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Background: Monoclonal antibody (palivizumab), intravenous immune globulin (IGIV), or respiratory syncytial virus (RSV)-polyclonal-hyperimmune-globulin (RSV-IG as Respigam®, RI-001, RI-002) are used with ribavirin in RSV-infected immunocompromised patients, with debated efficacy. Palivizumab-resistance (PR) can arise during treatment of persistent infections in this population. RSV-IG may confer benefit in PR-RSV infection., Methods: RSV-IG [RI-001] was provided for an immunocompromised infant with RSV-pneumonitis refractory to ribavirin and palivizumab. RSV-neutralizing antibody, respiratory RSV load (qPCR), and F-gene-sequence-detection of PR was determined. Prophylactic RSV-IG [RI-002] or palivizumab was administered in a cotton-rat model infected with wild-type and PR-RSV. Lung RSV load and neutralizing antibody were measured., Results: As protective RI-001-neutralizing antibody titers waned in the infant, a subpopulation of PR-escape mutants were detected with a fatal RSV-burden in the lungs. In PR-RSV-infected cotton rats, prophylactic RI-002 reduced RSV-load in the lungs (2.45 vs 0.28 log 10  PFU/g lung-tissue reduction, respectively, p < 0.05) and provided protective RSV-neutralizing antibody., Conclusions: RSV-IG and ribavirin use in immunocompromised patients requires further study., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Background: Respiratory syncytial virus (RSV) infection is a cause of substantial morbidity and mortality in young children. There is currently no effective therapy available., Methods: This was a Phase 2 study of the oral RSV fusion protein inhibitor AK0529 in infants aged 1-24 months, hospitalized with RSV infection. In Part 1, patients ( n  = 24) were randomized 2:1 to receive a single dose of AK0529 up to 4 mg/kg or placebo. In Part 2, patients ( n  = 48) were randomized 2:1 to receive AK0529 at 0.5, 1, or 2 mg/kg bid or placebo for 5 days. Sparse pharmacokinetic samples were assessed using population pharmacokinetics modelling. Safety, tolerability, viral load, and respiratory signs and symptoms were assessed daily during treatment., Results: No safety or tolerability signals were detected for AK0529: grade ≥3 treatment-emergent adverse events occurring in 4.1% of patients in AK0529 and 4.2% in placebo groups, respectively, and none led to death or withdrawal from the study. In Part 2, targeted drug exposure was reached with 2 mg/kg bid. A numerically greater reduction in median viral load with 2 mg/kg bid AK0529 than with placebo at 96 h was observed. A -4.0 (95% CI: -4.51, -2.03) median reduction in Wang Respiratory Score from baseline to 96 h was observed in the 2 mg/kg group compared with -2.0 (95% CI: -3.42, -1.82) in the placebo group., Conclusions: AK0529 was well tolerated in hospitalized RSV-infected infant patients. Treatment with AK0529 2 mg/kg bid was observed to reduce viral load and Wang Respiratory Score., Clinical Trials Registration: NCT02654171., Competing Interests: JZW is a co‐inventor of patents (WO2013020993A, 2012; CN105726488B, 2014) covering a compound targeting RSV diseases and a preparation method of the formula. ST and JZW are co‐inventors of a patent (WO2021083290A1; 2020) covering RSV fusion protein inhibitor composition and its use for the treatment and prophylaxis of RSV. FG, ST, GZ, and JZW are or were employees of and are shareholders in Ark Biosciences. JPD served as a compensated scientific consultant for Ark Biosciences and is a shareholder in the company. All other authors declare no competing interests., (© 2023 Shanghai Ark Biopharmaceutical Ltd Co. Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)

Respiratory syncytial virus (RSV) is the leading, global cause of serious respiratory disease in infants and is an important cause of respiratory illness in older adults. No RSV vaccine is currently available. The RSV fusion (F) glycoprotein is a key antigen for vaccine development, and its prefusion conformation is the target of the most potent neutralizing antibodies. Here, we describe a computational and experimental strategy for designing immunogens that enhance the conformational stability and immunogenicity of RSV prefusion F. We obtained an optimized vaccine antigen after screening nearly 400 engineered F constructs. Through in vitro and in vivo characterization studies, we identified F constructs that are more stable in the prefusion conformation and elicit ~10-fold higher serum-neutralizing titers in cotton rats than DS-Cav1. The stabilizing mutations of the lead construct (847) were introduced onto F glycoprotein backbones of strains representing the dominant circulating genotypes of the two major RSV subgroups, A and B. Immunization of cotton rats with a bivalent vaccine formulation of these antigens conferred complete protection against RSV challenge, with no evidence of disease enhancement. The resulting bivalent RSV prefusion F investigational vaccine has recently been shown to be efficacious against RSV disease in two pivotal phase 3 efficacy trials, one for passive protection of infants by immunization of pregnant women and the second for active protection of older adults by direct immunization.

The controlled human infection model and specifically the human viral challenge model are not dissimilar to standard clinical trials while adding another layer of complexity and safety considerations. The models deliberately infect volunteers, with an infectious challenge agent to determine the effect of the infection and the potential benefits of the experimental interventions. The human viral challenge model studies can shorten the time to assess the efficacy of a new vaccine or treatment by combining this with the assessment of safety. The newly emerging SARS-CoV-2 virus is highly contagious, and an urgent race is on to develop a new vaccine against this virus in a timeframe never attempted before. The use of the human viral challenge model has been proposed to accelerate the development of the vaccine. In the early 2000s, the authors successfully developed a pathogenic human viral challenge model for another virus for which there was no effective treatment and established it to evaluate potential therapies and vaccines against respiratory syncytial virus. Experience gained in the development of that model can help with the development of a COVID-19 HVCM and the authors describe it here., (© 2020 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.)

