Sequencing,Annotation, and Characterization of the Influenza Ferret Infectome

Ferrets have become an indispensable tool in the understanding of influenza virus virulence and pathogenesis. Furthermore, ferrets are the preferred preclinical model for influenza vaccine and therapeutic testing. Here we characterized the influenza infectome during the different stages of the infectious process in ferrets with and without prior specific immunity to influenza. RNA from lung tissue and lymph nodes from infected and naïve animals was subjected to next-generation sequencing, followed by de novo data assembly and annotation of the resulting sequences; this process generated a library comprising 13,202 ferret mRNAs. Gene expression profiles during pandemic H1N1 (pdmH1N1) influenza virus infection were analyzed by digital gene expression and solid support microarrays. As expected during primary infection, innate immune responses were triggered in the lung tissue; meanwhile, in the lymphoid tissue, genes encoding antigen presentation and maturation of effector cells of adaptive immunity increased dramatically. After 5 days postinfection, the innate immune gene expression was replaced by the adaptive immune response, which correlates with viral clearance. Reinfection with homologous pandemic influenza virus resulted in a diminished innate immune response, early adaptive immune gene regulation, and a reduction in clinical severity. The fully annotated ferret infectome will be a critical aid to the understanding of the molecular events that regulate disease severity and host-influenza virus interactions among seasonal, pandemic, and highly pathogenic avian influenzas.     

Hepatitis C Virus Driven AXL Expression Suppresses the Hepatic Type I Interferon Response

Treatment of chronic hepatitis C virus (HCV) infection is evolving rapidly with the development of novel direct acting antivirals (DAAs), however viral clearance remains intimately linked to the hepatic innate immune system. Patients demonstrating a high baseline activation of interferon stimulated genes (ISGs), termed interferon refractoriness, are less likely to mount a strong antiviral response and achieve viral clearance when placed on treatment. As a result, suppressor of cytokine signalling (SOCS) 3 and other regulators of the IFN response have been identified as key candidates for the IFN refractory phenotype due to their regulatory role on the IFN response. AXL is a receptor tyrosine kinase that has been identified as a key regulator of interferon (IFN) signalling in myeloid cells of the immune system, but has not been examined in the context of chronic HCV infection. Here, we show that AXL is up-regulated following HCV infection, both in vitro and in vivo and is likely induced by type I/III IFNs and inflammatory signalling pathways. AXL inhibited type IFNα mediated ISG expression resulting in a decrease in its antiviral efficacy against HCV in vitro. Furthermore, patients possessing the favourable IFNL3 rs12979860 genotype associated with treatment response, showed lower AXL expression in the liver and a stronger induction of AXL in the blood, following their first dose of IFN. Together, these data suggest that elevated AXL expression in the liver may mediate an IFN-refractory phenotype characteristic of patients possessing the unfavourable rs12979860 genotype, which is associated with lower rates of viral clearance.     

Slc15a4, a Gene Required for pDC Sensing of TLR Ligands,Is Required to Control Persistent Viral Infection

Plasmacytoid dendritic cells (pDCs) are the major producers of type I IFN in response to viral infection and have been shown to direct both innate and adaptive immune responses in vitro. However, in vivo evidence for their role in viral infection is lacking. We evaluated the contribution of pDCs to acute and chronic virus infection using the feeble mouse model of pDC functional deficiency. We have previously demonstrated that feeble mice have a defect in TLR ligand sensing. Although pDCs were found to influence early cytokine secretion, they were not required for control of viremia in the acute phase of the infection. However, T cell priming was deficient in the absence of functional pDCs and the virus-specific immune response was hampered. Ultimately, infection persisted in feeble mice. We conclude that pDCs are likely required for efficient T cell priming and subsequent viral clearance. Our data suggest that reduced pDC functionality may lead to chronic infection.     

