Search

SUMMARYThe currently expanding monkeypox epidemic is caused by a subclade IIb descendant of a monkeypox virus (MPXV) lineage traced back to Nigeria in 1971. In contrast to monkeypox cases caused by clade I and subclade IIa MPXV, the prognosis of current cases is generally favorable, but person-to-person transmission is much more efficient. MPXV evolution is driven by selective pressure from hosts and loss of virus–host interacting genes. However, there is no satisfactory genetic explanation using single-nucleotide polymorphisms (SNPs) for the observed increased MPXV transmissibility. We hypothesized that key genomic changes may occur in the genome’s low-complexity regions (LCRs), which are highly challenging to sequence and have been dismissed as uninformative. Using a combination of highly sensitive techniques, we determined a first high-quality MPXV genome sequence of a representative of the current epidemic with LCRs resolved at unprecedented accuracy. This effort revealed significant variation in short-tandem repeats within LCRs. We demonstrate that LCR entropy in the MPXV genome is significantly higher than that of SNPs and that LCRs are not randomly distributed.In silicoanalyses indicate that expression, translation, stability, or function of MPXV orthologous poxvirus genes (OPGs) 153, 204, and 208 could be affected in a manner consistent with the established “genomic accordion” evolutionary strategies of orthopoxviruses. Consequently, we posit that genomic studies focusing on phenotypic MPXV clade-/subclade-/lineage-/strain differences should change their focus to the study of LCR variability instead of SNP variability.

From November 2018 through February 2019, person-to-person transmission of Andes virus (ANDV) hantavirus pulmonary syndrome occurred in Chubut Province, Argentina, and resulted in 34 confirmed infections and 11 deaths. Understanding the genomic, epidemiologic, and clinical characteristics of person-to-person transmission of ANDV is crucial to designing effective interventions.Clinical and epidemiologic information was obtained by means of patient report and from public health centers. Serologic testing, contact-tracing, and next-generation sequencing were used to identify ANDV infection as the cause of this outbreak of hantavirus pulmonary syndrome and to reconstruct person-to-person transmission events.After a single introduction of ANDV from a rodent reservoir into the human population, transmission was driven by 3 symptomatic persons who attended crowded social events. After 18 cases were confirmed, public health officials enforced isolation of persons with confirmed cases and self-quarantine of possible contacts; these measures most likely curtailed further spread. The median reproductive number (the number of secondary cases caused by an infected person during the infectious period) was 2.12 before the control measures were enforced and decreased to 0.96 after the measures were implemented. Full genome sequencing of the ANDV strain involved in this outbreak was performed with specimens from 27 patients and showed that the strain that was present (Epuyén/18-19) was similar to the causative strain (Epilink/96) in the first known person-to-person transmission of hantavirus pulmonary syndrome caused by ANDV, which occurred in El Bolsón, Argentina, in 1996. Clinical investigations involving patients with ANDV hantavirus pulmonary syndrome in this outbreak revealed that patients with a high viral load and liver injury were more likely than other patients to spread infection. Disease severity, genomic diversity, age, and time spent in the hospital had no clear association with secondary transmission.Among patients with ANDV hantavirus pulmonary syndrome, high viral titers in combination with attendance at massive social gatherings or extensive contact among persons were associated with a higher likelihood of transmission. (Funded by the Ministerio de Salud y Desarrollo Social de la Nación Argentina and others.).

Summary The PALM trial in the Democratic Republic of the Congo identified a statistically significant survival benefit for two monoclonal antibody-based therapeutics in the treatment of acute Ebola virus disease; however, substantial gaps remain in improving the outcomes of acute Ebola virus disease and for the survivors. Ongoing efforts are needed to develop more effective strategies, particularly for individuals with severe disease, for prevention and treatment of viral persistence in immune-privileged sites, for optimisation of post-exposure prophylaxis, and to increase therapeutic breadth. As antibody-based approaches are identified and advanced, promising small-molecule antivirals currently in clinical stage development should continue to be evaluated for filovirus diseases, with consideration of their added value in combination approaches with bundled supportive care, their penetration in tissues of interest, the absence of interaction with glycoprotein-based vaccines, and filoviral breadth.

