Search

Significance Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are of concern, with the P.1 variants dominating in Brazil. Brazil is now seeing a record number of deaths. Here, we report that the pathogenicity in hamsters of a P.1 variant is similar to that of nonvariant SARS-CoV-2. However, it has an expanded host range as shown by its replication in mice. Prior infection with nonvariant SARS-CoV-2 strains efficiently prevented replication of the P.1 variant in the lower respiratory tract of hamsters upon reinfection. Convalescent sera from patients infected with nonvariants or sera from messenger RNA vaccinees showed comparable neutralization titers among the P.1 and previously circulating strains. These results suggest that previous SARS-CoV-2 infection and vaccines based on the original SARS-CoV-2 will provide some protection against P.1 infection., The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a key role in viral infectivity. It is also the major antigen stimulating the host's protective immune response, specifically, the production of neutralizing antibodies. Recently, a new variant of SARS-CoV-2 possessing multiple mutations in the S protein, designated P.1, emerged in Brazil. Here, we characterized a P.1 variant isolated in Japan by using Syrian hamsters, a well-established small animal model for the study of SARS-CoV-2 disease (COVID-19). In hamsters, the variant showed replicative abilities and pathogenicity similar to those of early and contemporary strains (i.e., SARS-CoV-2 bearing aspartic acid [D] or glycine [G] at position 614 of the S protein). Sera and/or plasma from convalescent patients and BNT162b2 messenger RNA vaccinees showed comparable neutralization titers across the P.1 variant, S-614D, and S-614G strains. In contrast, the S-614D and S-614G strains were less well recognized than the P.1 variant by serum from a P.1-infected patient. Prior infection with S-614D or S-614G strains efficiently prevented the replication of the P.1 variant in the lower respiratory tract of hamsters upon reinfection. In addition, passive transfer of neutralizing antibodies to hamsters infected with the P.1 variant or the S-614G strain led to reduced virus replication in the lower respiratory tract. However, the effect was less pronounced against the P.1 variant than the S-614G strain. These findings suggest that the P.1 variant may be somewhat antigenically different from the early and contemporary strains of SARS-CoV-2.

Background Representing biological networks as graphs is a powerful approach to reveal underlying patterns, signatures, and critical components from high-throughput biomolecular data. However, graphs do not natively capture the multi-way relationships present among genes and proteins in biological systems. Hypergraphs are generalizations of graphs that naturally model multi-way relationships and have shown promise in modeling systems such as protein complexes and metabolic reactions. In this paper we seek to understand how hypergraphs can more faithfully identify, and potentially predict, important genes based on complex relationships inferred from genomic expression data sets. Results We compiled a novel data set of transcriptional host response to pathogenic viral infections and formulated relationships between genes as a hypergraph where hyperedges represent significantly perturbed genes, and vertices represent individual biological samples with specific experimental conditions. We find that hypergraph betweenness centrality is a superior method for identification of genes important to viral response when compared with graph centrality. Conclusions Our results demonstrate the utility of using hypergraphs to represent complex biological systems and highlight central important responses in common to a variety of highly pathogenic viruses.

The 2013–2016 Ebola virus outbreak in West Africa was the largest and deadliest outbreak to date. Here we conducted a serological study to examine the antibody levels in survivors and the seroconversion in close contacts who took care of Ebola-infected individuals, but did not develop symptoms of Ebola virus disease. In March 2017, we collected blood samples from 481 individuals in Makeni, Sierra Leone: 214 survivors and 267 close contacts. Using commercial, quantitative ELISAs, we tested the plasma for IgG-specific antibodies against three major viral antigens: GP, the only viral glycoprotein expressed on the virus surface; NP, the most abundant viral protein; and VP40, a major structural protein of Zaire ebolavirus. We also determined neutralizing antibody titers. In the cohort of Ebola survivors, 97.7% of samples (209/214) had measurable antibody levels against GP, NP, and/or VP40. Of these positive samples, all but one had measurable neutralizing antibody titers against Ebola virus. For the close contacts, up to 12.7% (34/267) may have experienced a subclinical virus infection as indicated by detectable antibodies against GP. Further investigation is warranted to determine whether these close contacts truly experienced subclinical infections and whether these asymptomatic infections played a role in the dynamics of transmission., Author summary As the causative agent of an often lethal hemorrhagic fever disease in humans and nonhuman primates, Zaire ebolavirus typically causes high fever, severe diarrhea, and vomiting which results in case fatality rates as high as 90%. The 2013–2016 outbreak in West Africa was the largest and most devastating Ebola outbreak to date resulting in over 28,600 identified human cases and 11,300 deaths. Though our knowledge of virus transmission is incomplete, we do know that transmission occurs through direct contact with virus-contaminated body fluids (blood, secretions, or other body fluids), materials such as bedding contaminated with these fluids, and through the handling and preparation of contaminated food. Asymptomatic Ebola virus infections that result in seroconversion in the absence of disease symptoms have been observed both in humans and experimentally in animal models. In the present serology study, we determined a majority of Ebola survivors in our cohort had measurable antibody levels against at least one viral antigen, as expected. In our cohort of close contacts, relatives and health care workers who took care of Ebola-infected individuals during the outbreak, we observed a rate of seroprevalence of 12.7% as indicated by detectable GP antibody levels. Given that Ebola virus is typically associated with a highly lethal disease in humans, it is of great interest to determine the host-virus interactions and transmission dynamics associated with asymptomatic cases.

Ebola virus disease (EVD) often leads to severe and fatal outcomes in humans with early supportive care increasing the chances of survival. Profiling the human plasma lipidome provides insight into critical illness as well as diseased states, as lipids have essential roles as membrane structural components, signaling molecules, and energy sources. Here we show that the plasma lipidomes of EVD survivors and fatalities from Sierra Leone, infected during the 2014–2016 Ebola virus outbreak, were profoundly altered. Focusing on how lipids are associated in human plasma, while factoring in the state of critical illness, we found that lipidome changes were related to EVD outcome and could identify states of disease and recovery. Specific changes in the lipidome suggested contributions from extracellular vesicles, viremia, liver dysfunction, apoptosis, autophagy, and general critical illness, and we identified possible targets for therapies enhancing EVD survival.

