Megan C. Procario

A novel twelve class fluctuation test reveals higher than expected mutation rates for influenza A viruses.

Influenza virus’ low replicative fidelity contributes to its capacity for rapid evolution. Clonal sequencing and fluctuation tests have suggested that the influenza virus mutation rate is 2.7 × 10–6 - 3.0 × 10–5 substitutions per nucleotide per strand copied (s/n/r). However, sequencing assays are biased toward mutations with minimal fitness impacts and fluctuation tests typically investigate only a subset of all possible single nucleotide mutations. We developed a fluctuation test based on reversion to fluorescence in a set of virally encoded mutant green fluorescent proteins, which allowed us to measure the rates of selectively neutral mutations representative of the twelve different mutation types. We measured an overall mutation rate of 1.8 × 10–4 s/n/r for PR8 (H1N1) and 2.5 × 10–4 s/n/r for Hong Kong 2014 (H3N2) and a transitional bias of 2.7–3.6. Our data suggest that each replicated genome will have an average of 2–3 mutations and highlight the importance of mutational load in influenza virus evolution. DOI: http://dx.doi.org/10.7554/eLife.26437.001

Susceptibility to Acute Mouse Adenovirus Type 1 Respiratory Infection and Establishment of Protective Immunity in Neonatal Mice

ABSTRACT There is an incomplete understanding of the differences between neonatal immune responses that contribute to the increased susceptibility of neonates to some viral infections. We tested the hypothesis that neonates are more susceptible than adults to mouse adenovirus type 1 (MAV-1) respiratory infection and are impaired in the ability to generate a protective immune response against a second infection. Following intranasal infection, lung viral loads were greater in neonates than in adults during the acute phase but the virus was cleared from the lungs of neonates as efficiently as it was from adult lungs. Lung gamma interferon (IFN-γ) responses were blunted and delayed in neonates, and lung viral loads were higher in adult IFN-γ−/− mice than in IFN-γ+/+ controls. However, administration of recombinant IFN-γ to neonates had no effect on lung viral loads. Recruitment of inflammatory cells to the airways was impaired in neonates. CD4 and CD8 T cell responses were similar in the lungs of neonates and adults, although a transient increase in regulatory T cells occurred only in the lungs of infected neonates. Infection of neonates led to protection against reinfection later in life that was associated with increased effector memory CD8 T cells in the lungs. We conclude that neonates are more susceptible than adults to acute MAV-1 respiratory infection but are capable of generating protective immune responses.

Proinflammatory Effects of Interferon Gamma in Mouse Adenovirus 1 Myocarditis

ABSTRACT Adenoviruses are frequent causes of pediatric myocarditis. Little is known about the pathogenesis of adenovirus myocarditis, and the species specificity of human adenoviruses has limited the development of animal models, which is a significant barrier to strategies for prevention or treatment. We have developed a mouse model of myocarditis following mouse adenovirus 1 (MAV-1) infection to study the pathogenic mechanisms of this important cause of pediatric myocarditis. Following intranasal infection of neonatal C57BL/6 mice, we detected viral replication and induction of interferon gamma (IFN-γ) in the hearts of infected mice. MAV-1 caused myocyte necrosis and induced substantial cellular inflammation that was composed predominantly of CD3+ T lymphocytes. Depletion of IFN-γ during acute infection reduced cardiac inflammation in MAV-1-infected mice without affecting viral replication. We observed decreased contractility during acute infection of neonatal mice, and persistent viral infection in the heart was associated with cardiac remodeling and hypertrophy in adulthood. IFN-γ is a proinflammatory mediator during adenovirus-induced myocarditis, and persistent adenovirus infection may contribute to ongoing cardiac dysfunction. IMPORTANCE Studying the pathogenesis of myocarditis caused by different viruses is essential in order to characterize both virus-specific and generalized factors that contribute to disease. Very little is known about the pathogenesis of adenovirus myocarditis, which is a significant impediment to the development of treatment or prevention strategies. We used MAV-1 to establish a mouse model of human adenovirus myocarditis, providing the means to study host and pathogen factors contributing to adenovirus-induced cardiac disease during acute and persistent infection. The MAV-1 model will enable fundamental studies of viral myocarditis, including IFN-γ modulation as a therapeutic strategy.