Summary Background The 2018 Ebola virus disease (EVD) outbreak in Equateur Province, Democratic Republic of the Congo, began on May 8, and was declared over on July 24; it resulted in 54 documented cases and 33 deaths. We did a retrospective genomic characterisation of the outbreak and assessed potential therapeutic agents and vaccine (medical countermeasures). Methods We used target-enrichment sequencing to produce Ebola virus genomes from samples obtained in the 2018 Equateur Province outbreak. Combining these genomes with genomes associated with known outbreaks from GenBank, we constructed a maximum-likelihood phylogenetic tree. In-silico analyses were used to assess potential mismatches between the outbreak strain and the probes and primers of diagnostic assays and the antigenic sites of the experimental rVSVΔG-ZEBOV-GP vaccine and therapeutics. An in-vitro flow cytometry assay was used to assess the binding capability of the individual components of the monoclonal antibody cocktail ZMapp. Findings A targeted sequencing approach produced 16 near-complete genomes. Phylogenetic analysis of these genomes and 1011 genomes from GenBank revealed a distinct cluster, confirming a new Ebola virus variant, for which we propose the name “Tumba”. This new variant appears to have evolved at a slower rate than other Ebola virus variants (0·69 × 10−3 substitutions per site per year with “Tumba” vs 1·06 × 10−3 substitutions per site per year without “Tumba”). We found few sequence mismatches in the assessed assay target regions and antigenic sites. We identified nine amino acid changes in the Ebola virus surface glycoprotein, of which one resulted in reduced binding of the 13C6 antibody within the ZMapp cocktail. Interpretation Retrospectively, we show the feasibility of using genomics to rapidly characterise a new Ebola virus variant within the timeframe of an outbreak. Phylogenetic analysis provides further indications that these variants are evolving at differing rates. Rapid in-silico analyses can direct in-vitro experiments to quickly assess medical countermeasures. Funding Defense Biological Product Assurance Office .

Hantaan virus (HTNV) and Puumala virus (PUUV) are pathogenic hantaviruses found in Asia and Europe, respectively. DNA vaccines targeting the envelope glycoproteins of these viruses have been constructed and found to elicit neutralizing antibodies when delivered to humans by various technologies including intramuscular electroporation. Here, we report findings from a Phase 2a clinical trial of a combined HTNV/PUUV DNA vaccine delivered at varying doses and administration schedules using the Ichor Medical Systems TriGrid intramuscular electroporation delivery technology. The study was designed to characterize the effects of DNA vaccine dose and number of administrations on the frequency and magnitude of immunological response. Subjects (n = 120) were divided into four cohorts. Cohorts 1 and 2 received a dose of 2 mg of DNA (1 mg per plasmid), and cohorts 3 and 4 received a dose of 1 mg of DNA (0.5 mg per plasmid) each vaccination. Each of the four cohorts received a series of four administrations (days 0, 28, 56 and 168). For cohorts 1 and 3, the DNA vaccine candidate was delivered at each of the four administrations. For cohorts 2 and 4, in order to maintain blinding, subjects received the DNA vaccine on days 0, 56 and 168, but on day 28 received only the phosphate buffered saline vehicle rather the DNA vaccine. Sera were collected on days 0, 28, 56, 84, 140, 168, 196, 252 and 365 and evaluated for the presence of neutralizing antibodies by PUUV and HTNV pseudovirion neutralization assays (PsVNAs). Day 84 was also evaluated by a plaque reduction neutralization test (PRNT). Overall the PsVNA50 geometric mean titers (GMTs) and seropositivity rates among cohorts were similar. Cohort 3 exhibited the highest frequency of subjects that became seropositive to both PUUV and HTNV after vaccination, the highest peak GMT against both viruses, and the highest median titers against both viruses.

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., 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.