Significance Both highly pathogenic avian influenza virus and Middle East respiratory syndrome coronavirus (MERS-CoV) infections are characterized by severe disease and high mortality. The continued threat of their emergence from zoonotic populations underscores an important need to understand the dynamics of their infection. By comparing the host responses across other related respiratory virus infections, these studies have identified a common avenue used by MERS-CoV and A/influenza/Vietnam/1203/2004 (H5N1-VN1203) influenza to antagonize antigen presentation through epigenetic modulation. Overall, the use of cross-comparisons provides an additional approach to leverage systems biology data to identify key pathways and strategies used by viruses to subvert host immune responses and may be critical in developing both vaccines and therapeutic treatment., Convergent evolution dictates that diverse groups of viruses will target both similar and distinct host pathways to manipulate the immune response and improve infection. In this study, we sought to leverage this uneven viral antagonism to identify critical host factors that govern disease outcome. Utilizing a systems-based approach, we examined differential regulation of IFN-γ–dependent genes following infection with robust respiratory viruses including influenza viruses [A/influenza/Vietnam/1203/2004 (H5N1-VN1203) and A/influenza/California/04/2009 (H1N1-CA04)] and coronaviruses [severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV)]. Categorizing by function, we observed down-regulation of gene expression associated with antigen presentation following both H5N1-VN1203 and MERS-CoV infection. Further examination revealed global down-regulation of antigen-presentation gene expression, which was confirmed by proteomics for both H5N1-VN1203 and MERS-CoV infection. Importantly, epigenetic analysis suggested that DNA methylation, rather than histone modification, plays a crucial role in MERS-CoV–mediated antagonism of antigen-presentation gene expression; in contrast, H5N1-VN1203 likely utilizes a combination of epigenetic mechanisms to target antigen presentation. Together, the results indicate a common mechanism utilized by H5N1-VN1203 and MERS-CoV to modulate antigen presentation and the host adaptive immune response.

Influenza A viral ribonucleoprotein (vRNP) complexes comprise the eight genomic negative-sense RNAs, each of which is bound to multiple copies of the vRNP and a trimeric viral polymerase complex. The influenza virus life cycle centres on the vRNPs, which in turn rely on host cellular processes to carry out functions that are necessary for the successful completion of the virus life cycle. In this Review, we discuss our current knowledge about vRNP trafficking within host cells and the function of these complexes in the context of the virus life cycle, highlighting how structure contributes to function and the crucial interactions with host cell pathways, as well as on the information gaps that remain. An improved understanding of how vRNPs use host cell pathways is essential to identify mechanisms of virus pathogenicity, host adaptation and, ultimately, new targets for antiviral intervention.

Influenza A viruses (IAVs) cause epidemics and pandemics that result in considerable financial burden and loss of human life. To manage annual IAV epidemics and prepare for future pandemics, an improved understanding of how IAVs emerge, transmit, cause disease and acquire pandemic potential is urgently needed. Fundamental techniques essential for procuring such knowledge are IAV isolation and culture from experimental and surveillance samples. Here we present a detailed protocol for IAV sample collection and processing, amplification in chicken eggs or mammalian cells, and identification from samples containing unknown pathogens. This protocol is robust, and it allows for the generation of virus cultures that can be used for downstream analyses. Once experimental or surveillance samples are obtained, virus cultures can be generated and the presence of IAVs can be verified in 3-5 d via reverse-transcription (RT)-PCR or hemagglutination assay. Increased time frames may be required for less experienced laboratory personnel, or when large numbers of samples will be processed.

The outcome of respiratory virus infection is determined by a complex interplay of viral and host factors. Some potentially important host factors for the antiviral response, whose functions remain largely unexplored, are long non-coding RNAs (lncRNAs). Here we systematically inferred the regulatory functions of host lncRNAs in response to influenza A virus and severe acute respiratory syndrome coronavirus (SARS-CoV) based on their similarity in expression with genes of known function. We performed total RNA-Seq on viral-infected lungs from eight mouse strains, yielding a large data set of transcriptional responses. Overall 5,329 lncRNAs were differentially expressed after infection. Most of the lncRNAs were co-expressed with coding genes in modules enriched in genes associated with lung homeostasis pathways or immune response processes. Each lncRNA was further individually annotated using a rank-based method, enabling us to associate 5,295 lncRNAs to at least one gene set and to predict their potential cis effects. We validated the lncRNAs predicted to be interferon-stimulated by profiling mouse responses after interferon-α treatment. Altogether, these results provide a broad categorization of potential lncRNA functions and identify subsets of lncRNAs with likely key roles in respiratory virus pathogenesis. These data are fully accessible through the MOuse NOn-Code Lung interactive database (MONOCLdb).

Influenza infection of humans remains an important public health problem. Vaccine strategies result in a significant but only partial control (65–85%) of infection. Thus, chemotherapeutic approaches are needed to provide a solution both for vaccine failures and to limit infection in the unvaccinated population. Previously (Walsh et al., 2011; Teijaro et al., 2011) documented that sphingosine-1-phosphate 1 receptor (S1P1R) agonists significantly protected mice against pathogenic H1N1 influenza virus by limiting immunopathologic damage while allowing host control of the infection. Here we extend that observation by documenting S1P1R agonist can control pathogenic H1N1 influenza infection in ferrets. S1P1R agonist was more effective in reducing pulmonary injury than the antiviral drug oseltamivir but, importantly, combined therapy was significantly more effective than either therapy alone.