Prostaglandin E2 Induction during Mouse Adenovirus Type 1 Respiratory Infection Regulates Inflammatory Mediator Generation but Does Not Affect Viral Pathogenesis

Respiratory viruses cause substantial disease and are a significant healthcare burden. Virus-induced inflammation can be detrimental to the host, causing symptoms during acute infection and leading to damage that contributes to long-term residual lung disease. Prostaglandin E2 (PGE2) is a lipid mediator that is increased in response to many viral infections, and inhibition of PGE2 production during respiratory viral infection often leads to a decreased inflammatory response. We tested the hypothesis that PGE2 promotes inflammatory responses to mouse adenovirus type 1 (MAV-1) respiratory infection. Acute MAV-1 infection increased COX-2 expression and PGE2 production in wild type mice. Deficiency of the E prostanoid 2 receptor had no apparent effect on MAV-1 pathogenesis. Virus-induced induction of PGE2, IFN-γ, CXCL1, and CCL5 was reduced in mice deficient in microsomal PGE synthase-1 (mPGES-1-/- mice). However, there were no differences between mPGES-1+/+ and mPGES-1-/- mice in viral replication, recruitment of leukocytes to airways or lung inflammation. Infection of both mPGES‑1+/+ and mPGES-1-/- mice led to protection against reinfection. Thus, while PGE2 promotes the expression of a variety of cytokines in response to acute MAV-1 infection, PGE2 synthesis does not appear to be essential for generating pulmonary immunity.

Limited Effects of Muc1 Deficiency on Mouse Adenovirus Type 1 Respiratory Infection

Muc1 (MUC1 in humans) is a membrane-tethered mucin that exerts anti-inflammatory effects in the lung during bacterial infection. Muc1 and other mucins are also likely to form a protective barrier in the lung. We used mouse adenovirus type 1 (MAV-1, also known as MAdV-1) to determine the role of Muc1 in the pathogenesis of an adenovirus in its natural host. Following intranasal inoculation of wild type mice, we detected increased TNF-α, a cytokine linked to Muc1 production, but no consistent changes in the production of lung Muc1, Muc5ac or overall lung mucus production. Viral loads were modestly higher in the lungs of Muc1(-/-) mice compared to Muc1(+/+) mice at several early time points but decreased to similar levels by 14 days post infection in both groups. However, cellular inflammation and the expression of CXCL1, CCL5, and CCL2 did not significantly differ between Muc1(-/-) and Muc1(+/+) mice. Our data therefore suggest that Muc1 may contribute to a physical barrier that protects against MAV-1 respiratory infection. However, our data do not reveal an anti-inflammatory effect of Muc1 that contributes to MAV-1 pathogenesis.

Increased lethality and defective pulmonary clearance of Streptococcus pneumoniae in microsomal prostaglandin E synthase-1-knockout mice.

The production of prostaglandin E2 (PGE2) increases dramatically during pneumococcal pneumonia, and this lipid mediator impairs alveolar macrophage (AM)-mediated innate immune responses. Microsomal prostaglandin E synthase-1 (mPGES-1) is a key enzyme involved in the synthesis of PGE2, and its expression is enhanced during bacterial infections. Genetic deletion of mPGES-1 in mice results in diminished PGE2 production and elevated levels of other prostaglandins after infection. Since PGE2 plays an important immunoregulatory role during bacterial pneumonia we assessed the impact of mPGES-1 deletion in the host defense against pneumococcal pneumonia in vivo and in AMs in vitro. Wild-type (WT) and mPGES-1 knockout (KO) mice were challenged with Streptococcus pneumoniae via the intratracheal route. Compared with WT animals, we observed reduced survival and increased lung and spleen bacterial burdens in mPGES-1 KO mice 24 and 48 h after S. pneumoniae infection. While we found modest differences between WT and mPGES-1 KO mice in pulmonary cytokines, AMs from mPGES-1 KO mice exhibited defective killing of ingested bacteria in vitro that was associated with diminished inducible nitric oxide synthase expression and reduced nitric oxide (NO) synthesis. Treatment of AMs from mPGES-1 KO mice with an NO donor restored bacterial killing in vitro. These results suggest that mPGES-1 plays a critical role in bacterial pneumonia and that genetic ablation of this enzyme results in diminished pulmonary host defense in vivo and in vitro. These results suggest that specific inhibition of PGE2 synthesis by targeting mPGES-1 may weaken host defense against bacterial infections.

