Steroid Hormone Synthesis by Vaccinia Virus Suppresses the Inflammatory Response to Infection

The 3β-hydroxysteroid dehydrogenase (3β-HSD) isoenzymes play a key role in cellular steroid hormone synthesis. Vaccinia virus (VV) also synthesizes steroid hormones with a 3β-HSD enzyme (v3β-HSD) encoded by gene A44L. Here we examined the effects of v3β-HSD in VV disease using wild-type (vA44L), deletion (vΔA44L), and revertant (vA44L-rev) viruses in a murine intranasal model. Loss of A44L was associated with an attenuated phenotype. Early (days 1–3) after infection with vΔA44L or control viruses the only difference observed between groups was the reduced corticosterone level in lungs and plasma of vΔA44L-infected animals. Other parameters examined (body weight, signs of illness, temperature, virus titres, the pulmonary inflammatory infiltrate, and interferon [IFN]-γ levels) were indistinguishable between groups. Subsequently, vΔA44L-infected animals had reduced weight loss and signs of illness, and displayed a vigorous pulmonary inflammatory response. This was characterized by rapid recruitment of CD4+ and CD8+ lymphocytes, enhanced IFN-γ production and augmented cytotoxic T lymphocyte activity. These data suggest that steroid production by v3β-HSD contributes to virus virulence by inhibiting an effective inflammatory response to infection

Soluble Host Defense Lectins in Innate Immunity to Influenza Virus

Host defenses against viral infections depend on a complex interplay of innate (nonspecific) and adaptive (specific) components. In the early stages of infection, innate mechanisms represent the main line of host defense, acting to limit the spread of virus in host tissues prior to the induction of the adaptive immune response. Serum and lung fluids contain a range of lectins capable of recognizing and destroying influenza A viruses (IAV). Herein, we review the mechanisms by which soluble endogenous lectins mediate anti-IAV activity, including their role in modulating IAV-induced inflammation and disease and their potential as prophylactic and/or therapeutic treatments during severe IAV-induced disease

Vaccinia virus protein A46R targets multiple Toll-like-interleukin-1 receptor adaptors and contributes to virulence

Viral immune evasion strategies target key aspects of the host antiviral response. Recently, it has been recognized that Toll-like receptors (TLRs) have a role in innate defense against viruses. Here, we define the function of the vaccinia virus (VV) protein A46R and show it inhibits intracellular signalling by a range of TLRs. TLR signalling is triggered by homotypic interactions between the Toll-like-interleukin-1 resistance (TIR) domains of the receptors and adaptor molecules. A46R contains a TIR domain and is the only viral TIR domain-containing protein identified to date. We demonstrate that A46R targets the host TIR adaptors myeloid differentiation factor 88 (MyD88), MyD88 adaptor-like, TIR domain-containing adaptor inducing IFN-beta (TRIF), and the TRIF-related adaptor molecule and thereby interferes with downstream activation of mitogen-activated protein kinases and nuclear factor kappaB. TRIF mediates activation of interferon (IFN) regulatory factor 3 (IRF3) and induction of IFN-beta by TLR3 and TLR4 and suppresses VV replication in macrophages. Here, A46R disrupted TRIF-induced IRF3 activation and induction of the TRIF-dependent gene regulated on activation, normal T cell expressed and secreted. Furthermore, we show that A46R is functionally distinct from another described VV TLR inhibitor, A52R. Importantly, VV lacking the A46R gene was attenuated in a murine intranasal model, demonstrating the importance of A46R for VV virulence

Loss of a single N-linked glycan from the hemagglutinin of influenza virus is associated with resistance to collectins and increased virulence in mice

BACKGROUND: Glycosylation on the globular head of the hemagglutinin (HA) protein of influenza virus acts as an important target for recognition and destruction of virus by innate immune proteins of the collectin family. This, in turn, modulates the virulence of different viruses for mice. The role of particular oligosaccharide attachments on the HA in determining sensitivity to collectins has yet to be fully elucidated. METHODS: When comparing the virulence of H3N2 subtype viruses for mice we found that viruses isolated after 1980 were highly glycosylated and induced mild disease in mice. During these studies, we were surprised to find a small plaque variant of strain A/Beijing/353/89 (Beij/89) emerged following infection of mice and grew to high titres in mouse lung. In the current study we have characterized the properties of this small plaque mutant both in vitro and in vivo. RESULTS: Small plaque mutants were recovered following plaquing of lung homogenates from mice infected with influenza virus seed Beij/89. Compared to wild-type virus, small plaque mutants showed increased virulence in mice yet did not differ in their ability to infect or replicate in airway epithelial cells in vitro. Instead, small plaque variants were markedly resistant to neutralization by murine collectins, a property that correlated with the acquisition of an amino acid substitution at residue 246 on the viral HA. We present evidence that this substitution was associated with the loss of an oligosaccharide glycan from the globular head of HA. CONCLUSION: A point mutation in the gene encoding the HA of Beij/89 was shown to ablate a glycan attachment site. This was associated with resistance to collectins and increased virulence in mice

TaqMan real time RT-PCR assays for detecting ferret innate and adaptive immune responses

