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Adintrevimab is a human immunoglobulin G1 monoclonal antibody engineered to have broad neutralization against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants and other SARS-like coronaviruses with pandemic potential. In both Syrian golden hamster and rhesus macaque models, prophylactic administration of a single dose of adintrevimab provided protection against SARS-CoV-2/WA1/2020 infection in a dose-dependent manner, as measured by significant reductions in lung viral load and virus-induced lung pathology, and by inhibition of viral replication in the upper and lower respiratory tract.

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.

Airborne transmission is predicted to be a prevalent route of human exposure with SARS-CoV-2. Aside from African green monkeys, nonhuman primate models that replicate airborne transmission of SARS-CoV-2 have not been investigated. A comparative evaluation of COVID-19 in African green monkeys, rhesus macaques, and cynomolgus macaques following airborne exposure to SARS-CoV-2 was performed to determine critical disease parameters associated with disease progression, and establish correlations between primate and human COVID-19. Respiratory abnormalities and viral shedding were noted for all animals, indicating successful infection. Cynomolgus macaques developed fever, and thrombocytopenia was measured for African green monkeys and rhesus macaques. Type II pneumocyte hyperplasia and alveolar fibrosis were more frequently observed in lung tissue from cynomolgus macaques and African green monkeys. The data indicate that, in addition to African green monkeys, macaques can be successfully infected by airborne SARS-CoV-2, providing viable macaque natural transmission models for medical countermeasure evaluation.

Epidemics of emerging and re-emerging infectious diseases are a danger to civilian and military populations worldwide. Health security and mitigation of infectious disease threats is a priority of the United States Government and the Department of Defense (DoD). Next generation sequencing (NGS) and Bioinformatics (BI) enhances traditional biosurveillance by providing additional data to understand transmission, identify resistance and virulence factors, make predictions, and update risk assessments. As more and more laboratories adopt NGS and BI technologies they encounter challenges in building local capacity. In addition to choosing the right sequencing platform and approach, considerations must also be made for the complexity of bioinformatics analyses, data storage, as well as personnel and computational requirements. To address these needs, a comprehensive training program was developed covering wet lab and bioinformatics approaches to NGS. The program is meant to be modular and adaptive to meet both common and individualized needs of medical research and public health laboratories across the DoD. The training program was first deployed internationally to the Basic Science Laboratory of the US Army Medical Research Directorate-Africa in Kisumu, Kenya, which is an overseas Lab of the Walter Reed Army Institute of Research (WRAIR). A week-long workshop with intensive focus on targeted sequencing and the bioinformatics of genome assembly (n = 24 participants) was held. Post-workshop self-assessment (completed by 21 participants) noted significant median gains in knowledge domains related to NGS targeted sequencing, bioinformatics for genome assembly, and sequence quality assessment. The participants also reported that the information on study design, sample preparation, sequencing quality control, data quality assessment, reporting, and basic and advanced bioinformatics analysis were the most useful information presented in the training. While longer-term evaluations are planned, the training resulted in significant short-term improvement of a laboratory’s self-reported wet lab and bioinformatics capabilities. This framework can be used for future DoD laboratory development in the area of NGS and BI for infectious disease surveillance, ultimately enhancing this global DoD capability.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing an exponentially increasing number of coronavirus disease 19 (COVID-19) cases globally. Prioritization of medical countermeasures for evaluation in randomized clinical trials is critically hindered by the lack of COVID-19 animal models that enable accurate, quantifiable, and reproducible measurement of COVID-19 pulmonary disease free from observer bias. We first used serial computed tomography (CT) to demonstrate that bilateral intrabronchial instillation of SARS-CoV-2 into crab-eating macaques (Macaca fascicularis) results in mild-to-moderate lung abnormalities qualitatively characteristic of subclinical or mild-to-moderate COVID-19 (e.g., ground-glass opacities with or without reticulation, paving, or alveolar consolidation, peri-bronchial thickening, linear opacities) at typical locations (peripheral>central, posterior and dependent, bilateral, multi-lobar). We then used positron emission tomography (PET) analysis to demonstrate increased FDG uptake in the CT-defined lung abnormalities and regional lymph nodes. PET/CT imaging findings appeared in all macaques as early as 2 days post-exposure, variably progressed, and subsequently resolved by 6–12 days post-exposure. Finally, we applied operator-independent, semi-automatic quantification of the volume and radiodensity of CT abnormalities as a possible primary endpoint for immediate and objective efficacy testing of candidate medical countermeasures.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing an exponentially increasing number of coronavirus disease 19 (COVID-19) cases globally. Prioritization of medical countermeasures for evaluation in randomized clinical trials is critically hindered by the lack of COVID-19 animal models that enable accurate, quantifiable, and reproducible measurement of COVID-19 pulmonary disease free from observer bias. We first used serial computed tomography (CT) to demonstrate that bilateral intrabronchial instillation of SARS-CoV-2 into crab-eating macaques (Macaca fascicularis) results in mild-to-moderate lung abnormalities qualitatively characteristic of subclinical or mild-to-moderate COVID-19 (e.g., ground-glass opacities with or without reticulation, paving, or alveolar consolidation, peri-bronchial thickening, linear opacities) at typical locations (peripheral>central, posterior and dependent, bilateral, multi-lobar). We then used positron emission tomography (PET) analysis to demonstrate increased FDG uptake in the CT-defined lung abnormalities and regional lymph nodes. PET/CT imaging findings appeared in all macaques as early as 2 days post-exposure, variably progressed, and subsequently resolved by 6–12 days post-exposure. Finally, we applied operator-independent, semi-automatic quantification of the volume and radiodensity of CT abnormalities as a possible primary endpoint for immediate and objective efficacy testing of candidate medical countermeasures.

