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The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since late 2019 represented an unprecedented public health emergency, which included a need to fully understand coronavirus disease 2019 (COVID-19) across all ages and populations. In response, the US National Institute of Allergy and Infectious Diseases (NIAID) rapidly funded epidemiology studies that monitored COVID-19. However, the diversity and breadth of the populations studied in NIAID-funded COVID-19 observational cohorts were not easy to extrapolate because of siloed approaches to collect and report data within NIAID. Here, we describe the effort to develop a harmonized cohort-study reporting tool that includes common epidemiologic data elements as well as NIAID priorities. We report its implementation to analyze metadata from 58 COVID-19 cohort studies funded from February 2020 to June 2021, and we visualize key metadata, including geographic distribution, study duration, participant demographic characteristics, sample types collected, and scientific priorities addressed. A bibliographic analysis highlights the scientific publications and citations across these funded studies and demonstrates their enormous impact on the COVID-19 field. These analyses highlight how common data elements and reporting tools can assist funding agencies to capture the landscape and potential gaps during public health responses and how they can assist in decision making., (Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health 2023.)

On July 19th, 2023, the National Institute of Allergy and Infectious Diseases co-organized a workshop with the Society of Mathematical Biology, with the authors of this paper as the organizing committee. The workshop, "Bridging multiscale modeling and practical clinical applications in infectious diseases" sought to create an environment for mathematical modelers, statisticians, and infectious disease researchers and clinicians to exchange ideas and perspectives., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Biomedical datasets are increasing in size, stored in many repositories, and face challenges in FAIRness (findability, accessibility, interoperability, reusability). As a Consortium of infectious disease researchers from 15 Centers, we aim to adopt open science practices to promote transparency, encourage reproducibility, and accelerate research advances through data reuse. To improve FAIRness of our datasets and computational tools, we evaluated metadata standards across established biomedical data repositories. The vast majority do not adhere to a single standard, such as Schema.org, which is widely-adopted by generalist repositories. Consequently, datasets in these repositories are not findable in aggregation projects like Google Dataset Search. We alleviated this gap by creating a reusable metadata schema based on Schema.org and catalogued nearly 400 datasets and computational tools we collected. The approach is easily reusable to create schemas interoperable with community standards, but customized to a particular context. Our approach enabled data discovery, increased the reusability of datasets from a large research consortium, and accelerated research. Lastly, we discuss ongoing challenges with FAIRness beyond discoverability., (© 2023. The Author(s).)

The global emergence of many severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants jeopardizes the protective antiviral immunity induced after infection or vaccination. To address the public health threat caused by the increasing SARS-CoV-2 genomic diversity, the National Institute of Allergy and Infectious Diseases within the National Institutes of Health established the SARS-CoV-2 Assessment of Viral Evolution (SAVE) programme. This effort was designed to provide a real-time risk assessment of SARS-CoV-2 variants that could potentially affect the transmission, virulence, and resistance to infection- and vaccine-induced immunity. The SAVE programme is a critical data-generating component of the US Government SARS-CoV-2 Interagency Group to assess implications of SARS-CoV-2 variants on diagnostics, vaccines and therapeutics, and for communicating public health risk. Here we describe the coordinated approach used to identify and curate data about emerging variants, their impact on immunity and effects on vaccine protection using animal models. We report the development of reagents, methodologies, models and notable findings facilitated by this collaborative approach and identify future challenges. This programme is a template for the response to rapidly evolving pathogens with pandemic potential by monitoring viral evolution in the human population to identify variants that could reduce the effectiveness of countermeasures., (© 2022. Springer Nature Limited.)

Zika Virus (ZIKV) is a mosquito-borne flavivirus that the World Health Organization (WHO) declared a global concern due to the severity of infection. This study focuses on determining the level of detection of ZIKV RNA in human serum and urine. Known amounts of Zika virus were added to uninfected human serum and urine samples. Different reverse transcriptases were compared to select the optimal enzyme for this application. Zika RNA in these samples was then quantified with qRT-PCR to determine the lower limit of detection in these fluids and to construct a standard curve. Student's t-test of paired samples was used in order to identify statistical differences. The SuperScript III enzyme was able to produce more ZIKV cDNA when compared to PrimeScript. Zika virus RNA was found to be detectable at lower levels (2.5 PFU/mL) in urine than in serum (250 PFU/mL) when using SuperScript III. This study demonstrates how the selection of both the human clinical specimen, and the reverse transcriptase enzyme involved in the molecular detection of ZIKV by quantitative real-time polymerase chain reaction (qRT-PCR), play an important role in enabling improved detection of the virus., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

The recent emergence of Zika virus (ZIKV) has been concentrated in the Caribbean, Southeastern United States, and South- and Central America; resulting in travel-based cases being reported around the globe. As multi-disciplinary collaborations are combatting the ZIKV outbreak, the need to validate the sequence of existing strains has become apparent. Here, we report high-quality sequence data for multiple ZIKV strains made publicly available through the National Institutes of Health- (NIH) funded biorepository, BEI Resources (www.beiresources.org). Next-generation sequencing, 3' rapid amplification of cDNA ends (RACE), and viral genome annotation pipelines generated GenBank sequence records for 16 BEI Resources strains. Minor variants, consensus mutations, and consensus insertions/deletions were identified within the viral stocks using next-generation sequencing (NGS) and consensus changes were confirmed with Sanger sequencing. Bioinformatics analyses of the sequencing results confirm that the virus stocks available to the scientific research community through BEI Resources adequately represent the viral population diversity of ZIKV.

Enterovirus D68 (EV-D68) has historically been associated with respiratory illnesses. However, in the summers of 2014 and 2016, EV-D68 outbreaks coincided with a spike in polio-like acute flaccid myelitis/paralysis (AFM/AFP) cases. This raised concerns that EV-D68 could be the causative agent of AFM during these recent outbreaks. To assess the potential neurotropism of EV-D68, we utilized the neuroblastoma-derived neuronal cell line SH-SY5Y as a cell culture model to determine if differential infection is observed for different EV-D68 strains. In contrast to HeLa and A549 cells, which support viral infection of all EV-D68 strains tested, SH-SY5Y cells only supported infection by a subset of contemporary EV-D68 strains, including isolates from the 2014 outbreak. Viral replication and infectivity in SH-SY5Y were assessed using multiple assays: virus production, cytopathic effects, cellular ATP release, and VP1 capsid protein production. Similar differential neurotropism was also observed in differentiated SH-SY5Y cells, primary human neuron cultures, and a mouse paralysis model. Using the SH-SY5Y cell culture model, we determined that barriers to viral binding and entry were at least partly responsible for the differential infectivity phenotype. Transfection of genomic RNA into SH-SY5Y generated virions for all EV-D68 isolates, but only a single round of replication was observed from strains that could not directly infect SH-SY5Y. In addition to supporting virus replication and other functional studies, this cell culture model may help identify the signatures of virulence to confirm epidemiological associations between EV-D68 strains and AFM and allow for the rapid identification and characterization of emerging neurotropic strains. IMPORTANCE Since the EV-D68 outbreak during the summer of 2014, evidence of a causal link to a type of limb paralysis (AFM) has been mounting. In this article, we describe a neuronal cell culture model (SH-SY5Y cells) in which a subset of contemporary 2014 outbreak strains of EV-D68 show infectivity in neuronal cells, or neurotropism. We confirmed the difference in neurotropism in vitro using primary human neuron cell cultures and in vivo with a mouse paralysis model. Using the SH-SY5Y cell model, we determined that a barrier to viral entry is at least partly responsible for neurotropism. SH-SY5Y cells may be useful in determining if specific EV-D68 genetic determinants are associated with neuropathogenesis, and replication in this cell line could be used as rapid screening tool for identification of neurotropic EV-D68 strains. This may assist with better understanding of pathogenesis and epidemiology and with the development of potential therapies., (Copyright © 2018 Brown et al.)