Avian influenza A viruses rarely infect humans; however, when human infection and subsequent human-to-human transmission occurs, worldwide outbreaks (pandemics) can result. The recent sporadic infections of humans in China with a previously unrecognized avian influenza A virus of the H7N9 subtype (A(H7N9)) have caused concern owing to the appreciable case fatality rate associated with these infections (more than 25%), potential instances of human-to-human transmission, and the lack of pre-existing immunity among humans to viruses of this subtype. Here we characterize two early human A(H7N9) isolates, A/Anhui/1/2013 (H7N9) and A/Shanghai/1/2013 (H7N9); hereafter referred to as Anhui/1 and Shanghai/1, respectively. In mice, Anhui/1 and Shanghai/1 were more pathogenic than a control avian H7N9 virus (A/duck/Gunma/466/2011 (H7N9); Dk/GM466) and a representative pandemic 2009 H1N1 virus (A/California/4/2009 (H1N1pdm09); CA04). Anhui/1, Shanghai/1 and Dk/GM466 replicated well in the nasal turbinates of ferrets. In nonhuman primates, Anhui/1 and Dk/GM466 replicated efficiently in the upper and lower respiratory tracts, whereas the replicative ability of conventional human influenza viruses is typically restricted to the upper respiratory tract of infected primates. By contrast, Anhui/1 did not replicate well in miniature pigs after intranasal inoculation. Critically, Anhui/1 transmitted through respiratory droplets in one of three pairs of ferrets. Glycan arrays showed that Anhui/1, Shanghai/1 and A/Hangzhou/1/2013 (H7N9) (a third human A(H7N9) virus tested in this assay) bind to human virus-type receptors, a property that may be critical for virus transmissibility in ferrets. Anhui/1 was found to be less sensitive in mice to neuraminidase inhibitors than a pandemic H1N1 2009 virus, although both viruses were equally susceptible to an experimental antiviral polymerase inhibitor. The robust replicative ability in mice, ferrets and nonhuman primates and the limited transmissibility in ferrets of Anhui/1 suggest that A(H7N9) viruses have pandemic potential.

The continued emergence and spread of infectious agents is of great concern, and systems biology approaches to infectious disease research can advance our understanding of host-pathogen relationships and facilitate the development of new therapies and vaccines. Molecular characterization of infectious samples outside of appropriate biosafety containment can take place only subsequent to pathogen inactivation. Herein, we describe a modified Folch extraction using chloroform/methanol that facilitates the molecular characterization of infectious samples by enabling simultaneous pathogen inactivation and extraction of proteins, metabolites, and lipids for subsequent mass spectrometry-based multi-omics measurements. This single-sample metabolite, protein and lipid extraction (MPLEx) method resulted in complete inactivation of clinically important bacterial and viral pathogens with exposed lipid membranes, including Yersinia pestis, Salmonella Typhimurium, and Campylobacter jejuni in pure culture, and Yersinia pestis, Campylobacter jejuni, and West Nile, MERS-CoV, Ebola, and influenza H7N9 viruses in infection studies. In addition, >99% inactivation, which increased with solvent exposure time, was also observed for pathogens without exposed lipid membranes including community-associated methicillin-resistant Staphylococcus aureus, Clostridium difficile spores and vegetative cells, and adenovirus type 5. The overall pipeline of inactivation and subsequent proteomic, metabolomic, and lipidomic analyses was evaluated using a human epithelial lung cell line infected with wild-type and mutant influenza H7N9 viruses, thereby demonstrating that MPLEx yields biomaterial of sufficient quality for subsequent multi-omics analyses. Based on these experimental results, we believe that MPLEx will facilitate systems biology studies of infectious samples by enabling simultaneous pathogen inactivation and multi-omics measurements from a single specimen with high success for pathogens with exposed lipid membranes.

The West African outbreak of Ebola virus (EBOV) is largely contained, but sporadic new cases continue to emerge. To assess the potential contribution of fomites to human infections with EBOV, we tested EBOV stability in human blood spotted onto Sierra Leonean banknotes and in syringe needles under hospital and environmental conditions. Under some of these conditions, EBOV remained infectious for >30 days, indicating that EBOV-contaminated items may pose a serious risk to humans.

Viral pneumonia has been frequently reported during early stages of influenza virus pandemics and in many human cases of highly pathogenic avian influenza (HPAI) H5N1 virus infection. To better understand the pathogenesis of this disease, we produced nonlethal viral pneumonia in rhesus macaques by using an HPAI H5N1 virus (A/Anhui/2/2005; referred to as Anhui/2). Infected macaques were monitored for 14 days, and tissue samples were collected at 6 time points for virologic, histopathologic, and transcriptomic analyses. Anhui/2 efficiently replicated in the lung from 12 h to 3 days postinfection (p.i.) and caused temporal but severe pneumonia that began to resolve by day 14. Lung transcriptional changes were first observed at 6 h, and increased expression of vascular permeability regulators and neutrophil chemoattractants correlated with increased serum leakage and neutrophil infiltration in situ . Additional inflammatory, antiviral, and apoptotic genes were upregulated from 12 h, concurrent with viral antigen detection and increasing immune cell populations. A shift toward upregulation of acquired immunity was apparent after day 6. Expression levels of established immune cell molecular markers revealed remarkable similarity with pathological findings, indicating early and robust neutrophil infiltration, a slight delay in macrophage accumulation, and abundant late populations of T lymphocytes. We also characterized the putative mechanisms regulating a unique, pneumonia-associated biphasic fever pattern. Thus, this study is the first to use a comprehensive and integrative approach to delineate specific molecular mechanisms regulating influenza virus-induced pneumonia in nonhuman primates, an important first step toward better management of human influenza virus disease.