Immunological Characterization of the Teleost Adipose Tissue and Its Modulation in Response to Viral Infection and Fat-Content in the Diet

The immune response of the adipose tissue (AT) has been neglected in most animal models until recently, when the observations made in human and mice linking obesity to chronic inflammation and diabetes highlighted an important immune component of this tissue. In the current study, we have immunologically characterized the AT for the first time in teleosts. We have analyzed the capacity of rainbow trout (Oncorhynchus mykiss) AT to produce different immune mediators and we have identified the presence of local populations of B lymphocytes expressing IgM, IgD or IgT, CD8α⁺ cells and cells expressing major histocompatibility complex II (MHC-II). Because trout AT retained antigens from the peritoneal cavity, we analyzed the effects of intraperitoneal infection with viral hemorrhagic septicemia virus (VHSV) on AT functionality. A wide range of secreted immune factors were modulated within the AT in response to VHSV. Furthermore, the viral infection provoked a significant decrease in the number of IgM⁺ cells which, along with an increased secretion of IgM in the tissue, suggested a differentiation of B cells into plasmablasts. The virus also increased the number of CD8α⁺ cells in the AT. Finally, when a fat-enriched diet was fed to the fish, a significant modulation of immune gene expression in the AT was also observed. Thus, we have demonstrated for the first time in teleost that the AT functions as a relevant immune tissue; responsive to peritoneal viral infections and that this immune response can be modulated by the fat-content in the diet.     

Binding and entry of respiratory syncytial virus into host cells and initiation of the innate immune response

Respiratory syncytial virus (RSV) is the most common cause of severe lower respiratory tract infection in infants and the elderly. There is currently no effective antiviral treatment for the infection, but advances in our understanding of RSV uptake, especially the role of surfactant proteins, the attachment protein G and the fusion protein F, as well as the post-binding events, have revealed potential targets for new therapies and vaccine development. RSV infection triggers an intense inflammatory response, mediated initially by the infected airway epithelial cells and antigen-presenting cells. Humoral and cell-mediated immune responses are important in controlling the extent of infection and promoting viral clearance. The initial innate immune response may play a critical role by influencing the subsequent adaptive response generated. This review summarizes our current understanding of RSV binding and uptake in mammalian cells and how these initial interactions influence the subsequent innate immune response generated.     

Immune Mechanisms Responsible for Vaccination against and Clearance of Mucosal and Lymphatic Norovirus Infection

Two cardinal manifestations of viral immunity are efficient clearance of acute infection and the capacity to vaccinate against secondary viral exposure. For noroviruses, the contributions of T cells to viral clearance and vaccination have not been elucidated. We report here that both CD4 and CD8 T cells are required for efficient clearance of primary murine norovirus (MNV) infection from the intestine and intestinal lymph nodes. Further, long-lasting protective immunity was generated by oral live virus vaccination. Systemic vaccination with the MNV capsid protein also effectively protected against mucosal challenge, while vaccination with the capsid protein of the distantly related human Lordsdale virus provided partial protection. Fully effective vaccination required a broad immune response including CD4 T cells, CD8 T cells, and B cells, but the importance of specific immune cell types varied between the intestine and intestinal lymph nodes. Perforin, but not interferon gamma, was required for clearance of MNV infection by adoptively transferred T lymphocytes from vaccinated hosts. These studies prove the feasibility of both mucosal and systemic vaccination against mucosal norovirus infection, demonstrate tissue specificity of norovirus immune cells, and indicate that efficient vaccination strategies should induce potent CD4 and CD8 T cell responses.     