Ticks are vectors of a wide range of zoonotic viruses of medical and veterinary importance. Recently, metagenomics studies demonstrated that they are also the source of potentially pathogenic novel viruses. During the period from 2015 to 2017, questing ticks were collected by dragging the vegetation from geographically distant locations in the Republic of Korea (ROK) and a target-independent high-throughput sequencing method was utilized to study their virome. A total of seven viruses, including six putative novel viral entities, were identified. Genomic analysis showed that the novel viruses were most closely related to members in the orders Jingchuvirales and Bunyavirales. Phylogenetic reconstruction showed that the Bunyavirales-like viruses grouped in the same clade with other viruses within the Nairovirus and Phlebovirus genera, while the novel Jingchuvirales-like virus grouped together with other viruses within the family Chuviridae. Real-time RT-PCR was used to determine the geographic distribution and prevalence of these viruses in adult ticks. These novel viruses have a wide geographic distribution in the ROK with prevalences ranging from 2% to 18%. Our study expands the knowledge about the composition of the tick virome and highlights the wide diversity of viruses they harbor in the ROK. The discovery of novel viruses associated with ticks in the ROK highlights the need for an active tick-borne disease surveillance program to identify possible reservoirs of putative novel human pathogens.

Biosafety, biosecurity, logistical, political, and technical considerations can delay or prevent the wide dissemination of source material containing viable virus from the geographic origin of an outbreak to laboratories involved in developing medical countermeasures (MCMs). However, once virus genome sequence information is available from clinical samples, reverse-genetics systems can be used to generate virus stocks de novo to initiate MCM development. In this study, we developed a reverse-genetics system for natural isolates of Ebola virus (EBOV) variants Makona, Tumba, and Ituri, which have been challenging to obtain. These systems were generated starting solely with in silico genome sequence information and have been used successfully to produce recombinant stocks of each of the viruses for use in MCM testing. The antiviral activity of MCMs targeting viral entry varied depending on the recombinant virus isolate used. Collectively, selecting and synthetically engineering emerging EBOV variants and demonstrating their efficacy against available MCMs will be crucial for answering pressing public health and biosecurity concerns during Ebola disease (EBOD) outbreaks.

Summary Bundibugyo virus (BDBV) is one of four ebolaviruses known to cause disease in humans. Bundibugyo virus disease (BVD) outbreaks occurred in 2007–2008 in Bundibugyo District, Uganda, and in 2012 in Isiro, Province Orientale, Democratic Republic of the Congo. The 2012 BVD outbreak resulted in 38 laboratory-confirmed cases of human infection, 13 of whom died. However, only 4 BDBV specimens from the 2012 outbreak have been sequenced. Here, we provide BDBV sequences from seven additional patients. Analysis of the molecular epidemiology and evolutionary dynamics of the 2012 outbreak with these additional isolates challenges the current hypothesis that the outbreak was the result of a single spillover event. In addition, one patient record indicates that BDBV’s initial emergence in Isiro occurred 50 days earlier than previously accepted. Collectively, this work demonstrates how retrospective sequencing can be used to elucidate outbreak origins and provide epidemiological contexts to a medically relevant pathogen., Graphical abstract, Highlights In 2012, BDBV was circulating earlier than previously appreciated BDBV genomes from seven additional patients are sequenced Molecular analyses indicate that multiple spillover events fueled the BVD outbreak, Elucidation of the epidemiology underlying the 2012 Bundibugyo virus disease outbreak in the Democratic Republic of the Congo has been challenging. Hulseberg et al. acquire additional genomic and phylodynamic data indicating that multiple Bundibugyo virus spillover events, some of which occurred much earlier than previously known, contributed to the outbreak.

Summary: Bats asymptomatically harbor many viruses that can cause severe human diseases. The Egyptian rousette bat (ERB) is the only known reservoir for Marburgviruses and Sosuga virus, making it an exceptional animal model to study antiviral mechanisms in an asymptomatic host. With this goal in mind, we constructed and annotated the immunoglobulin heavy chain locus, finding an expansion on immunoglobulin variable genes associated with protective human antibodies to different viruses. We also annotated two functional and distinct immunoglobulin epsilon genes and four distinctive functional immunoglobulin gamma genes. We described the Fc receptor repertoire in ERBs, including features that may affect activation potential, and discovered the lack of evolutionary conserved short pentraxins. These findings reinforce the hypothesis that a differential threshold of regulation and/or absence of key immune mediators may promote tolerance and decrease inflammation in ERBs.