Previous studies reported associations of IFITM3 SNP rs12252 with severe influenza, but evidence of association and the mechanism of risk remains controversial. We prioritized SNPs in IFITM3 based on putative biological function and identified rs34481144 in the 5′ UTR. We found evidence of a novel association of rs34481144 with severe influenza in three influenza-infected cohorts characterized by different levels of influenza illness severity. We determined the role of rs34481144 as an expression quantitative trait loci (eQTL) for IFITM3, with the risk allele associated with lower mRNA expression. The risk allele was found to have decreased IRF3 binding and increased CTCF binding in promoter-binding assays, and risk allele carriage diminished transcriptional correlations among neighboring genes, indicative of CTCF boundary activity. Furthermore, the risk allele disrupts a CpG site that undergoes differential methylation in CD8 T-cell subsets. Carriers of the risk allele had reduced CD8 T-cells in their airways during natural influenza infection, consistent with IFITM3 promoting airway CD8 T-cell accumulation, indicating that a critical function for IFITM3 may be to promote immune cell persistence at mucosal sites. Our study identifies a new regulator of IFITM3 expression that associates with CD8 T-cell levels in the airways and a spectrum of clinical outcomes.

During the last decade, more than half of humans infected with highly pathogenic avian influenza (HPAI) H5N1 viruses have died, yet virus-induced host signaling has yet to be clearly elucidated. Airway epithelia are known to produce inflammatory mediators that contribute to HPAI H5N1-mediated pathogenicity, but a comprehensive analysis of the host response in this cell type is lacking. Here, we leveraged a system approach to identify and statistically validate signaling subnetworks that define the dynamic transcriptional response of human bronchial epithelial cells after infection with influenza A/Vietnam/1203/2004 (H5N1, VN1203). Importantly, we validated a subset of transcripts from one subnetwork in both Calu-3 cells and mice. A more detailed examination of two subnetworks involved in the immune response and keratinization processes revealed potential novel mediators of HPAI H5N1 pathogenesis and host response signaling. Finally, we show how these results compare to those for a less virulent strain of influenza virus. Using emergent network properties, we provide fresh insight into the host response to HPAI H5N1 virus infection and identify novel avenues for perturbation studies and potential therapeutic interventions for fatal HPAI H5N1 disease.

Pandemic influenza viruses modulate pro-inflammatory responses that can lead to immunopathogenesis. We present an extensive and systematic profiling of lipids, metabolites and proteins in respiratory compartments of ferrets infected with either 1918 or 2009 human pandemic H1N1 influenza viruses. Integrative analysis of high-throughput omics data with virologic and histopathologic data uncovered relationships between host responses and phenotypic outcomes of viral infection. Pro-inflammatory lipid precursors in the trachea following 1918 infection correlated with severe tracheal lesions. Using an algorithm to infer cell quantity changes from gene expression data, we found enrichment of distinct T cell subpopulations in the trachea. There was also a predicted increase in inflammatory monocytes in the lung of 1918 virus-infected animals that was sustained throughout infection. This study presents a unique resource to the influenza research community and demonstrates the utility of an integrative systems approach for characterization of lipid metabolism alterations underlying respiratory responses to viruses.

We show here that the varicella-zoster virus (VZV) open reading frame 66 (ORF66) protein kinase is one mechanism employed to reduce class I major histocompatibility complex (MHC-I) surface expression in VZV-infected cells. Cells expressing enhanced green fluorescent protein-tagged functional and inactivated ORF66 (GFP-66 and GFP-66kd) from replication-defective adenovirus vectors revealed that ORF66 reduced MHC-I surface levels in a manner dependent on kinase activity. Cells infected with recombinant VZV expressing GFP-66 exhibited a significantly greater reduction in MHC-I surface expression than that observed in cells infected with VZV disrupted in GFP-66 expression. MHC-I maturation was delayed in its transport from the endoplasmic reticulum through the Golgi in both adenovirus-transduced cells expressing only GFP-66 and in VZV-infected cells expressing high levels of GFP-66, and this was predominantly kinase dependent. MHC-I levels were reduced in VZV-infected cells, and analyses of intracellular MHC-I revealed accumulation of folded MHC-I in the Golgi region, irrespective of ORF66 expression. Thus, the ORF66 kinase is important for VZV-mediated MHC-I downregulation, but additional mechanisms also may be involved. Analyses of the VZV ORF9a protein, the ortholog of the bovine herpesvirus 1 transporter associated with antigen processing inhibitor UL49.5 revealed no effects on MHC-I. These results establish a new role for viral protein kinases in immune evasion and suggest that VZV utilizes unique mechanisms to inhibit antigen presentation.

Motivation: Identifying the shared and pathogen-specific components of host transcriptional regulatory programs is important for understanding the principles of regulation of immune response. Recent efforts in systems biology studies of infectious diseases have resulted in a large collection of datasets measuring host transcriptional response to various pathogens. Computational methods to identify and compare gene expression modules across different infections offer a powerful way to identify strain-specific and shared components of the regulatory program. An important challenge is to identify statistically robust gene expression modules as well as to reliably detect genes that change their module memberships between infections. Results: We present MULCCH (MULti-task spectral Consensus Clustering for Hierarchically related tasks), a consensus extension of a multi-task clustering algorithm to infer high-confidence strain-specific host response modules under infections from multiple virus strains. On simulated data, MULCCH more accurately identifies genes exhibiting pathogen-specific patterns compared to non-consensus and nonmulti-task clustering approaches. Application of MULCCH to mammalian transcriptional response to a panel of influenza viruses showed that our method identifies clusters with greater coherence compared to non-consensus methods. Further, MULCCH derived clusters are enriched for several immune system-related processes and regulators. In summary, MULCCH provides a reliable module-based approach to identify molecular pathways and gene sets characterizing commonality and specificity of host response to viruses of different pathogenicities. Availability and implementation: The source code is available at https://bitbucket.org/roygroup/mulcch Contact: sroy@biostat.wisc.edu Supplementary information: Supplementary data are available at Bioinformatics online.