Expression analysis of immune response genes in fish epithelial cells following ranavirus infection

Ranaviruses (family Iridoviridae) are a growing threat to fish and amphibian populations worldwide. The immune response to ranavirus infection has been studied in amphibians, but little is known about the responses elicited in piscine hosts. In this study, the immune response and apoptosis induced by ranaviruses were investigated in fish epithelial cells. Epithelioma papulosum cyprini (EPC) cells were infected with four different viral isolates: epizootic haematopoietic necrosis virus (EHNV), frog virus 3 (FV3), European catfish virus (ECV) and doctor fish virus (DFV). Quantitative real-time PCR (qPCR) assays were developed to measure the mRNA expression of immune response genes during ranavirus infection. The target genes included tumour necrosis factor α (TNF-α), interleukin-1β (IL-1β), β2-microglobulin (β2M), interleukin-10 (IL-10) and transforming growth factor β (TGF-β). All ranaviruses elicited changes in immune gene expression. EHNV and FV3 caused a strong pro-inflammatory response with an increase in the expression of both IL-1β and TNF-α, whereas ECV and DFV evoked transient up-regulation of regulatory cytokine TGF-β. Additionally, all viral isolates induced increased β2M expression as well as apoptosis in the EPC cells. Our results indicate that epithelial cells can serve as an in vitro model for studying the mechanisms of immune response in the piscine host in the first stages of ranavirus infection.     

Nrf2 expression modifies influenza A entry and replication in nasal epithelial cells

Influenza infection is a major cause of morbidity and mortality worldwide, especially during pandemics outbreaks. Emerging data indicate that phase II antioxidant enzyme pathways could play a role in virus-associated inflammation and immune clearance. While Nrf2-dependent gene expression is known to modify inflammation, a mechanistic role in viral susceptibility and clearance has yet to be elucidated. Therefore, we utilized differentiated human nasal epithelial cells (NEC) and an enzymatic virus-like particle entry assay, to examine the role Nrf2-dependent gene expression has on viral entry and replication. Herein, lentiviral vectors that express Nrf2-specific short hairpin (sh)-RNA effectively decreased both Nrf2 mRNA and Nrf2 protein expression in transduced human NEC from healthy volunteers. Nrf2 knockdown correlated with a significant increase in influenza virus entry and replication. Conversely, supplementation with the potent Nrf2 activators sulforaphane (SFN) and epigallocatechin gallate (EGCG) significantly decreased viral entry and replication. The suppressive effects of EGCG on viral replication were abolished in cells with knocked-down Nrf2 expression, suggesting a causal relationship between the EGCG-induced activation of Nrf2 and the ability to protect against viral infection. Interestingly, the induction of Nrf2 via nutritional supplements SFN and EGCG increased antiviral mediators/responses: RIG-I, IFN-β, and MxA at baseline in the absence of infection. Our data indicate that there is an inverse relationship between the levels of Nrf2 expression and the viral entry/replication. We also demonstrate that supplementation with Nrf2-activating antioxidants inhibits viral replication in human NEC, which may prove to be an attractive therapeutic intervention. Taken together, these data indicate potential mechanisms by which Nrf2-dependent gene expression regulates susceptibility to influenza in human epithelial cells.     

Bordetella pertussis infection exacerbates influenza virus infection through pertussis toxin-mediated suppression of innate immunity

Pertussis (whooping cough) is frequently complicated by concomitant infections with respiratory viruses. Here we report the effect of Bordetella pertussis infection on subsequent influenza virus (PR8) infection in mouse models and the role of pertussis toxin (PT) in this effect. BALB/c mice infected with a wild-type strain of B. pertussis (WT) and subsequently (up to 14 days later) infected with PR8 had significantly increased pulmonary viral titers, lung pathology and mortality compared to mice similarly infected with a PT-deficient mutant strain (ΔPT) and PR8. Substitution of WT infection by intranasal treatment with purified active PT was sufficient to replicate the exacerbating effects on PR8 infection in BALB/c and C57/BL6 mice, but the effects of PT were lost when toxin was administered 24 h after virus inoculation. PT had no effect on virus titers in primary cultures of murine tracheal epithelial cells (mTECs) in vitro, suggesting the toxin targets an early immune response to increase viral titers in the mouse model. However, type I interferon responses were not affected by PT. Whole genome microarray analysis of gene expression in lung tissue from PT-treated and control PR8-infected mice at 12 and 36 h post-virus inoculation revealed that PT treatment suppressed numerous genes associated with communication between innate and adaptive immune responses. In mice depleted of alveolar macrophages, increase of pulmonary viral titers by PT treatment was lost. PT also suppressed levels of IL-1β, IL-12, IFN-γ, IL-6, KC, MCP-1 and TNF-α in the airways after PR8 infection. Furthermore PT treatment inhibited early recruitment of neutrophils and NK cells to the airways. Together these findings demonstrate that infection with B. pertussis through PT activity predisposes the host to exacerbated influenza infection by countering protective innate immune responses that control virus titers.     