Prostaglandin E2 Production and T Cell Function in Mouse Adenovirus Type 1 Infection following Allogeneic Bone Marrow Transplantation

Adenovirus infections are important complications of bone marrow transplantation (BMT). We demonstrate delayed clearance of mouse adenovirus type 1 (MAV-1) from lungs of mice following allogeneic BMT. Virus-induced prostaglandin E2 (PGE2) production was greater in BMT mice than in untransplanted controls, but BMT using PGE2-deficient donors or recipients failed to improve viral clearance, and treatment of untransplanted mice with the PGE2 analog misoprostol did not affect virus clearance. Lymphocyte recruitment to the lungs was not significantly affected by BMT. Intracellular cytokine staining of lung lymphocytes demonstrated impaired production of INF-γ and granzyme B by cells from BMT mice, and production of IFN-γ, IL-2, IL-4, and IL-17 following ex vivo stimulation was impaired in lymphocytes obtained from lungs of BMT mice. Viral clearance was not delayed in untransplanted INF-γ-deficient mice, suggesting that delayed viral clearance in BMT mice was not a direct consequence of impaired IFN-γ production. However, lung viral loads were higher in untransplanted CD8-deficient mice than in controls, suggesting that delayed MAV-1 clearance in BMT mice is due to defective CD8 T cell function. We did not detect significant induction of IFN-β expression in lungs of BMT mice or untransplanted controls, and viral clearance was not delayed in untransplanted type I IFN-unresponsive mice. We conclude that PGE2 overproduction in BMT mice is not directly responsible for delayed viral clearance. PGE2-independent effects on CD8 T cell function likely contribute to the inability of BMT mice to clear MAV-1 from the lungs.

Loss of CCR2 signaling alters leukocyte recruitment and exacerbates γ-herpesvirus-induced pneumonitis and fibrosis following bone marrow transplantation

CCR2-expressing leukocytes are required for the progression of fibrosis in models of induced lung injury as well as models of bone marrow transplant (BMT)-related idiopathic pneumonia syndrome. Infection with murid γ-herpesvirus-68 (γHV-68) results in severe pneumonitis and pulmonary fibrosis following syngeneic BMT; however, the roles that various proinflammatory leukocyte populations play in this process remain unclear. Deletion of CCR2 in both non-BMT and BMT mice increased early lytic viral replication and resulted in a reduction in the numbers of lung-infiltrating GR1+,F4/80+ and CXCR1+ cells, while maintaining robust neutrophil infiltration. Similarly, in γHV-68-infected CCR2(-/-) BMT mice, recruitment of monocytes and lymphocytes were reduced whereas neutrophil recruitment was increased compared with wild-type (WT) BMT mice. Interestingly, levels of profibrotic IL-17 were increased in infected CCR2 BMT mice compared with WT BMT. Furthermore, an increase in lung-associated collagen was detected even though there was an overall decrease in the number of profibrotic CCR2+ fibrocytes detected in the lungs of CCR2(-/-) BMT mice. These data indicate that, contrary to most models of fibrosis, deletion of CCR2 offers no protection from γ-herpesvirus-induced pneumonitis and fibrosis, and, indeed, CCR2+ cells play a suppressive role during the development of pulmonary fibrosis following γ-herpesvirus infection post-BMT by limiting IL-7 and collagen production.

Contributions of CD8 T cells to the pathogenesis of mouse adenovirus type 1 respiratory infection

CD8 T cells are key components of the immune response to viruses, but their roles in the pathogenesis of adenovirus respiratory infection have not been characterized. We used mouse adenovirus type 1 (MAV-1) to define CD8 T cell contributions to the pathogenesis of adenovirus respiratory infection. CD8 T cell deficiency in β2m-/- mice had no effect on peak viral replication in lungs, but clearance of virus was delayed in β2m-/- mice. Virus-induced weight loss and increases in bronchoalveolar lavage fluid total protein, IFN-γ, TNF-α, IL-10, CCL2, and CCL5 concentrations were less in β2m-/- mice than in controls. CD8 T cell depletion had similar effects on virus clearance, weight loss, and inflammation. Deficiency of IFN-γ or perforin had no effect on viral replication or inflammation, but perforin-deficient mice were partially protected from weight loss. CD8 T cells promote MAV-1-induced pulmonary inflammation via a mechanism that is independent of direct antiviral effects.

Interferon-dependent immunoproteasome activity during mouse adenovirus type 1 infection

The immunoproteasome is an inducible host mechanism that aids in the clearance of damaged proteins. The immunoproteasome also influences immune function by enhancing peptide presentation by MHC class I and promotes inflammation via IκB degradation and activation of NF-κB. We used mouse adenovirus type 1 (MAV-1) to characterize the role of the immunoproteasome in adenovirus pathogenesis. Following intranasal infection of mice, immunoproteasome activity in the heart and lung was significantly increased in an IFN-γ-dependent manner. Absence of the β5i immunoproteasome subunit and pharmacological inhibition of β5i activity had minimal effects on viral replication, virus-induced cellular inflammation, or induction of cytokine expression. Likewise, the establishment of protective immunity following primary infection was not significantly altered by β5i deficiency. Thus, although immunoproteasome activity is robustly induced during acute infection with MAV-1, our data suggest that other mechanisms are capable of compensating for immunoproteasome activity to maintain antiviral immunity and appropriate inflammatory responses.