Intestinal microbiota controls graft-versus-host disease independent of donor-host genetic disparity.

Acute graft-versus-host disease (aGVHD) remains a major limitation of allogeneic stem cell transplantation (SCT), and severe intestinal manifestation is the major cause of early mortality. Intestinal microbiota control MHC class II (MHC-II) expression by ileal intestinal epithelial cells (IECs) that promote GVHD. Here, we demonstrated that genetically identical mice of differing vendor origins had markedly different intestinal microbiota and ileal MHC-II expression, resulting in discordant GVHD severity. We utilized cohousing and antibiotic treatment to characterize the bacterial taxa positively and negatively associated with MHC-II expression. A large proportion of bacterial MHC-II inducers were vancomycin sensitive, and peri-transplant oral vancomycin administration attenuated CD4+ T cell-mediated GVHD. We identified a similar relationship between pre-transplant microbes, HLA class II expression, and both GVHD and mortality in a large clinical SCT cohort. These data highlight therapeutically tractable mechanisms by which pre-transplant microbial taxa contribute to GVHD independently of genetic disparity.

A systems biology approach reveals that tissue tropism to West Nile virus is regulated by antiviral genes and innate immune cellular processes.

The actions of the RIG-I like receptor (RLR) and type I interferon (IFN) signaling pathways are essential for a protective innate immune response against the emerging flavivirus West Nile virus (WNV). In mice lacking RLR or IFN signaling pathways, WNV exhibits enhanced tissue tropism, indicating that specific host factors of innate immune defense restrict WNV infection and dissemination in peripheral tissues. However, the immune mechanisms by which the RLR and IFN pathways coordinate and function to impart restriction of WNV infection are not well defined. Using a systems biology approach, we defined the host innate immune response signature and actions that restrict WNV tissue tropism. Transcriptional profiling and pathway modeling to compare WNV-infected permissive (spleen) and nonpermissive (liver) tissues showed high enrichment for inflammatory responses, including pattern recognition receptors and IFN signaling pathways, that define restriction of WNV replication in the liver. Assessment of infected livers from Mavs(-/-) × Ifnar(-/-) mice revealed the loss of expression of several key components within the natural killer (NK) cell signaling pathway, including genes associated with NK cell activation, inflammatory cytokine production, and NK cell receptor signaling. In vivo analysis of hepatic immune cell infiltrates from WT mice demonstrated that WNV infection leads to an increase in NK cell numbers with enhanced proliferation, maturation, and effector action. In contrast, livers from Mavs(-/-) × Ifnar(-/-) infected mice displayed reduced immune cell infiltration, including a significant reduction in NK cell numbers. Analysis of cocultures of dendritic and NK cells revealed both cell-intrinsic and -extrinsic roles for the RLR and IFN signaling pathways to regulate NK cell effector activity. Taken together, these observations reveal a complex innate immune signaling network, regulated by the RLR and IFN signaling pathways, that drives tissue-specific antiviral effector gene expression and innate immune cellular processes that control tissue tropism to WNV infection.

Identification of fallopian tube microbiota and its association with ovarian cancer.

Investigating the human fallopian tube (FT) microbiota has significant implications for understanding the pathogenesis of ovarian cancer (OC). In this large prospective study, we collected swabs intraoperatively from the FT and other surgical sites as controls to profile the microbiota in the FT and to assess its relationship with OC. Eighty-one OC and 106 non-cancer patients were enrolled and 1001 swabs were processed for 16S rRNA gene PCR and sequencing. We identified 84 bacterial species that may represent the FT microbiota and found a clear shift in the microbiota of the OC patients when compared to the non-cancer patients. Of the top 20 species that were most prevalent in the FT of OC patients, 60% were bacteria that predominantly reside in the gastrointestinal tract, while 30% normally reside in the mouth. Serous carcinoma had higher prevalence of almost all 84 FT bacterial species compared to the other OC subtypes. The clear shift in the FT microbiota in OC patients establishes the scientific foundation for future investigation into the role of these bacteria in the pathogenesis of OC.

Isoflavone agonists of IRF-3 dependent signaling have antiviral activity against RNA viruses.