Respiratory syncytial virus (RSV) immunoprophylaxis (IP) has been shown to reduce RSV hospitalization rates in high-risk infants; however, it is unclear whether RSV IP is associated with increased risk of non-RSV disease, particularly non-RSV hospitalizations. We conducted a systematic literature review to understand the occurrences of non-RSV disease and/or non-RSV hospitalizations in published studies of RSV IP. Cochrane, Embase, and PubMed databases were searched and reviewed to summarize data regarding the incidence of RSV and non-RSV respiratory disease among RSV IP recipients and controls in randomized and non-randomized studies. Independent investigators screened and selected studies for inclusion. Risk-of-bias assessment was conducted to assess strength/validity of the data using the Jadad scoring system and Downs and Black quality assessment tool, where appropriate. Twenty studies were included for review (5 randomized controlled trials [RCTs]; 15 non-randomized studies). RCTs of RSV IP demonstrated reductions in RSV hospitalizations and all-cause hospitalizations, with no increase in hospitalizations for non-RSV disease. Non-randomized studies also demonstrated reduced RSV hospitalizations in RSV IP recipients but had mixed results in assessments of hospitalizations for non-RSV disease. When RSV IP recipients and controls were more similar in disease severity risk, results of non-randomized studies aligned more closely with RCTs. Observations of increased non-RSV hospitalization rates among RSV IP recipients in some non-randomized studies could be primarily explained by differences in the clinical characteristics between RSV IP recipients and controls., Competing Interests: Declaration of competing interest AB was an employee of AstraZeneca at the time this work was initiated; he is currently an employee of Sobi Inc. JPD has received research support and served on the Speakers Bureau of AstraZeneca; he has served as a consultant to AbbVie. CSA is an employee of AstraZeneca. LRK has received research grants and research support from AstraZeneca, Regeneron, Sanofi, and Pfizer; he has served as a consultant to Pfizer., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection in infants, and a need exists for prevention of RSV in healthy infants. Nirsevimab is a monoclonal antibody with an extended half-life that is being developed to protect infants for an entire RSV season with a single intramuscular dose., Methods: In this trial conducted in both northern and southern hemispheres, we evaluated nirsevimab for the prevention of RSV-associated lower respiratory tract infection in healthy infants who had been born preterm (29 weeks 0 days to 34 weeks 6 days of gestation). We randomly assigned the infants in a 2:1 ratio to receive nirsevimab, at a dose of 50 mg in a single intramuscular injection, or placebo at the start of an RSV season. The primary end point was medically attended RSV-associated lower respiratory tract infection through 150 days after administration of the dose. The secondary efficacy end point was hospitalization for RSV-associated lower respiratory tract infection through 150 days after administration of the dose., Results: From November 2016 through November 2017, a total of 1453 infants were randomly assigned to receive nirsevimab (969 infants) or placebo (484 infants) at the start of the RSV season. The incidence of medically attended RSV-associated lower respiratory tract infection was 70.1% lower (95% confidence interval [CI], 52.3 to 81.2) with nirsevimab prophylaxis than with placebo (2.6% [25 infants] vs. 9.5% [46 infants]; P<0.001) and the incidence of hospitalization for RSV-associated lower respiratory tract infection was 78.4% lower (95% CI, 51.9 to 90.3) with nirsevimab than with placebo (0.8% [8 infants] vs. 4.1% [20 infants]; P<0.001). These differences were consistent throughout the 150-day period after the dose was administered and across geographic locations and RSV subtypes. Adverse events were similar in the two trial groups, with no notable hypersensitivity reactions., Conclusions: A single injection of nirsevimab resulted in fewer medically attended RSV-associated lower respiratory tract infections and hospitalizations than placebo throughout the RSV season in healthy preterm infants. (Funded by AstraZeneca and Sanofi Pasteur; ClinicalTrials.gov number, NCT02878330.)., (Copyright © 2020 Massachusetts Medical Society.)

Objective: The SENTINEL1 observational study characterized confirmed respiratory syncytial virus hospitalizations (RSVH) among U.S. preterm infants born at 29 to 35 weeks' gestational age (wGA) not receiving respiratory syncytial virus (RSV) immunoprophylaxis (IP) during the 2014 to 2015 and 2015 to 2016 RSV seasons., Study Design: All laboratory-confirmed RSVH at participating sites during the 2014 to 2015 and 2015 to 2016 RSV seasons (October 1-April 30) lasting ≥24 hours among preterm infants 29 to 35 wGA and aged <12 months who did not receive RSV IP within 35 days before onset of symptoms were identified and characterized., Results: Results were similar across the two seasons. Among infants with community-acquired RSVH ( N  = 1,378), 45% were admitted to the intensive care unit (ICU) and 19% required invasive mechanical ventilation (IMV). There were two deaths. Infants aged <6 months accounted for 78% of RSVH observed, 84% of ICU admissions, and 91% requiring IMV. Among infants who were discharged from their birth hospitalization during the RSV season, 82% of RSVH occurred within 60 days of birth hospitalization discharge., Conclusion: Among U.S. preterm infants 29 to 35 wGA not receiving RSV IP, RSVH are often severe with almost one-half requiring ICU admission and about one in five needing IMV., Competing Interests: E.J.A., J.P.D., E.A.F.S., L.R.K., M.L.F., P.S.P., C.M.E., R.C.W., L.I.W., R.Y., P.A.C., J.B.D., and N.H. are independent investigators who have received research support from AstraZeneca/MedImmune. J.P.D., E.A.F.S., L.R.K., and M.L.F. also received travel support from AstraZeneca/MedImmune to present the results of this or other research studies at scientific meetings. J.P.D., M.L.F., and P.S.P. have served on the speakers’ bureau for AstraZeneca/MedImmune. E.J.A., E.A.F.S., and J.P.D. have served as consultants to AbbVie; P.A.C. has received research support from AbbVie. S.J.M. is an independent statistical consultant to AstraZeneca. K.K.M. and C.S.A. are employees of AstraZeneca and hold stock. V.R.K. and C.P.R. were employees of AstraZeneca at the time this research was conducted. C.P.R. is a current employee of Sobi Inc., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Many aspects of the respiratory syncytial virus (RSV) are still poorly understood. Yet these knowledge gaps have had and could continue to have adverse, unintended consequences for the efficacy and safety of antivirals and vaccines developed against RSV. Mathematical modelling was used to test and evaluate hypotheses about the rate of loss of RSV infectivity and the mechanisms and kinetics of RSV infection spread in SIAT cells in vitro. While the rate of loss of RSV integrity, as measured via qRT-PCR, is well-described by an exponential decay, the latter mechanism failed to describe the rate at which RSV A Long loses infectivity over time in vitro based on the data presented herein. This is unusual given that other viruses (HIV, HCV, influenza) have been shown to lose their infectivity exponentially in vitro, and indeed an exponential rate of loss of infectivity is always assumed in mathematical modelling and experimental analyses. The infectivity profile of RSV in HEp-2 and SIAT cells remained consistent over the course of an RSV infection, over time and a large range of infectivity. However, SIAT cells were found to be ∼ 100× less sensitive to RSV infection than HEp-2 cells. In particular, we found that RSV spreads inefficiently in SIAT cells, in a manner we show is consistent with the establishment of infection resistance in uninfected cells. SIAT cells are a good in vitro model in which to study RSV in vivo dissemination, yielding similar infection timescales. However, the higher sensitivity of HEp-2 cells to RSV together with its RSV infectivity profile being similar to that of SIAT cells, makes HEp-2 cells more suitable for quantifying RSV infectivity over the course of in vitro RSV infections in SIAT cells. Our findings highlight the importance and urgency of resolving the mechanisms at play in the dissemination of RSV infections in vitro, and the processes by which this infectivity is lost., Competing Interests: Two of the authors (QY, GC) were employed by AstraZeneca, and subsequently QY was employed by Rubius Therapeutics and GC was employed by Moderna, all while participating in this work. Three of the authors (YIK, JPDV, CAAB) received research funding as part of a sponsored research agreement with AstraZeneca Inc. (see Financial Disclosure). This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Respiratory syncytial virus (RSV) causes high morbidity, with mortality rates approaching or exceeding that of influenza in adult and infant patient populations, respectively. Lumicitabine (ALS-008176 or JNJ-64041575) is an oral nucleoside analogue prodrug in clinical development to treat RSV infections. This prodrug converts to plasma-circulating ALS-8112, and then to the 5'-active nucleoside triphosphate (NTP) form within host cells. We conducted an RSV-A challenge study in healthy adults to evaluate lumicitabine's activity during an active RSV infection., Objectives: To develop a semi-mechanistic mathematical model describing RSV kinetics, and the pharmacokinetics (PK) and pharmacodynamics (PD) of lumicitabine during treatment., Methods: Nasopharyngeal viral load and concentrations of ALS-8112 and ALS-8144 (uridine metabolite) were measured frequently over the study duration. Population viral kinetic and PK/PD models were developed using NONMEM. The RSV life-cycle was described using a target-cell-limited model that included a physiological delay., Results: The estimated clearances of ALS-8112 and ALS-8144 were 54.2 and 115 L/h/70 kg, respectively. A semi-physiological model was linked to predict ALS-8112 conversion to active intracellular NTP. Extensive and rapid RSV reduction occurred after lumicitabine treatment (EC50 = 1.79 μM), with >99% viral inhibition at 2 h after loading dose. Simulated NTP exposures and time to EC50 attainment suggested that rapid therapeutic effects and reduced dosing frequency are achievable in adult and paediatric patients., Conclusions: The semi-mechanistic model characterizes RSV kinetics and the antiviral effectiveness of lumicitabine in an adult challenge population. This model is applicable to guide dose selection in adult and paediatric patients.

