Cynthia A. Bonville

Recombinant Human Eosinophil-Derived Neurotoxin/RNase 2 Functions as an Effective Antiviral Agent against Respiratory Syncytial Virus

A dose-dependent decrease in infectivity was observed on introduction of eosinophils into suspensions of respiratory syncytial virus group B (RSV-B). This antiviral effect was reversed by ribonuclease inhibitor, suggesting a role for the eosinophil secretory ribonucleases. Recombinant eosinophil-derived neurotoxin (rhEDN), the major eosinophil ribonuclease, promoted a dose-dependent decrease in RSV-B infectivity, with a 40-fold reduction observed in response to 50 nM rhEDN. Ribonucleolytically inactivated rhEDN (rhEDNdK38) had no antiviral activity. Semiquantitative reverse transcriptase-polymerase chain reaction demonstrated loss of viral genomic RNA in response to rhEDN, suggesting that this protein promotes the direct ribonucleolytic destruction of extracellular virions. Ribonuclease A had no antiviral activity even at approximately 1000-fold higher concentrations, suggesting that rhEDN has unique features other than ribonuclease activity that are crucial to its effectiveness. These results suggest that rhEDN may have potential as a therapeutic agent for prevention or treatment of disease caused by RSV.

Functional Antagonism of Chemokine Receptor CCR1 Reduces Mortality in Acute Pneumovirus Infection In Vivo

ABSTRACT We present an antiviral-immunomodulatory therapeutic strategy involving the chemokine receptor antagonist Met-RANTES, which yields significant survival in the setting of an otherwise fatal respiratory virus infection. In previous work, we demonstrated that infection with the natural rodent pathogen pneumonia virus of mice involves robust virus replication accompanied by cellular inflammation modulated by the CC chemokine macrophage inflammatory protein 1α (MIP-1α). We found that the antiviral agent ribavirin limited virus replication in vivo but had no impact on morbidity and mortality associated with this disease in the absence of immunomodulatory control. We show here that ribavirin reduces mortality, from 100% to 10 and 30%, respectively, in gene-deleted CCR1−/− mice and in wild-type mice treated with the small-molecule chemokine receptor antagonist, Met-RANTES. As MIP-1α-mediated inflammation is a common response to several distantly related respiratory virus pathogens, specific antiviral therapy in conjunction with blockade of the MIP-1α/CCR1 inflammatory cascade may ultimately prove to be a useful, generalized approach to severe respiratory virus infection and its pathological sequelae in human subjects.

Animal Models for Studying Respiratory Syncytial Virus Infection and Its Long Term Effects on Lung Function

Background: Human respiratory syncytial virus (hRSV) infection causes a spectrum of illnesses ranging from mild infection to life-threatening bronchiolitis and respiratory failure. Human studies on the pathogenesis of RSV infection are invaluable, but animal models permit advances with the use of experimental strategies that would be inappropriate in human studies. Methods: We review the advantages and disadvantages of various animal models for the study of hRSV infection. Results: No animal model of hRSV infection replicates the complete spectrum of disease severity seen in humans. Available models differ in their ability to incorporate genetic technology and to allow the study of immunity, vaccine efficacy and treatment interventions. Although hRSV establishes disease in primates, this advantage is outweighed by the impracticalities and cost of using such models. The study of bovine RSV infection in calves is appealing because of parallels with human disease. Among rodent models, BALB/c mice have helped delineate the mechanisms underlying vaccine-enhanced RSV disease, and cotton rats have been used for preclinical testing. The single major disadvantage of studying hRSV in rodent models is the limited extent to which this host-restricted human pneumovirus replicates in mouse lung tissue. The rodent-specific Pneumovirus pathogen, pneumonia virus of mice, causes an infection that mirrors severe bronchiolitis and pneumonia in infants infected with RSV, including robust virus replication with profound inflammation. Conclusion: The recent development of the pneumonia virus of mice model has permitted exploration of the mechanisms of severe Pneumovirus disease in vivo with the use of sophisticated genetic tools and genetically manipulated mouse strains.