Early Virus-Host Interactions Dictate the Course of a Persistent Infection

Many persistent viral infections are characterized by a hypofunctional T cell response and the upregulation of negative immune regulators. These events occur days after the initiation of infection. However, the very early host-virus interactions that determine the establishment of viral persistence remain poorly uncharacterized. Here we show that to establish persistence, LCMV must counteract an innate anti-viral immune response within eight hours after infection. While the virus triggers cytoplasmic RNA sensing pathways soon after infection, LCMV counteracts this pathway through a rapid increase in viral titers leading to a dysfunctional immune response characterized by a high cytokine and chemokine expression profile. This altered immune environment allows for viral replication in the splenic white pulp as well as infection of immune cells essential to an effective anti-viral immune response. Our findings illustrate how early events during infection critically dictate the characteristics of the immune response to infection and facilitate either virus control and clearance or persistence.     

Host and viral factors in the immunopathogenesis of primary hepatitis C virus infection

Individuals infected with hepatitis C virus (HCV) have two possible outcomes of infection, clearance or persistent infection. The focus of this review is the host mechanisms that facilitate clearance. The interaction between HCV viral components and the immune system ultimately determines the balance between the virus and host. Strong evidence points to the aspects of cellular immune response as the key determinants of outcome. The recent discovery of viral evasion strategies targeting innate immunity suggests that the interferon-alpha/beta induction pathways are also critical. A growing body of evidence has implicated polymorphisms in both innate and adaptive immune response genes as determinants of viral clearance in individuals infected with HCV.     

Measles virus, immune control, and persistence

Measles remains one of the most important causes of child morbidity and mortality worldwide with the greatest burden in the youngest children. Most acute measles deaths are owing to secondary infections that result from a poorly understood measles-induced suppression of immune responses. Young children are also vulnerable to late development of subacute sclerosing panencephalitis, a progressive, uniformly fatal neurologic disease caused by persistent measles virus (MeV) infection. During acute infection, the rash marks the appearance of the adaptive immune response and CD8(+) T cell-mediated clearance of infectious virus. However, after clearance of infectious virus, MeV RNA persists and can be detected in blood, respiratory secretions, urine, and lymphoid tissue for many weeks to months. This prolonged period of virus clearance may help to explain measles immunosuppression and the development of lifelong immunity to re-infection, as well as occasional infection of the nervous system. Once MeV infects neurons, the virus can spread trans-synaptically and the envelope proteins needed to form infectious virus are unnecessary, accumulate mutations, and can establish persistent infection. Identification of the immune mechanisms required for the clearance of MeV RNA from multiple sites will enlighten our understanding of the development of disease owing to persistent infection.     