AbstractThe ferret is an excellent model for many human infectious diseases including influenza, SARS-CoV, henipavirus and pneumococcal infections. The ferret is also used to study cystic fibrosis and various cancers, as well as reproductive biology and physiology. However, the range of reagents available to measure the ferret immune response is very limited. To address this deficiency, high-throughput real time RT-PCR TaqMan assays were developed to measure the expression of fifteen immune mediators associated with the innate and adaptive immune responses (IFNα, IFNβ, IFNγ, IL1α, IL1β, IL2, IL4, IL6, IL8, IL10, IL12p40, IL17, Granzyme A, MCP1, TNFα), as well as four endogenous housekeeping genes (ATF4, HPRT, GAPDH, L32). These assays have been optimized to maximize reaction efficiency, reduce the amount of sample required (down to 1ng RNA per real time RT-PCR reaction) and to select the most appropriate housekeeping genes. Using these assays, the expression of each of the tested genes could be detected in ferret lymph node cells stimulated with mitogens or infected with influenza virus in vitro. These new tools will allow a more comprehensive analysis of the ferret immune responses following infection or in other disease states

Pandemic H1N1 Influenza A Viruses Are Resistant to the Antiviral Activities of Innate Immune Proteins of the Collectin and Pentraxin Superfamilies

Abstract Acquired immune responses elicited to recent strains of seasonal H1N1 influenza viruses provide limited protection against emerging A(H1N1) pandemic viruses. Accordingly, pre-existing or rapidly induced innate immune defenses are of critical importance in limiting early infection. Respiratory secretions contain proteins of the innate immune system, including members of the collectin and pentraxin superfamilies. These mediate potent antiviral activity and act as an initial barrier to influenza infection. In this study, we have examined the sensitivity of H1N1 viruses, including pandemic virus strains, for their sensitivity to collectins (surfactant protein [SP]-D and mannose-binding lectin [MBL]) and to the pentraxin PTX3. Human SP-D and MBL inhibited virus-induced hemagglutinating activity, blocked the enzymatic activity of the viral neuraminidase, and neutralized the ability of H1N1 viruses to infect human respiratory epithelial cells in a manner that correlated with the degree of glycosylation in the globular head of the hemagglutinin. Recent seasonal H1N1 viruses expressed three to four N-glycosylation sequons on the head of hemagglutinin and were very sensitive to inhibition by SP-D or MBL, whereas A(H1N1) pandemic viruses expressed a single N-glycosylation sequon and were resistant to either collectin. Of interest, both seasonal and pandemic H1N1 viruses were resistant to PTX3. Thus, unlike recent seasonal H1N1 strains of influenza virus, A(H1N1) pandemic viruses are resistant to the antiviral activities of innate immune proteins of the collectin superfamily

Role of Pentraxin 3 in Shaping Arthritogenic Alphaviral Disease: From Enhanced Viral Replication to Immunomodulation

10.1371/journal.ppat.1004649PLoS Pathogens11

Infection of mouse macrophages by seasonal influenza viruses can be restricted at the level of virus entry and at a late stage in the virus life cycle

Airway epithelial cells are susceptible to infection with seasonal influenza A viruses (IAV), resulting in productive virus replication and release. Macrophages (MΦ) are also permissive to IAV infection; however, virus replication is abortive. Currently, it is unclear how productive infection of MΦ is impaired or the extent to which seasonal IAV replicate in MΦ. Herein, we compared mouse MΦ and epithelial cells for their ability to support genomic replication and transcription, synthesis of viral proteins, assembly of virions, and release of infectious progeny following exposure to genetically defined IAV. We confirm that seasonal IAV differ in their ability to utilize cell surface receptors for infectious entry and that this represents one level of virus restriction. Following virus entry, we demonstrate synthesis of all eight segments of genomic viral RNA (vRNA) and mRNA, as well as seven distinct IAV proteins, in IAV-infected mouse MΦ. Although newly synthesized hemagglutinin (HA) and neuraminidase (NA) glycoproteins are incorporated into the plasma membrane and expressed at the cell surface, electron microscopy confirmed that virus assembly was defective in IAV-infected MΦ, defining a second level of restriction late in the virus life cycle

The Role of Neutrophils during Mild and Severe Influenza Virus Infections of Mice

Neutrophils have been implicated in both protective and pathological responses following influenza virus infections. We have used mAb 1A8 (anti-Ly6G) to specifically deplete LyG6high neutrophils and induce neutropenia in mice infected with virus strains known to differ in virulence. Mice were also treated with mAb RB6-8C5 (anti-Ly6C/G or anti-Gr-1), a mAb widely used to investigate the role of neutrophils in mice that has been shown to bind and deplete additional leukocyte subsets. Using mAb 1A8, we confirm the beneficial role of neutrophils in mice infected with virus strains of intermediate (HKx31; H3N2) or high (PR8; H1N1) virulence whereas treatment of mice infected with an avirulent strain (BJx109; H3N2) did not affect disease or virus replication. Treatment of BJx109-infected mice with mAb RB6-8C5 was, however, associated with significant weight loss and enhanced virus replication indicating that other Gr-1+ cells, not neutrophils, limit disease severity during mild influenza infections