Altered Pathogenesis of Severe Pneumovirus Infection in Response to Combined Antiviral and Specific Immunomodulatory Agents

ABSTRACT We report here the responses of mice with symptomatic pneumovirus infection to combined antiviral and specific immunomodulatory agents. Mice infected with pneumonia virus of mice, a natural mouse pathogen that replicates the signs and symptoms of severe infection with respiratory syncytial virus (RSV), responded to the antiviral agent ribavirin when it was administered in the setting of endogenous (gene deletion) or exogenous (antibody-mediated) blockade of the MIP-1α proinflammatory signaling cascade. Although neither treatment is effective alone, together they offer a dramatic reduction in symptoms and pathology, the most impressive of which is a significant reduction in morbidity and mortality. The findings presented are consistent with the notion of unique and independent contributions of virus replication and ongoing inflammation to the pathogenesis of severe respiratory virus infection, and they provide the impetus for the study of this treatment regimen in RSV-infected humans.

Enteroviral meningoencephalitis as a complication of X-linked hyper IgM syndrome

We describe 5 children from 2 families with mutations in the CD40 ligand (CD40L) gene leading to absent expression of CD40L on activated CD4 cells. All subjects presented with interstitial pneumonia with low serum IgG and normal serum IgM. One child had normal and one child had elevated serum IgA. Four had confirmed Pneumocystis carinii pneumonia. In spite of intravenous immunoglobulin treatment yielding therapeutic serum immunoglobulin levels, 3 children had enteroviral encephalitis. When assessed by flow cytometry, the 3 surviving affected male children had absent CD40L expression on activated CD4(+) T cells. The affected children from both families were shown to have the same single nucleotide insertion (codon 131) resulting in frameshift and early termination within exon 4 (extracellular domain). This observation demonstrates that persistent enteroviral infection is not only observed in X-linked agammaglobulinemia but may also occur in patients with X-linked hyper IgM syndrome.

Rapid Evolution of the Ribonuclease A Superfamily: Adaptive Expansion of Independent Gene Clusters in Rats and Mice

Abstract. The two eosinophil ribonucleases, eosinophil-derived neurotoxin (EDN/RNase 2) and eosinophil cationic protein (ECP/RNase 3), are among the most rapidly evolving coding sequences known among primates. The eight mouse genes identified as orthologs of EDN and ECP form a highly divergent, species-limited cluster. We present here the rat ribonuclease cluster, a group of eight distinct ribonuclease A superfamily genes that are more closely related to one another than they are to their murine counterparts. The existence of independent gene clusters suggests that numerous duplications and diversification events have occurred at these loci recently, sometime after the divergence of these two rodent species (∼10–15 million years ago). Nonsynonymous substitutions per site (dN) calculated for the 64 mouse/rat gene pairs indicate that these ribonucleases are incorporating nonsilent mutations at accelerated rates, and comparisons of nonsynonymous to synonymous substitution (dN / dS) suggest that diversity in the mouse ribonuclease cluster is promoted by positive (Darwinian) selection. Although the pressures promoting similar but clearly independent styles of rapid diversification among these primate and rodent genes remain uncertain, our recent findings regarding the function of human EDN suggest a role for these ribonucleases in antiviral host defense.

Clinical features, adenovirus types, and local production of inflammatory mediators in adenovirus infections.

Background: Adenovirus infection manifests in many ways, with respiratory and gastrointestinal symptoms predominating. Methods: We performed a retrospective chart review on children evaluated at our center who had a nasal wash culture positive for adenovirus. Archived nasal washes were retrieved. Polymerase chain reaction for 15 respiratory viruses was performed on these samples. Patients who were coinfected with another virus were excluded. Adenovirus typing was performed using polymerase chain reaction primers directed at the conserved hexon gene. Bead proteomics was used to measure concentrations of inflammatory mediators. Results: Seventy-eight patients were infected only with adenovirus. The clinical diagnosis was upper respiratory infection in 60%, pneumonia in 18%, febrile seizure in 8%, and bronchiolitis in 6%. Subgroup-C and B1 infections were most common. Seventy percent of patients with upper respiratory infection and all 5 patients with bronchiolitis had a subgroup-C infection; pneumonia was caused by subgroup-B1 and C viruses. Compared with asymptomatic control patients, adenovirus infected patients had higher nasal wash concentrations of interleukin (IL)-1α, IL-6, inducible protein-10, macrophage inflammatory protein-1α, tumor necrosis factor α, monokine induced by gamma interferon, and interferon-α (P < 0.05). In addition, we found that IL-8 and IL-1α (P < 0.05) were higher in the nasal washes obtained from hospitalized patients than in nonhospitalized patients. Conclusions: Adenovirus infection causes an array of clinical disease and is associated with local production of several proinflammatory cytokines. The observation that nasal wash IL-8 and IL-1α concentrations were higher in patients requiring hospitalization suggests that these mediators contribute to disease severity.