CD200 Receptor Controls Sex-Specific TLR7 Responses to Viral Infection

Immunological checkpoints, such as the inhibitory CD200 receptor (CD200R), play a dual role in balancing the immune system during microbial infection. On the one hand these inhibitory signals prevent excessive immune mediated pathology but on the other hand they may impair clearance of the pathogen. We studied the influence of the inhibitory CD200-CD200R axis on clearance and pathology in two different virus infection models. We find that lack of CD200R signaling strongly enhances type I interferon (IFN) production and viral clearance and improves the outcome of mouse hepatitis corona virus (MHV) infection, particularly in female mice. MHV clearance is known to be dependent on Toll like receptor 7 (TLR7)-mediated type I IFN production and sex differences in TLR7 responses previously have been reported for humans. We therefore hypothesize that CD200R ligation suppresses TLR7 responses and that release of this inhibition enlarges sex differences in TLR7 signaling. This hypothesis is supported by our findings that in vivo administration of synthetic TLR7 ligand leads to enhanced type I IFN production, particularly in female Cd200^−/− mice and that CD200R ligation inhibits TLR7 signaling in vitro. In influenza A virus infection we show that viral clearance is determined by sex but not by CD200R signaling. However, absence of CD200R in influenza A virus infection results in enhanced lung neutrophil influx and pathology in females. Thus, CD200-CD200R and sex are host factors that together determine the outcome of viral infection. Our data predict a sex bias in both beneficial and pathological immune responses to virus infection upon therapeutic targeting of CD200-CD200R.     

Dynamics of immune cell recruitment during West Nile encephalitis and identification of a new CD19+B220-BST-2+ leukocyte population

West Nile virus (WNV) is an emerging neurotropic flavivirus. We investigated the dynamics of immune cell recruitment in peripheral tissues and in the CNS during WNV encephalitis in an immunocompetent mouse model. In the periphery, immune cell expansion can successfully limit viremia and lymphoid tissue infection. However, viral clearance in the periphery is too late to prevent viral invasion of the CNS. In the CNS, innate immune cells, including microglia/macrophages, NK cells, and plasmacytoid dendritic cells, greatly expand as the virus invades the brain, whereas B and T cells are recruited after viral invasion, and fail to control the spread of the virus. Thus, the onset of WNV encephalitis was correlated both with CNS viral infection and with a large local increase of innate immune cells. Interestingly, we identify a new immune cell type: CD19(+)B220(-) BST-2(+), which we name G8-ICs. These cells appear during peripheral infection and enter the CNS. G8-ICs express high levels of MHC class II, stain for viral Ag, and are localized in the paracortical zone of lymph nodes, strongly suggesting they are previously unidentified APCs that appear in response to viral infection.     

Apoptotic Regulation of T Cells and Absence of Immune Deficiency in Virus-Infected Gamma Interferon Receptor Knockout Mice

Acute viral infections often induce a transient period of immune deficiency in which the host’s T cells fail to proliferate in response to T-cell mitogens and fail to make an antigen-specific memory recall response. This has been associated with the enhanced sensitivity of these highly activated T cells to undergo apoptosis, or activation-induced cell death (AICD), upon T-cell receptor ligation. Here we show that gamma interferon receptor-deficient (IFN-γ R^−/−) mice mount a T-cell response to lymphocytic choriomeningitis virus (LCMV) infection but fail to undergo the transient immune deficiency. Instead, their T cells were hyperproliferative and relatively, but not completely, resistant to AICD. The immune response returned to homeostasis, but with delayed kinetics, in parallel with delayed clearance of the virus. Wild-type mice receiving high doses of disseminating LCMV Clone 13 are known to undergo clonal exhaustion of their virus-specific cytotoxic T lymphocytes (CTL). To determine whether this process was mediated by AICD associated with IFN-γ or with Fas-Fas ligand interactions, LCMV-specific precursor CTL frequencies were examined in LCMV Clone 13-infected IFN-γ R^−/− or lpr (Fas-deficient) mice. In both instances, viral persistence was established and CTL precursors were greatly eliminated. This finding indicates that clonal exhaustion of CTL does not require IFN-γ or Fas, even though both molecules influence AICD and the transient immune deficiency seen in the LCMV infection.     