There is a growing need for novel antiviral therapies that are broad spectrum, effective, and not subject to resistance due to viral mutations. Using high-throughput screening methods, including computational docking studies and an interferon-stimulated gene 54 (ISG54)-luciferase reporter assay, we identified a class of isoflavone compounds that act as specific agonists of innate immune signaling pathways and cause activation of the interferon regulatory factor (IRF-3) transcription factor. The isoflavone compounds activated the ISG54 promoter, mediated nuclear translocation of IRF-3, and displayed highly potent activity against hepatitis C virus (HCV) and influenza virus. Additionally, these agonists efficiently activated IRF-3 in the presence of the HCV protease NS3-4A, which is known to blunt the host immune response. Furthermore, genomic studies showed that discrete innate immune pathways centered on IRF signaling were regulated following agonist treatment without causing global changes in host gene expression. Following treatment, the expression of only 64 cellular genes was significantly induced. This report provides the first evidence that innate immune pathways dependent on IRF-3 can be successfully targeted by small-molecule drugs for the development of novel broad-spectrum antiviral compounds.

Early transcriptional programming links progression to hepatitis C virus-induced severe liver disease in transplant patients.

UNLABELLED: Liver failure resulting from chronic hepatitis C virus (HCV) infection is a major cause for liver transplantation worldwide. Recurrent infection of the graft is universal in HCV patients after transplant and results in a rapid progression to severe fibrosis and end-stage liver disease in one third of all patients. No single clinical variable, or combination thereof, has, so far, proven accurate in identifying patients at risk of hepatic decompensation in the transplant setting. A combination of longitudinal, dimensionality reduction and categorical analysis of the transcriptome from 111 liver biopsy specimens taken from 57 HCV-infected patients over time identified a molecular signature of gene expression of patients at risk of developing severe fibrosis. Significantly, alterations in gene expression occur before histologic evidence of liver disease progression, suggesting that events that occur during the acute phase of infection influence patient outcome. Additionally, a common precursor state for different severe clinical outcomes was identified. CONCLUSION: Based on this patient cohort, incidence of severe liver disease is a process initiated early during HCV infection of the donor organ. The probable cellular network at the basis of the initial transition to severe liver disease was identified and characterized.

Proteome and computational analyses reveal new insights into the mechanisms of hepatitis C virus-mediated liver disease posttransplantation.

UNLABELLED: Liver transplant tissues offer the unique opportunity to model the longitudinal protein abundance changes occurring during hepatitis C virus (HCV)-associated liver disease progression in vivo. In this study, our goal was to identify molecular signatures, and potential key regulatory proteins, representative of the processes influencing early progression to fibrosis. We performed global protein profiling analyses on 24 liver biopsy specimens obtained from 15 HCV(+) liver transplant recipients at 6 and/or 12 months posttransplantation. Differentially regulated proteins associated with early progression to fibrosis were identified by analysis of the area under the receiver operating characteristic curve. Analysis of serum metabolites was performed on samples obtained from an independent cohort of 60 HCV(+) liver transplant patients. Computational modeling approaches were applied to identify potential key regulatory proteins of liver fibrogenesis. Among 4,324 proteins identified, 250 exhibited significant differential regulation in patients with rapidly progressive fibrosis. Patients with rapid fibrosis progression exhibited enrichment in differentially regulated proteins associated with various immune, hepatoprotective, and fibrogenic processes. The observed increase in proinflammatory activity and impairment in antioxidant defenses suggests that patients who develop significant liver injury experience elevated oxidative stresses. This was supported by an independent study demonstrating the altered abundance of oxidative stress-associated serum metabolites in patients who develop severe liver injury. Computational modeling approaches further highlight a potentially important link between HCV-associated oxidative stress and epigenetic regulatory mechanisms impacting on liver fibrogenesis. CONCLUSION: Our proteome and metabolome analyses provide new insights into the role for increased oxidative stress in the rapid fibrosis progression observed in HCV(+) liver transplant recipients. These findings may prove useful in prognostic applications for predicting early progression to fibrosis.

Optimizing metaproteomics database construction: lessons from a study of the vaginal microbiome.