Chikungunya virus (CHIKV) has been detected sporadically since the 1950s and includes three distinct co-circulating genotypes. In late 2013, the Asian genotype of CHIKV was responsible for the Caribbean outbreak (CO) that rapidly became an epidemic throughout the Americas. There is a limited understanding of the molecular evolution of CHIKV in the Americas during this epidemic. We sequenced 185 complete CHIKV genomes collected mainly from Nicaragua in Central America and Florida in the United States during the 2014-2015 Caribbean/Americas epidemic. Our comprehensive phylogenetic analyses estimated the epidemic history of the Asian genotype and the recent Caribbean outbreak (CO) clade, revealed considerable genetic diversity within the CO clade, and described different epidemiological dynamics of CHIKV in the Americas. Specifically, we identified multiple introductions in both Nicaragua and Florida, with rapid local spread of viruses in Nicaragua but limited autochthonous transmission in Florida in the US. Our phylogenetic analysis also showed phylogeographic clustering of the CO clade. In addition, we identified the significant amino acid substitutions that were observed across the entire Asian genotype during its evolution and examined amino acid changes that were specific to the CO clade. Deep sequencing analysis identified specific minor variants present in clinical specimens below-consensus levels. Finally, we investigated the association between viral phylogeny and geographic/clinical metadata in Nicaragua. To date, this study represents the largest single collection of CHIKV complete genomes during the Caribbean/Americas epidemic and significantly expands our understanding of the emergence and evolution of CHIKV CO clade in the Americas., Competing Interests: The authors have declared that no competing interests exist.

Non-retroviral integrated RNA viral sequences (NIRVs) potentially encoding ∼280 amino acid homologs to filovirus VP35 proteins are present across the Myotis genus of bats. These are estimated to have been maintained for ∼18 million years, indicating their co-option. To address the reasons for co-option, 16 Myotis VP35s were characterized in comparison to VP35s from the extant filoviruses Ebola virus and Marburg virus, in which VP35s play critical roles in immune evasion and RNA synthesis. The Myotis VP35s demonstrated a conserved suppression of innate immune signaling, albeit with reduced potency, in either human or Myotis cells. Their attenuation reflects a lack of dsRNA binding that in the filoviral VP35s correlates with potent suppression of interferon responses. Despite divergent function, evolution has preserved in Myotis the structure of the filoviral VP35s, indicating that this structure is critical for co-opted function, possibly as a regulator of innate immune signaling., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

We report here the whole-genome sequence of 11 Zika virus (ZIKV) samples from six pediatric patients in Nicaragua. Serum samples were collected, and ZIKV was isolated in tissue culture. Both serum and virus isolates were sequenced. The consensus ZIKV genomes are greater than 99% identical to each other., (Copyright © 2018 Oldfield et al.)

Background: The tick cell line ISE6, derived from Ixodes scapularis , is commonly used for amplification and detection of arboviruses in environmental or clinical samples. Methods: To assist with sequence-based assays, we sequenced the ISE6 genome with single-molecule, long-read technology. Results: The draft assembly appears near complete based on gene content analysis, though it appears to lack some instances of repeats in this highly repetitive genome. The assembly appears to have separated the haplotypes at many loci. DNA short read pairs, used for validation only, mapped to the cell line assembly at a higher rate than they mapped to the Ixodes scapularis reference genome sequence. Conclusions: The assembly could be useful for filtering host genome sequence from sequence data obtained from cells infected with pathogens., Competing Interests: Competing interests: SK has received travel support to speak at Oxford Nanopore conferences.

Background: The 50-year-old Aedes albopictus C6/36 cell line is a resource for the detection, amplification, and analysis of mosquito-borne viruses including Zika, dengue, and chikungunya. The cell line is derived from an unknown number of larvae from an unspecified strain of Aedes albopictus mosquitoes. Toward improved utility of the cell line for research in virus transmission, we present an annotated assembly of the C6/36 genome., Results: The C6/36 genome assembly has the largest contig N50 (3.3 Mbp) of any mosquito assembly, presents the sequences of both haplotypes for most of the diploid genome, reveals independent null mutations in both alleles of the Dicer locus, and indicates a male-specific genome. Gene annotation was computed with publicly available mosquito transcript sequences. Gene expression data from cell line RNA sequence identified enrichment of growth-related pathways and conspicuous deficiency in aquaporins and inward rectifier K+ channels. As a test of utility, RNA sequence data from Zika-infected cells were mapped to the C6/36 genome and transcriptome assemblies. Host subtraction reduced the data set by 89%, enabling faster characterization of nonhost reads., Conclusions: The C6/36 genome sequence and annotation should enable additional uses of the cell line to study arbovirus vector interactions and interventions aimed at restricting the spread of human disease.

Foot-and-mouth disease (FMD) is a severe infection caused by a picornavirus that affects livestock and wildlife. Persistence in ruminants is a well-documented feature of Foot-and-mouth disease virus (FMDV) pathogenesis and a major concern for disease control. Persistently infected animals harbor virus for extended periods, providing a unique opportunity to study within-host virus evolution. This study investigated the genetic dynamics of FMDV during persistent infections of naturally infected Asian buffalo. Using next-generation sequencing (NGS) we obtained 21 near complete FMDV genome sequences from 12 sub-clinically infected buffalo over a period of one year. Four animals yielded only one virus isolate and one yielded two isolates of different serotype suggesting a serial infection. Seven persistently infected animals yielded more than one virus of the same serotype showing a long-term intra-host viral genetic divergence at the consensus level of less than 2.5%. Quasi-species analysis showed few nucleotide variants and non-synonymous substitutions of progeny virus despite intra-host persistence of up to 152 days. Phylogenetic analyses of serotype Asia-1 VP1 sequences clustered all viruses from persistent animals with Group VII viruses circulating in Pakistan in 2011, but distinct from those circulating on 2008-2009. Furthermore, signature amino acid (aa) substitutions were found in the antigenically relevant VP1 of persistent viruses compared with viruses from 2008-2009. Intra-host purifying selective pressure was observed, with few codons in structural proteins undergoing positive selection. However, FMD persistent viruses did not show a clear pattern of antigenic selection. Our findings provide insight into the evolutionary dynamics of FMDV populations within naturally occurring subclinical and persistent infections that may have implications to vaccination strategies in the region.

