RSV Strains Research Articles

A fatal case associated with respiratory syncytial virus infection in a young child

BackgroundRespiratory syncytial virus (RSV) is the most common viral cause of pediatric bronchiolitis and pneumonia worldwide. Risk factors for high mortality and prolonged morbidity after RSV infection include premature birth, bronchopulmonary dysplasia, congenital heart disease, and Down syndrome. However, some previously healthy, full-term children who are infected with RSV also require hospitalization and even experience severe sequelae or death.Case presentationIn this report, we present the case of an RSV-associated death of a child who was born at full-term and developed normally up to the age of 2 years old. Cardiopulmonary arrest occurred within 3 days after the onset of symptoms, which included cough and high fever. Complete brain edema was prominent, and encephalopathy was developing. Viral antigen detection and microbiome analyses of oral swab and nasopharyngeal aspirate specimens verified an RSV infection, while bacterial culture of blood specimens yielded negative results. The RSV strain detected in this patient was subtyped as RSVB9, and no mutation was found in the six antigenic sites for targeted drugs or vaccines.ConclusionsThe patient had a severe infection associated with RSV, which was very likely the cause of her central nervous system infection and acute neurological complications.

Single-cycle respiratory syncytial virus infection induces robust adaptive immune responses and reduces disease severity in mice

Abstract Respiratory syncytial virus (RSV) is the leading cause of severe respiratory tract infection in infants and young children. However, despite extensive efforts, there remains no licensed RSV vaccine. Recently, a recombinant infectious RSV strain lacking the gene for the matrix (M) protein (M-null RSV) was developed as a potential vaccine candidate. The M protein is required for reassembly of the virion following infection and gene expression within a host cell. Infection with M-null RSV produces viral proteins but does not result in the generation of infectious virus progeny resulting in a single-cycle infection. To determine its immunogenicity in vivo, mice were infected with either M-null RSV or a wild-type (WT) RSV. M-null RSV-infected mice exhibited significantly reduced lung viral titers, weight loss, and pulmonary dysfunction compared to mice given WT RSV. Remarkably, M-null RSV infection induced CD4 and CD8 T cell responses in the lung and airways of similar magnitude to that of a WT RSV infection at acute time-points. Germinal center B cell numbers and serum antibody levels were also similar between M-null and WT RSV-infected mice. M-null RSV infection also generated robust memory immune responses. A substantial proportion of B cells, CD4 T cells, and CD8 T cells remained within the lung tissue of M-null RSV-infected mice 30 days post-infection. Importantly, RSV-specific memory CD8 T cells expressing CD69 and CD103, the canonical markers of tissue resident memory, were also generated by M-null RSV infection. Given the reduced disease severity and robust immune responses developed following single-cycle infection, M-null RSV may be an effective vaccine candidate.

Glucagon-like peptide 1 receptor signaling attenuates respiratory syncytial virus–induced type 2 responses and immunopathology

GLP-1R signaling, an emerging anti-inflammatory therapeutic 59 target, 60 attenuated type 2-associated immunopathology in mice infected with a strain of RSV that was 61 isolated from a hospitalized infant with severe lower respiratory tract infection and bronchiolitis.

Glucagon-like peptide-1 receptor signaling attenuates RSV-induced type 2 responses and immunopathology

Abstract Glucagon-like peptide-1 receptor (GLP-1R) agonists are a well-accepted and safe treatment for Type II diabetes. Although GLP-1R agonists mainly act to potentiate insulin and suppress glucagon secretion, recent evidence suggests that GLP-1R signaling also has anti-inflammatory effects. Severe RSV-associated illness is partially caused by type 2-associated immunopathology. We hypothesized that GLP-1R signaling inhibits type 2-mediated immunopathology during infection with RSV 12/12-6, a strain of RSV that was isolated from a hospitalized infant with severe lower respiratory tract infection and bronchiolitis. We show here that GLP-1R agonist treatment decreased airway inflammation, airway reactivity, and airway mucus production in RSV 12/12-6-infected mice. GLP-1R agonist treatment decreased total lung IL-13 and IL-33 levels, with concurrent decreases in lung IL-13-producing group 2 innate lymphoid cell (ILC2), CD4+ Th2 cell, and basophil numbers as well as IL-33-producing epithelial cells. The GLP-1R agonist prevented airway inflammation, and did not impact viral load, anti-viral interferon and antibody production during secondary RSV infection. Relative to the respective mock-infected groups, RSV-infected GLP-1R agonist treated mice did not have increased weight loss compared to vehicle treated mice. A phenome-wide association study (PheWAS) identified a link between GLP-1R signaling and acute bronchitis and bronchiolitis in humans. These studies demonstrate that GLP-1R signaling protects against type 2-mediated immunopathology during RSV infection. GLP-1R agonists are the first known FDA-approved agents to inhibit IL-33 and may represent a novel treatment strategy for RSV bronchiolitis.