Metaproteomics, a method for untargeted, high-throughput identification of proteins in complex samples, provides functional information about microbial communities and can tie functions to specific taxa. Metaproteomics often generates less data than other omics techniques, but analytical workflows can be improved to increase usable data in metaproteomic outputs. Identification of peptides in the metaproteomic analysis is performed by comparing mass spectra of sample peptides to a reference database of protein sequences. Although these protein databases are an integral part of the metaproteomic analysis, few studies have explored how database composition impacts peptide identification. Here, we used cervicovaginal lavage (CVL) samples from a study of bacterial vaginosis (BV) to compare the performance of databases built using six different strategies. We evaluated broad versus sample-matched databases, as well as databases populated with proteins translated from metagenomic sequencing of the same samples versus sequences from public repositories. Smaller sample-matched databases performed significantly better, driven by the statistical constraints on large databases. Additionally, large databases attributed up to 34% of significant bacterial hits to taxa absent from the sample, as determined orthogonally by 16S rRNA gene sequencing. We also tested a set of hybrid databases which included bacterial proteins from NCBI RefSeq and translated bacterial genes from the samples. These hybrid databases had the best overall performance, identifying 1,068 unique human and 1,418 unique bacterial proteins, ~30% more than a database populated with proteins from typical vaginal bacteria and fungi. Our findings can help guide the optimal identification of proteins while maintaining statistical power for reaching biological conclusions. IMPORTANCE Metaproteomic analysis can provide valuable insights into the functions of microbial and cellular communities by identifying a broad, untargeted set of proteins. The databases used in the analysis of metaproteomic data influence results by defining what proteins can be identified. Moreover, the size of the database impacts the number of identifications after accounting for false discovery rates (FDRs). Few studies have tested the performance of different strategies for building a protein database to identify proteins from metaproteomic data and those that have largely focused on highly diverse microbial communities. We tested a range of databases on CVL samples and found that a hybrid sample-matched approach, using publicly available proteins from organisms present in the samples, as well as proteins translated from metagenomic sequencing of the samples, had the best performance. However, our results also suggest that public sequence databases will continue to improve as more bacterial genomes are published.

Pandemic swine-origin H1N1 influenza A virus isolates show heterogeneous virulence in macaques.

The first influenza pandemic of the new millennium was caused by a newly emerged swine-origin influenza virus (SOIV) (H1N1). This new virus is characterized by a previously unknown constellation of gene segments derived from North American and Eurasian swine lineages and the absence of common markers predictive of human adaptation. Overall, human infections appeared to be mild, but an alarming number of young individuals presented with symptoms atypical for seasonal influenza. The new SOIV also showed a sustained human-to-human transmissibility and higher reproduction ratio than common seasonal viruses, altogether indicating a higher pathogenic potential for this newly emerged virus. To study the virulence of the SOIV, we used a recently established cynomolgus macaque model and compared parameters of clinical disease, virology, host responses, and pathology/histopathology with a current seasonal H1N1 virus. We here show that infection of macaques with two genetically similar but clinically distinct SOIV isolates from the early stage of the pandemic (A/Mexico/4108/2009 and A/Mexico/InDRE4487/2009) resulted in upper and lower respiratory tract infections and clinical disease ranging from mild to severe pneumonia that was clearly advanced over the mild infection caused by A/Kawasaki/UTK-4/2009, a current seasonal strain. Unexpectedly, we observed heterogeneity among the two SOIV isolates in virus replication, host transcriptional and cytokine responses, and disease progression, demonstrating a higher pathogenic potential for A/Mexico/InDRE4487/2009. Differences in virulence may explain more severe disease, as was seen with certain individuals infected with the emerged pandemic influenza virus. Thus, the nonhuman primate model closely mimics influenza in humans.

Viral replication rate regulates clinical outcome and CD8 T cell responses during highly pathogenic H5N1 influenza virus infection in mice.