IE62, the major transcriptional regulatory protein encoded by varicella-zoster virus (VZV), is nuclear at early times of VZV infection but then becomes predominantly cytoplasmic as a result of expression of the protein kinase encoded by open reading frame 66 (ORF66). Cytoplasmic forms of IE62 are required for its inclusion as an abundant VZV virion tegument protein. Here we show that ORF66 directly phosphorylates IE62 at two residues, with phosphorylation at S686 being sufficient to regulate IE62 nuclear import. Phosphotryptic peptide analyses established an ORF66 kinase-mediated phosphorylation of the complete IE62 protein in transfected and VZV-infected cells. Using truncated and point-mutated IE62 peptides, ORF66-directed phosphorylation was mapped to residues S686 and S722, immediately downstream of the IE62 nuclear localization signal. An IE62 protein with an S686A mutation retained efficient nuclear import activity, even in the presence of functional ORF66 protein kinase, but an IE62 protein containing an S686D alteration was imported into the nucleus inefficiently. In contrast, the nuclear import of IE62 carrying an S722A mutation was still modulated by ORF66 expression, and IE62 with an S722D mutation was imported efficiently into the nucleus. An in vitro phosphorylation assay was developed using bacterially expressed IE62-maltose binding protein fusions as substrates for immunopurified ORF66 protein kinase from recombinant baculovirus-infected insect cells. ORF66 kinase phosphorylated the IE62 peptides, with similar specificities for residues S686 and S722. These results indicate that IE62 nuclear import is modulated as a result of direct phosphorylation of IE62 by ORF66 kinase. This represents an interaction that is, so far, unique among the alphaherpesviruses.

A lot of research has been done on the spectrum of the sizes of maximal partial spreads in PG(3,q) [P. Govaerts and L. Storme, Designs Codes and Cryptography, Vol. 28 (2003) pp. 51--63; O. Heden, Discrete Mathematics, Vol. 120 (1993) pp. 75--91; O. Heden, Discrete Mathematics, Vol. 142 (1995) pp. 97--106; O. Heden, Discrete Mathematics, Vol. 243 (2002) pp. 135--150]. In [A. Gacs and T. Sz?nyi, Designs Codes and Cryptography, Vol. 29 (2003) pp. 123--129], results on the spectrum of the sizes of maximal partial line spreads in PG(N,q), N? 5, are given. In PG(2n,q), n ? 3, the largest possible size for a partial line spread is q2n-1+q2n-3+...+q3+1. The largest size for the maximal partial line spreads constructed in [A. Gacs and T. Sz?nyi, Designs Codes and Cryptography, Vol. 29 (2003) pp. 123--129] is (q2n+1?q)/(q2?1)?q3+q2?2q+2. This shows that there is a non-empty interval of values of k for which it is still not known whether there exists a maximal partial line spread of size k in PG(2n,q). We now show that there indeed exists a maximal partial line spread of size k for every value of k in that interval when q ? 9.

Summary The pathogenesis of human Ebola virus disease (EVD) is complex. EVD is characterized by high levels of virus replication and dissemination, dysregulated immune responses, extensive virus- and host-mediated tissue damage, and disordered coagulation. To clarify how host responses contribute to EVD pathophysiology, we performed multi-platform 'omics analysis of peripheral blood mononuclear cells and plasma from EVD patients. Our results indicate that EVD molecular signatures overlap with those of sepsis, imply that pancreatic enzymes contribute to tissue damage in fatal EVD, and suggest that Ebola virus infection may induce aberrant neutrophils whose activity could explain hallmarks of fatal EVD. Moreover, integrated biomarker prediction identified putative biomarkers from different data platforms that differentiated survivors and fatalities early after infection. This work reveals insight into EVD pathogenesis, suggests an effective approach for biomarker identification, and provides an important community resource for further analysis of human EVD severity.

A i>t-cover of a quadric {\cal Q} is a set {\cal C} of i>t-dimensional subspaces contained in {\cal Q} such that every point of {\cal Q} is contained in at least one element of {\cal C}. We consider (i>n − 1)-covers of the hyperbolic quadric i>Q+(2i>n + 1, i>q). We show that such a cover must have at least i>qi>n + 1 + 2i>q + 1 elements, give an example of this size for even i>q and describe what covers of this size should look like.

The domestic ferret (Mustela putorius furo) is an important animal model for multiple human respiratory diseases. It is considered the 'gold standard' for modeling human influenza virus infection and transmission. Here we describe the 2.41 Gb draft genome assembly of the domestic ferret, constituting 2.28 Gb of sequence plus gaps. We annotated 19,910 protein-coding genes on this assembly using RNA-seq data from 21 ferret tissues. We characterized the ferret host response to two influenza virus infections by RNA-seq analysis of 42 ferret samples from influenza time-course data and showed distinct signatures in ferret trachea and lung tissues specific to 1918 or 2009 human pandemic influenza virus infections. Using microarray data from 16 ferret samples reflecting cystic fibrosis disease progression, we showed that transcriptional changes in the CFTR-knockout ferret lung reflect pathways of early disease that cannot be readily studied in human infants with cystic fibrosis disease., Nature Biotechnology, 32 (12), ISSN:1546-1696, ISSN:1087-0156

The Systems Biology for Infectious Diseases Research program was established by the U.S. National Institute of Allergy and Infectious Diseases to investigate host-pathogen interactions at a systems level. This program generated 47 transcriptomic and proteomic datasets from 30 studies that investigate in vivo and in vitro host responses to viral infections. Human pathogens in the Orthomyxoviridae and Coronaviridae families, especially pandemic H1N1 and avian H5N1 influenza A viruses and severe acute respiratory syndrome coronavirus (SARS-CoV), were investigated. Study validation was demonstrated via experimental quality control measures and meta-analysis of independent experiments performed under similar conditions. Primary assay results are archived at the GEO and PeptideAtlas public repositories, while processed statistical results together with standardized metadata are publically available at the Influenza Research Database (www.fludb.org) and the Virus Pathogen Resource (www.viprbrc.org). By comparing data from mutant versus wild-type virus and host strains, RNA versus protein differential expression, and infection with genetically similar strains, these data can be used to further investigate genetic and physiological determinants of host responses to viral infection.