Broadly Reactive Anti-Respiratory Syncytial Virus G Antibodies from Exposed Individuals Effectively Inhibit Infection of Primary Airway Epithelial Cells.

Respiratory syncytial virus (RSV) causes severe respiratory disease in young children. Antibodies specific for the RSV prefusion F protein have guided RSV vaccine research, and in human serum, these antibodies contribute to >90% of the neutralization response; however, detailed insight into the composition of the human B cell repertoire against RSV is still largely unknown. In order to study the B cell repertoire of three healthy donors for specificity against RSV, CD27+ memory B cells were isolated and immortalized using BCL6 and Bcl-xL. Of the circulating memory B cells, 0.35% recognized RSV-A2-infected cells, of which 59% were IgA-expressing cells and 41% were IgG-expressing cells. When we generated monoclonal B cells selected for high binding to RSV-infected cells, 44.5% of IgG-expressing B cells and 56% of IgA-expressing B cells reacted to the F protein, while, unexpectedly, 41.5% of IgG-expressing B cells and 44% of IgA expressing B cells reacted to the G protein. Analysis of the G-specific antibodies revealed that 4 different domains on the G protein were recognized. These epitopes predicted cross-reactivity between RSV strain A (RSV-A) and RSV-B and matched the potency of antibodies to neutralize RSV in HEp-2 cells and in primary epithelial cell cultures. G-specific antibodies were also able to induce antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis of RSV-A2-infected cells. However, these processes did not seem to depend on a specific epitope. In conclusion, healthy adults harbor a diverse repertoire of RSV glycoprotein-specific antibodies with a broad range of effector functions that likely play an important role in antiviral immunity.IMPORTANCE Human RSV remains the most common cause of severe lower respiratory tract disease in premature babies, young infants, the elderly, and immunocompromised patients and plays an important role in asthma exacerbations. In developing countries, RSV lower respiratory tract disease has a high mortality. Without an effective vaccine, only passive immunization with palivizumab is approved for prophylactic treatment. However, highly potent RSV-specific monoclonal antibodies could potentially serve as a therapeutic treatment and contribute to disease control and mortality reduction. In addition, these antibodies could guide further vaccine development. In this study, we isolated and characterized several novel antibodies directed at the RSV G protein. This information can add to our understanding and treatment of RSV disease.

Molecular Epidemiology of Respiratory Syncytial Virus A G-Protein for 9 Years and Emergence of ON1 Genotype Having 72 Nucleotide Duplication in Korea

BackgroundRespiratory syncytial virus (RSV) is a major pathogen causing seasonal epidemics of lower respiratory tract infection in young children. The attachment (G) glycoprotein is a major virulence factor and a target of human neutralizing antibodies. The G protein shows the evidence of changes of amino acid over time, making its study important in vaccine development strategies. We aimed to explore the molecular epidemiology of G protein of RSV A in Korea.MethodsOne hundred and thirty-six RSV strains were obtained from children who were hospitalized at Seoul National University Children’s Hospital, from October 2005 to September 2014. The frozen stock viruses were propagated in HEp-2 cells and the infected cell lysates were collected when cytopathic effects were apparent. The polymerase chain reaction and sequencing were performed using cDNA constructed from viral RNA and primers from previous studies. Phylogenetic analysis and putative antigenicity plotting were performed using CLC Main Workbench ver. 6.6.5 software (CLC bio, Aarhus, Denmark).ResultsThe entire G-genes were sequenced from all 136 RSV A strains: most (n = 99, 72.8%) strains had 895 nucleotide-span, but the others (n = 45, 27.2%) had 72 nucleotide (nt) duplication, which are well-known ON1 genotype. On phylogenetic grouping, three different genotypes 1–3 except ON1 were additionally defined. During the 2005–2006 season, the prevalence of genotypes 1, 2, and 3 were comparable (36.4%, 27.3%, and 36.4%, respectively), but genotype 1 became predominate (69.2%) in the 2006–2007 season. The genotype 3 increased since 2007–2008 (80.0%) and was identified exclusively during three consecutive seasons thereafter (2009–2011). In the 2011–2012 season, the first ON1 strain was identified in Korea, and the prevalence increased to 91.7% in the 2012–2013 season, then to 100% thereafter (2013–2015). In spite of 72-nt duplication, the putative antigenicity patterns of G protein were comparable between ON1 genotype and the others.ConclusionOur findings highlight the genotype change of circulating RSV A in 3–4 years interval, which might explain the reason for annual local epidemics of RSV A. Also in Korea, RSV A ON1 genotype increased since 2011 and was circulating exclusively since 2013.Disclosures All authors: No reported disclosures.