Since the first recorded infection of humans with H5N1 viruses of avian origin in 1997, sporadic human infections continue to occur with a staggering mortality rate of >60%. Although sustained human-to-human transmission has not occurred yet, there is a growing concern that these H5N1 viruses might acquire this trait and raise the specter of a pandemic. Despite progress in deciphering viral determinants of pathogenicity, we still lack crucial information on virus/immune system interactions pertaining to severe disease and high mortality associated with human H5N1 influenza virus infections. Using two human isolates of H5N1 viruses that differ in their pathogenicity in mice, we have defined mechanistic links among the rate of viral replication, mortality, CD8 T cell responses, and immunopathology. The extreme pathogenicity of H5N1 viruses was directly linked to the ability of the virus to replicate rapidly, and swiftly attain high steady-state titers in the lungs within 48 hours after infection. The remarkably high replication rate of the highly pathogenic H5N1 virus did not prevent the induction of IFN-ß or activation of CD8 T cells, but the CD8 T cell response was ineffective in controlling viral replication in the lungs and CD8 T cell deficiency did not affect viral titers or mortality. Additionally, BIM deficiency ameliorated lung pathology and inhibited T cell apoptosis without affecting survival of mice. Therefore, rapidly replicating, highly lethal H5N1 viruses could simply outpace and overwhelm the adaptive immune responses, and kill the host by direct cytopathic effects. However, therapeutic suppression of early viral replication and the associated enhancement of CD8 T cell responses improved the survival of mice following a lethal H5N1 infection. These findings suggest that suppression of early H5N1 virus replication is key to the programming of an effective host response, which has implications in treatment of this infection in humans.

Temporal proteome and lipidome profiles reveal hepatitis C virus-associated reprogramming of hepatocellular metabolism and bioenergetics.

Proteomic and lipidomic profiling was performed over a time course of acute hepatitis C virus (HCV) infection in cultured Huh-7.5 cells to gain new insights into the intracellular processes influenced by this virus. Our proteomic data suggest that HCV induces early perturbations in glycolysis, the pentose phosphate pathway, and the citric acid cycle, which favor host biosynthetic activities supporting viral replication and propagation. This is followed by a compensatory shift in metabolism aimed at maintaining energy homeostasis and cell viability during elevated viral replication and increasing cellular stress. Complementary lipidomic analyses identified numerous temporal perturbations in select lipid species (e.g. phospholipids and sphingomyelins) predicted to play important roles in viral replication and downstream assembly and secretion events. The elevation of lipotoxic ceramide species suggests a potential link between HCV-associated biochemical alterations and the direct cytopathic effect observed in this in vitro system. Using innovative computational modeling approaches, we further identified mitochondrial fatty acid oxidation enzymes, which are comparably regulated during in vitro infection and in patients with histological evidence of fibrosis, as possible targets through which HCV regulates temporal alterations in cellular metabolic homeostasis.

Genomic analysis of increased host immune and cell death responses induced by 1918 influenza virus.

The influenza pandemic of 1918-19 was responsible for about 50 million deaths worldwide. Modern histopathological analysis of autopsy samples from human influenza cases from 1918 revealed significant damage to the lungs with acute, focal bronchitis and alveolitis associated with massive pulmonary oedema, haemorrhage and rapid destruction of the respiratory epithelium. The contribution of the host immune response leading to this severe pathology remains largely unknown. Here we show, in a comprehensive analysis of the global host response induced by the 1918 influenza virus, that mice infected with the reconstructed 1918 influenza virus displayed an increased and accelerated activation of host immune response genes associated with severe pulmonary pathology. We found that mice infected with a virus containing all eight genes from the pandemic virus showed marked activation of pro-inflammatory and cell-death pathways by 24 h after infection that remained unabated until death on day 5. This was in contrast with smaller host immune responses as measured at the genomic level, accompanied by less severe disease pathology and delays in death in mice infected with influenza viruses containing only subsets of 1918 genes. The results indicate a cooperative interaction between the 1918 influenza genes and show that study of the virulence of the 1918 influenza virus requires the use of the fully reconstructed virus. With recent concerns about the introduction of highly pathogenic avian influenza viruses into humans and their potential to cause a worldwide pandemic with disastrous health and economic consequences, a comprehensive understanding of the global host response to the 1918 virus is crucial. Moreover, understanding the contribution of host immune responses to virulent influenza virus infections is an important starting point for the identification of prognostic indicators and the development of novel antiviral therapies.

The NS1 protein of influenza A virus suppresses interferon-regulated activation of antigen-presentation and immune-proteasome pathways.

