Victoria S. Carter

Cellular transcriptional profiling in influenza A virus-infected lung epithelial cells: The role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza

The NS1 protein of influenza A virus contributes to viral pathogenesis, primarily by enabling the virus to disarm the host cell type IFN defense system. We examined the downstream effects of NS1 protein expression during influenza A virus infection on global cellular mRNA levels by measuring expression of over 13,000 cellular genes in response to infection with wild-type and mutant viruses in human lung epithelial cells. Influenza A/PR/8/34 virus infection resulted in a significant induction of genes involved in the IFN pathway. Deletion of the viral NS1 gene increased the number and magnitude of expression of cellular genes implicated in the IFN, NF-κB, and other antiviral pathways. Interestingly, different IFN-induced genes showed different sensitivities to NS1-mediated inhibition of their expression. A recombinant virus with a C-terminal deletion in its NS1 gene induced an intermediate cellular mRNA expression pattern between wild-type and NS1 knockout viruses. Most significantly, a virus containing the 1918 pandemic NS1 gene was more efficient at blocking the expression of IFN-regulated genes than its parental influenza A/WSN/33 virus. Taken together, our results suggest that the cellular response to influenza A virus infection in human lung cells is significantly influenced by the sequence of the NS1 gene, demonstrating the importance of the NS1 protein in regulating the host cell response triggered by virus infection.

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-1β, 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.

Global Host Immune Response: Pathogenesis and Transcriptional Profiling of Type A Influenza Viruses Expressing the Hemagglutinin and Neuraminidase Genes from the 1918 Pandemic Virus

ABSTRACT To understand more fully the molecular events associated with highly virulent or attenuated influenza virus infections, we have studied the effects of expression of the 1918 hemagglutinin (HA) and neuraminidase (NA) genes during viral infection in mice under biosafety level 3 (agricultural) conditions. Using histopathology and cDNA microarrays, we examined the consequences of expression of the HA and NA genes of the 1918 pandemic virus in a recombinant influenza A/WSN/33 virus compared to parental A/WSN/33 virus and to an attenuated virus expressing the HA and NA genes from A/New Caledonia/20/99. The 1918 HA/NA:WSN and WSN recombinant viruses were highly lethal for mice and displayed severe lung pathology in comparison to the nonlethal New Caledonia HA/NA:WSN recombinant virus. Expression microarray analysis performed on lung tissues isolated from the infected animals showed activation of many genes involved in the inflammatory response, including cytokine, apoptosis, and lymphocyte genes that were common to all three infection groups. However, consistent with the histopathology studies, the WSN and 1918 HA/NA:WSN recombinant viruses showed increased up-regulation of genes associated with activated T cells and macrophages, as well as genes involved in apoptosis, tissue injury, and oxidative damage that were not observed in the New Caledonia HA/NA:WSN recombinant virus-infected mice. These studies document clear differences in gene expression profiles that were correlated with pulmonary disease pathology induced by virulent and attenuated influenza virus infections.

Host genetic diversity enables Ebola hemorrhagic fever pathogenesis and resistance

Existing mouse models of lethal Ebola virus infection do not reproduce hallmark symptoms of Ebola hemorrhagic fever, neither delayed blood coagulation and disseminated intravascular coagulation nor death from shock, thus restricting pathogenesis studies to nonhuman primates. Here we show that mice from the Collaborative Cross panel of recombinant inbred mice exhibit distinct disease phenotypes after mouse-adapted Ebola virus infection. Phenotypes range from complete resistance to lethal disease to severe hemorrhagic fever characterized by prolonged coagulation times and 100% mortality. Inflammatory signaling was associated with vascular permeability and endothelial activation, and resistance to lethal infection arose by induction of lymphocyte differentiation and cellular adhesion, probably mediated by the susceptibility allele Tek. These data indicate that genetic background determines susceptibility to Ebola hemorrhagic fever. Intercrossed mice infected with Ebola virus show a spectrum of pathology from prolonged coagulation to total resistance. Variety of Ebola symptoms in mice Apart from monkeys, there are no animal models available that show the same symptoms of Ebola virus infection as those of humans. Rasmussen et al. tested the effects of Ebola virus in mice with defined genetic backgrounds in a series of pains-taking experiments performed under stringent biosafety conditions. Resistance and susceptibility to Ebola virus was associated with distinct genetic profiles in inflammation, blood coagulation, and vascular function. This panel of mice could prove valuable for preliminary screens of candidate therapeutics and vaccines. Science, this issue p. 987

Global Suppression of the Host Antiviral Response by Ebola- and Marburgviruses: Increased Antagonism of the Type I Interferon Response Is Associated with Enhanced Virulence