We performed epitope mapping studies on the major surface glycoprotein (GP) of Ebola virus (EBOV) using Chemically Linked Peptides on Scaffolds (CLIPS), which form linear and potential conformational epitopes. This method identified monoclonal antibody epitopes and predicted additional epitopes recognized by antibodies in polyclonal sera from animals experimentally vaccinated against or infected with EBOV. Using the information obtained along with structural modeling to predict epitope accessibility, we then constructed 2 DNA vaccines encoding immunodominant and subdominant epitopes predicted to be accessible on EBOV GP. Although a construct designed to produce a membrane-bound oligopeptide was poorly immunogenic, a construct generating a secreted oligopeptide elicited strong antibody responses in mice. When this construct was administered as a boost to a DNA vaccine expressing the complete EBOV GP gene, the resultant antibody response was focused largely toward the less immunodominant epitopes in the oligopeptide. Taken together, the results of this work suggest a utility for this method for immune focusing of antibody responses elicited by vaccination.

Summary Background The real-time generation of information about pathogen genomes has become a vital goal for transmission analysis and characterisation in rapid outbreak responses. In response to the recently established genomic capacity in the Democratic Republic of the Congo, we explored the real-time generation of genomic information at the start of the 2018 Ebola virus disease (EVD) outbreak in North Kivu Province. Methods We used targeted-enrichment sequencing to produce two coding-complete Ebola virus genomes 5 days after declaration of the EVD outbreak in North Kivu. Subsequent sequencing efforts yielded an additional 46 genomes. Genomic information was used to assess early transmission, medical countermeasures, and evolution of Ebola virus. Findings The genomic information demonstrated that the EVD outbreak in the North Kivu and Ituri Provinces was distinct from the 2018 EVD outbreak in Equateur Province of the Democratic Republic of the Congo. Primer and probe mismatches to Ebola virus were identified in silico for all deployed diagnostic PCR assays, with the exception of the Cepheid GeneXpert GP assay. Interpretation The first two coding-complete genomes provided actionable information in real-time for the deployment of the rVSVΔG-ZEBOV-GP Ebola virus envelope glycoprotein vaccine, available therapeutics, and sequence-based diagnostic assays. Based on the mutations identified in the Ebola virus surface glycoprotein (GP12) observed in all 48 genomes, deployed monoclonal antibody therapeutics (mAb114 and ZMapp) should be efficacious against the circulating Ebola virus variant. Rapid Ebola virus genomic characterisation should be included in routine EVD outbreak response procedures to ascertain efficacy of medical countermeasures. Funding Defense Biological Product Assurance Office.

Nucleoside analogues (NA) disrupt RNA viral RNA-dependent RNA polymerase (RdRP) function and fidelity for multiple viral families. The mechanism of action (MOA) of T-705 has been attributed alternatively or concurrently to chain termination and lethal mutagenesis depending on the viral species during in vitro studies. In this study, we evaluated the effect of T-705 on the viral population in non-human primates (NHPs) after challenge with Ebola virus (EBOV) or Marburg virus (MARV) to identify the predominant in vivo MOA. We used common virological assays in conjunction with deep sequencing to characterize T-705 effects. T-705 exhibited antiviral activity that was associated with a reduction in specific infectivity and an accumulation of low frequency nucleotide variants in plasma samples collected day 7 post infection. Stranded analysis of deep sequencing data to identify chain termination demonstrated no change in the transcriptional gradient in negative stranded viral reads and minimal changes in positive stranded viral reads in T-705 treated animals, questioning as a MOA in vivo. These findings indicate that lethal mutagenesis is a MOA of T-705 that may serve as an indication of therapeutic activity of NAs for evaluation in clinical settings. This study expands our understanding of MOAs of these compounds for the Filovirus family and provides further evidence that lethal mutagenesis could be a preponderant MOA for this class of therapeutic compounds.