Background: Herpes Simplex Virus encephalitis (HSVE) is a devastating disease of all ages. Rigorous studies correlating viral load with neuroradiological and clinical severity have not been performed, particularly in neonates. Understanding these relationships may improve therapies., Objectives: To correlate molecularly quantified HSV in cerebrospinal fluid (CSF) and disease severity., Study Design: HSV loads (VL) were evaluated by real-time PCR from the CSF of 33 patients (20 neonates, 5 children, 8 adults) with HSVE. We studied relationships between CSF VL and structural and volumetric brain abnormalities (MRI); hospital morbidity; and discharge and long-term (>3 month) clinical outcomes., Results: Initial CSF VL did not differ in neonates vs non-neonates (median 4.6 vs 5.1 log10 copies/mL, p = 0.75). Initial CSF VL was higher in neonates with HSV-2 vs HSV-1 (median 4.8 vs 3.2 log10 copies/mL, respectively, p = 0.02). Persistently detectable DNA in CSF despite acyclovir trended towards higher odds of unfavorable outcome at discharge for neonates [0.87 (CI 0.75-1), p = 0.07]. Initial VL correlated with higher CSF protein concentrations for the cohort and for neonates (p = 0.03 and 0.01, respectively), but not with lesion volume or subarachnoid exposure of involved brain (p all >0.05), hospital morbidity (p all >0.05), nor with higher odds of unfavorable discharge or long-term outcomes for the cohort [OR = 0.9(CI 0.5-1.6), p = 0.72; OR = 1.0(CI 0.5-1.8), p = 0.9] or for neonates [OR = 1.3(CI 0.5-3.3), p = 0.57; OR = 2.3(CI 0.7-8), p = 0.2]., Conclusions: Initial HSV VL did not predict neuroradiological or clinical outcomes in patients with HSVE, suggesting host inflammatory factors contribute to disease in treated patients with good viral clearance., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Antiviral therapy can lead to drug resistance, but multiple factors determine the frequency of drug resistance mutations and the clinical consequences. When chronic infections caused by Human Immunodeficiency Virus (HIV), Hepatitis C Virus (HCV) and Hepatitis B Virus (HBV) are compared with acute infections such as influenza virus, respiratory syncytial virus (RSV), and other respiratory viruses, there are similarities in how and why antiviral resistance substitutions occur, but the clinical significance can be quite different. Emergence of resistant variants has implications for design of new therapeutics, treatment guidelines, clinical trial design, resistance monitoring, reporting, and interpretation. In this discussion paper, we consider the molecular factors contributing to antiviral drug resistance substitutions, and a comparison is made between chronic and acute infections. The implications of resistance are considered for clinical trial endpoints and public health, as well as the requirements for therapeutic monitoring in clinical practice with acute viral infections., (Copyright © 2018. Published by Elsevier B.V.)

Objective: To describe an adenovirus outbreak in a neonatal intensive care unit (NICU), including the use of qualitative and semiquantitative real-time polymerase chain reaction (qPCR) data to inform the outbreak response., Design: Mixed prospective and retrospective observational study., Setting: A level IV NICU in the southeastern United States.PatientsTwo adenovirus cases were identified in a NICU. Screening of all inpatients with qPCR on nasopharyngeal specimens revealed 11 additional cases.InterventionsOutbreak response procedures, including enhanced infection control policies, were instituted. Serial qPCR studies were used to screen for new infections among exposed infants and to monitor viral clearance among cases. Changes to retinopathy of prematurity (ROP) exam procedures were made after an association was noted in those patients. At the end of the outbreak, a retrospective review allowed for comparison of clinical factors between the infected and uninfected groups., Results: There were no new cases among patients after outbreak identification. One adenovirus-infected patient died; the others recovered their clinical baselines. The ROP exams were associated with an increased risk of infection (odds ratio [OR], 84.6; 95% confidence interval [CI], 4.5-1,601). The duration of the outbreak response was 33 days, and the previously described second wave of cases after the end of the outbreak did not occur. Revisions to infection control policies remained in effect following the outbreak., Conclusions: Retinopathy of prematurity exams are potential mechanisms of adenovirus transmission, and autoclaved or single-use instruments should be used to minimize this risk. Real-time molecular diagnostic and quantification data guided outbreak response procedures, which rapidly contained and fully terminated a NICU adenovirus outbreak.

Background: Respiratory symptoms in RSV persist long after the virus is no longer detected by culture. Current concepts of RSV pathogenesis explain this by RSV inducing a long-lasting pathogenic immune cascade. We alternatively hypothesized that prolonged unrecognized RSV replication may be responsible and studied this possibility directly in a human wild-type RSV experimental infection model., Objective: The objective of the current report was to define the duration of true human RSV replication by studying it directly in immunocompetent adults experimentally infected with a clinical strain of RSV utilizing this previously established safe and reproducible model., Study Design: 35 healthy adult volunteers were inoculated with RSV-A (Memphis-37, a low11 passage clinical strain virus, manufactured from a hospitalized bronchiolitic infant) and evaluated over 12 days. Viral load by culture, parallel quantitative PCR (genomic, message) and RSV-specific IgA, were measured twice daily from serially collected nasal washes., Results: After inoculation, 77% (27/35) of volunteers became RSV infected. As expected, culture-detectable RSV ceased abruptly by the 5-6 t h 15 infection day. However, infected volunteers demonstrated prolonged RSV presence by both genomic and message PCR. RSV-specific IgA rose within respiratory secretions of infected volunteers during same time frame., Conclusions: RSV replication appears to continue in humans far longer than previously thought. The rise in nasal RSV-specific IgA shortly after infection likely neutralizes culture detectable virus producing misleadingly short durations of infection. Prolonged viral replication helps explain RSV's extended disease manifestations and increases the potential utility of antivirals., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Angiostrongyliasis is caused by infection and migration to the brain of larvae of the parasitic nematode Angiostrongylus cantonensis, or rat lungworm. Adult A. cantonensis reside in the lungs of the definitive wild rodent host, where they produce larvae passed in feces, which are then ingested by snails and slugs (gastropods). Human infection typically occurs when gastropods containing mature larvae are inadvertently ingested by humans. Although human infection often is asymptomatic or involves transient mild symptoms, larval migration to the brain can lead to eosinophilic meningitis, focal neurologic deficits, coma, and death. The majority of cases of human angiostrongyliasis occur in Asia and the Pacific Islands, including Hawaii, but autochthonous and imported cases have been reported in the continental United States (1,2), underscoring the importance of provider recognition to ensure prompt identification and treatment. The epidemiologic and clinical features of 12 angiostrongyliasis cases in the continental United States were analyzed. These cases were identified through A. cantonensis polymerase chain reaction (PCR) testing (3) of cerebrospinal fluid (CSF) submitted to CDC from within the continental United States. Six cases were likely a result of autochthonous transmission in the southern United States. All 12 patients had CSF pleocytosis and eosinophilia, consistent with eosinophilic meningitis. Health care providers need to be aware of the possibility of angiostrongyliasis in patients with eosinophilic meningitis, especially in residents in the southern United States or persons who have traveled outside the continental United States and have a history of ingestion of gastropods or contaminated raw vegetables., Competing Interests: Haidee Custodio reports grants from Allergan for a multicenter study outside the submitted work. No other conflicts of interest were reported.