Characterization of Circulating RSV Strains Among Patients In The Outsmart RSV Program During the 2015–16 Winter Viral Season in the United States

BackgroundRespiratory syncytial virus (RSV) is an established cause of serious lower respiratory tract disease in infants and young children. To better describe currently circulating strains, monitor their evolution and characterize the patient populations, theMethodsOUTSMART included 14 labs in 4 US regions and Puerto Rico that provided RSV-positive samples and matching demographic data during Dec 2015 to Mar 2016 for subtyping and descriptive analyses. To gauge the representativeness of the OUTSMART patient sample, results were compared with the Nationwide Emergency Department Sample (NEDS) and the National Inpatient Sample (NIS).ResultsParticipating labs reported 10,304 RSV-positive samples (10.9%) out of 94,710 tested, of which 525 samples were submitted for further analyses. The majority of samples came from children ≤ 2 years: 1–3 months (17.5%), 4–6 months (13.7%), 7–12 months (21.9%), and 1–2 years (17.5%). The distribution of samples from males (53.0%) and females (47.0%) were similar. Of those with determined RSV subtype (n = 392; 74.7%), 62.8% were subtype A and 38.3% were subtype B. Of the two co-circulating subtypes, A was more prevalent through age 5 while B was more common in those ages 6+. Hospitalizations >24 hours occurred among 36.6% of patients. The proportions of patients with subtype A were similar among hospitalized vs. non-hospitalized patients (62.3% and 63.2%, respectively). Subtype A predominated in all 4 regions, with the highest proportions of subtype A in the Midwest (65.1%) and the West (64.8%). Most RSV-positive samples were collected February- March. For all databases, the largest disease burden was among those <1 year old (OUTSMART: 45.1%, NEDS 62.3%, NIS 62.7%), followed by the 1–2 year age group (OUTSMART: 27.6%, NEDS 26.9%, NIS 21.8%). The databases were also similar by gender (% male–OUTSMART: 53.0%, NEDS 53.9%, NIS 53.9%).ConclusionThe OUTSMART program characterizes circulating RSV strains and monitors their temporal and geographic evolution in the US to inform the development of anti-RSV monoclonal antibodies and vaccines.Disclosures S. Pastula, AstraZeneca: Research Contractor, Research support; E. Levin-Sparenberg, AstraZeneca: Research Contractor, Research support; X. Jiang, AstraZeneca: Research Contractor, Research support; J. P. Fryzek, AstraZeneca: Research Contractor, Research support; J. Hackett, AstraZeneca: Employee, Salary; T. Villafana, Medimmune: Employee and Shareholder, Salary; L. Yu, Medimmune: Employee, Salary; M. T. Esser, MedImmune: Employee and Shareholder, Salary; A. Tovchigrechko, MedImmune: Employee and Shareholder, Long-term incentive stock grant and Salary; B. Lu, Medimmune: Employee, Salary; A. Ruzin, Medimmune: Employee and Shareholder, Salary

Respiratory syncytial virus infection activates IL-13–producing group 2 innate lymphoid cells through thymic stromal lymphopoietin