The NS1 protein of influenza virus counters host antiviral defences primarily by antagonizing the type I interferon (IFN) response. Both the N-terminal dsRNA-binding domain and the C-terminal effector domain are required for optimal suppression of host responses during infection. To better understand the regulatory role of the NS1 effector domain, we used an NS1-truncated mutant virus derived from human H1N1 influenza isolate A/Texas/36/91 (Tx/91) and assessed global transcriptional profiles from two independent human lung cell-culture models. Relative to the wild-type Tx/91-induced gene expression, the NS1 mutant virus induced enhanced expression of innate immune genes, specifically NF-κB signalling-pathway genes and IFN-α and -ß target genes. We queried an experimentally derived IFN gene set to gauge the proportion of IFN-responsive genes that are suppressed specifically by NS1. We show that the C-terminally truncated NS1 mutant virus is less efficient at suppressing IFN-regulated gene expression associated with activation of antigen-presentation and immune-proteasome pathways. This is the first report integrating genomic analysis from two independent human culture systems, including primary lung cells, using genetically similar H1N1 influenza viruses that differ only in the length of the NS1 protein.

The alpha/beta interferon receptor provides protection against influenza virus replication but is dispensable for inflammatory response signaling.

The innate immune response provides the first line of defense against foreign pathogens by responding to molecules that are a signature of a pathogenic infection. Certain RNA viruses, such as influenza virus, produce double-stranded RNA as an intermediate during the replication life cycle, which activates pathogen recognition receptors capable of inducing interferon production. By engaging interferon receptors, interferon activates the JAK-STAT pathway and results in the positive feedback of interferon production, amplifying the response to viral infection. To examine how deficiencies in interferon signaling affect the cellular response to infection, we performed influenza virus infections of mouse embryonic fibroblasts lacking the alpha/beta interferon receptor, the gamma interferon receptor, or both. In the absence of the alpha/beta interferon receptor, we observed increased viral replication but decreased activation of PKR, Stat1, and NF-kappaB; the presence or absence of the gamma interferon receptor did not exhibit discernible differences in these readouts. Analysis of gene expression profiles showed that while cells lacking the alpha/beta interferon receptor exhibited decreased levels of transcription of antiviral genes, genes related to inflammatory and apoptotic responses were transcribed to levels similar to those of cells containing the receptor. These results indicate that while the alpha/beta interferon receptor is needed to curb viral replication, it is dispensable for the induction of certain inflammatory and apoptotic genes. We have identified potential pathways, via interferon regulatory factor 3 (IRF3) activation or Hoxa13, Polr2a, Nr4a1, or Ing1 induction, that contribute to this redundancy. This study illustrates another way in which the host has evolved to establish several overlapping mechanisms to respond to viral infections.

Genomic profiling of tumor necrosis factor alpha (TNF-alpha) receptor and interleukin-1 receptor knockout mice reveals a link between TNF-alpha signaling and increased severity of 1918 pandemic influenza virus infection.

The influenza pandemic of 1918 to 1919 was one of the worst global pandemics in recent history. The highly pathogenic nature of the 1918 virus is thought to be mediated in part by a dysregulation of the host response, including an exacerbated proinflammatory cytokine response. In the present study, we compared the host transcriptional response to infection with the reconstructed 1918 virus in wild-type, tumor necrosis factor (TNF) receptor-1 knockout (TNFRKO), and interleukin-1 (IL-1) receptor-1 knockout (IL1RKO) mice as a means of further understanding the role of proinflammatory cytokine signaling during the acute response to infection. Despite reported redundancy in the functions of IL-1ß and TNF-α, we observed that reducing the signaling capacity of each of these molecules by genetic disruption of their key receptor genes had very different effects on the host response to infection. In TNFRKO mice, we found delayed or decreased expression of genes associated with antiviral and innate immune signaling, complement, coagulation, and negative acute-phase response. In contrast, in IL1RKO mice numerous genes were differentially expressed at 1 day postinoculation, including an increase in the expression of genes that contribute to dendritic and natural killer cell processes and cellular movement, and gene expression profiles remained relatively constant at later time points. We also observed a compensatory increase in TNF-α expression in virus-infected IL1RKO mice. Our data suggest that signaling through the IL-1 receptor is protective, whereas signaling through the TNF-α receptor increases the severity of 1918 virus infection. These findings suggest that manipulation of these pathways may have therapeutic benefit.