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.

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.

The lens epithelium-derived growth factor p75 (LEDGF/p75) is a chromatin-bound protein essential for efficient lentiviral integration. Genome-wide studies have located LEDGF/p75 inside actively transcribed genes where it mediates lentiviral integration. Although its role in HIV-1 integration is clearly established, the role of LEDGF/p75-associated proteins in HIV-1 infection remains unexplored. Using protein-protein interaction assays, we demonstrated that LEDGF/p75 complexes with a chromatin-remodeling complex facilitates chromatin transcription (FACT), a heterodimer of the structure-specific recognition protein 1 (SSRP1) and the human homolog of suppressor of Ty 16 (hSpt16). Detailed analysis of the interaction of LEDGF/p75 with the FACT complex indicates that LEDGF/p75 interacts with SSRP1 in an hSpt16-independent manner that requires the PWWP domain of LEDGF proteins and the HMG domain of SSRP1. Functional characterizations demonstrate a LEDGF/p75-independent role of SSRP1 in the regulation of HIV-1 replication. shRNA-mediated partial knockdown of SSRP1 reduces HIV-1 infection, but not Murine Leukemia Virus, in human CD4(+) T cells. Similarly, SSRP1 knockdown affects infection by HIV-1-derived viruses that express genes from the viral LTR but not from an internal immediate-early CMV promoter, suggesting a role of SSRP1 in LTR-driven gene expression but not in viral DNA integration. Together, our data demonstrate for the first time the association of LEDGF proteins with the FACT complex and give further support to a role of SSRP1 in HIV-1 infection.

The most recent Ebola virus outbreak in West Africa, which was unprecedented in the number of cases and fatalities, geographic distribution, and number of nations affected, highlights the need for safe, effective, and readily available antiviral agents for treatment and prevention of acute Ebola virus (EBOV) disease (EVD) or sequelae. No antiviral therapeutics have yet received regulatory approval or demonstrated clinical efficacy. Here we report the discovery of a novel small molecule GS-5734, a monophosphoramidate prodrug of an adenosine analogue, with antiviral activity against EBOV. GS-5734 exhibits antiviral activity against multiple variants of EBOV and other filoviruses in cell-based assays. The pharmacologically active nucleoside triphosphate (NTP) is efficiently formed in multiple human cell types incubated with GS-5734 in vitro, and the NTP acts as an alternative substrate and RNA-chain terminator in primer-extension assays using a surrogate respiratory syncytial virus RNA polymerase. Intravenous administration of GS-5734 to nonhuman primates resulted in persistent NTP levels in peripheral blood mononuclear cells (half-life, 14 h) and distribution to sanctuary sites for viral replication including testes, eyes, and brain. In a rhesus monkey model of EVD, once-daily intravenous administration of 10 mg kg(-1) GS-5734 for 12 days resulted in profound suppression of EBOV replication and protected 100% of EBOV-infected animals against lethal disease, ameliorating clinical disease signs and pathophysiological markers, even when treatments were initiated three days after virus exposure when systemic viral RNA was detected in two out of six treated animals. These results show the first substantive post-exposure protection by a small-molecule antiviral compound against EBOV in nonhuman primates. The broad-spectrum antiviral activity of GS-5734 in vitro against other pathogenic RNA viruses, including filoviruses, arenaviruses, and coronaviruses, suggests the potential for wider medical use. GS-5734 is amenable to large-scale manufacturing, and clinical studies investigating the drug safety and pharmacokinetics are ongoing.

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.

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

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.