Gene expression in epithelial cells in response to pneumovirus infection

Respiratory syncytial virus (RSV) and pneumonia virus of mice (PVM) are viruses of the family Paramyxoviridae, subfamily pneumovirus, which cause clinically important respiratory infections in humans and rodents, respectively. The respiratory epithelial target cells respond to viral infection with specific alterations in gene expression, including production of chemoattractant cytokines, adhesion molecules, elements that are related to the apoptosis response, and others that remain incompletely understood. Here we review our current understanding of these mucosal responses and discuss several genomic approaches, including differential display reverse transcription-polymerase chain reaction (PCR) and gene array strategies, that will permit us to unravel the nature of these responses in a more complete and systematic manner.

Novel inflammatory markers, clinical risk factors and virus type associated with severe respiratory syncytial virus infection.

Background: Virus-induced inflammation contributes to respiratory syncytial virus (RSV) pathogenesis. We sought to determine the specific mediators that are associated with more severe illness in young children. Methods: Children ⩽5 years of age seen in our emergency department for respiratory symptoms from September 1998 to May 2008 were eligible for enrollment. Nasopharyngeal wash samples were collected from all eligible patients, and clinical data were recorded. Individuals were included in this study if nasopharyngeal wash samples were positive for RSV only. Patients enrolled in the study were stratified by disease severity, defined as mild (not hospitalized), moderate (hospitalized) or severe (requiring intensive care unit stay). Concentrations of individual inflammatory biomarkers in nasopharyngeal wash fluids were determined using the Luminex human 30-plex assay. Results: Eight hundred fifty-one patients met study criteria: 268 (31.5%) with mild, 503 (59.1%) with moderate and 80 (9.4%) with severe illness. As expected, illness severity was directly associated with young age, prematurity, heart or lung disease, infection with RSV group A and elevated concentrations of interleukin (IL)-2R, IL-6, CXCL8, tumor necrosis factor-&agr;, interferon-&agr;, CCL3, CCL4 and CCL2. In addition, we report several novel and mechanistically important inflammatory biomarkers of severe RSV disease, including IL-1&bgr;, IL1-RA, IL-7, epidermal growth factor and hepatocyte growth factor. Conclusions: In a large, longitudinal study (10 years, 851 enrolled patients) limited to RSV infection only, in which well-known risk factors are confirmed, we identified 5 novel biomarkers specifically of severe disease. These markers may ultimately serve to elucidate disease mechanisms.

Local production of inflammatory mediators during childhood parainfluenza virus infection.

Objective: To describe the clinical manifestations of parainfluenza virus (PIV) infection and to characterize biochemical markers of PIV disease severity. Patients and Methods: We reviewed the medical records of 165 children who had a nasal wash culture positive for PIV at our institution between 1998 and 2008. Nasal wash samples were assayed for 26 inflammatory mediators using Luminex bead proteomics. Results: A total of 153 patients, ages 2 weeks to 12 years, with single virus infection were included in our final analysis. Fifty-two patients were infected with PIV1, 19 with PIV2, 74 with PIV3, and 8 with PIV4. Lower respiratory tract infection (LRTI) was diagnosed in 67 (44%) patients, 21 (14%) had laryngotracheobronchitis, and 49 (32%) had an upper respiratory infection other than laryngotracheobronchitis. LRTI was diagnosed in 54% of patients infected with PIV3, 35% of those infected with PIV1, 26% of those with PIV2, and 50% of those with PIV4. Compared with uninfected control patients, PIV-infected patients had higher nasal wash concentrations of interleukin-6, CX-chemokine ligand 8 (CXCL8 or interleukin-8), CCL3 (macrophage inflammatory protein-1&agr;), CCL4 (macrophage inflammatory protein-1&bgr;), CXCL9 (monokine induced by interferon gamma), and CCL5 (regulated upon activation, normal T cell expressed and secreted (RANTES). Patients with LRTI, moderate or severe illness, and PIV 1 or 3 (respirovirus) infection had higher nasal wash concentrations of CXCL8 when compared with patients with upper respiratory infection, mild illness, or PIV 2 and 4 (rubulavirus) infection (P < 0.05). Conclusions: PIV infection causes a spectrum of illnesses associated with the expression and release of several proinflammatory mediators. Of note, elevated concentrations of CXCL8 in nasal wash samples are associated with more severe forms of PIV disease.