Lethal dissemination of H5N1 influenza virus is associated with dysregulation of inflammation and lipoxin signaling in a mouse model of infection.

Periodic outbreaks of highly pathogenic avian H5N1 influenza viruses and the current H1N1 pandemic highlight the need for a more detailed understanding of influenza virus pathogenesis. To investigate the host transcriptional response induced by pathogenic influenza viruses, we used a functional-genomics approach to compare gene expression profiles in lungs from 129S6/SvEv mice infected with either the fully reconstructed H1N1 1918 pandemic virus (1918) or the highly pathogenic avian H5N1 virus Vietnam/1203/04 (VN/1203). Although the viruses reached similar titers in the lung and caused lethal infections, the mean time of death was 6 days for VN/1203-infected animals and 9 days for mice infected with the 1918 virus. VN/1203-infected animals also exhibited an earlier and more potent inflammatory response. This response included induction of genes encoding components of the inflammasome. VN/1203 was also able to disseminate to multiple organs, including the brain, which correlated with changes in the expression of genes associated with hematological functions and lipoxin biogenesis and signaling. Both viruses elicited expression of type I interferon (IFN)-regulated genes in wild-type mice and to a lesser extent in mice lacking the type I IFN receptor, suggesting alternative or redundant pathways for IFN signaling. Our findings suggest that VN/1203 is more pathogenic in mice as a consequence of several factors, including the early and sustained induction of the inflammatory response, the additive or synergistic effects of upregulated components of the immune response, and inhibition of lipoxin-mediated anti-inflammatory responses, which correlated with the ability of VN/1203 to disseminate to extrapulmonary organs.

Lethal influenza virus infection in macaques is associated with early dysregulation of inflammatory related genes.

The enormous toll on human life during the 1918-1919 Spanish influenza pandemic is a constant reminder of the potential lethality of influenza viruses. With the declaration by the World Health Organization of a new H1N1 influenza virus pandemic, and with continued human cases of highly pathogenic H5N1 avian influenza virus infection, a better understanding of the host response to highly pathogenic influenza viruses is essential. To this end, we compared pathology and global gene expression profiles in bronchial tissue from macaques infected with either the reconstructed 1918 pandemic virus or the highly pathogenic avian H5N1 virus A/Vietnam/1203/04. Severe pathology was observed in respiratory tissues from 1918 virus-infected animals as early as 12 hours after infection, and pathology steadily increased at later time points. Although tissues from animals infected with A/Vietnam/1203/04 also showed clear signs of pathology early on, less pathology was observed at later time points, and there was evidence of tissue repair. Global transcriptional profiles revealed that specific groups of genes associated with inflammation and cell death were up-regulated in bronchial tissues from animals infected with the 1918 virus but down-regulated in animals infected with A/Vietnam/1203/04. Importantly, the 1918 virus up-regulated key components of the inflammasome, NLRP3 and IL-1beta, whereas these genes were down-regulated by A/Vietnam/1203/04 early after infection. TUNEL assays revealed that both viruses elicited an apoptotic response in lungs and bronchi, although the response occurred earlier during 1918 virus infection. Our findings suggest that the severity of disease in 1918 virus-infected macaques is a consequence of the early up-regulation of cell death and inflammatory related genes, in which additive or synergistic effects likely dictate the severity of tissue damage.

P58(IPK): a novel "CIHD" member of the host innate defense response against pathogenic virus infection.

To support their replication, viruses take advantage of numerous cellular factors and processes. Recent large-scale screens have identified hundreds of such factors, yet little is known about how viruses exploit any of these. Influenza virus infection post-translationally activates P58(IPK), a cellular inhibitor of the interferon-induced, dsRNA-activated eIF2alpha kinase, PKR. Here, we report that infection of P58(IPK) knockout mice with influenza virus resulted in increased lung pathology, immune cell apoptosis, PKR activation, and mortality. Analysis of lung transcriptional profiles, including those induced by the reconstructed 1918 pandemic virus, revealed increased expression of genes associated with the cell death, immune, and inflammatory responses. These experiments represent the first use of a mammalian infection model to demonstrate the role of P58(IPK) in the antiviral response. Our results suggest that P58(IPK) represents a new class of molecule, a cellular inhibitor of the host defense (CIHD), as P58(IPK) is activated during virus infection to inhibit virus-induced apoptosis and inflammation to prolong host survival, even while prolonging viral replication.

