Background: Respiratory 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. Objective: We sought to determine the capacity of RSV infection to stimulate group 2 innate lymphoid cells (ILC2s) and the associated mechanism in a murine model. Methods: Wild-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. Results RSV 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. Conclusion: These 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.

Respiratory syncytial virus (RSV) has been reported to infect human mesenchymal stem cells (MSCs) but the consequences are poorly understood. MSCs are present in nearly every organ including the nasal mucosa and the lung and play a role in regulating immune responses and mediating tissue repair. We sought to determine whether RSV infection of MSCs enhances their immune regulatory functions and contributes to RSV-associated lung disease. RSV was shown to replicate in human MSCs by fluorescence microscopy, plaque assay, and expression of RSV transcripts. RSV-infected MSCs showed differentially altered expression of cytokines and chemokines such as IL-1β, IL6, IL-8 and SDF-1 compared to epithelial cells. Notably, RSV-infected MSCs exhibited significantly increased expression of IFN-β (∼100-fold) and indoleamine-2,3-dioxygenase (IDO) (∼70-fold) than in mock-infected MSCs. IDO was identified in cytosolic protein of infected cells by Western blots and enzymatic activity was detected by tryptophan catabolism assay. Treatment of PBMCs with culture supernatants from RSV-infected MSCs reduced their proliferation in a dose dependent manner. This effect on PBMC activation was reversed by treatment of MSCs with the IDO inhibitors 1-methyltryptophan and vitamin K3 during RSV infection, a result we confirmed by CRISPR/Cas9-mediated knockout of IDO in MSCs. Neutralizing IFN-β prevented IDO expression and activity. Treatment of MSCs with an endosomal TLR inhibitor, as well as a specific inhibitor of the TLR3/dsRNA complex, prevented IFN-β and IDO expression. Together, these results suggest that RSV infection of MSCs alters their immune regulatory function by upregulating IFN-β and IDO, affecting immune cell proliferation, which may account for the lack of protective RSV immunity and for chronicity of RSVassociated lung diseases such as asthma and COPD.

Background: RNA-dependent RNA polymerases of nonsegmented negative-strand RNA viruses (Mononegavirales) harbor multiple catalytic activities. Results: Domain screening and trans-complementation of Paramyxovirus polymerase fragments with dimerization tags identifies independent folding-competent domains. Conclusion: Paramyxovirus polymerases harbor at least two independent domains that lack high-affinity interfaces but require molecular compatibility for bioactivity. Significance: First demonstration of Mononegavirales polymerase trans-complementation sets the stage for structural analyses of folding domains.

Background: Early life acute respiratory infection (ARI) with respiratory syncytial virus (RSV) has been strongly associated with the development of childhood wheezing illnesses, but the pathways underlying this association are poorly understood. Objective: To examine the role of the nasopharyngeal microbiome in the development of childhood wheezing illnesses following RSV ARI in infancy. Methods: We conducted a nested cohort study of 118 previously healthy, term infants with confirmed RSV ARI by RT-PCR. We used next-generation sequencing of the V4 region of the 16S ribosomal RNA gene to characterize the nasopharyngeal microbiome during RSV ARI. Our main outcome of interest was 2-year subsequent wheeze. Results: Of the 118 infants, 113 (95.8%) had 2-year outcome data. Of these, 46 (40.7%) had parental report of subsequent wheeze. There was no association between the overall taxonomic composition, diversity, and richness of the nasopharyngeal microbiome during RSV ARI with the development of subsequent wheeze. However, the nasopharyngeal detection and abundance of Lactobacillus was consistently higher in infants who did not develop this outcome. Lactobacillus also ranked first among the different genera in a model distinguishing infants with and without subsequent wheeze. Conclusions: The nasopharyngeal detection and increased abundance of Lactobacillus during RSV ARI in infancy are associated with a reduced risk of childhood wheezing illnesses at age 2 years.

Background Respiratory 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/design The 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. Discussion In 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.

Molecular epidemiology studies have provided convincing evidence of antigenic and sequence variability among respiratory syncytial virus (RSV) isolates. Circulating viruses have been classified into two antigenic groups (A and B) that correlate with well-delineated genetic groups. Most sequence and antigenic differences (both inter- and intra-groups) accumulate in two hypervariable segments of the G-protein gene. Sequences of the G gene have been used for phylogenetic analyses. These studies have shown a worldwide distribution of RSV strains with both local and global replacement of dominant viruses with time. Although data are still limited, there is evidence that strain variation may contribute to differences in pathogenicity. In addition, there is some but limited evidence that RSV variation may be, at least partially, immune (antibody) driven. However, there is the paradox in RSV that, in contrast to other viruses (e.g., influenza viruses) the epitopes recognized by the most effective RSV-neutralizing antibodies are highly conserved. In contrast, antibodies that recognize strainspecific epitopes are poorly neutralizing. It is likely that this apparent contradiction is due to the lack of a comprehensive knowledge of the duration and specificities of the human antibody response against RSV antigens. Since there are some data supporting a group- (or clade-) specific antibody response after a primary infection in humans, it may be wise to consider the incorporation of strains representative of groups A and B (or their antigens) in future RSV vaccine development.