Dengue virus is endemic globally, throughout tropical and sub-tropical regions. While the number of epidemics due to the four DENV serotypes is pronounced in East Africa, the total number of cases reported in Africa (16 million infections) remained at low levels compared to Asia (70 million infections). The French Armed forces Health Service provides epidemiological surveillance support in the Republic of Djibouti through the Bouffard Military hospital. Between 2011 and 2014, clinical and biological data of suspected dengue syndromes were collected at the Bouffard Military hospital and analyzed to improve Dengue clinical diagnosis and evaluate its circulation in East Africa. Examining samples from patients that presented one or more Dengue-like symptoms the study evidenced 128 Dengue cases among 354 suspected cases (36.2% of the non-malarial Dengue-like syndromes). It also demonstrated the circulation of serotypes 1 and 2 and reports the first epidemic of serotype 3 infections in Djibouti which was found in all of the hospitalized patients in this study. Based on these results we have determined that screening for Malaria and the presence of the arthralgia, gastro-intestinal symptoms and lymphopenia < 1,000cell/ mm3 allows for negative predictive value and specificity of diagnosis in isolated areas superior to 80% up to day 6. This study also provides evidence for an epidemic of Dengue virus serotype 3 previously not detected in Djibouti., Author Summary Dengue virus is emerging worldwide, however, little is known about the burden of Dengue in Africa. Effectively, only sporadic cases and few epidemics have been reported in the last 30 years. This descriptive study reports clinical and biological data of Dengue-suspected cases analyzed in the Bouffard military hospital of Djibouti from 2011–2014. It also confirms the circulation of Dengue virus serotypes 1 and 2 and reports the first epidemic of Dengue virus serotype 3 infections in Djibouti. Directions for diagnosis are offered to practitioners working in resource limited settings and dealing with a Dengue-like syndrome of less than seven days and negative for malaria.

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.

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.

Lens epithelium-derived growth factor (LEDGF)/p75 is a cellular cofactor for HIV-1 DNA integration. It is well established that the simultaneous binding of LEDGF/p75 to chromatin and to HIV-1 integrase is required for its cofactor activity. However, the exact molecular mechanism of LEDGF/p75 in HIV-1 integration is not yet completely understood. Our hypothesis is that evolutionarily conserved regions in LEDGF/p75 exposed to solvent and harboring posttranslational modifications may be involved in its HIV-1 cofactor activity. Therefore, a panel of LEDGF/p75 deletion mutants targeting these protein regions were evaluated for their HIV-1 cofactor activity, chromatin binding, integrase interaction, and integrase-to-chromatin-tethering activity by using different cellular and biochemical approaches. The deletion of amino acids 267 to 281 reduced the cofactor activity of LEDGF/p75 to levels observed for chromatin-binding-defective mutants. This region contains a serine cluster (residues 271, 273, and 275) recurrently found to be phosphorylated in both human and mouse cells. Importantly, the conversion of these Ser residues to Ala was sufficient to impair the ability of LEDGF/p75 to mediate HIV-1 DNA integration, although these mutations did not alter chromatin binding, integrase binding, or the integrase-to-chromatin-tethering capability of LEDGF/p75. These results clearly indicated that serine residues 271, 273, and 275 influence the HIV-1 cofactor activity of integrase-to-chromatin-tethering-competent LEDGF/p75.

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.

We obtained the complete coding genome of an eastern equine encephalitis virus (EEEV) strain, EEEV V105-00210, and the complete genome of a Venezuelan equine encephalitis virus (VEEV) strain, VEEV INH-9813. They were obtained from human cases and are proposed as reference challenge strains for vaccine and therapeutic development in animal models.

Ebola virus (EBOV) was discovered in 1976 around Yambuku, Zaire. A lack of nomenclature standards resulted in a variety of designations for each isolate, leading to confusion in the literature and databases. We sequenced the genome of isolate E718/ME/Ecran and unified the various designations under Ebola virus/H.sapiens-tc/COD/1976/Yambuku-Ecran.

International audience; In 2014, Ebola virus (EBOV) was identified as the etiological agent of a large and still expanding outbreak of Ebola virus disease (EVD) in West Africa and a much more confined EVD outbreak in Middle Africa. Epidemiological and evolutionary analyses confirmed that all cases of both outbreaks are connected to a single introduction each of EBOV into human populations and that both outbreaks are not directly connected. Coding-complete genomic sequence analyses of isolates revealed that the two outbreaks were caused by two novel EBOV variants, and initial clinical observations suggest that neither of them should be considered strains. Here we present consensus decisions on naming for both variants (West Africa: "Makona", Middle Africa: "Lomela") and provide database-compatible full, shortened, and abbreviated names that are in line with recently established filovirus sub-species nomenclatures.

