Laurence Josset

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.

Attenuation and Restoration of Severe Acute Respiratory Syndrome Coronavirus Mutant Lacking 2′-O-Methyltransferase Activity

ABSTRACT The sudden emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 and, more recently, Middle Eastern respiratory syndrome CoV (MERS-CoV) underscores the importance of understanding critical aspects of CoV infection and pathogenesis. Despite significant insights into CoV cross-species transmission, replication, and virus-host interactions, successful therapeutic options for CoVs do not yet exist. Recent identification of SARS-CoV NSP16 as a viral 2′-O-methyltransferase (2′-O-MTase) led to the possibility of utilizing this pathway to both attenuate SARS-CoV infection and develop novel therapeutic treatment options. Mutations were introduced into SARS-CoV NSP16 within the conserved KDKE motif and effectively attenuated the resulting SARS-CoV mutant viruses both in vitro and in vivo. While viruses lacking 2′-O-MTase activity had enhanced sensitivity to type I interferon (IFN), they were not completely restored in their absence in vivo. However, the absence of either MDA5 or IFIT1, IFN-responsive genes that recognize unmethylated 2′-O RNA, resulted in restored replication and virulence of the dNSP16 mutant virus. Finally, using the mutant as a live-attenuated vaccine showed significant promise for possible therapeutic development against SARS-CoV. Together, the data underscore the necessity of 2′-O-MTase activity for SARS-CoV pathogenesis and identify host immune pathways that mediate this attenuation. In addition, we describe novel treatment avenues that exploit this pathway and could potentially be used against a diverse range of viral pathogens that utilize 2′-O-MTase activity to subvert the immune system. IMPORTANCE Preventing recognition by the host immune response represents a critical aspect necessary for successful viral infection. Several viruses, including SARS-CoV, utilize virally encoded 2′-O-MTases to camouflage and obscure their viral RNA from host cell sensing machinery, thus preventing recognition and activation of cell intrinsic defense pathways. For SARS-CoV, the absence of this 2′-O-MTase activity results in significant attenuation characterized by decreased viral replication, reduced weight loss, and limited breathing dysfunction in mice. The results indicate that both MDA5, a recognition molecule, and the IFIT family play an important role in mediating this attenuation with restored virulence observed in their absence. Understanding this virus-host interaction provided an opportunity to design a successful live-attenuated vaccine for SARS-CoV and opens avenues for treatment and prevention of emerging CoVs and other RNA virus infections.

Pathogenic Influenza Viruses and Coronaviruses Utilize Similar and Contrasting Approaches To Control Interferon-Stimulated Gene Responses

ABSTRACT 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 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.

European surveillance for enterovirus D68 during the emerging North-American outbreak in 2014

BACKGROUND In August and September 2014, unexpected clusters of enterovirus-D68 (EV-D68) infections associated with severe respiratory disease emerged from North-America. In September, the European Centre for Disease Prevention and Control (ECDC) asked European countries to strengthen respiratory sample screening for enterovirus detection and typing in cases with severe respiratory presentations. OBJECTIVES To provide a detailed picture of EV-D68 epidemiology in Europe by conducting a retrospective and prospective laboratory analysis of clinical specimens. STUDY DESIGN An initiative supported by the European Society for Clinical Virology (ESCV) and ECDC was launched to screen for EV-D68 in respiratory specimens between July 1st and December 1st 2014 in Europe and to sequence the VP1 region of detected viruses for phylogenetic analytic purposes. RESULTS Forty-two institutes, representing 51 laboratories from 17 European countries, analyzed 17,248 specimens yielding 389 EV-D68 positive samples (2.26%) in 14 countries. The proportion of positive samples ranged between 0 and 25% per country. These infections resulted primarily in mild respiratory disease, mainly detected in young children presenting with wheezing and in immuno-compromised adults. The viruses detected in Europe are genetically very similar to those of the North-American epidemic and the majority (83%) could be assigned to clade B. Except for 3 acute flaccid paralysis (AFP) cases, one death and limited ICU admissions, no severe cases were reported. CONCLUSIONS The European study showed that EV-D68 circulated in Europe during summer and fall of 2014 with a moderate disease burden and different pathogenic profile compared to the North-American epidemic.

