Human Epistatic Interaction Controls IL7R Splicing and Increases Multiple Sclerosis Risk.

Multiple sclerosis (MS) is an autoimmune disorder where T cells attack neurons in the central nervous system (CNS) leading to demyelination and neurological deficits. A driver of increased MS risk is the soluble form of the interleukin-7 receptor alpha chain gene (sIL7R) produced by alternative splicing of IL7R exon 6. Here, we identified the RNA helicase DDX39B as a potent activator of this exon and consequently a repressor of sIL7R, and we found strong genetic association of DDX39B with MS risk. Indeed, we showed that a genetic variant in the 5' UTR of DDX39B reduces translation of DDX39B mRNAs and increases MS risk. Importantly, this DDX39B variant showed strong genetic and functional epistasis with allelic variants in IL7R exon 6. This study establishes the occurrence of biological epistasis in humans and provides mechanistic insight into the regulation of IL7R exon 6 splicing and its impact on MS risk.

The N501Y spike substitution enhances SARS-CoV-2 infection and transmission.

The B.1.1.7 variant (also known as Alpha) of SARS-CoV-2, the cause of the COVID-19 pandemic, emerged in the UK in the summer of 2020. The prevalence of this variant increased rapidly owing to an increase in infection and/or transmission efficiency1. The Alpha variant contains 19 nonsynonymous mutations across its viral genome, including 8 substitutions or deletions in the spike protein that interacts with cellular receptors to mediate infection and tropism. Here, using a reverse genetics approach, we show that of the 8 individual spike protein substitutions, only N501Y resulted in consistent fitness gains for replication in the upper airway in a hamster model as well as in primary human airway epithelial cells. The N501Y substitution recapitulated the enhanced viral transmission phenotype of the eight mutations in the Alpha spike protein, suggesting that it is a major determinant of the increased transmission of the Alpha variant. Mechanistically, the N501Y substitution increased the affinity of the viral spike protein for cellular receptors. As suggested by its convergent evolution in Brazil, South Africa and elsewhere2,3, our results indicate that N501Y substitution is an adaptive spike mutation of major concern.

Parsing the roles of DExD-box proteins DDX39A and DDX39B in alternative RNA splicing.

DExD-box RNA proteins DDX39A and DDX39B are highly homologous paralogs that are conserved in vertebrates. They are required for energy-driven reactions involved in RNA processing. Although we have some understanding of how their functions overlap in RNA nuclear export, our knowledge of whether or not these proteins have specific or redundant functions in RNA splicing is limited. Our previous work has shown that DDX39B is responsible for regulating the splicing of important immune transcripts IL7R and FOXP3. In this study, we aimed to investigate whether DDX39A, a highly homologous paralog of DDX39B, plays a similar role in regulating alternative RNA splicing. We find that DDX39A and DDX39B have significant redundancy in their gene targets, but there are targets that uniquely require one or the other paralog. For instance, DDX39A is incapable of complementing defective splicing of IL7R exon 6 when DDX39B is depleted. This exon and other cassette exons that specifically depend on DDX39B have U-poor/C-rich polypyrimidine tracts in the upstream intron and this variant polypyrimidine tract is required for DDX39B dependency. This study provides evidence that despite a high degree of functional redundancy, DDX39A and DDX39B are selectively required for the splicing of specific pre-mRNAs.

Definition of germ layer cell lineage alternative splicing programs reveals a critical role for Quaking in specifying cardiac cell fate.

Alternative splicing is critical for development; however, its role in the specification of the three embryonic germ layers is poorly understood. By performing RNA-Seq on human embryonic stem cells (hESCs) and derived definitive endoderm, cardiac mesoderm, and ectoderm cell lineages, we detect distinct alternative splicing programs associated with each lineage. The most prominent splicing program differences are observed between definitive endoderm and cardiac mesoderm. Integrative multi-omics analyses link each program with lineage-enriched RNA binding protein regulators, and further suggest a widespread role for Quaking (QKI) in the specification of cardiac mesoderm. Remarkably, knockout of QKI disrupts the cardiac mesoderm-associated alternative splicing program and formation of myocytes. These changes arise in part through reduced expression of BIN1 splice variants linked to cardiac development. Mechanistically, we find that QKI represses inclusion of exon 7 in BIN1 pre-mRNA via an exonic ACUAA motif, and this is concomitant with intron removal and cleavage from chromatin. Collectively, our results uncover alternative splicing programs associated with the three germ lineages and demonstrate an important role for QKI in the formation of cardiac mesoderm.