BackgroundLongitudinal respiratory syncytial virus (RSV) dynamics have not been well studied despite the existence of factors favoring prolonged RSV replication including high mutation rates allowing rapid evolution and potential escape from immune control. We therefore measured viral load in previously RSV-naive infants over prolonged time spans.MethodsDuring 2014-2015, quantitative nasal aspirates were collected from 51 RSV-PCR+ infants. Multiple parallel assessments of viral loads were quantified at each collected time point using a well-validated real-time quantitative reverse transcriptase polymerase chain reaction assay. After observing viral load rebound phenomenon in some infants, the viral dynamics of 27 infants with sufficient longitudinal viral load data points were analyzed using the pre-defined criteria for viral rebound. Additional analyses were performed comparing age with viral rebound, viral clearance rates, and viral load area-under-the-curve (AUC VL ).ResultsThe 51 infants (303 nasal aspirate samples; mean of 5.9 per patient) exhibited slower than expected viral clearance. Lower age trended toward slower viral clearance and greater AUC VL . Six infants had detectable viral loads ≥1 month after symptom onset. Ten of twenty-seven evaluable subjects exhibited viral rebound and this rebound was age-dependent (P=0.0259). All but one rebounder were <70 days old.ConclusionInfants struggle to control primary RSV infections allowing prolonged viral replication and previously undescribed viral rebound; likely representing viral mutational immune escape.

Angiostrongylus cantonensis, the rat lungworm, is the most common infectious cause of eosinophilic meningoencephalitis worldwide. This parasite is endemic to Southeast Asia and the Pacific Islands, and its global distribution is increasing. We report A. cantonensis meningoencephalitis in a 12-month-old boy in Tennessee, USA, who had not traveled outside of southwestern Tennessee or northwestern Mississippi.

Although respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection in infants and young children, attempts to develop an effective therapy have so far proved unsuccessful. Here we report the preclinical profiles of PC786, a potent nonnucleoside RSV L protein polymerase inhibitor, designed for inhalation treatment of RSV infection. PC786 demonstrated a potent and selective antiviral activity against laboratory-adapted or clinical isolates of RSV-A (50% inhibitory concentration [IC 50 ], <0.09 to 0.71 nM) and RSV-B (IC 50 , 1.3 to 50.6 nM), which were determined by inhibition of cytopathic effects in HEp-2 cells without causing detectable cytotoxicity. The underlying inhibition of virus replication was confirmed by PCR analysis. The effects of PC786 were largely unaffected by the multiplicity of infection (MOI) and were retained in the face of established RSV replication in a time-of-addition study. Persistent anti-RSV effects of PC786 were also demonstrated in human bronchial epithelial cells. In vivo intranasal once daily dosing with PC786 was able to reduce the virus load to undetectable levels in lung homogenates from RSV-infected mice and cotton rats. Treatment with escalating concentrations identified a dominant mutation in the L protein (Y1631H) in vitro In addition, PC786 potently inhibited RSV RNA-dependent RNA polymerase (RdRp) activity in a cell-free enzyme assay and minigenome assay in HEp-2 cells (IC 50 , 2.1 and 0.5 nM, respectively). Thus, PC786 was shown to be a potent anti-RSV agent via inhibition of RdRp activity, making topical treatment with this compound a novel potential therapy for the treatment of human RSV infections., (Copyright © 2017 Coates et al.)

Background: Respiratory syncytial virus (RSV) may cause fatal lower respiratory tract infection (LRTI) in immunocompromised patients. Ribavirin with or without standard intravenous immunoglobulin (IVIG) is frequently given although efficacy is debated. Infusion of IVIG with high levels of neutralizing antibody against RSV may offer benefit in these patients., Methods: RI-001 contains standardized levels of high-titer anti-RSV neutralizing antibody and was provided for compassionate use to 15 patients with RSV LRTI who either failed conventional therapy or had significant risk of progression. Patients were treated on day 1 with RI-001 1500 mg/kg, followed 2 days later with 750 mg/kg. Pre- and post-infusion sera were measured for RSV neutralizing antibody. Patient data were analyzed for safety related to infusion of RI-001, and clinical outcomes., Results: Patients ranged in age from 2 months to 71 years and 80% had hematologic malignancy or were bone marrow or hematopoietic stem cell transplant recipients. Administration was well tolerated. Pre-infusion neutralizing titers ranged from 51 to 1765 geometric mean titer (mean 646±519) and all patients demonstrated at least a 4-fold rise (mean 6410±4470) 5-10 days post infusion. Eleven of 15 improved and were discharged from the hospital. Days from positive RSV test to RI-001 treatment was shorter in survivors compared to non-survivors (4.4±2.8 vs. 20.3±21.0 days, P=.02)., Conclusion: Administration of RI-001 was well tolerated and resulted in significant increases in serum neutralizing antibody titers to RSV. Our data suggest that early identification of RSV and treatment with RI-001 may offer benefit., (© 2017 The Authors. Transplant Infectious Disease Published by John Wiley & Sons Ltd.)

Objective  SENTINEL1 characterized U.S. preterm infants 29 to 35 weeks' gestational age (wGA) < 12 months old hospitalized for laboratory-confirmed respiratory syncytial virus (RSV) disease and not receiving RSV immunoprophylaxis during the 2014 to 2015 RSV season. Study Design  This is a noninterventional, observational, cohort study. Results  A total of 702 infants were hospitalized with community-acquired RSV disease, of whom an estimated 42% were admitted to the intensive care unit (ICU) and 20% required invasive mechanical ventilation (IMV). Earlier gestational age and younger chronologic age were associated with an increased frequency of RSV-confirmed hospitalization (RSVH), ICU admission, and IMV. Among infants 29 to 32 wGA and < 3 months of age, 68% required ICU admission and 44% required IMV. One death occurred of an infant 29 wGA. Among the 212 infants enrolled for in-depth analysis of health care resource utilization, mean and median RSVH charges were $55,551 and $27,461, respectively, which varied by intensity of care required. Outpatient visits were common, with 63% and 62% of infants requiring visits before and within 1 month following the RSVH, respectively. Conclusion  Preterm infants 29 to 35 wGA are at high risk for severe RSV disease, which imposes a substantial health burden, particularly in the first months of life., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Respiratory syncytial virus (RSV) is the most common cause of US infant hospitalization. Additionally, RSV is responsible for 10,000 deaths annually among the elderly across the United States, and accounts for nearly as many hospitalizations as influenza. Currently, several RSV vaccine candidates are under development to target different age groups. To evaluate the potential effectiveness of age-specific vaccination strategies in averting RSV incidence, we developed a transmission model that integrates data on daily infectious viral load and changes of behavior associated with RSV symptoms. Calibrating to RSV weekly incidence rates in Texas, California, Colorado, and Pennsylvania, we show that in all states considered, an infected child under 5 y of age is more than twice as likely as a person over 50 y of age to transmit the virus. Geographic variability in the effectiveness of a vaccination program across states arises from interplay between seasonality patterns, population demography, vaccination uptake, and vaccine mechanism of action. Regardless of these variabilities, our analysis showed that allocating vaccine to children under 5 y of age would be the most efficient strategy per dose to avert RSV in both children and adults. Furthermore, due to substantial indirect protection, the targeting of children is even predicted to reduce RSV in the elderly more than directly vaccinating the elderly themselves. Our results can help inform ongoing clinical trials and future recommendations on RSV vaccination., Competing Interests: A.P.G. and D.Y. report consulting for Sanofi Pasteur regarding modeling of infectious diseases unrelated to RSV and for Merck regarding RSV and other infectious diseases.