Implication of Inflammatory Macrophages, Nuclear Receptors, and Interferon Regulatory Factors in Increased Virulence of Pandemic 2009 H1N1 Influenza A Virus after Host Adaptation

ABSTRACT While pandemic 2009 H1N1 influenza A viruses were responsible for numerous severe infections in humans, these viruses do not typically cause corresponding severe disease in mammalian models. However, the generation of a virulent 2009 H1N1 virus following serial lung passage in mice has allowed for the modeling of human lung pathology in this species. Genetic determinants of mouse-adapted 2009 H1N1 viral pathogenicity have been identified, but the molecular and signaling characteristics of the host response following infection with this adapted virus have not been described. Here we compared the gene expression response following infection of mice with A/CA/04/2009 (CA/04) or the virulent mouse-adapted strain (MA-CA/04). Microarray analysis revealed that increased pathogenicity of MA-CA/04 was associated with the following: (i) an early and sustained inflammatory and interferon response that could be driven in part by interferon regulatory factors (IRFs) and increased NF-κB activation, as well as inhibition of the negative regulator TRIM24, (ii) early and persistent infiltration of immune cells, including inflammatory macrophages, and (iii) the absence of activation of lipid metabolism later in infection, which may be mediated by inhibition of nuclear receptors, including PPARG and HNF1A and -4A, with proinflammatory consequences. Further investigation of these signatures in the host response to other H1N1 viruses of various pathogenicities confirmed their general relevance for virulence of influenza virus and suggested that lung response to MA-CA/04 virus was similar to that following infection with lethal H1N1 r1918 influenza virus. This study links differential activation of IRFs, nuclear receptors, and macrophage infiltration with influenza virulence in vivo.

Increased Viral Loads and Exacerbated Innate Host Responses in Aged Macaques Infected with the 2009 Pandemic H1N1 Influenza A Virus

ABSTRACT In contrast to seasonal influenza virus infections, which typically cause significant morbidity and mortality in the elderly, the 2009 H1N1 virus caused severe infection in young adults. This phenomenon was attributed to the presence of cross-protective antibodies acquired by older individuals during previous exposures to H1N1 viruses. However, this hypothesis could not be empirically tested. To address this question, we compared viral replication and the development of the immune response in naïve young adult and aged female rhesus macaques infected with A/California/04/2009 H1N1 (CA04) virus. We show higher viral loads in the bronchoalveolar lavage (BAL) fluid and nasal and ocular swabs in aged animals, suggesting increased viral replication in both the lower and upper respiratory tracts. T cell proliferation was higher in the BAL fluid but delayed and reduced in peripheral blood in aged animals. This delay in proliferation correlated with a reduced frequency of effector CD4 T cells in old animals. Aged animals also mobilized inflammatory cytokines to higher levels in the BAL fluid. Finally, we compared changes in gene expression using microarray analysis of BAL fluid samples. Our analyses revealed that the largest difference in host response between aged and young adult animals was detected at day 4 postinfection, with a significantly higher induction of genes associated with inflammation and the innate immune response in aged animals. Overall, our data suggest that, in the absence of preexisting antibodies, CA04 infection in aged macaques is associated with changes in innate and adaptive immune responses that were shown to correlate with increased disease severity in other respiratory disease models.

Influenza A replication and host nuclear compartments: Many changes and many questions

It is over 40 years since investigations showed that influenza A, one of the rare nuclear replicating RNA viruses, induces marked remodeling of the host nuclear architecture. Influenza modifies and/or hijacks host nuclear machinery in order to replicate, express viral proteins and interfere with host antiviral response. Numerous interactions between constitutive nuclear proteins and viral factors are now characterized but less is known concerning their functional significance and their connection with viral-induced modifications of nuclear ultrastructure. Therefore, the purpose of this review is to summarize data and hypotheses about functional interplays between host nuclear compartments and influenza A during viral replication.

Annotation of long non-coding RNAs expressed in Collaborative Cross founder mice in response to respiratory virus infection reveals a new class of interferon-stimulated transcripts

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).

Transcriptomic Characterization of the Novel Avian-Origin Influenza A (H7N9) Virus: Specific Host Response and Responses Intermediate between Avian (H5N1 and H7N7) and Human (H3N2) Viruses and Implications for Treatment Options