BackgroundRespiratory syncytial virus (RSV) is a major health care burden with a particularly high worldwide morbidity and mortality rate among infants. Data suggest that severe RSV-associated illness is in part caused by immunopathology associated with a robust type 2 response.ObjectiveWe sought to determine the capacity of RSV infection to stimulate group 2 innate lymphoid cells (ILC2s) and the associated mechanism in a murine model.MethodsWild-type (WT) BALB/c, thymic stromal lymphopoietin receptor (TSLPR) knockout (KO), or WT mice receiving an anti-TSLP neutralizing antibody were infected with the RSV strain 01/2-20. During the first 4 to 6 days of infection, lungs were collected for evaluation of viral load, protein concentration, airway mucus, airway reactivity, or ILC2 numbers. Results were confirmed with 2 additional RSV clinical isolates, 12/11-19 and 12/12-6, with known human pathogenic potential.ResultsRSV induced a 3-fold increase in the number of IL-13–producing ILC2s at day 4 after infection, with a concurrent increase in total lung IL-13 levels. Both thymic stromal lymphopoietin (TSLP) and IL-33 levels were increased 12 hours after infection. TSLPR KO mice did not mount an IL-13–producing ILC2 response to RSV infection. Additionally, neutralization of TSLP significantly attenuated the RSV-induced IL-13–producing ILC2 response. TSLPR KO mice displayed reduced lung IL-13 protein levels, decreased airway mucus and reactivity, attenuated weight loss, and similar viral loads as WT mice. Both 12/11-19 and 12/12-6 similarly induced IL-13–producing ILC2s through a TSLP-dependent mechanism.ConclusionThese data demonstrate that multiple pathogenic strains of RSV induce IL-13–producing ILC2 proliferation and activation through a TSLP-dependent mechanism in a murine model and suggest the potential therapeutic targeting of TSLP during severe RSV infection.

A High Titer Anti RSV Polyclonal Antibody (RI-002) Prevents Infection with Palivizumab Resistant (PR) RSV in Cotton Rats and Achieves Greater Neutralizing RSV Activity As Compared to Palivizumab

James J. Mond, MD, PhD; Brian Gilbert, Baylor College of Medicine, ADMA Biologics, Ramsey, NJ. RATIONALE: Todeterminewhether themultiple antiRSVspecificities in RI-002 interact with a palivizumab resistant strain of RSVand to evaluate the RSV neutralizing activity of RI-002 as compared to Palivizumab. METHODS: A palivizumab resistant mutant of RSV/A Tracy was generated after serial passage in increasing concentrations of palivizumab. The resistant strain had a single amino acid substitution at position 262. Palivizumab was injected into cotton rats at 15mg/kg and RI-002 at 1500mg/kg followed one day later by intranasal inoculation with wild type or palivizumab resistant RSV. 4 days later lungs were lavaged and RSV quantified by a semi-quantitative plaque assay. RESULTS: RI-002 inhibited viral replication in the lung of rats infectedwith wild type and resistant RSV viruses (p<0.00001) while palivizumab had comparable inhibitory effects to RI-002 on thewild type virus but no effect on the resistant strain. Furthermore the titers of neutralizing antibody in the sera of cotton rats was greater for RI-002 as compared to palivizumab (P < 0.0039). CONCLUSIONS: There have been many reports of patients who have failed palivizumab treatment and who have been shown to harbor palivizumab resistant strains of RSV. Whether the emergence of resistant RSV strains accounts for the limited success of this monoclonal antibody is unknown. RI-002 has recently successfully completed a phase three trial in patients with primary immune deficiency disease with a very favorable outcome of primary and secondary endpoints. Whether this reflects the unique antibody profile of this IVIG remains to be determined.

DESIGN AND CHARACTERISTICS OF MONOCLONAL ANTI-BODIES SPECIFIC TO RESPIRATORY SYNCYTIAL VIRUS

An antibody panel that includes 6 MCA specifically interacting in ELISA with RSV strains of both existing antigenic gro-ups (A and B) has been designed. The new MCA are directed to 6 different conformation-dependent F protein epitopes, 3 of them inducing the synthesis of virus-neutralizing antibodies. Using real-time PCR with reverse transcription, immunoblotting, identification of MCA virus-neutralizing activity, competitive ELISA and cell-ELISA, it was shown that only the MCA who se synthesis was induced by variable RSV F protein determinants (unlike MCA directed to conservative determinants that are common for the viruses of various antigenic groups) have the virus-neutralizing activity. Differences in the interaction between RSV, MCA 4F2 and MCA 5F3 make it possible to identify by cell-ELISA of antigen group (RSV-A or RSV-B) of strains isolated from clinical samples.

Objectives, design and enrollment results from the Infant Susceptibility to Pulmonary Infections and Asthma Following RSV Exposure Study (INSPIRE).