Although Lloviu virus (LLOV) was discovered in the carcasses of insectivorous Schreiber&rsquo, s Bent-winged bats in the caves of Northern Spain in 2002, its infectivity and pathogenicity remain unclear. We examined the seroprevalence of LLOV in potentially exposed Schreiber&rsquo, s Bent-winged bats (n = 60), common serotine bats (n = 10) as controls, and humans (n = 22) using an immunoblot assay. We found antibodies against LLOV GP2 in all of Schreiber&rsquo, s Bent-winged bats serum pools, but not in any of the common serotine bats and human pools tested. To confirm this seroreactivity, 52 serums were individually tested using Domain Programmable Arrays (DPA), a phage display based-system serology technique for profiling filovirus epitopes. A serological signature against different LLOV proteins was obtained in 19/52 samples tested (36.5%). The immunodominant response was in the majority specific to LLOV-unique epitopes, confirming that the serological response detected was to LLOV. To our knowledge, this is the first serological evidence of LLOV exposure in live captured Schreiber&rsquo, s Bent-winged bats, dissociating LLOV circulation as the cause of the previously reported die-offs.

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.

A suspected case of sexual transmission from a male survivor of Ebola virus disease (EVD) to his female partner (the patient in this report) occurred in Liberia in March 2015. Ebola virus (EBOV) genomes assembled from blood samples from the patient and a semen sample from the survivor were consistent with direct transmission. The genomes shared three substitutions that were absent from all other Western African EBOV sequences and that were distinct from the last documented transmission chain in Liberia before this case. Combined with epidemiologic data, the genomic analysis provides evidence of sexual transmission of EBOV and evidence of the persistence of infective EBOV in semen for 179 days or more after the onset of EVD. (Funded by the Defense Threat Reduction Agency and others.).

The effects of EBOV evolution on diagnostic assays and therapeutic drugs appear to be low., To support Liberia’s response to the ongoing Ebola virus (EBOV) disease epidemic in Western Africa, we established in-country advanced genomic capabilities to monitor EBOV evolution. Twenty-five EBOV genomes were sequenced at the Liberian Institute for Biomedical Research, which provided an in-depth view of EBOV diversity in Liberia during September 2014–February 2015. These sequences were consistent with a single virus introduction to Liberia; however, shared ancestry with isolates from Mali indicated at least 1 additional instance of movement into or out of Liberia. The pace of change is generally consistent with previous estimates of mutation rate. We observed 23 nonsynonymous mutations and 1 nonsense mutation. Six of these changes are within known binding sites for sequence-based EBOV medical countermeasures; however, the diagnostic and therapeutic impact of EBOV evolution within Liberia appears to be low.

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.

The 2013-2016 West African epidemic caused by the Ebola virus was of unprecedented magnitude, duration and impact. Here we reconstruct the dispersal, proliferation and decline of Ebola virus throughout the region by analysing 1,610 Ebola virus genomes, which represent over 5% of the known cases. We test the association of geography, climate and demography with viral movement among administrative regions, inferring a classic 'gravity' model, with intense dispersal between larger and closer populations. Despite attenuation of international dispersal after border closures, cross-border transmission had already sown the seeds for an international epidemic, rendering these measures ineffective at curbing the epidemic. We address why the epidemic did not spread into neighbouring countries, showing that these countries were susceptible to substantial outbreaks but at lower risk of introductions. Finally, we reveal that this large epidemic was a heterogeneous and spatially dissociated collection of transmission clusters of varying size, duration and connectivity. These insights will help to inform interventions in future epidemics. ispartof: Nature vol:544 issue:7650 pages:309- ispartof: location:England status: published

Zika virus (ZIKV) is currently causing an unprecedented pandemic linked to severe congenital syndromes1,2. In July 2016, mosquito-borne ZIKV transmission was first reported in the continental United States and since then, hundreds of locally acquired infections have been described3. To gain insights into the timing, source, and likely route(s) of introduction into the United States, we tracked the virus from its first detection in Miami, Florida by direct sequencing of ZIKV genomes from infected patients and Aedes aegypti mosquitoes. We detected at least four distinct ZIKV introductions and estimate that 11-52 introductions contributed to the outbreak in Florida. Furthermore, our data suggests that ZIKV transmission likely started in the spring of 2016 - several months before initial detection. By analyzing epidemiological, surveillance, and genetic data, we discovered that several spatially distinct ZIKV transmission zones were likely portions of the same outbreak, rather than isolated events. Our analyses show that most introductions are linked to the Caribbean, which is supported by the high incidence rates and traffic, especially via cruises, from the region into Miami. By comparing mosquito abundance and travel capacity across the United States, we find that southern Florida is especially vulnerable to ZIKV introductions and at risk of repeat occurrences. By tracking the virus from its initial introduction into the United States, we provide a deeper understanding of how ZIKV initiates and sustains transmission in new regions.