RBFOX2 is required for establishing RNA regulatory networks essential for heart development.

Human genetic studies identified a strong association between loss of function mutations in RBFOX2 and hypoplastic left heart syndrome (HLHS). There are currently no Rbfox2 mouse models that recapitulate HLHS. Therefore, it is still unknown how RBFOX2 as an RNA binding protein contributes to heart development. To address this, we conditionally deleted Rbfox2 in embryonic mouse hearts and found profound defects in cardiac chamber and yolk sac vasculature formation. Importantly, our Rbfox2 conditional knockout mouse model recapitulated several molecular and phenotypic features of HLHS. To determine the molecular drivers of these cardiac defects, we performed RNA-sequencing in Rbfox2 mutant hearts and identified dysregulated alternative splicing (AS) networks that affect cell adhesion to extracellular matrix (ECM) mediated by Rho GTPases. We identified two Rho GTPase cycling genes as targets of RBFOX2. Modulating AS of these two genes using antisense oligos led to cell cycle and cell-ECM adhesion defects. Consistently, Rbfox2 mutant hearts displayed cell cycle defects and inability to undergo endocardial-mesenchymal transition, processes dependent on cell-ECM adhesion and that are seen in HLHS. Overall, our work not only revealed that loss of Rbfox2 leads to heart development defects resembling HLHS, but also identified RBFOX2-regulated AS networks that influence cell-ECM communication vital for heart development.

Ebola Virus Produces Discrete Small Noncoding RNAs Independently of the Host MicroRNA Pathway Which Lack RNA Interference Activity in Bat and Human Cells.

The question as to whether RNA viruses produce bona fide microRNAs (miRNAs) during infection has been the focus of intense research and debate. Recently, several groups using computational prediction methods have independently reported possible miRNA candidates produced by Ebola virus (EBOV). Additionally, efforts to detect these predicted RNA products in samples from infected animals and humans have produced positive results. However, these studies and their conclusions are predicated on the assumption that these RNA products are actually processed through, and function within, the miRNA pathway. In the present study, we performed the first rigorous assessment of the ability of filoviruses to produce miRNA products during infection of both human and bat cells. Using next-generation sequencing, we detected several candidate miRNAs from both EBOV and the closely related Marburg virus (MARV). Focusing our validation efforts on EBOV, we found evidence contrary to the idea that these small RNA products function as miRNAs. The results of our study are important because they highlight the potential pitfalls of relying on computational methods alone for virus miRNA discovery.IMPORTANCE Here, we report the discovery, via deep sequencing, of numerous noncoding RNAs (ncRNAs) derived from both EBOV and MARV during infection of both bat and human cell lines. In addition to identifying several novel ncRNAs from both viruses, we identified two EBOV ncRNAs in our sequencing data that were near-matches to computationally predicted viral miRNAs reported in the literature. Using molecular and immunological techniques, we assessed the potential of EBOV ncRNAs to function as viral miRNAs. Importantly, we found little evidence supporting this hypothesis. Our work is significant because it represents the first rigorous assessment of the potential for EBOV to encode viral miRNAs and provides evidence contrary to the existing paradigm regarding the biological role of computationally predicted EBOV ncRNAs. Moreover, our work highlights further avenues of research regarding the nature and function of EBOV ncRNAs.

Severe Acute Respiratory Syndrome Coronavirus 2 from Patient with Coronavirus Disease, United States.

The etiologic agent of an outbreak of pneumonia in Wuhan, China, was identified as severe acute respiratory syndrome coronavirus 2 in January 2020. A patient in the United States was given a diagnosis of infection with this virus by the state of Washington and the US Centers for Disease Control and Prevention on January 20, 2020. We isolated virus from nasopharyngeal and oropharyngeal specimens from this patient and characterized the viral sequence, replication properties, and cell culture tropism. We found that the virus replicates to high titer in Vero-CCL81 cells and Vero E6 cells in the absence of trypsin. We also deposited the virus into 2 virus repositories, making it broadly available to the public health and research communities. We hope that open access to this reagent will expedite development of medical countermeasures.