BackgroundRespiratory syncytial virus (RSV) lower respiratory tract infection (LRI) during infancy has been consistently associated with an increased risk of childhood asthma. In addition, evidence supports that this relationship is causal. However, the mechanisms through which RSV contributes to asthma development are not understood. The INSPIRE (Infant Susceptibility to Pulmonary Infections and Asthma Following RSV Exposure) study objectives are to: 1) characterize the host phenotypic response to RSV infection in infancy and the risk of recurrent wheeze and asthma, 2) identify the immune response and lung injury patterns of RSV infection that are associated with the development of early childhood wheezing illness and asthma, and 3) determine the contribution of specific RSV strains to early childhood wheezing and asthma development. This article describes the INSPIRE study, including study aims, design, recruitment results, and enrolled population characteristics.Methods/designThe cohort is a population based longitudinal birth cohort of term healthy infants enrolled during the first months of life over a two year period. Respiratory infection surveillance was conducted from November to March of the first year of life, through surveys administered every two weeks. In-person illness visits were conducted if infants met pre-specified criteria for a respiratory illness visit. Infants will be followed annually to ages 3-4 years for assessment of the primary endpoint: wheezing illness. Nasal, urine, stool and blood samples were collected at various time points throughout the study for measurements of host and viral factors that predict wheezing illness. Nested case-control studies will additionally be used to address other primary and secondary hypotheses.DiscussionIn the INSPIRE study, 1952 infants (48% female) were enrolled during the two enrollment years and follow-up will continue through 2016. The mean age of enrollment was 60 days. During winter viral season, more than 14,000 surveillance surveys were carried out resulting in 2,103 respiratory illness visits on 1189 infants. First year follow-up has been completed on over 95% percent of participants from the first year of enrollment.With ongoing follow-up for wheezing and childhood asthma outcomes, the INSPIRE study will advance our understanding of the complex causal relationship between RSV infection and early childhood wheezing and asthma.

P7.05 - Metal complexes of Meloxicam and Isoxicam decrease viability and proliferation of virus-transformed cancer cells

ABSTRACT Introduction: There is growing evidence supporting the potential antitumor properties of non-steroidal anti-inflammatory drugs (NSAIDs). They are among the most consumed drugs over the world, including patients with cancer - a fact which further enhances the interest in the influence of these preparations on the survival and proliferative activity of tumor cells. It is assumed that for the realization of their biological activity is important linking to metal ions in the body. The aim of our study was to evaluate the effect of metal (CuII, ZnII, CoII, NiII) complexes of selective (Meloxicam) and non-selective (Isoxicam) NSAIDs on viability and proliferation of virus-transformed cancer cells. Materials and Methods: Permanent cell lines established from transplantable tumors in chicken (LSCC-SF-Mc29 –E7 - liver cancer, induced by the myelocytomatosis retrovirus Mc29) and rat (LSR-SF-SR–sarcoma, induced by RSV strain Schmidt-Rupin) were used as model systems. The investigations were performed by MTT test, neutral red uptake cytotoxicity assay, crystal violet staining, double-staining with acridine orange and propidium iodide, colony forming method and FACS analysis. The compounds were applied at a concentration range of 10-500 µg/ml for 24-120 h (short-term assays with monolayer cultures) and 18 days (long-term assay with 3D colonies). Results: The compounds examined decrease significantly viability and proliferation of treated cells as well as their ability to grow in semisolid medium in a time- and concentration-dependant manner. Cytopathological changes (including chromatin fragmentation and apoptotic bodies) and lower percentage of cells in G2 / M phase were also found. Conclusions: Cu(II) complexes with Meloxicam and Isoxicam are found to be the most active cytotoxic and cytostatic agents. They are highly effective against LSCC-SF-Mc29 cells that contain v-myc gene – disregulation of its cellular analogues is involved in pathogenesis of > 80% of human cancers. Applied independently, the ligands express lower cytotoxic/cytostatic activity as compared to metal complexes. Acknowledgements: Supported by Grant DFNI - Б 02/30 from 12.12.2014, Bulgaria, and a bilateral project between Bulgarian Academy of Sciences and Romanian Academy

Molecular characterization of respiratory syncytial viruses infecting children reported to have received palivizumab immunoprophylaxis