Respiratory influenza virus infections represent a serious threat to human health. Underlying medical conditions and genetic make-up predispose some influenza patients to more severe forms of disease. To date, only a few studies have been performed in patients to correlate a selected group of cytokines and chemokines with influenza infection. Therefore, we evaluated the potential of a novel multiplex micro-proteomics technology, SOMAscan, to quantify proteins in the respiratory mucosa of influenza A and B infected individuals. The analysis included but was not limited to quantification of cytokines and chemokines detected in previous studies. SOMAscan quantified more than 1,000 secreted proteins in small nasal wash volumes from infected and healthy individuals. Our results illustrate the utility of micro-proteomic technology for analysis of proteins in small volumes of respiratory mucosal samples. Furthermore, when we compared nasal wash samples from influenza-infected patients with viral load ≥ 2(8) and increased IL-6 and CXCL10 to healthy controls, we identified 162 differentially-expressed proteins between the two groups. This number greatly exceeds the number of DEPs identified in previous studies in human influenza patients. Most of the identified proteins were associated with the host immune response to infection, and changes in protein levels of 151 of the DEPs were significantly correlated with viral load. Most important, SOMAscan identified differentially expressed proteins heretofore not associated with respiratory influenza infection in humans. Our study is the first report for the use of SOMAscan to screen nasal secretions. It establishes a precedent for micro-proteomic quantification of proteins that reflect ongoing response to respiratory infection.

In July 2014, the Committee on Infectious Diseases (COID) updated their guidance on the use of palivizumab, recommending against use in preterm infants 29 to 35 weeks' gestational age (wGA). A primary data source cited to support this significant change was the low respiratory syncytial virus (RSV) hospitalization rate observed in the subpopulation of preterm (<37 wGA) infants evaluated from 2000 to 2005 through the New Vaccine Surveillance Network (NVSN). Here we critically appraise the preterm infant data from the NVSN in the context of data regarding the use of palivizumab in this same time period. Data from the NVSN, an analysis of Florida Medicaid data, and a national survey of US in-hospital palivizumab administration demonstrated that during 2001 to 2007, palivizumab was administered to 59% to 83% of preterm infants born at <32 wGA and 21% to 27% of all preterm infants (<37 wGA). When the NVSN data regarding incidence of RSV hospitalization in preterm infant subgroups were evaluated as a function of chronologic age, preterm infants <32 wGA showed a paradoxical increase in RSV hospitalization with older age, with the highest risk of RSV hospitalization occurring at 18 to 23 months of age. This pattern is most consistent with a reduction in RSV hospitalizations in <32 wGA infants in the first 12 to 18 months of life due to high palivizumab use at these young ages. The NVSN data were not designed to and cannot accurately describe RSV disease burden in preterm infants given the small size of the analyzed subpopulation and the high use of palivizumab during the study period.

We studied preexisting respiratory syncytial virus (RSV)-specific serum and nasal antibodies and their correlation with infectivity, viral dynamics, and disease severity in a human experimental infection model. Higher preinoculation serum neutralizing antibody titers and nasal immunoglobulin (Ig) A predicted lower infectivity and lower measures of viral replication. However, once individuals were infected, no significant protective effect of preexisting antibodies was seen. Lack of correlation between serum and mucosal antibodies was observed, implying that they are independent co-correlates of protection against RSV infection. We suggest that protection from RSV infection is a function of a complex interplay between mucosal and serum humoral immune responses., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

BACKGROUND Respiratory syncytial virus (RSV) infection is a cause of substantial morbidity and mortality. There is no known effective therapy. METHODS We conducted a randomized, double-blind, clinical trial in healthy adults inoculated with RSV. Participants received the oral nucleoside analogue ALS-008176 or placebo 12 hours after confirmation of RSV infection or 6 days after inoculation. Treatment was administered every 12 hours for 5 days. Viral load, disease severity, resistance, and safety were measured throughout the 28-day study period, with measurement beginning before inoculation. The primary end point was the area under the curve (AUC) for viral load, which was assessed immediately before administration of the first dose through the 12th day after inoculation in participants infected with RSV. RESULTS A total of 62 participants received placebo or one of three ALS-008176 dosing regimens: 1 loading dose of 750 mg followed by 9 maintenance doses of 500 mg (group 1), 1 loading dose of 750 mg followed by 9 maintenance doses of 150 mg (group 2), or 10 doses of 375 mg (group 3). In the 35 infected participants (23 of whom were treated with ALS-008176), the AUCs for viral load for groups 1, 2, and 3 and the placebo group were 59.9, 73.7, 133.4, and 500.9 log10 plaque-forming-unit equivalents × hours per milliliter, respectively (P≤0.001). The time to nondetectability on polymerase-chain-reaction assay (P<0.001), the peak viral load (P≤0.001), the AUC for symptom score (P<0.05), and the AUC for mucus weight were lower in all groups receiving ALS-008176 than in the placebo group. Antiviral activity was greatest in the two groups that received a loading dose--viral clearance was accelerated (P≤0.05), and the AUC for viral load decreased by 85 to 88% as compared with the placebo group. Within this small trial, no viral rebound or resistance was identified. There were no serious adverse events, and there was no need for premature discontinuation of the study drug. CONCLUSIONS In this RSV challenge study, more rapid RSV clearance and a greater reduction of viral load, with accompanying improvements in the severity of clinical disease, were observed in the groups treated with ALS-008176 than in the placebo group. (Funded by Alios BioPharma; ClinicalTrials.gov number, NCT02094365.).

Respiratory syncytial virus (RSV) is the most common cause of infant hospitalizations and severe RSV infections are a significant risk factor for childhood asthma. The pathogenic mechanisms responsible for RSV induced immunopathophysiology remain elusive. Using an age-appropriate mouse model of RSV, we show that IL-33 plays a critical role in the immunopathogenesis of severe RSV, which is associated with higher group 2 innate lymphoid cells (ILC2s) specifically in neonates. Infection with RSV induced rapid IL-33 expression and an increase in ILC2 numbers in the lungs of neonatal mice; this was not observed in adult mice. Blocking IL-33 with antibodies or using an IL-33 receptor knockout mouse during infection was sufficient to inhibit RSV immunopathogenesis (i.e., airway hyperresponsiveness, Th2 inflammation, eosinophilia, and mucus hyperproduction); whereas administration of IL-33 to adult mice during RSV infection was sufficient to induce RSV disease. Additionally, elevated IL-33 and IL-13 were observed in nasal aspirates from infants hospitalized with RSV; these cytokines declined during convalescence. In summary, IL-33 is necessary, either directly or indirectly, to induce ILC2s and the Th2 biased immunopathophysiology observed following neonatal RSV infection. This study provides a mechanism involving IL-33 and ILC2s in RSV mediated human asthma.