Acupuncture Reduces Hypertrophy and Cardiac Fibrosis, and Improves Heart Function in Mice with Diabetic Cardiomyopathy.

PURPOSE: To assess the effects of electro-acupuncture (EA) on glycemic control, myocardial inflammation, and the progression of diabetic cardiomyopathy in mice with type 2 diabetes. METHODS: Db/Db mice received EA at PC6+ST36 (DM-Acu), non-acupoint simulation (DM-Sham), or no treatment (DM). EA was applied for 30 min per day, 5 days a week for 4 weeks. Heart function was assessed by echocardiography. Myocardium was assessed by RT-PCR, immunoblotting, and histology. Serum TNF-α, IL-1α, IL-1ß, IL-6, and IL-8 were measured. RESULTS: DM-Acu, but not DM-Sham, reduced fasting blood glucose without affecting body weight. DM decreased systolic function. DM-Acu, but not DM-Sham, attenuated the decrease in systolic function. Heart weight was significantly smaller in the DM-Acu than in the DM and DM-Sham groups. Percent fibrosis and apoptosis were reduced in the DM-Acu, but not the DM-Sham, group. Serum levels of IL-1α, IL-1ß, IL-6, IL-8, ICAM-1, MCP-1, and TNF-α were significantly lower in the DM-Acu than in the DM or DM-Sham groups. Protein levels of P-Akt and P-AMPK and mRNA levels of phosphoinositide-3-kinase regulatory subunit 6 (PIK3r6) were significantly higher in the DM-Acu group. Myocardial mRNA and protein levels of insulin-like growth factor 1 receptor (IGF1R) were significantly lower in the DM and DM-Sham groups compared with the DM-Acu group. CONCLUSIONS: EA reduced serum glucose; prevented DM-induced hypertrophy and deterioration of systolic function, inflammation, and fibrosis; and restored IGF1R, P-Akt, and P-AMPK levels in mice with type 2 diabetes mellitus.

Type II Epithelial-Mesenchymal Transition Upregulates Protein N-Glycosylation To Maintain Proteostasis and Extracellular Matrix Production.

Type II epithelial-mesenchymal transition (EMT) plays a vital role in airway injury, repair, and remodeling. Triggered by growth factors, such as transforming growth factor beta (TGFß), EMT induced a biological process that converts epithelial cells into secretory mesenchymal cells with a substantially increased production of extracellular matrix (ECM) proteins. Epithelial cells are not professional secretory cells and produce few ECM proteins under normal conditions. The molecular mechanism underlying the transformation of the protein factory and secretory machinery during EMT is significant because ECM secretion is central to the pathogenesis of airway remodeling. Here we report that type II EMT upregulates the protein N-glycosylation of ECMs. The mechanism study reveals that the substantial increase in synthesis of ECM proteins in EMT activates the inositol-requiring protein 1 (IRE1α)-X-box-binding protein 1 (XBP1) axis of the unfolded protein response (UPR) coupled to the hexosamine biosynthesis pathway (HBP). These two pathways coordinately up-regulate the protein N-glycosylation of ECM proteins and increase ER folding capacity and ER-associated degradation (ERAD), which improve ER protein homeostasis and protect transitioned cells from proteotoxicity. Inhibition of the alternative splicing of XBP1 or protein N-glycosylation blocks ECM protein secretion, indicating the XBP1-HBP plays a prominent role in regulating the secretion of ECM proteins in the mesenchymal transition. Our data suggest that the activation of XBP1-HBP pathways and elevation of protein N-glycosylation is an adaptive response to maintain protein quality control and facilitate the secretion of ECM proteins during the mesenchymal transition. The components of the XBP1-HBP pathways may be therapeutic targets to prevent airway remodeling.

The NFκB subunit RELA is a master transcriptional regulator of the committed epithelial-mesenchymal transition in airway epithelial cells.