BackgroundRespiratory syncytial virus (RSV) is a major cause of respiratory infections in children. Palivizumab (PZ) is the only RSV-specific immunoprophylaxis approved by the U.S. Food and Drug Administration. Mutations leading to amino acid substitutions in the PZ binding site of the RSV F protein have been associated with breakthrough RSV infections in patients receiving PZ. ObjectiveTo detect PZ resistance conferring mutations in RSV strains from children who received PZ. Study designChildren aged ≤24 months on October 31 who were hospitalized or had outpatient visits for respiratory illness and/or fever during October–May 2001–2008 in 3 US counties were included. PZ receipt was obtained from parent interviews and medical records among children subsequently infected with RSV. Archived nasal/throat swab specimens were tested for RSV by real-time RT-PCR. The coding region of the PZ binding site of the RSV F protein was sequenced using both Sanger and pyrosequencing methods. ResultsOf 8762 enrolled children, 375 (4.3%) were tested for RSV and had a history of PZ receipt, of which 56 (14.9%) were RSV-positive and 45 of these had available archived specimens. Molecular typing identified 42 partial F gene sequences in specimens from 39 children: 19 single RSV subgroup A, 17 subgroup B and 3 mixed infections. Nucleotide substitutions were identified in 12/42 (28.6%) RSV strains. PZ resistance mutations were identified in 4 (10.2%) of the 39 children, of which one had documented PZ receipt. ConclusionsAlthough RSV PZ resistance mutations were infrequent, most RSV-associated illnesses in children with a history of PZ receipt were not due to strain resistance.

Oral GS-5806 Activity in a Respiratory Syncytial Virus Challenge Study

BackgroundRespiratory syncytial virus (RSV) is a common cause of infant hospitalizations and is increasingly recognized as a cause of considerable morbidity and mortality. No accepted antiviral treatment exists.MethodsWe conducted a double-blind, placebo-controlled study of GS-5806, an oral RSV-entry inhibitor, in healthy adults who received a clinical challenge strain of RSV intranasally. Participants were monitored for 12 days. At the time of a positive test for RSV infection or 5 days after inoculation, whichever occurred first, participants were randomly assigned to receive GS-5806 or placebo in one of seven sequential cohorts. Cohorts 1 to 4 received a first dose of 50 mg of GS-5806 and then 25 mg daily for the next 4 days, cohort 5 received a first dose of 50 mg and then 25 mg daily for the next 2 days, cohort 6 received one 100-mg dose, and cohort 7 received a first dose of 10 mg and then 5 mg daily for the next 4 days. Dose selection for cohorts 5, 6, and 7 occurred after an interim analysis of data for cohorts 1 to 4. The primary end point was the area under the curve (AUC) for the viral load, which was assessed after administration of the first dose through the 12th day after inoculation. Secondary end points were mucus weight and symptom scores.ResultsAmong the 54 participants in cohorts 1 to 4 who were infected with RSV, active treatment was associated with a lower viral load (adjusted mean, 250.7 vs. 757.7 log10 plaque-forming-unit equivalents [PFUe] × hours per milliliter; P<0.001), lower total mucus weight (mean, 6.9 g vs. 15.1 g; P=0.03), and a lower AUC for the change from baseline in symptom scores (adjusted mean, −20.2 vs. 204.9 × hours; P=0.005). The results were similar in cohorts 5, 6, and 7. Adverse events, including low neutrophil counts and increased levels of alanine aminotransferase, were more common among participants receiving GS-5806.ConclusionsTreatment with GS-5806 reduced the viral load and the severity of clinical disease in a challenge study of healthy adults. (Funded by Gilead Sciences; ClinicalTrials.gov number, NCT01756482.)

Respiratory syncytial virus strains induce differential innate immune and CD4 T cell responses (P4039)

Abstract Respiratory syncytial virus (RSV) infects the respiratory tract causing acute pulmonary disease in both children and the elderly. The host immune response contributes to disease severity. However, it is not currently clear to what extent RSV strain differences influence the host immune response and disease severity. To determine the impact of viral strains on the severity of RSV-induced disease, we compared several different RSV A isolates. Following infection with the line 19 strain of RSV, we observed increased weight loss and decreased airway function between days 1-4 p.i. as compared to RSV strains A2 or 2-20. On day 2 p.i., the total number of macrophages and neutrophils was increased following RSV line 19 infection. In addition, IL-1β and IL-6 protein levels were increased at day 2 p.i.. Inflammasome activation leads to secretion of mature IL-1β following RSV infection. Our results suggest that there is increased priming and activation of the inflammasome following RSV line 19 infection. Coinciding with the increased priming and activation of the inflammasome, we observe an increased Th17 response following RSV line 19 infection as compared to RSV A2 and 2-20. Neutralization of IL-1β and IL-6 results in a decreased Th17 response following RSV line 19 infection. Overall, our results demonstrate that RSV strains differ in their capacity to elicit a robust innate immune response, and subsequent CD4 T cell response, contributing to differences in disease severity.