Respiratory syncytial virus (RSV) infection can result in severe disease partially due to its ability to interfere with the initiation of Th1 responses targeting the production of type I interferons (IFN) and promoting a Th2 immune environment. Epigenetic modulation of gene transcription has been shown to be important in regulating inflammatory pathways. RSV-infected bone marrow-derived DCs (BMDCs) upregulated expression of Kdm5b/Jarid1b H3K4 demethylase. Kdm5b-specific siRNA inhibition in BMDC led to a 10-fold increase in IFN-β as well as increases in IL-6 and TNF-α compared to control-transfected cells. The generation of Kdm5b^fl/fl-CD11c-Cre⁺mice recapitulated the latter results during in vitro DC activation showing innate cytokine modulation. In vivo, infection of Kdm5b^fl/fl-CD11c-Cre⁺mice with RSV resulted in higher production of IFN-γ and reduced IL-4 and IL-5 compared to littermate controls, with significantly decreased inflammation, IL-13, and mucus production in the lungs. Sensitization with RSV-infected DCs into the airways of naïve mice led to an exacerbated response when mice were challenged with live RSV infection. When Kdm5b was blocked in DCs with siRNA or DCs from Kdm5b^fl/fl-CD11c-CRE mice were used, the exacerbated response was abrogated. Importantly, human monocyte-derived DCs treated with a chemical inhibitor for KDM5B resulted in increased innate cytokine levels as well as elicited decreased Th2 cytokines when co-cultured with RSV reactivated CD4⁺T cells. These results suggest that KDM5B acts to repress type I IFN and other innate cytokines to promote an altered immune response following RSV infection that contributes to development of chronic disease.

Respiratory syncytial virus (RSV) causes severe lower respiratory tract infections, yet no vaccines or effective therapeutics are available. ALS-8176 is a first-in-class nucleoside analog prodrug effective in RSV-infected adult volunteers, and currently under evaluation in hospitalized infants. Here, we report the mechanism of inhibition and selectivity of ALS-8176 and its parent ALS-8112. ALS-8176 inhibited RSV replication in non-human primates, while ALS-8112 inhibited all strains of RSV in vitro and was specific for paramyxoviruses and rhabdoviruses. The antiviral effect of ALS-8112 was mediated by the intracellular formation of its 5'-triphosphate metabolite (ALS-8112-TP) inhibiting the viral RNA polymerase. ALS-8112 selected for resistance-associated mutations within the region of the L gene of RSV encoding the RNA polymerase. In biochemical assays, ALS-8112-TP was efficiently recognized by the recombinant RSV polymerase complex, causing chain termination of RNA synthesis. ALS-8112-TP did not inhibit polymerases from host or viruses unrelated to RSV such as hepatitis C virus (HCV), whereas structurally related molecules displayed dual RSV/HCV inhibition. The combination of molecular modeling and enzymatic analysis showed that both the 2'F and the 4'ClCH<inf>2</inf> groups contributed to the selectivity of ALS-8112-TP. The lack of antiviral effect of ALS-8112-TP against HCV polymerase was caused by Asn291 that is well-conserved within positive-strand RNA viruses. This represents the first comparative study employing recombinant RSV and HCV polymerases to define the selectivity of clinically relevant nucleotide analogs. Understanding nucleotide selectivity towards distant viral RNA polymerases could not only be used to repurpose existing drugs against new viral infections, but also to design novel molecules.

Vaccines are the most efficient tools to battle infectious diseases, with an estimated prevention of 2–3 million deaths per year [1]. Vaccine development, however, is costly and challenging, especially when the target pathogen can be subdivided into serologically distinguishable types (serotypes) that individually cause disease. Broad protection against serotypes can be achieved with either polyvalent vaccines of mixed serotype-specific immunogens or by discovery and use of a good immunogen conserved among serotypes. The latter is preferable but technically elusive. The poliovirus vaccine (containing three poliovirus serotypes) was first used as a polyvalent vaccine, beginning with the establishment of the Global Polio Eradication Initiative in 1988, reducing poliomyelitis by 99% [2]. Polyvalency has been arguably more useful than using conserved immunogens to target multiple serotypes, and polyvalency has steadily advanced despite complexity and barriers to manufacturing. Here, we review challenges and developments in polyvalent vaccines.

Immune tolerance is instituted early in life, during which time regulatory T (T reg ) cells have an important role. Recurrent infections with respiratory syncytial virus (RSV) in early life increase the risk for asthma in adult life. Repeated infection of infant mice tolerized to ovalbumin (OVA) through their mother's milk with RSV induced allergic airway disease in response to OVA sensitization and challenge, including airway inflammation, hyper-reactivity and higher OVA-specific IgE, as compared to uninfected tolerized control mice. Virus infection induced GATA-3 expression and T helper type 2 (T H 2) cytokine production in forkhead box P3 (FOXP3) + T reg cells and compromised the suppressive function of pulmonary T reg cells in a manner that was dependent on interleukin-4 receptor α (IL-4Rα) expression in the host. Thus, by promoting a T H 2-type inflammatory response in the lung, RSV induced a T H 2-like effector phenotype in T reg cells and attenuated tolerance to an unrelated antigen (allergen). Our findings highlight a mechanism by which viral infection targets a host-protective mechanism in early life and increases susceptibility to allergic disease.