Thanks to high-throughput sequencing technologies, genome sequencing has become a common component in nearly all aspects of viral research; thus, we are experiencing an explosion in both the number of available genome sequences and the number of institutions producing such data. However, there are currently no common standards used to convey the quality, and therefore utility, of these various genome sequences. Here, we propose five “standard” categories that encompass all stages of viral genome finishing, and we define them using simple criteria that are agnostic to the technology used for sequencing. We also provide genome finishing recommendations for various downstream applications, keeping in mind the cost-benefit trade-offs associated with different levels of finishing. Our goal is to define a common vocabulary that will allow comparison of genome quality across different research groups, sequencing platforms, and assembly techniques.

Background Non-human primates (NHPs) and humans share major biological mechanisms, functions, and responses due to their close evolutionary relationship and, as such, provide ideal animal models to study human diseases. RNA expression in NHPs provides specific signatures that are informative of disease mechanisms and therapeutic modes of action. Unlike the human transcriptome, the transcriptomes of major NHP animal models are yet to be comprehensively annotated. Results In this manuscript, employing deep RNA sequencing of seven tissue samples, we characterize the transcriptomes of two commonly used NHP animal models: Cynomolgus macaque (Macaca fascicularis) and African green monkey (Chlorocebus aethiops). We present the Multi-Species Annotation (MSA) pipeline that leverages well-annotated primate species and annotates 99.8% of reconstructed transcripts. We elucidate tissue-specific expression profiles and report 13 experimentally validated novel transcripts in these NHP animal models. Conclusion We report comprehensively annotated transcriptomes of two non-human primates, which we have made publically available on a customized UCSC Genome Browser interface. The MSA pipeline is also freely available. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-846) contains supplementary material, which is available to authorized users.

Bacterial proteins destined for the Tat pathway are folded before crossing the inner membrane and are typically identified by an N-terminal signal peptide containing a twin arginine motif. Translocation by the Tat pathway is dependent on the products of genes which encode proteins possessing the binding site of the signal peptide and mediating the actual translocation event. In the fully virulent CO92 strain of Yersinia pestis, the tatA gene was deleted. The mutant was assayed for loss of virulence through various in vitro and in vivo assays. Deletion of the tatA gene resulted in several consequences for the mutant as compared to wild-type. Cell morphology of the mutant bacteria was altered and demonstrated a more elongated form. In addition, while cultures of the mutant strain were able to produce a biofilm, we observed a loss of adhesion of the mutant biofilm structure compared to the biofilm produced by the wild-type strain. Immuno-electron microscopy revealed a partial disruption of the F1 antigen on the surface of the mutant. The virulence of the ΔtatA mutant was assessed in various murine models of plague. The mutant was severely attenuated in the bubonic model with full virulence restored by complementation with the native gene. After small-particle aerosol challenge in a pneumonic model of infection, the mutant was also shown to be attenuated. In contrast, when mice were challenged intranasally with the mutant, very little difference in the LD50 was observed between wild-type and mutant strains. However, an increased time-to-death and delay in bacterial dissemination was observed in mice infected with the ΔtatA mutant as compared to the parent strain. Collectively, these findings demonstrate an essential role for the Tat pathway in the virulence of Y. pestis in bubonic and small-aerosol pneumonic infection but less important role for intranasal challenge.

To identify polymorphic sites that could be used as biomarkers of Ebola virus passage history, we repeatedly amplified Ebola virus (Kikwit variant) in vitro and in vivo and performed deep sequencing analysis of the complete genomes of the viral subpopulations. We then determined the sites undergoing selection during passage in Vero E6 cells. Four locations within the Ebola virus Kikwit genome were identified that together segregate cell culture-passaged virus and virus obtained from infected non-human primates. Three of the identified sites are located within the glycoprotein gene (GP) sequence: the poly-U (RNA editing) site at position 6925, as well as positions 6677, and 6179. One site was found in the VP24 gene at position 10833. In all cases, in vitro and in vivo, both populations (majority and minority variants) were maintained in the viral swarm, with rapid selections occurring after a few passages or infections. This analysis approach will be useful to differentiate whether filovirus stocks with unknown history have been passaged in cell culture and may support filovirus stock standardization for medical countermeasure development.