The epithelial-mesenchymal transition (EMT) is a multistep dedifferentiation program important in tissue repair. Here, we examined the role of the transcriptional regulator NF-κB in EMT of primary human small airway epithelial cells (hSAECs). Surprisingly, transforming growth factor ß (TGFß) activated NF-κB/RELA proto-oncogene, NF-κB subunit (RELA) translocation within 1 day of stimulation, yet induction of its downstream gene regulatory network occurred only after 3 days. A time course of TGFß-induced EMT transition was analyzed by RNA-Seq in the absence or presence of inducible shRNA-mediated silencing of RELA. In WT cells, TGFß stimulation significantly affected the expression of 2,441 genes. Gene set enrichment analysis identified WNT, cadherin, and NF-κB signaling as the most prominent TGFß-inducible pathways. By comparison, RELA controlled expression of 3,138 overlapping genes mapping to WNT, cadherin, and chemokine signaling pathways. Conducting upstream regulator analysis, we found that RELA controls six clusters of upstream transcription factors, many of which overlapped with a transcription factor topology map of EMT developed earlier. RELA triggered expression of three key EMT pathways: 1) the WNT/ß-catenin morphogen pathway, 2) the JUN transcription factor, and 3) the Snail family transcriptional repressor 1 (SNAI1). RELA binding to target genes was confirmed by ChIP. Experiments independently validating WNT dependence on RELA were performed by silencing RELA via genome editing and indicated that TGFß-induced WNT5B expression and downstream activation of the WNT target AXIN2 are RELA-dependent. We conclude that RELA is a master transcriptional regulator of EMT upstream of WNT morphogen, JUN, SNAI1-ZEB1, and interleukin-6 autocrine loops.

Genomic characterisation of Cuiaba and Charleville viruses: arboviruses (family Rhabdoviridae, genus Sripuvirus) infecting reptiles and amphibians.

Viruses in the family Rhabdoviridae are ecologically very diverse, infecting mammals, birds, reptiles, fish, plants and a wide range of other terrestrial and aquatic invertebrates. The genus Sripuvirus currently comprises five viruses that appear to circulate in transmission cycles involving reptiles and sandflies. Here, we report an analysis of the complete coding sequences of Cuiaba virus (CUIV), isolated from an amphibian (Bufo marinus) collected in Brazil, and Charleville virus (CHVV), isolated from sandflies (Phlebotomus sp.) and lizards (Gehyra australis), collected in Australia. CUIV and CHVV cluster phylogenetically with the sripuviruses in maximum likelihood trees generated from complete L protein (RdRp) sequences. They also share with sripuviruses unique features in genome organisation, including an additional gene (U1) between the matrix protein (M) gene and glycoprotein (G) gene, and an alternative long open reading frame near the start of the G ORF that encodes a predicted transmembrane protein. In view of their phylogenetic relationships, similar genome organisations and similar ecological characteristics, we propose the assignment of CUIV and CHVV as novel members of the genus Sripuvirus.

Comparative Transcriptomics in Ebola Makona-Infected Ferrets, Nonhuman Primates, and Humans.

The domestic ferret is a uniformly lethal model of infection for 3 species of Ebolavirus known to be pathogenic in humans. Reagents to systematically analyze the ferret host response to infection are lacking; however, the recent publication of a draft ferret genome has opened the potential for transcriptional analysis of ferret models of disease. In this work, we present comparative analysis of longitudinally sampled blood taken from ferrets and nonhuman primates infected with lethal doses of the Makona variant of Zaire ebolavirus. Strong induction of proinflammatory and prothrombotic signaling programs were present in both ferrets and nonhuman primates, and both transcriptomes were similar to previously published datasets of fatal cases of human Ebola virus infection.

Terrestrial Bird Migration and West Nile Virus Circulation, United States.

Host migration and emerging pathogens are strongly associated, especially with regard to zoonotic diseases. West Nile virus (WNV), a mosquitoborne pathogen capable of causing severe, sometimes fatal, neuroinvasive disease in humans, is maintained in highly mobile avian hosts. Using phylogeographic approaches, we investigated the relationship between WNV circulation in the United States and the flight paths of terrestrial birds. We demonstrated southward migration of WNV in the eastern flyway and northward migration in the central flyway, which is consistent with the looped flight paths of many terrestrial birds. We also identified 3 optimal locations for targeted WNV surveillance campaigns in the United States-Illinois, New York, and Texas. These results illustrate the value of multidisciplinary approaches to surveillance of infectious diseases, especially zoonotic diseases.

Divergent Viruses Discovered in Arthropods and Vertebrates Revise the Evolutionary History of the Flaviviridae and Related Viruses.