Mannose binding lectin (MBL) generally plays a protective role during viral infection, yet MBL-mediated complement activation promotes Ross River virus (RRV)-induced inflammatory tissue destruction, contributing to arthritis and myositis. As MBL binds to carbohydrates, we hypothesized that N-linked glycans on the RRV envelope glycoproteins act as ligands for MBL. Using a panel of RRV mutants lacking the envelope N-linked glycans, we found that MBL deposition onto infected cells was dependent on the E2 glycans. Moreover, the glycan-deficient viruses exhibited reduced disease and tissue damage in a mouse model of RRV-induced myositis compared to wild-type RRV, despite similar viral load and inflammatory infiltrates within the skeletal muscle. Instead, the reduced disease induced by glycan-deficient viruses was linked to decreased MBL deposition and complement activation within inflamed tissues. These results demonstrate that the viral N-linked glycans promote MBL deposition and complement activation onto RRV-infected cells, contributing to the development of RRV-induced myositis., (Copyright © 2017. Published by Elsevier Inc.)

We report 26 complete genomes of Zika virus (ZIKV) isolated after passaging the Zika virus strain FLR in mosquito (C6/36) and mammalian (Vero) cell lines. The consensus ZIKV genomes we recovered show greater than 99% nucleotide identify with each other and with the FLR strain used as input., (Copyright © 2018 Moser et al.)

The human cell lines HepG2, HuH-7, and Jurkat are commonly used for amplification of the RNA viruses present in environmental samples. To assist with assays by RNAseq, we sequenced these cell lines and developed a subtraction database that contains sequences expected in sequence data from uninfected cells. RNAseq data from cell lines infected with Sendai virus were analyzed to test host subtraction. The process of mapping RNAseq reads to our subtraction database vastly reduced the number non-viral reads in the dataset to allow for efficient secondary analyses., Competing Interests: No competing interests were disclosed.

The human cell lines HepG2, HuH-7, and Jurkat are commonly used for amplification of the RNA viruses present in environmental samples. To assist with assays by RNAseq, we sequenced these cell lines and developed a subtraction database that contains sequences expected in sequence data from uninfected cells. RNAseq data from cell lines infected with Sendai virus were analyzed to test host subtraction. The process of mapping RNAseq reads to our subtraction database vastly reduced the number non-viral reads in the dataset to allow for efficient secondary analyses., Competing Interests: No competing interests were disclosed.

The human cell lines HepG2, HuH-7, and Jurkat are commonly used for amplification of the RNA viruses present in environmental samples. To assist with assays by RNAseq, we sequenced these cell lines and developed a subtraction database that contains sequences expected in sequence data from uninfected cells. RNAseq data from cell lines infected with Sendai virus were analyzed to test host subtraction. The process of mapping RNAseq reads to our subtraction database vastly reduced the number non-viral reads in the dataset to allow for efficient secondary analyses., Competing Interests: No competing interests were disclosed.

Type III IFNs are important mediators of antiviral immunity. IFN-λ4 is a unique type III IFN because it is produced only in individuals who carry a dG allele of a genetic variant rs368234815-dG/TT. Counterintuitively, those individuals who can produce IFN-λ4, an antiviral cytokine, are also less likely to clear hepatitis C virus infection. In this study, we searched for unique functional properties of IFN-λ4 that might explain its negative effect on hepatitis C virus clearance. We used fresh primary human hepatocytes (PHHs) treated with recombinant type III IFNs or infected with Sendai virus to model acute viral infection and subsequently validated our findings in HepG2 cell line models. Endogenous IFN-λ4 protein was detectable only in Sendai virus-infected PHHs from individuals with the dG allele, where it was poorly secreted but highly functional, even at concentrations < 50 pg/ml. IFN-λ4 acted faster than other type III IFNs in inducing antiviral genes, as well as negative regulators of the IFN response, such as USP18 and SOCS1 Transient treatment of PHHs with IFN-λ4, but not IFN-λ3, caused a strong and sustained induction of SOCS1 and refractoriness to further stimulation with IFN-λ3. Our results suggest unique functional properties of IFN-λ4 that can be important in viral clearance and other clinical conditions., (Copyright © 2017 by The American Association of Immunologists, Inc.)

We report here the complete genome of a Dezidougou virus (DEZV) isolated from a passaged culture of the Zika virus strain DAK AR 41524. The consensus DEZV sequence we recovered shows 99% nucleotide similarity using BLASTN to a previously reported DEZV (accession no. JQ675604.1). The current sequence has additional repeat regions as well as a deleted repeat region, which we confirmed by Sanger sequencing, that were not present in the originally published sequence, JQ675604.1., (Copyright © 2017 Shrivastava et al.)

Ebola virus and Marburg virus are members of the Filovirdae family and causative agents of hemorrhagic fever with high fatality rates in humans. Filovirus virulence is partially attributed to the VP35 protein, a well-characterized inhibitor of the RIG-I-like receptor pathway that triggers the antiviral interferon (IFN) response. Prior work demonstrates the ability of VP35 to block potent RIG-I activators, such as Sendai virus (SeV), and this IFN-antagonist activity is directly correlated with its ability to bind RNA. Several structural studies demonstrate that VP35 binds short synthetic dsRNAs; yet, there are no data that identify viral immunostimulatory RNAs (isRNA) or host RNAs bound to VP35 in cells. Utilizing a SeV infection model, we demonstrate that both viral isRNA and host RNAs are bound to Ebola and Marburg VP35s in cells. By deep sequencing the purified VP35-bound RNA, we identified the SeV copy-back defective interfering (DI) RNA, previously identified as a robust RIG-I activator, as the isRNA bound by multiple filovirus VP35 proteins, including the VP35 protein from the West African outbreak strain (Makona EBOV). Moreover, RNAs isolated from a VP35 RNA-binding mutant were not immunostimulatory and did not include the SeV DI RNA. Strikingly, an analysis of host RNAs bound by wild-type, but not mutant, VP35 revealed that select host RNAs are preferentially bound by VP35 in cell culture. Taken together, these data support a model in which VP35 sequesters isRNA in virus-infected cells to avert RIG-I like receptor (RLR) activation.

