Sequence optimized diagnostic assay for Ebola virus detection.

Rapid pathogen identification is a critical first step in patient isolation, treatment, and controlling an outbreak. Real-time PCR is a highly sensitive and specific approach commonly used for infectious disease diagnostics. However, mismatches in the primer or probe sequence and the target organism can cause decreased sensitivity, assay failure, and false negative results. Limited genomic sequences for rare pathogens such as Ebola virus (EBOV) can negatively impact assay performance due to undiscovered genetic diversity. We previously developed and validated several EBOV assays prior to the 2013-2016 EBOV outbreak in West Africa, and sequencing EBOV Makona identified sequence variants that could impact assay performance. Here, we assessed the impact sequence mismatches have on EBOV assay performance, finding one or two primer or probe mismatches resulted in a range of impact from minimal to almost two log sensitivity reduction. Redesigning this assay improved detection of all EBOV variants tested. Comparing the performance of the new assay with the previous assays across a panel of human EBOV samples confirmed increased assay sensitivity as reflected in decreased Cq values with detection of three positive that tested negative with the original assay.

Virus-encoded miRNAs in Ebola virus disease.

Ebola virus (EBOV) is a negative-strand RNA virus that replicates in the cytoplasm and causes an often-fatal hemorrhagic fever. EBOV, like other viruses, can reportedly encode its own microRNAs (miRNAs) to subvert host immune defenses. miRNAs are short noncoding RNAs that can regulate gene expression by hybridizing to multiple mRNAs, and viral miRNAs can enhance viral replication and infectivity by regulating host or viral genes. To date, only one EBOV miRNA has been examined in human infection. Here, we assayed mouse, rhesus macaque, cynomolgus macaque, and human samples infected with three EBOV variants for twelve computationally predicted viral miRNAs using RT-qPCR. Ten miRNAs aligned to EBOV variants and were detectable in the four species during disease with several viral miRNAs showing presymptomatic amplification in animal models. miRNA abundances in both the mouse and nonhuman primate models mirrored the human cohort, with miR-1-5p, miR-1-3p, and miR-T3-3p consistently at the highest levels. These striking similarities in the most abundant miRNAs during infection with different EBOV variants and hosts indicate that these miRNAs are potential valuable diagnostic markers and key effectors of EBOV pathogenesis.

Serosurveillance of viral pathogens circulating in West Africa.

BACKGROUND: Sub-Saharan Africa is home to a variety of pathogens, but disease surveillance and the healthcare infrastructure necessary for proper management and control are severely limited. Lassa virus, the cause of Lassa fever, a severe hemorrhagic fever in humans is endemic in West Africa. In Sierra Leone at the Kenema Government Hospital Lassa Diagnostic Laboratory, up to 70 % of acute patient samples suspected of Lassa fever test negative for Lassa virus infection. This large amount of acute undiagnosed febrile illness can be attributed in part to an array of hemorrhagic fever and arthropod-borne viruses causing disease that goes undetected and untreated. METHODS: To better define the nature and extent of viral pathogens infecting the Sierra Leonean population, we developed a multiplexed MAGPIX® assay to detect IgG antibodies against Lassa, Ebola, Marburg, Rift Valley fever, and Crimean-Congo hemorrhagic fever viruses as well as pan-assays for flaviviruses and alphaviruses. This assay was used to survey 675 human serum samples submitted to the Lassa Diagnostic Laboratory between 2007 and 2014. RESULTS: In the study population, 50.2 % were positive for Lassa virus, 5.2 % for Ebola virus, 10.7 % for Marburg virus, 1.8 % for Rift Valley fever virus, 2.0 % for Crimean-Congo hemorrhagic fever virus, 52.9 % for flaviviruses and 55.8 % for alphaviruses. CONCLUSIONS: These data exemplify the importance of disease surveillance and differential diagnosis for viral diseases in Sierra Leone. We demonstrate the endemic nature of some of these viral pathogens in the region and suggest that unrecognized outbreaks of viral infection have occurred.

Circulating microRNA profiles of Ebola virus infection.