Host responses to wild-type and attenuated herpes simplex virus infection in the absence of Stat1.

Humans and mice lacking the interferon signaling molecule Stat1 are sensitive to a variety of pathogens due to their presumed inability to mount a strong innate immune response. The herpes simplex virus type 1 (HSV-1) virion host shutoff (vhs) protein is a multifunctional immunomodulator that counteracts the innate immune response and viruses lacking vhs are attenuated and effective live vaccines in animal models. To investigate the interplay of viruses with an immunocompromised host, we performed functional genomics analyses on control and Stat1(-/-) mouse corneas infected with wild-type or vhs-null viruses. In control mice, correlative with viral growth, both viruses induced a transient increase in immunomodulators, followed by viral clearance. In contrast, infection of the Stat1(-/-) mice induced a heightened and prolonged induction of inflammatory modulators for both viruses, manifesting as a significant immune cell infiltrate and ocular disease. Moreover, while wild-type virus infection of Stat1(-/-) was always lethal, vhs-null infection was rarely lethal. There was a significant increase in Stat3- and interleukin-6 (IL-6)-dependent transcription in Stat1(-/-) mice, implicating the Stat3 and IL-6 pathways in the observed ocular pathology. Further, infected Stat1(-/-) mice showed phosphorylated Stat3 in the corneal epithelium. Our data show a role for vhs in evading innate host responses and a role for Stat1 in limiting virus infection and for facilitating an appropriate nonpathological inflammatory response.

Impaired cholesterol biosynthesis in a neuronal cell line persistently infected with measles virus.

Measles virus remains a substantial cause of morbidity and mortality, producing acute infection with a potential for development of viral persistence. To study the events underlying acute and persistent measles virus infection, we performed a global transcriptional analysis on murine neuroblastoma cells that were acutely or persistently infected with measles virus. In general, we found that acute infection induced significantly more gene expression changes than did persistent infection. A functional enrichment analysis to identify which host pathways were perturbed during each of these infections identified several pathways related to cholesterol biosynthesis, including cholesterol metabolic processes, hydroxymethylglutaryl-coenzyme A (CoA) reductase activity, and acetyl-CoA C-acetyltransferase activity. We also found that measles virus colocalized to lipid rafts in both acute and persistent infection models and that the majority of genes associated with cholesterol synthesis were downregulated in persistent infection relative to acute infection, suggesting a possible link with the defective viral budding in persistent infection. Further, we found that pharmacological inhibition of cholesterol synthesis resulted in the inhibition of viral budding during acute infection. In summary, persistent measles viral infection was associated with decreased cholesterol synthesis, a lower abundance of cholesterol and lipid rafts in the cell membrane, and inhibition of giant-cell formation and release of viral progeny.

The NS1 protein of the 1918 pandemic influenza virus blocks host interferon and lipid metabolism pathways.

The "Spanish influenza" of 1918 claimed an unprecedented number of lives, yet the determinants of virulence for this virus are still not fully understood. Here, we used functional genomics and an in vitro human lung epithelial cell infection model to define the global host transcriptional response to the eight-gene 1918 virus. To better understand the role of the 1918 virus NS1 gene, we also evaluated the host response to a reassortant 1918 virus containing the NS1 gene from A/Texas/36/91 (a seasonal isolate of human influenza virus), as well as the host response to a reassortant of A/Texas/36/91 containing the 1918 NS1 gene. Genomic analyses revealed that the 1918 virus blocked the transcription of multiple interferon-stimulated genes and also downregulated a network of genes associated with lipid metabolism. In contrast, the expression of genes encoding chemokines and cytokines, which serve to attract infiltrating immune cells, was upregulated. Viruses containing the NS1 gene from A/Texas/36/91 induced a significant increase in type I interferon signaling but did not repress lipid metabolism. The 1918 NS1 gene may therefore have contributed to the virulence of the 1918 pandemic virus by disrupting the innate immune response, inducing hypercytokinemia, and by blocking the transcription of certain lipid-based proinflammatory mediators that function as part of the host antiviral response.