The CP 96-1252 cultivar of sugarcane is a complex hybrid of commercial importance. DNA was extracted from lab-grown leaf tissue and sequenced. The raw Illumina DNA sequencing results provide 101 Gbp of genome sequence reads. The dataset is available from https://www.ncbi.nlm.nih.gov/bioproject/PRJNA345486/., Competing Interests: Competing interests: No competing interests were disclosed.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that is causally linked to severe neonatal birth defects, including microcephaly, and is associated with Guillain-Barre syndrome in adults. Dendritic cells (DCs) are an important cell type during infection by multiple mosquito-borne flaviviruses, including dengue virus, West Nile virus, Japanese encephalitis virus, and yellow fever virus. Despite this, the interplay between ZIKV and DCs remains poorly defined. Here, we found human DCs supported productive infection by a contemporary Puerto Rican isolate with considerable variability in viral replication, but not viral binding, between DCs from different donors. Historic isolates from Africa and Asia also infected DCs with distinct viral replication kinetics between strains. African lineage viruses displayed more rapid replication kinetics and infection magnitude as compared to Asian lineage viruses, and uniquely induced cell death. Infection of DCs with both contemporary and historic ZIKV isolates led to minimal up-regulation of T cell co-stimulatory and MHC molecules, along with limited secretion of inflammatory cytokines. Inhibition of type I interferon (IFN) protein translation was observed during ZIKV infection, despite strong induction at the RNA transcript level and up-regulation of other host antiviral proteins. Treatment of human DCs with RIG-I agonist potently restricted ZIKV replication, while type I IFN had only modest effects. Mechanistically, we found all strains of ZIKV antagonized type I IFN-mediated phosphorylation of STAT1 and STAT2. Combined, our findings show that ZIKV subverts DC immunogenicity during infection, in part through evasion of type I IFN responses, but that the RLR signaling pathway is still capable of inducing an antiviral state, and therefore may serve as an antiviral therapeutic target., Competing Interests: The authors have declared that no competing interests exist.

Since the India and Indian Ocean outbreaks of 2005 and 2006, the global distribution of chikungunya virus (CHIKV) and the locations of epidemics have dramatically shifted. First, the Indian Ocean lineage (IOL) caused sustained epidemics in India and has radiated to many other countries. Second, the Asian lineage has caused frequent outbreaks in the Pacific islands and in 2013 was introduced into the Caribbean, followed by rapid spread to nearly all of the neotropics. Further, CHIKV epidemics, as well as exported cases, have been reported in central Africa after a long period of perceived silence. To understand these changes and to anticipate the future of the virus, the exact distribution, genetic diversity, transmission routes, and future epidemic potential of CHIKV require further assessment. To do so, we conducted the most comprehensive phylogenetic analysis to date, examined CHIKV evolution and transmission, and explored distinct genetic factors associated with the emergence of the East/Central/South African (ECSA) lineage, the IOL, and the Asian lineage. Our results reveal contrasting evolutionary patterns among the lineages, with growing genetic diversities observed in each, and suggest that CHIKV will continue to be a major public health threat with the potential for further emergence and spread., Importance: Chikungunya fever is a reemerging infectious disease that is transmitted by Aedes mosquitoes and causes severe health and economic burdens in affected populations. Since the unprecedented Indian Ocean and Indian subcontinent outbreaks of 2005 and 2006, CHIKV has further expanded its geographic range, including to the Americas in 2013. Its evolution and transmission during and following these epidemics, as well as the recent evolution and spread of other lineages, require optimal assessment. Using newly obtained genome sequences, we provide a comprehensive update of the global distribution of CHIKV genetic diversity and analyze factors associated with recent outbreaks. These results provide a solid foundation for future evolutionary studies of CHIKV that can elucidate emergence mechanisms and also may help to predict future epidemics., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Several biosafety level 3 and/or 4 (BSL-3/4) pathogens are high-consequence, single-stranded RNA viruses, and their genomes, when introduced into permissive cells, are infectious. Moreover, many of these viruses are select agents (SAs), and their genomes are also considered SAs. For this reason, cDNAs and/or their derivatives must be tested to ensure the absence of infectious virus and/or viral RNA before transfer out of the BSL-3/4 and/or SA laboratory. This tremendously limits the capacity to conduct viral genomic research, particularly the application of next-generation sequencing (NGS). Here, we present a sequence-independent method to rapidly amplify viral genomic RNA while simultaneously abolishing both viral and genomic RNA infectivity across multiple single-stranded positive-sense RNA (ssRNA+) virus families. The process generates barcoded DNA amplicons that range in length from 300 to 1,000 bp, which cannot be used to rescue a virus and are stable to transport at room temperature. Our barcoding approach allows for up to 288 barcoded samples to be pooled into a single library and run across various NGS platforms without potential reconstitution of the viral genome. Our data demonstrate that this approach provides full-length genomic sequence information not only from high-titer virion preparations but it can also recover specific viral sequence from samples with limited starting material in the background of cellular RNA, and it can be used to identify pathogens from unknown samples. In summary, we describe a rapid, universal standard operating procedure that generates high-quality NGS libraries free of infectious virus and infectious viral RNA. IMPORTANCE This report establishes and validates a standard operating procedure (SOP) for select agents (SAs) and other biosafety level 3 and/or 4 (BSL-3/4) RNA viruses to rapidly generate noninfectious, barcoded cDNA amenable for next-generation sequencing (NGS). This eliminates the burden of testing all processed samples derived from high-consequence pathogens prior to transfer from high-containment laboratories to lower-containment facilities for sequencing. Our established protocol can be scaled up for high-throughput sequencing of hundreds of samples simultaneously, which can dramatically reduce the cost and effort required for NGS library construction. NGS data from this SOP can provide complete genome coverage from viral stocks and can also detect virus-specific reads from limited starting material. Our data suggest that the procedure can be implemented and easily validated by institutional biosafety committees across research laboratories.

Suppression of innate immune responses during filoviral infection contributes to disease severity. Ebola (EBOV) and Marburg (MARV) viruses each encode a VP35 protein that suppresses RIG-I-like receptor signaling and interferon-α/β (IFN-α/β) production by several mechanisms, including direct binding to double stranded RNA (dsRNA). Here, we demonstrate that in cell culture, MARV infection results in a greater upregulation of IFN responses as compared to EBOV infection. This correlates with differences in the efficiencies by which EBOV and MARV VP35s antagonize RIG-I signaling. Furthermore, structural and biochemical studies suggest that differential recognition of RNA elements by the respective VP35 C-terminal IFN inhibitory domain (IID) rather than affinity for RNA by the respective VP35s is critical for this observation. Our studies reveal functional differences in EBOV versus MARV VP35 RNA binding that result in unexpected differences in the host response to deadly viral pathogens., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