ABSTRACT We studied the effect of filovirus infection on host cell gene expression by characterizing the regulation of gene expression responses in human liver cells infected with Zaire Ebolavirus (ZEBOV), Reston Ebolavirus (REBOV), and Marburgvirus (MARV), using transcriptional profiling and bioinformatics. Expression microarray analysis demonstrated that filovirus infection resulted in the up-regulation of immune-related genes and the down-regulation of many coagulation and acute-phase proteins. These studies further revealed that a common feature of filovirus virulence is suppression of key cellular antiviral responses, including TLR-, interferon (IFN) regulatory factor 3-, and PKR-related pathways. We further showed that ZEBOV and MARV were more potent antagonists of the IFN response and inhibited the expression of most of the IFN-stimulated genes (ISGs) observed in mock-infected IFN-α-2b treated cells, compared to REBOV infection, which activated more than 20% of these ISGs. Finally, we examined IFN-related gene expression in filovirus-infected cells treated with IFN-α-2b. These experiments revealed that a majority of genes induced in mock-infected cells treated with type I IFN were antagonized in treated ZEBOV- and MARV-infected cells, while in contrast, REBOV infection resulted in a significant increase in ISG expression. Analysis of STAT1 and -2 phosphorylation following IFN treatment showed a significant reduction of STAT phosphorylation for MARV but not for ZEBOV and REBOV, indicating that different mechanisms might be involved in antagonizing IFN signaling pathways by the different filovirus species. Taken together, these studies showed a correlation between antagonism of type I IFN responses and filovirus virulence.

Integrated Molecular Signature of Disease: Analysis of Influenza Virus-Infected Macaques through Functional Genomics and Proteomics

ABSTRACT Recent outbreaks of avian influenza in humans have stressed the need for an improved nonhuman primate model of influenza pathogenesis. In order to further develop a macaque model, we expanded our previous in vivo genomics experiments with influenza virus-infected macaques by focusing on the innate immune response at day 2 postinoculation and on gene expression in affected lung tissue with viral genetic material present. Finally, we sought to identify signature genes for early infection in whole blood. For these purposes, we infected six pigtailed macaques (Macaca nemestrina) with reconstructed influenza A/Texas/36/91 virus and three control animals with a sham inoculate. We sacrificed one control and two experimental animals at days 2, 4, and 7 postinfection. Lung tissue was harvested for pathology, gene expression profiling, and proteomics. Blood was collected for genomics every other day from each animal until the experimental endpoint. Gross and microscopic pathology, immunohistochemistry, viral gene expression by arrays, and/or quantitative real-time reverse transcription-PCR confirmed successful yet mild infections in all experimental animals. Genomic experiments were performed using macaque-specific oligonucleotide arrays, and high-throughput proteomics revealed the host response to infection at the mRNA and protein levels. Our data showed dramatic differences in gene expression within regions in influenza virus-induced lesions based on the presence or absence of viral mRNA. We also identified genes tightly coregulated in peripheral white blood cells and in lung tissue at day 2 postinoculation. This latter finding opens the possibility of using gene expression arrays on whole blood to detect infection after exposure but prior to onset of symptoms or shedding.

Gene Expression Profiling of the Cellular Transcriptional Network Regulated by Alpha/Beta Interferon and Its Partial Attenuation by the Hepatitis C Virus Nonstructural 5A Protein

ABSTRACT Alpha/beta interferons (IFN-α/β) induce potent antiviral and antiproliferative responses and are used to treat a wide range of human diseases, including chronic hepatitis C virus (HCV) infection. However, for reasons that remain poorly understood, many HCV isolates are resistant to IFN therapy. To better understand the nature of the cellular IFN response, we examined the effects of IFN treatment on global gene expression by using several types of human cells, including HeLa cells, liver cell lines, and primary fetal hepatocytes. In response to IFN, 50 of the approximately 4,600 genes examined were consistently induced in each of these cell types and another 60 were induced in a cell type-specific manner. A search for IFN-stimulated response elements (ISREs) in genomic DNA located upstream of IFN-stimulated genes revealed both previously identified and novel putative ISREs. To determine whether HCV can alter IFN-regulated gene expression, we performed microarray analyses on IFN-treated HeLa cells expressing the HCV nonstructural 5A (NS5A) protein and on IFN-treated Huh7 cells containing an HCV subgenomic replicon. NS5A partially blocked the IFN-mediated induction of 14 IFN-stimulated genes, an effect that may play a role in HCV resistance to IFN. This block may occur through repression of ISRE-mediated transcription, since NS5A also inhibited the IFN-mediated induction of a reporter gene driven from an ISRE-containing promoter. In contrast, the HCV replicon had very little effect on IFN-regulated gene expression. These differences highlight the importance of comparing results from multiple model systems when investigating complex phenomena such as the cellular response to IFN and viral mechanisms of IFN resistance.