UNLABELLED: Viruses of the family Flaviviridae are important pathogens of humans and other animals and are currently classified into four genera. To better understand their diversity, evolutionary history, and genomic flexibility, we used transcriptome sequencing (RNA-seq) to search for the viruses related to the Flaviviridae in a range of potential invertebrate and vertebrate hosts. Accordingly, we recovered the full genomes of five segmented jingmenviruses and 12 distant relatives of the known Flaviviridae ("flavi-like" viruses) from a range of arthropod species. Although these viruses are highly divergent, they share a similar genomic plan and common ancestry with the Flaviviridae in the NS3 and NS5 regions. Remarkably, although these viruses fill in major gaps in the phylogenetic diversity of the Flaviviridae, genomic comparisons reveal important changes in genome structure, genome size, and replication/gene regulation strategy during evolutionary history. In addition, the wide diversity of flavi-like viruses found in invertebrates, as well as their deep phylogenetic positions, suggests that they may represent the ancestral forms from which the vertebrate-infecting viruses evolved. For the vertebrate viruses, we expanded the previously mammal-only pegivirus-hepacivirus group to include a virus from the graceful catshark (Proscyllium habereri), which in turn implies that these viruses possess a larger host range than is currently known. In sum, our data show that the Flaviviridae infect a far wider range of hosts and exhibit greater diversity in genome structure than previously anticipated. IMPORTANCE: The family Flaviviridae of RNA viruses contains several notorious human pathogens, including dengue virus, West Nile virus, and hepatitis C virus. To date, however, our understanding of the biodiversity and evolution of the Flaviviridae has largely been directed toward vertebrate hosts and their blood-feeding arthropod vectors. Therefore, we investigated an expanded group of potential arthropod and vertebrate host species that have generally been ignored by surveillance programs. Remarkably, these species contained diverse flaviviruses and related viruses that are characterized by major changes in genome size and genome structure, such that these traits are more flexible than previously thought. More generally, these data suggest that arthropods may be the ultimate reservoir of the Flaviviridae and related viruses, harboring considerable genetic and phenotypic diversity. In sum, this study revises the traditional view on the evolutionary history, host range, and genomic structures of a major group of RNA viruses.

Evolution of genome size and complexity in the rhabdoviridae.

RNA viruses exhibit substantial structural, ecological and genomic diversity. However, genome size in RNA viruses is likely limited by a high mutation rate, resulting in the evolution of various mechanisms to increase complexity while minimising genome expansion. Here we conduct a large-scale analysis of the genome sequences of 99 animal rhabdoviruses, including 45 genomes which we determined de novo, to identify patterns of genome expansion and the evolution of genome complexity. All but seven of the rhabdoviruses clustered into 17 well-supported monophyletic groups, of which eight corresponded to established genera, seven were assigned as new genera, and two were taxonomically ambiguous. We show that the acquisition and loss of new genes appears to have been a central theme of rhabdovirus evolution, and has been associated with the appearance of alternative, overlapping and consecutive ORFs within the major structural protein genes, and the insertion and loss of additional ORFs in each gene junction in a clade-specific manner. Changes in the lengths of gene junctions accounted for as much as 48.5% of the variation in genome size from the smallest to the largest genome, and the frequency with which new ORFs were observed increased in the 3' to 5' direction along the genome. We also identify several new families of accessory genes encoded in these regions, and show that non-canonical expression strategies involving TURBS-like termination-reinitiation, ribosomal frame-shifts and leaky ribosomal scanning appear to be common. We conclude that rhabdoviruses have an unusual capacity for genomic plasticity that may be linked to their discontinuous transcription strategy from the negative-sense single-stranded RNA genome, and propose a model that accounts for the regular occurrence of genome expansion and contraction throughout the evolution of the Rhabdoviridae.

Outbreak of Zika Virus Infection, Chiapas State, Mexico, 2015, and First Confirmed Transmission by Aedes aegypti Mosquitoes in the Americas.