While employing deep sequencing and de novo assembly to characterize the mRNA transcript profile of a cell line derived from the microbat Myotis velifer incautus, we serendipitously identified mRNAs encoding proteins with a high level of identity to herpesviruses. A majority were closely related to proteins of equine herpesvirus 2 (EHV-2), a horse gammaherpesvirus. We demonstrated by electron microscopy the presence of herpesvirus-like particles in the microbat cells. Passage of supernatants from microbat cells to Vero cells resulted in syncytium formation, and expression of viral genes and amplification of viral DNA were demonstrated by quantitative PCR. Susceptibility of human cell lines to productive infection was also demonstrated. Next-generation sequencing and de novo assembly of the viral genome from supernatants from Vero cells yielded a single contig of approximately 130 kb with at least 77 open reading frames (ORFs), predicted microRNAs (miRNAs), and a gammaherpesvirus genomic organization. Phylogenic analysis of the envelope glycoprotein (gB) and DNA polymerase (POLD1) revealed similarity to multiple gammaherpesviruses, including those from as-yet-uncultured viruses of the Rhadinovirus genus that were obtained by deep sequencing of bat tissues. Moreover, the assembled genome revealed ORFs that share little or no homology to known ORFs in EHV-2 but are similar to accessory proteins of other gammaherpesviruses. Some also have striking homology to predicted Myotis bat proteins. Cumulatively, this study provides the first isolation and characterization of a replication-competent bat gammaherpesvirus. IMPORTANCE Bats are of significant interest as reservoirs for zoonotic viral pathogens; however, tools to dissect bat-virus interactions are limited in availability. This study serendipitously identified, in an established bat cell line, a fully replication-competent gammaherpesvirus; determined the complete genome sequence of the virus; and generated a viral transcript map. This virus can replicate in select human and nonhuman primate cell lines. However, analyses of viral sequences support a bat origin for this virus; we therefore refer to the virus as bat gammaherpesvirus 8 (BGHV8). The viral genome contains unique open reading frames that likely encode modulators of bat innate and adaptive immune signaling pathways and expresses viral miRNAs. The virus and its gene products should provide a unique tool to dissect both bat and gammaherpesvirus biology.

During viral RNA synthesis, Ebola virus (EBOV) nucleoprotein (NP) alternates between an RNA-template-bound form and a template-free form to provide the viral polymerase access to the RNA template. In addition, newly synthesized NP must be prevented from indiscriminately binding to noncognate RNAs. Here, we investigate the molecular bases for these critical processes. We identify an intrinsically disordered peptide derived from EBOV VP35 (NPBP, residues 20-48) that binds NP with high affinity and specificity, inhibits NP oligomerization, and releases RNA from NP-RNA complexes in vitro. The structure of the NPBP/ΔNPNTD complex, solved to 3.7 Å resolution, reveals how NPBP peptide occludes a large surface area that is important for NP-NP and NP-RNA interactions and for viral RNA synthesis. Together, our results identify a highly conserved viral interface that is important for EBOV replication and can be targeted for therapeutic development., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Unlabelled: Deep sequencing of RNAs produced by Zaire ebolavirus (EBOV) or the Angola strain of Marburgvirus (MARV-Ang) identified novel viral and cellular mechanisms that diversify the coding and noncoding sequences of viral mRNAs and genomic RNAs. We identified previously undescribed sites within the EBOV and MARV-Ang mRNAs where apparent cotranscriptional editing has resulted in the addition of non-template-encoded residues within the EBOV glycoprotein (GP) mRNA, the MARV-Ang nucleoprotein (NP) mRNA, and the MARV-Ang polymerase (L) mRNA, such that novel viral translation products could be produced. Further, we found that the well-characterized EBOV GP mRNA editing site is modified at a high frequency during viral genome RNA replication. Additionally, editing hot spots representing sites of apparent adenosine deaminase activity were found in the MARV-Ang NP 3'-untranslated region. These studies identify novel filovirus-host interactions and reveal production of a greater diversity of filoviral gene products than was previously appreciated., Importance: This study identifies novel mechanisms that alter the protein coding capacities of Ebola and Marburg virus mRNAs. Therefore, filovirus gene expression is more complex and diverse than previously recognized. These observations suggest new directions in understanding the regulation of filovirus gene expression., (Copyright © 2014 Shabman et al.)

Bats harbor many viruses, which are periodically transmitted to humans resulting in outbreaks of disease (e.g., Ebola, SARS-CoV). Recently, influenza virus-like sequences were identified in bats; however, the viruses could not be cultured. This discovery aroused great interest in understanding the evolutionary history and pandemic potential of bat-influenza. Using synthetic genomics, we were unable to rescue the wild type bat virus, but could rescue a modified bat-influenza virus that had the HA and NA coding regions replaced with those of A/PR/8/1934 (H1N1). This modified bat-influenza virus replicated efficiently in vitro and in mice, resulting in severe disease. Additional studies using a bat-influenza virus that had the HA and NA of A/swine/Texas/4199-2/1998 (H3N2) showed that the PR8 HA and NA contributed to the pathogenicity in mice. Unlike other influenza viruses, engineering truncations hypothesized to reduce interferon antagonism into the NS1 protein didn't attenuate bat-influenza. In contrast, substitution of a putative virulence mutation from the bat-influenza PB2 significantly attenuated the virus in mice and introduction of a putative virulence mutation increased its pathogenicity. Mini-genome replication studies and virus reassortment experiments demonstrated that bat-influenza has very limited genetic and protein compatibility with Type A or Type B influenza viruses, yet it readily reassorts with another divergent bat-influenza virus, suggesting that the bat-influenza lineage may represent a new Genus/Species within the Orthomyxoviridae family. Collectively, our data indicate that the bat-influenza viruses recently identified are authentic viruses that pose little, if any, pandemic threat to humans; however, they provide new insights into the evolution and basic biology of influenza viruses.

The Ebola virus (EBOV) genome only encodes a single viral polypeptide with enzymatic activity, the viral large (L) RNA-dependent RNA polymerase protein. However, currently, there is limited information about the L protein, which has hampered the development of antivirals. Therefore, antifiloviral therapeutic efforts must include additional targets such as protein-protein interfaces. Viral protein 35 (VP35) is multifunctional and plays important roles in viral pathogenesis, including viral mRNA synthesis and replication of the negative-sense RNA viral genome. Previous studies revealed that mutation of key basic residues within the VP35 interferon inhibitory domain (IID) results in significant EBOV attenuation, both in vitro and in vivo. In the current study, we use an experimental pipeline that includes structure-based in silico screening and biochemical and structural characterization, along with medicinal chemistry, to identify and characterize small molecules that target a binding pocket within VP35. NMR mapping experiments and high-resolution x-ray crystal structures show that select small molecules bind to a region of VP35 IID that is important for replication complex formation through interactions with the viral nucleoprotein (NP). We also tested select compounds for their ability to inhibit VP35 IID-NP interactions in vitro as well as VP35 function in a minigenome assay and EBOV replication. These results confirm the ability of compounds identified in this study to inhibit VP35-NP interactions in vitro and to impair viral replication in cell-based assays. These studies provide an initial framework to guide development of antifiloviral compounds against filoviral VP35 proteins., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Kelch-like ECH-associated protein 1 (Keap1) is a ubiquitin E3 ligase specificity factor that targets transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) for ubiquitination and degradation. Disrupting Keap1-Nrf2 interaction stabilizes Nrf2, resulting in Nrf2 nuclear accumulation, binding to antioxidant response elements (AREs), and transcription of cytoprotective genes. Marburg virus (MARV) is a zoonotic pathogen that likely uses bats as reservoir hosts. We demonstrate that MARV protein VP24 (mVP24) binds the Kelch domain of either human or bat Keap1. This binding is of high affinity and 1:1 stoichiometry and activates Nrf2. Modeling based on the Zaire ebolavirus (EBOV) VP24 (eVP24) structure identified in mVP24 an acidic loop (K-loop) critical for Keap1 interaction. Transfer of the K-loop to eVP24, which otherwise does not bind Keap1, confers Keap1 binding and Nrf2 activation, and infection by MARV, but not EBOV, activates ARE gene expression. Therefore, MARV targets Keap1 to activate Nrf2-induced cytoprotective responses during infection., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Viral protein 35 (VP35), encoded by filoviruses, is a multifunctional dsRNA binding protein that plays important roles in viral replication, innate immune evasion, and pathogenesis. The multifunctional nature of these proteins also presents opportunities to develop countermeasures that target distinct functional regions. However, functional validation and the establishment of therapeutic approaches toward such multifunctional proteins, particularly for nonenzymatic targets, are often challenging. Our previous work on filoviral VP35 proteins defined conserved basic residues located within its C-terminal dsRNA binding interferon (IFN) inhibitory domain (IID) as important for VP35 mediated IFN antagonism and viral polymerase cofactor functions. In the current study, we used a combination of structural and functional data to determine regions of Ebola virus (EBOV) VP35 (eVP35) to target for aptamer selection using SELEX. Select aptamers, representing, two distinct classes, were further characterized based on their interaction properties to eVP35 IID. These results revealed that these aptamers bind to distinct regions of eVP35 IID with high affinity (10-50 nM) and specificity. These aptamers can compete with dsRNA for binding to eVP35 and disrupt the eVP35-nucleoprotein (NP) interaction. Consistent with the ability to antagonize the eVP35-NP interaction, select aptamers can inhibit the function of the EBOV polymerase complex reconstituted by the expression of select viral proteins. Taken together, our results support the identification of two aptamers that bind filoviral VP35 proteins with high affinity and specificity and have the capacity to potentially function as filoviral VP35 protein inhibitors.