Background: Viral culture plaque morphology in human cell lines are markers for growth capability and cytopathic effect, and have been used to assess viral fitness and select preattenuation candidates for live viral vaccines. We classified respiratory syncytial virus (RSV) plaque morphology and analyzed the relationship between plaque morphology as compared to subgroup, viral load and clinical severity of infection in infants and children., Methods: We obtained respiratory secretions from 149 RSV-infected children. Plaque morphology and viral load was assessed within the first culture passage in HEp-2 cells. Viral load was measured by polymerase chain reaction (PCR), as was RSV subgroup. Disease severity was determined by hospitalization, length of stay, intensive care requirement, and respiratory failure., Results: Plaque morphology varied between individual subjects; however, similar results were observed among viruses collected from upper and lower respiratory tracts of the same subject. Significant differences in plaque morphology were observed between RSV subgroups. No correlations were found among plaque morphology and viral load. Plaque morphology did not correlate with disease severity., Conclusion: Plaque morphology measures parameters that are viral-specific and independent of the human host. Morphologies vary between patients and are related to RSV subgroup. In HEp-2 cells, RSV plaque morphology appears unrelated to disease severity in RSV-infected children.

Background: There is a wide range of severity of respiratory syncytial viral (RSV) disease in previously healthy infants. Host factors have been well demonstrated to contribute to disease severity differences. However the possibility of disease severity differences being produced by factors intrinsic to the virus itself has rarely been studied., Methods: Low-passage isolates of RSV collected prospectively from infants with different degrees of RSV disease severity were evaluated in vitro , holding host factors constant, so as to assess whether isolates induced phenotypically different cytokine/chemokine concentrations in a human lung epithelial cell line. Sixty-seven RSV isolates from previously healthy infants (38 hospitalized for acute RSV infection (severe disease) and 29 never requiring hospitalization (mild disease)) were inoculated into A549, lung epithelial cells at precisely controlled, low multiplicity of infection to mimic natural infection. Cultures were evaluated at 48 hours, 60 hours, and 72 hours to evaluate area under the curve (AUC) cytokine/chemokine induction., Results: Cells infected with isolates from severely ill infants produced higher mean concentrations of all cytokine/chemokines tested (IL-1α, IL-6, IL-8 and RANTES) at all-time points tested. RSV isolates collected from infants with severe disease induced significantly higher AUCIL-8 and AUCRANTES secretion in infected cultures than mild disease isolates (p=0.028 and p=0.019 respectively). IL-8 and RANTES concentrations were 4 times higher at 48 hours for these severely ill infant isolates. Additionally, 38 isolates were evaluated at all-time points for quantity of virus. RSV concentration significantly correlated with both IL-8 and RANTES at all-time points. Neither cytokine/chemokine concentrations nor RSV concentrations were associated with RSV subgroup., Discussion: Infants' RSV disease severity differences may be due in part to intrinsic viral strain-specific characteristics.

Rationale: Despite relative antigenic stability, respiratory syncytial virus (RSV) reinfects throughout life. After more than 40 years of research, no effective human vaccine exists and correlates of protection remain poorly defined. Most current vaccine candidates seek to induce high levels of RSV-specific serum neutralizing antibodies, which are associated with reduced RSV-related hospitalization rates in observational studies but may not actually prevent infection., Objectives: To characterize correlates of protection from infection and the generation of RSV-specific humoral memory to promote effective vaccine development., Methods: We inoculated 61 healthy adults with live RSV and studied protection from infection by serum and mucosal antibody. We analyzed RSV-specific peripheral blood plasmablast and memory B-cell frequencies and antibody longevity., Measurements and Main Results: Despite moderately high levels of preexisting serum antibody, 34 (56%) became infected, of whom 23 (68%) developed symptomatic colds. Prior RSV-specific nasal IgA correlated significantly more strongly with protection from polymerase chain reaction-confirmed infection than serum neutralizing antibody. Increases in virus-specific antibody titers were variable and transient in infected subjects but correlated with plasmablasts that peaked around Day 10. During convalescence, only IgG (and no IgA) RSV-specific memory B cells were detectable in peripheral blood. This contrasted with natural influenza infection, in which virus-specific IgA memory B cells were readily recovered., Conclusions: This observed specific defect in IgA memory may partly explain the ability of RSV to cause recurrent symptomatic infections. If so, vaccines able to induce durable RSV-specific IgA responses may be more protective than those generating systemic antibody alone.

Infants and young children with acute onset of wheezing and reduced respiratory airflows are often diagnosed with obstruction and inflammation of the small bronchiolar airways, ie bronchiolitis. The most common aetological agents causing bronchiolitis in young children are the respiratory viruses, and of the commonly encountered respiratory viruses, respiratory syncytial virus (RSV) has a propensity for causing bronchiolitis. Indeed, RSV bronchiolitis remains the major reason why previously healthy infants are admitted to hospital. Why RSV infection is such a predominant cause of bronchiolitis is the subject of this review. By reviewing the available histopathology of RSV bronchiolitis, both in humans and relevant animal models, we identify hallmark features of RSV infection of the distal airways and focus attention on the consequences of columnar cell cytopathology occurring in the bronchioles, which directly impacts the development of bronchiolar obstruction, inflammation and disease., (Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children and is responsible for as many as 199,000 childhood deaths annually worldwide. To support the development of viral therapeutics and vaccines for RSV, a human adult experimental infection model has been established. In this report, we describe the provenance and sequence of RSV Memphis-37, the low-passage clinical isolate used for the model's reproducible, safe, experimental infections of healthy, adult volunteers. The predicted amino acid sequences for major proteins of Memphis-37 are compared to nine other RSV A and B amino acid sequences to examine sites of vaccine, therapeutic, and pathophysiologic interest. Human T- cell epitope sequences previously defined by in vitro studies were observed to be closely matched between Memphis-37 and the laboratory strain RSV A2. Memphis-37 sequences provide baseline data with which to assess: (i) virus heterogeneity that may be evident following virus infection/transmission, (ii) the efficacy of candidate RSV vaccines and therapeutics in the experimental infection model, and (iii) the potential emergence of escape mutants as a consequence of experimental drug treatments. Memphis-37 is a valuable tool for pre-clinical research, and to expedite the clinical development of vaccines, therapeutic immunomodulatory agents, and other antiviral drug strategies for the protection of vulnerable populations against RSV disease.

Background: Respiratory 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., Methods: We 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., Results: Among 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., Conclusions: Treatment 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.).