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.

We generated genome sequences from 218 cases of Ebola virus disease (EVD) in Sierra Leone (SLE) during 2014&ndash, 2015 to complement available datasets, particularly by including cases from a period of low sequence coverage during peak transmission of Ebola virus (EBOV) in the highly-affected Western Area division of SLE. The combined dataset was utilized to produce phylogenetic and phylodynamic inferences, to study sink&ndash, source dynamics and virus dispersal from highly-populated transmission hotspots. We identified four districts in SLE where EBOV was introduced and transmission occurred without onward exportation to other districts. We also identified six districts that substantially contributed to the dispersal of the virus and prolonged the EVD outbreak: five of these served as major hubs, with lots of movement in and out, and one acted primarily as a source, exporting the virus to other areas of the country. Positive correlations between case numbers, inter-district transition events, and district population sizes reaffirm that population size was a driver of EBOV transmission dynamics in SLE. The data presented here confirm the role of urban hubs in virus dispersal and of a delayed laboratory response in the expansion and perpetuation of the EVD outbreak in SLE.

SummaryThe 2013-2016 epidemic of Ebola virus disease in West Africa was of unprecedented magnitude, duration and impact. Extensive collaborative sequencing projects have produced a large collection of over 1600 Ebola virus genomes, representing over 5% of known cases, unmatched for any single human epidemic. In this comprehensive analysis of this entire dataset, we reconstruct in detail the history of migration, proliferation and decline of Ebola virus throughout the region. We test the association of geography, climate, administrative boundaries, demography and culture with viral movement among 56 administrative regions. Our results show that during the outbreak viral lineages moved according to a classic ‘gravity’ model, with more intense migration between larger and more proximate population centers. Notably, we find that despite a strong attenuation of international dispersal after border closures, localized cross-border transmission beforehand had already set the seeds for an international epidemic, rendering these measures relatively ineffective in curbing the epidemic. We use this empirical evidence to address why the epidemic did not spread into neighboring countries, showing that although these regions were susceptible to developing significant outbreaks, they were also at lower risk of viral introductions. Finally, viral genome sequence data uniquely reveals this large epidemic to be a heterogeneous and spatially dissociated collection of transmission clusters of varying size, duration and connectivity. These insights will help inform approaches to intervention in such epidemics in the future.

Surveillance of Ebola virus disease flare-ups uncovers a reduced rate of Ebola virus evolution during persistent infections., On 29 June 2015, Liberia’s respite from Ebola virus disease (EVD) was interrupted for the second time by a renewed outbreak (“flare-up”) of seven confirmed cases. We demonstrate that, similar to the March 2015 flare-up associated with sexual transmission, this new flare-up was a reemergence of a Liberian transmission chain originating from a persistently infected source rather than a reintroduction from a reservoir or a neighboring country with active transmission. Although distinct, Ebola virus (EBOV) genomes from both flare-ups exhibit significantly low genetic divergence, indicating a reduced rate of EBOV evolution during persistent infection. Using this rate of change as a signature, we identified two additional EVD clusters that possibly arose from persistently infected sources. These findings highlight the risk of EVD flare-ups even after an outbreak is declared over.