Antigen-presenting cells (APCs) are critical targets of Ebola virus (EBOV) infection in vivo. However, the susceptibility of monocytes to infection is controversial. Studies indicate productive monocyte infection, and yet monocytes are also reported to be resistant to EBOV GP-mediated entry. In contrast, monocyte-derived macrophages and dendritic cells are permissive for both EBOV entry and replication. Here, freshly isolated monocytes are demonstrated to indeed be refractory to EBOV entry. However, EBOV binds monocytes, and delayed entry occurs during monocyte differentiation. Cultured monocytes spontaneously downregulate the expression of viral entry restriction factors such as interferon-inducible transmembrane proteins, while upregulating the expression of critical EBOV entry factors cathepsin B and NPC1. Moreover, these processes are accelerated by EBOV infection. Finally, ectopic expression of NPC1 is sufficient to rescue entry into an undifferentiated, normally nonpermissive monocytic cell line. These results define the molecular basis for infection of APCs and suggest means to limit APC infection.

Ebolaviruses, highly lethal zoonotic pathogens, possess longer genomes than most other non-segmented negative-strand RNA viruses due in part to long 5' and 3' untranslated regions (UTRs) present in the seven viral transcriptional units. To date, specific functions have not been assigned to these UTRs. With reporter assays, we demonstrated that the Zaire ebolavirus (EBOV) 5'-UTRs lack internal ribosomal entry site function. However, the 5'-UTRs do differentially regulate cap-dependent translation when placed upstream of a GFP reporter gene. Most dramatically, the 5'-UTR derived from the viral polymerase (L) mRNA strongly suppressed translation of GFP compared to a β-actin 5'-UTR. The L 5'-UTR is one of four viral genes to possess upstream AUGs (uAUGs), and ablation of each uAUG enhanced translation of the primary ORF (pORF), most dramatically in the case of the L 5'-UTR. The L uAUG was sufficient to initiate translation, is surrounded by a "weak" Kozak sequence and suppressed pORF translation in a position-dependent manner. Under conditions where eIF2α was phosphorylated, the presence of the uORF maintained translation of the L pORF, indicating that the uORF modulates L translation in response to cellular stress. To directly address the role of the L uAUG in virus replication, a recombinant EBOV was generated in which the L uAUG was mutated to UCG. Strikingly, mutating two nucleotides outside of previously-defined protein coding and cis-acting regulatory sequences attenuated virus growth to titers 10-100-fold lower than a wild-type virus in Vero and A549 cells. The mutant virus also exhibited decreased viral RNA synthesis as early as 6 hours post-infection and enhanced sensitivity to the stress inducer thapsigargin. Cumulatively, these data identify novel mechanisms by which EBOV regulates its polymerase expression, demonstrate their relevance to virus replication and identify a potential therapeutic target.

Filoviruses, marburgvirus (MARV) and ebolavirus (EBOV), are causative agents of highly lethal hemorrhagic fever in humans. MARV and EBOV share a common genome organization but show important differences in replication complex formation, cell entry, host tropism, transcriptional regulation, and immune evasion. Multifunctional filoviral viral protein (VP) 35 proteins inhibit innate immune responses. Recent studies suggest double-stranded (ds)RNA sequestration is a potential mechanism that allows EBOV VP35 to antagonize retinoic-acid inducible gene-I (RIG-I) like receptors (RLRs) that are activated by viral pathogen-associated molecular patterns (PAMPs), such as double-strandedness and dsRNA blunt ends. Here, we show that MARV VP35 can inhibit IFN production at multiple steps in the signaling pathways downstream of RLRs. The crystal structure of MARV VP35 IID in complex with 18-bp dsRNA reveals that despite the similar protein fold as EBOV VP35 IID, MARV VP35 IID interacts with the dsRNA backbone and not with blunt ends. Functional studies show that MARV VP35 can inhibit dsRNA-dependent RLR activation and interferon (IFN) regulatory factor 3 (IRF3) phosphorylation by IFN kinases TRAF family member-associated NFkb activator (TANK) binding kinase-1 (TBK-1) and IFN kB kinase e (IKKe) in cell-based studies. We also show that MARV VP35 can only inhibit RIG-I and melanoma differentiation associated gene 5 (MDA5) activation by double strandedness of RNA PAMPs (coating backbone) but is unable to inhibit activation of RLRs by dsRNA blunt ends (end capping). In contrast, EBOV VP35 can inhibit activation by both PAMPs. Insights on differential PAMP recognition and inhibition of IFN induction by a similar filoviral VP35 fold, as shown here, reveal the structural and functional plasticity of a highly conserved virulence factor.