Early detection of Ebola virus (EBOV) infection is essential to halting transmission and adjudicating appropriate treatment. However, current methods rely on viral identification, and this approach can misdiagnose presymptomatic and asymptomatic individuals. In contrast, disease-driven alterations in the host transcriptome can be exploited for pathogen-specific diagnostic biomarkers. Here, we present for the first time EBOV-induced changes in circulating miRNA populations of nonhuman primates (NHPs) and humans. We retrospectively profiled longitudinally-collected plasma samples from rhesus macaques challenged via intramuscular and aerosol routes and found 36 miRNAs differentially present in both groups. Comparison of miRNA abundances to viral loads uncovered 15 highly correlated miRNAs common to EBOV-infected NHPs and humans. As proof of principle, we developed an eight-miRNA classifier that correctly categorized infection status in 64/74 (86%) human and NHP samples. The classifier identified acute infections in 27/29 (93.1%) samples and in 6/12 (50%) presymptomatic NHPs. These findings showed applicability of NHP-derived miRNAs to a human cohort, and with additional research the resulting classifiers could impact the current capability to diagnose presymptomatic and asymptomatic EBOV infections.

A parallel genome-wide RNAi screening strategy to identify host proteins important for entry of Marburg virus and H5N1 influenza virus.

BACKGROUND: Genome-wide RNAi screening has been widely used to identify host proteins involved in replication and infection of different viruses, and numerous host factors are implicated in the replication cycles of these viruses, demonstrating the power of this approach. However, discrepancies on target identification of the same viruses by different groups suggest that high throughput RNAi screening strategies need to be carefully designed, developed and optimized prior to the large scale screening. METHODS: Two genome-wide RNAi screens were performed in parallel against the entry of pseudotyped Marburg viruses and avian influenza virus H5N1 utilizing an HIV-1 based surrogate system, to identify host factors which are important for virus entry. A comparative analysis approach was employed in data analysis, which alleviated systematic positional effects and reduced the false positive number of virus-specific hits. RESULTS: The parallel nature of the strategy allows us to easily identify the host factors for a specific virus with a greatly reduced number of false positives in the initial screen, which is one of the major problems with high throughput screening. The power of this strategy is illustrated by a genome-wide RNAi screen for identifying the host factors important for Marburg virus and/or avian influenza virus H5N1 as described in this study. CONCLUSIONS: This strategy is particularly useful for highly pathogenic viruses since pseudotyping allows us to perform high throughput screens in the biosafety level 2 (BSL-2) containment instead of the BSL-3 or BSL-4 for the infectious viruses, with alleviated safety concerns. The screening strategy together with the unique comparative analysis approach makes the data more suitable for hit selection and enables us to identify virus-specific hits with a much lower false positive rate.

Role of EXT1 and Glycosaminoglycans in the Early Stage of Filovirus Entry.

UNLABELLED: Filoviruses, including both Ebola virus (EBOV) and Marburg virus (MARV), can infect humans and other animals, causing hemorrhagic fever with a high mortality rate. Entry of these viruses into the host is mediated by a single filoviral glycoprotein (GP). GP is composed of two subunits: GP1, which is responsible for attachment and binding to receptor(s) on susceptible cells, and GP2, which mediates viral and cell membrane fusion. Although numerous host factors have been implicated in the entry process, the initial attachment receptor(s) has not been well defined. In this report, we demonstrate that exostosin 1 (EXT1), which is involved in biosynthesis of heparan sulfate (HS), plays a role in filovirus entry. Expression knockdown of EXT1 by small interfering RNAs (siRNAs) impairs GP-mediated pseudoviral entry and that of infectious EBOV and MARV in tissue cultured cells. Furthermore, HS, heparin, and other related glycosaminoglycans (GAGs), to different extents, can bind to and block GP-mediated viral entry and that of infectious filoviruses. These results strongly suggest that HS and other related GAGs are attachment receptors that are utilized by filoviruses for entry and infection. These GAGs may have therapeutic potential in treating EBOV- and MARV-infected patients. IMPORTANCE: Infection by Ebola virus and Marburg virus can cause severe illness in humans, with a high mortality rate, and currently there is no FDA-approved vaccine or therapeutic treatment available. The ongoing 2014 outbreak in West Africa underscores a lack of our understanding in the infection and pathogenesis of these viruses and the urgency of drug discovery and development. In this study, we provide several pieces of evidence that demonstrate that heparan sulfate and other closely related glycosaminoglycans are the molecules that are used by filoviruses for initial attachment. Furthermore, we demonstrate that these glycosaminoglycans can block entry of and infection by filoviruses. Thus, this work provides mechanistic insights on the early step of filoviral infection and suggests a possible therapeutic option for diseases caused by filovirus infection.