Parasite-mediated upregulation of NK cell-derived gamma interferon protects against severe highly pathogenic H5N1 influenza virus infection

Outbreaks of influenza A viruses are associated with significant human morbidity worldwide. Given the increasing resistance to the available influenza drugs, new therapies for the treatment of influenza virus infection are needed. An alternative approach is to identify products that enhance a protective immune response. In these studies, we demonstrate that infecting mice with the Th1-inducing parasite Toxoplasma gondii prior to highly pathogenic avian H5N1 influenza virus infection led to decreased lung viral titers and enhanced survival. A noninfectious fraction of T. gondii soluble antigens (STAg) elicited an immune response similar to that elicited by live parasites, and administration of STAg 2 days after H5N1 influenza virus infection enhanced survival, lowered viral titers, and reduced clinical disease. STAg administration protected H5N1 virus-infected mice lacking lymphocytes, suggesting that while the adaptive immune response was not required for enhanced survival, it was necessary for STAg-mediated viral clearance. Mechanistically, we found that administration of STAg led to increased production of gamma interferon (IFN-γ) from natural killer (NK) cells, which were both necessary and sufficient for survival. Further, administration of exogenous IFN-γ alone enhanced survival from H5N1 influenza virus infection, although not to the same level as STAg treatment. These studies demonstrate that a noninfectious T. gondii extract enhances the protective immune response against severe H5N1 influenza virus infections even when a single dose is administered 2 days postinfection.     

Stochastic models for virus and immune system dynamics

New stochastic models are developed for the dynamics of a viral infection and an immune response during the early stages of infection. The stochastic models are derived based on the dynamics of deterministic models. The simplest deterministic model is a well-known system of ordinary differential equations which consists of three populations: uninfected cells, actively infected cells, and virus particles. This basic model is extended to include some factors of the immune response related to Human Immunodeficiency Virus-1 (HIV-1) infection. For the deterministic models, the basic reproduction number, R₀, is calculated and it is shown that if R₀<1, the disease-free equilibrium is locally asymptotically stable and is globally asymptotically stable in some special cases. The new stochastic models are systems of stochastic differential equations (SDEs) and continuous-time Markov chain (CTMC) models that account for the variability in cellular reproduction and death, the infection process, the immune system activation, and viral reproduction. Two viral release strategies are considered: budding and bursting. The CTMC model is used to estimate the probability of virus extinction during the early stages of infection. Numerical simulations are carried out using parameter values applicable to HIV-1 dynamics. The stochastic models provide new insights, distinct from the basic deterministic models. For the case R₀>1, the deterministic models predict the viral infection persists in the host. But for the stochastic models, there is a positive probability of viral extinction. It is shown that the probability of a successful invasion depends on the initial viral dose, whether the immune system is activated, and whether the release strategy is bursting or budding.     

Delayed Time-to-Treatment of an Antisense Morpholino Oligomer Is Effective against Lethal Marburg Virus Infection in Cynomolgus Macaques

Marburg virus (MARV) is an Ebola-like virus in the family Filovirdae that causes sporadic outbreaks of severe hemorrhagic fever with a case fatality rate as high as 90%. AVI-7288, a positively charged antisense phosphorodiamidate morpholino oligomer (PMOplus) targeting the viral nucleoprotein gene, was evaluated as a potential therapeutic intervention for MARV infection following delayed treatment of 1, 24, 48, and 96 h post-infection (PI) in a nonhuman primate lethal challenge model. A total of 30 cynomolgus macaques were divided into 5 groups of 6 and infected with 1,830 plaque forming units of MARV subcutaneously. AVI-7288 was administered by bolus infusion daily for 14 days at 15 mg/kg body weight. Survival was the primary endpoint of the study. While none (0 of 6) of the saline group survived, 83–100% of infected monkeys survived when treatment was initiated 1, 24, 48, or 96 h post-infection (PI). The antisense treatment also reduced serum viremia and inflammatory cytokines in all treatment groups compared to vehicle controls. The antibody immune response to virus was preserved and tissue viral antigen was cleared in AVI-7288 treated animals. These data show that AVI-7288 protects NHPs against an otherwise lethal MARV infection when treatment is initiated up to 96 h PI.