The broad range and diversity of interferon-stimulated genes (ISGs) function to induce an antiviral state within the host, impeding viral pathogenesis. While successful respiratory viruses overcome individual ISG effectors, analysis of the global ISG response and subsequent viral antagonism has yet to be examined. Employing models of the human airway, transcriptomics and proteomics datasets were used to compare ISG response patterns following highly pathogenic H5N1 avian influenza (HPAI) A virus, 2009 pandemic H1N1, severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome CoV (MERS-CoV) infection. The results illustrated distinct approaches utilized by each virus to antagonize the global ISG response. In addition, the data revealed that highly virulent HPAI virus and MERS-CoV induce repressive histone modifications, which downregulate expression of ISG subsets. Notably, influenza A virus NS1 appears to play a central role in this histone-mediated downregulation in highly pathogenic influenza strains. Together, the work demonstrates the existence of unique and common viral strategies for controlling the global ISG response and provides a novel avenue for viral antagonism via altered histone modifications., IMPORTANCE This work combines systems biology and experimental validation to identify and confirm strategies used by viruses to control the immune response. Using a novel screening approach, specific comparison between highly pathogenic influenza viruses and coronaviruses revealed similarities and differences in strategies to control the interferon and innate immune response. These findings were subsequently confirmed and explored, revealing both a common pathway of antagonism via type I interferon (IFN) delay as well as a novel avenue for control by altered histone modification. Together, the data highlight how comparative systems biology analysis can be combined with experimental validation to derive novel insights into viral pathogenesis.

This article discusses two problems on classical generalized quadrangles. It is known that the generalized quadrangle Q(4,q) arising from the parabolic quadric in PG(4,q) has a spread if and only if q is even. Hence, for q odd, the problem arises of the cardinality of the smallest set of lines of Q(4,q) covering all points of Q(4,q). We show in this paper that this set of lines must contain more than q2+1+(q−1)/3 lines. We also show that Q(4,q), q even, does not contain minimal covers of sizes q2+1+r when q⩾32 and 0

Background Influenza is a common infectious disease caused by influenza viruses. Annual epidemics cause severe illnesses, deaths, and economic loss around the world. To better defend against influenza viral infection, it is essential to understand its mechanisms and associated host responses. Many studies have been conducted to elucidate these mechanisms, however, the overall picture remains incompletely understood. A systematic understanding of influenza viral infection in host cells is needed to facilitate the identification of influential host response mechanisms and potential drug targets. Description We constructed a comprehensive map of the influenza A virus (‘IAV’) life cycle (‘FluMap’) by undertaking a literature-based, manual curation approach. Based on information obtained from publicly available pathway databases, updated with literature-based information and input from expert virologists and immunologists, FluMap is currently composed of 960 factors (i.e., proteins, mRNAs etc.) and 456 reactions, and is annotated with ~500 papers and curation comments. In addition to detailing the type of molecular interactions, isolate/strain specific data are also available. The FluMap was built with the pathway editor CellDesigner in standard SBML (Systems Biology Markup Language) format and visualized as an SBGN (Systems Biology Graphical Notation) diagram. It is also available as a web service (online map) based on the iPathways+ system to enable community discussion by influenza researchers. We also demonstrate computational network analyses to identify targets using the FluMap. Conclusion The FluMap is a comprehensive pathway map that can serve as a graphically presented knowledge-base and as a platform to analyze functional interactions between IAV and host factors. Publicly available webtools will allow continuous updating to ensure the most reliable representation of the host-virus interaction network. The FluMap is available at http://www.influenza-x.org/flumap/.

International audience; Respiratory infections stemming from influenza viruses and the Severe Acute Respiratory Syndrome corona virus (SARS-CoV) represent a serious public health threat as emerging pandemics. Despite efforts to identify the critical interactions of these viruses with host machinery, the key regulatory events that lead to disease pathology remain poorly targeted with therapeutics. Here we implement an integrated network interrogation approach, in which proteome and transcriptome datasets from infection of both viruses in human lung epithelial cells are utilized to predict regulatory genes involved in the host response. We take advantage of a novel ``crowd-based'' approach to identify and combine ranking metrics that isolate genes/proteins likely related to the pathogenicity of SARS-CoV and influenza virus. Subsequently, a multivariate regression model is used to compare predicted lung epithelial regulatory influences with data derived from other respiratory virus infection models. We predicted a small set of regulatory factors with conserved behavior for consideration as important components of viral pathogenesis that might also serve as therapeutic targets for intervention. Our results demonstrate the utility of integrating diverse `omic datasets to predict and prioritize regulatory features conserved across multiple pathogen infection models.

Background Influenza infection causes respiratory disease that can lead to death. The complex interplay between virus-encoded and host-specific pathogenicity regulators – and the relative contributions of each toward viral pathogenicity – is not well-understood. Results By analyzing a collection of lung samples from mice infected by A/Vietnam/1203/2004 (H5N1; VN1203), we characterized a signature of transcripts and proteins associated with the kinetics of the host response. Using a new geometrical representation method and two criteria, we show that inoculation concentrations and four specific mutations in VN1203 mainly impact the magnitude and velocity of the host response kinetics, rather than specific sets of up- and down- regulated genes. We observed analogous kinetic effects using lung samples from mice infected with A/California/04/2009 (H1N1), and we show that these effects correlate with morbidity and viral titer. Conclusions We have demonstrated the importance of the kinetics of the host response to H5N1 pathogenesis and its relationship with clinical disease severity and virus replication. These kinetic properties imply that time-matched comparisons of ‘omics profiles to viral infections give limited views to differentiate host-responses. Moreover, these results demonstrate that a fast activation of the host-response at the earliest time points post-infection is critical for protective mechanisms against fast replicating viruses.