Cynomolgus macaques (Macaca fascicularis) have become an important animal model for biomedical research. In particular, it is the animal model of choice for the development of vaccine candidates associated with emerging dangerous pathogens. Despite their increasing importance as animal models, the cynomolgus macaque genome is not fully characterized, hindering molecular studies for this model. More importantly, the lack of knowledge about the immunoglobulin (IG) locus organization directly impacts the analysis of the humoral response in cynomolgus macaques. Recent advances in next generation sequencing (NGS) technologies to analyze IG repertoires open the opportunity to deeply characterize the humoral immune response. However, the IG locus organization for the animal is required to completely dissect IG repertoires. Here, we describe the localization and organization of the rearranging IG heavy (IGH) genes on chromosome 7 of the cynomolgus macaque draft genome. Our annotation comprises 108 functional genes which include 63 variable (IGHV), 38 diversity (IGHD), and 7 joining (IGHJ) genes. For validation, we provide RNA transcript data for most of the IGHV genes and all of the annotated IGHJ genes, as well as proteomic data to validate IGH constant genes. The description and annotation of the rearranging IGH genes for the cynomolgus macaques will significantly facilitate scientific research. This is particularly relevant to dissect the immune response during vaccination or infection with dangerous pathogens such as Ebola, Marburg and other emerging pathogens where non-human primate models play a significant role for countermeasure development.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2124-x) contains supplementary material, which is available to authorized users.

Efficient T cell activation depends on the engagement of both TCR and CD28, although the molecular mechanisms that control this signal integration are not fully understood. Using fluorescence resonance energy transfer, we show that T cell activation can drive a reorientation of the cytosolic tails of the CD28 dimer. However, this is not mediated through CD28 ligand binding. Rather, TCR signaling itself mediates this conformation change in CD28. We also show that TCR signaling can induce CD28–ligand interactions. Although the CD28 dimer appears to bind ligand monovalently in solution, we show that both ligand binding sites are required to efficiently recruit CD28 to the immunological synapse. These results suggest, that analogous to the cross-talk from TCR that regulates integrin activation, TCR-initiated inside-out signaling may induce a conformational change to the extracellular domains of CD28, enabling ligand binding and initiating CD28 signaling.

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.

Single-molecule real-time (SMRT) sequencing technology with the Pacific Biosciences (PacBio) RS II platform offers the potential to obtain full-length coding regions (∼1100-bp) from MHC class I cDNAs. Despite the relatively high error rate associated with SMRT technology, high quality sequences can be obtained by circular consensus sequencing (CCS) due to the random nature of the error profile. In the present study we first validated the ability of SMRT-CCS to accurately identify class I transcripts in Mauritian-origin cynomolgus macaques (Macaca fascicularis) that have been characterized previously by cloning and Sanger-based sequencing as well as pyrosequencing approaches. We then applied this SMRT-CCS method to characterize 60 novel full-length class I transcript sequences expressed by a cohort of cynomolgus macaques from China. The SMRT-CCS method described here provides a straightforward protocol for characterization of unfragmented single-molecule cDNA transcripts that will potentially revolutionize MHC class I allele discovery in nonhuman primates and other species.

During T cell activation by APC, CD28 is colocalized with TCR in the central supramolecular activation cluster (cSMAC) region of the immunological synapse. CD28 signaling through PI3K results in the recruitment of protein kinase C (PKC)θ to the cSMAC, activation of NF-κB, and induction of IL-2 transcription. These results suggest that localized engagement of CD28 within the cSMAC may be required for CD28 activation and/or signal integration with TCR signals. To test this model we have examined the mechanism of CD28-mediated induction of IL-2 secretion when CD28 is engaged outside of the immunological synapse. CD4 T cells were stimulated with Ag presented by B7-negative APC and CD28 costimulation was provided in trans by anti-CD28-coated beads or by class II-negative, B7-positive cells. We show that induction of IL-2 secretion under these conditions did not require expression of PKCθ and did not induce NF-κB activation or IL-2 transcription. In contrast, CD28 costimulation in trans did induce IL-2 mRNA stability, accounting for the up-regulation of IL-2 secretion. These data indicate that the ability of CD28 to up-regulate IL-2 transcription requires colocalization of TCR and CD28 at the plasma membrane, possibly within the cSMAC of the immunological synapse. In contrast, the ability of CD28 to promote IL-2 mRNA stability can be transduced from a distal site from the TCR, suggesting that signal integration occurs downstream from the plasma membrane. These data support the potential role of trans costimulation in tumor and allograft rejection, but limit the potential functional impact that trans costimulation may have on T cell activation.