Bioenergetics of murine lungs infected with respiratory syncytial virus

BackgroundCellular bioenergetics (cellular respiration and accompanying ATP synthesis) is a highly sensitive biomarker of tissue injury and may be altered following infection. The status of cellular mitochondrial O2 consumption of the lung in pulmonary RSV infection is unknown.MethodsIn this study, lung fragments from RSV-infected BALB/c mice were evaluated for cellular O2 consumption, ATP content and caspase activity. The disease was induced by intranasal inoculation with the RSV strain A2 and lung specimens were analyzed on days 2–15 after inoculation. A phosphorescence O2 analyzer that measured dissolved O2 concentration as a function of time was used to monitor respiration. The caspase-3 substrate analogue N-acetyl-asp-glu-val-asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC) was used to monitor intracellular caspases.ResultsO2 concentration declined linearly with time when measured in a sealed vial containing lung fragment and glucose as a respiratory substrate, revealing its zero-order kinetics. O2 consumption was inhibited by cyanide, confirming the oxidation occurred in the respiratory chain. Cellular respiration increased by 1.6-fold (p<0.010) and ATP content increased by 3-fold in the first week of RSV infection. Both parameters returned to levels found in uninfected lungs in the second week of RSV infection. Intracellular caspase activity in infected lungs was similar to uninfected lungs throughout the course of disease.ConclusionsLung tissue bioenergetics is transiently enhanced in RSV infection. This energy burst, triggered by the virus or virus-induced inflammation, is an early biomarker of the disease and may be targeted for therapy.

Outbreak of Nosocomial Respiratory Syncytial Virus Infections in a Hematology and Transplant Unit.

AbstractAbstract 3032Respiratory syncytial virus (RSV) is a pathogen associated with bronchiolitis in infants and small children. However, immunocompromised adults might also be at risk for severe lower respiratory tract involvement induced by RSV. We report the rapid control of an outbreak of nosocomial RSV infections in an institution with special focus on allogeneic and autologous stem cell transplantations.Between November 2011 and March 2012 56 patients were diagnosed with RSV infection by RT-PCR at our institution. In order to identify possible risk factors for lower respiratory tract involvement in RSV infected patients, clinical course of infection as well as radiological and laboratory findings were evaluated. Characterization of RSV strains in respiratory specimens was achieved by sequence analysis of part of the RSV glycoprotein G gene and phylogenetic comparison of outbreak and community strains was performed.Out of 56 patients with RSV infection, 39 patients showed signs of lower respiratory tract involvement. 14 fatal outcomes were observed. In most of the lethal cases coinfections with other pathogens such as Aspergillus spp. or Pseudomonas aeruginosa were present. Therefore the exact impact of RSV on the fatal outcome remains difficult, if not impossible, to assess. Age, sex, underlying disease, disease control or transplant status did not differ significantly between severe and non-severe cases. However, hypogammaglobulinemia was associated with a higher risk for fatal infection (p = 0.03) in univariate analysis, whereas therapy with oral ribavirin might show a protective effect (p = 0.02). Prolonged viral shedding was frequently observed (median 22.5 [1 – 143] days), most pronounced in patients folllowing allogeneic transplantation (p = 0.04). The major mode of RSV transmission seemed to be direct patient-to-patient contact as suggested by evaluation of patient movements on the wards. Sequence analysis on respiratory specimens obtained from 46 patients revealed the presence of a particular outbreak strain in 40 patients, characterized by an identical nucleotide sequence of RSV GA2 subtype. Phylogenetic analysis showed low prevalence of the outbreak strain in the community. By implementation of rigorous isolation measures containment of outbreak was achieved.Highly contagious viral pathogens such as RSV are able to cause rapidly spreading outbreaks among immunocompromised patients. Hypogammaglobulinemia might be a risk factor for severe lower respiratory tract infection. Treatment with oral ribavirin might have a positive effect on outcome. Rigorous isolation measures are essential to the containment of outbreak. Disclosures:Off Label Use: ribavirin aerosol is FDA approved for severe cases of RSV in infants and young children.