Interactions of proteins regulate signaling, catalysis, gene expression and many other cellular functions. Therefore, characterizing the entire human interactome is a key effort in current proteomics research. This challenge is complicated by the dynamic nature of protein-protein interactions (PPIs), which are conditional on the cellular context: both interacting proteins must be expressed in the same cell and localized in the same organelle to meet. Additionally, interactions underlie a delicate control of signaling pathways, e.g. by post-translational modifications of the protein partners - hence, many diseases are caused by the perturbation of these mechanisms. Despite the high degree of cell-state specificity of PPIs, many interactions are measured under artificial conditions (e.g. yeast cells are transfected with human genes in yeast two-hybrid assays) or even if detected in a physiological context, this information is missing from the common PPI databases. To overcome these problems, we developed a method that assigns context information to PPIs inferred from various attributes of the interacting proteins: gene expression, functional and disease annotations, and inferred pathways. We demonstrate that context consistency correlates with the experimental reliability of PPIs, which allows us to generate high-confidence tissue- and function-specific subnetworks. We illustrate how these context-filtered networks are enriched in bona fide pathways and disease proteins to prove the ability of context-filters to highlight meaningful interactions with respect to various biological questions. We use this approach to study the lung-specific pathways used by the influenza virus, pointing to IRAK1, BHLHE40 and TOLLIP as potential regulators of influenza virus pathogenicity, and to study the signalling pathways that play a role in Alzheimer's disease, identifying a pathway involving the altered phosphorylation of the Tau protein. Finally, we provide the annotated human PPI network via a web frontend that allows the construction of context-specific networks in several ways., Author Summary Protein-protein-interactions (PPIs) participate in virtually all biological processes. However, the PPI map is not static but the pairs of proteins that interact depends on the type of cell, the subcellular localization and modifications of the participating proteins, among many other factors. Therefore, it is important to understand the specific conditions under which a PPI happens. Unfortunately, experimental methods often do not provide this information or, even worse, measure PPIs under artificial conditions not found in biological systems. We developed a method to infer this missing information from properties of the interacting proteins, such as in which cell types the proteins are found, which functions they fulfill and whether they are known to play a role in disease. We show that PPIs for which we can infer conditions under which they happen have a higher experimental reliability. Also, our inference agrees well with known pathways and disease proteins. Since diseases usually affect specific cell types, we study PPI networks of influenza proteins in lung tissues and of Alzheimer's disease proteins in neural tissues. In both cases, we can highlight interesting interactions potentially playing a role in disease progression.

Background Annually, influenza A viruses circulate the world causing wide-spread sickness, economic loss, and death. One way to better defend against influenza virus-induced disease may be to develop novel host-based therapies, targeted at mitigating viral pathogenesis through the management of virus-dysregulated host functions. However, mechanisms that govern aberrant host responses to influenza virus infection remain incompletely understood. We previously showed that the pandemic H1N1 virus influenza A/California/04/2009 (H1N1; CA04) has enhanced pathogenicity in the lungs of cynomolgus macaques relative to a seasonal influenza virus isolate (A/Kawasaki/UTK-4/2009 (H1N1; KUTK4)). Results Here, we used microarrays to identify host gene sequences that were highly differentially expressed (DE) in CA04-infected macaque lungs, and we employed a novel strategy – combining functional and pathway enrichment analyses, transcription factor binding site enrichment analysis and protein-protein interaction data – to create a CA04 differentially regulated host response network. This network describes enhanced viral RNA sensing, immune cell signaling and cell cycle arrest in CA04-infected lungs, and highlights a novel, putative role for the MYC-associated zinc finger (MAZ) transcription factor in regulating these processes. Conclusions Our findings suggest that the enhanced pathology is the result of a prolonged immune response, despite successful virus clearance. Most interesting, we identify a mechanism which normally suppresses immune cell signaling and inflammation is ineffective in the pH1N1 virus infection; a dyregulatory event also associated with arthritis. This dysregulation offers several opportunities for developing strain-independent, immunomodulatory therapies to protect against future pandemics.

Background Understanding host response to influenza virus infection will facilitate development of better diagnoses and therapeutic interventions. Several different experimental models have been used as a proxy for human infection, including cell cultures derived from human cells, mice, and non-human primates. Each of these systems has been studied extensively in isolation, but little effort has been directed toward systematically characterizing the conservation of host response on a global level beyond known immune signaling cascades. Results In the present study, we employed a multivariate modeling approach to characterize and compare the transcriptional regulatory networks between these three model systems after infection with a highly pathogenic avian influenza virus of the H5N1 subtype. Using this approach we identified functions and pathways that display similar behavior and/or regulation including the well-studied impact on the interferon response and the inflammasome. Our results also suggest a primary response role for airway epithelial cells in initiating hypercytokinemia, which is thought to contribute to the pathogenesis of H5N1 viruses. We further demonstrate that we can use a transcriptional regulatory model from the human cell culture data to make highly accurate predictions about the behavior of important components of the innate immune system in tissues from whole organisms. Conclusions This is the first demonstration of a global regulatory network modeling conserved host response between in vitro and in vivo models.

Prestimulation of the TLR4 pathway with lipopolysaccharide (LPS) protects mice from lethal infection with H5N1 influenza virus. Here, we reveal that the TLR4-TRIF pathway is required for this protective effect by using mice whose TLR4-related molecules were knocked out. Microarray analysis of primary mouse lung culture cells that were LPS pretreated and infected with an H5N1 virus indicated that TLR3 mRNA was upregulated. Primary lung culture cells of TLR3 knockout mice showed no response to LPS pretreatment against H5N1 virus infection, suggesting that TLR3 is also involved in the preventive effect of LPS. Our data suggest that the TLR4-TRIF axis has an important role in stimulating protective innate immunity against H5N1 influenza A virus infection and that TLR3 signaling is involved in this pathway.

