tissue culture. Each of the six mutations resulted in increased virus growth in monkey and human cell lines. All mutations are located in the GP2 fusion subunit and increase entry kinetics of EBOV virus-like particles (VLPs). The gain-of-entry function mapped to two mechanistic phenotypes. Mutations in heptad repeat 1 (HR1) decreased the requirement for cathepsin B activity for viral infection. Mutations directly within the fusion loop increased entry kinetics without altering the cathepsin B dependence. Several mutations in the fusion loop were substitutions of residues present in other ebolavirus glycoproteins, illustrating the evolutionary paths for maintaining an optimally functioning fusion loop under selection pressure.IMPORTANCEZaire ebolavirus (EBOV) is the causative agent of the highly lethal Ebola virus disease and poses a significant threat to the global health community. Approved antivirals against EBOV are lacking; however, promising therapies targeting the EBOV glycoprotein are being developed. Efficacy testing of these candidate therapeutics relies on EBOV laboratory stocks, which when grown in tissue culture may acquire mutations in the glycoprotein. These mutations can produce inaccurate results in therapeutic testing. Until recently, distinguishing between tissue culture mutations and naturally occurring polymorphisms in EBOV GP was difficult in the absence of consensus clinical GP sequences. Here, we utilize recombinant VSV (rVSV) pseudotyped with the consensus clinical EBOV Makona GP to identify several mutations that have emerged or have potential to emerge in EBOV GP during tissue culture passage. Identifying these mutations informs the EBOV research community as to which mutations may arise during preparation of laboratory virus stocks.

The Egyptian Rousette Genome Reveals Unexpected Features of Bat Antiviral Immunity.

Bats harbor many viruses asymptomatically, including several notorious for causing extreme virulence in humans. To identify differences between antiviral mechanisms in humans and bats, we sequenced, assembled, and analyzed the genome of Rousettus aegyptiacus, a natural reservoir of Marburg virus and the only known reservoir for any filovirus. We found an expanded and diversified KLRC/KLRD family of natural killer cell receptors, MHC class I genes, and type I interferons, which dramatically differ from their functional counterparts in other mammals. Such concerted evolution of key components of bat immunity is strongly suggestive of novel modes of antiviral defense. An evaluation of the theoretical function of these genes suggests that an inhibitory immune state may exist in bats. Based on our findings, we hypothesize that tolerance of viral infection, rather than enhanced potency of antiviral defenses, may be a key mechanism by which bats asymptomatically host viruses that are pathogenic in humans.

A conserved transcriptional response to intranasal Ebola virus exposure in nonhuman primates prior to onset of fever.

Ebola virus disease (EVD), caused by Ebola virus (EBOV), is a severe illness characterized by case fatality rates of up to 90%. The sporadic nature of outbreaks in resource-limited areas has hindered the ability to characterize the pathogenesis of EVD at all stages of infection but particularly early host responses. Pathogenesis is often studied in nonhuman primate (NHP) models of disease that replicate major aspects of human EVD. Typically, NHP models use a large infectious dose, are carried out through intramuscular or aerosol exposure, and have a fairly uniform disease course. By contrast, we report our analysis of the host response to EBOV after intranasal exposure. Twelve cynomolgus macaques were infected with 100 plaque-forming units of EBOV/Makona through intranasal exposure and presented with varying times to onset of EVD. We used RNA sequencing and a newly developed NanoString CodeSet to monitor the host response via changes in RNA transcripts over time. When individual animal gene expression data were phased based on the onset of sustained fever, the first clinical sign of severe disease, mathematical models indicated that interferon-stimulated genes appeared as early as 4 days before fever onset. This demonstrates that lethal EVD has a uniform and predictable response to infection regardless of time to onset. Furthermore, expression of a subset of genes could predict disease development before other host-based indications of infection such as fever.