Inclusion bodies are a characteristic feature of ebolavirus infections in cells. They contain large numbers of preformed nucleocapsids, but their biological significance has been debated, and they have been suggested to be aggregates of viral proteins without any further biological function. However, recent data for other viruses that produce similar structures have suggested that inclusion bodies might be involved in genome replication and transcription. In order to study filovirus inclusion bodies, we fused mCherry to the ebolavirus polymerase L, which is found in inclusion bodies. The resulting L-mCherry fusion protein was functional in minigenome assays and incorporated into virus-like particles. Importantly, L-mCherry fluorescence in transfected cells was readily detectable and distributed in a punctate pattern characteristic for inclusion bodies. A recombinant ebolavirus encoding L-mCherry instead of L was rescued and showed virtually identical growth kinetics and endpoint titers to those for wild-type virus. Using this virus, we showed that the onset of inclusion body formation corresponds to the onset of viral genome replication, but that viral transcription occurs prior to inclusion body formation. Live-cell imaging further showed that inclusion bodies are highly dynamic structures and that they can undergo dramatic reorganization during cell division. Finally, by labeling nascent RNAs using click technology we showed that inclusion bodies are indeed the site of viral RNA synthesis. Based on these data we conclude that, rather than being inert aggregates of nucleocapsids, ebolavirus inclusion bodies are in fact complex and dynamic structures and an important site at which viral RNA replication takes place.

Dengue virus (DENV) is a pathogen with a high impact on human health. It replicates in a wide range of cells involved in the immune response. To efficiently infect humans, DENV must evade or inhibit fundamental elements of the innate immune system, namely the type I interferon response. DENV circumvents the host immune response by expressing proteins that antagonize the cellular innate immunity. We have recently documented the inhibition of type I IFN production by the proteolytic activity of DENV NS2B3 protease complex in human monocyte derived dendritic cells (MDDCs). In the present report we identify the human adaptor molecule STING as a target of the NS2B3 protease complex. We characterize the mechanism of inhibition of type I IFN production in primary human MDDCs by this viral factor. Using different human and mouse primary cells lacking STING, we show enhanced DENV replication. Conversely, mutated versions of STING that cannot be cleaved by the DENV NS2B3 protease induced higher levels of type I IFN after infection with DENV. Additionally, we show that DENV NS2B3 is not able to degrade the mouse version of STING, a phenomenon that severely restricts the replication of DENV in mouse cells, suggesting that STING plays a key role in the inhibition of DENV infection and spread in mice.

The Zaire Ebola virus (EBOV) protein VP35 is multifunctional; it inhibits IFN-α/β production and functions as a cofactor of the viral RNA polymerase. Mass spectrometry identified the double stranded RNA binding protein 76 (DRBP76/NFAR-1/NF90) as a cellular factor that associates with the VP35 C-terminal interferon inhibitory domain (IID). DRBP76 is described to regulate host cell protein synthesis and play an important role in host defense. The VP35-IID-DRBP76 interaction required the addition of exogenous dsRNA, but full-length VP35 associated with DRBP76 in the absence of exogenous dsRNA. Cells infected with a Newcastle disease virus (NDV)-expressing VP35 redistributed DRBP76 from the nucleus to the cytoplasm, the compartment in which EBOV replicates. Overexpression of DRBP76 did not alter the ability of VP35 to inhibit type I IFN production but did impair the function of the EBOV transcription/replication complex. These data suggest that DRBP76, via its association with VP35, exerts an anti-EBOV function.

The Ebola virus (EBOV) protein VP24 inhibits type I and II interferon (IFN) signaling by binding to NPI-1 subfamily karyopherin α (KPNA) nuclear import proteins, preventing their interaction with tyrosine-phosphorylated STAT1 (phospho-STAT1). This inhibits phospho-STAT1 nuclear import. A biochemical screen now identifies heterogeneous nuclear ribonuclear protein complex C1/C2 (hnRNP C1/C2) nuclear import as an additional target of VP24. Co-immunoprecipitation studies demonstrate that hnRNP C1/C2 interacts with multiple KPNA family members, including KPNA1. Interaction with hnRNP C1/C2 occurs through the same KPNA1 C-terminal region (amino acids 424-457) that binds VP24 and phospho-STAT1. The ability of hnRNP C1/C2 to bind KPNA1 is diminished in the presence of VP24, and cells transiently expressing VP24 redistribute hnRNP C1/C2 from the nucleus to the cytoplasm. These data further define the mechanism of hnRNP C1/C2 nuclear import and demonstrate that the impact of EBOV VP24 on nuclear import extends beyond STAT1.

The Filoviridae family of viruses, which includes the genera Ebolavirus (EBOV) and Marburgvirus (MARV), causes severe and often times lethal hemorrhagic fever in humans. Filoviral infections are associated with ineffective innate antiviral responses as a result of virally encoded immune antagonists, which render the host incapable of mounting effective innate or adaptive immune responses. The Type I interferon (IFN) response is critical for establishing an antiviral state in the host cell and subsequent activation of the adaptive immune responses. Several filoviral encoded components target Type I IFN responses, and this innate immune suppression is important for viral replication and pathogenesis. For example, EBOV VP35 inhibits the phosphorylation of IRF-3/7 by the TBK-1/IKKε kinases in addition to sequestering viral RNA from detection by RIG-I like receptors. MARV VP40 inhibits STAT1/2 phosphorylation by inhibiting the JAK family kinases. EBOV VP24 inhibits nuclear translocation of activated STAT1 by karyopherin-α. The examples also represent distinct mechanisms utilized by filoviral proteins in order to counter immune responses, which results in limited IFN-α/β production and downstream signaling.

The ebolavirus (EBOV) VP35 protein binds to double-stranded RNA (dsRNA), inhibits host alpha/beta interferon (IFN-α/β) production, and is an essential component of the viral polymerase complex. Structural studies of the VP35 C-terminal IFN inhibitory domain (IID) identified specific structural features, including a central basic patch and a hydrophobic pocket, that are important for dsRNA binding and IFN inhibition. Several other conserved basic residues bordering the central basic patch and a separate cluster of basic residues, called the first basic patch, were also identified. Functional analysis of alanine substitution mutants indicates that basic residues outside the central basic patch are not required for dsRNA binding or for IFN inhibition. However, minigenome assays, which assess viral RNA polymerase complex function, identified these other basic residues to be critical for viral RNA synthesis. Of these, a subset located within the first basic patch is important for VP35-nucleoprotein (NP) interaction, as evidenced by the inability of alanine substitution mutants to coimmunoprecipitate with NP. Therefore, first basic patch residues are likely critical for replication complex formation through interactions with NP. Coimmunoprecipitation studies further demonstrate that the VP35 IID is sufficient to interact with NP and that dsRNA can modulate VP35 IID interactions with NP. Other basic residue mutations that disrupt the VP35 polymerase cofactor function do not affect interaction with NP or with the amino terminus of the viral polymerase. Collectively, these results highlight the importance of conserved basic residues from the EBOV VP35 C-terminal IID and validate the VP35 IID as a potential therapeutic target.