Influenza A virus uses cellular protein transport systems (e.g., CRM1-mediated nuclear export and Rab11-dependent recycling endosomes) for genome trafficking from the nucleus to the plasma membrane, where new virions are assembled. However, the detailed mechanisms of these events have not been completely resolved, and additional cellular factors are probably required. Here, we investigated the role of the cellular human immunodeficiency virus (HIV) Rev-binding protein (HRB), which interacts with influenza virus nuclear export protein (NEP), during the influenza virus life cycle. By using small interfering RNAs (siRNAs) and overexpression of a dominant negative HRB protein fragment, we show that cells lacking functional HRB have significantly reduced production of influenza virus progeny and that this defect results from impaired viral ribonucleoprotein (vRNP) delivery to the plasma membrane in late-stage infection. Since HRB colocalizes with influenza vRNPs early after their delivery to the cytoplasm, it may mediate a connection between the nucleocytoplasmic transport machinery and the endosomal system, thus facilitating the transfer of vRNPs from nuclear export to cytoplasmic trafficking complexes. We also found an association between NEP and HRB in the perinuclear region, suggesting that NEP may contribute to this process. Our results identify HRB as a second endosomal factor with a crucial role in influenza virus genome trafficking, suggest cooperation between unique endosomal compartments in the late steps of the influenza virus life cycle, and provide a common link between the cytoplasmic trafficking mechanisms of influenza virus and HIV.

Influenza A virus assembly is a complex process that requires the intersection of pathways involved in transporting viral glycoproteins, the matrix protein, and viral genomes, incorporated in the viral ribonucleoprotein (vRNP) complex, to plasma membrane sites of virion formation. Among these virion components, the mechanism of vRNP delivery is the most incompletely understood. Here, we reveal a functional relationship between the cellular Rab11 GTPase isoform, RAB11A, and vRNPs and show that RAB11A is indispensable for proper vRNP transport to the plasma membrane. Using an immunofluorescence-based assay with a monoclonal antibody that recognizes nucleoprotein in the form of vRNP, we demonstrate association between RAB11A and vRNPs at all stages of vRNP cytoplasmic transport. Abrogation of RAB11A expression through small interfering RNA (siRNA) treatment or disruption of RAB11A function by overexpression of dominant negative or constitutively active proteins caused aberrant vRNP intracellular accumulation, retention in the perinuclear region, and lack of accumulation at the plasma membrane. Complex formation between RAB11A and vRNPs was further established biochemically. Our results uncover a critical host factor with an essential contribution to influenza virus genome delivery and reveal a potential role for RAB11A in the transport of ribonucleoprotein cargo.

Introduction Successful respiratory viruses have developed means to overcome specific IFN and ISG effector functions. Yet globally, ISGs as a class have broad functions and impede both pathogenesis and viral replication. Therefore, highly successful respiratory viruses including SARS-CoV and Influenza A virus (IAV), likely attempt to short-circuit global ISG induction, potentially through diverse mechanisms. Methods In this work, global RNA (microarray) and proteomics data (mass spectrometry) are utilized to compare and contrast ISG control strategies employed by IAV H5N1, H1N1 2009, and SARS-CoV. Calu-3 cells, a human lung epithelial cell line, are treated with type I IFN to define a consensus list of IFN responsive genes. These genes are then examined in the context of virus infections to evaluate the global ISG response. Results SARS-CoV and IAV H5N1 utilize contrasting approaches to interfere with the global host ISG response. Whereas IAV H5N1 actively manipulates the ISG response with up and down regulation of ISG transcripts, SARS-CoV successfully limits ISG induction until late times during infection. Global proteomic analysis confirms the absence of ISG proteins following H5N1 infection; in contrast, SARS-CoV induces increased abundance of several ISG proteins, but not until after peak viral titers have been achieved. Together, the data illustrate that these two virulent respiratory viruses control the global ISG response. Further examination reveals ISG manipulation is absent in a less virulent IAV strain. H1N1 2009 infection results in robust ISG transcript expression with minimal down regulation. Similarly, proteomics analysis finds increased abundance of ISG proteins absent in H5N1 infection. Transcriptional factor analysis found no direct antagonism; however, truncation of the C-terminal portion of NS1 ablated ISG down regulation in H5N1 infections. Meta-analysis finds a similar ablation is observed in A549 cells infected H1N1 1918 when NS1 is manipulated. Together, the data strongly implicate a role for NS1 in mediating control of the entire global ISG response following virulent IAV infections. For SARS-CoV, the delayed ISG induction is maintained in both mutant and precursor viruses. DORF6, a SARS-CoV mutant lacking a major IFN antagonist, fails to induce ISG induction any earlier. Similarly, Bat-SRBD, a synthetic precursor virus, maintains the ability control the global ISG response, suggesting this trait is maintained in pre-cursors of the epidemic SARS-CoV. Additional work examines the ability of the SARS-CoV to camouflage its viral intermediates as well as replication within double membrane vesicles as mechanisms mediating delayed ISG induction. Conclusion Together, the work highlights the contrasting approaches used by SARS-CoV and IAV strains to limit global ISG induction. In addition, the results imply an important role for controlling ISG response in the context of virulent respiratory virus infection. Further examination of these results will likely produce novel approaches to treat important respiratory virus infection.

Since the beginning of the 20th century, humans have experienced four influenza pandemics, including the devastating 1918 'Spanish influenza'. Moreover, H5N1 highly pathogenic avian influenza (HPAI) viruses are currently spreading worldwide, although they are not yet efficiently transmitted among humans. While the threat of a global pandemic involving a highly pathogenic influenza virus strain looms large, our mechanisms to address such a catastrophe remain limited. Here, we show that pre-stimulation of Toll-like receptors (TLRs) 2 and 4 increased resistance against influenza viruses known to induce high pathogenicity in animal models. Our data emphasize the complexity of the host response against different influenza viruses, and suggest that TLR agonists might be utilized to protect against lethality associated with highly pathogenic influenza virus infection in humans.