BACKGROUND: After decades of obscurity, Zika virus (ZIKV) has spread through the Americas since 2015 accompanied by congenital microcephaly and Guillain-Barré syndrome. Although these epidemics presumably involve transmission by Aedes aegypti, no direct evidence of vector involvement has been reported, prompting speculation that other mosquitoes such as Culex quinquefasciatus could be involved. METHODS: We detected an outbreak of ZIKV infection in southern Mexico in late 2015. Sera from suspected ZIKV-infected patients were analyzed for viral RNA and antibodies. Mosquitoes were collected in and around patient homes and tested for ZIKV. RESULTS: Of 119 suspected ZIKV-infected patients, 25 (21%) were confirmed by RT-PCR of serum collected 1-8 days after the onset of signs and symptoms including rash, arthralgia, headache, pruritus, myalgia, and fever. Of 796 mosquitoes collected, A. aegypti yielded ZIKV detection by RT-PCR in 15 of 55 pools (27.3%). No ZIKV was detected in C. quinquefasciatus ZIKV sequences derived from sera and mosquitoes showed a monophyletic relationship suggestive of a point source introduction from Guatemala. CONCLUSIONS: These results demonstrate the continued, rapid northward progression of ZIKV into North America with typically mild disease manifestations, and implicate A. aegypti for the first time as a principal vector in North America.

Different Temporal Effects of Ebola Virus VP35 and VP24 Proteins on Global Gene Expression in Human Dendritic Cells.

UNLABELLED: Ebola virus (EBOV) causes a severe hemorrhagic fever with a deficient immune response, lymphopenia, and lymphocyte apoptosis. Dendritic cells (DC), which trigger the adaptive response, do not mature despite EBOV infection. We recently demonstrated that DC maturation is unblocked by disabling the innate response antagonizing domains (IRADs) in EBOV VP35 and VP24 by the mutations R312A and K142A, respectively. Here we analyzed the effects of VP35 and VP24 with the IRADs disabled on global gene expression in human DC. Human monocyte-derived DC were infected by wild-type (wt) EBOV or EBOVs carrying the mutation in VP35 (EBOV/VP35m), VP24 (EBOV/VP24m), or both (EBOV/VP35m/VP24m). Global gene expression at 8 and 24 h was analyzed by deep sequencing, and the expression of interferon (IFN) subtypes up to 5 days postinfection was analyzed by quantitative reverse transcription-PCR (qRT-PCR). wt EBOV induced a weak global gene expression response, including markers of DC maturation, cytokines, chemokines, chemokine receptors, and multiple IFNs. The VP35 mutation unblocked the expression, resulting in a dramatic increase in expression of these transcripts at 8 and 24 h. Surprisingly, DC infected with EBOV/VP24m expressed lower levels of many of these transcripts at 8 h after infection, compared to wt EBOV. In contrast, at 24 h, expression of the transcripts increased in DC infected with any of the three mutants, compared to wt EBOV. Moreover, sets of genes affected by the two mutations only partially overlapped. Pathway analysis demonstrated that the VP35 mutation unblocked pathways involved in antigen processing and presentation and IFN signaling. These data suggest that EBOV IRADs have profound effects on the host adaptive immune response through massive transcriptional downregulation of DC. IMPORTANCE: This study shows that infection of DC with EBOV, but not its mutant forms with the VP35 IRAD and/or VP24 IRAD disabled, causes a global block in expression of host genes. The temporal effects of mutations disrupting the two IRADs differ, and the lists of affected genes only partially overlap such that VP35 and VP24 IRADs each have profound effects on antigen presentation by exposed DC. The global modulation of DC gene expression and the resulting lack of their maturation represent a major mechanism by which EBOV disables the T cell response and suggests that these suppressive pathways are a therapeutic target that may unleash the T cell responses during EBOV infection.

A newly isolated reovirus has the simplest genomic and structural organization of any reovirus.

