Evolution and Spread of Ebola Virus in Liberia, 2014–2015

The 2013–present Western African Ebola virus disease (EVD) outbreak is the largest ever recorded with >28,000 reported cases. Ebola virus (EBOV) genome sequencing has played an important role throughout this outbreak; however, relatively few sequences have been determined from patients in Liberia, the second worst-affected country. Here, we report 140 EBOV genome sequences from the second wave of the Liberian outbreak and analyze them in combination with 782 previously published sequences from throughout the Western African outbreak. While multiple early introductions of EBOV to Liberia are evident, the majority of Liberian EVD cases are consistent with a single introduction, followed by spread and diversification within the country. Movement of the virus within Liberia was widespread and reintroductions from Liberia served as an important source for the continuation of the already ongoing EVD outbreak in Guinea. Overall, little evidence was found for incremental adaptation of EBOV to the human host.Organismic and Evolutionary Biolog

Genomic epidemiology reveals multiple introductions of Zika virus into the United States

Zika virus (ZIKV) is causing an unprecedented epidemic linked to severe congenital abnormalities. In July 2016, mosquito-borne ZIKV transmission was reported in the continental United States; since then, hundreds of locally acquired infections have been reported in Florida. To gain insights into the timing, source, and likely route(s) of ZIKV introduction, we tracked the virus from its first detection in Florida by sequencing ZIKV genomes from infected patients and Aedes aegypti mosquitoes. We show that at least 4 introductions, but potentially as many as 40, contributed to the outbreak in Florida and that local transmission is likely to have started in the spring of 2016-several months before its initial detection. By analysing surveillance and genetic data, we show that ZIKV moved among transmission zones in Miami. Our analyses show that most introductions were linked to the Caribbean, a finding corroborated by the high incidence rates and traffic volumes from the region into the Miami area. Our study provides an understanding of how ZIKV initiates transmission in new regions

De novo transcriptome reconstruction and annotation of the Egyptian rousette bat

Background The Egyptian Rousette bat (Rousettus aegyptiacus), a common fruit bat species found throughout Africa and the Middle East, was recently identified as a natural reservoir host of Marburg virus. With Ebola virus, Marburg virus is a member of the family Filoviridae that causes severe hemorrhagic fever disease in humans and nonhuman primates, but results in little to no pathological consequences in bats. Understanding host-pathogen interactions within reservoir host species and how it differs from hosts that experience severe disease is an important aspect of evaluating viral pathogenesis and developing novel therapeutics and methods of prevention. Results Progress in studying bat reservoir host responses to virus infection is hampered by the lack of host-specific reagents required for immunological studies. In order to establish a basis for the design of reagents, we sequenced, assembled, and annotated the R. aegyptiacus transcriptome. We performed de novo transcriptome assembly using deep RNA sequencing data from 11 distinct tissues from one male and one female bat. We observed high similarity between this transcriptome and those available from other bat species. Gene expression analysis demonstrated clustering of expression profiles by tissue, where we also identified enrichment of tissue-specific gene ontology terms. In addition, we identified and experimentally validated the expression of novel coding transcripts that may be specific to this species. Conclusion We comprehensively characterized the R. aegyptiacus transcriptome de novo. This transcriptome will be an important resource for understanding bat immunology, physiology, disease pathogenesis, and virus transmission

Error baseline rates of five sample preparation methods used to characterize RNA virus populations.

Individual RNA viruses typically occur as populations of genomes that differ slightly from each other due to mutations introduced by the error-prone viral polymerase. Understanding the variability of RNA virus genome populations is critical for understanding virus evolution because individual mutant genomes may gain evolutionary selective advantages and give rise to dominant subpopulations, possibly even leading to the emergence of viruses resistant to medical countermeasures. Reverse transcription of virus genome populations followed by next-generation sequencing is the only available method to characterize variation for RNA viruses. However, both steps may lead to the introduction of artificial mutations, thereby skewing the data. To better understand how such errors are introduced during sample preparation, we determined and compared error baseline rates of five different sample preparation methods by analyzing in vitro transcribed Ebola virus RNA from an artificial plasmid-based system. These methods included: shotgun sequencing from plasmid DNA or in vitro transcribed RNA as a basic "no amplification" method, amplicon sequencing from the plasmid DNA or in vitro transcribed RNA as a "targeted" amplification method, sequence-independent single-primer amplification (SISPA) as a "random" amplification method, rolling circle reverse transcription sequencing (CirSeq) as an advanced "no amplification" method, and Illumina TruSeq RNA Access as a "targeted" enrichment method. The measured error frequencies indicate that RNA Access offers the best tradeoff between sensitivity and sample preparation error (1.4-5) of all compared methods

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.Peer Reviewe

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

Informing the Historical Record of Experimental Nonhuman Primate Infections with Ebola Virus: Genomic Characterization of USAMRIID Ebola Virus/H.sapiens-tc/COD/1995/Kikwit-9510621 Challenge Stock "R4368" and Its Replacement "R4415".

The creation of licensed medical countermeasures against Select Agents such as Ebola virus (EBOV) is critically dependent on the use of standardized reagents, assays, and animal models. We performed full genome reconstruction, population genomics, contaminant analysis, and characterization of the glycoprotein gene editing site of historical United States Army Medical Research Institute of Infectious Diseases (USAMRIID) nonhuman-primate challenge stock Ebola virus Kikwit "R4368" and its 2014 replacement "R4415." We also provide characterization of the master stock used to create "R4415." The obtained data are essential to understanding the quality of the seed stock reagents used in pivotal animal studies that have been used to inform medical countermeasure development. Furthermore, these data might add to the understanding of the influence of EBOV variant populations on pathogenesis and disease outcome and inform attempts to avoid the evolution of EBOV escape mutants in response to current therapeutics. Finally, as the primary challenge stocks have changed over time, these data will provide a baseline for understanding and correlating past and future animal study results

Study sample diagram.

<p>Study sample diagram.</p

Percentage of errors by type of acquired diversity determined during sample preparation.

<p>(A) Percentage of error attributed to synonymous vs. non-synonymous variants. (B) Percentage of errors attributed to transitions vs. transversions. (C) Percentage of errors attributed to insertions or deletions vs. Intra-host single nucleotide variants (iSNV).</p