Ebolaviruses are causative agents of lethal hemorrhagic fever in humans and nonhuman primates. Among the filoviruses characterized thus far, Reston Ebola virus (REBOV) is the only Ebola virus that is nonpathogenic to humans despite the fact that REBOV can cause lethal disease in nonhuman primates. Previous studies also suggest that REBOV is less effective at inhibiting host innate immune responses than Zaire Ebola virus (ZEBOV) or Marburg virus. Virally encoded VP35 protein is critical for immune suppression, but an understanding of the relative contributions of VP35 proteins from REBOV and other filoviruses is currently lacking. In order to address this question, we characterized the REBOV VP35 interferon inhibitory domain (IID) using structural, biochemical, and virological studies. These studies reveal differences in double-stranded RNA binding and interferon inhibition between the two species. These observed differences are likely due to increased stability and loss of flexibility in REBOV VP35 IID, as demonstrated by thermal shift stability assays. Consistent with this finding, the 1.71-A crystal structure of REBOV VP35 IID reveals that it is highly similar to that of ZEBOV VP35 IID, with an overall backbone r.m.s.d. of 0.64 A, but contains an additional helical element at the linker between the two subdomains of VP35 IID. Mutations near the linker, including swapping sequences between REBOV and ZEBOV, reveal that the linker sequence has limited tolerance for variability. Together with the previously solved ligand-free and double-stranded-RNA-bound forms of ZEBOV VP35 IID structures, our current studies on REBOV VP35 IID reinforce the importance of VP35 in immune suppression. Functional differences observed between REBOV and ZEBOV VP35 proteins may contribute to observed differences in pathogenicity, but these are unlikely to be the major determinant. However, the high level of similarity in structure and the low tolerance for sequence variability, coupled with the multiple critical roles played by Ebola virus VP35 proteins, highlight the viability of VP35 as a potential target for therapeutic development., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Alphaviruses are mosquito-transmitted viruses that cause significant human disease, and understanding how these pathogens successfully transition from the mosquito vector to the vertebrate host is an important area of research. Previous studies demonstrated that mosquito and mammalian-cell-derived alphaviruses differentially induce type I interferons (alpha/beta interferon [IFN-alpha/beta]) in myeloid dendritic cells (mDCs), where the mosquito cell-derived virus is a poor inducer of IFN-alpha/beta compared to the mammalian-cell-derived virus. Furthermore, the reduced IFN-alpha/beta induction by the mosquito cell-derived virus is attributed to differential N-linked glycosylation. To further evaluate the role of viral envelope glycans in regulating the IFN-alpha/beta response, studies were performed to assess whether the mosquito cell-derived virus actively inhibits IFN-alpha/beta induction or is simply a poor inducer of IFN-alpha/beta. Coinfection studies using mammalian- and mosquito cell-derived Ross River virus (mam-RRV and mos-RRV, respectively) indicated that mos-RRV was unable to suppress IFN-alpha/beta induction by mam-RRV in mDC cultures. Additionally, a panel of mutant viruses lacking either individual or multiple N-linked glycosylation sites was used to demonstrate that N-linked glycans were essential for high-level IFN-alpha/beta induction by the mammalian-cell-derived virus. These results suggest that the failure of the mosquito cell-derived virus to induce IFN-alpha/beta is due to a lack of complex carbohydrates on the virion rather than the active suppression of the DC antiviral response.

A novel genetic vaccine that is based on a Venezuelan equine encephalitis virus (VEE) replicon launched from plasmid DNA is described. The plasmid encodes a VEE replicon under the transcriptional control of the cytomegalovirus immediate-early promoter (VEE DNA). The VEE DNA consistently expressed 3- to 15-fold more green fluorescent protein in vitro than did a conventional DNA vaccine. Furthermore, transfection with the DNA-launched VEE replicon induced apoptosis and type I interferon production. Inoculation of mice with VEE DNA encoding human immunodeficiency virus type 1 gp160 significantly increased humoral responses by several orders of magnitude compared to an equal dose of a conventional DNA vaccine. These increases were also observed at 10- and 100-fold-lower doses of the VEE DNA. Cellular immune responses measured by gamma interferon and interleukin 2 enzyme-linked immunospot assay were significantly higher in mice immunized with the VEE DNA at decreased doses. The immune responses induced by the VEE DNA-encoded antigen, however, were independent of an intact type I interferon signaling pathway. Moreover, the DNA-launched VEE replicon induced an efficient prime to a VEE replicon particle (VRP) boost, increasing humoral and cellular immunity by at least 1 order of magnitude compared to VEE DNA only. Importantly, immunization with VEE DNA, as opposed to VRP, did not induce any anti-VRP neutralizing antibodies. Increased potency of DNA vaccines and reduced vector immunity may ultimately have an impact on the design of vaccination strategies in humans.

Dendritic cells (DCs) are an important early target cell for many mosquito-borne viruses, and in many cases mosquito-cell-derived arboviruses more efficiently infect DCs than viruses derived from mammalian cells. However, whether mosquito-cell-derived viruses differ from mammalian-cell-derived viruses in their ability to induce antiviral responses in the infected dendritic cell has not been evaluated. In this report, alphaviruses, which are mosquito-borne viruses that cause diseases ranging from encephalitis to arthritis, were used to determine whether viruses grown in mosquito cells differed from mammalian-cell-derived viruses in their ability to induce type I interferon (IFN) responses in infected primary dendritic cells. Consistent with previous results, mosquito-cell-derived Ross River virus (mos-RRV) and Venezuelan equine encephalitis virus (mos-VEE) exhibited enhanced infection of primary myeloid dendritic cells (mDCs) compared to mammalian-cell-derived virus preparations. However, unlike the mammalian-cell-derived viruses, which induced high levels of type I IFN in the infected mDC cultures, mos-RRV and mos-VEE were poor IFN inducers. Furthermore, the poor IFN induction by mos-RRV contributed to the enhanced infection of mDCs by mos-RRV. These results suggest that the viruses initially delivered by the mosquito vector differ from those generated in subsequent rounds of replication in the host, not just with respect to their ability to infect dendritic cells but also in their ability to induce or inhibit antiviral type I IFN responses. This difference may have an important impact on the mosquito-borne virus's ability to successfully make the transition from the arthropod vector to the vertebrate host.

Mosquito-borne alphaviruses are a significant cause of both encephalitic and arthritic disease in humans worldwide. In contrast to the encephalitic alphaviruses, the pathogenesis of alphavirus-induced arthritic disease is not well understood. Utilizing a mouse model of Ross River virus (RRV) disease, we found that the primary targets of RRV infection are bone, joint, and skeletal muscle tissues of the hind limbs in both outbred CD-1 mice and adult C57BL/6J mice. Moreover, histological analyses demonstrated that RRV infection resulted in severe inflammation of these tissues. Characterization of the inflammatory infiltrate within the skeletal muscle tissue identified inflammatory macrophages, NK cells, and CD4+ and CD8+ T lymphocytes. To determine the contribution of the adaptive immune system, the outcome of RRV-induced disease was examined in C57BL/6J RAG-1(-/-) mice, which lack functional T and B lymphocytes. RAG-1(-/-) and wild-type mice developed similar disease signs, infiltration of inflammatory macrophages and NK cells, and muscle pathology, suggesting that the adaptive immune response does not play a critical role in the development of disease. These results establish the mouse model of RRV disease as a useful system for the identification of viral and host factors that contribute to alphavirus-induced arthritis and myositis.