Lens epithelium-derived growth factor (LEDGF) proteins p75 and p52 are transcriptional coactivators that connect sequence-specific activators to the basal transcription machinery. We have found that these proteins are posttranslationally modified by SUMO (small ubiquitin-like modifier)-1 and SUMO-3. Three SUMOylation sites, K75, K250, and K254, were mapped on the shared N-terminal region of these molecules, while a fourth site, K364, was identified in the C-terminal part exclusive of LEDGF/p75. The N-terminal SUMO targets are located in evolutionarily conserved charge-rich regions that lack resemblance to the described consensus SUMOylation motif, whereas the C-terminal SUMO target is solvent exposed and situated in a typical consensus motif. SUMOylation did not affect the cellular localization of LEDGF proteins and was not necessary for their chromatin-binding ability, nor did it affect this activity. However, lysine to arginine mutations of the identified SUMO acceptor sites drastically inhibited LEDGF SUMOylation, extended the half-life of LEDGF/p75, and significantly increased its transcriptional activity on the heat shock protein 27 promoter, indicating a negative effect of SUMOylation on the transcriptional activity of LEDGF/p75. Considering that SUMOylation is known to negatively affect the transcriptional activity of all transcription factors known to transactivate heat shock protein 27 expression, these findings support the paradigm establishing SUMOylation as a global neutralizer of cellular processes upregulated upon cellular stress.

Summary The 2013–2015 Ebola virus disease (EVD) epidemic is caused by the Makona variant of Ebola virus (EBOV). Early in the epidemic, genome sequencing provided insights into virus evolution and transmission and offered important information for outbreak response. Here, we analyze sequences from 232 patients sampled over 7 months in Sierra Leone, along with 86 previously released genomes from earlier in the epidemic. We confirm sustained human-to-human transmission within Sierra Leone and find no evidence for import or export of EBOV across national borders after its initial introduction. Using high-depth replicate sequencing, we observe both host-to-host transmission and recurrent emergence of intrahost genetic variants. We trace the increasing impact of purifying selection in suppressing the accumulation of nonsynonymous mutations over time. Finally, we note changes in the mucin-like domain of EBOV glycoprotein that merit further investigation. These findings clarify the movement of EBOV within the region and describe viral evolution during prolonged human-to-human transmission., Graphical Abstract, Highlights • In Sierra Leone, transmission has primarily been within-country, not between-country • Infectious doses are large enough for intrahost variants to transmit between hosts • A prolonged epidemic removes deleterious mutations from the viral population • There is preliminary evidence for human RNA editing effects on the Ebola genome, Ebola virus genomes from 232 patients sampled over 7 months in Sierra Leone were sequenced. Transmission of intrahost genetic variants suggests a sufficiently high infectious dose during transmission. The human host may have caused direct alterations to the Ebola virus genome.

SummaryMB-003, a plant-derived monoclonal antibody cocktail used effectively in treatment of Ebola virus infection in non-human primates, was unable to protect two of six animals when initiated 1 or 2 days post-infection. We characterized a mechanism of viral escape in one of the animals, after observation of two clusters of genomic mutations that resulted in five nonsynonymous mutations in the monoclonal antibody target sites. These mutations were linked to a reduction in antibody binding and later confirmed to be present in a viral isolate that was not neutralized in vitro. Retrospective evaluation of a second independent study allowed the identification of a similar case. Four SNPs in previously identified positions were found in this second fatality, suggesting that genetic drift could be a potential cause for treatment failure. These findings highlight the importance selecting different target domains for each component of the cocktail to minimize the potential for viral escape.

Background: Clonal expansion is a process in which a single organism reproduces asexually, giving rise to a diversifying population. It is pervasive in nature, from within-host pathogen evolution to emergent infectious disease outbreaks. Standard phylogenetic tools rely on full-length genomes of individual pathogens or population consensus sequences (phased genotypes). Although high-throughput sequencing technologies are able to sample population diversity, the short sequence reads inherent to them preclude assessing whether two reads originate from the same clone (unphased genotypes). This obstacle severely limits the application of phylogenetic methods and investigation of within-host dynamics of acute infections using this rich data source. Methods: We introduce two measures of diversity to study the evolution of clonal populations using unphased genomic data, which eliminate the need to construct full-length genomes. Our method follows a maximum likelihood approach to estimate evolutionary rates and times to the most recent common ancestor, based on a relaxed molecular clock model; independent of a growth model. Deviations from neutral evolution indicate the presence of selection and bottleneck events. Results: We evaluated our methods in silico and then compared it against existing approaches with the well-characterized 2009 H1N1 influenza pandemic. We then applied our method to high-throughput genomic data from marburgvirus-infected non-human primates and inferred the time of infection and the intra-host evolutionary rate, and identified purifying selection in viral populations. Conclusions: Our method has the power to make use of minor variants present in less than 1% of the population and capture genomic diversification within days of infection, making it an ideal tool for the study of acute RNA viral infection dynamics.