Unlabelled: Respiratory syncytial virus (RSV) infection is the number one cause of bronchiolitis in infants, yet no vaccines are available because of a lack of knowledge of the infant immune system. Using a neonatal mouse model, we previously revealed that mice initially infected with RSV as neonates develop Th2-biased immunopathophysiologies during reinfection, and we demonstrated a role for enhanced interleukin-4 receptor α (IL-4Rα) expression on T helper cells in these responses. Here we show that RSV infection in neonates induced limited type I interferon (IFN) and plasmacytoid dendritic cell (pDC) responses. IFN alpha (IFN-α) treatment or adoptive transfer of adult pDCs capable of inducing IFN-α prior to neonatal RSV infection decreased Th2-biased immunopathogenesis during reinfection. A reduced viral load and downregulation of IL-4Rα on Th2 cells were observed in IFN-α-treated neonatal mice, suggesting dual mechanisms of action., Importance: Respiratory syncytial virus (RSV) is the most significant cause of lower respiratory tract infection in infancy worldwide. Despite the dire need, we have failed to produce efficacious RSV vaccines or therapeutics. Part of the reason for this failure is our lack of understanding of how RSV interacts with the infant immune system to suppress the development of protective immunity. In the study described in the present paper, we used a neonatal mouse model, which more closely mimics human infants, to study the role of the innate immune system, particularly type I interferons (IFNs) and plasmacytoid dendritic cells (pDCs), in the pathogenesis of RSV infection. RSV infection in neonates induced limited type I IFN and pDC responses. IFN-α treatment or adoptive transfer of adult pDCs capable of producing IFN-α prior to neonatal RSV infection decreased Th2-biased immunopathogenesis during reinfection. These data suggest that IFN-α is a promising target for future RSV vaccine design., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: Adenoviruses (AdV) cause a variety of upper and lower respiratory tract infections, with the potential for severe outcomes, especially in persons with immune suppression or other underlying diseases. The ADENOVIRUS US R-gene (AdV R-gene, Argene/bioMérieux) is a FDA cleared real-time PCR assay that utilizes primers and fluorescent probes that target a conserved region of the hexon gene and an internal control DNA., Objectives: This prospective multi-center study evaluated the clinical performance of AdV R-gene for AdV detection in respiratory specimens from symptomatic patients of all ages., Study Design: Nucleic acids from nasopharyngeal washes/aspirates (NPW/A; n=393) and NP flocked swabs (NPS, Copan) (n=1183) were extracted using NucliSENS easyMAG (bioMérieux) and AdV R-gene PCR was performed using the SmartCycler (Cepheid). AdV R-gene results were compared to R-Mix culture (Quidel/Diagnostic Hybrids). For a subset of samples (n=946) AdV R-gene and R-Mix results were also compared to A549 cell culture., Results: In first intention analysis for NPS the AdV R-gene positive percent agreement (PPA), and negative percent agreement (NPA) were 91.7% and 96.2%, respectively, and for NPW/A were 100% and 94.4%, respectively, compared to R-Mix culture. In second intention analysis, discordant samples only were tested with an AdV real-time PCR assay (Viracor-IBT Labs) and amplicon sequencing. For NPS, the sensitivity, specificity, PPV and NPV for AdV R-gene were 98.9%, 100%, 100%, and 99.9%, respectively and for R-Mix culture were 51.7%, 99.7%, 93.8%, and 96.3%, respectively. For NPW/A, the sensitivity, specificity, PPV and NPV for AdV R-gene were 100%, 99.7%, 97.6%, and 100%, respectively, and for R-Mix culture were 52.5%, 100%, 100%, and 94.9%, respectively. Overall, AdV was detected by AdV R-gene and R-Mix in 7.4% and 4.1% NPS, respectively, and in 10.7% and 5.3% NPW/A, respectively. Children 5yr and younger had the highest rates of AdV infections. In a subset of specimens (n=946) the sensitivity of AdV R-gene, R-Mix, and A549 cell culture were 95.0%, 55.4% and 66.3%., Conclusions: AdV R-gene is sensitive and specific for the detection of AdV in NPW/A and NPS samples. AdV R-gene is simple to use and provides a rapid time to results (within 2.5-3h)., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Respiratory syncytial virus (RSV) infection is the major cause of bronchiolitis in young children. The factors that contribute to the increased propensity of RSV-induced distal airway disease compared with other commonly encountered respiratory viruses remain unclear. Here, we identified the RSV-encoded nonstructural 2 (NS2) protein as a viral genetic determinant for initiating RSV-induced distal airway obstruction. Infection of human cartilaginous airway epithelium (HAE) and a hamster model of disease with recombinant respiratory viruses revealed that NS2 promotes shedding of infected epithelial cells, resulting in two consequences of virus infection. First, epithelial cell shedding accelerated the reduction of virus titers, presumably by clearing virus-infected cells from airway mucosa. Second, epithelial cells shedding into the narrow-diameter bronchiolar airway lumens resulted in rapid accumulation of detached, pleomorphic epithelial cells, leading to acute distal airway obstruction. Together, these data indicate that RSV infection of the airway epithelium, via the action of NS2, promotes epithelial cell shedding, which not only accelerates viral clearance but also contributes to acute obstruction of the distal airways. Our results identify RSV NS2 as a contributing factor for the enhanced propensity of RSV to cause severe airway disease in young children and suggest NS2 as a potential therapeutic target for reducing the severity of distal airway disease.

Rationale: Children are an at-risk population for developing complications following influenza infection, but immunologic correlates of disease severity are not understood. We hypothesized that innate cellular immune responses at the site of infection would correlate with disease outcome., Objectives: To test the immunologic basis of severe illness during natural influenza virus infection of children and adults at the site of infection., Methods: An observational cohort study with longitudinal sampling of peripheral and mucosal sites in 84 naturally influenza-infected individuals, including infants. Cellular responses, viral loads, and cytokines were quantified from nasal lavages and blood, and correlated to clinical severity., Measurements and Main Results: We show for the first time that although viral loads in children and adults were similar, innate responses in the airways were stronger in children and varied considerably between plasma and site of infection. Adjusting for age and viral load, an innate immune profile characterized by increased nasal lavage monocyte chemotactic protein-3, IFN-α2, and plasma IL-10 levels at enrollment predicted progression to severe disease. Increased plasma IL-10, monocyte chemotactic protein-3, and IL-6 levels predicted hospitalization. This inflammatory cytokine production correlated significantly with monocyte localization from the blood to the site of infection, with conventional monocytes positively correlating with inflammation. Increased frequencies of CD14(lo) monocytes were in the airways of participants with lower inflammatory cytokine levels., Conclusions: An innate profile was identified that correlated with disease progression independent of viral dynamics and age. The airways and blood displayed dramatically different immune profiles emphasizing the importance of cellular migration and localized immune phenotypes.

Experimental and computational evidence suggests that HLAs preferentially bind conserved regions of viral proteins, a concept we term "targeting efficiency," and that this preference may provide improved clearance of infection in several viral systems. To test this hypothesis, T-cell responses to A/H1N1 (2009) were measured from peripheral blood mononuclear cells obtained from a household cohort study performed during the 2009-2010 influenza season. We found that HLA targeting efficiency scores significantly correlated with IFN-γ enzyme-linked immunosorbent spot responses (P = 0.042, multiple regression). A further population-based analysis found that the carriage frequencies of the alleles with the lowest targeting efficiencies, A*24, were associated with pH1N1 mortality (r = 0.37, P = 0.031) and are common in certain indigenous populations in which increased pH1N1 morbidity has been reported. HLA efficiency scores and HLA use are associated with CD8 T-cell magnitude in humans after influenza infection. The computational tools used in this study may be useful predictors of potential morbidity and identify immunologic differences of new variant influenza strains more accurately than evolutionary sequence comparisons. Population-based studies of the relative frequency of these alleles in severe vs. mild influenza cases might advance clinical practices for severe H1N1 infections among genetically susceptible populations.