UNLABELLED: A total of 2,691 mosquitoes representing 17 species was collected from eight locations in southwest Cameroon and screened for pathogenic viruses. Ten isolates of a novel reovirus (genus Dinovernavirus) were detected by culturing mosquito pools on Aedes albopictus (C6/36) cell cultures. A virus that caused overt cytopathic effects was isolated, but it did not infect vertebrate cells or produce detectable disease in infant mice after intracerebral inoculation. The virus, tentatively designated Fako virus (FAKV), represents the first 9-segment, double-stranded RNA (dsRNA) virus to be isolated in nature. FAKV appears to have a broad mosquito host range, and its detection in male specimens suggests mosquito-to-mosquito transmission in nature. The structure of the T=1 FAKV virion, determined to subnanometer resolution by cryoelectron microscopy (cryo-EM), showed only four proteins per icosahedral asymmetric unit: a dimer of the major capsid protein, one turret protein, and one clamp protein. While all other turreted reoviruses of known structures have at least two copies of the clamp protein per asymmetric unit, FAKV's clamp protein bound at only one conformer of the major capsid protein. The FAKV capsid architecture and genome organization represent the most simplified reovirus described to date, and phylogenetic analysis suggests that it arose from a more complex ancestor by serial loss-of-function events. IMPORTANCE: We describe the detection, genetic, phenotypic, and structural characteristics of a novel Dinovernavirus species isolated from mosquitoes collected in Cameroon. The virus, tentatively designated Fako virus (FAKV), is related to both single-shelled and partially double-shelled viruses. The only other described virus in this genus was isolated from cultured mosquito cells. It was previously unclear whether the phenotypic characteristics of that virus were reflective of this genus in nature or were altered during serial passaging in the chronically infected cell line. FAKV is a naturally occurring single-shelled reovirus with a unique virion architecture that lacks several key structural elements thought to stabilize a single-shelled reovirus virion, suggesting what may be the minimal number of proteins needed to form a viable reovirus particle. FAKV evolved from more complex ancestors by losing a genome segment and several virion proteins.

Analysis of the TGFß-induced program in primary airway epithelial cells shows essential role of NF-κB/RelA signaling network in type II epithelial mesenchymal transition.

BACKGROUND: The airway epithelial cell plays a central role in coordinating the pulmonary response to injury and inflammation. Here, transforming growth factor-ß (TGFß) activates gene expression programs to induce stem cell-like properties, inhibit expression of differentiated epithelial adhesion proteins and express mesenchymal contractile proteins. This process is known as epithelial mesenchymal transition (EMT); although much is known about the role of EMT in cellular metastasis in an oncogene-transformed cell, less is known about Type II EMT, that occurring in normal epithelial cells. In this study, we applied next generation sequencing (RNA-Seq) in primary human airway epithelial cells to understand the gene program controlling Type II EMT and how cytokine-induced inflammation modifies it. RESULTS: Generalized linear modeling was performed on a two-factor RNA-Seq experiment of 6 treatments of telomerase immortalized human small airway epithelial cells (3 replicates). Using a stringent cut-off, we identified 3,478 differentially expressed genes (DEGs) in response to EMT. Unbiased transcription factor enrichment analysis identified three clusters of EMT regulators, one including SMADs/TP63 and another NF-κB/RelA. Surprisingly, we also observed 527 of the EMT DEGs were also regulated by the TNF-NF-κB/RelA pathway. This Type II EMT program was compared to Type III EMT in TGFß stimulated A549 alveolar lung cancer cells, revealing significant functional differences. Moreover, we observe that Type II EMT modifies the outcome of the TNF program, reducing IFN signaling and enhancing integrin signaling. We confirmed experimentally that TGFß-induced the NF-κB/RelA pathway by observing a 2-fold change in NF-κB/RelA nuclear translocation. A small molecule IKK inhibitor blocked TGFß-induced core transcription factor (SNAIL1, ZEB1 and Twist1) and mesenchymal gene (FN1 and VIM) expression. CONCLUSIONS: These data indicate that NF-κB/RelA controls a SMAD-independent gene network whose regulation is required for initiation of Type II EMT. Type II EMT dramatically affects the induction and kinetics of TNF-dependent gene networks.

Itaya virus, a Novel Orthobunyavirus Associated with Human Febrile Illness, Peru.

Our genetic analyses of uncharacterized bunyaviruses isolated in Peru identified a possible reassortant virus containing small and large gene segment sequences closely related to the Caraparu virus and a medium gene segment sequence potentially derived from an unidentified group C orthobunyavirus. Neutralization tests confirmed serologic distinction among the newly identified virus and the prototype and Caraparu strains. This virus, named Itaya, was isolated in 1999 and 2006 from febrile patients in the cities of Iquitos and Yurimaguas in Peru. The geographic distance between the 2 cases suggests that the Itaya virus could be widely distributed throughout the Amazon basin in northeastern Peru. Identification of a new Orthobunyavirus species that causes febrile disease in humans reinforces the need to expand viral disease surveillance in tropical regions of South America.