ABSTRACT A novel avian-origin H7N9 influenza A virus (IAV) emerged in China in 2013, causing mild to lethal human respiratory infections. H7N9 originated with multiple reassortment events between avian viruses and carries genetic markers of human adaptation. Determining whether H7N9 induces a host response closer to that with human or avian IAV is important in order to better characterize this emerging virus. Here we compared the human lung epithelial cell response to infection with A/Anhui/01/13 (H7N9) or highly pathogenic avian-origin H5N1, H7N7, or human seasonal H3N2 IAV. The transcriptomic response to H7N9 was highly specific to this strain but was more similar to the response to human H3N2 than to that to other avian IAVs. H7N9 and H3N2 both elicited responses related to eicosanoid signaling and chromatin modification, whereas H7N9 specifically induced genes regulating the cell cycle and transcription. Among avian IAVs, the response to H7N9 was closest to that elicited by H5N1 virus. Host responses common to H7N9 and the other avian viruses included the lack of induction of the antigen presentation pathway and reduced proinflammatory cytokine induction compared to that with H3N2. Repression of these responses could have an important impact on the immunogenicity and virulence of H7N9 in humans. Finally, using a genome-based drug repurposing approach, we identified several drugs predicted to regulate the host response to H7N9 that may act as potential antivirals, including several kinase inhibitors, as well as FDA-approved drugs, such as troglitazone and minocycline. Importantly, we validated that minocycline inhibited H7N9 replication in vitro, suggesting that our computational approach holds promise for identifying novel antivirals. IMPORTANCE Whether H7N9 will be the next pandemic influenza virus or will persist and sporadically infect humans from its avian reservoir, similar to H5N1, is not known yet. High-throughput profiling of the host response to infection allows rapid characterization of virus-host interactions and generates many hypotheses that will accelerate understanding and responsiveness to this potential threat. We show that the cellular response to H7N9 virus is closer to that induced by H3N2 than to that induced by H5N1, reflecting the potential of this new virus for adaptation to humans. Importantly, dissecting the host response to H7N9 may guide host-directed antiviral development. Whether H7N9 will be the next pandemic influenza virus or will persist and sporadically infect humans from its avian reservoir, similar to H5N1, is not known yet. High-throughput profiling of the host response to infection allows rapid characterization of virus-host interactions and generates many hypotheses that will accelerate understanding and responsiveness to this potential threat. We show that the cellular response to H7N9 virus is closer to that induced by H3N2 than to that induced by H5N1, reflecting the potential of this new virus for adaptation to humans. Importantly, dissecting the host response to H7N9 may guide host-directed antiviral development.

H7N9 and Other Pathogenic Avian Influenza Viruses Elicit a Three-Pronged Transcriptomic Signature That Is Reminiscent of 1918 Influenza Virus and Is Associated with Lethal Outcome in Mice

ABSTRACT Modulating the host response is a promising approach to treating influenza, caused by a virus whose pathogenesis is determined in part by the reaction it elicits within the host. Though the pathogenicity of emerging H7N9 influenza virus in several animal models has been reported, these studies have not included a detailed characterization of the host response following infection. Therefore, we characterized the transcriptomic response of BALB/c mice infected with H7N9 (A/Anhui/01/2013) virus and compared it to the responses induced by H5N1 (A/Vietnam/1203/2004), H7N7 (A/Netherlands/219/2003), and pandemic 2009 H1N1 (A/Mexico/4482/2009) influenza viruses. We found that responses to the H7 subtype viruses were intermediate to those elicited by H5N1 and pdm09H1N1 early in infection but that they evolved to resemble the H5N1 response as infection progressed. H5N1, H7N7, and H7N9 viruses were pathogenic in mice, and this pathogenicity correlated with increased transcription of cytokine response genes and decreased transcription of lipid metabolism and coagulation signaling genes. This three-pronged transcriptomic signature was observed in mice infected with pathogenic H1N1 strains such as the 1918 virus, indicating that it may be predictive of pathogenicity across multiple influenza virus strains. Finally, we used host transcriptomic profiling to computationally predict drugs that reverse the host response to H7N9 infection, and we identified six FDA-approved drugs that could potentially be repurposed to treat H7N9 and other pathogenic influenza viruses. IMPORTANCE Emerging avian influenza viruses are of global concern because the human population is immunologically naive to them. Current influenza drugs target viral molecules, but the high mutation rate of influenza viruses eventually leads to the development of antiviral resistance. As the host evolves far more slowly than the virus, and influenza pathogenesis is determined in part by the host response, targeting the host response is a promising approach to treating influenza. Here we characterize the host transcriptomic response to emerging H7N9 influenza virus and compare it with the responses to H7N7, H5N1, and pdm09H1N1. All three avian viruses were pathogenic in mice and elicited a transcriptomic signature that also occurs in response to the legendary 1918 influenza virus. Our work identifies host responses that could be targeted to treat severe H7N9 influenza and identifies six FDA-approved drugs that could potentially be repurposed as H7N9 influenza therapeutics.