Pertussis is an under-recognized serious infection. Conventional cultures are insensitive and of limited utility after antibiotic exposure. We corroborated the utility of real-time polymerase chain reaction (PCR) as a diagnostic tool in pertussis and investigated its role as a prognostic tool by evaluating its benefit in the quantification of pertussis bacterial load. All pertussis-positive PCR tests (n = 104) submitted over 5 years were collected for retrospective study. PCR cycle threshold was compared to quantitative culture in 43. Compared to PCR, the sensitivity of culture was 41%. Our PCR assay reliably quantified bacterial load and was quantitatively reproducible. Higher bacterial load correlated with longer duration of hospitalization (P = 0.0003), and multivariate logistic regression models demonstrated this association to be independent. The study confirmed PCR as a superior diagnostic tool in pertussis. PCR quantification of bacterial load at initial diagnosis predicts later clinical disease severity, suggesting a potential benefit of PCR as a prognostic tool in pertussis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

RNA viruses are the causative agents for AIDS, influenza, SARS, and other serious health threats. Development of rapid and broadly applicable methods for complete viral genome sequencing is highly desirable to fully understand all aspects of these infectious agents as well as for surveillance of viral pandemic threats and emerging pathogens. However, traditional viral detection methods rely on prior sequence or antigen knowledge. In this study, we describe sequence-independent amplification for samples containing ultra-low amounts of viral RNA coupled with Illumina sequencing and de novo assembly optimized for viral genomes. With 5 million reads, we capture 96 to 100% of the viral protein coding region of HIV, respiratory syncytial and West Nile viral samples from as little as 100 copies of viral RNA. The methods presented here are scalable to large numbers of samples and capable of generating full or near full length viral genomes from clone and clinical samples with low amounts of viral RNA, without prior sequence information and in the presence of substantial host contamination.

Background: Antivirals reduce influenza viral replication and illness measures, particularly if initiated early, within 48 h of symptom onset. Whether experimental antivirals that reduce respiratory syncytial virus (RSV) load would also reduce disease is unknown. This study compares viral and disease dynamics in humans experimentally infected with influenza or RSV., Methods: Clinical strains of RSV-A and influenza A were inoculated intranasally into 20 and 17 healthy volunteers, respectively, on day 0. Symptom scores and nasal washes were performed twice daily, and daily mucus weights were collected. Viral loads in nasal washes were quantified by culture (plaque assay in HEp-2 cells for RSV and by end point dilution in Madin-Darby canine kidney cells for influenza)., Results: After influenza inoculation, influenza viral load and illness markers increased simultaneously until day 2. Within individual subjects, peak influenza load occurred 0.4 days (95% CI -0.4, 1.3) before peak symptoms. Influenza viral load and disease declined thereafter. After RSV inoculation, a longer incubation period occurred prior to viral detection and symptom onset. RSV load and disease increased together until day 5. Within individual subjects, peak RSV loads occurred 0.2 days (95% CI -0.7, 1.05) before peak symptoms, after which both illness measures and viral load declined together., Conclusions: Viral and disease dynamics in experimental human infections suggest that reducing RSV load, if timed similarly to clinically-effective influenza antivirals, might be expected to have a similar or greater window of opportunity for reducing clinical RSV disease.

Advances in the understanding of RNA biological processing and control are leading to new concepts in human therapeutics with practical implications for many human diseases, including antiviral therapy of respiratory viruses. So-called 'non-coding RNA' exerts specific and profound functional control on regulation of protein production and indeed controls the expression of all genes through processes collectively known as RNA interference (RNAi). RNAi is a naturally occurring intracellular process that regulates gene expression through the silencing of specific messenger RNAs (mRNAs). Methods are being developed that allow the catalytic degradation of targeted mRNAs using specifically designed complementary small interfering RNAs (siRNA). siRNAs are now being chemically modified and packaged into advanced delivery systems so as to acquire drug-like properties and the ability to deliver their effects systemically. Recent in vivo studies have provided proofs of the concept that RNAi may be useful therapeutically. Much of the design of these siRNAs can be accomplished bioinformatically, thus potentially expediting drug discovery and opening new avenues of therapy for many uncommon, orphan, or emerging diseases. Theoretically, any disease that can be ameliorated through knockdown of any endogenous or exogenous protein is a potential therapeutic target for RNAi-based therapeutics. Lung diseases in general are attractive targets for RNAi therapeutics, since the location of affected cells increases their accessibility to topical administration of siRNA, and respiratory viral infections are particularly attractive targets for RNAi-based drug discovery and development. RNAi therapeutics have been shown to exert potent antiviral effects against respiratory syncytial virus (RSV), parainfluenza, influenza, coronaviruses, measles and human metapneumoviruses in vitro and in vivo. Recently, a double-blind placebo-controlled clinical trial of an RNAi-based therapeutic against RSV demonstrated that this technology has therapeutic activity, representing the first proof-of-concept test of efficacy for RNAi's therapeutic effect in humans. This review discusses the science behind RNAi and the potential practical issues in applying this technology to various respiratory viral diseases.

Background. New aerosol drugs for infants may require more efficient delivery systems, including face masks. Maximizing delivery efficiency requires tight-fitting masks with minimal internal mask volumes, which could cause carbon dioxide (CO(2)) retention. An RNA-interference-based antiviral for treatment of respiratory syncytial virus in populations that may include young children is designed for aerosol administration. CO(2) accumulation within inhalation face masks has not been evaluated. Methods. We simulated airflow and CO(2) concentrations accumulating over time within a new facemask designed for infants and young children (PARI SMARTMASK(®) Baby). A one-dimensional model was first examined, followed by 3-dimensional unsteady computational fluid dynamics analyses. Normal infant breathing patterns and respiratory distress were simulated. Results. The maximum average modeled CO(2) concentration within the mask reached steady state (3.2% and 3% for normal and distressed breathing patterns resp.) after approximately the 5th respiratory cycle. After steady state, the mean CO(2) concentration inspired into the nostril was 2.24% and 2.26% for normal and distressed breathing patterns, respectively. Conclusion. The mask is predicted to cause minimal CO(2) retention and rebreathing. Infants with normal and distressed breathing should tolerate the mask intermittently delivering aerosols over brief time frames.

Background: Respiratory syncytial virus (RSV) disease severity was thought to be a result of host immunopathology but alternatively may be driven by high-level viral replication. The relationships between RSV load, viral clearance dynamics, and disease severity have not been carefully evaluated., Methods: Previously healthy RSV-infected children <2 years old were recruited. RSV load was measured in respiratory secretions by fresh quantitative culture over 3 hospital days. Measures of disease severity were hospital admission, duration of hospitalization, requirement for intensive care, and respiratory failure., Results: Multivariate logistic regression models revealed independent predictors of increased duration of hospitalization: male sex, lower weight, and higher viral load on any day. Viral loads at day 3 were more significantly associated with requirement for intensive care and respiratory failure than were viral loads at earlier time points. Faster RSV clearance was independently associated with shorter hospitalization., Discussion: These observations challenge the immunopathology-based pathogenesis paradigm. They also have major therapeutic implications, suggesting that application of antiviral agents early in the disease course, even at a time when viral replication is at its highest, might improve subsequent morbidity by significantly lowering viral load and direct viral cytopathic effects, and aborting the potential downstream immunopathology.