Integration of Clinical Data, Pathology, and cDNA Microarrays in Influenza Virus-Infected Pigtailed Macaques (Macaca nemestrina)

ABSTRACT For most severe viral pandemics such as influenza and AIDS, the exact contribution of individual viral genes to pathogenicity is still largely unknown. A necessary step toward that understanding is a systematic comparison of different influenza virus strains at the level of transcriptional regulation in the host as a whole and interpretation of these complex genetic changes in the context of multifactorial clinical outcomes and pathology. We conducted a study by infecting pigtailed macaques (Macaca nemestrina) with a genetically reconstructed strain of human influenza H1N1 A/Texas/36/91 virus and hypothesized not only that these animals would respond to the virus similarly to humans, but that gene expression patterns in the lungs and tracheobronchial lymph nodes would fit into a coherent and complete picture of the host-virus interactions during infection. The disease observed in infected macaques simulated uncomplicated influenza in humans. Clinical signs and an antibody response appeared with induction of interferon and B-cell activation pathways, respectively. Transcriptional activation of inflammatory cells and apoptotic pathways coincided with gross and histopathological signs of inflammation, with tissue damage and concurrent signs of repair. Additionally, cDNA microarrays offered new evidence of the importance of cytotoxic T cells and natural killer cells throughout infection. With this experiment, we confirmed the suitability of the nonhuman primate model in the quest for understanding the individual and joint contributions of viral genes to influenza virus pathogenesis by using cDNA microarray technology and a reverse genetics approach.

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

ABSTRACT 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.

Cell Host Response to Infection with Novel Human Coronavirus EMC Predicts Potential Antivirals and Important Differences with SARS Coronavirus

ABSTRACT A novel human coronavirus (HCoV-EMC) was recently identified in the Middle East as the causative agent of a severe acute respiratory syndrome (SARS) resembling the illness caused by SARS coronavirus (SARS-CoV). Although derived from the CoV family, the two viruses are genetically distinct and do not use the same receptor. Here, we investigated whether HCoV-EMC and SARS-CoV induce similar or distinct host responses after infection of a human lung epithelial cell line. HCoV-EMC was able to replicate as efficiently as SARS-CoV in Calu-3 cells and similarly induced minimal transcriptomic changes before 12 h postinfection. Later in infection, HCoV-EMC induced a massive dysregulation of the host transcriptome, to a much greater extent than SARS-CoV. Both viruses induced a similar activation of pattern recognition receptors and the interleukin 17 (IL-17) pathway, but HCoV-EMC specifically down-regulated the expression of several genes within the antigen presentation pathway, including both type I and II major histocompatibility complex (MHC) genes. This could have an important impact on the ability of the host to mount an adaptive host response. A unique set of 207 genes was dysregulated early and permanently throughout infection with HCoV-EMC, and was used in a computational screen to predict potential antiviral compounds, including kinase inhibitors and glucocorticoids. Overall, HCoV-EMC and SARS-CoV elicit distinct host gene expression responses, which might impact in vivo pathogenesis and could orient therapeutic strategies against that emergent virus. IMPORTANCE Identification of a novel coronavirus causing fatal respiratory infection in humans raises concerns about a possible widespread outbreak of severe respiratory infection similar to the one caused by SARS-CoV. Using a human lung epithelial cell line and global transcriptomic profiling, we identified differences in the host response between HCoV-EMC and SARS-CoV. This enables rapid assessment of viral properties and the ability to anticipate possible differences in human clinical responses to HCoV-EMC and SARS-CoV. We used this information to predict potential effective drugs against HCoV-EMC, a method that could be more generally used to identify candidate therapeutics in future disease outbreaks. These data will help to generate hypotheses and make rapid advancements in characterizing this new virus. Identification of a novel coronavirus causing fatal respiratory infection in humans raises concerns about a possible widespread outbreak of severe respiratory infection similar to the one caused by SARS-CoV. Using a human lung epithelial cell line and global transcriptomic profiling, we identified differences in the host response between HCoV-EMC and SARS-CoV. This enables rapid assessment of viral properties and the ability to anticipate possible differences in human clinical responses to HCoV-EMC and SARS-CoV. We used this information to predict potential effective drugs against HCoV-EMC, a method that could be more generally used to identify candidate therapeutics in future disease outbreaks. These data will help to generate hypotheses and make rapid advancements in characterizing this new virus.