Induction of IL-6 and CCL5 (RANTES) in human respiratory epithelial (A549) cells by clinical isolates of respiratory syncytial virus is strain specific

BackgroundRespiratory syncytial virus (RSV) is the major respiratory pathogen of infants and young children. During each seasonal epidemic, multiple strains of both subgroup A and B viruses circulate in the community. Like other RNA viruses, RSV genome replication is prone to errors that results in a heterogeneous population of viral strains some of which may possess differences in virulence. We sought to determine whether clinical isolates of RSV differ in their capacity to induce inflammatory cytokines IL-6 and CCL5 (previously known as RANTES [regulated upon activation, normal T-cell expressed and secreted protein]), which are known to be induced in vitro and in vivo in response to RSV, during infection of A549 cells.ResultsScreening of subgroup A and B isolates revealed heterogeneity among strains to induce IL-6 and CCL5. We chose two subgroup B strains, New Haven (NH)1067 and NH1125, for further analysis because of their marked differences in cytokine inducing properties and because subgroup B strains, in general, are less genetically heterogeneous as compared to subgroup A strains. At 12 and 24 hours post infection RSV strains, NH1067 and NH1125 differed in their capacity to induce IL-6 by an order of magnitude or more. The concentrations of IL-6 and CCL5 were dependent on the dose of infectious virus and the concentration of these cytokines induced by NH1125 was greater than that of those induced by NH1067 when the multiplicity of infection of NH1067 used was as much as 10-fold higher than that of NH1125. The induction of IL-6 was dependent on viable virus as infection with UV-inactivated virus did not induce IL-6. The difference in IL-6 induction most likely could not be explained by differences in viral replication kinetics. The intracellular level of RSV RNA, as determined by quantitative RT-PCR, was indistinguishable between the 2 strains though the titer of progeny virus produced by NH1125 was greater than that produced by NH1067 at 16, 24 and 36 hours but essentially equal at 48 and 72 hours. Full genome sequencing of the 2 strains revealed 193 polymorphisms and 4 insertions in NH1067when compared to NH1125 (2 single base insertions in non-coding regions and 2 duplications of 3 and 60 bases in the RSV G gene). Of the polymorphisms, 147 occurred in coding regions and only 30 resulted in amino acid changes in 7 of the RSV genes.ConclusionsThese data suggest that RSV strains may not be homogeneous with regard to pathogenesis or virulence. Identification of the genetic polymorphisms associated with variations in cytokine induction may lead to insights into RSV disease and to the development of effective antiviral agents and vaccines.

Identification of Deletion Mutant Respiratory Syncytial Virus Strains Lacking Most of the G Protein in Immunocompromised Children with Pneumonia in South Africa

Respiratory syncytial virus (RSV) G protein deletion mutants replicate effectively in vitro but have not been detected in nature. Subtyping of RSV strains in hospitalized children in South Africa identified G protein PCR amplicons significantly reduced in size in 2 out of 209 clinical specimens screened over 4 years. Sequence analysis revealed subtype B strains lacking nearly the entire G protein ectodomain in one HIV-positive and one HIV-exposed child hospitalized with pneumonia. The association of clinical strains lacking most of the G protein with lower respiratory tract infection in immunocompromised children may have implications for RSV vaccine development.

Human Respiratory Syncytial Virus in Children with Acute Respiratory Tract Infections in China

There are limited data on the prevalence and clinical and molecular characterization of human respiratory syncytial virus (HRSV) in children with acute respiratory tract infections (ARTIs) in China. From December 2006 to March 2009, 894 nasopharyngeal aspirates (NPAs) were collected from children under 14 years of age with ARTIs. Samples were screened for HRSV and genotyped by reverse transcription-PCR (RT-PCR) and sequencing. Demographic and clinical information was recorded. A total of 38.14% (341/894) of samples were positive for HRSV. Phylogenetic analysis revealed that 60.4% of the selected 227 RSV strains were GA2, 34.4% were BA, 4.8% were GB2, and 0.4% were GB3. A total of 40.47% of all of the RSV-positive samples were coinfected with other respiratory viruses, and adenovirus was the most common additional respiratory virus. No statistical differences were found in the frequency of diagnosis and symptoms between the coinfection group and monoinfection group. Additionally, no statistical differences were found in epidemiological characterizations or disease severity between genotype BA- and GA2-positive patients, except for a greater frequency of lower respiratory tract infections (LRTIs) (mostly bronchitis)with BA. HRSV is the most important viral pathogen in Chinese children with ARTIs. Four genotypes (i.e., GA2, BA, GB2, and GB3) circulate locally, and the predominant genotype may shift between seasons. Coinfection with other viruses does not affect disease severity. HRSV genotypes were not associated with different epidemiological characterizations or disease severity.